ABSTRACT THE RELATIONSHIP OF CHEMICAL STRUCTURE AND PLANT GROWTH REGULATOR ACTIVITY IN INDOL-3-YLACETAMIDES by Thomas Charles Hageman The plant growth regulating activity of indol—3- ylacetic acid is well established; numerous derivatives of indol-3-ylacetic acid also exhibit similar physiological activity. Indol-3-ylacetamide, a naturally~occurring deriv- ative of indol-B-ylacetic acid, exhibits definite auxin ac- tivity in many plant species. Recently, several other simple amides have shown plant growth regulating activity; among these are: N,N-dimethyl-2,2—diphenylacetamide, dimethylpropynylbenzamides, and the dimethylhydrazides of succinic and maleic acids. The purpose of this study was to determine the effects of N-substitution on the biological activity of indol-3-ylacetamide and to explore the.meehanism of actién of these compounds.‘ The following compounds were synthesized and bioassayed: Thomas Charles Hageman N-methylindol-3-ylacetamide N,N-dimethylindol-3-ylacetamide N-ethylindol-3—ylacetamide N,N-diethylindol-3-ylacetamide N-(2-chloroethy1)indol-3-ylacetamide N-propylindol-B-ylacetamide N,N-dipropylindol-3-ylacetamide N—(3-chloropropyl)indol-B-ylacetamide N-isopropylindol-B-ylacetamide N,N-diisopropylindolr3-y1acetamide N-cyclohexylindol-3-y1acetamide N-dimethylaminoindol-3-ylacetamide indol-3-y1acetanilide N,N-diphenylindol-3-ylacetamide N—methylindol-3-ylacetanilide N-(2-chlorophenyl)indol-3-ylacetamide N-(3-chlorophenyl)indol-3-ylacetamide N-(4-chlorophenyl)indol-3eylacetamide 'N-(2,4-dichlorophenyl)indol-3-ylacetamide N-(2,5-dichloropheny1)indol-B-ylacetamide N-(l-naphthyl)indol-3-ylacetamide N-benzylindol-3-ylacetamide NbN-dibenzylindol—3~ylacetamide N—benzyl-N-methylindol-3-ylacetamide N-(2-chlorobenzyl)indol-B—ylacetamide N-(3-chlorobenzyl)indol—Brylacetamide N-(4-chlorobenzy1)indol-B-ylacetamide N—(2,4-dichlorobenzyl)indol—B-ylacetamide :N—(3,49dichlorobenzyl)indol-39ylacetamide Thomas Charles Hageman The following bioassays were used: the 52222 straight growth assay, the cucumber root inhibition assay, and the cucumber epicotyl curvature assay. These compounds exhibited a wide range of activity in the bioassays; in general, the phenyl derivatives were the most-active, fol- lowed by the alkyl derivatives. The benzyl derivatives showed little or no activity. The addition of chlorine usually increased the activity of the compound. A correla- tion was found between the pKa's of the free primary amines and the activity of the corresponding amides. A metabolic study showed that these compounds are hydrolyzed to indol-B-ylacetic acid in vivo; there was a positive correlation between the amount of hydrolysis and the biological activity in the ézggg assay. _These compounds probably derive their activity from their conversion to. indol-3-ylacetic acid} N-dimethylaminoindol-3-ylacetamide may be an exception to this theory. Nf(3-chloropheny1)_ indol-3-ylacetamide is unusual in that it has high activity at extremely low concentrations in both the 53222 assay and the root inhibition assay. and yet it haS'very low activity in the cucumber curvature assay. This might be a means of localizing the effects of applied auxin solutions on plants. THE RELATIONSHIP OF CHEMICAL STRUCTURE AND PLANT GROWTH REGULATOR ACTIVITY IN> INDOL-B-YLACETAMIDES by Thomas Charles Hageman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1971 ACKNOWLEDGEMENTS The author expresses his sincere appreciation to Professor Harold M. Sell for the guidance and encouragement offered throughout the course of this work. He is grateful to Professor Martin J. Bukovac for his helpful suggestions concerning the biological assays. The author is also indebted to Maria Rojeski for her assistance with the laboratory work. ii TABLE OF CONTENTS LIST OF TABLES O O O O O O O O 0 LIST OF FIGURES . . . . . . . . INTRODUCTION. . . . . . . . . . HISTORICAL. . . . . . . . . . . BIOLOGICAL ASSAY. . . . . . . . METABOLIC STUDY . . . . . . . . EXPERIMENTAL. . . . . . . . . . Synthesis of Compounds. . . . Indol-3-y1acety1 chloride . N-methylindol-3-y1acetamide N-ethylindol-3-ylacetamide. N-(2-chloroethyl)indol-3-ylacetamide. N-(3-chloropropyl)indol-3-y1acetamide N-propylindol-3-y1acetamide N,N-dimethylindol-3-y1acetamide N,N-diethylindol-3-ylacetamide.n N ,N-dipropylindol-3-ylacetamide N-isopropylindol-3-ylacetamide. N ,N-diisopropylindol-3-ylacetamide. N ,N-dibenzylindol-3-ylacetamide . N-cyclohexylindol-3-ylacetamide . N-benzylindol-3-ylacetamide . N-benzyl-N-methylindol-3-ylacetamide. N-(2-chlorobenzyl)indol-3-y1acetamide N—(3-chlorobenzyl)indol-3-ylacetamide iii Page vi 10 12 12 13 14 15 15 15 15 16 16 16 16 16 17 17 17 18 18 18 TABLE OF CONTENTS (cont.) N-(4-chlorobenzy1)indol-3-ylacetamide . . N-(2,4-dichlorobenzyl)indol-3-ylacetamide N-(3,4—dichlorobenzyl)indol-3-ylacetamide Indol-3-ylacetanilide . . . . . . . . . . N-methylindol-3-y1acetanilide . . . . . . N-(2-chlorophenyl)indol-3-ylacetamide . . N-(3-chlor0pheny1)indol-3-ylacetamide . . N-(4-chlorophenyl)indol-3-ylacetamide . . N-(2,4-dichlorophenyl)indol-B-ylacetamide N-(2,5-dichlorophenyl)indol-34ylacetamide N-(1-naphthyl)indol-B-ylacetamide . . . . N,N-diphenylindol-3-ylacetamide . . . . . N-dimethylaminoindol-3-ylacetamide. . . . Biological Assays . . . . . . . . . . . . . Avena Straight Growth . . . . . . . . . . Cucumber Root Inhibition. . . . . . . . . Cucumber Epicotyl Curvature . . . . . . . Metabolic Study . . . . . . . . . . . . . . RESULTS AND DISCUSSION. . . . . . Effects of Alkyl Substitution . Effects of Phenyl Effects of Benzyl Metabolic Study . Discussion. . . . SUMMARY . . . . . . REFERENCES. . . . . ”PENDIXO O I O O . Substitution. Substitution. iv Page 18 18 18 18 18 18 18 l8 18 18 18 19 19 20 20 22 23 24 27 27 31 38 41 44 54 57 60 LIST OF TABLES Table Page I. ROOT INHIBITION ASSAY--ALKYL DERIVATIVES. . . . 30 II. ROOT INHIBITION ASSAY--PHENYL DERIVATIVES . . . 37 III. ROOT INHIBITION ASSAY--BENZYL DERIVATIVES . . . 42 IV. RESULTS OF METABOLIC STUDY. . . . . . . . . . . 