Entomologia Experimentalis et Applicata 104: 51–60, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Insect feeding deterrent activity of lignans and related phenylpropanoidswith a methylenedioxyphenyl (piperonyl) structure moiety

Juraj Harmatha1 & Jan Nawrot21Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo nam. 2, 166 10 Prague,Czech Republic; 2Institute of Plant Protection, Miczurina 20, 60-318 Poznañ, Poland

Accepted: April 11, 2002

Key words: Sitophilus granarius, Tribolium confusum, Trogoderma granarium, insect feeding deterrents, an-tifeedants, lignans, phenylpropanoids, phenolics, yatein, hinokinin, cubebin, podophyllotoxin, piperonylbutoxide

Abstract

The antifeedant activity of a series of lignan lactones, hemiacetals, ethers, and alcohols derived from yatein andcubebin, together with structurally related phenylpropanoids and phenolics possessing a methylenedioxyphenyl(piperonyl) moiety, was tested against selected stored products pests: Sitophilus granarius L. (Coleoptera: Cur-culionidae), Tribolium confusum Duv. (Coleoptera: Tenebrionideae), and Trogoderma granarium Ev. (Coleoptera:Dermestridae). The relation between molecular structure and antifeedant activity was examined and implicationof the piperonyl moiety is assessed. The compounds represent either natural substances isolated from plants (Li-bocedrus yateensis Guillaumin and Piper cubeba L.) or their structural analogues prepared by simple chemicaltransformations as well as compounds selected from commercially available sources. Natural lignan lactones withmethoxy and/or methylenedioxy substituents showed significant activity that is strong enough to affect plant -insect interactions. Presence of polar substituents, especially hydroxy or glycosyl groups, often reduce the activity.Non-polar substituents, such as methoxy or methylenedioxy groups, enhance the activity not only in lignans butalso in simple phenylpropanoids. The most active compound was synthetic piperonylbutoxide.

Introduction

Natural lignans belong to an immense chemical familyof biogenetically related compounds formed by oxida-tive dimerisation of two characteristic and variouslysubstituted phenylpropanoid units linked by the cen-tral carbon atoms of their propane side chains (C-8,C-8′ in Figure 1). All other linkages form structurescalled neolignans (see Whiting, 1985; Ayres & Loike,1990). Further transformations associated with thebiosynthesis can extend the structural variability byforming further related types, such as norlignans orconioids (Erdtman & Harmatha, 1979). Biosynthesisthat involves a combination of compounds of differentbiogenetic origin can form hybrid lignans or lignoids(Whiting, 1987).

The two phenylpropanoid units linked in lignansby the side chain C-8, C-8′ carbon atoms (Figure 1)can form a limited number of structural types (A

- C): one linear type A (dibenzylbutane type) andtwo cyclic types, namely, B (aryltetralin type, syn.2,7′-cyclolignan type) and C (dibenzocyclooctan type,syn. 2,2′-cyclolignan type). Further oxidation of thepropane chains, together with additional oxygen ringformation can lead to several hydroxy-, oxy-, or oxo-forms of each of the types A - C. They are: I (butaneform, i.e., non-oxidised form), II (butanediol form, orhydroxy form), III (oxy-butane form, syn. 9,9′-epoxyform, or cyclic ether form), IV (hemiacetal form, syn.‘lactol’ form), V (butyrolactone form, syn. butanolideform), VI (bis-epoxy form, syn. bis-perhydrofurane orfurofurane form), VII (7,9′ or 7′,9 -epoxy form), andVIII (7,7′-epoxy form). It is obvious that the formsVI - VIII can be derived only from the type A; formVI by 7,9′ and 7′,9 bis epoxidation, forms VII andVIII by a mono epoxidation of respective 7,9′ or 7′,9or 7,7′ positions. Forms VI - VIII, because of theirstructural specificity, can be regarded as independent

