This dissertation has been 64-2653microfilmed exactly as received

LEVAND, Oscar, 1927-PART I. SOME CHEMICAL CONSTITUENTSOF MORINDA CITRIFOLIA L. (NONI).PART IT. THE STRUCTURE OF THE NITRO­CAMPHOR ANHYDRIDES.

University of Hawaii, Ph.D., 1963Chemistry, organic

University Microfilms, Inc., Ann Arbor, Michigan

PART I. SO~lli CHEMICAL CONSTITu~NTS OF

MORINDA CITRIFOLIA L. (NONI)

PART II. THE STRUCTURE OF THE

NITRO CAMPHOR ANHYDRIDES

A THESIS SUBMITTED fro THE GRADUATE SCHOOL OF TI-IE

UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT

OF THE REQUIREr.'iENTS FOR 'rHE DEGREE OF

DOCTOR OF PHILOSOPHY

IN CHEMISTRY

JANUARY 1963

By

Oscar Levand

Thesis Committee:

Harold O. Larson, ChairmanDavid E. ContoisMichael M. FrodymaRichard G. InskeepPaul J. Scheuer

PART 1.

TABLE OF CONTENTS

SOME CHEMICAL CONSTITUENTS OF fl10RINDA CITRIFOLIA

L. (NONI)

LIST OF FIGURES •••••••••• 0 •••••••• l) ••••••••••••• v

A. INTRODUCrrION. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• 1.

1.

2.

Botanical

Medicinal

0 •••••••••••••••••••••••••••• 00

• ••• 0000 •••••••••••••••••••••• 0

1.

1.

3. Che mi ca 1 ••.••••••••.•••••••••••••••••••• 2.

4. Statement of the Problem •••••••••••••••• 4.

B. EXPERIMENTAL •••••••••••••••••••••••••••••••••••• 6.

1. Bacteriological Testing ••••••••••••••••• 7.

2. Procurement and Processing of

Noni Fruit ••••••• 0 •••••••••••••••••••••• 8.

3. Extraction of Fruit Pulp •••••••••••••••• 9.

4. Preliminary Work on the Crude

Hexane Residue .0 ••••••••••••••••••••••• 10.

5. Separation of Methanol Residue

into Two Fractions by a Darco-

Celite Adsorption Method ••••••••••••••• 12.

6. Acetylation of a Fraction Desorbed

from the Mixture of Darco and

Celi te •••• 0 ••••••••••••••••• 0.0 •••• 0 •• 0 14.

iii

7. Isolation of Compound A and B, and

a Hexane Soluble Fraction from the

Acetylated Material .................... 14.

8. Characterization of Compound A (~D­

Glucopyranose Pentaacetate) •••••••••••• 19.-

9. Preparation of ~-D-Glucopyranose

Pentaacetate ••••••••••••••••••••••••••• 19.

10. Characterization of Compound B

(Asperuloside Tetraacetate) ;; •••••••••• 21.

11. Preliminary Work on the Acetylated

Iiiqui d .••••••.•.•••.••••. 0 • • • • • • • • • • • •• 23 .

a. Degradation with Concentrated

Nitric Acid •••••••• 0 •••••••••• 0 •• 0. 26.

b. Degradation with Sodium Hydroxide •• 29.

c. At~empted Ozonization •••••••••••••• 29.

12. Isolation of Caproic and Caprylic Acid

from the Ripe Noni Fruit ............... 30 •

c. DISCUSSION ••••••••••••••••••••••••••••••••••••• 32.

1. Attempted Isolation of Chemical Consti-

tuents from the Methanol Residue ••••••• 32.

2. Isolation of an Unknown Liquid, 0-D­

Glucopyranose Pentaacetate and Asperu-

loside Tetraacetate from the Acety-

la ted Material ••••••••••••••••••••••••• 34.

3. Asperuloside as the Possible Anti-

biotic Substance in the Noni Fruit ••••• 38.

iv

• •••••••••••••••••• 0 ••• 0 •••••••• 0.

40.

42.

43.

••••••••••••••••• 0 ••••••

••••••••••• oo.o •• oo ••••• eMiscellaneous4.

Sm1IvIARY AND CONCLUSION

BIBLIOGRAPHY

D.

E.

LIST OF FIGURES

Fig. 1. Complete work-up of noni frui t pulp ••••••• 11.

Fig. 2. The infrared spectra of ~-D-glucopyranose

v

• • • • • • • • • • 0 ••••

pentaacetate isolated from the noni fruit

(upper), prepared by the method of

Wolfrom and Juliano (lower)

Fig. 3. The ul~raviolet spectrum of ~-D­

glucopyranose pentaacetate isolated

from the noni frui '[,. (c = 7.95 x 10-4!v'j

20 •

in abs. alcohol) •••••••••••••••••••••••• 0. 22.

Fig. 4. The. infrared spectra of asperuloside

tetraacetate isolated from ~he noni fruit

(upper), obtained from Dr. Briggs (lower) •• 24.

Fig. 5. The ultraviolet spectrum of asperuloside

tetraacetate isolated from the noni fruit

(c=8.6 x 10-5 fit in 95% alcohol) •••••••••• 25.

Fig. 6~ The infrared spectrum of the acetylated

liquid (liquid film) •••••••••••••••••• 0 ••• 27.

Fig. 7. The ultraviolet spectrum of the

ace tyla ted liquid (c =2.38 x 10-4 M in abs.

alcohol) 000000.0 ••• 0 •• 0 •••• 0 •••••• 28.

A. INTRODUCTION

1. Botanical

Morinda citrifolia L. belongs to the family of

Rubiaceae (1). The family has 4500 or more species and

about 350 genera widely distributed over Africa J Asia

and America. Polynesia possesses also a large number

of species~ In the Hawaiian Islands this family is

represented by 13 genera of which four (straussia; Bobea~

Gouldia and Kadua) are endemic.

The genus Morinda consists of about 46 species of

which only two J M. trimeria and Mo citrifolia J are found

in Hawaii.

M. citrifolia is a small tree about 15 feet in

height with a trunk of usually a few inches in diameter;

the leaves are broadly ovate 15 to 20 em. long and 10 to 15

em. wideo The fruit is 7 to 10 em. in diameter and it is

yellow when mature. The ripe fruit has an unpleasant odor

which becomes very fetid when decaying.

2. Medicinal

In Hawaii ~10rinda ci trifo lia.. is known as noni (2).

In the old days Hawaiians used noni fruit for medicinal

purposes and during the famine the fruit was a source of

food. From the mature fruit they extracted an oil of very

unpleasant odor and used it as a hair tonic; as a medicine

for broken bones, cuts J bruises and wounds; and the fruit

i tse If was used as a poultice. 'l'he leave s were used

medicinally against diarrhea and disturbances in men-

struation as well as for fever.

The medicinal value of the Doni fruit was scienti-

fically confirmed in vitro by Bushnell and co-workers (3)

who tested 101 Hawaiian plants for antibacterial activity.

The juice of the Doni fruit was found to be moderately

active against three strains of bacteria; Staphylococcus

aureus, Escherichia coli and Pseudomonas aeruginosa.

Antibacterial activity was also observe~ against five

different strains of enteric pathogens: Salmonella typhosa,

Sal. montevideo, Sal. schottmuelleri, Shigella paradysen­

teriae BH and Shig. paradysenteriae III-Z.

3. Chemical

Before the introduction of synthetic dyes, Doni roots

and bark prOVided yellow and red dyes respectively for

the coloring purposes. The chemical study of the color­

ing matter dates back to 1849 (4) by the discovery of

morindin and morindone. It remained for Thorpe and

Greenall (5) and Thorpe and Smith (6) to prove conclus­

ively that morindone has the formula C15Hl005. Anthra­

quinone structure was suggested for morindune by Simonsen

(7). This was later confirmed by Jacobson and Adams (8)

and Bhattacharya and. Simonsen (9) by the synthesis of

I

3.

The formula, C H 0 4' was only recently established27 30 1

for morindin, which was isolated from Coprosma australis

(10). Morindin was found to be a rhamnoglucoside of

morindone in which sugars are probably present as a

disaccharide attached ~o the ~-hydroxy group of

morindoneo

Besides morindin and morindone other anthraquinone

derivatives have been found in the bark and root of

N. citrifolia. Very recently Bowie and Cooke (11)

isolated nordamnacanthal (II), rubiadin (III) and

rubiadin-l-methyl ether (IV) as the principal products

and a minor component which was believed to be 1,6­

dihydroxy-2-methyl-anthraquinone (V), soranjidiol.

o OHII

f-/,c

oII

II

OH 0t It

C.H3~~

1- ~ II ~IHO~

IIo

III

4.

OHIIo

o1-\0

IV V

Theyalso isolated a compound with structure (VI) which

was presumed to be an artifact produced during the

extraction of damnacanthol (VII) from the plant with

acetone.

oc.H.3

·1o

VI VII

4. statement of the Problem

1~e systematic study of higher plants for the purpose

of detecting antibiotics in their tissues is of compar­

atively recent origin. The discovery of microorganisms

5.

as the causative agents of many infectious diseases of

man created interest in substances toxic to these organisms.

Although the most powerful antibiotic substances are

derived from bacteria, fungi or protozoa, the use of

plants and their extracts as drugs for the treatment of

human diseases has '-:len an age-old practice. Documents,

many of which are of great antiquity, reveal that plants

were used medicinally in China, Egypt and Greece long be­

fore the beginning of the Christian era. The search for

antibiotics in plants has stimulated the curiosity in

man to study their origin and synthesis.

The purpose of the investigation of noni fruits

was twofold. The main objective was to isolate and iden­

tify the antibacterial components of the fruit as indi­

cated by Bushnellfs research (3) and, secondly, if the

compounds were not bacteriologically active, they would

be iuvestigated in order to add some knowledge to the

chemical constituents present in the family Rubiaceae.

6.

B. EXPERIrlIENTAL *

1 0 Bacteriological Testing

The testing procedure was based on the Oxford Method

of penicillin assay (12) with two modifications. The

first variation followed that employed by Bushnell and

co-workers (3) when they originally screened a number of

native Hawaiian plants for antibiotic effect. In this

method, 0.5 ml. of a 24 hour broth culture of a test

organism was inoculated into 10 ml. of melted nutrient

agar which had been cooled to approximately 400 , mixed,

and poured into petri plate and allowed to harden.

* Melting points were taken with fully immersed

Anschutz thermometers. Ultraviolet spectra were measured

in absolute or 95% ethyl alcohol as indicated on a Beckman

DK 2A spectrophotometer. Infrared spectra were recorded

on a Beckman IR 5 instrument with the sample in a KBr

disk or as indicated. Chloroform was used as a solvent

for determining the optical rotation. Microanalyses and

molecular weight determinations (Rast camphor method)

were performed by A. Bernhardt, Mulheim (Ruhr), Germany.

standard porcelain cylinders (penicups) large enough

to accomodate 0.2 m1. of fluid were placed on the agar,

and pressed just deep enough below the surface to prevent

leakage when 0.2 m1. of a test solution was added. The

solution containing the antibiotic substance then diffuses

out into the agar in a circular area, the diameter of

which depends on a number of factors such as the viscosity

of the solution and its solubility in agar medlum. The

substance inhibits growth of the bacteria giving a zone

which was taken as an indication of its activity. The

plates were incubated at 37.50 for 24 hours, and examined

for presence of zones of inhibition.

In instance where a sample was very viscous or solid

and almost insoluble in water, a few milligrams were

placed directly onto the agar. The methods of testing

are indicated respectively as methods a and b.

All bacteriological tests are qualitative and no

especial efforts were made to determine quantitavie1y

the antibacterial potency of the tested samples.

Before the investigation of the noni fruit for

antibacterial activity, Bushnell's work was confirmed

by testing the fresh fruit juice in 1:1 dilution (method

a). The fresh juice showed antibacterial activity against

Salmonella typhosa, Shigella flexnerii, Shigella dysentry,

Pseudomonas aeruginosa, Proteus morganii, Staphylococcus

8.

aureus, Bacillus subtilis, and Escherichia coli, but it

was inactive against Salmonella schottmuelleri.

Since the isolation of any chemical constituent

from the natural sources involves in most cases extraction

with organic solvents at elevated temperature, it was

necessary to test the thermostability of extracted com­

pounds. Thus, the residues from the methanol extract of

dried fruit pulp and seeds were tested for antibacterial

activitYo

The methanol extract from the dried fruit pulp

(tested in 1:1 dilution, method a) was active against

all eight test organisms: Salmonella typhosa, Salmonella

schottmuelleri, Shigella flexnerii, Shigella dysentery,

Pseudomonas aeruginosa, Proteus morganii, Staphylococcus

aureus, Bacillus subtilis, and Escherichia coli.

The methanol 9xtract from the dried seeds was

tested against four test organisms. It was active (dis­

solving 0.9 g. of sample in 5.0 ml. of distilled water,

method a) against Shi5ella flexnerii, Staphylococcus

aureus, Bacillus subtilis, and inactive against Salmonella

txphosao

2. Procurement and Processing of Noni Fruit

The noni plant, M. citrifolia, was kindly identified

by Dr. Lamoureux of' the Department of Botany, University

of Hawaii.

A total of 266 kg. of ripe and half ripe noni

fruit was collected in the area of Waimea and Punaluu

on the island of Oahu. Since a large quantity of fruit

was not available during summer months, the time of

collection ranged from October 1960 to February 1961.

The half ripe fruit was allowed to ripen by placing them

on the floor in the sun light o The ripened fruit was

crushed and pressed through a fruit colander in order

to remove seeds. The fruit pulp and juice which passed

through the colander were separated by filtration. The

fruit pulp 1n small cakes and the seeds were dried at

60-700 in a ventilated oven and then ground for extraction.

From 99 kg. of fresh noni fruit, 4.3 kg Q of dried pulp

material and 3.7 kg. of dried seeds were obtained.

3. Extraction of Fruit Pulp

The complete scheme for extraction of noni fruit

pulp is given in Fig. 1. Dried and ground fruit pulp

(1 0 0 kg.) in 3.0 1. of hexane in 5 1. round-bottomed

flask provided with a condenser with caC12

drying tube and

a glass rod stirrer was refluxed with stirring for 24

hours after which titne the hot solution was filtered.

After removal of solvent, the filtrate afforded 39.0 g.

of liquid residue. The second extraction with hexane for

24 hours yielded 3.0 g. of a semisolid.

10.

Defatted pulp was then extracted three times with

3.0 1. of methanol. The first extraction after 5 hours'

of refluxing and stirring gave 289 g. of black residue l

the second after 8 hours 69 go and the third extraction

after 12 hours of refluxing and stirring yielded 25 g.

of residue o Thus 1 1 0 0 kg. of dried fruit pulp afforded

383 g. of methanol extract.

4. Preliminary Work on th~ Crude Hexane Residue

The crude liquid residue from the previous extraction

was active against Salmonella tyPhosa, Shigella flexnerii,

Bacillus subtilis, and inactive against Staphllococcus

aureUSj whereas the semisolid showed activity against

Shigella flexnerii, Staphylococcus aureus, Bacillus subti­

lis and inactivity against Salmonella typhosa.

The liquid residue (15 g.) was treated with saturated

sodium bicarbonate solution and extracted with ether.

After drying over anhydrous sodium sulfate, the ether

was evaporated to dryness to give 7 go of a neutral

fraction.

11.

