Phytoene desaturase inhibitors

Norfluorazon is an often-used inhibitor of carotenoid biosynthesis, interfering with the step ofphytoene desaturation (Sandmann, 1994). Norflurazon treatment of mustard seedlings showed that PS II assembly is more sensitive to reduced levels of carotenoids than PS I assembly (Markgraf and Oelmiiller, 1991). The assembly of D1 into the PS II reaction center appears to be dependent on /3-carotene. Upon treatment of Chlamydomonas reinhardtii with phytoene desaturase inhibitors, D1 degraded during photoinhibition caimot be replaced (Trebst and Depka, 1997). 

Synthesis and herbicidal activity of novel phytoene desaturase inhibitors ... 

Phytoene Desaturase Inhibitors j 197 Table 4.1.2 Structural evolution of phenoxypyridine ethers since 1994. 

Major Synthetic Routes for Phytoene Desaturase Inhibitors... 

Table 35.13 Chemically different classes of phytoene desaturase inhibitors (80-86) (see Fig. 35.20 for the basic structure).

A second class of herbicides primarily affects ( -carotene desaturase. These herbicides are apparent feedback inhibitors of PD as well. This class of compounds includes dihydropyrones like LS 80707 [90936-96-2] (56) and 6-methylpyridines (57,58). The third class consists of the ben2oylcyclohexane-diones, eg, 2-(4-chloro-2-nitroben2oyl)-5,5-dimethyl-cyclohexane-I,3-dione. This class of atypical bleaching herbicides induces phytoene accumulation when appHed either pre- or post-emergence. However, it does not inhibit phytoene desaturase activity in vitro (59). Amitrole also has been considered a bleaching herbicide, though its main mode of action is inhibition of amino acid synthesis. 

The carotenoid pathway may also be regulated by feedback inhibition from the end products. Inhibition of lycopene cyclisation in leaves of tomato causes increase in the expression of Pds and Psy-1 (Giuliano et al, 1993 Corona et al, 1996). This hypothesis is supported by other studies using carotenoid biosynthesis inhibitors where treated photosynthetic tissues accumulated higher concentrations of carotenoids than untreated tissues (reviewed by Bramley, 1993). The mechanism of this regulation is unknown. A contrary view, however, comes from studies on the phytoene-accumulating immutans mutant of Arabidopsis, where there is no feedback inhibition of phytoene desaturase gene expression (Wetzel and Rodermel, 1998). 

The molecular target site of triketone herbicides is the enzyme -hydroxyphenylpyruvate dioxygenase (HPPD). Inhibition of this enzyme disrupts the biosynthesis of carotenoids and causes a bleaching (loss of chlorophyll) effect on the foliage similar to that observed with inhibitors ofphytoene desaturase (e.g. norflurazon). However, the mechanism of action of HPPD inhibitors is different. Inhibtion of HPPD stops the synthesis of homogen tisate (HGA), which is a key precursor of the 8 different tocochromanols (tocopherols and tocotrienols) and prenyl quinones. In the absence of prenylquinone plastoquinone, phytoene desaturase activity is interrupted. The bleaching of the green tissues ensues as if these compounds inhibited phytoene desaturase. 

New agrochemicals introduced in the past five years include new chemistries with known modes of action, such as the protoporphyrinogen inhibitor bencarba-zone, the phytoene desaturase picolinafen and beflutamid, and sodium channel pyrethroids new chemistries with possibly new modes of action, such as flonic-amid and pyridalyl and new chemistries with established new modes of actions, such as flubendiamide, which activates ryanodine-sensitive intracellular calcium release channels, ryanodine receptors RyR, in insects. 

Inhibitors of carotenoid synthesis also lead to chlorophyll destruction by destabilizing the photosynthetic apparatus. Total carotenoid content decreased with increased (-)-usnic concentration (Fig. 1.4). Carotenoid biosynthesis can be interrupted by inhibiting the enzyme phytoene desaturase that converts phytoene to carotenes or by inhibiting the enzyme HPPD responsible for plastoquinone (required for phytoene desaturase activity) synthesis.14 Usnic acid possesses some of the structural features of the triketone HPPD inhibitors, such as sulcotrione (Fig. 1.1C).8 (-)-Usnic acid had a strong inhibitory activity on HPPD, with an apparent IC50 of 70 nM, surpassing the activity obtained with the commercial herbicide sulcotrione (Fig. 1.5). 

