- Email: [email protected]
Bleaching activity and chemical constitution of phenylpyridazinones
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
Bleaching
fiir
Physiologic
288-293 (1981)
Activity and Chemical of Phenylpyridazinonesl
G.SANDMANN, Lehrstuhl
15,
und Biochemie
K.-J. KUNERT, der Pjlunzen.
Universitiit
Constitution
AND P. BOGER Konstunz,
O-7750
Konstanz,
Germany
Received February 11, 1981; accepted April 14, 1981 The bleaching effect of 2-phenylpyridazinones substituted at the 4 or 5 position of the pyridazinone moiety or at the phenyl ring (position 9) was assayed using a greening Scenedesmus mutant after its transfer from heterotrophic to autotrophic growth. The following relationship between bleaching activities and Hammett electronic parameters of the various substituents could be demonstrated. The biological activity of the pyridazinone skeleton was enhanced with substituents showing (a) increasing (T, values in position 4, (b) increasing rrn or cp values in position 9, and (c) decreasing crp values in position 5. These findings could be corroborated by data on pigment bleaching and decrease of photosynthetic oxygen evolution of autotrophic (green) wild-type Scenedesmus after growth in the presence of sublethal concentrations of pyridazinones. There is no structure/activity relationship with direct inhibition of photosynthetic electron transport. Based on electronic parameters, the construction of phenylpyridazinone derivatives with bleaching activity is proposed. INTRODUCTION
Structure activity relationships were set up for herbicides inhibiting photosynthetic electron transport or photophosphorylation [see (1) for review]. In cell-free photosynthetic systems the inhibitory activity of, benzimidazoles (2), e-g., substituted triazines, asymmetric triazinones, or nitrophenols [data and refs. in (3)], pyrimidinones (4), or diphenylamines (5) could be correlated with their electronic or partition parameters. Up to now, there is no report to compare such parameters of certain herbicide classes with the bleaching of chlorophyll in a cellular system, although it is known that certain phenylpyridazinones effectively decrease the carotenoid and chlorophyll content of the cell (6). The unicellular alga Scenedesmus is used as a suitable model system. As reported (7), this allows for a most reliable quantitative determination as to the extent to which the pigment buildup is disturbed by different chemicals. In this paper, inhibition of greening is investigated with various 21 Dedicated to Prof. Dr. M. Seefelder. Ludwigshafen, on the occasion of his 60th birthday. 288 0048-3575/81/030288-06$02.00/o Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
phenylpyridazinones substituted at the 4, 5, or 9 position of the molecule according to the numbering used here. The differential 52
bleaching effects of the pyridazinone derivatives thus measured are compared with the Hammett electronic parameters a, and op of a series of different substituents. Apparently, a relationship can be set up, at least qualitatively, allowing the proposal as to which substituents are responsible for increasing the bleaching effect of phenylpyridazinones. MATERIALS
AND METHODS
The alga Scenedesmus acutus (Chlorophyceae), strain WDG, isolated in this laboratory from the wild strain 2763a (Algae Culture Collection, University of Gottingen, Germany) was grown in the dark in Fernbach flasks containing 1 liter of mineral medium according to (8) including 0.5% glucose and 0.25% Difco yeast extract. This leads to cells containing neither carotenoids nor chlorophyll (7). For greening experi-
BLEACHING
ACTIVITY
OF
ments, the cells were harvested from the stationary phase after at least 1 week of heterotrophic growth, washed with distilled water, and transferred to a medium containing inorganic salts only and adjusted to a density of 0.5 ~1 packed cell volume (pcv) per milliliter. The greening was terminated after 6 days of cultivation. Cultures of autotrophic (green) wild-type Scenedesmus were started with 0.2 ml pcv/ml using the mineral medium of (8). Greening and autotrophic cultivation was carried out in a growth apparatus (KnieseEdwards, Marburg, Germany) at 22-23°C with 250-ml vessels provided with sterile air enriched with 5% CO, (v/v). The cultures were illuminated by a bank of fluorescent lamps (Osram type L 65 W/32 and L 65 W/25) with an intensity of 8000 lux (equivalent to 30 W/m2. as determined with the Yellow Springs Radiometer, Model 65A). Herbicides dissolved in methanol were added immediately after the transfer of the algae from heterotrophic (dark) to autotrophic conditions; the concentration of the solvent was kept below 0.1%. Chlorophyll was measured by extraction with hot methanol (9), total carotenoids were determined after (7) using an extinction coefficient of 2500 for a 1% carotenoid solution. Chloroplast material was prepared according to (lo), and I,,, values of the phenylpyridazinones (Table 2, column 5) were determined in a system H,O + methylviologen (l ,l-dimethyl-4,4’-dipyridylium dichloride) (11) using the Clark-type electrode for oxygen gas exchange measurement (12). The density of the culture was measured as packed cell volume in calibrated microcentrifuge tubes of BO-~1 capacity. RESULTS
To compare structure with bleaching activity by the pyridazinones applied, their interference with the greening process in the light was measured after the dark-grown cells had been transferred into autotrophic medium containing the assay compounds.
