Abstract. A group of fungicidal antibiotics, b-methoxyacrylic acid derivatives (strobilurins, oudemansins, and myxothiazols), their producers, and mechanisms of action are considered. The fungi- cidal activity of these compounds is based on the suppression of cell respiration of fungi in the bc1-complex of cytochromes. They also manifest other biological activities that are not always coupled with inhibition of respiration.Studies of the structure of the natural methoxyacrylates has made it possible to create a novel class of synthetic agricultural fungicides with enhanced stability, high activity, and a broad spectrum of action. The main regularities of the structure ± activity relationship and meth- ods of synthesis of these compounds are discussed. The bibliog- raphy includes 159 references.I. Introduction Some antibiotics produced by various microorganisms (strobilur- ins, oudemansins, and myxothiazols) were found to inhibit the growth of a broad range of fungi.1 Mucidin or strobilurin A (1a) was the first to be discovered in this group of compounds. Later, several other compounds of similar structure that differed from mucidin only in the number of double bonds and the presence and position of substituents, were identified. These compounds were called strobilurins or oudemansins depending on the structure of their aliphatic chains.All of them are secondary metabolites of various fungi. Myxothiazol 2 has a bacterial origin and its struc- ture differs substantially from that of strobilurin A, although both compounds comprise analogous structural elements.The distinctive feature of this group of compounds is the presence of a fragment of b-methoxyacrylic acid (as methyl ester or amide) linked through an a- or b-carbon atom to the rest of the molecule. They possess identical mechanism of action, which consists in the inhibition of cell respiration at the region of the bc1 complex of electron-transport cytochromes (complex III).Such an inhibition mechanism made possible wide application of these compounds as a biochemical tool for the study of oxidative phosphorylation. These compounds also served as models for creation of a basically new class of pesticides currently known as `strobilurin analogues'. The electron-transport chain of respiratory enzymes plays a crucial role in supplying cells of all aerobic organisms (from bacteria, fungi, and plants to higher animals) with energy.The structure of components of the respiratory chain that are inter- mediates in the electron transfer from NADH to oxygen, varies insignificantly on passing from species to species. However, substantial differences are observed in the resistance of enzymes of the cytochrome system of various living organisms to specific inhibitors, which is used by certain organisms in the competition struggle.2, 3 Natural differences in the structure of respiratory enzymes can be used in the design of agrochemical preparations for achieving their high selectivity with respect to plants and low toxicity for mammals.2 Owing to the wide occurrence of redox processes involving the cytochrome system, the respiration inhibitors can be used for suppressing the vital activity of a great variety of organisms.Thus many synthetic strobilurin analogues possess not only fungicidal, but also insecticidal, acaridical, and nematocidal activities. Other natural compounds that incorporate a b-methoxya- crylic acid fragment as a structural element are also known.For instance, a b-methoxyacrylate fragment in dihydrokawain (5,6- dihydro-4-methoxy-6-phenylethyl-2H-pyran-2-one), which has been recently isolated from kava Piper methysticum 4 and pos- sesses fungicidal activity, includes a hydropyranone ring. How- ever, there is no evidence that this compound inhibits respiration. The recently discovered derivatives of mesoxalic acid amido- nitrile O-methyloxime (e.g., cymoxanil 3) are structurally similar R1=R2=H(a); R1=OMe, R2 = H (b), Cl (c); R1=H,R2=OMe (d); R1=OH, R2= H (e); R1=Me2C=CHCH2O, R2=H(f); R1=OH, R2=Me2C=CHCH2O (g).R1 R2 Me MeOOC OMe 1a7g Me Me Me S N N S OMe Me MeO CONH2 2 V V Zakharychev, L V Kovalenko D I Mendeleev Russian Chemico- Technological University, Miusskaya pl. 9, 125047 Moscow, Russian Federation.Fax (7-095) 200 42 04. Tel. (7-095) 948 54 63 (V V Zakharychev), (7-095) 496 58 37 (L V Kovalenko) Received 10 October 1997 Uspekhi Khimii 67 (6) 595 ± 605 (1998); translated by R L Birnova UDC 577.158.8.04 : 632.952 Natural compounds of the strobilurin series and their synthetic analogues as cell respiration inhibitors V V Zakharychev, L V Kovalenko Contents I.Introduction 535 II. Natural methoxyacrylate-type inhibitors 536 III. Biological activity of the natural compounds 537 IV. Synthetic strobilurin analogues and structure ± activity relationships 538 V. Synthesis of strobilurin analogues 541 VI. Conclusion 543 Russian Chemical Reviews 67 (6) 535 ± 544 (1998) #1998 Russian Academy of Sciences and Turpion Ltdto some strobilurin analogues.However, their fungicidal activity is associated with blocking of RNA synthesis in fungal cells.5 II. Natural methoxyacrylate-type inhibitors The high fungicidal activity of the new antibiotic mucidin (1a) isolated from the cultural medium and mycelium of the fungus Oudemansiella mucida was first discovered by Musilek et al.6 ± 8 in the mid-1960's. Two fungicidal antibiotics called strobilurins A and B were isolated from mycelium of the basidiomycete Strobi- lurus tenacellus in 1977.9 Structural elucidation of these com- pounds revealed that strobilurin A and mucidin are identical and represent methyl (2E,3Z,5E)-2-methoxymethylene-3-methyl-6- phenylhexa-3,5-dienoate.10, 11 The E,Z,E-configuration of the double bonds of strobilurins was confirmed by chemical and spectroscopic studies 12 as well as by stereospecific synthesis.13 The spectral properties of a synthetic E,E,E-isomer of stro- bilurin A13 differed from those of the natural compound.Later, several other secondary metabolites of various fungi were isolated and characterised. They are strobilurinAderivatives containing substituents in the benzene ring or in the aliphatic chain (compounds 1b ± g, 4 ± 8).Oudemansins (9a ± c) possess similar structure. Formally, they can be regarded as the products of addition of methanol at the C(3)7C(4) double bond of strobilurins. The known strobilurins and oudemansins together with their producers are listed in Table 1. Considering the trivial names of methoxyacrylate-type anti- biotics, it should be noted that the letter symbols A, B, andCin the names of strobilurins and oudemansins refer to substituents in the aromatic ring and their positions.The same letters are used for the same substitution pattern [cf. the structures of