Substituted benzanilides: Structural variation and inhibition of complex II activity in mitochondria from a wild-type strain and a carboxin-selected mutant strain of Ustilago maydis

PESTICIDE

BIOCHEMISTRY

AND

27, 249%260

PHYSIOLOGY

(19871

Substituted Benzanilides: Structural Variation and Inhibition of Complex II Activity in Mitochondria from a Wild-Type Strain and a Carboxin-Selected Mutant Strain of Ustilago maydis G. A. Received

May

WHITE

28, 1986: accepted

October

28. 1986

A number of substituted benzanilide compounds were tested for inhibition of Complex II tSDC1 activity in mitochondria isolated from a wild-type strain and a moderately carboxin-resistant mutant strain of corn smut (Ustilago tnqdis). Benzanilidea appear to inhibit the SDC at the same site as for other carboxanilides such as carboxin. While benzanilide was inactive. substitution of particular groups such as iodo. ethyl. or methyl at the 2-position of the benzene ring produced active compounds. The size or bulk of the substituent orrlro to the carboxanilido group seemed more critical than whether the group was electron donating or withdrawing. Certain groups such as i-butyloxy and n-pentyloxy at the 3’-position of c,-toluanilide increased inhibition 100.fold or more. Inhibition of the wild-type and mutant SDCs by a series of 3’-alkoxy substituted Z-methylbenzanilides showed that maximum potency was reached with a carbon chain length of 5. then decreased with further extension of the chain and increasing lipophilicity. A series of N-substituted derivatives of 3’-isopropoxy-2-methylbenzanilide showed anomalous results for the N-ethyl and N-npropyl analogs which had low inhibitory activity compared to longer alkyl chains. Alteration in the structure of the substituted benzanilide molecule also affected the sensitivity of the mutant SDC and certain types of substituent groups gave compounds which were selectively inhibitory to the carboxin-resistant mutant. For instance, the 4’.phenyl analog of +toluanilide was virtually noninhibitory to the wild-type SDC but 17 times more active than the latter compound toward the moderately carboxin-resistant mutant SDC. A pronounced degree of selectivity for the mutant SDC was shown also by the 4’-n-butyl analog of 2ethylbenzanilide and the N-n-pentyl and N-ndodecyl derivatives of 3’-isopropoxy-2-methylbenzanilide. The inhibition of the wild-type SDC ot U. mqdis by substituted benzanilides is paralleled in general by inhibition of mycelial growth of

where the compound Mepronil (3’-isopropoxy-2-methylbenzanilide) has been develThe first benzanilide-type compound in- oped as a new fungicide showing activity troduced as a greenhouse fungicide was the against rice sheath blight fungus (Rhixw wide-spectrum nonsystemic, salicylanilide tonin solnni Kuhn) and other diseases (1). Addition of chlorine in certain parts of caused by basidiomycetous fungi (4). A the molecule increased the toxicity to newer, nonphytotoxic compound. 3’-isospores of tomato leaf mold, Cl~dospoui~m propoxy-2-trifluoromethylbenzanilide. was f~lvunz, Cooke. Subsequently, Pommer (2) also discovered recently (5). found that replacement of the ortho-hyCarboxamides are known to inhibit the droxyl group by a methyl, trifluoromethyl, succinate-ubiquinone (Complex II) span in nitro, or amino group or by a -Cl or -Br the mitochondrial electron transfer chain substituent produced compounds with sys- (6, 7). White and Thorn (8) observed inhibitemic fungicidal activity toward basidiomytion by a few substituted benzanilides of cetous fungi. The 2-iodo analog of benzaniComplex II (SDC)’ activity in mitochondria lide offers good control of Puccinia striii Abbreviations used: SDC. succinate dehydrogeformis West. on wheat and barley and of P. nase complex: BSA. bovine serum albumin: DCIP: hordei Otth on barley (3). Interest in sub- 2,6-dichlorophenolindophenol: PMS. phenazine methstituted benzanilides has appeared in Japan osulfate. INTRODUCTION