PHENYLPYRIDAZINONES
289
After termination of the greening process, chlorophyll content was referred to packed cell volume of the culture which did not change during greening (7). Four groups of phenylpyridazinones were chosen, which were substituted at positions 4, 5, or 9. The u values of their substituents could be related to bleaching activity. The concentration had to be 0.1 ~)!4 for C-Psubstituted derivatives. and 1 PM for the pyridazinones with substituents at the C-4 and C-5 position. In group I of Table 1, the influence of four 4-chloro-9trifluoromethyl-3(2H)-pyridazinones was assayed, which differed in their substitution at C-5. The data demonstrate that with decreasing electronic parameters CT,,,and CJ~ of the substituent in question the bleaching effect of the pyridazinone increases. The compounds of group II neither have the trifluoromethyl group like those of group I, nor Cl at C-4. The important point for choosing this group lies in the fact that these pyridazinones have C-5 substituents representing higher (T,,, and m,, values than those of group I. Also, with an unsubstituted phenyl ring in the molecule. we have the same effect as in group I. The bleaching effect is inversely proportional to the (7P value. A relationship between bleaching activity and cr, value could not be found. Group III of Table 1 exhibits the influence of substituents at the 4 position of the pyridazinone ring. Obviously, the increase of the grn value increases the bleaching activity. In the last group (IV), the phenyl ring of 4-chloro-5-methoxy-2-phenyl-3(2H)pyridazinones is substituted at its position C-9 with ligands having increasing cr,,, or (Jp values. This increases the bleaching effect with Scenedesmus analogous to group III where the substituent is varied at the position C-4. In Table 2, three groups (I, III, IV) of phenylpyridazinones were checked by their influence on autotrophic wildtype Sc,etrcdesmus after a 48-hr growth period. The decreasing levels of carotenoids and chlorophyll due to the particular pyridazinones
SANDMANN,
290
Correlution
Group (concn
of Chlorophyll
Bleuching
KUNERT,
Group (concn
BdCER
TABLE 1 by Substituted 2-Phenylpyriduzinones Electronic Purumeters
with
Hummett
I 0.1 PM)
RI BAS SAN SAN SAN
AND
44521 9114 6706 9789 II 1 pLM)
~nl
-0CH:r -NH, -WWA -NHCH,
-0.21 -0.66 -0.83 -0.84
Chlorophyll
0.12 -0.16 -0.15 -0.30
content 0.94 0.71 0.39 0.06
OpR* 0 R2
BAS 138053 BAS 35668 BAS 78630 BAS 136831 Group (concn
-Br -Cl -CH, -OCH,
Chlorophyll 0.23 0.23 -0.17 -0.27
0.39 0.37 -0.07 0.12
Content 3.47 3.21 2.97 1.99
III 1 kLM)
Chlorophyll BAS BAS BAS BAS
136831 29095 13761 35337
-H -OCH, -Cl -Br
0.00 -0.27 0.23 0.23
0.00 0.12 0.37 0.39
content 2.25 1.52 0.90 0.88
Chlorophyll BAS BAS BAS BAS
13761 105108 100822 44521
-H -OCHF, -OCF,-CHF, -CF,
0.00 0.18 0.28" 0.54
0.00 0.31 0.35" 0.43
content 2.02 1.34 1.12 1.09
Note. Chlorophyll concentrations in mg chlorophyll/ml pcv; the control value was 3.5. up and o,,, values from (16). Analogs of group II have no Cl in position 4. ” Here the up and o,,, values for -OCF,-CHFCI were used, which are apparently close to the values of the substituent R, of BAS 100822.
generally exhibit the same tendency as found after the greening process using the WDG mutant. The differences in bleaching activity are, however, not so clear. As far as photosynthetic electron transport of cells
and chloroplasts is concerned, the structure/activity relationship as found relevant for bleaching has no bearing on inhibition of linear photosynthetic electron transport (Table 2, column 4, 5).
