Ann. appl. Bid. (1976), 84, 333-342 Printed in Great Britain

333

Investigations on fungicides XIX. The fungitoxicity and systemic antifungal activity of certain

pyrazole analogues of carboxin

BY G. A. CARTER, J. L. H U P P A T P AND R. L. WAIN Agricultural Research Council Plant Growth Substance and Systemic Fungicide

Unit, Wye College (University of London), Ashford, Kent, TN25 5AH

(Accepted 25 Muy 1976)

S U M M A R Y

A series of pyrazole derivatives, which are structural analogues of the syste- mic fungicide, carboxin (1j,6-dihydro-2-methyl-1,~-oxathiin-~-carboxani- lide), have been synthesized and their antifungal properties investigated. 3,5-dimethylpyrazole- I -carboxanilides, although active in vitro and in leaf disk tests, showed no systemic antifungal activity. Certain 3,s-dimethylpyra- zole-4-carboxanilides, however, and their corresponding I -methyl derivatives, showed good activity in spore germination tests and high activity against wheat and broad bean rusts in vivo. In several instances, systemic antifungal activity was of the same order as that of carboxin, although generally accom- panied by higher levels of phytotoxicity . I -Phenyl derivatives were essentially inactive. Substitution in the anilide ring by 3-methyl, 2-methyl or 3-chloro groups resulted in enhanced systemic activity, while 4-chlor0, 4-ethoxy, 2-nitro and g,4-dichloro substituents reduced activity.

I N T R O D U C T I O N

The oxathiins, an important class of systemic antifungal compounds, were first reported by Schmeling & Kulka (1966) and found to have a marked specificity for Basidiomycetes (Edgington & Barron, 1967; Snel, Schmeling & Edgington, 1970). The parent compound, carboxin (Ia), 5,6-dihydro-z-methyl- 1,4-oxathiin-3-carbox- anilide, and its sulphone derivative, oxycarboxin (Ib), have gained commercial acceptance for the control of many smut and rust diseases.

Structure-activity data currently available (Haken & Dunn, 1971 ; White & Thorn, 1975) suggest that a methyl group ortho to an anilide function (a cis-crotonanilide moiety) is the prime requirement for activity and that activity may reside in compounds of widely differing chemical type which incorporate this system. Thus, other groups of carboxanilide fungicides, all having a similar spectrum of antifungal activity to carboxin, have been discovered, notably the pyran (Ic) (Jank & Grossmann, 1971), 2,s-dimethylfuran (IIa) (Pommer, 1971), 2,s-dimethylthiophene (IIb) White & Thorn, 1975) and 2,4-dimethylthiazole (111) (Snel et at. 1970) analogues. Derivatives of o-toluanilide (IV) also possess systemic fungicidal activity specific towards Basidio- mycetes (Pommer & Kradel, 1969) indicating that a heterocyclic ring system is not an essential prerequisite for activity.

* On study leave from CSIRO, Division of Plant Industry, Canberra A.C.T., Australia.

334 G. A. CARTER, J. L. HUPPATZ AND R. L. WAIN The structural requirements for activity so far elucidated provide a basis for

synthesis of other potentially active molecules within this general group. Accordingly, we have investigated the fungicidal properties, hitherto unreported in the literature, of a series of pyrazole derivatives of general formulae V and VI (Fig. I). These compounds might be expected to provide further insight into the relationships between activity and chemical structure within the carboxanilide group of systemic fungicides.

I I1

(a) X=S (carboxin) (a) X=O @) x=so, (oxycarboxin) (b) X=S

I11 IV

I R

V VI Fig. I . Formulae of various carboxanilide compounds which have systemic properties.

M A T E R I A L S

The chemicals examined are listed in Table I. Compounds 1-3 were prepared from the appropriate pyrazole (Morgan & Ackerman, 1923) and phenyl isocyanate according to the method of Henry & Dehn (1949). Compounds 4-29 were prepared by reaction of hydrazine hydrate, methyl hydrazine or phenyl hydrazine with the appropriate diacetylacetanilide in boiling acetic acid. The required diacetylacetanilides were prepared from the reaction of acetylacetone and substituted phenyl isocyanates in

Fungitoxicity of pyraxole analogues of carboxin 335 dry benzene at room temperature in the presence of triethylamine. The structures of all new compounds were supported by analytical and nuclear magnetic resonance spectral data.

Technical grade samples of carboxin (La) and oxycarboxin (Ib) were included in the tests for comparison.

Table I. The pyraxole analogues of carboxin examined in this investkation F'yrazole-I-carboxanilides (V)

Compound no. X m.p. ("C) I

2

3

H 661' Br I O I - Z t

I 9 4 4

Compound no.