43 Figure I. II. III. IV. VI. LIST OF FIGURES Growth Curves of Avena Coleoptile Sections; Effects of Alkyl Substitution . . . . . . . Results of Cucumber Epicotyl Curvature Assay. Growth Curves of Avena Coleoptile Sections; Effects of Phenyl Substitution. . . . . . . Growth Curves of Avena Coleoptile Sections; Effects of Benzyl Substitution. . . . . . . Correlation of pK with Avena Straight Growth Activity . . . . . . . . . . . . . . Correlation of IAA in Tissue Extract with Avena Straight Growth Activity. . . . . . . Vi Page 29 33 35 40 46 50 INTRODUCTION INTRODUCTION A fundamental property of living organisms is their ability to control the processes of growth; development, and reproduction. In recent years biologists and bio- chemists have-taken an increasing interest in the control mechanisms of biological systems. In the plant kingdom growth and development are under the control of a highly complicated hormonal system. There are many types of compounds which are involved in the control processes; their activities; however, appear to be intricately interrelated; The first-class‘to be isolated and characterized were the auxins or indole compounds. Perhaps the most important auxin compound is indol-3- ylacetic acid (IAA). The role of IAA in controlling such widespread phenomenon as flowering,'apical dominance, cell elongation, and fruit setting is well established. Numerous derivatives of‘IAAthave"beenfsynthesized and studied in regard to~plant~growth regulating activity. A naturally occurring derivative of IAA is indol-3-ylacet- amide (IAAm). IAAm exhibits definite‘auxinaactivity in a number of plant species (1). In recent years the tremendous biological activity of some substituted amides has been discovered. N,N- dimethyl-Z.Z—diphenylacetamideu(diphanamid)wis~a powerful pre-emergent herbicide, it exhibits its activity through its effects on root tissue (2). Another class of pre- emergent herbicides*areathe‘dimethylpropynylbenzamides, in particular, 3,5-dichloro-N-(l,l—dimethyle2—propynyl)— benzamide (Kerb) (3). "More'recently, the inhibitory ef- fects of tflma dimethyl hydrazides of succinic-acid (3—995) and of maleic acid (C-Oll) have been reported*(4-6). These compounds, which are simple substituted amides; apparently interfere with the regulatory action of auxins and gibber- ellins (7)., This study was undertaken to establish a better understanding of the growth regulatory activity of substi— tuted amides and in particular indol-Béylacetamides. The study consists of three parts:' the chemical sysnthesis of a series of N-substituted indoleB—ylacetamidesg‘the characterization of their biological activity, and a brief study of the metabolism-of a few representative-compounds. In this way, we hope to establish the effects of structural modification on biological activity, to gain some understanding of the mechanism of action of indol-3-' ylacetamides, and to learn more about the metabolism of these compounds. HISTORICAL HISTORICAL Although indol-3-ylacetic acid was first prepared by Ellinger in 1904 (8), it was not until thirty years later that its effects on cell elongation of Aygna was shown by KBgl, Haagen-Smit, and Erxleben (9). Proof of its existence as a natural plant product did not come un- til 1946 when Haagen-Smit isolated-and characterized IAA from §_e_a_ gays (10). The amide of IAA was first synthesized in 1925 by treating indol-3-ylacetonitrile with zinc metal in acetic acid (11). “In 1940*Baker synthesized the amide through the pyrolysis of the ammonia salt of IAA (12).‘ Subsequent syntheses were devised by Snyder in 1948 (13) and Shaw in 1958 (14). The latter synthesis was the-reaction-of ammonia with indol-3-ylacetyl chloride, the acid chloride of IAA was first reported by Shaw and Wooley in 1953 (15). Very little-work is reported on the synthesis of N- substituted indol-3-ylacetamides.‘ In the 1950's most of the natural alpha amino acids were used to form*amide links 4 with IAA (16, 1?). Aside from these compounds only a few N-substituted amides of IAA have beenrmade; among these are: N,N-dimethylindol-3-ylacetamide (18), N,N-diethy- lindol-B-ylacetamide"(l9),‘NecyclohexylindoleBeylacetamide (20), N-benzylindol-3-y1acetamide (21), and indol-3-ylacet- anilide (21). The biological activity of"IAAm-was'first reported by Bentley and Housely in 1952, they proposed that IAAm was an intermediate in the interconversion of IAA and indol-3- ylacetonitrile (22). By the mid-1950's, IAAm was well established as a naturally occurring compound in plant tissue, IAAm was detected by-chromatography of extracts of tissue which had been incubated in-IAA solutions (23). By the chromatography of extracts of tissue which had been incubated in IAAm solutions, Wain and*Wightman showed that the amide is capable of being hydrolyzed'to“IAA in wheat, pea, tomato, and bean plants (1, 24-25). In 1960, the ac- tivities of indol-3-ylcarbonamide, indoléBeylpropionamide, indol-B-ylbutyramide, indol-3-ylvaleramide,-and indole3-' ylcaproamide were compared with those of the parent. acids (26). Studies were made on the metabolism of these compounds by Fawcett, Wain, and Wightman. By using similar incubation and chromatographic techniques as those employed in their previous studies, they found IAA in the extracts from compounds with an even-number of carbonatin the side chain, and they found indol-3-ylpropionic acid in those from compounds with an odd number of‘carbons71n the side chain. They concluded that the activitiesfof the amides were due to hydrolysis to the parent acid, followed by beta-oxidation to either IAA or indol-Bfiylpropionic acid. ‘ None of the N-substituted compounds previously listed were tested for activity as plant growth regulators. BIOLOGICAL ASSAY BIOLOGICAL ASSAY Investigators working with plant growth regulators have devised many biological assays to test the physio- logical activity of chemical compounds. These assays, using whole plants or excised plant parts, were designed to maximize the desired response and to minimize interfer- ing effects. In general these methods are quite sensitive and reproducible, thus making them a valuable tool in plant hormone studies. It is important to keep in mind, however, that these assays involve whole plant cells which makes it im- possible to distinguish between the primary effects of the compounds acting on the site of action and secondary ef- fects such as metabolism of the compound by the plant as well as factors of transport and penetrability. Bioassays are useful for what they do tell us, that is,-the effects of applications of chemicals on a plant, regardless of the- mechanism of action. Three bioassays were used in this experiment; they were: the A1223 straight growth, the cucumber root inhibi- tion, and the cucumber epicotyl curvature assay. The 53333 assay is a classic method of studying auxins, the effects of IAA on cell elongation was first established using Ayggg coleoptiles. In the straight growth assay the ability of the compound to cause cell elongation is measured, the limiting effects of transport and penetration are minimal. Oat coleoptile sections are floated