JPS6121088A - Novel anticancer antibiotic k-802-4 and preparation thereof - Google Patents

Abstract

NEW MATERIAL:An anticancer antibiotic K-802-4 having the following physico- chemical properties; Elementary analysis value; C; 59.0-59.2%; H; 4.3-4.5%; N, O%. Molecular weight; 372 [measured by field desorption mass spectrometry (FE-MAS)]. Molecular formula; C18-19H16-17O8-9. Specific rotatory power [delta]D<25>; -100 deg. (c=1, methanol). Melting point; 220-230 deg.C (decomposition). Ultraviolet absorption spectrum (methanol solution); Fig. 1. Infrared absorption spectrum (KBr method); Fig. 2. Solubility; Soluble in CH3OH, C2H5OH and acetone, somewhat slightly soluble in CHCl3 and aqueous acetic ester and insoluble in benzene, hexane and petroleum ether. Color reactions; Positive to copper sulfate, formaldehyde-o-dinitrobenzene, etc. Color and properties of the substance; Yellowish orange crystals. USE:An antibiotic for medical use, having antimicrobial activity against Gram- positive and Gram-negative bacteria, and exhibiting remedial effect on mouse Ehrlich ascitic cancer. PREPARATION:A species, belonging to the genus Streptomyces, and producing the anticancer antibiotic K-802-4 is aerobically cultivated in a culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel anticancer antibiotic -802-4 and a method for producing the same.

[Conventional technology]

Conventionally, antibiotics produced by microorganisms have been known.

[Problem that the invention seeks to solve]

The object of the present invention is to obtain a novel antibiotic useful for medical use.

[Failure to solve the problem]

While conducting research on substances produced by many naturally occurring microorganisms, the present inventor discovered that a specific microorganism produces -802-4, a novel anticancer antibiotic, and completed the present invention.

Anticancer antibiotic of the present invention (hereinafter simply referred to as "antibiotic") Bacterial strain producing K-802-4 (hereinafter referred to as "K-80
2-4 strain) has the following mycological properties.

(1) Morphotrophic hyphae develop well on both synthetic and natural media;
It simply branches, but never splits.

Aerial hyphae are relatively long and simply branch. The spore stalk is straight or gently curved, and chains of 20 or more spores are observed. Spore size is 0.5-0.6X
1. The size of the spore is 0~1.4μ, the shape is oval, and the surface of the spore is smooth.

Spore paste, whorled plates, flagellated spores, etc. are observed and strong, but knob-like knot plates with intertwined aerial mycelia are also observed.

(2) Growth status in various media E.B. Shearing et al. (Int, J, 5ys
t.

Bacteriol, vol. 16, p. 313, 196
The growth status after culturing at 28° C. for 7 to 14 days according to the method of 2010) is shown below.

(3) Physiological properties ■ Formation of melanin pigment Tryptone/Yeast Pross Positive Peptone/Yeast/Iron Agar Doubtful Tyrosine Agar Positive ■ Tyrosinase Positive ■ Reduction of nitrate Negative ■ Liquefaction of gelatin Positive ■ Hydrolysis of starch Positive ■ Cellulose Decomposition Negative ■ Coagulation of skim milk, peptonization Coagulation Positive Peptonization Positive ■ Growth temperature 15-40℃ ■ Growth pH 4.5-8.5 (4) Utilization of carbon source (Pridham-Godelive agar medium, l5P-9 ) D-glucose, L-arabinose, sucrose,
D-xylose, ynontol, D-mannitol, D
-7 Lactose, L-rhamnose, raffinose, D-
Make good use of galactose.

Use some salicin.

(5) Cell wall constituents Contains LL-diaminopimelic acid and glycine.

To summarize the properties of this strain, ■ No division is observed in the basal hyphae.

■ Aerial hyphae are simply branched, straight or gently curved, and have no whorled branches. Chains of 20 or more spores, sporangia, flagellated spores, etc. are not observed.

■ Cell wall composition is LL-diaminopimelic acid and belongs to the genus Guises.

Furthermore, bacteria similar to this strain were found in Niss ny Waxman's The Actinomycetes Volume 2 (S, A).

Waksman: The Actinomycete
s Vol, 1. ), E. B. Shirling and De Gottlieb's ISP description (E, B, Shi
riing and D,Gattlieb; In
t, J.

5yst, Bacteriol, 18.69-1
89.279~392; 19°391~512. Deafness, 265-394) and Berg's Manual of Determinative Bacteriology (Berg.
ey's manual of Determinat
iveBacteriology) 8th edition (1976
), this strain belongs to the gray series, and the sporophyte is Wax flexibilis (
Taking into account that Streptomyces cacao subspecies asoensis forms melanin pigment and has a smooth spore surface, a similar bacterium is Streptomyces cacao subspecies asoensis.
Stre-ptomyces cacaoi 5ubs
P, asoensis), Streptomyces griseorbiginosus (Streptomycesgri
seorubiginosua).