Sandman et. l. (8) have found the N-phenyl-2-phenoxynicotin-amides to be powerful inhibitors of phytoene desaturase. Several bleaching herbicides that inhibit the phytoene to phytofluene transformation have the same 3-trifluoromethylphenyl group (8, 9). This group includes norflurazon, metflurazon, fluridone, 7luometuron and fluorochloridone. In the nicotinamide series the same 3-trifluoromethylphenyl group gives optimum herbicide activity. 

As can be seen from Table IV, the nitrophenyl analogs of the nicotinamides are inactive. Interestingly, the 2-(3-trifluoromethylphenyl ) benzamides (compound IV minus the nitro group) were moderate herbicides (less than 50% weed control at 1 lb). These compounds are also bleaching herbicides as are the well known 3-phenoxybenzamides. The 3-phenoxybenzamides are also known to be inhibitors of phytoene desaturase (11). 

If our hypothesis is correct, this hypothetical binding site should also accommodate fluridone, norflurazon and difunone and some possible binding orientations of these molecules are compared with furanone 13 in Figures 7-9. Note that we have attempted to depict the molecules in such a way that key structural features, e.g., the CF3 phenyl and vinylogous amide subunits, occupy the same positions as nearly as possible. Finally, it should be emphasized that considerable further work is required to demonstrate that the furanones actually inhibit phytoene desaturase and to further probe the possibility of a common binding site for the proven inhibitors including those such as fluorochloridone (10) and the m-phenoxybenzamides (4), which do not incorporate the vinylogous amide substructure. 

Beflubutamid is a newly described carotenoid synthesis inhibitor, inhibiting phytoene desaturase. It has a very low toxicity to animals but is very toxic to algae and plants. It is not mutagenic or teratogenic in standard tests. 

Expression of an herbicide insensitive target has also been reported to provide resistance to diclofop and sethoxydim (ACCase inhibitors), various dinitroani-lines, and inhibitors of phytoene desaturase, lycopene cyclase and hydroxyphenyl-pyruvate dioxygenase. None of these traits are currently incorporated into commercial products. 

The mode of action of file phenylthiazolines has been investigated by looking for file accumulation of metabolic intermediates in chlorotic cress leaves. Treated plants showed an increase in levels of a compound that can not be observed in controls, identified by UV spectroscopy and HPLC cochromatography with an authentic phytoene. The latter has been produced by the application of file known carotenoid synthesis inhibitors such as flurtamon and identified independently by UV and mass spectroscopy. From these results, it can be assumed that chlorosis is caused by inhibition of carotenoid synthesis at the phytoene desaturase step. 

In order to exclude the possibility that inhibition of phytoene desaturation is caused by indirect or regulatory mechanisms, direct interaction of substituted phenylthiazolines with phytoene desaturase was demonstrated by in vitro studies. As a result, with increasing concentrations, more phytoene is retained which means that less phytoene was desaturated. Therefore, less 3-carotene is formed as the end product of this in vitro biosynthetic chain. Interaction of phenylthiazolines is very similar to inhibition of this enzyme by flurtamone. For the latter herbicide, it has been shown to be a reversible non-competitive inhibitor of phytoene desaturase (P). 

As a methodology to search for a new lead, we focused on the chemical approach using A,S-heterocycles. This approach led to the evolution of 5-methylene-thiazolidines as the lead to bleaching herbicides. Further structural modifications based on the parent compound resulted in the creation of a new family of bleaching herbicides, 2-(W-difluoroacelylimino)-S-methyl-3-(3-trifiuoromethylphenyl)-l,3-thiazoline, (S-3085) widi potent preemergence herbicidal activity and selectivity on cotton crops. On die basis of biochemical studies, 3-phenylthiazolines have been characterized as a new inhibitor of phytoene desaturase. Facile syndieses of 1,3-thiazolines and the manufacturing process toward S-3085 will be reported in future publications. 

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