JPH04290892A - New substance d329c and its production - Google Patents

Abstract

PURPOSE:To provide a new substance having antitumor activity and a method for efficiently producing the aforementioned substance. CONSTITUTION:A substance D329C expressed by the formula or its pharmaceutically permissible salt. Furthermore, a method for producing the above-mentioned substance D329C or its pharmaceutically permissible salt is characterized in that a microorganism, belonging to the genus Streptomyces and capable of producing the substance D329C expressed by the aforementioned structural formula is cultured in a culture medium and the above-mentioned substance is collected from the resultant culture.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a novel substance D329C (hereinafter abbreviated as D329C), a pharmaceutically acceptable salt thereof, and a method for producing the same.

0002

[Prior Art] It is already known that chartreusin has excellent antitumor activity [Progress in
Medicinal Chemistry (Progress i)
19, p. 247, 1982]. However, chartreusine has extremely low water solubility, so it has not been used clinically. It is also known that demethyl chartreusine, which is the demethyl form of chartreusine, has excellent antitumor activity (Japanese Patent Application Laid-Open No. 2-59596
issue). Various derivatives of chartreusine have been synthesized by chemical synthesis techniques, and some of these compounds are known to have antitumor activity superior to that of chartreusine (Japanese Patent Application Laid-open No. 174096/1983, JP-A-63-233996).

0003

[Problem to be solved by the invention] Many antitumor substances have been used clinically so far, but since their antitumor activity has a strong correlation with their chemical structure, it is desirable to have a novel chemical structure. There is a constant need to develop antitumor substances.

The present invention aims to solve the above problems by providing a new antitumor substance that can be used as a more effective medicine than known antitumor substances, and by establishing a method for producing the same.

[0005]

[Means for Solving the Problems] The present inventors have made repeated studies to solve the above problems, and have analyzed numerous products of mutant strains obtained by treating chartreusin-producing bacteria isolated from soil with nitrosoguanidine. As a result, D329-185
The present invention was completed by discovering that the strain produces D329C and clarifying its physicochemical properties.

That is, the present invention is based on the following structural formula:

000
7]

[Chemical formula 3]

[0008] In addition to providing D329C represented by [0008] or a pharmaceutically acceptable salt thereof, a microorganism that produces D329C belonging to the genus Streptomyces is cultured in a medium, and D329C represented by the above structural formula is collected from the culture. The present invention provides a method for producing D329C or a pharmaceutically acceptable salt thereof, which is characterized by the following.

[0009] The microorganism producing D329C of the present invention belongs to the genus Streptomyces and has the ability to produce D329C. One example is Chartreusin.
), which produces Streptomyces chartreusis (
Streptomyces chartreusis)
The mutant strain D329-185 obtained by treating the D329 strain (hereinafter referred to as D329 strain) with nitrosoguanidine (NTG) can be mentioned.

[0010] Strain D329 was isolated by the present inventors from soil collected in Omiya City, Saitama Prefecture, and its mycological properties are as follows. (1) Morphological characteristics Vegetative hyphae develop well on various media and do not undergo division. Aerial hyphae formed from branched vegetative hyphae are blue or gray in color, have chains of 10 to 50 or more spores, and are spiral-shaped. The spore surface is thorn-like, and its size is 0.7 x 0.9 to 1
．． It is 1 μm in diameter and has a cylindrical shape. No sclerotia, sporangia, or zoospores were found.

(2) Properties on various media Table 1 shows the culture properties on various media. In addition, as a color tone (
) The numbers shown in ) are from the ISCC-NBS Color Name Chart (ISCC-NBS Col.
or-Name chart),
These are the results of observation on each medium at 28°C for 3 weeks.

0012

[Table 1]

(3) Physiological properties ■Growth temperature range (yeast extract/malt extract agar, cultured for 14 days): 15-45°C ■Liquification of gelatin: Positive ■Hydrolysis of starch: Positive ■Coagulation of skim milk
: Positive ■ Peptonization of skimmed milk :
Positive ■ Nitrate reduction: Positive ■ Cellulose decomposition: Negative ■ Carbon source availability (Pridham-Gotlieb agar, 28°C, 14
Day-long culture): Use: L-arabinose, D-xylose, L-rhamnose, D-glucose, D-galactose, D-fructose, D-mannitol, salicin, sucrose, raffinose, inositol

