Insecticidal natural products: new rocaglamide derivatives from Aglaia roxburghiana

Extended Summaries : IUPAC Congress

tionality in the ‘left-hand’ side-chain can, of course, be written down. However, on the basis of the results above, we can conclude that there is much less scope for modiücation of the structure of methyl monates in the ‘left-hand’ side-chain than there is at the ‘right-hand’ side. Finally it is worth noting that the mixtures approach described above was useful here because of a speciüc set of circumstances : (1) most, if not all, of the analogues in the series were very weak inhibitors or inactive ; (2) there was an expectation, based on the potency of compounds with the natural ‘lefthand’ side-chain, that high activity could be achieved with a suitable synthetic compound ; (3) an enzyme assay, with sensitivity over at least four orders of magnitude, was in place ; and (4) the chemistry leading to the esters was high-yielding and sufficiently robust to work with mixtures of compounds (as, to an adequate extent, was the ether chemistry). Clearly, this approach is quite unsuitable for conventional series of analogues in which most or many have some activity. ACKNOWLEDGEMENTS We thank Ian Bryan, J oy Hughes and Stuart Ridley for testing the compounds described in this paper in enzyme and glasshouse assays. REFERENCES 1 Chain EB and Mellows G, Pseudomonic acid. Part 1. The structure of pseudomonic acid A, a novel antibiotic produced by Pseudomonas ýuorescens. J Chem Soc, Perkin Trans I 294–309 (1977). 2 Chain EB and Mellows G, Pseudomonic acid. Part 3. Structure of pseudomonic acid B. J Chem Soc, Perkin Trans I 318–322 (1977). 3 Clayton J P, O’Hanlon PJ and Rogers NH, The structure and conüguration of pseudomonic acid C Tet Letts 21 :881–884 (1980). 4 O’Hanlon PJ , Rogers NH and Tyler J W, The chemistry of pseudomonic acid. Part 6. Structure and preparation of pseudomonic acid D. J Chem Soc, Perkin Trans I 2655–2657 (1983). 5 Hughes J and Mellows G, Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Escherichia coli by pseudomonic acid. Biochem J 176 :305–318 (1978). 6 Barton J ED, Clinch K, O’Hanlon PJ , Ormrod J C, Rice MJ and Turnbull MD, Monic acid derivative herbicides. WO 93 19,599, published 14 Oct. 1993, priority date 7 April 1992. 7 Barton J ED, Clinch K, Clough J M, Ormrod J C and Rice MJ , Herbicidal activity of derivatives of pseudomonic acid. 8th International Congress of Pesticide Chemistry – Options 2000, Washington, DC USA, 4–9 J uly 1994. 8 Bryan IB, Rice MJ , Bartley MR, J utsum AR and Pastushok G, A monic acid derivative : evaluation as a cereal herbicide. Proc Brighton Crop Prot Conf – Weeds, 2 :725–730 (1995). 9 Coulton S, O’Hanlon PJ and Rogers NH, The chemistry of pseudomonic acid. Part 9. Reduction, inversion and replacement of the C–13 hydroxyl group. Tetrahedron 43 :2165– 2175 (1987). 10 Forrest AK, O’Hanlon PJ and Walker G, The chemistry of pseudomonic acid. Part 13. Modiücations at C-12 to C-14. Tetrahedron, 50 :10739–10748 (1994). 11 Luk K and Rogers NH, 13-Oxo-monic acid and its salts and esters and compositions containing them. EP 42,233, published 23 Dec 1981, priority date 14 J une 1980.

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12 Forrest AK, Osborne, NF and Pengelly D, Preparation of mupirocinsulfamates with antibacterial activity. WO 97 05,126, published 13 Feb 1997, priority date 29 J uly 1995. 13 Best DJ , Elder J S and Osborne NF, Preparation of sulfamoylcontaining alditols as bactericides and t-RNA synthetase inhibitors. WO 97 35,859, published 2 Oct 1997, priority 25 March 1996. 14 Keck GE, Kachensky DF and Enholm EJ , Pseudomonic acid C from L-lyxose. J Org Chem 50 :4317–4325 (1985). 15 Snider BB, Phillips GB and Cordova R, Formal total synthesis of (]/[)-pseudomonic acids A and C. The quasiintramolecular Lewis acid catalyzed Diels-Alder reaction. J. Org. Chem., 48 :3003–3010 (1983). 16 Hughes J and Mellows G, Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase. Biochem J 191 :209–219 (1980).