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from Ustilago maydis (D.C.) Cda and a basidiomycetous yeast, Cryptococcrrs lallrentii (Kufferath) Skinner. The present investigation is concerned with the structure-activity relationships among a wide variety of substituted benzanilides and inhibition of Complex II in mitochondria from a wild-type strain and a moderately carboxin-resistant mutant strain of U. rnaydis. With respect to the latter, it will be shown that specific molecular structures have been noted which are selectively active against the mutated SDC. MATERIALS

AND

METHODS

Chemicals. The various substituted benzanilides and structurally related compounds which were tested are shown in Tables 1 and 3. General methods for the synthesis and purification of carboxamide compounds in our laboratory were described earlier (8, 9). Commercially unavailable 2-ethylbenzoic acid was obtained from ethylbromobenzene by the general method of Gilman and Kirby (IO). The 2-hydroxy compounds were synthesized by reacting phenylsalicylate and the designated amine in hot methylnaphthalene (I I ). All benzanilides synthesized were verified by NMR spectroscopy. Carboxin was a gift from Dr. J. Wilson, Uniroyal Research Laboratory Ltd., Guelph, Ontario. Sigma Chemical Company supplied BSA, fraction V. and DCIP was purchased from BDH Chemicals. All chemical solutions were prepared in glassdistilled water. Corn smut strains. The genotypes of the wild-type strain, ATCC 14826, and the carboxin-resistant mutant strain, No. 77, were given by White et al. (9). Maintenance and liquid culture qf’ U. rnaydis. Wild-type and carboxin-resistant mutant strains of u. maydis were main-

tained at 15°C on Holliday’s complete solid medium (12). Sporidia were grown in liquid culture, harvested, and stored frozen as reported before (8). Gtwc,th inhihitiorl assays. R. solarli was

kept at 15°C on potato-dextrose agar slants. Toxicity assays with the benzanilides were done as described by White and Thorn (8) and the EC,, values were determined from log probability plots. Mitochondrial preparation. Mitochondria from wild-type and carboxin-resistant sporidia of U. maydis were isolated in sucrose-EDTA-BSA medium using acidwashed sea sand or small glass beads for cell disruption (9, 13). The final pellet of mitochondria was suspended gently in BSA-free sucrose-EDTA medium. Assay of Complex II (SD0 actiljity. The activity of the SDC was determined spectrophotometrically using DCIP as electron acceptor (8). Benzanilide compounds were added as ethanol solutions, 1.3% v/v final alcohol concentration. Rates of DCIP reduction and I,, (FM) values were calculated as reported by White et al. (9). The results shown are from typical experiments. Replicate assays were run with those substituted benzanilides having strong inhibitory activity. it should be explained that the method used for assaying succinate oxidation by U. rnaydis mitochondria essentially measures Complex II activity, i.e.. succinate-ubiquinone reductase. The results of Ulrich and Mathre (14) showed that inhibition of coenzyme Qg reduction by carboxin was equally reflected by the inhibition of DClP reduction. Soluble succinate dehydrogenase from U. mapdis is not inhibited by carboxamides and does not reduce DClP in the absence of PMS (7, 15). Current evidence suggests that reduction of DCIP per se is at the level of the coenzyme Q pool which, also, may be a second region of PMS reaction (7). More specifically, since succinate dehydrogenase itself does not reduce DCIP, the electrons pass catalytically via coenzyme Qlo or appropriate analogs such as 2,3-dimethoxy-S-methyl-6-pentyl1,4-parabenzoquinone (7) which, in their reduced state, react rapidly with DCIP. RESULTS

