Quantitative structure-activity relationships of the inhibition of photosynthetic electron flow by substituted diphenyl ethers

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  • Pestic. Sci. 1982, 13, 29-38

    Quantitative Structure-Activity Relatioiiships of the Inhibition of Photosynthetic Electron Flow by Substituted Diphenyl Ethers

    Gerard van den Berg and Jacobus Tipker

    Duphar B. V. Biochemistry Department, Crop Protection Division, PO Box 4, 1243 ZG 's-Graveland, The Netherlands

    (Manuscript received 30 September 1980)

    The effect of 41 substituted diphenyl ethers (derivatives of nitrofen and chloroxuron) on photosynthesis in isolated spinach chloroplasts has been studied. All the chemicals were found to be inhibitors of non-cyclic electron transport; the pI50 values varied from 3.17 to 7.16 (150 is the molar concentration causing 50% inhibi- tion; PISO= -log 150). Based on their structures, the compounds were divided into four groups; for most groups, a correlation between the inhibition of photosyn- thesis and physicochemical parameters was found. Lipophilicity proved to be the most important parameter; electronic effects did not play a role. Introduction of substi- tuents into the nitrophenyl ring of nitrofen lowered activity considerably. Nitrofen and and chloroxuron analogues seemed to inhibit at different sites in the electron transport chain. A relationship between inhibition of photosynthesis and herbicidal activity was not clear.

    1. Introduction

    In 1963, nitrofen (2,4-dichlorophenyl 4-nitrophenyl ether) was introduced as a selective pre- and post-emergence herbicide. Especially in Japan, this compound was widely used for pre-emergence weed control in transplanted rice. Many derivatives have been synthesised since, and a few have proved useful herbicides under field conditions.1

    It has been known for a long time that nitrofen requires light for ac t iva t i~n .~ - -~ White rice mutants were found to be tolerant whereas normal green and yellow mutants were susceptible.3 The same phenomenon has also been reported for other crop specie^.^ Probably light and pigments like chlorophylls and/or carotenoids are indispensable for herbicidal activity. The hypothesis that nitrofen itself is inactive but is converted into a toxic substance by light could never be proven.2 On the other hand, the light requirement was obvious only for para- and/or ortho-substituted diphenyl ethers; metu-substituted derivatives were also active in the dark and showed a different selectivity for Echinochloa cru~galli.~, Recent studies by Vanstone and Stobbe showed light of wavelengths between 565 and 615 nm to be the most active in activating oxyfluorfen.6 The relation between light and herbicidal effect became more obvious after the experiments by Moreland et ul.7 They showed nitrofen and two analogues to inhibit electron transport and non-cyclic phosphoryla- tion in isolated spinach chloroplasts, whereas cyclic phosphorylation was unaffected. The site of action appeared to be close to the DCMU-sensitive site. These results were recently confirmed by Bugg et al. although these authors concluded that the site of the action was between plastoquinone and cytochrome f, rather than at the DCMU site.s Contrariwise, Lambert et al. showed that, at least in the alga Bumilleriopsisfiliformis, inhibition of photophosphorylation is the most important feature of nitrofen a ~ t i o n . ~ Because of the successful use of substituted diphenyl ethers as herbi- cides, the diphenyl ether structure was used more than once to replace a phenyl ring in known herbicides. The herbicide chloroxuron is derived from monuron and the herbicide diclofop-methyl is based on 2,4-D.

    0031-613X/82/02o(Mo29 $02.00 8 1982 Society of Chemical Industry 29

  • 30 G. van den Berg and J. Tipker

    Chloroxuron appeared more active than monuron in inhibiting photosynthetic electron trans- port.1 Recently Trebst et al. showed that dinitrophenyl ethers of bromo- or iodothymol are also strong inhibitors of photosynthesis.ll9 l2 The objectives of this study were to investigate the relation- ship between chemical structure, inhibition of photosynthesis and the herbicidal effects of a series of diphenyl ethers. Chloroxuron analogues were also included.

