Pesric. Sci. 1984.15,303-309

Synthesis and Fungicidal Activity of a Series of 1, 1-Disubstituted-but-3-yn-l-olsa Anna Arnoldi, Eliseo Bettob, Gandolfina Farinab, Attilio Formigoni', Remo Galli and Lucio Merlini

lsrituto di Biochimica Generale and blstiruro di Patologia Vegerale, Facolrd di Agraria, Universira degli Srudi, Via Celoria2,1-20133 Milano. and'SIPCA M S.p .A. , Via Gian Galeazzo3,1-20136 Milano. holy

(Revised manuscript received 1 August 1983)

Twenty-three aromatic or heterocyclic l,l-disubstituted-but-3-yn-l-ols were prepared by the reaction between prop-2-ynylmagnesium bromide and suitable ketones. They were tested in vivo for antifungal activity against eight phytopathogenic fungi of different taxonomic classes. Four compounds had noteworthy activity on Sphaerothe- cu fuliginea in protectant, systemic and eradicant assays. The relationship between structure and activity is discussed on the basis of the recent hypotheses on the mechanism of action of ergosterol biosynthesis inhibitors.

1. Introduction

The fungicidal activity of some acetylenic and halovinyl heterocyclic carbonyl compounds' and some 1, 1-disubstituted-3-phenylpropyn-1-ols2 has been reported. As some 3-pyridyl derivatives of the latter class had interesting activity against Sphaerothecafuliginea on Cucumissativur, other 3-pyridyl derivatives containing different acetylenic groups were prepared. In particular, a series of 1-(3-pyridy1)-1-substituted-but-3-yn-1-ols3 were synthesised and tested in vivo for fungicidal activity. These compounds contained the prop-2-ynyl moiety and therefore it was possible that they could act as irreversible enzyme inhibitors.' As several of these compounds had a wider spectrum of activity and were more effective against Xfuliginea than the previous two series. the introduction of heterocyclic substituents other than 3-pyridyl was planned.

Among the many heterocyclic nuclei that could have been chosen, some were selected because they contained structures present in natural or synthetic fungicides. Thus benzofuran appears in vignafuran' (a phytoalexin), pyridine is present in parino16 [4,4'-dichloro-a-(3-pyridyl)benzhydryl alcohol], and pyrimidine in fenarimol' [2,4'-dichloro-a-(pyrimidin-5-yl)benzhydryl alcohol], whereas imidazole* and 1 .2,4-triazole9 appear in some fungicides, more recently introduced, that act as inhibitors of sterol biosynthesis." The compounds prepared had the general structure A (Figure 1) and are shown in Table 1.

Heterocycle I I

HO-C-CH2-C=CH HO-C!-CH,-C-CH

6 2

I R

(A)

(B) Figure 1. General structures A and B.

"Based on a poster presented at the symposium Ergosrerol biosynrhesic inhibirors on 22-24 March 1983, organised by the Physiochemical and Biophysical Panel and the Pesticides Group. Society of Chemical Industry.

303

304 A. Arnoldi el al.

Table 1. Compounds tested of general structure A

General structure A Yield M.p.

Compound Heterocycle R (%) ("(3

I 2-Fury1 Phenyl 95 Oil II 2-Thienyl Phenyl 88 Oil Ill 2-Benzofuryl Methyl 77 IV 2-Benzofuryl Phenyl 98 Oil

-

V 1 -Methylimidazol-2-yl Phenyl 96 168-170 VI Imidazol-1 -ylme thy1 Phenyl 75 122-125 VII 1,2.4-Triazol-l-ylmethyl Phenyl 45 93 - 95

IX 3-Pyridyl Phenyl 98 102b X 4-Pyridyl Phenyl 36 83-87b

XI1 Phenyl Cyclohexyl 95 Oil

VlII 2-Pyridyl Phenyl 41 Oil

XI Pyrimidin-5-yl Phenyl 77 Oil

"B.p.=120-125"C (0.1 mmHg) Recrystallised from ethanol.