Insecticidal natural products : new rocaglamide derivatives from Aglaia roxburghiana Louis -Pierre Molleyres ,1* Alfred Rindlis bacher,1 Tammo Winkler1 and Vijaya Kumar2 1 Novartis Crop Protection AG , PO Box 4002 Bas el , Switzerland 2 Univers ity of Peradeniya , Sri Lanka

Abstract : In the course of the screening for novel, naturally occurring pesticides from the plant family Meliaceae, an extract of the stem bark of Aglaia roxburghiana was found to exhibit signiücant insecticidal activity. In addition to rocaglamide, a known insecticide isolated from several species of the genus Aglaia, 15 new natural products were isolated from this plant. Isolation and structure elucidation of the natural products is described. The outstanding insecticidal activities of some of the compounds as well as a structure–activity relationship study are presented. ( 1999 Society of Chemical Industry

Keywords : Aglaia roxburghiana ; meliaceae ; plant metabolites ; benzofurans ; rocaglamide analogues ; aglaroxins, insecticidal activity 1 INTRODUCTION Plants provide an abundant source of secondary metabolites possessing biological activities in the crop protection area. In a few cases, either the natural products themselves (pyrethrin, rotenone, azadiracthin) or synthetically modiüed, but closely related, structures (pyrethroids) have reached the market place. In recent years, the most detailed studies of the eþects of a natural product on insect behaviour and physiology have been those carried out on the limonoid, azadirachtin, from the neem tree, Azadirachta indica J uss (Meliaceae). Rocaglamide was isolated from Aglaia elliptifolia by King et al1 by following the antileukemic activity against P388 lymphocytic leukemia in CDF1 mice. Simultaneously, Pachlatko and Kumar2 discovered that rocaglamide, isolated from Aglaia congylos, * Corres pondence to : Louis -Pierre Molleyres , Novartis Crop Protection AG, PO Box 4002 Bas el, Switzerland. E-mail : louis -pierre.molleyres =cp.novartis .com (Received 5 Augus t 1998 ; accepted 16 December 1998 )

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Figure 1. Furan analogues .

showed herbicidal activity. The potent insecticidal activity of rocaglamide, isolated from Aglaia odorata, was reported in 1992.3h5 Recently, several novel rocaglamide derivatives, isolated from diþerent Aglaia species, have been reported to show insecticidal,6h8 cytotoxic,9h12 platelet activating (PAF)13,14 or fungicidal properties.15 Studies on the bioefficacy of crude extracts of Aglaia species have also been published.16,17 In the course of the screening for novel, naturally occurring pesticides from the plant family Meliaceae, an extract of the stem bark of Aglaia roxburghiana (syn A elaeagnoidea) was found to exhibit signiücant insecticidal activity. In addition to the known insecticide rocaglamide (Fig 1, 1) 15 new analogues were

isolated in our laboratory. Their identiücation and biological activities are described below. 2 METHODS 2.1 Isolation A large-scale collection (500 kg) of the stem bark of Aglaia roxburghiana was organized in Sri Lanka. The bark was ünely ground and stirred at room temperature with dichloromethane ] methanol (1 ] 1 by volume). After ültration and concentration, the crude extract was puriüed on silica gel with a dichloromethane ] methanol gradient (atmospheric pressure), then with medium pressure chromatography (ethyl acetate ] dichloromethane ] methanol) and ünally with ýash chromatography

Figure 2. Pyrimidinone analogues .

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Figure 3. Pyran analogues .

(hexane ] ethyl acetate ] methanol). After concentration of the diþerent fractions, a total of 20 g of greyish-white powder was obtained. Further separation of 7 g of this powder by means of high pressure chromatography on RP silica gel with a water ] acetonitrile gradient yielded rocaglamide (1) and a series of new derivatives named aglaroxins [2 to 16 ; (Figs 1–3)]. Yields of the compounds were 1 : 110 mg ; 2 : 340 mg ; 3 : 240 mg ; 4 : 195 mg ; 5 : 55 mg ; 6 : 860 mg ; 7 : 11 mg ; 8 : 49 mg ; 9 : 9 mg ; 10 : 9 mg ; 11 : 7.2 mg ; 12 : 8 mg ; 13 : 275 mg ; 14/15 (mixture): 7.3 mg ; 16 : 1.8 mg. 2.2 Identiücation The structures have been elucidated using [1H] and [13C]NMR as well as FD-mass spectroscopy. The fully assigned proton-correlated [13C]NMR spec-

