Vol. 41 December, 1992 No. 12

Weed Abstracts

Herbicide safeners: chemistry, mode of action, applications

Tamas Komives

Plant Protect ion Institute, Hungarian Academy of Science, H-1525 Budapest P.O. Box 102, Hungary

Summary

The successful introduction of herbicide safeners into agricultural practice to protect maize against thiolcarbamate and chloroacetanilide herbicide injury has stimulated rapid progress and opened new possibilities for further research and development in the last decade. Active ingredients with new chemistry, increased efficacy, and a broader selectivity spectrum have been synthesized, developed for agricultural use and commercialized. Research on their biological activity under different conditions, together with structure-activity relationship and mode of action studies helped to optimize their chemical properties and to understand their biological effects. Several examples indicate close similarity between chemical structures possessing herbicidal and safener properties. In some cases this differentiation may be marginal, as shown in crops pretreated with low herbicide doses leading to safening effects. In other examples, however, structural optima for safening and herbicidal efficacy can be clearly separated. Elevated substrate levels, enzyme activities and detoxication rates are involved in safener action.

I. Introduction

In order to increase food production for an increasingly hungry world, agricultural scientists working in the area of herbicide research are concerned with at least two means of providing greater amounts of food: 1) reducing current losses due to weed infestation, and 2) preventing crop damage by herbicides.

The object of this review is to cover the subject of protection of crops against herbicide damage by chemicals known as "herbicide safeners" (formerly referred to as "herbicide antidotes" or "crop pro-tectants"). By definition, a herbicide safener is a chemical which has limited phytotoxicity and selectively protects crop plants against herbicide damage without protecting any weed species (Hatzois, 1989). Several such agents are marketed presently for different crop/herbicide combinations under field conditions.

Traditionally, herbicide safeners have been considered to have the ability to counteract phytotoxic effects of thiolcarbamates and chloroacetamides to maize (Zea mays L.) and sorghum (Sorghum bicolor L.) (Hatzios, 1989; Stephenson and Yaacoby, 1991). However, recently developed compounds have changed this simplistic view dramatically, expanding the spectrum of safener efficacy to a variety of active ingredients (including phytotoxic fungicides) and to other crops, like rice Oryza sativa L.) and wheat ('Triticum aestivum L.) (Amrein et al., 1989; Bieringer et al., Peek et al., 1988; Stephenson and Yaacoby, 1991). Higher specificity and efficacy have been achieved by the design and synthesis of safeners with rather complex chemical structures (Komives and Hatzios, 1991). This paper is an attempt to focus upon the chemical characteristics contributing to their biological activity, their biological modes of action and areas of application.

©C.A.B. International 1992

554 Weed Abstracts 1992 Vol. 41 No. 12

II. Chemical classification

Crop safeners for herbicides currently marketed belong to the following chemical classes: 1) dichloroacetamides 2) naphthopyranones 3) oxime ethers 4) quinolyloxycarboxylic acid esters 5) substituted phenyl pyrimidines 6) derivatives of 2,4-disubstituted 5-thiazolecarboxyIates 7) substituted phenyl triazoles 8) dichloromethyl acetals and ketals 9) thiolcarbamates 10) diaryl ketones 11) haloalkl aryl sulfones.

Chemical structures of representative safeners of these classes (arranged in the above order) together with the herbicides against which they give protection in a particular crop are shown in Figure 1.

N A (II)

CI-N - 0

F F F

C G A - 1 3 3 2 0 5 (V)

C G A - 1 8 5 0 7 2 (VII)

d i m e p i p e r a t e (XIV)

Br

BCS (XVI)

Safeners

E P T C

vernolate

CGA-184927

bensul furon methyl

fenclorim (VIII)

flurazole (IX)

0 J

f e n c h l o r a z o l e - e t h y l (XII)

0 -

M G - 1 9 1 (XIII) butylate

m e t h o x y p h e n o n e ( X V ) thiobencarb

0

thiobencarb

Fig. 1. Represen ta t ive s t ruc tures of d i f ferent chemica l groups of safeners and of the herbic ides against which they are appl ied .

