The effect of acifluorfen-methyl on tetrapyrrole synthesis in greening chloroplasts of Cucumis sativus was examined. Formation of Mg-proto-porphyrin IX from 6-aminolevulinate was reduced 98% by 10 micromolar acifluorfen-methyl. Conversion of protoporphyrin IX to Mg-protoporphyrin IX was unaffected, but protoporphyrin IX synthesis from 6-aminolevulinate was blocked, indicating a site of inhibition prior to the Mg-chelatase. The enzymic oxidation of protoporphyrinogen IX to protoporphyrin IX was highly sensitive to acifluorfen-methyl, indicating that the site of action of the herbicide is the protoporphyrinogen oxidase. (© 1989 FMC Corporation. All rights reserved.) MATERIALS AND METHODS ChemicalsProto IX was purchased from Porphyrin Products (Logan, UT) and purified as outlined by Fuesler et al. (7). Protogen IX was freshly prepared by reduction of Proto IX with a Na/ Hg amalgam as outlined by Jacobs and Jacobs (1 1), utilizing the purified Proto IX at a concentration of 300 Mm. Mg Proto IX and AFM were synthesized at the FMC Princeton Research and Development facility. The Bio-Rad protein determination kit, which employs the Bradford method (1), was purchased from Bio-Rad Laboratories. All other reagents were purchased from Sigma or Aldrich.Recently a number of laboratories have reported dramatic accumulations of Proto IX' in plant tissues treated with the diphenyl ether herbicide AFM, methyl-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate. The amount of Proto IX accumulated correlates with the extent of herbicide induced injury observed; further, inhibitors of tetrapyrrole synthesis such as levulinic acid, gabaculine, and dioxoheptanoic acid prevent or reduce the membrane disruption which normally results following illumination of AFM treated tissues (12,13,20). Proto IX is recognized as a potent photosensitizer (4, 18). Therefore, these observations have led to the hypothesis that Proto IX is the photosensitizing agent initiating lipid peroxidation in AFM treated plant tissues. Analyses of the pigment profiles in extracts of in vivo systems treated with AFM have shown an inverse relation between chlorophyllide and Proto IX levels; as herbicide dose increased, Proto IX levels also increased with a concomitant reduction in chlorophyllide and no increase in Mg-Proto IX suggesting that the Mg insertion step was inhibited (12,20). Based on these observations in vivo we conjectured that the primary site of phytotoxic action for AFM might be the . The purpose of the present study was to determine experimentally the specific portion of the tetrapyrrole biosynthetic pathway affected by AFM in isolated, developing chloroplasts.

Treatment with acifluorfen-methyl (AFM), methyl 542-chloro-4-Itrifluoromethyll phenoxy)-2-nitrobenzoate, inhibited protochlorophyllide synthesis in dark-held, 5-amino levulinic acid-fed, excised cotyledons of cucumber (Cucumis sativus L.). Protochlorophyllide and protoporphyrin IX levels in AFM-treated cotyledons were inversely related and dependent on AFM concentration; as the herbicide dose increased, protoporphyrin IX levels also increased with a concomitant loss of protochlorophyllide. Significant protoporphyrin IX accumulation was induced by concentrations of AFM from the linear region of the membrane disruption dose response curve. The patterm of precursor accumulation seen in HPLC chromatograms from extracts of AFM-treated tissue indicate that Mg insertion into the tetrapyrrole ring is inhibited, suggesting interference with Mg-chelatase. An inhibitor of 5-amino levulinic acid synthesis, gabaculine (3-amino-2,3-dihydrobenzoic acid), completely blocked the membrane disruption activity of AFM in illuminated cotyledons. Protoporphyrin IX accumulating in AFM-treated tissues may serve as the primary photosensitizer for initiating lipid peroxidation.As a consequence of the rapid induction of lipid peroxidation, illumination ofplant tissues treated with p-nitro DPEI herbicides results in the disruption ofa wide range ofbiological constituents and functions (18,26). This broadly destructive activity by the DPE herbicides has made it difficult to monitor specific interactions between these compounds and light-dependent in vivo physiological processes. These effects lead to one of the most readily visible manifestations of the treatment of whole plants with DPE herbicides: the bleaching of the foliar tissue as a consequence of pigment photooxidation.In an earlier study (15) greening was induced in AFM-treated cucumber cotyledons without triggering a loss in plasmalemma membrane integrity under a regime oflow intensity, intermittent illumination. Yet, even under these conditions, we observed significant reductions in the levels of Chl accumulated in AFMtreated tissue. These results suggested the possibility that, in addition to the pigment bleaching in DPE-treated plant tissue which occurs under continuous illumination, AFM might also directly inhibit pigment biosynthesis in a manner not related to photooxidation.We have since found further evidence that the DPE herbicides directly inhibit Chl synthesis prior to the formation of Pchl(ide).' Abbreviations: DPE, diphenyl ether; AFM, acifluorfen-methyl; ALA, 6-amino levulinic acid; Pchl(ide), a mixture of Pchlide and Pchlide ester; proto IX, protoporphyrin IX; Mg-proto IX, Mg-protoporphyrin IX; Mgproto IX Me, Mg-protoporphyrin IX monomethyl ester; LA, levulinic acid: LSD (0.05), least significant difference at the 5% probability level.

A factorially designed set of 3‐substituted 2‐aryl‐4,5,6,7‐tetrahydroindazoles was synthesized and their herbicidal properties were evaluated using regression analysis. For optimal activity, the substituent at position 3 should be neither strongly hydrophobic nor hydrophilic and should have a small minimum radius. A study of the mechanism of action indicated that the tetrahydroindazoles are inhibitors of the enzyme protoporphyrinogen oxidase.

No abstract

The process of sorting leads identified via screening can be made more efficient by the application of experimental design strategies. Such strategies were applied during the course of optimization of a series of 4-aryl-l,2,4triazole-5(1H)-thiones which were initially identified as being herbicidal in greenhouse screens. Through application of a Free-Wilson strategy and subsequent sequential simplex optimization, potency improvements of 25 000-fold over the unsubstituted phenyl compound have been realized in a hydroponic cucumber assay. Synthesis of these analogues will be discussed as well as the development of the QSAR model which relates structural modifications to potency.