AND NOLOGCAL ACnViTY OF ETHYL ACETATE LUORENGNE THE SYNTHES5 SOME ALL‘I’L, AC- ETHESLS OF FLU ETIC AC“) AND GRENE AND F Thesis for the Dam!“ o§ Ph. D. MXCHIGAN STATE UNNERSETY Charms Richaré Bar: 1960 L: :wfiARY gran Scan: 1 THESIS ,p /. . L ‘ 9")“ ~ \ i” I; \ J 9,1: V (‘I’Sit’ I memem Sm: mwxmswv THE SYNTHESIS AND BIOLOGICAL ACTIVITY OF SOME ALLYL, ACETIC ACID AND ETHYL ACETATE ETHERS OF FLUORENE AND FLUORENONE By CHARLES RICHARD BARR AN ABSTRACT Submitted to the School of Advanced Graduate Studies Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1960 Approved P a me Charles Richard Barr A number of ethers with an aromatic nucleus and acetic acid side chain have been shown to possess growth-regulat— ing activity. The relative biological activity of these substances has been shown to depend upon the position of the side chain in the aromatic ring. In the present study procedures were devised for the incorporation of allyl, acetic acid and ethyl acetate ether groups into the l, 2, and 3 positions of fluorenone and into the 2 position of fluorene. The fluorenol and fluoren- onol intermediates were synthesized by procedures given in the literature. The ethers were prepared by employing a modified Williamson synthesis; the fluorenol or fluoren- onol was allowed to react with ethyl bromoacetate or allyl bromide in anhydrous acetone over anhydrous potassium carbonate. The composition of these compounds was verified by elemental analysis for carbon and hydrogen. The melting points and absorption spectra were determined as physical constants. All of the ethers were tested for growth-regulating properties by applying them in a lanolin paste to the emasculated flowers of tomato plants. The compounds show- ed no positive stimulation of parthenocarpy seven days after treatment of the tomato flowers. Abscission of ovaries occurred in some flowers treated with ethyl l- fluorenonoxy acetate, 1-fluorenonoxyacetic acid, 2-alloxy- Charles Richard Barr fluorene, ethyl 3-f1uorenonoxy acetate, 5-fluorenonoxy- acetic acid, and fluorene-Q-acetic acid. The water soluble compounds, the fluorenonoxyacetic acids and fluorene-Q-acetic acid, were also tested for biological activity by measuring their effect on the root growth of cucumber seeds. Measurement of the root length three and one-half days after germination of the seeds showed that 2-fluorenonoxyacetic acid and 3-f1uorenonoxy acetic acid stimulated root growth, whereas f1uorene-9- acetic acid showed a marked inhibitory effect. VITA CHARLES RICHARD BARR Candidate for the degree of DOCTOR OF PHILOSOPHY Dissertation: The Synthesis and Biological Activity of Some Allyl, Acetic Acid and Ethyl Acetate Ethers of Fluorene and Fluorenone. Outline of Studies: Major Subject: Biochemistry Minor Subjects: Organic Chemistry and Plant Physiology Biographical: Born: Dakota, Illinois, May 16, 1932. School: Dakota Public Schools, Diploma, 1950. Undergraduate Studies: North Central College, B.A., 1954. Graduate Studies: lichigan State University, M.S., 1957. Experience: Undergraduate Laboratory Assistant, North Central College, 1952-1954. Special Graduate Research Assistant, Michigan State University, 1954-1958. Assistant Professor, Department of Chemistry, University of Dubuque, 1958- . Membership in Professional Societies: American Chemical Society Society of the Sigma Xi lidwest Association of Chemistry Teachers in Liberal Arts Colleges. THE SYNTHESIS AND BIOLOGICAL ACTIVITY OF SOME ALLYL, ACETIC ACID AND ETHYL ACETATE ETHERS OF FLUORENE AND FLUORENONE By CHARLES RICHARD BARR A THESIS Submitted to the School of Advanced Graduate Studies Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1960 TABLE OF CONTENTS INTRODUCTION 0 . O O O O O O . HISTORICAL . . . . . . . . . EXPERIMENTAL . . . . . . . . . Synthesis of Compounds . Fluorene-Q-acetic acid . . . Ethyl l-fluorenonoxy l-Fluorenonoxyacetic l-Alloxyfluorenone . acetate acid . Ethyl 2-f1uorenoxy acetate . 2-Fluorenoxyacetic acid . . 2-Alloxyf1uorene . . Ethyl 2-f1uorenonoxy 2-Fluorenonoxyacetic 2-Alloxyfluorenone . Ethyl 5-fluorenonoxy S-Fluorenonoxyacetic 5-Alloxyf1uorenone . Biological Assay . . . . Tomato parthenocarpy acetate acid . acetate acid . test . Cucumber seed root test . . iii RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 33 SUMMARY 0 O O O O O O O O O O O O O O 0 O O O O O O 43 BIBLIOGRAPHY O O O O O O O O O O O O 0 O O O O O O O 45 LIST OF TABLES TABLE PAGE I. Melting Points and Analysis for Carbon and Hydrogen for Fluorene Compounds . . . . 55-56 II. Absorption Characteristics of the Fluorene COMPOUUOS o o a o e e o e e a o e o a a 37-58 Ill. Stimulation of Parthenocarpy in the Tomato by Fluorene Compounds in Lanolin Paste 59-40 IV. Stimulation of Root Growth in Cucumber Seeds by Fluorene Compounds . . . . . . . . . 