on the solution to be tested, the elongation after twenty-four hours is a measure of activity. The cucumber root inhibition assay measures the capacity of the compound to inhibit root growth. In all tissue low auxin concentrations stimulate growth and higher than optimal concentrations cause inhibition of growth. Because root tissue has a low optimal auxin concentration they are easily inhibited, thus providing a sensitive test for auxin activity. The cucumber curvature assay measures the secondary factors of transport and absorption as well as the ability to induce cell elongation in intact plants. Solutions are applied to one cotyledon of a young cucumber plant, for activity to be observed the compound' must be absorbed and transported to the main stem, where active compounds induce a curvature away from the side of application. Details of the bioassays will be given in the experimental section. METABOLIC STUDY METABOLIC STUDY As waS-mentioned before, it is impossible to de- termine from the bioassays alone whether the observed activity is due to the applied compound or if it is an artifact, due to the metabolism of the compound to another more active substance. ‘In particular, Wain and Wightman claim that the activity of IAAm is derived from its hy- drolysis to IAA. In their experiments they incubated plant tissues in solutions containing 1000 micrograms of IAAm per 50 m1. of water. When an extract from this solu- tion was chromatographed they obtained a distinct spot corresponding to IAA; a bioassay of a similar chromato- graph confirmed the presence of IAA. While Wain and Wight- man did show that IAA and IAAm are capable of-being metabol- ically interconverted, their evidence does-not-conclusively prove that the amount of IAA formed from IAAm is sufficient to evoke the response elicited by the IAAm. In our study we have compounds having activity greater than IAAm and others having almost no activity 10 11 at all. If the activity of the amides is due to their hydrolysis to IAA, then the amount of IAA formed should vary with the activity of the amide tested. Our study, a modification of the procedure used by Wain-and Wightman, is an attempt to correlate the amount of IAA in plant tissue with the activity of various amides.~ This study includes chromatographs which were examined by both chem- ical developing reagents and biological assays. Six compounds were chosen for this study; they were picked to give a wide range of activity and type of substitution. The compounds studied are: -IAAm, N-methyl IAAm, N,N-dimethyl IAAm,.N—methylamino IAAm,-N-(2—chloro- ethyl) IAAm, and N-(3-chlorophenyl) IAAm. EXPERIMENTAL EXPE RIMENTAL Synthesis of Compounds N-substituted indol-B-ylacetamides were prepared by the reaction of indol-3-ylacetyl chloride with the appropriate primary or secondary amines. The indolylacetyl chloride was prepared by treating IAA with phosphorous pentachloride according to the method of Shaw and Wooley (15). This compound is somewhat unstable;-it was always reacted with the amines immediately after it was prepared. The reaction with the amines proceeds rapidly in ether solutions at 0°C. The free amines were reacted in a 2:1 molar ratio with the acid chloride. When the~amine hydro- chlorides were used, they were reacted in-a water-ether mixture using sodium hydroxide to absorb the HCl. Pyridine, which is commonly used to absorb HCl in reactions, could not be used because it reacts with indol-3~ylacetyl chloride in ethereal solutions to form an insoluble orange tar (27). The greatest difficulty encountered involved the purification and crystallization of the products. The 12 13 aromatically substituted amides crystallized from methanol, aqueous ethanol, or ethyl acetate. The lower molecular weight aliphatically substituted amides were much more dif- ficult to crystallize; they tend to form an oil from most solvents rather than to crystallize. This problem was overcome by first crystallizing the compounds from the oils, then using some of these crystals to seed-the solutions from which the recrystallizations were made. Indol—31ylacetyl chloride Four and 38 hundredths of a gram (0.025 mole) of IAA was dissolved in 150 ml. of anhydrous-ethyl ether and the solution was cooled to -5°C in a salted-ice-water bath. Five and 73 hundredths of a gram (0.0275 mole) of phos- phorous pentachloride was added with stirring in five or six portions over a period of twenty minutes;vthe reaction was stirred for an additional fifteen minutes.~ The ether was decanted from the unreacted phosphorousepentachloride and was concentrated to about forty ml. by vacuum, the. solution was poured into 400 ml. of petroleum ether (b.p. 30-60’C) which had been previously cooled to -20°C.- 14 After fifteen minutes the precipitate was-collected on a Bfichner filter and was washed with 50 ml. of petroleum ether. The product was used immediately without further purification. The product melts at 58-60°C-with decompo- sition. Yields ranged from 2.5 to 4.0 gm. (.0129-.0206 mole, 52-83%). The product first forms as-white flakes; they quickly turn pink on standing when exposed to atmos- pheric moisture at room temperature. N-methylindol-3eylacetamide One hundred and fifty ml. of ether, 10 m1. of water, and 2.00 gm. (.0296 mole) of methylamine hydrochloride were mixed and cooled to 0-5°C in a flask equipped with a mag- netic stirrer. One and 85 hundredths of a gram (.0280 mole) of potassium hydroxide (85%) was added. With maximum stir- ring, 3.00 gram (.0155 mole) of indol-3-ylacetyl chloride was added over a period of twenty minutes; the solution was allowed to warm to room temperature as it was stirred for an additional hour. Fifty ml. of water was added, and the solutions were separated. The water fraction was extracted with 100 m1. of ether. The ether fractions were combined, dried over anhydrous sodium sulfate, and evaporated to an 15 oil residue. The oil was crystallized by cooling in an ice bath and scratching it with a glass rod. The product was recrystallized from methanol and water giving 1.86 gm. (.0099 mole, 64% yield) of yellow prisms. ‘The final pro- duct melts at 104-105°C. Analysis: calculated-- 14.94% N.;-found-- 14.81% N. The following compounds were synthesized in a manner similar to N-methyl IAAm; Analysis (%N.) Compound M.P. Yield Calculated Found N-ethylindol-B-yl 67-69° 35% '13485 13.76 acetamide N-(2-chloroethy1) 93-94° 19% 111.84 11.93 indol-3-ylacetamide N-(3-chloropropyl) 78° -15% -11.17 11.22 indol—3-ylacetamide N-propylindol-3-y1acetamide A-solution of 2.20.gm.:(.01l4vmo1e) of indol-3- ylacetyl chloride in 75 ml. of ether was cooled to -10° C and mixed with a solution of 1.35 gm. (.0228 mole) of propylamine in 75-m1..of ether, which had-also been cooled to -10°C. The reaction was stored in thewfreezer overnight, then the ether.was decanted from.the preeipitate and l6 evaporated to an oil. The oil was crystallized by scratch- ing at 0°C. The product was recrystallized from methanol and water giving 0.73 gm. melting at 79-82°C.