DEGRADATIVE and SYNTHETIC STUDIES on PIGMENTS of the OSAGE ORANGE % DISSERTATION Presented in Partial Fulfillment of the Require

DEGRADATIVE AND SYNTHETIC STUDIES ON

PIGMENTS OF THE OSAGE ORANGE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

OSCAR MICHAEL WINDRATH,^ B.S., M.S.

The Ohio State University

195^

Approved by:

Adviser TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

STATEMENT OF THE PROBLEM ...... 6

EXPERIMENTAL ...... 8

DISCUSSION OF RESULTS ...... 6]

A. Investigation Toward the Synthesis of Osajin and Pomiferin ...... 63

B. Root Bark Pigments ...... 80

C. Detection of the Catechol and Hydro- quinone Structure in Organic Molecules ,.., 96

SUMMARY ...... 101

SUGGESTIONS FOR FURTHER STUDY ...... 10?

CHRONOLOGICAL BIBLIOGRAPHY ...... Ill

ACKNOWLEDGMENTS ...... II6

AUTOBIOGRAPHY ...... 117

A 11

TABLE OF CONTENTS

OF THE

EXPERIMENTAL SECTION

Page

Preparation of Phloroglucinaldehyde ...... 8

Preparation of 5»7-Dlacetoxycoumarin ...... 9

Preparation of ^,7-Dlhydroxycoumarln ..... 11

Acétylation of 9,7-DIhydroxyconmarIn ...... 11

Preparation of 5,7-Dlmethoxycoumarln ...... * 12

Preparation of 5,7-Dlbenzyloxycoumarln ...... 13

Reaction Between Méthylmagnésium Iodide and

Substituted Coumarlns ...... l4

Preparation of 5-Hydroxy-7-benzyloxy-2,2-dlmethyl-

chroman-4-one ...... l6

Meerwein-Ponndorf Reduction of Substituted

Chromanones ...... 17

Sodium Amalgam and Ethanol Reduction of 5j7-Dl-

methoxy-2,2-dlmethylehroman-^— one ...... l8

DeetherifIcatlon of !)-Benzyloxy-7-"methoxy-2,2-dl-

methyl- -chromene ...... 19

Preparation of 2-Chloro-^-methylphenol ...... 20

Preparation of Phenylacetyl Chloride ...... 20

Preparation of 2-Chloro-4-methylphenyl Phenyl

Acetate ...... 21 ill

Page

Preparation of 2-Hydroxy-3-chloro-5-methyl-OC-

phenylacetophenone by a Fries Reaction ...... 22

Preparation of 6-Chloro-8-methyli3oflavone ..... 23

Phloroglucinol Trimethyl Ether ...... -...... 2^ sym-Trimethoxybromobenzene ...... 25

Preparation of 2,4,6-Trlmethoxytriphenylcarbinol ... 26

Preparation of p-Nitrobenzyl Cyanide ...... 28

Preparation of p-Aminobenzyl Cyanide ...... 28

Preparation of p-Hydroxybenzyl Cyanide ...... 29

Houben-Hoesch Reaction Between 2 ,^,6-Trimethoxy-

bromobenzene and p-Hydroxybenzyl Cyanide .... 30

Homoanisic Acid ...... 31

Homoanisoyl Chloride ...... 32

A Friedel-Crafts Reaction Between 2,^,6-Trimethoxy-

bromobenzene and Homoanisoyl Chloride ...... 33

X-ray Powder Diffraction Patterns of the Root Bark

Pigment Substance I of the Osage Orange ...... 3^

Action of Dilute Alkali on Substance I ...... 35

Action of Potassium Hyd r ox id e-D i oxane on Tetra-

hydro-Substance I Trimethyl Ether ...... 37

Substance I Dimethyl Ether ...... 38

Tetrahydro-Substance I Dimethyl Ether ...... 39

Attempts to Methylate Tetrahydro-Substance I ...... 40

Alkaline Peroxide Oxidation of Substance I ...... 40 Iv

Page

Isolation of a Dinitrophenylhydrazone from

Alkaline Peroxide Oxidation of Substance I .... 4-2

Isolation of a 2,^-Dinitrophenylhydrazone Through

the Action of Alkali on Substance I ...... 44

Action of Potassium Hydroxide on Substance I

Dimethyl Ether ...... 49

Alkaline Potassium Permanganate on Substance I ...... 50

Action of Dilute Nitric Acid on Substance I ...... 51

Chromic Acid in Acetic Acid on Substance I ...... 52

I Potassium Permanganate in Acetone on Substance I

1 Trimethyl Ether ...... 53

I Action of Hydrobromic Acid on the Acid Obtained on 1 I Oxidation of Substance I Trimethyl Ether ..... 54 4 i Action of Dilute Base on Unknown Acid ...... 55

1 Ethylmagnesium Bromide on Substance I Trimethyl Ether. 55

I Potassium Hydroxide on Substance III ...... 56

I Méthylation of Substance III ...... 56

1 Color Tests on Substance III and Derivatives...... 57 I I Action of N-Bromosuccinimide on Pomiferin

I Trimethyl Ether ...... 58 i ] Infrared and Ultraviolet Absorption Spectra ...... 59 ■i I Separation of Substance II from a Mixture with I Substance I ...... 59 INTRODUCTION

The osage orange tree (M a d u r a pomlfera Raf.), called by the French hois d'arc, by the Indians bow wood and hedge- apple tree in the vernacular, is a native of Arkansas where it rises in beautiful proportions to the height of sixty feet,

It has been called one of the most beautiful of our native trees. The wood is, perhaps, the most durable in the world and has been used on a limited scale in shipbuilding and furniture manufacture. It is remarkably tough, strong and elastic and was preferred by the Indians to all other woods for bows.

Most widely arranged in the form of hedge-rows, from whence the common name of the fruit, hedge-apple, originated, it was first planted in Missouri as such as early as I8OO and during the next eighty years thousands of bushels of seeds were distributed to tne northeasty^and west ern states. The advantage of this tree as a hedge was wide ly recognized and commercially exploited so that before many years had elapsed the osage orange hedge-row was a familiar sight throughout the central plain states (1).

(1) Report of the Comm, of Agriculture, I8 6 8,

G.P.O., Washington, I8 6 9, p. 19^? 247-8.

The osage orange is the only species of the genus ! Madura of the plant family of dicotyledons called Moraceae

I which belongs to the order Urticales. It is a member of the i same family as the fig, mulberry, india rubber plant and

' breadfruit tree.

As a result of investigation under the direction of

Professor M, L. Wolfrom conducted in the laboratories of the

Department of Chemistry at the Ohio State University, two

crystalline pigments, osajin and pomiferin, have been iso

lated from the fruit of the osage orange and their chemical

constitution determined (2).

' (2) M. L. Wolfrom, W. D. Harris, G. F. Johnson,

J. E. Mahan, S. M, Moffett and B. Wildi, J. Am, Chem, Soc,,

4o6 (19^6)

CH. OH OH

CH^ CH^ Osajin

Pomiferin contains, in addition, an hydroxyl group

i in the 3* position, Î •3

In 19^1 , Wolfrom and Dickey (3) attempted to iso-

(3) E, E. Dickey, "A Study of the Isolation of the

Pure Substances from the Wood and Root Bark of the Osage

Orange (Ma d u r a pomifera Raf,)," M. Sc. Thesis, The Ohio

State University (194-1).

late a crystalline pigment from the wood and bark of the

osage orange. Further improvements in the isolation of the yellow pigment, m.p. l8l-l8l.7°, were devised by MeWain (4-),

(4-) P. McWain,"Root Bark Pigments of the Osage

Orange (Ma d u r a pomifera Raf,)," M, A, Thesis, The Ohio

State University (194-7) •

Through chromatography he isolated a new yellow pigment, m.p, 264— 265.5° which he called Substance II. The pigment

isolated earlier was designated Substance I. Investigation by McWain and Looker (5) did much to develop the structures

(5) J. H. Looker, "Root Bark Pigments of the Osage

Orange (Mad ura pomifera Raf.) and Their Structures," Ph,

D. Dissertation, The Ohio State University (194-9). of these two root-bark pigments. Looker improved the method

of isolation and as a result of acétylation and méthylation

experiments the following points of structure were deter

mined. Substance I contains three hydroxyl groups, ons in

a hindered position and the other two probably adjacent to

each other. A positive Perkin test indicates the presence

of a ^-pyrone ring while a positive Wilson boric acid test

is indicative of the unit -C— C-C-Cs • OH 0 j The presence of two easily reducible double bonds has been

Î determined. The absence of formic acid on degradation with j \ alkali (4) apparently rules out the possibility of an iso-

] flavone nucleus. Only méthylation and acétylation studies

Ij with elementary analysis were carried out on Substance II. I Two hydroxyl groups are indicated, I i Dr. A. Thompson, a Research Associate in the Depart- I i ment of Chemistry, The Ohio State University, was assigned J I the problem by Dr. Wolfrom of increasing the supply of these y 1 pigments whose scarcity greatly hampered the structural I I studies. He (6) developed an improved method for the iso- i i ■Î

j (6) A. Thompson, "Report on the Osage Orange Root

Bark Pigments," The Ohio State University (1950).

lation of Substance I and isolated a third pigment designated Substance III, Structural studies on this compound were in conclusive.

Further elaboration on the work of Looker and

Thompson is unnecessary since Mundell (7) has included an

(7) P. M. Mundell, "Structural Studies on Root

Bark Pigments of the Osage Orange," Ph. D. Dissertation,

The Ohio State University (1953).

excellent, detailed review of their work in his thesis. STATEMENT OF PROBLEM

The object of the present investigation was two

fold. The original problem as assigned was a confirmation

of the structures of the fruit pigments osajin and pomi

ferin by a synthesis of these two compounds. Previously,

Wolfrom and Wildi (8) had succeeded in the preparation of

(8) M. L. Wolfrom and Bernard S. Wildi, J. Am.

Chem. Soc., 235 (195D.

the derivatives dihydro-iso-osajin and dihydro-isopomi-

ferin.

A continuation of the degradative studies on the

root-bark pigments of the osage orange initiated by

McWain, Looker and extended by Mundell was to be attempted.

Evidence thus far indicated uncertainty as to the basic

structure of these pigments. Degradative studies utilizing

various concentrations of alkali and standard oxidizing I i agents, methods which proved successful when applied to

i many other natural products in structure determination, "KèJ 1 -ëeve resulted in producing either nothing or only insig-

I nifleant amounts of crystalline material; amounts too small .1 1 for further study. Therefore, it was desirable to develop 1 I methods of degradation by which sufficient amounts of I material could be obtained without the necessity of sacri ficing unnecessarily large amounts of the pigments. An identification of these degradation products was to be attempted in the hope that this would culminate in the determination of the basic structures of the root-bark pigments. As an aid to this conclusion various spectral measurements involving ultraviolet and infra-red were to be made on osajin, pomiferin and the root-bark pigments for the purposes of comparison with existing data. -8

EXPERIMENTAL

All melting points recorded are uncorrected and ex

pressed in centigrade units. They were taken on a Fisher-

Johns melting point block. All analyses were made by the

Huffman Microanalytical Laboratory except where noted.

Preparation of Phloroglucinaldehyde

CHO OH

This substance was prepared essentially according to

the directions of Shriner and Kleiderer (9).

(9) R. L. Shriner and E, C. Kleiderer, J. Am Chem.

Soc., 1269 (1 9 2 9). See also T. Malkin and M. Nierenstein,

ibid., H , 2hl (I9 3I).

Dry hydrogen chloride gas was passed into a vigorous

ly stirred mixture of 30 g. of phloroglucinol (made anhydrous

by heating on a water bath for 5 hrj and 30 g. of anhydrous

zinc cyanide (10) dissolved in 250 ml. of anhydrous ether. il (10) R. Adams and Edna Montgomery, J. Am. Chem,

Soc., M , 1518 (1924).

A clearly defined oily lower layer formed after about 1 hr. which on continued addition of hydrogen chloride gas solid ified after 15 min. The gas was passed in for about 1.5 hr. longer to insure complete reaction. The imide hydrochloride was separated by suction filtration and dissolved in about

200 ml. of water and heated to boiling. The hot solution was filtered and cooled whereupon orange red crystals of phloroglucinaldehyde appeared. Repeated crystallization from water with decolorizing carbon gave cream colored needles of no definite melting point; yield 46^ (recorded

(9)} 49^). Identity was established through preparation of the phenylhydrazone with a melting point of 120-122° (re corded (9), 120°).

Preparation of 5.7-Diacetoxycoumarin

HO HO OH

This is an improvement over a previous procedure using the method of Heyes and Robertson (11). They used 10

(11) R. G. Heyes and Alexander Robertson, J. Chem,

Soc., 1832 (1 9 3 6).

pyridine as a catalyst. In our work this method gave a 30^ yield. The authors do not report a yield.

A mixture of 3,6 g. of phloroglucinaldehyde, 3.6 g,

of anhydrous sodium acetate and I8 ml. of acetic anhydride

together with 0,25 g. of iodine were heated on an oil bath

at 185° to 1 9 0° for 6 hr, in a three-necked round-bottomed flask equipped with moisture guarded reflux condenser and a mercury-seal stirrer. After the reaction time was completed,

the mixture was poured into 50 ml. of water. Part of the material crystallized out immediately and was separated by décantation. The residue, a brown colored mass, was added

to 50 ml. of water and stirred until pulverized. Repeated

extraction with boiling water and cooling of the filtrate

resulted in complete crystallization. A yield of 52^ or

3 ,2 g. of pale tan crystals with a melting point of 136-37° was obtained (recorded (11), 1^0°), 11

Acétylation of 5.7-Dlhydroxycoumarln

This was accomplished using acetic anhydride and pyridine, A mixture of 2.5 g. of 5,7-dihydroxycoumarin

(see following preparation), 20 g. of acetic anhydride and

2 ml. of pyridine was refluxed for 0.5 hr. and while still hot the solution was poured, with stirring, into 150 ml. of cold water. A white crystalline precipitate resulted.

This was 5,7-diacetoxycoumarin; m.p. 137-9°. A yield of

^2% was obtained.

Preparation of 5.7-Dihydroxycoumarin

By using the method of Heyes and Robertson (11)

(dilute alkali heated with compound in alcoholic solution) a 10/Ü yield was obtained. The authors do not report a yield.