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Figure 1. Types and forms of lignans. Types (A–C) are derived from the coupling mode and degree at the two phenylpropane units. A:dibenzylbutane type, B: aryltetralin (or 2,7′-cyclolignan) type, and C: dibenzocyclooctane (or 2,2′-cyclolignan) type. Forms I–V reflect theoxidation degree at positions 9 and 9′ feasible at all three types (A–C); I: butane form, II: butanediol form, III: oxy-butane form, IV: hemiacetalform, and V: butyrolactone form. Forms VI–VIII reflect the mode of oxy- ring formation at the propane moieties, feasible only at type A; VI:bis-epoxy form, VII: 7,9′ or 7′,9 -epoxy form, and VIII: 7,7′-epoxy form. Forms VI - VIII are regarded as independent structural types D - F,respectively.

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structural types (D - F). Further structural variability ispossible by the addition of one or two double bonds inthe butane moiety and by various substituents (mostlyhydroxy, methoxy, methylenedioxy, or glycosyl) onone or on both aromatic rings and/or aliphatic carbonatoms.

Lignans have attracted much interest over the yearsbecause of their widespread occurrence in nature,mainly in the plant kingdom, and because of theirbroad range of biological effects (MacRae & Tow-ers, 1984). Neolignans and lignoids, although morediverse in structure, are less numerous than lignansand are obviously more specific to certain plant fam-ilies. Much is known about the effects of lignans ondifferent organisms, including man, at the molecu-lar, enzymatic, physiological, pharmacological, andeven clinical level. However, not so much is knownabout their role and function in the plants producingthem. The biogenetic relation of lignans to lignin isof considerable interest. The same phenylpropanoidprecursors serve as basic units in the biosynthesis ofboth lignans and lignin and they are present in all or-gans of the plant, as well as in plant tissue cultures.The biogenetic origin and the widespread distributionof lignans throughout many plant families suggest thatthey play an important role in plant evolution. At theecological level, they play an important role in theinteraction of plants with other organisms (mainly be-cause of their defence function) and in protection ofplants against physical damage (Hartmann, 1996).

Information concerning effects of lignans on in-sects and their role in plant - insect interactions arestill limited (MacRae & Towers, 1984; Harmatha &Nawrot, 1988; Ayres & Loike, 1990; Bernard et al.,1995). The first reports about lignan activity againstinsects in 1942 concern synergistic action of sesaminand related compounds, asarinin and pinoresinol (allof type D in Figure 1) with pyrethrum insecticides(Haller et al., 1942a,b). The proposal to use sesameoil as a synergist or activator combined with insecti-cides is even older (Eagleson, 1940). More than twodecades later a series of research results on synergisticactivity of dibenzylbutyrolactone type of lignans (typeA,V; Figure 1) were published (Yamashita & Matsui,1961; Matsubara, 1972). Insecticide synergists of thesimple phenylpropanoid type (Lichtenstein & Casida,1963) as well as the lignan type have also been de-scribed and reviewed by Casida (1970). In that review,the mode of synergistic action was discussed at thebiochemical level, concerning synergism specificityand resistance mechanisms. Although most structural

types were described as synergists, they were indi-vidually inactive. Other lignans, structurally relatedto podophyllotoxin (type B,V) have been describedas insecticides (Kozawa et al., 1982). Also lignansof dibenzylbutyrolactone type (type A,V) have beendescribed as feeding inhibitors (Wada & Munakata,1970), feeding deterrents (Harmatha & Nawrot, 1984,1988; Nawrot et al., 1986a,b, 1991; Krecková et al.,1988; Bloszyk et al., 1990; Harmatha et al., 1991), orlarval growth inhibitors (Russell et al., 1976). Some ofthe lignans were tested for juvenile hormone activity,but they were nearly inactive (Bowers, 1968). Certainstructural features, such as the methylenedioxyphenyl(piperonyl) moiety of many lignans became of spe-cific interest and were used as models for selectingnatural (Neil, 1989; Bernard et al., 1995) or design-ing synthetic (Yamauchi & Taniguchi, 1991, 1992a,b)insecticides or insecticide synergists. Also, bioassay-guided isolations afforded new lignans with insectgrowth inhibitory effects (Miyazawa et al., 1994). Theactivity of lignans and related phenylpropanoids hasbeen investigated also at the enzymatic level (Casida,1970; Bernard et al., 1989).

