JPS6360747B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anthracycline antibiotic, 2-hydroxy aclacinomycin A (2-hydroxy
aclacinomycin A) and acid addition salts thereof, their production methods, and anticancer agents as pharmaceuticals containing these as active ingredients.More specifically, the present invention relates to anticancer agents belonging to the genus Streptomyces and expressed by the following formula by microbial conversion. - Cultivating a strain capable of producing anthracycline glycoside from hydroxyaclavinone under appropriate culture conditions,
2-Hydroxyaclavinone (formula) 2-hydroxyaclacinomycin A is produced by adding and culturing 2-hydroxyaclacinomycin A, which is represented by the structural formula, and is collected from the culture or made into an acid addition salt by a salt-forming reaction. The present invention also relates to a method for producing acid addition salts thereof, and anticancer agents as pharmaceuticals containing these as active ingredients. The substance of the present invention 2-hydroxyaclacinomycin A of the above formula has a strong therapeutic effect on murine leukemia (L1210), but also exhibits excellent anticancer effects on various other animal experimental tumors, Moreover, because of its low toxicity, it can be advantageously used as an anticancer agent. For the production of the compounds of the present invention, for example, aclacinomycin (Japanese Patent No. 86485 and Japanese Patent Publication No.
38099, J. Antibiotics 28 , 830, 834, (1975),
32 , 791-800, 801-819 (1979)).
A mutant strain isolated from MA 144-M1 (ATCC31133, FERM-2455) that is non-antibiotic producing and has the ability to convert aclavinone to aclacinomycin A is used; for example, KE303 is one of them.
(Feikoken Bibori No. 4808) is useful. Regarding the isolation method and bacterial description of this mutant strain, please refer to the patent application filed in
89552 (Special Publication No. 60-23679) and Patent Application No. 54-29820
(Japanese Unexamined Patent Publication No. 55-120786), and is entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
I have obtained the above bacterial infection number. The present invention is based on the fact that the present inventors, while researching the biosynthesis of anthracycline glycoside, discovered that the genes governing the biosynthesis were found to be linked to the aglycone moiety and the sugar chain moiety, which are its constituent components.
In other words, by appropriate mutation treatment, a mutant strain lacking one or both of them and unable to produce the completed anthracycline glycoside can be obtained.In other words, a mutant lacking the biosynthetic function of the aglycone moiety can be obtained. Although no antibiotic production is observed in this strain, active antibiotics (e.g. aclacinomycin A) can be produced by adding an appropriate anthracyclinone (e.g. akravinone) to the fermentation medium from the outside and culturing it. The present invention was completed based on this discovery. A general method for isolating mutant strains that are non-pigment producing (non-antibiotic producing) and have conversion ability from these strains will be described below. Mutation treatment The anthracycline non-producing mutant strain KE303 is, for example, the parent strain ( Streptomyces galilaeus ) MA144−
M1, from Microtechnical Research Institute No. 2455, NTG as an example
It can be obtained by the following method using drug treatment. YS slant medium (yeast extract 0.3%, soluble starch 1.0
%, PH7.0) at 28°C for one week .
The spores scraped from the surface of one slant culture of galilaeus MA 144-M1 were suspended in 5 ml of 0.2 M trisumarate buffer at pH 7.5, and sonicated twice for 15 seconds each (with an ultrasonic distruptor model 1UR). −
200p type, 20KHz, Tomy Seiko KK). Pass the treated solution through a pre-sterilized absorbent cotton filter tube (diameter 0.8 x layer thickness 2 cm), and remove the spore suspension (4 ml, approx.
A solution of N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (10 ^mg /ml) dissolved in ethanol was added to a final concentration of 1000 μg/ml).
ml and shake for 60 minutes at 30°C in the dark (usually has a sporicidal effect of 75-80%).
Centrifuge the NTG-treated spore solution at 300 rpm for 10 minutes, suspend the spores in 0.85% physiological saline, and dilute appropriately.
YS agar medium (yeast extract 0.3%, soluble starch 1.0
%, agar 1.5% PH7.0).
Cultivate at 28°C for 5 days to form colonies. Method for isolating mutant strains: Colonies formed on YS agar as described above are
Pick up on a slant medium and culture at 28°C for one week.
One platinum loopful from each slant was added to the seed medium (yeast extract
0.1%, soluble starch 1.0%, pH 7.0) into a test tube containing 4 ml, and cultured with shaking at 28°C for 2 days. Next, in a 250 ml Erlenmeyer flask, add 25 ml of production medium (glucose 1%, soluble starch 1.5%, yeast extract 0.1
%, soy flour 3.0%, K ₂ HPO ₄ 0.1%, MgSO ₄ .