Herbicide safeners: chemistry, mode of action, applications 555

III. Chemical and physical properties

The large amount of information published on the routes for the chemical synthesis of the majority of the safeners in Figure 1 have been reviewed by Hatzios (1983) and will not be repeated here. Further information on the preparative methods of different safeners is available in the literature: Riden (1976) for NA; Pallos et al. (1977) and Stephenson et al. (1978) for the preparation of dichloroacetamide analogues; Martin (1978) for oxime ether derivatives; Howe and Lee (1980) and Sacher et al. (1980) for 2,4-disubstituted thiazoles; Dutka and Komives (1987) for dichloromethyl ketals and acetals; and Quadranti and Ebner (1984) for the phenylpyrimidine safener fenclorim.

Safeners introduced into agricultural practice are characterized with widely different chemical and physical properties (Table la and lb). With the exception of MG-191 and dichlormid, which are liquids with moderate water solubility and relatively high volatility, most of the safeners are solids with low water solubility, high lipophilicity and low vapour pressure.

IV. Structure-activity relationships

Research findings published on the structure-activity relationships of herbicide safeners have been recentéy reviewed (Komives and Hatzios, 1991). Therefore, only general patterns emerging following an investigation of the chemical structures and the physical properties of the safeners in Figure 1 and in Table 1 will be discussed here:

a) Similarities between the chemical structure of a herbicide and its safener (e.g., in the case of EPTC and dichlormid in Figure 1) are sometimes evident. This observation gained further support from structure-activity studies (Stephenson et al., 1978; Dutka et al., 1980), molecular graphics investigations (Yenne and Hatzios, 1990), and also from findings that crops pretreated with low herbicide doses become less susceptible to damage by the same herbicide (Ezra et al., 1985). The simplest explanation for this apparently beneficial similarity may be a possible competitive antagonism between the thiolcarbamate herbicide and the safener molecules for a common target site (Stephenson et al., 1978; Stephenson et al., 1979). In contrast, structural optima of safener (Rubin and Kirino, 1989) and herbicidal imides (Hamada et al., 1989) are markedly different (Komives and Hatzios, 1991). In this context, it is interesting to note that herbicidal thiolcarbamates (like dimepiperate and thiobencarb in Figure 1) may function as safeners against injury by a structurally unrelated sulfonylurea derivative, bensulfuron methyl (Matsunaka and Wakabayashi, 1989).

b) Several pairs of data in Table 1 suggest that similarities in the physical properties of a safener to those of the herbicide against which it gives protection may also be beneficial. Though the scarcity of data in this respect does not allow a firm conclusion, it is interesting to note that the "outliers" in Table 1 (MG-191 and dichlormid, liquids of relatively low lipophilicity (indicated by low log P values, high water solubility and vapour pressure) are safeners tailored to give maximum protection against injury by EPTC and related thiolcarbamates. Indeed, dichlormid and MG-191 have physical properties quite similar to those of the aliphatic thiolcarbamates. These herbicides and their safeners may act at the same site of action (Fedtke and Trebst, 1987) and are thought to be taken up by the same crop plant tissue from the vapour phase (Gray and Joo, 1978).

c) All maize safeners, with the exception of NA, contain a dichloromethyl group, show protective action against thiolcarbamates and chloroacetanilides, and are applied in a mixture with the herbicide.

d) In sorghum only seed safeners proved to possess sufficient activity. Chemically they belong two different classes, which, unlike those active in maize, do not seem to have common features.

e) Safeners developed to protect wheat are solids of remarkably high lipophilicity, low water solubility, and low vapour pressure.