In this paper, we present results of testing ninedibenzylbutane type lignans (type A, varied in forms II- V) for their antifeedant activity, tested against a num-ber of stored products insects. They are directly com-pared with podophyllotoxin, an aryltetraline (B type)lignan and with structurally related phenylpropanoids.

Materials and methods

Insects. Three species of stored products insect pestswere selected for testing: adults of grain weevil beetle(Sitophilus granarius L.), adults and larvae of con-fused flour beetle (Tribolium confusum Duv.), andlarvae of khapra beetle (Trogoderma granarium Ev.).They came from laboratory colonies that were rearedin a chamber maintained at 26 ± 1 ◦C and 60 ± 5% r.h. on wheat grain or whole-wheat meal diet.

Feeding deterrent activity test. The laboratory as-say was conducted according to the method developedand standardised for storage insects feeding activity(Nawrot et al., 1986a): Wheat wafer discs (1 cm indiameter) were used as food and as a test substrate.The discs were dipped in ethanol (control disc C) or inethanol solution of the test compound at a concentra-tion of 10 mg/ml (treated disc T). After evaporation ofthe solvent in air for 30 min, the discs were weighed

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Figure 2. Structures of lignans tested for antifeedant activity: yatein (1), tribromoyatein (2), tetrahydroyatein (3), podophyllotoxin (4),hinokinin (5), cubebin (6), dihydrocubebin (7), deoxycubebin (8), tracheloside (9), and carthamoside (10).

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Figure 3. Structures of phenylpropanoids (11–14): latifolon (11), demethyllatifolon (12), safrol (13), piperonylideneacetic acid (14); singlephenolics (15–17): acetopiperone (15), piperonal (16), sezamol (17); and related compounds: piperonylbutoxide (18), and β-thujaplicin (19).

and offered to the insects in plastic boxes as a sole foodsource for five days. Feeding of insects was recordedunder three conditions: (a) on two control discs CC,(b) on a choice between one treated disc T and onecontrol disc C (choice test), (c) on two treated discsTT (no-choice test). Each of the three experiments wasrepeated five times with three beetles of S. granarius,20 beetles of T. confusum, or 10 larvae of T. granariumor T. confusum. After five days, the discs were re-weighed and the average weight of eaten food wascalculated. On the basis of amounts of consumed foodin control (CC), choice test (C,T) and no-choice test(TT), three feeding deterrent activity coefficients werecalculated:(1) Absolute coefficient of deterrency:A = (CC-TT)/(CC+TT) ×100 (calculated from theno-choice test).(2) Relative coefficient of deterrency:R = (C-T)/(C+T) ×100 (calculated from the choicetest).(3) Total coefficient of deterrency: T = A + R

The values of the total coefficient T represent theindex of antifeeding activity expressed on a scalefrom 0 to 200, where 0 means an inactive compound,and 200 means maximum activity. Strong antifeedants

have indices of 150–200. In certain cases, a value ofless than 0 can be achieved for compounds demon-strating feeding stimulation (feeding attractants).

The data from the experiments have been statisti-cally analysed by an analysis of variance. The leastsignificant differences at the 5% level of significancewere calculated for the data given in Tables 1–4. Thescheme of experiments and statistical analysis are de-pendent on insect species, its food, and behaviour. Wetake into account the total coefficient (T) as an addi-tional confirmation of usually good activity describedby the relative coefficient (R).