Hexane residue

Dried Fruit Pulp

r-- f Extraction with hexane

IDefa tted pulp

Extraction withmethanol

Pulp

Treated with Darcoand Celite

Methanol residue

IChloroform soluble

fraction

Liquid Solid~ (m.p. 226-2700

)

~ rl-------..IAcidic Neutral Unadsorbed Adsorbed

fraction fraction fraction

IAcetylation

IWater soluble

fraction

Chromatography onsilica gel G

Liqgid(b.p. 183 ) at 0.6 mm.)

con. HN03or

20% NaOH

Compound AIII .

~-D-GlucopyranosePen taace ta te(m.p. 132-133~

Compound BIII

AsperulosideTetraacetate(m.p. 152-155')

Phthalic acid

Fig. 1. - Comp Ie te work-up of noni fruit pulp

12.

The aqueous layer was acidified with dilute hydro­

chloric acid and extracted with ether. The ether layer

was dried over anhydrous sodium sulfate and removed under

reduced pressure. The acidic fraction weighed 8 g. No

attempts were made to investigate these two fractions.

A small amount of hexane was added to the semisolid

and then allowed to stand overn~ght in the refrigerator.

A white solid was filtered and crystallized four times

from ethyl alcohol. The amorphous solid melted at 266-2700

and analyzed for C, 78,25; H, 10 0 18; 0, 11,52. The com­

pound was bacteriologically inactive and was not inves­

tigated further.

5. Separation of Methanol Residue into Two Fractions

by a Darco-Ce1ite Adsorption Method *

A suspended black solution of 140 g. of methanol

residue in 200 m1. of water was added to 1 1. of hot water

containing 250 g. Darco. Before the addition, the system

was boiled for a short time to expel air. The mixture

was heated gently to boiling and then allowed to stand at

* The adsorbents used were Darco a-60, an active

carbon from Atlas ~owder Co., and Celite, a high quality

diatomic filter-aid from Johns-Manville Co.

13.

room temperature for 20 minutes with occasional stirring.

Celite (60 g.) was added, mixed thoroughly, heated to

boiling and filtered on a Buchner funnel. The Darco­

Celite cake was carefully washed with water to remove

the unadsorbed material. After removal of water with a

vacuum evaporator at 60-700 there was obtained 74 g. of

colorless viscouse residue which turned brown on standing.

The Darco-Celite cake was added to 800 mI. of ethyl

alcohol in which the big lumps were broken up and then

heated gently to boiling. After heating for about 5

minutes, the mixture was filtered and washed several times

with hot ethyl alcohol. Removal of solvent with a vacuum

evaporator at 60-700 yielded 33 g. of slightly brown

residue which also darkened on standing.

The first fraction which was not adsorbed by Darco

and Celite in water solution showed antibacterial activity

against Salmonella typhosa, Shigella flexnerii, Staphy­

lococcus aureus, and had a stimulating effect on Bacillus

subtilis; whereas the second fraction desorbed from Darco

and Celite was active against Shigella flexnerii, Staphy­

lococcus aureus, Bacillus sUbtilis, and was not active

against Salmonella typhosa.

14.

6. Acetylation of a Fraction Desorbed from the Mixture

of Darco and Celite

A solution of 70 g. of residue dissolved in 550

mI. of anhydrous pyridine was cooled to 00• Acetic

anhydride (500 ml.) was then added portion-wise to the

cold solution while the temperature was maintained be­o

tween 5 to 10. The resulting reaction mixture was

kept in ice-water for 10 more minutes and then allowed

to come to room temperature. After standing for 2 days

at room temperature, the reaction mixture was poured

into 6 1. of ice-water and extracted with. chloroform.

The combined chloroform extracts (2 1.) were washed with

sulfuric acid (10%), water, sodium bicarbonate solution

(10%) and again with water. After drying over anhydrous

magnesium sulfate, the chloroform was removed on a vacuum

evaporator to give 70 g. of acetylated material.

7. Isolation of Compound A and B, and a Hexane Soluble

Fraction from the Acetylated Material

Two preliminary separations were carried out with a

smaller amount of acetylated material from which hexane

soluble fractions and a small amount of a glassy solid

were obtained. The larger run only will be reported.

15.

All hexane soluble fractions obtained throughout the

chromatography of acety1ated material were combined l

dissolved in hexane and filtered. The solvent was removed

and the residue was distilled under reduced pressure.

(See experiment 11).

Acety1ated material (70 G.) from the previous

experiment was chromatographed under slight pressure on

a column of 500 g. of silica gel G with the following

results.

Fraction Eluant Eluate Weight(v/v) (m1. ) (g. )

1 Benzene 1000 1.52 " 1000 1.83 " 500 0.74 II 500 2.05 II 1000 2.4

6 10% ethyl ace ta te90% benzene 800 1,,9

7 II 800 10.08 II 1000 7.69 II 950 2.0

10 II 1000 002

20% ethyl acetate11 80% benzene 800 0.612 " 500 1.113 " 800 305

20% ethyl acetate14 80% benzene 900 4.915 " 1000 3.216 II 800 1.7

16.

Fraction Eluant Eluate Weight(v/v) (ml. ) (g. )

50% ethyl acetate17 50% benzene 900 0.918 II 1000 3.219 II 500 2.020 II 1000 1.221 II 450 0.5

22 ethyl acetate 1500 2.6

23 methyl alcohol 1000 106

Fraction 1 was soluble in hexane. Fraction 2 was

partly soluble J whereas other fractions were insoluble

in hexane. When anhydrous ether was poured over fractions

3 to 7 and the ether allowed to evaporate to dryness J a

white solid formed which was triturated in cold ether

and filtered. The combined solidJ referred to as compound

AJ weighed 6.0 g. and melted at 90-1310 (See experiment 8).

Although the elution with methyl alcohol was not suitable

because calcium sulfate J which is present in the adsor-

bentJis partly soluble in methanolJ nevertheless J the

eluate (fraction 23) was evaporated to dryness under

reduced pressure. Ethyl acetate was added to the residue J

warmed and filtered in order to remove calcium sulfate.

After removal of ethyl acetate the residue was treated

six times with Darco in methanol to remove colored

impurities o The solvent was removed to give 1.6 g. of

viscous residue. The ,\:;otal weight of eluted residues

was 54.2 g.

The second chromatography of separated :ractions, ­

The residues from the filtrates 3-7 and fractions 8-10

were combined (21 g.) and chromatographed on 300 g. of

silica ge 1 G.

Fraction Eluant Eluate Weig11t(v/v) (m1. ) (g. )

1 benzene 800 Owl2 II 750 1.03 II 700 0 0 14 II 700 0.2

10% ethyl acetate5 90% benzene 800 0 0 16 II 800 10 0 97 II 600 3 0 88 II 500 1.7

50% ethyl acetate9 50% benzene 1000 1.6

Fractions 1-4 were soluble in hexane. Attempts to

solidify fractions 5-9 by the addition of ether failed

and only a trace of white solid was obtained melting at

70-1300 • Upon the distillation under reduced pressure,

combined fractions 5-8 decomposed with evolution of gas.

Fractions 11-16 were combined (15 g.) and chroma-

tographed on a column of 300 g. of silica gel G.

Fraction

12

3

Eluant Eluate Weight(v/v) (m1. ) (g. )

10% ethyl acetate90% benzene 900 2.0

" 1 {v",n 0=7.. v.....,_

20% ethyl acetate80% benzene 1200 1.0

Fraction

4567

89

18.

Eluant Eluate Weight(v/v) (m1. ) (g. )

20% ethyl acetate80% benzene 800 3.2

II 420 1.0II 800 2.3II 700 1.4

ethy,l acetate 700 3.. 0700 0.1

Fractions 1 and 2 were soluble in hexane. The

addition of a small amount of anhydrous ether to fractions

4-9 caused viscous residues to solidify. Solids were

triturated in cold ether and filtered. Their melting

points ranged from 130 to 1500• The combined solid

weighed 3.3 g. and was referred to as compound B. (See

experiment 10).

Fractions 17-21 were combined (7.8 g.) and chroma-

tographed on a column of 170 g. of silica gel G.

Fraction Eluant Eluate Weight(v/v) (m1. ) (g. )

20% ethyl acetate1.61 80% benzene 1500

40% ethyl ace ta te2 60% benzene 1000 2.33

11 500 1.04 II 350 0.1

5 50% ethyl acetate50% benzene 250 0.7

6 II 500 0.4

7 ethyl ace ta te 1000 1.6

Fraction 1 was soluble in hexane. Attempts to

solidify other fractions failed.

19.

8. Characterization of Compound A ( -D-Glucopyranose

Pe ntaace ta te )

Compound A (6.0 g.) obtained from the chromatography

of the acetylated material was crystallized five times

from ethyl acetate-hexane. White crystals melted at

132-1330 and weighed 1.6 g. The infrared spectrum (Fig.

2) showed major bands at 5.72 and 8015f (combined acetyl

groups) and 10.92)J (glucopyranose ring) (15). The ultra­

violet spectrum (Fig. 3) exhibited a maximum at 209 mjU

(log E 2.45) in abs. alcohol. [.J-j28 +4.43 (~5.0,D

ch loroform ) 0

On the basis of the chemical analysis and of the

mixture melting point with an 'authentic sample of ~-D-

glucopyranose pentaacetate and by comparison of their

infrared spectra (Fig. 2), compound A was identified as

~-D-glucopyranose peDtaacetate. Lit. values (16):

m.p. 133.5-1340, L.....J 22 +2 0 (~0.9, chloroform);

D

GJ..]~O 1-3.9 (~5.25, chloroform) (17).

Anal. Calcd. for C16H22011: C, 49.23; H, 5.68;

0, 45.09. Mol. wt., 390.34; acetyl (5 groups), 55.12;

C-methyl (due to 5 acetyl groups), 19.25. Found: C,

49.26; H, 5.77; 0, 44.93; Mol. wt., 370; acetyl, 57.68;

C-methyl, 19.68.

9. Prepara tion of i~ -D-Glucopy L'i::W0S8

"":"i !. i 'IE I ! I

I

,I

L.,

I

J,---J

7 " ':':!

,OCO .c::::J =0 2~OO 2'Y.:lO I~CJ '4UO 'JC:'

WA'/EN,. "'er~ eM.~ ');:. '':~': :OJ';

100 v

:: tiS:@ :t+-j~~~j .

i

l·

Ithe

::~G:~!';c~k' m-s~]WJ;~;U'H"fE ~i~rt;~j~~t1~~'- hi~.- ··~1=j j, roJ .. .5 6 7 B ';) 10 if 12 13 14 I) '--'6 0

Fig. 2. - The infrared spectra of ~-D-glucopyranose pentaacetate isolated from •noni fruit (upper), prepared by the method of Wolfrom and Juliann (lower).

21.

r-D-Glucopyranose pentaacetate was prepared accord­

ing to the method of Wolfrom and Juliano (16). A mixture

of 4.1 g. (0.0228 mole) of D-glucose and 10 g. (0.122

mole) of sodium acetate in 70 mI. of acetic anhydride

was gently refluxed for 10 to 15 minutes. The reaction

mixture was slightly cooled, poured into 400 mI. of ice-

water, stirred for 3 hours at room temperature, and ex-

tracted with four 60 mI. portions of chloroform. The

combined chloroform layers were washed twice with water

and dried over anhydrous sodium sulfate. After removal

of chloroform, the residue was dissolved in anhydrous

ether, filtered and crystallized by the addition of

petroleum ether (b.p. 30-600 ) to incipient cloudiness.

The crystals were treated with Darco and crystallized

three times from ethyl acetate-hexane. White crystals

weighed 2.0 g. and melted at 132-1330 • Lit. m.p. 13305­

1340 (16).

10. Characterization of Compound B (Asperuloside Tetra­

acetate)

Compound B (3.3 g.) obtained from the chromatography

of the acety1ated material was crystallized four times

from ethyl acetate-hexane. White crystals melted at

152-1530 and weighed 0045 g. (yield: 2.5 x 10-3%based

on the fresh fruit). If] 28 -133.8 (£.5.0, chloroform).D

2.5 r

2.1

1,7

1.5

210 220 230 240

1V'A VE LENGTH (mp)

Fi~. ~. - The ultravinlet spectrum of

~-D-glucopyranose pertaacetate isolated from

-4the noni fruit. (c= 7.PS x 10 M in abs.

alcohol)

23.

The infrared spectrum (Fig. 4) showed prominent bands

at 5.66f (d.,(~-unsaturated t-lactone) (18); 5.74,8.08

and 8.22ft (combined acetyl groups) and 6001/U (enol

ether) (19, 20). The ultraviolet spectrum (Fig. 5)

exhibited a maximum at 232 mf (log E 3.89) in 95% alcohol.

On the basis of the chemical analysis and of the

mixture melting point with an authentic sample* kindly

provided by Dr. L. H. Briggs, and by comparison of their

infrared spectra (Fig. 4), Compound B was identified

as asperuloside tetraacetate. Lit. values (21): m.p.

154.5-155; the ultraviolet spectrum, 234.5 m~ (log E

3.92) in ~. M/8500 alcoholic solution.

Anal. Calcd. for C26H30015: C, 53.61; H, 5.19;

0, 41.20. Mol. wt., 582.50; acetyl (5 groups), 36.94;

C-methy1 (due to 5 acetyl c;roups), 12.88. Found: C,

53.80; H, 5.19; 0, 41.23. Mol. wt., 537; Acetyl, 36.30;

C-me thyl, 12.00.

11. Preliminary Work on the Acety1ated Liquid

The hexane soluble fraction (24 g.) obtained by

chromatography of the acetylated material was distilled

three times under reduced pressure to give about 10 g.

of colorless liquid boiling at 190-1920 at 1.2 mm. of

pressure ,£7 1. 4851-1. 4853).

* The sample obtained from Dr. L. H. Briggs melted

ol

·1o~fj

:1 1 ,700.:5000 4.000 JCQO 2500 leaD 1500 J.(~ t~CO 1200 1100 1000 900 800

m,-F/UHHlFJ'HIIHhLIFl npJ[':':E!l"'" ll' I' ""1'1,:': olr.f.!:I~I';lt-I,'fl:flf[':L~lloll/,I! ~~f !-l fL!f-tPd2lJP~',f\}c;.I;:-t If; L,lq .1pIt~;i+_r;tTEI

80~;ttirtttt~~fS1f~+==t~tlfSli':~04-~ ~y_cj~"

~.

~--

oIL::'::-:i~,~~r:-<4·::~'f.lt:: !~'c;l:c-· --I: ::O·~lli',~l ',~, n····+' :·:n·: :-:cl.:.::-:- L:;·'kL to :~·~4:-~l·"I:'.f>:",+:;1#?f~::.. " " ~ . '! 12 I] .. : L.-.:.-J L.

"!

