This dissertation has been 64-2659microfilmed exactly as received

WERNY, Frank, 1936-THE ALKAWIDS OF PLATYDESMACAMPANULATA MANN.

University of Hawaii, PhoD., 1963Chemistry, organic

University Microfilms, Inc., Ann Arbor, Michigan

THE ALKALOIDS OF PLATXDESMA CAMPANULATA

MANN

A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF THE

UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

JULY 1962

by Frank Werny

THESIS COMMITTEE

Paul J. Scheuer, Chairman

Har old O. Lar son

Albert H. Banner

John J. Naughton

Kerry T. Yasunobu

To

Inez and Mark

List of Figures

List of Tables

TABLE OF CONTENTS

v

vii

Chapter I. Introduction

A. Botanical 1

B. Chemical 2

Chapter II. Experimental

A. Procurement of Plant Material 6

B. Isolation of Alkaloids 7

1. Root and Bark (KauaH 7

2 0 Root and Bark (H awaii) 11

3. Leaves (all collections) 17

C. Characterization of the Alkaloids 21

1. The Mixture of the Bases A and B 21

2. Base B 21

3. Base C 27

4. Base D 30

5. Base E 32

6. Base F 37

D. Syntheses 43

Chapter III. Results and Discussion

A. Base B 54

B. Ba se C 57

C. Base A 58

D. Base D 60

TABLE OF CONTENTS (continued)

E. Base E

F. BaseF

Chapter IV. Conclusion and Summary

Bibliography

Acknowledgements

iv

65

67

73

77

86

Fig. 2"

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 1 0

Flg. 8.

Fig. 9.

Fig. 100

Fig. 11.

Fig. 12.

Fig. 13.

v

LIST OF FIGURES

Scheme for extraction of bark and root wood

(Kauai> • 8

Scheme for extraction of bark and root wood

(Hawaii). 12

Scheme for extraction of leaves. 18

Ultraviolet spectrum of base B in 95% ethanol. 23

Infrared spectra of base B (upper) and base C

<lower) (Chloroform). 24

Ultraviolet spectrum of 6-methoxyisodictamnine. 26

Infrared spectra of the iso-compound of base B

(upper) and 6-methoxyi sodictamnine (lower)

(C hI orof orm) • 28

Ultraviolet spectrum of kokusaginine in 95%

ethanol. 29

Ultraviolet spectra of base D. 31

Infrared spectra of base D (upper) and base F

<lower) (Chloroform).. 33

Ultraviolet spectra of base E. 35

Infrared spectra of base E (upper) ~iid 1,2,3-

trimethyl-4-quinolone (lower) (Chloroform). 36

Nuclear magnetic resonance spectra of base E

(upper) and 1,2,3-trimethyl-4-quinolone

(lower). 3e

Fig. 15.

Fig. 16.

vi

LIST OF FIGURES (continued)

Infrared spectra of base F picrate (upper) and

the picrate of the oxidation product of base F

<lower) (KBr). 39

Ultraviolet spectra of base F. 41

Ultraviolet spectra of 1.2,3-trimethyl-4-quino-

lone. 47

Fig. 17. Ultraviolet spectrum of maculosidine in 95%

ethanol. 56

Fig. 18. Ultraviolet spectra of dihydrodictamnine. 61

Table I.

Table II.

Table 1110

LIST OF TABLES

Results of Work-up

Chromatography of the Mixture of the

Bases A and B

The Alkaloids of Platydesma eampanulata

Mann

vii

16

21

5

CHAPTER I

INTRODUCTION

A. Botanical

Platydesma campanulata MannI is a member of the

plant f~mily Rutaceae, which is a prominent contributor to

the flora of the warmer regions of the earth. 2 Three endemic

genera of this family are found in the Hawaiian Islands.

They are Platydesma, PeIea, and Fagara, all of which are

classified by Engler and Prantl 3 in the Xanthoxylae group of

the subfamily Rutoideae. In this group Platydesma is placed

between the Mexican genus Choisya and the New-Caledonia ge­

nus Dutaillyea. 3 Stone,l however, considers Platydesma most

closely related to Medlcosma, which is an Australian genus.

If Stone is correct, the origin of Platydesma is in the Old

World. A study of the alkaloids occurring in Platydesma may

shed some light on this pOint since the alkaloids of Medicos­

ma4 and Choisya 5 have been investigated. A comparison of the

alkaloids found in these two genera with those to be isolated

from Platydesma may point to a relationship of Platydesma

with Medicosma or Choisya.

Platydesma campanulata is usually found as a small

tree in the rain forests of the Hawaiian Islands. It occurs

most commonly at an elevation of 2000-5000 feet but is never

R frequent member of the vegetation. Its most outstanding

visible feature are the large lush leaves which may be as

wide as twenty centimeters and 8S long as fifty centimeters. l

2

When crushed, these leaves emit an odor of essential oils and

the bark emits 8 semeniferous odor. Cuttings of the plant

when left to dry in the laboratory were found to be strong

attractants for the male Oriental fruitfly, Dacus dQrsalis

Hendel.

The Hawaiian name of Platldesma campanpJata is

Pilo kea,6 but the plant is apparently not mentioned in the

sparse literature on medicinal uses of Hawaiian plants.

B. Chemical

Of the approximately 1300 species of the Rutaceae

fewer than twenty percent have been investigated for the

presence or absence of alkaloids. According to a survey

published in 1955,1 one hundred and seventy-three species

had been examined; of these. seventy-four gave a positive

test for alkaloids while ninty-nine gave a negative test.

By 1959 the number of rutaceous species in whicb alkaloids

bad been detected bad risen to one hundred and eighty-one. 8

On the basis of these surveys it would seem that the family

Rutaceae is a moderately promising source of alkaloids.

Normally, a given plant family will produce alka­

loids of a certain structural type. For example, alkaloids

which have been isolated from Apocynaceae are structurally

related to indole (I) while those from Papaveraceae are re­

lated to isoquinoline (II).

3

The behavior of the Rutaceae is in sharp contrast

to this norm. Among the molecular species which have been

isolated are evodiamine (indole type, I), berberine (iso­

quinoline type, II), melicopine (acridone type, III), dic­

tamnine (furoquinoline type, IV), and arborine (quinazolone

type, V). It therefore was of interest to investigate an

Hawaiian representative of the family.

I II

oI I

III

IV

o

(JC111

N-H

~ JN

v

In a preliminary survey the presence of alkaloids

was detected in all three Hawaiian genera. 9 One species of

the widely distributed genus Fegara has been investigated

more closely.lO The genus Platydesmo was chosen for closer

scrutiny because it is wholly endemic to the Hawaiian Islands

4

and the species campaDU)ata W as selected since it promised

a reasonable supply of raw msterial o

Some genera which are closely related to Platydes-

~ have been found to contain alkaloids. A species of ~­

cosma yielded medicosmine (VI) ,4 and a species of ChoiSY8

yielded skimmianine (VII), evoxine (VIII), and an alkaloid

(C1<YI210SN) of undetermined structure. 5

CHP

)

VI VII

~{ ifCH3

o

C.H P3 VIII

All three alkaloids are elaborations of the dictamnine skele-

ton (IX) and PJatydesma might possibly yield alkaloids which

are related to dictamnine. About a dozen other substituted

dictamnines have been isolated from Rutaceous plants. li

5

Furoquinolines constitute perhaps the most characteristic

group of alkaloids isolated from Rutaceae.

IX

Dictamnine itself is physiologically active

against urogenital diseases l2 and has pharmacochemical pro­

perties. 13 Derivatives of this base may therefore be reason­

ably expected to exhibit physiological activity.

An investigation of the alkaloids of Plat1desma

campanulata is tberefore of interest to the chemist because

of the variety of alkaloid types which occur in Rutaceaei to

the botanist because chemical knowledge may assist him in

~ttP.~pts to correlate chemical structure of plant constitu­

ents with plant morphologyl4 and taxonomYi 15 and to the

pharmacologist because PlatJdesmg campapulata may produce

physiologically active alkaloids. 16

The object of this research was to isolate pure

alkaloids from PJat,desmg campapulata and to determine mole­

cular structures of the principal constituents.

,. ............"Ptl ......~nArlr..n J.J.

EXPERIMENTAL

All melting points were determined on 8 Fisher-

Johns melting point apparatus and are uncorrected.

Elemental and functional group analyses were per-

formed by Dr. A. Bernhardt, Mulbeim, Germany.

Infrared absorption spectra were obtained with 8

Beckmann IR-5 double beam instrument either in chloroform

solution or as potassium bromide pellets. Ultraviolet ab-

sorption spectra were obtained on a Beckmann DK 2 spectro-

photometer.

The N.M.R. Spectra were measured by Dr. Leon

Mandell, Emory University, Atlanta, Georgia and a mass spec­

trum was determined by Dr. Klaus Biemann, Massachusetts In-

stitute of Technology, Cambridge, Massachusetts.

Alumina Woelm of activity grade I was used and all

other adsorbents were used as supplied by tbe manufacturers.

Alkaloid tests were considered positive, if Mayer's

and Dragendorff's reagents gave a precipitate. 19

A. Procurement and Preparation of Plant Material

Plant material for a preliminary investigation was

collected in the Koolau Range on OahU, mostly along the

Ridge Trail in the Pupukea area. Material for the major

On Kauai a sufficient supply of ~. campapuJata

1

was found in the Kokee area sOuth of the Ranger Station.

Taxonomic identification was made by Dr. B. Co Stone. On

Hawaii £. eampanulata was collected in the Kohala mountains

south of Hawi and taxonomic identity was confirmed by Mr. I.

Eo Lane.

All plant material was prepared for extraction by

drying in a forced draft oven for 48 br. at 60 0 , followed by

grinding in a Wiley mill to pass 16 mesh.

Bo Isolation of Alkaloids

The alkaloids were isolated by conventional

metbods.17, 18, 19

The bark and whole root from Kauai e that from

Hawaii and the combined leaves, were worked up separately.

For eacb of the three work-ups a different scheme was used

(Figs. 1,2,3,). An attempt was made to correct shortcomings

of a scheme during a subsequent work-up.

I. Root and Bark (Kaua!)

The entire extraction scheme is summarized in a

flowsbeet (Fig. 1).

Dried and ground stem bark <6.4 kg.) and whole

root \4.5 kg.) was extracted with hexane under reflux for 36

hr. Tbe bexane extract gave a positive alkaloid test, but

attempts to isolate alkaloids failed.

Tbe plant material was next extracted with reflux-

8

E,~tlVlt1,)l Extrlli·-T------- -----

AqlWOUS 'rartaric Acid- -------~/~---

9~1l5~ Aqueous Solution(discarded) ~

Chloroformr-------.-----"

,/ "

Hood

Aqueon §u.091 i It ·1! ,! ':(di scard <'}' ~ )

Aci~

~tanol Solution(discarded')--

Chloroform Extract

\Florisil m1romatography

I

,tract

~ChlQrQfQr~

Aqueous Solution(discarded)

Bases A, B, and G

Fig o 1'. Scheme for extraction of bark and ro&t wood (Kauai)~

9

Ing methanol for 48 hr. The methanolic extract was concen­

trated to 4 1. in a steam-jacketed vacuum evaporator. To

the concentrated methanolic extract 4 1. of 5% aqueous tar­

taric acid was added and the mixture was filtered using

Kenite filter aid and sand. The resulting solid was washed

with 5% tartaric acid, followed by 5% HCl. The washings

were combined with the original filtrate. This combined

aqueous acidic solution was then extracted with chloroform

in a continuous liquid-liquid extractor for 24 hr. Removal

of the chloroform in a rotary evaporator under water pump

vacuum yielded 119 g. of brown oil. Further extractions of

the aqueous fraction at pH 1 and pH 10 yielded solutions

giving positive alkaloid tests, but subsequeot chromatogra­

phy did not yield crystalline alkaloids. The brown oil was

now taken up in 1 1. of butanol and extracted with 20 X 100

mI. portions of 5% HCl. The acidic extract was again extrac­

ted continuously with chloroform for 48 hr. Upon concentra­

tion of the chloroform extract a brown oil, ~. 12 g., was

obtained.