Chemicals. The compounds (Figures 2 and 3) wereisolated from plant sources or prepared by simplechemical transformations from natural substances, andidentified by MS and 1H-NMR spectroscopy. Thedibenzylbutane lignans were prepared from yatein(1) isolated from the heartwood of a tropical tree Li-bocedrus yateensis Guillaumin (Erdtman & Harmatha,1979) or from cubebin (6) isolated from seeds of Pipercubeba L. (Jensen et al., 1993). The derivatives 2and 3 were prepared from yatein (1) by brominationand by complex hydride reduction, respectively (Har-matha et al., 1982). Similar reduction of cubebin (6)

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Table 1. Feeding deterrent activity coefficients of lignans for adults of Sitophilus granarius and Tribolium confusum

Compound Sitophilus granarius Tribolium confusum

Type Form Coefficients

Relative Absolute Total Relative Absolute Total

1 Yatein A V 100.0 e 47.2 bc 147.2 de 100.0 c 46.6 de 146.6 c

2 Tribromoyatein A V 27.8 a 23.7 ab 51.5 b 48.6 a 12.1 ab 60.9 a

3 Tetrahydroyatein A II 49.5 c 21.6 ab 71.1 bc 91.4 bc 13.5 ab 104.9 bc

4 Podophyllotoxin B V 61.4 bcd 0.8 a 62.2 b 100.0 c 37.2 cd 137.2 c

5 Hinokinin A V 94.5 de 95.7 c 191.2 e 100.0 c 37.7 cd 137.7 c

6 Cubebin A IV 100.0 e 92.6 c 192.6 e 74.2 b 52.9 e 127.1 bc

7 Dihydrocubebin A II 75.7 cde 47.9 b 123.6 cd 84.0 bc 27.5 bc 111.7 bc

8 Deoxycubebin A III 72.2 cde 38.3 b 110.8 cd 100.0 c 36.8 cd 136.8 c

9 Tracheloside A V 62.3 bcde −0.9 a 61.4 b 98.9 c 47.3 de 146.2 c

10 Carthamoside A V −38.0 a −0.6 a −38.6 a 83.6 bc 27.8 bc 111.4 bc

Least signif. difference (LSD0.05) 37.8 29.6 48.3 20.7 15.3 38.1

Chemical structures of lignans 1–10 are shown in Figure 2. For types and forms see Figure 1. Values followed by the same letterin each column are not significantly different.

Table 2. Feeding deterrent activity coefficients of lignans for larvae of Tribolium confusum and Trogoderma granarium

Compound Tribolium confusum Trogoderma granarium

Type Form Coefficients

Relative Absolute Total Relative Absolute Total

1 Yatein A V 83.0 c 81.4 d 164.7 e 100.0 c 66.0 b 166.0 c

2 Tribromoyatein A V −0.7 a 16.5 ab 16.5 a 10.2 a 8.7 a 18.9 a

3 Tetrahydroyatein A II 40.8 b 21.9 abc 62.7 b 71.1 bc 28.9 a 100.0 b

4 Podophyllotoxin B V 100.0 c 29.1 bc 129.1 d 62.9 bc 19.0 a 81.9 b

5 Hinokinin A V 82.2 c 23.3 abc 115.5 cd 64.0 bc 83.9 b 147.9 c

6 Cubebin A IV 100.0 c 31.0 abc 131.0 d 92.2 bc 81.0 b 173.2 c

7 Dihydrocubebin A II 89.3 c 5.2 a 94.5 c 54.5 b 27.7 a 83.9 b

8 Deoxycubebin A III 75.7 c 21.7 abc 97.4 c 5.4 a 25.7 a 83.1 b

9 Tracheloside A V 94.8 c 83.9 d 178.7 e 67.3 bc 12.5 a 78.8 b

10 Carthamoside A V 86.2 c 33.9 cd 120.1 cd 54.3 b 17.7 a 72.0 b

Least signif. difference (LSD0.05) 25.4 23.4 31.0 39.9 28.3 44.3

Chemical structures of lignans 1–10 are shown in Figure 2. For types and forms see Figure 1. Values followed by thesame letter in each column are not significantly different.

yielded dihydrocubebin (7), which was transformedby dimethylsulfoxide dehydration to deoxycubebin(8), or when oxidised by silver carbonate absorbedon the celite surface, it produced hinokinin (5) (Har-matha et al., unpubl.). Lignan glycosides 9 and 10were isolated from seeds of a medicinal plant en-demic to Siberia, but cultivated also in Europe, Leuzeacarthamoides DC [syn. Rhaponticum carthamoides(Wild) Iljin] (Harmatha et al., unpubl.). Podophyl-lotoxine (4), phenylpropanoids 11–14, and phenolics15–18 were obtained from commercial sources, fromAldrich or from Sarsyntex (Merignac, France).