;l~'~

.r ;" - I I700800900'000

WAVELENGTH IN MICRONS

1200 11001300..0015002000'2>00JOOO.owLr: L',

100 F~=i'-P':-=~b"~~~E==i:-=~='l:""'=='i'-=-"'-;.t""'''-;''=-f~'f''=t-f''~+"",",''='+'7;..;.-r~"""r-'''i=,r.~--'-:iF~4,",,,,,'7R~=7'rr~-:--+~$.=t=~.;,.;+~~+,-;,-;-,--r;-"-''-'-

I I t

~•

I"

"

,.--;

-- i--

""

::tl"tY,I~:ii;i'-;;~e-=~~-~"tt~i~Vi~~;~tfi~;1f,,~1!;i _'~ ~;i~:i'L~~:' .'; ,i" ,:;tt=20 p~i-.,-,- -··-.1~~~~~~H-'-'r~ '-"-J~t-d~-t:-1--'---;- \--L~j ... L :-7-Y"-f~-,,.f~- ~ ·;~·-l' ::.: : : 1'-::' ." :'~~'" F?-+-'-~ c,..c-c!"::':'~:~ ~~~-f~-'-.l·~~:-+·-J~--i'·c r~"~ '--":l~ ~:..:.!- ~i *:~8, .:~' :~::;'>:'j~..;~~ .._. ..O~~~_-L \ . ,.. '! .• 'L~_ : .: ~Lc_L__.....L_-,--.L~ . i.. .' i' . "L'''' I.', ,,~+".o;: '.;.,;:L'-"::il ::>iT'u; ~~

J .. !. ~ 7 tt 9 !J 11 12 I)

Fig. 4. - The infrared spectra of asperulaside tetraacetate isolated Dram therani fruit (upper), ahtained from Dr. Briggs (lower).

3.894 r-

3.890

:i.RR6

r:J

v 3.882c--:l

8.878

3.874

231 233 2311 2:i7

25.

WA VE LENGTH (mf' )

Fi~. 11. - The uJtravi0let spectrum of

asperuloside tetraacetate isolated from the

-5non; frui t. (c= 8.6 x ln 'r i n Clf)~t alcohol)

26.

A middle fraction, b.p. 1830 a~ 0.6 mm. (n~7

1.4844), from the second distillation analyzed for

C17H2603. The infrared spectrum (Fig. 6) showed major

peaks at 3.4, 5.7~, 6.8, 7.7-7.8, 8.85 and 9. 26)A .

'['he uH;ra'/iolet spectrum (Fig. 7) exllibited two maxima:

225 mi,{ (log E 3.78) and 275 ml.< (log E 2097) in absoI /

alcohol. rJ-l ~7 t 0 00260 C~. 5.0, chloroform).

Anal. Calcd. for C17H2603: C, 73.34; H, 9.42, 0, 17.24.

Acetyl (one group), 15.46; C-methyl (two groups, one

due to acetyl), 10.8 Found: C, 73059; H, 9.47; 0, 17.28.

Acetyl, 12.08; C-methyl, 10.23.

A middle frac~ion from the ~hird distillation did

not give the same results for ~he elemenGs: C, 73.53;

H, 9.67; 0, 16.83.

a. Degrada~ion with Concentra~ed Nitric Acid. - A

mixture of 1.0 g. of acetylated liquid in 10 ml. of con-

cen'~ra ted ni "tric acid was refluxed for 2.5 hours after

which time ehe reaction mixture was cooled in ice-water.

Tile solid which formed was filtered, trea~ed wi~h Darco

and crystallized two times from water. Crystals melted

at 206.0-206.50 (dec.) and weighed 0.2~. The mixture

melting point with an authenGic sample of phthalic acid

was not depressed. The degradation product was further

pro~ed to be phthalic acid by the chemiual analysis

by comparison of the infrared spectrum with that of

y'!':'"

.5'>:'::: .4:..:~ J:O-,}:1 2~:'C 2:':0 U:>.J '.tCO l:!OO 12C~ 1100 1000 9C'J 800 700 6~:

.5 6 7 ; 9 1·:i 11 12 13 1.1 IS

I\<."Vfl.i!'-;GTli lS MICi'C~S

I

Fig. 6. - The infrared spectrum of the acetylated linuid (linuid film).

[\)

~•

3.7

3. Pi

woo~ 3.3

3. 1

2.~

280 2~O 270 2Sl0

lvAVE LEKGTTf (m p)

Fig. 7. - The ultraviolet spectrum of the

acetylated liouid. (c= 2.:111 x 10- 4~f in abs.

alcohol)

28.

29.

phthalic acid.

Anal. Calcd. for C8

H604: C, 57.83; H, 3.64; 0,

38.52. Neut. equi. J 83.06. Found: CJ 58.05; H, 3.61;

0, 38.39. Neut. equiv. 87.5.

b. Degradation with Sodium Hydroxide. - A mixture of

3.0 g. of acetylated liquid in 50 mI. of 20% sodium hydrox­

ide solution (25 mI. of methanol) was refluxed for 3

hours. The methano~ was distilled and the residue was

extracted with ether. The aqueous layer was acidified

with dilute hydrochloric acid and extracted with ether.

After drying over anhydrous sodium sulfate, the ether

was evaporated to dryne ss. The re sidue was tl'ea ted with

Darco and crystallized twice from ethyl acetate. The

crystals melted at 204.5-205.50 (dec.) and weighed

0.5 g. The mixture melting point with an authentic

sample of phthalic acid was not depressed and the infra-

red spectrum was identical with that of phthalic acid.

c. Attempted Ozonization. - A stream of ozone was passed

through a solution of 3.0 g. of acetylated liquid in

50 mI. of methanol for 10 minutes at 00• Water was

added and methanol was removed under reduced pressure.

The residue was extracted with ether and the ether layer

was washed with 5% sodium hydroxide solution and water.

After drying over sodium sulfate, the ether was removed

to give 2.9 g. of residual liquid. The infrared spectrum

30.

of the crude liquid was identical with that of the start-

ing material.

12. Isolation of Caproic and Caprylic Acid from the Ripe

Noni Fruit

Ripe yellow fruit (6 kg.) was sliced with a knife,

water was added and steam distilled. The distillate

was extracted with 2 1. of ether. The combined ether

layers were concentrated to 500 mI. and washed with sat-

urated sodium bicarbonate solution. After drying over

anhydrous sodium sulfate, the ether was evaporated to

dryness to give 2.0 g. of neutral residue which was not

investigated further.

The aqueous bicarbonate solution was acidified with

dilute hydrochloric acid and extracted with ether. After

drying over anhyrous magnesium sulfate, the ether was

evaporated to dryness and the residue was distilled under

reduced pressure.

Fractions boiling between 115 and 1180 at 8.7 mm.

(n~8 1.4237-1.4239) were combined (1.8 g.) and converted

in the usual manner to the corresponding amide, m.p.

51-530 and anilide, m.p. 103-1050 • On the basis of

chemical analysis and by comparison of their respective

melting points with those in the literature, 550 and 1060

(13), the acidic portion was characterized as caprylic

acid.

Anal. Calcd. for C8H17NO: C, 67.08; H, 11.97;

N, 9.78. Found: C, 67.37; H, 11.94; N, 9.87.

Calcd. for C 4H NO: C, 76.66; H, 9.65; N, 6.39; Found:1 21CJ 76.47; H, 9.49; N, 6.52.

31.

Similarly, fractions boiling between 890 and 95°

at 8.7 mm. (n~8 1.4149-1.4168) were combined (1.0 g.)

and converted to the corresponding anilide, m.p. 93-95°,

It analyzed for caproic acid anilide and its melting

point was in agreement with that of the literature

value, m.p. 96°, (13).

Anal. Calcd. for C H NO: C, 75.35; H, 8.96;12 17

N, 7.32. Found: C, 75.52; H, 9.10; N, 7.20.

32.

C. DISCUSSION

1. Attempted Isolation of Chemical Constituents from

the Methanol Residue

In view of antibacterial activity and by choice,

the investigation of the methanol residue from the ex­

traction of dried fruit pulp was undertaken. The residue

was found to be active against nine tested microorganisms:

Salmonella typhosa, Salmonella schottmuelleri, Shigella

flexnerii, Shigella dysentery, Pseudomonas aeruginosa,

Proteus marganii, Staphylococcus aereus, Bacillus subtilis

and Escherichia coli.

Two approaches to the isolation of antibacterial

substances were considered. The first approach was to

separate the methanol residue into smaller distinct

fractions by column chromatography using a variety of

adsorbents if necessary, and to test their antibacterial

activitYo The loss of activity in fractions or the

failure of isolation of chemical constituents would un­

doubtedly lead to the second approach. The latter was

to stabilize the antibiotic substances by conversion to

their acetylated derivatives in the hope that the deri­

vatives could be later hydrolyzed to their parent sub­

stances as they are found in the fruit. Since most of

the acetylated derivatives are insoluble in water" the

33.

acetylation would also enable us to separate the acetyl

derivatives from those which do not react with acetic

anhydride and which are soluble in water.

Although the second approach seemed to be practical

and attractive, the first approach was reluctantly chosen

for two reasons. Acetylation would perhaps deactivate

antibiotic substances and consequently the tracing of

antibiotics during the isolation would be extremely

difficult. Secondly, the acetylated substances might

be unstable to the conditions of hydrolysis of the

acetyl groups. Unfortunately, however, all attempts to

isolate chemical constituents by the first approach

were unsuccessful. Elution of the crude methanol residue

with chloroform in which the methanol content was

gradually increased and with methanol over silica gel,

florisil and neutral alumina gave bacteriologically active

fractions in which the same activity was observed. The

rechromatography of fractions did not change the appear­

ance of the dark brown fractions nor their bacteriological

activity. The solubility of the methanol residue in

water limited the use of a variety of organic solvents

in the chromatography and in the attempted crystalli­

zations.

Preliminary ion exchange chromatography using

Dowex 50-x8 and Amberlit IR-4B did not seem to be promising.

34.

It became apparent that the procedure for the isolation

of antibiotic substances had to be modified or changed

completely.

2. Isolation of an Unknown Liquid, ~-D-Glucopyranose

Pentaacetate and Asperuloside Tetraacetate from the

Acetylated Material

It was realized that the presence of sugars in the

methanol residue might be mostly responsible for the

above-mentioned failures, and methods by which sugars

can be successfully removed were sought. Whistler and

Durso (22) had successfully separated monosaccharides

from disaccharides by charcoal column chromatography

where the ethyl alcohol content was gradually increas~d

in the water eluant. Furthermore, Trim and Hill (14)

had successfully separated glycosides from crude plant

extracts by a charcoal adsorption method. This involved

heating the plant extract with a large amount of charcoal

and Kieselguhr in water solution and desorbing the

glycosides from the mixture of charcoal and Kieselguhr

with ethyl alcohol. It was hoped that most of the sugars

and materials which are very soluble in water would not

be adsorbed on charcoal, whereas the organic materials

which are less soluble would be adsorbe~ and later re-

covered by heat1ug the charccnl in boiling alcohol:

The separation of the methanol residue into two

35.

fractions was successfully accomplished by the charcoal

adsorption method using Darco and Ce1ite. The separation

was judged by bacteriological testing and by a glycoside

test. The fraction which was not adsorbed on Darco and

Ce1ite showed antibacterial activity against Salmonella

typhosa, Shi~e11a f1exnerii, Staphylococcus aureus and

had a stimulating effect on the growth of Bacillus sub­

ti1is. The fraction deadsorbed from the mixture of Darco

and Ce1ite was active against Shigella f1exnerii, Staphy­

lococcus aureus, Bacillus subti1is and was inactive

against Salmonella typhosa. The desorbed fraction gave

a deep blue color with Trim's reagent* (14) which is

characteristic for aucubin and its related glycosides.

It was finally decided to investigate the desorbed

fraction because it was expected to contain no sugars.

The absence of sugars would undoubtedly facilitate the

isolation of chemical constituents from a mixture.

Although there are only a few references in the liter­

ature for glycosides possessing antibacterial properties

(23,24), it was decided to isolate the glycoside which was

detected by Trim's reagent.

* Trim's reagent: glacial acetic acid, 10 vo1s.; 0.2%

CuS045H20, 1 vol.; conc. hydrochloric acid, 0.5 vol.

36.

Several methods of glycoside separation cited in

the literature (25) were tried unsuccessfully. Therefore,

it seemed necessary to acetylate the fraction in the hope

that the free glycoside as it occurs in the fruit could

be recovered by removal of the acetyl groups. The

complete procedure by which the acetylated compounds were

isolated is given in Fig. 1.

The acetylation was accomplished with acetic anhy­

dride in pyridine. The acetylated material showed anti­

bacterial activity against all three test organisms:

Bacillus subtilis, Staphylococcus aureus and Escherichia

coli.

Elution of the crude acetylated material with benzene

over silica gel G gave mostly a hexane soluble liquid

which was bacteriologically inactive. The liquid could

not be purified by simple distillation. This was judged

by the inconsistent elemental analyses. Treatment with

concentrated nitric acid and alcoholic sodium hydroxide

yielded phthalic acid, but the liquid did not react with

ozone.

Elution with benzene and with a mixture of 10%

ethyl acetate and 90% benzene gave an acetylated sugar,

0-D-glucopyranose pentaacetate, m.p. 132-1330 , which was

also bacteriologically inactive. The identity of the

sugar acetate was established by chemical analyses, by a

mixture melting point with an authentic sample prepared

37.

by a known method (16) and by comparison of their infra­

red spectra (Fig. 2). With the isolation of 0-D-g1uco­

pyranose pentaacetate~ it is apparent that the separation

of sugars by the charcoal adsorption method was not

complete.

Elution with a mixture of 20% ethyl acetate and

80% benzene yielded a glycoside~ m.p. 152-1530~ which

gave a deep blue color with Trim's reagent. [J..l~8 -133.80

(£ 5.0~ chloroform). The empirical formula was found to

be C26H300l5. It showed the presence of 5 acetyl groups

and 5 C-methyl groups (due to acetyl groups). The infra­

red spectrum (Fig. 4) showed bands at 5.66f' ((~'0-unsat­

urated i-lactone) (18), 5074, 8.08 and 8.22jU (combined

acetyl groups)~ and 6.0l? (enol ether) (19,20). The

ultraviolet spectrum (Fig. 5) exhibited a maximum at 232

mjU (log E 3.99) in 95% ethyl alcohol. The identity of

the glycoside as asperuloside tetraacetate (VIII)

VIII

was established by chemical analyses, by a mixed melting

point with an authentic sample obtained from Dr. L. H.

Briggs and by comparison of their infrared spectra (Fig.4).

38 •.

3. Asperuloside as the Possible Antibiotic Substance

in the Noni Fruit

Although the chemical and physical properties of

asperuloside have been known for years, its correct

structure was determined only recently by Grimshaw (26).

The glucoside, asperu10side (IX), has been regarded as a

0- c.:.o

¢OCH1..0Ac. OGluC05e-

IX

characteristic product of plants in the family Rubiaceae

(21,26-37) until it was isolated for the first time in

1951 from Daphniphyl1um macropodum, a Chinese plant of

uncertain affinity placed in the family Euphorbiaceae

(38,39). A large scale isolation of asperuloside from a

variety of plants was undertaken by P10uvier (40) in 1956

who isolated ~he glucoside from ten species and hybrids

of Esca11onia.

Asperuloside is chemically related to aucubin, a

glucoside (X) (14). Its structure was only recently

determined (41,42) and confirmed (43,44) by several

workers. Aucubin is an antibacterj.al substance (24)

39.OH

roC\-\:IPH OGluC05C2..

X

and active against Staphylococcus aureus, Escherichia

coli, Bacillus sUbtilis, Mycobacterium ph1ei, Ophiostoma

paradoxum, Usti1ago nuda and Penici11um ita1icum.

Asperu10side tetraacetate, however, was found to be in-

active against three tested organisms: Bacillus sUbtilis,

Staphylococcus aureus· and Escherichia coli. The inact-

ivity to three tested microorganisms may be due to the

fact that the acetyl derivative of asperu10side is not

soluble in water. According to Briggs and Cain (21)

asperu10side tetraacetate cannot be hydrolyzed to its

original structure as it is found in the fruit because

both acid and base hydrolysis of the acetyl groups

leads to the decomposition of asperu1oside. Because of

the labile nature of asperu10side structure, bacterio10-

gica1 tests on asperu10side itself could not be carried

out as was originally intended.