This oil was dissolved in benzene and chromatograph­

ed in a column containing 500 g. of Florisil. Thirty frac­

tions were collected upon successive elution with the follow­

ing solvents: 2 1. of benzene, 1 1. of 1:1 chloroform-ben­

zene, 1 1. of chloroform, 1 1. of 1:1 chloroform-acetone, 1

1. of acetone, 2.5 1. of methanol. Only fractions 1-14 show­

ed promising spots on ascending paper chromatograms using

ethyl a cetate. pyridine, and water in the ratio 1.5 : 2.3

: 1.65 as developing 80lution. 20 These 14 fractions were

therefore combined and rechromatographed in a column con-

taining 100 g. of Florisil. Elution was started with 300

mI. of benzene and continued as follows: 200 mi. of 3:1

benzene-chloroform, 100 mI. of 1:1 benzene-chloroform. 300

mI. of chloroform, 200 mI. of 1:1 chloroform-acetone. 200

mI. of acetone. and 500 mI. of methanol. Twenty-two frac­

tions were collected. The first 6 fractions gave a positive

alkaloid test, and fractions 4, 5, and 6 crystallized.

Fractions 1-3 were extracted with hot petroleum ether (bop.

30_600). UPon cooling the combined extracts 30 mg. of a

crystalline substance, m.p. 115-116°0 was isolated.

After extraction with hot petroleum ether the

fractions 1-3 were taken up in hot benzene. Upon addition

of petroleum ether (b.p. 30-60°) to cloudiness and subse­

quent cooling slightly yellow rosettes crystallized. Those

from fractions 1 and 2 melted at 124-128° and those from

fraction 3 melted at 134-135°.

Thin-layer chromatography on aluminum oxide G in

1:1 benzene-chloroform of the 3 crystalline fractions thus

obtained showed that the crystals melting at 134-135 0 were a

pure base, labelled B. Those melting at 115-116 0 and those

meltin~ at 124-1280 a~~eared to be mfYtuT~~ of two ~!k~!9!~~

base B and a base, labelled Ao

Altogether 252 mg. of chromatographically pure

11

base B was obtained from fraction 3 and by recrystallization

of the mixed crystals from benzene-petroleum ether (b.p.

30-60°). Attempts to obtain pure base A from the mixture

of the two bases failed.

Trituration of fractions 4-6 with absolute ethanol

yielded 18 mg o of a white crystalline solidi mQpO 164-167 09

which was labelled base Ce

2. Root and Bark (Hawaii)

The entire extraction scheme is summarized in a

flowsheet (Fig. 2)0

A total of 12 kg. of stem bark and whole root was

extracted with refluxing methanol for 48 hr. The extract was

concentrated to 4<1., to whicb 7.6 1. of 5% HCI was added.

A .olid precipitated whicb was filtered off and repeatedly

extracted with 5%. 10%, and 20% HCl in succession. The a-

cidic extracts were combined with the acidic filtrate and

the solid was discarded.

The aqueous acidic solution was neutralized witb

cooling using concentrated ammonium hydroxide and then ex-

tracted with chloroform for 24 hr. in a liquid-liquid ex-

tractor. The chloroform solution was evaporated to near-

dryness in a rotary evaporator under water pump vacuum and

then triturated with etber. The original chloroform extract

~h"~ ~~n~TA~~d into An ~~hpT_~nl"hl~ 'TA~tfnn_ wpinhfnn------ --r------ ---- -- ------ --.----- ---- -- --. ·--·v·----~

190 g., and an ether-Insoluble fraction, weighing 158 g.

12

Bark and Root WoodI

Methanol

Methanol Extract Bark and Root WoodI (discarded)

2-20% Hydrochloric Acid

Solid / ~Cid:f.C Solution(discarded) :

Ammonium Hydroxide to pH 6I

Chloroform

Aqueous Solution /" 'ahloroform Extract(discarded) I

Ether

Ether Soluble Ether rnso uble

5-10% HYdrodhloric Acid 5% HYdrOC~loric AcidI I

Ammonium Hydroxide to pH 6 ChloroformI I

Chloroform 80% Methanol

./ '" IChloroform Aqueous Solution CarbonEXtract (discarded) /\Tetrachlorider '\

Chromatoeraphy 80 '0 Hethanol Solution Carbon Tetra-I discarde chloride Solution

Bases A, B, C IChromatography

IBases A, B, C, D, E

Fig. 2. Scheme for extraction of bark and root wood (Hawaii)o

13

a. The Ether-Soluble Fraction

The ether was removed on a rotary evaporator under

water pump vacuum and the residual oil was taken up in I 1.

of benzene and subsequently extracted, first with 1 I. of

5% HCl and then with 1 10 of 10% HCI. The acidic extracts

were brought to pH 6 with solid sodium bicarbonate and ex­

tracted w!tb chloroform for 24 hr~ About 30 go of a viscous

oil remained after distillation of the chloroform on a rota­

ry evaporator under water pump vacuum. The viscous oil was

dissolved in 50 mI. of benzene and chromatographed in a

column containing 500 g. of Florisil. Elution was started

with benzene, gradually changed to chloroform. then to

ethanol and finally to methanol. Fractions of 25 mI. each

were collected with an automatic fraction collector. The

first 50 fractions gave positive alkaloid tests. Thin-layer

chromatography on aluminum oxide G showed that the 50 frac­

tions were resolved poorly. The fractions were therefore

combined, dissolved in 2:1 benzene-carbon tetrachloride and

rechromatographed on basic alumina (Woelm). The column was

eluted with the following solvents: 1 1. of 2:1 benzene­

carbon tetrachloride, I 1. of benzene, 1 I. of 9:1 benzene­

chloroform, 2 1. of 4:1 benzene-chloroform, 2.5 1. of 1:1

benzene-chloroform, 1 1. of chloroform, 1 I. of ethyl ace-

25 mI. each were collected. Only fractions 150-299 gave a

positive alkaloid test. These were combined in benzene and

14

chromatographed on 100 g. of silica gel Ge Elution with

chloroform-benzene mixtures (1:9. 1:4. 2:3, 1:1, 3:1)

brought down several crystalline fractions. Fractions 1090

124 of the silica gel G column could be shown by thin-layer

chromatography (1:1 benzene-chloroform on aluminum oxide G)

to contain alkaloids with Rf ~alues of bases Ao Bo and Co

They were again combined and ehromatographed in a column

containing 50 g. of silica gel G. The alkaloids were eluted

with 1:9 chloroform-benzene followed by 1:6 chloroform-ben­

zene. Fractions 31-62 and 87-110 contained the alkaloidso

Fractions 87-110 were combined and recrystallized from etha~

nol to yield 67 mg. of base C, m.p. 169-169.5 0 •

Bot petroleum ether extracted 27.7 mg. of base B

from fractions 31-62. Upon recrystallization from bot pe­

troleum ether, it melted at 134-135°. An examination of the

mother liquors by thin-layer chromatography showed the pre­

sence of bases A and B. Attempts to separate the two were

unsuccessful.

b. The Ether-Insoluble Fraction

The ether-insoluble oil was mixed with 4 lbs. of

sand, filled into a column and extracted in succession with

3 1. of 5% BCI, 2 1. of 10% BCl, and 4 I. of 20% BCl. The

e~t~~~t5 ~!~~ ~~utTAlized with solid sodium bicarbonate,

combined, and extracted with chloroform for 24 hr. The

chloroform was distilled off and the residual oil was taken

15

up in 80% methanol which was extracted with carbon tetra­

chloride for 3 days. The methanol solution was evaporated

to near-dryness and triturated with benzene. Upon evapora­

tion of the solvent the combined carbon tetrachloride and

benzene fractions gave 12 g. of a viscous brown oil. The

oil was separated into two fractions& one which was eluted

from 500 g. aluminum oxide G with chloroform, and one which

was eluted with ethanol. The ethanol eluate yielded no al··

kaloids. The chloroform eluate was dissolved in benzene and

applied to a column containing 150 g. of silica gel Go The

alkaloids were eluted with 500 mI. of 3:1 benzene-chloroform,

I 1. of 7:3 benzene-chloroform, 500 mI. of 3:2 benzenechlo­

roform, 500 mI. of 1:1 benzene-chloroform, 500 mI. of 1:3

benzene-chloroform, 1 1. of chloroform, and I 1. of 5% me­

thanol in chloroform. Three hundred and twenty-five frac­

tions were collected. Fractions 1-38 and 274-325 contained

no alkaloids. It could be shown be thin-layer chromatography

(aluminum oxide G in chloroform) that fractions 39-130 con-

"tained non-alkaloidal substances and an alkaloid of Rf 0.6­

0.7. Similarly, fractions 269-273 could be shown to contain

an alkaloid with Rf 0.2. In order to purify the fractions

picrates of the alkaloidal components were prepared by dis­

solving the fractions in a minimum amount of hot ethanol and

then adding 1-2 mI. of concentrated picric acid solution in

methanol. The results of this work-up are summarized in

table I.

16

Table I. Results of Work-up

Fraction Melting Weight of M.P. after After 2ndPoint Picrate 1st recryst. recryst.

from EtOH

1-38

39-65 160-1700 48 mge Dot cryst.

66-69 172-1760 39 mg. 177...1600 193-195°(dec. )

70-89 171-172 0 91 mg. 185-185.50

90-130 205-207° 54 mg. ""

131-210

211-216 92-96 0 168 mg. 98-99 0 107-1090

217-268

269-273 234-245 0 105 mg. 234-235 0

(dec. ) (dee.)274-325

By thin-layer chromatography, comparison of ultra­

violet and infrared absorption spectra, and melting points

it was established that 1) the picrates from fractions 39-65

and 66-69 were mixtures of the picrates of th~ bases A and B

isolated previously; 2) the picrate from fractions 70-89 was

a mixture of picrates of previously found bases A, B, and C;

3) the picrate from fractions 90-130 was identical with the

picrate of base C; 4) the picrate from fractions 211-216 was

the picrate of a new base. labelled D; and 5) the picrate

from fractions 269-273 was the picrate of another new base,

labelled E.

17

3. The Leaves

The entire extraction scheme is summarized in a

flowsheet (Fig. 3).

A total of 4Q 8 kg. of dried and milled leaves was

extracted with refluxing methanol for 48 hr. The methaDolic

extract was concentrated to a volume of 4 1. OD a steam-jac­

keted vacuum ev,porator. Four liters of 5% HCI was added to

the concentrate. The resulting oily precipitate was fil­

tered and washed with 2 I. of 5% HCI. The combined acidic

extracts had a pH of 2 and were extracted with chloroform for

48 hr. to yield 175 g. of 8 viscous brown oil upon evapora­

tion of the chloroform. The aqueous solution was now adjus­

ted to pH 8 with concentrated ammonium hydroxide and again

extracted with chloroform for 48 hr. to yield ~O g. of a

brown oil upon evaporation of the chloroform. The pH of the

aqueous solution was now raised to 13-14 with solid sodium

hydroxide and once more extracted with chloroform for 48 hr.