Results and discussion

The data and the evaluation of feeding deterrent activ-ity of a series of lignans (Figure 2) and structurally re-lated phenylpropanoids or simple phenolics (Figure 3)against the three species of insects are summarised inTables 1–4. Each of the compounds inhibited feedingwith a different specificity.

Of the lignans 1–10 (Figure 2), yatein (1) and hi-nokinin (5), the two dibenzylbutyrolactone (type A,V;Figure 1) lignans, exhibited strongest average activityagainst all tested pests (Tables 1 and 2). Only cubebin(6) with the five member hemiacetal group (type A,IV)

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Table 3. Feeding deterrent activity coefficients of phenylpropanoids and related phenolics for adults of Sitophilusgranarius and Tribolium confusum

Compound Sitophilus granarius Tribolium confusum

Type Coefficients

Relative Absolute Total Relative Absolute Total

11 Latifolon PP 87.1 a 50.2 d 137.3 c 100.0 d 22.8 b 122.8 c

12 Demethyllatifolon PP 25.1 a 14.4 ab 39.5 a 82.8 cd 10.0 a 92.8 b

13 Safrol PP 100.0 c 100.0 e 200.0 f 25.7 a 47.6 cd 73.3 ab

14 Piperonylideneacetic acid PP 66.0 b 7.6 a 73.6 b 39.0 ab 23.9 b 62.9 ab

15 Acetopiperone SP 100.0 c 48.2 d 148.2 cd 39.7 ab 26.0 bc 65.7 ab

16 Piperonal SP 100.0 c 31.7 c 131.7 c 66.9 bc 14.9 ab 81.8 ab

17 Sesamol SP 100.0 c 26.4 bc 126.4 c 36.4 ab 12.0 a 48.4 a

18 Piperonylbutoxide CP 100.0 c 100.0 e 200.0 d 100.0 d 100.0 e 200.0 d

19 β-Thujaplicin TD 100.0 c 86.9 e 186.9 ef 77.8 cd 55.6 d 134.3 c

20 Azadirachtin TT 100.0 c 70.3 d 170.3 de 100.0 d 92.2 e 192.2 d

Least signif. difference (LSD0.05) 26.4 15.6 26.8 31.1 21.4 38.4

Chemical structures of compounds 11–19 are shown in Figure 3. Types of compounds: phenylpropanoid (PP), singlephenolic compound (SP), complex phenolic compound (CP), tropolone derivative (TD) and triterpenoid (TT). Valuesfollowed by the same letter in each column are not significantly different.

Table 4. Feeding deterrent activity coefficients of phenylpropanoids and related phenolics for larvae of Tribolium confusum andTrogoderma granarium