Relationship

het\zleerz the structure

of

SUBSTITUTED

BENZANILIDES:

substituted benzanilides and inhibitiotl of Complex II activity in mitochondria from a wild-type strain of U. maydis. Table 2 presents structure-activity results for a wide variety of substituted benzanilides and Complex II activity in mitochondria from the wild-type strain of U. maydis. Although the basic anilide structure required for inhibition of the enzyme complex is well delineated (8, 16), it has been modified recently to include pyrazole carboxanilide compounds in which the significant methyl and carboxanilide groups are separated by a formal carbon-to-carbon or carbon-to-nitrogen single bond (17, 18). The results in Table 2 show that benzanilide (I) was totally inactive and inhibition increased with the type of substituent group at the ‘-position of the benzene ring. As noted previously (8), the size or bulk of the group ortho to the carboxanilido function seems to be more critical than whether the group is electron donating or withdrawing. In the series I-VIII, the order of inhibitory activity was I = ethyl > CH, > Br > Cl = OH > F > H, which correlates with the size of atomic radius for the elements, I > Br > Cl > F > H, and for the substituents, CH,CH, > CH, > OH. A methyl substituent in the 3- or 4-position (IX, X) greatly diminished activity while the 2,4-methyl analog (XI) was as active as the parent compound o-toluanilide (VII). Certain substituent groups on the aniline ring of the parent molecule, particularly at the 3’-carbon, markedly increased inhibition of the enzyme complex. For example, the 3’-i-butyloxy (XXIX) and 3’-n-pentyloxy (XXX) analogs were 100 and 147 times, respectively, more potent than VII while the 3’-t-butyl (XXII), 3’-n-butyloxy (XXVII), and 3’-s-butyloxy (XXVIII) analogs were 2% to 44-fold more active than o-toluanilide (VII). Increasing the carbon chain length above C5 gradually lowered activity and the 3’-n-dodecyloxy analog (XxX111) was approximately 160 times less inhibitory than the 3’-n-pentyloxy derivative (XXX). Figure I shows a plot of inhibition values for a series of 3’-alkoxy-substi-

INHIBITION

OF

COMPLEX

II

251

tuted 2-methylbenzanilides. The inhibitory activity toward Complex II increased with the length of the carbon chain, reaching a maximum at the n-pentyloxy analog (XXX) and decreasing progressively as the carbon chain increased in length. Only three 3’ alkyl substituted compounds were tested (XIX, XXI, and XXII): however, inhibitory activity also increased as the carbon chain extended (Table 2). Substitution of an npropyloxy (XXV) or an i-propyloxy (XXVI) group at the 3’-position increased activity 11- to 14-fold relative to VII. The 3’methoxy (XXIII) and 3’-trifluoromethyl (XX) analogs were about as active as otoluanilide (VII). The 3’-ethoxy (XXIV) and 3’-methyl (XIX) analogs had intermediate inhibitory activity. A chloro (XVII) or nitro (XVIII) group at the 3’-position produced weak inhibition. Except for the nitro and chloro compounds, monosubstitution in the 3’-position generally gives equal or enhanced activity to the parent anilide (VII). Benzanilides substituted with a methyl (XII), methoxy (XIII), or i-propoxy (XIV) group at the 2’-position were not as active as their counterpart 3’-substituted analogs (XIX, XXIII, and XXVI, respectively). The bulk of the substituent group at the 2’-position did not appear to affect activity significantly (cf. compound XV). The 2’,3’-dimethyl derivative (XVI) had intermediate activity. Introduction of a polar 4’-carboxy (XXXIV) or a nonpolar 4’-phenyl (XXXV) group into VII severely reduced inhibition. However, the 4’-n-butyl analog (XLIV) of 2-ethylbenzanilide was about as active as o-toluanilide but 3-fold less inhibitory than its parent anilide (VIII). Addition of a methyl group to the phenyl ring of 2-hydroxybenzanilide (XL) or 2-ethylbenzanilide (XLI, XLII) produced about the same effects as did mono- or dimethyl substitution with o-toluanilide, i.e., increased activity by a 3’-methyl group and the reverse by 2’-substitution. A 3’-i-propyloxy derivative (XXXVIII) of 2-chlorobenzanilide (III) was as active as VII. Replacement of the phenyl ring of VII