--Sixty-three hundredths of a gram melting at 76-80°C were obtained from the precipitate by adding water and extracting it with ethyl acetate. The total yield was 55% (.0063 mole); the final melting point is 93-94°C. AnaZysis: calculated—- 12.92% N.;. found-- 12.83% N. The following compounds were synthesized in a manner similar to N-propyl IAAM: »Analysis (%N.) Compound. M.P. . 31222 Calculated . Found N,N-dimethyl IAAm 115-116°1 92% Lit., 116-7° 126-8°# N-N-diethyl IAAm 101° 73% Lit., 101° (19) N,N-dipropyl IAAm 79-80° 71% 10.84 10.79 N-isopropyl IAAM 115° 57% 12.92 12.80 N,N-diisopropyl IAAm 211-213° 83% 10.84 10.75 #Fish, Johnson, and Horning synthesized dimethyl IAAm and reported the melting point as 126-8°C; however, they also reported that several crops of dimethyl.IAAm melted re- peatedly at 116-117°C. The I.R. spectrum of the two, samples with different melting point were identical. Our product was recrystallized to-a constant melting point using aqueous ethanol and also ethyl acetate (18). 17 N,N-dibenzylindol-3fy1acetamide One and five-tenths gram.(.00775 mole) of indol-3- ylacetyl chloride was dissolved in 50 m1. of ether and 3.07 gm. (.0156 mole) of dibenzylamine was dissolved in ten m1. of ether. Both-solutions were.cooled—to ~20°C and then they were mixed. The reaction was stirred for three hours at 0°C. Then 50 m1. of water was added andwthe solution was poured into a round-bottom flask; the ether-was removed by vacuum, leaving crystals in the water solution. The pro- duct was collected on a Bfichner filter.v The crystals were dissolved in hot ethanol, and water was added. ~Three grams (.0846 mole, 100% yield) of colorless prisms formed on cool- ing. Recrystallization was done from.ethyl acetate, the final melting point is 155-156°C. Analysis: calculated-- 7.90% N.; found-- 7.73% N. *The following compounds were preparedrin a manner similar to N,N-dibenzy1 IAAm: Analysis (% N.) Compound M.P. Yield - Calculated . Found N-cyclohexyl IAAm 157° 100% Lit.,-155-6° (20) N—benzyl IAAm 154-5° 100% Lit., 152.5 ~153.5° (21) 18 Analysis (% N.) Compound M.P. Yield Calculated Found N-benzyl—N- 150-1° 93% 10.06 9.92 methyl IAAm N-(2-chlor0r 161-2% 33% 9.38; 9.43 benzyl) IAAm N-(3-chloro- 121° 38% 9.38 9.30 benzyl) IAAm N-(4-chloro- 176-7° 41% 9.38 9.30 benzyl) IAAm N-(2,4-dichoro- 169-7l° 54% 8.41 8.54 benzyl) IAAm N-(3,4-dichloro- 167-8° 55% 8.41 8.46 benzyl) IAAm Indol-3-y1acet- 153-4° 100% Lit.,l49.5 anilide -150.0° (21) N-methylindol-3- 134-5° 53% 10.60' 10.57 ylacetanilide N-(2-chloro- ll7-8° 45% 9.84 9.98 phenyl) IAAm N-(3-chloro- 98° 48% 9.84 9.90 phenyl) IAAm N-(4-chloro— 170-2° 67% 9.84 9.88 phenyl) IAAm N-(2,4-dichloro- 152-3° 54% 8.78 8.91 phenyl) IAAm ' phenyl) IAAm N—(l-naphthyl) IAAm 164 83% 9.33 9.22 19 N,N-diphenylindol-3-ylacetamide A solution of 2.15 gm. (.0111 mole) of indol-3- ylacetyl chloride in 50 m1. of ether was mixed with 3.75 gm. (.0222 mole) of diphenylamine at room.temperature. Unlike the other reactions this reaction is very slow at this temperature; the reaction mixture was left in the dark at room temperature for 48 hours. The ether was de- canted from the crystals and evaporated by vacuum. Both fractions were dissolved in ethanol and combined; water was added and 2.24 gm. (.0068 mole, 69% yield) of crystals were obtained. Recrystallization was done with ethyl ace- tate, the final melting point is l73-175°C. Analysis: calculated—- 8.58% N.; found-- 8.45% N. N-dimethylaminoindol-31y1acetamide One and 59 hundredths of a gram (.0082 mole) of indol—3-ylacety1 chloride was slowly added to a solution of 0.99 gm. (.0165 mole) of 1,1-dimethyl-hydrazine in 150 ml. of ether and 10 ml. of water. The addition was made at 5°C over a period of twenty~minutes; the reaction was stirred for an hour afterward. The ether was separated from the water and was dried over anhydrous sodium sulfate. 20 The ether was evaporated by vacuum leaving an oil. On the addition of methanol a white powder precipitates (0.20 gm., m.p.l41-44°C); the solution was filtered and the methanol was removed by.vacuum. -The oil was dissolved in ethyl ace- tate and petroleum ether was added to a slight cloudiness. After two days at -5°C, five-tenths of a gm. (.0023 mole, 28% yield) was obtained; the final melting-point is 123-4°C. Analysis: calculated-- 19.34% N.;. found-- 17.95% N. Biological Assays Avena Straight Growth (28,29) Solutions: Solutions of 0.01 molaraconcentration of the compounds were made by.dissolving the compounds in 100% ethanol (8.0% final concentration) and diluted to volume with a pH 5 citrate-phosphate buffers: The buffer was prepared from 20 gm. of sucrose, 1.794 gm; of dipotas- sium phosphate, 1.019 gm. of citric acid monohydrate, and one ml. of Tween 80 diluted to one liter with glass di- stilled water. At a 0.01 molar concentrationsmost of the solutions quickly form a milky suspension; the dilutions 21 were made rapidly before precipitation occurred. The fol- lowing concentrations were prepared using the citrate— phosphate buffer and were used in the assay: lO-3M, 10-4M, lO—SM, lO—6M, 10-7M, and 10-8M, The buffer—was used as the control; 0.8% ethanol in buffer has no effect on the assay results. Plant Material: Oat seeds (var.-Torch) were washed in the dark under running tap water for two.hours. They were planted on moist vermiculite and exposed to weak red light for twenty-four hours. They were-then covered with one cm. of moist vermiculite and grown in—the-dark for 48 hours.- At this time 5 mm. segments were cut 5 mm. below the tip, a cutting jig was used to insure uniform sections. The coleoptiles were floated for two hours on a solution of one mg. of magnesium sulfate monohydrate per liter of water; only the coleoptiles still floating at this time were used in the assay. . -3 Treatment: One ml. of each solution (10 M to -8 . . . 