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First Symposium on Chemical-Biological Correlation, May 26-27, 1950 (1951)

Chapter: Relation of Structure of Diphenyl Compounds to Fungitoxicity

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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 217
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 218
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 219
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 220
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 221
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 222
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 223
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 224
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 225
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 226
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 227
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 229
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
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Page 230
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 231
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 232
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 233
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 234
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 235
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 236
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 237
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 238
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 239
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 240
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 241
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 242
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 243
Suggested Citation:"Relation of Structure of Diphenyl Compounds to Fungitoxicity." National Research Council. 1951. First Symposium on Chemical-Biological Correlation, May 26-27, 1950. Washington, DC: The National Academies Press. doi: 10.17226/18474.
×
Page 244

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RELATION OF STRUCTURE OF DIPHENYL COMPOUNDS TO FUNGITOXICITY by James G. Horsfall, R. A Chapman and Saul Rich Department of Plant Pathology and Botany The Connecticut Agricultural Experiment Station New Haven, Connecticut

210 INTRODUCTION The literature of microbiology is full of data on the toxicity of benzene derivatives. By contrast diphenyl has been neglected. Nevertheless, diphenyl seems to be an architecturally significant nucleus in fungicides. During a span of 12 years we have worked sporadically on diphenyl derivatives. The time seems ripe to exhume the data from the catacombs of the litera- ture and our file drawers, to correlate it, and to work it into as reasonable a set of conclusions as possible. Bateman and Henningsen in 1923 demonstrated that diphenyl retarded somewhat the growth of Fomes annosus in culture. In 1938 work in our laboratory began with the finding that bis (4- aminophenyl) methane was fungitoxic. A few bits of data were published in 194513. The latest development is the demonstration of the practical significance for plant disease control of bis (2-hydroxy-5-chlorophcnyl) sulfide'*. We have investigated approximately one hundred forty analogues. The action of eighty-six of these will be discussed below. In the interim, Goodavage disclosed the mildew proofing action on cotton cloth of bis (2-hydroxy-5-chlorophenyl) methane and of bis {2-hydroxy-3, 4, 5-trichlorophenyl) methane. Marsh and/co-workers' ' have published excellent papers on these compounds and their relatives. Technique Used Since results depend upon techniques used, these will be indicated briefly. The details have been printed elsewhere^. What is called fungitoxicity often depends upon the method of assaying a test compound. Briefly, we may say that two basic techniques are employed; (1) spores suspended in distilled water are germinated overnight on dried deposits of the test chemical, (2) the fungus is grown for several days on treated food. Some investigators, notably Goldsworthy and co-workers, 9.'" modify technique (1) by placing the non-germinated spores on a clean food supply and note recovery. Results from the three methods vary. Diphenyl itself, for example, is a good inhibitor of growth - a poor inhibitor of spore germination. In visualizing the effect of test chemicals we should remember that a germinating spore carries its own rations. It merely needs to mobilize them quickly to form a short germ. tube. A test chemical can only prevent this mobilization and it must penetrate and act within a very few hours if it is to be effective. On the other hand, a growing hypha must obtain its nourishment from the ambient medium A test chemical has additional possibilities in this case. It can alter the food outside the spore as well as inside. It can prevent the food from entering. Goldsworthy's technique permits the test compound to be removed from the spore by the media. In our experiments, fungitoxicity of the test compound has been assayed by the spore germination method. The two test organisms were Stemphylium (formerly Macrosponum) sarcinaeforme and Sclerotinia fructicola. These are very common test fungi. Two techniques have been used for applying the test compounds; (A) a solution in a suitable solvent is applied with a graduated one milliliter syringe to the flat bottom of a cylindri- cal depression in a thick glass slide (cavity slide), (B) a suspension is applied by spraying to a cellulose-nitrate coated glass slide. Multiple doses are used normally differing by a factor of 10 for technique A and a factor of the square root of 2 for technique B. Spores of the test fungi are added to the dried deposits. Results for technique A are recorded as the least dose that prevents germination. Results for technique B are recorded as percentage of spores not germinating - usually without correction for the usual one or two percent of natural mortality.

211 Criteria of Action Data on spore inhibition for technique B are plotted against dose, using a logarithmic- probability grid, and a curve is fitted by inspection in accordance with the suggestion of Wilcoxon and McCallan'8. Such a curve has two qualities - position and slope. Position indicates potency of the test substance. It is usually expressed as ED SO, i. e. , the effective dose for 50 per cent inhibition. In other words, compounds can be compared in terms of the amount required for a given response. Slope is calculated as the change in probit mortality with unit change in log. dose. Hence, the larger the number, the steeper the slope. Slope may be expressed by the well-known function of ^ Dr. A. E. 1nn.mud of this laboratory has suggested that slope can be derived from the dosage-response curve as plotted on log. -probit paper. The formula simply is: Slope = log ED 84 - log ED 50 Slope is usually accepted as an indication of mode of action. If compounds show different slopes, they presumably differ in the manner in which they inhibit spore germination. Of course, similar slopes need not necessarily indicate similar modes of toxic action. In the tables included herewith the test compounds are listed in terms of both slope and ED 50 and ED 100 expressed in micrograms and molality per cmZ. It must be remembered, of course, that an ungerminated spore is not necessarily dead. Possibly, the toxicant could be removed by Goldsworthy 's technique or otherwise and the spore would recover. Toxicity at 1000 micrograms per cm' is considered essentially non-toxic. A compound must be toxic in the range of 1 to 1 0 micrograms per cmZ before it can be considered highly toxic. The compounds were used as received from the manufacturer or from Eastman Kodak Company. In general, they were laboratory samples and presumably they were reasonably pure. Data are presented in Tables 1 to 6. Table 1 comprises the names of the test compounds arranged alphabetically and the corresponding structural formulae. The toxicity data are given in Tables 2 to 6. TABLE 1 CHEMICAL NAMES AND STRUCTURES OF TESTED DIPHENYL DERIVATIVES 4-Amino-2-chloro diphenyl ether 2-Amino diphenyl H2r 4-Amino diphenyl 4-Amino-2-phenyl phenol

212 TABLE 1 (Cont. ) Cl Cl Cl , etc. Arochlor 1248 Mixture of trichlorodiphenyls, position of chlorines uncertain HC1 a-Benzyl-a-phenyl hydrazine hydrochloride H3C Auramine base hydrochloride CH3 QH2 C- Benzyl sulfone "*> H3C Auramine O .C NH2 Benzyl thiobenzoate Benzyl benzoate /—^ r-?H "\Je\J OH OH 2,3',4.5',6-Biphenylpentol Benzyl disulfide 4,4'-Bisphenol O9 c-o-c -/ ^ Benzyl-4-hydroxy benzoate Bis(2-aminophenyl) diaulfide Benzyl-2-hydroxy benzoate V—/cVyN" Bis(4-aminophenyl)methane