    The following abbreviations are used in the text: DBIMB 2,5-dibromo-3-isopropyl-6-methyl-p- benzoquinone (also known as dibromothymoquinone); DCMU 3-(3,4-dichlorophenyI)-l,l- dimethylurea ; FeCy hexacyanoferrate(II1) ; MV methylviologen (paraquat dichloride) ; PD p- phenylenediamine; PMS 5-methylphenazinium methyl sulphate; and TMPD N,N,N,N-tetra- methyl-p-phenylenediamine.

    2. Experimental methods 2.1. Chemicals All inhibitors used were synthesised in the Organic Synthesis Department according to known methods.

    2.2. Photosynthesis measurements Chloroplasts were isolated from spinach leaves by grinding the leaves in a medium containing 0.3~-sorbitoI, SOmM-HEPES/sodium hydroxide (pH 7.6), and 5mM-magnesium chloride. HEPES is 2-[4-(2-hydroxyethyl)piperazin-I -yl]ethanesulphonic acid. After filtering through four layers of cheese cloth the homogenate was centrifuged at 5000g for 90 s; the pellet was resuspended in grind- ing medium, and the chlorophyll content was determined as described by Bruinsma.l3 Unless stated otherwise, photosynthetic processes were measured in a medium (4 ml) containing: tricine/ sodium hydroxide buffer (pH 8.0; 200 pmol) and magnesium chloride (4 pmol). Tricine is N- [2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine. The medium also contained chloroplasts equiva- lent to 50 pg of chlorophyll and appropriate electron donors or acceptors such as FeCy (3 pmol), MV (0.04 pmol) plus sodium azide (0.8 pmol), TMPD (0.8 pmol) plus ascorbic acid (30 pmol) or PMS (0.08 pmol) (under nitrogen). Photosystem I1 activity was blocked by DCMU (40 nmol). Phosphorylating systems contained in addition : adenosine 5-diphosphate (0.8 pmol), inorganic phosphate (4 pmol), glucose (80 pmol) and hexokinase (Sigma type 111; 6 units). Inhibitors were added as methanolic solutions, the final methanol concentration always being 2 % by volume. Incubation mixtures were illuminated by a projector lamp producing a light intensity of 200 W m-2 and incubations were carried out at 25C.

    Electron transport was measured spectrophotometrically or by using a Clark electrode (Gilson Medical Electronics Inc., Middleton, Wisconsin, USA). Adenosine 5-triphosphate (ATP) forma- tion was determined by colorimetric measurement of the disappearance of inorganic phosphate, or by determining the production of glucose-6-phosphate en~ymical1y.l~

    2.3. Herbicide tests The compounds were tested for post-emergence herbicide activity in the greenhouse. The tests included the grass species Avena fatua, Alopecurus myosuroides and Panicum miliaceum, and the broadleaf species Chenopodium album, Galium aparine, Galinsoga parvipora, Polygonum convolvulus and Senecio vulgaris. Plants were sprayed with acetone solutions of the test chemicals, 10 days after germination, and kept in the greenhouse for 3 weeks. Chemicals were applied at dosages of 0.5, 1,3, 5 and 10 kg a.i. ha-l and dose-response curves were drawn for total activity on mono- or dicotyle- don weeds. In this way, the minimum rate producing severe damage (nearly fatal) could be calcula- ted. When this minimum rate was 10 kg ha-, a herbicide score of 6, 5, 4, 3, 2, 1 or 0, respectively, was recorded. Of the two figures given in the tables, the first represents the score on monocotyledones, and the second, the score on dicotyledones.

    2.4. Multiple regression analysis Biochemical inhibition data were correlated with physicochemical parameters by a multiple regres- sion analysis. Hydrophobic (T) and electronic (u) parameters were taken from Hansch and Leo.15

  • Inhibition of photosynthetic electron flow by substituted diphenyl ethers 31

    Two types of steric parameters were used: Taft's E, parameter,15 and Sterimol parameters from Verloop et aZ.16 Regression equations are presented in section 3.3; in these equations, n is the number of compounds, r is the correlation coefficient, s is the standard error of the estimate, and F represents the overall statistical significance of the equation. The Student's t-test values of the regression coefficients are placed in parentheses.