f) Importance of the presence of at least one electrophilic centre in known safener molecules is also apparent. This centre may be a carbon atom of an oxo function (in NA, dichloroacetamides, CG A-185072, flurazole, dimepiperate, and fenchlorazole-ethyl) or of a haloalkyl group (in BCS, dichloromethyl group-containing safeners), an aromatic carbon in a heterocylic ring (in fenclorim, CGA-185072, fenchlorazole-ethyl), or a carbon-carbon double bond bridging two carbonyl groups (in derivatives of maleic acid). Possible reaction partners attacking these electrophilic centres may be (macro) molecules with sulfhydryl group(s) (Breaux et al., 1989; Lay and Casida, 1976; Komives and Dutka, 1989). Though in some cases such chemical transformations of safeners ("conjugation reactions") were clearly demonstrated (Breaux et al., 1989), information on such nucleophilic substitutions by sulfhydryls on dichloromethyl group-containing safeners (with or without oxidative bioactivation) are only circumstantial (Komives and Dutka, 1989). The products of these transformations ("conjugates") may have direct or indirect roles in the regulation of the defense mechanisms of plants against phytotoxic chemicals (Breaux et al., 1989; Komives and Dutka, 1989).

556 Weed Abstracts 1992 Vol. 41 No. 12

Table l a . List of safeners introduced into agricultural practice

Common name Chemical name Molecular formula

MW Physical Melting state point, °C

I. Dichlormid N,N-diallyl-2,2-dichloroacetamide C 8 H n C I 2 N O 208.09 Liquid

II. NA Naphthalene-1,8-dicarboxylic acid anhydride c I 2 h 6 o , 198.18 Solid 270-274

III. Cyometrinil (Z)-alpha-(cyanomethoxy)-imino-benzeneacetonitrile

c , 0 h 7 n 2 o 2 185.19 Solid 5 5 - 5 6

IV. Oxabetrinil a - ( 1,3-Dioxolan-2-y 1-methoxy)-iminobenzeneacetonitrile

c , 0 h 1 2 n 2 o , 232.24 Solid 7 7 - 7 9

V. C G A - 1 3 3 2 0 5 0 - [ 1,3-Dioxolan-2-yl-methyI]-2,2,2-trifluoro-4'-chIoroacetophenone-oxime

C12H11CIF3NO3 309.67 Solid

VI. Benoxacor 4-Dichloroacetyl-3,4-dihydro-3-methyl-2 H-1,4-benzoxazine

c , , h , , c i 2 n o 2 260.12 Solid 107.6

VII. CGA-185072 l-Methylhexyl-([5-chloro-8-quinolyl]-oxyacetate

c , 8 h 2 2 c i n o 3 335.83 Solid 59.3

VIII. Fenclorim 4,6-Dichloro-2-phenylpyrimidine C i 0H 6C1 2N 2 225.08 Solid 96.9

IX. Flurazole Phenylmethyl-2-chloro-4-trifluoro-methyl-5-thiazolecarboxylate

c I 2 h 7 c i f , n o 2 s 321.71 Solid 5 6 - 5 8

X. B A S - 1 4 5 1 3 8 l-Dichloroacetyl-hexahydro-3,3,8-a-tri-methylpyrrolo-[ 1,2a]-pyrimidin-6-(2H)-one

c 1 2 h i 8 c i 2 n 2 o 2 293.20 Solid

XI. AD-67 N-Dichloroacetyl-l-oxa-3-aza-spiro-4,5-decane c i 0 h i 5 c i 2 n o 2 252.14 Solid 106

XII. Fenchlorazolethyl Ethyl-1-2(2,4-dichlorophenyl)-5-tri-chloromethyl-1H-1,2,4-triazole-3-carboxylate

c i 2 h 8 c i 5 n 3 o 2 403.48 Solid 1 0 8 - 1 1 2

XIII. MG-191 2-Dichloromethyl-2-methyl-l ,3-dioxolane c 5 h 8 c i 2 o 2 171.02 Liquid

XIV. Dimepiperate N-( 1,1 -Dimethylbenzylthio-thiocarbonyl)-piperidine

c I 5 h 2 , n o s 263.40 Solid 3 8 . 5 - 3 9 . 3

XV. Methoxyphenone 3,3'-Dimethyl-4-methoxy-benzophenone C | 6 H I 6 ° 2 240.30 Solid 6 2 . 0 - 6 2 . 5

XVI. BCS Chloromethyl 4-bromophenyl sulfone C7H 6 BrC10 2 S 269.55

Table l b . List of safeners introduced into agricultural practice (continued)

Method of application Protected Vapor pressure plant Hg mm)

Water solubility at 20° (mg/L)

log P* Patented by

I . Tank mix Maize 6 . 0 X 1 0 - 1 (25°) 5000 2 .063 Stauffer Chemical Co.