001
4) (4) Cell wall tissue Analysis of diaminopimelic acid in the whole bacterial cell revealed that it was type LL. To summarize the mycological properties of strain D329,
It has LL-diaminopimelic acid, the shape of the aerial mycelia is spiral, and the surface of the spores is spiny. The color tone of aerial mycelia ranges from blue to gray. Soluble pigments produce yellow to brown pigments on various media, and melanin-like pigments on peptone/yeast extract agar media. Based on these properties, the D329 strain is a Streptomyces strain.
It is considered to be a bacterial strain belonging to the genus P. omyces, and is listed in Bergey's Manual of Determinative Bacteriology.
Terminative Bacteriology
)” 8th edition and ISP report “International Journal of Systematic Bacteriology (
International Journal of
Systematic Bacteriology)
Vol. 18, p. 69, p. 279 (1968), Vol. 19, p. 391 (1969), Vol. 22, p. 265 (1969)
As a result of a search from 1972), Streptomyces chartreusis was listed as a closely related species. Therefore, D3
29 strains and Streptomyces chartreuse JCM
Table 2 and Table 3 summarize the results of comparison with 4570.
become that way.

[0015]

[Table 2]

[0016]

[Table 3]

As is clear from the table, the spores have a thorn-like surface, and the spore chains are spiral-shaped. The two closely match in terms of producing melanin-like pigments and assimilating sugar. In addition, there is a difference in the production of soluble pigment between the two, but this is due to D.
This is thought to be due to the fact that strain 329 produces a large amount of chartreuse (yellow to olive color). Therefore,
Strain D329 was transformed into Streptomyces chartreusis (
Streptomyces chartreusis)
was identified.

[0018] The mutant strain D329-185 obtained by treating the D329 strain with nitrosoguanidine is a strain of Streptomyces chartreuse.
No. 3269 (FER
MBP-3269).

D329C of the present invention can be obtained by culturing microorganisms, but it can also be obtained by chemical modification using similar compounds as raw materials, or by total synthesis using chemical synthesis techniques. In the case of culturing microorganisms, the strains used include, for example, naturally isolated strains such as those mentioned above, and D.
D329 obtained by mutation treatment of strain 329 by the present inventors
-185 strains are listed. As observed in many other actinomycetes, the D329-185 strain is prone to change in properties, so the D329-185 strain is recommended as a D329C-producing strain.
In addition to the 9-185 strain, natural mutant strains, artificial mutant strains (by ultraviolet irradiation, cobalt-60 irradiation, addition of chemical mutagenic agents, etc.), and even phenotypic and genetically modified strains,
Anything that has the ability to produce D329C can be used in the present invention.

D329C can be produced by aerobically cultivating the D329-185 strain in an appropriate medium and isolating it from the culture. Here, the medium is D32
Any nutrient source available to the 9-185 strain may be used. Specific examples include glucose, fructose, glycerin, starch, dextrin, molasses, animal and vegetable oils, and depending on the assimilation ability of bacteria, hydrocarbons, alcohols, etc. can be used alone or in combination as carbon sources. On the other hand, nitrogen sources include soybean flour, peptone, wheat germ, meat extract, yeast extract, cottonseed meal, corn steep liquor, oatmeal, Pharmamedia (registered trademark), ammonium sulfate, sodium nitrate, ammonium chloride,
Natural products such as urea and sodium glutamate, or organic and inorganic nitrogen compounds can be used alone or in combination. In addition, if necessary, inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, sulfuric acid, phosphoric acid, and other ions, including sodium chloride, calcium carbonate, copper sulfate, and manganese chloride, may be added singly or in combination. It's okay. Furthermore, in order to suppress foaming during culture, commonly used antifoaming agents such as silicone KM73 (registered trademark), Adekanol (
(registered trademark) etc. may be added as appropriate.

[0021] As the culture method, aerobic liquid culture is most suitable, similar to the general antibiotic production method. The culture conditions are
The culture may be carried out at a temperature of 25 to 37°C, preferably 28 to 30°C, and under aerobic conditions. D32 by this method
The production of 9C is usually highest on the 8th day of culture in shaking culture. When the amount of D329C accumulated in the culture reaches the maximum, the culture is stopped, and the target product is collected and purified from the culture. To obtain D329C from a culture, a method commonly used for purifying and isolating antibiotics from a microbial culture,
Particularly, as shown in the Examples below, this can be carried out taking into consideration the property that the present substance is fat-soluble. That is, the substance is extracted from the culture using various solvents (acetone, chloroform, ethyl acetate, etc.). D329C transferred to the solvent can be isolated and purified using various techniques such as those described below. Namely, synthetic adsorbent (Diaion HP-20/manufactured by Mitsubishi Kasei Corporation, Amberlite XAD-2)
/ manufactured by Rohm and Haas, etc.), gel filtration agents (Sephadex LH-20 / manufactured by Pharmacia, Toyopearl HW-40 / manufactured by Tosoh, etc.), and silica gel,
This is effectively carried out by using carriers such as cellulose, alumina, and chemically modified silica gel alone or in appropriate combinations.

