$YN‘FHESIS AND P‘HYSIQLQGICAL ACTIVITY OF SEVERAL bSWSTETUTED {NQC'LE‘fi-ACETEC ACEDS AND ESTERS Thesis {3m- Hm Dogma of DH. D. fiECHEGkN STATE URE‘IEESITY Robert: Louis Franklin 1967 {HESIS This is to certify that the thesis entitled SYNTHESIS AND PHYSIOLOGICAL ACTIVITY OF SEVERAL l-SUBSTITUTED INDOLE-B-ACETIC ACIDS AND ESTERS presented by Robert Louis Franklin has been accepted towards fulfillment of the requirements for __.B};Il'__degree ithry “W MESEQQ/ Major profesén' ’ Date 0-169. f. Hiking“. #- Byliclfigan Sq; 3. University :1 —J- I .9 I....‘w---MW’; ' . as]: Z 193*; at??? E z 3- E #24 l’ Q n T I LEE—4. .. ”J 0f 1: cans in; Whic ABSTRACT SYNTHESIS AND PHYSIOLOGICAL ACTIVITY OF SEVERAL 1-SUBSTITUTED INDOLE-B-ACETIC ACIDS AND ESTERS by Robert Louis Franklin Much scientific activity has followed the isolation of indole-B-acetic acid and the discovery of the re8ponse caused by this auxin in plants. Current research is attempt- ing to discover the aSpects of this and similar structures which may be reSponsible for biological activity. The present investigation was undertaken to study the effect on plant growth reSponse when substituents are attached to the nitrogen atom of indole-B-acetic acid. The compounds possessed a range of steric properties and lipophilicities as well as a range of electronic effects. Each of the following compounds was synthesized and biologically assayed: 1-methylindole-3-acetic acid l-ethylindole-B-acetic acid 1gnypropylindole-B-acetic acid 1-;gggpropylindole-3-acetic acid lengbutylindole-B-acetic acid 1-i§grbutylindole-3-acetic acid 1aggggbutylindole-B~acetic acid 1-tert-butylindole-B-acetic acid 1-gppentylindole-3-acetic acid lggrdecylindole-B-acetic acid Ia-vwn-BI-n n. Ultra mente poun: r81a' SQUE apps tior tute 111g act: the Robert Louis Franklin 1ggroctadecylindole-B-acetic acid ethyl 1~2ybenzylindole-3-acetate ethyl 1-pychlorobenzylindole-B-acetate ethyl 1-p¢bromobenzylindole-3-acetate ethyl 1-pgmethy1benzylindole-B-acetate ethyl 1-prmethoxybenzylindole-3-acetate ethyl 1-pynitrobenzylindole-3-acetate methyl 1-BéD-glucopyranosylindole-3-acetate Ultraviolet and infrared spectra were used to supplement ele- mental analysis in the characterization of the preceding com- pounds. The synthesis and characterization of 51 other indole related compounds and their intermediates are also reported. All of the alkyl substituted compounds had a level of activity relative to indole-B-acetic acid of less than 10% in four biological assays. These were: 飧2§_straight growth, buckwheat root inhibition, bean petiole abscission, and tomato ovary growth assays. However, in the cucumber curva- ture assay, 1-i§gypropylindole-3-acetic acid was outstanding in its activity. The physiological response was approximately equal to that of indole-3-acetic acid, but was slower in appearance and much more persistent. A qualitative correla- tion existed between the £2222 activity of the acids substi- tuted with lower alkyl groups and the Taft E8 values, indicat- ing that steric factors are important in decreasing the activity of these derivatives. A methyl group attached to the nitrogen atom lowers the activity normally associated with indole-B-acetic acid to approximately 10%, but larger groups gradually reduce the If" ' 3.7" .u—-— ”L. act: butyl Shows Value bKCkw effec Subst made 88393 a hya that Syste Robert Louis Franklin activity to the point of considerable inhibition. Thus, £333- butylindole-B-acetic acid at 10-” 3 concentration was a strong inhibitor of growth in the Azgna straight growth assay. Several of the substituted benzyl esters had activity approaching that of indole-3-acetic acid in both the 51223 and bean petiole abscission assays. Other tests involving intact plants did not respond well to these derivatives, an indication that they are not easily tranSported through an intact membrane. The benzyl derivatives substituted with halogens showed a qualitative correlation between the Hammett Up values and the activity in both Azggg.straight growth and in buckwheat root inhibition. Factors other than electronic effects may be involved in the reSponse of the different substituted benzyl esters as no obvious correlation could be made between activity and thetrp value of the substituent. Methyl 1-B-D-glucopyranosylindole-B-acetate had essentially no activity in any test as would be expected of a hydrophilic compound. The low activity is good evidence that this N-glucoside is not degraded in any of the plant systems investigated. SYNTHESIS AND PHYSIOLOGICAL ACTIVITY OF SEVERAL 1-SUBSTITUTED INDOLE-BnACETIC ACIDS AND ESTERS By Robert Louis Franklin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1967 Harold ; throng? , by Prof .L J 1081 8C ‘ and Er. assists Hrs. Sh Joan, f the Nat 0f Heal ciated. ACKNOWLEDGMENTS The author eXpresses his appreciation to Professor Harold M. Sell for the guidance and encouragement offered throughout the course of this work. The direction given by Professor Martin J. Bukovac in determination of biolog~ ical activity is sincerely appreciated. Mrs. Magda Emodi and Mr. Abbas Al-Jamali are offered thanks for their able assistance with the biological assays. Thanks are due Mrs. Shirley Randall. Miss Sandra Sandell, and my wife, Jean, for their help in preparing the manuscript. Both the National Science Foundation and the National Institute of Health provided financial support which is much appre- ciated. *************** ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . HISTORICAL O C O O I O O I O O O SYNTHESIS OF l-SUBSTITUTED INDOLES CHEMICAL STRUCTURE AND PHYSIOLOGICAL ACTIVITY BIOLOGICAL ASSAY . . . . . . . . EXPERIMENTAL . . . . . . . . . . Synthesis of Compounds . . lnAlkyl Indolines . . . . . l-Methylindoline . . . i-Ethylindoline . . . l-n-Propylindoline . . 1- iso-Propylindoline . l-n-Butylindoline . . 1-iso-Butylindoline . l-sec-Butylindoline . l-t—eT't-Butylindoline . i-n-Pentylindoline . . l-n-Decylindoline . . l-n-Octadecylindoline i—Alkyl Indoles . . . . . . l-Methylindole . . l-EthylindOle e o 1-n-Propylindole . l-iso-Propylindole i-n-Butylindole . 1-iso-Butylindole -sec-Butylindole 1-‘EEFt-Buty11ndo1e l-nyPentylindole . l-nyDecylindole . 1-3:Octadecylindole HP 111 . O O O O O O O O O C O ' O O O O O O O O O O O O 0000,0000... E1 E1 1- 1.. Ethyl 1-Alky11ndole-3—Acetates . . . . . . . Ethyl diazcacetate . . . . . . Ethyl l-methylindole-B-acetate . . Ethyl l-ethylindole-B-acetate . . . Ethyl i-g-prcpylindole-3-acetate . Ethyl 1-i§gepropylindole-3-acetate Ethyl 1-ggbutylindole-3-acetate . . Ethyl 1- Lso-butylindole-3-acetate . Ethyl 1- sec-butylindole-B-acetate . Ethyl 1- te ert-butylindole-B-acetate Ethyl 1-gypentylindole-3-acetate . Ethyl 1-ggdecylindole-3-acetate . . Ethyl 1-groctadecylindole-B-acetate O C O O O O O O I O O O O O O O O I O O O O O O l-Alkyl IndOle-B-Acetic‘ ACidS o o o o o 1-Methylindole-3-acetic acid . . . 1-Ethylindcle-3-acetic acid . . . . 1-n-Propylindcle-3-acetic acid . . 1~Lso-Propylindole-3-acetic acid . l-n-Butylindole-B-acetic acid . . . -Lso-Butylindole-3-acetic acid . . E—Butylindole-B-acetic acid . . ert-Butylindole-B-acetic acid . -grPentylindole-3-acetic acid . . -grDecylindole-3-acetic acid . . . -gyOctadecylindole-3-acetic acid . O O O O O O O O I O O O O O O O O O C O O O HHHHHH Ethyl IHdOle-B-Aoetate o o o c o o o o o c o Ethyl l-Bara-Substituted Benzylindole-B-Acetates Ethyl l-benzylindole-B-acetate . . . . Ethyl 1-Erfluorobenzylindole-3-acetate Ethyl 1-2ychlorobenzylindole-3-acetate Ethyl 1figpbromobenzylindole-B-acetate . Ethyl 1-Ermethylbenzylindole-B-acetate Ethyl 1~2rmethcxybenzylindole-3—acetate l-E-Substituted Benzylindole-B-Acetic Acids i-Benzylindole-B-acetic acid . . . . 1-ErFluorobenzylindole-3-acetic acid 1-2rChlorobenzylindole-3-acetic acid 1-2yBromcbenzylindole-3-acetic acid . 1-27Methylbenzylindole-B-acetic acid 1-2rMethcxybenzylindole-B-acetic acid 1-(2',3',4',6'-Tetra-Ochetyl-B-D-Gluco- pyranosyl) Indoline . . . . . . . . . . iv fr! {I} 1-(2',3',n',6'-Tetra-O-Acetyl-B-D-Gluco- pyranosyl) Indole . . . . . . . . . . . . . 79 Ethyl 1-(2',3',4',6'-Tetra-0-Acety1-B-D— G1ucopyranosyl)-3-Indole Acetate . . . . . 8O Methyl l-(B-D-Glucopyranosyl) Indole-B-Acetate . 81 igEgSubstituted Benzyl Indolines and Indoles . . 82 l-ErChlorobenzylindoline . . . . . . . . . 83 leg-Chlorobenzylindole . . . . . . . . . . 84 l-E-NitrObenzy11nd01ine o o o o o o o o o o 814’ 1-R-N1tr0benzy11nd013 o o o o o o o o o o o 85 Ethyl 1-2yNitrobenzylindole-3-Acetate . . . . . 85 EpSubstituted Benzyl Indole-3chetates } . . . . 87 E-MGthyleHZYl 1nd01e-3-acetate o o o o o o 87 ‘EgChlorobenzyl indole-3-acetate . . . . . . 88 EpBromobenzyl indole-B-acetate . . . . . . 88 EgNitrobenzyl indole-B-acetate . . . . . . 88 Characterization of Compounds . . . . . . . . . 89 Biological Assays . . . . . . . . . . . . . . . 9O Tomato ovary growth . . . . . . . . . . . . 9O Buckwheat root inhibition . . . . . . . . . 91 Cucumber seedling curvature . . . . . . . . 92 Bean petiole abscission . . . . . . . . . . 93 Avena straight growth . . . . . . . . . . . 94 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 97 Physical Methods . . . . . . . . . . . . . . . . 98 Synthesis . C O O C C O C C . O C C O O C C O O 11 7 Biological Activity . . . . . . . . . . . . . . 138 Effect of 1-alkylation of biological aCt1v1tYooooooooo o coo Effect of para-substitution on the benzyl moiety of ethyl 1-benzylindole—3-acetate 163 . 138 SUMMARY 0 O O O O O O O C O O O O O O O O O O O O O O 1 76 REFERENCES 0 O O O O O O O O O O O O O O O O O O O O 1 8 0 APPENDIX 0 O O O O O O O O O O O O O O O O O O O O O 1 8 8 Table Physical Data Physical Data Physical Data Acetate . . . Physical Data ACidS o o o 0 Physical Data LIST OF TABLES for l‘Alkyl INdOlines o o o i-Alkyl Indoles . . . . Ethyl l-Alkyl-Indole-B- iqukyl-Indole-B-Acetic Ethyl-i-Eara-Substituted Benzylindole-B-Acetates . . . . . . . . . Physical Data for 1-2ara-Substituted Benzylindole-B-Acetic Acids . . . . . . . Physical Data for Several Indole and Indoline Intermediates . . . . . . . . . Physical Data for para-Substituted Benzyl IHdOle-B-Acetates o o o o o o o o o o o 0 vi 101 103 105 127 129 131 133 r a. 10 11 12 13 11+ 15 Figure 10 11 12 13 14 15 LIST OF FIGURES Infrared Spectra of Indoline and 1-nrDecy1— 1nd011ne o o o o o o o o o o o o o o o o o o o 107 Infrared Spectra of Indole and 1-nyDecyl- 1nd013 o o o o o o o o o o o o o o o o o o o o 109 Infrared Spectra of Ethyl Indole-3-Acetate and Ethyl l-nrDecyl Indole-3-Acetate . . . . . 111 Infrared Spectra of Indole-3-Acetic Acid and legrDecylindole-3-Acetic Acid . . . . . . 113 Infrared Spectra of ppChlorobenzylindoline and BfChlorObenzylind01e o o o o o o o o o o o 123 Infrared Spectra of Ethyl 1-27Chlorobenzyl- indole-3chetate and ppChlorobenzyl Indole- BHAcetate o o o o o o o o o o o o o o o o o o 125 Infrared Spectra of Glucosyl Derivatives . . . 135 Tomato Fruit-Set Assay, Effect of 1~A1kyla- tion 0 o o o o o o o o o o o o o o o o o o o o 139 Tomato Ovary Abscission Assay, Effect of 1-AJ—kylation I O O O O O O O O O O O O O 0 O O 1 “'1 Bean Petiole Abscission, Effect of l-SUbStitUtion o o o o o o o o o o o o o o o o 143 Buckwheat Root Inhibition Assay, Effect of 1-sub8t1tUtlon o o o o o o o o o o o o o o o o 145 Buckwheat Root Inhibition Assay, Effect of lqukYIatlon o o o o o o o o o o o o o o o o o 1u8 Cucumber Curvature Assay, Kinetics of Curvature o o o o o o o o o o o o o o o o o o 150 Cucumber Curvature Assay, Kinetics of curvature O O O O O I 0 O O O O O O O 0 O O O 152 Avena Straight Growth.Assay, Effect of lézlkylation o o o o o o o o o o o o o o o o o 157 vii 20 Figure Page 16 Avena Straight Growth Assay, Effect of ylation C C C C O O O O C O C C O O O O O 159 17 ‘Avena Straight Growth.Assay, Effect of 1-Imylationooooo‘oooooooooooo 161 18 Avena Straight Growth Assay, Effect of para Substitution . . . . . . . . . . . . . . 166 19 Correlation of Taft's ES Values with Avena Straight GrOWth ACtiVlty o o o o o o o o o o o 168 20 Tomato Ovary Growth Assay, Effect of para SUbStltutionooooooooooooooooo 172 viii IN TRODUCT I 0N “L 4" A. . . “fl._tl~lh W—S INTRODUCTION In higher plants indole-3-acetic acid exerts control over a variety of essential functions such as flowering, apical dominance, fruit-setting, and root extension. Obvi- ously, the existence of indole-3-acetic acid is of great importance in the coordination of plant growth. There exists a pressing need to learn the mode of action of plant growth regulators, both natural and synthetic, before fur- ther advances can be made in the intelligent use of these substances for controlling the size and yield of plants. The control of plant growth presents man with two contra- dictory challenges: first, plant growth must be encouraged to meet the demands of the rapidly eXpanding population: and second, plants that interfere with beneficial plants must be selectively destroyed. Therefore, both growth- stimulating and growth-inhibiting compounds are of impor- tance. Moreover, the problem is also of interest in under- standing the mechanism by which a chemical signal or hormone can coordinate and control diverse cellular functions. Ramifications of the solution to this problem extend through- out biochemical systems and encompass vital functions not well understood in biology and medicine (17, 28, 111). In animal systems, a given hormone usually acts on a specific target organ; however, in plants, a hormone may '« ‘HK "-01.41 4" N-‘éZI' 3 elicit responses in a number of different organs. Consider- able interplay exists between the known plant regulators as they often exhibit synergism in their action (107). Such phenomena complicate the study of plant hormones and neces- sitate the use of several different bioassays in an attempt to isolate the diverse reSponses in cellular activity. While indole-3-acetic acid may not be the chemical entity that functions at each site which reSponds to the stimulus of this and related substances, the fact remains that a multitude of biological responses can be elicited by these compounds. Therefore, as one of the compounds most deeply implicated in growth responses, indole-3-acetic acid is a worthy candidate for chemical modification and observa- tion of the changes wrought by this alteration. Thus, the quest continues for new compounds which are more active than the parent acid as well as those which will open new avenues of research into the mechanism by which molecules may inter- act intermolecularly or intramolecularly and with subcellu- lar organelles. Studies by Bitzert, Sell and Bukovac (81) have shown that ethyl indole-B-acetate bearing a para-substituted benzoyl group attached to the nitrogen has a reasonably high degree of activity in tomato fruit-set and Aggng straight growth assays. However, the question remains unanswered as to whether this compound acted pg;_§§_or was degraded to an active compound. Although the benzyl radical is similar in steric F'" _ '4". ' IVA-'1'“ Y ngfla Lt requirement to the benzoyl radical, the latter is more sus- ceptible to hydrolysis. In this work, substituted benzyl derivatives of ethyl indole-B-acetate were made by a known synthetic route (65) which would yield a compound approxi- mately isosteric with the benzoyl analog. Porter and Thimann's (75, 76) hypothesis that the degree of auxin activity is proportional to the extent of partially posi- tive charge on the indolic nitrogen, leads one to eXpect that benzyl derivatives would not exhibit as high a degree of activity as the aroyl compounds of Ritzert gt 2;. (81). The activity of the 1-aroyl derivatives is enhanced by the additional electron withdrawing effect of the amide carbonyl. An investigation was made to determine whether or not benzyl compounds have significant activity and whether or not such activity could be correlated with the Hammett sigma constants of the substituents attached to the benzyl ring. Hansch ggflgi. (27) indicated a correlation of lipo- philicity with activity in both plant growth regulators and in derivatives of the antibiotic chloromycetin. Mitchell and Linder (67) demonstrated that d-methoxylation and N-acetylation enhance absorption and translocation of the derivatives in comparison to indole-3-acetic acid. Alpha- methylation of indole-3-acetic acid was shown to enhance the fruit-setting activity of tomato plants (9). Stowe and his coworkers (72, 9h, 95, 96) demonstrated a growth reSponse which can be attributed to long-chain fatty acid esters and related compounds. The reSponse is 5 most obvious in pea plants and is usually seen as a synergism involving the lipid as well as indole-3-acetic acid and gibberellic acid. These considerations as well as the obser- vation of Baskakov and Mel'nikov (u) that i-ethyl and 1-propyl indole-3-acetic acids are active in bean root growth add impetus to the hypothesis that 1-alkyl derivatives of indole- 3-acetic acid are worthy of further study. A series of i-alkyl derivatives of indole-3-acetic acid were prepared which exhibit a range of steric requirement as well as a range of lipophilicity while maintaining a fairly constant electronic effect. These analogs allow an evalua- tion of the effect of steric bulk and lipophilicity of the group on activity unimpeded by electronic considerations. Diametrically opposed in solubility to the lipophilic alkyl groups lies the hydrophilic glucosyl moiety. There- fore, the methyl ester of 1-B-D-glucopyranosylindole-3-acetic acid was synthesized and analyzed. The series of 1-derivatives of indole-3-acetic acid in this investigation exhibit a Span of solubility ranging from the highly lipophilic to the highly hydrophilic. The range of steric requirement encompasses carbon chains from one through eighteen atoms and includes two different ring sys- tems. Electronic variations were induced in the indole ring by substituting the benzyl moiety with groups ranging in electronic effects from nitro to methoxy. An attempt was made'to eXplain the observed biological effects of these derivatives with their structure. 6 In addition to the physiological considerations and their relationship to structure, part of this work was con- cerned with synthesis. A novel route for the synthesis of 1-substituted indole-3-acetic acids is reported which involves a sequence of known syntheses. Several different pathways were investigated for the synthesis of similar com- pounds. The use of dichlorodicyanobenzoquinone was found to be generally favored over chloranil for aromatization of the indoline nucleus. The synthesis of glycosyl indole compounds is of interest as a possible model for other biochemically important indole analogs. HISTORICAL acid ; Cant . HISTORICAL Indole-3-acetic acid was first considered as a con- stituent of higher plants in 1909 (31) when Herter suggested its presence. It remained for Haagen-Smit and coworkers (23) to isolate and characterize the crystalline compound from immature §§§_M§y§ kernels in 1946. However, Kggl, Haagen- Smit and Erxleben (50) had shown the importance of the acid in plant growth in 1934, when they isolated this crystalline material from human urine. The isolated indole-3-acetic acid was then shown to cause cell elongation in Azgng coleop- tiles. Many instances of the occurrence of indole-3-acetic acid in higher plants have been reported (7). It is signifi- cant that evidence for its presence is almost always based on chromatographic data and bioassays. Indole compounds, that can be degraded to indole-3- acetic acid, have also been reported. In 1944, Larsen (55) reported a compound that was tentatively identified as indole-B-acetaldehyde. Proof of this tentative structural proposal was later presented by the same investigator (56) from evidence gathered through comparison studies with authentic indole-3-acetaldehyde. This neutral growth sub- stance was isolated from etiolated epicotyls of beans, peas, and sunflower plants. L ’-‘!‘r:_.m ‘3 ji'rmig .. Sui-3c- fivh' 0? th tiOna fOr t Droiu & theSe 9 In 1953, Henbest, Jones, and Smith (30) isolated a substance that exhibited the same infrared Spectrum as did authentic indole-3—acetonitrile. Both the aldehyde and the nitrile probably owe their activity to their degradation to indole-3-acetic acid AE.E£EQ (54, 102, 122). Acidic analogs of indole-3-acetic acid have also been reported. These include indole-3—carboxylic acid, which has been shown to be present in plants such as pea, tomato, and Brassicae (18). Linder and coworkers (59) presented tenta- tive evidence for the presence of B-indole-3-propionic acid in white cabbage and Brussel sprouts. Potato tubers during the dormancy period have been shown to contain.XLindole-3- butyric acid (8). Recently, Zenk (127) has reported the isolation and characterization of a carboxy-bound glucose derivative of indole-3-acetic acid. Andreae and Good (1) found a similar compound linked through the indole-3-acetic acid carbonyl to the amino group of aSpartic acid. Still more complex deriva- tives have been isolated from mature corn kernels (gga.flay§) by Bandurski and coworkers (25. 53). I The occurrence pattern of the glucose derivative and of the aSpartate conjugate is such as to suggest an evolu- tionary pattern of development of the systems reSponsible for their synthesis (127). Some plants have the ability to produce both compounds. The physiological significance of these conjugates is not clear. While no 1-substituted derivatives of indole-3-acetic 10 acid have been reported as occurring naturally in plants, several examples of synthetic derivatives showing Signifi- cant activity are found in the literature (81, 86). The degree of activity, absorption, or translocalizability sometimes equals or exceeds that of the parent acid. Ritzert gtmgl. (81) reported the synthesis and activity of several ethyl 1-acylindole-3-acetates some of which had activity greater than the parent acid. Alpha-methoxylation and N-acetylation was shown by Mitchell and Linder (67) to enhance absorption and translocation. However, activity remained at approximately the same level as indole-B-acetic acid. Several workers (76, 107) have reported that nitrogen methylation of indole-3-acetic acid greatly decreases its activity. Porter and Thimann (76) indicate that the activity is 13% that of indole-3-acetic acid. 1-Ethyl- and 1-propy1- indole-3-acetic acid were synthesized and tested in a bean root growth assay by Baskakov and Mel'nikov (3). They reported enhancement of root growth by these compounds. SYNTHESIS OF 1-SUBSTITUTED INDOLE DERIVATIVES A 1?.