4 5 6 7 8 9

10 I1 I2

13 I4 15 16 I7 18 I9 20 21 22

23 24 25 26 27 28 29

Pyrazole-~-carboxanilides (VI) R R

CH&XaOH H CH2COOEt H H H H 2-cH3 H 3-cH3 H 3-Cl H 4 x 1

H 4 - 0 ~ t H z-NOS CH3 H CHa z-CH~ CH, 3 - m a CH, 3-Cl CH3 4-Cl CH3 394-cla ma 4 - 0 ~ t

H 3,4-c1a

2-NOa H 2-CH3 3 - m a 3-c1 4-Cl

4 - 0 ~ t 3,4-cla

z-NOB

m.p. ("C)

162-4 172-4 244-5 230-2 209-1 I

234-5 271-2 259-60 234-5 224-5 1613f 152-3 159-61 155-6 2 0 6 1 168-70 163-4 16870 I 82-3 144-6 148-9 143-5 218-9 212-3 193-4 141-2

* Literature m.p. 69" (Wheeler & Norton, 1928). t Literature m.p. 100-1" (Wheeler & Norton, 1928). f Literature m.p. 159' (Rojahn & Kiihling, 1926).

METHODS

Because of the specificity of carboxin and structurally-related compounds towards Basidiomycetes, special emphasis was placed upon activity in vitro against the fungi causing rust of wheat and broad bean and loose smut of barley and activity in mko against the two rust diseases. Direct antifungal activity was assessed by spore germina- tion tests in vitro and by application of protective sprays to wheat and bean seedlings. Therapeutic activity was measured using inoculated leaf disks and eradicant sprays

13 APB 84

336 G. A. CARTER, J. L. HUPPATZ AND R. L. WAIN whilst systemic activity was assessed using excised broad bean shoots and by applying the test compounds to the roots of wheat seedlings.

Spore germination test Each compound was incorporated at IOO mg/l into 10 ml Oxoid Czapek Dox agar

which was then poured into a 6 cm diameter sterile Petri dish. Plugs 8 mm in diameter (vol. c. 0.2 ml) were then cut from the agar layer and placed on microscope slides contained in humid boxes. One drop (c. 0.005 ml) of a suspension of spores of a test fungus in sterile distilled water was applied to the top of each plug. After incubation for 18 h at 25 "C each agar plug was examined microscopically and counts made of the percentage germination of the spores.

The test organisms used were Altemaria brassicicola (Schw.) Wiltshire, Botrytis civzerea Pers. ex Fr., Septoria nodorum Berk., Uromyces viciae-fabae (Pers.) Schroet. and Cladosporium cununerinum Ell. & Arth. All compounds inhibiting the germination of U. viciae-fabae uredospores at IOO mg/l were retested at 20 and 4 mg/l against this organism and also against Puccinia recondita Rob. ex Desm. f. sp. tritici (Erikss. & Henn.) and UstiZago nuda (Jens.) Rostr.

Protectant and eradicant activity All compounds were formulated in 50% aqueous acetone containing 0.05 yo v/v,

Tween 20 and applied as foliar sprays to wheat seedlings immediately prior to inoculation with either powdery mildew (Erysiphe graminis DC f. sp. tritici em. Marchal) or wheat rust (P. recondita) and to broad bean seedlings immediately prior to inoculation with rust (U. viciae-fabae). Eradicant activity was assessed by applying foliar sprays to broad bean seedlings 48 h after inoculation with U. viciae-fabae. Complete details of the above assays have been reported previously (Lehtonen, Summers & Carter, 1972). The development of all three diseases was graded on an arbitrary scale of c-5 based upon the percentage of leaf area showing disease symptoms.

Leaf disk test Leaf disks, 12 mm diameter, were cut from leaves of cucumber, previously dusted

with conidia of powdery mildew (Sphaerotheca fulE;einea (Schecht) Salm.), and floated, inoculated side uppermost, upon solutions or suspensions of the test compounds at 500, 100,20 or 4 mg/l in distilled water. Cucumber leaf disks, inoculated with droplets of a dense suspension of spores of the anthracnose pathogen, Colletotrichum lagenarium (Passer) Ell. & Halst., and broad bean leaf disks, dusted with a 5 : I mixture of talc and uredospores of U. vicim-fabae, were treated similarly. Subsequent disease development on the disks and phytotoxicity symptoms were noted 7-10 days later.

