Pyrazole carboxanilide fungicides

PESTICIDEBIOCHEMISTRYAND PHYSIOLOGY25, 163- 168 (1986)

Pyrazole Carboxanilide I. Correlation

of Mitochondrial Electron Transport and Anti-fungal Activity

G. A. WHITE,* J. N. PHILLIPS,? J. L. HUPPATZ,? *Agriculture

Canada KSIRO.

Research Dhhiorz

Fungicides

Centre, of Plant

University Industry,

B. WITRZENS,~

Sub Post Office, London, GPO Box 1600. Canberra

Inhibition AND S. J. GRANT*

Ontario, Canada City, 2601 AusrraIia

N6A5B7;

aad

Received January 2. 1985: accepted April IO. 1985 A series of carboxin-like compounds. the N-methylpyrazole carboxanilides and their mono- and dimethyl derivatives have been assayed as inhibitors of succinate dehydrogenase enzyme complexes (SDCs) isolated from Ustilago maydis, Rhizoctonia solani, Gneamannomyces gramink. and Fasariccm oxysporum. The pattern of inhibitory activity within the series was broadly similar for each of the fungi although minor differences indicated some structural variation between the enzyme complexes. There was a general correlation between inhibition of the SDCs isolated from R. solani and G. graminis and inhibition of mycelial growth of these same organisms which was consistent with the primary mode of action of these compounds being interference with mitochondrial electron transport. No such correlation was evident with F. oxysporum. where some of the compounds showed activity against the SDC but none had any effect on fungal growth. This suggests that if SDC inhibitory activity is the primary determinant of the anti-fungal activity of these compounds it does not necessarilv determine their anti-fungal specificity: some possible explanations are offered. 0 1986 Academic Pres. In<

carboxanilides and their mono- and dimethyl derivatives by comparing their effectiveness against mycelial growth of Rhizoctonia solani with their ability to protect cotton seedlings from root rot disease caused by that organism (4). This series of pyrazoles (see Fig. 1) seemed appropriate for studying the relationship between molecular structure and inhibition of both mitochondrial succinate dehydrogenase activity and of mycelial growth in the one organism. Carboxanilide fungicides are known to be selectively active against Basidiomycete fungi (5, 6) and the species chosen for this comparative study included a Basidiomycete, Rhizoctonia solani and two non-Basidiomycetes, Fusarium oxyspot-urn and Gaeumannomyces graminis. In addition SDC inhibition data relating to mitochondria from the Basidiomycete Usti/ago maydis have been included to facilitate comparison of the relative activities of the pyrazole derivatives with other carbox-

INTRODUCTION

The mode of action of carboxin and related carboxanilide fungicides has been attributed to their ability to interfere with mitochondrial electron transport by inhibiting the succinate dehydrogenase enzyme complex (SDC; Complex II; succinate-ubiquinone reductase) (1, 2). A broad range of aryl and heteroaryl carboxamide derivatives have been investigated and the structural features required for optimum enzyme inhibitory activity and for anti-fungal activity have been shown to be similar (3). However, there has been no detailed comparative study of the inhibitory activities of a closely related series of carboxamide derivatives against both mitochondrial SDC and mycelial growth using the same organism . Recently, a good correlation between in vitro and in vivo anti-fungal activity was observed for a series of N-methylpyrazole 163

004%3575/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

164

WHITE

ET

AL.

0

P C-N-@ ‘mH

’ ‘:

P

vlll’

CH3

FIG.