_7H2O 0.1%, NaCl 0.3%, Minerals 0.125%, PH
7.4) Dispense the above seed culture into the sterilized one.
ml inoculation and culture with shaking on a rotary shaker at 28°C for 2 days. Centrifuge 5 ml of the culture solution, add 5 ml of acetone to the precipitated bacterial cells, and mix well to obtain an acetone layer. The yellow pigment (absorption at OD 430 nm of akravinone) extracted into acetone is measured with a spectrophotometer, and anthracycline non-producing colonies are selected. Next, the ability of this anthracycline non-producing strain to produce glycosides by adding anthracyclinone is confirmed by the following method. One platinum loopful from the slant of the non-producing strain was inoculated into the seed medium, shaken at 28°C for 2 days, and 2.5 ml of this seed medium was added to a 250 ml Erlenmeyer flask containing 25 ml of the production medium. After shaking culture on a rotary shaker for 20 hours at 28℃, add 0.5 ml of akravinone solution (dissolved in methanol, 1 mg/ml).
(final concentration 20 μg/mg) and continue culturing for an additional 24 hours. 5 ml of the culture solution was taken and centrifuged to obtain bacterial cells, and the product was extracted with 5 ml of acetone and concentrated under reduced pressure. 0.1ml to this concentrate
of toluene and 1 ml of 0.2M Tris-HCl buffer (PH
7.5) was added, and after shaking and stirring, a toluene layer was obtained. A 20μ to 50μ toluene layer was spotted onto a silica gel thin layer plate (F ₂₅₄ , manufactured by Merck & Co.) alongside standard aclacinomycin A, and developed in a methanol:chloroform (1:20) mixture. Through this operation, a strain that has been found to produce aclacinomycin A is defined as a mutant strain that can be used in the present invention. That is, in the above method, an anthracycline pigment-forming ability, that is, an anthracycline glycoside non-producing mutant strain is first selected, and then the anthracyclinone donated therefrom is combined with sugar residues produced in the medium. Mutant strains having the ability to form anthracycline glycosides can be selected and bred. Moreover, according to this method, according to the above-described mutation treatment and mutant strain breeding and isolation methods, the mutant strain ( Instead of the KE303 strain, anthracycyl glycosides whose sugar chain moieties are the same as aclacinomycin, such as cinerubin, galerubin, and pyromycin, were isolated from the following strains that produce them. It goes without saying that mutant strains that can be used may also be used. In that case, the strains used to obtain the mutant strain of the present invention include, for example, St. galilaeus ATCC 14969,
Streptomyces cinereolvar ( St.
cinereoruber) ATCC 19740, St. antibioticus
ATCC 8663, St. nivereoruber ( St. nivereoruber ) ATCC 14971, etc. The substrate 2-hydroxyacrabinone used in the present invention is Streptomyces galilaeus MA 144-
M1 (ATCC 31133), an aglycone-accumulating mutant strain, ANR-58 (Feikoken Bacterial Serial No. 5081)
The details can be obtained and used by culturing Japanese Patent Application No. 115520 (1983).
7905). Note that the substrate used in the present invention may be a crude preparation, such as ANR-58.
After concentrating the acetone extract containing 2-hydroxyaclavinone from the culture, it may be dissolved in methanol and added as is. The compound of the present invention is produced by first using an agar slant medium (yeast extract 0.3%, soluble starch 1.0%, agar 1.5%,
A strain (e.g., KE303 strain) having the above-mentioned conversion ability, cultured at pH 7.2) and stored at 6 to 7°C, is inoculated into a normal liquid medium consisting of, for example, starch, glucose, an organic nitrogen source, and inorganic salts. , 1~ at 25~32℃
Culture with shaking for 3 days to prepare seed mother. Next, 1 to 3% of the above seed mother was inoculated into a normal liquid medium, such as a medium consisting of sucrose, glucose, soybean flour, and inorganic salts, and cultured with shaking at 25 to 32°C for 15 to 48 hours. To the culture solution that has reached the growth phase,
Add a methanol solution of 2-hydroxyaclavinone to a final concentration of 10 to 200 μg/ml,
Continue incubation for an additional 15-72 hours to complete microbial conversion. In order to suppress foaming during fermentation, antifoaming agents such as Adekanol (trademark of Asahi Denka Kogyo Co., Ltd.) and silicone (trademark of Shin-Etsu Chemical Co., Ltd.) can be added as appropriate. The culture solution is separated into bacterial cells and liquid, and the crude pigment containing the compound is extracted and purified from the bacterial cells and liquid. Acetone, methanol, chloroform, ethyl acetate, toluene, dilute mineral acid, acidic buffer, etc. are used for extraction. For purification, silica gel (manufactured by Wako Pure Chemical Industries, Ltd. and Merck & Co., Ltd.), cross-linked dextran gel (Sephadex LH-20,
Pharmacia Co., Ltd. trademark), columns and thin layer chromatography using weakly acidic ion exchange resins, etc.