42 ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Professors R. U. Byerrum and H. M. Sell for their interest and guidance throughout the course of this work. The writer appreciates the financial support granted by the National Science Foundation, Washington 25, D.C., which made this study possible. Grateful acknowledgement is due Mr. Roger Ritzert and Mrs. Carolyn Titus for aid in the determination of physical properties of the compounds. INTRODUCTION INTRODUCTION A large number of synthetic compounds have been evalu- ated for growth-regulatory properties, however, relatively few of these have been found to possess biological activity. The synthesis and testing of compounds structurally related to substances occurring naturally in plants which are thought to be involved in their natural growth is the logical ap- proach to selective active compounds. A number of ethers composed of an aromatic nucleus and acetic acid side chain have been shown to have growth- regulatory properties, of these substances phenoxyacetic acid analogs have been most studied. Quite recently con- siderable attention has been focused on the unusual bio- logical activity of gibberellic acid, a naturally occur- ring plant growth regulator with a decahydrofluorene nucleus. Another fluorene derivative, f1uorene—9-acetic acid, was found to modify growth in plants (1). Since fluorene derivatives have been shown to possess plant growth-regulatory activity, a number of ether deriva- tives of fluorene and fluorenone were synthesized to deter- mine what if any physiological effect they have on plant growth. The problem consisted of two parts. The object of the first part of the problem was to devise a satisfactory method for the preparation of ethers from fluorenols, since the chemical procedures used for the preparation of the corresponding phenyl and naphthyl ether analogs gave yields of 5% or less when used for the synthesis of fluorene ethers. The second part of the work had a two fold purpose: 1) to ascertain whether the fluorene ethers possessed biological activity, either wsgrowth promotors or inhibitors; and 2) to correlate the activity with the structure of the compounds. HISTORICAL and». .u...H.H-|l1. . J'lwfilll U! 4.3a”...r1l.wd mdhwd. HISTORICAL A large number of chemical compounds have been found to have growth-regulating properties. Since 2,4-dichloro- phenoxyacetic acid is one of the frequently used herbicides it is not surprising that extensive studies of related compounds have been made. A large number of selected com- pounds related to 2,4-dichlorophenoxyacetic acid have been synthesized (2,3). A study of the effects of nuclear sub- stitution in phenoxyacetic acid on cell elongation was systematically investigated by Muir gt_gl, (4). The effect of substitution in the side chain of aryl- oxyacetic acids was studied by Osborne and Wain (5). Using the pea test and the straight growth assay these workers observed that substitution of an alkyl group in the acetic acid side chain had little effect upon activity in most cases. However,‘quédisubstituted compounds were inactive in straight growth, but some of them were slightly active in the pea test. The authors suggested that a chemical reaction involving a hydrogen atom of the carbon adjacent to the carboxyl group operates in the growth response. Applying the pea test and using a large number of active compounds, Koepfli st 21* (6) formulated the fol- lowing five structural requirements for compounds which are capable of inducing cell elongation: l. a ring system nucleus 2. a double bond in the ring 5. a side chain 4. a carboxyl group or a structure readily converted to a carboxyl on the side chain at least one carbon removed from the ring 5. a particular space relationship between the ring and the carboxyl group. The five structural requirements for cell elongation have more recently been condensed into two by Veldstra (7): l. a basal ring system (nonpolar part) with high interface activity 2. a carboxyl group (polar part) in such a spatial position with respect to the ring system that this functional group will be situated as periph- erally as possible to a boundary on adsorption of the active molecule. Veldstra (8) has found that the degree of fat sol- ubility as influenced by the ring structure and the water solubility as influenced by the side chain structure could be correlated with growth-regulating activity. He con- cluded that activity was greatest when the lipophilic and the hydrophilic preperties were balanced. Thus, he thought of the auxin action as being something of a physical bond- ing of some lipoidal material to some non-aqueous phase. The work described in this thesis consists of the methods of synthesis and the biological assay of acetic acid ether and its analogs substituted in different posi- tions in the fluorenone nucleus. EXPERIMENTAL EXPERIMENTAL Synthesis g§_Compounds The ethers of fluorene and fluorenone were prepared by employing a modified Williamson type of synthesis. The appropriate hydroxyfluorene or hydroxyfluorenone was re- fluxed with ethyl bromoacetate in anhydrous acetone over powdered anhydrous potassium carbonate to form the ethyl esters of the acetic acid ethers. Hydrolysis with acid or base yielded the corresponding free acids. The allyl ethers were prepared by substituting allyl bromide for the ethyl