trum of rocaglamide, including the H/D isotope eþects, as well as the proton carbon coupling constants (Fritz H, 1983, pers comm), was used as the starting point. All new structures were then elucidated using HSQC, HMBC, and ROESY experiments together with exhaustive NOE diþerence spectroscopy. The absolute conüguration of 1 has been proven by X-ray analysis,1 (Rihs G, 1983, pers comm). CD and optical rotation data of 1 and 4 are similar to those reported in the literature.6 In the course of the isolation work, the derivatives 46 and 1210 have been published. All the other analogues are either new or have been patented by Novartis.18 The structures of furan pyrimidinone and pyran analogues are given in Figs 1, 2 and 3 respectively.

Table 1. Ins ecticidal activities

Name

Compound a

Dos e (mg litre É1) giving 80 –100 % mortality Heliothis vires cens Spodoptera littoralis Spo .L3 b Hel .e /l b Hel .L1 b Hel .L3 b

Rocaglamide Aglaroxin A Aglaroxin B Aglaroxin E Aglaroxin F Aglaroxin C Aglaroxin D Aglaroxin G Aglaroxin H Aglaroxin I Aglaroxin J Aglaroxin K Aglaroxin L

1 2 3 4 5 6 7 8 9 10 11 12 13

3 0.8 3 12.5 25 3 D100 25 25 25 [100 [100 [100

3 3 3 12.5 25 50 [100 [100 [100 [100 [100 [100

12.5 12.5 50 12.5 50 [100 [100 [100 [100 [100 [100 [100

3 12.5 12.5 12.5 50 12.5 D100 [100 [100 [100 [100 [100 [100

Plutella xylos tella Plu .2 /3 b 3 3 12.5 12.5 50 12.5 [100 100 [100 [100 [100 [100

Diabrotica balteata Dia .L2 b 3 3 [100 [100 12.5 [100 D100 50 [100 12.5 [100 [100 [100

a See Figs 1–3 for s tructures . b See text.

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2.3 Biological activities Rocaglamide and the analogues 2–13 have been tested against the following insects : Heliothis virescens F – Hel.e/l egg-larval on artiücial diet – Hel.L1 ürst instar on soybean – Hel.L3 third instar on soybean Spodoptera littoralis Boisd – Spo.L3 third instar on soybean Plutella xylostella L – Plu.L2/3 second/third instar on cabbage Diabrotica balteata Lec – Dia.L2 second instar on maize seedlings The compounds were tested either as a 500 g litre~1 emulsiüable concentrate or in acetone ] water solutions. 3 RESULTS 3.1 Structure–activity relationship The compounds 1 to 5, bearing the cyclopentatetrahydrobenzofuran ring (Fig 1) show potent insecticidal activities, rocaglamide being the most active (Table 1). This cyclopentatetrahydrobenzofuran moiety is also present in the pyrimidinone analogues 7 to 10, (Fig 2). However, due to the planar pyrimidinone ring, these compounds display a completely diþerent steric conüguration, but are still active. The cyclopentatetrahydrobenzopyran derivatives 11 to 16 (Fig 3) are inactive. They may be formed by addition of a ýavone (kaempferol ?) and odorine, a natural diamine also found in Aglaia species. 4 CONCLUSION We have isolated 15 new analogues of rocaglamide. Rocaglamide and the furan analogues 2 to 5 are the most active compounds isolated. However, the insecticidal activity of rocaglamide could not be improved upon. Meaningful information on structure–activity relationships can be uncovered by the detailed study of the complete spectrum of natural products occurring in biologically active extracts. REFERENCES 1 King ML, Chiang CC, Ling HC, Fujita E, Ochiai M and McPhail AT, X-ray crystal structure of rocaglamide, a novel antileukemic 1H-cyclopenta[b]benzofuran from Aglaia elliptifolia. J Chem Soc, Chem Commun 1150–1151 (1982). 2 Pachlatko J P, Natural products in crop protection. Chimia 52 :29–47 (1998). 3 Satasook S, Isman MB and Wiriyachitra P, Activity of rocaglamide, an insecticidal natural product against the variegated cutworm Peridroma saucia. Pestic Sci 36 :53–58 (1992). 4 J anprasert J , Satasook S, Sukumalanand P, Champagne DE, Isman MB, Wiriyachitra P and Towers GHN, Rocaglamide, a natural benzofuran insecticide from Aglaia odorata. Phytochemistry 32 :67–69 (1993). 5 Ishibashi F, Satasook C, Isman MB and Towers GHN, Insecticidal 1H-cyclopentatetrahydro[b]benzofurans from Aglaia odorata. Phytochemistry, 32 :307–310 (1993).

