JPS615090A - Novel anthracycline tmf518 and its preparation - Google Patents

Abstract

NEW MATERIAL:Antibiotic TMF-518 of formula I (R1 is formula II; R2 is formula II, H): having following physicochemical properties (R1 and R2 are both formula II): molecular weight: 1164; molecular formula: C60H80N2O21; melting point: 190-200 deg.C; solubility: insoluble in water, hexane, soluble in methanol, ethyl acetate, chloroform; basic; yellowish powder; elementary analyses (%): C 61.95, H 6.54, N 2.51, O 29.00. USE:Antibacterial, antitumor agent. PREPARATION:Streptomyces cosmosus FERM-P-7074 (AJ9450) is aerobically cultured at a pH of 4.0-9.0 and 15-40 deg.C and extraction is effected with n-butanol. The compound of formula I where R2 is H is also obtained by catalytic reduction of the compound of formula I where R2 is formula II, preferably at normal temperature and normal pressure in the presence of a Pb/BaSO4 catalyst for 0.5-3hr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anthracycline substance TMF 518 C having antibiotic activity and anti-inflammatory activity.
It is abbreviated as 18. ) and its manufacturing method.

Antibiotics such as adriamycin, daunomycin and aclacinomycin have been known as anthracycline substances produced by microorganisms of the genus Streptomyces. The present inventors discovered that TMF518 in the same anthracycline substance was found in the Murlne Sarcoma Virus strain (Murrne Sarcoma Virus).
Mouse fibroblasts λ1-MSV Batb3T3 transformed with US) (Motoney)
It was discovered for the first time that the plant has a strong effect on the growth of plants. Therefore, TMF518 of the present invention can be used as an antitumor agent. In addition, this 9-point weight exhibits strong antibacterial properties against Sartina lutea, Bacillus subtilis, etc., so it can be expected to be used as a biological negative for cells.

The anthracyclines and T↑l4F518 of the present invention are represented by the following general formula.

Here R1 is and R2 is It is.

Specific examples of TMF 518 of the present invention include the anthracycline substance T'' 518C (hereinafter abbreviated as TMF518C) (compound of formula 2), and the anthracycline substance TMF 518D (hereinafter abbreviated as TMF 518D).
There is (2■ combined middle power).

These TMF may exist in the form of a salt, and examples of the salt include i such as hydrochloric acid, hydrochloric acid, phosphoric acid, and acetic acid. It is also possible to consider the form of a metal complex made of copper, iron, etc.

TMF 518C and TMF 518D of the present invention have the following physical and chemical properties.

* Determined by FD-MS, T-518C belongs to the Strezotomyces genus, and is produced by culturing a microorganism capable of producing T-518C, producing it in a culture solution, and then using known isolation and purification methods to produce it. It can be obtained by collecting TMF518D.
It can be obtained by subjecting TMF518C obtained in this manner to catalytic reduction according to the n method.

The conditions for the catalytic reduction are not particularly limited, but it is preferably carried out at room temperature and pressure using Pb/BaSOa as a catalyst and reacting H2 gas for about 30 minutes to 3 hours. Also TMF5
18D can also be isolated directly from the culture fluid of the above-mentioned microorganisms capable of burying TMF 518C.

The medium used for culturing the above microorganisms to produce TMF 518 is a conventional liquid medium containing a carbon source, an organic source, an inorganic salt, and, if necessary, organic micronutrients.

Examples of carbon sources include glucose, fructose, maltose, sucrose, and star f.

Carbohydrates such as dextrin, starch hydrolyzate, and blackstrap molasses, organic acids such as citric acid, succinic acid, fumaric acid, and acetic acid, and alcohols such as glycerin are used. As a nitrogen source, for example, bre ammonium, jA ammonium, ammonium phosphate.

Ammonium salts such as ammonium nitrate and ammonium acetate, nitrates such as sodium nitrate and potassium nitrate, urea,
Ammonia water, ammonia gas, amino acids, peptone, soybean whey, soybean flakes, and their hydrolysates are used. ◇Other inorganic salts include manganese salts and phosphates, and organic trace nutrients are used as appropriate. Amino acids, vitamins, peptones containing these, yeast extracts, and the like can be used as appropriate.

Culture conditions vary depending on the medium composition and other factors, but are usually pH 4, Q to 9.0. Culture is carried out under aerobic conditions such as shaking culture 1 and aeration stirring culture at a temperature of 15 to 40°C.

