JPS59232093A - Anthracycline-type substance tmf-518 and its preparation - Google Patents

Abstract

NEW MATERIAL:The anthracycline-type substance TMF-518 having the following properties: Color, red; nature of the substance, basic,; molecular weight (mass-spectrometry), 755; molecular formula C40H53NO13; melting point, 139- 143 deg.C; solubility, soluble in methanol, ethanol and chloroform, and hardly soluble in water and n-hexane; UV-adsorption spectrum and IR-absorption spectrum, as shown in the figures. USE:An antitumor agent and antibacterial agent. PREPARATION:A microbial strain belonging to Streptomyces genus and capable of producing the anthracycline-type substance TMF-518 (Streptomyces cosmosus, FERM-P No.7074) is cultured in a medium at 4.0-9.0pH and 15-40 deg.C under aerobic condition, and the organic solvent extract of the cultured liquid and the microbial cell is treated by a proper combination of conventional separation and purification processes to collect the objective TMF-518.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anthracycline substance TMF-518 having antibiotic activity and antitumor activity and a method for producing the same.

The present inventors have conducted a wide range of searches on microbial cultures for the purpose of searching for new antibiotics and anti-HA active substances.
As a result, it was discovered that actinomycetes belonging to the genus Streptomyces isolated from soil produce anthracycline-based substances that have antibiotic and antitumor activities. Recognized as an anthracycline substance, this substance is T! v! It was named F-518 (hereinafter often simply abbreviated as TMF-518).
Conventionally, antibiotics such as ayliaisin, daunoisin, and aclacinomycin have been known as anthracycline-based substances produced by microorganisms of the genus Streptomyces. τλf-518 in the substance is Murindercoma virus Moloney strain (Mrin* Sarcoma Virus) (M
It has been discovered for the first time that this compound has a strong growth inhibitory effect on M-MSV Ba1b 3T3 mouse fibroblasts transfected with M-MSV Ba1b 3T3.

TMF-518 has a strong growth inhibitory effect on M-MSV Balb 3T3 and can be used as an antitumor agent. Furthermore, TMF-518 exhibits strong antibacterial properties against Zarcina lutea, Bacillus zuzotilus, etc., and can also be used as an antibiotic.

That is, the present invention provides a novel anthracycline substance having the molecular formula C4°H5!1NO13m and having antitumor and antibacterial activities. And, a method for producing TMF-518 comprising culturing actinomycetes that belong to the Streptomyces parent family and have TMF-518-producing ability, producing TMF-518 in the culture solution, and collecting the TMF-518.
-518 may be present in salt form. (3) Examples of salts are hydrochloric acid, acetic acid, and sulfur! 12 e') Examples include salts such as phosphoric acid. Furthermore, metal complexes made of copper, iron, etc. are also conceivable.

The microorganism that produces TMF-518 of the present invention belongs to the Streptomyces parent and is an actinobacterium that has the ability to produce TMF-518. For example, a new bacterial species Strezotomyces cosmosus FP that belongs to the Streptomyces parent 'RM P-7
074 (AJ 9450).

The medium used to produce TMF-518 using this microorganism is a conventional liquid medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, organic nutrients.

Examples of carbon sources include dulcose, fructose, maltose, sucrose, starch, dextrin,
Hydrolyzed starch, carbohydrates such as blackstrap molasses, organic acids such as citric acid, succinic acid, fumaric acid, and acetic acid, and alcohols such as glycerin are used. Examples of nitrogen sources include ammonium chloride, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonium (4) such as [1 ammonium], nitrates such as lamb salt, sodium nitrate, potassium nitrate, urea, aqueous ammonia, ammonia gas, amino acids, and more. Proteins such as peptone, soybean whey, soybean flakes and their hydrolysates, rice bran, etc. are used. As the geothermal salt, for example, manganese salt and phosphate are used as appropriate, and as organic micronutrients, amino acids, vitamins, and the like are used (deton, yeast extract, etc. are used as appropriate).Culture conditions are determined by the culture medium composition. Although the conditions vary, for example, culture is usually carried out under aerobic conditions such as p) f 4.0 to 9.0 and a temperature of 15 to 40° C. with shaking or aeration and agitation.