Deacetylation was accomplished more successfully using a general procedure for carbohydrate deacetylation suggested by Bates and associates (12). An amount of 3.0 g. 12

(12) F, J, Bates and associates, "Polarimetry and

Saccharimetry", Circular C440, United States Department of

Commerce, National Bureau of Standards, p, I+94-.

of 5J7-diacetoxycoumarin was dissolved by heating in 90 ml. of dry methanol and 18 ml. of dry methanol containing 0.5 g. of sodium per 100 ml. of methanol were added to the mixture.

The solution, contained in an Erlenmeyer flask mounted with a reflux condenser, was heated on a steam bath for 0.5 hr., then allowed to stand overnight. The solution was filtered, evaporated to dryness on the water bath and the cake which formed was recrystallized from 250 ml. of water. Pale tan needles (1.6 g.) in a yield of 80r^ was obtained. The melt ing point is 2 8 6-8° (recorded (11), 285-6°).

Preparation of 5.7-Dimethoxycoumarin

OCH:

This compound was prepared according to the direct ions of Heyes and Robertson (11). An amount of 0,7 g. of

5,7-dihydroxycoumarin was dissolved in 30 ml. of dry acetone and refluxed with stirring with 3 g. of methyl iodide and

9 g. of anhydrous potassium carbonate for 15 hr. The s 13 i solution was filtered and the potassium carbonate washed thoroughly with more acetone. The combined filtrate was

evaporated to dryness, taken up in hot methanol and par

tially recrystallized into white needles. On allowing the

methanol solution to stand, a low melting substance preci

pitated which gave no ferric chloride test for hydroxyl

groups. On washing well with water and cold methanol the

impurities were removed and 5j7-dimethoxycoumarin remained

as the residue. Crystals (0,4 g,) of m,p, l44-5° were ob

tained in 50;o yield (recorded (1 1 ), 147° and authors re

port no yield).

Preparation of 5.7-Dibenzvloxycoumarin

HO C6H5CH90 ••00. C6H5CH20

An amount of 0,7 g. of 5,7-dihydroxycoumarin was

dissolved in 30 ml, of dry acetone and 3 g. of benzyl

chloride and 10 g, of potassium carbonate were added and

the solution stirred and refluxed for 24 hr. Then an add

itional 3 g, of benzyl chloride, 10 g, of potassium carbon

ate and 30 ml, of acetone were added and the solution re

fluxed for an additional 24 hr. The yellow solution was

then filtered and the potassium carbonate in the residue

was washed thoroughly with acetone and the combined fil- I trates were evaporated to dryness on the water bath. The oily residue, containing excess benzyl chloride, was shaken with N sodium carbonate solution and ether. Crystals appeared at the interface and were filtered, washed with ether and cold water. Recrystallization from ethanol gave colorless rods of m.p, 136-7°, A negative ferric chloride test was obtained, A yield of 32^; 0.^5 g» was obtained.

Anal, Calcd. for C22 H 18O1+: C, 77.16; H, 5.03,

Found: C, 77,10; H, 5,25,

Reaction Between Méthylmagnésium Iodide and Sub stituted Coumarins

+ CH^Mgl

In the hope of obtaining a crystalline product numerous variations of the reaction between these two sub stances were attempted. The following alterations were tried,

1) The solvents used in various runs were: ether, anisole, benzene, toluene, petroleum ether and dioxane; the latter forms an insoluble addition product with the Grignard reagent,

2) The conditions were varied. The reaction was run at 0°, at room temperature and at reflux temperature of 15 the various solvents used.

3) The mode of addition was changed. The coumarin was added to the Grignard reagent in a number of runs and the rate of addition was varied. The reverse addition was also attempted.

h) Reaction time was varied between 2 and 2h hr., with stirring and without stirring.

5^ Methods of decomposition of the Grignard add ition product involved use of concentrated ammonium chloride, dilute and concentrated hydrochloric acid and refluxing with water containing a trace of hydrochloric acid.

6) Runs were made with the following coumarins.

(a) 7-Dihydroxycoumarin

(b) 7-Diacetoxycoumarin

(d) 5j7-Dibenzyloxycoumarin

As a general procedure, the method of Shriner and

Sharp was followed (13). These workers prepared a series of

(13) R. L. Shriner and A. G. Sharp, J. Org. Chem., h, 575-82 (1939).

2 ,2-dialky1-1,2-benzopyrans. An amount of 0,0125 mole of the coumarin in 20 ml. of ether was added to a Grignard solution prepared from 0.04l mole of magnesium and 0.04 mole of the 5 I 16 I alkyl halide in 15 to 20 ml. of ether over a period of about

I 1 hr. The mixture was decomposed with 20 ml. of 22$^ ammonium 1 \ chloride solution containing a trace of concentrated hydro- .'I I chloric acid. The ether layer was separated, dried with I 1 calcium chloride and evaporated in a stream of dry air. A

Î yellow oil which soon darkened with résinification was Invar- { I iably obtained. 1 I Preparation of 5-Hÿdroxy-7-benzyloxy-2.2-dimethyl- I chroman-^— one

HO P, HG 0

“°00cch3 "

A mixture of 0.5 g. of 5,7-dihydroxy-2,2-dimethyl-

chroman-4-one (1^), 0.5 ml. of benzyl chloride (added in two

(l4) W. D. Harris, "The Structure of Osajin and Pom

iferin; Nature of the Chromene Ring Portion," Ph. D. Disser

tation, The Ohio State University (194^4).

portions), 3 g. of potassium carbonate (also added in two

portions) and 25 ml. of dry acetone was refluxed for h hr.

The solution was filtered, the potassium carbonate was wash

ed well with acetone and the filtrates combined and evapor-

' 17

a ted to dryness on the water hath. The residue was taken up

in ethanol which on cooling deposited a layer of fine white

needles. A yield of 0.4 g or 57% of the compound having

m.p, 130-1° was obtained. The literature (15) records the

(15) S. W. George and Alexander Robertson, J. Chem.

Soc., 1539 (1937).

m.p. 134 °. The substance gives a positive ferric chloride

test.

Meerwein-Ponndorf Reduction of Substituted Chro-

manones 1 cvOCk;: —' m-OCk:' I ^ ^ J Harris (l4) had succeeded in preparing 5-henzyloxy- i 7-methoxy-2,2-dimethylehromene utilizing a Meerwein-Ponndorf

reduction of the appropriately substituted chroman-4-one,

In the hope of utilizing this method toward the

solution of the problem, a Meerwein-Ponndorf reduction was

carried out on the following chroman-4-ones.

(a) 5,7-Dihydroxy-2,2-dimethylchromanone

(b) 5,7-Dimethoxy-2,2-dimethylchromanone

manone

(d) 5(7)-Hydroxy-7(5)“benzyloxy-2,2-dimethyIchro-

manone

(e) 5-A.cetoxy-7-methoxy-2,2-dime thy Ichromanone

These reactions were carried out following the same

general procedure reported in Harris' thesis. The results

are identical, A yellow resinous material was obtained

which resisted all attempts at crystallization.

Sodium Amalgam and Ethanol Reduction of 7-Di

me thoxy-2 ,2-dime thy lehr oman-4-one

The procedure used was similar to that of Bachmann

(16) who successfully reduced benzophenone by this method,,

(16) W. E, Bachmann, J, Am. Chem. Soc., 772

(1933).

An amount of 6 g. of 2/o sodium-mercury and 30 ml. of absolute

ethanol was added to 0.5 g. of the chromanone and the mixture

was shaken vigorously. A mild reaction ensued and the amal

gam became liquid. After 3 hr. the mixture was hydrolyzed.

The alcohol was evaporated and the aqueous mixture was ex

tracted with ether. On drying and evaporation an oil was

again obtained. When this oil was taken up in benzene and

chromatographed on Magnesol-Cellte (1:5), an indistinct smear

re.sulted. I 19

DeetherifIcatlon of 5-Benzyloxy-7-methoxy-2.2-dl-

I metbyl- A'^■.chromene

It Is often possible to prepare a compound of a cer

tain structure by utilizing a deetherlficatlon reaction when

I the presence of hydroxyl groups lend Instability to the mol I ecule. Three different means were used here: Hydrlodlc acid

and acetic acid; potassium hydroxide and ethylene glycol; and

aluminum chloride and benzene. The procedures for each method i were essentially similar as were the results, so only the first method, that utilizing hydrlodlc acid and acetic acid, will be

detailed here.

An amount of 0.5 g. of 5-benzyloxy-7-methoxy-2,2- di

me thy lehr omene (1^) was dissolved In 50 ml, of glacial acetic

acid and 10 ml. of hydrlodlc acid (57*5^ aqueous) was added

and the mixture refluxed 3 hr. Addition of water on cooling,

followed by extraction with ether, gave on evaporation a

small amount of yellow oil. This oil gave a positive test for

a phenolic group but could not be crystallized.

The following series of preparations Involve Inter-

mediates In the synthesis of a chlorolsoflavone. 20

Preparation of 2-Chloro-^-methvl-'phenol

I SO2 CI2

This compound was prepared according to the direct

ions of Sah and Anderson (17). To 27 g. of _p-cresol was

(1 7) P. P. T. Sah and H. H, Anderson, J, Am, Chem,

Soc,, 6 3 , 3164-7 (1941),

added, with stirring, 33.75 g. (20 ml.) of sulfuryl chloride

(SOgClg). The reaction began at room temperature and hydro

gen chloride gas was emitted. Following completion of the

reaction by heating on a water bath, the material was dis

tilled and the fraction boiling between 190 and 2 0 5° was

collected. This was then redis tilled and the fraction boil

ing between 194 and 1 9 8° was collected, kn amount of 15.5 g>

of a pale yellow oil was obtained. This is a 44;^ yield.

It is reported (17) as a colorless oil, b.p, 195 to 197°,

and obtained in 77fo yield.

Preparation of Phenylacetyl Chloride

A mixture of 10,4 g, of phenylacetic acid and 10 ml,

of thionyl chloride was refluxed on the water bath. After 21

0.5 hr,, 5 ml. more of thionyl chloride was added down the fi || condenser and the mixture heated for 1 hr, on the water hath.

After standing overnight, the yellow liquid was subjected to Ï distillation at reduced pressure. The majority of the liquid boiled at 130 to 150° and a yield of 83/u or 9.7 g. was ob

tained, The literature value (18) is 100° at 12 mm,

(18) R, Adams and L. H, Ulich, J, Am. Chem, Soc,,

hg, 599 (1920),

Preparation of 2-Chloro-4-methylohenyl Phenvlacetate

Cl Q Cl OH -fClCCHg^ ^ — aH^C ^ ^ o6-C%-^ ^

This compound was prepared according to the proced

ure of Chakravarti and Bera (19). An amount of 7.1 g. of

(19) D, Chakravarti and B, C, Bera, J. Ind. Chem,

Soc,, 21, 44 (1944),

(2“chloro-4-methylphenol) in 20 ml, of anhydrous carbon

tetrachloride was kept under reflux while 7.7 g. of phenyl-

acetyl chloride was added slowly down the condenser. The

mixture was refluxed on an oil bath about 4 hr. at which 22

point the evolution of hydrogen chloride gas had ceased.

The solvent was removed by distillation and the residue was

then distilled under reduced pressure (15 mm.) to give a

yellow oil with a boiling range of I90 to 205°. The com

pound, which did not give a test for a phenolic group, was

obtained in 86^ yield or 11.1 g. The boiling point at 6.5

mm. is reported (19) at 201°.

Preparation of 2-Hydroxy-l-chloro-5-methvl- Ql-

phenylacetophenone by a Fries Rearrangement

ÿÿ=^0CCH2-^~~\

-- 0 fa This compound was prepared by a variation of the

method of Chakravarti and Bera (19). An intimate mixture I of 10 g. of 2-chloro-4-methylphenyl phenylacetate and 8.7 g. of anhydrous aluminum chloride was put in a flask mounted

with a reflux condenser. The mixture was heated on a gly

cerol bath while the temperature was gradually increased to

130 to 135 ° and maintained at that temperature for 0.5 hr.

On cooling, the glassy mass was decomposed by the addition

of a mixture of 50 g . of ice and 30 ml. of ice-cold con

centrated hydrochloric acid. The solution was extracted

with ether, the ether layer separated and washed with aqueous

sodium carbonate solution until the washings were colorless 23

and then the ether was evaporated. The residue was distill

ed at 15 mm. pressure with the majority coming over at 205°.

After standing for a few minutes, the liquid distillate be

gan to crystallize in yellow needles of m.p. 107-8°. Liter

ature (19) reports yellow needles of m.p. 110°. The sub

stance gave a positive test for a phenolic group, and a pos

itive test for halogen by sodium fusion and a positive test

for a ketone group on the addition of 2 ,4-dinitrophenylhydra-

zone,

Preparation of 6-Methyl-8-chloroisoflavone

An amount of 200 mg. of the ketone prepared above was dissolved in 10 ml. of freshly distilled ethyl formate,

cooled to 0° and the solution added drop-wise to an ice-

hydrochloric acid cooled flask containing 0.5 g. of thinly

sliced sodium under a nitrogen atmosphere. The mixture

was placed in the refrigerator overnight and then poured

into 200 ml. of an ice-water mixture and allowed to stand

for 12 hr. At the end of this time the solution was cloud

ly with a small amount of oil on the bottom of the beaker.

It was extracted twice with ether and the ether evaporated

in a stream of air. The yellow oily residue was taken up 24- in ethanol and the ethanol reduced in volume to about 5 ml. by warming on a water bath. Water was added to incipient opalescence. On standing, white needles formed which had a melting point of 128-9°. These crystals gave a negative phenolic test, a negative Wilson boric acid test, a positive halogen test and a positive Perkin test for the y -pyrone structure.

Anal. Calcd. for C^^H^^02C1: C, 71.11; H, 4.07;

01, 12.95. Found: C, 70.97; H, 4.4-2; 01, 13.18.

(A substandard size sample was sent for analysis)

The following series of reactions are involved in attempts to prepare appropriately substituted desoxybenzoins and exploratory reactions utilizing lithium phenyls.

Synthesis of Phloroglucinol Trimethyl ether.