In view of the fact that asperu10side is an

aucubin-type glucoside and it contains an unsaturated

lactone which is present in many antibacterial substances

(23), such as protoanemonin (XI), anemonin (XII), kawain

(XIII), parasorbic acid (XIV), patulin (XV), penici11ic

acid (XVI), etc., (Lf5) it is reasonable to believe that

40.

oIol o1'l. 0 0

0/' h- ~ ~

XI XII 0

< }C~:CHO~OXIII

;~~o

C~JOHoXV

C.4II 10H

CH - c.--t--lr0 C.H 3

J OJit

XVI 0

asperuloside as it occurs in the fruit has some anti-

bacterial properties. According ~o Briggs and Cain (21),

extensive bacteriological testing of asperuloside has

revealed. no outs~anding antibacLerial properties.

4. Miscellaneous

Steam distillation of ripe yellow fruit yielded

caproic and caprylic acid. The acids are probably re-

sponsible for an unpleasant odor in the ripe fruit.

Caprylic acid was bac1eriologicallf inactive.

Hexane extraction of dried noni fruit pulp afforded

a liquid re sidue and a high me 1ting solid. rrhe crude

41.

liquid residue was active against Salmonella typhosa,

Shigella flexnerii, Bacillus subtilis and inactive against

Staphylococcus aureus; whereas the solid was bacterio­

logically inactive and was not investigated further.

The liquid residue, l10wever, was separated into acidic

and neutral fraction with sodium bicarbonate, but no

attempGs were made to investigaGe these fractions.

42.

D. SUMMARY AND CONCLUSION

The methanol residue from the extraction of dried

noni fruit was investigated for antibacterial substances.

Preliminary experiments~o isolate chemical substances

directly from Ghe residue by column and ion exchange

chromatography were unsuccessful. Acetylation of the

methanol residue followed by chromatography over silica

gel G yielded three compounds: an unknown liquid, b.p.

183° at 0.6 mm., ~-D-glucopyranose pentaacetate, m.p.

132-1330 and asperuloside tetraacetate, m.p. 152-153°.

The unknown liquid was bacteriologically inactive and

it was not identified. Inconsisten0 elemental analyses

indicated that the liquid could not be purified by dis­

tillation. On treatment with concentra~ed nitric acid

and with sodium hydroxide, phthalic acid was isolated,

but the liquid was resistanc to ozone.

The acetylated sugar, ~-D-glucopyranose penta­

acetate, was also bacteriologically inactive and no

attempts were made to hydrolyze it. The third compound,

asperuloside te~raacetaGe, exhibited no activity either.

Asperuloside itself could not be tes~ed for antibacterial

activity because its structure is very labile to the

condition of hydrolysis of the acetyl groups in the sugar

moiety. In view of the fact that asperuloside is an

aucubin-type glucoside which is bacteriologically active,

and it contains an unsaturated lactone which is presentI

in many antibiotics, it is reasonable to believe that

asperuloside as il occurs in the frui~ migh0 have some

antibacterial properties.

42a.

E. BIBLIOGRAPHY

1. Josep F. Rock" "The Indigenous Trees of the HawaiianIslands,," Published Under Patronage" Honolulu"1913" p. 467.

2. Otto Degener, "Planes of Hawaii National Park,,"Honolulu Star-Bulletin, Ltd." Honolulu" Hawaii"1930 pp. 282-286.

3. The antibacGerial properties of some plants foundin Hawaii. O. A. Bushnell, Mitsuno Fukuda" andTakashi Makinodan. Pacific Sci. ~, 167-83 (1950)

4. Ueber den Farbstoff der Morinda citrifolia. Th.Anderson. Ann.·, 71, 216-24 (1849).

5. On morindin and morindon. T. E. Thorpe and T. H.Greenall. J. Chem. Soc., 21, 52-8 (1887).

6. On morindon. T. E. Thorpe and William J. Smith.ibid. 53, 171-5 (1888).

7. Morindone o John Lionel Simonsen. ibid." 113, 766-74(1918) •

8. Trihydroxy-methylanthraquinones. V. Synthesis ofmorindone. R. A. Jacobson with Roger Adams. J.Am. Chem. Soco, 47" 283-90 (1925).

9. Synthesis of morindone. Ramkanta Bhattacharya andJ. L. Simonsen. J. Indian Inst o Sci." lOA, 6-9(1927) •

10. Chemistry of the Coprosma genus. Part I. The colour­ing mat~ers from Coprosma australis. Lindsay H.Briggs and Jack C. Dacre. J. Chem. Soc." 564-8(1948).

11. Colouring matters of Australian plants. IX. Anth­raqunones from Morinda species. J. H. Bowie andR. G. Cooke. AustralIan J. Chem., 15" 332-5 (1962).

12. Further observations on penicillin. E. P. Abraham"E. Chain" Co M. Fletcher, A. D. Gardner, N. G.HBRtley~ M. A. Jennings. Lancet, 241" 177-88 (1941).

13.

14.

15.

17.

18.

19.

44.

Samuel M. McElvain, "The Characterization of OrganicCompounds," 2nd Ed., The MacMillan Co., New York,N. Y., 1953, p. 191.

The preparation and properties of aucubin, asperu­loside and some related glycosides. A. R. Trimand R. Hill. Biochem. J., 2.Q., 310-6 (1952).

Melville L. Wolfrom, "Advances In CarbohydrateChemisi~ry," Vol. 12, Academic Press Inc., Publishers,New York, N. Y., 1957, p. 23.

Chondroitin sulfate modifications. I. Carboxyl­reduced chondroitin and chondrosine~ M. L. Wolfromand Bienvenido O. Juliano. J. Am. Chern. Soc., 82,1673-7 (1960). -

A comparison of the optical rotatory powers of thealpha and beta forms of certain acetylated deriva­tives of glucose. C. S. Hudson, J. K. Dale. ibid.,37, 1264-70 (1915). ----

L. J. Bellamy, "The Infra-red Spectra of ComplexMolecules," 2nd Ed., John Wiley and Sons, Inc.,New York, N. Y., 1959, p. 187.

Infra-red adsorptions of vinyl and isopropenyl groupsin polar compounds. W. H. T. Davison and (in part)G. R. Bates. J. Chern. Soc., 2607-11 (1953)0

20. The infrared adsorption of vinyl ethers. G. D.Meakins. ibid., 4170-2 (1953).

21.

22.

23.

.24.

Chemistry of the Coprosma genus. Part IX, Theconsti tU-Gion of asperuloside. Lindsay H. Briggsand B. R. Cain. ibid., 4182-93.

Chroma tographic separation of sugars OrJ Charcoa 1.Roy Lo Whistler and Donald F. Durso. J. Am. Chern.Soc., ~' 677-9 (1950).

K. Paech and M. V. 'rracey, "Modern Me thods of PlantAnalysis," Vol. III, Springer-Verlag, Berlin, Germany,1955, pp. 626-725 and references cited therein •

The chemical nature of uhe antibacterial substancepresent in Aucuba javonica Thunbg. J. E. Romboutsand J. Links •. Experientia, 12,78-80 (1956).

26.

28.

29.

30.

31.

32.

33.

34.

35.

36.

structure of asperuloside. J. Grimshaw. Chem.& Ind. (London), 403-4 (1961).

C. A. 19, 29702 (1925). Asperu1osid, a new gluco­side extracted from Asperula odorata. H. Herissey.Compt. rend., 180, 1695-7 (1925).

C. A. 20, 16467 (1926). The chemical compositionof Asperula odorata. Extraction and proyertiesof a new glucoside, asperu1oside. H. Herissey.Bull. soc. chim. bioI., 7, 1010-6 (1925).

C. A. 20, 2182 5 (1926). Detection of asperu10sidein plants. Extraction of this glucoside from Caliuma arine L. H. Herissey. Compt. rend., 182, 865-7

192 • ---

C. A., 27, 51489 (1933). Extraction of asperulosidefrom Coprosma baueriana Hook. H. Herissey. J.pharm. chim., 17, 553-6 (1933).

C. A. 27, 58909 (1933). Extraction~of asperulosidefrom Coprosma baueriana Hook. H. Herissey. Bull.soc. chim. bioI., 15, 793-5 (1933).

C. A. 21, 30694 (1927). Extraction of asperu10sideof GalIUm verum L. Probably presence of this gluco­side in a number of species of Rubiaceae. H. Herissey.Compt. rend., 18L~, 1674-5 (1927).

C. A. 22, 11761 (1928). Extraction of asperulosideof Gallium vernum L. Probably presence of thisglucoside in many of the species of Rubiaceae. H.Herissey. Bull. soc. chim. bioI., 9, 953-6 (1927).

c. A. 21, 1148 (1927). Asperuloside in plants.Extraction of this glucoside from Galium aparine L.H. Herissey. ibid., 8, 489-96 (1926).

c. A. 3s, 70769 (1938). Extraction and localizationof asperuloside noted in Crucianella martima L. andC. angustifolia L. A. Jui1let, J. Susplugas andV. Massa. J. pharm. chim., 27, 56-62 (1938).

Chemistry of the Corposma genus. VIII. The occurrenceof asperuloside. Lindsay H. Briggs and G. A. Nicholls.J. Chem. Soc., 3940-3 (1954).

46.

37. 'rne accumula tion and utiliza tion of auperulosidein the Rubiaceae. A. R. Trim. Biochem. J., 50,319-26 (1952). --

38. Occurrence of asperuloside in Daphniphyllum macropodum(Euphorbiaceae) and a closely related ~lucoside inMonotropa~ Walt. (Pyrolaceae). A. R. Trim.Nature, 161, 4~5 (1951).

39. The preparation and properties of aucubin, asperulo­side and some related glycosides. A. R. Trim andR. Hill. Biochem. J.,. 50, 310-9 (1952).

40. C. A. ~ 10985b (1956). The presence of asperulo­side in Escallonia and of dulicitol in Brexia mada­gascariensis {Saxifrage). Victor Plouvier. Compt.rend., 242, 1643-5 (1956).

410 Structure of aucubin. J o Grimshaw and H. R. Juneja.Chem. & Ind. (London), 656=7 (1960).

42. Structure of aucubin. S. Fujise, H. Obara and Ho

Uda. ibid•. 289-90 (1960).

43. Aucubin. A. J. Birch, J. Grimshaw and H. R. JunejaoJ. Chem. Soc., 5194-8 (1961).

44. Die Struktur des Aucubins. W. Haegele, F. Kaplanand H. Schmid. Tetrahedron Letters, 110-8 (1961)0

45. Alfred Burger, "r~edicinal Chemistry, II 2nd Ed.,Interscience Publishers, Inc., New York, N. Y.,1960, pp. 951-952.

TABLE OF CONTENTS

PART II. 'l'HE STRUCTURE OF THE NITROCAr~PHOR ANHYDRIDES

LIST OF FIGURES •••••••••• 0.00 ••••••• 0•••••••••••••••• iii

A. INTRODUC1'ION. • • • • • • • • • • • . • • • • • • • • • • • • • • • • • • • • • • •• 1.

B. EXPERIMENTAL •••••• o ••••••••••• o.e ••• o •••• G ••••••• 4.

10 Preparation of 3-Nitrocamphor •••••••••••• 4.

2. Prepara~ion of the Nitrocamphor

Anhydrides ••••••..•••••••••••• 0 •••••••••• 6.

30 Attempted Ozonization of

Nitrocamphor Anhydride ••••••••••••••••••• 22.

4. A~tempGed Oxidation of Nitrocamphor

Anhydride with Potassium Permanganate ... 22 .

5. Preparation of Phenylni~romethane ••••••• 23.,-o. Action of Formamide on Phenylnitro-

methane •. e 0 ••••• 24.

7. Prepara 'cion of Diphenyl Urea ............ 24.

8. Preparation of Benzoyl Cyanide .......... 25 .

9. Prepara tion of ~-NiGroacetophenone ..... 26.

10. Action of Hydroxylamine on

~ -Nitroace~ophenone ••••••••••••••••••• 26.

11. Preparation of Benzohydroxamic

Acid •••..•••..•••••.••.•. 0000 ••••••••••• 27.

C. DISCUSSION • 0 •••••••••••••••••••••• 0 •••••••••••••• 290

ii

1. ~::8 Strue l,ure of the Nicrocamphor

Anhydrides •• 0.0 ••• 0 ••••••••••••••••••••••• 29.

2. The Possible Nitro-Nitroso

Intermediate in ~he Conversion

of Nitro Compounds to Furoxanes •• 0 ••••••••

3. Miscellaneous ••...•• 0 •••••••• 0 •• 0 ••••• 0 •••

••• 0 ••••• 00.0 ••••••••••••••Do

E.

SUM~~RY AND CONCLUSION

BIBLIOGRAPHY ... o •••••••• • ••• 0 ••••••••••••••• 0 ••••

34.

46.

49.

52.

LIST OF FIGURES

Fig. 1. The nuclear maGnetic resonance

spectrum of 3-nitrocamphor,

m. p. 105-107° 0 ••• 0 • • • • • • • • • • • r( •

Fig. 2. The infrared spectrum of 3-nitrocamphor,

m.p. 105-107° 0 •••••••• 0 ••••••••••••• 8.

Fig. 3. The nuclear magnetic resonance spectrum

of nitrocamphor anhydride, 170.5-

Fig. 4. The infrared spectrum of niGrocamphor

anhydride, m.p. 170.5-171.50 (dec.) ••••••••• 11.

FiG' 5. The ultraviolet spectrum of nitrocamphor

iii

anhydride, m.p. 170.5-171.50 (dec.)

(c= 1.135 x 10 -3 M in abs. alcohol) ........ 12 •

F', ~

lb. o. The ultraviolet spectrum of nitrocamphor

anhydride, m.p. 170.5-171.50 (dec.)

(c:: 1.135 x 10-4 N in abs. alcohol) • • 0 •••••• 13 •

Fig. 7. The infrared spectrum of nitrocamphor

anhydride, m.p. 158-1600 (dec.) ••••••••••••• 15.

Fig. 8. The ultravio1e~ spectrum of nitrocamphor

anhydride, m.p. 158-1600 (dec.)

(c =-1.135 x 10-3 M in abs. alcohol) • • • • • • • 0 • 16•

Fig. 9. The ultraviolet spectrum of nitrocamphor

anhydride, m.p. 158-1600 (dec.)• I.

(c= 2.27 X 10-'-!- f-1 in abs. alcohol) •••••••••• 17.

...........Fig. 10. The infrared specl:.rum of nitrocamphor

anhydride, m.p. 190.5-1920 (dec.)

Fig. 11. The ultraviolet spectrum of

iv.

19•

nitrocamphor anhydride, m.p. 190.5-1920

(dec.) (c ~1.135 x 10-3 M in abs. alcohol) •• 20.

FiC. 12. The ultraviolet spectrum of nitrocamphor

anhydride, m.p. 190.5-1920 (dec.)

(c ::-1.135 x 10-4 M in abs. alcohol) ••••••••• 21.

A. INTRODUCTION

A nitroca~phor 2~hydride, C20H28N205' was first

prepared by u)wry (1) in 1898 by heating an alcoholic

solution of 3-nitrocamphor to dryness on a steam bath.

