Fungicidal -Methoxyacrylates - ACS Publications - American Chemical

Chapter 34

Fungicidal β-Methoxyacrylates

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From Natural Products to Novel Synthetic Agricultural Fungicides John M. Clough, Paul J. de Fraine, Torquil E. M. Fraser, and Christopher R. A. Godfrey ICI Agrochemicals, Jealott's Hill Research Station, Bracknell, Berkshire RG12 6EY, United Kingdom A knowledge of the structures and properties of the fungicidal natural products strobilurin A and oudemansin A has led to the discovery of a new class of agricultural fungicides containing the (E)-methyl β-methoxyacrylate group with high levels of broad spectrum fungicidal activity and a good persistence of effect.

The quest for new agricultural fungicides is motivated by several important factors. Firstly, it is becoming increasingly desirable to replace existing products with compounds of lower toxicity to non-target species and with acceptable levels of persistence in the environment. Secondly, in order to remain competitive, agrochemical companies are obliged to look for new compounds which show marketable advantages over existing products in terms of efficacy and breadth of spectrum. These chemicals can often be protected with patents which prevent access by competitors for a limited period. Finally, the development of fungicides with novel modes of action is an important strategy in the search for ways to overcome cross-resistance to established products. The discovery of new fungicide leads has been achieved in several different ways, including the random screening of compounds, the design of inhibitors of vital biochemical processes and the exploitation of loop-holes in competitors' patents. Natural products with fungicidal activity represent a pool of structurally diverse compounds which can offer the chemist an attractive starting point for synthesis. However, with a few notable exceptions [such as pyrrolnitrin (/)], examples which have led to the development of new fungicides are rare. This account describes how a consideration of the chemical and physical properties of the natural products strobilurin A and oudemansin A has led to the discovery of a new class of synthetic fungicides containing the (£)-methyl β-methoxyacrylate group with high levels of broad spectrum activity and a good persistence of effect. A more detailed account of the initial stages of this work has recently been published (2). 0097-6156/92/0504-0372$06.00/0 © 1992 American Chemical Society

In Synthesis and Chemistry of Agrochemicals III; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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Natural β-Methoxyacrylates as Leads for Synthesis Strobilurin A and oudemansin A (Figure I) are the simplest members of a family of fungicidal natural products (now totalling sixteen compounds) which contain the (£)-methyl β-methoxyacrylate group, most of which are derived from the mycelia of various Basidiomycete fungi of European origin. We first became interested in these compounds in 1981 following a publication by Steglich and co-workers, which drew together for the first time the structures of three β-methoxyacrylates (strobilurins A and Β and oudemansin A) and the related compound myxothiazol (J). In spite of the obvious structural differences of myxothiazol, these compounds all share a common mode of action and can therefore be said to be biochemically equivalent. Indeed, it has now been established that they inhibit fungal respiration by binding strongly to a spécifie site on cytochrome b, thereby preventing electron transfer between cytochromes b and c The modest fungicidal activity reported for these compounds, coupled with their potentially resistance-breaking mode of action, combined to make this an attractive area for synthesis. Furthermore, a knowledge of the mode of action meant that it would be possible to set up an in vitro assay which could be used to direct the synthesis of analogues. The potential of a respiration inhibitor for mammalian toxicity was also recognised at the outset, but this was not considered to be a fatal negative at this early stage. Later results supported this conclusion (see below). However, before committing ourselves to any synthetic work, we felt that it was necessary to confirm the reported fungicidal activity of the natural products on our own biological screens. Although we were initially unable to obtain a sample of strobilurin A, Prof. Anke (University of Kaiserslautern, FRG) and Dr. Reichenbach (GBF, Braunschweig, FRG) kindly furnished us with samples of oudemansin A and myxothiazol, respectively. Both compounds subsequently showed good in vivo activity in the glasshouse at 33ppm against a range of commercially important fungi. v