1. N-Methylpyrazole

amide fungicides 7, 8). MATERIALS

reported AND

carboxanilides

earlier (1, 2, 3, METHODS

Chemicals. The pyrazole carboxanilide derivatives were prepared and characterized as described previously (4, 9, 10). Carboxin was obtained from Dr. J. Wilson, Uniroyal Research Laboratory Ltd., Guelph, Ontario, Canada. Bovine serum albumin (BSA, fraction V) was purchased from Sigma Chemical Company. BDH Chemicals Ltd. supplied 2,6-dichlorophenol-indophenol dye (DCIP). Peptone, yeast extract and potato dextrose broth were obtained from Difco Laboratories. Fungal cells were disrupted with glass beads (0.1-0.11 mm; B. Braun Apparate Melsungen AG, Germany) acquired from Oxford Laboratories of Canada Ltd., London, Ontario, Canada. All chemical and nutrient solutions were prepared in glass-distilled water. Maintenance of fungi. The wild-type strain of Ustilago maydis (D.C.) Cda, ATCC 14826, was maintained on slants of Holliday’s complete medium (11). Rhizoc-

and their

mono-

and dimethyl

CH

derivatives.

tonia solani Kuhn and F. oxysporum

f. lycopersici (Sacc.) Snyder and Hansen DAR 34716 were kept on potato-dextrose agar (PDA). Gaeumannomyces graminis (Sacc.) Arx et. Oliver var. tritici DAR 17916 was maintained on the basal medium of Coursen and Sisler (12) which was modified as follows: glucose, 25 g; peptone, 5 g; yeast extract, 4 g; NaNO,, 0.3 g; KH2P0,, 2 g; K,HPO,, 1 g; (NH,),SO,, 1 g; trace element and vitamin stock solutions, 1.0 mg each (2); additional biotin and thiamine, 1.0 mg each and 15 g of agar per liter of distilled water, pH 7.5. Stock biotin and thiamine solutions (1 mg/ml) were autoclaved separately before addition to the medium. All fungi were grown at 28°C on solid medium and then stored at 15°C. Inhibition of mycelial growth. Agar disks (approx 0.7 cm*) overgrown by mycelium were placed in the centre of an 8.5-cm petri dish containing PDA medium in which the test compound had been incorporated at the required concentration. There were two replicates per treatment and control. The dishes were then incubated at 25°C until the untreated controls had just covered the

PYRAZOLE

CARBOXANILIDE

FUNGICIDES,

STRUCTURE-ACTIVITY

STUDIES

165

plate. The ratio of the treatedand untreated days at 24°C. Such liquid cultures were colony diameter was plotted against the blendedfor 20 set and poured into 450 ml molar concentration of the test compound of medium. Mycelia were grown by shaking and the EC,, value determined. at 150 rpm for 24 hr at 24°C. The fungus Liquid culture of fungi. Sporidia of U. maydis were cultured and harvested as re-

ported previously (3), then stored as frozen pellets in centrifuge tubes. For growth of F. oxysporum f. lycopersici, conidia and mycelial fragments were gently scraped with a glass rod into sterile distilled water from the surface of five, 2-week-old PDA plate cultures (28°C) and placed in a 2-liter Erlenmeyer flask containing 500 ml of basal liquid medium (12) supplemented with 1 g/ liter of yeast extract. Flasks were shaken at 150 rpm (rotary shaker, New Brunswick Scientific Co., New Brunswick, N.J.) for 24 hr at 24°C. Cells were then collected by vacuum filtration on Whatman No. 3 paper. The filtered cell mat was broken up either in ice-cold distilled water or an ice-cold solution of 0.25 M sucrose, 0.005 M EDTA, and 0.15% BSA, pH 7.0 (3) and refiltered. The washing process was repeated once. Cell mats were cut into 6 g lots, wrapped in aluminium foil, and frozen at - 18°C. Rhizoctonia solani was grown at 28°C on plates of PDA medium. Approximately one-third of a plate culture was blended (stainless steel Waring Blendor) for 20 set under sterile conditions in 50- 100 ml of potato dextrose broth containing 0.1% CaCO, at pH 7.6. The homogenized mycelial fragments were shaken at 150 rpm for 4-5 days at 24°C. Subsequently, the clumps of myCelia were blended and inoculated into 450 ml of the potato-dextrose-CaCO, broth. Flasks were shaken as above for 24 hr. The mycelia were collected by centrifugation and washed as for U. maydis except that the fungal tissue was blended briefly in wash medium prior to centrifugation. Cell pellets were stored frozen. Gaeumannomyces graminis was grown at 28°C on plates containing the modified solid medium described before. Pieces of agar culture were placed in 50 ml of modified liquid medium and shaken at 150 rpm for 3-4

