Note: Descriptions are shown in the official language in which they were submitted.
SY-1143
10~3953 USSN 990, 994
NEW ANTIE~IOTIC COMPLEX
This invention relates to a new anthracycline anti-
hiotic complex and to its production and recovery.
There is provided by the present invention a
new anthracycline antibiotic comple~ designated
herein as figaroic acid complex, said complex being
prepared by cultivating a new strain of Strepto-
sporan~ium designated StreptosporanRium sp. strain
C-31,751, A.T.C.C. No. 31129, in an aqueous nutrient
medium containing assimilable sources of nitrogen
and carbon under submer~ed aerobic conditions until
a subst~ntial amount of figaroic acid complex is
produced by said organism in said culture medium and
optionally recovering the figaroic acid complex from
the culture medium. The invention embr~ces the
unresolved mixture of anthracycline antibiotics
designated as figaroic acid complex in dilute solution,
as crude concentrates or in solid form.
FIG.l shows the infra-red absorption spectrum of
figaroic acid complex (KBr pellet).
FIG.2 shows the ultraviolet absorption spectra
of figaroic acid complex in 0.1N HCl in methanol
(solid line) and in O.lN MaOH in methanol (dotted
line).
1063953
The new strain of Streptosporan~ium designated
Streptosporangium sp. strain C-31,751 was obtained
from a soil sample taken from Seelyville, Indiana.
A culture of the organism has been deposited in the
American Type Culture Collection, Washington, D.C.,
and added to its permanent collection of micro-
organisms as A.T.C.C. 31129.
Figaroic acid complex inhibits growth of various
Gram-positive bacteria, for example, StaphYlococcus
aureus and MYcobacterium tuberculosis, and various
protozoa and yeasts, for example, Candida albicans,
Histoplasma_~R~l,atu~, Trichomonas vaginalis and
Trichomonas faetus. The substance exhibits phage
inducing properties and inhibits growth of various
lymphatic and solid tumor systems in rodents in-
cluding Sarcoma 180, L-1210 lymphatic leukemia,
Walker 256 carcinosarcoma, P-388 lymphatic leukemia
and B-16 melanoma. The figaroic acid complex may
be used alone or in combination with other anti-
bacterial agents to prevent the growth of, or
reduce the number of, the sensitive Gram-positive
bacteria, yeasts and protozoa mentioned above.
It is useful in wash solutions for sanitation
purposes, e.g., for washing hands and disi~fecting
various laboratory, dental and medical equipment
or other contaminated materials and as a bacterio-
static rinse for laundered clothes. It is also
useful in treating the above-mentioned tumor
systems in mice and rats.
10~i3~53
Tl~e strain C-31,751 has the ~ollowing mor-
phologica~ characteristics: Scant aerial mycelium
are formed. During the early growth p~ase, a spore
chain which is short, compact and irregularly
coiled appears at the tip of the sporophore. The
coiled spore chain develops in~o a real sporangium
which is spherical in shape and 4-12~u in diameter.
Most of the s~oranglophores measure ~-lO~u in length.
Looped or short flexuous spore chains are occasionally
co-produced with the fiporangia. The substrate
mycelium is branched, often curved and probably
non-septated. The sporangiospore is non-motile,
spherical to oval in shape and 0.7-0.9~1 in size.
The spore-surface structure has not yet been
determined,
Table I reports the cultural properties ob-
tained on different media, the observations beingafter 1-2 months culture at 28C. The organism
forms aerial mycelium slowly on sucrose-nitrate
agar3 inorganic salts-starc,h agar, yeast extract-
- malt extract agar and oat meal agar. Mass color
of the aerial mycelium is whitish pink to pink.
Aerial mycelium was not formed on asparagine
agars, tyrosine agar, nutrient agar and peptone-
yesst extract-iron agar.
;3953
The sporangium is formed on inorganic salts-
starch agar, yeast extract-malt extract agar and
oat meal agar. Numerous sporangia were seen on
the latter two media after incuhation for seven
weeks at 28C. The m~ss of substraee mycelium
shows granular shape on macro- and microscopic
observation. The princi~al color of the sub-
strate mycelium is reddish orange and reddish
purple on glucose-asparagine agar and yeast
extract-malt extract agar, respectively. Light
yellowish diffusible pigment is produced in
glucose-asparagine agar and yeast extract-malt
extract agar~ A trace amount or no melanoid
pigment is produced in tyrosine aga~ an~ peptone-
yeast extract-iron agar.