strobilurin B (1c) and oudemansin B (9b)]. The early name of strobilurin A, mucidin, was preserved in the biochemical literature. Strobilurin X was termed by analogy with oudemansin X,1 because the investigators who first discovered this compound did not give it any trivial name.Two strobilurins F were identified and named almost simultaneously by independent groups of investigators; therefore, they bear additional numerical indices (in the order of publication).1 Myxothiazol 2 (or myxothiazol A) mentioned above produced by the myxobacteria Myxococcus fulvus is also a derivative of b-methoxyacrylic acid.14, 26, 27 In contrast with strobilurins and oudemansins, the b-methoxyacrylate fragment of the myxothiazol molecule is represented by an amide rather than by an ester and is substituted at the b-carbon (but not at the a-carbon) atom.Nevertheless, the mechanism of biological action of this antibiotic is close to that of the natural esters of methoxyacrylic acid.28, 29 For a long time, myxothiazol had no analogues among b-methoxyacrylate-type compounds.However, a great number of related compounds were discovered recently. At present, 33 myxothiazols have been isolated, and the structures of 24 of them have been given in a review.1 Unfortunately, comprehensive information about the structure of myxothiazols is still absent in the current literature.It is known only that in myxothiazols B7I, K7O (10a) and Q, X, and Y (10b), R2=MeCO, MeCH(OH), or an (E,E)-nonadienyl residue containing hydroxy, carbonyl, and epoxide groups. Myxothiazol J does not exist. Myxothiazol P (11) has only one thiazole ring. In myxothiazols R7W (12), the b-methoxyacrylic fragment is absent and R is an oxygenated three-, five-, or six-carbon-atom chain.1 3 O EtNHCONH CN NOMe X=H (a), OH (b).O O X MeOOC OMe Me Me O Me Me 4a,b O O Me MeOOC Me O O Me Me Me Me 5 O O Me Me Me Me 6 OMe 7 OMe O O Me Me O H2C Me Me 8 Me MeOOC OMe Me MeOOC OMe Me MeOOC OMe R1=R2=H (a); R1=OMe, R2=Cl (b); R1=H, R2=OMe (c). OMe Me MeOOC OMe R1 R2 9a7c Table 1. Natural strobilurins and oudemansins.Struc- Name Producing organism Ref. ture 1a Strobilurin A Oudemansiella mucida, 6 ± 9, (mucidin) Strobilurus tenacellus, 15 ± 17 Bolinea lutea etc. 1c Strobilurin B S. tenacellus 9 1f Strobilurin C Xerula longipes, 16 X. melanotricha 1d Strobilurin X O. mucida 18 4b Hydroxystrobilurin D Mycena sanguinolenta 19 5 Strobilurin E Crepidotus fulvotomentosus 20 4a Strobilurin D Cyphellopsis anomala 21 1e Strobilurin F-1 Cyphellopsis anomala 21 1g Strobilurin F-2 Bolinea lutea 15, 22 6 Strobilurin G B.lutea 15, 22 1b Strobilurin H B. lutea 15, 22 7 9-Methoxystrobilurin Aa Favolaschia spp. 23 8 9-Methoxystrobilurin Ka Favolaschia spp. 23 9a Oudemansin A O. mucida 24 9b Oudemansin B Xerula longipes, 16 X. melanotricha 9c Oudemansin X O. radicata 25 a Trivial nomenclature is retained. 536 V V Zakharychev, L V KovalenkoStrobilurins A and B, all of oudemansins,1 and myxothia- zol A30 have been synthesised. The generalised data concerning natural methoxyacrylates can be found in a comprehensive review.1 The conformer of strobilurin A (1a) with a minimum energy was calculated using the molecular mechanics method and the molecular orbital theory.It was found that the phenylpentadienyl and methyl b-methoxyacrylate fragments are planar, but are arranged in perpendicular planes. However, according to these calculations, the energy barrier to rotation around the C(2)7C(3) bond is low, therefore enantiomers cannot be isolated at room temperature.31 The structure of oudemansin A 9a in the crystalline state was determined by X-ray diffraction analysis.24 It was found that its conformation closely resembles the conformer of strobilurin A with the minimum energy.31 Apparently these conformations of strobilurin and oudemansin are close to the forms of these molecules bound to cytochrome.31 III.Biological activity of the natural compounds Low concentrations of strobilurins, oudemansins, and myxothia- zols inhibit the growth of diverse fungi.Their mechanism of action is associated with violation in electron transport in complex III of the mitochondrial membrane, which results in the inhibition of cell respiration.28, 29, 32 ± 34 Complex III (ubiquinone ± cytochrome c oxidoreductase or the bc1-complex) is an intermediate link in the chain of respiratory enzymes of bacteria and mitochondria (plant chloroplasts contain a structurally homologous b6 f-complex).It catalyses the electron transfer from hydroquinone to cytochrome c. The energy of the redox reaction is thereby converted into a chemiosmotic mem- brane potential through the translocation of 2e7 and 4H+ across the membrane. According to the Mitchell Q-cycle concept,3, 35, 36 complex III has two quinone reaction centres localised on opposite sides of the membrane.One of them, the so-called Qo-centre (also designated asQp orQz), is the site of hydroquinone oxidation by the reaction: 2QH2 2Q+4e7+4H+. Two electrons are transferred by a high-potential route via the FeS protein and cytochrome c1 to reduce cytochrome c. The other two electrons move to the opposite side of the membrane by a low- potential route formed by cytochromes bH and bL, to the second quinone centre, the so-called Qi-centre (also designated as Qn, Qc, and Qr) where the quinone is reduced: Q+2H++2e7 QH2 .The inhibitors binding to the Qi-centre induce changes in the absorption spectrum of cytochrome bH (b562). Antimycin is the most specific of these inhibitors. Methoxyacrylates were among the first to be discovered in a group of compounds that block respiration by binding to the Qo-centre.This binding results in a shift in the absorption band of cytochrome bL (b566).29, 32, 33, 37 ± 42 Since all tissue respiration inhibitors of this type contain a residue of b-methoxyacrylic acid (MOA) as a structural fragment, which seems to be the key element for manifestation of biological activity, they were termed MOA-inhibitors.33 The binding site of MOA-inhibitors is not identical with the quinone site, since in the presence of these compounds ubiquinone remains bound with cytochrome b.This may be accompanied by changes in the relative position of the ubiquinone molecule at the reaction centre due to a conformational rearrangement of the cytochrome and the resulting violation of the electron transport to the FeS protein.43 ± 45 Thus the concentration of myxothiazol required for 50% inhibition of bovine heart cytochrome b activity is 0.58 mol per mol of cytochrome.29 At high concentrations, MOA-inhibitors can displace the quinone from the site of its binding with the Rieske FeS-protein.41 Apparently, the binding of inhibitors is reversible, since they can replace one another in the Qo-centre.39 Like