Table 2. Compounds tested of general structure B

General structure B Yield M.P

Compound X Z (%) ("C)

Xlll XIV xv XVI XVlI XVlIl XIX xx XXI xxll XXIlI

H H H H H H 4 - 0 H H H H

H 4-CH, 4-F 2-CI 4-CI 2,4-CI2 4-CI 4-OH 4-0-CH3 4-O-CO-CH3 4-0-CO-Phenyl

84 88 88 86 90 94 78 82 94 74 75

6'4-66" 48-50 Oil Oil Oil Oil Oil Oil Oil Oil 97 - 98"

"B.p. 125-130°C (0.3 mmHg); recrystallised from light petroleum

The analogue with a phenyl instead of a heterocyclic ring (XIII) was also synthesised and found active. Compounds of this class are more easily prepared, and therefore an investigation of the effects of substituents on the phenyl group was undertaken. These compounds have general structure B (Figure 1, Table 2).

All the compounds were tested in vivo against eight phytopathogenic fungi of different taxonomic classes in protectant tests. In the most interesting cases, systemic and eradicant activities were also investigated. The results of the tests on l-phenyl-l-(3-pyridyl)but-3-yn-l-ol (IX) from the previous paper,3 are cited for comparison.

2. Experimental methods

2.1. General chemistry Most of the compounds tested were obtained by the reaction of prop-2-ynylmagnesium bromide with the appropriate ketone. In the case of phenyl pyrimidin-5-yl ketone, good yields could be achieved only by using the Barbier-Grignard method recommended for allylmagnesium bromide," probably because this procedure avoids high concentrations of the organometallic reagent during the addition of the ketone.

Fungicidal activity of l,l-disubstituted-but-3-yn-l-ols 305

N(CH&

Figure 2. Synthesis of phenyl pyrimidin-5-yl ketone from 3-phenylpropionaldehyde.

Some of the ketones used were commercial products; some were known and were prepared using standard procedures involving acylation of substituted benzenes. Benzofuran-2-yl phenyl ketone'* was prepared by condensation of salicylaldehyde with 2-bromoacetophenone; imidazol-1-ylmethyl phenyl ketoneI3 and phenyl 1,2,4-triazol-l-ylmethyl ketoneI4 were prepared by the alkylation at room temperature with 2-bromacetophenone, of imidazole in dimethylformamide, and of 1,2,4-triazole in acetonitrile, respectively, using potassium carbonate as the base.

Phenyl pyrimidin-5-yl ketone was prepared from 3-phenylpropionaldehyde by a sequence of reactions involving acetal formation with ethanol and hydrochloric acid, condensation with dimethylformamide and phosphoryl trichloride to obtain a masked derivative of 2- ben~ylmalonaldehyde,'~ cyclisation with formamide and formic acid to 5-benzylpyrimidine, and oxidation with potassium permanganate with phase transfer catalysis (Figure 2).

Compounds XXII and XXIII were prepared by esterification of the phenolic group of XX. The yields reported in Table 2 refer to this step.

Purification of the compounds was accomplished by distillation or recrystallisation whenever possible, o r by column chromatography on silica gel with the appropriate mixture of hexane and ethyl acetate (in general, ranging from 9 : 1 to 1 : 1 by vol) followed by recrystallisation in the case of solids.

Thin-layer chromatography (t.1.c.) on silica gel, and gas-liquid chromatography (g.1.c.) on a DAN1 3600 gas graph using columns 2 m x mm i.d. packed with 3% OV-17 or 3% Versamid on Chromosorb W AW-DMCS, were used as criteria of the purity of the compounds. Melting points were uncorrected. For all the compounds, the infrared, nuclear magnetic resonance and mass spectra were in agreement with the expected structures.