BLEACHING
ACTIVlTY
OF
2
TABLE
Different
Inhibitory
Effects
(2)
(1) Growth (~1 pcv/ml)
Code Control
Chlorophyll (mgml
4452 1 9174 6106
SAN
9789
BAS BAS BAS
136831 29095 13761
BAS
35337
BAS BAS
13761 105108
BAS BAS
100822 4452 I
Note. Herbicide ” System H,O oxygen uptakeimg
(3) Carotenoids (mg/ml pcv)
Cellular (~mol/~I
OH Scenedesmuh, (4) 0, evolution pcv x h)
Wild
Type
(5) I-,,, (M) of electron transport”
0.65
1.42
I.5
10.5
0.33
18.9 5.0
0.65 0.13
1.00 1.13 0.27
4 y IO ”
3.1 0.9
3.0
0.13
0.31
9 ’
3.1 3.0
19.9 17.5
0.68 0.70
6x IO ’ 5 x IO ‘i
3.5 3.0 Group
17.4 16.9
0.64 0.66
1.42 I .40 I .29 1.39
3 x IOI”
0.7 Group
-
I 9 g IO i 5 x 10 ‘i IO i
III
6 x 10 Ii
IV
1.4
17.4
0.64
3.5 0.5
16.4 12.8 10.5
0.60 0.41 0.33
I.5
+
pcv)
Phenylpyriduzinones
19.2
3.5 Group
BAS SAN SAN
of Substituted
191
PHENYLPYRIDAZINONES
concentration: methylviologen chlorophyll
As pointed out previously (7), the unicellular green alga Scenedesmus again proves to be a good assay system to quantitatively investigate the biological activity of certain compounds. Pyridazinone concentrations of 0.1 to 1 pM were sufficient to determine their influence on chlorophyll content of the cell during greening and buildup of the photosynthetic apparatus. During the greening period assayed here photosynthesis is not substantial. So, additional effects (e.g., on photosynthetic electron transport) are excluded. Furthermore, the I,, value for bleaching during the greening period is generally lower than the corresponding figure to inhibit photosynthetic electron transport. SAN 9789 has an I,, of 8.8 nM to inhibit greening (7), an Iso of 0.35 mM for photosynthetic oxygen evolution of the cell (12), and an Iso of about 0.1 mM for inhibition of cell-free electron transport (Table 2). The same pyridazinone series were applied to autotrophic Scenedesmus, i.e.,
IO”
3 x 10~” 3 x IO ;
0.72 1.00
1 PM (columns I-4) during a 48-hr autotrophic (spinach chloroplasts) with control rates x hr in the presence of 1 mM NH,CI.
DISCUSSION
2x
1.29 1.05
9 x IO I’ growth of 90-100
period. pmol
light-induced
cells which already had a complete photosynthetic apparatus (Table 2). Their effect on the latter system was found to be identical with one exception. SAN 9774 is stronger in the greening assay than BAS 44521, whereas the latter pyridazinone is more effective on Scenedesmus cells which are already green and grow autotrophically. Together with previous findings (6) this indicates that active pyridazinones apparently exhibit two effects: on the one hand, they inhibit the buildup of the photosynthetic pigments, particularly carotenoids, and on the other hand pigment inhibition is followed by peroxidative destruction of existing carotenoids and subsequently of chlorophyll (13). The latter effect is accompanied by a decrease of (cellular) photosynthetic oxygen evolution (cf. Table 2, column 4). The first effect is mainly seen with the greening assay, the latter one with green cells after autotrophic growth with pyridazinones present. Apparently, a stronger pigment destruction occurs with BAS 44521 (6), which cannot be seen so clearly in the greening assay. The strong
292
SANDMANN,
KUNERT,
bleaching effect of SAN 9774 on the greening Scenedesmus mutant is similar to that reported for germinating wheat seedlings (14). The series of pyridazinones in the groups I and II of Table 1 with regard to their bleaching activity in the greening assay is in accordance with the series of the u’p values of the substituents at position 5. It is apparent that a substituent at C-5 having more negative up values and thereby shifting electrons toward the pyridazinone ring increases the bleaching activity of this derivative in our assay. The opposite is true with the ligands at position 4 as well as 9 (groups III, IV). With those, the bleaching effect is favored by decreased electron density at the C atoms 4 and 9, respectively. In group III, the activity of the phenylpyridazinones matches with the a;, values of the substituent of C-4, and in group IV bleaching increases with increasing u,,, and mp values of the C-4 substituents. Also in the case of N-phenyl-3,4,5,6-tetrahydrophthalimides an electron-withdrawing substituent in meta position at the phenyl ring increases the herbicidal action of these compounds on higher plants (15). We do not intend to speculate on how inductive and mesomeric effects of the different ligands contribute to the charge distribution along the complete molecule. However, it appears that the ligands at the carbon positions 4, 5, and 9 stabilize a resonance structure which-due to appropriate charge delocalization-could lead to a positive charge at the N-atom 2 and at the substituent of position C-5. Further, these ligands cause negative partial charges at the oxygen of carbon-atom 3, and at the ligands of C-4 or C-9. The resulting effect over the complete pyridazinone molecule and the stabilization of the partial charges at the positions mentioned appears to be a prerequisite for the bleaching effect of these compounds. According to our data presented the following scheme indicates the charge distribution at the phenylpyridazinone molecule leading to optimum bleaching:
AND
BijCER
At the moment-comparing all the pyridazinones we have in our hands-SAN 9789 (norflurazon) best fulfills these conditions in the greening assay as well as in the system under exclusively autotrophic conditions. According to our data, the bleaching effect of SAN 9789 may be reached or even increased by substituting the CF, group at C-atom 9 by substituents with more positive (TV and u, values and the Cl at C-4 by groups having more positive v,,, values. For example such ligands for C-9 may be a cyano or nitro group. As a further possibility, an additional electron-withdrawing substituent introduced in meta position of C-l 1 of the phenyl ring should stabilize the charge distribution indicated in the formula. These proposed derivatives of phenylpyridazinones should exert good bleaching activity in our algal model system. Presumably the two rings form a dihedral angle (unpublished results of Dr. F. R. Rittig, Limburgerhof). A prerequisite is, therefore, that electronic influences are not counteracted by a change of the angle due to different substituents. Furthermore, a substituent should not introduce a lack of lipophilicity nor penetration or breakdown problems. Finally, the question as to whether these compounds are herbicides will, of course, have to be checked with higher plants. ACKNOWLEDGMENTS This study was supported by the Deutsche Forschungsgemeinschaft (Grant Bo 310/l 1 to P.B.) and by the Fonds der Chemischen Industrie. The authors are grateful to Landwirtschaftliche Versuchsstation Limburgerhof and Hauptlabor of BASF AG. Ludwigshafen. Germany, for efficient cooperation and supply of the phenylpyridazinone derivatives. REFERENCES 1. K. H. Btichel. ture activity photosynthesis,
Mechanisms of action and strucrelations of herbicides that inhibit Pestic. Sci. 3, 89 (1972).