bromoacetate. The hydroxyfluorene and hydroxyfluorenone intermediates were prepared according to procedures found in the litera— ture. A summary of the reactions utilized in the synthesis of these compounds is outlined below. Fluorene-9-acetic acid ' N-bromgsgcgimmige 9502020 JCH-COZCQHQ Na"L \ / ,//'\\ H5C202C COZCZHS ___11_NSOH \_ heat \ 2) HCl /' IIIIII ’ H H02C’/§C\\602H H H2~C02H 9-Bromof1uorene (9). A solution of 50.0 g. (0.50 mole) of fluorene in 225 m1. of carbon tetrachloride was reflux- ed with 55.0 g. (0.50 mole) of N-bromosuccinimide for three hours. After reaction, succinimide floated on the surface and was removed by filtration. The solvent was removed by distillation under reduced pressure and the residue crystal- 1ized from dry benzene. One recrystallization yielded 55.5 g. of white needles with melting point of 100-1050 C. The crystals of 9-bromof1uorene decomposed quite rapidly on storage. Fluorene-9-acetic acid (10). A solution of 4.5 g. (0.018 mole) of 9-bromof1uorene in 15 ml. of dry benzene was added to a chilled solution of ethyl sodiomalonate prepared from 10 0.8 g. (0.055 mole) of sodium, 5.2 g. (0.041 mole) of di- ethyl malonate and 50 m1. of absolute ethanol under an atmosphere of nitrogen gas. The mixture was refluxed for one hour and the solvents removed by distillation under reduced pressure. The residual ester was saponified by refluxing for one hour with an excess of an aqueous 40% potassium hydroxide solution. The alkaline solution was acidified by the addition of hydrochloric acid and the precipitate collected by filtration. The crude fluorene- 9-malonic acid was dried and then heated at 200° C. for 20 minutes. The solid product was dissolved in hot ace- tone, refluxed with Norite and filtered through filter-eel. The fluorene-9-acetic acid was obtained by removing the solvent under reduced pressure. The acid was recrystal- lized from dilute acetic acid, yielding 2.5 g. of white needles, m.p. 151.5-152.5° C. Bachmann and Sheehan (10) give 151.5-152.5° C. as the melting point of fluorene-9- acetic acid. Ethyl l-fluorenonoxy acetate CI‘O; \ ' 7 11 ' SOClg A i H H 0 CFO 0 $20 01 C1 NaNg \ ’// HC1 \ 7 ) heat 7 \\\ H O 1:0 | + .- NZNZN N8N02 > HCl Br-CH2-002C2H5 heat 7 K2003, acetone 0 -CH2-00202H5 Fluorenone-l-carboxylic acid (11). A solution of 175 g. (1.75 moles) of chromic anhydride in 150 ml. of water and 100 m1. of glacial acetic acid was added to 50 g. (0.25 mole) 12 of fluoranthene dissolved in 400 m1. of glacial acetic acid at such a rate that the reaction mixture was kept just be- low its boiling point. The mixture was allowed to stand for two hours, poured into two liters of water and the pre- cipitate collected on a filter. The crude acid was extract- ed with a cold 10% solution of sodium hydroxide and re- precipitated with hydrochloric acid. The acid was further purified by extraction with a hot aqueous suspension of barium carbonate; acidification of the filtrate with hydro- chloric acid and isolation of the precipitate by filtration yielded 27.5 g. of orange fluorenone-l-carboxylic acid which melted at 190-1920 C. Elugrenone-l-carboxylic azigg_(l2). Ten grams (0.045 mole) of fluorenone-l-carboxylic acid and 9.0 ml. (0.09 mole) of thionyl chloride were warmed at 40° C. in a water bath for three hours. The excess thionyl chloride was distilled from the flask under reduced pressure. The residue was dissolved in 200 ml. of dry acetone, cooled to 5° C. in an ice bath and a solution of 4.0 g. (0.06 mole) of sodium azide in 16 ml. of water was added from a dropping funnel as the reaction mixture was stirred. Stirring was con- tinued for one hour and the solution diluted to 400 ml. with water and the precipitate collected by filtration. The crude azide weighed 10.5 g. and melted at 89-91° C. after recrystallization from ethanol. 15 1:Aminofluorenone. Ten grams of fluorenone-l-carboxylic azide was covered with 250 m1. of concentrated hydrochloric acid and the suspension refluxed for three hours. The re- sulting solution was diluted with 200 m1. of water and re- fluxed for an additional hour. The solution was cooled, made alkaline by the addition of ammonium hydroxide, stirred for one hour and the precipitate collected on a filter. The l-aminofluorenone was recrystallized from ethanol yielding 4.0 g. of fine golden needles, m.p. 115-115° C. l-Hydroxyfluorenone. Five grams (0.025 mole) of 1-amino- fluorenone was dissolved in a solution composed of 100 m1. of concentrated sulfuric acid, 100 ml. of glacial acetic acid and 200 ml. of water by heating nearly to boiling. The hot solution was filtered through a sintered glass filter, cooled to 5° C. in an ice bath and a solution of 1.7 g. (0.025 mole) of sodium nitrite in 50 m1. of water was added to the stirred solution from a dropping funnel. The solution was kept cold for an additional one-half hour and then 5 g. of urea was added and the solution again stirred for one-half hour. The diazotized solution was placed in a dropping funnel and added to one liter of boiling water containing 25 ml. of sulfuric acid over a