Cut shoot test Excised shoots of broad bean, decapitated and trimmed to two pairs of fully expanded

leaflets, were stood in 10 ml of an aqueous solution or suspension of a test compound. Further water was added as necessary to maintain turgidity in the shoots. Twenty- four hours after treatment commenced, all leaflets were inoculated on their lower

Fungitoxicity of pyraxole analogues of carboxin 337 surface with U. viciae-fabae and the shoots placed under high humidity overnight. When leaflets of the control shoots bore mature sporulating uredosori (c. 10 days), the rust infection present on each leaflet was assessed on an arbitrary scale of 0-5. Any phytotoxic symptoms on stems and leaves were also assessed.

Root application test Batches of five 3-day-old seedlings of wheat (cv. Eclipse) were planted into waxed

paper cups containing c. 70 ml of either sand or John Innes No. I compost. Each pot was immediately treated with 20 ml of a test solution as a drench; sand-grown plants also received a mineral nutrient solution. Four days later each pot received a further 2oml of test solution. On the fifth day, the seedlings were sprayed with a dense suspension of the uredospores of P. recondita and placed under high humidity at 21 "C for 16 h. When the control plants bore mature sporulating uredosori (12-14 days after inoculation) rust infection was graded on an arbitrary scale of 0-5. Phytotoxicity was also assessed.

RESULTS

All compounds were examined in the spore germination tests but only those which, at 100 mg/l, caused greater than 50 yo inhibition of spore germination of at least one test organism are included in Table 2.

Table 2. Activity of s m e pyrazole carboxanilides in spore germination tests yo Inhibition of germination

I *

\

I 00 mg/l 20 mg/l 4 mg/l I

A \ --

Compoundno. A b Bc Sn Uf Cc Uf Pr Un Uf Pr I 2 3 6 8

14 I5 16 17 I8 19 24

I 2

o o 100 66 I00 0 100 I00

0 0 I00 100 0 0 0 I 0 0 0 0 0 81 0 0 0 58 0 0 0 78 0 0 0 I 0 0 0 0 100 I 0 0 0 0 0 100 0 0 0 I00 0 0 0 I00 0 0 0 78

Ia (carboxin) Ib (oxycarboxin)

100 I00 I00

0

0

0

0

0

I 0 0 I00

64 76 0

0 0

100 100 1 0 0 I00

84 0 0 0 0 0

0 0

22 72 48 76 40 0 0 0

0 0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

I00 0 0

I00 0 0

I 0 0 0 0

I 0 0 0 0

0 0 0

0 0 0

0 0 0

1 0 0 I00 100 100 100

80 98 IOO 0 0

Ab, Altemaria brassicicola; Bc, B6trytis c i w e a ; Sn, Septork nodorum; Uf, Uromyces viciae-fabae; Cc, Cladospwium nmunerinum; Pr, Pwcinia recondita; Un, Ustilago nu&.

Un 0

0

0

0

0 0

0

0

I00 0

0

0

0

I 0 0

0

Carboxin (Ia) proved to be very active, causing complete inhibition of the germina- tion of spores of U. viciae-fabae, P. recondita and U. nuda at 4 mg/l. Only one of the pyrazoles (16) showed any activity at this level and then only against U. nu&. The

13-2

338 G. A. CARTER, J. L. HUPPATZ AND R. L. WAIN pyrazole-I-carboxanilides (1-3) tended to be more inhibitory towards rusts than towards the smut, whilst the reverse was true for the pyrazole-4-carbodides (14-17). At 100mg/l activity was not confined to Basidiomycetes since the more active test compounds also inhibited S. nodorum and C. cucumminum and, in one case, A. brasskicola.

Table 3. Protectant and eradicant activity of some pyraxole carboxmilides

Compound no.

6 7 8 9 I4 15 16 I7 18 I9 20 21

Ia (carboxin) Ib (owcar- boxin)

against rust &eases of wheat and bra bean Protectant activity*

Ptlccinia r e e d & (Wheat) Uromyces viciae-fabae (bean)

IOO mg/l 25 mg/l 10 mg/l IOO mg/l 25 mg/l 10 mg/l *

f \ ( \

38

48 48

27

< I

4 0

I

7 42

27 22

- - - - I 0

45 16 35 18 66 51 52

26 46

45

3

22

0

2 0

3 7

4 I

0 < I < I 2 4 40

9 7' 82 8 14 86

* The figures given are infection grades as a percentage of control.

Eradicant activity" Uromyces

viciae-fabae 1000 mg/l

I00 I00 I00 I00 0

<I 2

I 0 0 I00 I00 I00 I00

0

< I

The results obtained against rust diseases in the protectant and eradicant assays are shown in Table 3 (only those compounds which reduced infection by greater than 50 yo are listed).