213 BrOso2O Br Bis(4-bromophenyl) sulfone COOH HOOC Bis(2 -ca rboxypheny l)dis ulf ide H3C' \ f s f v^n3 Bis (4-dimethyl aminophenyl) methane Bis (4-chlorophenyl) phenone Bis (4-chlorophenyl) sulfide Bis (4-chlorophenyl) sulfoxide ^ H2 s-^ c — so2— c — / Vi Bis (2,4-chlorobenzyl) sulfone TABLE 1 (Cont. ) Bis (2-diphenyl) monophenyl phosphate CL OH c — \ / C1N - 'Cl Bis (2-hydroxy-3, 5, 6-trichlorophenyl) methane C - l C1N - 'Cl Bis (2-hydroxy-S-chlorophenyl) methane Bis (2-hydroxy-5-chlorophenyl) sulfide H0( i-7cv s >OH CH3 CH3 Bis (4-hydroxyphenyl) isopropane H0QS02Q OH Bis (4-hydroxy phenyl) sulfone

214 TABLE 1 (Cont.) p-O H,CO( )—C ( >OCH3 W vV Bis (4-methoxyphenyl) benzylimido methane CH3 Bis (4-methyl phenyl) sulfonic acid HO 6-Chloro-4-(3-chloro-4-hydroxy-a, a-di- methylbenzyl)-1 -phenol-2-sodium sulfonatc A2 O-C-COOH Cl 4-Chloro-2-cyclohexylphenoxy acetic acid a-02 s- N02 Bis (2-nitrophenyl) disulfide C^s-s-ONo2 Bis (4-nitrophenyl) disulfide / \ O H H / \ Br/ Vc—C = C-/ \ 4-Bromo-4'-chloro chalcone 2-Bromo-4-phenyl phenol ci BP Cl 4-Chloro-2-phenyl phenol 6-Chloro-2-phenyl phenol (OH Cl 6-Chloro-2-phenyl phenol) 4-Chloro-2-phenyl phenol J mi mixture 2-Chloro-4-phenyl phenol Cl Cl Cl 2-Bromo-3, 5-dichloro-pentachlorophenyl benzoate

215 TABLE 1 (Cont. ) 2-Cyclohexylphenol "CKD 4-Cyclohexylphenol H3COCHN/ ^6O2NH/ VlHSO/ \ 4,4' -Dinitro-N, N' -4 -phenylenebis benzene. sulfonamide Diphenyl amide NHCOCH3 , N'-4-phenylenebis sulfanil- 010 Diphenyl am me 2,4-Diammo diphenyl ammr Diphenyl benzidine HC1 . H2N/ \ - / ) \ _ / \ _ / HC1 4,4'-Diamino diphenyl dihydrochloride (C4H9)2N02S/~~\OCH2CH2C/^S02N(C4H9)2 / \H H O H H ( VN — N— C— N— N \ / s-Diphenyl carbazide o Off \ / )NHNHCONHNH \ / sulfonamide 0 s -Diphenyl carbazone N - ( S Dicyclohexyl amine Diphenyl disulfide

216 H N-H t Diphenyl guanidine TABLE 1 (Cont. ) •N- O N-NH, 4,4-Diphenyl semicarbazide Diphenyl sulfide 0*0 Diphenyl sulfone Diphenyl guanidine phthalate N-NH2 . HC1 AS -Diphenyl hydrazine hydrochloride Diphenyl methane C—( 6 Diphenyl phthalate Q-O Diphenyl sulfoxide / \ H H S H H / V / ^—N—N—C—N —N-/ \ Diphenyl thiocarbazide aH H S / V. N-N-C-N=N-( J Diphenyl thiocarbazone ¥ s. N—C— Diphenyl thiourea

217 TABLE 1 (Cont. ) C2H5 Ethylbenzyl aniline OH Phenyl -2 -hydroxybenzoate CH2 \ - / Ethylidene aniline Ethyl-2-benzoyl benzoate ,OH 2-Phenyl phenol HO 4-Phenyl phenol 4-HydrcKy diphenyl amine J yU^U^M3 > w 2-Phenyl phenol acetate Isopropyl diphenyl ether 2-Phenyl sodium phenate H3C V Cl, \ - / Cl ci Pentachlorophenyl-4-toluenesulfonic acid CJtrO Salicylanilide Phenyl benzoate Triphenyl guanidine

TABLE 1 (Cont. ) (CH3)2N 2,4,6-Tris (4-dimethyl amino phenyl)-£-trithiane EXPERIMENTAL We introduced this paper with the statement that diphenyl seems to be an architecturally significant nucleus for a fungicide. Diphenyl itself is a curious compound. It is a very weak inhibitor of spore germination. In fact, it is adjudged ineffective for the spores of our two test fungi. It is a moderate to good inhibitor of mycelial growth, however, as Bateman and Hennings,n4 nrst reported and as Heiberg and Ramsey'' hsLVf confirmed. Heiberg and Ramsey demonstrated diphenyl to be a vapor-phase fungistat, which appeared to act chiefly on the plasma- membrane. The apparent hiatus between spore germination and mycelial inhibition data may perhaps be explained on the basis of "availability"1*. Diphenyl may enter the protoplast so slowly that there is time for a germ tube to develop before the fungistat is sufficiently concentrat- ed within the fungus to produce its effect. This growth of 50 to 100^i of mycelium would not be apparent when measuring mycelial colonies macroscopically. Diphenyl itself is simply two benzene rings joined end to end. Sometimes the two rings are joined through bridges of various types. We have studied the fungitoxic effect of different bridges and also the effect of hydroxyl, carboxyl, amino, and halogen substitutions. Effect of Bridges The effect of bridges will be discussed first. Data are given in Table 2 and Table 3. Some of these bridges are simple; others are very complex. In general, bridges by themselves added no toxicity to diphenyl. The following bridged compounds were non-toxic: TABLE 2 LIST OF NON-TOXIC BRIDGED COMPOUNDS Benzyl disulfide Dicyclohexylamine Diphenyl amine Diphenyl benzidine s-Diphenyl carbazide Diphenyl guanidine phthalate Diphenyl methane Diphenyl phthalate 4,4-Diphenyl semicarbazide Ethyl benzyl aniline Ethylidene aniline Bis (2-diphenyl) monophenyl phosphate Triphenyl guanidine