10 M) was put in a test tube With ten coleoptiles. The tubes were put in a near horizontal position on a drum ro- tating at one revolution per minute; they were incubated 22 in the dark for 20-24 hours. The coleoptiles were measured by placing them in a-photographic enlarger, the shadows were enlarged five times and their lengths were measured to the nearest mm- Each series contained two -3 -8 buffer controls and an IAA standard (10 M to 10 M). The data are expressed as percent of control growth and are the mean of three replications. Cucumber Root Inhibition (30) Solutions: Solutions of 0.001 molar concentration of the compounds were prepared by dissolving them in a minimal amount of 100% ethanol and diluting them to volume with glass-distilled water. From these solutions the fol- lowing concentrations were prepared with glass-distilled 514.: 10'6M. 10492. and-1078M. When the water: 10-413, 10" ethanol in the 0.001 molar solution exceeded-0.8%, ethanol solutions were included-with the controls, which were glass- distilled water. Eight-tenths-of a percent of ethanol had no measurable effects on-the results-of the assay. Plant Material and Treatment: Cucumber seeds were germinated in the dark for twenty-four hours on filter 23 paper moistened with distilled water. Ten seeds with uni- form radicals were placed on a filter paper—in each Petri dish with 4.5 ml. of the solution to be tested.- The dishes were placed in the dark at 25°C, after 48 hours the root length was measured to the nearest mm. Two water blanks and a series of-IAA standards were included in each run. The data is-the mean of three replications and is expressed as percent inhibition of control. Cucumber Epicotyl Curvature (29) Solutions: Solutions of 0.001-molar concentration were prepared by dissolving the compounds in five m1. of 100% ethanol and diluting to 25 ml. with a 1.25% Tween 80 water solution. Plant Material: Cucumber seeds were—grown on moist vermiculite for nine days under fluorescent lights in the laboratory. The plants were then removed from the vermicu- lite and their roots were washed free from non-plant ma- terial with distilled water. They were~c1amped between two strips of balsa wood (1/8" x l/8'-x 12“)-held together by rubber bands cut from rubber tubing. The roots were 24 placed in a 1/3 Hoagland's nutrient solution; they were allowed to equilibrate for at least two hours before treatment. Treatment: The initial angle of the stem was measured with a protractor equipped with acrotating wire. Ten microliters of the 0.001 molar solutions-were placed in the center of one of the cotyledons, aftervthree hours the curvature was-again measured in degrees.--The control plants were treated with 0.001-M IAA solution;r20% ethanol in one percent Tween 80 does not cause any curvature. The ethanol lessens-the.response»of*IAA.by.1ess-than ten per- cent. ~The treatmentrconsists-of-four~rep1ications of five plants each; the data are the mean and are reported as per- cent of IAA curvature. Metabolic-Study4(24), Oat seeds were soaked and grown-under-red light as for the Avena assay. They were covered withomoist vermicu- lite and grown for 52 hours atrZSPC in thesdarkit Then, one cm. sections were cut.five mm. below the tip,~100 of these 25 sections were floated on 50 m1. of a 10-4M solution (0.8% ethanol) made with glass-distilled water.--Controls included tissue incubated in 50 m1. of glass-distilled water and 50 ml. of the solutions incubated without tiSSHE$v The tissue were incubated in the dark for 48 hours at 25°C, then, they were frozen overnight. The next day they were thawed, the tissue was removed, ground to a paste, and recombined with the solution. The pH was adjusted to 12.00 with sodium hydroxide; the solution was extracted with 508ml. of ethyl acetate. The pH was raised to 2.50 with-sulfuric acid and the solutions were.extracted three times with 50 m1. por- tions of ethyl acetate.- The ethyl acetatesfrom the acidic extractions was dried over anhydrous sodium sulfate and evaporated to dryness by vacuum. The residue was taken up with a small amount of ethyl acetate and-was spotted on a TLC plate (Merk,-Silica Gel, F-254, 0.25 mms):- The plate was chromatographed in all glass tanks with n-butanol, ammonia, and water (100:3:18,v/v). Afterrthexsolvent had moved 15 to 20 cm., the plateswas air driedsand sprayed with Ehrlich's~reagent-(1%-pedimethylaminobenzaldehyde in 50% alcoholic HCl). 26 When the chromatographs were to be examined by bio- assay, they were prepared and treated in the same way ex- cept they were not sprayed with Ehrlich's reagent. Instead, a section of the silica corresponding to the location of IAA was scraped off of the plate and placed in-a test tube. The section included 1.25 cm. either side ofra point cor- responding to the Rf of the standard of IAA which was chromatographed on the same plate. Five m1. of the ézggg buffer was added to the tubes containing the-silica. One ml. of this solution and one m1. of a one-insten dilution were assayed in the 52223 straight growth bioassay. These assays included IAA standards and.buffer controls. RESULTS - AND- DISCUSSION RESULTS AND DISCUSSION Effects of Alkyl Substitution The alkylated derivatives of indol-3-ylacetamide exhibit a wide range of activity. The results of the 53222 straight growth assay are summarized in Figure I. IAAm itself has moderate activityain this assay; methyl, ethyl, propyl, or cyclohexyl substitution reduces the activity of the amide. The isopropyl derivative has ac- tivity about equal to IAAm. The addition of chlorine to the ethyl or propyl derivatives greatly increases the ac- tivity of these compounds. Dimethylamino IAAm-has activ- ity which-is much greater than IAAm. The di-substituted methyl and ethyl derivatives are inactive, while the di-substituted propy1~and isopropyl compounds are more active than IAAm. In therroot inhibition assay, summarized in Table I, IAAm shows significant activity at IOIAM and lO-SM concen- trations, the two chlorinated compounds anva-cyclohexyl 27 28 Figure I.--Growth Curves of Avena Coleoptile Sections Effects of Alkyl Substitution—- 1) IAA, 2) IAAm, 3) N-methyl IAAm, 4) N,N-dimethyl IAAm, 5) N-ethyl IAAm, 6) N,N-diethy1 IAAm, 7) N-(2-chloro- ethyl) IAAm, 8) N-propyl IAAm,A 9) N,N—dipropyl IAAm, 10) N-(3-chloropropyl) IAAm, 11).N-isopropyl IAAm, 12) N,N-diisopropyl IAAm, 13) N-cyclohexyl IAAm, and 14) N-dimethylamino IAAm. 29 zoo» 150» 100» 50 '- du- , J 4v 3 i :7 : ; : + : i 1 : : 0 b I O 200 *- dr- M 0 b ‘E o m 0 ’50 _ dr- $’ II 100» 1 l3 s o — q- l 1 J 1 1 1 1 1 1 1 10" IO“ 10" 10" lo" 10“ Molar Contchtros-t \on FIGURE I 30 TABLE I. ROOT INHIBITION ASSAY--ALKYL DERIVATIVES 10 M 10 M 10 M 10 M 10 M IAA 66 19 6 4 -l 11 IAAm 51 20 3 1 3 6 N-methyl IAAm 23 4 2 -4 3 6 N,N-dimethyl IAAm 12 -4 -l -3 3 6 N-ethyl IAAm 9 -5 O -2 -3 ll N,N diethyl IAAm l4 5 2 l -2 11 N-(2-chloro- 73 31 15 8 1 11 ethyl) IAAm N-propyl IAAm 15 -4 -4 2 12 ll N,N-dipropyl IAAm 20 -3 -8 2 -2 11 N-(3-chloro- 60 29 4 -5 O 11 propyl) IAAm N-isopr0py1 IAAm 19 l 4 2 -3 10 N,N-diisopropy1 IAAm 15 3 6 2 4 10 N-cyclohexyl IAAm 32 11 1 1 5 10 #This level is significantly different from zero at the 99.5% confidence level. 