- " l '. ‘— IT.— Maid-1w»! with t0 be tiOn: SYNTHESIS OF l-SUBSTITUTED INDOLE DERIVATIVES Indole derrvatives may be synthesized from a variety of precursors (82). Equally numerous are the synthetic approaches to indole compounds. However, reported syntheses of 1-substituted indoles are somewhat less numerous. The Fischer indole synthesis technique is a classical method for most synthetic attempts at indole compounds. This route involves closure of the pyrrole ring and allows for a variety of substituents including those attached to the nitrogen. The starting materials are phenylhydrazine or a substituted phenylhydrazine (i) and a carbonyl compound (ii) with carbon atoms arranged in such a manner as to allow them to be incorporated into the nascent indole nucleus at posi- tions 2 and 3. 0112-8" I clzsz-Ii' ”C-R' R“ + _.9 __> I ’NHLNHZ c m N-N H 0 R' (R'") . I: \ (R )H n (3' )- t. 3(3) 0 R': 3(a) - 3(3) (1) (11) (iii) (iv) (After formation of the intermediate arylhydrazone (iii), the ring is closed by treatment with an acid reagent such as zinc chloride or Sulfuric acid. Recently pyrophos- phate ester has been investigated as a catalyst (41). Although Fischer fused an excess of zinc chloride with the 12 “i 1 . --:‘m_'na-iuli'u Ana—oi: ~ reagv boil. 2028 such the p prepa steri: Siierl the a 13 reagents, the procedure can be improved by the use of a high boiling solvent such as methylnaphtalene and a small amount of the catalyst (2). While the technique has wide applica- bility, the simplest case involving acetaldehyde phenylhydra- zone does not give the eXpected indole. Activated ketones such as pyruvic acid react very well (82). The limitations of the reaction are few but real. For the preparation of i-substituted indoles it is necessary to prepare the correSponding phenylhydrazine. The possibility of steric hindrance caused by the substituent must also be con- sidered: IAssymetrically substituted phenylhydrazones offer the additional complication of two modes of ring closure: H R ((11241 R C R. / . R' ”\R' ——> 0 I . + Cl II . ‘NHN , N R R N R I I H H (V) (v1) (v11) Another route to l-substituted indoles is the Bischler synthesis which Julia and coworkers have investigated exten- Sively (38, 39). An aniline (viii) substituted on the nitro- ISen.is reacted with an d-haloketone (ix) in a two step c 0‘ \\ II b_Ru c—R ' CH 2 + [032 ——9 / 2-—>0 1' I N N (R')H in X ‘B ’H I am . R B R (viii) (ix) (x) (xi) Dr 0171 14 reaction. The second step is often enhanced by using an acid reagent such an anhydrous zinc chloride. As with the Fischer method, the intermediates may be difficult to prepare. Another disadvantage of the Bischler method is the uneconom- ical use of an additional mole of the aniline to trap the evolved hydrogen halide. Nevertheless, the technique has the advantage of allowing for attachment of both 1- and 3-sub- stituents simultaneously. By using ethyl-F-bromoacetoacetate (xiii) it is possible to make 1-substituted ethyl esters of indole-3-acetic acid (xv) without careful purification of the intermediates (xiv) (40). 0\ c-cszcocczn5 2 [IlllN + ,CH "“‘;> I 2 R Br (xiii) I R 0 (xii), ‘t—CHZCOOCZH5 | CHZCOOCZH5 /CH2 211012 \ | N A If ——7\ I R B (xiv) (xv) Several other synthetic procedures are available for providing indoles with 1-substituents. Some of these result in products which have an oxindole or isatin nucleus while others yield compounds with altered phenyl rings. In the Nenitzescu Synthesis (5, 6, 15), benzoquinone (xvi) is treated with an N-substituted unsaturated-F-amino ester (xvii). By this route a series of very important chemicals are r... AT usefi 15 are produced in the form of serotonin analogs. CH-COOC H 0 ll 25 + ’03 H0 cochH 0 EN ---€> N' 5 I R R (xvi) (xvii) (xviii) Alkylated anilines (xix) and "glyoxol bisulfite" (xx) yield products hydrolyzable to 1-substituted oxindoles (xxi). HOCHSOBH + H0 HSO H \\ [::1~N-H 3 ” [::]:;:lc ' I R R (xix) (xx) (XXi) A versatile synthetic route which is particularly useful for either 4-substituted or 4,5-disubstituted indoles has been reported by Cornforth and coworkers (13). The synthetic intermediates, 4-keto-4,5,6,7-tetrahydroindoles (xxii), can be prepared by the reaction of 1,2-cyclohex- anediones with d-haloketones followed by cyclization with ammonia or primary amines (57). ‘\/ it a; O 2— (xxiii) The product of the cyclization (xxii) can be converted 16 to various other derivatives by manipulation of the groups at positions 4 and 5. Final aromatization of the ring to produce compound (xxiii) is best accomplished with 10% palladium on charcoal in refluxing cumene (13). Dehydro- genation with dichlorodicyanobenzoquinone is not successful unless the 5-position bears a hydroxymethylene group (80). Depending on whether ammonia or a primary amine is used in the ring closure step, 4-ketotetrahydroindoles with the ring nitrogen either free or alkylated, reSpectively, are obtained. Direct alkylation is often employed to afford the substituted indoles (33, 82, 125). However, it is necessary to make an intermediate N-metal salt in order to get the desired nitrogen substitution.. Reaction of indole with an alkyl halide gives a variety of carbon alkylations in addi- tion to nitrogen alkylation (82). Excess methyl iodide yields tetramethylindolenium iodide (xxiv) when it is reacted with indole (82). CH3 @331an N cm3 I CH3 I (xxiv) Several partially methylated intermediates have been charac- terized which react further to yield the tetramethyl deriva- tive. To minimize these complications, alkylations are I _ ‘ ~ In.“ A‘— ' A- \‘Z‘. i w.“ I I 115118 soii has ever as t; proi‘ Whiz? reflu 3 0 ‘. 112': 511311 i tent VETSa 17 usually carried out in basic solution in the presence of sodium or potassium (126). Other cations have been employed including lithum and tetramethylammonium cations (57). How- ever, with direct alkylation, liquid ammonia is often employed as the solvent and so necessitates cryogenic apparatus. The product from such a reaction is often obtained in good yield, but suffers from contamination by unreacted starting material which is not always easy to remove (71, 89). Prolonged refluxing with sodium is sometimes used to effect separation of unreacted starting material (33). Unfortunately, groups such as Aggypropyl and‘tggtebutyl with a large steric require- ment have not been attached by this technique even though repeated attempts have been made (126). Reactive halides such as benzyl halides will react with the N-sodium salt of indole compounds in dimethylformam- ide (65) soluticn. However, since this reaction is incomplete, it requires careful fractionation of the products. It is very versatile and numerous derivatives have been reported (60, 61, 62, 63) including many 1-acy1 compounds. Still another technique for preparing i-substituted indoles involves using a substituted indoline as an inter- mediate (97, 98). A distinct advantage of this procedure is the possibility of removing the unaltered indoline from the product. (Acetic anhydride or benzene sulfonyl chloride (Hinsberg's reagent) may be employed to reduce the basicity of indoline through reaction to yield the amide. The former reagent has been used in a similar synthesis of N-methyl, Ans-iris" ""1 LIED ' 18 Ngtgrtgbutyl aniline (126). The substituted indoline can then be extracted from the amide with acid. Chloranil (97, 98) or dichlorodiCyanobenzoquinone (78) can be used to aromatize the substituted indoline ring. The Madelung-Verley synthesis involving another ring closure can also yield i-substituted indoles (xxiii) (58, 82, 118). A basic condensing agent such as potassium.§§gtr butoxide is employed to cyclize the substituted aniline (XXV); @ZHS W> CU NH / N \ l B R (xxv) (XXVi) Other less useful routes to N-alkylated products exist. For example isogramine (xxvii) can be made by con- trolling carefully the conditions of the Mannich reaction (99) in Which gramine (xxviii) is usually produced. 9 CH N(CH ) 2 3 2 GE 0;] l I CH2 H N(CH3)2 (xxvii) (xxviii) A reaction involving ring closure by still another route was reported by Piper and Stevens (73). In their syn- thetic scheme, substituted anthranilic acids (xxix) were [M\ C) F“ \ A ‘3 19 cyclized to 1-acetyl-chloro-2—methylindoxyl acetates (xxx). coon CH COOH I 000033 NHCH~COOHZ 3 :> CH3 C1 I I Cl CH3 COCH3 (xxix) (xxx) 0 BrCH cooc H - I HZCOOCZHS - H 2 2 5:> H Cl I CH3 Cl I 3 COCH3 00033 (xxxi) (xxxii) After removal of the 3-acetyl group with sodium sulfite in dioxane and water, the Reformatsky product (xxxii) was obtained by the action of ethyl bromoacetate.' Several observations are pertinent to the considera- tions of a synthetic sequence involving i-substituted indoles. One-alkyl indoles will react well with oxalyl chloride to yield the 3-substituted compound; a glyoxyly- chloride (xxxiii) (28). O O O O N H H H I n C-C-Cl I n -C-NR R I I I B R (xxxiii) (xxxiv) 3H H—CH Nfi R" LiAIH u 2 u.:> [::1\N I R (xxxv) 1-a1} forms “ #9. P. Ou‘u With 20 After amidation, however, the glyoxylylamide will not undergo the usual reduction to a saturated side-chain which is typical of indoles with a free i-position, but yields instead the alcohol (xxxv) (31). The importance of this synthetic tech- nique lies in its usefulness for preparing tryptamine analogs. Another anomalous reaction has been observed with l-alkyl indoles. In the Mannich reaction of indole with formaldehyde and dimethylamine, gramine (xxxvii, R = H) is produced in excellent yield (92). ' CH N(CH ) 2 3 2 + 3030 + NH(CH3)2—> I I B (B = H; alkyl) R (xxxvi) (xxxvii) The dimethylamine moiety of gramine may be replaced with cyanide by direct reaction with sodium cyanide (92) to yield a mixture of indole-3-acetic acid and indole-3- acetonitrile. However, when i-methyl gramine (xxxvii, R = CH3) is reacted under similar conditions only starting material can be isolated (91). Nevertheless, if the methiodide salt of 1-methyl gramine is used in the reaction with cyanide, the expected product is obtained. Signifi- cantly, this product is accompanied by a 2-substituted nitrile (#2, 90). CHEMICAL STRUCTURE AND PHYSIOLOGICAL ACTIVITY CHEMICAL STRUCTURE AND PHYSIOLOGICAL ACTIVITY Compounds which elicit a certain set of plant reSponses similar to that produced by indole-3-acetic acid are known as auxins. An acceptable definition for growth regulators was presented in 1951 by a committee of the American Society of Plant Physiologists (112) who defined them as "organic com- pounds, other than nutrients which in small amounts promote, inhibit, or otherwise modify any physiological process in plants.” Auxins as a subclass of the above were defined as "a generic term for compounds characterized by their capacity to induce elongation in shoot cells. They resemble indole-3- acetic acid in physiological action." Unfortunately, cell elongation is not the only observed effect of real auxins. Auxins are in some way involved in differentiation, fruit-set, abscission, and apical dominance, and indole-3-acetic acid as the reference compound can alter all of these processes. The growth regulators are employed in agriculture for such pur- poses as weed control, fruit-thinning, root induction, and control of flowering. It is not surprising that synthetic derivatives of indole-3-acetic acid are similar in their action to the parent acid, but it is less obvious that compounds like phenoxyacetic and naphthyleneacetic acids should give a physiological response similar enough to indole-3-acetic acid to be classi- fied as auxins. 22 biol 3H5 Hoe; '0'}. / Vow 23 The structural requirements which are necessary for biological activity have been sought for many years. Early attempts to enumerate the essential factors were made by Koepfli, Thimann, and Went in 1938 (#7) who listed five structural requirements: (a) a ring system as nucleus (b) a double bond in this ring (0) a side-chain (d) a carboxyl group (or a structure readily con- verted to a carboxyl) on the side-chain at least one carbon removed from the ring (e) a particular Space relationship between the ring and the carboxyl group. As new compounds were discovered and assayed, it became apparent that these requirements were too confining. The activity exhibited by the halogenated benzoic acids removed the requirement for a side-chain. Only two general requirements were considered necessary by Veldstra and Booij (116), who studied the relationship of chemical structure to biological activity in many compounds and proposed the follow- ing requirements for activity: (a) a basal ring system (non-polar part) with high interface activity (b) a carboxyl (polar part)--in general a group of acidic character--in such a Spatial position with respect to the ring system, that on absorption of the active molecule to a boundary (the non-polar part playing the most important role) this func- tional group will be situated as peripherally as possible. Numerous exceptions to these condensed rules, however, have been observed. Among these are the observations by J14 :- I id. §- '6' twang; Veli auzin Carba; tomatc Struci So in q L‘.‘ “ ‘Vn 1 J- V 24 Veldstra (114, 117) that primary nitro or sulfonic acid groups will substitute for the carboxyl as will phOSphonous and phos- phonic acid derivatives. The tetrazole analog of indole-3— acetic acid, 5-(3'-indole-methyl)-tetrazole, also exhibits slight auxin activity. Because tetrazoles of this type have approximately the same pKa values as do aryl carboxylic acids (30), this could account for a similar character. In each of the cases mentioned, the group that replaced the carboxyl had some degree of anionic charge indicating the important aSpect of this structure. Significantly, anionic groups other than the carboxyl moeity impart a much lower degree of activity than do the carboxylic acids. Even certain compounds without a ring structure show auxin activity. Thus, 5-(carboxymethyl)-dimethyldithio- carbamic acid was observed to cause growth of cucumber, tomato, and pea plants (113). Compounds with this unique structure may assume a planar configuration and probably do so in the resonance hybrid: s5 3" CH H CH + | 35‘; <———> 3\ _ /N c\ /N—C\ CH3 S-—CH2COOH CH3 S-—CHZCOOH (A) (13) According to Pitzer (74) only the first row elements produce strong multiple bonds. The formation of such bonds with heavier elements is hampered since they lack the capacity to bond in this way due to the "inner shell repulsion" of the completed inner octets. Therefore, the resonance contributor 25 (B), with its trigonally hybridized Sp2 nitrogen atom, would 3 configuration. be more highly favored than the non-planar Sp In addition to possessing the planar structure thought to be necessary for an auxin response, the contributor (B) would have the double bond and complement of pi electrons required for high surface activity as Specified by Veldstra. Appar- ently the demand for an unsaturated ring must be modified as a requirement for a planar unsaturated structure. In 1938, Thimann and Bonner (106) showed that the activity of indole alkanoic acids alternated with side-chain extension. Even numbered carbon chains always showed a higher activity than the odd numbered counterparts. The even numbered chains are degraded to indole-B-acetic acid by B-oxidation while the odd numbered chains yield the inactive indole-B-carboxylic acid. At the time of the investigation, however, the process of B-oxidation in fatty acid metabolism remained to be eluci- dated. Wightman (12#) investigated the B-oxidation of indole- 3-alkanoic acids with chains up to seven carbons in length. He was able to demonstrate that acids with even numbered chains were degraded to indole-3-acetic acid. The longer even members also gave appreciable quantities of indole-B-butyric acid, an indication of B-oxidation. Odd numbered acids gave only indole-B-propanoic acid in measureable quantities and none of the eXpected indole-B-carboxylic acid could be detected. Thimann and his coworkers (76, 105) proposed that a critical property of auxins is the Spacial relationship 26 between the anionic charge on the carboxy group and a par- tially positive charge located 5.5 angstroms from the car- bonyl group. In the indole auxins the cationic center is the nitrogen of the pyrrole ring. The position of the charge centers and their orientation is shown in Figure A (105). Indole-B-acetic acid is compared with the most active auxins of three other classes. HN / 4+ c/ :+ 0710? C__ _. . // 4 \ Cl 0 Ci——f;__Cl CH _CH '+ /O H \ __.c __r — Figure A More recently, Porter and Thimann (76) have postulated the importance of the extent of partially positive charge on the degree of auxin activity. Indole-B—acetic acid substituted 27 in the 2-position with either chlorine or bromine has an auxin activity of 350% and 160% reSpectively, relative to the parent compound. Significantly, similar halogenation of lysergic acid diethylamide greatly increases its activ- ity as a serotonin antagonist in animal systems (111). Other work seems to support Thimann's hypothesis. Sell ggflgl. (86) and Ritzert gt El: (81) have assayed 1-benzoyl derivatives of indole-3-acetic acid and found them to be quite active in tomato fruit-set. The latter workers also noted considerable 53333 straight growth activity. 1-Benzoyl derivatives would be eXpected to delocalize the lone pair of electrons on the nitrogen and enhance the partially positive charge. In Opposition to this enhancement would be the steric requirement of the 1-substituent. With the data at hand, one cannot be sure that the derivatives acted p§£_§§ and were not degraded to indole-3-acetic acid. Veldstra (115) showed that the nitrogen atom of indole-3-acetic acid was not essential for activity. The carbocyclic analog of indole-3—acetic acid, indene-B-acetic acid, as well as the oxygen heterocyclic analog, coumaran- 3-acetic acid, show some activity in the Aggga straight growth assay. Lower activity was seen.in.A£§n§_curvature assays, an indication that the synthetic material was not well transported. Substitution of the nitrogen atom with a sulfur atom lowers the activity considerably but does not destroy it (#5). Hellman 23.9l. (29) found that insertion pr 0p lea s 28 of an additional nitrogen in the 2 position did not alter activity in Ag§g§_straight growth, tomato fruit-set, or cucumber root inhibition assays. Substituents on the carbon atoms of the rings can cause drastic alteration in activity. The high activity of the Z-halo-derivatives of indole-B-acetic acid has already been mentioned. Chlorination on any of the carbon atoms of indole-3-acetic acid enhances the activity of the molecule (75). Methyl groups, on the other hand, generally lower activity when substituted on the rings. This is particularly true of a methyl group on positions 1, 2, or 7,--positions closely associated with the ring nitrogen atom. With the exception of direct methyl substitution on the nitrogen, it seems unlikely that the electronic effect of this group is of much importance. Insofar as electronic properties are concerned, the methyl group is one of the least powerful donating groups usually encountered (35). Steric blockage is then the most likely explanation for reduction in activity. Even though the nitrogen is not an absolute necessity and in fact does not exist in some of the most potent auxins, it seems to be implicated as an important center in indole-3-acetic acid. Polar substituents attached to the auxin molecule almost always decimate the activity. Thimann (107) has shown that two carboxyls on an auxin always destroy activity in the pea test. Among the indoles, indolemethylenemalonic acid showed no activity. Invariably introduction of an ~ 7'" o'“ 9‘ is 29 hydroxyl group drastically lowers activity. Bearing on this is the demonstrated low activity of the four indole auxins: 5-hydroxy-indole-3-acetic acid, 7-hydroxy-indole-3-acetic acid, indole-B-lactic acid and indole-B-glycolic acid. A related compound, indole-B-glyoxylic acid containing an d-carboxyl group, also showed very low Azgn§.activity. Perhaps the anionic nature of the substituents causes them to compete for an attachment site with the side-chain carboxyl group. The low activity of the hydrophilic hydroxyl group may be explained by the hypothesis that mem- branes are somewhat lipoidal and prone to reject hydrotg philic entities. Certainly of major importance in auxin I action are the phenomena of transport and translocation involving lipid solubility and partitioning, which were pointed out by Hansch 22.229 (26, 27) in their rather exhaustive work on phenoxyacetic acids. Hydrophilic indole- acetylglucose was proposed by Zenk as a detoxification form of indole-3-acetic acid. Hydrolysis of this conjugate to yield indole-B-acetic acid may explain its activity. . When Smith and Wain (88) investigated the erreet of various substituents on the side-chain, they proposed that three factors are important in binding the active compounds to an active site: (a) an unsaturated ring, (b) a polar side group, and (c) at least one hydrogen atom on the carbon in the side-chain. Veldstra (117) found that d-methylene and d-isopropylidene phenylacetic acids, however, were more active in the split pea test than was the parent acid. attac 30 Because the hydrogenation product of the latter compound, d-isopropylphenyl acetic acid is inactive, obviously activity cannot be attributed to hydrogenation. This find- ing would tend to negate the importance of an d-hydrogen atom. In addition, d-dimethylindole-B-acetic acid has been reported to be active in high concentrations (103) even though it is devoid of an d-hydrogen. Although Fredga and.Aberg (19) reviewed the effects of sterioisomerism in auxins, they indicated that very little had been done with indoles. K5g1,.th first researcher to study the optically active forms of d-methyl-indole-B-acetic acid, found considerable