was harvested,

washed, and stored as per

R. solani. Preparation

of mitochondria. Mitochondria from cells of the four fungi were isolated essentially as described earlier for I/. maydis (3, 8) with the exception that a 55 ml tissue grinder (Wheaton Scientific, Millville, N.J.) fitted with a Teflon pestle was employed. Varying quantities of frozen fungal cell pellets were disintegrated with glass beads, depending upon enzyme activity. The weight of glass beads and volume of isolation medium was adjusted to give a medium-thick grinding mixture. Succinate dehydrogenase assays. Mitochondrial succinate dehydrogenase activity was measured by recording the rate of DCIP reduction at 600 nm and 22°C using a double beam recording spectrophotometer. Reactions were started by the addition of mitochondria to the assay system used by White et al. (7). All carboxamides were added as ethanol solutions; final alcohol concentration, 1.3% v/v. Ethanol was included in the control runs. Rates of DCIP reduction were calculated using an extinction coefficient (C) of 2.1 x IO4 [M- lcm ‘1 at 600 nm. Where needed, rates were corrected for small endogenous activities without succinate. The I,, values for inhibition were determined from semi-log plots as reported previously (7). RESULTS

Table 1 records inhibitory activities expressed in terms of I,, @Ml values for the series of mono- and dimethyl N-methylpyrazole carboxanilides shown in Fig. 1 in relation to electron transport catalysed by the mitochondrial SDC prepared from various fungi, viz., R. solani, G. graminis, F. oxysporum, and U. maydis. Data for carboxin are also included for comparison. Compounds II, VI, VIII, IX, X, XI, and XII showed measurable inhibitory activity

166

WHITE

ET AL.

TABLE Inhibitory

Effects

of N-Methylpyrazole

I II III IV V VI VII VIII IX X

a I,, ((*M)

and Mycelial

FO

UM

GG

d d d d

d d d d d

20

23

d

d

22 160 28 71 24 0.68

250 200 300 200 300 1.84

40 21 50 100 25 5.9

data for SDC isolated from R. solani

Growth

(EC,,)

EC,,? (PM)

RS 120

XI XII Carboxin

(I,,)

hoa (PM)

Compound No.

1

Carboxanilides on SDCs of Various Fungi

RS’

GG

FO

d d d d d

d d d d d d d d d d d d d

d

d

d

84

48

320

d d d

d d d

d d d

9.8

5.7

20

d

d

d

d

10 120 13 7.4 6.2 0.22

8 200 40 80 40 0.4

100 400 40 350 200 5

(RS); G. graminis

(GG);

F. oxysporam

12

(FO); and U. maydis

WM).

b EC,, (PM) data for mycelial growth of R. solani c Ref. (4). d I,, or ECSo > 500 FM.

(RS); G. graminis

while I, III, IV, V, and VII were inactive at the highest concentration tested (500 $l4) against all four fungal SDCs. 1,3-Dimethylpyrazole 4-carboxanilide (VI) appeared to be the best inhibitor overall. Interestingly, its closely related isomer, 1,4dimethylpyrazole 3-carboxanilide (II), in which the positions of the methyl and carboxanilide substituents on the pyrazole ring are reversed, was significantly less active on all four fungal enzymes. As Table 1 shows, the level of SDC inhibitory activity of the pyrazole derivatives varied according to the nature of the organism, the enzyme complex from U. may& being the most sensitive and that from G. gruminis least sensitive. However, the most active of the pyrazoles was generally an order of magnitude less active than carboxin on any of the SDCs studied. Table 1 also records toxicity data expressed in terms of EC,,-&M) values for inhibition of mycelial growth of G. graminis, F. oxysporum, and R. solani on agar nu-