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220 The bridge seemed to have importance in a few cases. The bridge seemed to be more important in the toxicity of the compounds to Sclerotinia than to Stemphylium. For example, Stemphylium was inhibited by very few bridged compounds with otherwise unsubstituted rings. The most effective of these was diphenyl guanidine, 0 - NHCNHNH - 0. This has an exposed =NH group joined by its double bond to the central carbon in the chain. Its action will be discussed below in the section on - Sclerotinia. on the other hand, responded to several bridged compounds with unsubstituted rings. Compounds with sulfide or sulfoxide bridges were more effective against Sclerotinia than Stemphylium. Compounds with sulfone bridges were not toxic to either. Effect of -CO-O- and Related Bridges Sclerotinia also reacted to nearly all compounds with a -CO-O- bridge between the rings, but Stemphylium did not. (Data in Table 3). This was the situation with benzyl benzoate, benzyl - 4-hydroxy benzoate, benzyl -2 -hydroxy benzoate (benzyl salicylate), phenyl benzoate. 2-bromo- 3, 5-dichloropentachlorophenyl benzoate and phenyl-2-hydroxy benzoate (phenyl salicylate). Benzylthiobenzoate was not toxic to either organism. Cl.iyton ej al. 5 report that benzyl salicylate is able to prevent the downy mildew disease of tobacco, although it was not very effective in inhibiting spore germination. The -CO- bridge in bis (4-chlorophenyl) phenone and in 4-bromo- 4'-chloro chalcone was also toxic to Sclerotinia but not to Stemphylium. If, however, the two chlorines are removed from bis (4-chlorophenyl) phenone and -COOC^H^ is put on in the 2- position, it is toxic to neither (ethyl 2-benzoylbenzoate). Goldsworthy and Gertler'" report a -CO- bridged compound that is non-fungicidal, bis (4-aminophenyl) ketone. This must be viewed in the light of their technique, however. They put treated spores on agar. There is one other interesting bridge effect. As just noted, phenyl-2-hydroxybenzoate (phenyl salicylate) is toxic to Sclerotinia only. Salicylanilide is toxic to both organisms. The compounds are alike except for the bridge In the former the bridge is -CO-O-. In the latter the bridge is -CO-NH-. The action of this compound will be discussed below under the section on -OH attached to the bridge. Effect of Substituting -OH Ever since Lord Lister and carbolic acid, we have known that the addition of an -OH to benzene induces toxicity to microorganisms. Likewise the addition of an -OH to diphenyl will increase fungitoxicity. We have studied more -OH compounds than any other type. For convenience these will be divided on the basis of substitution on one ring or on both in the 4- and 2- positions or on the bridge. Data are given in Table 4. The well-known effect of -OH substitution on fungitoxicity shows up. Some other less well-known relations also appear. Among other things, fungitoxic -OH compounds in general have very flat dosage -response curves. They range from about 3. 0 to 6. 0. Effect of -OH in the 4-Position 4-phenyl phenol is an easy point of departure. It may be visualized as having an -OH on one end of the diphenyl long axis. 4-Phenyl phenol is moderately toxic to Stemphylium, quite toxic to Sclerotinia. We tested two similar compounds in which the phenyl portion of the molecule had been changed: 4-cyclohexyl phenol and benzyl-4-hydroxy benzoate. The toxicity to Stemphylium was completely knocked out and the toxicity to Sclerotinia was greatly reduced by both changes. These two substitutions steepened the dosage-response curve for Sclerotinia which suggests that the mode of action was also changed. 4-Hydroxydiphenyl amine was moderately toxic to Stemphylium. Appraisal of changes in ED 50 must be cautious, however, if the slope is changed simultaneously. ^ Effect of -OH in the 2-Position Passing to 2-phenyl phenol, we have a compound with the -OH along the side of the

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224 diphenyl long axis and near the junction of the two rings. This change in position of the OH from that in 4-phenyl phenol weakens the fungitoxic action and may possibly flatten the slope. In this case we have examples also of tampering with the -OH- free ring. The insertion of a -CO-O-CH2- bridge and even a -CO-O- bridge (benzyl-2-hydroxy-benzoate and phenyl-2- hydroxy benzoate) reduced the potency for 2-phenyl phenol just as much as for 4-phenyl phenol. This effect is particularly striking in view of the fact discussed below that -SOZ- bridge also reduced the potency of a dihydroxy compound. Using cyclohexane for the second ring gave a strikingly opposite effect for 2-phenyl phenol than for 4-phenyl phenol. It increased rather than reduced the potency of 2-phenyl phenol to both organisms and flattened the slope. Since all four tests involved were made on the same day, this can hardly be a result of biological variation. This obviously would warrant further study. Although the data on halogenation of 2-phenyl phenol are incomplete, the evidence is that 6-chloro-2-phenyl phenol and 4-chloro-2-phenyl phenol are more fungitoxic and have steeper slopes than 2-phenyl phenol alone. It seems significant that halogenation of both 4-phenyl phenol and 2-phenyl phenol should steepen the slope of the dosage-response curve. Effect of -OH on Both Rings We have tested several compounds with -OH on both rings. These are often called biphenols or bisphenols. We tested four bisphenols with the -OH in the 4-position and three with the -OH in the 2-position. The most striking result is that of the seven compounds only three are toxic to Stemphylium. Of course, only a few of the phenyl phenol series were toxic to Stemphyl- ium. Sclerotinia was more sensitive than Stemphylium to the bisphenols just as it was more sensitive to the phenyl phenols. In the case of 4 . 1' bisphenol it was some hundredfold more sensitive. The insertion of an isopropane bridge seems to quench what little toxicity that 4, 4'-bisphenol shows toward Stemphylium, and it reduces the toxicity to Sclerotinia. The insert- ion of a sulfone ( -SO2- ) bridge quenches the toxicity to both. It seems significant that the insertion of a -CO-O- bridge reduces or even quenches the toxicity of 2-phenyl phenol and 4-phenyl phenol. The interest in one compound of the 4, 4'-bisphenol series equals the length of its name, 6-chloro-4-(3-chloro-4-hydroxy-o, a-dimethylbenzyl)-l-phenol-2-sodium sulfonate. It is non- toxic to Sclerotinia. The problem is why? Of course the compound has so many substituents that one cannot be positive which one is basically responsible. This compound may be considered as a chlorinated 4, 4'-bisphenol compound with an isopropane bridge and a sodium sulfonate group in the 2-position to the hydroxyl on one ring. For the following reasons, we must conclude, tentatively at least, that the sulfonate group is mainly responsible for quenching the toxicity: (a) although an isopropane bridge reduced toxicity of 4, 4'-bisphenol to Sclerotinia, it did not quench it; (b) chlorination has not quenched toxicity elsewhere; (c) since chlorination in the 5-position does not quench the toxicity of -OH in the 2-position, it presumably does not do so for -OH in the 4-position. We have then two cases in which a sulfone grouping quenches the toxicity - in the bridge of bis (4-hydroxy phenyl) sulfone and on the ring in 6-chloro-4-(3-chloro-4-hydroxy-a, a-dimethyl- benzyl)-l-phenol-2-sodium sulfonate. This brings to mind the report by Horsfall and Rich15 who found that a sulfonic acid in the 5-position quenches the fungitoxicity of 8-quinolinol. Here is a good case for further research. It is of further interest to note that although slope data are meager, the slope of the Sclerotinia curve for bis (4-hydroxyphenyl) isopropane is 4. 22, very close to that for 4-phenyl phenol. Apparently, this suggests that the -OH group inhibits the fungus by the same mechanism whether it rides on one ring or on both. There is a slight further confirmation of the effect of halogenation on slope already noted under the phenyl phenols. The slope of bis (2-hydroxy-5-chlorophenyl) sulfide for Sclerotinia is steeper than that for bis (4-hydroxyphenyl) isopropane. Although again one cannot ascribe this for sure to the chlorine, this is reasonable because of the effect of chlorine to steepen the slope of the monohydroxy diphenyls.