252

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” Eastman Kodak Co., Rochester, NY. b E.-H. Pommer, Bad&he Anilin-und Soda-Fabrik A.G.. Limburgerhof, FRG. ’ P. R. Wallnofer, Bayer Landesanstalt fur Bodenkulture Pflanzenbau und Pflanrenshutz. d Synthesized by G. D. Thorn, Agriculture Canada. Research Centre. London. Ontario, e 1. Shimazaki. K-I Chemical Research Institute Co.. Ltd.. Shizuoka. Japan. /S. Tsuchiya, Kumiai Chemical Industry Co., Ltd., Tokyo. Japan. 7 P. ten Haken and C. L. Dunn, Woodstock Agricultural Research Centre. Sittingbourne h J. Wilson, Uniroyal Research Laboratory Ltd.. Guelph. Ontario. Canada.

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Wild-type Compound NO. I II III IV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII XVIII XIX xx XXI XXII XXIII XXIV xxv XXVI XXVII XXVIII XXIX xxx xxx1 xxx11 xxx111 XXXIV XXXV XXXVI XXXVII XXXVIII XXXIX XL XL1 XL11 XL111 XLIV XLV XLVI XLVII XLVIII XLIX L LI LII LIII LIV LV

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>500.0(0)’ 250.0 100.0 32.0 8.X 100.0 25.0 Y.0 >500.0(4OJ >115.0(10~ 3.5 250.0 150.0 76.0 Y3.0 67.0 3?0.0 I Il.0 x.0 22.0 I.8 0.8h 18.0 4.0 1.3 1.X 0.9 0.57 0.25 0.17 0.5x 1.7 27.0 ~375.Ot48) >75.0( I?) 5.1 350.0 75.0 143.0 87.0 163.0 4.8 Ih.0 30.0 140.0 25.0 5.2 475.0 >500.0143) >500.0~40) >500.0( 18) >500.0(29) >100.0(0) >?50.0(33) 0.36

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” I,, (FM): concentration of compound inhibiting the rate of DCIP reduction by SOR a\ found from aemilog plots of percentage Inhibition vs concentration. b Relative sensitivity: ratio of concentration of compound VII to the analog concentration giving 50% inhibition of DCIP reduction. L Resistance level: ratio of the concentration needed for 5OTJ inhibitmn of the mutant SDC to the conccntl-ation requksd for 5O”r inhibition of the wild-type SDC. ’ EC,, ()*A41 value: effective concentration which gives 50% inhibition of radial mycelial growth. e Relative wnsitivity: ratio of concentration of compound VII to the analog concentration giving 504 inhibition of mycelial growth. ‘Value in parenthews denote\ the percentage of inhibition of SDC actlwty or growth at the lqo (~Lnf) or EC,, ((*MJ values shown in the table. 254

SUBSTITUTED

BENZANILIDES:

INHIBITION

OF

COMPLEX

II

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30 -

20 -

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c 0

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with N-alkyl groups produced active analogs, in particular, the N-n-decyl compound (XLVII). Reduction of carbon chain length below C, led to lower inhibition (cf. XLV and XLVIII). The results observed for compounds XLIX and L show that the carbonyl group could not be replaced with sulfoxide and the carbonyl oxygen is essential for full inhibition and not substituted effectively with sulfur. Replacement of the phenyl ring of VII with a pyridyl (LI, LII), thiazoline (LIII), or a thiazole ring (LIV) nearly abolished activity. Relcttionship het,r?een the structure of substituted henzunilides and Complex II activity in nlitochondria from N moderately cnrho.uin-resistant mutant strain of U. mapdis. The inhibitory effect of substituted