Analyses and molecular weight determination indicated a

structure derived from the condensation of two molecules

of 3-nitrocamphor with the elimination of water to which

Lowry assigned structure (I), mop. 1900, GLl ~l +1870

~ 5.0, benzene).

In 1903 Lowry (2) reported another compound, m.p.

220°, ~J~5t26.4° (£ 2.9, acetone), which showed the

composition of a nitrocamphor anhydride. This compound

was a by-product from the preparation of camphoryloxime

resulting from the treatment of nitrocamphor with concen­

trated hydrochloric acid. structure (II) was assigned to

the new compound, camphoryloxime anhydride.

Twelve years later in 1915 Lowry (3) reported a

third compound, m.p; 1840

, ~]5761 _60 and ~]5780 _4 0

(£ 1.3, benzene), which analyzed for a nitrocamphor anhy­

dride, C20H28N205and to whicll he assigned structure (III).

........... CSH14""" .CSH14-:----CO C:N-O-N=C.... _ ................ CO

-........0............... "o~

II

i,:fc N-0-6~I%JC8~.c{ ~H14

.~ ~

'0 O'

III

2.

The new isomer was prepared either by refluxing nitro-

camphor or its ammonium sa 1 t in ethyl ace ta 1.;e in the

presence of formamide. Lowry recognized that the differ-

ence between the two nitrocamphor anhydrides (I) and (III)

was due to stereoisomerism and not to a difference in

skeletal struc~ure. In view of the low optical activity

of s~ructure (III) he suggested that the twO nitro groups

are acting in opposition, whereas in I they are both con-

tribuc.ing toe-he large dex·,~roro~atory power of the compound.

Although the aliphatic nitro compounds have been

known since lS72 (4), their structure was only recently

established by Kornblum and his co-workers (5). Lowry's

structures of the nitrocamphor anhydrides must be consid-

ered as derivatives of nitronic acids which decompose

rapidly on being heated (6). The stability of the nitro­

camphor anhydrides suggests then that formulas (I) and

(III) do not correctly represent their strucGures.

In the preliminary stage of the present study

structures (IV) and (V) seemed to be reasonable alterna-

tlves to wwry:s pr'uputjals.

IV V

3.

4.

B. EXPERIMENTAL*

1. Preparation of 3-Nitrocamphor

Lowry's method was used for the preparation of

3-nitrocamphor (1). A mixture of 260 g. (1.125 moles)

of d-bromocamphor in 750 ml. of concentrated nitric acid

in a 3 1. three-necked round-bottomed flask was refluxed

with stirring for 25 hours. The resulting dark brown

mixture was added to 2 1. of ice-water and extracted with

three 500 m1. portions of ether. The combined ethereal

extracts were washed with water and poured into a 3 1.

~hree-necked round-bottomed flask provided with a stirrer.

* Melting points were ~aken with fully immersed

Anschutz thermometers. Ultraviolet spectra were measured

in absolute or 95% ethyl alcohol as indicated on a Beckman

DK 2A Spectrophotometer. Infrared spectra were recorded

on a Beckman IR 5 instrument with the sample in a KBr

disk. Benzene was used as a solvent for determining the

optical rotations. Microanalyses were performed by A.

Bernhardt, Mulheim (Ruhr), Germanyo The nuclear magnetic

resonance spectra were obtained on an A-60 High Resolution

Spectrometer with tetramethylsilane as an internal re­

ference.

5.

Saturated sodium bicarbonate solution was added cautiously

to the slowly stirred echerea1 solution until the evolution

of carbon dioxide ceased. The ether layer was washed

once more with saturated bicarbonate solution and then

three times with water. After drying over anhydrous

magnesium sulfate, the ether was removed under reduced

pressure to give 149.5 g. of yellow semisolid, 3-bromo-

3-oi trocamphor.

The crude bromonitrocamphor (149.5 g.) 0.543 mole)

was dissolved in 370 m1. of hot absolute e~hyl alcohol

and poured into a 3 1. three-necked round-bot~omed flask

fitted with a stirrer and condenser with CaC12

drying

tube. Sodium ethoxide prepared from 25 g. (1.09 moles)

of sodium dissolved in 300 m1. of hot absolute e~hy1 ~lco­

ho1 was added to the solutimof bromonitrocamphor. The

addition was exothermic and it was necessary to control

the cemperature by adding sodium ethoxide solution in

small portions. After the addition was completed, the

resulting thick reaction mixture was stirred for an addi-

tiona1 hour at room temperature, cooled and filtered.

The sodium salt of 3-nitrocamphor was dissolved in water

and extracted with ether. The aqueous layer was cooled

to 5°, acidified with dilute hydrochloric acid and extrac­

~ed with ether (600 m1.). The combined ether portions

were washed with water and dried over anhydrous magnesium

6.

sulfate. Upon removal of ether there was obtained 65 g.

of crude yellow semisolid which after two crystalliza-

tions from ethyl alcohol-water gave 37 g. of 3-nitrocamphor,

m.p. 99-1000, (overall yield: 17%)

Fur~her crystallization from a mixture of ethyl

alcohol and water, and final crystallization from petro-

point to

-108.4 C£. 5.0,

(£ 5.0, benzene).

leum ether (b.p. 60-1000) raised the melting

r 128105-1070 (lit. m.p. 1020 (1), L~ D

benzene), lit. value ~J21_104° (1)D

The nuclear magnetic resonance spectrum (Fig. 1) showed

peaks at d~5.1, 2.75'. 1.07, 0.98, and 0.91. 'l'he infra­

red spectrum (Fig. 2) showed absorption at 5.71jU (car­

bonyl) and 605~(nit~o); the second band assigned to the

nitro group in the 7.0-7.~jU region appeared as a shoulder

(7.35)J ) on the me l.;hyl band.

Anal. Calcd. for CIOH15N03: C, 60.89; H, 7.67;

N, 7.10. Found: C, 61.13; H, 7.55; N, 7.28.