.
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10f~3953
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10~;3953
The physiological characteristics and carbo-
hydrate utilization of stràin C-31,751 are shown
in Tables 2 and 3, respectively. The organism
reduces nitrate to nitrite in a natural organic
medium but not in an inorganic medium. Like
most Micromonospora species, it is considerably
sensitive to sodium chloride. It is a mesophilic
organism. Certain carbohydrates such as sucrose,
raffinose, soluble starch and D-mannitol are
utilized after a long lag time.
.
10~3953
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~ i3953
Table 3
CarbohYdrate-utilization Of Strain C31751*
- -- .
I** II ' I II
,
" D(-)-Arabinose - - D(-)-Melibiose t+A tA
L(+)-Arabinose tt tt Trehalose tt +t
D-Xylose t+A tt Raffinose tA
D-Ribose +t ++ D(-)-Melezitose
L-Rhamnose ~+ I+ Soluble starch +
D-Glucose tt ++ Cellulose t
D(+)-Galactose +t ~t Glycerol tt ~+
D-Fructose + +t Inositol +t ~+
D-Mannose l~ ~ D-Mannitol tA tA
L(-)-Sorbose ~ - ~-Sorbitol
Sucrose +A tA Dulcitol
Lactose I + Salicin -
Cellobiose ++ +t No sugar
~,...
~; * Observations after 1-2 months' culture at 28C,
*~ Basal medium I: Pridham-Gottlieb medium plus 0.1%
Difco yeast extract.
II: Leudemann's organic medium, composed
of 0.5% yeast extract, O ,1% CaC0
and 1,5% agar in distilled water,
A: Aerial mycelium poorly formed. No aerial mycelium on
,~ the other sugar media.
,
10~3953
Strain C-31,751 contains meso-diaminopimelic acid
(meso-DAP) as a characteristic amino acid component in
the cell wall. Diagnostic carbohydrate was not present.
Summarizing the above characeeristics, strain
C-31,751 forms whitish pink (shell pink) aerial mycelium
and spherical sporangium. The sporflngiospore is not
motile. The sporangiophore is short, usually less than
10JU in length. The mass color of the substrate mycelium
is orange to violet. Distinct diffusible pigment
(including melanin) is no~ produced. Almost all of the
usual csrbohydrates are utilized for the growth, The
cell wall of the strain contained meso-DAP but no
diagnostic sugar component.
, .
These major characteristics indicate that strain
:.~
:j C-31,751 is a species of the genus StreptosporanRium.
.
According to the taxonomic classification of Strepto-
sporan~ium species by H. Nonomura and Y. Ohara (J.
Ferment. Technol. 47 (11): 701-709, 1969 and 52 (2):
71-77 (1974), sixteen spccies are described. Among
them, eight species have pinkish aerial mycelium and
short sporangiophore; they are Streptosporan~ium rubrum
Potekhina 1965, S. longisporum Schaffer 19fi9, S. roseum
Couch 1955, S. amethysto~enes Nonomura et Ohara 1960,
S. amethystogenes var. nonreducens Prauser et Eckerdt
- 1967, S. vulgare Nonomura et Ohara 1960, S. pseudovulgare
Nonomura et Ohara 1969 and S. nondiastaticum Nonomura et
Ohara 1969. Subsequently, S. violaceochromo~enes
MK-49 was added to the same species-group (Japanese
Patent 49-42896 of April 4, 1974).
10~3953
Strain C-31,751 differs ~rom Streptosporan~ium
ameehysto~enes S. roseum and S. vul~are in its
positive growth at 42C.; from S. lonp~isporum in
its glubose spore; from S. nondiastaticum in its
positi~e utilization of rhamno~e, inositol and starch
and from S. Pseudovul~are in its positi~e utiliza-
tion of rhamnose and inositol and its orange or
reddish purple substrate mycelium, S. violaceochromo-
~enes is differentiated from Strain C-31,751 in
its colorless or gold substrate mycelium and its
negative or doubtful utili~ation of inositol and
rhamnose. Strain C-31,751 shares several char-
acteristics in common with Streptosporangium
rubrum described by L. L. Potekhina ~n Mikrobiologiya,
34, 292 (1965) such as the aerial mass color, color
of substrate mycelium, soluble pigment and spore-
shape. However, the descriptions on S. rubrum
presently available are not sufficient to make a
definite conclusion about the identity of the two
organisms, and strain C-31,751 will therefore be
considered an undetermined s~ecie~s of
Streptosporan~ium until further data is available.