225 One compound demonstrates the significance of the -OH in the bisphenols. Arndtl 1 showed that bis (2-hydroxy-5-chlorophenyl) methane was effective as a curative for cottonseed that is infected with Colletotrichum. His data reveal that replacing the -OH groups with acetoxy groups quenched the toxicity. Effect of Multiple -OH Substitutions The effect of multiple -OH substitutions is interesting. We have one compound with five -OH groups, 2, 31, 4, 51, 6-biphenylpentol. It was essentially non-toxic to both test organisms. It is of interest here to look at the molar toxicities of one, two, and five -OH groups on a diphenyl nucleus. The molar ED 50 values were 0.41, 4.91, and infinity (not toxic) for 4-phenyl phenol, bis (4-hydroxy phenyl) isopropane, and 2,3',4,5',6 biphenylpentol; respectively. Horsfall^ reports data showing that adding extra -OH units to phenol, phenyl phenol, and naphthol usually reduces the fungitoxicity. Effect of -OH Attached to the Bridge Dion and Lord6 show that the addition of an -OH to the methane bridge of bis (4-dimethyl - aminophenyl) methane lends toxicity to this compound. At this point we must refer back to the interesting anomaly between salicylanilide and phenyl-2-hydroxy benzoate mentioned earlier. The bridges are -CO-NH- and -CO-O-, respect- ively. The former is toxic to Stemphylium, the latter is not. We offer the following tentative explanation to explain it: we assume that the double bond to the carbon in the bridge of salicyl- 9H 9 ? anilide can resonate between the oxygen and nitrogen to give an equilibrium -C = N^±-C-N-. This should give an -OH compound which then should have the flat slope characteristics of -OH compounds. Salicylanilide has a very flat slope. Finally, such resonance cannot occur in a -CO-O- bridge because no mobile hydrogen is available. Effect of Substituting -NH? -NH2, like -OH, lends toxicity to the diphenyl nucleus. Of course, -NH2. like -OH, may be substituted on the ring or on the bridge. Data are given in Table 5. Fungitoxic -NH2 compounds in general have steep dosage-response curves. They range from about 5. 0 to 9. 0. Effect of -NH2 on One Ring Here also it is of interest to contrast the two rings of diphenyl with the single ring of benzene. The addition of an amine group to diphenyl enables it to prevent spore germination. The addition of an amine group to benzene (to form aniline) does not'3. This result can be stated in other words. Benzene does not prevent spore germination. It cannot be made to do so by.the addition of a ring (to form diphenyl) or the addition of an -NHZ group (to form aniline). It can be made to prevent spore germination, however, by the addition of both a ring and an -NH/ group to form aminodiphenyl. How can this be ? Since we are studying structure, we can make substitutions on the ring or on the -NHZ group to see which is the more important. The -NH2 is the simpler. Therefore, diphenyl amine is of interest because in it the second ring merely replaces one of the hydrogens of the amine. Except for the loss of one hydrogen atom, diphenyl amine has the same molecular weight as aminodiphenyl, but diphenyl amine is not fungitoxic. If, however, amino groups are added to diphenyl amine to form 2, 4-diaminodiphenyl amine, the toxicity is restored. Goldsworthy et al. ' first reported the fungitoxicity of this compound. Barry et al. Z showed that 2-aminodiphenyl amine was toxic to tuberculosis bacteria. For these data we can devise one conclusion. Toxicity appears to require 2-rings plus a primary ammo group. Of the two, the amino group seems to be the more important because (a) an amino group makes a diphenyl nucleus toxic, (b) substitution on the amino group quenches the toxicity. Without a more detailed study, we cannot be sure about the comparative toxicities of the 2-substitution, the 4-substitution and the Z, 4-substitutions except to say that the double