2. Preparation of the Nitrocamphor Anhydrides

A solution of 37.0 g. (0.188 mole) of 3-nitrocam­

phor and 9.25 g. (0.206 mo le) of formamide in 95 m1. of

ethyl acetate was maintained Qt the reflux temperature

for 61 hours, and the volatile solvent was removed on a

vacuum evaporator. The resulting yellow residue was

rT.:r

.Y50

~ ,

~~~ V -.J

11: I:: :. ~;r j' ':[; : I,11 ;1 JlIi III, . ,IIi I "I : flI· .'I' I,;;; :

I.' '

1;1 ;

J~~i',;~~~

8.0 7.0 6.0 5. 0 ppi'f lcf' 4.'0 3.0 2.0 1.0 o

Fig. 1. - The nuclear magnetic resonance spectrum of~nitrocamphor, m.p. 105-107°.-...;]

•

.......VENU,y.6f;; C'"

------! -

---------I.i

--- -----.j ,

I---,\:::?-:!:. -. -l-'--r~"-F nL ~ __ L _: _I _:- .. _: ~~:c..-":J.: __ l __ J_"":'" ...J. '------' _:~ 1) I.l

5C::!O .£000 3000 2'00 2000 I~QC LtC) I' l]v:.; n.:iJ . X~ ::::: ilC'!J l00

'::~~rc~'!E:"'o~!~/ ~:::~F-"~.".Y-j,r~"=~l==~T':Ef}~E'~I-Fl~"~I! ";'~R.j:--~-:-~t--V _.. <t:,--oC:~<-~~---:t--:'A - r:---/~V\!;~~ /~--\/lJl-:-'-j:J.j__ --_i--_----- J ;l:, \--'- L r~' ,

::S-:+~:fc-lH~'-~~;-'H\},~;1'1"f;i!\!J ~'j,-=---CII- L ! i_ ' i "Ii,.--'-7-f'-----o-~~.+__-,- ,--H--'- - -J, ~!--=--llf--';T 1__ - -,- - -- -1-: -- 1_ -- ;__1. - .----- ! r--t --7- -

·_":--t 1-': III V t· ,'! 1 I· r ,

,., ... ' .. ~' ... :.::1 I" "\00,;·:;10:-; ~

Fig. 2. - The infrared spectrum of 3-nitrocamphor, m.p. 10~-107°.

co

9.

dissolved in ether and washed with water to remove

formamide. After drying over anhydrous magnesium sulfate,

the e~her was removed 10 give 31.0 g. of crude semisolid.

Ethyl alcohol was added and the solution was allowed to

~tand overnigh~ in the refrigera~or. After filtration

there was obtained 7.1 g. of crude solid, m.p. 135-1450

(dec.). It was partly dissolved in hexane and filtered.

rrhe solid which was insoluble in hexane was crystallized

twice from ethyl alcohol to give 3.0 g. (8.5%) of analy­

tically pure nitrocamphor anhydride, m.p. 170.5-171.50

(dec), ~l ~7 + 9.670 (£. 5.0, benzene). The molecular

weight by isothermal distillation was found to be 362

(calcd. 376) •. The nuclear magnetic resonance spectrum

(Fi s . 3) showed peaks at J~3.75, 2.72, 1.07, 0.98, and

0.91. The infrared spectrum (Fig. 4) snowed bands a~

5.65 and 5.71? (carbonyl), 6.12f (nitroso), 6.45 and

7.4l)U (nitro). The ultraviolet spectrum (Fig. 5 and 6)

showed absorption a"c A 240 and 381 m , log E 3.89max

and 1.98 respectively in absolute ethyl alcohol.

Anal. Ca1cd. for C20H28N205: C, 63.81; H, 7.50;

N, 7.44; 0, 21.25. Found: C, 63.85; H. 7.63; N, 7.33;

0, 21.38.

The first two filtrates were combined and evaporated

to dryness. The yellow residue (29.~ g.) was treated with

,-",rTT

X

~- --

~Ii ,I' .Il _'

~r'oLO

.------- ------ ---- - - ,I

:\

l : il, .

I'1"

~ ~2.03.07.0 6.• 0 5.0 PPH (d) 4.0

:----rI

R.O

....,..,..r._JpNowro~~-r-.,.,....~~ ..........~v"-,..,....~ ........-..,.~~~.....,.-~1{"'t""'rcO

Fi~. 3. - The nuclear magnetic resonance spectrum of nitrocarnphor anhydri~e, ~.p. 170.~-171.~n.

~

o•

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I'< I'i " :' "~I' .11. ,j" '";;,,' 'II. :' 'I "",1:1 :'/1 '~''II' ''I III" ',';"1-· -~I" ...-II! . "I "1' III I ' , ,~+' .... I'••d ol ,,",,',,"'" ."" ,",~; :I' ' "I ~:~: ' II d: '1 ' :-,' II! .il' I ~i: j,,'tff;. ~,..:. "I~I

:", :~iU: .... '1:' *IJ: :: ::; lil"{lliiil:i!! ·:ijf~II!JI'iir, 'Ir':I:'; . 'I·· ...., , , . I :'1.1

"" 1:1,. '1' 1"1 11':'111" H ':1'1'"~. I. 'I, Ill. 'I" 'I 11'1:1'1 III ilil '1' , I" " I;l 1-;' :...., ,: :11 I, J1::P ,I , " ' I ~.;l,.JL::.L .., " ..;.; I ' I" I I ! ill I " , 'II" 1:'1 I 'Iii : II I ,

~. ' , ,."" 1,1 '::'", I ,i 'i':,III'·,!1 "il;";11 ,

'1 " 'II I" I'll " I, 'il 'I' I,., 'j I~ .:... ,.! ': ·~I:; ''':'I~,i '.; Ij.: ::.~::~: ~..:: i:!]:'~.:J[.J";!! II. !

I • I ,I, !' , I 'I l/ 1 I I 'I 1 ,I.' 101 I

II ' ~ 'I': I I" "I':' I": '1'1 ! '. I'" '.I' I ", ': l~tll',' Iithll" 1'1' ,', 1,1, ,I +J '

~!~. I-';...·~JI "I .,' .,' ::i', I : 'I ,.: 't'::-~'-~"" I" '''r-','~, I I ~ ''''I I .' II I :,'~ ! :1\ ,'I I. "11,\ " '... !" ,., " 1'1 I ".::: I, II 'I' 1 I II '::: "I' .. ..·":",:r"··, , .:. ." . '::'I~: d<l.I.: .."::'

:':.: ~~ .;- .+- ,:;- ..:;"':'/':", ", ',:'" ; 'I ,:~ ',.. 'I', It': ,,;, :1

1, t 'I::' ,', Ii' I) ,; ,

h.-. _J_, .. - "y' ' .:.1- -r' ,..)1- •. "1-' - eo -

[c j'" ,<4.: ,:Ir', ;'!,1" "1 i'l, 1/ ":, I,ll.,'

~ U:~·Jl1ri....'i '::"JIl:'"'iil:: ::'I11i::: :.. il'''m·:':·l':Ell':'".'! iffi:/'~:I/, j::l, I I ", .. I I' I L' I I', ,H"" !? "p' '.. 'II. .' ,., ':::' , 'N_

2: ~. ." ~ :.\ r:> ~.; g 'l

2.1

w'-'0~

1.9

820 360 400 440

12.

Fig-. 5. - The ul travi olet spectrum of

nitrocamphor anhydrirle, m.p. 170.~-l7l.5° (rlec.).

(c= l.l8~ x 10- 3 M in 8bs. alcohol)

13.

230 250 270 290

IvA VB LENGTH (mf)

Fig. 6.- The ultraviolet spectrum of

nitrocamphor anhydride, m.p. 170.5-171.5° (dec.).

( -4c= 1.185 x 10 M in abs. alcohol)

14.

120 ml. of 5% sodium hydroxide solution which was then

extracted with ether. Acidification of the alkaline

solution yielded 12.4 g. of 3-nitro-camphor. The combined

ether extracts were washed wi~h water and dried over

anhydrous sodium sulfate. Upon removal of ether there

was obtained 13.9 g. of semisolid. It was dissolved in

80 ml. of hexane and 5 ml. of benzene and kept overni6ht

in ehe refriGerator. After filtration ehe solid was crys­

tallized from hexane-benzene to give 1.4 g. of greenish

yellow crystals, m.p. 156-1580 (dec.). Further crystal-

lization from methanol and twice from ethyl alcohol afforded

0.7 g. (2.0%) of analy~ically pure nitrocamphor anhydride,

m.p. 158-1600 (dec.), /j.J~8 +66.60 (c 5.0, benzene).

The infrared spec~rum (Fig. 7) showed bands at 5.67 and

5.7~)U (carbonyl), 6.10~(ni~roso), 6.46 and 7.42;U

(nitro~. The infrared spectrum was essentially identical

wi~h ~hat of the isomer melting au 170.5-171.50 (dec.).

The ultraviolet spectra (Fig. 8 and 9) exhibited two

maxima: 238 rr;? (log E 3.82) and 379 m!' (log E 1.69)

in absolute eGhyl alcohol.

Anal. Calcd. for C H 1'1 0: C, 63.81; H, 7.50;20 28 2 5

1'1, 7.44; 0, 21.25. Found: C, 64.07; H, 7.35; 1'1, 7.46;

0, 21.35.

The filtrates from the last isolation consisting

of hexane-benzene were combined and l.,be solvents VIere

3:

i':'

"

:~

~---~')

,'-~:_.. --~-_::. =

•WAVRENGTliI'H MICRONS

d::-:"'~

~

7

fl'AVENUMBER C,-\"

15,]0 1.100 )JeD 120') 1100 100:,) 9,)0 c'::; 700

=-~:..:. . _~~-Li::L' ',-~~rO:P2:.-r::~-r:- r;-T'FT'

·if::~"tt~~~~§ ~~~::~~~t ;;~'=d:: ~~f~"/ -~~~l~'~ ~-~~~j§:; O';:~ --;:-;i: ~-:~:-~~- _~~ _~~ ~~~~: _~~=..=-,;~~~ ~E-; h; ,~~-:F

~==+~~-'-:o__=-_I--'---T---+-----;--""--+--'---i--------!j·1 ._. i

-----+-=~-A-~.~~

. , ~u

- ::-;-- :<.. : l

~'-:y.- ",

i-i-I'

==='-"-'-=--L-===-=--'-"-=:=c.L_=L.=,-,- '. I

10 l.l ~.\

I

-'~F"~C._ .l..

==.".;: --

~

~

-

.•~='r="-

so

60

;;0

I

20

1'10 a==o ~

..,

SO.' J~

'~I-

]~ig. 7. - The infrared spectrum of nitrocamphor anhydrjde, m.p. 158-160° (dec.).

I-'IJl

2 . fi

16.

2. 1

1.7

1. 5 \320 360 4()() 440

1vA VE LE1\(~ TII (mf )

fig. R. - The ultraviolet spectrum of

nitrocamphor anhydride, m.p. lfi8-160o (dec.).'J

(c= 1, 13;:; x 10-" N in ahs. alcohol)

17.

4.2

..3.8

2702S02801...-- --'- -1.- ..1..- ---'-__1

290 300

-\'iA VE LEKGTH (m,U)I

Fig. 9. - The ultraviolet spectrum of

nitrocamphor anhydride, m. p. 158-l60o (dec.).

-4(c= 2.27 x 10 N in abs. alcohol)

18.

removed with a vacuum evaporator. At~empts to crystallize

the residue (8.8 g.) failed. Finally it was dissolved in

a mixture of hexane and benzene and chromatographed on a

column of silica gel G. Elution with benzene gave a solid

which af~er five crystallizations from ethyl alcohol

afforded 0.1 6. (0.28%) of white crystals, nitrocamphor

anhydride, m.p. 190.5-1920 (dec.). The infrared spectrum

(Fi,,;. 10) showed absorption at; 5.64 and 5.71 P (carbonyl),

6.09)l (ni troso), 6.46 and 7. 3~)J (nicro). 'l'here were

slight differences in the infrared spectrum from the

specGrum of the isomer melting a~ 170.5-171.50 (dec.).

The ultraviolet spectra (Figs. 11 and 12): ~ 238n'\lJ..max r

log E 3.92 in absolute ethyl alcohol.

Anal. Ca1cd. for C20H28N205: C, 63.81; H, 7.50;

N, 7.44. Found: C, 64.03; H, 7.50; N, 7.40.

In order to show the homogeneity of isolated products

0.3 g. of anhydride, m.p. 170.5-171.50 (dec.) was chroma-

tographed on a column of 10 g. of silica gel G. Elution

with e~hyl acetate afforded three fractions which, after

cryscallization from ethyl alcohol, melted at 170.5-1720

(dec.). The homogeneity was further proved by taking the

mixture melting points wi~h three isomers. The anhydride,

m.p. 170.5-171.50 mixed with ~he isomer, m.p. 158-1600

melted a~ 159-1640 and with uhat, m. p. 190.5-1920

melted at 1G6°. Admixture of isomers, m.p. 158-1600

19.

,i ~

.0...E

o0:~

.,..,t:

I 1I .

1"'1 :

1.1

····1

~-~-+~~:.-~. .'..I --1-':: n-I- J-- -- -'·-'1· ~

.1] .' ·;-,1 •J ~._ ._J___ I__ ..L._

.:::.i., '

i:

20.

300 340 3HO 420

fiG. 11. - The u1 t r nvi 018 t s pee t I' Lin] 0 f

nitrocClmphor anhydride, r:l.p. lS().:"-Ei2() (dec.).

-~(c= 1.135 x In M in abs. a1cnbol)

21.

4.2

3.8

8. 1-1

3.0

2.'3

2802GO240

~ -L- -L- ........ I

300

HAIlE LENGTH (TTl,?)

fig. 12. - The ultraviolet spectrum of

nitrocamphor anhydride, m.p. 190.5- 192 0 (dec.).

(c= 1.135 x 10--1 ?'[ in a 1)8. alcohol)

22.

and 190.5-1920 melted at 161-1620• All admixtures, like

individual isomers, decomposed at the melting point.

3. Attempted Ozonization of Nitrocamphor Anhydride

A stream of ozone was passed through a solution of

nitrocamphor anhydride, m.p. 170.5-171.50 (dec.) (1.0 g.)

in 100 ml. of chloroform for 35 minutes at 00 • Water

was added and the chloroform was removed by steam dis~il-

la~ion. The yellow solid was isolated by filtration and

amounted to 0.94 g. and melted at 170.5-171.50 (dec.).

After crystallization from ethyl alcohol, the yellow

crystals (0.83 g.) melted at 170.5-171.50 (dec.) and

a mixture melting point with the nitrocamphor anhydride

was not depressed.

4. At0empted Oxidation of Nitrocamphor Anhydride with

Potassium Permanganate

A solution of nitrocamphor anhydride, m.p. 170.5-

171.50 (dec.). (1.0 g.), and 0.42 g. of potassium perman-

gana ·ce in 100 m1. of acetone was maintained a t~he reflux

temperature for 4.5 hours. After standing for 16 llours

at room temperature, tlle excess permanganate was destroyed

witll 5 ml. of etllyl alcohol. After filtration and removal

• _l'l ).,.__

Ul 0Ilt:: solvent there was obtained 0.92

23.o

m.p. 166-167 (dec.). Crystallization from ethyl alcohol

afforded 0.8 g. of crystals, m.p. 170.5-171.50 (dec.).

A mixture me It-ing poin t wi lh ·Ghe ni trocamphor anhydride

was not depressed.

5. Preparation of Phenylnitromethane

Phenylnitromethane was prepared according to the

me0hod of Kornblum (7). Benzyl bromide (51.3 g., 0.30

mole) was poured into a slirred mixture of 600 011. of

dimetllyl formamide (DlYlF), 36 g. (0.52 mole) of sodiumo

ni~ri~e and 40 g. of urea maintained a~ -20 to -15 •

Af;.:;er 5 hours of stirring a ~ this temperature, the reac-

~ion mix0ure was poured into 1.5 1. of ice-water layered

over wi th 200 011. of ether. rU1e aqueous phase was extracted

four times with 250 011. portions of ether which was washed

with four 100 011. portions of water and dried over anhy-

drous magnesium sulfate. After removal of ether, the

residue was distilled with the following results.

26Head Bath Pressure Weight n

DFract. (G C) (t C) (0101. ) (g. )

1 34-38 80-110 5 13.0 1. 49762 38-39 110-115 5-2 1.5 1. 5244,., 39-59 115-130 2 2.5 1.5380.)

4 59-65 130 2 2.0 1. 53385 65-73 130-136 2 13.0 1. 53146 73 136 2 2.0 1. 5318

24.

Fractions 5 and 6 were combined to give 15.0 g.

(36.5%) of phenylnitromethane (lit. b.p. 76~a~ 2 mm.,

n;O 1.5316) (7).

6. Action of Formamide on Phenylnitromethane

A solution of 2.74 g. (0.02 mole) of phenylnitro­

me thane and 1.1 60 (0.024 mole) of formamide in 8 ml. of

ethyl ace~a~e was gently refluxed for 68 hours. After

removal of solvent on a vacuum evaporator the residual

yellow semisolid was dissolved in 80 mI. of ether and

washed with waGer. The e~her was dried over anhydrous

sodium sulfate and then evaporated to dryness to give

0.5 G. of slightly yellow solid. Two crystallizations

from ethyl acetate afforded 0.3 g. of white crystals, m.p.

238-2400 • It has the same empirical formula and the

melting point as ~-diphenyl urea, but their infrared

spectra were distinctly differen~ and their mixture

melting point was depressed; m.p. 220-2360•

Anal. Calcd. for C13H12N20: C, 73.56; H, 5.70;

N, 13.20. Found: C, 73.51; H, 5.70; N. 13.28.

7. Preparation of Diphenyl Urea

The procedure of Davis and Blanchard (8) was slightly

modified in the synthesis of diphenyl urea. Thus, a

solution of 7.6 g. (0.125 mole) of urea and 15.6 g.

(0.121 mole) of aniline hydrochloride in 200 ml. of water

was refluxed for 4 hours. The no~ solution was fil'Gered

and 'Ghe solid on the Buchner funnel was washed several

times with ho~ water. Crystallization from ethyl alcohol

yielded 5.0 g. (39.4%) of diphenyl urea, m.p. 239-2410 ,

(lil;o m.p. 2350) (8).

8. Preparation of Benzoyl Cyanide

Benzoyl cyanide was prepared according to the me~hod

of Oakwood and Weisgerber (9). Cuprous cyanide (110 g.,

1.2 moles) and 143 g. (118 m1., 1.02 moles) of freshly

distilled benzoyl chloride were placed into a 500 ml.

three-necked round-bottomed flask fitted wi~h a thermo­

meter, condenser, and a CaC12 drying tube. The flask

was shakenl.-o mois'Gen almos'G all the cuprous cyanide and

was placed in a Wood's metal bath which had been previously

heated to 145-150°. The tempera~ure of the bath was raised

to 220-2300 and ~aintained between these limits for 1.5

hours. During the hea -c;inc; "~he flask was fre que n"l;ly removed

from the bath and the contents were thoroughly mixed by

vigorous shaking. At the end of 1.5 hours the benzoyl

cyanide was distilled under reduced pressure by the aid

26.

Head Bath Weigh\" 28Fract. Cc C) (t C) (g. ) n

D1 102-104 to 150 20.02 104-105 150 18.0 1.54423 105-106 150 14.0 1.54544 106 150-156 16.2 1.54685 106 156-170 18.2 1.54726 106-107 170 16.2 semisolid

Fractions 4, 5 and 6 were combined to sive 50.6 g.

(38%) of benzoyl cyar;ide (Ii '0. b.p. 208-2090 aG 745

mm. ) (9) •

9. Prepara 'Gion of LJ -Ni 'croace '~ophenone

0-Nitroacetophenone was prepared according to the

procedure of Bachman and Hokama (10). Benzoyl cyanide

(30.0 g., 0.25 mole) was added in one hour to a mixture of

30.6~. (0.5 mole) of nitrome'chane and 53.0 g. (0.5

mole) of anhydrous pyridine (dried over calcium hydride),

and Jvhe reac tion mix'Gure VlaS stirred for 5 hours. The

suspension was filtered and the precipitate was washed

with 300 ~lo of water and acidified with dilu~e hydro­

chloric acid at 00'Go 50. HiGroacetophenone was filtered

and crystallization from heptate afforded 12.0 g. (29%)

of white crystals, m.p. 105-1070, (lit. m.p. 105-1060 (10).

10. Action of Hydroxylamine on U) -Ni troace tophenone

A mixture of 2.78 g. (0.04 mole) of hydroxylamine

hydrochloride and 3.36 g. (0.04 mole) of sodium bicar-

27.

bonate in 40 ml. of water was added to a solution of 3.3 g.

(0.02 mole) of w-nitroacetophenone dissolved in 100 ml.

of ethyl alcohol and the resulting reaction mixture was

heated on a steam bath for one hour. Ethyl alcohol was