benzanilides on Complex II activity of a moderately carboxin-resistant mutant strain of U. maydis (o.rr-1A mutant No. 77) is shown in Table 2. Similar to oxathiin and thiophene carboxamides (9, 19), the effec-

tiveness of the substituted benzanilides on the wild-type and mutant SDCs was compared to that of the parent anilide, in this case, o-toluanilide (VII), by computing values of relative sensitivity. The values for resistance level (9) show the effect of mutation on the inhibitory activity of each benzanilide. Values under 1.0 indicate that the mutant SDC is more sensitive to the inhibitor than is the wild-type SDC. As seen in Table 2, as with the wild-type SDC, alterations in the structure of the substituted benzanilide molecule can also affect the sensitivity of the SDC from mutant No. 77. Certain types of substituent groups may be selectively inhibitory to the carboxin-resistant mutant. This is clearly demonstrated by the 3’-nitro (XVIII) and 4’-phenyl (XXXV) analogs of o-toluanilide (VII) and the 4’-n-butyl derivative (XLIV) of 2-ethylbenzanilide (VIII). The 4’-phenyl analog (XXXV) of the parent anilide (VII) is virtually noninhibitory toward the wildtype SDC and 17 times more active than

256

G.

A.

WHITE

VII on the mutant SDC. Analog XLIV was about as active as VII on the wild-type SDC and 60 times more active than VII toward the mutant SDC. The substitution of particular groups at the 3’-position, such as the i-butyloxy (XXIX), n-pentyloxy (XXX), and n-octyloxy (XxX11) derivatives, increased I,, values to both the wild-type and the mutant SDC, with generally weaker inhibition of the mutated enzyme complex. An electrophilic 3’-chloro group (XVII) gave no such effect. An iodo group (V) on the ~-POsition of benzanilide raised the relative sensitivity 10 times for the mutant SDC but only 3 times for the wild-type enzyme complex. The resistance level, however, was above 1.0. Selectivity for the SDC of mutant No. 77, as indicated by low resistance levels of about 1.0, was also shown by such diverse structures as the 2,4-dimethyl (XI), 3’-trifluoromethyl (XX), and 3’-n-dodecyloxy (XxX111) analogs of o-toluanilide (VII) and the 3’-methyl derivative (XL) of 2-hydroxybenzanilide. On the other hand. a high degree of nonselectivity for the mutant SDC (resistance levels %I .O) was exhibited by the 3’-t-butyl (XXII), 3’-i-butyloxy (XXIX), 3’-n-pentyloxy (XXX), and 3’-n-hexyloxy (XxX1) analogs of compound VII. None of the benzanilides in Table 2 were found to be more inhibitory to the mutant SDC than carboxin is to the wild-type SDC (not negatively correlated to carboxin). A plot, similar to Fig. 1, of inhibitory activity as related to carbon chain length for the 3’-alkoxy analogs of 2-methylbenzanilide and the mutant No. 77 SDC was somewhat scattered compared to the results with the wild-type enzyme complex. However, the same trend of increased activity with carbon chain length up to C, and decrease beyond C, prevailed. Reltrtiomhip het,c~ec~n tllr strrrctrlw of’Zatld N-substituted 3’-isopropo.ughett~~ttlilides md inhibition o.f’ Comple.\- II trc,ti\zit> iti tnitockmdria frotn (I ,t,ild-type sttvtitl Lttld (I modertrtrly ~crrho.~in-t.rsi.~t~ttlt strctin of U. tnuydis. Table 3 shows structure-ac-