TMF 518C, TMF 518 produced by culture
D is present in the culture solution. TMF 518C from culture solution
, methods for decomposing and collecting TMF 518 D include extraction with organic solvents such as n-butanol, normal phase and reverse phase silica gel,
Adsorption chromatography using adsorbents such as cellulose.

This is carried out by appropriately combining known fractionation and purification methods such as the gel filtration method and the method that utilizes the difference in solubility in Tani JJf solvents. Preparative and purified TMF 518C, TMF518
D is obtained as a yellow powder by removing the solvent. In addition, a more appropriate method can be devised by taking into account the above-mentioned medium composition, culture conditions, rice producing the target substance, etc.

Both TMF 518C and Th1F 518D of the present invention are new substances, and they have antimicrobial and antitumor activities.

Next, the present invention will be explained in detail by examples.

Example 1 (a) Production of TPiiF 518C Soluble starch 2.0, glucose 1.0%, soytone 07%, yeast extract 0.2%, KM2PO40.1.

MgSO4・7H200,1%, NaCtO,1
% (p) 17.0) 10. Sterilize the 500+++l dissolution flask in which ON was dispensed at 120°C for 20 minutes, and add Streptomyces cosmosus FERM.
-P7074 (AJ9450) culture solution 1 was inoculated and cultured at 27°C for 4 days.

On the other hand, a 30A' capacity stainless steel jar fermentor
Put the above medium in 181 and sterilize it, then add the above i
(Inoculating 1 + Mother 21, 4fi stirring (350 rpm),
Aeration ('7vvm) L The culture was continued at 27°C for 2 days.

Furthermore, 28 ol of the above-mentioned medium was placed in a 3,001-volume stainless steel tank as a secondary compound, and then sterilized and glazed.
m) L Cultured at 27°C for 3 days. The obtained culture solution of 2801 was centrifuged to obtain mycelium and supernatant.

This supernatant was concentrated to 1 #301, 6 ml of n-butanol was added, and the mixture was shaken vigorously, then shaken to separate the layers, and then the n-butanol layer containing the target substance was concentrated to 31. Chloroform 31 was added to this mixture to separate the layers. Among these, ooform Hd 300 rn containing the target substance
Reduce l to 100 power, LH-20 (manufactured by Pharmacia)
Dull filtration was carried out using methanol as a solvent.

The obtained fractions were subjected to thin layer chromatography (silica gel plate F254, Merck, solvent chloroform:methanol 19:1) and fractions having a yellow spot with an Rf of about 0.6 were collected. These fractions were concentrated and then subjected to silica column chromatography (Wako gel C-2).
00, chloroform to methanol (30:1). The obtained fractions were each subjected to the above-mentioned thin layer chromatography, and fractions having yellow spots were collected. Concentrate this and
Silica gel plate for separation and separation (20X 20X O,
2m) was used for purification by scraping.

Development was carried out using a solvent system of 11 to 1 benzene to methanol. Rf=0 obtained by developing with this solvent system
．． The yellow band of No. 4 was scraped off and eluted with a 1/1 mixture of chloroform/methanol, and the eluate was concentrated to dryness. This is dissolved in ethyl acetate, washed with water three times, and hexane is added to the ethyl acetate layer containing the target substance to form a yellow precipitate. After removing the molten W and drying the precipitate, TMF5
An ink is produced that yields a yellow powder of 18C. TMF 518C
has the above-mentioned physical and chemical properties and is a new substance that does not fall under any of the known anthrazycline substances. Also, TMF 518C has a 0. 8 with IN HCl
When hydrolyzed for 30 minutes at 5°C, rhodosamine, 2-deoxyfucose, and citromidinone sugars are produced as aglycones.
Synel o −, x, B'c given L le. An anthracycline with citromidinone as the aglycone has never been found before (TMF 518C is the first anthracycline with citromidinone as the aglycon).

(b) Manufacture of TMF 51SD I7:1 New anthracycline substance TMF 518C obtained by the above method
was dissolved in methanol, and Pd/B a SOa was added in an amount twice the weight of Tken' 518C for 30 minutes.
By pumping hydrogen gas for 1 hour and causing a reaction, TF/I
You can get F518D. The same substance as TMF 518D thus obtained can also be obtained from the culture medium of 'l'MF 518C using the same method as for isolation of TMF 518C. Next, the physiological activities of TMF 518C and TMF 518D of the present invention will be shown.

a) Antibacterial activity Pact Annapaiodic Medium 3 (manufactured by Difco) 1.75 cm, Gaidai 〇, 8% medium was heated at 120°C.
Heat sterilize for 15 minutes. This is kept in a constant temperature bath at 42℃,
When the temperature of the medium reaches 42°C, add 1 m7 of the above medium to each of the Mycobacterium strains shown in the first rock, which have been incubated at 37°C for 20 hours. Inside 1
Prepare so that there are 08 cells. These media 2
Dispense 0 mtf into each petri dish to prepare a plate.