(GG); and F. oxysporum

(FO).

trient medium. There is a broad correlation between the relative inhibitory activity of the different pyrazoles on the enzyme complexes isolated from R. sofuni and G. graminis and their relative inhibitory effectiveness on the mycelial growth of these organisms. However, this does not apply in the case of F. oxysporum where the enzyme complex is inhibited by several of the pyrazoles but mycelial growth is unaffected by any of the compounds even at the highest concentration tested (500 $l4). DISCUSSION

The relative inhibitory activities of the mono- and dimethyl-N-methylpyrazole carboxanilides on electron transport catalyzed by the mitochondrial SDC isolated from R. solani are consistent with the structure-activity relationships found for the same series of compounds as inhibitors of myCelia1 growth of that organism (4). The more effective inhibitors of mycelial growth of R. soluni tend to be the more potent inhibitors

PYRAZOLE

CARBOXANILIDE

FUNGICIDES,

of its enzyme complex and their relative activities follow a similar pattern (VIII and VI > X and XII > XI > II and IX) (Table 1). Moreover, derivatives (I, III, V) which lack a methyl group adjacent to the carboxanilide function are relatively inactive inhibitors of both the enzyme complex and of mycelial growth as are derivatives (IV, VII) in which there is only one adjacent methyl group and that group is flanked by a second methyl group. This broad correlation between the ability of the pyrazoles to inhibit electron transport catalyzed by the enzyme complex obtained from R. soluni and their ability to inhibit mycelial growth of the organism supports the hypothesis that the primary mode of action of carboxanilide fungicides involves interference with the mitochondrial electron transport process. Furthermore, derivatives which are inactive against the enzyme complex isolated from R. solani mitochondria, i.e., I, III, IV, V, and VII are also inactive against the corresponding complexes from U. maydis, G. graminis, and F. oxysporum while those (II, VI, VIII, IX, X, XI, and XII) which are active against the R. solani enzyme complex are also active against the other fungal enzyme complexes. However, the relative order of SDC inhibitory effectiveness of the pyrazole compounds differs among the four fungi tested and the activity of particular pyrazoles against the SDCs of the four different species can vary by up to 50-fold (Table 1). This indicates that while the SDCs of the various fungi are all susceptible to inhibition by some of the pyrazoles there are obviously subtle differences in their molecular architecture which can profoundly influence the inhibitor-receptor site interaction. Such variations in the selectivity behaviour of an inhibitor against SDCs isolated from different fungi are similar to those noted in studies involving U. maydis and Aspergillus nidulans where it was observed that certain carboxin analogs were more active against the enzyme complex isolated from carboxin-resistant mu-

STRUCTURE-ACTIVITY

STUDIES

167

tants than from the carboxin-sensitive wildtype species (7). The pyrazole derivatives reported here appear to be relatively weak inhibitors of mitochondrial electron transport as judged by their I,, values as compared with I,, values for other carboxanilide fungicides. For example, the most active pyrazole (VI) against U. maydis SDC has an I,,, value of 5.7 PM while carboxin and the more active furan, thiophene, and thiazole carboxanilides have I,, values ~0.3 (LM for the same enzyme complex (3, 7, 8). This may reflect the fact that the pyrazole nucleus is a less effective “carrier” of the significant methyl and carboxanilide groups than other heterocyclic nuclei perhaps resulting from subtle differences in the spatial arrangement of these groups in the different series (4). Carboxanilide fungicides are known for their activity against diseases caused by Basidiomycete fungi (5, 6) and a good correlation between inhibition of mycelial growth of the Basidiomycete, R. solani. and control of Rhizoctonia root rot in cotton seedlings has been reported for this series of pyrazoles (4). As Table 1 shows there is also a correlation between mycelial growth and SDC inhibition for R. solani so that for this organism a close relationship appears to exist between inhibition of the enzyme complex, inhibition of fungal growth and disease control. In the non-Basidiomycetes a correlation between SDC and mycelial growth inhibition is observed for G. graminis but not for F. oxysporum. In this latter case the enzyme complex is relatively sensitive to the pyrazole inhibitors but mycelial growth is unaffected indicating possibly that the mitochondrial respiratory pathway in some species may bypass succinate oxidation or that membrane permeability or metabolic degradation may be involved. The relatively poor activity of the pyrazole derivatives as compared with carboxin both as inhibitors of mitochondrial electron transport and as inhibitors of mycelial