removed under reduced pressure and the residue was dissolved

in ether and washed with water. After dryinG over anhy-

drous sodium sulfate, the ether was evaporated to dryness

to give 0.5 g. of viscous residue. ~JO crystallizations

from ethyl acetate-peLroleum ether (b.p. 60-1000) and

then from a mixture of ethyl acetate and benzene afforded

0.2 g. of white crys~als, m.p. 130-131.50 • On the basis

of the chemic al analysis and of the mixed mel·~ing point

with an authenLic sample of benzohydrosamic acid and by

comparison of their infrared spectra, the reaction product

was identified as benzohydroxamic acid.

Anal. Calcd. for C7

H7

N02 : C, 61.31; H, 5.14;

N, 10.22. Found: C, 61.54; H, 5.23; N, 10.30.

11. Preparation of Benzohydroxamic Acid

The procedure of Jones and Hurd was employed in the

synthesis of benzohydroxamic acid (11). A mixture of 4.2 g.

(0.06 mole) of hydroxylamine hydrochloride and 6.4 g.

(0.06 mole) of anhydrous sodium carbonate was suspended

in 200 ml. of ether. When 8.4 g. (7.0 ml., 0.06 mole)

28.

of benzoyl chloride was added there was little reaction~

but i~ became more vigorous when 7 ml. of water was added.

After 2.5 hours of stirring, 100 ml. of water was added

and the ether layer was dried over anhydrous sodium

sulfate. The ether was removed and the residue was CI'y­

stallized from ethyl acetate-benzene to give 3.0 g.

(36.0%) of white crystals, m.p. 125-127°. Further

crystallization from ethyl aceta~e-hexane raised the

melting point to 129-130° (lit. m.p. 124-125° (11) and

125-128° (12.)

29.

C. DISCUSSION

1. The structure of the Nitrocamphor Anhydrides

Condensa tion of 3-nitrocamphor did no"i:; occur as

easily as the published reports (1,3) indicated, and mod­

ification of "Che experimental procedures was necessary

to obtain adequate amounts of products. The reac1;ion

did not proceed when 3-nitrocamphor was heated on a

steam bath for 12 hours, and prolonged heating gave a

reaction mixture which did not crys~allize. Failure to

obtain crys~als was no~ surprising because 3-nitrocamphor

i~self, if n01; analytically pure, decomposes spontaneously

on s1;anding to a variety of ill-defined products (2).

However, three isomeric compounds corresponding to the

empirical formula, C20H2oN205' were isolated when 3-niGro­

camphor was refluxed in ethyl acetate in tne presence of

formamide for bl hours. The isomer, m.p. 1'(0.5-171.50

(dec.), was isolated in larges~ amounts and provided a

basis for the presen"C study.

In order to distinguish between structures (IV) and

(V) as the correcL. scructure for the a[Jhydrides, attampL;s

were made to oxidize a possible ethylenic double bOlld

with ozone or por"'ssium permanganate. Ozolle would cleave

only the double bond in V, whereas potassium permanganate

viOuld oxidize the nl troso gl'OUp 1,1 IV L,U Ghe carre sponding

IV V

30.

ni~ro group. In botn cases oxidation of the anhydride

failed and the orgaCJic material \'las almost quantitavely

recovered. The unreactivi~y of the anhydride to both

oxidizing agents indicates thal it does not co~tain a

double bo~d. The absence of ethylenic double bond will

therefore tenl~aL;ively eliminate structure (V). 1'he

resistance of the nitroso group to the oxida~ion by potas­

sium permangana~e may be due to the protection provided

by bulky neighboring groups. The alkaline degradation of

the ni trocamphor anhydride described by wwry (3) seemed

too extensive to provide decisive information about the

structure of the anhydride, itself.

The molecular weight deGermination and the spectral

studies indicate that the nitrocamphor anhydride exists

as the monomer which is unusual for C-nitroso compounds

(13). Primary and secondary nitroso co~pounds easily

~automerize to the corresponding oximes and ~erLiary

ni 'croso compounds in which oxime forma don is impossible

31.

dimerize readily. The unusual property of nitroso com-

pounds to dimerize has made at temp 'GSGO de Germine the

infrared absorption of monomeric nitroso groups qui~e

difficul'c. Luttke (14) s'cudied~,he changes in the infra-

red spectrum which occurred with time when primary and

secondary nitroso compounds were volatilized and studied

'i:.erLiary ni'Groso compounds in dilute solution and in the

vapor state in which ~hey exist in 'eha monomeric form.

Wich niGrocamphor anhydride, however, the nitroso group

can be direcG1y meas~red in the solid s~ate. The exis'cence

of vhe anhydride as the monomer is readily explained by

Gfle steric environment of 'Ghe ni Groso group which prevents

dimeriza-vion.

The infrared spectrum of the nitrocamphor anhydride,

m.p. 170.5-1~1.5° (dec.), (Fig. 4) contained two bands

in the carbonyl region a0 5.65 and 5.71u. Carbonyl absor~-I

tion in 3-ni trocamphor (Fig. 2) occurred a'", 5. 71/{,(, and a

similar structural fea~ure is indica~ed for the anhydride.

l'he band in "he anhydride at; 5.65u mUSG be due ',,0 \;he/

carbonyl on the adjacent carbon atom bearing the nitroso

group. 'rhe parent ke tone, camphor, showed carbonyl absorp-

tion a~ 5.74~(. The very slight shift cowards shorter

#ave length for the absorption of the carbonyl group due

to Lhe nitro group, and the substantial change in the

0a~honyl absorption due to the nit;roso 0rouP must be

ana10;ous to the shifGing of the carbonyl absorption of

32.

cyclic ketones in their ~-halo,:seil derivatives which has

been studied extensively (15). The band at 6.12;U was

assiGned to the nii.:.roso group in accordance with sugges-

0ions by Bellamy and Williams (16), and Jander and Haszel-

dine (17). The bands at, 6.45 and 7.41fl corresponded to

Ghe niGro group (18). The infrared spectrum clearly

supports structure (IV) for the nitrocamphor anhydrides.

Absorption in the carbonyl region is not in accord with

s ·,-,ruc 'cure (V).

The nitrocamphor anhydride, m.p. 170.5-171.50

(dec.), showed absorption in the ultraviolet region at

240 m,p (log E 3.89) and 381 m,U (log E 198) (Fig. 5 and 6).

The spectrum appears to be consistent with the absorption

reported for C-nitroso compounds (13) and the bathochromic

shift of both bands in the spectrum of the anhydride may

be due to the proximity of the carbonyl group.

The nuclear magne0ic resonance spectrum of 3-nitro­

camphor (Fig. 1) showed a peak at <.r.=. 5.1 which was split

in~o a doublet and musL:. be due to the hydrogen on the

carbon atom bearing the nitro group. The resonance of the

neighboring proton occurred a~ or: 2.75, and the 1hree

methyl groups gave peaks at ~ 0.91, 0.98, and 1.07.

The spectrum of the nitrocamphor anhydride, m.p. 170.5­

171.50 (dec.) showed the absence of the proton on the

carbon bearing the nicro group (Fig. 3). A peak ac

J~ 2.72 corresponds closely to the peak at 2.75 in

33.

3-ni trocamphor. 'rhe peak observed at cr-=3.28 may be due

to the bridgehead proLon adjacen0 to the nitroso group.

The nuclear magnetic resonance spectral data are consistent

with structure (IV), but do not exclude V.

The nitrocamphor anhydride, m.p. 158-1600, gave

an infrared spectrum (Fig. 7) which was essentially iden-

tical with the speccrum of the isomer melcing a~ 170.5­

171.50 (dec.), and 0he ultraviolet spec~ra (Fig. 8 and

9) of the two isomers were exceedinsly similar. The

substantial difference in optical rotations of the two

compounds and the similarity of their spectra indicate

Ghe anhydrides to be s0ereoisomers.

The nitrocamphor anhydride, m.p. 190.5-1920 (dec.),

gave an infrared spectrum (Fig. 10) in which there

were slight differences from the spectra of the two iso-

meric anhudrides. The well defined absorption in the

carbonyl re~ion establishes the structural similarity of

the three anhydrides. In 0he ultraviolet spectra (Figs.

11 and 12) at 380 m,u a clear maximum was not shown; the

absorption gradually increased to the maximum a L 238 m((./

The nitrocamphor anhydride, m.p. 190.5-1920 (dec.), was

probably described by Lowry (1). The small amount isolated

in the present sLudy did not permit a comparison of its

optical rotation with the value reported by Lowry.

The co~de~s~tio~ of

occurred to form a nitrone, structure (VI). The closest

34.

analogy (VII) has spectral properties completely different

from the spectra of the nitrocamphor anhydrides (19) •

VI

.0::- 0

t/.

N·,0-

VII

The two new anhydrides, m.p. 158-1600 and 170.5­

171.50 in the present study and the two anhydrides, mop.

18~·0 and 1900 , describQld by Lowry (1,3) represen'c the

four possible stereoisomers for structure (IV). In the

present investigation the stereochemical structures have

not been assigned to the anhydrides, but possibly the

assignment could be done by optical rotatory dispersion

or X-ray analysis.

2. ~he Possible Nitro-Nitroso Intermediate in che

Conversion of Nitro Compounds to Furoxanes

The condensation of two molecules of 3-nitrocamphor

to give nitrocamphor anhydrides, nitro-nitroso compounds,

demonstrates a new reaction which is feasible for nitro

alkanes. In the case of primary nitro alkanes the anhydro

35.

intermediate would be expected to proceed further to form

a furoxane.

Cr-H -C - C-C,~H

b 5 II II 0 5i'J N

EO/' '0 'OH

) C,~H5CH - CHCI'H5

+ H20o I I t)

N02 NO

\C6H

5C=N-0 -t C6H

5CH2N0

2

/C:::~~N-O

C6H5- n- ~-C6H5

N,O)~'O

The sequence of reactions finds support in Wieland's

research which demonstrated a new route to furoxane from

nitro-nitroso compounds (20).

1

36.

The cleavage of nitro oxime tc the corresponding nitrile

oxide (VIII) and nitro alkane would also lead to the

expected product because nitrile oxide readily dimerizes

to furoxane (21).

Recently Parker and his co-workers (22) obtained

dicyanofuroxane from the nitration of cyanoacetic acid

with nitric acid in the presence of sulfuric acid) but

the me~hanism of its formation was not investigated.

2 NC-C - C-CNII IIN N,"01 0

The mechanism can readily be rationalized as proceeding

through the nitro-nitroso intermediate.

The first produclJ of Lhe niGrat;ion of cyanoacetic

acid would be nitrocyanoacetic acid (IX) which would

then decarboxylate to the corresponding nitroacetonitrile

(X). The decarboxylation of :x.. -ni trocarboxylic acid

occurs readily (23) 24, 25). Analogous to the decarbo­

xylation of ~-ketocarboxyliC acid which leads directly

to the enol forn~ of tIle reaction product (24), the decar-

boxyla tion of '=" -ni trocarboxylic acid leads to aci-

form, of the ni tro compound. The aci-form would be favorable

for the condensation reaction to give the corresponding

nitro-nitroso intermediate (XI). The formation of furoxane

(XIII) can occur then ei ther by tautomeriza tion U1 lJ.l tl'0

and nitroso group (route a), or by ~he decomposition of

nitro oxime intermedia~e (XII) to nitrile oxide (XIV)

and nitroacetonitrile (route b). Both reaction routes

find support in Wieland's research (20,21).

37.

NCCH COOH2

+ HNO3

NC-CHCOOHi\T02

I\rC-CH::'lJ::::~H

+

NC-CH='N/~H

NC-CH-NOI

NC-CH-N02

---~;>

)

IX

lW_CR.::WiOH'"0

X

NC-CP.-HO

I

XI

11C-C=N"I OH

NC-CH-NO2

XII

+

I'\C-~':'N 'OH

NC-C-NOH 2

!(routeHC-C;:N-O

XIV+

NC-C,~N'OH(route a)---~') NC-C=N/OH

....0

b)NC-C-::N-O

>

XIII

rrhe condensa i-ion of ni tronic an acid (~~) is somewha t

similar ~o the Nef reaction (26). in which ni~ronic acid

is an intermedia~e. The Nef reaction involves conversion

of primary or secondary nitro compounds to the correspond-

ing aldehydes or ketones by addin~ Ghe alkali salv of the

former to aqueous mineral acid. An excellen~ mechanism

for Nef reaction has been proposed by van Tamelen and

Thiede (27).

Feuer and Nielsen (28), however, found tha~ 2-nitro-

oc~ane can be converted ~o 2-ocLanone without first formin~

an alkali salt of 2-nitrooctane. In other words, the

sauuomerism of 2-nitrooc0ane to the corresponding nitronic

acid is acid ca~alyzed. Acid catalysis of the nitro compound

Where B is base such as water or chloride ion.

to ~he nitronic acid and, furthermore, the activating

39.

cyano i~roup in ni troacetoni. ~rile strongly support ehe

hypothesis thaL Lhe condensation to the nitro-nitroso

intermediate proceeds via nitronic acid in the formation

of dicyanofuroxane.

'The condensation of nitro compounds to furoxanes

via a ni0ronic acid intermedia~e in acid medium is further

demonstraced by Alexander, Kinter and McCollum (29)0

'rhey ob"cained dibenzoylfuroxane by trea ting phenylme c.hyl-

carbinol, acetophenone, isonitrosoacetophenone or

LO-nitroacetophenone wi~h red fuming nitric acid in boiling

"_;lacial ace tic acid so lu tion. The proposed me chanism for

the conversion of phenylmethylcarbinol to dibenzovlfuroxane

suffers only one weakness in thau the condensation of

nitronic acid involves the carbonium ion which is adjacent

to the partially positive carbonyl carbon.

OHC6HSCHCH3

°C6HSCCH3 HONO)

ti°C6Hs6CH =-NOH

q +/OHC,HSCCH:::N,

o OH

1~ + _ /OH

C6H CCH-N,S OH

\ )

J -

, J0-° .. OH" e:-- '"C....-HSCCH::N

o '0\

Q +_/OHC....-HSCCH-N,

o OH

As itl the formatiotl of dicyatlofuroxatle, tbe con-

version of phenylmethylcarbitlol to the correspotlding

furoxane may have an alterna~ive mechatlism itlvolvitlg the

ni~ro-nitroso intermedia~e (XV).

oCbHsCCH-NO

) 01C6H

SCCH2N02

XV

40.

A reasonable mechatlism for the formation of diphenyl­

furoxane was proposed by Kortlblum and Weaver (30) for

the reac1iotl of benzyl bromide with sodium nitrite in

dimethyl formamide (DMF) at -160 •

41.

HN02 +

C-l-i~9H-N02 ---7 HN02 '1- C6-HSC::N-O

o -'NO

XVII

) C,-HS-C - C-C,-HSOn.. 0

N, / N....

° °XVIII

The proposed mechanism was jusGified by the evidences

-chat the intermediaGe, nitro1ic acid (XVI), under the

same reaction conditions gave the correspondin; furoxane

(XVIII), and that benzonitri1e oxide (XVII) is known to

dimerize to form dipheny1furoxane (21).

~he presence of an intermediate, benzonitrile oxide,

in the forma~ion of diphenylfuroxane from phenylnitro-

methane was confirmed by Mukaiyama and Hoshino (31).

In their studies of the reaction of primary nitropar-

affins with isocyanate and trialkylamine, they were able

to trap nitrile oxide with vinyl aceta~e to form isoxa-

zoline (XIX). The proposed mechanism by which diphenyl-

furoxane is formed is almost similar to ~he mechanism of

CH2~ CHOAc

+o

C6HSNHCNHC6HS +

AcO-CH-CH2I IO'N~O 'R

XIX

Kornblum and Weaver. Ins~ead of nitrite ion, phenyl

isocyanate reac~s wi~h nitronate ion to form an additio~

compound which wi 11 de compose 'co ni "cri le oxide and the

dimerization of the latLer will give the corresponding

furoxane.

oI q I

RNHCON:::CHRI

----7 RNHCOOHI !-

RI'JH 2

1FiNoC =0

1 01RNHCNHR

RC=.N-O

iRC=N-O

R-C - C-RN I~'0/ '0

The proposed mechanism by Mukaiyama and Hoshino is excellent,

however, an alternavive mechanis~' seems ~o be reasonable

via the nitro-ni~roso intermediate (XXI).

-RCH-NO

2

J -~ 0

RCH=N~ _o

+

TI

RN=C=O

Q q I

RCH'::N-OCNHR

+-RCH-N02

)

o 0 I

RCH:;I~-O~m-IR

XX

R-CH-NOI

R-CH-NO2

XXI

I

RNHCOOH

RCH-NOI

RCH-N02>

(route b)>

RC::;N'OH

IRCH-N02

1(route a)

R-C",N'OH

R-C::.N"OH'0

> R-C - C-R.~ ~.

l~ N'0/ '0

IRHNCOOH -~) RNH2 1-

L~J·c,oo

I U I

RNHCNHR

44.

Simi lar to 1-he me chanism of Mukaiyama and Hoshino, "I:he

ni~rona~e ion is formed in the presence of a basic

catalysL and one of lhe oxygen atoms of nitronaGe ion

combines with the positively charged carbon of ~he iso-