difference (30 times) between the D(+) and L(—) antipods with the D form more active in the cat coleoptile curvature test (48). However, the cat cylin- der test gave identical results with both isomers (100). The general trend is the same in non-indole auxins in that the D form is more active (107). These findings lend support to the theory proposed by Wain (88) concerning three point attachment to the auxin active site. The contradictory results from the investigations con- cerning the importance of an dehydrogen indicate the tenta- tive nature of the present knowledge. In view of the most recent findings, however, Thimann's hypothesis concerning the requirement for a positive center associated with an electron rich planar structure and separated by a given distance from a negative center seems at this time to be the most limiting demand which can be placed on the structure of an auxin. BIOLOGI CAL ASSAY I“ BIOLOGICAL ASSAY A variety of biological assays have been investigated. These assays were designed to examine a particular phenomenon or to minimize side effects caused by certain aSpects of plant structure. Thus, the Aygng_curvature assay (122) was designed to investigate basipetal tranSport of an applied auxin down one side of an.Azgg§ coleoptile and depends upon cell elongation for the resultant curvature. This and other curvature assays such as the Split pea assay (116) have been widely used even though the response is more complex than simple cell elongation. In this work, five assays were used. The Ay§g§_ straight growth assay (70) is widely employed for testing auxins. This assay has the advantage that penetration and transport effects are held to a minimum. The increase in length of coleoptiles floated on a test solution as compared to those floated on buffer is a measure of auxin activity. Boot inhibition by test compounds also serves as an index of activity. Buckwheat root inhibition was shown by rVitou and Wain (119) to be a reliable and valuable assay for the auxin reSponse. This assay depends upon the inhibition of root growth caused by supraoptimal concentrations of auxin. Wittwer and Tolbert (125) showed that gibberellins 32 A ‘ 3-H .1 ’5. . r a“ l '- .l’: 4-'\ an: 0f t1 an 12 trea1 P0811 from 33 and auxins are involved in tomato fruit-set. Active members of either series will cause parthenocarpic growth. Lanolin solutions of the test compounds in question were applied to the ovaries of emasculated flowers and the ovary diameter was measured a few days later. Bean petiole abscission is a long term assay which is a measure of the ability of the test compound to replace the factor in the intact leaf which maintains a healthy petiole. Lanolin solutions were applied to the severed petiole. The degree of activity was judged by the length of time necessary for abscission to occurr. The cucumber curvature test (83) serves as a measure of the secondary properties, absorption and translocation in an intact plant. One cotyledon of a cucumber seedling was treated with a lanolin solution of the test substance. A positive response was noted when the plant stem curved away from the treated cotyledon. Complete details of each assay are presented in the appropriate section. EXPERIMENTAL enc~ pre: indc prer ch10 extr Stit dich Whic much When PUri: EXPERIMENTAL Synthesis of the Compounds Reaction of indoline with an alkyl iodide in the pres- ence of anhydrous, powdered sodium carbonate (8?) was used to prepare intermediates in the synthesis of several 1-alkyl indoleacetic acids. In this way a 1-a1kyl indoline could be prepared which was purified by the action of benzenesulfonyl chloride in base on the unreacted indoline, followed by extraction, and vacuum distillation. Aromatization of the substituted indoline to a sub- stituted indole was easily accomplished by reacting it with dichlorodicyanobenzoquinone (DDQ). The reaction mixture which employed dichlorodicyanobenzoquinone was found to be much more easily purified and afforded better yields than when chloranil was used. Distillation ig_zggug.gave the purified indole derivative. Attachment of the side-chain to yield the i-substituted ethyl ester of indole-3-acetic acid was accomplished by reac- tion with ethyl diazoacetate (69, 16). Vacuum distillation always gave a fore-run of unreacted N-substituted indole as well as some lower boiling material. Saponification in alcoholic aqueous sodium hydroxide yielded the appropriate acids in 25-85% yields. Recrystallization from benzene- hexane improved the color in most cases. 35 F . *éflrun‘nm m was sol sod cool dry p-SL stir liza prep for isol benz. indo. Cong. for ' even The J Bethc Compc aCeti HSing (97) 36 Benzylation of the indolic nitrogen was effected using the method reported in a Merck patent (64) for similar com- pounds. Using this procedure, a dimethylformamide (DMF) solution of the N-Sodium salt of ethyl indole-B-acetate (EIA) was prepared at room temperature by adding a dimethylformamide solution of ethylindole-B-acetate to a stirred slurry of sodium hydride in dimethylformamide. The salt solution was cooled to the temperature of either an ice water bath or a dry ice-acetone bath and a dimethylformamide solution of the p-substituted benzyl halide was added Slowly with continual stirring. Careful fractional crystallization and recrystal- lization gave the desired N-substituted products. However, preparative layer chromatography was found most effective for isolation of pure material. A principle by-product was isolated and shown to be the correSponding p-substituted benzyl ester of indole-3-acetic acid. Ethyl 1-pynitrobenzyl- indole-B-acetate could not be prepared by this method, and consequently another synthetic route analogous to that used for the preparation of 1-alkyl derivatives was found suitable even though workup was tedious and the yield was minimal. The prchlorobenzyl derivative was also synthesized by this method and was used to verify the structure of the chloro compound previously described. Glucose was attached to the 1-position of indole-3- acetic acid (IAA) by preparing i-(B-D-glucopyranosyl) indole using the synthetic sequence of Suvorov and Preobrazhenskaya (97) except that dichlorodicyanobenzoquinone was employed 37 instead of chloranil. Ethyl diazoacetate proved satisfactory for attachment of the ethyl acetate side-chain, but purifica- tion had to be carried out with column chromatography followed by crystallization. The acetate blocking groups were removed with methanolic barium methoxide (34) and the resulting methyl 1-glucosyl-indole-3-acetate crystallized from methanol- water. Saponification of the methyl ester with barium hydrox- ide gave an amorphous precipitate which could be separated from the starting material with preparative layer chromato- graphy (PLC) but could not be crystallized. l-Alkyl Indolines H H N 2 I 2 R | H I II III i-Methylindoline (III, R = methyl) (87) Twenty-three and eight tenths grams (0.2 mole) of indoline was mixed under stirring with 21.8 g of anhydrous, powdered sodium carbonate and then approximately 100 ml of dimethylformamide was added. While cooling to 0°, the slurry was stirred with a magnetic stirrer. Twenty-five grams (0.2 mole) of methyl iodide was added and stirring was con- tinued overnight. The mixture was heated to 1000 for 6 hours and cooled. Then water was added and the organic material was extracted with chloroform. The chloroform layer was washed twice with water, dryed with anhydrous calcium chloride, 38 filtered, and evaporated to an oil on a Rinco rotary flash evaporator. The impure oil containing unchanged indoline was shaken in a 5% sodium hydroxide suSpension with excess benzenesulfonyl chloride until a negative test for the halide was attained. The desired substituted indoline was removed by extraction with ethyl ether from the reaction mixture. The ether solution was extracted with 1 Nlhydro- chloric acid several times, and the acid extract neutralized with 5% sodium hydroxide. (The solution clouded noticeably and turned from violet-brown to yellow at the end point.) Be-extracting of the product from the alkaline solution with ether, washing twice with water, drying with anhydrous calcium chloride, filtering, and evaporating gave an oil that distilled at 0.08 mm Hg and 40-44°. Purity was indi- cated by lack of the characteristic N-H stretching absorbance (2.9 u) in the infrared Spectrum. The yield was 11.1 g (44%). Ultraviolet absorption showed a bathochromic shift from the parent compound of 5-8 mm with peaks at 249 mu and 297 mu, the former being more prominent. The refractive index was n35 1.5670. 1-Ethzllndollne (III. R = ethyl) (87) Ethyl iodflie (23.9 g, 0.15 mole) was dropped into an ice-cold, stirred Slurry of 14.8 g (0.14 mole) anhydrous, powdered sodium carbonate and 16.6 g (0.14 mole) of indoline. The reaction mixture was stirred for 3 hours and permitted to warm to room temperature. After the mixture was heated for 6 hours on a boiling water bath, it was cooled. Then (’3 wit ext Aft re-¢ Chl< disi repc 1.56 inf: Were 39 water was added, and the product was extracted twice with ethyl ether. The ether layer was first washed twice with water and then evaporated to an oil which was reacted in 5% sodium hydroxide with excess benzenesulfonyl chloride until its odor disappeared. This reaction mixture was extracted twice with ether. The ether was washed twice with water and extracted with 1 fl_hydrochloric acid to remove the product. After the solution was neutralized with 5% sodium hydroxide, re-extracuai with ether, and dryed with anhydrous calcium chloride, the solvent was evaporated. Then the product was distilled at 108-110° and 13 mm Hg. Skeinkman and Kost (87) report 99-102° at 8 mm Hg and the refractive index n35 1.5603. Ten grams of product lacking NeH absorption in the infrared was isolated in a yield of 41%. Physical constants were n35 1.5580 and lgzga?;:)(95%) 252 and 300. i-nePro lindole III R = n-propyl) Eleven and nine-tenths grams (0.1 mole) of indoline (11.9%) was stirred with 10.6 g (0.1 mole) of anhydrous sodium carbonate at 0° while 17 g (0.1 mole) of appropyl iodide was added dropwise. The slurry was stirred overnight, then heated to the temperature of a boiling water bath for 6 hours. After addition of water, extraction with ethyl ether, and evaporation of the solvent, the reaction mixture was reacted with excess benzenesulfonyl chloride in 5% sodium hydroxide until the odor of the halide disappeared. This mixture was extracted with ether and then the ether was I‘- ’1 m was DOW 131‘0 Sti hou Str EXC mix Was; hm: (I) O {)1 Wit] anh. 40 washed twice with water. This product was extracted from the ether with 1 N_hydrochloric acid. Neutralization of the acid solution, re-extraction of the product with ether, dehydra- tion of the solvent over calcium chloride, and evaporation afforded an oil which distilled at 50—54° at 0.1 mm Hg. The yield of product devoid of N-H stretch in the infrared was 47%. Dimethylformamide could be used as a solvent with little ethanol (95%) max (mu) 254 and 302. The refractive index was ”$5 1.5478. change in yield. Ultraviolet absorption showed 1 1-isoéPropylindoline (III, R = iso-propyl) (87) Thirteen and eight-tenths grams (0.2 mole) of indoline was stirred together with 21.2 g (0.2 mole) of anhydnpus, powdered sodium carbonate at 00 while 24.6 g (0.2 mole) iggg propyl bromide was added drop by drop. The mixture was stirred for 2 days and was finally heated to 100° for 6 hours. Water was added and the mixture was extracted with ethyl ether. After washing the ether twice with water and stripping the solvent, the resulting oil was reacted with excess benzenesulfonyl chloride in 5% sodium hydroxide until the reaction mixture was void of the chloride odor. This mixture was extracted twice with ether. The ether was washed twice with water and then extracted twice with i N hydrochloric acid. After the acid was neutralized with sodium hydroxide (litmus paper), the product was re-extracted with ether, and then washed 2 times with water, dryed over anhydrous calcium chloride, and evaporated to yield an oil. Di sh bei but the the 'hat 6X1 41 Distillation at 46-48° and 0.1 mm Hg gave the product which showed no N-H stretch in the infrared Spectrum. The yield ethanol (95%) max (mu) Sheinkman and Kost (87) give the boiling range as 112-115° was 36% with n35 1.5501 and l 254 and 302. 2 at 10 mm Hg and the refractive index as ”D5 1.5585. legrButylindoline (III, R = nebutyl) (87) While 15 grams (0.136 mole) of indoline and 13.4 g (0.136 mole) of anhydrous, powdered sodium carbonate were being stirred vigorously at 0°, 26.0 g (0.136 mole) of 2: butyl iodide was added drop by drop. After 3 hours at 0°, the mixture was permitted to warm to room temperature and the solution was heated for 6 hours at 1000 in an oil bath. Water and ethyl ether were added. The ether phase was extracted twice with water, and evaporated to dryness. The resulting oil was treated with benzenesulfonyl chloride in 4% sodium hydroxide solution until no benzenesulfonyl chlor- ide odor remained. This mixture was extracted twice with ether which was in turn extracted 2 times with water. One normal hydrochloric acid was used to remove the product from the ether layer. After the acid extract was neutra- lized with 4% sodium hydroxide (litmus paper), ether was used to re-extract the product. The washed and dryed ether solution (anhydrous calcium chloride) was evaporated to dryness and the residual oil distilled ig_zgggg at 130-1310 and 13 mm Hg. Sheinkman and Kost (87) report the boiling range of 128-1320 at 12 mm Hg. Their value for the refrac- tive index was “£5 1.5429. No N-H band was seen in the inf rar ed 254 and j iimethylf A; NI 7.79%. l-isg-But m EQi 8) and an} stirred tc butfl iodi stirred a; hours. 00c phase. Tv Were follc °f the res sodium hyc EXtraCtioy 1’18 of the Product 1.} acid 801111 extracted water and tion 013‘ t} 0,1 mm Hg The infra; ViOlet abs 42 ethanol (95%) infrared region. Ultraviolet absorbancy was Xmax (mp) 254 and 302. Yield was 45% (10 g) with n35 1.5407. Use of dimethylformamide as solvent gave 61%. Analysis: Calcd. for C12H17N: N, 7.99%; Found: N. 7.79%- 1- so-Butylindoline (III, R = iso-butyl) Equimolar quantities (0.144 mole) of indoline (17.2 g) and anhydrous, powdered sodium carbonate (16.3 g) were stirred together at 00 while (0.144 mole, 26.5 g) of igg- butyl iodide was added slowly. After the mixture was stirred approximately 4 hours, it was heated to 1000 for 6 hours, cooled, and extracted with chloroform from the water phase. Two washings of the chloroform extract with water were followed by evaporation of the chloroform and reaction of the residue with excess benzenesulfonyl cnloride in 5% sodium hydroxide until the odor of the reagent disappeared. Extraction of the basic solution with ethyl ether and wash— ing of the extract was followed by re-extraction of the product from the ether with 1 N_hydrochloric acid. The acid solution was neutralized with 5% sodium hydroxide and extracted with ether. The ether was washed twice with water and dryed with anhydrous calcium chloride. Evapora- tion of the ether gave an oil which distilled at 58-600 at 0.1 mm Hg for a yield of 9.4 g (37%) and with an n35 1.5379. The infrared showed no N-H band. The compound showed ultra- ethanol (95%) violet abs r ti A o p on max (mp) 256 and 305. I" ‘4... v iu~nvv.r N, 7.93 1-§§ggB line an carbons mole) g ture wa 100° f0 mixture form we oil whi Sodium had die with et aCid, hydrOXi of the 43 Analysis: Calcd. for C12H17N: N, 7.99%; Found: N. 7.93%. 1-sec-Butylindoline (III, B = sec-butyl) Twenty-four and one-half grams (0.205 mole) of indo- line and 22 g (0.207 mole) of anhydrous, powdered sodium carbonate were stirred together at 00 while (38.4 g, 0.207 mole) §ggrbutyl iodide was added carefully. After the mix- ture was stirred approximately 4 hours, it was heated at 1000 for 6 hours. Upon cooling, water was added and the mixture was extracted twice with chloroform. The chloro- form was extracted twice with water and evaporated to an oil which was reacted with benzenesulfonyl chloride in 5% sodium hydroxide. When the odor of benzenesulfonyl chloride had disappeared, the reaction mixture was extracted twice with ethyl ether and the ether in turn with 1 N_hydrochloric acid. Neutralization of the acid extract with 5% sodium hydroxide and re-extraction with ether yielded a solution of the product which could be isolated by drying and evapor- ating the solvent and then distilling the residue at 64-660 at 0.2 mm Hg. This product had no N-H stretch band in its infrared spectrum. Yield was 12.5 g (35%) with iethan°1 (95%) max (mu) 257 and 308. Its refractive index was n35 1.5379. Analysis: Calcd. for 012H17N: N, 7.99%; Found: N, 7.88%. I-tert-Butylindoline (III, R = tert-butyl) One hundred grams (0.54 mole) of tert-butyl iodide l (1‘. Aflydfitfl’ iTI'I‘EL _ I. 5011 ' was ad (0.5 m anhydr temper heatin until was ex ings v was re benzei the m was e' mater aCid I‘EB-ex tion Yield Strei purij 881 < 256 ; 44 was added dropwise to a cooled (0°), stirred slurry of 32.5 g (0.5 mole) of indoline and 53 g (0.5 mole) of finely divided anhydrous sodium carbonate. The solution was warmed to room temperature and the reaction was brought to completion by heating approximately 6 hours on a hot water bath (60-800) until a solid mass formed. Water was added and the mixture was extracted twice with ethyl ether. After several wash- ings with water, the ether was evaporated and the residue was reacted with i 1 of 5% sodium hydroxide and 60 ml of benzenesulfonyl chloride. When the reaction was complete, the mixture was extracted twice with ether and the ether was extensively washed and decanted to remove a solid material. Two extractions of the ether with 5% hydrochloric acid and neutralization with 5% sodium hydroxide followed by re-extraction of the aqueous phase with ether and evapora- tion gave an oil which boiled at 125-127° and 13 mm Hg. Yield was 5.5 g (11.5%) of product having only a little N-H stretch in its infrared Spectrum. The analytical sample was purified on preparative layer chromatography (2 mm silica ethanol (95%) gel G in 18% butanone-hexane). Physical data were Amax (mu) 256 and 299: n35 1.5431. Analysis:, Calcd. for chH N, 7.99%: Found: ‘ 17N: N. 7.97%- iegfiPentylindoline (III, R = nrpentyl) Thirty and six-tenths grams of 1-iodopentane (0.154 mole) was allowed to drip into a stirred suSpension of 13 g a}? g i 1 4 (0.15 powde room for 6 chlor water sodiL the 1 washe anhy< drynq gave and ' Show chlc °hlc h’a 8 an( 45 (0.154 mole) of indoline and 15.7 g (0.154 mole) anhydrous, powdered sodium carbonate at 0°. After being warmed to room temperature, the reaction mixture was heated to 1000 for 6 hours, cooled, diluted with water, and extracted with chloroform. The organic liquid phase was washed twice with water and evaporated to an oil which was reacted in 5% sodium hydroxide with excess benzenesulfonyl chloride. Then the product was extracted with ethyl ether, the ether was washed twice with water, and the solvent was dryed with anhydrous magnesium sulfate. Subsequent evaporation to dryness followed by distillation at 75-81° and 0.2 mm Hg gave a 13 g yield (45%). Observed constants were n35 1.5318 ethanol (95%) max (mu) Showed no N-H stretch. - and A 254 and 302. Infrared Spectral analysis Analysis: Calod. for C13H19N: N, 7.40%: Found: N: 7.29%- i-nfDecylindoline (III, B = nrdecyl) Twenty-three and eight-tenths grams (0.2 mole) of indoline was stirred together with 21.1 g (0.2 mole) of dry, powdered, sodium carbonate at 0° while 53.6 g (0.2 mole) 1-iododecane was added dropwise. The resulting mixture was heated to 100° for 6 hours, cooled, and extracted with chloroform: Water soluble material was removed from the chloroform extract by two washings with water. This extract was dryed with anhydrous magnesium sulfate and evaporated to an oil Which was distilled at 132° and 0.3 mm Hg. The yield of prod was seen eth was 1 max A; N. 5.51% leg-Dots * S indoline of anhyd decyl 10 heated t t10H nix Chlorofc Was 8coc Solvent Solid um and nece Cleanin8 209° and etha X n01 max (mJ 10495“. g N’ 3-94z 46 of product was 48.3 s (80%) with n35 1.5164. No N-H band was seen in the infrared Spectrum. The ultraviolet Spectra 256 and 305. Analysis: Calcd. for 018H29N: N, 5.40%: Found: N. 5-51%- '1“ l-ngctadecylindoline (III, R = geoctadecyl) Seven and eighty-five hundredths grams (0.066 mole) indoline was vigorously stirred with an equimolar quantity of anhydrous, powdered sodium carbonate (7 g) and grocta- decyl iodide (25 g) at room temperature for 9% hours and heated to 100° for 6 hours. Water was added and the reac- tion mixture was extracted twice with chloroform. The chloroform solution was washed twice with water. Drying was accomplished with anhydrous sodium sulfate and the solvent removed in.y§ggg, Unreacted indoline and some solid material was recovered in the lower boiling fractions and necessitated dismantling the distillation apparatus for cleaning before the final distillation of the product at 209° and 0.25 mm Hg. The product crystallized in the receiver, mp 33.5-34.50. Yield was 63% (1.52 g) with Aethanol (95%) max (mu) 1.4954: No N-H stretch was noted in the infrared spectrum. 254 and 300. The refractive index was n35 Analysis: Calod. for C26H45N: N, 3.77%: Found: N: 3.94%- fl 47 1-Alk 1 Indoles //\ N I H III IV l-Methylindole (IV. R = methyl) Ten and six-tenths grams (0.08 mole) of methyl indoline was dissolved in approximately 250 ml of dry xylene and an equimolar quantity of dichlorodicyanobenzoquinone was added. The addition of dichlorodicyanobenzoquinone caused consider- able heating. When solution was complete, the mixture was refluxed for 6 hours, cooled, and filtered. The solvent was removed under reduced pressure. The residue was distilled at 58-650 under 0.6 mm Hg and yielded 6.6 g (63%). The refractive index was n35 1.5945. The infrared spectrum was devoid of N-H stretching absorbance. Ultraviolet absorbance ethanol (95%) max (mu) report boiling point 720 at 0.85 mm Hg for the product showed A 274, 282 and 294. Noland 23,2;, (71) obtained from the reaction of methyl iodide and sodium amide with indole in liquid ammonia. Gray and Archer (21) report n35 1.6038. 