The Total Synthesis of Strobilurin A In view of its perceived importance as the simplest member of the group, the decision was then taken to prepare a sample of strobilurin A. Initial work aimed at the preparation of the compound with the published (all-E)- configuration established that the structure of the natural product had been incorrectly assigned and that the correct assignment was probably (£,Z,E), as shown in Figure I. The geometry was finally confirmed by an unambiguous synthesis starting from a dienoate of known configuration (4). Conformational analysis of the structure of strobilurin A using a combination of molecular mechanics and molecular orbital calculations showed its minimum energy conformation to be comprised of two planar portions (the phenylpentadienyl and the β-methoxyacrylate groups) positioned orthogonal to each other. Interestingly, although the central unit of oudemansin A is rather different, in that it has two adjacent chiral sp centres in place of the (Z)-olefinic bond of strobilurin A, the published single crystal X-ray structure indicates that the overall shape is very similar (5). It is therefore not necessary to postulate that oudemansin A is a biological precursor (via loss of methanol) to strobilurin A. By contrast, (û//-£)-strobilurin A is radically 3



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different in terms of overall shape, thus providing further evidence for the (£,Z,E)-geometry of the natural product. With a sample of strobilurin A in hand, we were able to test for fungicidal activity. The result turned out to be very disappointing, bearing in mind the earlier results obtained for oudemansin A and myxothiazol, since the compound was essentially inactive against fungi growing on whole plants in the glasshouse. By contrast, in vitro tests carried out in subdued light on fungi growing on agar were positive and biochemical studies using mitochondria isolated from beef heart confirmed that the compound was a potent inhibitor of respiration. Following laboratory tests using thin films of the compound on glass plates and a xenon lamp to simulate sunlight, we concluded that photochemical instability was the reason for the inactivity of strobilurin A in vivo. Later work established that loss through volatilisation was also a contributing factor. Analogues of Strobilurin A and Oudemansin A This information prompted us to pursue the synthesis of analogues with improved photostability. It seemed reasonable that the characteristic β-methoxyacrylate group, present in virtually all of the natural products, was important for activity ("the toxophore") and that the rest of the molecule (the phenylpentadienyl moiety in the case of strobilurin A) might be functioning as a "carrier group" for the toxophore and so would be amenable to further modification. If this was the case, we felt that it should be possible to replace the carrier group with other lipophilic groups of greater photochemical stability. The activity of oudemansin A reassured us that the β-methoxyacrylate group was unlikely to be of limiting photochemical stability. Figure II illustrates some of the compounds which were made to test this hypothesis. The first idea was to remove some or all of the unsaturation in the carrier group. The resulting compounds 1 and 2 were clearly flexible enough to adopt the shape of the natural products, but neither compound was active in the glasshouse (although the styrene 2 did show some activity in vitro). A second approach was to replace the central unit with a relatively stable amide bond to produce an analogue such as 3. In this case, the conformer required to mimic the shape of the natural products (as shown) was calculated to be the preferred one. This was more successful in terms of observed in vivo activity, but scope for further structural modification turned out to be limited. Of far greater promise was the idea of freezing the geometry of the potentially labile (Z)-double bond of strobilurin A by fixing it within a benzene ring. The resulting stilbene 4 was readily prepared and was shown to have very good broad spectrum activity in the glasshouse (6). Consistent with this result was the improved photostability of this compound in comparison with strobilurin A in our laboratory tests. The results of the tests indicated that strobilurin A decomposed very rapidly in artificial sunlight [photochemical T^ (film) = 1 minute] , whereas stilbene 4 was significantly (but not dramatically) longer lived [photochemical T^ (film) = 3 minutes] (Figure ΙΠ; T is the time taken for the loss of the first 50% of the material under test). This also supported the idea that the reduced volatility of the stilbene 4 with respect to the natural product was also contributing to its much better performance in the glasshouse. 50