II. Seed dressing Maize < 2 2.438 Gulf Chemical Co.

III. Seed dressing Sorghum 95 1.161 Ciba-Geigy Ltd.

IV. Seed dressing Sorghum 5 . 2 x l 0 - 4 (25°) 20 1.018 Ciba-Geigy Ltd.

V. Seed dressing Sorghum 10 3.292 Ciba-Geigy Ltd.

VI. Tank mix Maize 4 . 4 X 1 0 - 6 (20°) 20 3.296 Ciba-Geigy Ltd.

VI. Tank mix Wheat 2.5 x l O - 6 (20°) 0 .8 7.312 Ciba-Geigy Ltd.

VIII. Tank mix Rice 9 .Ox 10- 5 (20°) 2 .5 3.055 Ciba-Geigy Ltd.

IX. Seed dressing Sorghum 3 . 8 x l 0 - 5 (25°) 5 .226 Monsanto Co.

X. Tank mix Maize 30 1.789 BASF Ltd.

XI. Tank mix Maize 328 3.625 Nitrokemia Co.

XII. Tank mix Wheat 6 . 7 X l O - 9 (20°) 0.9 7 .144 Hoechst Ltd.

XIII. Tank mix Maize 3 .2X10-1 (20°) 9730 1.503 Nitrokemia Co.

XIV. Tank mix Rice 3.469 Du Pont de Nemours Co.

XV. Tank mix Rice 4 .397 Kumiai Co.

XVI. Tank mix Rice 2 .366 Kumiai Co.

•Estimated by computer program ProLogP 4.1 (CompuDrug Ltd, Budapest. Hungary) (Bordas, 1991)

Herbicide safeners: chemistry, mode of action, applications 557

V. Biological activity

Depending on their chemical structure and methods of application and plant development, herbicide safeners antagonize phytotoxic effects of a number of herbicides on several plant species in addition to the crop-herbicide combination for which they were developed (Parker, 1983). Although in general it may be stated that known safeners are applied exclusively as protectants of monocotyledonous crops (Hatzios, 1989), dichlormid, for example, shows significant protective activity in beans (Phaseolus vulgaris, L.) as well (Blair, 1979). In a few experiments detectable though marginal protection of some weeds by safeners was also observed (Parker, 1983).

There are examples of biological data for safeners indicating effects opposite to "safening" on plants too. Fenclorim has been shown to reduce the growth rate of the weed species Echinochloa crus-galli (Komives and Dutka, 1989a), and dichlormid (at least in the high concentration range of 2 to 8 kg/ha) acted as a synergist of the herbicide acifluoren and of inhibitors of photosystem II in Ipomoea hederacea (Fedtke and Strang, 1990).

Herbicide safeners have also been shown to interfere with membrane and surface lipid metabolism in plants, by causing significant alterations in the ratios of their saturated/unsaturated and long-chain lipids, and possibly as a result of these changes, with water utilization of the affected plants (Barta et al., 1983).

Due to the ability of some herbicide safeners to influence monooxygenase enzymes in insects, they may act as synergists of insecticides known to be metabolized by these enzymes in insects and plants (Ketchersid et al., 1985).

VI. Mode of action

With such biological specificity there is much activity in a search for the mechanism of action of herbicide safeners. In theory (Hatzios, 1989) safener treatment of crop plants may lead to: A) exclusion of herbicides from reaching their site of action as a result of

1) chemical antagonism or 2) biochemical antagonism, by:

a) reduction of herbicide uptake and/or translocation, or b) enhancement of herbicide detoxication.

B) Interactions of herbicides and safeners at the site of action, by

1) competitive antagonism, or 2) physiological antagonism.