31 IAAm are the only other compounds to cause~significant in- hibition of root growth at the-10-5M_concentration. The activity of the alkyl derivatives-in the cucum- ber epicotyl curvature assay parallels their activity in the £2223 assay (Figure II). There are two notable excep- tions, both IAAm and N,N-diisopropyl IAAm-exhibit less ac- tivity in this assay than would be expected from their ac- tivity in the éygga assay. N-cyclohexyl IAAm, which is active in the inhibition of cucumber root elongation, does not exhibit activity in this assay. Effects of Phenyl Substitution The addition of an aromatic nucleus—next to the amide nitrogen of IAAm greatly increases—the auxin activ- ity of these compounds. The results of the A2223 assays are given in Figure III. Except for N,N-dipheny1 IAAm and N-methylindol-3-y1acetani1ide the activity of these compounds is greater than IAAm and in some cases it sur- passes the activity of IAA at low concentrations. The diphenyl IAAm is unusual in that its response is 32 Figure II.--Results of Cucumber Epicotyl Curvature Assay The curvature induced by each compound is pre- sented as percent of the curvature induced by IAA. Solid bars represent values significantly different from zero with 99% confidence. With the exception of IAA and IAAm, the compounds are designated by-the substitution on the amide nitrogen of IAAm. 33 Curvature- % of IAA (0 .a o- O> 0’ (o '0 l 1 r IAA IAAm methyl- dimethyl - ethyl diethyl I chloroethyl prepyl diprOpyl chlorOprOpyl isopropyl diisoprOpyl cyclohexyl III dimethylamino phenyl diphenyl N-pnenyl-Nemethyl 2-chloropheny1 3-chlor0pheny1 4-chlorophenyl 2,4-dichlor0pheny1 2,5-dichlorophenyl 1-naphthy1 benzyl- di benzyl — N-benzyl-N-methyl 2-chlorobenzy1 3-chlorobenzyl — 4-chlorobenzyl 2,4-dichlorobenxy1' 3,4-dichlorobenzy1 FIGURE II. a¢TO filNDI 34 Figure III.--Growth Curves of Avena Coleoptile Sections Effects of Phenyl Substitution-v-l) IAA, 15) indol-B-ylacetanilide, 16) N,N-dipheny1 IAAm, 17) N-methylindol-3-ylacetanilide, 18) N-(2-chlor0phenyl) IAAm, l9) N-(3-chlorophenyl) IAAm, 20) N-(4-chloro- phenyl) IAAm, 21) N-(2,4-dichlorophenyl) IAAm, 22) N-(2,5-dichloropheny1) IAAm, and N-(l-naphthyl) IAAm. 35 200 I50 [00 5O 200 ISO °/o Con‘H-ol Growth [00 so— ~~ 1 J l L 1 l 1 10" lo" 10" In" Molar Concentrahon FIGURE III . IO " Io" 36 significantly below the control level. Four of the five chlorinated compounds have activity that is greater than 150% of control at the lo-gM concentration. (In the mono- chlorinated compounds the position of the chlorine has little effect, but—in the dichloro derivatives the posi- tion of the chlorine does affect activity. The lack of inhibition at the 10-3M concentration is probably due to the fact that many of these compounds precipitated from solution at this concentration. The increased-activity of the phenyl derivatives also occurs in the root inhibition assay,‘the-data is pre- sented in Table II. Again N,N—diphenyl IAAmmand N-methyl- indol-3-ylacetanilide have—comparatively-low activity, surprisingly N-(2,5-dichlorophenyl) IAAm is inactive in this assay. All of the other compounds cause significant inhibition at the 10-6M concentration; their-activity is generally greater than IAA. The position-of the chlorine in the mono-chlorinated compounds has-little affect in this assay. 37 TABLE II. ROOT INHIBITION ASSAY--PHENYL DERIVATIVES Percent Inhibition Compound -4 —5 -6 -7 —8 Level Of 10 M 10 M 10 M 10 M 10 M Significance# IAA 65 21 12 5 5 9 Indol-3—ylacet- 84 69 38 8 3 8 anilide N,N-dipheny1 IAAm 3 7 3 4 3 8 N-methylindol-3-yl 37 10 7 O 1 8 acetanilide N-(2-chloro- 83 71 32 10 -4 9 phenyl) IAAm N-(3-chloro- 82 55 11 4 2 9 phenyl) IAAm N-(4-chloro- 81 74 24 4 -8 9 phenyl) IAAm N-(2,4-dichloro- 78 64 26 6 -1 9 phenyl) IAAm N-(2,5-dichloro- -8 2 1 -2 -3 9 phenyl) IAAm N—(l-naphthyl) IAAm 62 63 21 7 -3 11 #This level is significantly different from zero at the 99.5% confidence level. 38 In the curvature assay, N,N-dipheny1 IAAm once again has low activity. However, N-methylindol-3-ylacet- anilide has much more activity than would be expected. Also unexpected is the low activity of N~(3-chloropheny1) IAAm, apparently the-chlorine in the three position blocks the transport of this compound. The other~compounds elicit responses which parallel those of the Avena assay. Effects ofrBenzyl Substitution The placing of a methylene bridge~between the aro- matic ring and the amide nitrogen of IAAm greatly reduces the activity of these compounds. This reduced-activity is evident in the Mzggg growth curves, Figure IV. Only two compounds have significant activity at concentrations below 10-3M. These are N-benzyl-N-methyl IAAm and N-(4-choro- benzyl) IAAm. In the-benzyl derivatives the-position of the chlorine does alter the pattern of what-little activity that there is. 39 Figure IV.--Growth Curves of Avena Coleoptile Sections Effects of Benzyl Substitution-- l) IAA, 24) N-benzyl IAAm, 25) N,N-dibenzyl IAAm, 26) N-benzyl-N- methyl IAAm, 27) N-(2-chlorobenzy1) IAAm, 28) N-(3- chlorobenzyl) IAAm,- 29) N-(4-chlorobenzy1) IAAm, 30) N-(2,4-dichlorobenzyl) IAAm, and 31) N-(3,4-dichloro- benzyl) IAAm. °/o Control Gr north 200 I50 [00 50 4O Molar C. oncerrhrahon FIGURE IV. T I r l I I I h— +- '- q)- 29 \ V 2 7 28 L 5 _ I 1 L 1 l 1 1 + + : : : i lo" 10" to" zoo» ’ ISO #- SI 30 I00 '- 0 so - \ I 1 L J l lo" 10" 10'4 41 The results of the root inhibition assay are given in Table III, the same low level of activity is observed again. N-benzyl IAAm is active as well as the 3-chloro and 4-chloro derivatives. Only N-benzyl IAAm has signifi- cant activity at lO-IM concentration. The position of the chlorine is also important-in this assay. In the curvature assay an approximate parallel can again be drawn-to the Avena assay. Metabolic Study. The chromatographs, which were developed by Ehr- lich's reagent, showed spots corresponding-to IAA in all solutions incubated with tissue, including the water con- trols.- None of the solutions incubated without tissue showed any trace of IAA. The spots were~s1ight1y larger and darker in the solutions which contained an amide than those from the water control. However, it was impossible to determine the relative amounts of IAA-present from . these plates. 