lib substitution is probably not more toxic than a single substitution. The 4-substitution is probably a little more toxic than the 2-. The slopes of the three compounds are probably identical, suggesting that they inhibit the spores by the same mechanism. Effect of -NH2 on Both Rings We obtained data on several compounds that show the effect of making a substitution of an amino group on each ring. All were toxic unless the amino hydrogen was substituted. Everitt and Sullivan' showed that bis (2-aminophenyl)sulfide was quite toxic in cultures of fungi that attack human skin, and we show that the same compound with a disulfide bridge was toxic to our spores. Benzidine (HC1) may be considered as the basic compound for 4, 4' -substitution. It is 4,4'-diaminodiphenyl. Its toxicity curve has about the same slope as 4-aminodiphenyl which suggests that the two compounds kill by the same technique. The toxicity of the benzidine may be blocked by substitution of a benzene ring for one hydrogen in each amine group. (See diphenyl benzidine). The hydrogens are similarly blocked in s-diphenyl carbazone and the toxicity is blocked for Sclerotinia. ^-Diphenyl carbazone, however, has a double bonded nitrogen and a carbonyl group to account for the toxicity to Stemphylium which it exhibits. Another 4,4'-diamino substitution occurs in bis (4-aminophenyl) methane. This compound is very close to 4,4'-diaminodiphenyl in toxicity and slope, which suggests also that the methane bridge plays no role in the toxicity. The toxicity of this compound also was quenched by substituting both hydrogens with methyl groups as in bis (4-dimethylaminophenyl) methane 2,4,6 Tris (4-dimethylaminophenyl)s-trithiane is a basically similar compound to bis (4-dimethylaminophenyl) methane and it is also non-toxic, presumably because the amino hydrogens have been substituted. Goldsworthy and Gertler^ have shown that bis (4-aminophenyl) ketone is non-fungitoxic. This is another case where oxygen attached by a double bond to the bridge is associated with low toxicity. Effect of -NHz Attached to the Bridge The -NH2 group adds toxicity when added to the bridge as well as when added to the ring, just as in the case of -OH. as-Diphenyl hydrazine (HC1) is such a compound which consists of an -NH2 group added to the nitrogen bridge of diphenyl amine. Thus, diphenyl amine is made toxic. a-Benzyl a-phenyl hydrazine is also toxic. This differs only in having an extra -CH2- in the bridge. Of course, phenyl hydrazine is also fungitoxic. One can consider that this is aniline with an -NH2 group attached to the amine group. Horsfalll3 has suggested that, through the Hinsberg reaction, phenyl hydrazine binds up the glucose in the spore thus depriving it of energy. Presumably, these two diphenyl hydrazines act also to deprive the spore of energy. The addition of an -NH2 group to a methane bridge also imparts toxicity just as the addition to an amine bridge. We have said above that bis (4-dimethylaminophenyl) methane was not toxic. If an t..'Hj group is added to the methane bridge to form auramine (HC1) toxicity is added. In fact, the toxicity and slope are almost precisely identical with that of the bis (4-amino- phenyl) methane before it is substituted with methyl groups. Dion and Lord^ obtained toxicity to Fusarium by adding a hydroxyl group to the methane bridge to form bis (4-dimethylaminophenyl) methanol. In fact, this compound was more toxic in their tests than the -NH > analogue. If now the primary amine on the methane bridge is reduced to a secondary amine, as in bis (4-methoxyphenyl) benzylimido methane, the toxicity is blocked again. From all this study of -NH2, we can conclude that toxicity of an amino diphenyl compound depends upon the action of a primary amine. It matters not whether the primary amine is attached to the ring or to the bridge. Another point is that the primary amine should be in connection with two rings. One ring is not enough. The biologist wonders if the mode of toxicity is the same when the compound is activated by -NH2 as it is by -OH. This may be studied by comparing the slopes of the dosage-response curves. Perusal of the data in Table 2 shows that the amine substituents give steeper slopes in

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231 all cases than the hydroxyl substituents. The best example perhaps is to be found in the com- parison of 2-phenyl phenol and 2-aminodiphenyl. The slope of the dosage-response curves of the amine compound is 7. 80 and the slope of the hydroxyl curve is 3. 80, much flatter. We interpret this to mean that the two types of compounds act on the fungus at different loci and hence the mode of action is probably different. Effect of =N- and -NH- in the Bridge We tested twenty-one compounds with =N- and -NH- in the bridge. Of this number, nine were recorded as non-toxic to the spores of the test organisms. With only a single exception, the nitrogen in these non-toxic compounds occurred either in a secondary or in a tertiary amine The exception was 4,4-diphenyl semicarbazide. In this compound the -NHj occurs at the end of a short chain attached to an amine bridge, but this chain also has a -C=O- grouping which is associated in several compounds with low toxicity. Except for the carbonyl group, diphenyl semicarbazide is not too dissimilar to as -diphenyl hydrazine which is fungitoxic. Of the twelve toxic compounds with nitrogen in the bridge, we have already discussed the toxic action of the primary amines on the two hydrazines and 2, 4-diaminodiphenyl amine. We have discussed the hydroxyl of 4-hydroxyl diphenyl amine and salicylanilide. The N4.N4'-di- acetyl compound is only very weakly fungitoxic; the toxicity of the 4,4'-dinitro compound is probably due to the -NO2 group. This accounts for the toxicity of seven of the twelve. The eighth compound that we might discuss is diphenyl guanidine. It appears to be a key compound. It has no toxic substituents on the ring. It has a secondary amine attached to the bridge, but most available evidence indicates that secondary amines are not very fungitoxic. We offer the following explanation which parallels that for salicylanilide already discussed. The bridge is as follows: H NH H -N-C—N- Presumably the double bond can resonate as follows: H NH H H NH2 -N-C N-^=i-N-C N- If so, a primary amine exists at equilibrium, and it can be fungitoxic. If it is, it should show a steep dosage-response curve and it does. The resonance discussed can be prevented by substituting to give triphenyl guanidine or diphenyl guanidine phthalate. If the resonance theory is sound, these compounds should be non-toxic and they are non-toxic. This leaves only the carbazide-carbazone, thiocarbazide-thiocarbazone series to be discussed. These are -CO- and -CS- analogues and should not be fungitoxic to Stemphylium and only weakly toxic to Sclerotinia if the quenching action of -CO- groups is general. The facts are in good agreement. This series is not fungitoxic at all or only negligibly fungitoxic to Stemphylium, and they are only weakly toxic to Sclerotinia. Effect of Sulfur As we have already suggested in the section on bridges, the introduction of sulfur into a diphenyl molecule imparts fungitoxicity. We have studied twenty-four sulfur-containing com- pounds. Data are given in Table 6. The sulfur in all cases was either a part of a bridge or attached to the bridge. Fourteen of the compounds were more or less toxic to Sclerotinia. None was toxic to Stemphylium unless the compound contained a toxic grouping other than the sulfur. Presumably, this is because of the well-known fact that Sclerotinia is sensitive to sulfur. Stemphylium is not. Five types of sulfur-containing bridges were tested - sulfide, disulfide, sulfoxide, sulfone, and sulfate. No compound with a sulfate bridge was toxic unless it contained some toxic substitu- tnt on the ring. Also the fact is noteworthy that no sulfone of the five tested was toxic to either