The procedure of Freudenburg was used (20). On the

(20) K. Freudenburg, Ber., 1425 (1920).

water bath, 25 g. of anhydrous phloroglucinol (dried by heat ing on a steam bath for 3 hr.) was dissolved in 250 ml. of anhydrous methanol. The solution, under cooling, was satur ated with dry hydrogen chloride gas, allowed to stand for

48 hr. and then, under reduced pressure, concentrated to a 25 thick syrup. This residue was dissolved in 100 ml. of di methyl sulfate and the flask, mounted with an air condenser, was warmed and within 15 min., 300 ml. of 7.5 N potassium hydroxide was added with shaking at such a rate that the tem perature remained between 60 and 90°. As soon as the heat of reaction subsided, the flask was heated for 10 min. long er at 90° with shaking. The reaction product was separated by steam distillation. The oil in the distillate soon solid ified to a white crystalline solid that could be removed by filtration. Recrystallization from ethanol gave an 80% yield of colorless needles with a m.p. 51-2 °.

Preparation of 2,4,6-Trimethoxybromobenzene

CH.O

A mixture of 2 g. of 1,3,5-trimethoxybenzene and

2.12 g. of N-bromosuccinimide in 60 ml. of carbon tetra chloride was refluxed for 20 hr. The solution was cooled and the crystals of succinimide were filtered. The filtrate was evaporated to dryness on the water bath and the crystal line residue was taken up in hot ethanol. On cooling, white crystals of m.p. 98° were deposited. A yield of 81.5^ amounting to 2,h- g. was obtained. This procedure is a dis tinct improvement over the method offered by Hesse (21). 26

(21) 0. Hesse, Ann., 226, 329-30 (1893).

He used bromine in chloroform and the product, obtained in

56,^ yield, was contaminated with substantial amounts of the dibromo derivative.

Preparation of 2 ,^.6'-Trimethoxytrinhenylcarbinol

Li OCH

CH30 ^ 0CH3 OCH OCH3

To a flask containing 0.88 g. of trimethoxybromobenzene, 6 ml. of a solution of phenyl lithium (22 ) in ether

(22) C. Fe H, Allen and J. Van Allan, Organic

Syntheses, 22, 83 (19^3).

(0.3 ^ g. of phenyl lithium per 1 g. of the bromo derivative on a mole to mole basis) was added and the mixture put under a nitrogen atmosphere and shaken for 0 .5 hr. during which, time a fine precipitate formed. Then 0.65 g. of benzophenone was added and the contents of the flask refluxed briskly while the precipitate gradually disappeared. After 0,5 hr, the reaction mixture was shaken with water and the ether evaporated. An oil separated which on scratching and wash- 27 ing with low boiling petroleum ether crystallized. It was recrystallized from ethanol and had a m.p. 110-11°. This is identical with the compound as prepared through a Grignard reaction between phenylmagnesium bromide and the substituted benzophenone by Kaufmann and Kieser (23). The yield amount-

(23) H. Kaufmann and F. Kieser, Ber., }+6, 3788-

3802 (1913).

ed to 0.95 g. or 80.5;^ of the theoretical based on benzo phenone. The influence of the halogen on the yield can easily be seen through comparison of the above amount with the percentage yields obtained by Wittig and Fuhrmann (24-).

(24-) G. Wittig and G. Fuhrmann, Ber., 73B. 1197-

1218 (194-0).

They reacted the unhalogenated trimethoxybenzene with phenyl lithium followed by benzophenone. Their yields of the same carbinol are recorded as; after 60 hr., 86^; after 5 hr., 67.^. Thus, when a halogen is present the rate of lithium interchange is increased and the reaction approaches completion in a shorter period of time. Here the yield has been increased by over 13 ^ while the time nec essary was only one-tenth of the former. 28

Preparation of tt-Nitrobenzyl Cyanide

An amount of 117 g . of benzyl cyanide was added dropwise with vigorous stirring to 700 g. of fuming nitric acid previously cooled to below 5°. During the addition, the temperature was not allowed to go above 7°. After the addition was complete, the cooled solution was poured onto cracked ice and after standing for 12 hr. the precipitate which had formed was separated by filtration. To elimiate the ortho and meta isomers, the substance was twice re crystallized from alcohol. The pale yellow crystals (87.8 g. or 5^.2:^) gave a melting point of 115-6°. The literature

(25) reports ll6-7°.

(25) R. Pschor, 0. Wolfes and W. Buckow, Ber., 88. 170 (1900).

Preparation of p-Aminobenzyl Cyanide

P - -vx ri "t — To a mixture of 32.4 g. o^benzyl cyanide and 44 g. of mossy tin covered by 400 ml. of ethyl alcohol, 200 ml. of hydrochloric acid was added gradually. The mixture was stirr ed during the addition and kept at a temperature below 25°.

After the addition of the acid, the solution was stirred about 3 hr. until almost all of the tin had reacted. The alcohol was evaporated at reduced pressure and the liquid 29 neutralized slowly, while cooling in ice, with sodium hydrox

ide pellets and left overnight. Silver colored platelets

formed in the liquid. These were filtered and recrystallized,

by air evaporation, from a 35/^ aqueous ethanol solution, A

yield of 72.7^ (19.2 g.) of crystalline material melting at

4-4-^° was obtained. The literature (25) value is recorded

as 46°,

Preparation of n-Hydroxybenzyl Cyanide

This compound was prepared according to the proced

ure of Pschorr and co-workers (25). An amount of 9 g. of p-

aminobenzyl cyanide was added to a boiling mixture of 4-00 ml,

of water and 200 ml, of dilute (25^) sulfuric acid and to

this solution 7 g. of sodium nitrite, dissolved in hot water

(25 ml,), was added dropwise with vigorous stirring. The tip

of the dropping funnel was about one inch below the surface

of the liquid. The solution was then cooled rapidly, fil

tered from the resin and the filtrate extracted four times with ether. The ether was allowed to evaporate and a hard

yellow-red cake resulted. This was taken up in a large vol

ume of water (about 4-00 ml.), made slightly acid by the add

ition of hydrochloric acid and decolorized with decolorizing

carbon. On air evaporation, silvery white crystals appeared,

some of which were in the form of hexagonal and octagonal e-

longated plates. The authors (25) report 70°. Actual yield was 4- g, of material with a melting point of 70-72°. 30

Â Houben-Hoesch Reaction Between. 2.4-.6-TrImethoxy-

bromobenzene and •p-Hvdroxybenzvlcyanlde

Br V” t^«W-/>,-«3 0-CH OH

A. mixture of 2 g. of 2 ,^,6-trimethoxybromobenzene

and 1,09 g. of ^-hydroxybenzyl cyanide was dissolved in

^0 ml, of dry ether and cooled in an ice bath and 1,0 g, of

fused zinc chloride was placed in a three-necked flask

mounted with a moisture guarded reflux condenser and a

hydrogen chloride gas inlet. With the flask in an ice-salt

bath, dry hydrogen chloride gas was bubbled in until the

ether solution became saturated (about 3 hr,). The flask

was then placed in the freezer nart of the refrigerator

overnight. The mixture was poured with stirring into 150 ml,

of ice water and the aqueous solution was extracted once

immediately with 50 ml, of ether. The aqueous solution was

almost neutralized with sodium bicarbonate solution and di

gested for 1,5 hr. During this time the solution became

clear and then later cloudy and eventually crystals appeared.

The solution was cooled and filtered. Crystals in the amount

of 0,18 g, (6^ yield) were obtained. On recrystallization

from ethanol, the crystals melted at l68°. The results of

a sodium fusion test indicated no bromine oresent in the 31

molecule. The physical constants are identical with that

ketone obtained from the unbrominated phenyl ether (26).

(26) G. Zempl^, R, Bognar and L. Farkas, Ber.,

JZ6, 267 (1943 ).

We were thus led to the conclusion that the nitrile had pre

ferentially attacked the halogen on the phenyl ether, which

halogen had then been replaced by the benzyl-imino structure,

Consequently this was not a satisfactory method for the pre

paration of the desired halogen-containing ketone.

Preparation of Homoanisic Acid

This compound was prepared according to the dir

ections of Schwenk and Papa (27). A mixture of 52 g. of

(27) E. Schwenk and D. Papa, J. Org. Chem., 11.

800 (1946). i

p-methoxyacetophenone, 13.5 g. of sulfur and 29.8 g. of dry

morpholine was refluxed for 5 hr. The reaction mixture

was slowly poured into water and allowed to stand in the 32 refrigerator until solid. The dark mass was then hydro lyzed by boiling with hydrochloric acid on the water bath, allowed to cool and the crystals in the solution were removed by filtration, kn amount of 26 g, (78%) of material melting at 83-5° was obtained. The authors report a similar constant.

Preparation of Homoanisovl Chloride

Homanisic acid (1+.2 g.) and thionyl chloride (9 g. ) were gently refluxed on the water bath for 2 hr. Then,

10 ml. of benzene was added and the mixture was distilled under reduced pressure. A yield of 3.9 g. or 82^ of the chloride was obtained.

A Friede1-Crafts Reaction between 2.4,6-Trimethoxv- bromobenzene and Homoanisovl Chloride

OCH.

OCH3

Anhydrous aluminum chloride (6,6 g.) was dissolved in 25 ml. of nitrobenzene. Then, with stirring and cooling in a hydrochloric acid-ice bath, 2.82 g. of homoanisoyl chloride in 15 ml. of ^-tetrachloroethane was added dropwise. The solution turned deep blue-black. This was followed by the dropwise addition of g. of 2,4,6-tri- 33 methoxybromobenzene in 20 ml. of ^-tetrachloroethane. The solution was stirred for 2 hr., heated on the water bath for 1 hr. and then stirred for 30 hr. at room temperature.

The mixture was poured into ice water containing l6 ml. of concentrated hydrochloric acid. The total volume was now equal to 2 50 ml. This was stirred overnight at room temper ature and then allowed to separate into two layers. The aqueous layer (top) was decanted off and the lower layer was steam distilled until nitrobenzene and _s-tetrachloroethane stopped coming over (the product is also steam distillable). While still hot, the aqueous solution was poured from the oily residue and allowed to cool. A yield of 320 mg. of white needles that were sublimable and melted at 17^-5° was obtained. Sodium fusion indicated the pre sence of halogen in the product so the material was sent out for analysis.

Anal. Calcd. for C^gH^gO^Br: C, 5^.7; H, 4.8l;

Br, 20.12. Calcd. for C2 yH2 yOyBr; C , 59.7; H, ^.97;

Br, 1^.72. Calcd. for C35H 35O 9: C, 70.7; H, 5.9.

Pound: C, 6l.4l; H, 5.^2; Br, 3.52.

Thus it appears that a mixture of di- and tri-acyl-substi tuted phenyl ether had been formed. The procedure was im practical also because of the low yield combined with the necessity of separating the isomers. 3^

X-ray Powder Diffraction Patterns of the Root Bark

Pigment Substance I of the Osage Orange

All data were obtained with Cu-Kq^ radiation, X - 1 .5^18 S.; the exposure time was 3 hr. The measurements were made on the high melting dimorph.

Position of intensity Radius of circle d-spacing (28)

1 (strongest) 6 .6O mm. 13.392 2 8.40 10.526 8 10.75 8.2106 9 12.05 7.3780 4 15.40 5.7535 5 19.575 4.5343 10 20.40 4.3532 6 22.70 3.9171 3 24.625 3.6151 7 32.50 2.7549

(28) U. S. Dept, of Interior, Geol. Survey Circ. 29

Aug. 1948. Table of d-Spacings for Angle 2 0 Cu-E% . 35

The following series of experiments deal with the degradative studies carried out on pigments isolated from the root bark of the Osage orange and on their derivatives,

There are numerous examples in the literature wherein the investigators have used alkali in some form as a means of de gradation of natural products for the purposes of structure determination. Our initial attempts also followed this gen eral method of attack. The extent and nature of the degrada tion depends, largely, on the concentration of the alkali used and the duration of contact of the alkali with the mater ial. In addition, the type of derivative of the natural pro duct which is used must also be taken into consideration.

On the proper balance of all these factors depends the ul timate success or failure of an experiment of this nature.

Frequently, fission of a substance can also be achieved through the use of various oxidizing agents, either alone or in conjunction with alkali. Procedures of this type were al so utilized in this investigation.

Action of Dilute Alkali on Substance I

To 25 ml. of a solution of aqueous 10% potassium hy droxide was added 1 g, of Substance I. The material readily dissolved when the mixture was heated and the solution turned the characteristic dark maroon color. After refluxing for

5 hr., the liquid was cooled and extracted with ether. This 36 ether extract was designated the neutral fraction. The basic solution was then acidified, with cooling, with 4N hydrochloric acid and the amorphous precipitate which appeared was extract ed thrice with ether. The combined ether extracts were evap orated in a stream of dry air to a volume of 50 ml. and ex tracted twice with 25 ml. of 5^ sodium carbonate solution.

The ether solution was evaporated to dryness and this portion was designated the phenol portion. The sodium carbonate solution was neutralized with dilute hydrochloric acid and then an additional 5 ml. of acid was added to insure complete conversion of the organic acid. This acid solution was then extracted with ether and the ether solution evaporated to dry ness. This part was termed the acid fraction. The amount of neutral fraction was negligible, amounting to less than 10 mg. of amorphous, colored material which did not invite further work. Both the acidic and phenolic fractions were isolated as semi-oily material which could not be brought to crystal lization. The acid fraction was taken up in a minimum amount of a cetone and placed on a column of Magnesol-Celite and devel oped with petroleum ether (low boiling) containing a trace of benzene. Using this method at least four zones could be dis tinguished on the column, each zone giving insufficient pure material for further characterization. This method could per haps be used successfully if larger amounts of the pigment, perhaps up to 10 g., were used. These results are substan- 37

tially the same as those obtained by Looker (5)«

Action of Potassium Hvdroxide-Dioxane on Tetrahvdro-

Substance I Trimethyl Ether

To 10 ml. of ethanol floating on a mixture of 3*5 g. of potassium hydroxide in 4 ml. of water was added 1 g. of tetrahydro-Substance I trimethyl ether. The mixture was con tained in a beaker covered with a watch glass and digested on the hot plate. The ethanol evaporated leaving a semi-solid yellow mass floating on top of the alkaline solution. This was allowed to stand overnight at room temperature and then

20 ml, of unpurified dioxane was added. The solid mass dis solved in it while the potassium hydroxide layer became a light maroon color. This was similarly heated for 2 hr. dur ing which time the dioxane vaporized and the mixture in the beaker became frothy. Water was added and a yellow solid separated which was extracted with ether. The ether was evap orated and the residue taken up in absolute ethanol which on cooling deposited yellow needles with a melting point of 160- o ^ 2 . This product gave a positive test for the j -pyrone nu cleus as well as a positive test for a phenolic group (green).