2.2. Synthesis of phenyl pyrimidin-5-yl ketone 2-Benzyl-3-(dimethylamino)acrylaldehyde5 (60 g, 0.31 mol) in formamide (40 ml) was added in drops over 2 h to a solution of formamide (200 ml) and formic acid (16 ml) at 180°C. The mixture was refluxed for 6 h, poured into iced water and extracted with dichloromethane (3x100 ml). After the extract had been dried, concentrated. and subjected t o column chromatography with hexane+ethyl acetate (8+2 by volume), 5-benzylpyrimidine was obtained as an oil (28 g, 45%). This compound (25 g, 0.15 mol) in dichloromethane (200 ml) and potassium permanganate (70 g) in water (150 ml) were vigorously stirred together for 40 h in the presence of benzyltriethylammonium chloride (5.5 g). The solution was filtered on Celite, the layers were separated and the aqueous phase was extracted with dichloromethane ( 2 ~ 100 ml). The solution was dried and the solvent evaporated under vacuum. Phenyl pyrimidin-5-yl ketone was obtained by chromatography with hexane+ethyl acetate (7+3 by volume) (16g, 59%; m.p. 92-93°C from hexane).

2.3. General procedure for the synthesis of compounds I-XXI 3-Bromoprop-1-yne (78 mmol) in diethyl ether (50 ml) was dropped into a slurry of magnesium (75 mmol) and mercury (11) chloride (30U mg) in diethyl ether (50 ml) at a temperature between -10 and 0°C. Then the appropriate ketone (30 mmol) in diethyl ether or dichloromethane (50 ml) was

306 A. Arnold ct al.

added in drops below 0°C. The mixture was kept at about 0°C for 2 h, and then hydrolysed by adding a 10% aqueous solution of ammonium chloride (100 ml) or by pouring it into a mixture of solid ammonium chloride and ice. The layers were separated and the aqueous phase was extracted with dichloromethane (2X 100 ml). The organic layers were removed, washed with water (2x100 ml), combined and dried. Evaporation of the solvent gave a crude residue which was purified by crystallisation or by column chromatography. Yields and chemical characteristics of the compounds are shown in Tables 1 and 2.

2.4. Yhysicochemical characterisation of the compounds Infrared spectra were recorded in liquid film or in Nujol mull on a Perkin-Elmer Model 21 infrared spectrometer. N.m.r. spectra were recorded in deuterochloroform using tetramethylsilane as internal standard on a Varian EM-390 spectrometer at 90 MHz. Mass spectra were obtained on a Hitachi Perkin-Elmer RMU-6D mass spectrometer or a Finnigan 4021 gas chromatographlmass spectrometer equipped with an INCOS Data System.

2.5. Biological assays

The pathogenlhost combinations used were the following: Sphaerotheca fuliginealCucumis sativus, Erysiphe graminisiTriticum aestivum, Uromyces appendiculatuslPhaseolus vulgaris, Puccinia reconditalTriticum aestivum, Phytophthora infestanslSolanum lycopersicum, Colletotrichum 1indemuthianumiPhaseolus vulgaris, Cercospora beticolalBeta vulgaris, and Rhizoctonia solanil Phaseolus vulgaris.

The procedures used for cultivation of the plants and pathogens and for inoculation have been reported previously.' Compounds were tested as aqueous suspensions; solids were suspended, with a Potter homogeniser, in the minimum of water containing Tween 20 (0.1 g litre-') as surfactant, and then diluted to the required concentration; stirring gave fairly stable suspensions which, if used immediately, could be applied easily to the plants. Liquid compounds, on the other hand, were suspended after formulation, obtained by the addition of micronised kaolin mixed with 2% of dioctyl sulphosuccinate. sodium salt.

2.5.1. Direct protectant activity Both surfaces of the plant leaves were sprayed to run-off with suspensions of the compounds, and allowed to dry before being inoculated. Inoculation was performed 24 h after treatment. With reference to the R . solanilP.vulgaris test, the treatment was applied as a soil drench (20 ml per pot) immediately after inoculation.