Compound Tribolium confusum Trogoderma granarium

Type Coefficients

Relative Absolute Total Relative Absolute Total

11 Latifolon PP 84.3 cd 51.2 d 135.5 b 63.7 b 19.2 ab 82.9 b

12 Demethyllatifolon PP 34.9 ab 11.2 abc 46.1 a −10.4 a 14.3 ab 3.9 a

13 Safrol PP 100.0 d 61.2 de 161.2 bc 100.0 c 92.0 d 192.0 c

14 Piperonylideneacetic acid PP 58.6 abc −12.1 a 46.5 a 53.6 b 55.9 c 109.5 b

15 Acetopiperone SP 38.2 ab 19.0 bc 57.2 a 64.4 b 37.9 bc 102.3 b

16 Piperonal SP 21.1 a 4.3 ab 25.4 a 0.1 a −2.4 a −2.3 a

17 Sesamol SP 20.8 a 33.2 cd 54.0 a 57.9 a 58.6 c 116.5 b

18 Piperonylbutoxide CP 100.0 d 100.0 ef 200.0 c 100.0 c 100.0 d 200.0 c

19 β-Thujaplicin TD 66.8 bcd 56.2 d 123.0 b 100.0 c 78.5 cd 178.5 c

20 Azadirachtin TT 100.0 d 88.6 ef 188.6 c 100.0 c 94.8 d 194.8 c

Least signif. difference (LSD0.05) 37.8 28.7 43.6 32.2 35.3 52.1

Chemical structures of compounds 11–19 are shown in Figure 3. Types of compounds: phenylpropanoid (PP), single phenoliccompound (SP), complex phenolic compound (CP), tropolone derivative (TD), and triterpenoid (TT). Values followed by thesame letter in each column are not significantly different.

had high activity, similar to that of the structurally re-lated lactones. Deoxycubebin (8) with the five membercyclic ether moiety (type A,III) as well as butanedioltypes (type A,II) represented by dihydrocubebin (7)and tetrahydroyatein (3), syn. yateindiol (Harmathaet al., 1982), showed only medium activity. Very lowactivity was obtained for the tribromoyatein (2), aderivative with Br substituents located on aromaticrings. Surprisingly, podophyllotoxin (4), a B,V type oflactone with a structure related to yatein (1), but with

a different spatial arrangement and more rigid con-formation (Harmatha et al., 1982) was considerablyless active, especially against S. granarius and T. gra-narium. However, for T. confusum adults and larvae ithad quite high activity, similar to that of dibenzylbu-tyrolactones 1 and 5 (type A,V) or to the hemiacetal 6(type A,IV).

The activity of lignans was compared with thatof structurally related phenylpropanoids 11–14 (Fig-ure 3), the assumed biogenetic precursors of lignans.

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Those compounds, containing a methylenedioxy sub-stituent (Tables 3 and 4), varied from not to highlydeterrent, but were generally less active than the lig-nans. Only safrol (13) was strongly active against allinsects with the exception of the T. confusum adults.Safrol was selected as a substitute in our tests for theclosely related dilapiol, which is known as an efficientinsecticidal defence compound of Piperaceae plantsand as a mono-oxygenase activity inhibitor (Bernardet al., 1989, 1995). However, Piperaceae plants con-tain also piperonyl amides, another type of compoundswith a methylenedioxyphenyl group, which were de-scribed as active insecticides (Su & Horwat, 1981;Bernard et al., 1995). The safrol and dilapiol activ-ities turned our attention to asarones, another groupof closely related phenylpropanoids. A series of sixsynthetic alpha-asarone isomers were tested using thesame test model (Poplawski et al., 2000), with resultssimilar to ours with safrol. Contact toxic and fumiganteffects of safrol and isosafrol against S. zeamais andT. castaneum were also described (Huang et al., 1999).Isosafrol was generally more toxic than safrol. In con-trast to our results, safrol had in a flour disc bioassayonly very slight or no feeding deterrent activity againstthese two species. Stronger feeding deterrence wasmanifested by isosafrol.

The presence of methylenedioxy and/or tri-methoxy groups in our tested compounds had a gen-erally positive and almost equal effect on the activity.This was not the case when the methoxy group was re-placed by a more polar hydroxy group, as in demethyl-latifolon (12), which in all cases was less active thanthe latifolon (11). However, the structurally relatedmethylenedioxy-containing simple phenolics 15–17had only low activities (Tables 3 and 4). In this case,the methylenedioxy substituent apparently could notsubstitute for the missing propanoid side chain. Nev-ertheless, in lignans and phenylpropanoids, the pres-ence of a methylenedioxyphenyl (piperonyl) struc-tural moiety clearly resulted in increased antifeedantactivity. To learn more about the influence of thismoiety, a complex methylenedioxyphenyl-containingcompound, piperonylbutoxide (18) was also tested.This is a commercially available synthetic compoundand a well-known insecticide synergist generally usedin pest control, often together with pyrethroids. Thiscompound showed a maximum activity in both choiceand no-choice tests, with a total index of 200. It provedto be the strongest antifeedant in this series, whenapplied at lower than lethal doses.