Upon evaporation of the solvent 30 g. of dark brown oil was

obtained.

Bo The pH 2 Extract

The oil (175 g.) was taken up in 300 mle of metha­

nol and 1200 mI. of 5% HCl was added. An oily precipitate

formed which was filtered off on a Buehner funnel. The BciG­

ic filtrate was extracted with chloroform for 48 hr. Evapo­

ration of the chloroform gave 35 g. of a brown oil. This

18

Base C

Leaves

THethjOl

5~roCDloriCAcid.~

Dark Oil Aqueous Solution(discarded) (pH 2)

~./ 1 r ,orm"'-,

Aqueous Chloroform

~ ~tAmmonium Hydroxide ChrQmatQgranhY~. I

7'Aqueous Solution ChlQroform Extract

I rSodium Hydroxide Chromatography

~ IBase E

Aqueous Solution(di scarded)

Chloroform Extract

IChromatography. I

Bases E, F

Fig. 3. Scheme for ext~action of leaves.

19

oii was now dissolved in a minimum amount of chloroform and

cbromatographed in a column containing 500 go of basic alu­

mina (Woelm). Elution was started with chloroform and grad­

ually changed to ethanol. Only early fractions which were

eluted with pure chloroform gave positive alkaloid test.

Upon thin-layer chromatography of these fractions on alumi­

num oxide G in 1:1 benzene-chloroform only one alkaloidal

spot was noticed. The alkaloidal fractions were therefore

combined, dissolved in 100 mI. of ethanol, and 1 g. of pic­

ric acid was added. The mixture was heated to boiling.

Upon cooling, a non-crystalline yellow picrate was collecte~

Crystallization from ethanol yielded 11 mg. of fine yellow

needles, melting 205-207.5 0 • Its infrared absorption spec­

trum and a melting point showed this picrate to be identical

with the picrate of base C isolated from the root and bark.

b. The pH 8 Extract

The oil (20 g.) was dissolved in 50 mI. of chloro­

form and chromatographed on 600 g. of basic alumina (Woelm).

Fractions 1-58 resulted from elution with pure chloroform.

Fractions 59-160 were eluted with 20% ethanol in chloroform,

and the remainder with ethanol. Only fractions 14-14 gave

alkaloidal spots on a thin-layer chromatogram. These frac­

tions were therefore combined and dissolved in 50 mI. of 95%

ethanol and 5 mI. of concentrated methanolic picric acid was

added. The solution was hrougni iu in~ uulliny puiut aucl

20

then slowly evaporated to drynesso Upon trituration with

acetone. a yellow picLate &emained. Reery~talli6ation irom

ethanol yielded 145.5 mg. of yellow needleso The infrared

absorption spectrum and a mixture melting point showed this

base to be identical with base Eo which had been isolated

from the root and bark.

Co The pH 14 Extract

The oil (30 g.) was dissolved in 50 mI. of cbloro-

from and chromatographed in a column containing 500 g. of

basie alumina (Woelm). Chloroform eluted two bands. MOre

polar solvents eluted no further alkaloids. The fractions

eluted with chloroform were combined and dissolved in ben-

zene. The benzene solution was chromatographed in a column

containing 250 g. of aluminum oxide G. Elution was started

with benzene, continued with 1:1 benzene-chloroform, and

then with chloroform. The fractions eluted with the 1:1

benzene-chloroform sbowed one alkaloidal spot on a thin-

layer chromatogram. They were combined in chloroform and

about 4 mI. of concentrated methanolic picric acid was added.

The solution was brought to the boiling point and then

cooled resulting in precipitation of a yellow picrate. By

fractional crystallization from ethanol the picrate could

be separated into the picrates of tbe previously found base

E and a new base F. Base F picrate began to soften at 195 0

and decomposed from 203-216 0 • There was 71 mg. of this pic

21

Co Characterization of the alkaloids

1. The Mixture of the Bases A and B

The mixture of the bases A and B which was obtain-

ed from both extractions of root and bark was chromatogra-

phed on a thin layer of aluminum oxide G in 1:1 benzene-

chloroform concurrently with pure base B and an authentic

sample of evolitrine. 21 Development with modified Dragen­

dorff's reagent 22 yielded results which are summarized in

Table II.

Table II. Chromatography of the Mixture ofThe Bases A and B

Pure Authentic MixtureBase B EvoU tri ne Spot 1 Spot 2

Hf Value 0.58 0.61 0.58 0.61

Color Dark Purple Tan Dark Purple Tan

Since attempts to separate bases A and B failed, base A

could not be further characterized.

2. Ba se B

Base B could be recrystallized by dissolving it

in a minimum amount of benzene, adding petroleum ether to

appearance of cloudiness, and subsequent cooling. It sub­

limed readily at 107°/3 X 10-2 mm. The analytically pure

base melted 134-1350. It was very sQl~hle fa ~hluruiorm.

alcohol. benzene and carbon tetrachloride; sparingly soluble

22

in acetone and petroleum ether (b.p. 30-600); and insoluble

in water. and aqueous base, but it did dissolve slowly in 5%

HCI. The base was chromatographically pure. Tbe Rf values

on aluminum oxide G were 0.58 in 1:1 benzene-chloroform and

0.44 in 3:1 benzene-chloroform.

Anal. Calcd. for C13HII03N: C. 68.11; He 4.84;

2 OCRa• 27.08. Found: C. 68.37. 68.24; H. 5006. 40 86;

OCH3. 25.77.

The ultra~!o!et absorption spectrum (Figo 4) in

4.4 X 10-5 M 95% ethanol solution showed the following max­

ima and minima: ). max. 350 mf<-<Iog €. 4.68). 333 (4.75),

307.2 (5.04), 295 0 5 (4.99) 284 sh (4.81), 260.7 (4.91).

248.8 (6.oll, 238 (6.10); Amin. 342 (4.63), 324.2 (4.68) t

267 (4.62), 220 (5.85).

The major peaks in the infrared absorption spec­

trum (Fig. 5) occurred at the following wavelengths:\ chloroformA maxo 3.40~(m). 6.18 (s), 6.33 (s), 6.49 (m), 6.64

(s). 6.83 (s). 7.07 (m). 7.23 (s). 7.67 (s)o 7.92 (m).

8.11 (s), 8 0 23 (S). 8.67 (s)o 9.01 (8),9$15 (S). 9.68 em).

10.10 (m). 10.20 (m), 11.78 (w). 12.06 (m), 14.20 (w).

A picrate of base B was prepared by dissolving 4.5

mg. of the b~se in 1 mI. of absolute ethanol. Upon addition

of 2 drops of concentrated methanolic picric acid. the pic­

rate precipitated immediately as fine yellow needles. Re­

crystallization from ethanol gave fine needles melting with

decomposition at 195-196°.

6.0

5.0 -

3.0

23

200 240 280 320 360 l.}-OO

vIAVELENGTH. (mfA-) 0

Fig. 4.-Ultraviolet spectrum of base B in 95% ethanol"

~;n""l • '. C.. rLd'>h~: r=i 'd':i~"~lI '('.i I 1......-:=.--,!,1 ~_l.: j./ 'I f·: t. . \~ r .-; I

. _ 1 I iJtt=! 0 ! l-

.IUHl\---lilJlI- ILlr\l!,,--I'\I-:-j~ I ::--1- r: _I I I :- t -I ' !"'. i - , - - ! i, 1-1'

! r

j

".;: ~ ",':~·~ .."l::·~:~-=!J• .1. L_

nco . _~TII·FI4·I.Ftiiq·i-tF-i ,It-

J I I I I I ,_..~~~;" MOO'> J ! iii I i_.... ..... _. __ •••••__.' k'._ •••

I~ ,..."0 .-.- ''I'''''''' II.:" .... r.=:J

l_jIi~J 1:~~~..J;;i~;!~~;·':r _ i

Fig. 5. Infrared spectra of base B (upper and base C (lower)(Chloroform) •

I\:)

~

25

The Iso Compound. - Base B (112 mg.) was sealed in

a Pyrex tube with 2 mI. of methyl iodide and left overnight

at 100°. After opening of the tube the solvent was evapora-

ted and the residue extracted with three 5 mI. portions of

chloroform. The extract was concentrated to 5 mI. in a

stream of nitrogen and applied to a column containing 10 g.

of basic Alumina (Woelm). Three successive colored frae-

tions could be eluted with chloroform gave a crystallln~

residue, m.p. 206-2090• The residue Nas recrystallized by

dissolving it in a minimum amount of cold absolute ethanol,

warming the solution, and then adding water until the 50lu-

tion appeared cloudy. Colorless crystals separated over-

night in the refrigerator. This compound melted from 214-

216°. The crystals slowly turned p.ink on standing.

Anal. Caled. for CI3HII03N: C, 66.11i H, 4.84.

Found: C, 67.82i H, 4.82.

Upon melting, cooling, and remelting the iso-com­

pound melted from 208-2100. An authentic sample of 6-metho­

xyisodictamnine23 melted at 211.5-212.5°. Upon cooling and

remelting it melted from 208-210 0 • Two samples when mixed

by fusion followed by cooling, had on remelting, a melting

point of 208~210.5°.

The ultraviolet absorption spectrum (Fig. 6) in

4.39 X 10-5 M 95% ethanol solution showed the following max-\

Ima and mi nima: 1\ 361 M)4 (log E 3.66), 344 (3.64), 329max.

(3.38>, 302 (3.07), 290 (3.19), 264.5 (4.15), 255 (4.03),

26

5.0

3.. 0

\i'--------'------'------'---------'---------,

200 240 280 320 360

~'rAV2LEIrGTH (m}-4) 0

F'ig o 6 o -Ultraviolet spectrum of 6-methoJCY1sodictaJTIl1ine in

95% ethanol o

21

243 (4.11), 216.5 (3.91); Amin. 350 (3.53), 310 (2 0 88),

260 (3.99), 250 (3.94), 225 (3.86).

The infrared absorption spectrum (Fig. 7) gave the

following peaks: Achloroform 3.39 fA- (m) , 6.11 (s) , 6 0 28 (s) ,max.

6 0 49 (s) , 6.63 (s) , 6.80 (m) • 6 0 98 (m) ; 1.42 (w) • 1.65 (s) I

8,,08 (s) Il 8,,31 (m) Il 8 0 44 (m) 0 8,,60 (m) 0 8 0 84 (w) 0 9 0 02 (m) 0

9 0 63 (w) 0 9 0 88 (w) t 11 .. 12 (w) t 11.34 (w) " 12.32 (w) •

3. Base C

Recrystallization of base C from absolute ethanol

gave needles melting 169-169.50 • The base was found to be

soluble in chloroform, ethanol, and methanol. It was slight­

ly soluble in benzene, ether, and 5% BCl. It was insoluble

in water, aqueous base, and petroleum ether.

Anal. Calcd. for C12H130 4 N: C, 64.36; H, 5.05,.

Found: C, 65.12, 64 0 86; H, 5.39, 5.20.

The ultraviolet absorption spectrum (Fig. 8) in

3.86 X 10- 5 M 95% ethanol showed the follOWing maxima and mi~

ima: Amax. 334 m)L <log E 3.53), 323 (3.64), 352 (4.23), 246

(4.11); A i 315 (3.60),263 (2.71>.m D.