232 organism. Goldsworthy and GertlerlO have shown the non-fungitoxic quality of the sulfone bridge. How about sulfides and sulfoxide ? Data of Marsh e.t al.'7 show that the sulfide compound is more toxic than sulfoxide and sulfone. Combining their data and ours, it appears that the order of fungicidal value is -S- > -SO- > -SO2-. If so, the more oxidized the sulfur, the less its fungi - toxicity. This suggests that the sulfide compound must compete unfavorably with the fungus as an oxygen consumer and thus reduce its respiration. This needs testing. The disulfide appears to be the most toxic of the unsubstituted diphenyl compounds with sulfur in the bridge. Also, it provides the nucleus for several interesting substitutions. If the compound is changed from an -S-S- bridge to a -CH2-S-S-CH2- bridge, the toxicity disappears, reason unknown. Several 2, 2' -substitutions on diphenyl disulfide were studied. -COOH and -NO2 in the 2-position quench or seriously hamper the toxicity of the sulfur in the bridge. On the other hand, -NH2 in the same position provides one of the most, perhaps the most, fungitoxic compound in the whole series here reported. In fact, it is the only compound in the sulfur series that is significantly toxic to Stemphylium. Goldsworthy and Gertler'O reported the fungitoxicity of bis (4-aminophenyl) sulfide. The effect of bis (2-aminophenyl) disulfide is not particularly unexpected. It possesses -NH2 groups that are known by other evidence to lend toxicity to both organisms. The difficulty is with the analogues that contain active carboxyl and nitro groups. Normally they lend toxicity if anything, not take it away. In fact, bis (4-nitrophenyl) disulfide is fungitoxic. Perhaps, the explanation is as follows. The oxidized carboxyl and nitro groups compete with the reduced sulfur so that neither can inhibit spores. If the nitro group is removed spatially to the far-end of the molecule, the effect is not noticeable. Effect of Substituting Halogen Chlorination of diphenyl has not improved its fungitoxicity because Arochlor, a commer- cial mixture of chlorinated diphenyls, was no more toxic in our tests tha.n diphenyl itself. Bis (4-chlorophenyl) phenone also was non-toxic to Stemphylium but slightly toxic to Sclerotinia. We have already shown that diphenyl sulfone was non-toxic. The addition of bromine in the 4-position on each ring of diphenyl sulfone did not make it toxic. Chlorination of diphenyl sulfide to form bis (4-chlorophenyl) sulfide reduced its toxicity. The same was true of the Chlorination of di- phenyl sulfoxide to form bis (4-chlorophenyl) sulfoxide. Horsfall' 3 has reported that DDT is non- fungitoxic. This is bis (4-chlorophenyl) trichloroethane. Chlorination or bromination seemed to reduce the fungitoxicity of phenyl phenol. A mixture of 4-chloro-2-phenyl phenol and 6-chloro-2-phenyl phenol was less toxic than 2-phenyl phenol. 2-Chloro-4-phenyl phenol was less toxic than 4-phenyl phenol and 2-bromo-4-phenyl phenol was even less toxic than 2-chloro-4-phenyl phenol. This latter evidence that a bromine compound is less potent than its chlorine analogue is confirmed by the fact that bis (2-hydroxy-5-bromophenyl) sulfide is very much less toxic than bis (2-hydroxy-5-chlorophenyl) sulfide to both Chaetomium and Aspergillus (see Marsh et al.*7). From the evidence of Marsh e^ a.l. it is clear that the result is not due merely to the larger atomic size of bromine. Hatfield'' made a study of the effect of chlorinating phenyl phenols on toxicity to Fomes annosua in culture. He found that 2-chloro-orthophenyl phenol was slightly more toxic than orthophenyl phenol. 4-Chloro-orthophenyl phenol was less toxic and 2,4-dichloro-orthophenvl phenol was still less toxic. These compounds are practical wood preservatives. The only case so far found where chlorination increases toxicity is bis (2-hydroxyphenyl) methane. Marsh, Butler, and Clark'7 say that Chlorination in the 5 and 5' positions increases fungitoxicity of that compound. On the other hand, their data show that in the case of a similar bisphenol with a sulfur bridge, the 5, S'-dichloro substitution was less toxic to Chaetomium tha- the 5, 5'-dimethyl compound.

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236 As reported in the section on -OH derivatives, halogenation steepened the slope of the dosage-response of both 4-phenyl phenol and 2-phenyl phenol. Chlorination appeared to steepen the slope of hydroxyphenyl sulfide. Effect of Position of Substituent on the Ring The comparative ranking of 2 and 4 -OH substituents are as follows: 2-cyclohexyl phenol > 4-cyclohexyl phenol, 4-phenyl phenol > 2-phenyl phenol, 2-chloro-4-phenyl phenol > 4-chloro- 2-phenyl phenol, benzyl-4 -hydroxybenzoate > benzyl-2-hydroxybenzoate. In the case of amino substituents 4-amino diphenyl > 2-amino diphenyl and bis {4-mtro- phenyl) disulfide > bis (2-nitrophenyl) disulfide. Out of the six comparisons the 4-position was more active than the 2-position in five cases. Apparently, slope was not influenced by the position of the substituent group. Presumably then position has no influence on mode of reactivity merely, but on the amount of activity Effect of -CH=CH- Bonding We have a little evidence on the reactivity of the -CH=CH- bond. Marsh et alJ7 have clearly shown that if a bisphenol has a -CH I 1f- bridge it is more fungitoxic than if it has a -CHj- bridge. Likewise, auramine O is more toxic than auramine itself. The chief difference is that one ring of the former is quinoid with opposite double bonds. We have some data on cyclohexyl phenol versus phenyl phenol. In the case of the ortho position, the unsaturated phenyl ring was less toxic to both organisms than the saturated cyclo- hexyl ring. On the other hand, the reverse was true for both organisms for the para position. There can be little doubt as to'the accuracy of the result because all our comparisons were made on the same day. Of course, it is possible that impurities could account for it, but it seems unlikely. Synergism Between Diphenyl and Acenaphthene Acenaphthene is no more toxic to our spores than diphenyl. If acenaphthene is added to diphenyl, however, the mixture is fungitoxic as Bateman3 first showed. This is a strange phenomenon, the data for which appear in the graph in Figure 1. It was of some interest to ascertain the proportion of acenaphthene and diphenyl to give maximum fungitoxicity. Two tests were made and both agreed that 75 percent by weight of diphenyl and 25 percent of acenaphthene gave maximum response against Stemphylium. Sclero- tinia was not tested. Synergism is not uncommon between two toxicants or between a toxicant and a non- toxicant. It is a great rarity between two non-toxicants. Of course, probably neither compound is as bland as water, and if so, we can only say that synergism of a higher order is displayed by two weak toxicants. It is difficult to visualize how acenaphthene and diphenyl could react to produce a new toxic molecule. We can speculate that each acts on a different locus in the cell. If only one is applied and only one locus is affected, perhaps the organism can function by a different route. If, how- ever, both are blocked, it cannot continue the struggle. Obviously, the problem needs additional research. DISCUSSION We should not close out this paper without some discussion of the biological implications of the data. Some of these have been scattered along in the text. More effort should be given to correlating the work on physiology of living fungi with the work on the physiology of dying fungi. Various possibilities exist for accounting for the fungitoxicity of various chemicals.