168

WHITE ET AL.

growth in the fungi studied (see Table 1) might suggest that these compounds would be ineffective disease control agents. However, 1,3,5-trimethylpyrazole-4-carboxanilide (VIII) has been shown to be superior to carboxin in controlling root rot disease caused by R. solani in cotton (4, 13). This has been attributed to improved systemic behaviour within the plant (13) possibly due to the greater metabolic stability of the pyrazole nucleus and demonstrates the difficulty of correlating fungicidal activity in vitro with performance in vivo. REFERENCES 1. G. A. White, A potent effect of 1,Coxathiin systemic fungicides on succinate oxidation by a particulate preparation from Ustilago maydis, Biochem. Biophys. Res. Commun. 44, 1210 (1971). 2. J. T. Ulrich and D. E. Mathre, Mode of action of oxathiin systemic fungicides V. Effect on the electron transport system of Ustilago maydis and Saccharomyces cerevisiae, J. Bacterial. 110, 628 (1972). 3. G. A. White and G. D. Thorn, Structure-activity relationships of carboxamide fungicides and the succinic dehydrogenase complex of Cryptococcus laurentii and Ustilago maydis, Pestic. Biochem. Physioi. 5, 380 (1975). 4. J. L. Huppatz, J. N. Phillips, and B. Witrzens, Structure-activity relationships in a series of fungicidal pyrazole carboxanilides, Agric. Biol. Chem.

48 45 (3984).

5. B. von Schmeling and M. Kulka, Systemic fungicidal activity of 1,4-oxathiin derivatives, Science

(Washington,

D.C.)

153, 659 (1966).

6. M. Snel, B. von Schmeling, and L. V. Edgington, Fungitoxicity and structure-activity relationships of some oxathiin and thiazole derivatives, Phytopathology 60, 1164 (1970). 7. G. A. White, G. D. Thorn, and S. G. Georgopoulos, Oxathiin carboxamides highly active against carboxin-resistant succinic dehydrogenase complexes from carboxin-selected mutants of Ustilago maydis and Aspergillus nidulans, Pestic. Biochem. Physiol. 9, 165 (1978). 8. G. A. White and G. D. Thorn, Thiophene carboxamide fungicides: Structure-activity relationships with the succinate dehydrogenase complex from wild type and carboxin resistant mutant strains of Ustilago maydis. Pestic. Biochem. Physiol. 14, 26 (1980). 9. G. A. Carter, J. L. Huppatz, and R. L. Wain, The fungitoxicity and systemic anti-fungal activity of certain pyrazole analogues of carboxin. Ann. Appl. Biol. 84, 333 (1976). 10. J. L. Huppatz, Systemic fungicides. The synthesis of certain pyrazole analogues of carboxin, Aust. J. Chem. 36, 13.5 (1983). 11. R. Holliday, The genetics of Ustilago maydis, Genet. Res. Camb. 2, 204 (1961). 12. B. W. Coursen and H. D. Sisler, Effect of the antibiotic, cycloheximide on the metabolism and growth of Saccharomyces pastorianus. Amer. J. Bot. 47, 541 (1960). 13. J. L. Huppatz, J. N. Phillips and B. Witrzens, Laboratory and glasshouse studies of the activity of carboxanilide derivatives against Rhizoctonia solani in cotton, Plant Dis. 67, 45 (1983).