period of 20 minutes. The resulting solution was refluxed for one hour, cooled and the precipitate isolated by fil- tration. After recrystallization from 50% acetic acid the fine yellow needles weighed 4.7 g. and melted at 210- 211° C. 14 Ethyll:fluorenonoxy acetate. To a solution of one gram (0.005 mole) of 1-hydroxyf1uorenone in 20 ml. of anhydrous acetone was added 1.25 g. (0.0075 mole) of ethyl bromo- acetate and 1.04 g. (0.0075 mole) of powdered anhydrous potassium carbonate. The suspension was refluxed for 12 hours, cooled and poured into 200 ml. of ice water. The yellow precipitate was collected on a filter and re- crystallized from 80% ethanol giving 1.08 g. of long lemon- yellow needles, m.p. 92-95.5° C. EtOH Absorbance ,X 257 mu (log a = 4.817) max 250 mu (log 5 = 4.746) Anal. Calcd. for 017H14O4: C, 72.5; H, 5.00. Found: C, 72.5; H, 4.95. l-Fluorenonoxyacetic acid H20 H2304 Twenty-eight hundredths gram (0.001 mole) of ethyl 1- fluorenonoxy acetate was dissolved in a solution of 50 m1. of ethanol, 50 ml. of water and 10 ml. of concentrated sulfuric acid and the solution refluxed for three hours. The solu- tion was cooled and poured into 150 m1. of water and the precipitate collected by filtration. The acid was separated from unhydrolyzed ester by extraction with 50 ml. of a 10% 15 sodium hydroxide solution and precipitation from the extract by acidification with hydrochloric acid. Recrystallization from 70% ethanol gave 0.10 g. of fine yellow crystals, m.p. 220-2240 C. 0H t 258 mu (log a 4.792) E Absorbance ,Amax 251 mu (10g 8 ; 4.752) Anal. Calcd. for 015H1004: C, 70.9; H, 5.95. Found: C, 71.23 H, 5.92. l-Alloxyfluorenone Br-CHz-CH20H2 \ ' K2003, acetone ’ H 0 OH O O - GHQ-CH=CH2 A solution of 2.75 g. (0.014 mole) of l-hydroxyfluoren- one in 50 m1. of anhydrous acetone was refluxed for 24 hours with 2.55 g. (0.021 mole) of allyl bromide over 2.90 g. (0.021 mole) of finely powdered anhydrous potassium carbon- ate. The reaction mixture was cooled and added to an ice and water slurry and the precipitate collected by filtration. A yield of 5.24 g. of yellow platelets with a melting point of 67-68° C. was recovered after recrystallization from 80% ethanol. Dioxane Absorbance A 505 mu (log 5 = 5.290) max 291 mu (log £:= 5.550) 281 mu (log £«= 5.569) 259 mu (log a = 4.851) -251 mu (log a = 4.805) 16 Anal. Calcd. for C16H12O2‘ C, 81.5; H, 5.12. Found: C, 81.4; H, 5.26. Ethyl 2-f1uorenoxy acetate HN03 g ' / N02 H H Zn > / | I \ NaN02 \ / HCl 7 \ / NH2 H H BP'CH2-00202fl5‘;> K2003, acetone O-CHg-COZCQHS 2-Nitrof1uorene (15). Twenty grams of fluorene was dis- solved in 160 m1. of warm glacial acetic acid in a three- necked flask equipped with thermometer, mechanical stirrer and dropping funnel. The flask was placed in a water bath and the temperature raised to 50° C., and 50 ml. of concen- trated nitric acid was added with stirring during 15 minutes. 17 The water bath was then slowly heated so that the tempera- ture reached 65° C. after 20 minutes. Stirring and heating were continued so that the reaction temperature reached 80° C. after another 25 minutes. After stirring for five minutes at 80° C. the water bath was removed and the prod- uct filtered, washed with two 10 m1. portions of cold glacial acetic acid each containing 0.2 g. of potassium acetate and then several times with water. The crude 2- nitrofluorene was recrystallized from glacial acetic acid giving 17.7 g. of fine yellow needles, m.p. 156° C. 2-Aminof1uorene (15). A thin paste was prepared from 6.5 g. (0.03 mole) of dried and powdered 2-nitrof1uorene and 225 m1. of 75% ethanol. A solution of 2.4 g. of calcium chlor- ide in 4 ml. of water and 69 g. (1.05 moles) of zinc dust were added to the thin paste and the suspension was reflux- ed for two hours. The sludge of zinc dust and zinc oxide was removed from the hot solution by filtration and extract- ed with 20 m1. of hot 75% ethanol. The combined filtrates were diluted to one liter with water and the white floc- culent precipitate was collected on a filter. Recrystal- lization from 50% ethanol yielded 4.4 g. of white needles, m.p. 127-1290 C. 2-Hydroxyf1uorene. A solution of 2-aminofluorene was pre- pared by heating 10.0 g. (0.055 mole) of the amine in 550 m1. of 2.5 N hydrochloric acid. The solution was cooled to 50° C. and an aqueous solution of 5.8 g. (0.055 mole) 18 of sodium nitrite was added from a dr0pp1ng funnel with stirring. The resulting 2-diazofluorene was decomposed by diluting the solution to 500 ml. with water and reflux- ing until the evolution of nitrogen ceased. The solution was cooled and the solidified mass was filtered off. The crude product was recrystallized from 50% acetic acid yield- ing 6.0 g. of white platelets, m.p. 168-1690 C. EthylL2-fluorenoxy acetate. Five grams (0.027 mole) of 2-hydroxyfluorene was dissolved in 100 m1. of anhydrous acetone and 5.5 g. (0.04 mole) of anhydrous powdered po- tassium carbonate and 6.7 g. (0.04 mole) of ethyl bromo- acetate were added. The suspension was refluxed for 24 hours, the reaction mixture cooled and poured into one liter of ice water. The powdery white precipitate was collected by filtration and recrystallized from 90% ethanol yielding 5.0 g. of fine white needles, m.p. 110.5-lll.5o C. tOH E Absorbance .A 511 mu (log a = 5.827) max 501 mu (log 8 = 5.849) 269 mu (log 5 = 4.598) An‘le CEICde for 017111603: C, 76.1; H, 6.01. Found: C, 75.9; H, 5.10. 