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C0 Me 2

Revised

Geometry

X=Y=H, Strobilurin A X=MeO, Y=C1, Strobilurin Β


OMe

Myxothiazol Figure I : Natural Fungicidal Derivatives of β-Methoxyacrylic

Figure II : Analogues of Strobilurin A and Oudemansin A


Acid

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Disappointingly, limited trials with the stilbene 4 showed that it was much less active in the field than was forecast from the glasshouse studies and this was attributed to the fact that its photostability was still less than ideal. The incorporation of an adjuvant which could function as an ultraviolet filter in the formulation was consequently examined as a possible means of further stabilising the stilbene 4 under field conditions. Of about 10 such compounds tried, Tinuvin 327 was found to be the most effective at increasing the stilbene's persistence in the laboratory (Figure HI), but translation of this effect to the field proved to be impracticable. Further Photochemical Degradation Studies Clearly, the addition of a filter was not the way forward and so we looked in more detail at the fate of the stilbene 4 on irradiation. Since the use of thin films gave no identifiable products, we turned our attention to the photolysis of solutions of the compound in dichloromethane. Under these conditions, irradiation with a "Hanau suntest" lamp led to the formation of the naphthalene derivative 5, which is presumably formed via the electrocyclic process shown in Figure IV. This result suggested the synthesis of analogues such as 6 and 7 possessing extra substituents designed to disfavour the conformation required for decomposition by this pathway. In fact, the dichlorinated analogue 6 was even less stable than the parent stilbene 4 and poorly active, whereas the methylated derivative 7, although more stable photochemically, suffered a significant drop in activity when compared to stilbene 4. A far better approach was again to freeze the olefinic bond of the stilbene in the required (E)-configuration within a benzene ring. We therefore prepared the corresponding phenyl substituted naphthalene derivative 8, which proved to be much more stable to light and highly fungicidal (7). Replacement of the Styryl Group with a Phenoxy Group A much simpler idea, however, both conceptually and synthetically, was to replace the offending styryl group of stilbene 4 with groups which could not participate in the observed decomposition pathway. In practice, many different side-chains can be used (see Figure V, for example) and in some cases fungicidal activity is substantially improved. More surprising was the observation that the readily prepared diphenyl ether 9 (Figure VI) (6), which maps much less effectively onto strobilurin A than the stilbene 4, showed dramatically increased stability to light [photochemical T^ (film) = 30 hours] and good levels of fungicidal activity against a range of commercially important and taxononomically diverse fungi (6). Some of the key physical properties of the diphenyl ether 9 are shown in Figure VI, while Table I shows the results of 24 hour protectant tests (foliar spray/root drench) vs. Puccinia recondita (brown rust on wheat, a Basidiomycete), Plasmopora viticola (vine downy mildew, a Phycomycete), Venturia inaequalis (apple scab, an Ascomycete) and Pyricuiaria oryzae (rice blast, a Deuteromycete). In the last case the observed activity was as good as the commercial standard. Prior to field testing, the diphenyl ether 9 was also put through a battery of simple tests to detect mammalian toxicity and the results were very favourable. The acute oral toxicity (median dose, male and female rat) was greater than SOOmgkg" and 1



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Q


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I

1

1

1

1

1

1

1

1

Ι

Ο

10

20

30

40

50

60

70

80

90

TIME (MINUTES) Figure III : Photochemical Degradation of Strobilurin A and Stilbene 4

Figure IV : Photochemical Degradation of Stilbene 4



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Τ so (film) = 30 hours 9 Figure V : Replacements for the Styryl Group



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OK

Cl" K0 C 2

55%

i,

BuLi,

ii,

Me0 C.C0 Me, THF 2

Et 0; 2

2

i, C u C l , Δ ; H, H 3 0

+

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;