It did not dissolve in boiling 10^ sodium hydroxide solution.

These facts indicated a déméthylation of the hydroxyl group

"peri" to the carbonyl group. 38

Anal, Calcd. for C, 70.37; H, 7.03. Found: 0, 70.10; H, 6 .9 8 .

In order to substantiate the identity of this degra dation product, a synthesis of the dimethyl ether of tetra hydro-Substance I was initiated.

Preparation of Substance I Dimethyl Ether

In a test tube was dissolved 300 mg. of pure Sub stance I in 4 ml. of N sodium hydroxide solution. This re sulted in a solution with the usual deep red color. Then

0.8 ml. of dimethyl sulfate was added and the tubs stoppered and shaken until a finely divided amorphous yellow precipitate developed. This was filtered and the residue washed well with cold water. Crystallization from ethanol, using decolorizing carbon, gave clusters of yellow needles with a melting point of 162-3°. The substance gave an olive-green color with ferric chloride and was insoluble in cold N sodium hydroxide solution.

The compound was analyzed as the dimethyl ether of Substance I.

Looker (5) has reported the preparation of a dimethyl ether of

Substance I through the action of diazomethane on Substance I.

His compound had a recorded melting point of 219° (dec.) and analyzed satisfactorily. A sample of his product, when recent ly tested, gave a melting point spread of l60-200°.

Anal. Calcd. for C, 71.07; H, 6.20; OCH3 ,

1^.69 . Found; C, 71.00; H, 6.27; OCH3 , l4.^4. 39

Preparation of the Dimethyl Ether of Tetrahydro-

Substance I

An amount of 50 mg. of the dimethyl ether as prepared above was dissolved in 25 ml, of ethanol containing 100 mg. of palladium-on-carbon catalyst. Through the use of a water bath the temperature was raised to 6o° and hydrogen gas was blown through the solution from a dispersing tube for 0,5 hr.

The catalyst was then removed from the solution by filtration and the filtrate was evaporated to dryness. The pale yellow cake which resulted was taken up in ethanol which on cooling deposited 4^- mg. of yellow needles with a melting point of l60-l°. A depression in the melting point occurred on ad mixture of the product with starting material. The mixture melted over range l57-l6l^. There was no noticable depres sion of the melting point when this substance was mixed with crystals of the degradation product obtained on page 3 7 .

The ferric chloride color test was also identical.

Before the procedure for the synthesis of tetrahy dro-Substance I dimethyl ether, as outlined above, was ap plied successfully, the following attempts were found to be unfruitful. 40

Attempts to Methvlate Tetrahydro-Substance I

An amount of 200 mg. of tetrahydro-Substance I was dissolved in 10 ml. of methanol and while kept under a ni trogen atmosphere with cooling to 0 °, a solution of 0.5 g. of potassium hydroxide in 8 ml. of methanol was added to it with vigorous stirring. To this mixture was added

3.6 ml of dimethyl sulfate and the solution was allowed to stand for 1 hr. and poured into ice water. The precipitate which formed was removed by filtration and twice crystallized from ethanol. The material had a melting point similar to o that of the starting material; that is 204-5 , mixed melt ing point was undepressed.

A solution of 100 mg. of tetrahydro-Substance I in

20 ml. of dry ether was made and to it was added 30 ml. of ethereal diazomethane solution containing about 350 to

400 mg. of diazomethane. This was a calculated large excess.

The solution was placed in an ice bath for 3 hr. and was then allowed to stand at room temperature overnight. The ether was evaporated and the yellow cake was taken up in ethanol from which crystals of the starting material melting at 203-205° (mixed melting point undepressed) were deposited.

Alkaline Peroxide Oxidation of Substance I

In a solution of 5 g. of sodium hydroxide in 150 ml. 4 l of water was dissolved 1 g, of Substance I and then 50 ml. of 7.5^ hydrogen peroxide was added. The solution changed from a dark red to an opaque yellow. After standing for

20 hr. at room temperature with occassional shaking, the yellow solution containing an oily material was heated on the water bath until the excess hydrogen peroxide was de stroyed as evidenced by a cessation of gas evolution. The solution was filtered and the filtrate was acidified with

4 N hydrochloric acid. On acidification there was a notic able evolution of gas which turned lime water milky. This was probably carbon dioxide. The clear liquid was then ex tracted twice with ether and the combined ether extracts were extracted with 5^ sodium bicarbonate solution. This solution was then acidified with k- N hydrochloric acid and, after cooling, extracted twice with ether. The ether layer on evaporation gave a crystalline material suspended in an oily layer.

The crystalline substance sublimed above 130° to give colorless hexagons with a melting point of 162-3°. A nega tive ferric chloride test was obtained which indicated the absence of phenolic groups. The compound was definitely acidic since it dissolved instantly in a 5% solution of sodium bicarbonate. A positive Baeyer*s test (29) (alkaline potas sium permanganate) for an unsaturated acid was obtained and the compound also gave a positive benzenoid test (yellow) with k2

(29) F, Schneider, "Qualitative Organic Micro analysis," John Wiley and Sons, New York, N. Y . , 19^6, p. 1^9.

aluminum chloride and chloroform.

When this procedure was applied to crystalline Sub stance I obtained from another batch of root bark, repeated runs produced none or only a trace of this material. This acid may actually be a degradation product of a contaminant.

Anal. Calcd, for 68,70; H, 6.87,

Calcd, for C23H22O 5: C, 6 7 .99; H, 6,92, Found: C, 68,66,

68,35; H, 7.01, 6 ,92 , The yellow oily material had a rancid odor which closely resembled that of propionic or isobutyric acid.

Attempts to derivatize this failed at the acyl chloride stage,

Isolation of a Dinitrophenylhvdrazone from Alkaline

Peroxide Oxidation of Substance I

After the alkaline solution of Substance I, which contained the hydrogen peroxide, had stood overnight, the excess hydrogen peroxide was destroyed by the addition of sodium bisulfite and the acid, which in this case separated in the form of crystals after acidification, was filtered.

The solution was heated under reflux; using a Newman take-off 43

column, 25 ml. of distillate was collected and to this was

added a saturated solution of 2,4-dinitrcphenylhydrazine in

10% sulfuric acid, A yellow precipitate formed which coagu

lated on shaking and was removed by filtration. The materi al was recrystallized from methanol to give yellow plates with m,p, 118-9°. This compound proved identical with the

2,4-dinitrophenylhydrazone of acetone through a comparison of X-ray diffraction patterns. Infra-red spectra were also used.

X-ray Diffraction Patterns of the 2.4-dinitro- nhenvlhydrazone Derivatives of Acetone (A) and the Degra dation Product (B)

(A) (B) Figure 1, hk

In Figure 1 is shown a comparison between the X-ray diffraction patterns for acetone 2 ,4-dinltrophenylhydrazone and the 2 ,4-dinitrophenylhydrazone derivative of the ketone obtained as a degradation product of Substance I. The

"d" values of the most intense lines of the degradation pro duct dinitrophenylhydrazone are similar to those of litera ture (30) "d" values for acetone dinitrophenylhydrazone.

( 3 0 G, L. Clark, W, I, Kaye and T. D, Parks, Ind.

Eng, Chem,, Anal. Ed., IS, 310 (19^6),

Comparison of "d" Values of Most Intense Lines

of Dinitrophenylhydrazones

I II III IV (innermost line)

Acetone 3.27 5.70 9.3 9.3 Degradation Product 3.27 5.78 9.2 9.2

Isolation of a 2.^-Dinitrophenylhydrazone Through the Action of Alkali on Substance I

A solution of 0.5 g. of Substance I in 30 ml. of 30# potassium hydroxide was refluxed for 2 hr. in a flask mount ed with a Newman take-off condenser. Then 20 ml. of water was added and 20 ml. of liquid was removed by distillation. On the addition to the distillate of a saturated solution of

2,4-dinitrophenyIhydrazine in 10# sulfuric acid a precipitate ^5 formed. This coagulated on standing and was removed by filtration and the residue was recrystallized in needles from methanol. The crystalline material had a melting point of 116-8° hut the X-ray diffraction pattern did not corre spond to that previously obtained for acetone 2,4-dinitrophenylhydrazone. The homogeneity of the dinitrophenylhy drazone was proved by placing 1-2 mg. of the sample in chloroform on a column of silicic acid pre-wet with benzene and the column was developed with a 10^ solution of ether in "Skellysolve B" (b.p. 68-72°) (31). Only one zone

(31) B. E. Gordon, F. Wopat, Jr., H. D. Burnham and L. C. Jones, Jr., Anal. Chem., 22, 175^ (19^1).

appeared. Analysis of the material gave a composition ident ical with that of acetone.

Anal. Calcd. for C^HioN^O^.: C, ^5*37; H, 4.20;

N, 23.51. Found: C, 45.35, 45.49; H, 3.96, 4.23; N, 23.40,

23 .44.

However, the X-ray diffraction pattern gave "d" values which did not compare favorably with those of either dimorph of acetone dinitrophenylhydrazone as reported by

Clark and co-workers (30). if6

Comparison of "d” Values of Most Intense Lines

of Dinitrophenylhydrazones

I II III IV (innermost line) Acetone I 3.27 5.70 9.30 9.30 Acetone II 9.^5 11.15 3.03 11.15 Degradation Product 9.80 4.86 10.84 10.84

The infra-red patterns for acetone 2,4-dinitrophenyl* hydrazone as obtained from alkaline peroxide degradation and the dinitrophenylhydrazone from alkali degradation are reproduced in Figure 2 on page 4?. A comparison of the position of wave length bands as recorded (32) for acetone

(32) J. H. Ross, Anal. Chem., 2j, 1288 (1953).

dinitrophenylhydrazone and this unknown dinitrophenylhydra zone show a close similarity. WAVE WAVI ION too 100 4_L--

t,V4ninofHmjBauMi -r •va

WAVE IBWTH M MOONS WAVE UN»TH M MOONS

WAVE WAVE NUMMRS M C M ' <000 MOO I MO IMO I MO 1100 1000 lOOt-T

tiV^amenBsiUDuiaB

WAVE UNOTH M MOONS WAVE UNOTH M MOONS h8

Wavelength Bands from Infra-red Patterns

of Dinitrophenylhydrazones

(Mulls in Nujol; oil bands at 6 «85 and 7.26 microns)

Acetone Acetone Unknown (Ross) (degradation product)

6.17 microns 6.15 microns 6.12 microns 6.27 6.22 6.21 6.50 6.55 6.6o 6.58 6.62 6.68 6.85 6.85 6.83 7.02 7.0 MM 7.26 7.25 7.25 7.38 7.35 7.32 7.51 7.48 7.49 7.69 7.60 7.60 7.80 7.8 7.78 8.0 8.0 8.0 8.22 8.2 8.2 8.82 8.8 8.8 9.09 9.1 9.09 9.^+2 9.4 9.4 9.73 9.7 9.7 10.18 10.2 —— 10.79 MM 10.85 10.85 10.88 10.95 11.62 11.65 11.72 11.91 11.93 11.97 12.02 12.02 12.02 13.11 13.1 13.1 13.^^ 13.45 13.45 13.86 13.9 13.88 14.16 MM 14.66 14.72 MM 15.08 15.07 4-9

All solutions show a doublet in the vicinity of 6.2 and 6.25 microns due to the phenyl group and 11.9 and 12.0 microns due to a 1,2,4— trisubstituted phenyl group.

The alkaline residue from which the distillate was removed, as above, was neutralized with 4- N hydrochloric acid while in an ice bath and the mixture was extracted with ether. The residue from the evaporation of the ether was an amorphous dark colored material. A portion of it was placed on a Fischer-Johns melting point block and on heat ing, colorless needles sublimed above 200®. This material decomposed at 272-3° and gave a positive ammonium phosphomolybdate test for a phenolic group.

Action of Potassium Hydroxide on Substance I Dimethyl Ether

An amount of 100 mg. of Substance I dimethyl ether was refluxed for 2 hr. with 1$ ml. of 30^ potassium hydrox ide solution and after the addition of 20 ml. of water, 20 ml. of solution was removed by distillation through a Newman take-off condenser. The addition of an acid solution of

2,4— dinitrophenylhydrazine gave no precipitate and the alka line solution in the flask had no odor. The dimethyl ether is practically insoluble in alkali and there was no indica tion of a reaction. About 85 mg. of starting material was recovered. 50

Action of Alkaline Potassium Permanganate on

Substance I

A 2,2-dimethichromene structure was indicated by the formation of acetone as a degradation product of alka line treatment of Substance I. Very often (33) -hydroxy-

(33) J. A. Lamberton and J, R, Price, Australian J.

Chem., 6, 66 (1953); see also E. 8 . Spath, P. K. Bose,

J. Matzke and N, 0, Guha, Her., 72 B . 21 (1939) •

isobutyric acid can be obtained from this unit through attack with alkaline potassium permanganate.

In 35 ml. of 5/^ sodium hydroxide was dissolved 1,2 g. of Substance I. To this solution was added 200 ml. of water and then with stirring 180 ml. of potassium permanganate was added in 20 ml, portions every 20 min. The mixture was allowed to stir for 2 hr. after the final addition and then the excess permanganate was destroyed by the addition of

10 ml. of hydrazine followed by heating the mixture on the water bath for 1 hr. The solution was filtered and the fil trate made strongly acid with concentrated hydrochloric acid.

The mixture was saturated with sodium chloride and extracted overnight with ether by means of a continuous extraction apparatus. The ether solution was shaken with dilute ammon ium hydroxide solution and oxalic acid was precipitated as 51

the calcium salt through the addition of a saturated calcium

chloride solution. This precipitate was removed by filtra

tion and the filtrate was acidified with 4- N hydrochloric

acid and extracted with ether. The amorphous residue which

was obtained on evaporation of the ether could not be sub

limed at 100° at 0,8-1 mm. The residue only resinifled.

If OC -hydroxyisobutyric acid had been present as a degrada

tion product, it should have appeared as a sublimate under

these conditions. Therefore it must be concluded that none

was present.