tivity results for a group of 2- and N-substituted 3’-isopropoxybenzanilides. With the wild-type enzyme complex, substitution of an iodo group (II) for methyl at the 2-position of the benzene ring increased activity relative to o-toluanilide (VII, Table 2) by 3% and 532-fold, respectively, compared to the parent 3’-isopropoxy-2-methylbenzanilide (III) and o-toluanilide (VII, Table 2). A methoxy group at the 2-position (IV) reduced activity slightly relative to compound III. The 2-fluoro analog (I) gave the same inhibition as o-toluanilide but was 14 times less active than the 3-methyl compound (III). Low inhibition values were found with the N-ethyl (V) and N-n-propyl (VII) analogs as compared, for example, to the N-ally1 (VI), N-n-butyl (VIII), and N-ibutyl (IX) derivatives of III. The latter analogs were about as active as o-toluanilide but were weaker inhibitors than 3’-isopropoxy-2-methylbenzanilide (III). Extension of the alkyl carbon chain beyond C, decreased inhibition and the N-n-dodecyl analog (XI) was almost inactive. A relatively bulky N-benzyl group (XII) lowered activity 0.5 to 37 times, respectively, compared to o-toluanilide and the parent anilide (III). Particular substituent groups on the carboxamide nitrogen of 3’-isopropoxy-2methylbenzanilide (III) produce analogs selectively inhibitory to the mutated enzyme complex (resistance level d hen~ani1ide.s and gro,~~tl? itlllihitiotl c?f‘R. solani. The effect of selected

substituted benzanilides on the mycelial growth of R. solotli is shown in Tables 3 and 3. In general, the inhibition of the wildtype SDC of CJ. rtznydis is paralleled by a

SUBSTITUTED

BENZANILIDES:

\

X

o=o -

* n\

/

INHIBITION

OF

COMPLEX

II

258

G.

A.

WHITE

similar inhibition of R. solani growth. Some exceptions were encountered with the 3’-n-octyloxy (XxX11) analog of VII and the 4’-n-butyl derivative (XLIV) of VIII which had little effect on growth but were inhibitory to SDC (Table 2). Similar results with other types of carboxamides have indicated that, in these cases, cell permeability factors may be involved in the poor inhibition of growth (9, 13, 19). In contrast (Table 3) the N-ethyl and N-n-propyl analogs (V, VII) of 3’-isopropoxy-2-methylbenzanilide (III) were fairly inhibitory to the growth of R. soluni var. sasrlkii and weak inhibitors of the wild-type and mutant SDCs of U. mcrydis. DISCUSSION

The structure-activity results with substituted benzanilides are consistent with the hypothesis that carboxamide fungicides are primary inhibitors of mitochondrial electron transport. In fact, the genera! correlation observed in Table 2 between inhibition by benzanilides of the SDC of wildtype U. maydis and the mycelial growth of R. solani would strongly agree with an oxidative process. such as electron transport, being the site of attack by this type of inhibitor. Kawada et rll. (4) found that, as with carboxin. 3’-isopropoxy-2-methylbenzanilide (Meproni!) inhibited glucose oxidation by cells of R. solnni at the same concentration as required for growth inhibition and the oxidation of pyruvate and oxaloacetate was affected likewise. Our enzyme and growth inhibition results (Table 2) coincide closely with the growth inhibition tests of Kawada et crl. (4) using R. solani. These workers observed that 3methylbenzanilide substituted on the aniline ring with 3’-alkyl or 3’-alkoxy groups exhibited stronger activity than 2’- or 4’substituted and nonsubstituted compounds. Thus, growth inhibitory activity increased with the length of the carbon chain of alkoxy or alky! groups at the 3’position of o-toluanilide; the increase.