237 Figure 1. Synergisra between diphenyl and acenaphthene. Depression slide teat using Stemphyllu.il sarc.naeforme. (B) 100 (A) - Percent of Diphenyl in mixture. (B) m Percent of Acenaphthene in mixture. Total concentration for each mixture was 625 PP».

238 Permeation Most compounds presumably must permeate the cell wall before they can do damage to the spore. One is struck in checking over the data by the fact that effective compounds can be considered as having lipophilic and hydrophilic components, polar and non-polar groups. This point is being discussed also in a collateral paper on nitrogen heterocycles*5. The non-polar group in many cases must have about twelve carbon atoms as we have discussed elsewhere'3. For example, in this series, -NH2 on one ring is not sufficient. It must have two rings - twelve carbon atoms. The classical theory, of course, is that of Overton-Meyer. They postulated many years ago that the semi-permeable membrane around the cell is composed of a fat and water emulsion. Hence, a compound that penetrates readily should be soluble in both. Of course, if the fat solubility is too high, the compound may not be water-soluble enough to react once inside the cell. Analogues of Cell Constituents None of these compounds seems sufficiently similar to be acting as a deleterious analogue of a vitamin, sugar, or amino acid in the cell. Of course, phenylalanine, thyroxine. and iodo- gorgoic acid are ring-containing amino acids. These amino acids do not contain double rings, however. Reaction with Cell Constituents As mentioned earlier the diphenylhydrazine compounds may be interfering with glucose utilization by reacting to formlglucoseazone by the Hinsberg reaction. The diphenylhydrazine compounds may react with ketone groupings in the cell as well. According to Eastman's reagent catalogue bis (4-aminophenyl) methane is useful for reacting with aliphatic acids. Since several of these occur in cells perhaps this is the mechanism of its action. Similarly, 2-hydroxydiphenyl is said to react with lactates and pyruvates and 4-hydroxydiphenyl is said to react with lactates. Interference with Energy Systems Since benzidine reacts with phosphates, one is tempted to suggest that it and perhaps some of its relatives may interfere with the phosphorylation enzymes that are involved in sugar metab- olism. Of course, energy is developed in the spore by a series of oxidation reactions. One finds himself tempted to speculate on the position that fungicides occupy in the oxidation-reduction system of the fungus cell. Little, that is concrete, has yet come from such speculations. It seems reasonably clear that the more reduced the sulfur, the more fungitoxic are the diphenyl compounds with sulfur bridges. If so, we deduce that the reduced sulfur must compete for oxygen with the spore and thus interfere with its respiration. This deduction is confirmed somewhat by the fact that carboxyl, carbonyl, and nitro groups seem to quench toxicity that seems due to reduced sulfur or to -NHZ. This needs studying with a respirometer. One of the characteristics of diphenyl is that the two rings tend to rotate about the point of juncture. Dion and Lord pointed out that this could be stabilized with a second bridge. In general, they found that if the second bridge contained nitrogen or oxygen, toxicity is only slightly reduced. If the second bridge contains sulfur, their data show a striking reduction in potency. In our studies £-hydroxydiphenyl was toxic, but 3-hydroxyphenothiazine was not toxic.

23* SUMMARY 1. Diphcnyl seems to offer an interesting architectural nucleus for making fungitoxic compounds. 2. The technique of testing was to apply the test compound to spores in the absence of extraneous matter like agar or nutrients. The test organisms were Stemphylium sarcinaeforme and Sclerotinia fructicola. 3. Toxicity was measured as percentage of non-germinated spores. When dose is plotted against spore inhibition on a logarithmic-probability grid, a linear curve is usually generated. Such a curve has two characters that are useful - position and slope. Position is expressed as ED 50 - the effective dose for 50 percent response. This is a measure of potency. Slope is a measure of mechanism. If two compounds have different slopes, we assume that they are acting by different mechanisms. 4. Unsubstituted rings show very limited fungitoxicity. 5. if the two rings are separated by bridges, toxicity is occasionally enhanced. A -CO-O- or an -S- bridge may lend toxicity to Sclerotinia not to Stemphylium. 6. If -OH is added to one of the rings, to both, or to a bridge, toxicity is added. The slope of the dosage-response curve of -OH compounds is usually flat. 7 As the number of -OH groups in the molecule increases the toxicity falls. 8. Likewise, if -NH2 is added to one of the rings, to both, or to the bridge, toxicity is added. The slope of the dosage-response curve of -NH2 compounds is usually steep. 9. An increase in the number of -NH2 groups in the molecule does not increase the toxicity and they do not change the slope of the curve which presumably means that they do not change the mode of biological action. 10. Slope data suggest that -OH and -NH2 substitutions inhibit spores by different mechanisms. 11. Fungitoxicity of amino diphenyl compounds seems to depend upon the presence of a primary .amine. If the primary amine is converted to a secondary or tertiary amine by replacing the hydrogens, toxicity disappears. 12. Moreover, a primary amine attached to benzene is not toxic. It must be attached to diphenyl. In other words, toxicity rests on a molecule as big as diphenyl plus a primary amine. The data available suggest that the primary amine is the toxic grouping; the rings serve as vehicles to carry it into the spore. 13. In the diphenyl compounds, as in many others, the addition of sulfur adds toxicity to Sclerotinia not to Stemphylium. The former, of course, is sulfur-sensitive. The latter is not. 14. Halogenation of non-toxic diphenyl compounds did not make them toxic. Halogenation of toxic phenolic types of compounds in general reduced their toxicity to spores. Hatfield* ^ reported that chlorination and phenyl phenols increased toxicity to growing mycelia. We had no halogenated amino derivatives for study. 15. Out of six comparisons made 4-substitution imparted more fungitoxicity than 2-substitution in five cases. On the other hand both substitutions gave the same slope, which suggests that they inhibited the fungus by similar mechanisms. 16. Compounds with double bonds in the bridge or in substitutions seemed to be more fungitoxic than others.

240 17. There is a curious case of synergism between diphenyl and acenaphthene. Neither inhibits spores alone. A mixture inhibits the spores very well indeed. Nothing is known about the mechanism. 18. Some possible modes of action on the fungus are discussed. Some of the compounds may react with cell constituents, as for example, diphenylhydrazine with glucose. Diphenyl benzidine may react with essential metals. Most of the active compounds contain replaceable hydrogen. They lose their toxicity if they lose their replaceable hydrogen.