D32 obtained by the above method
The physicochemical properties of 9C are shown below. (a) Molecular weight and molecular formula 786, C38H42O18 (b) Mass spectrum (FD-MS) m/z 8
09 (M+sodium cation) (c) High performance liquid chromatography capsule pack C18 column (φ4.6mm x 1
50 mm (manufactured by Shiseido) and eluted in 7.5 minutes using a system of deionized water/acetonitrile = 7/3 at a flow rate of 1 ml/min.

(iv) Ultraviolet absorption spectrum The spectrum measured in methanol solution is as shown in FIG. (e) Infrared absorption spectrum The spectrum measured by the KBr disk method is as shown in FIG. (f) Proton nuclear magnetic resonance spectrum The 400 MHz proton nuclear magnetic resonance spectrum measured in deuterated pyridine at 60°C is as shown in FIG.

(g) Carbon-13 nuclear magnetic resonance spectrum The 100 MHz carbon-13 nuclear magnetic resonance spectrum measured in heavy methanol is shown in FIG. (H) Soluble dimethyl sulfoxide, soluble in methanol, water, acetone, pyridine, poorly soluble in chloroform, benzene, n
- Insoluble in hexane (li) Color reaction p - Positive for anisaldehyde reagent, positive for phosphorus-molybdic acid reagent, negative for ninhydrin reagent (nu) Distinction between acidic, neutral and basic acidic appearance yellow powder

Based on the above-mentioned physicochemical properties, as a result of further structural research, the chemical structure of D329C was determined as shown in the above-mentioned structural formula. Since there are no known compounds that match the above physicochemical properties, D329C is recognized as a novel compound. Specific examples of the salt of D329C of the present invention include sodium, potassium, lithium, etc. as alkali metals.

[0026]

[Effect] (1) Antitumor activity ■ Inject mouse fibrosarcoma Meth-A cells into the peritoneal cavity of mouse 1
D329 dissolved in a 10% dimethyl sulfoxide aqueous solution was administered to CDF/1 mice transplanted with 1 x 106 cells per mouse, once a day starting 24 hours after cell transplantation, for a total of three times.
The therapeutic effect when C was administered intraperitoneally was measured by the survival effect, and the results are shown in Table 4. In addition, the survival effect in the table is expressed as a percentage of the ratio of the average survival days (T) of the treated group to the average survival days (C) of the untreated group.

[0027]

[Table 4]

[0028] Cytotoxic activity against cell line W98Vac obtained by tumor transformation of human fetal lung cells with SV40 virus was investigated in the following manner. In other words, the cytotoxicity of W98Vac cells when D329C is added to the culture solution as a methanol solution in a 10% methanol solution for the growth of W98Vac cells in the culture solution is W.
It was measured as a 100% growth inhibition concentration for 98Vac cells. As a result, 100μ
It was effective at g/ml.

(2) Acute Toxicity In an acute toxicity test using BALB/c mice, no toxicity was shown even at 400 mg/kg when administered intraperitoneally. (3) Formulation When D329C of the present invention is actually formulated, it consists of a compound represented by the above structural formula and a pharmaceutically acceptable excipient. These may be for oral administration or parenteral administration.

[0030] Orally administered preparations usually take the form of powders, tablets, emulsions, capsules, granules, liquids, etc. Specific examples of excipients for oral preparations include powders and other powders for internal use. Examples of excipients in liquid formulations include lactose, starch, dextrin, calcium phosphate, calcium carbonate, synthetic and natural aluminum silicate, magnesium oxide, aluminum hydroxide, magnesium stearate, sodium bicarbonate, etc. is, water,
Examples include glycerin and simple syrup. Parenteral preparations include injections, which include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of aqueous solutions and suspensions include distilled water for injection and physiological saline. Examples of non-aqueous solutions and suspensions include propylene glycol,
polyethylene glycol, vegetable oil such as olive oil,
Alcohols such as ethanol, polysorbate 80
(registered trademark) etc. Such compositions may further contain adjuvants such as lubricants, emulsifiers, and dispersants. These are sterilized, for example by filtration through bacteria-retaining filters or by irradiation. They can also be prepared as sterile solid compositions and dissolved in sterile water or sterile injectable solvents before use.