showing a peak at C, to C, and then decreasing steadily from C, to C,?, was also observed with inhibition of the wild-type SDC from U. rnu?,di.s (Fig. I). Note for the wild-type SDC in Table 2 that the branched chain i-butyloxy analog (XXIX) was approximately as active as the n-pentyloxy compound (XXX). Pommer and Kradel’s (20) studies with benzanilides and R. solani and rust fungi demonstrated the potential use of these carboxanilides to control diseases caused by basidiomycetous fungi. Benzanilide and the 2-hydroxy derivative. salicylanilide, 2methoxy and 4-methoxy analogs proved inactive. The 3-methyl analog was slightly active as shown also in Table 2. Fungitoxicity was noted with o-toluanilide and derivatives in which the 2-methyl group was replaced by a -Cl or -Br function or a -NO, or -CF, group. Iodobenzanilide (V) was more active than o-toluanilide in controlling Puccinia rusts on wheat and barley (3). The inhibition value for iodobenzanilide and the SDC of wild-type U. maydis was 8.8 pM compared to 25.0 pM for o-toluanilide (V, VII, Table 2). The order of inhibitory activity of 2-substituted benzanilides toward the enzyme complex was I > CH, > Br > Cl = OH > F which agrees reasonably well with the spectrum of activity against certain basidiomycetous fungi (2, 3). Previous papers (9, 13, 19) have reported that specific types of molecular structures of oxathiin and thiophene carboxamides exhibited selective activity toward particular mutated SDCs of carboxin-selected mutants of Ii. maydis and the ascomycete, Asprrgillus nidulclns. Thus, certain compounds could apparently affect the phenotypic expression of mutation(s) for carboxin resistance by “identifying” alterations in the carboxin binding site of the mutated Complex II and, in effect, alleviate resistance to carboxin. With oxathiin and thiophene carboxamides and moderately carboxin-resistant U. mrrvdis mutants,

SUBSTITUTED

BENZANILIDES:

such as No. 77, specific types of molecular structures (4’-substituted compounds) showed a strong selectivity for the mutated SDCs and actually reversed the effect of carboxin-selected mutation in the SDCs. A pronounced selectivity for the mutant No. 77 SDC was shown by the 4’-phenyl analogs of carboxin (9) and of 2-methylthiophene-3-carboxanilide (19). In Table 2. it is seen that the 4’-phenyl analog (XXXV) of o-toluanilide (VII) is at least three times less inhibitory than VII to the wild-type SDC but 17 times more effective than VII on the SDC from mutant No. 77. While the number of 4’-substituted derivatives of Otoluanilide tested was somewhat limited it could be expected, on the basis of results with 4’-substituted analogs of carboxin and 2-methylthiophene-3-carboxanilide (9, 19). that 4’-substituent groups other than a phenyl would give low resistance levels with the mutant enzyme complex. The 4’-n-butyl analog (XLIV) of 2-ethylbenzanilide did give such a resistance level. In addition to selectivity for the mutant SDC by specific 4’-substituted o-toluanilides, several N-substituted analogs (VII, X, XI) of 3’-isopropoxy-2-methylbenzanilide (III) were more active on the mutant than the wild-type SDC (Table 3). The weak inhibitions noted for the N-ethyl and N-n-propyl analogs of 3’-isopropoxy-2-methylbenzanilide could be related to the more hydrophilic nature of the smaller n-alkyl groups compared to n-butyl and n-pentyl (Table 3). For comparison, the N-ethyl analog of Otoluanilide was synthesized and found to be a weak inhibitor of the wild-type SDC (I,,. 180 p/W.

To date, there have been few instances of naturally occurring resistance to carboxamide fungicides. Abiko et nl. (21) reported oxycarboxin-resistant strains of chrysanthemum rust (P. ho~iana). Kawada et al. (4), following the frequency distribution of Mepronil (3’-isopropoxy-2-methylbenzanilide) sensitivity in R. sohi isolates after 5 years of exposure to the compound in the