methoxyacrylates, other compounds can also bind with the Qo-centre.Pyricidine (which inhibits also complex I), 6-hydroxy-5-n-undecyl-4,7-dioxobenzothiazole,2 dibromothy- moquinone,2, 41 and the chromone antibiotics stigmatellins A and B2, 41, 44, 46 also possess inhibitory activity. Despite the recip- rocal competition of these compounds, their binding sites in the Qo-centre are other than those of MOA-inhibitors.1, 2 The structure of the bc1-complex and the Qo-centre as well as the mechanisms of resistance to MOA-inhibitors are discussed in a number of publications.3, 35, 36, 47 Strobilurins and oudemansins manifest nearly identical high activity in vitro against a wide array of fungi,9, 15, 16, 18, 19 ± 21, 23 ± 25, 48, 49 but are inactive against bacteria.Strobilurin F-1 (1e), which manifests much lower activity, is an exception.21 For example, the minimum inhibitory concentrations of strobilurin A (1a) for Candida albicans, C. crusei, C. para- poilosic, C. tropicalis, Cryptococcus neofarmans, Trichophyton mentagrophytes, Epidermophyton êoccosum, and Microsporum canis lie in the range 0.1 ± 12.8 mg ml71, whereas the inhibitory concentrations for Aspergillus spp.and Scopulariopsis spp. range from 100 to 1000 mg ml71 (see Ref. 48). Owing to its antimycotic activity, strobilurin A has been used in clinical and veterinary medicine under the commercial name of Mucidermin Spofa.1 Myxothiazol A 2 is one of the most active MOA-inhibitors.32 It manifests not only fungicidal properties, but inhibits also the growth of some Gram-positive bacteria,1 reversibly inhibits the late G1/S phase of the cell cycle of lymphoblastic T-cells,50 and possesses insecticidal activity.1 In addition, myxothiazol 2 can bind to the phylloquinone site in photosystem I (Kd= 9.561076 mol litre71).However, this inhibitor does not mani- fest any activity in vivo due to the higher affinity of phylloquinone for this site.51, 52 Myxothiazol 2 inhibits also complex I of mitochondria.2 Other myxothiazols rank below myxothiazol A in fungicidal activity.1 OudemansinA(9a),24 strobilurinsA(1a), B (1c),9 E (5),20, 49D (4a),21 and G (6)15 inhibit the growth of human tumour cells, the latter three compounds manifesting the highest activity. 9-Methoxystrobilurins (7, 8)23 and strobilurin G (6)15 also display cytostatic activity.Strobilurins D (4a)21 and E (5)20, 49 possess antiviral properties. Strobilurins A (1a) and B (1c) as well as oudemansin A (9a) inhibit chitin synthase.53 Strobilurins A (1a), B (1c), C (1f), and X (1d) as well as oudemansins A (9a) and B (9b) are relatively non-toxic for mice.According to different data, the peroral lethal dose, LD50, for strobilurin A is 500 mg kg71 (see Ref. 8) or 825 mg kg71 (see Ref. 18). Intraperitoneal LD50 is 250 mg kg71 (see Ref. 8); that for oudemansin A exceeds 300 mg kg71 (see Ref. 31). However, myxothiazol 2 is highly toxic for all animals tested (peroral LD50 for mice is 2 mg kg71).27 R1=H (a), Me (b).N S R1 OMe 10a,b MeO Me CONH2 N S R2 OMe N S O H2N 11 MeO Me CONH2 Me Me Me S N 12 S N R Natural compounds of the strobilurin series and their synthetic analogues as cell respiration inhibitors 537Despite the variety of useful properties, natural MOA-inhib- itors have found only limited use as medicinal drugs or biochem- ical tools for the study of cell respiration. They cannot be used as agricultural pesticides because they have some serious disadvan- tages.For example, strobilurin A, which possesses high activity against a wide variety of fungi in vitro, is fairly inactive in greenhouse studies. This may be due to the low photochemical stability and a relatively high volatility of this compound. Hence, it rapidly evaporates from the surface of leaves of the treated plants.The time of the photochemical loss of the first 50% of strobilurin A in thin film experiments (t50) is 1 min.31 Never- theless, this group of antibiotics presents substantial interest. The relative simplicity of their structure, their ability to retain high activity irrespective of significant changes in their structure, and a basically new mechanism of their action, which entails the absence of cross-resistance in pathogens insensitive to the currently used fungicides, stimulate the synthesis of biologically active analogues of the natural substances.Although respiration inhibitors may be hazardous for homoiothermal organisms, the low toxicity of individual strobilurins and oudemansins suggests that synthetic analogues may exist which differ substantially in toxicity with respect to fungi and homoiothermal organisms.1 IV.Synthetic strobilurin analogues and structure ± activity relationships Although the first attempts to synthesise analogues of natural MOA-inhibitors were undertaken only in the early 1980's, over 200 patents for the synthesis of these compounds, their practical applications, and composite mixtures, have been pub- lished.31, 54, 55 Synthetic analogues of strobilurins have been patented as broad-spectrum agricultural and industrial fungicides, nemato- cides, insecticides and acaricides, plant growth regulators, anti- tumour and antiviral preparations.The undeniable leaders in this area are the British company ICI and the German company BASF who have succeeded in creating experimental fungicides ICI-5504 (13) and BAS-490F (14).Two fragments can be distinguished in the molecules of natural MOA-inhibitors and their synthetic analogues, viz., a toxophore or a pharmacophore, and a carrier group 56 (or a ballast group 54 or backbone 47). Thus in strobilurins, the b-methoxyacrylate fragment plays the role of a toxophore and a substituted (E,E)-phenylpentadienyl residue is the backbone.It is the toxophore that is responsible for the binding with the methoxyacrylate site of the enzyme, although it is inactive if it is not bound with the backbone. In turn, the backbone not only imparts lipophilicity to the compounds, which is needed for the transport of a pharmacophore into the quinone centre, but also enables matching of the shape of the inhibitor molecule and that of the Qo-centre cavity.47 Both factors must be taken into consideration in design of strobilurin analogues.The structure of strobilurins can be modi- fied in two independent ways: (1) substitution of the (E,E)- phenylpentadienyl chain of strobilurins for various aryl, hetero- cyclic, and other groups; (2) substitution of the toxophore element, a b-methoxyacrylic acid fragment, for isosteric fragments of crotonic, methoxyiminoacetic, and analogous acids.Synthetic analogues of the natural MOA-inhibitors are compounds with the general formula 15. In synthetic analogues, Q usually stands for a bulky hydro- phobic substituent, which mimics a labile phenylpentadienyl residue (the backbone). This largely determines the biological activity, photostability, selectivity, and systemic properties of a compound.The rest of the molecule is a toxophore. The elements V, X, Y, and Z can vary rather independently. More or less active are esters, amides, and thioamides of appropriately substituted acrylic 57 and crotonic 58 acids, b-alkoxy-,59 b-alkylthio,60 b-ami- noacrylic acids,61 O-alkyloximes,62, 63 S-alkylthiooximes,64 and alkylhydrazones of glyoxalic acids 65 and unsaturated acids of analogous structure.Toxophore groups with different structures are also known. For example, compounds 15 devoid of ester or amide groups (R1=H, Me, X=CH2, Y=O, Z=N, V=O, R2=Me) are known to inhibit respiration.47 However, these compounds rank below esters and amides of the above-mentioned acids in activity.After the discovery of a large variety of compounds that are not derivatives of b-methoxyacrylic acid but are able to inhibit cell respiration, the term `synthetic MOA-inhibitors' is no longer a correct definition for this group of biologically active substances. It was therefore suggested that such compounds be called `syn- thetic strobilurin analogues'.66 The pioneering studies in the field of synthesis of strobilurin analogues were aimed at increasing their photostability.The effects of structural changes on the photostability of strobilurin analogues were considered by some authors more comprehen- sively.31, 56 Greenhouse studies of oudemansin activity revealed that the presence of a b-methoxyacrylate group did not decrease the photochemical stability of these compounds.31, 56 The simplest strobilurin A analogue 16 with partially hydro- genated double bonds and the oudemansin A analogue 17 did not manifest any useful activity in hothouse studies, although com- pound 17 shows fungicidal properties on agar cultures of fungi.31 One of the first synthetic analogues of natural MOA-inhib- itors was the so-called MOA-stilbene 18.57, 67 ± 70 This compound possesses fungicidal and insecticidal properties and surpasses its natural prototypes in activity.1 X-Ray analysis of a monocrystal of compound 18 showed that the planar methyl b-methoxyacry- late and stilbene fragments are nearly perpendicular to each other as is the case with the strobilurin molecule.31 Stilbene 18, like strobilurin A, is a potent inhibitor of mitochondrial respiration (I50 for stilbene is 0.04 mM, that for strobilurin A is 0.11 mM31).This compound is photochemically more stable than its natural analogue (t50 for irradiation in thin film is 3 min) and less volatile.56 The photostability of MOA-stilbene increases consid- erably in the presence of photostabilisers.56 In the series of b-alkoxy-a-arylacrylates 19, even methyl (E)-b- methoxy-a-phenylacrylate 19a (without substituents in the ben- zene ring) manifests a pronounced fungicidal activity (Table 2). N N O O CN MeOOC OMe 13 14 O Me N MeOOC OMe Y R1X Z Q VR2 X=O, S, NR3; Y=O, S; Z=CH, N; V=CR4, O, S, NR5; R17R5=H, Alk; Q =Ar, Het, ArX7, HetX7, etc. 15 MeOOC OMe 16 MeOOC OMe Me 17 538 V V Zakharychev, L V KovalenkoOn the whole, phenylacrylates 19 are more active for R1=R2=Me than in those cases where R1 or R2 are represented by other alkyl substituents or hydrogen atoms.E-Isomers are usually more active than Z-isomers; it is probable that the latter, being very weak inhibitors of mitochondrial respiration, acquire the fungicidal activity following isomerisation.31 Substituents in the nucleus can both strongly enhance the activity and decrease it.Compounds having a bulky substituent similar to the styryl fragment in the strobilurin molecule at position 2 of the phenyl ring are usually more active. The activity of naphthylacrylates strongly depends on the position of a toxophore: a-naphthylacry- late 20 (X=H) is much more active than the b-isomer 21 (see Table 2). Although substituted MOA-stilbenes manifest high fungicidal activity in hothouses, in field tests they are moderately active due to their low photostability.1 However, owing to their availability MOA-stilbenes, together with natural compounds, have found wide use as tools in biochemical studies.Of particular interest are stilbenes 22, which are analogues of strobilurin E and possess antiviral and antitumour properties.71 In order to increase the photostability of such compounds, it was suggested to hydrogenate the double bond between two benzene rings 72 and to replace it by other spacer groups, such as CH2O, SCH2, O, S,57, 73 ± 75 SO, SO2,57 CH2SO, CH2SO2,76 etc.56, 77 Methyl (E)-methoxyimino-[2-(2-methylphenoxymethyl)- phenyl]acetate (14) (BAS-490F), an experimental fungicide pro- duced by BASF and possessing a protective, curative, and eradicating activities is an example. Its standard doses vary from 50 to 350 g ha71 (see Ref. 66). Acrylate 23a, a diphenyl ether derivative, is also highly efficient against a great number of fungi.57, 78 This compound is sufficiently resistant to light (t50 for irradiation in thin film is 30 h).56 In addition, this compound possesses systemic fungicidal activity.1, 54 Compound 23b, which contains three benzene rings, is even more active,54, 77 but is devoid of systemic properties.Besides, a serious disadvantage of this derivative is that it caused severe damages to some cultures in field tests.54 The reasons for phytotoxicity of such compounds have not been finally estab- lished.79 A change in the position of a phenoxy group in the molecule results in a sharp decrease in the fungicidal activity.Thus the isomer 23c is a weak fungicide. In heterocyclic analogues of the ester 23b, the high level of fungicidal activity is often associated with their ability to migrate in the plant. However, the biological properties of compounds change in an unpredictable manner depending on the number and position of heteroatoms in the rings: sometimes this activity drops drastically, and the action spectrum is narrowed or the phytotoxicity is enhanced.54 Among derivatives of the heterocyclic series, methyl (E)-2-{2-[6-(2-cyano- phenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate (13) (ICI-A5504), turned out to be the most efficient. This compound is an experimental fungicide and exerts systemic and translaminar activities against asco-, basidio-, deutero-, and oomycetes.80, 81 MeOOC OMe 18 R1OOC OR2 X 19a7j X=H, Ph, Hal, Alk etc.X MeOOC OMe MeOOC OMe 20 21 22 O O MeOOC OMe O O R3 R2 R1 R=H (a), 3-PhO (b), 4-PhO (c). O MeOOC OMe R 23a7c Table 2. Comparative activities of acrylates 19, 20, and 21.57 Com- R1 R2 X Subject of study a pound P.r.b E.g.c V.i.d P.o.e C.a.f P.v.g E-19a Me Me H 4 4 4 0 2 4 Z-19b Me Me H 2 0 0 0 0 0 E-19c Et Me H 0 0 0 0 0 4 E-19d Me Et H 0 0 1 0 0 0 E-19e Et Et H 0 0 0 0 0 1 E-19f Me Me 2-(E-PhCH=CH) 4 4 4 3 h 4 4 E-19g Me Me 3-(E-PhCH=CH) 0 0 0 0 0 3 E-19h Me Me 4-(E-PhCH=CH) 0 0 0 0 0 2 Z-19j Me Me 2-(¦-PhCH=CH) 4 4 4 3 h 4 4 E-19k Me Me 2-Cl 0 4 0 0 4 4 20 7 7 H 4 3 4 4 4 4 21 7 7 7 3 0 2 2 7 4 Note.The infected plants were sprayed with emulsions (the concentration of active substance was 100 ppm). a Designations: 4�undamaged plants; 3�up to5%damage; 2�6%± 25% damage; 1�26% ±59% damage; 0�60% ± 100% damage in comparison with nontreated plants. b P.r.