The infrared absorption spectrum (Fig. 5) in 0.13

M chloroform showed the follOWing peaks: Amax • 3.36?-(m),

3.52 (w), 6.13 (m), 6.43 (w), 6.62 (5).6.11 (5),6.81 (m),

6.90 (m), 1.53 (s), 1 0 91 (s), 8.38 (w), 8.50 (S). 8.60 (S)II

8.12 (m), 9.09 (S), 9.46 (m) 0 9.60 (w) , 9.82 (s>, 10.04 \mJ.

10.52 (m), 11.63 (m), 14.22 (w), 15.13 (w).

!r

}.:;oc .or...o ~"";, 1i'IC! I:')'; IS"'" I.::', 1,:"J :::-1 .:~

~[T~~I~_=f~m,~~~rr~"1~;i~['~'~Ar~ED'".'1':1r 'i='f'" ,,~~f7~:',: '"-+,c~t~";',~l:', I~±L:,: JI:V1

Y+}J~h, c:oHt:\L~'f"'; ·L-'/;' " . ; ",)

:llE~~l ~C~:·~~ l~~ ~I-~ I~~C~'~~ :~"}:i~.r-k-:-:~= H; ~-_ ..

~;~~ ~t,r-~~ ,{:~¥-r1=~~~i:~,b;l~:r-", F'-'~i

;~~r~~~;~fH:c-;: ~'h+~~. !

.:-; ~~~l..- :~,;.~ ~-~t~ i ~_:-_-; ~~:~~k~

:~'::ct,F"U,"iO'h:::~d· O"d~£d·; ~'I· 0:01 ,::-,01 >:qc~' r', L~h-=r:U'-::-' Lo':"-j:- •L:'-.l-----'~L., i e., '-J.:...'--, __.....L-_<__ ,

J j iii j i .4m~o+ .'a~ 'f 'f 'f 'l=J..1 I

WAVD«J,oomc..-·

1'COO IJOe) !I:l') 1)00 1100 1100 1('(.'0 900 100 1n

~~_.~.~--tugp.1rrJP:fJ!~n~t:~~ljlp:~-~f_!:t;FI': 1~;t_-fF; =:t,.~iVfl~r:-'r~qy'Ji;:t Hyrfl;~}fP:!tHUf~t f r~-tJil;~:~P):rrn'-1~:~"-r;r;.!Tr:T1-:-1-'lT -r.T-T-!·f ;- -; ,

';~;;!0;' ,~~;~;,~t~~~~~f+,,~~~~~~:ffi-tHf~: ~~~ ~,' :T: -~*I~ ~ir:="-, :'Sf> ~:-f~~;~~:':_:'+ ; ': .

:~~~~/~~~;;tE~j~~-_:~!Ct,,~'_-t·,'~tTFb~?i:~-~2 ] • , 6 ~ , 9 ')

I"'~'I~LM'-;~'" "1(."""'''

Fig. 7. Infrared spectra of the iso-compound of base B (upper) and

6-metboxyisodictamnine (lower) (Chi oroform) •NC\')

29

5.0

oaH

2.0

L- --L- ----ll-- _"_ --J"--- ..

200 240 280 320 360

\'IAVELENGTH (m,M) 0

Fig. 8'.-Ultra.violet spectrum of kokUsaginine in 95% eth~mol.

30

The base (5 mg.) was dissolved in 10 drops of me-

thanol and 3 drops of concentrated methanolic picric acid

were added. A precipitate formed immediately. After recry­

stallization from ethanol it melted 205-207 0 (dec.). An

authentic sample of kokusaginine picrate23 had m.p. 204-206 0

(deco).and the melting point of the mixed compounds was 205­

208 0 (dec.). The infrared absorption spectrum of base C

picrate and that of kokusaginine picrate were identical.

4" Base D

Base D Picrate. - The base was isolated as the

picrate. The picrate after recrystallization from methanol

softened around 93 0 and melted 107-1090.

AnaLCalcd. for CI5H1703N.C6"307N3: C, 51 0 64; H,

4.13. Found: C~ 51.55, 51.75; "~ 4.24, 4.11.

Base D. - The free base was obtained from the pic­

rate by absorbing 50 mg. on 2 g. of aluminum oxide G and

placing this mixture O~ tcp of 10 g. aluminum oxide G in a

column. Elution with chloroform and evaporation of the sol-

vent gave 21.3 mg. of crystalline base.

Base D dissolved readily in benzene, chloroform,

and ethanol; it was slightly soluble in 5% HCI; and insol­

uble in petroleum ether. It was recrystallized from ben-

zene-petroleum ether, yielding white rosettes of needles,

Its ultraviolet absorption spectrum (Fig. 9) show-

31

6,,0

rw Io 4.0 - Io ,rl ,

2.0

200 280 320 360 400

WAVELENGTH (m~.

FiG. 9.-Ultrav101et spectra of base D: -; in 95% ethanol;

---, in 0.01 N hydrochloric acid in 95% ethanol o

32

ed the fullowing maxima and minima. In 3.86 X 10=5 M 95%

ethanol: Amax • 320.5 mjk<logE 3.55),307.2 (3.49), 294.5

sh (3.24), 283 (3.65),272 (3.73), 262.3 (3.65), 253.5

(3.50), 238.1 (4.43), 229.3 (4.57); ~ min. 314 (3.38, 292

(3.23), 278.3 (3.59), 266.5 (3.64), 248.5 (3.43), 236.5

(4042). 3 0 86 X 10- 5 M in 0.01 N HCl in 95% ethanol: AmaXe

315 (3.69), 302.5 (3.97), 291.5 (3.89), 240.2 (4.33), 236

(4 040),215 0 8 (4 co 39); Amin • 312 (3.68),253 (3.24), 226

(4.23) •

The mass spectrum of the ba se showed two princi­

pal peaks at 259 and 200 m/e. 24

The infrared absorption spectrum (Fig. 10) of the

base gave the following peaks: 1\ chloroform 2 79 p- (w) 3.36max. 0 ,

(s) , 6.11 (s) , 6.29 (s) , 6.59 (s) , 6.!:H (m), 7.00 (s), 7.16

(s) , 7.31 (s) , 7.49 (m) • 7.64 (m) , 7.72 (m) , 8.08-8.31 (m) ,

8.47 (m) , 8.58 (m) • 8.72 (w) • 8.91 (s) • 9.08 (s) , 9.82 (s) ,

10.04 (m). 10.50 (m).

5. Base E

Base E Picrate. - The picrate of base E was solub~

in acetone and 95% ethanol. Recrystallization from 95% etha-

Dol yielded yellow needles m.p. 234-236°, dec. 2370.

Anal. Caled. for CI1HI10N·C6H307N: Co 50.75; H,

3.51; N, 13.93; OCH3' O. Found: C, 51.19, 51.39; H, 3.79,

3.73; N~ 13~78; OCH S; 0:

The picrate of base E (60 mg.) was absorbed on 2 g.

I , , '!f!'"1'-' ,

i __

:~.;:_,••:. .. ""c::-;..:t .; 'J.H.r: _ t- j --0-------

'r'

I ----- ------.

. _iiii .-----.-

1~·.>; L-rr, ....;:

,i~'"',~,~~:m'inn'T"" J'" ":,~ ,.,.':', .. ,' "",'T',' ' _ .

"! ;;:Get'%< l~ff''3i~ '--J. !! ~i ~~4 .I .' • C L·

I ' 'I1 I ~ - r I I::

__ L

~r·--··: :~~~T ~~;-~-l.!-;T:' T

..

i-'.)r­

li

::l,n:~1=lrl :n,;'! 'il _! ,I': n: i·m ..,.. i ~=-.,It---,·,·1 Ie ·11 I !,-~: ,? • j: I I I I I ! ""::-~I'e..:l .,...=r'4 =~ ':.'19). T ·f~•• ,,;:-:'f:.:-.::: •

:l_ ;--\ : _~__ L.L_ '-_u. ;u L_. L._,._J _·.. ·~.Lu __:_ .:

Fig. 10. Infrared spectra of base D (upper) and base F (lower)(Cbloroform) • tI)

CIo)

34

of aluminum oxide G by dissolving the picrate in acetone.

adding the adsorbent and subsequently evaporating the sol-

vent on a rotary evaporator under water pump vacuum. The

resultant mixture was placed on top of a column cODtaining

10 g. of aluminum oxide G. The base was eluted with 10%

methanol in chloroform. Evaporation of the solvent gave 23

mg. of slightly yellow needles of base E.

Base E. - The free base was soluble in chloroform;

hot benzene, ethanol. methanol. and 5% Hel. It was recry-

stallized from chloroform-petroleum ether or from benzene.

It crystallized as fine needles which turned to prisms at

174 0 and melted sharply at 178-179°.

Anal. Calcd. for CIIHIION: C. 76.27; H, 6.40.

Found: C. 75.96. 76.10; H, 6.51. 6.46.

The ultraviolet absorption spectrum (Fig. 11)

showed the following maxima and minima. In 5.78 X 10-5 M

95% ethanol: ~ 335.5 m..« <1og E 3.994), 321.5 (3.96).max. I

310 (3.72). 290 (3.29), 279 (3.17).266 (2.97). 239.5 (4.20).

206.7 (4.44); A i 327.5 (3.89), 261 (2.92).222.5 (3.97);m n.

5.78 X 10- 5 M in 0.01 N Hel in 95% ethanol: \ 302.8max.

(3.95), 247.1 (2.95) t 233.1 (4.70); \ i 257.5 (2.84. 219m n.

The infrared absorption spectrum (Fig. 12) in 0.21

M chloroform showed the following peaks: A 3.25 jA (w).max.

3.34 (s), 6.23 (5), 6.32 (ml. 6.65 (5), 6.18 (m). 6.42 (m),

6.97 (w) t 7.05 (m). 7.22 (w) 9 7.41 (w) t 7.56 (m) t 7.86 (m).

\1J

0 4•.0 1'\0 I \H

I \L \

3.. 0 \

\\\

2.0

35

200 240 280 320 380 400

WAVELENGTH (mp) It'

Fig. 11 .--Ultraviolet spectra of base E: --~ in 95% ethanol;

---, in 0.01 N hydrochloric acid in 95% ethanol.

........t~·c ...