However, Streptomyces cacao subspecies asoensis has its original description CAgr, B
iol, Chem, 29.848 (1965)
), the shape of the sporophyte is an open spiral, only a small amount of arabinose and xylose are used, aerial hyphae are not formed on tyrosine agar medium but aerial hyphae are formed on ordinary agar medium, and nitrate This strain differs from this strain in that it has positive reducing ability.

Next, Streptomyces griseorbiginosus IF
When we conducted a comparative experiment between strain O13047 (ISP5469) and this strain, the two strains matched well in terms of color series of aerial mycelia, morphological properties such as the shape of sporophores, sugar utilization, and various physiological properties. In addition, both strains had knot-like knot-like bodies (Int, J, 5ys
T, Bacteriol, Su, 437. (196
9)) was observed on yeast malt shishishi, oatmeal agar, and glycerol asparagine agar media. but,
Comparing both strains with respect to production of soluble pigments, this strain does not produce pigments on oatmeal agar or starch inorganic salt agar medium, whereas IF013047
The strain did not produce pale pink and light brown-gray pigments on these media, respectively, and the strain produced light brown and reddish-brown pigments on glycerol asparagine agar and sucrose nitrate agar, respectively, but IFo
The 13047 strain was different in that no pigment production was observed in any of the media.

As stated in the paper, this strain produces red to brown soluble pigments and is similar to IF in terms of morphological and physiological properties.
o It is similar to strain 13047, but differs in its ability to produce soluble pigment on agar medium. Therefore, the present inventor identified this strain as belonging to Streptomyces griseorbiginosus, and identified it as belonging to Streptomyces griseorbiginosus.
ubiginosus) K-802-4, and was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as the 71st
No. 91 (FERM P-7191).

The antibiotic -802-4 of the present invention is produced by inoculating the above strain into a nutrient-containing medium and culturing it aerobically at 20 to 40°C for 2 to 5 days. Antibiotics-802-4
The production strains listed above are the 802-4 strain, and even its artificial or natural mutant strains cannot be used as antibiotics.
Any strain capable of producing the 802-4 strain can be used in the present invention.

A normal actinomycete culture method is used to culture the antibiotic-802-4 producing strain. As the nutrient medium, any of a limited medium, a synthetic medium, a semi-containing medium, or a natural medium containing an appropriate amount of assimilable carbon sources, nitrogen sources, inorganic substances, etc. can be used.

Examples of carbon sources include glucose, shakerose,
Fructose, xylose, galactose, starch, glycerin, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used.

As nitrogen sources, inorganic or organic nitrogen compounds, such as peptone, dried yeast, ammonium sulfate, ammonium chloride, ammonium nitrate, casamino acids, etc., and natural products, such as meat extract, soybean flour, cottonseed flour, cornstap liquor, etc., may be used alone. Or they can be used in combination. Examples of inorganic substances include sodium chloride, calcium carbonate,
Potassium chloride, dipotassium phosphate, magnesium sulfate,
Ferrous sulfate and the like can be used alone or in combination.

Isolation of the antibiotic -802-4 from the culture can be carried out by using various methods alone or in appropriate combinations, taking into consideration the physicochemical properties of this antibiotic, as shown in Examples 2 to 4 below. It is done.

That is, in order to collect antibiotic -802-4 from a culture, the culture is separated into bacterial cells and culture F solution by centrifugation or filtration, and then the bacterial cells and culture F solution are separated using normal separation means. Separate and purify -802-4 as a good antibiotic.

Preferred separation and purification methods include, for example, the following methods. First, the bacterial cells are extracted with an organic solvent such as acetone or methanol, and the culture P solution is extracted with n-
Extract with a water-immiscible organic solvent such as butanol, ethyl acetate, chloroform, or
1. Obtain a crude substance by adsorbing it onto a carrier such as Diaion HP-2 or activated carbon and eluting it with 70% methanol.

Next, this group of substances is isolated by a method normally used for purifying fat-soluble substances, for example, by concentrating the extract and subjecting it to silica gel column chromatography, octadecylsilane-treated silica gel column chromatography, etc.

The antibiotic -802-4 obtained as described above has the following physicochemical properties.

Physical and chemical properties ■Elemental analysis value C+59.0~59.2ch 1(:4.3~4.5ch N: 0chi ■Molecular weight 372 (by FD-MAS8) ■Molecular formula %formula% ■Ultraviolet absorption spectrum (methanol solution) Figure 1 ■Infrared absorption spectrum (KBr method) Figure 2 ■Solubility Soluble in methanol, ethanol and acetone.

Slightly insoluble in chloroform, acetate, and water. Insoluble in benzene, hexane and petroleum ether.