2-F1uorenoxyacetic acid KOH \ O-CH -CO C H H H 2 2 2 5 0-CH2-002H 19 Four grams of ethyl 2-f1uorenoxy acetate was added to a solution composed of 25 m1. of ethylene glycol, 4 m1. of water and 2.0 g. of potassium hydroxide and the solution heated at 125° C. for two hours. The solution was cooled, diluted to 200 ml. with water and acidified by the addition of hydrochloric acid. The precipitate was collected on a . filter and recrystallized from dilute acetic acid yielding 5.45 g. of white crystals, m.p. 178° C. (softened at 155° C.) EtOH Absorbance ,Xm 512 mu (log 5 = 5.907) 502 mu (log 5 4 5.927) 270 mu (log g = 4.454) Anal. Calcd. for 015H1203: C, 75.0; H, 5.05. Found: C, 74.82; H, 5.26. 2-Alloxyfluorene (14) Br-CH2 -CH=C 3% K8003, acet O-CH2-0H=CH2 A solution of 2.0 g. (0.011 mole) of 2-hydroxyf1uorene in 50 ml. of anhydrous acetone was refluxed for 24 hours with 2.06 g. (0.017 mole) of allyl bromide over 2.55 g. (0.017 mole) of finely powdered anhydrous potassium carbon- ate. The reaction mixture was cooled and added to an ice and water slurry which precipitated a clear oil. The ice was allowed to melt and the water decanted from the oil. Crystallization from dilute acetic acid yielded 1.75 g. of 20 white platelets, m.p. 96.5-97.5o C. (Reported m.p. 95-96° / K CO acetoné , 2 5' ll 0H \\\ . '3 .-CHZCH=CH2 A solution of 8.0 g. (0.04 mole) of 2-hydroxyfluorenone in 50 ml. of anhydrous acetone was refluxed for 24 hours with 7.5 g. (0.06 mole) of allyl bromide over 8.5 g. (0.06 mole) of finely powdered anhydrous potassium carbonate. The reaction mixture was poured into 200 ml. of ice water and the orange red precipitate collected by filtration. The 2- alloxyfluorenone was recrystallized from 80% ethanol yield- ing 9.75 g. of long yellow-orange needles, m.p. 80° C. Dioxane Absorbance .X 510 mu (log 5 = 5.714) max 294-298 mu (10g 5 = 5.808) 57 mu (log a = 4.955) 24 Anal. Calcd. for 015H1202: C, 81.5; H, 5.12. Found: C, 81.5; H, 5.12. Ethyl 5-f1uorenonoxy acetate P015 > TNH-SO2C >CH5 \ COCl p.101;5 ‘ C) 002H N82C05 + - 2%f‘ NEN H80?- NaNQg > \:fl:—C /( D\\ heat \ H2504 ” \\\ 0 CH3 / /\ OCH5 A101 H 3 \ " / (fusion) 7 25 YO‘CHZ‘COZCZHS gEToluenesulfonanthranilic acid (16). A 150 ml. solution of 20% sodium carbonate was heated to 80° C. and 25.2 g. (0.18 mole) of anthranilic acid was dissolved in the warm solution. The solution was maintained at this temperature as 34.2 g. (0.18 mole) of prtoluenssulfonyl chloride was added in small portions, making certain that the solution remained alkaline throughout the reaction. After the re- action was complete, indicated by a clear light brown so- lution, the reaction mixture was added slowly with mechanic- al stirring to a dilute hydrochloric acid solution.. The nearly colorless precipitate was recovered by filtration and recrystallized from 70% ethanol yielding 40.0 g. of colorless needles, m.p. 213° C. prTolusnesulfon-2-amino-4'-methoxybenzophenone (16). Thirty grams (0.10 mole) of powdered Retoluenesulfonanthranilic acid (dried at 120° C.) was covered with 600 m1. of carbon disulfide, 25 g. (0.12 mole) of phosphorus pentachloride was added and the mixture warmed for 20 minutes to produce the acid chloride. The solution was cooled and 16.0 g. (0.12 mole) of aluminum chloride was added to the reaction vessel followed by the addition of 53 g. (0.30.mole) of 26 anisole from a dropping funnel. After addition of all of the anisole the solution was refluxed until the red bis- aluminum complex precipitated. Then the solution was cool- ed and an additional 16.0 g. of aluminum chloride was added and the refluxing continued until the evolution of hydrogen chloride ceased. The carbon disulfide was decanted off and the solid red mass was disintegrated by the addition of ice water and the resulting white precipitate collected on a filter. The crude product was redissolved in a dilute so- lution of sodium hydroxide, treated with charcoal, filtered, cooled and precipitated by the addition of hydrochloric acid. after recrystallization from.benzene-ligroin a yield of 36.0 g. of yellow prisms was obtained which melted at 139-1420 C. S-Methoxyfluorenone (16). A solution of 20.0 g. of 2: toluenesulfon-2-amino-4'-methoxybenzophenone in 80 ml. of glacial acetic acid and sulfuric acid (1:1) was heated on a steam bath for one hour to remove the tosylate group. The solution was diluted with 500 ml. of water, treated with charcoal and filtered. The clear filtrate was cooled to 10° C. and 2-diazo-4'-methoxybenzophenone produced by the addition of 60 ml. of a 6% solution of sodium nitrite from a dropping funnel. The diazotized solution was fil- tered and the filtrate slowly heated until the evolution of nitrogen gas ceased. The solution was cooled