42 TABLE III. ROOT INHIBITION ASSAY--BENZYL DERIVATIVES _ Percent Inhibition Level of Compound 10-412- lO-SM 10-6fl 10-7£1 10-8£ S1gn1f1cance# IAA 52 16 9 -2 -10 8 N-benzyl IAAm 55 12 0 -6 -l 8 N,N-dibenzyl IAAm 6 1 4 O l 8 N-benzyl-N-methyl IAAm 0 3 1 —3 —1 8 N-(2-chloro- 7 8 1 1 3 11 benzyl) IAAm N-(3-chloro- 27 l l 3 4 11 benzyl) IAAm N-(4-chloro- 15 6 1 4 0 11 benzyl) IAAm N-(2,4-dichloro— 9 2 5 -1 -5 ll benzyl) IAAm N-(3,4—dichloro- O 1 0 -2 -2 11 benzyl) IAAm #This level is significantly different from zero at the 99.5% confidence level. 43 The results of the biological-examination of the plates are given below. TABLE IV. RESULTS OF METABOLIC STUDY Response of Avena Assay (% control growth) Treatment of Tissue - Originaleluant 1:10 Dilution Water 65 98 IAAm 79 142 N-methyl IAAm 29 111 N,N-dimethyl IAAm 93 108 N-(Z-chloroethyl) IAAmu 75 196 N-(3-chlorophenyl) IAAm 90 158 N-dimethylamino IAAm 89 156 The original eluant seems to contain some impurity which obscured the assay results; perhaps this was a trace of the solvent which was absorbed on the silica. The dilu- tion evidently lessened the effects of the impurity so that the relative amounts of IAA could be determined. 44 Discussion In these amides there does not appear to be any correlation between steric hinderance about the nitrogen and the activity of the amide. In N,N—dipropy1 IAAm and N,N-diisopropyl IAAm the amide nitrogens are very hindered sterically, yet these compounds are more active than IAAm whose nitrogen is completely unhindered. Another compound, N-benzyl-N-methyl IAAm, also has very large groups surround- ing the nitrogen; this compound also has more activity than IAAm. This would seem to indicate that the active center of these compounds is not the amide nitrogen but is at least one atom removed. A significant correlation can be found between the pKa of the free primary amines and the activity of the cor- responding amide in the 52233 assay. Figure V is a plot of the response in the 52222 assay at lO-SM concentration ver- sus the pKa of the free amine (31). The pKa is the negative logarithm of the equilibrium constant defined by the following equation: K _ [B:1[ n+1 ‘ [BH+1 45 Figure V.--Correlation of pK with fizggg Straight Growth Activity A plot of the response of the amides in the Mygga assay at the lO-SM concentration versus the pKa of the corresponding amines. The points are labeled such that the amide is designated by the substitution on the nitrogen (eg. 2,4-0-C12 means N-(2,4-dichloro- phenyl) IAAm). The line was determined by a least squares analysis, the slope is 8.92; the intercept is 204. 46 V\ 05ft or: no cvl Nx \\ Ox 0 0 h 0 h. _ d . _ q _ a m ”PVOFfi o , rid} .. 0450 o . 13..., inside . 1:10 . —>‘..r‘ .3 O I»)!!! o (g. . 01,336 7:10.: .w 0 V — n. . Mn. 00 00\ ONs OVx OWx 00\ CON H+W°J9 I°J+V°D ‘79 FIGURE V. 47 The pKa can be used as a measure of the electron density around the nitrogen in the amine. A high pKa indicates that the amine is a strong base, that is, the nitrogen is able to easily donate its electrons to bind a proton. Electron donating groups such as alkyl groups strengthen the basicity of the amines. A low pKa indicates a weak base, the electron density at the nitrogen is lowered by electron withdrawing groups. Phenyl groups are electron withdrawing by induction, chlorine is also highly electro- negative. Figure V clearly shows the relationship between pKa and activity, the pKa's of chloroethylamine and chloro- propylamine could not be found; however, from inductive effects, their pKa's would be expected to be higher than the parent amines. This would correspond to the higher activity of these compounds in the bioassays. Since inductive effects are short range, extending only over two or three atoms, it is obvious that the ef- fects are probably altering the character of the carbonyl of the amide. 48 Amides can be stabilized by resonance between forms (I) and (II). _ e o '0' R-t-fi-R’ +--+ R-¢=§-R’ _ 'R” R4 (I) (II) This effect is well known to protein chemists; it plays an important role in the structure of polypeptides. This type of resonance probably occurs in all amides to some degree, depending on the type of N-substitution. Form II would be stabilized by electron-donating groups and destabilized by electron-withdrawing groups. This seems to indicate that the most active amides are those where resonance stabiliz- ation is minimal, and the electrons are pulled away from the carbonyl by electron-withdrawing functions. The results of the metabolic study clearly indicate .what the mechanism of action of the amides is and why the effect of pKa is so pronounced. When the chromatographic plates were examined using a bioassay it was possible to quantitate the IAA which was extractedand to correlate it to the activity of the com- pounds. Figure VI shows that there is a very high degree 49 Figure VI.--Correlation of IAA in Tissue Extract with the Activity in the M3333 Assay A plot of the amount of IAA extracted from 53222 tissue, which had been incubated in amide so- 1utions, as a function of the activity of the amide in the 53223 assay at 10-4M concentration. The re- sponse of an M1223 bioassay is the measure of the amount of IAA. The points are labeled as in Figure V. 50 0‘.£6J>J*It . w 0 JrabLn aOL+£Od 0‘0 I 11750$£OU «+0 >*.>.+d< OON OO\ 00‘ 0.x Oux _ q q . . $140,! .1 o OO\ 09 1 . 00 QOx ONx OVx OO\ OQs OON W*M°Jg ‘oaiuoo o/o .. 15161113 thsu‘i \M VV' FIGURE VI . 51 of correlation between these two variables. It is obvious then, that the amides of IAA are not active per se, but their activity is derived fron1the hydrolysis of the amide to IAA. The apparent activities would be a function of two rates; first, the rate of entry of the amide into the cell, secondly, the rate of hydrolysis of the amide to the acid. Since these amides are all rather non-polar com- pounds, their diffusion into the cell would probably be a fairly rapid process. The activity limiting factor would Seem to be the rate of hydrolysis. This would agree with the correlation of pKa to activity; the active center in the hydrolysis step is the carbonyl functionality, not the nitrogen. The development of a partial positive charge on the carbonyl would greatly increase the susceptibility of the amide‘to hydrolysis. The high activity of the chlorophenyl IAAm com- pounds can be readily explained by this model. If the assumption is made that the diffusion of the non-polar. amides is more rapid than the diffusion of IAA, which is a polar molecule because of