    3. Results 3.1. Effects of nitrofen on photosynthesis The effects of 3, 10, 30 and 100pM-nitrofen (compound 12) on different photosynthetic reactions were studied and the molar concentrations causing 50% inhibition (150) were determined. The results are presented in Table 1 in which p h = -log 150. The effect of nitrofen on basal electron transport seemed pH-dependent and therefore this effect was studied over a pH range of 6 to 8.5

    Table 1. Effect of some substituted diphenyl ethers on various photosynthetic reactions in chloroplasts

    pI50 values for following inhibitorsn ___ ___. - Measurement

    Electron transport pathway made 1 2 12 21 26 35 - ~ ~ ~ ~ _ _ -~

    HzO+FeCy, pH 7.5, basal Reduction 3 . 5 3 . 5 4.05 4.40 3 .7 6.64 Hz0-t FeCy, 2m~-ammonium chloride Reduction 3.85 4 .34 4.55 4.19 4.01 6.68 HzOjFeCy, pH 8.0, basal Reduction 4.26 4 .75 4.92 4.57 4 . 5 2 6.97 Hz0-r FeCy, phosphorylating Reduction 4.25 4.72 4.92 4.49 4.37 7.13

    ATPformation 4.26 4.72 4.98 4 .59 4.42 7.12 HzO-tMV, basal Oxygenuptake 3.94 4.43 4.67 4.32 (38) 6.98

    TMPD/ascorbate+ MV PMS (nitrogen)

    Oxygen uptake (0) (0) (0) (0) (0) (0) ATPformation (35) (22) (11) (0) (18) 4.38

    a See Tables 2-5 for the structures of the inhibitors. 150 is the molar concentration causing 50% inhibition; PISO= -log 150. Data in parentheses are the percentage inhibition at 0 . ImM. Except for the first reaction, all the activities were measured at pH 8.0.


    4 300 e

    8 I 0

    I " I I I I I I I 6 6.5 7 7.5 8 8 . 5


    I I 1 I I 2 3 4 5 6 7

    PH Log P

    Figure 1. The effect of pH on the inhibition of hexacyanoferrate(ll1) reduction (Hill reaction) (pmol mg-1 of chlorophyll h-I), using HEPES buffers of pH 6 .O, 6.5, 7 . 0 and 7.5, and tricine buffers of pH 7.5, 8 .O and 8 . 5 : 0, control; +, IOpM-nitrofen; and 0 , 0 . 3 p ~ - D C M U .

    Figure 2. Relationship between the inhibition of the reduction of hexacyanoferrate(II1) (Hill reaction) and log P (the log of the octan-I-ol/water partition coefficient): 0, compounds from Table 2 by equation 7; and 0 , compounds from Table 5 by equation 8.

  • 32 G. van den Berg and J. Tipker

    as can be seen in Figure I . At higher pH values, greater inhibition was found for nitrofen as well as for DCMU.

    3.2. Effects of related compounds The effects of five different series of substituted diphenyl ethers on basal electron transport at pH 8.0, using FeCy as acceptor, were studied at three different concentrations, and the 150 values were determined graphically. The negative logarithms of these values (PISO) are presented in the tables. Because of the limited solubility of the compounds O.lmM was the highest concentration used.

    At first, a study was made of the effect of altering the substitution pattern of the halogenated ring of nitrofen. Many para-substituted-phenyl 4-nitrophenyl ethers were studied in addition to some nitrofen isomers having chloro atoms at different positions (see Table 2). The second series of compounds consisted of nitrofen analogues in which the nitro group was replaced (see Table 3). In this series, the herbicide diclofop-methyl (HOE 23 408; compound 21) was included. Nitrofen derivatives having another substituent in the nitro-substituted ring are listed in Table 4. Compound 26 is the herbicide chlormethoxynil (X-52).