TMF-518 of the present invention exists in the culture solution obtained by culturing in this way and in the bacterial cells,
Methods for separating and collecting -518 include organic solvent extraction using n-butanol, adsorption chromatography using adsorbents such as normal-phase and reverse-phase silica gel, and cellulose, gel filtration, and the use of differences in solubility with respect to various mKs. This method is carried out by appropriately combining known separation and purification methods. On the other hand, (5) TMF-518 produced within the bacterial cells was extracted with an organic solvent such as chloroform, and then purified and separated using the above method. The separated and purified TMF-518 is obtained as a red powder by removing the solvent.

A more appropriate method can be devised depending on the medium composition, culture conditions, production amount of TMF-518, etc.

Next, a manufacturing example will be shown.

Cultivation was carried out as follows.

Soluble Denzone 2.0%, Glue 1.01. Soyton 0.7%, yeast extract 0.2 #KH2PO40,
1ch.

MgSO4”7H200,1%, NaC10,I
5. Dispense 100 d of medium (pH 7,0) consisting of
Sterilize the 00 at molten lafrus ff at 120°C for 20 minutes, and add strepto ζ sescosmosanu FERM-P.
7074 (AJ 9450) culture solution IItl was inoculated and cultured at 27°C for 4 days. On the other hand, put 181 of the above medium into a 30J capacity stainless steel Schafer Mentor, sterilize it, inoculate the above seed mother 21, and stir (350
rpy+), aeration (A mam), and continued culturing at 27°C for 2 days. Furthermore, 300 using 201 as a secondary glaze mother! Inoculate the medium 2807 with the above composition into a sterilized tank, stir (310 rpm), and aerate (%4).
vvm ) L was cultured at 27°C for 3 days. Obtained 2
The culture solution of 80j was centrifuged to obtain hyphae and supernatant. First, to obtain hyphae and TMF-518, the following method may be followed.

13 kg of this mycelia, chloroform; methanol (1:
Add mixture 601 of 1) and stir at room temperature for 4 hours until 7'21
．． The mycelia were removed by post-filtering. Includes 〒8-518 tr
The chloroform:methanol mixture was condensed to give a red oily substance.

This was applied to a silica gel (Merck Co., Ltd. 1g) column (35X1
50 w) K was poured and elution was performed stepwise with a chloroform-methanol solvent. First, both fractions containing free aglycone were eluted with chloroform, and then the solvent was switched to chloroform:methanol=1.5:IK1 for elution, and both fractions containing the target substance were concentrated.

Next, this was spotted on 6064 cellulose, TLC (manufactured by Jusei Kagaku Co., Ltd.), and ethyl ether:n-hex? y:
It was developed with opening solution B having a composition of methanol:pyridine=10:4:0.5:0.1. Two red spots will appear. The residue with a larger Rf value (S/,) was extracted with ethyl ether and concentrated under reduced pressure. This is dissolved in a small amount of chloroform, and n-hexitin is added to precipitate it. Centrifuge, remove precipitate, and dry to obtain red powder TM
Obtained F-518.

The above operation was repeated to obtain 15 μm of TMF'-518.

A purified sample of Zero MF'-518 was purified by silica dull thin layer chromatography (chloroform:methanol concentration 10:1).
Alternatively, they were confirmed to be a single substance (butanol:acetic acid:water (4:1:1)) and cellulose (thin layer of cellulose) (ethyl ether acetic acid:water - 10:1:0.5).

Next, TMF'-518 in the culture solution was tested as follows. This culture medium 2801 is added to 301t P2i L
, n-butanol 601 was added, and the mixture was shaken vigorously and left to stand. The n-butanol layer was concentrated using 31FC. 3 for this! ! After processing and separating the chloroform into layers, it was concentrated to obtain a red oily substance containing 200 mjoT8/y-51. After O, follow the method for obtaining bacterial cells described above and use TR.
Obtained 6 das of IIF-518.