I , ~.:-+- I--+~-+--~- H··+--4- t- :.jL+_+__ j_ - ~t-- IJ-~...--+-+

\I U 'II~'_ .. H f \/1 '~I r: I \ 'I~~ l--111~lIV-,rrt~=t=J-·_·- --,--1 ---I .. ---r--!

l. -.,,+

=Eci-~~'-~'-~$~~~~~~~~8'00 r=;::'i.. h-'~-b';.:.J\J.il¥+'-":""'+~4"'"'-'-'4-,..:..jf----\-'--+-,-~+".."r+--'--+-J-\l-,'-7-.l1-+J.i<--I-+--+-

.- _. ::- -.~:; ~

_.I

~H: ;[H '~;:~H[~~;':' ,.:~~~. ~;l~:r1~·'=~'[:__ X~=~~~~:!,~,_~,· "_~Hin 'i_,,-:: ,,::~ ~:-c-;I .._.i \---, '---T \ ; I 1

I. ,:c: I: :.' '1-;' 'I'"'~.L. ':.! ,. , I __ .:.L_--,-__c:::::r:.__~r==:I--.l. _-,--_I .i.I • 11 ";; II

w .....flfHGfH ...~

'W'Art-u. (.01'

>000 ""'" -.:rTIT."• XXI 1700 llCO '000 "00 ~ J-1 ,',v

(I' ;1: !I ;fI;l:ilr.rrnr.TI-rp~:~~fJl[n"'II"i '~.t~-i 11] .:..rrrlp-~r--rr-1 -rli -r---,-r--,-,-.-r: l-- r l

: I,'iq:,;"b~""h,=-:;f"'cd"'"~V';I-II- :L:...L~~J I Il-L.__L~.:...:_L I _.1-=-.1.__'- __.---' ,"

~ 1iI!j·'·~[i. j~~ ~;;:ij ." '. l'·';d~?2~~:;~,t:£:~,~t~~_! ::~'~1:!"": !~•H.i:~T;':¥ tt:.C ~~-L"~ ~_,-L ~;"-h ~1.:-~--~-,-t--- --+-:- t- ~- ---i ---J-i:=l---f-- --~-j-- -- +-- \

:'.I

Fig. 12 0 Infrared spectra of base E (uppe~ and 1,2,3-trimethyl-4­qUinolone <lower) (Chloroform). C.o'

0-

37

8.45 (m), 8.61 (w), 8.92 (w), 9.24 (m), 9.61 (m), 9.73 (w),

11.08 (m), 11.86 (m).

The N.M.R. spectrum of base E (Fig. 13) showed a

3-proton peak at 140 c.p.s., a 3-proton peak at 215 c.p.s.,

a I-proton peak at 363 c.pos •• a split 3-proton peak at 443

c.p.s •• and a split I-proton peak at 499 CopoSo Tetramethy~

silane was the standard and deuteriochloroform the solvent.

6. Base F

Base F Picrateo - Base F was isolated as the pic­

rate. It could be recrystallized from absolute ethanol.

The picrate softened at 194°, began to turn brown at 203°.

and was decomposed by 216°.

Anal. Calcd. for CI6H2103N.C6H307N3: C, 52.38;

H, 4.80; 1 OCH3 , 6.16; I NCH 3 , 5.75. Found: C, 52.51, 52.56;

H, 4. 35 • 4. 39; OC H3' 5. 69 ; NC H3 • 9. 48.

The infrared absorption spectrum is shown in

Fig. 14.

The picrate <0.11 g.) was adsorbed on 2 go of alu­

minum oxide G and added to the top of a column containing

10 g. aluminum oxide G. Elution with chloroform brought

down the free base. Fifty milligrams of the free base was

collected o

Bas e F. - Bas e F c ° u1d not be 0 bt a i ned cry stall i ne.

Attempts were made to crystallize base F from benzene, 95%

ethanol acetate. ether, benzene-petroleum ether,

38

THS

-------~) '-

100200300

_----'- ,__-'-- ----JI-- ---l. J.-

o

r---- , ,--,-, ----.,- .----.-

TMS

500 400 300 200 100 o

?j.r:;~ 13 .-Nucle.3r ma.gnetic resonance spectra of base E

(upper) and 1,2,3-trimethyl-4-quinolone (lovrer) in deu­

t.p.r~,n~hlnroform at 60 mc .. The frequency is relative to

tetramethylsilane o

.........~~c .... '

lO

,.

''''' ~-- '-p- --- • --

-~irr~rrn'~~:c~I\J-JJ~~~li~d~~~:-rTT':-'f,l

IW·~"'IIN •.lCIIl:.>lS I--

>000

T'-''-'frnnT1:O....'TfJ,-,.

+----"'"

y~

1000

t_~~En~

c-I-c -+-:---,' ci ci~.' H~';' I·.:~i 'i~:K;k_~, rIITFF~;+-:+-~,8f=H~~i-:-~-hLb;': hd:~~ i~-T:--,.ct~ 't----j-- -- ; I

!

-~+,,+:: i' :;tu+~£:c.~~HMn;F+F:'~F-:I-~=F':0-3:.;.¥-fH4:cH+--R-:-+- --r---+- ~:~-,~ . "'~I~~ ,--, c~~ "~'-"-'~F0+'::C+-14. /"'1;:- -/t!\ ':-'~clnt=FFT7:

i-'-'-·--1-1--f_-4-~--'--+-_.' 4.'-'-:-r-:-:-"~- ~+~I--,-+--+f~~---- --~f+-+-'- -t-:.- ---M-i/·; :" ·,f. F- c".""" :: ~"Ti.:. -'+s-H- t'hl-f}'-~ --=-- }J~~-~ ~-j Htl--1-'+1=r AA- \V- -' ,I-c'J:Tl~:p -<~Y~~~';:ie~~~~IIJ---: ;V:~+~_: c-l-:\'f~~:-:_ >. -~~~-:~f7 -1 -~i~:-·

·:-"t~:'-:~:-:l:-C: ;,~> :.";- >'+-:~I ~~"'oHH:"':-I--:+-I- t.:.'~,_iL-i'V -f-- ~ 3, -+-- 1-

--,

Fig. 14. Infrared spectraof the oxidation

of base F picrateproduct of base F

(upper)<lower)

aod the(KBr) •

picrateI:,)'.0

40

chloroform-petroleum ether, ethanol-water, and ethyl ace-

tate-petroleum ~tber. The base always appeared as a whitish

opaque oil.

Tbin-layer chromatograpby of the base on aluminum

oxide G with chloroform as the developer gave a single spot

of Rf 0 0 35 0

The ultraviolet absorption spectra (Fig. 15) of

this oil had the following maxima and minima. In 4.76 X

10-5 M 95% ethanol: A 336.5 mU. <logE 4.39),324.8max. !.

(4 0 34) 0 291 0 6 sh (3~ 75). 281 sh (3 0 66) 0 248 (4 0 72) 0 242

(4.74), 230.8 sh (4.63). 215 (4.68); ~ min. 276 (3.64),

223.4 (4.61), 205 (4.62); 4.76 X 10-5 M in 0.01 N HCl in

95% ethanol: ~ max. 336 (3.94), 322 (4.01), 308.7 (3.96).

248 sh (5.34),234 (5.51). 215 (4.30); Amin. 275.4 (3.47).

220.5 (4.28), 210 (4.27).

The infrared ab sorpt i on spec trum (F ig. 10) showed

the following peaks: ~ ~~~~roform 2.73jA-(w), 3.34 (d, 3.48

(sh), 3.53 (sh), 6.14 (s), 6.22 (s), 6.37 (m), 6.79 (m),

6.90 (w), 7.06 (w), 7.41 (w), 7.53 (ll'l), 7.60 (w), 7.85 (w),

8.46 (w), 8.61 (w), 9.23 (w), 9.68 (w), 10.29 (w), 11.56

(w). 11.71 (w).

Oxidation of Base F.25 - The base (50 mg.) was

dissolved ib 6 mI. of glacial acetic acid and a solution of

100 mg. of chromic anhydride in 10 mi. of 90% acetic acid

was added dropwise. No immediate change in color was noted.

Upon standing, the solution turned dark. After one hour at

6.0

t···... \f "\

41

\\

\

IJIo 4.0o~

2.0

I--- ......_~.-.-

1.--J

, , \\ \

'..

\' /'_' .-\. ..' \\ ~<.~._.- -- .\\ .. -;/ -._\

~:;.- \\/- \,

\\\\

200 280 ~~2.0 .:) (:'n... ) - -' 400

HAVELEHGTII (m,P-) co

Fig.15.--Ultraviolet spectra of base F • -. ,---, in 0.01 N hydrochloric acid in 95% et;;(i.ilOJ.

42

room temperature the solution was slowly pOured iute 30

of concentrated ammonium hydroxide with cooling. The basic

solution was extracted with 25 mI. portions of chloroform.

The chloroform solution was washed with 100 mI. of water and

dried by pouring it through a bed of sodium sulfate on a fu~

nel. The solution was evaporated to dryness on a rotary

evaporator under water pump vacuum. The resulting brown oil

was dissolved in 10 mI. of 95% ethanol and treated with 15

mI. of 2,4-dinitrophenylhydrazine reagent. 26 After refri-

geration overnight the SOlution became cloudy but no cry­

stals could be detected. The cloudy solution was slowly

neutralized with 5~ sodium bicarbonate solution. At pH 1.5

the salt of unreacted 2.4-dinitrophenylhydrazine precipita-

ted. Neutralization was continued with potassium hydroxide

pellets and the solution was thus brought to pHIO and extrac­

ted with three 50 mI. portions of chloroform. Upon evapora­

tion of the chloroform the residual solid was taken up in 10

mI. of absolute ethanol, and 2 mI. of concentrated methanolic

picric acid was added. No picrate precipitated. Addition

of 5 mI. of 10% acetic acid and cooling overnight yielded a

crystalline picrate. Recrystallization from absolute etba-

nol gave fine yellow needles. melting without decomposition

from 187-1900. The ultraviolet absorption spectrum of this

picrate was identical with that of base F picrate, but their

43

Anal. Calcd. for C16H1903N-C6H307N3: C, 52_58;

H, 4.42. Found: C. 52.57, 52.66; H 4.32, 4.31.

D. Syntbeses

1. Synthesi s of 2,3-Dimethyl-4-quinolone

a. By the Niementowski Reaction27

Butanone-2 (1.35 moles) and anthranilic acid (0.73

wvle), 100 g. of each. were beated in a 500 mI. 3-neck flask

equipped with a thermometer and a downward condenser above

an air cooled reflux condenser. Tbe mixture was first

beated to 90 0 and held tbere 0.5 hr. and tben to 190-2000

for 1.5 hr. On cooling a white precipitate appeared wbicb

was washed with boiling benzene. Recrystallization from 95%

ethanol yielded fine colorless needles (7 g., 5.5%), m.p. ~

295°. After two additional recrystallizations from 95% eth­

anol, the melting point was 305-307 0 (dec.).

Anal. Calcd. for CIlHl10N: C, 76.28; H, 6.40; N,

8.09; act. H, 0.58. Found: C, 76.45, 76.52; H, 6.31, 6.53;

N, 8.23; act. H, 0.58.

Tbe ultraviolet absorption spectrum showed tbe

following maxima and minima. In 2.89 X 10-5M 95% ethanol:

Amax • 335.4 mjA--<logE. 4.09),327.4 (4.08), 292.4 ISh (3.44),

279 sb (3.27), 245.8 (4.46). 238.2 (4.49) i ~ mi n. 328.7 (4.03),

256.8 (3.14), 221 (4.17); 2.89 X 10-5M in 0.01 N HCl in 95%

th 01 • \ ~~5" h C'). c:.n\ "1')1 ('1 01\ .,,,,n n t., 0., ...e =an __ -/1 ..._~ ..._;_ s..... ;...._,." ......... ","'.V.l.I, VU"'.7 ''''.U~/.max.

281.7 sh (3.97), 267.2 sh (3.40), 246 sh (4.19), 232 (4.62),

212 (4.37); ~ 256.4 (3.24), 219.4 (4 0 33).mi n.

44

b. From o-AcetamidopropiopAenone

o-NitropropioPhenone. 28• To 425 mI. of fuming ni·

tric atid (d. 1.5 g./ml.) in a 3-neck round bottom flask

equipped with a stirrer o thermometer and dropping funnel was

added 67 g. of freshly distilled propiophenone <0.5 mole).

