JPS63203695A - Novel substance k818 - Google Patents

Abstract

NEW MATERIAL:A substance K818, expressed by the formula and having the following physico-chemical properties. White powder. Specific rotatory power; +2.4 deg. (methanol, c=0.5). Solubility; Soluble in methanol, ethanol, acetone and ethyl acetate and sparingly soluble in chloroform, water and hexane. Melting point; 202 deg.C. Elementary analysis (%); C, 61.62; H, 6.54; O, 28.00; Cl, 3.64. Molecular weight; 971.5, etc. USE:An antitumor agent. PREPARATION:For example, Streptomyces sp. K818 strain (FERM-P No.9028) is cultivated at 25-30 deg.C for about 5 days, preferably by an aerobic cultivation method to collect the aimed substance from the resultant cultivation filtrate.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] The present invention relates to a new substance of the macrolide type, and more particularly to 818, a novel substance having antitumor properties that can be produced by the 818 strain belonging to Streptomyces. .

Many antitumor substances have already been put into practical use as medicines. In general, the physiological activity of a chemical substance largely depends on its chemical structure, so it can be said that there is a constant desire for new compounds with antitumor properties.

[Summary of the invention]

The present invention meets the above needs.

That is, the novel substance 818 according to the present invention is represented by the following chemical formula (I).

The present invention also relates to the base addition salts of 818 to this new substance.

[Detailed description of the invention]

Novel Substance 818 1) Chemical Structure The novel substance 818 according to the present invention has a chemical structure represented by the above formula (I). These chemical structures were determined as follows.

K818's proton cough ri! L gas resonance spectrum (3rd
) and carbon-13 nuclear magnetic resonance spectra (Figure 4).
l.

11elv, Cheap, Acta 52; 12
7-142 (I989)). From a more detailed examination of the spectrum, K
In 818, it was suggested that the hydrogen atom at the 2″′ equatorial position of chlorothricin was replaced with a hydroxyl group.Moreover, the molecular weight determined from the S1MS spectrum was 97.
0 is also 16 more than chlorothricin, and other instrumental analysis data match well with chlorothricin, so K81
8 was determined to be the 2″-hydroxyl form of chlorothricin.

Since this compound has a carboxylic acid, a base addition salt exists. In this case, the salt may be any suitable salt;
For example, there are inorganic salts such as sodium, potassium, calcium, and the like.

2) Physicochemical properties (+) Appearance White powder (2) Specific optical rotation +2.4 degrees (in methanol) (cO, 5) (3) Solubility Soluble in methanol, ethanol, acetone, ethyl acetate, chloroform , sparingly soluble in water and hexane.

(4) Rf value (using Merck's "Silica Gel 60F254") Chloroform-methanol (9: 1) 0.62 (5) 51MS
Spectrum (m/z) 993 (M+Na)” (6) Ultraviolet absorption spectrum Figure 1 λ (
e) 222nm (I0000)ala! (in methanol) 258nm (3700) 284nm
(I600) (7) Infrared absorption spectrum Figure 2 (KBr
(8) Proton nuclear magnetic resonance spectrum Figure 3 (500
MHz, in deuterated chloroform) (9) Carbon-13 nuclear magnetic resonance spectrum Figure 4 (I25 MHz, in deuterated methanol) (I0) Melting point 202°C (I1) Elemental analysis analysis value (%) CHOCl B1.88 8.78 27.52 3.9
5 Calculated value (%) Molecular formula” 50H83CIO17CH
OC1 81,828,542g, 00 3.64 (I2)
Molecular weight (calculated value) 971.5 Production summary of compound 818 At present, compound 818 can only be obtained by culturing microorganisms, but it can also be produced by synthetic chemical modification or microbiological modification of related compounds. It could also be produced completely synthetically.

In the case of culturing microorganisms, a strain belonging to the genus Streptomyces and having an ability to produce 818 is used. Specifically, the present inventors isolated Streptomyces sp. strain 818 (SLrepLoIlyces
Although the present inventors have revealed that crab 818 is produced by crab sp. It is possible to do so. Furthermore, there is still room to increase the K818 production ability by subjecting Streptomyces sp., including the 818 strain, to irradiation or other mutagenic treatments. Furthermore, with the development of genetic engineering, other microorganisms have been introduced into this 818 strain to produce 818.
It is also possible to produce 8.

The K818 strain, which the present inventors have discovered as a Streptomyces strain capable of producing 818, has the following content.

1) Strain 818 in terms of origin and deposit number was isolated from soil collected in Kuragano-cho, Takasaki City, Gunma Prefecture, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on November 5, 1985, and was classified as a “microbial strain”. We have obtained the number J (FERMP-9028).

2) Mycological properties The mycological properties of the 818 strain are as follows.

(I) Aerial hyphae extend long from well-branched substrate hyphae and form simple branches. The tip is mainly straight or wavy, but hook-shaped or incompletely spiral-shaped tips are also often observed.