1-Ethylindole (Iv, R = ethyl) 1-Ethylindoline (8.9 8: 0.060 mole) and 13.7 g (0.060 mole) of dichlorodicyanobenzoquinone were dissolved in approximately 250 ml dry xylene and brought to reflux. After heating 6 hours, the suSpension was cooled, filtered to remove the by-; tor. Ti Hg t0 yi xethanol max (mr. attribui (22) re] index 6; i-g-Pro ‘ Quantit Solved Solid m 6 hours the by. at 64° in the 48 the by-product quinone, and evaporated on a flash evapora- tor. The resulting product distilled at 114-120° and 13 mm Hg to yield 4.3 8 (48%) of the product with n35 1.5860 and Aggga?g&)(95z) 274, 282 and 294. No infrared absorbance attributable to the N-H group was observed. Gray gghgl. (22) report a boiling point of 82-85° and a refractive index n36 1.5889. i-nfiPropylindole (IV, R = ngpropyl) gfiPropylindoline (8.05 g, 0.05 mole) and an equimolar quantity (11.4 g) of dichlorodicyanobenzoquinone were dis- solved in approximately 200 ml of dry xylene. After all solid material had dissolved, the solution was refluxed for 6 hours. Upon cooling, the solution was filtered to remove the by-product and evaporated. The resulting oil distilled at 64° and 0.15 mm Hg. No N-H stretch absorbance was seen in the infrared region. The typical bathochromic shift was observed in the ultraviolet spectrum (126) with Aggga?gfi)(95%) 276, 282, and 294. The refractive index was n35 1.5704 on a product obtained in 45% yield (3.6 g). 1-iso-Propylindole (IV, R =‘iso-propyl) A solution of 9.8 g (0.061 mole) of iggepropyl indoline with 14.9 g (0.061 mole) chloranil in approximately 250 m1 dry xylene was brought to reflux and heated for 6 hours with stirring. After the solution was cooled and filtered, it was washed once with dilute sodium hydroxide to remove some of the substituted hydroquinone, twice with dilute hydro- chloric acid to remove unreacted substituted indoline, and finally £2229: again w rous me was the mm Hg. Ultravi N0 N-H (66) g. l-Q-Bui \ 13410111 Quinone Was re] an oil 49 finally with water. When the solvent had been removed‘yn 22222, ethyl ether was added and the solution was washed again with dilute sodium hydroxide and with water. Anhyd- rous magnesium sulfate was used to dry the solution which was then evaporated to an oil and distilled at 69° and 0.08 mm Hg. Yield was 6.1 g (48%) of an oil with 0%5 1.5755. ethanol (95%) 276, 283, 293. Ultraviolet absorption showed Amax (mu) No N-H band was seen in the infrared Spectra. Michaelis (66) gave the boiling point as 250°. i-grButylindole (IV, R = nabutyl) Nine and one-half grams (0.054 mole) of 1-nrbutyl- indoline plus 12.3 g (0.541 mole) of dichlorodicyanobenzo- quinone in a solution of approximately 250 ml dry xylene was refluxed 6 hours, cooled, filtered, and evaporated to an oil which was distilled at 139-145° at 13 mm Hg. The product was obtained in 60% yield (5.8 g) and showed no N-H stretch in the infrared Spectrum. The 0:5 was 1.5628 ethanol (95%) With Amax (ml-1) 275, 282, and 294. 1- soeButylindole (IV, R = iso-butyl) A solution of 13.1 g (0.0535 mole) chloranil and 9.25 g (0.0530 mole) 1-ig2rbutylindoline was refluxed 8 hours. After cooling, filtering, and washing successively several times with dilute sodium hydroxide, dilute hydrochloric acid, and water, reSpectively, the solution was dryed (magnesium sulfate), filtered with a little Norit A through Hiflow Supercel and finally evaporated. The residual oil distilled at 75° 5 1.5626. and 294. Spectrur I N: 7.91? l-Sec-Bl ‘— mately 1 dichlorl the res. filters: distill; a produ 1:5636 ; absorpt 50 2 at 75° and 0.5 mm Hg for a yield of 5.05 g (54%) with nDS ethanol (95%) max (mp) 276, 283 and 294. No N-H stretch was observed in the infrared 1.5626. Absorption Spectra showed A Spectrum. Analysis: Calod. for C12H15N: N, 8.08%; Found: N: 7.91%: 1-sec-Butylindole (IV, R = sec-butyl) To 11.7 8 (0.067 mole) gggebutylindoline in approxi- mately 250 ml dry xylene was added 15.2 g (0.067 mole) of dichlorodicyanobenzoquinone. When solution was complete, the reaction mixture was refluxed for 6 hours, cooled, and filtered. The filtrate was evaporated to an oil. After distillation at 690 and 0.08 mm Hg, it gave 6.5 g (54%) of a product free of N-H stretch in the infrared. The n35 was 1.5636 after preparative layer chromatography. Ultraviolet absorption showed Agzga?gfi)(95%) 273, 282 and 294. Analysis: Calcd. for C12H15N: N, 8.08%; Found: N, 7.70%. i-tertLButylindole (IV, R = tert-butyl) tégggButylindoline (1.6 g, 0.0103 mole) and 2.34 g (0.0103 mole) of dichlorodicyanobenzoquinone were dissolved in 25 ml xylene and refluxed 6 hours. After the solution was cooled, it was filtered and evaporated. The product was distilled from the residue to yield 1.3 8 (67%). Boil- ing point was 75° at 0.08 mm Hg. The A;::°?;:)(95%) was 274, 281, 292. The n35 was 1.5708 after preparative layer chromatography. Only a very small N-H band was noted in the infrar N, 7.9 i-g-Pe and 10 about soluti the Sc tilled 4.3 g the as kethar max ( N. 70L l-a-D: \ 51 infrared region. Analysis: Calcd. for C12H15N: N, 8.08%; Found: N: 7.97%- 1-nfiPentylindole (IV, R = nepentyl) A solution of 8.4 g (0.0444 mole) of gypentylindoline and 10.1 g (0.0446 mole) of dichlorodicyanobenzoquinone in about 200 ml dry xylene was refluxed for 6 hours. After the solution was cooled and filtered to remove the by-product, the solvent was evaporated. The resulting product was dis- tilled at 82-95° and 0.07-0.08 mm Hg. The yield was 53% or 4.3 g with an n§5 1.5537. Infrared data was consistent with the assigned structure. Ultraviolet absorption was ethanol (95%) Amax (mu) 273, 283, and 294. Analysis: Calcd. for 013H17N: N, 7.48%; Found: N. 7.41%. i-nrDeoylindole (IV, R = grdecyl) After dissolving 11.45 g (0.0443 mole) of nrdecylindo- line in 300 ml dry xylene, 10 g (0.0443 mole) of dichloro- dicyanobenzoquinone was added. When solution was complete, the reaction mixture was refluxed for 6 hours. The solution was cooled, filtered, and evaporated, yielding an oil which distilled at 136-143° and 0.1 mm Hg. Six g (52%) of 25 product was obtained with an “D 1.5264. No N-H band was present in the infrared Spectrum. In the ultraviolet, absorbance was AethanOI (95%) f max (mu) Analysis: Calcd. for 018H27N: N, 5.44%; Found: N: 5.39%- 270, 283, and 294. sole) 0 in 500: in a va and fin: was fil' oil gavi to that l-Q-Ootz \ 1 g-ootadi beHZOQu‘ tion of tion of an 011 . procedu: Still b. filtere, t1lled With Xe I; H: 3.77‘ 52 A similar procedure was followed using 28 g (0.108 mole) of nadecylindoline and 26.6 g (0.108 mole) chloranil in 500 ml xylene. The xylene filtrate was washed extensively in a vain attempt to remove the very dark colored materials and finally dryed with anhydrous magnesium sulfate. Next it was filtered and evaporated. Distillation of the resulting oil gave 8.25 g (29%) of product having properties Similar to that of the material made with dichlorodicyanobenzoquinone. i-nrOctadecylindole (IV, R = neoctadecyl) Hefluxing a solution made of 15.2 g of (0.0412 mole) neoctadecylindoline and 9.35 g (0.0412 mole) dichlorodicyano- benzoquinone in 200 ml xylene for 6 hours resulted in oxida- tion of the substituted indoline. Isolation involved filtra- tion of the cooled slurry and evaporation of the filtrate to an oil which distilled at 210-225° and 0.1 mm Hg. During the procedure, some solid by-product had to be removed from the still before distillation could be completed. The oil was filtered to remove a small amount of solid and was redis- tilled for a net yield of 11 g (59%). The n35 was 1.5108 ethanol (95%) max (mu) Analysis: Calcd. for 026Hy3N: N, 3.79%: Found: N. 3.77%. with A 276, 283, and 294. Ethyl ieAlkyl Indole-3-Acetates O N I + N20H00202H5 ——_>°uC1 CHZCOZCZHS + N2 | R I R IV V VI Ethyl salt < acetai an 10: mole) follo: 20° N] Slowl; react; Separz returi immed. five 1 ture : ether caI‘bo: m1 of °°mbii Sodlu: used . ins d: trace; 381101 53 Eghyl-Diazoacetate (y)_ A solution of 210 g (1.5 mole) of the hydrochloride salt of ethyl glycine ester and 1.05 g (0.0129 mole) sodium acetate in 220 ml of water was cooled in a large beaker in an ice bath. While the solution was stirring, 157.5 g (2.3 mole) of sodium nitrite in 220 ml water was slowly added, followed by 125 ml of ether. The bath was maintained below 20° while 25 ml of a 10% solution of sulfuric acid was added slowly with continued stirring. In about 30 minutes the reaction was complete and the liquid was then drawn into a separatory funnel with vacuum. The aqueous layer was returned to the reaction beaker. The ether solution was immediately washed with cold 10% sodium carbonate. Twenty- five m1 of 10% sulfuric acid was added to the reaction mix- ture and it was re-extracted with 100 ml of ether. This ether extract was also washed with the cold aqueous sodium carbonate. The entire procedure was then repeated with 75 ml of 10% sulfuric acid solution and 100 ml of ether. The combined washed ether extracts were washed with a saturated sodium chloride solution. Anhydrous sodium sulfate was used for drying and the ether was distilled in yggug, Blow- ing dry nitrogen through the solution removed the remaining traces of ether. The yield was 144 g (84%) of a light yellow oil which could be stored in the cold in a dark bottle for over 1 year without loss of activity. Ethyl 1-methylindole—3racetate (VI, R = methyl) A solution of 2.1 g (0.016 mole) 1-methylindole in 54 approximately 15 ml of dry benzene was heated to boiling on a steam bath and a few milligrams of cuprous chloride was added. A reflux condenser was fitted to the flask and a small dropping funnel placed in the top of the condenser. The dropping funnel was charged with an equimolar quantity (1.83 g) of ethyl diazoacetate in approximately 10 ml of dry benzene. Careful addition of the ester solution resulted in steady evolution of nitrogen. When addition was completed, refluxing was continued for 3 to 4 hours, and the resulting brown solution was filtered. The filtrate was evaporated to an oil which was distilled at 125-130° and 0.09 mm Hg. Yield was 1.6 g (52%) and the “:5 1.5580. Ultraviolet absorption ethanol (95%) max (mu) N-H stretch but had intense C=O (5.80 p) and ROC=O (8.65 p) was A 277s, 287 and 2988.. The product Showed no bands in the infrared region. King and L'Ecuyer (46) give a boiling point of 165° at 1 mm Hg: Julia and Tchernoff (40) report 155-60° at 1 mm. Ethyl i-ethylindoleq3-acetate (VI, H = ethyl) A Solution of 2.66 g (0.0183 mole) 1-ethylindole in approximately 15 ml dry benzene was heated to boiling on a steam bath and a few milligrams of cuprous chloride was added. Two and one-fourth grams (0.0197 mole) ethyl diazo- acetate in 10 ml dry benzene was added dropwise through a reflux condenser at such a rate as to allow the evolved nitrogen to escape without undue foaming. When addition was completed, the solution was allowed to continue reflux- ing for another 4 hours. Cooling, filtering, evaporating gave a Yield includ (C=0): (40) r ~thyl l-a-pz reflui catalz mole) of 10 that reflu Then an oi yielc V101: and ; band 55 gave an oil which distilled at 129-140° and 0.1 mm Hg. 25 Yield was 1.6 g (38%) with nD 1.5492. Absorption Spectra ethanol (95%) . neat max (m) 2778, 288 and 297s, Amax (u) 509 (Cso), 8.69 (ROC=O), and no N-H (2.9). Julia and Tchernoff included: A (40) report the boiling point as 165-170° at 1 mm Hg. Ethyl 1fin-propylindole-3-acetate (VI, R = nrpropyl) Two and thirty-eight hundredths grams (0.15 mole) of ifin-propylindole dissolved in dry benzene and brought to reflux on a steam bath was reacted in the presence of a catalytic amount of cuprous chloride with 1.71 g (0.015 mole) of ethyl diazoacetate. Thus, the ester in a solution of 10 ml of dry benzene was added dropwise at such a rate that the nitrogen formed was easily evolved through the reflux condenser. Refluxing was continued for 4 hours. Then the solution was cooled, filtered, and evaporated to an oil which was distilled at 130-140° at 0.12 mm Hg. The yield was 1.25 g (34%) of an oil with n§5 1.5455. Ultra- violet and infrared data were: A$§§°?g&)(95%) 1783, 288, neat max (0 band around 2.9. and 2988: A ) 5.79 (C=O), 8.69 (ROC=O) with no N-H Analysis: Calcd. for C15H19N02: N, 5.71%: Found: N. 5.76%- Ethyl 1-iso-propylindole-3eacetate (VI, H = iso-propyl) To_a refluxing solution of 4.5 8 (0.0282 mole) 1-iSo- propylindole in dry benzene (15 ml) was added a catalytic amount of cuprous chloride. Dropwise addition of 3.18 g (0.027 lowed which produc after ficati matogz showed (0:0) 3‘ inf: and 56 (0.0279 mole) ethyl diazoacetate in 10 ml dry benzene fol- lowed by a reflux period of 4 hours resulted in a solution which was filtered, evaporated, and distilled to yield the product. Three and four—tenths grams (44%) was collected after distillation at 128-1380 and 0.15 mm Hg. After puri- fication of an analytical sample on preparative layer chro- matography, the n:5 was 1.5462 and the absorption Spectra Showed: Aeth°?°1)(95%) 277s, 287, and 2988: Aneat 5.79 max mu max (h) (C=O), 8.64 (ROC=0), with no N-H band near 2.9. Analysis: Calcd. for C15H19N02: N, 5.71%: Found: N, 5.67%. Ethyl 1enebutylindole-3—acetate (VI, H = grbutyl) Ethyl diazoacetate (5.6 g, 0.049 mole) in 15 ml dry benzene was added dropwise to a reflux dry benzene solution of 5.7 8 (0.033 mole) of ifinrbutylindole containing a few mgs of cuprous chloride. The rate of addition allowed for a Slow steady evolution of nitrogen. After addition was completed, the solution was refluxed for 4 more hours. The oil which resulted after the solution was filtered and evaporated distilled at 159-165° and 0.5 mm Hg. A yield of 2 1‘D 278s, 287, and 2973: the 3.9 g (45%) was obtained with 5 1.5384. Ultraviolet ethanol (95%) max (mu) neat infrared region showed Amax (u and no band at 2.9 (N-H). absorption was A ) 5.79 (C=O), 8.68 (ROC=0), ' Analysis: Calcd. for Cl6H21N02: N, 5.40%: Found: N. 5.59%. thyl 2 acetati 2.45 8 gave a: tion w: filter was f1: 2 ligh ethan max (; 8.62 (j N! 505 Ethifl, : \ bUtYIii bath a) slow a: ethYl c Such a tion we and Cor distill (41%). and 298 2'9 (r. 7 57 Ethyl 1eigggbutylindole-3-acetate (VI, R = gggybutyl) Slow addition of 1.61 g (0.0141 mole) of ethyl diazo- acetate in 10 ml of dry benzene to a refluxing solution of 2.45 g (0.0141 mole) 1-lgg—butylindole in 10 ml dry benzene gave after isolation 1.5 g (41%) of the product. The solu- tion was fully reacted by refluxing for 4 hours, cooling, filtering, and evaporating to a small volume. The product was finally distilled at 130-145° and 0.3 mm Hg to yield aslight oil. Physical constants found were: n35 1.5390; ethanol (95%) . neat _ Amax (mu) 277s. 287. and 297s. kmax (H) 5.79 (0-0). 8.62 (ROC=O) with no band near 2.9 (N-H). Analysis: Calod. for C16H21N02: N, 5.40%; Found: N: 5.54%- ' Ethyl 1-secabutylindole-3-acetate (VI, R = sec-butyl) Five and eight-tenths grams (0.0333 mole) of 1-§§gr butylindole in 15 ml dry benzene was refluxed on a steam bath and a catalytic amount of cuprous chloride was added. Slow addition of a solution of 3.82 g (0.0333 mole) of ethyl dizaoacetate in benzene was begun and continued at such a rate that nitrogen was smoothly evolved. The solu- tion was heated for 4-6 hours longer, cooled, filtered, and concentrated in ygggg_to an oil. The resultant oil distilled at 135-145° and 0.1 mm Hg. The yield was 3.6 g ethanol (95%) (41%). Absorption spectra showed Amax (mu) 2773, 288, and 298s; A3::t(u) 5.78 (C=O), 8.61 (ROC=O), no band near 25 2.9 (N-H). The refractive index was "D 1.5416. GSter the ar SaVe a Cthar (max ( and 8, aCetat l‘fl‘pe: as to 1 Four hi 58 Analysis: Calod. for C16H21N02: N, 5.40%; Found: N, 5.78%. Ethyl 1-tert-butylindole-3-acetate (VI, R = tert-butyl) A dry benzene solution (10 ml) of 1-Egggrbutylindole (2.13 g, 0.0123 mole) was brought to boiling on a steam bath and a few mgs of dry cuprous chloride were added. To this mixture was carefully added a solution of 10 ml of benzene in which was dissolved 2.1 g (0.0185 mole) ethyl diazoacetate. After addition was completed, refluxing was continued for 2 hours. The solution was cooled, filtered, evaporated, and the residue distilled at 127-140° at 0.09 mm Hg. One and six-tenths grams (67%) of the substituted ester was obtained with an n35 of 1.5450. Purification of the analytical sample on preparative layer chromatography gave a product with ”:5 1.5431. Absorption data include: ethanol (95%) , neat max (m) 2778. 2859 and 2958, Amax (H) 5.78 (C=O) and 8.65 (BOC=O). No band at 2.9 (N-H) was seen. A 'Analysis: Calcd. for 016H21NOZ: N, 5.40%: Found: N: 5.55%- Ethyl 1~gfpentylindole-3-acetate (VI, R = nrpentyl) DrOpwise addition of 2.15 g (0.0189 mole) ethyl diazo- acetate in 10 ml of dry benzene to a refluxing solution of 1-nppentylindole (4 g, 0.0214 mole) containing a catalytic amount of dry cuprous chloride was continued at such a rate as to allow for constant evolution of the nitrogen by-product. Four hours after all of the ester had been added, the solution was coc was fol pressu1 yellow 2773, 2 and no 1053260 N. 5.21 EthYl 1 \— (0.019 quantit 2-6 a ( was beg refluxe ation 0 residUe (50%). t1Ve la inchide 5'79 (c: J I N. 4.19; Ethyl 1. ~~.\~‘~~ l 59 was cooled and filtered. Removal of the benzene ;n_y§ggg was followed by distillation at 140-160° under 0.09 mm pressure to yield 2.6 g (44%) of the product. The clear yellow oil showed absorption as follows: A;;:°?g&)(95%) 2778, 288, and 298s: A:::t(u) 5.79 (C=O), 8.70 (BOC=O), and no N-H band near 2.9. The refractive index was n§5 1.5326. Analysis: Calcd. for C17H23N02: N, 5.12%; Found: N. 5.21%. Ethyl 1ggydecylindole-3-acetate (VI, R = nedecyl) To a refluxing dry benzene (15 ml) solution of 6 g (0.019 mole) of iegedecylindole was added a catalytic quantity of anhydrous cuprous chloride. Then addition of 2.6 g (0.023 mole) of ethyl diazoacetate in 10 ml benzene was begun. After addition of the ester, the solution was refluxed for an additional 3 hours. Filtration and evapor- ation of the solution was followed by distillation of the residue at 166-1880 and 0.1 mm Hg for a yield of 3.25 g (50%). The refractive index was 0:5 1.5164 after prepara- tive layer chromatography purification. Absorption data include: xgzgangfi)(95%) 277s, 287, and 297s; A:::t(u) 5.79 (C=O), 8.70 (ROC=O), and no N-H near 2.9. Analysis: Calcd. for C22H33N02: N, 4.08%; Found: N, 4~19%. Ethyl 1-n_-octadecylindole-3-acetate (VI, R = _r_1_-octadecyl) 1gn¢0ctadecylindole (8.5 g, 0.023 mole) dissolved in approxi a few 1 Slow a: diazoa( a rate being filter at 0.1 water The ab 298s; observ N. 3.c C 60 approximately 20 ml of dry benzene was heated to reflux and a few milligrams of cuprous chloride were placed in the flask. .Slow addition of an equimolar quantity (2.63 g) of ethyl diazoacetate in 10 ml benzene was begun and continued at such a rate as to allow for constant evolution of nitrogen. After being heated 8 hours, the dark brown solution was cooled, filtered, and evaporated to an oil which distilled at 222-260° at 0.11 mm Hg. This oil could be crystallized from methanol- water for a mp 38°. The yield was 6 g (58%) with n35 1.5069. ethanol (95%) max (mm) 78 (0:0) and 8.68 (ROC=0). No N-H band was The absorption spectra was A neat max (u) 5’ 2788, 287, and 2988: A observed. Analysis: Calcd. for C N, 3.05%. 30°49N°2: N, 3.07%: Found: 1-Alkyl Indoleg3-Acetic Acids ' CH 0 0 $329009sz + Na0H V m 2 0 H a N l | R R VI VII i-Methylindole-3-acetic acid (y;;. R = methyll Five grams (0.023 mole) of ethyl 1-methylindoleJ3- acetic acid was saponified in 100 ml of approximately z‘y aqueous methanolic sodium hydroxide by refluxing for 6 hours. The solution was extracted with ether after evaporation of the solvent and re-addition of water. Neutralization of the aqueous phase was followed by extraction of the free acid into at chloride talline anol by gave an values v showed ) (cooH) : l-Ethyl l-methy With 2 the So] SOIUtic with c( re~ext "as'wa talliz tal112 61 into ether. After the ether was dryed with anhydrous calcium chloride and evaporated, the yield was 3.7 g (85%) of a crys— talline solid which was then recrystallized from cold meth- anol by addition of water. Recrystallization from benzene gave an analytical sample with mp 127-128°. Literature values were 127-128° (40) and 128° (4). Absorption Spectra showed A°th°n°l)(95%) 2773, 287 and 298s: Agpr (g) 3-4 max (mu ax (COOH) and 5.89 (acid C=O). 