2.5.2. Systemic protectant activity This activity was tested only against S. fuliginea on C. sativus, either by root application or by leaf spray. For the root application assay, cucumber plants were grown in plastic beakers containing sand, and watered once a week with a nutrient solution. A suspension (20 ml) of the compounds was poured on the sand when the plants were about 20 days old. After 72 h , the leaves were inoculated by spraying a suspension of S. fuliginea spores on the adaxial surface.

The spray application assay was performed as for the direct protectant test. except that the compounds were applied only to the abaxial surface of cucumber leaves. Two days after treatment. the adaxial surfaces of the same leaves were inoculated with a suspension of S. fuliginea spores. In this way, translaminar activity was evaluated.

2.5.3. Eradicant activity The eradicant activity was tested only against S. firliginea on C. sativus. The test was performed as for the direct protectant test, except that the compounds were applied to cucumber leaves 48 h after inoculation.

2.5.4. Disease rating and the calculation of EDSO values The area of the inoculated leaves that was covered by disease symptoms was assessed on a six-point scale (from 0 to 5) as follows: 0, no symptoms visible to the naked eye; 5, 100% of the leaves covered. The assessment of the R. solanilP. vulgaris test was made on the hypocotyls.

Activity was estimated on the basis of the percentage of inhibition of the disease in comparison with the inoculated untreated plants. EDSO values were estimated by plotting the percentage of inhibition of the disease, against concentration on logarithmic probability paper. constructing the best straight line, and recording the concentration at which 50% inhibition of disease occurred. l6

3. Results

The compounds were tested in direct protectant assays on the eight phytopathogenic fungi at a concentration of 2000 or 500 mg litre -', Those which showed a high degree of phytotoxicity were tested again at lower concentrations, together with the compounds that had an inhibitory effect higher than 75% in the first assay. The results at selected concentrations are reported in Table 3. As can be seen, several compounds were phytotoxic, even at low concentration on C. sativus and P . vulgaris. A response higher than 75% at 2000 mg litre-' and higher than 50% at 500 or 250 mg litre-' was considered to be an indication of activity. S. fuliginea, E . graminis, C . beticola and R. solani were

Table 3. Biological activity of 1.1-disubstituted-but-3-yn-1-01s in protectant assays

Activitf at selected concentrations (mg litre-')

S.1C.b

Compound 500 250

I 3 3 U 0 u1 3 0 lv Ph 0

0 V Vl 3 3 Vll 3 3 vln O+ M 3 3 X 2 1 XI 0 -

1 XI1 XIU 3 3 XIV 1 - xv Ph 1

0 XVl - XVlI Ph 0 XVIII Ph 1 XIX Ph 0 xx 0

0 XXI XXII 0 Xxlll 2 1

-

-

-

u. i P. E.IT. ~ P. IT. P . is. 2000 500 250 2000 2000

C.IP. C.iB.

500 250 2000 R.IP. 2000

1 1 0 0 2 Ph 0 Ph 1 0 Ph 1 0 0 3 1 o + + 2 3+ 0 0 3 3 0 0 Ph 0 0 1 0 Ph 0 0 Ph 0 0 Ph 0 0 Ph 1 0 Ph 0 0 Ph 2 0 0 0 2 2 0 1 0 0

0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 O+ + 0 2 0 1 0 1 0 0 0 1 0 2 0 2 2 0 0 1' 1 0 0 0 2 3 0 0 0 0 0

- - 0 1 0 Ph O+ 0 Ph 0 Ph 0 1 0 0 0 3 1 0 1 3

3 0 0 0 Ph 0 3 Ph 0 0 Ph 0 0 Ph 0 0 2 + + 0 Ph 0 2 Ph 0 0 1 0 Ph 0 0 1 0 0 0

- -

3 0 1 3 3 0 2 0 0 0 2 2 1 2 0 7

7 - - 3 2 7 7 - - 0 2

"The infection on the test plant. calculated as a percentage of that on the untreated control plants. is expressed on the scale: 0 = 2 5 9 . 1=25-50%. 2=50-75%. 3=75-100%. ++ and + indicated decreasing degree of phytotoxicity. Ph indicates that phytotoxicity did not allow inoculation of the plants or evaluation of the results.