This can be advantageously carried out by combining conventional methods such as liquid chromatography using an appropriate solvent and countercurrent distribution method. For example, the product is separated from unconverted aglycones by gel filtration of the crude extract on a cross-linked dextran gel (Sephadex LH-20) column, followed by a thin layer of preparative silica gel (PF ₂₅₄ , Merck & Co.). The purified compound can be easily obtained by repeating the chromatography while changing the solvent. The obtained compound has ultraviolet, visible light absorption spectra (hereinafter referred to as UV), and infrared absorption spectra (IR).
and 100MHz high-resolution proton and C-13 nuclear magnetic resonance spectra (PMR and CMR), mass spectrometry and elemental analysis, and after separation into the aglycone part and sugar part by hydrolysis, each was subjected to instrumental analysis or thin layer analysis. From the results of qualitative analysis by chromatography, it was determined that the substance of the present invention is 2-hydroxyaclacinomycin A represented by the above structural formula. That is, L-rhodosamine, 2-deoxy-L-fucoce, and L-cinerulose are added to the C-7 position of 2-hydroxyaclavinone, which is the added substrate aglycone. Aclacinomycin A (J.
Antibiotic 32 , 801-819 (1979)) was found to have a structure in which one hydroxyl group was introduced at the 2-position.
In addition, aglycone components obtained by hydrolysis:
The identification of 2-hydroxyaclavinone was made in a patent application filed in 1984.
This was done because it completely matched the analysis values regarding various physical and chemical properties described in detail in 115520. On the other hand, the determination of the sugar component, the order of the sugar chain, and the bonding position with the aglycon is described in the above-mentioned document (J.
Antibiotic 32 , 801-819 (1979)), the compound was found to have the same sugar structure as aclacinomycin A. revealed. The compounds of the invention are also available as free bases or non-toxic salts. The free base can be prepared using a non-toxic acid such as sulfuric acid, hydrochloric acid, hydrochloric acid, nitric acid,
It is recovered as addition salts with phosphoric acid, acetic acid, propionic acid, maleic acid, oleic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, lauryl sulfonic acid, methanesulfonic acid, naphthalene sulfonic acid, etc. Its therapeutic effect is substantially equivalent to that of the free salt. That is, the addition salt can be formed by reacting the free base with the non-toxic acid mentioned above in a suitable solvent and lyophilizing it, or by precipitating it using a solvent in which the acid addition salt is only slightly soluble. I can do it. The physicochemical properties of 2-hydroxyaclacinomycin A, the compound of the present invention, will be described below. Shape Yellowish brown powder Melting point 165-167℃ Molecular weight 827.9 Elemental analysis: C ₄₂ H ₅₃ NO ₁₆ Calculated value (%) 60.93 6.45 1.69 30.93 Experimental value 60.27 6.20 1.64 - Specific optical rotation [α] ²³ _D +42.3゜(c 0.04, MeOH) UV-visible absorption spectrum λ90%MeOH max nm (E1% 1cm): 222 (375),
256 (235), 295 (207), 450 (110) λ90%MeOH-0.1NHCl max nm (E1% 1cm): 226 (448), 254 (238), 268
(245), 291 (251), 440 (158) λ90%MeOH−0.1NNaOH max nm (E1% 1cm): 240s (370), 297 (252), 330s
(196), 540 (143) IR (KBr) cm ^-1 : 3450, 2975, 2940, 1735, 1675, 1620,
1610, 1450, 1400, 1380, 1300, 1255,
1230, 1170, 1120, 1010 PMR spectrum (100Hz, CDCl ₃ ): δppm: (See Figure 3) Next, the usefulness of the chemical substance according to the present invention will be described. The compound of the present invention significantly inhibits the proliferation and nucleic acid synthesis of mouse leukemia cultured cells (L1210).