Two dibenzylbutyrolactone (type A,V) lignan gly-cosides, tracheloside (9) and carthamoside (10), wereincluded in this series, as representative analogues thatlack the piperonyl moiety. They exhibited differentialeffects on the insects varying from high feeding de-terrent activity (e.g., tracheloside (9) for T. confusumlarvae) to significant feeding stimulant activity (e.g.,carthamoside (10) for S. granarium adults). This indi-cates that conjugation with the carbohydrate moietycan modify the effect on feeding in the lignan se-ries even more than any so far examined variation inhydroxy, methoxy, and methylenedioxy groups.

β-Thujaplicin (19), a tropolone analogue, whichis considered from the chemotaxonomical point ofview as an alternative constituent to phenylpropanoidsin Cupressaceae (Erdtman & Harmatha, 1979), andwhich is known as a fungitoxic compound, was alsotested. Its activity was similar to that of the mostdeterrent lignan lactones.

The well-known and potent (but structurally unre-lated) triterpenoid antifeedant azadirachtin from theinsect resistant neem tree (Mordue & Blackwell,1993), was selected as a standard compound for com-paring activities against the same insects. Its totalactivity coefficients (Tables 3 and 4) were slightlyhigher than those of the most active lignans, yatein (1),hinokinin (5), and cubebin (6), but not higher than theabsolute antifeedant, piperonylbutoxide (18).

The chemical mechanism of antifeedant activityagainst stored products insect pests is so far only verylittle understood. The fact that a wide variety of struc-tures are effective as feeding deterrents (reviewed inNawrot & Harmatha, 1994), would indicate that sev-eral different mechanisms may be involved. Therefore,the relationship between chemical structure and an-tifeedant activity can be compared only for closelyrelated types of compounds. Such fine structural varia-tion can be achieved either by small gradual chemicaltransformations or by the isolation of structurally re-lated metabolites from taxonomically related naturalsources. Lignans represent a convenient model group,because of their specific biogenetic origin, which hasresulted in close structural relations (Figure 1). Ourpresent results were obtained with only dibenzylbu-tane (type A) lignans, that varied in forms (II–V).These were compared with only one aryltetraline (Btype, form V) lignan, podophyllotoxin (4), as a repre-sentative of more than 200 natural and semisyntheticderivatives (Ward, 1999). The biological effects ofpodophillotoxin on other unrelated insect species canbe more complex, interfering not only with feeding,

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but also with ecdysis or diuresis (Garcia et al., 2000).However, in the Drosophila melanogaster BII cellbioassay for ecdysteroid agonist and antagonist ac-tivities, podophillotoxin was inactive and cytotoxic(> 10−5 M), equally as azadirachtin (Dinan et al.,2001).

To achieve the best structure–activity relationshipin our system, selected compounds were limited to aminimum of substituent variations (mostly methoxyand methylenedioxy derivatives). This selection wasbased on our previous experience with coumarins andfurocoumarins (Harmatha et al., 1991) and with otheraromatic compounds (Nawrot & Harmatha, 1994), inwhich these two substituents increased activity to agreater degree than did the more polar phenolic hy-droxyls. Future efforts will be focused on preparationand testing of dibenzocyclooctane (type C) deriva-tives of yatein and cubebin, using suitable methodsfor chemical transformation as reported by Pelter et al.(1992) and Ward & Hughes (2001).

Acknowledgements

This work was supported by the Grant Agency of theAcademy of Sciences of the Czech Republic, grantNo: A455404, in part also by the research project Z4055 905. We thank Professor Wolfgang Kraus, Univ.Hohenheim, Stuttgart, FRG, for providing us with thesample of azadirachtin. This paper is dedicated to thememory of the late Professor Holger Erdtman on theoccasion of the 100th anniversary of his birthday.

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