Carboxin (Ia) provided complete control of powdery mildew (E. gramink) on wheat at IOO mg/l; oxycarboxin (Ib) and all test compounds were completely ineffec- tive. Carboxin was also very effective as a protectant against wheat rust but was rather less active against broad bean rust. A number of the pyrazole-4-carboxanilides (14-18) all showed considerable activity against wheat rust although none was as effective as carboxin. These compounds, and 20, gave good protection against broad bean rust and here their activity was equal to, or better than, that of carboxin. Carboxin and oxycarboxin were also effective eradicants of broad bean rust. This type of activity was restricted to only three (14, 15 and 16) of the pyrazole-4-carboxanilides.

Both carboxin and oxycarboxin were highly active in the leaf disk tests (Table 4) completely inhibiting the development of bean rust at 4 mgll. At IOO mg/l, carboxin was also active against powdery mildew and anthracnose of cucumber. At higher rates of application it was, however, toxic to the leaf disks. The majority of the test com- pounds showed some activity against the rust pathogen although much less activity was shown against powdery mildew and anthracnose. The pyrazole- I -carboxandides

Fungitoxicty of pyraxole analogues of carboxin 339 (1-3) effectively inhibited rust development, whilst certain of the pyrazole-4- carboxanilides (notably 16, but also 14, 18 and 19) showed activity similar to that of the two commercial compounds.

Table 4. The actiwity of some pyrazole carboxanilides in leaf disk and cut shoot tests

Minimum inhibitory cone.' (mg/l) Leaf disk test Cut shoot test

Broad bean

Compound no.

I

2

3 6 7 8 9

1 0 I1

14 15 16 17 18 19 20 22

23 24

Ia (carboxin)

Ib (oxycarboxin)

I ,

1

Cucumber Broad bean

I OOP r00P

Rust

I 0 0 I 0 0 20

I 0 0 - I OOP

500 500

4

-

I00

4p I 0 0

4 4

500 I 0 0 - 500

4p

4

Toxicity$ Rust

infection+ Stem - 33

I00 I 0 0

0 - - 6

0 k 44 -

I 0 0 I 0 0

§ 0

0

0

0

I00 0

I00 I 0 0 I00

0 -

0 -

Leaf

+

+ + + +++ + +

+++ + ++ + + + + +

+ +

* Concentration required to give complete inhibition of disease symptoms. -, indicates that the

t Infection grade as a percentage of control. 1 Toxicity expressed on a scale: -, -+, +, + +, + + +. 9 Plant tissue too damaged to permit disease assessment. P, Phytotoxicity evident at concentrations less than 100 mg/l.

compound was ineffective at 500 mg/l.

Results obtained in cut shoot tests are recorded in Table 4 (only those compounds which reduced infection by greater than 50% at 100 mg/l, or were active in leaf disk tests, are given). Of the nine pyrazole-4- carboxanilides active in leaf disk tests at ~oomg/ l or less, six were also active against broad bean rust when applied to cut shoots at 100 mg/l, two (19, 22) were inactive and one (14) proved lethal to cut shoots at this concentration. Two additional compounds (7,20) were also active in cut shoots at IOO mg/l, even though inactive (7) or showing only marginal activity (20) in leaf disk tests. Most active compounds caused moderate to severe phytotoxicity to the leaves of treated shoots but the absence of toxic symptoms in stem tissues suggests a ready transport to the leaves with little accumulation in the stem.

Compounds exhibiting systemic activity in the root application tests against wheat

340 G. A. CART^, J. L. HUPPATZ AND R. L. WAIN rust are recorded in Table 5 . Generally, those compounds which were active in the cut shoot tests were also effective following root application. Of the eleven compounds active when applied to sand, only one (21) was ineffective when applied to compost. The most active compounds (8, 15, 16 and 17) show a level of systemic activity at least equal to that of carboxin and oxycarboxin but they appear to be more phytotoxic, 16 and 17 being markedly so.

Table 5 . Systemic activity of certain pyraxole carboxanilides against wheat rust following root application*

Com- pound

no.