ìPuccinia recondita (on wheat); c E.g.ìErysiphe graminis f. sp. hordei (on barley); d V.i.ìVenturia inaequalis (on apple- tree); e P.o.ì Pyricularia oryzae (on rice); f C.a. ì Cercospora arachidicola (on peanut); g P.v. ì Plasmopara viticola (on grapes); h spraying with an emulsion with the concentration of the active substance of 25 ppm. Natural compounds of the strobilurin series and their synthetic analogues as cell respiration inhibitors 539In a series of compounds of the formula 24 the toxophoric group of which is attached to an aryl or a heterocyclic residue not directly, but through O and S atoms or through NR or CR2 groups, the derivatives with meta-substituents in the aromatic ring display the highest activity (Table 3).A vast array of structurally diverse synthetic analogues of strobilurin are presently known. Thus biological activities of a great number of compounds with the general formula 15 (whereQ designates various substituted carbo- and hetero-cycles), such as derivatives of indole,83 pyrazole,84 ± 86 pyrrole 87 ± 89 (methoxya- crylate derivatives of pyrrole have been described in the review 55), triazine,90 dibenzo[b,e]-1,4-dioxin,91 thiazole,92 ± 94 isoxazole,92 pyridine,95 ± 98 pyrimidine,99 etc., have been assayed.The structur- e ± activity relationships for this group of compounds are nearly the same as those for a-phenylacrylates.Thus the activity of monocyclic compounds increases as certain substituents are introduced at the position adjacent to the toxophoric group. As in the case of a-phenylacrylates, the b-styryl fragment and the radicals having similar bulk and shape, enhance the activity.Methyl methoxyacrylates derived from amides of aromatic and heteroaromatic acids 25 possess fungicidal properties.100, 101 In some cases, esters 26 manifested higher activity than the corresponding derivatives of glyoxylic acid.102 Fungicidal activity was also found in compounds 27 with an unusual structure of the toxophoric group.103, 104 Certain oximes 28105, 106 and 29 107 ± 119 show enhanced fungi- cidal activity in comparison with their analogues that do not contain an oxime fragment in their backbone.The spatial isomer- ism about the C=N bond may be crucial for the manifestation of activity of these compounds.120 In the series of synthetic analogues of the natural MOA- inhibitors, the experimental fungicides ICI-A5504 (13) and BAS- 490F (14) have been studied in most detail.Like the natural compounds, these analogues bind with the Qo-centre of cyto- chrome b and actively inhibit cell respiration.66, 80, 81, 121, 122 Both substances have low toxicity in rats (LD50>5000 mg kg71). The reason for such a selectivity are the peculiarities of their biokinetic properties rather than their selective affinity for the molecular target.Thus the inhibition constants of complex III from various living organisms were determined using various methoxyacrylates and their analogues.121 The pI50 values for MOA-stilbene (18), BAS-490F (14), and myxothiazol (2) are given in Table 4. The oxidase from cereals was the least sensitive to all of the inhibitors tested. The difference in the pI50 values is less pronounced for enzymes from other species.Structural changes in compounds that enhance their activity against fungi, induced analogous changes in the activities against other species. Analysis of biokinetic properties of a series of compounds revealed that such properties as absorption, transport, and metabolism are more important for their selective toxicity. For example, the high activity of compound 14 against surface-located powdery mildew is due to its diffusion through the gaseous phase and the low level of its absorption by leaves in which it is rapidly metabolised.In contrast, an analogue of this preparation in which the ester group is replaced by the methylamide group is expected to be highly active against endoparasitic fungi.123 A recent report deals with N-methyl-(E)-methoxyimino-(2- phenoxyphenyl)acetamide (code SSF-126) 124 (30), a new prepa- ration of this series, which possesses a broad spectrum of fungicidal activities and is being tested.It is assumed that the Y MeOOC OMe X 24a7i T is the toxophore R=Ar, Het. O N R Me MeOOC OMe 25 X R 26 X 27 O T R2 R1ON 28 Het is oxazol-2-yl, 1,3,4-oxadiazol-2-yl, etc. NOMe MeOOC NOMe Het T 29 O N R2 R1 Table 3.Activities of acrylates 24a ± i.82 Com- X Y Object of study a pound P.r. E.g. V.i. P.o. C.a. P.v. 24a 2-Ph CH2 1 0 3 0 0 0 24b 3-Ph CH2 4 3 4 0 3 4 24c 4-Ph CH2 0 0 0 2 0 0 24d H NMe 0 4 4 4 1 4 24e 3-Ph NMe 4 4 1 3 4 4 24f H O 0 0 2 0 0 0 24g 3-Ph O 4 0 4 3 3 4 24h H S 0 0 0 3 0 3 24i 3-Ph S 4 4 4 2 4 4 Note. The infected plants were sprayed with emulsions (the concentration of the active substance of 100 ppm).a For designations see footnote to Table 2. Table 4. pI50 Values for inhibitors with respect to complex III of various organisms.121 Compound Yeast Botrytis Fly Rat Corn MOA-stilbene (18) 7.3 7.4 7.8 7.6 6.3 BAS-490F (17) 7.9 7.8 6.9 6.3 6.0 Myxothiazole (2) 8.0 7.9 8.3 7.8 6.5 54arychev, L V Kovalenkomain area of its application is the fight against paddy rice diseases.125 Some strobilurin analogues can also bind with the b6 f-com- plex of chloroplasts in the Qo or Qi centre.Thus compounds 18 and 23a,b bind with the Qi centre, whereas compounds 24b,e,g, with the Qo centre. No binding was observed for compounds 20 (X=Ph) and 25 (Ar=2- or 4-ClC6H4).126 V. Synthesis of strobilurin analogues Syntheses of b-alkoxyacrylates are usually based on esters of substituted acetic acid 31.Condensation with formates in the presence of a base gives aldehydoesters 32.31, 54, 56, 81 The reaction with dimethylformamide dimethylacetal gives compounds 34, which also yield aldehydoesters 32 following hydrolysis or alco- holysis. Alkylation of enolates (compounds 32) results in b-alkox- yacrylates 33 (Scheme 1).Scheme 1 b-Methoxyacrylates are often synthesised from glyoxalates 36 by the Wittig reaction 31, 56 (Scheme 2). Scheme 2 Synthesis of the corresponding derivatives of acetic (31) or glyoxylic (36) acids is carried out by conventional methods. Alkylation of nitrogen-containing heterocycles with methyl bromoacetate in the presence of bases was used, e.g., in the synthesis of pyrrole derivatives 37.55, 