Action of Dilute Nitric Acid on Substance I

A mixture of 1 g. of Substance I and 25 ml, of V?%

nitric acid was refluxed overnight. The nitric acid solu

tion was brought to a boil on a glycerol bath and 1 g, of

Substance I was added. The solution immediately became

brown and frothed. The mixture was refluxed 29 hr. at which

time the solution was pale yellow with yellow particles

floating in it. Following extraction with ether the ether

solution was extracted with 0,5 N sodium carbonate solution.

The aqueous layer became a dark red color characteristic of

a solution of Substance I in base. On slow neutralization with h N hydrochloric acid the solution turned yellow, al

most chartreuse, and after standing for a few minutes a fine

precipitate developed. This was taken up in ether which was 52

evaporated in a stream of dry air to give a cake of non-

crystallizable material. The acidified aqueous layer was

evaporated to dryness and washed with hot acetone. The re

sidue from evaporation of the ether washing partially sub

limed above 100°. The sublimed material melted at 189-90®

and was identical in physical properties with oxalic acid

(mixed melting point undepressed).

Action of Chromic Acid in Acetic Acid on Substance I

Oxidation with chromic acid usually converts an

aliphatic side chain on an aromatic nucleus into a carboxylic

acid group. With this purpose the following reaction was

undertaken.

In 20 ml. of glacial acetic acid was dissolved

300 mg, of Substance I triacetate. Substance I is relative

ly insoluble in acetic acid. To this solution at 0° was

added 0,7 g. of chromic acid in 5 ml, of 80^ acetic acid, with stirring at the rate of one drop per sec. After 2 hr,

of stirring the excess chromic acid was destroyed by the

addition of sodium bisulfite and the solution was poured in

to water. The acetic acid was removed by steam distillation

and the residue was extracted with ether. The ether extract was washed with a solution of sodium carbonate. This

solution was neutralized and washed with ether. Only an

amorphous material was obtained. 53

Action of Potassium Permanganate In Acetone on

Substance I Trimethyl Ether

To a solution of 130 mg. of the trimethyl ether of

Substance I in 10 ml, of acetone was added 0,325 mg, of pow

dered potassium permanganate and the mixture was refluxed for 10 min., then cooled and filtered. The residue was sus pended in 15 ml, of water and into this suspension was bub bled sulfur dioxide gas. The brown manganese dioxide dis solved and an oil separated which was removed by extraction with ether. The ether washings were shaken twice with 10^ sodium carbonate solution. On neutralization with N hy drochloric acid an oil separated which solidified on stand ing overnight. Clusters of needles melting at 187-9° were obtained on slow crystallization from methanol. The yield was 75 mg, The product was an acid since it was soluble in

5% sodium bicarbonate solution. It did not give a positive test for a phenolic hydroxyl group (ferric chloride test),

Anal. Calcd. for ^22^22^10 ^ ^HgO; C, 5*+.77» H,

5,39; mol, wgt, ^82; 30CH^, 19.31- Found: 0, 54.46, 54,63;

H, 5.32, 5.25; neut. equiv, 240, 234; OCH^,

An amount of 16.8 mg, of the acid (dried to constant weight in the Abderhalden over methanol) was heated with toluene for 2 hr. The weight then was 15.8 mg,; a loss of

5.9^. A dihydrate with a molecular weight of 482 contains

7,4^ water. 5^

Action of Hydrobromic Acid on the Acid Obtained

on Oxidation of Substance I Trlmethvl Ether

An amount of 100 mg. of the acid obtained as above was refluxed for 2 hr, with 15 ml. of hydrobromlc acid

(density 1.42). The solution was cooled and 30 ml. of water was added and the residue filtered. On air drying, an

amorphous olive-green colored material was obtained that

partially sublimed above 200°. The material was Insoluble

In 10^ sodium bicarbonate solution but dissolved readily

In % sodium hydroxide solution. A greenish blue color was

obtained with alcohllc ferric chloride. This amorphous mate rial was dissolved In 15 ml. of 2 , % sodium hydroxide solu

tion to give a deep maroo.i color. The solution was extract

ed with ether which on evaporation gave a trace of white material. The sodium hydroxide solution was neutralized with

10% hydrochloric acid and an amorphous precipitate was form

ed which appeared pale yellow In color. This seemed to be a mixture of at least two substances. On addition of acetone, part of the material dissolved Immediately. The residue was removed. It was a white, non-acldlc substance which gave a blue-green color with ferric chloride and sub limed above 200° with eventual decomposition above 300°.

The acetone-soluble portion when placed on the melting point block darkened gradually up to 200°. It does not sublime and gives a deep blue-black color with ferric chloride 55 solution.

Action of Dilute Base on Unknown Acid

A solution of 100 mg. of the unknown acid was re fluxed with 10 ml. of 15^ potassium hydroxide for 1 hr. and then extracted with ether. The aqueous portion was acidi fied and extracted with ether. The residue from the evap oration of this ether was dissolved in 1 0^ sodium carbon ate and extracted with ether. The basic solution was acidified to give an oily material which solidified on standing overnight and proved to be identical with the starting material.

The Action of Ethylmagnesium Bromide on Substance I

Trimethvl Ether

Xanthones when reacted with Grignard reagents give xanthydrols which on the addition of dilute sulfuric acid give a solution of green fluorescence (34). To an ether solu-

(34) Richard Meyer and Erich Saul, Ber., 26, 1276

(1893).

tion containing 25 mg. of Substance I trimethyl ether was added an ether solution of ethylmagnesium bromide. This mixture was hydrolyzed with dilute hydrochloric acid and the 56 ether layer was separated and evaporated to give a pseudo- crystalline material which turned red on exposure to con centrated acid. The addition of dilute sulfuric acid gave a solution of yellow color which had a slight greenish fluorescence.

The Action of Potassium Hvdroxide on Substance III

Substance III undergoes a partial decomposition when allowed to stand in contact with air. This is evi denced by an appearance of a deep red coloring on the sur face of the crystal. An amount of 0.5 g. of this partially decomposed Substance III was dissolved in 30 ml. of 30^ po tassium hydroxide solution and the mixture was refluxed for

2 hr. After the addition of 20 ml. of water, 20 ml. of distillate was removed through a Newman take-off condenser.

The addition of a solution of 2,^-dinitrophenylhydrazine in

10^ sulfuric acid to the distillate gave a yellow precipitate which was separated and recrystallized from ethanol. The needles, which melted at ll6-8°, gave an X-ray diffraction pattern identical with that obtained by a similar reaction on Substance I (see page !+5).

Méthylation of Substance III

The partially decomposed Substance III was purified by chromatography on a column of Magnesol-Celite and 50 mg. of this purified material was dissolved in 4 ml. of N sodium 57 hydroxide solution to give a red colored mixture. To this was added dimethyl sulfate in 0,5 ml, amounts with shaking after each addition. Finally an oil separated; in the oil were colorless needles which were isolated by pouring the oil on a porous plate. The oil absorbed and the needles remained on the surface of the plate. They had a melting point of 148-50° and did not give a positive ferric chlo ride test for a phenolic hydroxyl group. By comparison, through mixed melting point, these crystals proved identi cal with the completely methylated Substance III as pre pared by Thompson (6),

Color Tests on Substance III and Derivatives

A phenolic hydroxyl group which is flanked by groups large enough to hinder its activity will frequently not re spond to the standard ferric chloride test. In these in stances its presence can often be detected through the use of ammonium phosphomolybdate (35). A few crystals of the

(35) G, H, Stillson, D, W, Sawyer and C, K, Hunt,

J, Am, Chem, Soc,, 303 (1945). methylated Substance III, as prepared above, were dissolved in ethanol and a drop of a 2 % aqueous solution of phospho- molybdic acid and 2 drops of concentrated ammonium hydroxide were added. The blue color of a positive test did not appear. 58 ■

Glücksmann test for pyrogallol structure. A trace of Substance III In 1 ml. of glacial acetic acid and 5 drops of paraldehyde was heated to boiling and a few drops of con centrated hydrochloric acid was added to the hot solution.

A red color, indicative of this structure, did not appear.

Action of N-bromosuccinimide on Pomiferin Trimethvl

Ether

Halogenated derivatives of insecticides have usually proved more potent than the compounds themselves. With this purpose, an attempt was made to brominate pomiferin trimethyl ether (36). A mixture of 462 mg. of pomiferin tri-

(36) M. L. Wolfrom, F. L. Benton, A. S. Gregory,

W. W. Hess, J. E. Mahan and P. W. Morgan, J. Am. Chem.,

Soc., 61, 2832 (1939).

methyl ether and IBO mg. of N-bromosuccinimide in 4o ml, of dry carbon tetrachloride was refluxed for 48 hr. The solu tion, which had turned yellow, deposited crystals of succinimide on being cooled. These were filtered and the filtrate was evaporated to a yellow oily residue which gave a green- black color in the ferric chloride test. It could not be crystallized from ethanol. 59 •

Infrared and Ultraviolet Absorption Spectra

The infrared spectra of Substance I, Substance I trimethyl ether and the completely methylated Substance III were made to be used in structural studies. The infrared spectra of the fruit pigments osajin and pomiferin and their completely methylated derivatives were made for the purpose of comparison with those of the root-bark pigments.

The measurements were made with a Baird Infrared Spectro photometer, using a sodium chloride prism. All compounds were run as mulls in Nujol, a refined mineral oil. These are shown in Figures !+ through 10.

The ultraviolet absorption spectrum for the com pletely methylated Substance III was made with a Beckman

Spectrophotometer, model DU, cell length 1 cm. This is shown in Figure 11.

Separation of Substance II from a Mixture with

Substance I

Substance I (1 g.) as isolated by Thompson (6) was dissolved in 40 ml. of ethanol and allowed to evaporate slowly in a beaker covered with a watchglass. The first crystals deposited were in clusters of m.p. 263-*+°. Ad mixture with authentic Substance II crystals showed no de pression. About 20 mg. were isolated in this manner.

Substance I came out later in individual crystals. 60 .

W A V t M WAVI IM MM I tM

WAV# IM IH M WAV# LM TH M

WAV#

f l M t t 5

WAV# UNMTH M

A» , ," P , MM

W A V # amm m 61

WAV! NUMHaS Oé> W AVk N U M K a s C M < MOO 7000 TOO

i i nuB» N m u

W A V I U N » T H MICaONS W A V I U N 6 TH M MfCOONS

WAVI NUMKas IN CM* WAVt NUMKas IN CM* MOO 700

rioou • oouta D t M t m

W A V I LIN 9 TH M W A V I LIN6TH M MICaONS

WAVICM' WAVI NUMKas M CM' s i e o TOO

W A V I U m i H M WAVI LM TH M MCaONS

WAVI WAVI MOO 14001100 1000 700

nti -A.

I M T HAV I W A V I I M T HWA r

iLlli

29 63

DISCUSSION OP RESULTS

Investigation Toward the Synthesis of Osajin and Pomiferin

The structures which have been established for

osajIn and pomiferin are shown on page 2, A variety of

syntheses for compounds containing the basic Isoflavone nu cleus have been reported. It seems pertinent to review the

Important procedures which have been applied.

Isoflavone

The first generally useful method was developed In

1930 by Spath and Lederer (37) and was greatly Improved In

(37) E, Spath and E, Lederer, Ber,, 7^3 (1930).

193*+ by Mahal, Ral and Venkataraman (38) who carried out a

(38 ) H, 8, Mahal, H, S. Ral and K, Venkataraman,

J, Chem, Soc,, 1120, I769 (193^).

condensation at 0° between sodium, ethyl formate and a deoxybenzoln to obtain yields of about 30^ of the Isoflavone. 61+

This reaction may be illustrated by the following general equation*

0->o0

In this and in other cases the only free hydroxyl group in the deoxybenzoin was that required for ring closure. This is a severe limitation since many natural isoflavones con tain such hydroxyl groups as substituents. In 1953» Baker and co-workers (39» 40) succeeded in devising a successful

(39) W. Baker, J, Chadderton, J. B. Harborne and

W. D. Ollis, J. Chem. Soc., 1852 (1953).

(40) W. Baker, J. B. Harborne and W. D. Ollis,

J. Chem. Soc., i860 (1953).

method of synthesis which overcomes this difficulty.

Benzyl-o-hydroxyphenyl ketones are reacted with n + 1 moles of ethoxalyl chloride (n - number of free hydroxyl groups) in pyridine at room temperature to give ethyl isoflavone-

2-carboxylates which on hydrolysis and decarboxylation give high overall yields of isoflavones. The scheme of the re action can be indicated by the following specific equations, 65

HO % % ftQ ClCCOCgH^

o COOH H O f ^ ^hydrolysis) F JR

Baker, Chadderton, Harborne and Ollis (39) studied the mechanism of this general reaction and presented evi dence that it proceeded by the following steps.

1) Ethoxylation of all phenolic hydroxyl groups except one ortho to the carbonyl group.

2) C-Ethoxylation of the reactive methylene group of the deoxybenzoin.

3) Cyclization to the hydroxyisoflavanone car boxylic ester:

^ 4cOOEt

OR o •» ^

k) Loss of a molecule of water to give the iso flavone carboxylic ester which is followed by removal of the ethoxalyl groups and ethyl radical by reaction with dilute acid.

5) Decarboxylation by heating slightly above the melting point. 66

Ko useful synthesis of an isoflavone has appeared

which does not require a deoxybenzoin. Such compounds can

be prepared by a number of different methods of which the

most applicable is the Houben-Hoesch reaction (^1). The

(^1) E. Spoerri and A. 8, DuBois in "Organic

Reactions", Vol. V, John Wiley and Sons, New York, K. Y.,

19^5, p. 387.

Friedel-Crafts reaction and the Fries rearrangement have

also been applied to this synthesis.