BLEACHING
ACTIVITY
OF PHENYLPYRIDAZINONES
2. K. H. Btichel. W. Draber, A. Trebst. and E. Pistorius. Zur Hemmung photosynthetischer Reaktionen in isolierten Chloroplasten durch Herbizide des Benzimidazol-Typs und deren Struktur-Aktivitats-Beziehung unter Berticksichtigung des Verteilungskoeffizienten und des pk,-Wertes. Z. Nnrurforsch. 21b, 243 (1966). 3. A. Trebst and W. Draber, Structure activity correlation of recent herbicides in photosynthetic reactions, in “Advances in Pesticide Science” (H. Geissbuhler. Ed.). Part 2. pp. 223-234. Pergamon. Oxford/New York, 1979. 4. L. K. Gibbons, E. F. Koldenhoven. A. A. Nethery, R. E. Montgomery. and W. P. Purcell. Quantitative structure-activity relationships among selected pyrimidinones and Hill reaction inhibition. J. Agr. Food Chem. 24, 203 (1976). 5. W. Oettmeier. Quantitative structure activity relationship of diphenylamines as inhibitors of photosynthetic electron transport and phosphorylation. Z. Naturforsch. 34c, 1024 (1979). 6. K.-J. Kunert and P. Boger, Influence of bleaching herbicides on chlorophyll and carotenoids. Z. Narurforsch. 34~. 1047 (1979). 7. G. Sandmann. K.-J. Kunert, and P. Boger, Biological systems to assay herbicidal bleaching, Z. Nuiurfb-sch. 34c, 1044 (1979). 8. K. -J. Kunert, H. Bohme. and P. Boger, Reactions of plastocyanin and cytochrome 553 with photosystem 1 of Scenedesmus. Biochrm. Bioph.vs. Acfa 449. 541 (1976).
293
9. P. Boger. Das Strukturproteid aus den Chloroplasten einzelliger Grtinalgen und seine Beziehung zum Chlorophyll, Norcr (Jentrt 154. I74 (1964). 10. H. Bohme and W. A. Cramer. Plastoquinone mediates electron transport between cytochrome h-559 and cytochrome f in spinach chloroplasts. FEBS Lutt. 15. 349 (1971) 11. P. Boger and K. -J. Kunert, Phytotoxic action of paraquat on the photosynthetic apparatus. 1. Ntrfu$iwhc,h. 33c, 668 ( 1978). 12. P. Boger and U. Schlue, Long-term effects of herbicides on the photosynthetic apparatus. I. lnfluence of diuron. triazines and pyridazinone\. Weed Res. 16. 149 (1976) 13. G. Sandmann and P. Boger. Mode of action of bleaching herbicides. in “Biochemical Rrsponses Induced by Herbicides” tD. E. Moreland. Ed.), Amer. Chem. Sot. Symp. Serie\. Washington. D.C., 1981. in press. 14. J. B. St. John, Manipulation of galactolipid fatty acid composition with substituted pyridazinones, /%trzf Physiol. 57. 38 (1976). 15. K. Wakabayashi. K. Matsuya. H. Ohta. and T. Jikihara. Structure-activity relationship of cyclic imide herbicides. in “Advances in Pesticide Science” (H. Geissbuhler. Ed.). Part 2. pp. 2566260. Pergamon. Oxford/New York. 1979. 16. C. Hansch. A. Leo. S. H. Unger. K. H. Kim, I). Nikaitani. and E. J. Lien. Aromatic substituent constants for structure-activity correlation. J. Med. Chem. 16. 1207 (1973).