Despite high variability in the physiology and fate of herbicides in plants and in our environment (Hatzios and Penner, 1985) it is remarkable that herbicide safening is achieved by a relatively small number of mechanisms. Research findings using a variety of plant-herbicide-safener systems showed that the overwhelming majority of safeners exert their protective action by increasing the crop plant's capacity to detoxify the herbicide (Gronwald, 1989; Hatzios, 1989; Komives and Dutka, 1989; Yenne et al., 1990).

— HX GSH + X - R GS - R

GSH = glutathione

X - R = herbicide (X = leaving group)

GS - R = glutathione conjugate

The most important routes of (bio)chemical transformations of herbicides in plants leading to inactive products or derivatives of significantly lower phytotoxicity are oxidative and conjugation processes. Importance of the conjugation reaction (Equation 1) with glutathione in the detoxication of a number of herbicides (e.g. chlorotriazines, thiolcarbamates and chloroacetanilides, Fig. 2) has been demonstrated (Breaux etal., 1989; Lay and Casida, 1976; Komives and Dutka, 1989). Increased levels of the enzyme glutathione transferase (EC 2.3.4.5) and its cofactor glutathione have been detected in safener-treated maize (Lay and Casida, 1976; Mozer et al. 1983; O'Connell et al., 1988; Ezra et al., 1985; Wiegand et al., 1986), sorghum (Dean et al., 1990; Ebert and Gerber, 1989; Gronwald, 1989), and rice plants (Komives and Dutka, 1989a).

Involvement of cytochrome P-450 containing monooxygenases catalyzing oxidative transformations of herbicides in plants has also been implicated in safener mode of action; induction (Komives and Dutka, 1980; Komives et al., 1985), as well as inhibition of the enzyme by herbicide safeners (Fedtke and Trebst, 1987) have been proposed. Although the net cytochrome P-450 content in safener-treated maize seedlings showed only a 2-fold increase (Fonne-Pfister et al., 1990) or no increase at all (Komives et al., 1985), activity of the enzyme to hydroxylate the aryl rings of primisulfuron (Fonne-Pfister, et al., 1990) and the ring-methyl group of chlorotoluron (Fonne-Pfister and Kreuz, 1990) was increased at least 15-fold by CGA-154281.

558 Weed Abstracts 1992 Vol. 41 No. 12

CI CI F 0 C!

0 o CI

t r idiphane S - 23142

acetochlor EPTC - sulfoxide

0 H H /

N /

H-N

F

acif luorfen atrazine

Fig . 2. C h e m i c a l s t ruc tu res of se lec ted herb ic ides tha t are de tox i f ied by con juga t ion with g lu ta th ione in p lan ts ( the as ter isk d e n o t e s the chemica l b o n d in the he rb ic ide molecule which is split by g lu ta th ione dur ing the c o n j u g a t i o n r eac t i on ) .

Herbicide-metabolizing enzymes induced by safeners play an important role in reducing the intracel-lular concentration of the herbicide. Differences in base levels and inducibilities by the safener of these enzymes and their cofactors in crops and weeds result in biological selectivity (Komives and Dutka, 1989). Interestingly, certain herbicides (e.g., thiolcarbamates) require to be metabolized by these enzymes before they exert their phytotoxic effect (Lay and Casida, 1976; Komives and Dutka, 1980). The expression of herbicide-metabolizing enzymes can therefore either potentiate or reduce the phytotoxicity of herbicides, so changes in both the activation and deactivation pathways are important variables that can result in safening action.

Several lines of evidence indicate the existence of an essential control mechanism that is responsible for coordinating the expression of xenobiotic-metabolizing enzymes and the other proteins handling xenobiotics in plants, as well as providing an elevation in some of the enzyme cofactors like glutathione. This protection mechanism presumably evolved to enable plant cells to combat a wide range of genotoxic and cytotoxic compounds (Komives and Dutka, 1989; Rennenberg, 1982). The existence of this defence process is supported by the remarkable diversity of safener-induced changes and their consistency in several plant-safener systems, as well as by the fact that many of the changes observed in the plant cell are apparently not involved in overcoming the toxic actions (if any) of the actual safeners employed. This safener- (occasionally herbicide-) induced response can be clearly differentiated from the nonspecific response of plants to environmental (chemical, microbial, and mechanical) stress that begins with the evolution of ethylene and ultimately leads to membrane lipid peroxidation reactions and plant cell death (Komives and Casida, 1983).