its acid functionality, and 52 if the hydrolysis of the chlorophenyl IAAm is a rapid pro- cess, then the concentration of IAA will be greater in the cells exposed to the amide than the cells exposed to the same level of IAA. This effect would be especially notice- able at low concentrations where the rate of diffusion of IAA approaches the rate of destruction of IAA by metabolic processes. The activity of the dimethylhydrazide of IAA might not be completely explained by the hydrolysis theory. In both Figure V and Figure VI this compound deviates substan- tially from the line. In both cases the activity is higher than that predicted by the plot. It is possible that this compound has a different mechanism of action. The most interesting compound is N-(3-chlorophenyl) IAAm, while it exhibits high activity in both the 53222 assay and the root inhibition assay it has very little ac- tivity in the cucumber curvature assay. Either it is not transported or it blocks the transport of the IAA produced. Whatever the mechanism, it might have valuable applications in agriculture. Not only is it active at extremely low concentrations, but its effects can be localized to one 53 section of the plant where it is applied. Further tests would be needed to determine whether this is a general phenomenon and to determine whether the compound is toxic. S UMMARY S UMMARY A series of indol-3-y1acetamides were synthesized and the physiological activity of these compounds in plants was determined. The amides were prepared by the reaction of indol-3-ylacety1 chloride with the appropriate amines. A list of the amides follows: N-methylindol-3-y1acetamide N,N-dimethylindol-3-ylacetamide N-ethylindol-3-ylacetamide N,N-diethylindol-3-ylacetamide N—(2-chloroethyl)indol-3-y1acetamide N-propylindol-3-y1acetamide N,Nédipropylindol-3-ylacetamide N-(3-chloropropyl)indol-3-y1acetamide N-isopropylindol-3-y1acetamide N,N-diisopropylindol-3-ylacetamide N-cyclohexylindol-3-ylacetamide N-dimethylaminoindol-3-ylacetamide indol—3-y1acetanilide N,N-diphenylindol-3-ylacetamide N-methylindol-3-y1acetanilide N-(2-chloropheny1)indol-3-y1acetamide N-(3-chlorophenyl)indol-3-y1acetamide N-(4-chlorophenyl)indol-3-ylacetamide 54 55 N—(2,4-dichlorophenyl)indol-3-ylacetamide N-(2,5-dichlorophenyl)indol-3-y1acetamide N-(l-naphthyl)indol-3-y1acetamide N-benzylindol-3-ylacetamide N,N—dibenzylindol-3-y1acetamide N-benzyl-N-methylindol-3-ylacetamide N-(2-chlorobenzy1)indol-3-ylacetamide N-(3-chlorobenzyl)indol-3-ylacetamide N-(4-chlorobenzyl)indol-3-ylacetamide N-(2,4-dichlorobenzyl)indol-3-ylacetamide N-(3,4-dichlorobenzyl)indol-3-ylacetamide The method of synthesis, the yield, and the melting point are given for each compound. A nitrogen analysis was used to characterize those compounds that were not re- ported in the literature. Biological assays were used to characterize the physiological activity of the compounds. Three bioassays were used: the Mygga straight growth, the cucumber root inhibition, and the cucumber epicotyl curvature assay. The N-alkyl amides displayed a wide range of activity; the most active of these is N-(2-chloroethyl) IAAm. The N-phenyl derivatives generally had very high activity;- N-(3-chlor0pheny1)IAAm was unusual in that it was highly 56 active at extremely low concentration in the M2233 and root inhibition assays but it was relatively inactive in the cucumber curvature test. These properties might make this compound useful in agriculture. Most of the benzyl deriva- tives had very low activity in all three assays. A correlation was found between the pKa's of the free primary amines and the Avena activity of the corre- sponding amide. No correlation was readily apparent be- tween activity and steric effects. A metabolic study showed that these compounds are hydrolyzed to IAA, a correlation was found between the amount of hydrolysis and the activity of the amide. It was postulated that the amides derive their activity from their conversion to IAA; N-dimethylamino IAAm may be an exception to this mechanism. A model was given to explain the high activity of the chlorophenyl derivatives. REFERENCES 10. 11. 12. REFERENCES Seeley, R. C., Fawcett, C. H., Wain, R. L., and Wight- man, F., The Chemistryéand Mode of Action of Plant Growth Substances, Wain, R. L. and Wightman, F. (eds.), Butterworth Sci. Publ., London (1956). P. 234. Alder, E. F., Wright, W. L., and Soper, O. F., Proc. Northeast. Weed Control Conf. 12,69 (1961). Swithenbank, C., McNulty, P. J., and Viste, K. L., J. Agr. Food Chem., 12, 417 (1971). Bose, H., Sci. Cult., 24, 464 (1968). Sebanek, J. and Hradilik, J., Biol. Plant., 11, 356 (1969). Jain, M. 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D., Nature, 169, 452 (1952). Sonntag, N. O. V., Chem. Rev., 53, 237 (1953). Leopold, A. C., Auxins and Plant Growth, Berkeley, University of Calif. Press (1955). APPENDIX Chemical Name Indol-3-ylacetic acid Indol-3-y1acetamide N-methylindol-B-yl- acetamide N,N-dimethylindol-3-yl- acetamide N-ethylindol-B—yl- acetamide APPENDIX 60 Structure 9 | CHZCOH /\ 0 Cl I CHZCNHCH3 P} 9 C. I CHZCNCHB fl CH 0 II “H “C ZCNHCHZCH3 E 61 Chemical Name Structure 0 N,N-diethylindol-B-yle ' l CHZENCH CH3 acetamide a CHZCH3 O N-(2-chloroethyl)- ' CH2CNHCH2CH2C1 indol-3-y1acetamide E I O N-propylindol-3-yl- . CH CNHCH CH CH l 2 2 2 3 acetamide H 9 N,N-dipropyl- CH CNCH CH CH indol-B-ylacetamide I 2 ' 2 2 3 B CH CH CH 2 2 3 O N-(3-chloropropyl)- C. CHZ-‘CNHCH CH CH C1 indol-3-ylacetamide 3 2 2 2 62 Chemical Name N-isopropyl— indol-3-y1acetamide N,N—diisopropyl- indol-3-ylacetamide N-cyclohexyl- indol-3-ylacetamide N-dimethylamino- indol-B-ylacetamide- (indol-B-ylacetic acid- 2,2-dimethylhydrazide). Indol—3-ylacetanilide Structure 0 on ENHCHCH 3 Chemical Name N,N-diphenyl- indol-3-ylacetamide N-methylindol-B-yl- acetanilide N-(2-chlorophenyl)- indol-B-ylacetamide N-(3-chlorophenyl)- indol—B-ylacetamide N-(4-chlorophenyl)- indol-B-ylacetamide 63 Structure 8. on on, Cl so CI I c1112 H 11 64 Chemical Name N-(2,4-dichlorophenyl)- indol-B-ylacetamide N-(2,5-dichlorophenyl)- indol-e-ylacetamide N-(l-naphthy1)- indol-3-ylacetamide N-benzylindol-3-yl- acetamide N,N-dibenzyl indol-3-ylacetamide Structure Chemical Name N-benzyl-N-methyl- indol-3-y1acetamide N-(2-chlorobenzyl)— indol-3-ylacetamide N-(3-chlorobenzyl)- indol-3-y1acetamide N-(4-chlorobenzyl)— indol-3-y1acetamide N-(2,4-dichlorobenzyl)- indol-B-ylacetamide 65 Structure C1 C1 Chemical Name N-(3,4-dichlorobenzyl)- indol-B-ylacetamide 66 Structure C1 0 I mCHZCNHCHZ M'Tlfl'lTIflfitfljM!fliflflflffllfllflfljfifluflmfi