and the brown oil was extracted with ethyl ether. The ether was removed by distillation and the oily residue was covered 27 with a dilute solution of sodium hydroxide and warmed to remove unreacted material. The solid residue was collect- ed by filtration and recrystallized from benzene-ligroin yielding 4.85 g. of lemon-yellow needles, m.p. 98-990 C. S-Hydroxyfluorenone (16). A solid mixture of 5.65 g. (0.017 mole) of 3-methoxyfluorenone and 4.65 g. (0.055 mole) of aluminum chloride was placed in a nine inch test tube and heated at 150° C. in a sulfuric acid bath until the evolu- tion of methyl chloride ceased. The dark fusion product was cautiously decomposed by the addition of dilute hydro- chloric acid, boiled with water and the residue collected on a filter. The 5-hydroxyf1uorenone was recrystallized from benzene yielding 2.05 g. of yellow crystals, m.p. 229° C. Ethyl S-fluorenonoxy acetate. Two grams (0.01 mole) of S-hydroxyfluorenone was dissolved in 40 ml. of anhydrous acetone and 2.08 g. (0.015 mole) of powdered anhydrous potassium carbonate and 2.50 g. (0.015 mole) of ethyl bromo- acetate were added and the suspension refluxed for 24 hours. The reaction mixture was cooled and poured into 400 ml. of ice water and the precipitate collected by filtration. The ester was recrystallized from 70% ethanol yielding 2.5 g. of fine yellow crystals, m.p. 110-1110 C. tOH 273 mu (log 5 4.708) Absorbance ,XE max 254 mu (log 5 = 4.754) Anal. Calcd. for 017H1404: C, 72.3; H, 5.00. Found: c, 72.6; H, 5.08. 28 5-Fluorenonoxyacetic acid \\\ O-CHZ-COQCQH 0-0H2_002H H20 \ H H o 0 Five-tenths gram of ethyl 2-fluorenonoxy acetate was A. dissolved in 50 ml. of ethanol and 40 ml. of water and 10 m1. of concentrated sulfuric acid were added. The solution was refluxed for eight hours, cooled and poured into 150 m1. of cold water. The precipitate was isolated by fil- tration, extracted with 50 ml. of a 10% sodium carbonate solution and reprecipitated by addition of hydrochloric acid. Crystallization from dilute acetic acid yielded 0.22 g. of yellow crystals which softened at 211° and melted at 2500 C. EtOH Absorbance Am 2'75 mu (log 5 4.556) 41 253 mu (log a 5 4.596) Anal. Calcd. for C15H1004: C, 70.9; H, 5.96. Found: C, 71.08; H, 4.25. o—CHZ-CH—z-CHB // KMnO4 .\ acetone 7'\\\.. 29 A solution of 2.0 g. (0.0085 mole) of 5-alloxyf1uoren- one in 20 m1. of anhydrous acetone was heated almost to boiling in a three—necked flask equipped with condenser and sealed mechanical stirrer, and 4.0 g. (0.025 mole) of potassium permanganate was added in portions so that the solution boiled gently. After all of the permanganate had been added to the solution it was refluxed with stirring for four hours. The solvent was distilled off and the residue extracted with a 10% solution of sodium carbonate and filtered. The filtrate was acidified with hydrochloric acid and the yellow precipitate collected by filtration. The acid was recrystallized from dilute acetic acid yield- ing 0.65 g. of yellow crystals, m.p. 250° c. A mixed melt- ing point of the acids prepared by the two different methods gave no depression of the melting point. 5-Alloxyf1uorenone 0-CH2-CH=CH2 Br-CngCH=CHg>\ K2C03, aceton37\\\_. To a solution of 2.0 g. (0.01 mole) of 5-hydroxy- fluorenone in 50 m1. of anhydrous acetone were added 1.8 g. (0.015 mole) of allyl bromide and 2.1 g. (0.015 mole) of finely powdered anhydrous potassium carbonate and the re- sulting suspension refluxed for 24 hours. The reaction mixture was cooled and poured into 400 m1. of ice and water 30 slurry which precipitated a yellow solid. The precipitate was collected by filtration and crystallized from 80% ethan- ol giving 2.58 g. of fine yellow platelets, m.p. 81.5-85° C. Dioxane Absorbance ,X 525 mu (log g = 5.560) max 511-515 mu (10g 5 = 5.456) 5 mu (log g = 5.886) 275 mu (log a = 4.645) 255 mu (log a 2.4.614) Anal. Calcd. for 016H1202: C, 81.3; H, 5.12. Found: C, 81.2; H, 5.240 51 Biological Assay Tomato Parthenocarpy Test Lanolin test solutions. A known quantity of the compound to be tested was dissolved in anhydrous, peroxide-free ethyl ether and diluted to the desired concentration. An aliquot of this solution containing the desired amount of the compound was added to one gram of anhydrous lanolin and'stirred until solution was effected. The ether was removed by immersing the containers of the solutions in a hot water bath. The lanolin pastes were prepared in this manner to contain the following concentrations of fluorene compounds: 41:10"5 E, 4x10"6 .111, 4x10"7 )3, and 41:10"8 E, or approximately 1%, 0.1%, 0.01% and 0.001% respectively. Elant material. Tomato plants were grown to the stage where each of the first two fruit clusters had nearly open flowers and the flowers were emasculated by removing the stamens. About four blossoms from the first flower clusters of two tomato plants of comparable physiological and nutri- tional status were employed for each dilution. All other flowers were removed from the tomato plant. Application of lanolin solutions. Fifteen milligrams of lanolin