BIOCHEMISTRY
AND
PHYSIOLOGY
Bleaching
fiir
Physiologic
288-293 (1981)
Activity and Chemical of Phenylpyridazinonesl
G.SANDMANN, Lehrstuhl
15,
und Biochemie
K.-J. KUNERT, der Pjlunzen.
Universitiit
Constitution
AND P. BOGER Konstunz,
O-7750
Konstanz,
Germany
Received February 11, 1981; accepted April 14, 1981 The bleaching effect of 2-phenylpyridazinones substituted at the 4 or 5 position of the pyridazinone moiety or at the phenyl ring (position 9) was assayed using a greening Scenedesmus mutant after its transfer from heterotrophic to autotrophic growth. The following relationship between bleaching activities and Hammett electronic parameters of the various substituents could be demonstrated. The biological activity of the pyridazinone skeleton was enhanced with substituents showing (a) increasing (T, values in position 4, (b) increasing rrn or cp values in position 9, and (c) decreasing crp values in position 5. These findings could be corroborated by data on pigment bleaching and decrease of photosynthetic oxygen evolution of autotrophic (green) wild-type Scenedesmus after growth in the presence of sublethal concentrations of pyridazinones. There is no structure/activity relationship with direct inhibition of photosynthetic electron transport. Based on electronic parameters, the construction of phenylpyridazinone derivatives with bleaching activity is proposed. INTRODUCTION
Structure activity relationships were set up for herbicides inhibiting photosynthetic electron transport or photophosphorylation [see (1) for review]. In cell-free photosynthetic systems the inhibitory activity of, benzimidazoles (2), e-g., substituted triazines, asymmetric triazinones, or nitrophenols [data and refs. in (3)], pyrimidinones (4), or diphenylamines (5) could be correlated with their electronic or partition parameters. Up to now, there is no report to compare such parameters of certain herbicide classes with the bleaching of chlorophyll in a cellular system, although it is known that certain phenylpyridazinones effectively decrease the carotenoid and chlorophyll content of the cell (6). The unicellular alga Scenedesmus is used as a suitable model system. As reported (7), this allows for a most reliable quantitative determination as to the extent to which the pigment buildup is disturbed by different chemicals. In this paper, inhibition of greening is investigated with various 21 Dedicated to Prof. Dr. M. Seefelder. Ludwigshafen, on the occasion of his 60th birthday. 288 0048-3575/81/030288-06$02.00/o Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
phenylpyridazinones substituted at the 4, 5, or 9 position of the molecule according to the numbering used here. The differential 52
bleaching effects of the pyridazinone derivatives thus measured are compared with the Hammett electronic parameters a, and op of a series of different substituents. Apparently, a relationship can be set up, at least qualitatively, allowing the proposal as to which substituents are responsible for increasing the bleaching effect of phenylpyridazinones. MATERIALS
AND METHODS
The alga Scenedesmus acutus (Chlorophyceae), strain WDG, isolated in this laboratory from the wild strain 2763a (Algae Culture Collection, University of Gottingen, Germany) was grown in the dark in Fernbach flasks containing 1 liter of mineral medium according to (8) including 0.5% glucose and 0.25% Difco yeast extract. This leads to cells containing neither carotenoids nor chlorophyll (7). For greening experi-
BLEACHING
ACTIVITY
OF
ments, the cells were harvested from the stationary phase after at least 1 week of heterotrophic growth, washed with distilled water, and transferred to a medium containing inorganic salts only and adjusted to a density of 0.5 ~1 packed cell volume (pcv) per milliliter. The greening was terminated after 6 days of cultivation. Cultures of autotrophic (green) wild-type Scenedesmus were started with 0.2 ml pcv/ml using the mineral medium of (8). Greening and autotrophic cultivation was carried out in a growth apparatus (KnieseEdwards, Marburg, Germany) at 22-23°C with 250-ml vessels provided with sterile air enriched with 5% CO, (v/v). The cultures were illuminated by a bank of fluorescent lamps (Osram type L 65 W/32 and L 65 W/25) with an intensity of 8000 lux (equivalent to 30 W/m2. as determined with the Yellow Springs Radiometer, Model 65A). Herbicides dissolved in methanol were added immediately after the transfer of the algae from heterotrophic (dark) to autotrophic conditions; the concentration of the solvent was kept below 0.1%. Chlorophyll was measured by extraction with hot methanol (9), total carotenoids were determined after (7) using an extinction coefficient of 2500 for a 1% carotenoid solution. Chloroplast material was prepared according to (lo), and I,,, values of the phenylpyridazinones (Table 2, column 5) were determined in a system H,O + methylviologen (l ,l-dimethyl-4,4’-dipyridylium dichloride) (11) using the Clark-type electrode for oxygen gas exchange measurement (12). The density of the culture was measured as packed cell volume in calibrated microcentrifuge tubes of BO-~1 capacity. RESULTS
To compare structure with bleaching activity by the pyridazinones applied, their interference with the greening process in the light was measured after the dark-grown cells had been transferred into autotrophic medium containing the assay compounds.