    The compounds 32 and 35 of Table 5 can be regarded as being derived from nitrofen and, consequently, they should have been reported in Tables 2 and 3, respectively. However, because of the rather high activity of these compounds, they do not fit well in these series. As will be discussed later, these compounds are members of a totally different group of photosynthesis inhibitors,

    Table 2. Inhibition of the reduction of hexacyanoferrate(II1) (Hill reaction) and the herbicidal effects of a series of substituted 4-nitrophengl phenyl ethers



    1 2 3 4 5 6 7 8 9

    10 11 12 13 14 15 16 17 18

    R x .~ .~

    H 0.00 4-CI 0.74 4-0-CO-NH-CH3 - I .21 4-CH.1 0.51 4-0-cH3 0.05 4-SOz-CH3 - 1-67 4-CO-CHs -0.48 4-NHz -1.10 4-S-CH3 0.61 4-0-CzHj 0.56 4-C(CH3)3 2.05 2,4-CIs 1.48 2,3-Cla 1.48 2,6-C1z 1.48 2,5-Clz I .48 3,4-Ch 1.48

    2,3,4,6-C14 2.96 2,6-(CHa)z 1.02

    Observed -

    4.26 4.15 3.61 4.63 4.48 3.17 4.11 3.65 4 .95 4.19 5 05 4.92 4.71 4.89 4.72 4.89 4.63 5.42

    pled' ~


    Equation I Equation 2 Herbicide


    4.44 4.34 4.79 4.68 3.59 3.62 4.69 4.58 4.47 4.31 3.17 3.29 4. I4 4.08 3.68 3.69 4.74 4.63 4.12 4.60 5.08 5.09

    4.94 4.94 4.94 4.94 4.94 4.79 5.23

    a Is0 is the molar concentration causing 50% inhibition; pIso= -log 150. 0 Defined in section 2.3.

  • Inhibition of photosynthetic electron flow by substituted diphenyl ethers 33

    Table 3. Inhibition of the reduction of hexacyanoferrate(lI1) (Hill reaction) and the herbicidal effects of a series of 2,4-dichlorophenyl 4-substituted-phenyl ethers

    ('I /

    Compound ____ .~ ~ ~ -_- Herbicide Number R PIsoa scorch

    12 -NO2 4.92 616 19 -OH 4.39 20 -0-CO-N H-CHs 4.54 010 21 -O--CH(CHs)--COO--CH3 4.57 413

    a 150 is the molar concentration causing 50% inhibition; p150= -log 150. b Defined in section 2.3.

    Table 4. Inhibition of the reduction of hexacyanoferrate(lI1) (Hill reaction) and the herbicidal effects of a series of nitrofen analogues

    Compound Plsob Herbicide ___ _____ _.

    Number X B4" Observed Calculatedc scored

    12 H I .o 4.92 4.87 616 22 -NHe 1.8 4.61 4.68 216 23 c 1 1.8 4.69 4.68 415

    25 -NH-(CH&-CH3 4.4 4.12 4.06 415 26 -0-CHs 2.9 4.52 4.42 616 27 -CH(CH& 3.2 4.26 4.34 010

    ____ ~ ~ .______ -___- __

    24 -S -CHs 3.3 4.24 4.32 215

    a Sterimol parameter. h 150 is the molar concentration causing 50% inhibition; PISO= -log 150. c Calculated from equation 3. d Defined in section 2.3.

    namely analogues of chloroxuron (compound 29). Many derivatives of chloroxuron were studied and the results are presented in Table 5. A few analogues of nitrofen, in which the oxygen bridge between the two phenyl rings has been changed, are included in Table 6.

    Some representatives of each series were studied in more detail in order to ascertain if all the chemicals act in the same way. Table 1 shows the effects on FeCy reduction under basal, uncoupled, and phosphorylating conditions. Moreover, the effects on typical photosystem I activity, such as PMS phosphorylation and electron transport using MV as acceptor, are reported. Further experi- ments to characterise the site of action showed that the inhibition of the MV reduction by the compounds listed in Table 1 could not be abolished by TMPD alone. However, the inhibition of the FeCy reduction by compounds 1, 2, 12, 21 and 26 could be partially reversed by O.lmM-PD, whereas the inhibition by compound 35 and by DCMU was not.


  • 34 G . van den Berg and J. Tipker

    Table 5. Inhibition of the reduction of hexacyanoferrdte(lI1) (Hill reaction) and the herbicidal effects of a series of...


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