Standards of weed control will increase still further, and correspondingly the demand for safer and more dependable technologies will also continue to rise. The biggest opportunity for introducing new safeners probably lies with the appearance of new, highly effective herbicidal compounds with novel characteris-tics that may lack sufficient selectivity, or may have a soil life longer than optimum.

Agricultural use of a number of highly efficient fungicides is hindered because of phytotoxic side-effects. The selectivity of these compounds may be increased to acceptable levels by applying them in combination with appropriate safeners to reduce their phytotoxicity.

The ability of safeners to induce glutathione-related enzymes was used in the characterization of ATP-sulfurylase in maize by Farago and Brunold (1990). Though the base level of this rather labile

VII. Prospects for the future

Herbicide safeners: chemistry, mode of action, applications 5 5 9

e n z y m e in m a i z e p lan ts is ve ry low, a f te r t r ea tment wi th the d i c h l o r o a c e t a m i d e s a f ene r benoxaco r , it increases m o r e than ten- fo ld , thereby fac i l i ta t ing its i sola t ion, pu r i f i ca t ion and charac te r iza t ion .

T h e un ique abi l i ty of herb ic ide sa feners to i n f l uence g e n e exp res s ion wi thou t phy to tox i c s ide e f fec t s has in i t ia ted research and d e v e l o p m e n t in the area of mo lecu l a r b io logy . P r o m o t e r f r a g m e n t s inducib le by sa fene r s h a v e been isolated, and ope rab ly l inked to a g e n e s equence c o d i n g fo r a se lec ted g e n e p roduc t . A s a resul t , i nc reased express ion of se lec ted g e n e s b e c a m e poss ib le b y e x p o s u r e of t r an s fo rmed p lan ts to sa fene r s (Du Pon t de N e m o u r s Co. , 1990) .

VIII. Conclusions

Our k n o w l e d g e on the chemis t ry and b iochemica l m o d e ( s ) of ac t ion of he rb ic ide s a fene r s has e x p a n d e d great ly in recent years . T h e impor t ance of s t ructura l s imi lar i ty b e t w e e n a he rb ic ide and its sa fener (s ) has been s h o w n to be a d v a n t a g e o u s bu t no t an abso lu te necess i ty fo r e f f i c i en t p ro t ec t ive ac t ion. R e c e n t s tudies demon s t r a t e d the r equ i r emen t of at least one e lec t rophi l ic site in m o l e c u l e s of he rb ic ide sa feners ava i lab le for poss ib l e nuc leoph i l i c d i sp l acemen t reac t ions . I n v o l v e m e n t of the e n z y m e s ca ta lyz ing the ox ida t ive t r ans fo rma t ions of the herb ic ides and e n z y m e s d e t e r m i n i n g the g lu ta th ione c o n j u g a t i o n abi l i ty of the c rop plants in the s a f e n i n g act ion has been c lear ly es tab l i shed . A m a j o r ques t ion n e e d i n g fu r the r c lar i f ica t ion is the poss ib l e role of sa fener c o n j u g a t e s w i th g lu ta th ione and /o r o the r e n d o g e n o u s su l fhydry l con ta in ing ( m a c r o ) m o l e c u l e s in the sa fene r ac t ion , and the e luc ida t ion of s t ructura l r equ i r emen t s fo r o p t i m u m activity of such con juga t e s .

A t the m o l e c u l a r level it is ev iden t that m u c h r e m a i n s to be learnt abou t the cont ro l of express ion of he rb i c ide -me tabo l i z ing genes .

S i n c e a n u m b e r of c o m p a n i e s are ac t ive ly s ea rch ing f o r n e w s a f e n e r s wi th nove l p roper t ies and d i f fe ren t m o d e s of act ion, there is little doub t that the d i scove ry of n e w ac t ive ingred ien t s will con t inue in the nea r fu tu re .

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