Application of these methods to the syntheses of

osa j in and pomiferin is complicated by the 2,2-dimethylpyran

grouping appended to the isoflavone nucleus. This 2,2-di

methylpyran structure, present as a chromeno-chromone unit

A Chromeno-chromone unit

(one isomer shown) o

is no longer as unique as once believed and each year addi

tional natural products are isolated which contain it. A

discussion of methods for its detection through degradation

is included on page 8^-. Among natural products which con

tain this structure are evodione (42) from the volatile oil

of Evodia ellervana. a leguminous fish-poison from plants of 67 the Perris malacensis (43), luvangetin (44) from the roots and fruit of Luvanga scandeus. sesel in (45) from seeds of

Sesell indicum. lonochocarpine (46) from the seeds and roots of Lonochocarnus sericeus. jacareuhin (47) from the heartwood of Calonhyllum hrasiliense Camb., xanthyletin

(48) and xanthoxyletin (48) both from the bark of

Zanthoxvlum americanum. and deguelin (49), tephrosin (50) and toxicarol (5D from extract of Perris root,

(42) S. E. Wright, J. Chem. Soc., 2005-8 (1948).

(43) S. H. Harper, J. Chem. Soc., 1178 (1940).

(44) E. Spath, P. Bose, H. Schmid, E. Pobrovolny, and A. Mookerjee, Ber . , 73B. 136 I-8 (1940).

(45) E. Spath, P. K. Bose, J. Matzke and N, C.

Guha, Ber. %2B, 821-30 (1939).

(46) J. Baudrenghien, J. Jadot and R. Huls, Bull, soc. roy. sci. Liege, 3^, 52-9 (1949); C.A., hh, 3982 (1950).

(47) F. E. King, T. J. King and L. C. Manning, J.

Chem. Soc., 3932 (1953).

(48) J. C. Bell, W. Bridge and Alexander Robertson,

J. Chem. Soc., 1542 (1937).

(49) Alexander Robertson and T. S. Subramanian, J.

Chem. Soc., 286 (1937).

(50) E. P. Clark, J. Am. Chem. Soc., Ü , 313 (193D.

(51) M. Hanriot, Compt. rend., l44. 150 (1907). 68

The structural formulas determined for these sub stances are shown below. H3 C Q ^ 3 OCH3 CCH3 H3 C

OCH^ OCH Evodione Harper’s fish poison

Luvangetin Seselin

OH H3 C C C H 2 ^ ^ ,0H H 3 C

Lonchocarpine Jacareubin

OCH.

Xanthyletin Xanthoxyletin 69

X Y Deguelin H H

Tephrosin H OH

Toxicarol OH H H- OCH

It is significant that only in chromeno-coumarin type com pounds has a successful synthesis of a natural product con taining the 2 ,2 -dimethylpyran side chain been accomplished.

Spâth and co-workers (52) have prepared luvangetin and

(52) E. Spâth and H. Schmid, Ber., 74b . 193-6

(1941).

seselin (53) by the Interaction of the appropriately sub-

(53) E. Spath and R. Hillel, Ber., 2BB, 963-5

(1939). stituted coumarin and 2-methyl-3-butyn-2-ol as illustrated for luvangetin in the following equation.

OCH. 70

+-HC5CC (CHt

The yields are, however, extremely low; 0,8/^ for seselin and

1 .5^ for luvangetin

The usual proof of structure by synthesis Involves the preparation of the dihydro derivative which is then com pared with the compound obtained by catalytic reduction of the natural substance. The unsaturation in this ring is quite susceptible to hydrogenation and readily adds a molecule of hydrogen. If a method could be devised which would lead to the inclusion of this elusive structure in a synthetic approach to natural product preparation, it undoubtedly could be ap plied toward the synthesis of many of these compounds whose structures appear on pages 68 and 69.

The original proposed method for the synthesis of the fruit pigments osajin and pomiferin is shown by the following series of equations.

H^C H3C H3 C OH U ^ N C C H ^ ^ C-CH2 OH

(Compound A.) 71

1, HCOgEt Compound A 2. HOH OH

(Compound B)

Compound B / BrCHp=C(CH^)2 — — Osajln ^ Ether

This last reaction is an adaptation from the work of Riedl

(5^) who prepared lupulone in the manner shown in the follow-

(5^) W. Riedl, Brauwissenschaft, 133 (195D.

ing equation. 72

0 9 CCHgCHCCHgig ^H2CH(CH3)2 OH OH

/ MepC-CHCH^Br H2CH=CMe2 Me2C=CHCH2

The first intermediate whose preparation was to he attempted was $,7-dihydrozy-2,2-dimethyl-Zl^-chromene (I).

Coumarins react with Grignard reagents to give pro ducts, the nature of which is dependent upon the position of the substituents. Unsubstituted coumarins, in which the

3,^ positions are occupied only by hydrogens, react with ex cess n-alkyl magnesium halides to give 2,2-dialkyl-l,2-benzopyrans (13, 55).

(55) L. I. Smith and P. M. Ruoff, J, Am, Chem, Soc,,

6 2 , m-5 (1940).

+ RMgX CQ R

In the work of this thesis, méthylation of 5,7-dihydroxycoumarin, prepared by hydrolysis of 5?7-diacetoxycoumarin, which in turn resulted from a Perkin reaction on phioroglueinaldehyde, formed $,7-dimethoxycoumarin. Methyl 73 magnesium iodide was prepared and to it was added a solution of the coumarin. Ether extraction of the hydrolyzed product invariably gave a yellow colored oil which turned amber and hardened a short time after evaporation of the ether.

Since different investigators used varied procedures in synthesizing 2,2-dialkyIchromenes containing other ring substituents, the following alterations were tried.

1) The solvents used in various runs were: ether, anisole, benzene, toluene, petroleum ether and dioxane; the latter forms an insoluble addition product with the Grignard reagent.

2) The conditions were varied. The reaction was run at 0°, at room temperature and at reflux temperature of the various solvents used.

3) The mode of addition was changed. The coumarin was added to the Grignard reagent in a number of runs and the rate of addition was varied. The reverse addition was also attempted.

4) Reaction time was varied between 2 and 2*+ hr., with stirring and without stirring.

5) Methods of decomposition of the Grignard addition product involved use of concentrated ammonium chloride, dilute and concentrated hydrochloric acid and refluxing with water containing a trace of hydrochloric acid.

6) Runs were made with the following coumarins. 7^

(a) 5<)7“Ditiydroxycoumarin

(b) 5)7-Diacetoxyc;ouraarin

(d) 5,7-Dlbenzyloxycoumarin

The isolable product was, invariably, a yellow oil that re sisted all attempts at crystallization such as scratching, cooling, dissolving in base and neutralizing.

This approach to the compound was abandoned,

Harris (1*+) had succeeded in preparing 5-benzyloxy-

7-methoxy-2,2-dime thy lehr omene utilizing

C é H ^ C H ^

H^CO

The chroman-4-ol, which may be presumed as an intermediate, was not isolated since immediate loss of a molecule of water resulted in the formation of the ^-chromene.

In the hope of utilizing this method toward the solu tion of the problem, a Meerwein-Ponndorf reduction was carried out on the following chroman-4-ones,

(a) 5,7-Dihydroxy-2,2-dimethylehromanone

(b) 5,7-Dimethoxy-2,2-dimethylchromanone

(d) 5(7)-Hydroxy-7(5)”ben2yloxy-2,2-dimethylchro-

manone

(e) 5-Acetoxy-7-niethoxy-2,2-dimethylchromanone

procedure, resulted. Reduction of the chromanone to a chromene using sodium amalgam and ethanol was also attempted.

The results were similar,

Deetherification of 5-benzyloxy-7-methoxy-2,2-dimethylchromene through the use of standard ether splitting

reagents: hydroiodic and acetic acids, potassium hydroxide

and ethylene glycol, and aluminum chloride in benzene was

tried. All resulted in the formation of the usual reddish

oils.

Apparently the presence of nucleophilic substituents

in the 5 and 7 positions renders the product unstable,

Whalley (56), who has attempted preparation of compounds con-

(56) W, B. Whalley, private communication made dur

ing visit to Department of Chemistry, The Ohio State Univer

sity, May 30, 1952.

taining structures of a similar nature has postulated the formation of an unstable benzopyrylium salt which readily undergoes decomposition.

Another approach to this problem was an attempt to 76

prepare the appropriate isoflavone nucleus and then Insert

the dimethylpyran side chain. A method by which it was be

lieved this could be accomplished was through the reaction

of an 8-lithium isoflavone with senicaldehyde, followed by

a rearrangement of the allyl side chain to a propenyl group

with subsequent cyclization.

(?%)2 M e O f ^ / (CH3)2C=CHCH0 (!

OMe

9H3 0.01 M HCl in dioxane

OH OMe

The reaction of this product with isopentenyl bromide and sodium would introduce the required side chain. Here, there is the possibility of two isomers being produced; one by introduction of the side chain into the 3 ’ position and the desired substance by attack at the 6 position. These could be separated chromatographically. The rearrangement indica ted is a postulated extension of the work of Braude and co- 77 workers (57558) who worked with phenylpropenols.

(57) E, A, Braude and C. J. Timmons, J, Chem. Soc., 2000-8 (1950).

(58) E. S, Weight and E. A. Braude, J. Chem. Soc.,

419-24 (1953).

^ ^^HCH=CH2 ------^ ^ ^ CH^CHCHgOH

The indicated direction of reaction between the organolithium compound and senicaldehyde is to be expected since aryl li thiums tend to react with conjugated systems by a 1,2 rather than a 1,4 addition (59, 60).

(59) H. Gilman and R. H. Kirby, J. Am. Chem. Soc.,

Ü , 2046-8 (1941).

(60) A. Michael and C. M. Saffer, Jr., J. Org. Chem.,

8 , 60-3 (1943).

That an aryl lithium flanked by methoxyl groups will still be sufficiently unhindered so that the normal attack on a carbonyl function will occur is a direct application of the work of Wittig and Fuhrmann (24). 78

The cyclization reaction is similar in result to the ex periments of Smith and Ruoff (55).

To initiate these series of reactions necessitated the preparation of 8-lithium isoflavone. It was believed that this could be best achieved through replacement of a halogen in the 8 position by reaction with phenyl lithium.

An examination of the literature failed to reveal the pre paration of an isoflavone containing a halogen in a known position. To affect the synthesis of a halogenoisoflavone it would be necessary to determine whether the halogen sub stituted on a deoxybenzoin could remain uneffected during a sodium ethyl formate cyclization. An exploratory inves tigation of this reaction utilizing a known (19) chlorodeoyy- benzoin, (2-hydroxy-3-chloro-5-methyl)-phenyl benzyl ketone, produced the desired halogeno-isoflavone on cyclization.

The identity of the product, 6-methyl-8-chloroisoflavone, was established by color tests (Perkin test for y -pyrone structure) and by elemental analysis.

Since it did not seem possible to prepare an appro priately substituted benzene ring which would contain an hydroxyl group adjacent to the halogen and methoxy groups 79 ortho-para to it, the Fries rearrangement was not included as a method to he used in preparing the ketone intermediate.

OH /}— Ketone

OH Intermediate OCH3

Both the Houhen-Hoesch and the Friedel-Crafts re actions were run between 2,4,6-trimethoxybromobenzene (pre pared in our work in excellent yield by the reaction of N- bromosuccinimide with the trimethylether of phloroglucinol) and ^-hydroxyphenylacetonitrile in the former reaction and

^-hydroxyphenylacetylcbloride in the latter case. In both reactions, the halogen was preferentially displaced and the ketone, 2,4,6-trimethoxyphenyl-_p-hydroxybenzyl ketone, was isolated. The identity of this product was established by analysis and comparison of the physical constants with the molecule of known structure (26 ).

H^COi OCH CHg-^ ^ OH

OCH

As a consequence of the unsatisfactory results of these exploratory investigations, this method of approach was also abandoned. A more feasible method is included in the section on Suggestions for Further Study. 80

ROOT BARK PIGMENTS

Substance I . The molecular formula for Substance I has been established by Looker (5) as ' By reaction and color tests, various functional groups have been deter mined to be present in the molecule. Substance I gives a positive Perkin test and therefore contains a y -pyrone structure. The positive Wilson boric acid test obtained is indicative of the structural unit: I II II

I ti OH 0

A positive ferric chloride test has shown a phenolic hydroxyl group to be present in the compound. Acétylation under mild conditions produced a yellow diacetate which gave a positive

Perkin and Wilson boric acid test. Under vigorous conditions a triacetate is isolated. Mild méthylation gave a compound which gave a positive Perkin and Wilson boric acid test, A negative ferric chloride test was obtained with the com pletely methylated and acetylated compounds. They also gave a negative Wilson boric acid test.

The following conclusions can be based on the above information. Substance I contains three hydroxyl groups, either phenolic or enolic. The color tests show that the free hydroxyl group of the disubstituted compound is probably 81 located in position "peri" to the carbonyl of the ^ -pyrone structure. The hydroxyl group is in a hindered position and therefore resists acétylation and méthylation when mild con ditions are employed. The absence of formic acid as a degra dation product precludes the possibility that the basic nu cleus is of an isoflavone nature. Reduction studies have shown two easily attacked non-aromatic points of unsatura tion. No new hydroxyl groups are formed by this action.

Likewise the y -pyrone structure was not affected. The double bonds also are not conjugated since no maleic an hydride adduct was formed.

Studies by Looker were inconclusive in establishing the relative positions of the two remaining hydroxyl groups.

Application of the periodate test appears to indicate their location as being vicinal,

^C-OH

’12^15^ oI %

A difficulty in degradative studies on Substance I was its apparent extraordinary resistance to attack by dilute alkali and also the tendency for the molecule to furnish only traces of crystalline material when stronger alkali was used.

It has been suggested that Substance I possesses a flavone or flavonol nucleus. Compounds containing these structures 82 are not unusually resistant to clean degradation by alkali.

There are numerous examples of successful structure deter mination of natural products containing these structures.

The fission occurs in a definite manner as is illustrated by the following equations.

Flavone; Lotoflavin

HO OH HO HO lOH OH OH

Flavonol: Fisetin

OH __ QH HO OH HO OH 0“

Flavanone: Hesperitin OH iMe [I / OH C O nH Isoflavone: Genistein

HCO qH/ I f , „çO»- H 02C 83

The lines through the compounds indicate the direction of fission.

Methoxyl groups are usually resistant to fission under the conditions employed. Yet, when tetrahydro-Sub- stance I trimethyl ether was reacted with potassium hydroxide-dioxane mixture déméthylation occurred preferent ially to a nuclear fission. The flavanone nucleus is defi nitely excluded since the compound does not form an oxime or phenyIhydrazone. A positive Perkin test for the if -pyrone nucleus also omits this structure.