bS.iC. =Sphaerotheca fuliginealCucumis sariws: €.IT. = Erysiphe graminislTriticum aesriwm; U.IP. = Uromyces appendicularuriPhaseoluc vulgaris; P. IT. = Puccrnia recondiral Trirrcum aesrivum: P . IS. = Phyrophrhora infesransiSolanrim lycopersicum; C . iP. = Collerorrichum IindemurhianumlPhaseofrcs vulgaris; C. /B. = Cercosporo bericolalBera vulgaris; R.l P. = Rhirorronia solanilPhaseohcs vulgaris.

'Compound tested at a concentration of 500 mg litre-'.

308 A. Arnoldi. el d.

Table 4. Biological activity of compounds VI, VII, I X and XI11 against Sphoerorheca fuligrnea in protectant, systemic and eradicant assays

LDso (mg litre-')

Systemic assays

Protectanr Root Leaf Eradicant Compound assay application application assay

VI 15 VII 15 I X 19 XI11 35

<I0 6 c 10 10 < 10 220

33 25

<I0 <l 35

<10

the diseases most often controlled. No compound was active against P. infestans. Except for VII and IX, most of the compounds had a quite narrow spectrum of activity.

At concentrations lower than 250 mg litre-'* activity was retained only against S . fuliginea, which was therefore chosen for more detailed biological studies. Only compounds VI, VII, IX and XI11 were active at concentrations lower than 100mg litre-' and were therefore tested for systemic activity by root and leaf application, and for eradicant activity. The results are reported in Table 4 expressed as the EDSO values. In these assays, the four compounds had high activities.

To complete the data reported in Table 4, further systemic tests by root application showed that compounds VI, VII and IX retained 100% activity a t 10 mg litre-', while by leaf application, VI, VII and XI11 had good activity, and IX had only moderate activity. In eradicant tests, VI, VII and XI11 had 70% activity a t 10, 7.8 and 10 mg litre-', respectively.

4. Discussion

Inspection of Table 3 shows that in structure A (Figure l), the fungicidal activity depends strongly on the heterocyclic nucleus, the most active compounds being the 1,2,4-triazole, imidazole and pyridine derivatives. Among the imidazole and pyridine derivatives, the effect of the position of the ring substitution is clear.

The high activity of IX on S. fuliginea was retained only by three compounds (VI, VII and XIII), and indeed, they were better in systemic assays by leaf application and also in eradicant assays. The slightly lower activity of XI11 in systemic assays by root application was probably connected with the higher lipophilicity of this compound; the log(octan-1-ollwater partition coefficient) (log P ) is 2.38 for XIII, against 1.68 for IX. Contrary to expectations, the substituted derivatives of compound XI11 (Structure B, Table 2) showed that neither lipophilic nor hydrophilic groups had any good effect on activity; in fact, even on S. fuliginea, these compounds had only very weak activity. Moreover almost all lipophilic substituents increased the phytotoxicity of the compounds.

The similarity of the structures of VI, VII and IX (and also XIII; see later) to those of compounds" that are known to act as ergosterol biosynthesis inhibitors (EBIs), and the common activity against powdery mildews, lead to the supposition that they share the same mechanism of action. With this assumption, some of the above results can be rationalised on the basis of the most recent hypotheses on the mechanism of action of EBIs (Marchington, A. F.; paper presented at the symposium Ergosterol biosynthesis inhibitors, and ref. 17). High activity is indeed shown only by compounds with a heterocyclic group (VI, VII, IX), that can act as a ligand for the Fe atom of cytochrome Pr450 of mixed function oxidases. The lack of activity of compounds V and VIII can be attributed to the steric hindrance of the substituents (a to the N atom) towards the N-Fe interact i~n,"- '~ but n o explanation can be offered here for the inactivity of the fenarimol analogue XI.