58 Reaction of benzyl halides and their ortho-substituted deriv- atives 38 with sodium cyanide results in phenylacetonitriles. Their subsequent hydrolysis and esterification yield esters of the corre- sponding phenylacetic acids.128 Esters of glyoxylic acids 40 were obtained by oxidation of alkyl mandelates 39 with sodium hypochlorite.129 Alternatively, they are synthesised from isatins 41.130 To this end, substituted isatins are hydrolysed with a solution of NaOH on heating; the amino group is then substituted for iodine by treatment of the corresponding diazo compound with potassium iodide and copper bronze.The resulting substituted o-iodophe- nylglyoxylic acids 42 are esterified with methyl chloroformate in the presence of triethylamine.In compounds 43, iodine can be exchanged for other substituents to give a wide range of sub- stituted glyoxylic acids. Synthesis of compounds of the type 31 or 36 was also carried out by other methods.65, 74, 131 ± 133 Thus MOA-stilbene 18 was synthesised from o-bromobenzaldehyde. This was coupled with benzylmagnesium bromide. The resulting alcohol was dehydrated in the presence of an acid to give 2-bromostilbene (44).The Grignard reagent prepared from the latter was slowly added to an excess of dimethyl oxalate. The ester arylglyoxylic acid 45 that formed was introduced into the Wittig reaction with (methox- ymethylene)triphenylphosphorane. The target product 18 con- tained a small admixture of the Z,E-isomer.57 Yet another method to obtain compound 18 is the bromina- tion of (E)-2-(2-methylphenyl)-b-methoxyacrylate (46) with N-bromosuccinimide. The reaction of the resulting bromomethyl derivative with trialkyl phosphite and subsequent condensation of methoxyacrylate 47 with benzaldehyde by the Wittig ± Horner ± Emmons reaction results in stilbene 18.70 O N MeNHOC OMe 30 a c QCCOOR1 CHOH 32 e (a) HCOOR, NaH; (b) Me2NCH(OMe)2, Py .TsOH; c) H2O, H+; (d) MeOH, H+; (e) B, R2X (X=Hal, SO3OR2); R1, R2=Alk; Q=Ar, Het, ArX7, HetX7, etc. 34 Q CHCOOR1 CH(OMe)2 35 c d QCH2COOR1 31 31 QCCOOR1 HCNMe2 b 34 QCCOOR1 CHOR2 33 QCOCOOR MeOCH2P+Ph3Cl7, B7 QCCOOR CHOMe 36 N H BrCH2CO2Me, NaH DMF N CH2COOMe 37 X=H, Hal, OAr; X CH2Br (a) NaCN, EtOH, H2O; (b) HCl; (c) MeOH. 38 X CH2COOMe a, b, c X=Alk, Ar, Het.CH(OH)CO2Me X COCO2Me X NaOCl 39 40 N O O H X a b X COCO2H I 41 42 (a) 1. aq. NaOH; 2. NaNO2, H2SO4, H2O; 3. KI, Cu; (b) ClCO2Me, Et3N, CH2Cl2, 108C; X=H, Alk, AlkO, Hal, O2N, NC. X COCO2Me I 43 2-BrC6H4CHO a Br Ph b 44 (a) 1. PhCH2MgCl; 2. aq. HCl; 3. H3PO4, D; (b) 1. Mg; 2. (CO2Me)2; (c) MeOCH2P+Ph3Cl7, NaH. COCO2Me Ph 45 MeOOC OMe Ph 18 c (a) 1.NBS; 2. (RO)3P; (b) NaH, PhCHO. Me OMe MeOOC a b (RO)2PCH2 OMe MeOOC O 18 46 47 Natural compounds of the strobilurin series and their synthetic analogues as cell respiration inhibitors 5417-Substituted 1-naphthylacrylates 20 that are promising fun- gicides are synthesised in several steps from substituted benzenes.131, 134 ± 136 First, benzene or its derivative is condensed with succinic anhydride under conditions of the Friedel ± Crafts reaction. The 40-substituted 4-oxo-4-phenylbutyric acid 48 thus obtained is esterified and the keto group is reduced.After hydrolysis of the ester, 4-phenylbutyric acid 49 is converted into an acid chloride, which is further cyclised into a 7-substituted a-tetralone 50. Its condensation with methyl bromoacetate in the presence of zinc gives a mixture of isomeric methyl esters 51a,b, which is dehydro- genated with sulphur on heating to give 7-substituted methyl naphthylacetates 52.The latter are converted into the target compounds 20 by condensation with ethyl formate followed by methylation with dimethyl sulfate. It was proposed to synthesise compound 13 from methyl a-(o- benzyloxyphenyl)-b-methoxyacrylate 53,137 which is first hydro- genated to remove the protective benzyl group. The resulting phenol is introduced into the reaction with 4,6-dichloropyrimi- dine in the presence of potassium carbonate to give compound 54.An alternative procedure is as follows: 3-methoxymethylene- benzo-2-(3H)-furanone (55)132 is introduced into reaction with 4,6-dichloropyrimidine in the presence of sodium methoxide.The chlorine atom in pyrimidine 54 synthesised by either of these methods is replaced by heating with 2-cyanophenol inDMFin the presence of potassium carbonate to give compound 13. Since the synthesis of b-methoxacrylates according to Schemes 1 and 2 usually gives a mixture of E- and Z-isomers, it was proposed to use isomerisation of the double bond by treat- ment with gaseous HCl, Hg(OAc)2,138 some radical reagents (halogens, N-halosuccinimide),139 various O-, S-, C-, Hal-, or N- nucleophiles 140 to yield (E)-b-methoxyacrylates possessing higher biological activity.Methylthioacrylates 56 can be obtained from the correspond- ing b-hydroxyacrylates 57, e.g., via tosyl derivatives, which are introduced in the reaction with NaSMe.141 It was also proposed to synthesise methylthioacrylates 56 from esters of glyoxalic acids 58 by the Wittig reaction, i.e., by analogy with the synthesis of methoxyacrylates.133 Esters of acrylic and crotonic acids and their homologues are obtained in a similar way from esters of glyoxalic acids 58 by the Wittig reaction 60, 61, 142, 143 or by condensation with paraformaldehyde.60, 143, 144 Esters and amides of methoxyaminoacetic acids are prepared by the reaction of O-methylhydroxylamine with the correspond- ing esters or amides of glyoxylic acid.62, 63, 145 The target E-isomer is formed from a mixture of isomeric esters in the presence of acids.146 Similarly, alkylhydrazones of glyoxylates are obtained from alkylhydrazines.65 It was proposed to synthesise glyoxylic and methoxyimino- acetic acid amides by aminolysis of the corresponding acid chlorides,147 ± 149 esters,97, 150 ± 153 or nitriles (with subsequent deamination of the amidines formed with nitric acid).154 The synthesis of N-methylamides of substituted phenylglyoxylic acids 59 has been patented.155 The latter are used as intermediates in the synthesis of N-methylamides of methoxyiminoacetic acids.This method consists in the reaction of substituted benzoyl chlorides with methylisocyanide followed by hydrolysis of the product formed. C6H5X a b X O (CH2)2COOH 48 c X (CH2)3COOH 49 X O 50 d X CHCOOMe + 51a X CH2COOMe 51b X CH2COOMe 52 f 20 (a) AlCl3, ; (b) 1. EtOH; 2. CF3CO2H, Et3SiH; O O O 2. Me2SO4; X=H, Ph, Hal, Alk. 3. KOH, aq. EtOH; 4.HCl; (c) 1. SOCl2; 2. CF3SO3H; (d) BrCH2CO2Me, Zn, THF; (e) S, 2208C; ( f ) 1. HCO2Et, NaH; e PhCH2O MeOOC OMe 53 O CHOMe O 55 a b O N N Cl MeOOC OMe 54 c O N N PhO MeOOC OMe 13 (a) 1. H2, Pd/C; 2. 