INHIBITION

OF COMPLEX

159

II

field, found no significant appearance of resistance. From detailed studies (9, 13, 19) with oxathiin and thiophene carboxamides and different categories of carboxin-selected mutants of U. maydis and A. nidufarzs it seems feasible that, with a fungal SDC, any type of mutation for resistance which arises due to selection by carboxin or other carboxamide compounds can be overcome, in terms of inhibition, by subtle alteration in the molecular structure of the parent carboxamide. Thus, there could be the possibility of overcoming mutational resistance to commercially used carboxamide fungicides by appropriate group substitution in the phenyl ring of the parent anilide or even by particular carboxamides with different types of heterocyclic rings (13). Pertinent to these possibilities is the recent discovery (22) of a carboxin-resistant strain of Ustilrtgo rzudcr which, in teliospore germination tests, was cross-resistant to different carboxamides but negcrtil,ely cross-resistant to 3’-isopropoxy-2-methylbenzanilide. In this case it would be of interest to isolate mitochondria from carboxin-resistant sporidia of U. rzrrdtr and test 3’- or 4’-substituted analogs of carboxin or other heterocyclic carboxanilides for inhibition of Complex II activity. ACKNOWLEDGMENT The author appreciates sistance of Sandra Grant.

the competent

technical

as-

REFERENCES 1. A. J. Smith and W. H. Read, Investigations on fungitoxic derivatives of salicylaldehyde. I. The 3-. 5- and 3.5.chlorinated derivatives of salicylanilide and salicyl-para-chloroanilide. Ann. Appt. Bid. 49, 102 (19611. 2. E.-H. Pommer, Substituierte benzosaureanilide als fungizide mit selektiver wirksamkeit.

Meded. R(jks.foc. Lundholrn~~~~etr~~s~~~~. Gent. 33, 1019 (1968). 3. A. J. P. Frost, K. U. Jung, and J. L. Belford. The timing of application of benodanil (BAS 3170F) for the control of cereal rust, Meded. Rijksfkc. LNndho~r~rrrens~~~. Gerir. 1, I I1 (1973). 4. S. Kawada. A. Sakamoto and 1. Shimazaki. De-

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of a new fungicide. Mepronil. J. lot?). 315 (1985). F. Araki. Moncut tflutolanil), a new systemic fungicide. Jrcprrn Pcstic,. Iufivw~. 47, 23 ( 1985). P. C. Mowery. B. A. C. Ackrell. T. P. Singer. G. A. White. and G. D. Thorn. Carboxins: Powerful selective inhibitors of succinate oxidation in animal tissues, Bioclrern. Biophys. Rrs. Covmrrn. 71, 354 t 1976). P. C. Mowery. D. J. Steenkamp. B. A. C. Ackrell. T. P. Singer. and G. A. White. Inhibition of mammalian succinate dehydrogenase by carboxins. Arch. Biochenl. Biopllys. 178, 495 (1977). G. A. White and G. D. Thorn, Structure-activity relationships of carboxamide fungicides and the succinic dehydrogenase complex of Cr.ypro~OC’~IIS larrrentii and Ustiltrgo mrr~tii.c, Pcstic,. Biochenz. P/1y.\io/. 5. 380 ( 1975). G. A. White. G. I>. Thorn. and S. G. Georgopoulos. Oxathiin carboxamides highly active against carboxin-resistant succinic dehydrogenase complexes from carboxin-selected mutants of Cistilcrpr, mrryclis and Aspergillru uitlrrltr~s. Pcstic. Bioc~llem. Plrwird. 9, I65 ( 1978). H. Gilman and R. H. Kirby. in “Organic Syntheses” (H. Gilman. Ed.). Coil. Vol. I. 2nd ed.. p. 361. Wiley. New York. 1941. C. F. H. Allen and J. Van Allen, irr “Organic Syntheses” (E. C. Horning. Ed.). Coil. Vol. 3. p. 765. Wiley, New York. 1955. R. Holliday. The genetics of (/stil~rxo rr~trytli.v. &net. Rcs. 2, 204 (1961). G. A. White and S. G. Georgopoulos. Thiophene carboxamide fungicides: Structure-activity relationships with the succinate dehydrogenase complex from wild-type and carboxin-resistant mutant strains of A.spcrgi//u.\ nidr//m.t , Pev/ic. Biocher~. Physiol. 25, I88 t 1986). J. T. Ulrich and D. E. Mathre. Mode of action of systemic fungicides. V. Effect on the electron Pestic.

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