■Color reaction Positive for copper sulfate and formaldehyde-〇-dinitrobenzene. Negative for ninhydrin, Molish, and Fehling's reagents.

■Color and properties of the substance Yellow-orange crystals 0 heat stability (aqueous solution) As antibiotics similar to the antibiotic of the present invention having physical and chemical properties such as the following:
No. 439), deoxyfrenolicin (U.S. Pat. No. 345)
2051), Kara 7 Anjin (J.

Antibiotics, 21.454. (196
8)), O8-4742 (Special Publication No. 58-42760)
, and Raimaikun (Japanese Patent Publication No. 39-14496).

However, although frenolicin B1 carafungin has a five-membered ring lactone absorption at 1770 crn-' in its infrared absorption spectrum, the antibiotic of the present invention does not have a corresponding absorption and has a carbonyl absorption of 1720 crn-'. In addition, frenolicin B1 deoxyfrenolicin B
In the ultraviolet absorption spectrum, 1kara7 anjin and raimycin have an absorption of 310n, which is absorbed by the antibiotic of the present invention.
No absorption band of m is observed. Furthermore, O8-4H42 has a molecular weight of 878 to 952, and an optical rotation [α): +xo
4, which is different from the antibiotic of the present invention. For the above reasons, antibiotic -802-4 was determined to be a new antibiotic.

[Effect]

Antibiotic-802-4 has the following biological properties.

Biological properties> (1) Acute toxicity■ ICR mice (body weight 23-28f!, male, 5 mice per group) were given antibiotic -802-4 dissolved in physiological saline by intravenous injection into the tail vein. Or once administered intraperitoneally, 2 times after administration.
The animals' general condition and whether they were alive or dead were observed every week. Also, L
D, was calculated by the Probit method. Results first
Display the table.

As a result of the test, no noteworthy toxic symptoms were observed in the case of intravenous administration, and mild piloerection was observed 24 hours after intraperitoneal administration. Deaths occurred within 24 hours after intravenous administration and between 24 and 48 hours after intraperitoneal administration.

■ Antibiotic -802-4 dissolved in physiological saline was administered once into the tail vein of Wistar rats (body weight 160-180 z, 1 male, 5 rats per group), and the general condition was evaluated for 2 weeks after administration. and observed whether they were alive or dead. Also, LDv
was calculated by the Probit method. The results are shown in Table 2.

As a result of the tests in Table 2, no noteworthy toxic symptoms were observed after administration, and death occurred within 24 hours.

(2) Antibacterial effect Table 3 shows the minimum inhibitory concentration (MIC) of antibiotic -802-4 against various microorganisms.

Table 3 (3) Antitumor effect antibiotics -802-4 Mouse Ehrlich (Ehr)
(lich) The therapeutic effect on ascites cancer was tested by the following method. The results are shown in Table 4. The life extension rate in the table was calculated using the following formula.

Test method: 1X10' tumor cells were intraperitoneally transplanted into ICR mice (male), and 24 hours later, antibiotic -802-4 was intraperitoneally administered once a day for a total of 10 times.

Table 3 [Effects of the invention] It is clear from these results that antibiotics -802-
4 has antibacterial activity against Durham-positive bacteria and Durham-negative bacteria. It also shows a therapeutic effect on murine Ehrlich ascites cancer. In view of these antibacterial and antitumor activities, antibiotic -802-4 is a useful substance as a medical antibiotic.

〔Example〕 Next, the present invention will be explained with reference to Examples.

Example 1 3 glucose, 0.5% pear, 0 meat extract
．． A medium (pf(7,0)70-500
After dispensing and sterilizing the mixture into an R1 Yosaka flask, a loop of slant culture medium of Streptomyces griseorbiginosus strain -802-4 was inoculated thereto, and cultured with shaking at 28°C for 24 hours. This culture was grown in the same medium containing 200-1! The cells were transplanted into Yosaka Rof flasks for 1.5 days and cultured with shaking at 28°C for 24 hours.

This seed culture was treated with 3 t of glucose, 0.5 t of peptone, 0.2 n of ticazamino acid, 0.1 t of dipotassium phosphate, 0.05% potassium chloride, 0.05 t of magnesium sulfate, 0.2 n of ferrous sulfate, ooi*, calcium carbonate 0.2t (pH
200 Jl of medium consisting of 6,6)? 2 was inoculated into a stainless steel tank containing the following, and cultured with aeration and stirring at 28°C for 70 hours by blowing sterile air at a rate of 100 m/min.

This culture solution contains Ni-802-4 substance at 120 mc7.
/vLl was included.

Example 2 The culture solution obtained in Example 1 was filtered using a filter press using diatomaceous earth as an auxiliary agent to obtain culture solution 180 containing the K-802-4 substance.