cyanate. Since the nitronate ion is an ambidGn~ ion,

the carbon of the nitronate ion would also combine with

\:,he isocyana te -co form """ -ni tro-amide (XXII). However,

the oxygen addition would be favored because of greater

electronegativi0y of oxy~en relative ~o carbon, and also

due to less steric hindrance (10), or if 1-he ~-nitroamide

is formed it would readily dissociate to its components

in ~he presence of trialkylamine (31). The formation of

nitro-nitroso inLermediate (XXI) would occur directly

~hrough the interaction of carbon of the niGrona~e ion

and compound (XX). The conversion of the nitro-nitroso

intermediate GO diphenylfuroxane would follow route a

or be as mentioned earlier in the mechanisms for the

formation of dicyano- and diphenylfuroxane. As according

to Mukaiyama and Hoshino, the decomposi tion of "~he

carbamic acid would lead to the corresponding diphenyl

urea and carbon dioxide.

o I

RCH~-NHR

IN02

XXII

It is interesting to note tha~ secondary nitro-

paraffins, such as 2-ni tropropane and --" -phenylni "croe­

thane react with isocyanate in the presence of ~riethyla-

mine ~o ~ive sym- diphenyl urea wi~h evolution of carbon

dioxide, but Mukaiyama and Hoshino were no~ able to

isolate ~he corresponding dehydrated produc~. Since

secondary nitro compounds canno~ form furoxane, the de-

hydrated product would be undoubtedly the corresponding

nicro-nicroso compound (XXIII).

RR-C-N02

HT R'l\[=-C=O

RR-C-NO

IR-C-NO

I 2

R

XXIII

I q I

mmC-NHR

With the discovery tha~ the condensa~ion of 3-

nitrocamphor leads to the ni~rocamphor anhydrides, which

are nitro-nitroso compounds, it is imperative to re-examine

critically the mechanisms involved in the formation of

furoxanes from ni~ro compounds.

46.

3. Miscellaneous

In connection with other problems it was of interes0

~;o prepare Ul -ni troace tophenone oxime. The compound

was previously prepared as early as in 189S by Sommer

(32) by acGion of arsenic and concentrated ni "ric acid

on styrene and in 1903 by Wieland (33) on trea0ment of

styrene in glacial acetic acid with concentrated sodium

nitrite. Hurd and Patterson (34) who studied 'uhe addition

of hydroxylamine to various unsaturated nitro compounds

prepared W -ni troace tophenone oxime by trea tin[:; u,)-

ni 'ero styrene wi th hydroxylamine. The re su1 ting reaccion

product was uhen oxidized wi~h chromic acid to the corres-

ponding nitro-nitroso dimer which in hot chloroform dis-

socia ted andcau tomerize d to w -ni troace tophenone oxime

(XXIV) •

---')') C6HSQH-CH2N02NHOH

2 C6HS

?H-CH2N02NHOH

H2

S04

+ Na2cr

207)

-- ~) 2 C6HSS-CH2N02NOH

XXIV

31r1(;8 w -ni tronce tcphCDcnc "v·JD.G

connection with the study of nitro ketones, it was hoped

L~'7 •

tha t perbaps Lv -ni troace tophenone oxime could be direc ely

prepared by treating ~be correspondins nitro ketone witb

hydroxylamine. However, the bond cleavage occurred between

~he carbonyl carbon and tbe carbon to which the nitro

group is at~ached and benzohydroxamic acid (XXV) was

obtained. Feuer and Anderson (35) have taken advanGage

of tbis type of bond cleavage by preparing

alkanes from the corresponding mono-potassium

c:ljw -dini tro-I

o<.tl-

dinitrocyclar.ones either in basic or acidic media.

NaHC03 °NH20H BCI ~ C6H5~-NHOH + CH3N02

xxv

Since ~he condensa~ion of two molecules of 3-nitro-

camphor ~o Ghe nitro camphor anhydrides i~ slightly basic

medium demonstra~es a new reaction which is feasible for

nitroalkanes, at~empts were made to condense phenylnitro-

me the.ne under ;,f"l8 same reac don conditions to tbe corre s-

ponding nitro-nitroso compound or to diphenylfuroxane.

Even thouGh there is a variety of methods described in

the literature for the preparation of diphenylfuroxane

(21, 30, 36-44), nevertheless, nobody has ever reported

attempts 'GO condense phenylnitromethane which misht lead

to furoxane. Preliminary experiments, however, were

inconclusive. A crystalline reaction product was isolated

which had the same empirical formula and the melting

point as sym- diphenyl urea, but their infrared spectra

were distinctly different and their mixture meltins

48.

point was depressed. No further efforts were made to

characterize the reaction product. However, isolation of

an unidentified reaction product and the interpretation

of the formation of furoxanes inspired by the structure of

the nitrocamphor anhydrides maJ be the basis for additional

re search.

As mentioned earlier in the introduction Lowry (2)

isolated a compound, m.p. 2200 , as a by-product in the

prepara tion of camphoryloxime from the trea tmen'(, of ni tro-

camphor with concentra~ed hydrochloric acid. The compound

was named camphoryloxime anhydride to which Lowry assigned

struc~ure (II). In the structural investigation of

II

camphoryloxime, Edward Wat (45) in this Laboratory did

not observe the presence of camphoryloxime anhydride in

the reaction mixture. Since Lowry preferred structure

(XXVI) for camphoryl oxime which is not correct on the

basis of new eVidence, it is obvious that structure (II)

does not correctly represent the camphoryloxime anhydride.

XXVI

D. SUMIvlARY AND CONCLUSION

Lowry's structures must be regarded as improbable

on the basis of the spectral and chemical evidence ob~ained

in the structural studies of the nitrocamphor anhydrides.

Since the structure of camphor and the reactions of

ni tro compounds were not known at l.,ha 1~ time, Lowry's

proposals coulr:'l. not be expected GO represent the structure

of the nitrocamphor anhydrides.

On the basis of spectral as well as of chemical

studies, structure (IV) was assigned to the nitrocamphor

anhydrides and not the alternative (V). The appearance

of two bands in the carbonyl region at S.64-S.67~ in

N04. NO

IV V

the infrared spectra clearly demonstrate ~hat ~he two

carbonyl groups have a diffe~2nt environment. Carbonyl

absorption in 3-ni trocamphor occurred a'l., S.711--1 and the

additional band in the anhydrides at S.64-S.67jA is due

to carbonyl adjacent to the nitroso group. The parent

ketone, camphor, showed carbonyl absorption at S.74;U •

50.

The very slight shift ~owards shorter wave length for the

absorption of the carbonyl group due to the nitro group

and the substantial change in the carbonyl absorption due

to the nitroso group is analogous to the shifting of the

carbonyl absorption of cyclic ke tones in their 'J\ -halogen

derivatives which has been studied extensively. The

appearance of two carbonyl bands, a nitro and a nitroso

band in Ghe infrared spectrum of nitrocamphor anhydrides

which agree closely with those in che literature favors

structure (IV). The ultraviolet spectra of the nitro­

camphor anhydrides are consistent wi~h ~he ultraviolet

spec~ra of nitroso compounds in which a bathocromic shift

has occurred.

The unreactivity of the nitrocamphor anhydride to

ozone and potassium permanganate gives additional support

in favor of structure (IV). The resistance of the nitroso

group to potassium permanganate oxidation may be due to

protecuion provided by bulky neighboring groups. The

steric environment of the nitroso group also accounts for

its existence as the monomer which is indicated by ~he

molecular weight and is unusual for C-nitroso compounds.

Although there are numerous publications on nitro

and nitroso compounds in the literature, only a few

publications deal with nitro-nitroso compounds (20, 29,

33, 46-48). The scarcity of the latter is due to the fact

51.

thaG nitro-nitroso compounds are hard to prepare and to

isolate from the reac·~ion mixture. Upon heating secondary

nitro-nitroso compotinds are readily converted to the

corresponding nitro oxime or furoxane. The recognition

that the nitrocamphor anhydrides are nitro-nitroso

compounds miGht supplement the existing mechanisms proposed

for the formation of furoxanes from niGro compounds and

might open a new general route to the syn~hesis of nitro­

nivroso compounds.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

52.

E. BIBLIOGRAPHY

Studies of the ~erpenes and allied compounds.Nitrocamphor and its derivatives. 10 Stereoisomericch loro- and brorno- ni trocamphors. II. Pseudo-ni tro­camphor. III. CampllOryloxime (camphonitrophenol).Martin Lowry. J. Chem. Soc., 73, 986-1006 (1898).

Nitrocamphor and iL,s deriva~ives. V. Sesquicamphory­lamine, a product of the spontaneous decomposition ofnitrocamphor. VI. Camphoryloxime anhydride. VII.

(3 -Bromo- r:;;;..' -ni trocamphor. P and jj' Bromocamphoryl­oxime. T. Martin Lowry. ibid.~ 83, 953-68 (1903).

Nit,rocamphor and i'cs derivatives. Part VIII. Theaction of formamide on nitrocamphor. Thomas r~rtin

Lowry and Victor Steele. ibid., 107, 1038-43 (1915).

N. V. Sidgwick, "rrhe Organic Chemistry of Ni trogen,"2nd Ed., Clarendon Press, Oxford, Great Britain,193'(, p. 228.

The basis for the reported optical ac~ivity of thesalt of aliphatic ni~ro compounds: 2-nitrooctane.Na than Kornblum, Norman N. Lich'cin, John 1'. Pattonand Don. C. Iffland. J. Am. Chem. Soc., 69,307-13 (1947). -

Relations between acidi~y and ~automerism. Part III.rrhe ni'ero-group and the ni'~ronic esters. Fri tz Arndtand John D. Rose. J. Chem. Soc., 1-10 (1935).

A new method for the syn~hesis of aliphatic nitrocompounds. N. Kornblum, Harold O. Larson, RobertK. Blac~~ood, David D. Moorberry, Eugene P. Oliveto,and Galen E. Grahm. J. Am. Chem. Soc., 78,1497-501 (1956)~ -

The urea dearran~ement, II. Tenny L. DaVis and KennethC. Blanchard. ibid., 45, 1816-20 (1923).

Na~han L. Drake, EJitor-in-Chief. T. S. Oakwood andC. A. Weisgerber, "Organic Syntheses," VoL 24,John Wiley and Sons, Inc., New York, N. Y., 19L~4,

pp; 14-15.

Preparation of ni~roketones. C-Alkylation of primarynitroparaffins. G. Bryant Ba~hman and Takeda Hokama.J. Am. Chem. Soc., 81, 4882-5 (1959).

53.

11. RearrangemenGs of some new hydroxamic acids related toheterocyclic acids and to di-phenyl- and Griphenylacetic acids. launder W. Jones and Charles Do Hurd.ibid., 43, 2422-54 (1921).

12. John R. Johnson, Editor-in-Chief o C.R. Hauser andW. B. Renfrow, "Organlc Syntheses," Vol. 19,· JohnWiley and Sons, Inc., New Y)rk~ N. Y., 1939, pp. 15-17.

13. Struc l,ure and propertie s of C-ni~roso-compounds.

B. G. Gowenlock and W. Luttke. Quar. Revs. (London),12, 321-40 (1958).

14. Spektroskopische Untersuchungen an Ni~rosoverbindungen.

1. Mitteilun~: Die charakteristischen Infrarotban­den der monomeren Nitrosoverbindungeno Wolf~ang

Luttke. Z. Elel{crocr.em., 61, 302-13 (1957).

15. L. J. Bellamy, "'l'he Infra-red Spectra of Complexf'i101ecules, II 2nd Ed., John \'liley and Sons, Inc., NewYork, N. Y., 1958, pp. 139-141.

16. Infrared spectra and polar effects. Part VI. Internaland external spectral relationship. L. J. Bellamyand R. L. Williams. J. Chem. Soc., 863-8 (1957).

Ii. Reactions of flurocarbon radicals. Part XIV. Hexa­fluroazoxymethane. J. Jander and R. N. Haszeldine.ibid., 919-25 (1954).

18. The infrared absorption spectra of nitroparaffins andalkyl ni'crates. Na'l~han Kornblum, Herbert E. Unt!;nade,and Robert A. Smiley. J. Ori.£. Chem., 21, 377-9 (1956)

19. Experimentsl.:;owards '~11e synthesis of Corrins. Part IV.rrhe oxidation and ring expansion of 2,4,4-trimethyl- d ­pyrroline-l-oxide. R. F. C. Brown, V. M. Clark andSir Alexander Todd. J. Chern Soc., 2105-8 (1959).

20. Zur Kenntniss der Pseudonitrosite. Heinrich Wieland.Ann., 326, 225-68 (1903).

21. Zur Kenntniss der Nitriloxyde. Heinrich Wieland.Ber., 40, 1667-76 (1907).

22. Chemistry of dinitroacetonitrile-I. Preparation andproperties of dinitroacetonitrile and its salts.Charles 0 0 Parker, William D. E. Emmons, Henry A.Rolewicz and Keith S. McCallum. Tetrahedron, 17,70_~7 ('Oh0) --1;;'-'-'1 \ ... ./".... ,.

54.

23. An improved synthesis of esters of nl~roace~lC acidoHo Feuer; Ho B. Hass and Ko S. Warren. J. Am. Chern.Soc., 71, 3078-9 (1949).

2Lf. The decomposition of ~ -nitrocarboxylic acids. KaiJulius Pedersen. J. Phys. Chern., 38, 559-71 (1934).

25. 'rhe ve loci ty of the decomposition of ni troace (·ic acidin aqueous solution. I~i Julius Pedersen o Trans.Faraday Soc., 23, 316-28 (1927).

26. Uber die Constitution der Salze der NiL;roparaffine.J. U0 Nef. Ann., 280, 263-91 (1894).

27. The synthe~ic application and mechanism of the Nefreaction. Eugene E. van Tdmelen and Rober~ J. Thiede.J. Am. Chern. Soc., 74, 2615-8 (1952).

28. Direct Nef reaction by acid-catalyzed hydrolysis of2-nitrooctane to 2-octanone. Henry Feuer and ArnoldT.' Nielsen. ibid., 84, 688 (1962).

29. A mechanism for 0he formation of dibenzoylfurozaneoxide from phenylmethylcarbinol o EllioL R. Alexander,Mark R. Kinter and John D. McCollum. ibid., 72, 801-3(1950). - -

30. The reaction of sodium nl~rl~e with eL;hyl bromoacetateand benzyl bromide. Nathan Kornblum and William M.Weaver. ibid., 80, 4333-7 (1958).

31. The reactions of primary nitroparaffins with isocyanates.Teruaki Mukaiyama and Toshio Hoshino. ibid., 82,5339-42 (1960). -- -

32. Ueber die Einwirkung der ~alpetrigen Saure auf Styrol.E. A. Sommer. Ber., 28, 1328-31 (1895).

33. Zur Kenntniss der sogen. Styrolnitrosite. Ubereine neue Bi ldungsvleise der untersa lpe trigen ' Saure.H. Wieland. ibid., 36, 2558-67 (1903).

34. 'l'he addi tion of hydroxylamine to l0 -ni 'eros tyrene,furylnit~oethylene and nitroolefins. Charles D.Hurd and John Pa tcerson. J. Am. Che m. Soc., 75,285-8 (1953). -

35. A new synthesis of o:.<,UJ -dinir,roalkanes. Henry Feuerand Roy Scott Anderson. ibid., 83, 2960-1 (1961).

55.

36. Uber die Reaktionsweise des Hi trosy1ch10rids. II.Einwirkung von Nitrosy1ch10rid auf aromatischeAldoxime. Heinrich Rheinbo1dt. Ann., 451, 161-78(1927). -

37. Zur Kenntniss der Benznitro1saure. Heinrich Wielandand Leopold Semper. Ber., 39, 2522-6 (1906).

38. Zur Isomerie der Benza1doxime IV. Ernst Beckmann.ibid., 22, 1588-97 (1889).

39. Ueber das drit~e Benzi1dioxime. Karl Auwers andVictor Meyer. ibid., 48, 705-20 (1915).

40. Uber Nitrosoverbindunzen, I. Mittei1.: Bildunggemina1er Ch10r-nitroso-Verbindungen durch Radika1­reaktionen. Eugen Muller and Horst Metzger. Chern.Ber., 87, 1282-93 (1954 ).

41. C.A. 47, 2688e (1953). Action of oxides of nitrogenand nitric acid on mercury-paraffin compounds. Theapplication of the reaction to the study of thenitration of paraffins. A. I. Titiv and D. E.Rusanov. Dok1ady Akad. Nauk S.S.S.R., 82, 65-8(1952). --

42. Dehydrogenation of glyoximes. J. H. Boyer and U.Tog5weiter. J. Am. Chern. Soc., 79, 895-7 (1957).

43. A1doximes and dinitroben tetroxide.H. A1u1. ibid., 81, 4237-9 (1959).

J. H. Boyer and

44. Reactions of dinitroolefins with nucleophilic reagents.William D. Emmons and Jeremiah P. Freeman. J. Org.Chern., 22, 456-7 (1957).

45. Unpublished work of Edward Wat.

46. The infrared spectra of nitro and other oxidizednitrogen compounds. John F. Brown, Jr. J. Am. Chern.Soc., 77, 6341-51 (1955).

The reaction of nitric oxide with isobuty1ene.F. Brown, Jr. ibid., 79, 2480-8 (1957).-- --

John

48. Mononitrohydrocarbons. Ch~r1es A. Burkhard and JohnF. Brown, Jr. US Patent 2,867,669. C. A. 53,11225c (1959).

ACKNOW LEDGEy,lENT

'The author wishes to express his gratitude to

I. Lynus Barnes for the help in taking and interpreting

the ultraviolet spectra, to Mrs. Vira Walker for typing

the manuscript and to the National Institute of Health

for financial assistance.