Figaroic acid complex is produced by cultivating a
figaroic acid-producing strain of streptosporangium having
the characteristics of A.T.C.C. 31129 or a mutant thereof
under submerged aerobic conditions in an aqueous nutrient
medium. The organism is grown in a nutrient medium
containing an assimilable carbon source, ~or example
an assimilable carbohydrate Examples of preferred
carbon sources include lactose, glycerol, sucrose, corn
starch, glucose, mannose and fructose. When starch is
10~39S3
used as the carbon source in the nutrient medium, amylase may
be added to the broth before harvest to reduce any emulsion
problems which may occur. The nutrient medium should also
contain an assimilable nitrogen source such as, for example,
fish meal, peptone, soybean flour, peanut meal, cotton seed
meal and corn steep liquor. Nutrient inorganic salts may also
be incorporated in the medium, and such salts may comprise
any of the usual salts capable of providing sodium, potassium,
ammonium, calcium, phosphate, sulfate, chloride, bromide,
nitrate, carbonate or like ions.
Production of the figaroic acid complex can be
effected at any temperature conducive to satisfactory growth
of the organism, i.e., room temperature up to about 43C., and
is conveniently carried out at a temperature of around 27C.
Ordinarily optimum production is obtained after incubation
periods of about 170-210 hours. The medium normally is slightly
alkaline, but the exact pH may vary according to the particular -
~media used. The fermentation may be carried out in Erlenmeyer
flasks and in laboratory or industrial fermenters of various
capacities. When tank fermentation is to be carried out, it is
desirable to produce a vegetative inoculum in a nutrient broth
by inoculating the broth culture with a slant or soil culture
or a lyophilized culture of the organism. After obtaining
an active inoculum in this manner, it is transferred aseptically
to the fermentation tank medium for large scale production of
the antibiotic complex. The medium in which the vegetative
inoculum is produced can be the same, as, or different from,
that utilized in the tank for the production of the new complex,
as long as it is such that a good growth of the microorganism is
obtained.
--11--
1 ~ ~ 39 5 3
When the fermentation is complete, the antibiotic
complex is extracted from the whole broth with a suitable
water-immiscible organic solvent, the organic extract
concentrated, and the solid complex precipitated by
dilution of the concentrated extract with a suitable
antisolvent. Extraction may be carried Oue with water-
immiscible organic solvents varying in polarity from
methylene chloride to n-butanol and in a pH range of
from about 3.5 to 8.5. Solvents in the intermediate
polarity range such as ketones and esters are preferred
since they are found to be more selective than alcohols
but polar enough to give good distribution character-
istics. Examples of especiallv preferred extraction
solvents are methyl isobutyl ketone and ethyl acetat-e.
The most preferred solvent is methyl isobutyl ketone.
Extraction is conveniently done either under weakly
acidic conditions, i.e. pH of 4.0-5.0 effected by
addition of a mineral acid such as HCl or H2S04, or
under weakly alkaline broth pH conditions, i.e. pH
of ~-8.5. Maximum yields are obtaill~d at pH 4.5-5.0
wi~h methyl isobutyl ketone. Filter aid is preferably
added to the extraction mixture and the mixture then
filtered. The organic phase is concentrated and
diluted with an appropriate antisolvent, e.g. ether,
to precipitate out the figaroic acid complex. If
recovered under alkaline conditions, the purple figaroic
acid complex may be converted to the red-orange free
acid form by dissolving the complex in water and
acidifying the solution to precipitate out the acid
complex which can then be recovered hy filtration or
extracted into organic solvents.
~063953
Properties of Figaroic Acid Complex
The antibiotic complex designated herein as
figaroic acid complex is an orange-red amorphous
solid in the free acid state. It is insoluble in
relatively non-polar solvents such as ether, henzene
and aliphatic hydrocarbons, e g., n-hexane, mostly
soluble in lower alcohols tmethanol, ethanol, n-butanol,
2-propanol), acetone, tetrahydrofuran and dioxane,
and totally soluble only in very polar solvents such
as dimethylformamide and climethylacetamide. Upon
recovery under alkaline conditions, the complex is
deep purple in color indicating conversion of the acid
form to the anionic state.