A feature that is perhaps worth further investigation is the possible importance of the optimum distance between the ligand N atom and the 0 atom of the polar -OH (or -OR, or=CO) group (see structures of Figure 3. The relevant data are becoming available from studies of the crystal structures

Fungicidal activity of 1.l-disubstitutd-but-3-yn-l-ols

OH I

ny C-R’ Q \

I RI-C-OH I OH

(1X) R’

I OH

(XI)

(X)

Figure 3. Structures of the compounds referred to in section 4; R1=phenyl. R’= -CH2 -C rCH

of such compounds as the p-fluoro analogue of IX, fenarimol, and triadimefon (Poster communication by A. Albinati and the present authors at the symposium Ergosterol biosynthesis inhibitors and ref. 19).

Again, on the basis of the EBI hypothesis, the high activity of the carbocyclic compound XIII, which lacks the N atom, is striking. Experiments are in progress to check whether compound XI11 could have a different mechanism of action.

Acknowledgement This research was supported by the ‘Progetto Finalizzato Fitofarmaci e Fitoregolatori’ of the Italian National Research Council (C.N.R.).

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Arnoldi. A . ; Betto. E.: Bizzozero. N.: Farina. G.: Formigoni. A.; Galli. R. Pesric. Sci. 1983. 14, 191-198. Arnoldi. A. ; Betto. E.: Farina. G . ; Formigoni. A,: Galli. R.: Griffini. A. Pesric. Sci. 1982. 13, 670-678. Arnoldi. A . ; Betto. E. ; Ceresa. L.: Farina. G.: Formigoni. A.: Galli. R.: Scaglioni, L. Pesric. Sci. 1983. 14, 576-586. Walsh, C. Terrohedron 1982. 38, 871-909. Preston. N. W.: Chamberlain. K.: Skipp. R. A. Phyrochemisrc cO.rf.) 1975. 14, 1843-1844 Van Heyningen, E. M.; Taylor. H. M.. British Patent 1 175 693. Davenport. J . D.; Kackler. R. E.; Taylor. H. M., French Patent 1 569 940. Biichel. K. H.; Draber. W.; Regel. E.: Plempel. M. Armeim. Forschung 1972. 22, 1260-1272. Kramer. W.; Biichel, K. H. ; Meiser. W.; Brandes, W.: Haspers. H.: Scheinpflug. H. Advances in PesricideScience Part 2. (Geissbuhler. H.; Ed.) Pergamon Press. Oxford and New York. 1979. 274-279. Siegel, M. R. Plonr Dis. 1981. 65, 986-989. Dreyfuss. P. M. J. Org. Chem. 1963. 28. 3269. Freudenberg. K.: Fikentscher, H.; Harder. M. Jusrus Liebigs Ann. Chem. 1925, 441, 175-!80. Bogatkov. S. V.: Kormanskaya. B. M.: Mochalin. V. B.: Cherkasova. E. M. Khrm. Gererorsikl. Soedin. 1971.662-661. Kramer. W.: Biichel. K. H.; Timmler. H.: Haller. 1.: Plempel. M. Ger. Olyen. 2816816; Chem. Absrr. 1980.92, 1J6776d. Arnold. 2.: Sorm. F. Czech Patent 90 045: Chem. Absrr. 1962. 56, 4621d. Martin. Y. C. Quanrirarive Drug Design; A Crirical Inrroducrion Marcel Dekker. New York, 1978. pp, 126-135. Mercer. E. I . Perfic. Scr. 1984. 15, 133-155. Rogerson. T. D.: Wilkinson, C. F.; Heterski. K. Eiochem. Pharmocol. 1977. 26, 1039-1042, Now*ell. I . W.: Walker. P. E.; Anderson. N . H. Acra Ccsrogr. Secr. E 1982, 38, 1857-1859.

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