4,6-dichloropyrimidine, K2CO3, DMF; (b) NaOMe, 4,6-dichloropyrimidine; (c) 2-NCC6H4OH, K2CO3, DMF. (a) 1. TsCl; 2. NaSMe; (b) MeSCH2P+Ph3Cl7, ButOK. QCCOOMe CHOH 57 QCOCOOMe 58 a b QCCOOMe CHSMe 56 R1, R2=H, Alk.QCOCOOMe R1R2C PPh3 58 QCCOOMe CR1R2 QCCOOMe CH2 59 (CH2O)n, K2CO3, Bu4NI QCH2COOMe 542 V V Zakharychev, L V KovalenkoSome S-methylmethoxyiminothioacetates that are sometimes more active in trials than compound 14 can be obtained by hydrolysis of methoxyiminoacetic acid esters and subsequent reaction of free acids with sodium methanethiolate 156 in the presence of carbonyldiimidazole.Methyl O-esters and amides of methoxyiminothioacetic acids are obtained by the reaction of esters and amides of metoxyimino- acetic acids with Lawesson's reagent.157 ± 159 VI. Conclusion The modern approach to the synthesis of pesticides that makes use if the natural compounds for identification of biological targets and for modelling new biologically active substances is well known and has often been employed, e.g., for elaboration of pyrethroid insecticides, nereistoxin analogues, juvenoids, neon- icotinoids, 4-hydroxycoumarine derivatives, etc.Strobilurin ana- logues also provide an illustrative example of how bioisosterism can be applied for directed enhancement of properties of natural compounds. Despite high biological and particularly fungicidal activity of methoxyacrylate-type antibiotics, their application for plant protection is impeded due to their high sensitivity to light.Nevertheless, it is on the basis of the natural methoxyacrylates that synthetic agrochemical preparations with a basically new mechanism of action have been obtained. Analogues of the natural MOA-inhibitors have indisputable advantages over other systemic fungicides because of the lack of natural resistant microbial strains.Thus, ICI-A5504 (compound 13) efficiently inhibits fungi that are resistant to inhibitors of C-14- demethylase, phenylamides, dicarboxyimides, and benzimida- zoles.80 Strobilurins and their analogues constitute a large group of compounds that are hardly inferior to triazole fungicides in structural diversity.They represent a new class of plant-protecting agents that meet all the demands that are made nowadays for pesticides. Intensive studies aimed at a search for novel bio- logically active pesticides are currently under way by different manufacturers. However, these studies are still in their infancy and so far only three fungicides have been produced by ICI, BASF, and Shionogi.Probably, original products will be offered very soon by Bayer and Roussel UCLAF. Interest in this group of compounds is increasing with every passing year as can be evidenced from the number of patent applications. The place occupied by strobilurin analogues on the world pesticide market will be evident in due course. References 1. J M Clough Nat.Prod. Rep. 10 565 (1993) 2. M D Esposti,M Crimi, A Ghelli Biochem. Soc. Trans. 22 209 (1993) 3. B M Geier, U Haase, G von Jagow Biochem. Soc. Trans. 22 203 (1993) 4. R Haensel, D Weiss, B Schmidt Arch. Pharm. (Weinheim) 301 369 (1968) 5. N M Golyshin Fungitsidy (Fungicides) (Moscow: Kolos, 1993) 6. Fr. P. 1 505 738; Chem. Abstr. 70 18 900 (1969) 7. Fr. Med. P. 6638; Chem.Abstr. 74 123 689 (1971) 8. Czech. P. 136 495; Chem. Abstr. 75 4029 (1971) 9. T Anke, F Oberwinkler,W Steglich, G Schramm J. Antibiot. 30 806 (1977) 10. P Sedmera, V MusõÂ lek, F Nerud, M Vondra'c Ï ek J. Antibiot. 34 1069 (1981) 11. G von Jagow, G W Gribble, B L Trumpewer Biochemistry 25 775 (1986) 12. T Anke, G Schramm, B Schwalge Liebigs Ann. Chem. 9 1616 (1984) 13. K Beautement, J M Clough Tetrahedron Lett. 28 475 (1987) 14. W Trowitzsch, G Reifenstahl, V Wray, K Gerth J. Antibiot. 33 1480 (1980) 15. A Tredenhaden, A Kuhn, H H Peter, V Cuomo, U Giuliano J. Antibiot. 43 655 (1990) 16. T Anke, H Besl, U Mocek, W Steglich J. Antibiot. 36 661 (1983) 17. F Nerud, P Sedmera, Z Zouchova, V MusõÂ lek, M Vondra'c Ï ek Collect. Czech. Chem. Commun. 47 1020 (1982) 18.M Vondra'c Ï ek , J Vondra'c Ï ek, P Sedmera, V MusõÂ lek Collect. Czech. Chem. Commun. 48 1508 (1983) 19. S Backens, W Steglich, J BaÈ uerle, T Anke Liebigs Ann. Chem. 5 405 (1988) 20. W Weber, T Anke, B Steffan, W Steglich J. Antibiot. 43 207 (1990) 21. W Weber, T Anke, M Bross,W Steglich Planta Med. 56 446 (1990) 22. A Tredenhaden, P Hug, H H Peter J. Antibiot. 43 661 (1990) 23.S Zapf, A Werle, T Anke, D Klostermeyer, B Steffen,W Steglich Angew. Chem., Int. Ed. Engl. 34 196 (1995) 24. T Anke, H J Hecht, G Schramm,W Steglich J. Antibiot. 32 1112 (1979) 25. T Anke, A Werle,M Bross,W Steglich J. Antibiot. 43 1010 (1990) 26. W Trowitzsch, G HoÈ fle, W S Sheldrick Tetrahedron Lett. 22 3829 (1981) 27. K Gerth, H Irschik, H Reichenbach,WTrowitzsch J. Antibiot. 33 1474 (1980) 28. G Tierbach, H Reichenbach Antimicrob. Agents Chemother. 19 504 (1981) 29. G Tierbach, H Reichenbach Biochim. Biophys. Acta 638 282 (1981) 30. B J Martin, J M Clough, G Pattenden, I R Waldron Tetrahedron Lett. 34 5151 (1993) 31. K Beautement, J M Clough, P J de Fraine, Ch R A Godfrey Pestic. Sci. 31 499 (1991) 32. G von Jagow, W F Becker Bull. Mol. Biol. Med. 7 1 (1982) 33.W F Becker, G von Jagow, T Anke, W Steglich FEBS Lett. 132 329 (1981) 34. J Subic, V Covacova, G Takacsova Eur. J. Biochem. 73 275 (1977) 35. T Ohnishi, V D Sled, N I Rudnitzky, S V Meinhardt, T Yagi, Y Hatefi, T Link, G von Jagow, A S Saribas, F Daldal Biochem. Soc. Trans. 22 191 (1993) 36. T A Link, H Wallmeier, G von Jagow Biochem. Soc. Trans. 22 197 (1993) 37. I Kucera, R Hedbavny, V Dadak Biochem.J. 252 905 (1988) 38. G von Jagow, W D Engel FEBS Lett. 136 19 (1981) 39. Yu Kamenskii, A A Konstantinov, V S Kunts, S Surkov FEBS Lett. 181 95 (1985) 40. Yu A Kamenskii, A A Konstantinov, V S Kunts, S A Surkov Biol. Membrany 2 56 (1985) 41. O V Oleskin, V D Samuilov Biokhimiya 53 1803 (1988) a 42. G von Jagow Instrum. Forsch. 9 26 (1982) 43. U Brandt, H Schraegger, G von Jagow Eur.J. Biochem. 173 499 (1988) 44. J P Di Rago, J Y Coppee, A M Colson J. Biol. Chem. 264 14543 (1989) 45. G von Jagow, P O Ljungdahl, P Graf, T Ohnishi, B L Trumpewer J. Biol. Chem. 259 6318 (1984) 46. G Thierbach, B Kunze, H Reichenbach, G HoÈ fle Biochim. Biophys. Acta 765 227 (1984) 47. T E Wiggins Biochem. Soc. Trans. 22 221 (1993) 48. J Kejda Pract.Lek. 60 26 (1980) 49. BRD P. 3 815 484; Chem. Abstr. 113 189 792 (1990) 50. P Conradt, K E J Dittmar, H Schliephacke, WTrowitzsch-Kienast J. Antibiot. 42 1158 (1989) COCl RZ 1. MeNC, 60 8C, 6 h 2. CaCO3, Me2CO±H2O RZ O NHMe O 59 R=H, Alk, Hal, Ar, Het; Z=CH2, O, S, CHOH, CO, CH2CH2, CH=CH. QCCOOH NOMe QCCOSMe NOMe 1. Im2CO 2. NaSMe Im2CO= NCON N N Natural compounds of the strobilurin series and their synthetic analogues as cell respiration inhibitors 54351.Sh Itoh,M Iwaki FEBS Lett. 250 441 (1989) 52. Sh Itoh,M Iwaki, in Current Research on Photosynthesis (Proceedings of the 8th International Conference on Photosynthesis) (Dordrecht: Kluwer Academic, 1990) Vol. 2, p. 651; Chem. Abstr. 