Figaroic acid complex readily forms salts with
bases and pharmaceutically acceptable salts of the
complex, ~or ex~mple, the ?harmaceutically acceptable
alkali metal and alkaline earth salt~J are included
within the scope of the present invention.
The complex contains approximately 2.5% nitrogen
by analysis. It is soluble in aqueous NaHCO3 and
Ba(OH)2 giving respectively red-violet and blue solu-
tions. It gives a deep red solution having red
fluorescence with aicoholic magnesium acetate and a
black solution (orange brown with violet fluorescence
on dilution) with alcoholic ferric chloride. The
complex gives a positive Tollens test but carbazole
1063953
and ninhydrin tests are mask~d by the color of the
pigment. It gives no color change with acid zinc dust,
sodium bisulfite and hydro~en peroxide, There is a
slight fading of the color from violet to red with
alkaline zinc dust and a rapid change to red with
alkaline sodium bisulfite. Alkaline hydrogen peroxide
gives no e~fect except in large excess whereupon the
color fades from violet to pink.
The infra-red and ultraviolet absorption spectra
of figaroic acid complex indicate that the complex
is a mixture of anthracycline components. The infra-
red spectrum (KBr pellet) of FIG.l shows major bands
at 2.94 (broad), 3.4, 6.04-6.13, h.l~, 6.3, 6.95
(broad), 7.1 (broad), 8.1, 9.3, 9.5 and 9O7Ju. The
ultraviolet absorption spectra of the complex under
acidic and ~asic conditions are shown in FIG.2.
Adsorptivity in FIG.2 is ~efined by the e~uation
d - bc
where A is the adsorbance, b is the cell ~idth in
cm. and c is the sample concentration in ~./1. At
a concentration of 50 ~ug./ml. in 0.1N HCl in nethanol,
figaroic acid complex shows absorptioll peaks (solid line)
at 233, 253, 287-288 (shoulder), 467 (shoulder), 4~0
(shoulder), 490, 511 (shoulder) and 524 (shoulder) m
In 0.1N NaOH in methanol the comple~ sho~s absorption
peaks (dotted line) at 238, 266-26~ (shoulder) and 553 m ~.
1~3953
Biological ActivitY Data
The in vitro minimum inhibitory concentrations
~MIC) of figaroic acid complex were determined for
a number of microorganisms using the standard tube
dilution procedure~ The results shown in Table 4
indicate that several gram-positive organisms,
yeast and three protozoans were sensitive to the
antibiotic. Gram-negative organisms were insensitive.
Tahle 4
Antimicrohial Spectrum of Figaroic Acid Comp~ex
Test organism MIC,~ /ml
Bacteria:
Staphylococcus aureus A9537 1.6
Mycobacterium tuberculosis BCG A9579?5
Escherichia coli A15119 50
Pseudomonas aeruginosa A9843 >50
Proteus mirabilis A9900 ~50
Salmonella enteritidis A9531 >50
Yeasts:
Candida albicans A9~40 50
Trychophyton mentagrophytes A9870 ~50
Microsporum canis A9872 >50
Protozoa:
Histoplasma capsulatum A1505~ 6.3
Trichomonas vaginalis A?0074 1.~
Trichomonas faetus A~00750.31
1063953
Figaroic acid complex was tested for its ability
to induce bacteriophage production in th~ lysogenic
strain of Escherichia coli W 1709. Significant in-
dùction was observed down to 0.8 ~g/ml. Tul-e clilution
protein tests to determine cytoxic efects on He La
cells in tissue culture gave a ~07O end-poin~ (ED50)
of 0,004 ~Ig/ml (method described in ~n~imicrohial
Agents and Chemotheropy; l9fi6: f~l3-618, 1967).
The effect of figaroic acid complex on several
rodent tumor systems was also studied. ~etails of
the methods used have been described ;n Cancer
Researcl~ 22: 167-173, 1962 and Cancer Chemoth.Repoxts
3: 1-87(Part 3), 1972. Treatment of mice having
Sarcoma 180 implanted subcutaneously as a solid
tumor with figaroic acid complex fermentation broth
caused 37% inhibition of tnmor ~iameter increase
(estimated 75% inhibition in tumor weight increase).
Treatment with the ~ame broth also increased the life
span of mice bearing L-1210 leukemia by 29% over
control animals. The ~igaroic acid complex broth was
found to be active against Walker 25fi carcinosarco~a
(intramuscular), P-388 lymphatic leukernia and B-16
melanoma in rodents. Solid figaroic acid complex was
also tested and found active on various tumor systems.