For example, L1210 cells were inoculated at 5 x ¹⁰ cells/ml into RPMI 1640 medium (Rothwell Park Institute 1640) containing 20% calf serum, and at the same time, the compound of the present invention was added at concentrations of 0.1 and 0.5 μg/ml. The cells were cultured in a carbon dioxide gas incubator at 37°C, and the 50% growth-inhibiting concentration compared to the control group was determined. Furthermore, 5×10 ⁵ of the above L1210 cultured cells were added to RPMI 1640 medium containing 10% calf serum.
After 1 to 2 hours of culture in a carbon dioxide gas incubator at 37°C, the compounds of the present invention were added at various concentrations, and after 15 minutes they were further ^{incubated with 14C-}
Uridine (0.05 μCi/ml) and ¹⁴ C-thymidine (0.05 μCi/ml) were added and cultured at 37° C. for 60 minutes. Add 10% trichloroacetic acid solution to the reaction solution,
At the same time as stopping the reaction, acid insoluble matter is precipitated,
After further washing three times with 10-5% trichloroacetic acid, it was dissolved in formic acid, the radioactivity in the acid-insoluble material was measured, and the 50% uptake inhibition concentration was determined from the radioactivity uptake rate relative to the control group. The effect on experimental tumor animals was determined by intraperitoneally injecting mouse leukemia L1210 cells into CDF1 mice at 1×10 ⁵ cells/cell.
After 24 hours of transplantation into mice, 2-hydroxyaclacinomycin A was intraperitoneally administered every day for 10 days.
It was expressed as a survival rate calculated in comparison with the control group (physiological saline administration). Table 1 shows the antitumor effects and acute toxicity values against L1210 leukemia. [Table] From the above results, 2-hydroxyaclacinomycin A of the present invention kills the proliferation of murine leukemia L1210 cells at low concentrations, exhibits an excellent survival effect on leukemia cell-transplanted mice, and shows that 2-hydroxyaclacinomycin A of the present invention kills the proliferation of murine leukemia L1210 cells and exhibits an excellent survival effect on mice with leukemia cell transplantation. It is far less toxic than known anthracycline antibiotics such as daunomycin, and even more so than aclacinomycin A, which has been researched and developed as an anthracycline antibiotic with the lowest toxicity and low cardiotoxicity. It has low toxicity and an antitumor effect equal to or greater than that of aclacinomycin A. Furthermore, the concentration range in which it exhibits antitumor effects is approximately twice that of aclacinomycin A, and it has excellent properties as an anticancer drug. have. Furthermore, regarding nucleic acid synthesis, which is considered to be the direct point of action of anthracycline antibiotics, this substance has the characteristic of inhibiting RNA synthesis more specifically, and its inhibitory effect is similar to that of aclacinomycin. and the rhodomycin group of antibiotics. Next, the details of the present invention will be explained with reference to Examples. Example 1 Soluble starch 1.5%, glucose 1%, soy flour 1
%, yeast extract 0.1%, salt 0.3%, dipotassium phosphate 0.1%, magnesium sulfate (MgSO ₄ 7H ₂ O)
0.1%, copper sulfate ( _CuSO4.5H2O ) 0.007%, iron sulfate ( _FeSO4.7H2O ) 0.001%, manganese chloride ( _MnCl2.4H2O ) _0.0008 %, _zinc sulfate ( _{ZnSO4 .}
Dispense 100 ml of a medium consisting of 7H ₂ O) 0.0002%, pH 7.4 into a sterilized 500 ml Erlenmeyer flask, and add Streptomyces galilaeus .
One platinum loop was inoculated from the slanted agar culture of strain KE303, and cultured with shaking on a rotary shaker at 28°C for 48 hours to prepare seeds. Next, 50ml of fermentation production medium with the above medium composition increased to 2% soybean flour and 0.2% yeast extract was dispensed and sterilized to 500ml.
1 ml of the above seed culture was inoculated into 1000 Erlenmeyer flasks, and cultured with shaking on a rotary shaker (210 rpm) at 28°C for 17 hours. To this, 2-hydroxyaclavinone (total amount
0.5 ml (final concentration: 20 μg/ml) of methanol solution (2 mg/ml) of 1.0 g) was added per flask, and the culture was continued for an additional 24 hours. In order to check the conversion production rate of 2-hydroxyaclacinomycin A at the end of fermentation, 5 ml of the culture solution was collected, and chloroform-methanol (3:
2) 5 ml of the mixed solution was added, and the mixture was shaken and stirred using a thermomixer, and the product was extracted into the chloroform layer. After concentrating this to dryness, it was dissolved again in 0.2 ml of chloroform, and 20μ of it was spotted on a thin layer of silica gel F ₂₅₄ (manufactured by Merck & Co., Ltd.), and chloroform:methanol:
Developed with a developing solvent of concentrated aqueous ammonia (50:10:0.5) and air-dried, showing an R value of 0.51. 2-hydroxyaclacinomycin A and R value of 0.30.