115 68 677 (1991) 53. W Pfefferle, H Anke, M Bross,W Steglich Agric.Biol. Chem. 54 1381 (1990) 54. J M Clough, D A Evans, P J de Fraine, T E M Fraser, Ch R A Godfrey, D Youle Am. Chem. Soc. Symp. Ser. 551 37 (1994) 55. K Beautement, J M Clough, P J de Fraine, Ch R A Godfrey Am. Chem. Soc. Symp. Ser. 584 326 (1995) 56. J M Clough, P J de Fraine, T E M Fraser, Ch R A Godfrey Am. Chem. Soc. Symp. Ser. 504 372 (1992) 57. Eur. Appl. 178 826; Chem.Abstr. 105 78 670 (1986) 58. Eur. Appl. 206 523; Chem. Abstr. 106 98 100 (1987) 59. Eur. Appl. 310 954; Chem. Abstr. 111 92 323 (1990) 60. Eur. Appl. 348 766; Chem. Abstr. 113 5946 (1989) 61. BRD Appl. 3 705 389; Chem. Abstr. 109 230 562 (1988) 62. Eur. Appl. 254 426; Chem. Abstr. 109 37 610 (1988) 63. BRD Appl. 3 623 921; Chem. Abstr. 108 182 217 (1988) 64. Eur. Appl. 336 211; Chem.Abstr. 112 216 453 (1990) 65. Eur. Appl. 331 061; Chem. Abstr. 112 118 461 (1990) 66. E Ammermann, G Lorentz, K Schelberger, B Wenderoth, H Sauter, C Rentzea Brighton Crop. Prot. Conf.ìPests. Dis. (1) 403 (1992) 67. Eur. Appl. 474 042; Chem. Abstr. 117 69 578 (1992) 68. BRD Appl. 3 519 280; Chem. Abstr. 106 101 890 (1987) 69. BRD Appl. 4 029 192; Chem. Abstr. 116 230 226 (1992) 70.Eur. Appl. 203 606; Chem. Abstr. 106 101 890 (1987) 71. BRD Appl. 3 905 911; Chem. Abstr. 114 81 853 (1991) 72. BRD Appl. 3 545 318; Chem. Abstr. 107 193 008 (1987) 73. BRD Appl. 3 620 860; Chem. Abstr. 109 6228 (1988) 74. Eur. Appl. 278 595; Chem. Abstr. 109 210 725 (1988) 75. BRD Appl. 3 519 282; Chem. Abstr. 106 80 390 (1987) 76. Eur. Appl. 335 519; Chem. Abstr. 112 231 326 (1990) 77.Eur. Appl. 307 103; Chem. Abstr. 111 114 869 (1989) 78. Eur. Appl. 307 101; Chem. Abstr. 111 114 868 (1989) 79. T E Wiggins, B J Jager, J D Scholes. Biochem. Soc. Trans. 22 69S (1993) 80. J R Godwin, V M Anthony, J M Clough, Ch R A Godfrey Brighton Crop. Prot. Conf.�Pests. Dis. (1) 435 (1992) 81. J A Frank, P L Sanders Brighton Crop. Prot. Conf. �Pests. Dis. (2) 871 (1992) 82.Eur. Appl. 212 859; Chem. Abstr. 107 6939 (1987) 83. Eur. Appl. 274 825; Chem. Abstr. 110 57 507 (1989) 84. Jpn. Appl. 05 201 980; Chem. Abstr. 120 134 461 (1994) 85. Eur. Appl. 571 326; Chem. Abstr. 120 164 166 (1994) 86. Eur. Appl. 483 851; Chem. Abstr. 117 90 275 (1992) 87. Eur. Appl. 273 572; Chem. Abstr. 109 190 240 (1988) 88. Br. Appl. 2 198 726 ; Chem. Abstr. 109 170 228 (1988) 89.Eur. Appl. 532 126; Chem. Abstr. 119 49 381 (1993) 90. Eur. Appl. 503 436; Chem. Abstr. 118 22 256 (1993) 91. Eur. Appl. 270 252; Chem. Abstr. 110 39 009 (1989) 92. Eur. Appl. 4 223 357; Chem. Abstr. 120 323 576 (1994) 93. Eur. Appl. 508 901; Chem. Abstr. 118 147 552 (1993) 94. Eur. Appl. 509 857; Chem. Abstr. 118 124 528 (1993) 95. Eur. Appl. 312 243; Chem.Abstr. 111 232 578 (1989) 96. Eur. Appl. 312 221; Chem. Abstr. 112 7376 (1990) 97. BRD Appl. 4 328 385; Chem. Abstr. 122 239 551 (1995) 98. Jpn. Appl. 02 121 970; Chem. Abstr. 113 191 173 (1990) 99. Eur. Appl. 634 405; Chem. Abstr. 122 265 393 (1995) 100. Eur. Appl. 178 808; Chem. Abstr. 105 37 476 (1986) 101. Eur. Appl. 402 246; Chem. Abstr. 114 164 213 (1991) 102. Eur. Appl. 407 891; Chem.Abstr. 115 8319 (1991) 103. WO PCT 94-22 884; Chem. Abstr. 122 31 529 (1995) 104. WO PCT 96-25 406; Chem. Abstr. 125 221 882 (1996) 105. Eur. Appl. 386 561; Chem. Abstr. 114 228 542 (1991) 106. Eur. Appl. 579 124; Chem. Abstr. 122 55 720 (1995) 107. Eur. Appl. 463 488; Chem. Abstr. 116 151 325 (1992) 108. Eur. Appl. 472 300; Chem. Abstr. 116 255 332 (1992) 109. WO PCT 92-18 487; Chem.Abstr. 118 124 558 (1993) 110. WO PCT 92-18 494; Chem. Abstr. 118 124 548 (1993) 111. WO PCT 92-13 830; Chem. Abstr. 118 6744 (1993) 112. Jpn. Appl. 05 201 946; Chem. Abstr. 119 264 648 (1993) 113. Jpn. Appl. 05 294 948; Chem. Abstr. 120 270 454 (1994) 114. WO PCT 94-08 968; Chem. Abstr. 121 108 822 (1994) 115. WO PCT 94-08 948; Chem. Abstr. 121 157 307 (1994) 116.WO PCT 94-14 322; Chem. Abstr. 121 127 881 (1994) 117. WO PCT 94-14 761; Chem. Abstr. 121 133 714 (1994) 118. Eur. Appl. 370 629; Chem. Abstr. 114 23 556 (1991) 119. WO PCT 90-07 493; Chem. Abstr. 114 6032 (1991) 120. M Watanabe, T Tanaka, Sh Yokoyama, H Kobayashi Biochem. Soc. Trans. 22 67S (1993) 121. F Roehl, H Sauter Biochem. Soc. Trans. 22 63S (1993) 122. T E Wiggins, B J Jager Biochem. Soc. Trans. 22 68S (1993) 123. H Kohle, R E Gold, E Ammermann Biochem. Soc. Trans. 22 65S (1993) 124. Eur. Appl. 398692 Evropa; Chem. Abstr. 114 246946 (1991) 125. H Hayase, T Kataoka, M Masuko,M Niikawa,M Ichinari, H Takenaka, T Takahashi, Y Hayashi, R Takeda Am. Chem. Soc. Symp. Ser. 584 343 (1995) 126. P R Rich, S A Madgwick, T E Wiggins Biochem. Soc. Trans. 22 217 (1993) 127. WO PCT 91-07 385 Chem. Abstr. 115 135 691 (1991) 128. Eur. Appl. 260 832; Chem. Abstr. 109 88 201 (1988) 129. Eur. Appl. 422 597; Chem. Abstr. 115 158 721 (1991) 130. Br. Appl. 2 273 499; Chem. Abstr. 121 204 959 (1994) 131. Eur. Appl. 267 734; Chem. Abstr. 109 88 207 (1988) 132. WO PCT 92-08 703; Chem. Abstr. 117 131 219 (1992) 133. Eur. Appl. 244 077; Chem. Abstr. 109 37 611 (1988) 134. Eur. Appl. 538 097; Chem. Abstr. 119 116 984 (1993) 135. Eur. Appl. 566 455; Chem. Abstr. 120 163 737 (1994) 136. WO PCT 94-23 576; Chem. Abstr. 122 25 866 (1995) 137. Eur. Appl. 382 375; Chem. Abstr. 114 81 870 (1991) 138. Br. Appl. 2 248 613; Chem. Abstr. 117 111 148 (1992) 139. Br. Appl. 2 248 614; Chem. Abstr. 117 111 149 (1992) 140. Br. Appl. 2 248 615; Chem. Abstr. 117 111 150 (1992) 141. BRD Appl. 3 732 093; Chem. Abstr. 111 214 234 (1989) 142. BRD Appl. 3 816 577; Chem. Abstr. 113 5943 (1990) 143. Br. Appl. 2 225 011; Chem. Abstr. 113 190 939 (1990) 144. BRD Appl. 3 821 503; Chem. Abstr. 113 5946 (1990) 145. BRD Appl. 3 827 361; Chem. Abstr. 113 131 748 (1990) 146. BRD Appl. 4 042 272; Chem. Abstr. 117 170 986 (1992) 147. BRD Appl. 4 030 038; Chem. Abstr. 116 255 338 (1992) 148. Eur. Appl. 535 928; Chem. Abstr. 119 159 886 (1993) 149. Eur. Appl. 617 011; Chem. Abstr. 122 9681 (1995) 150. Eur. Appl. 468 775; Chem. Abstr. 116 173 786 (1992) 151. Eur. Appl. 596 254; Chem. Abstr. 121 230 489 (1994) 152. BRD Appl. 4 318 917; Chem. Abstr. 122 80 889 (1995) 153. Jpn. Appl. 04 182 461; Chem. Abstr. 118 59 429 (1993) 154. Eur. Appl. 644 183; Chem. Abstr. 122 265 042 (1995) 155. Eur. Appl. 547 825; Chem. Abstr. 119 159 900 (1993) 156. BRD Appl. 3 938 054; Chem. Abstr. 115 158 731 (1991) 157. WO PCT 94-26 700; Chem. Abstr. 122 160 286 (1995) 158. Eur. Appl. 432 503; Chem. Abstr. 116 58 972 (1992) 159. Eur. Appl. 528 681; Chem. Abstr. 119 8509 (1993) a�Biochemistry (Engl. Transl.) 544 V V Zakharychev, L V Kov