Results on L-1210 leukemia and B-16 melanoma in mice
are shown in Table 5.
~3953
Table 5
Effect of fiR~roic acid complex on transplanted mouse tumors
L-1210 leukemia .'B 16 melanoma
.
Avg, Wt T/C A~g. Wt T/C
Dose difference percent Survivor di~ference percent Survivors
u~ /day (T~C,~ MST Day 5 (T-C,~ MST Day 5
128 -5.2 Tox 1/6 ~3.4 Tox 6/6
64 0 Tox~ 6/6
32 -2.8 150 6/6 ~3 4 2s4(1) 6/6
16 _3 7 ~400(2) 6/6
8 -2.6 129 6/fi -0.9 206 ~;o
4 t3.6 160 6/6
2 -0.7 11~ 6l~ ~0.9 126 6/6
1 -1.7 129 6/6
Treatment: Once daily for 9 days, i.ntraperitoneally
Evaluation: T/C percent MST ~ median s-~rvival time in days:
Treated MST/Control MST X 100.
Criteria: T/C~ 125 considered significant tumor inhibition
(prolongation of host survival)
(1) 2/6 survivors at 60 days
(2) ~/6 survivors at 60 days
~o63953
The following examples serve to illustrate the
lnvention without limiting it. Skellysolve B is a
petroleum ether fraction of b.p. 60-68C. consisting
essentially of n-hexane. MIBK is methyl isobutyl
ketone.
Example 1
Shake-flask fermentation
The organism Streptosporan~ium sp. strain C-31,751
is grown on an agar slant medium consisting of 2 g.
D-glucose, 20 g, oatmeal, 2 g. soy peptone and 20 g.
.
agar made up to one liter with distilled water. After
at least 6 days growth at 27C., spores are transferred
to a 500 ml. Erlenmeyer flask containing 100 ml. of
sterile medium consisting of 50 g. corn starch, 10 g.
soy flour, 10 g. peanut meal and 3 g. CaC03 made up to
one liter with distilled water. This vegetative culture
is incubated at 27C. on a rotary shaker (Gyrotory tier
shaker, Model G53, New Brunswick Scientific Co., Inc.)
set at 210 rev./min. describing a circle with a 5.1 cm.
diameter. After 48 hours 4 ml. of culture is transferred
to a 500 ml. Erlenmeyer flask containing 100 ml. of
sterile production medium consisting of 50 ~. sucrose,
-18-
3953
20 g. soy flour, 20 g. peanut meal and 3 g. CaC03
made up to one liter with distilled water. The
culture is incubated at 27C. on a shaker,set at
230 rev./min. for 170 hours. At this time antibioeic
activity consisting of the figaroic acid complex is
found in the culture filtrate and mycelium.
Example 2
Tank fermentation
A tank fermentor with 37.8 liters of sterile
production medium (as in Example 1) is inoculated
with 1.89 liters of vegetative culture prepared
according to Example 1, agitated with an impeller
speed of 375 rev./min., aerated at a rate of 76.5
liters/min. and incubated at 27C. After 190 hours
the antibiotic complex is isolated.
Example 3
Tank fermentation
A tank fermentor with 3030 liters of production
medium (as in Example 1) is inoculated with 152 liters
of vegetative culture (as prepared by Example 1),
agitated with an impeller speed of 155 rev./min.,
aerated at a rate of 1420 liters/min. and incubated at
27C. The figaroic acid complex is isolated after
210 hours.
10~;3953
Example 4
Extraction of Broth and MYcelia at broth pH (sli~htlY
alkaline) with n-butanol
Filtration of a broth from a shake flask fermen-
tation using a total of 5 liters of starting
medium gave 2.5 liters filtrate, pH 8.5. Of this 1.5
liters was extracted two times with 1 liter batches
of n-butanol, the phases separated, and the combined
organic phases concentrated. Dilution of the concen-
trate with ether gave 1.4 g. of an amorphous purple
solid, active against L-1210 leulcemia in mice at
2 mg./kg./day. The mycelial cake from broth filtration
was stirred for 30 min. with enough methanol to obtain
a fluid slurry and then filtered. The filtrate was
concentrated until most of the alcohol had been removed
and the aqueous residue extracted as above to afford
6.35 g. of figaroic acid complex. Thc product, an
amorphous purple solid, was found to be active vs.