Under an electron microscope, the spores form a chain of 10-50 or more, the spore surface is smooth, the shape is cylindrical or oval, and the size is 0.6-0.8 x 1.0-1.3μ It is.

Specialized forms such as sporangia, flagellated spores, and sclerotia are not observed.

(2) Growth on various media The results of culturing 818 strains on various media at 27° C. for 3 weeks are shown in Table 1.

(3) Physiological properties The physiological properties of strain 818 are shown in Table 2.

(4) Carbon source utilization The carbon source utilization capacity of strain 818 (on Bridham-Gotlieb agar medium) is as shown in Table 3.

(5) Analysis of diaminopimelic acid As a result of analyzing diaminopimelic acid, which is one of the amino acids constituting the cell wall, LL-diaminopimelic acid was detected.

From the above mycological properties, strain K818 was determined to belong to the genus Streptomyces, and has the following characteristics.

(a) Aerial hyphae are straight, wavy, hook-shaped, or incompletely spiral, and spores are cylindrical or oval with a smooth surface.

(b) Forms aerial mycelia of yellowish to brownish to light olive-gray colors in various media.

(c) The color of the back side is yellowish to light yellow to yellowish brown. “ (d) No soluble dye is produced.

(e) Produces melanin-like pigment.

Based on the above results, the present inventors injected 818 strains into Streptomyces sp. K 818 (Strept-tay
cas sp, K818).

Table 2 Table 3 +: Used: Use is questionable m: Not used Cultivation / 818 produced compounds 818 belong to the genus Streptomyces
It can be produced by aerobically cultivating 8-producing bacteria in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrient source that can be utilized by the K818-producing bacteria. Specifically, for example, glucose, sucrose, maltose, starch, fats and oils can be used as carbon sources, and soybean flour, soybean flour and nitrogen can be used as nitrogen sources.
Organic materials such as cottonseed meal, dried yeast, yeast extract and corn staple liquor and inorganic materials such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride can be utilized. In addition, sodium chloride, potassium chloride, phosphate,
Inorganic salts such as heavy metal salts can be added. In order to suppress foaming during fermentation, a suitable antifoaming agent,
For example, silicone oil can also be added.

The most suitable culture method is the aerobic liquid culture method, which is the same as the commonly used antibiotic production method. The culture temperature is suitably 20-37°C, preferably 25-30°C. In this method, the production amount of 818 was determined by shaking culture,
Both the aeration and agitation culture reached the maximum after 5 days of culture.

In this way 818 accumulated cultures are obtained. In culture, K818 is present mostly in the culture filtrate.

Any convenient method can be used to harvest 818 from such cultures. One method is based on the principle of extraction, and specifically, for example, for Ni818 in the culture filtrate, it is extracted with a water-immiscible Ni818 solvent (see above), such as ethyl acetate. There are ways to do this. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

Another method for collecting 818 from cultures is based on the principle of adsorption, in which 818 is already in liquid form.
18-containing substances, such as the culture filtrate or the extract obtained by performing the extraction operation as described above, an appropriate adsorbent such as silica gel, activated carbon,
``818 is adsorbed on the target using Diaion HP-20J (manufactured by Mitsubishi Kasei Corporation), and then 818 is adsorbed on the target by elution with an appropriate solvent.
Then, by elution with a suitable solvent, 81
You can get 8. Thus obtained 81
8 solution is concentrated to dryness under reduced pressure to obtain a crude sample of K818.

In order to further purify the crude sample of 818 obtained in this way, the extraction method and adsorption method described above may be combined with gel filtration method, high performance liquid chromatography, etc. as necessary, and the procedure may be performed as many times as necessary.

For example, high-performance liquid chromatography using adsorbents such as silica gel, column chromatography using gel filtration agents such as Sephadex LH-20J (manufactured by Pharmacia Co., Ltd.), and rYMC Pack (manufactured by Yamamura Kagaku Co., Ltd.). The flow distribution methods can be combined as appropriate. Specifically, for example, K818 crude sample is dissolved in a small amount of methanol, and "Sephadex LH-2O
The active fraction was applied to a J column, eluted with methanol, and concentrated to dryness to obtain pure product 818.

The thus obtained 818 of formula (I) can be converted into a base addition salt thereof by treatment with a base addition salt thereof, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, etc., according to a method known per se. can be changed to

Uses of K818 The compounds according to the present invention, 818, are useful in that they have antitumor activity.

Biological activity 1) Cytostatic activity Mouse P388 leukemia cells were suspended at 5 x 10 cells/ml in RPM11640 medium containing 10% heat-inactivated calf serum and 50 μM mercaptoethanol, and incubated with various concentrations of Ni-818 at 37°C. ■C5o value after 2 days of culture was 5.7 μg/ml.