1-Ethylindole-3-acetic acid (VII. B = ethyll One and four-tenths grams (0.0061 mole) of ethyl 1-methylindole-3-acetate was saponified by boiling the ester with 2 N_solution of methanol-water (50:50). After 8 hours, the solvent was removed AE.Y§222 and water was added. This solution was extracted twice with ethyl ether and neutralized with concentrated hydrochloric acid. The aqueous phase was re-extracted with ether to remove the product. The ether was washed, dryed, and evaporated. Then the acid was crys- tallized from methanol-water to yield 0.75 8 (63%). Recrysu tallization from benzene-hexane gave a product melting at 107-108°. The literature gives a range of values: 102° (40), 106° (4). Infrared and ultraviolet Spectra showed KBr . ethanol (95%) Amax (p) 3-4 (COOH), 5.89 (801d C=0), Amax (mu) 2788, 288, and 298s. 1-nyPropylindole-3-acetic acid (VII, R = nepropyl) Three grams (0.0122 mole) of ethyl i-nrpropylindole- 3-acetate dissolved in methanol to which an equal volume of 6 hot and t lizat was U twice evapo metha lizat The 1 xetha max 5.87 62 4 N sodium hydroxide had been added was heated at reflux for 6 hours. Upon evaporation of the solvent, water was added and the solution extracted twice with ether. After neutra- lization of the aqueous solution with concentrated HCl, ether was used to extract the acid. This ether extract was washed twice with water, dryed with anhydrous calcium chloride and evaporated to produce a residue which was crystallized from methanol-water to yield 1.65 g (63%) of product. Recrystal- lization from benzene-hexane gave a melting point of 80-81°. The literature value was 85° (4). Absorption data were ethanol (95%) KBr max (mu) 278s, 288, and 298s; Amax (H) 3 5087 (0:0) 0 A -L|’ (801d. 0:0), 1-iSo-Propylindole-3-acetic acid (VII, R = iso-propyl) Two normal sodium hydroxide in 50% methanol contain- ing a suSpension of 1 g (0.0041 mole) ethyl igigg-propyl- indole-3-acetate was refluxed 6 hours and evaporated in’ ygggg.and the residue was redissolved in water. After the solution was extracted twice with ethyl ether, it was neutralized with concentrated hydrochloric acid and re- extracted with ether. Washing of the ether extract and dry- ing with anhydrous calcium chloride was followed by evapor- ating the ether and dissolving the residue in methanol. Gradual addition of water to the cold methanol solution caused precipitation of 0.4 g (45%) of crystals, mp 104.5- 105.5°. The melting point was not changed upon recrystal- lization from benzene-hexane. Absorption data were ethanol (95%) KBr max (mu) 2793, 288, and 2988; A A max (u ) 3-4 (COOH), 63 5.88 (acid C=O). Analysis: Calcd. for C13H15N02: N, 6.45%; Found: N, 6.46%. legrButylindole-3-acetic acid (VII, R = nabutyl) Saponification of 2 g (0.077 mole) ethyl 1-nrbutyl- indole-3-acetate with 2 g sodium hydroxide in 50% methanol for 6 hours at reflux gave the title acid in 78% yield (1.4 g); After the solvent was evaporated and water was added, the basic solution was extracted with ethyl ether, which was then washed with water. When the ether had been dryed with anhydrous calcium chloride, it was evaporated to a residue which was crystallized from cold methanol by care- ful addition of water. Recrystallization of this material from benzene-hexane mixture gave an analytical sample with ethanol (95%) max (mu) 3-4 (COOH), 5.88 (acid C=O). melting point 75-760. Absorption showed A KBr 6 , max (0) Analysis: Calod. for °14HI7N°2: N, 6.06%: Found: 2783, 288, and 2988; A N, 6.10%. 1-iso-Butylindole-3-acetic acid (VII, R = iso-butyl) A methanol solution of 1.38 (0.0053 mole) ethyl leggg— butylindole-3-acetate was treated with an equal volume of 4 §_sodium hydroxide and refluxed 6 hours. The solvent was removed yg.y§ggg_and water was added again. Extraction of the aqueous solution with ethyl ether was followed by neutra~ lization with concentrated hydrochloric acid and ether extraction of the product. The ether was evaporated and the res! (57? not com; foll refl 64 residue was crystallized from methanol-water giving 0.7 g (57%) material with a melting point of 93-94°, which was not improved by recrystallization from benzene-hexane. The compound absorbed infrared and ultraviolet radiation as KBr 3 ethanol (95%) max (0) max (mu) 278s, 288, and 2988. follows: A -4 (COOH), 5.87 (acid C=O); A Analysis: Calcd. for C14H17N02: N, 6.06%: Found: N. 5.98%. 1-Sec-Butylindole-3-acetic acid (VII, R = sec-butyl) Saponification of a methanol solution of 1.4 g (0.0054 mole) ethyl 1-§ggybutylindole-3-acetate was effected by refluxing it with an equal volume 4 N sodium hydroxide for 6 hours. Evaporation in,ygggg_and readdition of water produced a solution which was then extracted with ethyl ether, neutra- lized with concentrated hydrochloric acid, and re-extracted with ether. The latter extract was dryed with anhydrous calcium chloride and evaporated to a mass which was crystal- lized from methanol-water and recrystallized from benzene- hexane for a yield of 0.75 g (60%). Its melting point was ethanol (95%) 64-64.5°, and it showed absorption as follows: A max (mu) KBr 278s, 288, and 298s, Amax (u) 3-4 (COOH), 5.87 (acid 0:0). Analysis: Calcd. for C14H17N02: N, 6.06%; Found: N, 6.02%. 1-tert-Butylindole-3-acetic acid (VII, R = tert-butyl) Ethyl 1-tert-butylindole-3-acetate (1.4 g, 0.0054 mole) was dissolved in methanol and an equal volume of 4 N sodium and eva twice w; extract extrac ti solvent by the z slightl; Solvent 1128131 01 104,5-11 ) Ethane: ‘max (m; 5:37 (a: f 1-n-P . Ken- I I an°1 (5< 3-aceta1 agid in 65 sodium hydroxide added. The solution was refluxed 6 hours and evaporated. Water was added and the solution extracted twice with ethyl ether. Neutralization of the aqueous extract with concentrated hydrochloric acid was followed by extraction of the product with ether. Evaporation of the solvent gave a residue which was crystallized from methanol by the addition of water for a yield of 0.3 s (25%). The slightly green crystals were recrystallized from the same solvent system by using Norit A charcoal. Final crystal- lization from benzene-hexane gave white crystals with mp 104.5-105.5°. Ultraviolet and infrared absorption were: KBr max (W) 3-4 (COOH) Afi§£°?g&)(95%) 276s, 286, and 2958; A 5087 (301d. C=O) 0 Analysis: Calcd. for 014H17N02: N, 6.06%; Found: N. 6.27%. 1anyPentylindole-3-acetic acid (VII, R = nrpentyl) Saponification in 2 y_sodium hydroxide aqueous meth- anol (50%) of 2 g (0.0073 mole) of ethyl l-gypentylindole- 3-acetate by refluxing for 6 hours gave the correSponding acid in 81% yield (1.46 g). Isolation of the acid was effected by removing the solvent from the basic mixture, redissolving the salt in water, extracting with ethyl ether to remove neutral impurities, and finally neutralizing to free the acid. Ether extraction of this solution followed by evaporation of the ether gave a solid which was crystal- lized (Norit A) from methanol-water. The melting point was 63-64°. and 298 N, 5.66 i-g-Dec u ethyl 1 sodium the est Was add neutral re-extr the Sol liZed f benZene ab80rpt )KBI‘ max ((1 3“. 4.1.4 1~n~oct \ 66 63-64°. Absorption data were: A:::°?;&)(95%) 278s, 288, KBr max (H) 3-4 (COOH) 5.87 (acid C=O). Analysis: Calcd. for 015H19N02: N, 5.71%: Found: N. 5.66%. and 2988; A i-nrDecylindole-3-acetic acid (VII, R = nrdecyl) Refluxing a methanol solution of 2.3 g (0.0067 mole) ethyl 1-nydecylindole-3-acetate with an equal volume of 4 N sodium hydroxide for 6 hours resulted in saponification of the ester. After the solvent was removed iniygggg, water was added. Extraction with ethyl ether was followed by neutralization with concentrated hydrochloric acid and re-extraction with ether to remove the product. Removal of the solvent gave a solid (1.75 g. 83%) which was crystal- lized from methanol and water. It was recrystallized from benzene and hexane to give a product with mp 51-51.5°. The ethanol (95%) max (mu) -4 (COOH) and 5.88 (acid C=O). absorption Spectra showed A 2783, 288, and 298s; KBr max (1:) 3 Analysis: Calcd. for CZOH29N02: N, 4.44%; Found: N, 4.44%. A 1-g¢Octadecylindole-3-acetic acid (VII, R = groctadecyl) Saponification of 4 g (0.0093 mole) ethyl 1-nrocta- decylindole-B-acetate was effected by refluxing a methanolic solution of the ester in a like volume of 4 N_sodium hydrox- ide. After removal of the solvent ;n_ygggg.and readdition of water, the basic solution was extracted with ethyl ether. Neutralization with concentrated hydrochloric acid was- Luau It‘s!” folloWE The at? a solid Norit A point w” 2785, 2: 1 N. 3.31;» which he ProcedUr of abSOl tOluenes on an e]. was I‘emo and the with mag] tion. Va After St' m"Herbal Hater gav 67 followed by extraction of the carboxylic acid with ether. The ether was washed and evaporated to give 3.35 8 (90%) of a solid which was crystallized three times from methanol. Norit A was used on the final crystallization. Melting ethanol (95%) max (mu) 278s, 288, and 298s: AKBr 3-4 (COOH) and 5.85 (acid 0:0). max (0) Analysis: Calcd. for C28H45N°2: N, 3.28%; Found: N, 3.31%. point was 75.5-76.5° with absorption as follows: A Ethyl Indole-3-Acetate Seventy-five grams of crude indole-3-acetic acid, which had been prepared by the high temperature autoclave procedure of Johnson and Crosby (89), was dissolved in 2 l of absolute ethanol to which had been added 4 g of pr toluenesulfonic acid. The solution was refluxed 16 hours on an electric heating mantle and cooled, and the ethanol was removed in.yggug, Ethyl ether was added to the residue and the solution was extracted with dilute sodium bicarbon- ate. After washing the ether layer with water and drying it with magnesium sulfate, the solvent was removed by distilla- tion. Vacuum distillation of the residual oil at 1550 at 0.8 mm Hg afforded a clear liquid in 75.8% yield (65.8 g). After standing in a refrigerator for several hours, the material crystallized. Recrystallization from methanol and water gave white needles mp 42-43°. 68 Ethyl i-para-Substituted Benzylindole-3chetates I 0H2 C::I:—ETCHZCOOC2H5 DMF ; ,/\| 0H200002H5 N NaH .\/LN” I Y I H 9H2 Ix x [::] XI Y Ethyl 1-benzylindole-3yacetate (XI, Y =.Hl Eight and fifty-four hundredths grams (0.042 mole) of ethyl indole-3-acetate dissolved in about 50 ml dry dimethyl- formamide was added slowly to a stirred dimethylformamide (109) suSpension of 2.12 g (0.044 mole) sodium hydride (50% in oil). After the ester had been added, stirring was con- tinued for 30 minutes. The solution was cooled to 0° and 5.31 g (0.042 mole) of benzyl chloride in approximately 50 ml of dimethylformamide was added Slowly while the reaction mixture was stirred. The reaction mixture was allowed to warm to room temperature overnight and was then heated at 60° for 3 hours. After removal of the solvent ;g_y§ggg, the remaining oil was distilled at 170° and 0.1 mm Hg. Julia and Tchernoff (40) report 180-200° at 0.8 mm Hg but do not report a melting point. Careful addition of water to a methanol solution of the oil resulted in crystallization. The melting point was 44-44.5°. The yield was 4.55 g (39%). A similar yield was obtained when the oil prepared as described above was subjected to preparative layer 69 chromatography rather than distillation. An analytical 1 d Ab ti t ethanol (95%) as e was 8 re are . sor on s ec ra were NP 0 P P KBr P P max (mu) 277s, 285, and 296: Amax (u) 5.78 (0:0), 8.70 (ROC=O). Analysis: Calcd. for €19H19N02: N, 4.77%: Found: N, 4.77%. It was then evident that a significant amount of by-product, which had been isolated in the chromatographic procedure, was present. This by-product was characterized as the benzyl ester of indole-3-acetic acid through consider- ation of its ultraviolet and infrared Spectra. Ethyl 1fiprfluorobenzylindole-3-acetate (XI, Y = F) Ethyl indole-3-acetate (12.8 g, 0.063 mole) in approx- imately 75 ml of dry dimethylformamide was added dropwise to a stirred dimethylformamide suSpension of 3.18 g (0.66 mole) of 50% sodium hydride in oil. The mixture was stirred for 20 minutes and then was cooled to 0°. A dimethylformamide solution of 9.64 g (0.067 mole) of Effluorobenzyl chloride was added slowly while the reaction mixture was stirred and its temperature maintained at 0°. When addition of the halide was complete, the mixture was allowed to warm to room temperature and was stirred for 16 hours longer. (After chloroform and water were added and the chloroform was removed, the aqueous solution was extracted a second time with chloroform. The combined chloroform extracts were washed extensively with water, dryed with anhydrous calcium chloride, and evaporated to yield an oil. The oil distilled at 170-180° and 0.01 mm Hg and yielded 8.5 s (44%). The oil 70 could be crystallized from methanol-water. However, in order to get a pure product devoid of N-H stretch in the infrared spectrum, it was necessary to purify the material on prepara- tive layer chromatography. The melting point was 69°. Absorption spectra showed Aggga?gfi)(95%( 267. 272, 285. and 295: A§2§ (0 Analysis: Calod. for 019H18N02F: N, 4.50%: Found: N, Ll'gu7zO ) 5.79 (C=O). 8.64 (H00:0). Ethyl prchlorobenzylindole-3-acetate (XI, Y = Cl) To a stirred suspension of 1.06 g (0.022 mole) of sodium hydride (50% in oil) in dry dimethylformamide was added 4&26 g (0.021 mole) of ethyl indole-3-acetate. After addition of the ester was completed, the mixture was stirred for 20 minutes and cooled to the temperature of a dry ice- acetone bath. Three and fifty-eight hundredths g (0.0222 mole) of pychlorobenzyl chloride in approximately 50 ml dimethylformamide was added dropwise to the cooled solution. Stirring was continued as the solution was allowed to warm overnight to room temperature. After water was added, the solution was extracted twice with chloroform. The combined chloroform extracts were washed well with water and dryed with anhydrous calcium chloride. After the solvent was evaporated, the residual oil was fractionally crystallized from methanol-water. The first fractions were the title compound which was present in 43% yield (3 g). Recrystal- lization from methanol gave a product with improved color. 71 Preparative layer chromatography was employed for purifica- tion of the analytical sample. The sample melted at 96-98°. Absorption Spectra were consistent with the assigned struc- ethanol (95%) . KBr max (mp) 277, 285, and 2959 Xmax (u) 5079 (0:0), 8.60 (300:0). ture: A Analysis: Calod. for C19H18N02Cl: N, 4.27%; Found: N, 4.22%. Ethyl 1epybromobenzylindolee3-acetate (XI, Y = Br) One hundred six milligrams (0.0022 mole) of sodium hydride (50% in oil) was stirred in 10 ml dry dimethylfor- mamide while 0.43 g (0.0021 mole) of ethyl indole-3-acetate was added dropwise. After the green solution was stirred for 20 minutes and then cooled to 0°, drOpwise addition of 0.557 g (0.00222 mole) of prbromobenzyl bromide was begun. Stirring was continued overnight as the solution warmed to room temperature. Water was added and the solution was extracted twice with ethyl ether. The ether solution was washed extensively with water and then dryed with anhydrous calcium chloride. Next the ether was removed ;QDy§gug_and a little methanol and water were added. After sitting in the cold for a day, 0.24 g (31%) of the product was iso- lated. (Additional material could be obtained from the fil- trate by preparative layer chromatography. ethanol (95%) The absorption data were Amax (mu) 274, 283, and 296s; AKBr 5.79 (C=O), 8.45 (ROC=O). The melting max (u) point was 104-104.5°. 72 Analysis: Calcd. for C19H18N02Br: N, 3.76%: Found: No 3.81%9 Ethyl 1~prmethylindole-3-acetate (XI, Y = CH3) To a stirred suspenSion of 0.106 g (0.0022 mole) of sodium hydride in 10 ml dry dimethylformamide was added 0.46 g (0.0021 mole) of ethyl indole-3-acetate in 10 ml dimethylformamide. After all the ester was added, the mix- ture was stirred for 20 minutes and then was cooled to 0°. To the cooled solution 0.313 g (0.00222 mole) of prmethyl- benzyl chloride in 10 ml dimethylformamide was added slowly with stirring. The mixture was stirred and allowed to warm to room temperature overnight. Water was added and the product extracted with two portions of ethyl ether. The pooled ether extracts were washed several times with water, dryed with anhydrous calcium chloride, and evaporated to give an oil. Five milliliters of acetone was added. One and one-fourth ml of this solution was streaked on a pre- parative layer chromotography plate and developed in 18% butanone-hexane. The product moved at a higher Rf (0.4) than did the chief by-product--an ester described below. Yield was 80 mg (41%). The melting point was 65.5°. ethanol (95%) . Absorption data included Amax (mu) 275, 285, and 296s, KBr max ((1) Analysis: Calcd. for C20H21N°23 N, 4.56%: Found: N, 4.61%; A 5.79 (C=O), 8.70 (ROC=O). 73 Ethyl 1sprmethoxybenzylindole-3-acetate (XI, Y = CHBO) Eight and fifty-four hundredths grams (0.042 mole) of ethyl indole-3-acetate in 100 ml dimethylformamide was added dropwise to a stirred dimethylformamide suspension of 2.12 g (0.1044 mole) sodium hydride (50% in oil). Having been stirred for 20 minutes, the solution was cooled to 0°. Seven grams (0.0444 mole) of prmethoxybenzyl chloride (which had been synthesized from anisyl alcohol and hydrogen chlor- ide in benzene solution at -10°) in 50 ml dimethylformamide was added drop by drop while maintaining vigorous agitation. The mixture was allowed to stir for 24 hours as it warmed to room temperature. Water was added and the solution was extracted twice with chloroform. The chloroform extracts were pooled and washed freely with water. After drying the extract with anhydrous calcium chloride, the solvent was removed gg.ygggg to yield approximately 50% (6.1 g) of crude product; Attempts were then made to crystallize the residual oil. While it was not possible to effect crystallization at this point, a sample of the saponified material was later re-esterified and purified with preparative layer chromato- graphy to yield the analytical sample with the melting point ethanol (95%) 0 being 38.5-40 . The absorption showed Amax (mp) 278, 284, and 297s; AKBr 5.79 (C=O), 8.60 (ROC=O). max (0) Analysis: Calcd. for C20H21N03: N, 4.33%: Found: N, 4.25%. 74 1fip-Substituted Benzylindole-3-Acetic Acids CHZCOOH CHZCOOH ‘ Na0H gy l I’ I N CH2 CH2 0 XI 0 XII Y Y 1-Benzylindole-3-acetic acid (XII, Y = H) One gram (0.00341 mole) of crystalline ethyl i-benzyl- indole-3-acetate was dissolved in approximately 25 ml of methanol. An equal volume of 19% sodium hydroxide was added and the mixture was refluxed for 6 hours. After the solvent was distilled ig,ygggg, the residue was taken up in methanol. Water was added to the salt and the basic solution extracted with ethyl ether. Hydrochloric acid was used to neutralize the aqueous extract, which was then extracted with ether. After the ether phase was washed with water and evaporated to dryness, the residue was dissolved in methanol. Water was added to precipitate the product as white crystals. mp 155-156°. Julia and Tchernoff (40) reported mp 149°. The ultraviolet and infrared Spectra showed these features: ethanol (95%) KBr "max (mu) max (0) 3 ‘5.88 (C=O). 2778, 286, and 296s: A -4 (O-H). 1eerluorobenzylindole-3-acetic acid (XII, Y = F) One hundred milligrams (0.00032 mole) of ethyl p- 75 fluorobenzylindole-3-acetate, which had been purified with preparative layer chromatography, was treated with a 50% methanolic solution of 3.5 N_sodium hydroxide. The solution was heated at reflux for 2 hours and then evaporated to dry- ness ;n_ygggg. Water was added to the residue and the solu- tion extracted with ether. After the solution was neutra- ! lized with concentrated hydrochloric acid, the product was extracted with ether. The extract was washed twice with l water and the ether was removed by distillation. Then the " residue (39 mgs, 45%) was dissolved in methanol and water was added to the point of turbidity.’ Crystals formed after the flask was allowed to stand in the cold overnight. The melting point was 159.5-160.5°. Absorption of the compound ethanol (95%) max (mu) (043). 5.86 (C=O). was A 267, 273, 289, and 296s; AEZ: (H) 3-4 Analysis: Calod. for C17H14N02F: N, 4.94%; Found: N, 4u96%. 1gp¢Chlorobenzylindole-3-acetic acid (XII, Y = Cl) A methanol solution of 135 mg (0.00041 mole) of ethyl 1eprchlorobenzylindole-3~acetate, which had been purified with preparative layer chromatography, was combined with a like volume (10 ml) of 7 N_sodium hydroxide. Saponification of the ester was effected by refluxing the solution 2 hours. After the solvent was evaporated, the residue was dissolved in water and the solution extracted with ethyl ether. Hydro- chloric acid was employed to neutralize the base and the 76 product was then extracted into ethyl ether. The product crystallized from benzene diluted with hexane. The yield was 106 mg (85%) of white crystals, mp 145.5-147.5°. ethanol (95%) max (mu) -4 (801d OH), 5.88 (C=O). Absorption data of the compound showed A 277, KBr max (0) 3 Analysis: Calcd. for C17H14N02Cl: N, 4.67%: Found: N, 4.68%. 285, and 2958; A 1-p:Bromobenzylindole-3-acetic acid (XII, Y = Br) Ten milliliters of 7 y sodium hydroxide was added to an equivalent volume of methanol containing 35 mg (0.000094 mole) of ethyl 1epybromobenzylindole-3—acetate which had been purified previously with preparative layer chromato- graphy. .After the solution was heated at reflux for 2 hours and evaporated to dryness, water was added to the residue and the basic solution extracted twice with ethyl ether. Concentrated hydrochloric acid was used to neutralize the solution, which was then extracted with ether. The ether was washed with water, dryed with anhydrous calcium chloride, and removed from the residue by distillation $n,y§gug, The product crystallized from benzene in a yield of 17 mg (54%). The absorption was A322°?g:)(95%) 277, 285, 295: AEE: (M) 3-4 (acid OH). 5.87 (C=O), and a small band at 2.9 (H20 or N-H). The melting point was 152°. Analysis: Calcd. for C17H1yN02Br: N, 4.07%: Found: II, 4.06%. 77 1eprMethylbenzylindole-3-acetic acid (XII, Y = CH3) After ethyl 1-p¢methylbenzylindole-3-acetate (90 mg) was dissolved in 10 ml methanol, 10 ml N sodium hydroxide was added. The solution was refluxed 2 hours then evapor- ated to dryness. Water was added and the solution was extracted with ethyl ether. The aqueous solution was neutralized with concentrated hydrochloric acid and extracted with ethyl ether. After the ether extract was washed with water and dryed with anhydrous calcium chloride, the ether was removed by distillation. Benzene served as a useful solvent for crystallization. The melting point was 125-126°. A yield of 56 mg (68%) was obtained with ethanol (95%) . max (mu) 274, 286, 2963, -4 (acid-OH), 5.86 (0:0). absorption data as follows: A KBr 3 max (0) Analysis: Calcd. for °18H17N°2: N, 5.01%: Found: N. 5.00%. A 1-p:MethoxybenZylindole-3-acetic acid (XII, Y = CHBO) First six grams (0.0176 mole) of crude ethyl l-py methoxyindole-3-acetate was dissolved in methanol: an equal 'volume of 4 N'sodium hydroxide was then added. The solution 'was refluxed 6 hours and evaporated to dryness ig,y§ggg. Water was added and the solution was extracted with ethyl ether. After the alkaline solution was neutralized with concentrated hydrochloric acid, the product was extracted ‘with.ether; Distillation of the ether ;n_y§ggg yielded a :residue which was crystallized from methanol and water. 78 Recrystallization of the product from methanol and finally from benzene gave the analytical sample in 51% yield (2.8 g). The compound melted at 136-137°. The following absorptivity KB 322393;)(955) 278, 284 and 296s: Ana: (0) 3-4 (acid OH), 5.87 (0:0). was noted: A Analysis: Calod. for C18H17N03: N. 4.74%: Found: N. 485%. 