[0031] Each of the above formulations can be prepared according to conventional methods. When the compound represented by the above structural formula of the present invention is used as a preparation, it is usually administered in an amount of 0.2 to 400 mg/dose for adults.
1 day is administered orally or parenterally.

[0032]

[Examples] The present invention will be explained in detail by examples below, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded. Example 1 Dry yeast 1.0%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.1%, mannitol 0.5%, ferrous sulfate 10 ppm, manganese chloride 10 ppm, zinc sulfate 10 ppm, copper sulfate 10 ppm , a liquid medium consisting of 10 ppm cobalt chloride was adjusted to pH 8.0,
A 500 ml Erlenmeyer flask containing 100 ml of this medium was sterilized at 121°C for 15 minutes, and D329-185 strain (
FERM BP-3269) was inoculated onto the agar slant medium into the Erlenmeyer flask.

[0033] A primary seed culture solution was prepared by culturing with shaking at 30°C for 4 days. The above primary seed culture solution was inoculated at 4% each into 17 3-liter Erlenmeyer flasks containing 600 ml of a medium having the same medium composition, and cultured with shaking at 30° C. for 8 days. After completion of the culture, 10 liters of n-butanol was added to 10.2 liters of the culture solution, and extraction was performed at room temperature for 3 hours. The obtained organic layer was concentrated under reduced pressure to obtain crude extract 14.
．． 9g was obtained. 14.9 g of the obtained crude extract was treated with chemically modified silica gel (YMC GEL ODS manufactured by Yamamura Chemical Co., Ltd.).
-AQ) column (φ4 cm x 130 cm) and developed with a system of deionized water and acetonitrile (7:3). The D329C fraction was concentrated to dryness to obtain 194 mg of crudely purified D329C. 194 mg of the crude product was subjected to high performance liquid chromatography (Column Waters, NOV
A-PAK HR C-18 φ25mm×10
0 mm), deionized water/acetonitrile (7:3
) was eluted in the system. The D329C substance fraction was concentrated to dryness,
19 mg of pure D329C was obtained.

Example 2 The culture solution obtained in Example 1 was separated into a cake containing bacterial cells and a filtrate using a Sharpless centrifugal filter. The resulting cake containing bacterial cells was immersed in 5.0 liters of methanol, and after stirring at room temperature for 3 hours, solids such as bacterial cells were removed by filtration to obtain a methanol extract. Methanol was removed from the extract under reduced pressure to obtain 6.4 g of a crude extract. 10 liters of n-butanol was added to the filtrate obtained using a Sharpless centrifugal filter, and extraction was performed at room temperature for 3 hours. The obtained organic layer was concentrated under reduced pressure to obtain 7.6 g of crude extract. The two crude extracts were combined and treated as in Example 1 to obtain 17 mg of pure D329C.

[0035]

EFFECTS OF THE INVENTION D329C of the present invention is considered to be useful as an anticancer agent because it exhibits antitumor activity, and is expected to be used as a medical anticancer agent. Also, D329C
corresponds to a structure in which one additional sugar is bonded to demethyl chartreusine. As is clear from this structure, D
329C exhibits higher water solubility than chartreusine and demethylchartreucine. As mentioned above, one of the problems with chartreuse and demethyl chartreuse is that they have low water solubility. D329C of the present invention, which has improved this problem, is expected to further improve its activity by synthesizing derivatives by chemical synthesis techniques, and has high value as a synthetic intermediate.

[0036]

[Brief explanation of the drawing]

FIG. 1 is an ultraviolet absorption spectrum diagram of D329C measured in methanol.

FIG. 2 is an infrared absorption spectrum diagram of D329C measured by the KBr disk method.

FIG. 3 is a proton nuclear magnetic resonance spectrum diagram of D329C measured in deuterated pyridine at 60°C.

FIG. 4 is a carbon-13 nuclear magnetic resonance spectrum of D329C measured in heavy methanol.

Claims (3)

[Claims] Claims: 1. D329C substance represented by the following structural formula: [Image Omitted] or a pharmaceutically acceptable salt thereof. 2. A novel D329C substance or its pharmaceutical product, characterized in that a microorganism that produces a D329C substance belonging to the genus Streptomyces and having the following structural formula is cultured in a medium, and the D329C substance is collected from the culture. Acceptable salt manufacturing methods. [Case 2] Claim 3: The microorganism that produces the D329C substance is Streptomyces chartreusis D329-185.
Streptomyces chartreusi
3. The manufacturing method according to claim 2, wherein the method is as follows.