241 LITERATURE CITED 1. Arndt, C. H. . Phytopath. . 38, 978-987(1948). I. Barry. V. C. , Belton, J. G. . Conalty, M. L. , and Twomey. D. , Nature. 162. 622-623 (1948). 3. Bateman. E. , U.S. Dept. Agri. Tech. Bui. 346. l-54 (1933). 4. Bateman. E. and Henningsen, C. . Airier. WoodPreserv. Assoc. 19th meeting, 136-143 (1923). 5. Clayton, E. E. , Smith, T. E. , Shaw, K. J. . Gaines, J. G. , Graham, T. W. , and Yeager, C. C. Jour. Agr. Res.. 66, 261-276(1943). 6. Dion, W.M. and Lord, K. A. . Ann. Appl. Biol. . 31., 22l-231 (1944). 7. Everitt, E. L. and Sullivan, M. X. , Jour. Wa»h. Acad. Sci. , 30, 125-131 (1940). 8. Goodavage, J. E. , Amer. Dyestuffs Rep." 32, 265-270(1943). 9. Goldsworthy, M. C. , Green, E. L. and Halle r, H. H. , Jour. Agr. Res., 64, 667-678(1942). 10. Goldsworthy, M. C. and Gertler, S.I. , Plant Disease Rep. Supp. , 182. 82-109 (1949). II. Hatfield. Ira., Amer. WoodPreserv. Assoc. 31st meeting, 57-66(1935). 12. Heiberg, B.C. and Ramsey, G. B. . Phytopath. 36, 887-891 (1946). 13. Horsfall, J. G. , Fungicides and their action. 239 pgs. Chronica Bot. Co. , Waltham, Mass. (1945). 14. Horsfall. J.G. and Rich, S. , Phytopath. , 40, 13 (1950). 15. Horsfall, J.G. and Rich, S. , Contrib. Boyce-Thompson Inst. , 1.6 (1951 in press). 16. Hwang. L. and Klotz, L. J. , Hilgardia, 1£. 1-38(1938). 17. Marsh, P. B. , Butler, M. L. and Clark, B. S. , Indus, and Eng. Chem. , 41, 2176-2184 (1949). 18. Wilcoxon, F. and McCallan, S. E. A. , Contr. Boyce-Thompson Inst. , 1.0, 329-338(1939).

242 DISCUSSION DR. LOUIS F. FIESER (Harvard University, Cambridge, Massachusetts): I want to compliment Dr. Horsfall on a very interesting paper and, particularly, on the application of exact methods for analysis of the bio-assay results. Use of dose-response curves and attention to both the LD50 and the slope of the curve is, I think, of distinct advantage. It would seem to me that a further advance in correlating the data might be to use not the toxicity on the weight basis, but to compare molar toxicities. That might iron out some of the results which appear a little irregular on a purely empirical basis; that is, comparing a bromo compound with a chloro compound. On a weight basis, the chloro compound may seem a good bit more active, but on a molar basis they come out to be about the same. As an organic chemist, I want to say I was a little bit unhappy about the discussion of structure with relationship to biological activity. The compounds discussed are all regarded as diphe-nyls; as a matter of fact, that is the term used in the title of the paper. Yet, to an organic chemist, diphenyl is a compound with two rings joined together; if the rings are separated by <:Hr, . you have diphenylmethane, which is something quite different. Separation of the rings by the group NH gives diphenylamine. Diphenylmethane and diphenylamine are not diphenyl com- pounds. To compare the effect o? substituents in one series, where the two rings are joined together, with that in another series, where they are separate is, I think, taking a little bit of liberty with organic chemistry. However, I must say that organic chemists or anybody else who has tried to work out relationships between structure and biological activity have not progressed very far, so that this is merely a personal comment on the paper; and I say that, if Dr. Horsfall can disregard some of the principles which appeal to a chemist, and still make some sense out of the correlation, I wish to congratulate him. DR. HORSFALL: Mr. Chairman, that is an excellent comment, with respect to comparing diphenyl with diphenylmethane. I realize that diphenylamine is not diphenylmethane, and that diphenylmethane is not diphenyl. I realize that a chemist would not compare directly an amino substitution on diphenyl- methane with an amino substitution on diphenyl. Back in the nervous part of my speech, before I got going, I neglected to say that we made an extensive study of the effect of bridges on toxicity. In general the bridges had little influence on the toxicity. Hence we tried to compare substituents irrespective of the bridge concerned. DR. RICH: These were all converted on a molar basis, weren't they? DP. HORSFALL: Oh, yes; in the manuscript which will be printed, the data are all expressed on a molar basis. DR. McKEEN CATTELL (Cornell University Medical College, New York, New York): I fully agree with what Dr. Horsfall has said regarding the differences in slope of the dosage- response curve as indicating a qualitatively different action. There is, however, a consideration which, it seems to me, needs emphasis. We are concerned about the fundamental action of these compounds in the final solution. It is the goal of the pharmacologist to determine what reaction these compounds influence. The differences in slope may well be related to subsidiary factors, such as the retention of the compound in the body, the speed with which it is broken down and its absorption; factors could conceivably throw us off the track in our reasoning in relation to the actual mechanisms of action. I am wondering whether, in these closely related diphenyl compounds when there was an ammonium or a hydroxyl substitution, if there might not have been an influence on the stability of the molecule, or a difference in its absorption or penetration.

243 DR. HORSFALL: I dare say you have a point there. Dr. Cattell. We have made an extended study of what slope means in these compounds (not these compounds exclusively, but compounds in general). We plant pathologists are apt to spray surfaces to protect them against disease. We have the problem of what we call coverage; it is analogous to the greased pig problem whether it is better to put on concentrated mixtures thinly spread, or weak mixtures with a lot of water. In other words, if you are permitted in the country fair to have two thousand pounds of human flesh to catch the pig, is it better to have ten 200-pounders or twenty 100- pounders? You are more apt to catch him with twenty 1 00-pounders, but you are more apt to hold him when you catch him, with ten 200-pounders. That makes a difference in slope in the response curve. The ten 200-pounders come out with a flatter slope than the twenty 100-pounders. That is a case of distribution, you see; in your case, it is excretion from the body. It is admitted that slope does differ with other factors than mere mechanism of action but, if the slopes are different, it shows us things about the compounds which we would not have known if we had not had slope; therefore, it makes it a worth-while point for advance of further research.

244

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First Symposium on Chemical-Biological Correlation is a summary of a symposium held on May 26-27, 1950 by the Chemical-Biological Coordination Center. The purpose of the symposium was to bring together scientists trained in chemistry and biology for discussion of problems concerned with the effect of structure of chemicals on their biological activity and the mechanism of such actions.

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