1-(2',3',4',6'-Tetra-O:Acetyl-B-D—Glucopyranosyl) Indoline (XIV) AoO-CH2 0 H N 32 Ac Br . > N H2 l ' OAc H ADD-CH2 o I XIII Ac XIV Ac Ab Three grams (0.025 mole) of indoline was reacted with 10.3 g (0.025 mole) of 1-(2,3,4,6-tetra-O-acetyl~a-Dr glucopyranosyl) bromide in the presence of 2.6 g (0.025 mole) anhydrous, powdered sodium carbonate at 0°. Anhydrous ethyl ether was added to make a slurry and this mixture was stirred while it warmed to room temperature. After the solution was refluxed for 3 days, water was added to the mixture and the product was extracted twice with ether. Then the organic layer was washed twice with water and dryed over anhydrous magnesium sulfate. Removal of the solvent ;n_y§ggg,was followed by crystallization of the product from 95% ethanol. Thin layer chromatography on Eastman sheet 6060 showed a 79 single Spot with R 0.39 when develOped in 25% butanone- f hexane. The dimethylaminocinnamaldehyde Spray reagent gave a red Spot on the chromatogram. The yield was 7.38 g (66%) 1 with a melting point of 117-118°, [d]§5 4.0° (0H013, 0:1). The literature (98) gives 117.8-118.5° and [s]§° 5.5° (0014). 1-(2'.3'.4'.6r-Tetra-o-Acetyl-g-Glucopyyanosyl) Indole XV 0 OH H Cl CN Cl N :17?- + ——> Cm + N H2 Cl CN N. Cl CN 0 ‘ OH AcO-CH AGO-032 AC XIV AC XV Ac AcO 0A0 Ac 1-(2',3',4',6'-Tetra-O-acetyl-8-D-glucopyranosyl) indoline (7.35 g. 0.016 mole) was dehydrogenated with 3.72 g (0.0164 mole) dichlorodicyanobenzoquinone (DDQ) in a solu- tion of 150 ml xylene by refluxing for 6 hours. This solu- tion soon lost the dark red-violet color, and the substituted hydroquinone by-product precipitated. After filtering the warm solution, the solvent was removed ;g_ygggg and the residue crystallized frOm methanol. This material moved as one Spot at R 0.26-0.28 in 25% butanone-hexane on Eastman f sheet 6060 thin-layer chromatography. The dimethylamino- cinnamaldehyde reagent slowly turned a blue color. Yield was 5.85 g (79%): melting point 148-149.5°. The literature value 1Optical rotations were determined by using a Bendix Automatic Digital Polarimeter. 80 for the melting point was 148.5-149.5O (98). The optical rotation was found to be [d];5 1.30 (CHCl3, 0:1). The reported optical rotation was [9150 1.5° (CHClB). Ethyl 1-(2'43',4',6'~Tetra-O-Acetyl-g-D-Glucopyyanosyl)- 3gIndole Acetate (XVI) Qj .:Nj.CHZCOOCZH5 N + N20H00002H5 -—9 A00- H AGO-CH2 A XV V A XVI AcO AbO OAc OAc After a solution of 0.85 g (0.0019 mole) 1-(2',3',4', 6i-tetra-O-acetylglucopyranosyl)-indole in 10 ml xylene was brought to reflux on a wax bath, a catalytic amount of anhydrous cuprous chloride was added: To the refluxing solution was added dropwise a solution of 1.2-2.5 g (0.0073- 0;02 mole) ethyl diazoacetate (V) in 10 ml xylene. Addition was continued at such a rate as to allow slow evolution of the nitrogen gas which formed. When addition of ethyl diazoacetate was completed, the reaction mixture was allowed to reflux for 8-12 hours. The dark yellow solution was cooled and the solvent was evaporated, leaving an oil. Puri- fication of the product was effected by chromatography on a 3u x 4.5” column of silicic acid powder (Mallinckrodt 2844). After silicic acid was added to the residue along with acetone to make a slurry, the solvent was evaporated to yield a powder containing the product. This was layered on the 81 column, then eluted with 30% butanone-hexane. Fractions of approximately 13 ml were collected and the product was found in tubes 95-116. Then the eluate from tubes 96-106 was pooled and evaporated 22.23222: and methanol was added. After distillation and readdition of methanol, the product crystallized when left for 3 hours at -15°. The yield of crystalline material from tubes 96-106 was approximately 280 mg. An additional crop was obtained from the filtrate of the first crop as well as from the fractions following tube 106 by addition of water. Total yield was 40% (395 mg). The Rf was 0.20-0.23 in 25% butanone-hexane on Eastman 6060 sheet thin-layer chromatography. The material slowly gave a magenta Spot when Sprayed with the dimethyl- aminocinnamaldehyde reagent. The melting point was 138-139° with [UJSS -9.8 (CHClB, C=1). Absorption data were: $2; (u) 5.74 (0:0). 7.95-8.25 (300:0): i;::°?;fi)(95%) 267, 279. and 290. A Analysis: Calcd. for 026H31N011: c, 58.53%: H, 5.856%: N, 2.625%: Found: 0, 58.19%: H, 5.83%: N, 2.87%. Meth l 1- -D-Gluco ranos l Indole- :Acetate XVII 0H200002H5 H.2\—N_J'ZCH 0000H3 N Ba(OCH 3>)2 AGO-CH8 0" AcO .Ac 82 To an ice cold solution of 250 mg ethyl 1—(2',3',4', 6'-tetra-O-acetyl-8-D-glucopyranosyl)indole-3-acetate in 25 m1 methanol was added 3.2 ml of 0.04 N.barium methoxide solution. The solution was allowed to stand 24 hours at 5°. Approximately 100 mg of damp IR 120 Amberlite resin was added and then the mixture was stirred for 1 hour and filtered. After evaporation and readdition of methanol, the pnoiuct crystallized on standing at -5° for 1 to 2 days. A Slowly developing magenta Spot appeared when treated with dimethylaminocinnamaldehyde. The R was 0.15 using Eastman f sheet 6060 thin layer chromatography: HCC13:EtOAc:HCO2 5:5:1. Yield was 110 mg, (67%). The melting point when H. the material was crystallized from methanol was 163-164°. Absorption data were: A ) 2.75-3.2 (OH), 5.83 (ester KBr max (0 0:0). 8.1-8.55(H00:0):i$::°?gfi)(95%) 272, 279, and 290. The optical rotation was [e335 -8.3° (0H30H, 0:1). Analysis: Calcd. for C17H21NO7: C, 58.11%, H. 6.024%; N. 3.986%. Found: C. 57.73%; H, 6.17%; N. 3.96%. 1-prSubstituted Benzyl Indolines and Indoles CH -X 2 (I732 —: (1 H2 CU + -———€> l 1‘ ;H2 [::j I“ 2 N I Y I l H CH2 CH2 I XVIII XIX XX 83 l-prhlorobenzylindoline (XIX, Y = Cl) prChlorobenzyl chloride (16.1 g, 0.1 mole) was added dropwise with stirring to a slurry containing 5.83 g (0.055 mole) of anhydrous, powdered sodium carbonate and 11.9 g (0.1 mole) of indoline in 100 ml of xylene at 0°. The mix- ture was stirred overnight and then heated to 1000 for about 0.5 hours. The solvent and a small amount of other distill- able material was removed lg,y§ggg. Distillation of the product was not possible because a thixotropic material formed in the condenser. Therefore, the residue was dis- solved in ethyl ether and extracted twice with 1 N,hydro- chloric acid. Neutralization of the acidic solution with 7 N sodium hydroxide, extraction with ether, and evapora- tion of the solvent gave an oil which could not be crystal- lized from methanol and water. Reaction of the residue with excess benzene-sulfonyl chloride in base freed the mixture of unsubstituted indoline. After extraction of the basic solution with ether, the product was extracted from the ether layer with 1 N_hydrochloric acid. Neutralization of the acid fraction and re-extraction with ether gave a jproduct which crystallized from methanol-water upon stand- ing in a refrigerator overnight. The yield was 1.73 g (7.2%) of crystals mp 26-27°. Absorption data were consis- ethanol (95%) max (mu) 253 and 'tent with the assigned structure: A 2953 kgzthl) 3039 (O’H)o Analysis: Calcd. for C15H14NCI: N, 5.75%: Found: N, 5.91%. ‘ . 84 leEEChlorobenzylindole (XX, Y = Cl) A xylene solution of 1.5 g (0.0062 mole) of 1727 chlorobenzylindoline was reacted with 1.4 g (0.0062 mole) of dichlorodicyanobenzoquinone. When the quinone was com- pletely dissolved, the solution was heated to reflux tem- perature and maintained there for 5 hours. Filtration of the product was followed by evaporation of the solvent lg. y§£22_to yield 0.95 g (64%) of crude oil. Crystallization of the oil from methanol-water was unsuccessful and hence, the product was used directly for the next synthetic step. An analytical sample was purified by preparative layer chromatography. The absorption of the compound was as ethanol (95%)» . neat max (mu) 270 and 292, Amax (u) 3.3 (C-H). 'Analysis: Calcd. for 015H12N01: N, 5.80%: Found: N. 5.66%. follows: A lfiprNitrobenzylindoline (XIX, Y = N02) An ethyl ether solution of 2.16 g (0.01 mole) of Ernitrobenzyl bromide and 1.19 g (0.01 mole) of indoline was stirred together with 1.06 g (0.01 mole) of anhydrous, powdered sodium carbonate at 0°. The stirred ether solution was permitted to warm to room temperature overnight and was then extracted with water. After drying of the ethereal solution over anhydrous calcium chloride, it was filtered and concentrated to give a crystalline mass. The product 'was dissolved in hot methanol and upon cooling yielded 2 g (79%) of orange colored crystals mp 98.5-99.5°. The ultra- 'violet Spectra was not typical of an indoline and showed 85 A3§2°?g&)(95%) 258. The infrared data were: Amelt ) 6 6 max (u 7.42 (N02). Analysis: Calod. for C15H14N202: N, 11.02%; Found: N, 10.95%. i-pyNitrobenzylindole (XX, Y = N02) Four grams (0.0157 mole) of 1-prnitrobenzylindoline was dissolved in 150 ml of dry xylene. An equal molar quantity of dichlorodicyanobenzoquinone (3.55 g) was added to the solution. When solution was complete, it was heated to the boiling point and refluxed for 6 hours. The solution was cooled and filtered. The xylene was removed yegggEQIand the residue was crystallized from methanol. The yield was 1.95 8 (49%) of crude product. Recrystallization from methanol gave an analytical sample with mp 102°. The observed absorption values were: Aggg°?g&)(95%) 269 and 292; A::;t(u) 6.6, 7.42 (N02). Analysis: Calcd. for C15H12N202: N, 11.11%: Found: N, 11.10%. Ethyl ieprNitrobenzylindole-3-Acetate (XXI) CH 0000 H + NCH2 00001: | 2 25 N 2 2 5 N | C C CH2 u 1 N02 86 One and three-fourths grams (0.0067 mole) of 1-23 nitrobenzylindole was dissolved in about 20 ml of dry benzene and the solution heated to boiling. A few milli- grams of anhydrous cuprous chloride was added, and addi- tion of 1.53 g (0.0134 mole) of ethyl diazoacetate in a benzene solution was then begun at such a rate as to allow for smooth evolution of nitrogen. After all the ester had been added, heating was maintained for an addi- tional 4 hours. The catalyst was removed by filtration and the solvent by distillation. The residue was dis- solved in acetone and a few grams of the silicic gel (Mallinckrodt 2844) was added. After removal of the Solvent, the residue was placed on top of a silica gel adsorbent in a 3 inch (diameter) by 4 inch (length) column. Elution of the adsorbent in the column with 30% butanone-hexane gave a peak of indolic material which moved at a lower Rf on thin layer chromatography than did the starting material. All tubes containing the indolic substance were pooled and the solvent concentrated in_y§ggg, The substance crystallized from methanol-water upon standing for several days in the cold. The yield of the product was 88 mg (4%) and had a mp of 101°. The following absorbancy properties were shown: Azzga?;:)(95%) 273 and 295: Afigi (u) 5.79 (0:0) and 8.47 (ROC=O). A small band around 2.8 to 3.1 u nearly disappeared after the sample had been melted thus indicating the presence of moisture: 87 Analysis: Calcd. for C19H18N20u: N, 8.28%: Found: N. 8.01. prSubstituted Benzyl Indole-3-Acetates Several by-products of the reaction of a prsubsti- tuted benzyl halide with the N-sodium salt of ethyl indole- 3-acetate were characterized. Elemental analysis and spectral data showed these to be the corresponding benzyl ester of indole-3-acetic acid. Yields were varible but approached that of the main product as determined by the intensity of the band on the preparative layer chromato- graphy plate. Spraying the chromatogram with the dimethyl- aminocinnamaldehyde reagentz immediately gave a blue spot in each case. The by-product of each reaction moved at a lower Rf (0.30-0.35) than did the 1-substituted ester. pyMethylbenzylhindole-3nacetate (XIII, Y = CH3) The major by-product was isolated by preparative layer chromatography from the reaction mixture of pgmethyl- benzyl chloride and the N-sodium salt of ethyl indole-3- acetate. This material was found to be the title compound ethanol (95%) ‘ max (mu) (u) 2.99 (N-H), 5.81 (ester C=O), from consideration of the following data: A KBr max 8.55 (ROC=0). A melting point of 70-71° was found. 273, 280, and 290: A Analysis: Calcd. for 018H17N02: N, 5.01%: Found: II, 4.87%. 2Dimethylaminocinnamaldehyde reagent was made from 50 ml 6 N HCl, 50 ml 95% ethanol, and 1 g dimethylamino- c innamaldenyde . 88 p:Chlorobenzyl indole-3-acetate (XIII, Y = Cl) At 0° the reaction of pechlorobenzyl chloride with sodium hydride and ethyl indole-3-acetate was unpredictable. In certain attempts only the title compound could be iso- lated from the reaction mixture while under apparently similar conditions a mixture of this compound and 1-prchloro- benzylindole-3-acetate was isolated. In the latter case, this mixture was separated with preparative layer chromato- graphy and the lower band was eluted and recrystallized from methanol-water. The structure was indicated by these data: A:::°?;:)(95%) 279 and 290: AKB: (H) 2.94 (N-H), 5.79 (ester 0:0). 8.65 (ROC=O); mp m95.5°. Analysis: Calcd. for °17H14N°2 Cl: N, 4.67%: Found: N, 4.67%. pyBromobenzyl indole-3-acetate (XIII, Y = Br) The products of the reaction of pebromobenzyl bromide with the N—sodium salt of ethyl indole-3-acetate were separated by preparative layer chromatography. The lower band was isolated and shown to have the following properties ethanol (95%) max (mu) 2.94 (N-HO). 5.79 (ester 0:0). 8.64 ‘which characterized the structure: A 273, 280, KBr max (0 ) (H00:0): mp 102.5-104.° and 290: A Analysis: Calcd. for C17H1uN02Br: N, 4.07%: Found: N, “028%. IérNitrobenzyl indole-3-acetate (XIII, Y : N02) A dimethylformamide slurry of 1.06 g (0.022 mole) of 89 sodium hydride (50% in oil) was vigorously stirred while 4.27 g (0.021 mole) of ethyl indole-3-acetate was added. After 20 minutes, the solution was cooled with a dry ice- acetone bath and 4.75 g (0.0222 mole) p—nitrobenzyl bromide in approximately 30 ml of dimethylformamide was added drop- wise. The mixture was stirred overnight and water was added. The aqueous suspension was extracted with ethyl ether, the ether dryed with anhydrous sodium sulfate, and the solvent distilled. After dissolving the residue in hot methanol, the solution was diluted with water and allowed to stand in the cold. The crystals which formed were recrystallized from methanol-water to yield 3.5 g (53%) and melted at 114°. The following data indicate that the compound is not the expected 1-substituted ethyl ester but rather the title compound. Ultraviolet and infrared data were: A:::a?;:)(95%) 270 and 289: l§§§ (u) 2.98 (N-H), 5.79 (ester 0:0), 8.56 (ROC=O). Analysis: Calcd. for C17H1uN204: N, 9.03%: Found: N, 8.87%. Characteggzation of Compounds The infrared absorbancy of liquid compounds was determined as films (neat) on sodium chloride plates. Solid compounds were analyzed in the form of potassium 'bromide pellets made from 410 mg of potassium bromide and one to two mg of the sample. Each solid sample was 'thoroughly mixed with the salt in a dental amalgamator (Cresent Dental Manufacturing Company) using glass beads 90 as a muller. AfiBeckman pellet press under a force of 20,000 lbs. was used to form the pellet ;n_y§ggg, Determination of the Spectra was done on a Beckman Model Ir-5 double beam recording spectrophotometer. All samples were dissolved in 95% ethanol for ultra- violet spectral analysis. One centimeter Silica cuvettes were used in a Beckman Model DK-2 ratio-recording Spectro- photometer. Melting points, which were obtained on a Fisher-Johns apparatus, are reported uncorrected. The refractive indexes were reported on all liquid samples using an Abbe/refracto- meter. Spang Microanalytical Laboratory of Ann Arbor, Michigan, and Micro-Tech Laboratory of Skokie, Illinois, performed the elemental analyses. Biolo ical Assa s Tomato gvary growth assay' Solutions: A sufficient quantity of each alklecom- pound to make a 10"3 M solution was added to 5 ml of lanolin (Merck) and alternately heated on a steam bath and mixed ‘with a Vortex Jr. tube shaker several times. One-half ml of this solution was added to 4.5 ml of lanolin and the above heating and shaking procedure was repeated to prepare a 10- 21 solution. This method was repeated for the other dilu- tions of 10"5 y and 10"6 g. The benzyl derivatives and the glucose derivatives were dissolved in absolute ethanol and appropriate volumes cxf the solutions were added to the tubes containing 5 ml 91 lanolin to make final concentrations of 10"3 M, 10'“ M, 6 .10'5 M, and 10- MIin lanolin. The ethanol was removed by alternately heating on a steam bath and shaking with a Vortex Jr. mixer. All samples were stored in a refriger- ator until ready for use. Egant materiaI: Two varieties of tomato plants (Lycopersicum esculentum, Michigan-Ohio hybrid and WR-7) were grown in the greenhouse. The first flower cluster of each plant was prepared for bioassay by trimming back all of the immature flowers except two, which were emasculated before anthesis. The calyx was trimmed flush with the surface of the ovary. Later flower clusters were treated in a similar manner for replications. Treatment: Lanolin paste containing various concen- 6 trations (10"3 MIto 10' M) of each compound was applied to the ovary in such a manner as to assure complete coverage and in sufficient quantity to fill the cavity formed by the truncated calyx. The bioassays of the first replication indicated that all of the compounds had biological activity less than did IAA: and therefore, subsequent replications were made with 10"3 M_solutions. Ovary diameter was mea- sured after 6 to 10 days and compounds exhibiting low :activity were evaluated by recording delay of absission :relative to the controls. Buckwheat root inhibition assay Solutions: All solutions were made up in 0.05% Tween 80 solution in glass distilled water. A sufficient 92 quantity of each compound to make 100 ml of 10'“ MIsolution was dissolved by alternately heating gently on a steam bath and agitating vigorously with a sonifier (Branson S-125) until all the material was in solution. From this stock solution, dilutions were made just before treatment. Five ml of stock solution was placed in a test tube and 0.5 ml was removed for addition to the next tube in the series which contained 4.5 ml of glass distilled water. After mixing, the process was repeated for the remaining dilutions. Egant material: Japanese buckwheat seeds (Fagopyrum esculentum) were planted approximately 400 per 10 inch cul- ture dish on filter paper which overlay a perforated proce- lain plate. Distilled water was added to a level just below the seeds. The seeds were allowed to germinate at 25° in the dark for 24 hours. Treatment: Ten seeds with radicals approximately 1-3 mm in length were selected and placed on filter paper in a Petri dish. To each dish was added 4.5 ml of the test solutions. The roots were allowed to grow in the dark at 25° for 48 hours. The mean length in millimeters was determined for each compound and each concentration. Four replications were made on each compound. Cucumber seedling curvature assay Solutions: Lanolin solutions of previous experi- ments were used for this assay employing only 10'3 M concentrations. Plant materiaI: Cucumber seeds (Cucumis sativa, 93 variety MSU 736) were soaked for 8 hours under cold running tap water in a large beaker covered with cheesecloth. These seeds were planted in moist vermiculite and allowed to germinate in the dark for 48 hours. The trays were then placed in the laboratory under a bank of fluorescent lights until the plants were erect and the cotyledons extended. The plants were removed from the vermiculite, the root system washed free from non-plant material, and the stems clamped carefully between %" x %" x 15" strips of wood, one of which had a kerf cut at 1" intervals to accept the stems. The roots were placed in distilled water and allowed to equilibrate 2 to 5 hours before treatment. Treatment: A quantity of lanolin paste sufficient to form a drop approximately 2 mm in diameter was placed on one cotyledon 1 mm from the stem. In different replica- tions, the front and back leaves were treated so as to cancel any phototropic effects of stray lighting. Four replications were made to determine the angle of curvature and its time course. A plastic goniometer was used to measure the angles of the upper surfaces of the cotyledons in degrees every 15 minutes after the beginning of the initial curvature. No reSponse was noted in the control plants treated with lanolin. Beanpetiole abscission assay Solutions: Lanolin paste solutions of the test com- pounds prepared as previously described under tomato ovary 3 growth assay were used for this test. Only 10' fl_solutions 94 were employed for this assay. Plant material: Bean plants (Phaseolus vulgaris, variety Contender) were planted four to a pot and allowed to grow in a greenhouse until the primary leaves were well formed. Before the plumule had emerged, two uniform plants were selected in each pot and the other two were cut out. Petioles were cut off both primary leaves about 1 cm from the stalk with a razor blade. Treatment: The eXposed stub was treated immediately after cutting with lanolin paste containing the compounds to be assayed. Enough paste was applied to cover just the severed area and overlap the edge slightly to form a cap. After several days, when the first petioles began to abscise, observations were made thrice daily-~in the morning, mid-day, and evening. Abscission was indicated when the petiole fell off under the influence of a device employing a spring brass blade which deflected to exert a force of approximately 10 g when the blade was pressed downward against the severed petiole. Time of abscission was recorded to the nearest hour. Five replications involving h petioles each were ‘made of this assay. .Ayena straight growth assay Solutions: Fifty ml of the Tween 80 solutions of each.compound prepared as described for the buckwheat assay *was treated with sufficient dipotassium phoSphate (89.7 mg), (zitric acid monohydrate (51 mg), and sucrose (1.00 g) to nuzke a pH 5 buffered solution. A stock solution of buffer 95 was made from 20 g sucrose, 1.794 g dipotassium phosphate, 1.019 g citric acid monohydrate, and 1 m1 Tween 80 diluted to 1,000 ml with glass distilled water. This buffer was -4 -5 - used to dilute the 