TMF-518 has the following physical properties.

a) Molecular weight: 755 (Manunuiktor method) b)
Molecular formula: C4QH5! No□ C) Melting point: 139-143℃ d) Color of substance: Red ・) Solubility: Soluble in methanol, ethanol and dichloroform, slightly soluble in water and hexane f) Basic, Fa, neutral Distinction: Basic property g) Ultraviolet absorption spectrum (methanol 111 figure h) Infrared shark absorption nuictor (knee)): 92 figure 1) 74-ru sodic, n-mas suictle: Physical properties shown in Figure 3 and above ) Identify TMF-518. In the ultraviolet absorption spectrum, TMF-518 has λmax: 23
8nm @256nm *295nm, 496nm
# It has a characteristic absorption at 532 nm, which is very similar to anthracyclines such as daunorubicin, carminoid (9) and B1 carminoid, retamycin. In addition, in the infrared green absorption spectrum, TMF'-518 has a broad absorption of 3460i', a strong absorption of 2936ffi-', and a strong absorption of 2812m-'.
Weak absorption of s2758m-', strong absorption of 1601ffi-1.

These are roseolcine A and B, lovirubin A and B.

? It is thought that the absorption characteristic of anthracycline-based substances is 4B. In conclusion, the present substance TMF-51B has been identified as an anthracycline substance. In addition, field desodic, mass spectra (FD-MS) all measurements.

In addition, T'MEP-518 showed a molecular ion peak in 1756, but in the field method, a molecular ion peak may be observed at a location that is 1 larger than the molecular weight. From, T Nuto 51
There are several anthracycline-based substances whose zero molecular weight is around 750, where the molecular weight of 8 is 755.

Demethoxical? Nil MA 144 Gl (
Molecular weight 753) (10) 10-0-decarpomethoxy aclacinomycin h
(molecule 1753) Daunomycinone K
(molecule 1769) γ-rhodomycin A
(Molecular weight: 769) Since the molecular weight of TMF-518 is 755, it is clearly different from these substances.

The above data is real 5! iTFM-518 was identified as a novel anthracycline %rgJ.

TMF-518 has the following physiological activities.

a) Antibacterial activity Pact Antibiodi, Coumedeum 3 (manufactured by Difco) 1.75cm, 51! A medium consisting of 0.8 liters of 12
Heat sterilize at 0°C for 15 minutes. This is kept in a constant temperature bath at 42°C, and when the temperature of the medium reaches 42°C, it is heated to 37°C in advance.
The various bacterial strains shown in Table 1 were cultured for 20 hours in the above medium i.
Prepare so that there are 10 cells in rnl.

A plate is prepared by dispensing 20M of these medium into each petri dish.

1 Then, TM at a concentration of 0 to 11R9/Tnl
Soak F'-518-50μ into the E/mother disc. This peno-arm disc was placed on the above plate and incubated at 37°C for 20 hours, and the size of the inhibition hole formed by TMF-518 was measured, and the minimum growth inhibitory concentration (MI
C) was obtained.

The results are shown in Table 1.

Table 1 ATCC9341 No+tilus spuptilus 2.9 A'I'CC6633 b) M-MSV Ba1b transformed by virus
Effect on 3T3 ■M Darui, Ko powder medium (product of Gibeo) 1g
After dissolving in 00d of double-distilled water, 0.14.9 of Na
) Add ICOs and dissolve, milli? afilter(0,2
2μ) and added fetal bovine serum (manufactured by Flora Latry Co., Ltd.) to a culture medium at 37°C in advance.
Cultured for 3 days in the presence of 5% CO2 and subjected to 7'j M-MS
VBalb 3T3 to 8. The cells were dispersed to a concentration of OX 10 cells/M, and 0.2 d of each medium was dispensed into Microplate (96-well manufactured by Nunc) and cultured at 37° C. in the presence of 5% carbon dioxide gas for 3 hours. To this, O~50μ9
TMF'-518 at a concentration of /rnl was added and cultured for 3 days. Thereafter, the growth amount was measured, and the growth-inhibiting concentration (concentration that inhibits growth of 50q6) IC5o was determined.

I.C. =0.25 μm1/Id In addition, the Stredetomyces spmosus FERMP-7
074 (AJ 9450) shows the following mycological properties.