2) 818 for $1 cancer activity is EhrlichJl$2 1nv for water cancer
lvo anticancer activity was observed. In other words, ICR mouse (
Ehrlich tumors I x 10B were intraperitoneally transplanted to a 5-week-old male (day 0), and samples were injected (intraperitoneally) six times in a row from day 1 to day 6, and the number of surviving mice was observed.

Number of viable mouths ± SD n Control 15.9 ± 3.2104 mg
/kg 12.8 ± 4.4 640mg/
kg >40.0"±12.6 6 * 3 out of 6 animals survived for more than 50 days As mentioned above, it was revealed that the present invention's 818 exhibits antitumor properties. Therefore, the present invention's 818 can be used as an antitumor agent.

3) Due to acute toxicity, LD5o was 295a after intraperitoneal administration of 818 to mice.
+g/kg.

Antitumor Agent It has thus been revealed that 818 of the present invention exhibits antitumor properties against animal tumors, particularly malignant tumors.

Therefore, the compounds of the present invention can be used as antitumor agents or tumor therapeutic agents.

The compounds of the present invention as antitumor agents can be administered by any convenient route of administration and in a dosage form determined by the route of administration employed. The drug is usually in a diluted form with a pharmaceutically acceptable carrier or diluent.

When actually administering the compound of the present invention as an antitumor agent, one typical method is to dissolve the compound in distilled water for injection or physiological saline and inject it. Specifically, in the case of animals, injection methods such as intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration are used, and in the case of humans, injection methods include intravascular injection into a vein or artery or local administration. There is a method of injection.

The dosage of the compound of the present invention is determined in consideration of the results of animal tests and various circumstances so that the total dosage does not exceed a certain amount when administered continuously or intermittently. The specific dosage may vary depending on the method of administration, the circumstances of the patient or treated animal, such as age, weight, sex, sensitivity, diet, time of administration, concomitant drugs, and the degree of the patient or his or her disease. Needless to say, the appropriate dose and frequency of administration under certain conditions must be determined by a specialist's appropriate dose determination test based on the above guidelines.

Experimental example In the following, "%" is "rw/v%".

Example 1 1) Preparation of Seed Mother The medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.2.

Glucose 1g Polypeptone 1g Meat extract 1g Dispense 100ml of the above medium into a 500ml Erlenmeyer flask with warts, and after sterilization, Streptomyces sp.
One platinum loop of the strain was inoculated from a slant and cultured with shaking at 27°C for 3 days, which was used as a seed mother.

2) The culture medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.5.

Glycerol 3.0g Cohn-Steebly force - 1. or Dried yeast 0.3g Calcium carbonate 0.35° Sodium chloride 0.5g Dispense 25 liters of the above medium into 50 liter jar fermenters and add 300ml of the above seed mother to the sterilized ones.
was added and heated at 27°C for 5 days, 20Orpm, 0.4V
VM was cultured with aeration and agitation.

3) Collection of K818 After culturing under the above conditions, the culture solution was filtered to obtain a culture filtrate. 50 liters of the culture filtrate was adsorbed onto a Diaion HP-20 (manufactured by Mitsubishi Kasei) column (I0 x 50 cm), and
After washing with 25 liters of 5% methanol, 25 liters of methanol
It was eluted with a little bit.

After concentrating the eluate (2.5 liters), the pH was adjusted to 3 with hydrochloric acid.
, 0 and extracted with 3 liters of butyl acetate. After extracting the extract with 3.0 liters of sodium bicarbonate water, the pH was adjusted to 3.0 liters.
0, extracted again with 3.0 liters of butyl acetate, and concentrated to dryness. After dissolving this in 50ml of chloroform and removing insoluble materials, a column (4cm+φX20CI+
), washed with chloroform, and then chloroform-
Elution was performed with methanol (40:1). The eluate was concentrated and dissolved in 10 ml of chloroform, and then 100 ml of hexane was added. Remove the precipitate and concentrate to dryness to obtain K818.
A crude specimen 2O was obtained.

Example 2 20 g of the crude sample obtained in Example 1 was dissolved in a small amount of methanol, and the mixture was added to a Sephadex LH-20 column (4X60
cm) and developed with methanol.

Concentrate and dry K818 to obtain 10 mg of K818 purified product.
I got it.

[Brief explanation of the drawing]

FIG. 1 is a reproduction of the ultraviolet absorption spectrum of K818 in methanol. FIG. 2 is a reproduction of the infrared absorption spectrum of K818 obtained by the KBr disk method. Figure 3 shows K818 at 500% in deuterated chloroform.
This is a copy of the megahertz proton nuclear magnetic resonance spectrum. FIG. 4 is a reproduction of the 125 MHz carbon-13 nuclear magnetic resonance spectrum of K818 in heavy methanol. Applicant's agent Sato - Male Procedural Amendment December 1986/Date

Claims (1)

[Claims] A novel substance K818 represented by the following formula (I) or a base addition salt thereof. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)