containing the desired chemical and concentration was applied to each ovary. Ovary diameters were measured 52 seven days after application to the test plants to deter- mine fruit development. Cucumber Seed Root Test Test solutions. A weighed sample of the acid to be test- ed was added to water and a small excess of solid sodium bicarbonate was added to effect solution. The solution was adjusted to a pH of 7 by the addition of dilute acetic acid and diluted to give the desired concentration of com- pound. The solutions used in this test had the following concentrations of fluorene compounds: 4le M, 4x10"8 191, and 4x10'9 ‘E, or approximately 0.01%, 0.001%, and 0.0001% respectively. Distilled water was used as the control solution. Seed material. Ten cucumber seeds selected for vigor of growth and uniformity of size were placed on filter paper in a Petri dish for each test and one milliliter of test solution was added. The seeds were allowed to germinate in the covered dishes in the laboratory, and the length of the roots measured three and one-half days after germ- ination. RESULTS AND DISCUSSION 54 RESULTS AND DISCUSSION In Table I are tabulated the melting points and ana- lyses for carbon and hydrogen together with the calculated values for these elements for each of the new compounds synthesized. Analysis of compounds for carbon and hydro- gen was done by a micro-combustion method according to the directions described by Steyermark (l7) employing a Sargent automatic micro-combustion apparatus or by the Spang Labora- tories, Ann Arbor, Michigan. The melting points of the compounds were determined on a Fisher-John's melting point block and are uncorrected. The molecular weight and absorption maxima for each of the compounds are tabulated in Table II. The absorp- tion curves were obtained using a Beckman DK-2 ratio- recording double-beam spectrophotometer. The compounds were dissolved in 95% ethanol or dioxane and matched silica cells of one centimeter diameter were employed in obtaining the spectra. The diameter of the tomato ovaries in.millilsters seven days after treatment with varied concentrations of fluorene compounds are given in Table III. The sizes tabu- lated represent the average values of each of two replicates. Only those ovaries greater than 4.51nm. in diameter showed growth significant to indicate that the compounds induced parthenocarpy in the tomato. "The growth of the ovary is 55 mfl.m m.Hm ma.m n.~m om oaocosoeachxoflae-m mo.e no.0e mm.n m.oe m.oma-eoa ease oapoosaxococoeosam-m wa.m mm.mb oc.m n.mb m.pmumm unmoved uneconoaosamuw Hunum me.o mv.em an.e 6.0m m.pm-m.om ecososaehwofiae-m ew.m mm.ee no.n o.me mmea ease capoosmxoaoeosam-m oa.m m.me Ho.6 H.0e m.HHH-m.OHH oneness haocoeosaunm Henna 6m.m 6.Hm ma.m n.Hm mo-mo oaocoeocfiuaxoaa<-a m6.n m.He em.n m.oe emmuomm once oauooeaeococoeosaesa no.4 m.me 00.6 n.me m.nm-mm essence axococoeosfie-a flagsm u--- u:uu nun: :c:u m.mnaam.ana whoa cupboaumnocoaosam m o m o .o «seamen pcaoa 0:509500 ecsoe .eoauo chfiuaoz lll||l I); mQZDomsoo mzmmODub mom zmwomawm 02¢ Zommmo mom mHmMuws pecmflampcpn .ucobaom mu pom: use ecuxoaa m .zauoonm noduQAOmnw no cofipocfisampmo pop unobaom as mom: was Amway moswnum . H ea0.e 0mm nee.e new 000.0 000 0nw.n mH0-HHn 00m.n 000 0m.0nm monocwsosaehxoflfle-n 00m.e new emm.e mam mm.emm ease oauooaexocoeoaosam-n ene.e emm 00e.e new 00.m0m 6060660 axoeoeosoefie-n Hanan 000.e emm 000.0 000-v00 eae.n 0H0 0m.0nm mmcocoeosdeaxoflaeum e0m.6 new 0m.emm 0006 assessaxococueosfimam H0H.0 new Hen.6 nemmuamm 06.00m assumes emococmsosfic-m Harem 000.m Hem Hmn.0 e00 0H0.m wee mm.mmm «cameosfleaxoaae-m 59 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0000 000000000000000000-0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0000000 000000000000-0 00000 0.0 0.0 0.0 0.0 0.0 0.0 00.0 00.0 00000000000000-0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0000 0000000000000000-0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0000000 0000000000-0 00000 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0000000000000000-0 0.0 0.0 0.0 0.0 0.0 0.0 00.0 00.0 0000 000000000000000000-0 00.0 00.0 0.0 0.0 0.0 0.0 00.0 00.0 0000000 000000000000-0 00000 0.0 0.0 0.0 0. 00.0 00.0 0.0 0.0 0000 000000-0-00000000 0 H 0 H 0 H 0 H (I- .m 0-0000 _m 0-0000 .0 0-0000 0 mT0000 00000000 mCOfipS How .HO COHUGFUCOOCOU mem00 mpounEHHHHE :0 00000>o mo 00005009 0 0.00 0.0 0.0 0.0 0.00 0.00 00000 000000-0-0000000 00.0 00.0 00.0 00.0 00.0 00.0 0000000000000000-0 00.0 00.0 00.0 00.0 00.0 00.0 0.0 0.0 0000 000000000000000500-0 00.0 00.0 00.0 .0.0 00.0 00.0 0000000 000000000000-0 00000 0.0 0.0 0.0 0.0 00.0 0.0 000:000000000000-0 0.0 41 proportional to the logarithm of the concentration of the growth regulator within the limits of 0.1 to 10 micrograms per flower" (18). None of the fluorene compounds tested showed a posi- tive stimulation of parthenocarpy in the tomato. A number of compounds caused the abscission or ovaries to indicate damage to the plant structure. Ethyl 3-f1uorenonoxy acet- ate, 3-f1uorenonoxyacetic acid, and l-alloxyfluorenone caused