Flavone and substituted flavones are represented by a characteristic two band system (6l). It is, of course,

(61) F. C. Chen and C. H. Lin, J. Formosan Science,

6, 81-124 (1952).

possible that the presence of additional large groups append ed on the basic nucleus would change this into a three or four band system. Substance I has three maxima at 2280 fi.

2825 S, and 3375 &. The wavelengths for maxima in flavones are in the region of 2900 to 30OO £. and 3700 to 4o502 , For flavonols an extra band appears at 3300 to 3400 2. How ever, even in flavonols, with two hydroxyl groups on the benzene side chain the spectrum reverts to two maxima at

3000 and 4000 2, From a consideration of the ultraviolet absorption spectra the probability is that a flavone or fla- 84 vonol nucleus is not favored. This tends to substantiate

the poor results from degradative action of alkali,

A ring system similar to that present in many nat ural coloring matters is xanthone or dibenzo-^-pyrone. It

is present in the anthoxanthins as a cationic nucleus and in other pigments as euxanthone^ the color principle in Indian yellow, and gentisein, the yellow dye abtained from gentian roots, as a neutral molecule, Xanthones are truly resistant to strong alkali and only after persistent heating are traces of a degradation product found. These are usually phenolic in nature. The ultraviolet spectra of xanthone and its de- rivitives is in a range with maxima at 2500 fi,, 2800 5, and

3300 S, (6 2 ,63). This compares quite favorably with the

(62 ) R, A, Morton and W, T, EarIan, J, Chem, Soc,,

159-169 (1941), (63) R. P. Mull and F, F, Nord, Arch, Biochem,, 4

419-33 (1944), spectra for Substance I, A test indicative of a xanthydrol was obtained when the product from the reaction of Substance I with a Grignard reagent was dissolved in sulfuric acid. This would suggest a xanthone nucleus for Substance I,

Acetone was identified as a degradation product from the attack of 30^ potassium hydroxide on Substance I, It al so was formed by alkaline peroxide oxidation of Substance I, 85

The formation of acetone is good proof for a 2,2-dimethy1- chromene structure; not all give acetone under these condi tions (33) but the majority do. Frequently -hydroxyisobutyric acid can be isolated from permanganate oxidation of compounds containing this structure. This, however, could not be achieved with Substance I, The yield is always low and this failure may be due to the small amount of material employed in the reaction.

There is a strong indication, therefore, that Sub stance I is a compound which possesses a xanthone nucleus to which are attached three hydroxyl groups (one in the five position, two adjacent on a benzene ring), and a 2 ,2-dimethyl* chromene structure. This would account for a molecular for mula of The difference between this formula and that of Substance I, is C^Hg. This is an isoprene unit; a grouping of carbon and hydrogen that is found in numerous natural products.

King and co-workers (^7) have isolated and ident ified a natural product jacareubin from the heartwood of

Calonhvllum brasiliense. They assign to it the following structure; this is shown on page 68 .

This is precisely the formula mentioned above. All the requirements of structure (except the rela tive positions of the hydroxyl groups) are present in this compound. The addition of an isoprene unit would make its 86

molecular formula similar to that of Substance I. An exam

ination of the ultraviolet spectra of jacareubin reveals a

favorable comparison with that of Substance I, They are

listed in the following table.

Ultraviolet Spectra of Substance I

and Jacareubin*

Compound ^Max. % a x . % a x . Substance I 2280 26,600 2825 45,000 3375 19,500 Jacareubin 2400 12,200 2790 40,200 3340 18,200

Difference -120 /35 /35

* In 95^ ethanol.

It should be noted that the last two maxima differ by exactly /35 This is significant because it has been established (64, 65) that a shift to the right approximating

(64) R. N. Jones, Chem. Revs., 32, 37 (1943)

(65) H. Gilman, "Organic Chemistry", John Wiley

and Sons, New York, N. Y . , (1953)? Vol. 3? P« 168.

50 S. units occurs in the ultraviolet spectrum if an allylic

side chain is attached to a polynuclear compound. This would

tend to substantiate the suggestion that an isoprene unit is 87

appended to a xanthone nucleus whose structure is similar

to that of jacareubin.

A similarity between these two compounds Is also

encountered in the reductive color reactions, colors with

ferric chloride, concentrated mineral acid and alkali.

The trimethyl ether of jacareubin when oxidized in

acetone with potassium permanganate undergoes oxidation

caused by rupture at the point of unsaturation in the

chromene ring,

- OCH3

The resulting dibasic acid decomposes at 2^9°. The acid

can also be decarboxylated and demethylated by heating with

hydrobromic acid to give pale yellow needles of tetrahydroxy-

xanthone, m.p. 310° (decomp.); the ferric chloride test is

green. This same xanthone is given by fusion of jacareubin with alkali. The trimethyl ether of Substance I when ox

idized under identical conditions gives a crystalline acid without déméthylation. The neutralization equivalent (234,

24o) indicates it to be dibasic. It decomposes at 187-9°.

The action of hydrobromic acid on this compound effected,

similarly, decarboxylation and déméthylation. The product

decomposed above 300° and gave a green color with ferric 88 chloride. Fusion of Substance I with alkali gave traces of a crystalline phenolic material which melted with decom position at 272-3 °. The sequences of these degradation pro ducts bear a close resemblance. This acid has probably been formed by an attack similar to that shown on page 8 7. On the basis of a molecular formula ^22^22*^10 this acid and a consideration of degradative work and previous data it is possible to illustrate this oxidation by the follow ing equation.

H;C/r ,CO_H O ' OMe OMe

C(CHg)2

Substance II. Due to scarcity of this pigment no additional work has been done with it. However, a reconsid eration of available information has led to a structure proposal.

Looker’s (5) investigations have fairly well estab lished the molecular formula as Color tests have indicated the presence of a ^-pyrone nucleus as well as pwo acetylatable hydroxyl groups one probably "peri" to the car bonyl group of the ^-pyrone structure. The ultraviolet absorption spectra of Substance II showed maxima at 2850 89

3400 î, and 3800 A.

A difference of one oxygen appears between the mole cular formula for jacareubin and Substance II and this could be accounted for by the presence of three hydroxyl groups in jacareubin while only two are in Substance II. Following this line of reasoning, Substance II would have a formula similar to jacareubin but with one less hydroxyl group.

However, difficulty is encountered when the ultraviolet spectra is considered. Here there is a substantial varia tion that would not be apparent if there was a difference of only one hydroxyl group.

Ultraviolet Spectra of Substance II

and Jacareubin*

Compound A X A Substance II 2850 3400 3800

Jacareubin 2400 2790 3340

* In 95^ ethanol

A study (6 l) of ultraviolet absorption in the flavone series has shown that flavonol (3-hydroxyflavone) possesses a three band spectra with maxima at 2900 2 ,, 3300 £, and

4o50 A. The addition of another hydroxyl group in various positions gives compounds some of whose patterns bear a 90 striking similarity to that of Substance II. An example is

3 ‘-hydroxyf lavonol with maxima at 2800 X., 3350 X. and o 3900 A, In most cases the upper and lower maxima are shift ed toward lower wavelengths, A simple method of proof for the postulation that Substance II possesses a flavonol structure would be a study of the pattern for the completely methylated derivative. Méthylation of flavonols convert their spectra into that of the corresponding flavone; that is the three band spectra reverts to a two band spectra.

Substance III. Thompson (6) developed an improved method for the isolation of Substance I and also isolated a third pigment, Substance III, by chromatography of the ether- soluble fraction from Substance I extraction on Magnesol - / o Celite (3:1). Yellow crystals were obtained, m.p. l55-o .

The pigment is quite unstable and becomes highly colored on standing. It gave a dark green color with ferric chloride. Substance III is not a hydrate since no loss of weight resulted on heating at 80° for 6 hr. over phosphorus pentoxide.

Acétylation under mild conditions (acetic anhydride and pyridine at 0°), vigorous conditions (acetic anhydride and sodium acetate at 135°) or reductive conditions (acetic anhydride, sodium acetate and zinc dust at 130°) gave the same yellow crystalline acetate, m.p. 209-11°. Méthylation with diazomethane in ethyl ether and with alkali and di- 91 methyl sulfate gave the same product; pale yellow crystals

of the methyl ether, m.p, 150-1®, This compound gave no

test for a phenolic hydroxyl group.

Substance III acetate had a hydrogen uptake to give a crystalline compound the analysis of which indicated a tetrahydro derivative and no new hydroxyl group was form ed in this reduction.

Substance III dimethyl ether and its tetrahydro- acetate derivative each gave a positive Perkin test indica tive of a if -pyrone unit, A negative Wilson boric acid test on Substance III methyl ether indicated the absence of an hydroxyl group "peri" to the carbonyl in the if -pyrone structure, A hydroxyl group, if it were originally in this position, would not be affected by méthylation under the mild conditions employed and consequently méthylation would not have destroyed the groups necessary for a positive

Wilson test. The conclusion: there is no hydroxyl group

"peri" to the carbonyl of the ^-pyrone structure,

A negative antimony pentachloride test (7) shows the absence of a chaleone structure. The yellow flocks obtained are indicative of a flavonoid structure.

The ultraviolet absorption spectra for derivatives of Substance III are compiled in the following table. 92

Absorption Characteristics of Substance III

Methyl Ether, Acetate and Reduced Acetate

Molar Cone, Maximum Maximum Maximum Cornuound in 95 % EtOH I II III

Substance III 3 o o o Methyl Ether 9 x lO”"^ 2500 A. 2610 A. 3200 A. (inflection)

Substance III _ Acetate 2.7 x 10'^ 2390 2580 3 IIO

Substance III Tetrahydro- t Acetate 3.2 x lO"^ 2390 2580 3350

A positive periodate test for the catechol grouping in Substance III indicated the hydroxyl groups are adjacent,

Through alkaline degradation of Substance III a 2 ,4-dinitro- phenyIhydrazone was obtained which was identified as iden tical with that obtained from acetone. The formation of acetone is good proof (66) that a 2 ,2 -dimethylchromene

(66) 8 . Wawzonek in "Heterocyclic Compounds",

Vol. 2, John Wiley and Sons, New York, N. Y . , 1952, p. 329.

structure is present.

In a search for a basic nucleus to which the above information could be incorporated we were naturally led to a consideration of the following classes all of which are 93

represented by natural products.

Flavone. The absorption of flavones are represented by a characteristic two band system at wave n’ombers 3^50 S., and 40$0 A, which appear in most members of this series.

The only alteration in this rule appears on the intro duction of an hydroxyl group into the 3 position; that is the formation of a flavonol. A distinct three band system is formed at 2900 S., 3300 2., and 4-0^0 2 , This effect is noticably annuled if the 3 hydroxyl is methylated with the absorption resuming that of the parent flavone.

Acétylation, however, ir'^luences very little the spectra for flavones and flavonols. The 2 ,3-dihydro-fla vone s (flavanones) show a spectra similar to that of fla vone with bands at 3100 A. and 4-050 2.

1.4-Nanthoguinones. Compounds of this class usu ally have a well defined band at 334-0 2 , with inflexion in the 2560 2 . and 3900 to 4-600 A. region. Hydroxylation re sults in a displacement of the inflexions to higher wave length.

Anthraguinone. While monohydroxylated anthroquinones show maxima in the 26OO 2 ,, 2800 2 , and 3300 A. region the addition of another hydroxyl group adds an upper limit in the area of 4-000 to 4-300 S. Xanthone. This compound shows a maxima at 2500 2 » and 3000 f. due to the effect of ; a ..

In addition there are maxima at 2833 and 2762 X, These latter bands are absent in hydroxylated xanthone which has maxima at 2500 , 3390 and 36^0 S, Alkylation of hy droxylated xanthones lowers all bands and seems to be char acterized by the appearances of a maxima in the 2700 to

2800 A. region.

The inability to induce further acétylation in the presence of a reducing agent indicates the absence of a structure related to napthoquinone, anthraquinone or a car bonyl in a side chain. The ultraviolet absorption bands of

Substance III derivatives fail to correspond with those of flavones or flavonols. One indication that does appear cer tain is the presence of the structure:

a 0 characterized by bands at 2500 2. and 3200 A.

The addition of new analytical data has made possi ble a more thorough study and a new molecular formula has been derived whose composition corresponds closely with the observed values. The data are presented in the following 95

table.

Compound Calculated values Observed values*

Substance III C, 69 ,51; H, 6,10; C, 69,31; H, 6.12: (C19B20O5) mol, wgt,, 328 mol, wgt,,(Rast)384

Substance III C, 70,78; H, 6,74: C, 70,96 ; H, 6 ,78 ; dimethyl ether 20CH3 ,17 .4 3 ;m.w,356 OCH3 ,i7 ,55;m,w,4lO (62182^05)

Substance III C, 66,99; H, 5 .83; C, 66,80; H, 5.97; diacetate 2C-CH:», 13.12; mol. C-CH3 , 13.3 ; mol, (G23B24O7) wgt,, 412, wgt.tRast),496; OCH, less than 0,3

Tetrahydro- C, 66,34; H, 6.72, C, 66,44; H, 6 ,68 , Substance III diacetate (C23H28O7)

♦The analysis of Substance III was done by Mrs, Klotz and

that of tetrahydro-Substance III diacetate by Mr. Warfel,

Now, on the basis of the facts presented above we must include the following points in any formula which is proposed for Substance III,

1, The nucleus contains a ^-pyrone ring,

2, Two adjacent phenolic hydroxyl groups are pre sent but none is "peri" to the carbonyl in the -pyrone structure. There is no methoxyl group in the compound,

3, Two easily reduced double bonds are present,

k, A 2 ,2-dimethylchromene structure is present. 96

5. The formula is

A possible formula to which the above facts apply is :

The presence of a group as^CzCH-CHg- is indicated by infra red absorption bands at 10 and 11,2 microns. A (05^)20= group is suggested by bands at 8.57 and 8.78 microns.

Detection of the Catechol and Hydroauinope Structure in Organic Molecules

The position of one of the three hydroxyl groups in

Substance I has been established (5) on the basis of acéty lation and méthylation studies combined with certain color tests to be located in a hindered position, probably "peri" to a carbonyl group. Numerous color tests were applied to determine the relative positions of the remaining hydroxyl groups. Substance I formed a lead salt following treatment with lead acetate, indicating an ortho relationship between the two hydroxyl groups. The addition of one drop of neutral aqueous silver nitrate to an ethanolic solution of Substance I produced a red color in thirty seconds and when the test so- 97

lution was permitted to stand overnight, a silver mirror was formed. This is a positive test for the same group ing (67).