L-1210 leukemia in mice at a concentration of 8 mg./kg./
day.
1063953
Example 5
Extraction of whole broth at broth pH with n^butanol
Whole broth, 1.5 liters at pH 8.6, was stirred
with about an equa~ volume of n-butanol. The thick
mass was filtered through a Celite ttrademark of
diatomaceous earth produced by Johns-Manville Products
Co.) cake, the phases separated, and the organic phase
concentrated to a small volume. This was diluted
with excess ether to precipitate 621 mg. of figaroic
acid complex. The amorphous purple solid is found to
be active against L-1210 leukemia in mice at a dosage
of 0.2 mg./kg./day. The solid does not melt but
decomposes above 200C.
Example 6
Extraction of whole broth at acid pH with n-butanol
The general procedure of Example 5 was repeated
except that pH of the whole broth was adjusted to pH
4.0 with HCl and maintained there during extraction.
Four liters of whole broth yielded 1.7 g. of figaroic
acid complex in the free acid state as an orange-red
solid. The solid is toxic to mice at a concentration
of 0.25 mg./kg./day and shows phage inducing properties
down to a dilution of 1.5 lug./ml.
-21-
10~3953
Example ?
Extractio _of whole broth ~sli~htly alkal~ne) with
n-butanol on a lar~e scale
Whole broth (2788 liters st pH 8.6) was stirred
with 1357 liters n-butanol. The organic phase was
separated to gi~e 783 liters of rich extract which
was concentrated to 13 liters. Addition of 80 liters
"Skellysolve B" gave 728 g. of the crude purple figaroic
acid complex.
Exam~le 8
Acid extraction of whole broth with methyl isobutyl
ketone (MIBK)
` . Whole broth (10 liters) which had been stored
frozen was thawed and stirred with 10 liters MIBK for
20 minutes after adjustment of the pH to 4.5. Filter
aid was stirred into the mixture and the latter was
then filtered on a filter aid pad. The phases in the
filtrate were separated and the organic phase concen-
trated to a small volume. Dilution of thls with excess
"Skellysolve B" gave 4 g. of the orange-red figaroic
acid complex free acid having phage inducing activity
at a dilution of 6.2 ~g/ml.
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~o~3953
Example 9
Acid extraction of whole broth with methyl isobutyl
ketone on a lar~e scale (MIBK~
Whole broth (3095 liters) was adjusted from pH
8.3S to 3.35 at 10C. by addition of 49 liters 30%
H2S04 with stirring and cooling. Two volumes (6412
liters) MIBK were stirred with the broth followed by
addition of excess filter aid (diatomaceous earth).
The mixture was filtered across a precoated vacuum
filter using 2554 liters additional MIBK as rinse.
The rich MIBK extract (7555 liters) was separated
and concentrated to 10 liters. During the concen-
tration 1.285 k~. of wet solids precipitated and were
collected. Addition of 100 liters "Skellysolve B"
to the concentrate afforded an additional 365 g. of
crude oily material. The first crop of solids was
dried to 858 g. and divided into three batches. ~ach
of these was stirred in 4 liters acetone and insoluble
matter filtered off. The latter proved to be filter
aid and, when combined and dried, weighed 367 g. The
acetone solution was concentrated and diluted with
excess ether to give 294.5 g. of crude figaroic acid
complex, Evaporation of the ether gave 19.2 g. of
inactive material. The oily "Skellysolve B" precipitate
was treated also with ether, but proved inactive.
.,
~0~3953
.
Example 10
Conversion of figaroic acid comDlex salt to its free
acid form
The crude purple solid (~5 g.) obtained hy
alkaline n-hutsnol extraction as describe~ in Example
7 was stirred in 1 liter H20 at 25 until it dissolved.
The purple solution was adjusted downward in pH from
8.3 to 1.1 by dropwise addition of concentrated HCl
with stirring. Figaroic acid complex free acid
formed as a fine, silt-]ike brick-red precipitate and
was collected by centrifugation. After drying it
weighed 13.h g. and was active against the L-l?ln
tumor system in mice down to 0.? mg. !Icg. /day dosage.
The sunernatant liquor was lyoPhili7e~l to yiek1
lO.O ~. of totally inactive amor~hous solids con-
taining 7.8~/~ ash by ignition. The restllts of the
comb-lstion analvsis indicate that the cationic
material bound to the purple anionic figaroic acid
complex is largely organic.
: `:
. ~ .
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