PHENYLPYRIDAZINONES
289
After termination of the greening process, chlorophyll content was referred to packed cell volume of the culture which did not change during greening (7). Four groups of phenylpyridazinones were chosen, which were substituted at positions 4, 5, or 9. The u values of their substituents could be related to bleaching activity. The concentration had to be 0.1 ~)!4 for C-Psubstituted derivatives. and 1 PM for the pyridazinones with substituents at the C-4 and C-5 position. In group I of Table 1, the influence of four 4-chloro-9trifluoromethyl-3(2H)-pyridazinones was assayed, which differed in their substitution at C-5. The data demonstrate that with decreasing electronic parameters CT,,,and CJ~ of the substituent in question the bleaching effect of the pyridazinone increases. The compounds of group II neither have the trifluoromethyl group like those of group I, nor Cl at C-4. The important point for choosing this group lies in the fact that these pyridazinones have C-5 substituents representing higher (T,,, and m,, values than those of group I. Also, with an unsubstituted phenyl ring in the molecule. we have the same effect as in group I. The bleaching effect is inversely proportional to the (7P value. A relationship between bleaching activity and cr, value could not be found. Group III of Table 1 exhibits the influence of substituents at the 4 position of the pyridazinone ring. Obviously, the increase of the grn value increases the bleaching activity. In the last group (IV), the phenyl ring of 4-chloro-5-methoxy-2-phenyl-3(2H)pyridazinones is substituted at its position C-9 with ligands having increasing cr,,, or (Jp values. This increases the bleaching effect with Scenedesmus analogous to group III where the substituent is varied at the position C-4. In Table 2, three groups (I, III, IV) of phenylpyridazinones were checked by their influence on autotrophic wildtype Sc,etrcdesmus after a 48-hr growth period. The decreasing levels of carotenoids and chlorophyll due to the particular pyridazinones
SANDMANN,
290
Correlution
Group (concn
of Chlorophyll
Bleuching
KUNERT,
Group (concn
BdCER
TABLE 1 by Substituted 2-Phenylpyriduzinones Electronic Purumeters
with
Hummett
I 0.1 PM)
RI BAS SAN SAN SAN
AND
44521 9114 6706 9789 II 1 pLM)
~nl
-0CH:r -NH, -WWA -NHCH,
-0.21 -0.66 -0.83 -0.84
Chlorophyll
0.12 -0.16 -0.15 -0.30
content 0.94 0.71 0.39 0.06
OpR* 0 R2
BAS 138053 BAS 35668 BAS 78630 BAS 136831 Group (concn
-Br -Cl -CH, -OCH,
Chlorophyll 0.23 0.23 -0.17 -0.27
0.39 0.37 -0.07 0.12
Content 3.47 3.21 2.97 1.99
III 1 kLM)
Chlorophyll BAS BAS BAS BAS
136831 29095 13761 35337
-H -OCH, -Cl -Br
0.00 -0.27 0.23 0.23
0.00 0.12 0.37 0.39
content 2.25 1.52 0.90 0.88
Chlorophyll BAS BAS BAS BAS
13761 105108 100822 44521
-H -OCHF, -OCF,-CHF, -CF,
0.00 0.18 0.28" 0.54
0.00 0.31 0.35" 0.43
content 2.02 1.34 1.12 1.09
Note. Chlorophyll concentrations in mg chlorophyll/ml pcv; the control value was 3.5. up and o,,, values from (16). Analogs of group II have no Cl in position 4. ” Here the up and o,,, values for -OCF,-CHFCI were used, which are apparently close to the values of the substituent R, of BAS 100822.
generally exhibit the same tendency as found after the greening process using the WDG mutant. The differences in bleaching activity are, however, not so clear. As far as photosynthetic electron transport of cells
and chloroplasts is concerned, the structure/activity relationship as found relevant for bleaching has no bearing on inhibition of linear photosynthetic electron transport (Table 2, column 4, 5).