10 fl stock solutions to 10 n, 10 6 7 E, 10- fl, and 10-8 fl.concentrations. Buffer solution was used as the control. giant materygl: Oat seeds (Agggg,sativa, variety Torch) were placed in a vacuum flask under tap water in the dark. A vacuum was drawn on the flask with a water aSpira- tor and released after a few minutes and then immediately repeated. After intervals of 30 minutes, #5 minutes, and 45 minutes, the process was repeated. The seeds were planted on moist vermiculite in rectangular glass dishes after the final rinse. After 2h hours in the dark, red light was allowed to shine on the seeds for 2 hours. Then they were lightly covered with vermiculite which was moistened slightly. After #8 additional hours in the dark, 0.5 mm sections were cut 3 or 4 mm below the tip. These sections were floated for 2 hours on a glass distilled water solution containing 1 mg magnesium sulfate monohy- drate per liter. All operations except red light treatment were carried out in the dark or under green light. Treatment: Ten coleptile sections were placed in each six-inch test tube which contained 1.8 ml of each solu- tion to be assayed. Assays were made on five concentrations 5 6 7 8 (10'4 a, 10- £1, 10" _F_I_, 10- _1V_I_, 10- ll) of each compound by jplacing the charged tubes in a drum rotating at 1 rpm. The 96 drum was placed in a dark incubator at 26°. After 22 hours, the sections were removed and placed in a photographic, enlarger (Federal Manufacturing and Engineering Corporation). The image, enlarged 5X, was measured in millimeters. Two to four replications were made of each eXperiment. The data for each concentration were averaged and presented as percent of control growth. RESULTS AND DISCUSSION RESULTS AND DISCUSSION Physical methods The melting points or boiling points, results of elemental analyses, absorption data, and other physical constants of the various 1-alkylindole-3-acetic acids are given in Table 4. CorreSponding data are tabulated in Tables 1, 2, and 3 for the intermediates employed in synthesizing these acids. Infrared absorption spectra of a representative series of compounds are shown in Figures 1, 2, 3, and 4. The Spectra include that of indoline which is contrasted with that of 1-gfdecylindoline (Figure 1). In the latter case, the most conspicuous feature is the lack of N-H stretching absorption near 2.9 u. That substitution has occurred on the indolic nitrogen is apparent by comparing 'the Spectrum of the substituted indole with indole itself in Figure 2. Likewise, the lack of N-H stretch in the spectra of ethyl 1egrdecylindole-B-acetate and i-nrdecyl- indole-3-acetic acid in Figures 3 and 4 is good evidence for N-substitution: In the latter two cases, carbonyl stretching vibra- 'tions are evident in the infrared Spectra. Absorption :near'8.0 and 8.6 u is eXpected of esters and can be attributed to the BOC=0 grouping in the ester (11) (Figure 3). All normal saturated esters except acetates absorb in 98 99 mm.m mom . osaHoWha as.s mm.m om.m 1:: 3mm poem.a ma no anatoma mm.msa adapsmusua mz. : mm.m mom osfimmwsd mm.w :u.m Mm.m sun emm Homm.a H.o pm mane: mm.oma nasaonmuomflna . om.m _ mom _ . msaabwsa as.m ee.m mm.m nu: 3mm ween.” H.o pm :mnom mm.owH aaaaosmasufl omwm N:.m com mQHHopsa sun Hm.m mm.m In: Nmm ommm.a ma pa oafinmoa Nm.sea nasspmna mm.m mm.m Nam . , msaaopsd nun mm.OH mm.m an: mam ospm.a mo.o pa 3: om.mma uaaspmznfi Nam II: mu.HH mm.m mm.m Him mamm.a mo.o pm dine: ma.maa moCHHopSH 1 1 18 Q mm SE pad. 2 R 2 R mno mlz abs m : NopsH o momawon unmaoz endow .poamo m ob pomamom chammohm can Haddocaoz pQSoaaoo madam: sowmmmmmm¢ pedom medaaom mmsflaoesHuHaaH5 mm: NodsH o mmohwoa pnmamz osSom .ooamo o> pedagom mndmmonm and Adadoodoz dadomaoo maaxmz noapanomp4 psaom mnaaaom moaoeqHuassaduH 000 0009 00000000 .0 00909 102 .mopwam od0noano 800000 00 maaaw so dosdahmpou mums mhpoomm .mpamSopsa 00 00000 ws0mdonood £0 Qo>0m ohm Amvdsmn psms0aoha pmoa 0:» mo 00500» 0:90 .Hosmzpm Rmm was pambaomH 00.0 000 0000.0 . 00 0a 000w00 00.0 00.0 00.0 -- 000 0mc_ 0.0 00 000-000 00.000 -000000000-z-0 00hr 00.0 000 m0pwz0 00.0 00.0 00.0 -- 000. . 0000.0 0.0 00 000-000 00.000 -00omq-:-0 Rm . i . w 00.0 000 . 00.0 000000 00.0 00.0 00.0 -- mm0 0000.0 -00.0 pm 00-00 00.000 -00psmm-:-0 {-0 mom . oHOdGH 00.0 00.0 00.0 -- 000 0000.0 00.0 00 00 00.000 -00psm-pnop-0 F000. 4 00.0 000 . 0mmwn0 00.0 00.0 00.0 -- 000 0000.0 00.0 00 00 00.000 -00psm-0mm-0 00L. 0.0.0! 00.0 000 mwmms0 00.0 00.0 00.0 -- 000 0000.0 0.0 00 00 00.000 00000-000-0 00m 103 00mm opmpooMIM 00.0 o0.m m0.m II: 00m 0wmm.0 m.o pm 000lmm0 0m.mmm IoHWUs0ampsn 0000 -0-0 00000 mmam mpmpmomlm 00.0 00.0 00.0 :1: 0mm 0000.0 00.0 p0 meImNH mm.mdm I0HNM¢0009000 0000 -o00-0 00000 . mmmm . mpwpmomlm 00.0 00.0 00.0 -- 000 0000.0 00.0 00 000-000 00.000 -00fim0000mo00 mm0m Isl0 Hmzpm 0000 . 0000000-0-000000 :II 00.0 00.0 III mmm mm0m.0 0.0 pd 000-000 00.0mm I003¢0|0 flagpm MIAM- 0000 opdpmodnmumaods0 In: 00.0 00.0 III 000 0000.0 00.0 90 om0nmm0 00.000 :00Spma-0 00£pm 0000- com mpwpmmmlm -- 00.0 00.0 00.0 000 ..- 0.0 0.0 000 00.000 .6035 00.00.00 000 1 1 10 mm 08 cum 0 0 0 0 one 0-0 00: a: 00000 0 0000000 000003 0:500 .00000 N mMmpoahmmm whammmhm 0:0 000500002 undomaoo 080M02 so0pahomnd 00000 0000000 00000000-0-000000-00000-0 00000 000 0000 00000000 .0 00000 10H .000000 00000000 a50000 .000005000 0000000 no 08000 no 00050008 0003 0000000 000000000 R00 00 0000500000 0003 0000090 000O0>00002 0 0000 0000.0 00 as 000000010 00.0 00.0 00.0 -- 000 m: 00.0 00 000-000 00.000 -00o000m0om000oo 0000 0 -0-0 00000 0000 000000010 00.0 00.0 00.0 -- 000 0000.0 0.0 00 000-000 00.000 -00m00000oo0 0000 -0-0 00000 000 . 000000010 00.0 00.0 00.0 -- 000 0000.0 00.0 00 000-000 00.000 -000000000000 0000 -0-0 00000 0000 000000010 00.0 00.0 00.0 -- 000 0000.0 0000 00 000-000 000000 -00o00000000 0000 -0000-0 00000 0000 .\ 0000000-0 00.0 00.0 00.0 -- 000 0000.0 0.0 00 000-000 00.000 JWHM00000000 0000 100010 thpm 0000 000000010 00.0 00.0 00.0 -- 000 0000.0 0.0 00 000-000 00.000 -wmm00000000 0000 -o00-0 00000 «1. ~.. 105 mmmm daoa oapmownm oa.o wo.o mm.m sun wmm wmpmw om.an~ uoaodndampsmumra mmnm mmmm . cdoa oapooaum 03.0 m:.o mm.m nun mmm m.moaum.:oa mm.mam nmaodnfiamgonmwomaua m m mmmm caow odpmoMIm nun m:.o mm.m us. new stom um.mfim umaousdammoamwzafl mmmm mmmm ddom oapoom an: mm.o mm.m In: mmm moauuoa :m.mom umumaocsHHmSpmnfi mmmm mmmm uaom oapooaum sun 30.5 mm.m sn- mmm wmanmmfl mm.mma uoaovsaflmspmzsfi mumml . omm nu- oo.m om.m wm.m owm wmaumwa mfi.mua ddom oapmoaamtoaoonH sum ‘11 z & 2 R 1 1 13 o mmmnwma pswaoz @250m .voawo ouo muz Nbfi szaom msapamz hadfiomaoz omdomaoo mmadwmz Soap homnd mvdo< oapmo¢nmamaodnHuammH4na Hogapmm agedmhsm .: wanme 106 .mnoapsaom Hosanna Rmm :« cthmmma mama mnpommm pmaoabmapap .dopomnhoosfi ohm can mzpmnmmn¢.mn30hlmmsmam a no donwahmpod whoa mpnaon wnapamz .1 w.m. op o.m mwzaa asp ad camp ddom omega Haoamhp 0:» umzo:m mcaom umpspdpmnsm man go Hadm N H mmmm udoa oapmnmwm am.m mm.m mm.m nu: mmm m.ouum.mu no.5m: nmaovsaahomumpooazna mmmm mmmm ‘ cdom capwom ::.: ::.: mm.m nun mmm m.amuflm m:.mfim nmuoaocndahoonnnnfl mm N mmmm daom capopmwm ww.m H5.m um.m --- mmm :m-mm Nme:m -oflocndflhpnmm1z-fi II. mmwm i. mmmm daod oapmodlm um.o mo.m nm.m nu: 0mm m.moaam.:oa om.amm nmaoundampamnpnmpufi mmuw mmmm vdoa oapwmmwm «0.0 00.0 mm.m nun mmm m.:mu:m om.Hmm umaodcdahpsmnoomna mmnm . . mama ado» odpwwmum mm.m mo.o um.m sun mam :mnmm om.Hmm umHoonaahpsmuoma-H mum 107 FIGURE 1 Infrared Spectra for: Upper: Indoline Lower; grDecylindoline «ZO- Ui Z. EOZuau)(3 o. n. v. 2 ~— : o. o O 0. ca On 0! On 00 )0 OK on 00 ‘ ,_ , _, _ h _... r. r. _; .LL .__ LIL—.2: 22.2.2. 2:3... :._ _ .I V? I: 59:11 :::____. Ono con 8. 00. 80. 00: 8h. 80. 8: 8n. oOO~ Sou coon 09.; on. _\ .10 coogzgdi O 0.. 1 a _ . .7; 7.777 ‘. . b » P7. P. _‘_ L? C::..‘ I: 3:52p: _.::..;_._.__:Z__ L. S. : .._ _ _-:.‘: 2.1.2.3123: :2: I: 03 8k 8. 8.. 89 8: 82 82 8‘ ~ 82. 89 can. 00... 00a. 80.. . 1U c§320><3 .. - , Hun; @‘V‘S‘ 109 FIGURE 2 Infrared spectra for: Upper; Indole Lower; l-ggDecylindole 110 g z E§£(’ . 8. an, __ T: :23. :___:___I if... 03 8» oo- 8. 03. 8: c2. Sn. 8: 82 82 82 82 3? 3,1 l1|l ~ _ r L 111 FIGURE 3 Infrared Spectra for: Upper: Ethyl indole-B-acetate Lower; Ethyl l-gydecylindole-B— acetate filo-U3 l £01m§><3 2 n. z 2 2 : o. . a s o a . n a ‘ P: I: I _:__p :CZ: FL at: Z. ,2: :‘:_‘::_Z .;_:_____: 03 8s 8. 8. 08. oo: 82 82 8! 8m. 82 82 coon 0.8. 5? _ ‘U ngzg<3 ‘1!— 4!. 0n 0. o. 9.. . .h p P Fe. .p 77 .L. .L FL. Tarpc: LESI: Z.— Z... 2::___.__..:_:.:__ L. ”I-.. #7:..-»P 7:52: :2: :L. ___:_.:.. 3. 8K 8. 8o 08. 8: 82 82 8! 82 82 82 88 08‘ 82 _ lu italg<3 e dole-1' L U a? v 113 FIGURE u Infrared Spectra for: Upper: Indole-B-acetic acid _ Lower: l-grDecyllndole-B- acetic acid '. 4" 4|. SK _—~ w§¥ 1. £2?(’ 22 222.12.: __.__.__:___ 89 8: 86. 10 social—>43 00»; 001. I. 82 _ ~— _ 22:22:21.1: 82 82 88 2.1:: 5.5:... 80m 00, a nooa o. on Co 0. 0.. 0, oo. :2... I: 801 000’ L 000' _... (.0 115 the 8.26 to 8;62 u range (110). Katritzky gt 2;, (#3. 4#) report that a peak near 8.“ u is to be eXpected with esters having chains two or more atoms in length attached to each side of the ester grouping (ROC=O). The typical broad absorption band in the range of 3-4 u is characteristic of the carboxylic acids (68) and serves as definite evidence for the structure assigned in Table 4 and Figure 4. Carbon-hydrogen stretching vibra- tions are buried beneath the broad carboxy absorbance band and-contribute to the high frequency absorbance. An acid dimer band near 10.5-11 u was seen in each spectrum of the acids. This absorption is attributable to the OH out of plane deformation (24); The strong ester band near 8.6 u either disappeared or significantly changed shape upon saponification‘ Infrared absorbance near 2.9 u was the first indica- tion of an impure product obtained by benzylation of the nitrogen of ethyl indole-B-acetate. Silica gel thin-layer chromatography with either Eastman sheet 6060 or Brinkman precoated glass plates in 18% butanone-hexane was incon- clusive because the Rf values of the reactant ester and that of the by-product prsubstituted benzylindole-B-acetate were indistinguishable. Hence, the thin-layer chromato- graphic method of monitoring the reaction indicated the presence of only the starting material (ethyl indole-3- acetate) and the expected N-substituted indole analog. However, the melting points and elemental analyses of the 116 products revealed that the reaction mixture contained the N-substituted indole compound and the correSponding benzyl ester of indole-3-acetic acid. A bathochromic shift in the ultraviolet Spectra of 1-substituted indoline and indole derivatives was very useful in characterization of reaction products (Tables 1, 2, 3, #). The latter phenomenon was utilized in the isola- tion and characterization of the by-product prsubstituted benzyl esters of indole-3-acetic acid. Although no exten- sive work has been done on the ultraviolet Spectra of indole derivatives, several reports of a similar bathochro- mic shift are quoted in the literature. For example, Yamada gtflgl. (126) observed this shift on alkylation of tryptophan. They reported that the peak near 290 u changes to a shoulder which is shifted toward longer wavelengths by approximately 5 to 6 u. Hinman and Lang (65) reported a similar value for i-methylindole. Substitution in the 3-position could also be verified by ultraviolet data. This is evident by considering the absorption maxima of indole-3-acetic acid relative to indole (Figures 2 and 4). In this case, the bathochromic shift is only about 2-3 p. An additive effect is seen when a 1, 2- disubstituted indole is used. Thus, i-alkyl indole-B-acetic acids or esters of the acids are shifted about 9 u relative to indole and only about 3 u relative to a i-substituted indole. Many of the products and intermediates in the synthe- tic pathways were purified for analysis on Brinkman fluores- 11? cent 2 mm silica gel preparative layer chromatography plates. One hundred to 150 mg of a compound or a reaction mixture could be separated at one time. The absorbent containing each product was then scraped from the plate and eluted with methanol. Liquid samples isolated in this manner were distilled in a side-arm test tube attached to a cold finger condenser. Solid compounds were recrystallized from methanol-water or from benzene-hexane. Synthesis Indoline is a more nucleophilic compound than is indole; This characteristic can be attributed to the aromaticity of the pyrrole ring of indole molecule which makes the lone pair of electrons less available for reac- tion. The reactivity of indoline is similar to that of aniline. In agreement with this fact was the finding that indoline reacts with a Egggybutyl halide to approximately the same extent as does N-methylaniline. In the present work, indoline was chosen as a start— ing material for some of the 1-substituted indole-B—acetic acids, since it was possible to make derivatives containing bulky iggrpropyl or‘tggtpbutyl moieties. The literature reports indicate that direct alkylation of the indole nucleus with large halide moieties has not been successful even when the N-sodium salt was used (126). Purification of the substituted indoline before conversion to the substi- tuted indole was easily accomplished by'uping benzenesul- fonyl chloride. Acid extraction of the product from the 118 amide was then possible. A short path "Kontes" still equipped with a fraction- ating receiver was used for distillations because it had very little holdup. The solution in the distilling flask was stirred vigorously with a magnetic stirring bar while it was heated in a sand filled electric heating mantle. This Simple still was adequate for separating indoline from the alkylated product when the chain was 5 carbons or more in length. Dichlorodicyanobenzoquinone was a satisfactory dehydrogenating agent for aromatizing the substituted indoline nucleus. However, heating in boiling xylene was necessary for the dehydrogenation reaction to go to comple- tion. Approximately one-half as much product was isolated when a solution of the reactants was merely mixed and allowed to stand at room temperature. An exothermic reac- tiOn was observed when dichlorodicyanobenzoquinone was added to a xylene solution containing indoline. Comparative studies demonstrated that chloranil gave a reaction mixture which was difficult to purify and afforded poorer yields than did dichlorodicyanobenzoquinone. Neither the use of excess chloranil nor refluxing mesitylene (bp 165°) as the solvent proved as effective for aromatization. Jansen gt 2;. (36) found Attenburrow manganese dioxide the best reagent tried for the dehydrogenation of a tricyclic indoline. Their yield of 64% was approximately the same as those_reported herein. Russian investigators (97) employed chloranil for the aromatization of a glucosyl 119 indoline, but later utilized dichlorodicyanobenzoquinone for a similar reaction (78). In the present study, the reaction worked as well for ppsubstituted benzylindolines as it did for the alkylindolines. Snyder and Eliel (90) and Katritzky (#2) indicated that whereas 1-methylindole underwent a normal Mannich reaction and formed the eXpected methiodide salt, the alkylation with cyanide proceeded in an anomalous manner. In addition to the dimethylamino substitution product, 1~methylindole-3-acetonitrile, a significant amount of isomeric by-product was formed. Snyder and Eliel (90) demonstrated this phenomenon to be an aILylic rearrangement to produce 2-cyano-1,3~dimethy11ndole. As a representative compound, igngdecylindole was chosen for the investigation of this synthetic route. The side-chain was successfully attached to the indole ring by the Mannich reaction employing formaldehyde and dimethyl— amine (51). The structure of the reaction product was verified by infrared and ultraviolet Spectra. ignfiDecyl- gramine had the typical bathochromically diSplaced shoulder at 297 mu? In the infrared region, the presence of a dimethylamine moiety was indicated by a doublet at 3.58 and 3.6% u (12). Although the distilled product had no N-H stretch, it did have the intense aliphatic C-H bands characteristic of the nrdecyl chain. ‘ Stowe (93) indicated a preference of the methosulfate of gramine over the methiodide as an intermediate in the 120 alkylation of gramine. In deference to Stowe's study, the methosulfate of legydecylgramine was reacted with excess dimethyl sulfate and a purified tetrahydrofuran solution of the alkyl gramine. White crystals of the salt formed after the solution stood overnight in the cold. The product was characterized only by its melting point which was 99-101°. To determine whether two compounds were produced in the alkylation step, the methosulfate salt was reacted with a 3-fold excess of potassium cyanide by refluxing in a dimethylformamide solution according to a method outlined by Hill (32); After extraction with ethyl ether and removal of the solvent, the product was purified by distil- lation and the infrared Spectrum examined. The most strik- ing features of the Spectrum were the absence of a N-dimethyl doublet and the presence of a nitrile band at n.35 u. The latter material was purified by preparative layer chromato- graphy. Two distinct bands were observed which were separately eluted and examined with ultraviolet Spectrometry. One band contained material which had the expected Spectrum of 1anydecylindole-3-acetonitrile whereas that from the other band showed a series of peaks at longer wavelengths,; which are not typical of indOle compounds. The latter_band’ contained a compound which was assumed to be the Z-nitrile homolog of the material isolated by Snyder and Eliel (90). Katritzkfij(42) found that a potassium hydroxide solu- tion perferentially hydrolyzed 1-methylindole-3-acetonitrile 121 to the corresponding acid when the isomeric mixture was saponified. However, with the mixture investigated herein the base extractable material from the saponification melted at a lower temperature than did the l-nydecylindole- 3-acetic acid synthesized by employing ethyl diazoacetate. Therefore, the synthetic sequence for attachment of the side chain involving the Mannich base was abandoned in favor of ethyl diazoacetate. Nametkin §§,§;. (69) investigated the effect of various conditions on the reaction of ethyl diazoacetate with indole. They concluded that cuprous chloride was the most effective catalyst and on the basis of their finding the same catalyst was used in the present work. Several Merck investigators (60to 65) have synthe- sized a large number of 1-benzylated indole derivatives. Among these are several compounds closely related to l-pp benzylindole-B-acetic acid. In their synthetic scheme these investigators used the N-sodium salt of the indole compound in question. Dimethylformamide, a highly polar aprotic solvent which solvated the newly formed salt, was used for the reaction medium. Reaction of the N-sodium salt with a benzyl halide yielded the 1-benzyl derivative of the indole. They made no reference to any by-products. This procedure was followed in the preparation of lip- substituted benzyl derivatives of ethyl indole-B-acetate. Significantly, a considerable quantity of by-product was also obtained by this method. The by-product was inseparable 122 from the desired compound by simple vacuum distillation. Fractional crystallization from methanol-water of either the reaction mixture or the distillate afforded partial purification of the product. To obtain chromatographi- cally pure material, preparative layer chromatography was utilized and the by-product was separated from the main product. Elemental and Spectral analysis revealed that the by-product was the correSponding ppsubstituted benzyl indole-B-acetate (Figure 6). r-w The structure of ethyl ppchloroindole-B-acetate was verified by synthesizing it by another route. Indoline was reacted with.pgchlorobenzyl chloride and the resultant product (Figure 5) was dehydrogenated. Reaction of the. substituted indole with ethyl diazoacetate yielded the same material as that made from ethyl indole-B-acetate. Superimposition of the infrared Spectra was possible as can be seen in Figure 6. Tables 5 through 8 list the physical data of the benzyl derivatives and the intermedi- ates used in the syntheses. The synthesis of methyl-1-glucosylindole-3-acetate involved the same general route as outlined for the l-alkyl indole-B-acetic acids. Several attempts to repeat the synthesis of 1-(2',3',h',6'~tetra-O-acetyl-BAD-glucopyran- osyl)indole by the procedure of Suvorov and Preobrazhenskoyz (97, 98) were unsuccessful. Although reasonable yields of the intermediate 1-glucosyl indoline were obtained, diffi- culty was encountered in dehydrogenation of the indoline 123 FIGURE 5 Infrared Spectra for: Upper; p—Chlorobenzylindoline Lower; p—Chlorobenzylindole 1,223+ ate-v.1 z. xsozaJl-I o. s r a 9 a. ( 2.. 2.. I; 2: ~31: 1U I3!)1u)(’ o, 2 v. n. 2 : o. o - s o n v ~ S x s. . a. . 9 3 . B a. , co 8. ,. _._ 2.. 2.. .2. 2.2.1: ._:____ I. ______. .2322: :2 co. 89 8., co: 3: R... 3: 83 2.. 2.2 a}: p. lu 13319:} aline >le L 3'.- v-w vocally-m 125 FIGURE 6 Infrared spectra for: Upper; Ethyl l-pychlorobenzylindole- 3—acetate Top, Synthesized from indoline Bottom, Synthesized from ethyl indole-3-acetate Lower: 27Chlorobenzylindole-B-acetate H (\J- (\ ’zylmiclf' 1000 on in“? from 915- WAVINUMII CIA ‘ Um w‘vlvduulfl (A ‘ 8.34mi I700 uoo ISOO 2300 HI llll WAVEIENG'N IN uICIONS 127 momm. .- opopoos Hm.m ms.m ms.m ma.m mmm m.:oau:oa s~.msm unusaossdaassop :SN sofionnlmra ahspm mam mpspoow mm.: sm.s oo.w ma.m mmm mmumm Hm.smm umnmaosssaausmp sum uosoasoLMuH, Hanan mmm mmm «passes as.s om.s ss.w ma.m mam so mm.HHm umnmaosssaasssn sow nososacpmrfi Hanna 0mm mumpoomnmlofloocd as.s as.s os.m ma.m mmm m.s:u:s sm.mmm uaassonna Hanna m m 2 R z N 1 1 18 o mooawmn unwaoz oszom .ooamo oauam ouo Nb: annaom wadpaoz amazooaos oSSoaaoo $8.“wa no.3. Homfld mapssoosrmnsaossaaassom sensuapmnsmusssmuauaaspm soc span awedmasm .m manta 128 .Anmwoaohs .nom mv m.