The ketone was added so as to keep the temperature of the

ice-cooled mixture around 10 0• After addition of the ketone

the mixture was stirred for 10 min. and then poured into

2 1. of water. After cooling to room temperature, the mix-

ture was filtered. The filtrate was extracted with three

200 mI. portions of benzene and the benzene extract was

warmed and used to dissolve the product on the filter. The

resulting benzene solution was then concentrated on a rotary

evaporator under water pump vacuum. When no more benzene

distilled, the remaining oil was taken up in 500 mI. of 95%

ethanol and cooled overnight. The white crystalline precip­

itate (m-nitropropiopbenone) was filtered and washed with 95%

ethanol. The filtrate as well as the washings were evapo­

rated to dryness. A yellow oil remained. It was distilled

at 180°/20 mm. About 15 mI. was c~llected at this tempera-

ture and pressure. Upon addition to the distillate of ~

small amount of benzene crystals appeared.

o-Acetamidopropiopbenone.- The benzene solution of

o-nitropropiophenone was diluted to 100 mI. with benzene.

45

To this solution. 4 g. of palladium-charcoal (10%) was added

and the mixture was placed under 42 lbs. of hydrogen pres-

sure for 5 hr. with shaking. After separation of the cata-

lyst by filtration the benzene solution was combined with 60

ml. of acetic anhydride and 60 ml. of glacial acetic acid

and heated for 2 hr. on a steam bath. which removed the ben-

zene. The reaction mixture was poured into 900 mI. of water.

The green oil which separated crystallized overnight in the

refrigerator. It was recrystallized from aqueous ethanol to

yield 7.4 g. of crystals, m.p. 70.5-71.5°. (Literature m.p.

71 0.29)

Anal. Calcd. for CllH1302N: C, 69.11; H. 6.85.

Found: C, 69.00. 69.06; H. 6.75, 6.69.

2,3-Dimethyl-4-quinolone.- A solution of 7 g.

(0.037 mole) of o-acetamidopropi ophenone, 450 mI. of water,

150 ml. of ethanol, and 4.5 ml. of 40% aqueous sodium hy-

droxide was refluxed for 3 hr. on a steam bath. The ethanol

was then distilled and the solution cooled. On cooling

crystals (3.7 g., 58%) separated which had a melting point

and infrared absorption spectrum identical with the com-

pound which was synthesized by the Niem~ntowski reaction

(See part a.).

2. Synthesis of 1,2,3-Trimethyl-4-quinolone

a. Using Methyl Iodide

2,3-Dimethyl-4-quinolone (2 g•• 0.012 mole) was

46

mixed with 0.3 g. (0.013 mole) of sodium and 6 mI. of metha­

nol and warmed slightly until solution was complete. The

solution was then sealed in a pyrex tube with 7 mI. of

methyl iodide. After 20 hr. at 100 0 the solvent was evapora­

ted and the residue chromatographed on 50 g. of basic alumi­

na (Woelm) 0 Upon evaporation of the solvent fraction 1 gave

a white crystalline residue m.p. 187-189°. Only 5 mg. (0.2%)

of this substance was obtained in addition to the unchanged

starting material.

b. Using Dimethyl Sulfate31

To a solution of 0.5 g. (0.009 mole) of potassium

hydroxide in 30 ml. of met ha nol 1 g. (0.006 mol e) of 2, 3-di­

methyl-4-quinolone was added. The solvent was evaporated

and an excess of dimethyl sulfate (about 10 mI.) was added

slowly. The solution was then heated for 0.5 hr. on a steam

bath. Excess dimethyl sulfate was destroyed by slowly add­

ing 40% potassium hydroxide to a pH of 10. The aqueous so­

lution was then extracted with chloroform. The chloroform

solution was dried over sodium sulfate and evaporated to

dryness. The residue was recrystallized twice from benzene

to yield 59 mg. (53%) of colorless prisms m.p. 188-1890•

Anal. Calcd. for C12HI30N: C, 76.99; H, 7.00.

Found: Ce 76.51, 76.64; He 7.61, 6.96.

The ultraviolet absorption spectra (Fig. 16) gave

the following maxima and minima. In 5.4 X 10-5 M 95% etha-

47

6•.0

5.·0

\U

o 4.0oH

2.0 -

200 240 280 320 380 400

WAVELENGTH (mf4J.

F1g.16~--U1traviolet spectra of 1,2,3-trimethyl-4-quinolone:

--, in 95% ethanol;---, in Og01 N hydrochloric acid

in 95% ethanol.

following

6036 (s).

48

Dol: "A max. 344.5 mf'- <I og E 4.06), 331.6 (4.04), 293.6 sh

(3.18),281.9 (3.0S), 269.7 (2.97),249-243 (4.39-4.42), 214

(4.36)IA min. 337.3 (4.00), 264.2 (2.87).224.4 (4.02); S.4 X-S \10 M in 0.01 N HCl in 9S% ethanol: {\ 344.9 (3.72), 327max.

(3.87), 317.4 (3.8S), 247.2 (4.29), 236.7 (4.50). 214 (4.30)i

Amin • 339.7 (3.72), 323.3 (3.85), 260.8 (2.87), 223 (3.91).

The infrared absorption spectrum (FigG12) had the

peaks: ~ chloroform 3.36 /A (s), 6018 (s). 6.2S (s~max.

6046 (S). 6.79 (5), ·6.87 (m), 6.97 (m). 7.04 (m),

7.29 (m), 7.40 (m), 7.64 (s). 7.72 (s), 8.34 (m), 8.89 (m),

9.88 (m), 10.31 (m), 14.38 (m), IS.12 (m).

3. Synthesis of 1.2-Dimethyl-4-quinolone by the Conrad­

Limpaeh Method32

a. 2-Methyl-4-quinolone

To a mixture of SI.5 g. (0.5S mole) of aniline and

66 g. (O.SI mole) of ethyl acetoacetate methylene chloride

was added to a volume of 2S0 mI. After 4 d. at room tempera­

ture the solution was washed successively with 100 mI. of 2%

HC1, 100 mI. of water. 100 mI. of 0.5 N sodium hydroxide and

again with 100 mI. of water. Subsequently, the solution was

dried over magnesium sulfate and the solvent evaporated.

The residual light brown oil was added to SOO mI. of paraffin

it was formed. After the addition the temperature was

raised to 240 0 and kept there for 10 min. The mixture was

then cooled with stirring. A solid separated on cooling. It

was filtered and washed with benzene. Washing with boiling

chloroform removed the final traces of color and lefto 33

6.29 g. (7.8%) of a white solid m.p. 234-235.5. The

ultraviolet absorption spectrum was identical with that pub­

lished by Ewing and Steck34 for 2-methyl-4-quinoloneo

b. 1,2-Dimethyl-4-quinolone

To a solution of 0.71 g. <0.013 mole) of potassium

hydroxide in 50 mI. of hot methanol 2 g. <0.013 mole) of 2-

methyl-4-quinolone was added. The methanol was then di6­

tilled off on a rotary evaporator under water pump vacuum

and 5 mI. of dimethyl sulfate was added to the residue. The

reaction mixture was refluxed for 0.5 hr. and then taken up

in an excess of aqueous potassium hydroxide. The resulting

purple solution was extracted with chloroform and the ex-

tract was passed through a column containing 50 g. of alumi­

num oxide G. The eluant was evaporated to dryness and then

taken up in a minimum of chloroform. Upon addition of 3-4

times as much petroleum ether as there was chloroform purple

crystals appeared. These were sublimed at 120%.3 mm.

A total of 0.3 g. <13%) of white crystals. m.p. 179 0 (Lit.

174-1750 35). was collected. Two recrystallizations from

o~p.~~~~~ r~i~~d the m.~. to 179.5-180.5 •

Anal. Calcd. for C11HIION: C, 76.27; H. 6.40; 0.

9.24; Nt 8.09; NCH 3 • 8.66;· OCH3. O.

50

Found: C, 76.22, 75.99; Ho 6 0 42, 6.44; 0, 9.78; N, 8.15;

NCH3' 7.94; OCH 3 , o.The ultraviolet and infrared absorption spectra

were identical with those of base E shown in Figs. 11 al1d 12.

40 Results of Other Syntheses

a. The Reaction of Anthranilic Acid and Propanal

A mixture of 100 g. (1.7 mole) of propanal and 100

g. (0.73 mole) of anthranilic acid was beated for 0.5 hr. at

about 1000. The temperature was then slowly raised to 2100.

Tbe beating was discontinued when no more water was dis-

tilled. The solution was left to cool to about 1500 and

then poured into 200 mI. of benzene. About 35 g. of light

yellow precipitate was collected. It could be crystallized

from benzene, chloroform, metbanol, or ethanol. Recrystal-

lizatlon from ethanol resulted in colorless needles m.p.

225-226°.

Found: C, 73.04, 73.22; H, 6.24, 6.36; N, 6.49.

The ultraviolet 8bso~ption spectra sbowed the fol­

lowing maxima and minima. In 6.34 X 10-5 M 95% ethanol:

Amax • 326.2 mfl-<Iog f 3.84), 312.1 (3.77),298.1 (3.72),

287.2 (3.64), 222 (3.94); Amin • 312.4 (3.63),307.3 (3.68),

252.8 (3.04); 6.34 X 10-5 M in 0.01 N Hel in 95% ethanol:

A 325.6 (3.94). 321.5 (3.84). 299.4 (3 0 73). 220.6ut8A.

(3.93) i f\ min. 321.2 (3 0 79}·o 255.6 (3.04).

The infrared absorption spectrum showed the

51

following peaks: A~:~ 3.26~(w), 3.36 (5), 3 0 42 (m), 3.46. .

(w) • 4.25 broad (m). 5.27 (m). 5.90 (s), 6.12 (s), 6.31 (s) ,

6.52 (s) • 6.71 (m) , 6.88 (s) , 6.96 (m) • 7.08 (s) , 7.23 (s) ,

7038 (m) • 7.48 (m) , 7.58 (m) , 7.88 (m) , 8.19 (s) , 8.31 (s) ,

8e 67 (w) , 8.89 (w) , 9.35 (m) , 9.56 (m) , 10.01 (s) , 10.19 (m),

10.96 (s), 12.46 (m), 12.72 (s), 13.29 (s).

b. Attempted Synthesis of 2-Methyl-4-quinolone Using

the Procedure of Stark36 .

A mixture of 51..5 g.. (0.55 mole) of freshly dis-

tilled aniline and 66 g. <0.51 mole) of ethyl acetoacetate

was dissolved in 250 mI. of methylene chloride and warmed

on a steam bath to 40 0 for 48 hr. Four milliliters of wa-

ter was separated with a separatory funnel. The solution

was then dried over magnesium sulfate. Methylene chloride

was distilled on a rotary evaporator under water pump vacuum

and the resulting light brown oil was heated very rapidly

to 240 0 • Heating was stopped to allow the temperature to

drop to 235 0 when it was again raised to 240 0 and then left

to cool. When the temperature had dropped to 1000. the red-

brown oil was extracted with 300 mI. of boiling water. No

crystals appeared on cooling of the aqueous solution. The

brown oil was now triturated with two 200 mI. portions of

benzene and then two 200 mI. portions of ether. A solid re­

mained. It was filtered and washed again with ether. Upon

recrystallization from chloroform, 3.6 g. of white crystal-

line needles, m.p. 244-245°, was isolated. The ultraviolet

52

absorption spectrum showed a maximum at 256 m~

c. Attempted Synthesis of l,2-Dimethyl-4-quinolone

o-Acetomidoacetophenone. - A mixture of 10 go

<0.074 mole) of o-aminoacetophenone, 50 mI. acetic acid and

50 mI. of acetic anhydride was heated for 2 hr o on a steam

batho It was poured into 500 mI. of water, which was then

neutralized with sodium bicarbonate. Upon neutralization.

the amide precipitated. Recrystallization from ethanol gave

7 0 9 g. (63%) of crystals, map. 75-76 0 <Lito 76-77 0 37)e

Condensation of the Amide. - To a solution of 3.5

g. (0.02 mole) of o-acetamidoacetopbenone. in 325 mI. of

water and 110 mI. of ethanol was added 3.25 mI. of 40%

aqueous sodium hydroxide. The solution was refluxed on a

steam bath for 3 hr. The ethanol was allowed to distil and

the aqueous solution was placed in 8 refrigerator for cool-

ing. A crystalline white solid precipitated. Filtration

and washing with water yielded 1.65 g. (52%) of crystals.

m.p. 223-224 0 (4-methylcarbostyril, Lit. m.p. 223 0 38).