(67) B. S. Wildi, Science, 112, 188 (1951).

However, since the addition of dilute ammonium hy droxide to the deep green solution resulting from the addi tion of alcoholic ferric chloride to Substance I failed to produce a red-violet color which is a characteristic re action of most catechol groupings present as a part of a larger molecule and the reaction of Substance I with Millons reagent (mercurous nitrate in nitric acid) did not give a colored precipitate characteristic of this structure, there was a possibility that Substance I did not possess two ad jacent phenolic hydroxyl groups.

The action of periodic acid on vicinal dihydroxy groupings results in a fission of the molecule at this point, converting each hydroxyl group into a carbonyl group as il lustrated in the following series of equations (68):

(68) E, L, Jackson jja "Organic Reactions", Vol. II,

John Wiley and Sons, New York, N. Y,, 1944, p. 341. 98

I -C-OH -C-CIOfHh ' / HflCU --- — ^ ^ ^ / HpO -C-OH 5 6 -C-OH;i ^ -0—0 V HlOj / HgO / 2 0=0 ^ / H2O -»C—0

In the belief that a fission might result with ad jacent dihydric groups, if they existed, in Substance I, sodium metaperiodate solution was added to a methanol solu tion of Substance I. An unexpected crystalline precipitate formed within one-half hour. Suspecting that this precipi tate could be due to the presence of adjacent hydroxyl groups, the same procedure was applied to various other com pounds known to contain this structure and also applied to some which did not. It appears significant that only those substances with a catechol grouping in the molecule gave the identical reaction. The results are shown in the following table. 99

Action of Sodium Metaperiodate on Compounds Containing

Catechol Groupings and Hydroquinone

Compound amount used form, of ppt. color rotenone 0.00985 g. colorless osajin 0.0101 pale yellow pomiferin 0.0105 / red quercitin 0.00846 / red, then fading morin 0.00846 did not dissolve catechol 0.00275 / pale yellow Substance I 0.00985 / red Substance I trimethylether 0.0109 colorless Substance III 0.0099 / pale red hydroquinone 0.00275 / colorless blank colorless

To each of the above compounds dissolved in ten milliliters of dry methanol in the amount of 0.00025 moles was added 0.00025 moles of sodium metaperiodate. (1 ml. of

0.25M aq. solution). The crystalline material was deposited within one-half hour except in the case of hydroquinone.

Here an additional hour and one-half was required.

Under the same conditions ethylene glycol and glyc erol deposited a white amorphous precipitate immediately and with glucose, needles were deposited overnight.

A new method has, therefore, been developed to de tect the presence of ortho and para dihydroxy groups using relatively small amounts of material. It is also possible to distinguish between these two groupings.

The substance deposited did not melt at 350^, con- 100 tained iodine (potassium hydroxide-starch test) and a neg ative carbon, hydrogen content was indicated upon analysis for these elements. A solution of the material in water tested basic. These facts and a consideration of the equations on page 98 indicated the compound could be sodium iodate. A simple test was performed to confirm this belief.

The addition of acid and sodium iodide to sodium iodate pro ceeds by the following equation:

NalOj/^Nal/éHCl = ôNaCl/HIO^/^HI = 6NaCl/3H20/3l2

The liberation of iodine indicates the presence of this originally in an oxidized state; the only possibility here being the iodate form. The test was carried out and the solution turned red, consequently the crystalline deposit was undoubtedly sodium iodate. 101

SUMMARY

1. An improved procedure has been developed for the preparation of 5j7~diacetoxycoumarin. This has result ed in doubling the percentage yield obtained from the re action.

2. The yield of 5,7-dihydroxycoumarin has been in creased 8 fold through the application of a different meth od of deacetylation of 5?7-diacetoxycoumarin than that used by previous investigators.

3. A new derivative of coumarin, 5^7-dibenzyloxycoumarin has been synthesized and the roposed structure is evidenced by color tests and elemental analysis.

Numerous variations of the reaction between a

Grignard reagent and a coumarin has shown that a 2,2-dimethylchromene with hydroxyls in the 5 and 7 positions is unstable. Other methods that were used were also unsuccess ful in that no pure substance could be isolated.

5. A halogenated isoflavone, 6-methyl-8-chloroisoflavone has been prepared. This is unique in that there is no record in the literature of an isoflavone with a nuclear halogen. Color tests and elemental analysis support the proposed structure.

6. A distinct improvement has been made in the method for the synthesis of 2,4,6-1rimethoxybromobenzene by the utilization of N-bromosuccinimide as the brominating 102 agent. The yield is an improvement over the reported pre paration by another method. In addition the possibility of contamination of the product with the dibromo derivative has been eliminated,

7. The presence of a halogen diortho substituted on an aromatic nucleus has been shown to exert a profound influence in the attack of phenyl lithium on the ring. It is shown that the time of reaction necessary for satisfac tory yield is considerably less when a halogen is present.

Also, the point of attack is preferentially at the position occupied by the halogen.

8. A X-ray powder diffraction pattern of Substance I is recorded containing a greater number of lines in the pat tern than that reported previously,

9. Dilute alkali on Substance I resulted in the isolation of traces of degradation material,

10, Partial déméthylation of tetrahydro-Substance I trimethyl ether was affected by the action of alkali-dioxane mixture. Elemental analysis and color tests indicate this déméthylation occurred in the "5'* position on the nucleus.

This is preferential to any ring fission,

11, A new isomer of Substance I dimethyl ether, not identical in physical constants with the compound reported by Looker, has been prepared. The aid of elemental analy sis and color tests were used to support the proposed 103 structure,

12, Reduction of Substance I dimethyl ether pro duced a compound whose physical properties are identical with the product reported in paragraph 10

13. Alkaline peroxide oxidation of Substance I has produced a small amount of crystalline acid with the pro bable molecular formula In addition, acetone has been isolated and identified through comparison with the

2,^--dinitrophenylhydrazone of acetone. Use of the X-ray dif fraction patterns and infrared absorption spectra were made in this comparison. These are reproduced in the disserta tion, A ik. Aj> appnron^ dimorph of acetone 2,4-dinitro- phenyIhydra2onelii y -\mvtpoi'ted^ has been obtained as a product from the attack of 3>0% potassium hydroxide on Sub- stance I, It has been^characterized through X-ray diffrac tion and infrared spectroscopy.

15. A small amount of a high melting crystalline phenolic compound was also isolated in amount too small for analysis.

16. No detectable degradation was noted following the action of potassium hydroxide on Substance I dimethyl ether.

17. The action of alkaline potassium permanganate on Substance I did not reveal any o(-hydroxyisobutyric acid 104 as a degradation product thus failing to substantiate the presence of a 2 ,2-dimethylehromene ring. It is believed that the quantity requirements were not met for the suc cessful utilization of this degradation experiment.

18. The action of chromic acid in acetic acid on Substance I failed to produce any crystalline degradation material.

19. Only oxalic acid was isolated and identified from oxidation of Substance I with dilute nitric acid.

20. A substantial amount of crystalline acid was obtained through the action of potassium permanganate in acetone on Substance I trimethyl ether. Elemental analysis has shown this compound to be possibly C22H220io^2H20. The neutralization equivalent indicates a dibasic acid. A neg ative test for phenolic hydroxyl groups was obtained. This acid is not affected by the action of dilute base.

21. Déméthylation and decarboxylation of the acid can be accomplished by the use of hydrobromic acid

(dens. 1.42).

22. The action of ethylmagnesium bromide on Sub stance I trimethyl ether produced a compound whose color in sulfuric acid is indicative of a xanthydrol structure.

23. Substance III can also be degraded to produce acetone by the action of alkali in a manner similar to that applied to Substance I . 105

2^. Substance III has been completely methylated when a method was used (alkali and dimethyl sulfate at room temperature) which did not attack a hydroxyl group "peri" to the -pyrone structure in Substance I. Color tests have been applied.

25. Pomiferin trimethyl ether has been partially demethylated by the use of N-bromosuccinlmide.

26. Infrared spectra are recorded for Substance I,

Substance I trimethyl ether and Substance III dimethyl ether as well as for the fruit pigments osajin and pomiferin and their completely methylated derivatives.

27. Ultraviolet absorption spectra for completely methylated Substance III is presented.

28. Substance II has been isolated from,Substance I by fractional crystallization in ethanol.

29. Methods for the syntheses of isoflavones have been reviewed and the possibility of their application to the preparation of the fruit pigments, osajin and pomiferin considered. Exploratory investigation has eliminated three possible approaches.

30. A novel structure for Substance I has been pro posed based on consideration of previous data, evidence ob tained from degradative work and comparative studies with ultraviolet and infrared spectra.

31. Reconsideration of available information has 106 led to a structure proposal for Substance II

32. Based on additional analytical data, a molecular formula for Substance III has be rived. A study of ultraviolet data for natural prod has been combined with degradative and substituent d for the purpose of suggesting a structural formula

Substance III,

33. A method has been developed for the det of a catechol and hydroquinone structure in large mo through the use of sodium periodate. This has been ; to Substance I and Substance III as well as other coj 106

led to a structure proposal for Substance II

32, Based on additional analytical data, a new

molecular formula for Substance III has been de

rived. k study of ultraviolet data for natural products

has been combined with degradative and substituent data for the purpose of suggesting a structural formula for

Substance III.

33. A method has been developed for the detection

of a catechol and hydroquinone structure in large molecules through the use of sodium periodate. This has been applied to Substance I and Substance III as well as other compounds. 107

SUGGESTIONS FOR FURTHER STUDY

The ability of Substance I and its derivatives to resist degradation under mild alkaline conditions has proved to be a major obstacle to structure determination.

The small amount of material obtained on attack with more concentrated alkali has made this approach practically worthless. Now that a method has been found from which a degradation product can be isolated in relatively large amounts the logical extension would be a study of degrada tive attack on this material. Déméthylation and decarboxy lation resulted when the material was treated with hydrobromic acid but the conditions employed gave only a trace of crystalline product. A study of this procedure with large amounts of material would be advisable. Also, a var iation of conditions employed may lead to more satisfactory results.

It would be of interest to obtain an ultraviolet absorption pattern for the dimethyl ether of Substance II.

The results would most likely determine whether the materi al is a hydroxylated flavonol since it has been established that a definite change in pattern results for methylated flavonols. Further degradative work is hampered by lack of material.

Synthetic studies for the preparation of osajin and pomiferin have met with limited success. A number of meth- 108

ods have been examined and discarded. A novel approach is

outlined below with pertinent references at each step.

A Proposed Method for the Synthesis of Osajin.

(CH^)^C-C=GH ----- — ------=“ (CH^)gC-C=CH (69) ^ CuCl & NHxCl ^ OH (Favorski method) 01

(69) G. F. Hennion, J. J. Sheehan and D. E. Maloney,

J. Am, Chem. 80c., 2Â, 35^2 (1950).

HO (CH,)2Q-C5CH + I II *■ ^ '^O-CCCHjjjCSCH (7°) Cl H(k Np=/ 2om*ound A

(70) A. N. Pudovik, Zhur. ObscheY Khim., (J. Gen.

Chem.), 21, 1^62-71 (195D; C. A., k6, kk67e (1952).

H 3 C > ^ H^C

Compound A +CICCH 2 // ^ ^ O H 0 \zzz/ N. Compound A^

Compound A2 109

Aluminum chloride is known to catalyze the addi

tion of acetylenes to phenols (hydroxystyrene from the ac

tion of acetylene on phenol in the presence of AlCl^) as

well as the standard Friedel-Crafts reaction.

Cyclization of A^ and Ag

iMe

Na & Et formate A 1 Compound A-.A

OMe OH

Compound A-,B

Na & Et formate A, OMe

Compound AgA 110

The two isomers and Ag could undoubtedly be sepa rated chromatographically and then reacted individually with sodium and ethyl formate to cyclize into the isoflavone iso mers. The compounds A^^A and A^B could easily be distin guished from their isomer AgA since the former have hindered hydroxyl groups while the latter does not and therefore the latter could be expected to undergo such reactions as acéty lation with acetic anhydride and pyridine in the cold as well as méthylation with diazomethane in ether solvent,

A comparison of the ease of coupling with a diazon ium salt should enable one to distinguish between A^A and

A^B since the latter should react faster in this reaction.

Compound A^A could then be reacted with isopentenyl bromide and sodium which should put the desired grouping adjacent to the ring hydroxyl group.

Compound A^A / (CH^)2C = CHCHgBr — ^ Osajin Ill

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ACKNOWLEDGMENTS

The author wishes to express his appreciation to Dr. M. L. Wolfrom for his suggestion of this problem and for his helpful advice and consistent guidance during the course of the investigation,

I am indebted to Dr. A. Thompson through whose work a supply of Substance I and Substance III were made available and also for his assistance in furnishing the ultraviolet and infrared patterns for Substance III dimethyl ether.

The interest and suggestions offered by Dr. Bernard

S. Wildi are gratefully acknowledged.

I wish to express my indebtedness to The Ohio

State University for the assistantships and fellowships which enabled me to carry out this research.

Finally, I owe a special debt of gratitude to my wife without whose encouragement and assistance my work would have seemed more difficult and less meaningful. 117

AUTOBIOGRAPHY

I, Oscar Michael Windrath., was horn in Buffalo,

New York, on February 10, 1924. I received my secondary school education in the public schools of the city of

Buffalo. Mr undergraduate training was obtained at

Canisius College, from which I received the degree of

Bachelor of Science in July, 1944. From August, 1944 to

September, 1945 I was on active duty in the United States

Army. In February, 1946 I enrolled in the graduate school of the University of Detroit from which I received the degree of Master of Science in August, 194?. I was en rolled in the graduate school of the University of Buffalo for the academic year 1947-1948, For the years 1949-1951

I was an instructor in Chemistry at Niagara University.

In June, 1951, I enrolled in the graduate school of The

Ohio State University. While completing requirements for the degree Doctor of Philosophy I held the position of graduate assistant during the year 1951-1952 and was a recipient of a Monsanto Fellowship for the year 1951-1953.

Since September, 1953 I have been a member of the Depart ment ol Chemistry at Niagara University.