As a result of quantifying the remaining 2-hydroxyaclavinone spots using a Shimadzu thin layer chromatography scanner (model CS-910), it was determined that 90% of the remaining 2-hydroxyaclavinone was present.
The above was converted, and the total amount of 2-hydroxyaclacinomycin A produced was 680 mg. The main culture solution (50) was collected, bacterial cells were obtained by centrifugation, and the product was extracted with 8 acetone. After concentrating the acetone extract to 1/3 under reduced pressure, the product was dissolved in about 3 portions of chloroform and concentrated to dryness to obtain a crude extract. Example 2 This crude converted extract was dissolved in 50 ml of methanol, and after removing insoluble matter by centrifugation, the supernatant was applied to a Sephadex LH-20 column (40 x 5.0 cm) and eluted with methanol. The first yellow pigment fraction was separated, concentrated to dryness, and then dissolved in a small amount of chloroform.
) (Kieserguhr, 60PF ₂₅₄ , manufactured by Merck).
0.3). Scraping off the main band consisting of 2-hydroxyaclacinomycin A, which has an R value of approximately 0.68,
Approximately 200ml of chloroform:methanol (4:
1) Extract with a mixed solution, add an appropriate amount of distilled water, shake and wash, separate the chloroform extract layer, and concentrate to dryness. This was dissolved again in a small amount of chloroform, and the preparative silica gel thin layer (25
The mixture was spotted in a linear manner on a sheet of paper as described above, and developed with chloroform:methanol:glacial acetic acid (80:10:0.5). Scrape off the yellow dye band showing an R value of 0.22 and add approximately 200 ml of chloroform:methanol:
Extracted with concentrated aqueous ammonia (40:10:0.5). The extract was washed with distilled water, and the chloroform layer was fractionated and concentrated to dryness to obtain 388 mg of a purified sample. This sample was dissolved in 30 ml of methanol, passed through a Sephadex LH-20 column (40 x 5.0 cm), the relevant fractions were pooled, concentrated to dryness, and the contents were dissolved in 20 ml of 0.2 M acetate buffer (PH3.5). dissolved in After removing a small amount of insoluble material by centrifugation, the supernatant was neutralized with 4N NaOH in ice water at low temperature, and extracted with chloroform. The chloroform extract was 0.01M EDTA (PH
6.0) and then water, and the chloroform layer was dried with Glauber's salt and concentrated under reduced pressure. to concentrated liquid
-Add excess hexane to form an orange precipitate, accumulate by over-operation, and vacuum dry.
-Hydroxyaclacinomycin A pure product 293mg
I got it.

[Brief explanation of drawings]

The figure is a PMR spectrum diagram of 2-hydroxyaclacinomycin A of the present invention.

Claims (1)

[Claims] First-order general formula Anthracycyl glycoside antibiotic, 2-hydroxyaclacinomycin (2-
hydroxyaclacinomycin) A and its acid addition salts. 2 Belongs to the genus Streptomyces and is represented by the following formula, A bacterial strain capable of converting 2-hydroxyaclavinone to 2-hydroxyaclacinomycin A was grown aerobically at 20-40°C in an actinomycete nutrient medium containing carbon sources, nitrogen sources, inorganic components, and trace components. The 2-hydroxyacrabinone was added to the medium during the culture, and the culture was continued to carry out the above conversion, resulting in the following formula: 2-hydroxyaclacinomycin A represented by
A method for producing 2-hydroxyaclacinomycin A or a salt thereof, which comprises producing and accumulating 2-hydroxyaclacinomycin A or a salt thereof, which is isolated and purified by a conventional method, or further subjected to a salt-forming reaction to obtain an acid addition salt. 3 From the above 2-hydroxyaclavinone to 2-
Bacterial strains capable of converting to hydroxyaclacinomycin A are Streptomyces galilaeus MA 144-M1,
ATCC 31133, Streptomyces galilaeus
ATCC 14969, St. cinereoruber ATCC 19740, St. niveoruber ,
ATCC 14971 is a type of mutant strain isolated from the culture of St. antibioticus ATCC 8663 that is non-antibiotic producing and has the conversion ability as described in claim 2. A method for producing 2-hydroxyaclacinomycin A or a salt thereof.