The ultraviolet absorption spectrum in 9.5 X 10- 5

M 95% ethanol had the following maxima and minima: ~ max.

339.8 !!!r-<1og E: 3047); 326 0 2 (3 0 62); 314,,1 sh (3.53),276.7

(3.55), 267.6 (3.62).259.3 sh (3.54), 245 (3.84, 230

(4.34), 224.9 (4.31> i A i 335.1 (3.47), 288.3 (2.97),m n.273.3 (3.55), 254.5 (3.50). 242.6 (3.84).

53

N-Methyl-o-acetamidQacetophenone. - To a solution

of 3.54 g. m.02 mole) of o-acetamidoacetophenone in 50 mI.

of benzene was added 0.8 g. (00035 mole) of sodium ribbono

The mixture was warmed on a water bath till no more sodium

was evident. Two milliliters (d. 1.33 g./ml.) of dimethyl

sulfate was added slowly and the solution was refluxed for

005 hr. on a steam bath. A reddish-purple solution resulted.

which was washed by extracted it with three 50 mI. portions

of water. The benzene solution was dried over magnesium

sulfate and evaporated to dryness. About 2 g. (52%) of cry­

stalline N-methyJ-o-acetamidoacetophenone was COllected.

1.4-Dimethylcarbostyril. - To a solution of 2 go

(0.01 mole) of N-methyl-o-acetamidoacetophenone in 100 mI.

of water and 70 mI. of ethanol was added 2 mI. of 40% aque­

ous sodium hydroxide. This solution was refluxed on a steam

bath for 3 hr. Ethanol was distilled on a rotary evaporator

under water pump vacuum. The remaining aqueous solution

was extracted with 3 X 50 mI. chloroform. The chloroform

extract was dried over magnesium sulfate and evaporated to

dryness. The resulting solid was purified by chromatography

on aluminum oxide G in chloroform. Recrystallization from

benzene gave a crystalline SOlid, m.p. 130-132 0 • Thin-layer

chromatography gave no indication of the pre6ence of 1,2­

dimethyl-4-quinolone.

CHAPTER III

RESULTS AND DISCUSSION

A. Base B

Base B was isolated as a weak base from the root

and bark collections on Hawaii and Kauai but not from the

leaves. From the plant material collected on Kauai two hun-

dred and fifty-two milligrams (0.0023 percent) of base B

was isolated o The yield from the plant material from Hawaii

was smaller.

The ultraviolet (Fig. 4) and the infrared absorp­

tion spectra (Fig. 5) of base B were typical of furoquino­

line alkaloids. 39 ,40.41,42 Elemental and functional group

analyses suggested a composition of C13HII03N with two meth­

oxyl groups but without methyl bound to nitrogen. Thus, a

methoxydictamnine was indicated. Two such compounds, 1­

methoxydictamnine (evolitrine)43 and 8-methoxydictamnine

(j-fagarine) ,44 had been reported previously. Direct com­

parison of the base with an authentic sample of r-fagarine45

by infrared absorption and melting point of the mixed com­

pounds showed that base B was not 8-methoxydictamnine. Its

physical properties obviously differentiated it from evoli-

trine. Thus. base B had to be one of the two hitherto unre-

ported monomethoxydictamnines, 5-methoxydietamnine or 6-

methoxydictamnine.

A comparison of the ultraviolet absorption spec­

trum of base B (Fig. 4) with spectra of other known metho­

xy-substituted dictamnines39 showed that the spectrum of

55

base B strongiy resembled that of 6:8-dimethoxydictamnine

(masculosidine) (Fig. 17);46 this similarity suggested that

base B might be 6-methoxy- rather than 5-methoxydictamnine o

If this assumption was correct, then the iso-com-

pound of base B should be identical with 6-methoxyisodictam­

nine isolated by Lamberton and Price4 as a degradation pro-

duct of medicosmine o Isomerisation of base B (X) with

Mel 1100 0

x XI

methyl iodide at 1000 yielded the expected iso-compound (XI).

This was shown by comparing the absorption spectra (Fig. 7)

with those of an authentic sample,23 and by the melting

point of the mixed compounds. This constituted proof that

base B was 6-methoxydictamnine, an alkaloid which had not

been isolated preViously.

This brings to three the number of known mono-

methoxydictamnines. 5-Methoxydictamnine remains u~known.

It is somewhat surprising that 6-methoxydietamnine has not

been isolated from other species of the Rutaceae, since it

is so closely related to kOkusaginine and may even be a

oiugcuctie ~re~~rsor of kokusaginine.

Evolitrine has now been isolated from four species

3.·0

5.·0

56

200 240 280 320 360

WAVELENGTH (m~.

Fig.17.--Ultraviolet spectrum of macu!osidine in 95% ethanol.

57

and 6-methoxydictamnine only from PlatJdesma eampanulata,

while kokusaginine has been isolated from twelve species

in Rutaceae. 8 In three of the eases where kokusaginine was

isolated a monomethoxydictamnine was also isolated and dic­

tamnine was isoiated in only five cases. At the present

time no taxonomic or biogenetic significance can be attach­

ed to this since it is not known whether a monomethoxydie­

tamnine or dictamnine itself is a necessary biogenetic pre­

cursor of the more highly oxygenated compounds.

B. Base C

Base C was isolated from all parts of the plant

and was found to occur in ~. ~Qanulata from Kauai and from

Hawaii. Only eighteen milligrams was isolated from the Kauai

root and bark, but the root and bark from Hawaii yielded ~.

one hundred milligrams (0.00083 percent) of base C. Combin­

ed with the thirty-eight milligrams which was obtained from

the leaves the total yield was 0.0016 percent of base C,

which therefore may be considered to be the second major al­

kaloid of Platydesma campanulata.

The melting point of the free base and of its pic­

rate as well as the ultraviolet absorption spectrum clearly

suggested ~hat base C was identical with the known alkaloid

kokusaginine (6,7-dimethoxydictamnine). Direct comparison

of the picrate with an authentic sample23 by infrared spec-

LLU~~UPY aud hy m~lti"g pviut uf the mixed compounds proved

58

identity.

Kokusaginine was first isolated by Terasaka from

Orixia Japonica Tbnnb. 47 Its structure (XII) was determined

by Hughes, Ritchie and coworkers 48 through degradation to

2,4-dihydroxy-6,7-dimethoxyquinoline, which had been syn­

thesized previously.49

XII

In Platydesma campann]ata this alkaloid was found

to occur together with evolitrine and 6-methoxydictamnine

only in the root and bark, while in the leaves it was not

accompanied by the two monomethoxy compounds.

C. Base A

Base A was isolated from the root and bark but not

from the leaves of £. campanu]ata. It was found in the same

fractions as were bases B and Co It was possible to isolate

the latter two in crystalline form, but attempts to separate

pure base A gave only pure base B and a mixture of bases A

and B. Since base B was proven to be 6-methoxydictamnine

an~ b~sc C ~,7-riimethoxydictamnine, base A might well be

7-methoxydictamnine (evolitrine). XIII.

59

XIII

Evolitrine had been isolated previously. first by

Cooke and Haynes. 43 later by Briggs and Cambie,50 and by

Rapoport and Tian Gwan Hiem. 51 Ohta and Mori 52 proved its

structure by synthesis. Interestingly enough kokusaginine

(base C) was encountered by both Cooke 43 and Briggs 50 in

their work.

Chromatography of an authentic sample of evoli­

trine2l parallel with the mixture of bases A and B on a thin

layer of aluminum oxide G with benzene-chloroform (1:1) as

developer showed the following results. The mixture yielded

two spots. one with the Rf and the color of evolitrine, and

the other with the Rf and color of base B. The origin of

the color upon development with Dragendorff's reagent is not

known but it does seem to be a criterion for the differen-

tiation of the two alkaloids.

Although this evidence is not conclusive, there

is little d~~bt that e~o!itri~~ is ~ minor alkaloid of

Platydesma campanuJata. It is the fourth recorded occurence

of the alkaloid all of which have been in the same subfamily

60

of the Rutaceae.

D. Base D

This base occurred in the root and bark of

Platydesma campanulata which was collected on Hawaii. It

was not isolated from the plant material collected on Kauai.

A total of one hundred and sixty-eight milligrams of Base D

picrate was isolated, which corresponds to a yield of 0.00073

percent.

The elemental and functional group analyses of the

picrate best agree with an empirical formula of C15H17N03.

This was further supported by a molecular weight of 259 as

determined by mass spectrometry.

The ultraviolet absorption spectra (Fig. 9) in

ethanol and in 0.01 N hydrochloric acid were identical with

those of dihydrodictamnine (Fig. 18).53

The mass spectrum of the free base 24 had only two

prominent peaks, the molecular weight peak at 259 mle units

and a peak at 200 mle units. This latter peak is caused by

a fragment after a loss of 59 m/e units. A dihydrodictam­

nine radical, XIV. has a molecular weight of 200; since the

presence of this moiety was also suggested by the ultravio­

let absorption spectrum, it may be assumed that the alkaloid

is a substituted dihydrodictamnine.

The substitution of the dihydrodictamnine nucleus

must be on the non-aromatic part of the molecule, since

61

5.0

I/

II

\ /""

I

\

200 230 260 290 320

WAVELENGTH (ny4.

Fig.18o --Ultraviolet spectra of dihydrodictamnine:---, in

95% ethanol;---, in 0.01 N hydrochloric acid in

95% ethanol.

62

XIV

cleavage during electron bombardment occurs most readily at

aliphatic or alicyclic bonds. 54 Infrared evidence excluded

the possibility that the fragment of weight 59 arose from a

C2H302 unit. Furthermore, subtraction of the elements

C12Hl002N corresponding to dihydrodictamnine from the molec­

ular formula leaves a C3H10 fragment. A CSH10 radical can

only be an ether or an alcohol. A band at 2.19 fA- in the in­

ira red ab sorpti on spec t rum (F ig. lOj indicate s that a n hydro­

xy group is present in the free base D. There is no evi-

dence for an ether linkage other than the known methoxy

group. The 2.19)A- band is at a sufficiently long wavelength

to have arrisen from a tertiary alcohol. 55

If the alcohol function in the C3H10-fragment is

indeed tertiary, its nature would be as in XV.

/CH3

..,- OH-c\

xv

63

A choice of two structures, XVI or XVII, remains

for alkaloid D.