some abscission of ovaries at all levels of con- centration. Abscission of ovaries at the higher concen- trations occurred with: ethyl l-fluorenonoxy acetate, l-fluorenonoxyacetic acid, 2-alloxyf1uorene, S-alloxy- fluorenone and fluorene-Q-acetic acid. In Table IV is tabulated the average length in centi- meters of cucumber seeds treated at varied concentrations of the fluorene acids and measured three and one-half days after germination. The lengths given in the table repre- sent the average values of 10 roots at each concentration. B-Fluorenonoxyacetic acid and 3-f1uorenonoxyacetic acid showed stimulation of root growth in cucumber seeds, with the former showing the greatest activity. Optimum concen- tration for stimulation was 4x10"8 M in the acid. 1- Fluorenonoxyacetic acid showed no significant stimulation of root growth and fluorene-Q-acetic acid strongly in- hibited growth. ab (\3 TABLE IV STIMULATIONI or ROOT GROWTH IN CUCUMBER SEEDS BY FLUORENE COMPOUNDS Concentration of solutions__ C°mp°und 4x10’7 y; 4::10’8 M 4x10‘9 g Fluorene-Q-acetic acid u.3:t0.6 8.3::O.S 8.5i?l.0 l-Fluorenonoxyacetic acid 8.2i:0.9 lO.9i:O.9 lO.lj:O.6 2-Fluorenonoxyacetic acid lloOJtl-l 1203311-4 1009130-7 S-Fluorenonoxyacetic acid lO.hi:O.8 110613006 9.1i:1.1 Control (distilled water) --------- (9.71:0.5) ---------- 1 Root length in centimeters three and one-half days after germination. SUMMARY SUMMARY A method of synthesis is given for a number of allyl, acetic acid and ethylacetate ethers of fluorene and fluor- enone. The synthesis of the following compounds have not been reported in the chemical literature. Ethyl l-rluorenonoxy acetate l-Fluorenonoxyacetic acid l-Alloxyfluorenone Ethyl 2-Fluorenoxy acetate 2-Fluorenoxyacetic acid Ethyl 2-fluorenonoxy acetate 2-Fluorenonoxyacetic acid Z-Alloxyfluorenone Ethyl 3-fluorenonoxy acetate B-Fluorenonoxyacetic acid S-Alloxyfluorenone The melting points and absorption spectra were deter- mined for the ethers. Elemental analysis for carbon and hydrogen was determined for all new compounds. The compounds were tested for biological activity using the tomato parthenocarpy test and the cucumber seed root test (the latter for the acids only). None of the fluorene ethers showed a positive stimulation of partheno- carpy in the tomato. A number of the compounds, however, caused abscission of ovaries in the tomato test plants. Root growth in cucumber seeds was stimulated by 2-f1uoren- onoxyacetic acid and 3-fluorenonoxyacetic acid, whereas fluorene-Q-acetic acid caused inhibition of growth. BIBLIO CHULPHY 5. 4. 5. 10. BIBLIOGRAPHY P. W. Zimmerman and F. Wilcoxan, Several Chemical Growth Substances which Cause Initiation of Roots and Other Responses in Plants, Contrib. Boyce Thompson Inst. 1, 209 (1935); C.A. 39, 1431 (1936). H. E. Thompson, C. P. Swanson and A. G. Norman, New Growth-Regulating Compounds. I. Summary of Growth Inhibitory Activities of Some Organic Com- pounds as Determined by Three Tests, Botan. Gaz., lgz, 476 (1945). R. L. Weintraub, J. W. Brown, J. C. Nickerson and K. N. Taylor, Studies on the Relation Between Mole- cular Structure and Physiological Activity of Plant Growth-Regulators. I. Abscission Inducing Activity, Botan. Gaz., 113, 348 (1952). R. M. Muir, C. H. Hansch and A. H. Gallup, Growth Regulation by Organic Compounds, Plant Physiol., g3, 359 (1949). D. J. Osborne and R. L. Wain, Plant Growth—Regulating Activity in Certain Aryloxyalkylcarboxylic acids, Science, 114, 92 (1951 . J. B. Koepfli, K. V. Thimann and F. W. Went, Phyto- hormones: Structure and Physiological Activity, J. Biol. Chem., 122, 763 (1938). H. Veldstra and C. vand de Westeringh, Researches on Plant Growth Regulators. XIX. Structure/Activity. IV. Partially Hydrogenated Naphthoic Acids and drAlk l-phenylacetic Acids, Rec. trav. chim., 19, 1113 1951). H. Veldstra, The relation of Chemical Structure to Biological Activity in Growth Substances, Ann. Rev. Plant Physiol., 3, 151 (1953). G. Wittig and G. Felletschin, fiber Fluorenylide und die Stevenssche Unlagerung, Ann., 555, 133 (1944). W. E. Bachmann and J. C. Sheehan, The Synthesis of 2-, 4- and 9-Fluoreneacetic Acid, J. Am. Chem. Soc., gg, 2687 (1940). ll. 12. 13. 14. 15. 16. 17. 18. 47 L. E. Fieser and A. M. Seligman, Cholanthrene and Related Hydrocarbons, J. Am. Chem. Soc., 51, 2174 1935 . Elizabeth K. Weisburger and J. H. Weisburger, An Improved Synthesis of l-Aminofluorene, J. Org. Chem., ;§, 854 (1955). W. E. Kuhn, 2-Nitrof1uorene and 2-Aminoflu0rene, Organic Syntheses, Coll. Vol. II, New York, John Wiley and Sons, Inc. (1943), p. 447. W. C. Lothr0p, The Structure of Fluorene, J. Am. Chem. Soc., 51, 2115 (1939). O. Diels, Zur Kenntniss des Fluorens, Ber. 33, 1758 (1901). F. Ullmann and H. Bleier, Zur Darstellung von o-Amino- benzophenonderivaten, Ber. 35, 4275 (1902). A. Steyermark, Quantitative Organic Microanalysis, McGraw-Hill Book Co. (1951), p. 105. A. C. Leoplod, Auxins and Plant Growth, University of California Press (1955), p. 45. NOV I 6 ‘60 GIEMISTRY um TY LIIBIRARIES 7343 MICIITIIWWHHTIIWITll‘EW (WI)! 3 12 9 3 0 3 012