6 7 8 9

1 0

14 15 16 I7 18 20 21 22

Ia Ib

Application to sand-grown seedlings Dosage (mg/pot)

Application to soil-grown seedlings

Dosage (mg/pot)

4 O +

O+

O+ 0

55+t O+ + O+ + +++§ + + + I

O f + O+

62 + O+ + O f

0

2

O f O+

O+

0

5 I t O+

O+ + O+ + O f

O+

87 k O+

O f

+++§

0

I

5+t O+

O+ 0

91 O+

O +

O + + +++§

9t 0

89

0

0

0.4

54t 15 k

O+

50

6t O f O +

O+

71 104 98

8 0

0'2

95 62 O f

I 0 2

8t

O+

O+

19t

98

38

0

I 0 0

3t

4 2

4+t 8ft O+ %k O f O f 0 85 t

O+ 3ft o f %+ O + O f o++ of 4ft 7t O f 49 t 92

0 0

O + 0

I

47 t 58t

% I08

st % O f

IZt 0

94

0

Zt

* Figures are infection grades as a percentage of control. t Development of rust infection retarded compared with control. 1 Chlorotic flecks present which may be sites of fungal infection. 8 Plants too damaged to permit disease assessment. &, + , + + , + + + indicate increasing levels of phytotoxicity.

D I S C U S S I O N

A number of the new compounds tested proved highly active against rust fungi when applied as foliar sprays or as root drenches to seedlings of wheat or broad bean. Moreover, certain compounds (8, 15, 16 and 17), when applied to the roots of wheat seedlings grown in either sand or compost, showed systemic activity of the same order as that of carboxin or oxycarboxin.

Within the series of pyrazole derivatives, definite relationships between structure and activity emerge. The three pyrazole-I-carboxanilides ( I-3), although significantly active in spore germination and leaf disk tests, are quite ineffective in both cut shoot and root application assays. Pyrazole-I-carboxanilides are relatively unstable, the carboxanilide function being removed merely by boiling in water or ethanol (Wheeler & Norton, 1928), and the poor systemic performance of these compounds may well be a reflexion of the labile nature of the anilide group in plant tissue. It is, however, note- worthy that these compounds are intrinsically antifungal, although they possess a

Fungitoxicity of pyrazole analogues of carboxin 341 carboxin-related structure in which the carboxanilide moiety is attached to the heterocyclic ring through nitrogen rather than carbon, i.e. they do not contain the crotonanilide structure.

In the pyrazole-4-carboxanilide series, compounds containing an N-phenyl substituent (22-29) show no systemic activity other than the weak activity shown by 22 following application to sand-grown seedlings. Both 22 and 24 showed some inhibitory activity against bean rust in leaf disk tests but essentially this group of compounds was inactive both in vivo and in vitro.

A comparison of the pyrazole-4-carboxanilides unsubstituted on nitrogen (6-13) with their N-methyl derivatives (14-21) reveals that the latter group of compounds show greater systemic activity although they are frequently more phytotoxic (e.g. 8, cf. 16; 9, cf. 17). Moreover, the N-methyl pyrazoles (14-21) showed significantly better protectant activity and were generally more active in leaf disk tests and spore germination assays. However, only three (14, 15, 16) of these compounds were effective as eradicant sprays against broad bean rust and lack of eradicant activity in many compounds highly effective against this disease in leaf disk tests suggests that these chemicals cannot penetrate intact leaf cuticles in effective concentrations.

The nature of ring substituents in the anilide function is of considerable importance in determining systemic activity. The relative activity in root application tests was of the order: 3-CH3 > 2-CH3 > 3-Cl > H > 4-Cl > 4-OEt, 2-NO2 or 3,4-C12 for the N-unsubstituted group of pyrazole-4-carboxanilides (6-13) and of the order: 3-CH3, 2-CH3 or 3-Cl > H 2 4-Cl > 4-OEt > 2-NO, > 3,4-C12 for the N-methyl compounds (14-21). Clearly, both groups show similar changes in activity with changes in ring substituents. Thus, the activity of both parent compounds (6 and 14) is enhanced by methyl substitution at positions 2 or 3 or by a chloro substituent at position 3. A chloro substituent at position 4, however, reduces activity, as does substitution with nitro or ethoxy groups. Both the 3,4-dichloro substituted com- pounds (I I and 19) are devoid of any systemic activity, although the latter compound is highly active against bean rust in leaf disk tests.

The observed effects of phenyl substituents in the pyrazole series are in reasonable agreement with those shown by substituted oxathiins in tests for eradicant activity against bean rust (Snel et al. 1970). However, in other carboxanilide fungicides, e.g. the 2,s-dimethylfuran (IIa) (Pommer, 1971) and the 2,q-dimethylthiazole (111) (Hardison, I 971), substituents in the anilide group seriously reduce or eliminate systemic activity. In general, the systemic activity of the pyrazole derivatives described herein appears to be less sensitive to phenyl substitution than has previously been the case in analogous carboxanilide series.

A grant from the Agricultural Research Council to one of us (J.L.H.) is gratefully acknowledged.

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