This culture solution was adjusted to pH 4.5 with hydrochloric acid and passed through a column filled with Amberlite XAD-■ 20n for adsorption. After adsorption, the mixture was washed with 40A water, eluted with 70A water, and the active fraction was collected, concentrated to dryness under reduced pressure, and then sequentially dissolved in ethyl acetate and acetone to remove insoluble materials. The extract thus obtained was concentrated under reduced pressure to produce 26fP of brown oil containing the -802-4 substance.
I got it.

Example 3 The culture obtained in Example 1 was filtered using a filter press using diatomaceous earth as an auxiliary agent to separate the bacterial cells and liquid f. After adding methanol 404 to the bacterial cells and stirring for 30 minutes, the mixture was filtered through a Bunner funnel and the methanol layer was collected. Return the liquid to the extraction tank, add 7 liters of ethyl acetate, stir for 30 minutes, let stand overnight, and separate the ethyl acetate layer. Combine the methanol extract from the bacterial cells and the ethyl acetate layer and reduce the pressure. The mixture was concentrated to dryness to obtain 30p of a brown oily substance.

Example 4 Brown oily substance obtained in Example 2 5? was applied to a silica gel column (Merck, Kiesselgel 60, 6002) and developed with chloroform:methanol (10+1).

Each 7 lactation (lQml) was assayed using E. coli (E, Co11) NIHJ as a test bacterium, and the active fraction was collected and dried under reduced pressure, then applied again to the same silica gel column (400 g)' and chloroform:methanol ( The active fraction was concentrated by developing with a ratio of 50:1 to 10:2) to obtain needle-shaped crystals.

The crystals were recrystallized from chloroform to obtain yellow-orange needle crystals. The ward amount is 420! It was n9.

Example 5 The brown oily substance 5D obtained in Example 3 was dissolved in 30 timeethanol, and this was added to 100rnl of octadecylsilane-treated silica gel (silica gel 0DS-Qa manufactured by Fuji Gel Co., Ltd.) equilibrated with the same 30 timeethanol. , developed with the same solvent and fractionated into 10 d portions. Each fraction was analyzed for visible absorption at 410 nm and N. coli NI.
The antibacterial activity by HJ was checked, and the active fraction was collected and concentrated under reduced pressure to obtain crude crystals. The crystals were recrystallized using isopropanol to obtain 510 mg of yellow-orange columnar crystals.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the antibiotic -802-4, and FIG. 2 shows the infrared absorption spectrum of the same substance. or more % Figure 1 Ihat Sanku Written procedural amendment (voluntary) August 16, 1980 Manabu Shiga, Commissioner of the Patent Office Suit 1 Indication of the case 1981 Patent Application No. 143841 2 Name of the invention Novel anti-cancer antibiotic Substance -802-4 and its manufacturing method r3.
Relationship with the case of the person making the amendment Applicant Address No. 5, 1-24 Minamiikebukuro, Toshima-ku, Tokyo Name
Pharmaceutical Antibiotics Research Co., Ltd. Deputy Representative Representative Tadashi Mochizuka 4 Agent 6, “Detailed Description of the Invention” column 7 of the specification subject to amendment, Contents of the amendment (1) In the specification, page 5, bottom In the second line, ``trypton'' should be corrected to ``trypton''. (2) Same, page 6, line 15, "inositol" is corrected to "inositol". (3) Same, page 9, line 13, ``Do not generate,'' is corrected to ``Generate,''. t4) Same, page 16, first line [08-4H42J is corrected to [08-47424].

Claims (1)

[Claims] 1. Novel anticancer antibiotic K-8 having the following physical and chemical properties
02-4. (1) Elemental analysis value C: 59.0-59.2% H: 4.3-4.5% N: 0% (2) Molecular weight 372 (according to FD-MASS) (3) Molecular formula C_1_8_-_1_9H_1_6_-_1_7O_8
＿～＿9(4) Specific optical rotation [α] ^2^5_D-100° (c = 1 methanol) (
5) Melting point: 220-230°C (decomposition) (6) Ultraviolet absorption spectrum (methanol solution) Figure 1 (7) Infrared absorption spectrum (KBr method) Figure 2 (8) Solubility Soluble in methanol, ethanol, acetone. Slightly insoluble in chloroform, acetate, and water. Insoluble in benzene, hexane and petroleum ether. (9) Color reaction positive for copper sulfate and formaldehyde-o-dinitrobenzene. Negative for ninhydrin, Moritschew, and Fehling's reagent. (10) Color and properties of the substance Yellow-orange crystal 2, belongs to the genus Streptomyces, and is an anticancer antibiotic -80
An anticancer antibiotic K-802-, which is characterized by culturing a bacterial strain that produces K-2-4 aerobically in a medium, accumulating K-802-4 in the medium or in the bacterial cells, and collecting the K-802-4. 4 manufacturing method.