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6 Nugroho BW, Edrada RA, GuŽ ssregen B, Wray V, Witte L and Proksch P, Insecticidal rocaglamide derivatives from Aglaia duppereana. Phytochemisty 44 :1455–1461 (1997). 7 Nugroho BW, GuŽ ssregen B, Wray V, Witte L, Bringmann G and Proksch P, Insecticidal rocaglamide derivatives from Aglaia elliptica and A harmsiana. Phytochemistry 45 :1579– 1585 (1997). 8 GuŽ ssregen B, Fuhr M, Nugroho BW, Wray V, Witte L and Proksch P, New insecticidal rocaglamide derivatives from ýowers of Aglaia odorata. Z Naturforsch 52c :339–344 (1997). 9 Ohse T, Ohba S, Yamamoto T, Koyano T and Umezawa K, Cyclopentabenzofuran lignan protein synthesis inhibitors from Aglaia odorata. J Nat Prod 59 :650–652 (1996). 10 Dumontet V, Thoison O, Omobuwajo OR, Martin M-T, Perromat G, Chiaroni A, Riche C, Pa•Ž s M and Sevenet T, New nitrogenous and aromatic derivatives from Aglaia argentea and A forbesii. Tetrahedron 52 :6931–6942 (1996). 11 Heebyung BC, Santisuk T, Reutrakul V, Farnsworth NR, Cordell GA, Pezzuto J M and Kinghorn AD, Novel cytotoxic 1H-cyclopenta[b]benzofuran lignans from Aglaia elliptica. Tetrahedron 53 :17625–17632 (1997). 12 Kokpol U, Venaskulchai B, Simpson J and Weavers RT, Isolation and X-ray structure determination of a novel pyrimidinone from Aglaia odorata. J Chem Soc, Chem Commun 773–774 (1994). 13 Ko F-N, Wu T-S, Liou M-J , Huang T-F and Teng C-M, PAF antagonism in vitro and in vivo by aglafoline from Aglaia elliptifolia. European J Pharmacol, 218 :129–135 (1992). 14 Wu T-S, Liou M-J , Kuoh C-S, Teng C-M, Nagao T and Lee K-H, Cytotoxic and antiplatelet aggregation principles from Aglaia elliptifolia. J Nat Prod 60 :606–608 (1997). 15 Fuzzati N, Dyatmiko W, Rahman A, Achmad F and Hostettmann K, Triterpenoids, lignans and a benzofuran derivative from the bark of Aglaia elaeagnoidea. Phytochemistry, 42 :1395–1398 (1996). 16 Ewete F, Nicol RW, Hengsawad V, Sukumalanand P, Satasook C, Wiriyachitra P, Isman MB, Kahn Y, Duval F, Philoge` ne BJ R and Arnason J T, Insecticidal activity of Aglaia odorata extracts and the active principle, rocaglamide, to the european corn borer, Ostrinia nubilalis. J Appl Ent 120 :483– 488 (1996). 17 Koul O, Shankar J S, Mehta N, Taneja SC, Tripathi AK and Dhar KL, Bioefficacy of crude extracts of Aglaia species and some active fractions against lepidoptera larvae. J Appl Ent 121 :245–248 (1997). 18 Molleyres L-P and Kumar V, Insecticidal polycyclic compounds. PCT Int. Appl. WO 96/04284 A (1994). 1

Bioactive compounds from neem tissue cultures and screening against insects Athanas s ios K Zounos ,1 Eunice J Allan2 and A Jennifer Mordue(Luntz)1 1 Department of Zoology , Univers ity of Aberdeen , Tillydrone Avenue , AB24 2TZ , UK 2 Department of Agriculture , Univers ity of Aberdeen , 581 King Street , AB24 5UA , UK

Abstract : Hairy root cultures have been derived from neem (Azadirachta indica A Juss, Family Meliacceae) using Agrobacterium rhizogenes and

* Corres pondence to : Athanas s ios K Zounos , Department of Zoology, Univers ity of Aberdeen, Tillydrone Avenue, AB24 2TZ, UK. Contract/grant s pons or : Commis s ion of the European Union. Contract/grant no : FAIR-CT-96-5014. (Received 4 Augus t 1998 ; accepted 16 December 1998 )

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