iii, M e O H , H

+

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2

3

2

4

OMe

Melting Point = 41-3 C Log Ρ (octanol/water) = Water-Solubility

3.3

(room temp.) = 30ppm

Figure V I : Synthesis of Diphenyl Ether 9


SYNTHESIS AND CHEMISTRY O F A G R O C H E M I C A L S


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III


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the compound was neither a strong skin sensitiser nor a skin irritant. Furthermore, it gave a negative result in the Ames' test. In the field, the diphenyl ether 9 showed encouraging activity at 750gha \ equalling the standards (at their commercial rates) against Puccinia recondita, Erysiphe graminis (powdery mildew on wheat and barley) and Septoria nodorum (glume blotch on wheat) and showing some activity against Rhynchosporiwn secalis (leaf blotch on barley). However, in several (but not all) of the trials some crop damage was also observed. These promising results represented both the culmination of our efforts to improve the photostability of the natural products and the starting point for all of our subsequent work on the optimisation of physical and biological properties of synthetic analogues in the diphenyl ether area. The main objectives of this work were to attain higher levels of activity in the field and to reduce the phytotoxicity to an acceptable level. Diphenyl Ether Analogues First of all, in order to confirm that the ortho-phtnoxy substituent of the diphenyl ether 9 was in the optimum position, we prepared the two possible regioisomers 10 and 11. The results from our screens clearly demonstrated that fungicidal activity falls off very sharply as the phenoxy group is moved around the ring, which was consistent with the modelling studies that we had carried out earlier. The validity of the model was further supported by the results of testing the corresponding stilbene analogues (regioisomers of the stilbene 4). We next looked at the effect of chlorination of the phenoxy substituent and discovered that whereas meta- and /wra-substitution retained activity, an ortho-subsûtutnt caused a significant drop in activity (Table I). Similarly, introduction of a chlorine substituent into the ring bearing the β-methoxyacrylate group at the position ortho to the phenoxy-substituent reduced activity sharply. The most obvious explanation for this is that these orrAo-substituents induce a conformational change in the diphenyl ether system which makes binding at the active site less favourable. Another consequence of introducing these substituents was that the (octanol/water) log P's were increased by an estimated 0.5 to 0.8 and this (at least in part) led to loss of activity via root uptake. Since systemic activity is desirable in a commercial fungicide, we went to some lengths to prepare further analogues with lower partition coefficients. Introduction of a Ring Nitrogen Apart from the introduction of substituents with negative π values, we found that an effective way of lowering the log Ρ in this series was to replace one of the benzene rings of the diphenyl ether with a nitrogen heterocycle. The pyridine 12, which was prepared according to the steps shown in Figure VII, is an example of such a compound (#). As predicted, the partition coefficient of pyridine 12 (measured log Ρ = 2.6) was significandy lower than the benzene analogue 13 (estimated log Ρ = 4.1) and as a consequence very good levels of systemic activity were achieved (Table I). Later work established that the position of the nitrogen atom in the ring and the nature of the In Synthesis and Chemistry of Agrochemicals III; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.



382

Figure VII : Synthesis of Pyridine 12


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substituents have a crucial bearing on the physical and biological properties of compounds of this type. The results of these studies will be published elsewhere.


Conclusions Synthetic work around the natural products strobilurin A and oudemansin A has led to the discovery of a new class of synthetic fungicides with high levels of activity, a novel mode of action and physical properties which have been optimised for systemicity. The large number of patent applications published by over 10 different companies during the last five years demonstrate the considerable interest in this area of chemistry within the agrochemical industry. Acknowledgements We wish to thank our many colleagues at ICI Agrochemicals who have participated in this project and have contributed to its success. Literature Cited 1.

2. 3. 4. 5. 6. 7. 8.

Nevill, D., Nyfeler, R., and Sozzi, D., Brighton Crop Protect. Conf., Pests and Diseases-1988, vol. 1, British Crop Protection Council, Thornton Heath, U K , 1988, pp. 65-72. Beautement, K., Clough, J. M . , de Fraine, P. J., Godfrey, C. R.A., Pestic. Sci., 1991, 31, 499. Becker, W. F., von Jagow, G., Anke, T., and Steglich, W., FEBS Letts., 1981, 132, 329. Beautement, K. and Clough, J.M., Tet. Letts., 1987, 28, 475. Anke, T., Hecht, H . J., Schramm, G . and Steglich, W., J. Antibiot., 1979, 32, 1112. EP178,826 (1984, ICI). EP267,734 (1986, ICI). EP242,081 (1986, ICI).

RECEIVED June 29,

1992


Fungicidal -Methoxyacrylates - ACS Publications - American Chemical

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