BLEACHING
ACTIVlTY
OF
2
TABLE
Different
Inhibitory
Effects
(2)
(1) Growth (~1 pcv/ml)
Code Control
Chlorophyll (mgml
4452 1 9174 6106
SAN
9789
BAS BAS BAS
136831 29095 13761
BAS
35337
BAS BAS
13761 105108
BAS BAS
100822 4452 I
Note. Herbicide ” System H,O oxygen uptakeimg
(3) Carotenoids (mg/ml pcv)
Cellular (~mol/~I
OH Scenedesmuh, (4) 0, evolution pcv x h)
Wild
Type
(5) I-,,, (M) of electron transport”
0.65
1.42
I.5
10.5
0.33
18.9 5.0
0.65 0.13
1.00 1.13 0.27
4 y IO ”
3.1 0.9
3.0
0.13
0.31
9 ’
3.1 3.0
19.9 17.5
0.68 0.70
6x IO ’ 5 x IO ‘i
3.5 3.0 Group
17.4 16.9
0.64 0.66
1.42 I .40 I .29 1.39
3 x IOI”
0.7 Group
-
I 9 g IO i 5 x 10 ‘i IO i
III
6 x 10 Ii
IV
1.4
17.4
0.64
3.5 0.5
16.4 12.8 10.5
0.60 0.41 0.33
I.5
+
pcv)
Phenylpyriduzinones
19.2
3.5 Group
BAS SAN SAN
of Substituted
191
PHENYLPYRIDAZINONES
concentration: methylviologen chlorophyll
As pointed out previously (7), the unicellular green alga Scenedesmus again proves to be a good assay system to quantitatively investigate the biological activity of certain compounds. Pyridazinone concentrations of 0.1 to 1 pM were sufficient to determine their influence on chlorophyll content of the cell during greening and buildup of the photosynthetic apparatus. During the greening period assayed here photosynthesis is not substantial. So, additional effects (e.g., on photosynthetic electron transport) are excluded. Furthermore, the I,, value for bleaching during the greening period is generally lower than the corresponding figure to inhibit photosynthetic electron transport. SAN 9789 has an I,, of 8.8 nM to inhibit greening (7), an Iso of 0.35 mM for photosynthetic oxygen evolution of the cell (12), and an Iso of about 0.1 mM for inhibition of cell-free electron transport (Table 2). The same pyridazinone series were applied to autotrophic Scenedesmus, i.e.,
IO”
3 x 10~” 3 x IO ;
0.72 1.00
1 PM (columns I-4) during a 48-hr autotrophic (spinach chloroplasts) with control rates x hr in the presence of 1 mM NH,CI.
DISCUSSION
2x
1.29 1.05
9 x IO I’ growth of 90-100
period. pmol
light-induced
cells which already had a complete photosynthetic apparatus (Table 2). Their effect on the latter system was found to be identical with one exception. SAN 9774 is stronger in the greening assay than BAS 44521, whereas the latter pyridazinone is more effective on Scenedesmus cells which are already green and grow autotrophically. Together with previous findings (6) this indicates that active pyridazinones apparently exhibit two effects: on the one hand, they inhibit the buildup of the photosynthetic pigments, particularly carotenoids, and on the other hand pigment inhibition is followed by peroxidative destruction of existing carotenoids and subsequently of chlorophyll (13). The latter effect is accompanied by a decrease of (cellular) photosynthetic oxygen evolution (cf. Table 2, column 4). The first effect is mainly seen with the greening assay, the latter one with green cells after autotrophic growth with pyridazinones present. Apparently, a stronger pigment destruction occurs with BAS 44521 (6), which cannot be seen so clearly in the greening assay. The strong
292
SANDMANN,
KUNERT,
bleaching effect of SAN 9774 on the greening Scenedesmus mutant is similar to that reported for germinating wheat seedlings (14). The series of pyridazinones in the groups I and II of Table 1 with regard to their bleaching activity in the greening assay is in accordance with the series of the u’p values of the substituents at position 5. It is apparent that a substituent at C-5 having more negative up values and thereby shifting electrons toward the pyridazinone ring increases the bleaching activity of this derivative in our assay. The opposite is true with the ligands at position 4 as well as 9 (groups III, IV). With those, the bleaching effect is favored by decreased electron density at the C atoms 4 and 9, respectively. In group III, the activity of the phenylpyridazinones matches with the a;, values of the substituent of C-4, and in group IV bleaching increases with increasing u,,, and mp values of the C-4 substituents. Also in the case of N-phenyl-3,4,5,6-tetrahydrophthalimides an electron-withdrawing substituent in meta position at the phenyl ring increases the herbicidal action of these compounds on higher plants (15). We do not intend to speculate on how inductive and mesomeric effects of the different ligands contribute to the charge distribution along the complete molecule. However, it appears that the ligands at the carbon positions 4, 5, and 9 stabilize a resonance structure which-due to appropriate charge delocalization-could lead to a positive charge at the N-atom 2 and at the substituent of position C-5. Further, these ligands cause negative partial charges at the oxygen of carbon-atom 3, and at the ligands of C-4 or C-9. The resulting effect over the complete pyridazinone molecule and the stabilization of the partial charges at the positions mentioned appears to be a prerequisite for the bleaching effect of these compounds. According to our data presented the following scheme indicates the charge distribution at the phenylpyridazinone molecule leading to optimum bleaching:
AND
BijCER
At the moment-comparing all the pyridazinones we have in our hands-SAN 9789 (norflurazon) best fulfills these conditions in the greening assay as well as in the system under exclusively autotrophic conditions. According to our data, the bleaching effect of SAN 9789 may be reached or even increased by substituting the CF, group at C-atom 9 by substituents with more positive (TV and u, values and the Cl at C-4 by groups having more positive v,,, values. For example such ligands for C-9 may be a cyano or nitro group. As a further possibility, an additional electron-withdrawing substituent introduced in meta position of C-l 1 of the phenyl ring should stabilize the charge distribution indicated in the formula. These proposed derivatives of phenylpyridazinones should exert good bleaching activity in our algal model system. Presumably the two rings form a dihedral angle (unpublished results of Dr. F. R. Rittig, Limburgerhof). A prerequisite is, therefore, that electronic influences are not counteracted by a change of the angle due to different substituents. Furthermore, a substituent should not introduce a lack of lipophilicity nor penetration or breakdown problems. Finally, the question as to whether these compounds are herbicides will, of course, have to be checked with higher plants. ACKNOWLEDGMENTS This study was supported by the Deutsche Forschungsgemeinschaft (Grant Bo 310/l 1 to P.B.) and by the Fonds der Chemischen Industrie. The authors are grateful to Landwirtschaftliche Versuchsstation Limburgerhof and Hauptlabor of BASF AG. Ludwigshafen. Germany, for efficient cooperation and supply of the phenylpyridazinone derivatives. REFERENCES 1. K. H. Btichel. ture activity photosynthesis,
Mechanisms of action and strucrelations of herbicides that inhibit Pestic. Sci. 3, 89 (1972).
