CA1063953A - Figaroic acid antibiotic complex from streptosporangium - Google Patents
Figaroic acid antibiotic complex from streptosporangiumInfo
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- CA1063953A CA1063953A CA255,684A CA255684A CA1063953A CA 1063953 A CA1063953 A CA 1063953A CA 255684 A CA255684 A CA 255684A CA 1063953 A CA1063953 A CA 1063953A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P29/00—Preparation of compounds containing a naphthacene ring system, e.g. tetracycline
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/62—Streptosporangium
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Abstract
New Antibiotic Complex Abstract of the Disclosure A novel anthracycline antibiotic complex designated herein as figaroic acid complex is produced by fermentation of Streptosporangium sp.
strain C-31,751, A.T.C.C. 31129. Figaroic acid complex inhibits the growth of various micro-organisms, e.g., Staphylococcus aureus, exhibits phage inducing properties and inhibits the growth of various tumors in rodents, e.g., Sarcoma 180, L-1210 lymphatic leukemia, B-16 melanoma, Walker 256 carcinosarcoma and P-388 lymphatic leukemia.
strain C-31,751, A.T.C.C. 31129. Figaroic acid complex inhibits the growth of various micro-organisms, e.g., Staphylococcus aureus, exhibits phage inducing properties and inhibits the growth of various tumors in rodents, e.g., Sarcoma 180, L-1210 lymphatic leukemia, B-16 melanoma, Walker 256 carcinosarcoma and P-388 lymphatic leukemia.
Description
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).
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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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.
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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.
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,
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).
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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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.
.
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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.
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 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 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
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 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 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
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, 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.
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.
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.
~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.
: `:
. ~ .
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, 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.
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.
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.
~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.
: `:
. ~ .
Claims (6)
1. A process for producing the antibiotic complex designated figaroic acid complex which comprises cultivating a figaroic acid-producing strain of Streptosporangium having the characteristics of A.T.C.C. 31129 in an aqueous nutrient medium containing assimilable sources of nitrogen and carbon under submerged aerobic conditions until a substantial amount of figaroic acid complex is produced by said organism in said culture medium, and recovering the figaroic acid complex.
2. The process of claim 1 in which the strain is A.T.C.C.
31129 or a mutant thereof.
31129 or a mutant thereof.
3. The process of claim 1 wherein the whole cultural broth is extracted with a water-immiscible organic solvent, the organic phase concentrated, and the solid figaroic acid complex precipitated by dilution of the concentrated organic extract with an antisolvent.
4. The process of claim 3 wherein the whole broth is extracted with n-butanol and the solid complex precipitated from the concentrated organic extract with an ether.
5. The process of claim 1 wherein the whole broth is adjusted with acid to a pH of about 4.5-5.0, the broth is extracted with a water-immiscible organic solvent, the organic phase is concentrated, and the solid figaroic acid complex is precipitated by dilution of the concentrated organic extract with an antisolvent.
6. The process of claim 5 wherein the acidified broth is extracted with methyl isobutyl ketone and the solid complex precipitated from the concentrated organic extract from an ether.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US59099475A | 1975-06-27 | 1975-06-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1063953A true CA1063953A (en) | 1979-10-09 |
Family
ID=24364592
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA255,684A Expired CA1063953A (en) | 1975-06-27 | 1976-06-25 | Figaroic acid antibiotic complex from streptosporangium |
Country Status (12)
Country | Link |
---|---|
BE (1) | BE843517A (en) |
CA (1) | CA1063953A (en) |
DE (1) | DE2628487A1 (en) |
DK (1) | DK141782B (en) |
ES (1) | ES449163A1 (en) |
FR (2) | FR2316965A1 (en) |
GB (1) | GB1541473A (en) |
HU (1) | HU171233B (en) |
IL (1) | IL49912A (en) |
NL (1) | NL166983C (en) |
SE (1) | SE7607279L (en) |
YU (1) | YU155276A (en) |
-
1976
- 1976-06-23 ES ES449163A patent/ES449163A1/en not_active Expired
- 1976-06-23 NL NL7606836.A patent/NL166983C/en not_active IP Right Cessation
- 1976-06-24 SE SE7607279A patent/SE7607279L/en unknown
- 1976-06-25 HU HU76BI00000542A patent/HU171233B/en unknown
- 1976-06-25 CA CA255,684A patent/CA1063953A/en not_active Expired
- 1976-06-25 IL IL49912A patent/IL49912A/en unknown
- 1976-06-25 DK DK289676AA patent/DK141782B/en unknown
- 1976-06-25 YU YU01552/76A patent/YU155276A/en unknown
- 1976-06-25 DE DE19762628487 patent/DE2628487A1/en not_active Ceased
- 1976-06-28 FR FR7619574A patent/FR2316965A1/en active Granted
- 1976-06-28 BE BE168402A patent/BE843517A/en not_active IP Right Cessation
- 1976-06-28 GB GB26833/76A patent/GB1541473A/en not_active Expired
-
1978
- 1978-08-31 FR FR7825228A patent/FR2391731A1/en active Granted
Also Published As
Publication number | Publication date |
---|---|
NL166983B (en) | 1981-05-15 |
ES449163A1 (en) | 1977-08-01 |
YU155276A (en) | 1982-06-30 |
GB1541473A (en) | 1979-03-07 |
IL49912A (en) | 1978-10-31 |
DK289676A (en) | 1976-12-28 |
NL166983C (en) | 1981-10-15 |
HU171233B (en) | 1977-12-28 |
SE7607279L (en) | 1976-12-28 |
FR2316965A1 (en) | 1977-02-04 |
DK141782C (en) | 1980-11-03 |
DK141782B (en) | 1980-06-16 |
DE2628487A1 (en) | 1977-01-20 |
BE843517A (en) | 1976-12-28 |
NL7606836A (en) | 1976-12-29 |
FR2391731B1 (en) | 1981-07-17 |
IL49912A0 (en) | 1976-08-31 |
FR2391731A1 (en) | 1978-12-22 |
FR2316965B1 (en) | 1980-11-07 |
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