Then TMF 518C at a temperature of 0-1 Cape/IILt
and 50 μl of TMF 518D into a hard disk. This Pegnog disc was placed on the above plate and cultured at 37°C for 20 hours.
The size of the inhibition circle formed by TMF518D was measured, and the minimum growth succession (MIC) was determined.

The results are shown in Table 1.

Table 1', -)'%恕'=” >1000 >100
0ATCC10145 b) M-MSV Ba1b transformed by virus
Effect on 3T3 MEM Dulbecco powder medium (Gibco product) III
was dissolved in 100 mJ of double-distilled water, 0.14.9
of NaHCo 5 was added and dissolved, and filtered with a Millidea filter (
0.22μ) and added beef tallow blood (Yoi (Flora made by Latry) 11 times) to a medium in advance at 37°C.
M-MSVBalb cultured for 3 days in the presence of 5% CO2
3T3 was dispersed at 8.0 x 10' cells/me, and each medium Q and zae was dispensed into a microplate (manufactured by 96-well Nunc Co., Ltd.) and incubated for 3 hours in the presence of 5% charcoal gas. Cultured at ℃. Toreni, O~50μIV
Add TlviF 518C,D at a concentration of 7 ml.
．． It was cultured for 3 days.

Thereafter, the growth amount was measured, and the growth inhibition level (the degree of inhibition of growth by 50%) IC5o was determined. The results are shown in Table 2 of Asahi.

Table 2 TMF 518CO, 15 TMF 518D 0.25

[Brief explanation of drawings]

FIG. 1 is an ultraviolet absorption spectrum (in methanol) of TIviF 518C. FIG. 2 is the ultraviolet green absorption spectrum of TMF 518D (in methanol). FIG. 3 is an electrolytically deoxidized enzyme spectrum of TMF 5180. FIG. 4 shows the proton nuclear magnetic resonance spectral of TMF 518C. FIG. 5 is a 41B''C-nuclear magnetic resonance spectrum of T■''518C. Figure 6 is the electrolytic desorption mass spectrum of T''518D. Applicant: Ajinomoto Co., Inc. Date 1, July 1982, Case Indication, Patent Application No. 126258 of 1982, Title of the Invention, New Anthracycline Substance rMr51a and its Manufacturing Process, 3, Person Making the Amendment, Case Relationship, Address of Patent Applicant No. 4, 5M8, Kyobashi-chome, Chuo-ku, Tokyo, Date of amendment order Vol.
4 Correct the sentence, ``cyto [] midinone, sugar.''"1-7Clicquot" on page 15, line 5 of the specification is "7'Guri"
” he corrected. In the 7th line from the bottom of page 15 of the specification, ``for 30 minutes'' is deleted. (Procedural amendment writing method) % formula % 2. Name of the invention: New insulin-based substance TMF518 and its manufacturing method 3. Person making the amendment Relationship with the case Patent applicant address: 5-8 Kyobashi-chome, Chuo-ku, Tokyo issue (shipment date)
September 25, 1980) 5. Increased due to correction η
Number of inventions: None The title of the invention in the description, "Anthracycline Monogacho MF518 and its manufacturing method" is corrected to "New Anthracycline-based substance TMF518 and its manufacturing method."

Claims (3)

[Claims] (1) General formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (here R_1 is ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ and R_2 has ▲mathematical formula, chemical formula, table, etc.▼ or H Anthracycline substance TMF518 represented by (2) It is characterized by culturing a microorganism belonging to the genus Streptomyces and having the ability to produce the anthracycline substance TMF518, accumulating the anthracycline substance TMF518, collecting it, and performing catalytic reduction as necessary. , General formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) Here, R_1 is ▲There are mathematical formulas, chemical formulas, tables, etc.▼, and R_2 is ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or H. Anthracycline substance TMF518
manufacturing method. (3) A microorganism that belongs to the genus Streptomyces and has the ability to produce the anthracycline substance TMF518 is Streptomyces cosmosus FERM-P-7074 (AJ945).
0) The manufacturing method according to claim 2.