BLEACHING
ACTIVITY
OF PHENYLPYRIDAZINONES
2. K. H. Btichel. W. Draber, A. Trebst. and E. Pistorius. Zur Hemmung photosynthetischer Reaktionen in isolierten Chloroplasten durch Herbizide des Benzimidazol-Typs und deren Struktur-Aktivitats-Beziehung unter Berticksichtigung des Verteilungskoeffizienten und des pk,-Wertes. Z. Nnrurforsch. 21b, 243 (1966). 3. A. Trebst and W. Draber, Structure activity correlation of recent herbicides in photosynthetic reactions, in “Advances in Pesticide Science” (H. Geissbuhler. Ed.). Part 2. pp. 223-234. Pergamon. Oxford/New York, 1979. 4. L. K. Gibbons, E. F. Koldenhoven. A. A. Nethery, R. E. Montgomery. and W. P. Purcell. Quantitative structure-activity relationships among selected pyrimidinones and Hill reaction inhibition. J. Agr. Food Chem. 24, 203 (1976). 5. W. Oettmeier. Quantitative structure activity relationship of diphenylamines as inhibitors of photosynthetic electron transport and phosphorylation. Z. Naturforsch. 34c, 1024 (1979). 6. K.-J. Kunert and P. Boger, Influence of bleaching herbicides on chlorophyll and carotenoids. Z. Narurforsch. 34~. 1047 (1979). 7. G. Sandmann. K.-J. Kunert, and P. Boger, Biological systems to assay herbicidal bleaching, Z. Nuiurfb-sch. 34c, 1044 (1979). 8. K. -J. Kunert, H. Bohme. and P. Boger, Reactions of plastocyanin and cytochrome 553 with photosystem 1 of Scenedesmus. Biochrm. Bioph.vs. Acfa 449. 541 (1976).
293
9. P. Boger. Das Strukturproteid aus den Chloroplasten einzelliger Grtinalgen und seine Beziehung zum Chlorophyll, Norcr (Jentrt 154. I74 (1964). 10. H. Bohme and W. A. Cramer. Plastoquinone mediates electron transport between cytochrome h-559 and cytochrome f in spinach chloroplasts. FEBS Lutt. 15. 349 (1971) 11. P. Boger and K. -J. Kunert, Phytotoxic action of paraquat on the photosynthetic apparatus. 1. Ntrfu$iwhc,h. 33c, 668 ( 1978). 12. P. Boger and U. Schlue, Long-term effects of herbicides on the photosynthetic apparatus. I. lnfluence of diuron. triazines and pyridazinone\. Weed Res. 16. 149 (1976) 13. G. Sandmann and P. Boger. Mode of action of bleaching herbicides. in “Biochemical Rrsponses Induced by Herbicides” tD. E. Moreland. Ed.), Amer. Chem. Sot. Symp. Serie\. Washington. D.C., 1981. in press. 14. J. B. St. John, Manipulation of galactolipid fatty acid composition with substituted pyridazinones, /%trzf Physiol. 57. 38 (1976). 15. K. Wakabayashi. K. Matsuya. H. Ohta. and T. Jikihara. Structure-activity relationship of cyclic imide herbicides. in “Advances in Pesticide Science” (H. Geissbuhler. Ed.). Part 2. pp. 2566260. Pergamon. Oxford/New York. 1979. 16. C. Hansch. A. Leo. S. H. Unger. K. H. Kim, I). Nikaitani. and E. J. Lien. Aromatic substituent constants for structure-activity correlation. J. Med. Chem. 16. 1207 (1973).