¢a pd zoom ome mm: mama muodbno ad: .mpmaaoa moaaoan adammmpoa no penummoa one: mapooam consumaHm .mnoapaaom Hocsspo &mm ca donaaaopoo one: mapooam poaodboapabm .oopooaaoosd was end mapsnsaas masonlaosmam a no coma one: mpnaoa wsdpaoza it. mom opsuoos Ho.m mm.m as.m ma.m mam Hos mm.mmm umuoaossaaauson nossdsLMuH Hanna mama opspoos mm.: mm.: oo.m ma.m 3mm osum.mm mm.m~m unsoaomsaaasssp mam nawosposu as Hanna mwmm endpood so.: on.: os.m ma.m mam m.mm mm.som umuoflo sdaassop mum sasspss. us Hanna 129 mam odes mo.s so.s sm.m sum mmm was s~.ssm oapoosumumaosss mum IHhNaonoaonmLmra mmmm sass mw.: no.3 mm.m sum mmm m.s:Hnm.m:H os.mmm oapoosnmcmao sa mum Iamnzopoaoasuu 3H meow mam Sass 00.: :m.: ww.m :Im mum m.omaum.mma om.mwm capoomtnloaoona new IdhunonoaosamLmvH _ momm odes odpmom tun mm.m mm.m dun mmm omalmma Hm.mwm Imnoaoonaamucomla m N . 1 1 1a 0 mooawom pswaoz endow .ooamo ono mno «>5 Humaom wadpaoz HSHSooHoz psaoaeoo madam: Soapmaompd Jl asses oapmosrmumaoesaaassmm smpspameSmusssmuH soc span Headmasm .m manta .L w 130 mm.: .mpoHHoa moaaoan asHmmdpoa no ooHSmSoE who: «anomam pmamamch .onapfiaom Hosanna Rmm ad dondahopod ohms mapooam pmaoabaapao N .oopomaaooss mam osm mandammas manoblaoSmdm a so coma cams mpnaoa wadpamza momm 35.: sm.m :nm swm mam mmaawma daom capoomIMIoHo ad IHhNSoDhSOSpozI Ia oo.m mwmm Ho.m om.m sum own sum emanmma odes capooStmamHo ad IahuzothSpoZI Ia 131 . 0mm mpdpooosmuoaooaa mm.m mm.m mm.m ms.mh N.m mam assumes mm.Hmm aaamosssaaossaw :ms.~ New Iowans Haspmz evapoomlmsoaoosd omm AHhmonwahaooSlemrm mm.m mmo.m :m.m mm.m In: mum mmaumma mm.mmm Iahpoosuonsapoau.o sow ..s..m..~vua Hanna oaoosa mmm Adamozmaamoosawtn --- ms.n as.m ms.m --- ssm m.sss-msH m:.sss -m-sssoos-o-sssms mom n.0..:..m..mvna madaooca mom Adamonmah9005HMIQ uuu NH.m as.m mm.m nu- wsm mssussa ss.mss unnaspsosnousssoa I.@..#..M..NVIH z N z & 1 1 *1 1a 0 mooawom pnmamz canon .ooaso ouo mno Mo Nba Hanaom wadpaoz asadooaoz dadoaaou mmaawmz Soap Hound mmpmavoaaopsH ocHHoosH ps6 mHooQH HoSpo Mom mama awosmssm .s sassy 132 .mapooam dogmawzd mo maaopoo you axon comm .msoapsaom HoSdSpo Rmm ad moms ohms mapooam poaoabsapasm .oopooaaooss was was mapmhmaas manobIHoSmam s so dodafihmpmu who? mpmaoa wnapflmsa Nam oaodna oa.aa Ha.HH In: mm.m In: mom Ned mm.mmm IHhusmnoapaZLmrH ossaosss no.0“ mo.HH It: mm.m III mmm m.mmlm.mo omJSmN Iahunopoapazmmrfl mom oaossd ms.m om.m In: m.m an: osm as: as.flsm uaassonoaoasonmrs mom ocaaooza om.m ms.m 1-: m.m nun mnm smumm ms.msm uaassonosoasonmrs 133 .mpoaaoa moaaoan agammmpoa no dehummoa cams mapooam ooasamsHm .mSoadeom HosmSpo Rmm ma docaaaopoo who: sapoomm poaoabmhpas N .oopooanoozd was was mandamaam unschnwmnmam a no donaaaopod mama upmaoa wnapaoza mmm evapoomIMIoHodzd mm.m mo.m om.m mm.m mm.m cum :HH am.oam :HmNSonoauaZLm omm mumpooQIMIoHooSa mm.: 50.: :m.m mm.m :m.m owm denim.moa HN.::M ,HhuzopoaoHMLm lI, AMMN omm . opspoomuMIoHocnd no.3 no.3 mo.m ma.m :m.m mum m.mm om.mmm ;HhN2onoHOH£0Lm omm mumpoowlmnodoond sm.s Ho.m mm.m Hm.m mm.m 0mm Hates sm.msm .Hsssopaaspssnm mum 2 R z R 1 1 1 1a 0 mooawoa pswdmz USSom .ooamo onoom ouo mtz «>5 Humaom waapaoz amazooaoz USSoaaoo mdaawsz soap Homnw mopspoosrmnoaossdasssom smpspapmSSmnsssm soc span asofimssm- .m tapas 134 ring with chloranil. The melting point of the isolated reaction product was variable and the ultraviolet Spectrum indicated a mixture of an indoline and an indole. Dichloro- dicyanobenzoquinone, however, proved completely satisfac- tory for this and all other aromatizations investigated. The by-product, a substituted hydroquinone, was simply filtered from the cooled xylene solution. Care must be exercised so that the solution is not cooled too much because the indole-sugar derivative will crystallize from cold xylene. Figure 7 shows the infrared Spectra of the intermediates and the product of this synthetic scheme. Attachment of the side-chain to the glucosyl-indole went smoothly only if xylene was used as the solvent in preference to benzene. The ethyl ester prepared from ethyl diazoacetate was purified by column chromatography and crystallized from methanol-water. The acetate blocking groups were effectively removed by treating a cold methanol solution of the ester with barium methoxide as described for similar compounds by Isbell (34). Deacetylation was attended by transesterification of the side-chain to yield the deacylated methyl ester. Attempts to hydrolyze the methyl ester resulted in a product that had a lower Rf value on thin-layer chromatography than did the ester. The hydrolytic product did not crystallize. The Spectra of Figure 7 serve to verify that each reaction step took place. The center Spectrum has a wider carbonyl band than the starting material (upper Spectrum) v ’- ' .‘ Lv': 511-]. $31!“ 135 FIGURE 7 Infrared Spectra of glucosyl derivatives: Upper; 1-(2',3',4',6'-Tetra-O-acetyl-B-D- glucopyranosyl)indole Center: Ethyl 1-(2',3',u',6'-tetra-O-acetyl- glucopyranosyl)indole-B-acetate Lower: Methyl i-B-D-glucopyranosyl- indole-B-acetate res: rl-B-D' .ossetxl- :ate 136 “nun-cw. r‘ “III! III I v 1 um..- (- nun um— (- rum-ov- — noo- 137 which was Shifted to a slightly longer wavelength. Increased C-H absorption is also noted and a new ester band appears at 8.7 u: A large hydroxyl band is evident in the deacetylated product as shown in the lower Spectrum. In this case the carbonyl band at about 5.85 is shifted still farther and the broad ester peak at 8.1 u is not present. An alternate route for synthesis of the glucosyl derivative proved unsuccessful. Thus, 1-(2',3',h',6'- tetra-O-benzyl-B«D-glucopyranosyl)indole was prepared by the laborious route of Preobrazhenskaya and Suvorov (77). Begin- ning with benzylation of methyl-a-D-glucoside and treatment of the product with hydrochloric acid in acetic acid one obtains a glucose derivative blocked in all but the 1-posi- tion with benzyl groups (8h). A methylene chloride-pyridine solution of this material was treated with Ernitrobenzoyl chloride and the ester product was treated in turn with hydrobromic acid in methylene chloride. The product of the latter reaction, 2',3',4',6'-tetra-O-benzyl-d-D-gluco- pranosyl bromide, was reacted with indoline in ethyl ether to yield a i-substituted indoline. Finally, dichlorodicyano- benzoquinone was employed in the dehydrogenation step. Several attempts were made to react the i-glucosyl indole with ethyl diazoacetate but to no avail. Molecular models of the compound indicate that considerable steric hinderance is encountered in an attempted reaction at the 3-position of the indole moiety. An equally fruitless effort was made to react the N- mercury Salt of indole with acetobromoglucose in analogy to 138 the synthesis of thymine derivatives reported by Fox 23.91. (20). Biological Activity Effect of l-alkylation on biological activity The effect of alkyl substitution on the nitrogen atom of indole-3-acetic acid was measured by different assays. The i-alkyl compounds were not active enough to cause sig- nificant parthenocarpic activity in the tomato ovary assay as depicted in Figure 8. EXperiments indicate that fruit- setting effectiveness decreased as the chain length and size were increased. The time required for abscission of the treated tomato ovary is a measure of activity. These data are given in Figure 9. The indole compounds were assayed at 4, and 10'5 n, three different concentrations--10'3, 10- Significantly, all of the derivatives, even at 10-5 fl, showed some degree of activity in this assay. However, the relative effectiveness of these derivatives cannot be assigned on the basis of either the fruit-set or ovary abscission time assay. Bean petiole abscission was also investigated as a means of determining the effect of alkylation at the nitro- gen atom. Figure 10 gives the results of this method. Unfortunately, the relative activity could not be estimated by these data because all of the alkyl indole derivatives showed very little response. Figure 11 demonstrates the striking decrease in L11. 139 FIGURE 8 Tomato Fruit-Set Assay Effect of 1-alkylation on tomato ovary growth at 10 days fol- lowing treatment with 10 fl lanolin solutions of the test compounds. The control abscissed at 6 days when the diameter was 3.5 mm. Methyl Ethyl gePropyl iso-Propyl n—Butyl iso-Butyl. seg-Butyl Egrthutyl anentyl ngDecyl grOctadecyl EEEEEEEEEEEE Ovary Diameter (mm) 140 O\'\1 I (n I \o I H O "ZI "CI _,n-[ ' cu. 1H1 FIGURE 9 Tomato Ovary Abscission Assay Abscission time of tomato fruit treated with 10"3 M_lanolin solutions of the test compounds. Starred (*) compounds at the indicated concentrations did not abscise during the course of this experiment. Methyl Ethyl ngropyl igggPropyl ‘anutyl iggrButyl gggrButyl Eggthutyl n—P entyl _r_1_-Decyl n¢Octadecyl 1#2 Abscission Time (Hrs) Percent of Control SSSSEHHHHHHHPH , tummmwx‘loooo omomomomomomom AIIITTIIIIIIIFI IAA.-%é ID‘ llllll \\\\ \\ IAA I-‘HH °.°.°. Kit-Pb) xxx-w Islam \‘\\\\\\ \ \\\\ \ \\\\\\A.\S\ \\\XVI ‘ I \\ \\ \\\\X .\\\\\ \\\ \ \\\\ X\\ \ \\\L\\I xx \ \\\\\\x'\\xxx\ \\Y\\\\VXJ E (d.- \\\Y\A\\\\\X\\\\\QX\\\}\1 fiwrfl-m T Ilr" ‘ "to . ' small k 1' “ ‘Vu‘ 3w; 1&3 FIGURE 10 Bean Petiole Abscission Assay Effect of 1-substitution on abscission time of debladed petiole following treatment with 10"3 M_lanolin solutions of the test compounds. w» p’ 170 0 6 1 b no 5.4 1. 130 Hoapsoo wo psooaom made coammaomnd 3233333333333 HhoooMpoOLm Anacondaxua.h Hancomtm H35.» .8» HmpsmLmMm Hapsmummw. thsmlm. Haaoammew Haaoamnm. Essa H33: 170 160 F. A similar cor- relation was also observed in the buckwheat root inhibition assay (Figure 11). Secondary effects were obviously involved in the £1222 reSponse of the other benzyl derivatives. Neither the activ- ity nor Hammett 0p values are very different for the methyl 166 FIGURE 18 Avena Straight Growth Assay Growth curves of Avena coleOptile sections, effect of para- substitution on the Benz 1 moiety of ethyl i-benzylindole- 3-acetate: (1) IAA,"(14 Benzyl EIA, (15) p-Me EIA, (16) 2" F—Benzyl EIA, (17) p—Cl-Benzyl EIA, (18) p—Br-Benzyl EIA, (19) p-MeO-Benzyl EIA, (20) p-NOZ-Benzyl EIA. Percent of Control Growth 167 200t- 180)— 160- 140- 120- ': u 17 {A 100" ‘ \2 En BOF' A ) gis )( 60— 40 J n l __ | J ~10‘8 10'7 10'"6 10'5 10'“ Molar Concentration .V' '.' ‘i 1 li“ “I,“ IV 168 FIGURE 19 Correlation of Taft ES values with Alena Straight Growth.Activity Avena straight growth activity of various 1-alkyl indole-3- acetIc acids as compared with the Taft E8 values of the sub- stituents. b.H m.s s.a m.a ~.H H.H o.” o.o m.o a.o b.o m.o :.o m.o m.o duo 169 m m o Hmod _ q q emanate .O/ a a smaoom d _ smlom fix _ A _ 1‘ _ q fi‘ — mm VVI qqnoxg toxiuoo queoxed punodmoo 439$ qqmoxg toxquoo queoxeg OOT X 170 and methoxy substituents, but the activity of both is dis- proportionally large if the electronic effect is acting as suggested by Thimann (75). In contrast the nitro substitu- ent, which would be expected to have among the strongest electron withdrawing properties, imparts no higher activity than did the methyl and methoxy substituents. Hansch and Muir (26) demonstrated that nitro-substi- tuted auxins often have low levels of activity. This may be attributable to polar groups which reduce the lipophilic character of the ring (117). Another possibility is that the anionic character of the nitro group causes the auxin to complete with the attachment site that normally would be occupied by the carboxyl. The latter possibility is in agreement with the finding that nitro groups can replace the carboxyl groups in certain synthetic auxins (117). These results may explain the discovery that the ppnitrobenzyl derivative had no higher activity than did methyl or methoxy compounds. However, the reason for the low activity of the fluoro- compound is not obvious. The rather high activity observed for ethyl 1-benzyl- indole—3-acetate is noteworthy. paparSubstituents do not contribute much to the relative size of the benzyl radical and hence are not expected to interfere significantly with activity on the basis of steric factors alone. None of the withdrawing groups enhanced the activity in this experiment nor did any of the donating groups deduct significantly from that of the unsubstituted benzyl compounds. Therefore, the reasons for the observed activity are not clear. 171 The finding that ethyl 1-benzylindole-3-acetates did Show a level of activity in Azggg straight growth approach- ing that of indole-3-acetic acid lends support to the pos- sibility that the substituted benzoyl analogs assayed by Ritzert g1; 51. (81) are active Egg §_e_. A given substituted benzoyl moiety has greater electron withdrawing power than does the correSponding benzyl group and would thus be more active according to Thimann's theory. The latter analog of ethyl 1ndole-3-acetate would not be eXpected to hydrolyze the linkage at the nitrogen atom as easily as would the former and thus, would be more likely to remain intact in the plant. Both the benzyl and benzoyl groups may resist hydrolysis Since compounds bearing both groups had reason- ably high activity in the Agggg test with the acyl compound being more active. In addition, a much more labile compound, methyl-B-D-glucopyranosylindole-3-acetate, had essentially no activity. If the latter compound were hydrolyzed, it would give the high level of activity associated with methyl indole-3-acetate. The low level of activity noted for all of the benzyl analogs in intact plants may be eXplained by the inability of these large compounds to penetrate to the site of action. Another assay on intact plants, the cucumber curvature test, gave a minimal reSponse as is evident in Figure 13. Nevertheless, some tomato ovary growth was noted with this series (Figure 20) indicating that the compounds were trans- ported through an intact membrane to some extent. 172 FIGURE 20 Tomato Ovary Growth Assay ' Effect of various ara substituents on the benzyl radical of ethyl 1-benzylindoIe-3-aceta5e on tomato ovary growth ten days following treatment with 10' M lanolin solutions of test comp pounds. Benzyl er-Benzyl pyCl-Benzyl pyBr-Benzyl prCH -Benzyl 3 prCH O-Benzyl 3 [pyNOZ-Benzyl EIA EIA EIA EIA EIA EIA EIA EIA 173 H' H~ l4 u: #— \n 0x -q x) c: +9 h) "I *I r I I I I I I *j l _] ;:2 ._ 174 In both the 21222 straight growth and bean petiole abscission assays, in which the compounds need not pass through a membrane, a higher level of activity was observed. Figure 10 records the data of the latter experiment. Secon- dary factors, other than the electronic considerations, must be involved. In this case the nitro group appears to exert an effect that could be attributed to electron withdrawal and an attendant enhancement of the partially positive charge on the indolic nitrogen (75, 76). Additional evidence of an essentially negative nature was gathered to support the premise that the substituted 5 compounds are active pgrflgg. When a 10- M'solution of 1-benzylindole-3-acetic acid was allowed to stand overnight with a few small slices of green tomato fruit no material which migrated as indole-3-acetic acid on thin-layer chromatography could be seen. This is not definite evidence against hydrolysis because the free acid may be degraded issue. To ascertain whether or not 1-substituted derivatives of indole-3-acetic acid acted as antiauxins or antagonists, a 10.5 M'solution of indole-3-acetic acid was diluted with 5 an equal volume of either 10-“ M.or 10- M'solutions of several representative indole derivatives. In one experi- 5 ment, (2.5 m1 of 10" 91 indole-3-acetic acid was combined with a like volume of 10'” M,i-methylindole-3-acetic acid and in another eXperiment with 2.5 ml of a 10"5 M_solution of the substituted acid. Solutions of 1-iso-propylindole- 3-acetic acid and ethyl 1-benzylindole-3-acetate were 175 prepared in a Similar manner. Each solution was assayed in the buckwheat root inhibition test. In all cases the level of activity was very nearly that expected of 5 x 10"6 M_ solution of indole-3-acetic acid, thus indicating that the substituted acid or ester did not function as an auxin antagonist by reversing the inhibition established by the free acid. SUMMARY SUMMARY A series of 1-alkyl indole-3-acetic acids were synthe- sized and assayed for physiological activity. These com- pounds were made from indoline by way of a substituted indoline: The substituted indoline was dehydrogenated to the correSponding indole with dichlorodicyanobenzoquinone. Attachment of the Side-chain ester group to indole was effected with ethyl diazoacetate. Finally the free acids were prepared by saponification of the esters. A list of the indole acids so prepared follows: 1-methylindole-3-acetic acid 1-ethylindole-3-acetic acid 1gnrpropylindole-3-acetic acid 1eiggrpropylindole-3-acetic acid iggpbutylindole-3-acetic acid 1-1ggybutylindole-3-acetic acid 1egggybutylindole-3-acetic acid 1etggtrbutylindole-3-acetic acid 1eggpentylindole-3-acetic acid iggrdecylindole-3-acetic acid 1farcetadecylindole-B-acetic acid Methyl 1-BéD-glucopyranosylindole-3-acetate was pre- pared by an analogous synthetic route and then assayed for biological activity. Several ethyl 1fi2§BUDSt1tUted indole-3-acetates were synthesized from ethyl indole-3-acetate. The latter com- pounds were assayed by the same methods as were the alkyl analogs.=‘ The members of this series are: 177 178 ethyl 1-benzylindole-3-acetate ethyl 1-prfluorobenzylindole-3-acetate ethyl 1~p:chlorobenzylindole-3-acetate ethyl 1-prbromobenzylindole-3-acetate ethyl 1-prmethylbenzylindole-3-acetate ethyl 1-pymethoxybenzylindole-3-acetate ethyl 1~prnitrobenzylindole-3-acetate The correSponding acids were prepared from all of the above esters except the nitro-compound. Ultraviolet and infrared Spectroscopy was used to supplement elemental analysis in the characterization of the compounds. Refractive indexes, boiling points, and melting points are reported for all compounds including the synthetic intermediates. Biological assays were employed to evaluate the activity of the new compounds.. A3923 straight growth test was the most sensitive measure of activity investigated. Low activity of each compound was noted in the buckwheat root inhibition and tomato fruit-setting assays. The cucumber curvature assay showed essentially no reSponse to any of the compounds tested with the exception of 1-iggr propylindole-3-acetic acid. The latter compound caused delayed bending which was much more persistent than that caused by indole-3-acetic acid. The bean petiole abscission and the £2222 straight growth assays do not require compounds to penetrate intact membranes. Benzyl derivatives were the most active in these tests, indicating that the larger molecules do possess a certain level of activity when they are allowed to reach the Site of action. 179 A correlation was noted between Taft's E8 values and the activity of the lower alkyl compounds. Thus, 1-methyl- indole-3-acetic acid has some activity in the Ayggg straight growth test and 1gtggtebutylindole-3-acetic acid showed definite inhibition. Each of the alkyl compounds showed only a very low level of activity in all of the other assays. A correlation apparently exists between activity in !. §y§n§_or buckwheat and Hammett's 6p values for the three 3 halo-compounds assayed. Secondary effects apparently over- i. shadowed the electronic contribution of other papa substitu- ents associated with the benzyl esters. Methyl 1-B-D-glucopyranosylindole-3-acetate was almost entirely inactive in all assays. Such a reSponse is to be eXpected of very hydrophilic entities. REFERENCES 7. 8. 9 0) 10. 11. 12. 13. 14. 15. 16. 17. 18. REFERENCES Andreae,)W. A., and N. E. Good, Plant Physiol. 0, 380 1955 . Arbusow,.A: E., and w. M. Tichwinsky, Chem. Ber. 23. 2301 (1910). Baskakov, Y? A., and N. N. Mel'nikov, F. Rastenil, Acad. Nauk SSSR 3, 208 (1956). Baskakov, Y. A., and N. N. Mel'nikov, Sbornik Statei Obshch. Khim., Acad. Nauk SSSR ;, 712 (1953). Beer, R: J. 8., K. Clarke, H. F. Davenport, and A. Robertson, J. Chem. Soc. 1251, 2029. Beer, R. J. S., K. Clarke, H. F. Davenport, and A. Robertson, J. Chem. Soc. 1222, 1262. Bentley, J. 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APPENDIX Chemical Name Indole-3-acetic acid 1-Methylindole-3- acetic acid 1-Ethylindole-3- acetic acid l-normal-Propylindole-3- acetic acid l-iso-Propylindole-3- acetic acid APPENDIX I Abbreviation IAA Me IAA E1 IAA g-Pr IAA so-Pr IAA 189 Structure I I H 03200011 N I CH3 N I $32 CH3 CHZCOOH \N I (CHZ)Z I 033 HBC-fi~CH3 H Chemical Name 1-normal-Butylindole-3- acetic acid 1-iso-Butylindole-3- acetic acid 1-secondarygButylindole- 3-acetic acid 1-tertiarz-Butylindole- 3-acetic acid l-normal-Pentylindole- 3-acetic acid 190 Structure .UJHZCOOH N I (CH) I23 CH3 ficazcoon N s32 Abbreviation g-Bu IAA so-Bu IAA H N I H-C-C33 sec-Bu IAA l 032 I 033 - CHZCOOH tert-Bu IAA O I N I H C-C-CH 3,3 CH3 320008 n-P entyl IAA N l I ($32)” CH3 Chemical Name i-normal-Decylindole-B- acetic acid 1~norma1-Octadecylindole- 3-acetic acid Ethyl indole-3-acetate Ethyl 1-benzylindole43- acetate 191 Abbreviation Structure (.1 CHZCOOH N I (Cflz)9 I CH ggDecyl IAA 3 08200011 IAA | N I (CH2)17 1., 01120000211 5 N I H CHZCOOCZH5 N I ggOCtadecyl EIA Benzyl EIA C32 CH2C00C2H5 Ethyl 1-(para-f1uorobenzyl)- I indole—3-acetate N EfF-Benzyl EIA I CH F N 192 Chemical Name Abbreviation Structure CHZCOOCZH5 Ethyl 1-(para-chlorobenzyl)- I indole-3-acetate prCl-Benzyl EIA I OH 2 Cl CHchOCZHS Ethyl 1- (para-bromobenzyl)- N indole-3-acetate EyBr-Benzyl EIA | C32 Br flCHzcooc Ethyl 1-(para-methylbenzyl)- N indole-3-acetate prMe-Benzyl EIA 6H] ' 2 0 tn u) 193 Chemical Name Abbreviation Structure / CHZCOOCZH5 Ethyl 1-(Eara-methoxy- -p_-MeO-Benzyl EIA O N benzyl)-indole-3-acetate I CH2 0 I CHZCOOCZH5 Ethyl 1-(para-nitro- ‘ErNOZ-Benzyl EIA N I benzyl)-indole-3-acetate 032 0 N02 1-(2' ,3' ,4' ,6'-Tetra- Indoline TAG . I H2 - N H 0-acety1-8nD-gluco- AcO-CH 2 pyranosyl) indoline Ac Ac .OAc 194 Chemical Name .Abbreviation Structure 1-(2: ,3: 1’1}! ,6!..Tetra- Indole TAG O l N O-acetyl-B-D-gluco- AcO-CHZ pyranosyl) indole OAc Ac OAc .:——J- 0 C0 0 H Ethyl 1-(2'.3'.4'.6'- EIATAG O NI HZ 2 2 5 Tetra-O-acetyl-B-D- AcO-CH glucopyranosyl) Ac indole-B-acetate ,Ac OAc CHZCOOCHB Methyl 1-8-D-glucopyran- Glucosyl MIA 0 I N osylindole-B-acetate HO'CHZ OH H H OH "IIIILIIIIZIIIIIIIIIIIJIIIIIIIIIIIIIIII“