"fOHXVI XVII

Biogenetic reasoning supports structure XVI o Re-

action of 4-methoxyquinoline with an isoprene unit as sug­

gested by Ruzicka,56 with the aid of an operator as suggest­

ed by Robinson 57 would allow the following biogenetic scheme:

6"3 f

0'" + C c-c ~:".. /' \ c Operat orN

ox.

OH

XVI

64

If the 'operator' placed the isoprene unit in a

position other than the 3-position of methoxyquinoline, a

dihydrodictamnine nucleus would not arise. Furthermore, two

alkaloids with a skeleton as proposed in XVI have been iso-

lated from BalfourodendrQn riedelianum58 and from Lun8sia

amara Blanco. 59 Both have structure XVIII and are enantio-

merie. Balfourodine is the dextrorotatory and hydroxyl una-

crine is the levorotatory antipode.

2t

CQL-:I /OH

CH3'-o ClH3 ·U· IXVIII

An oxidation analogous to the one that is proposed

in the biogenetic scheme has precedents in the alkaloids

maculosine, XIX,46 and hydroxylunacridine, XX. 59

XIX

OH

OH

Both compounds are oxidized to the glycol stage in the iso-

prene portion of the molecule.

65

Thus structure XV .for base D appears to be reaSOD-

able on biogenetic and chemical grounds.

The alkaloid has been named platydesmine. It is

the first dihydrodictamn!ne derivative which has been iso-

lated from natural sources. Its occurrence is therefore, if

Dot surprising, biogenetically interesting i since the loss

of an isopropyl alcohol unit would lead to dictamnine o

)

This may well be a clue to the origin of the furan group in

furoquinoline alkaloids, which has not been explained to

date. 57

E. Base E.

Base E was isolated from the root, bark, and

leaves from Hawaii, but not from those from Kauai. The to-

tal yield was one hundred and seven milligrams <0.0017 per­

cent) •

Elemebtal analysis of the picrate and of the free

base established CI1H1ION as the empirical formula of the

Dh~",,"1'.-- ..,_ .......

The ultraviolet absorption spectrum of the base

66

Wig. 12) is typical of 4_quinolones. 34 ,60 It shows a bi­

furcated peak between 320 and 340 mf4in 95% ethanol, which

is shifted to lower wavelength and appears as a single peak

in 0.01 N ethanolic hydrochloric acid. This is typical of

2-methyl substituted 4-quinolones.

A N.M.R. spectrum (Fig. 13) established the pre­

sence of two methyl groups. Since no quinoline alkaloids

bearing methyl substituents on the benzene ring had been

found previously, it could be assumed that the second methyl

group was either in the I-or 3-position of the molecule.

The N.M.R. spectrum of a synthetic sample of 1,2,3-trime­

thyl-4-quinolone (Fig. 13) showed one additional three­

proton peak at lower chemical shift. This indicated that

the third methyl group, the one not present in base E, must

be in the 3=position, since these hydrogens would show the

smallest chemical shift with respect to the internal stan­

dard, tetramethylsilane. Base E was therefore proven to be

1,2-dimethyl-4-quinolone. 6l

2-Methyl-4-quinolone was synthesized by the Conrad­

Limpach method. 32 Methylation with dimet~yl sulfate led to

the expected 1,2-dimethyl-4-quinolone. 31 The synthetic base

was identical with the natural product in every respect.

The presence of 1,2-dimethyl-4-quinolone in £lA­

tydesma campanulata came as a surprise, since there has only

been one other occurrence of such a simple substituted qui­

nolone in the Rutaceae. Greshoff isolated the alkaloid

67

echinopsine, XXI, from the seeds of EcbiDnps ritras. 62

Substitution in the two-position, however, is not

new. Among the alkaloids of Angostura bark 2-substituted

quinolones and quinolines are quite common o63 but none of

them are 2-methylquinolones.

F • Ba se F •

Base F could not be isolated in crystalline form.

It was isolated as the picrate in a yield of 0.00081 percent.

It was found only in the leaves.

Its empirical formula rests solely on the analyses

of its picrate. C22H22010N4 agrees best, but C22H24010N

fits well enough to merit consideration. Subtraction of the

element of picric acid leaves a free base of formula C16H19­

03N or CI6"2103N. Functional group analysis showed the pre­

sence of one methoxy group and one methyl group linked to

oi t roge n.

The ultraviolet absorption spectrum (Fig. 15) of

thiS base was comparea With those 1n the

64 This comparison suggests that base F is a substituted 1-

68

methyl-8-methoxy-4-quinolone. The shift of its peaK to

lower wavelength in 0.01 N acid has been suggested to be due

to the substituent on the benzene ring. It was noted by

Goodwin ~ A1., that an 8-methoxy substituent causes a hyps~

chromic shift, while a 7,8-methylenedioxy group gives rise

to a bathochromic shift of the long wavelength peaks in 0001

N acid. 59 The infrared absorption spectrum is also consis-

tent with a 4-quinolone structure. A partial structure of

I-methyl-8-methoxy-4-quinolone, XXII, may therefore be pro-

posed ..

XXII

This leaves 8 CSH9_lJ O fragment to be placed. The

nature of this tragment could not be ~etermined with certai~

ty, but it may be assumed that the five carbons constitute

an isoprene unit 8S has been observed in mapy other c~ses.

The presence of 'operators' in the plant as suggested by

Robinson57 would make the attachment of such an isoprene

unit on the aromatic nucleus possible. A survey of other

alkaloids would at first indicate that attachment of such a

fragment could be possible almust - - .....- ... - - - '" _ + h ~UUI'"'U"'&''' Via .... _mnllt>I'nllt>.--------.

However, all 4-quinolones containing an isoprene unit which

69

have been isolated to date are substituted in the 3-posi­

tion. Part structure XXII may therefore be expanded to part

structure XXIII.

R

XXIII

Presence of a furano group may be excluded since

none of the characteristic bands mentioned in the work by

Briggs and Colebrook 4l are present in the infrared absorp­

tion spectrum. Furthermore, only one oxygen atom remains

to be placed. Tbis oxygen atom is clearly part of an alco­

hol function and gives rise to a peak at 2.72fA in the in­

frared absorption spectrum (Fig. 10).

An attempt was made to determine the position of

the hydroxy group on the side chain. The peak at 2.72~

would indicate that the alcohol is primary or secondary,55

but this cannot be taken for granted since the accuracy of

the spectrometer is ± 0.05~. Thus, a tertiary alcohol is

not excluded.

A small amount of the free base was oxidized with

chromic anhydride in acetic acid. A picrate of the product

identical with that of the picrate of the base and exhibited

70

the three major peaks of base F. The infrared absorption

spectrum (Fig. 13) and the melting point of the picrate of

the oxidation product were different from those of the pic­

rate of free base F.

The elemental analysis agreed well with a formula­

tion of C16H1903N• If the empirical formula of base F is

C16H2103N. the formula of the oxidation product would indi­

cate a loss of two hydrogen atoms corresponding to the oxi­

dation of an alcobol to a ketone. Because of the limited

accuracy of a hydrogen determination this argument is open

to question. No doubt an oxidation took place since the

oxidizing agent was reduced. It was an oxidation which was

accompanied by a very slight change in percentage composi­

tion. This adds some substance to the above argument.

Since the ultraviolet spectrum remained unchanged~

the oxidation must have taken place more than one carbon

atom away from the aromatic system. This leaves only one

likely position for a secondary alcohol. The possibility of

a primary alcohol cannot be excluded, but since there are no

analogies for it, it must be considered less likely than a

secondary alcohol.

The following tentative structure may be proposed

for base F. XXIV.

71

XXIV

This alkaloid has been named pilokeanine. The

name is derived from the Hawaiian name of the planto

Lunacrine, XXV, and hydroxylunacrine,XXVI, are two

alkaloids which have been isolated from Lunasia amara

BlancQ. 59 They differ from each other only by an hydroxyl

group in the aliphatic side chain.

XXV XXVI

If one accepts the tentative structure XXIV for

pilokeanine, analogous though more remote relationship

exists in the aliphatic side chains of pilokeanine (XXIV)

XXIV XIV

72

and platydesmine (XVI).

Proposed structure XXIV is consistent with all

chemical and biogenetic evidence but it cannot be consider­

ed definitely established.

CHAPTER IV

CONCLUSION AND SUMMARY

It was shown that at least six bases occur in

Platydesma campanuJata: evolitrine (XIII), 6-methoxydietam-

nine (X) 0 kokusaginine (XII), platydesmine (XVI), 1,2-di­

methyl-4-quinolone (XXVII), and pilokeanine (XXIV).

XIII

XII

XXVII

x

XVI

XXIV

74

The presence of evolitrine was demonstrated by

comparison chromatography. The structure of 6-methoxydic­

tamnine was established by isomerization to 6-methoxyiso­

dictamnine, which was a known compound. 4 The presence of

kokusaginine was proven by direct comparison of base C pic­

rate with an authentic sample of kokusaginine picrate o The

proof of structure of platydesma was based on the use of

physical methods and biogenetic principles. Base E was

shown to be 1,2-dimethyl-4-quinolone by instrumental methods

and by synthesiso A structure has been proposed for pilo­

keanine. It has not been proven beyond doubt, but it appears

to be consistent with all chemical evidence and with cur­

rently held biogenetic theories.

6-Methoxydictamnine, platydesmine, 1.2-dimethyl-4­

quinolone, and pilokeanine are new natural products. Evoli­

trine and kokusaginine were isolated preViously. The sali­

ent data pertaining to these alkaloids are summarized in

Table III.

The taxonomic question which was posed in the in­

troduction as to the position of the genus Platydesma among

the other genera of the subfamily Xanthoxyleae cannot be

answered on the basis of the new information. The isolated

alkaloids clearly confirm that Platydesma is a member of

the subfamily Rutoideae. A relationship to the genus Lunasia

is suggested, but the alkmloids of MedicQsma cunninghamij

are not more closely related to those of Platydesma than

TABLE III. The Alkaloids of Platydesma Campaoulata Mann.

,II.R. lPicrateBases M.p. Yield UoV o OccT.&rence

spectra in plant m.p.-

Base A 114-115 0 ? - .. Root and 191-192°

evol i tJ· i ne bark

Base B 134..135 0 203 X 10-3% Fig. 4 F Ig o 5 Root and 195-196 0

6-methoxydictamnlne bark

Base C 169-169.5° 1.6 X 10-3% Fig. 8 Fig. 5 Leaves, root 205-208 0

kokusauinine and bark (dec.)

Base D 137-138 0 7.3 X 10-4% Fig. 9 F 19. 10 Leaves, root 107-109°

platydHsmine and bark-----,Base E 178-1790 1.7 X 10-3% Fig. 11 Fig. 12 Leaves, root 234-236 0

1,2-dimethyl-4-quinolone and bark (dec. )

Base F oil 7.0 X 10- 4% Fig. 15 Fig. 10 Leaves 216 0

pilokennine (dec.)

-.til

76

are those of ChQisya ternata.

Pharmacological studies could not be carried out

because of the small amount of pure bases which were avail­

able. However, the isolation of alkaloids which are related

to those isolated from Lllnasia Amara Blanco59 is of poten­

tial interest, since the bark of that plant has been used in

preparing arrow poisons by the nati~es of Luzon Island. 65

BIBLIOGRAPHY

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83

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85

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to his

wife for her patience and her help in typing the first

drafts of this thesis and to Mr. W. G. Wright for the typ­

ing of the final copies of this thesis o

This research was supported financially through

grants to the University of Hawaii from the National

Science Foundation and from the National Institute of

Health.