(a) Morphology 1,) Branching method of spore-forming hyphae: Simple (no axle-like shape) 2,) Morphology of spore-forming hyphae: R@ctifle
xlbiles 4,) Presence or absence of flagellar spores: None 5,) Presence or absence of sporangia 6,) Epiphytic position of sporophyte: Aerial hyphae 1 佽) Growth status in each medium (13) 1.) Sucrose/Hyphae Growth on Poshio agar medium: Poor (Pals yellow) Soluble pigment: None 2) Growth on Glucose/Anunogline agar medium
: Middle F (PILI@red purple) Soluble pigment: Pa1e purplish pink pigment production 3,) Glycerol-asparagine agar medium (21
Day) Soluble pigment: Slightly produced (pink pigment) 4゜) Starch-free salt agar medium (21 days) Growth: Good (
Pal@broW11) Soluble pigment: Table 5,) Tyrosine University of Tokyo medium (21 days) (14) Growth: Moderate (Pal@brown) Aerial hyphae; Good settlement (array) Soluble pigment: None 6,) Nutrient agar Medium growth: Medium s [(Pale brown) Soluble pigment: None 7,) Yeast extract-malt No. 2 agar medium growth: J! Good (
BroWn) Soluble pigment - Tashi 8,) Oatmeal agar medium Soluble e31: Purpl@keishiro Shisei 9,) Tsuix R, Kusugo Ten medium) Growth: pale broWn) Soluble color fan: None c) Physiology 1.) Growth temperature range: 2-40℃, does not grow at 45℃ 2.) Growth-range: 26-9 4) Production of melanin pigment Tyrosine agar medium: Negative Tryptone-yeast extract liquid medium: Negative 5.) Hydrolysis of nutarch (starch agar medium): #j property 6.) Coagulation of skim milk, itonization coagulation: Negative peptonization: Positive 7.) Decomposition of cellulose; Negative (d) Assimilability of carbon source (Derridham-Godliep agar medium) As mentioned above, this strain branches in the medium and into the air, forms well-developed true hyphae, and forms a long chain of spores at the tip of the aerial hyphae. Furthermore, since no spores or pen hair spores were observed, this strain belongs to the genus Streptomyces. When searching for the taxonomic position of this strain according to the 8th edition of the Seeds Manual Otsu Determinative Bacteriolotor, this strain was found to be CY: RF:
C-S 8M Series (Percy's Manual of Determinative Pacteriology 8th Edition (1974)
762-e-C) Belongs to FC. 17 bacterial species belonging to this series Streptomyces omyaensis is a bacterial species whose intermediate sugar assimilation z4 turn matches that of this bacterial strain. Therefore, a description of the mycological properties of this strain and Streptomyces omyaensis (Int, J, 8ysL Baet,
22 e 33L1972) and found that: ■ The basal hyphae of this fungus show a reddish-purple color, whereas the basal hyphae of Streptomyces omyaensis show a grayish-yellow color. ■ This fungus produces pink to reddish-purple soluble pigments. On the other hand, Ses omyaensis (Nutrep) does not produce soluble pigments (17), which is a clear difference in its mycological properties.

As mentioned above, Internasi, Nal Streptomyces
The color tone of the basal hyphae and the thermophilicity of production of soluble pigments, which are taken up as mycological characteristics that distinguish species belonging to the genus Streptomyces in the project, clearly distinguish this strain from Nutrede. It's different. Therefore, the present inventors recognized this strain as a new bacterial species belonging to the genus Streptomyces and named it Cosmosus in Strezotomyces.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet m@ collection quittle of TMF-518. FIG. 2 shows an infrared absorption nucleator of TMF'-518. FIG. 3 shows field desorption of TMF-518. It is a mass swictor. Applicant Aji no Hana Co., Ltd. (18)

Claims (2)

[Claims] (1) Anthracycline substance TMF-518 with the following physical and chemical properties (,) Molecular weight: 755 (w Nunuiktor method) (b)
Molecular formula” 040H55NOf!i (,) Melting point: 13
9 to 143°C (d) Ultraviolet absorbing squictor: As shown in Figure 1 (,)
Infrared absorption spectrum: as shown in Figure 2 (f) field desociation; h) Distinction between basic, acidic and neutral: Basic (i) Color of substance: Red (2) Anthracycline TMF is produced by culturing a microorganism belonging to the genus Streptomyces and having the ability to produce anthracycline Th9'-518 in a medium.
A method for producing an anthracycline 5fTMF-518, which comprises producing -518 and collecting the same.