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.
~
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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
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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).
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pp; 14-15.
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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).
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13. Struc l,ure and propertie s of C-ni~roso-compounds.
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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).
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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).
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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).
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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.