KR900005056B1 - Substance 4181-2and its derivatives and antitumor agent with its compounds - Google Patents

Abstract

Antileukaemia compsn. comprises substance 4181-2 deriv. of formula (I), where R1 and R2 are each H, alkyl, or acyl, and a pharmaceutically acceptable carrier. (I) can be prepd. by cultivating microorganism strain 4181 of streptomyces genus, and opt. alkylating or acylating. (I) have good antitumor activity.

Description

Method for preparing substance 4181-2 and derivatives thereof and antitumor agent containing these compounds

1 shows the ¹ H-NMR spectrum (DMSO-d ₆ ) of Compound 1,

2 shows the ultraviolet absorption spectrum (CHCI ₃ ) of Compound 1,

3 shows the infrared absorption spectrum (KBr) of compound 1,

4 shows the ¹ H-NMR spectrum in CDCI ₃ of Compound 2,

The fifth turning depicts the ¹ H-NMR spectrum in the compound 3 in CDCI _3,

FIG. 6 shows the ¹ H-NMR spectrum in CDCI ₃ of Compound 4. FIG.

The present invention relates to the preparation and use of the novel material 4181-2 and its derivatives.

The material 4181-2 according to the present invention is a formula that has never been described in the literature.

Is a novel compound.

The present inventors studied the substances produced by various kinds of microorganisms and the characteristics of such substances, and when cultivating any microorganism belonging to Streptomyces, the above-mentioned substance 4181-2 was produced and the substance was Gram-positive and It has been found that not only has a broad spectrum of antibiotics, including gram-negative bacteria and several types of fungi, but also excellent anti-tumor activity. In addition, the inventors found that derivatives obtained by substituting both two hydrogen atoms of two hydroxyl groups of two hydroxyl groups of substance 4181-2 with acyl groups have similar antitumor and other activities.

Therefore, the present invention is a general formula

In which R is a hydrogen atom or an acyl group.

The inventors also found that compounds obtained by chemically modifying one or both of the two hydrogen atoms represented by R of substance 4181-2 with alkylating and / or acylating agents also possess antitumor activity. Therefore, the present invention is a general formula

An anti-tumor agent is provided in which R ₁ and R ₂ are the same or different and each contains an effective amount of a compound which represents a hydrogen atom, an alkyl group or an acyl group together with a pharmaceutically acceptable carrier.

The invention also provides for the treatment of tumors in mammals, including humans, characterized in that the mammal is administered an effective amount of a compound of formula (II).

The term "acyl" refers to an acyl group having from 2 to 5 carbon atoms, such as acetyl, propionyl, butyryl, valeryl, and the like, unless otherwise indicated as recited herein by formulas (I) and (II). The term "alkyl" is used to mean straight or branched chain alkyl groups having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl and t-amyl.

The 106th General Assembly of the Japanese Pharmaceutical Society held on April 2-4, 1986, on pages 207 and 489 of the Proceedings (March 10, 1986) describes compounds similar to general formula (I) according to the present invention. It is. This compound has the following structural formula

And is called serbinomycin A ₂ . The compound obtainable by reducing its xanthone nucleus is called serbinomycin A ₁ . The Journal is a Sergio unexposed erythromycin A compounds substituted the hydrogen of the hydroxyl groups of A ₂ a methyl group or a group acetyl, and Sergio unexposed erythromycin A ₁ of the 3 hydroxyl groups of each of Sergio unexposed erythromycin A ₁ triacetate is the Substituted that of hydrogen atoms are acetyl It also describes. And Japanese Patent Application Publication No. 102897/1982, European Patent Application No. 54601 and The Journal of Antibiotics Vo1. XXXV, No 6, June, 1982 describe the above-mentioned serbinomycin A ₁ and A _{2 not} by structural formula but by physical and chemical properties. However, the document only states that Serbinomycin A ₁ and A ₂ and the derivatives thereof mentioned above have antimicrobial activity and do not mention at all that the compounds have antitumor activity.

The manufacturing method of the compound of general formula (I) and (II) is described below.

Among the compounds of formula (I) and (II), substituent R of formula (I) or substituent R ₁ of formula (II), and compound wherein R ₂ is hydrogen (i.e., substance 4181-2) may be cultured by It can manufacture. As such, the microbial strain capable of producing Substance 4181-2 is cultured under appropriate conditions and the Substance 4181-2 generated from the mycelia cakes or the culture filtrate is collected.

Acyl derivatives or alkyl derivatives of substance 4181-2 can be synthesized by conducting conventional acylation or alkylation reactions with substance 4181-2. For example, the acyl derivatives of material 4181-2 may be prepared by treating the material 4181-2 with or without organic solvents and organic bases or metal hydroxides such as ammonia, pyridine, triethylamine and the like (e.g. sodium hydroxide, potassium hydroxide, etc.), In the presence or absence of inorganic bases such as inorganic salts (e.g. sodium carbonate, potassium hydrogen carbonate, etc.), metal oxides (e.g. silver oxide, etc.) between about -50 ° C and about 100 ° C, preferably between room temperature and the boiling point of the solvent used It can be prepared by reacting with an acyl halide or acid anhydride at a temperature of. The alkyl derivative of substance 4181-2 may be prepared by reacting substance 4181-2 with an alkyl halide or alkyl ester of sulfonic acid, sulfonic acid or phosphoric acid under such conditions.

As for the acylating agent, examples of acyl halides having 2 to 5 carbon atoms include acetyl chloride, acetyl bromide, propionyl chloride, propionyl bromide, butyryl chloride, butyryl bromide, valeryl chloride, valeryl bromide and the like. Examples of acid anhydrides include fatty acid anhydrides having 2 to 5 carbon atoms, such as acetic anhydride, propionic anhydride, butyric anhydride and valeric anhydride.

With respect to the above alkylating agent, examples of alkyl halides having 1 to 5 carbon atoms include methyl iodide, methyl bromide, methyl chloride, ethyl iodide, ethyl bromide, propyl chloride, propyl bromide, isopropyl iodide, Butyl iodide, isobutyl bromide, s-butyl chloride, t-butyl bromide, pentyl bromide, isoamyl chloride, t-amyl chloride, 2-bromopentane, and the like. Examples of the alkyl esters include alkyl esters of sulfuric acid, Dimethyl sulfate and diethyl sulfate; As alkyl ester of sulfonic acid, methyl p-toluene sulfonate, ethyl p-toluene sulfonate, methyl benzene sulfonate, ethyl benzene sulfonate, butyl p-toluene sulfonate, butyl benzene sulfonate, propyl p-toluene sulfonate, etc .; And alkyl esters of phosphoric acid include methyl phosphate, ethyl phosphate, propyl phosphate n-butyl phosphate, t-phosphate and the like.

If an acylation or alkylating agent is used in an amount of about 1 mole per mole of material 4181-2, this reaction generally produces a mono-acyl or mono-alkyl derivative as the main component and further reacts with the acylation or alkylating agent as desired. In formula (II), a compound in which one of R ₁ and R ₂ is an acyl group and the other is an alkyl group, or both R ₁ and R ₂ are the same or different alkyl or acyl groups can be prepared. On the other hand, if an acylation or alkylating agent is used in an amount of at least about 2 moles, preferably about 2 to 10 moles per mole of material 4181-2, the reaction is mainly the same in formula (II) with R ₁ and R ₂ being acyl Or the compound which shows an alkyl group is manufactured.

As the organic solvent, hydrocarbons such as hexane, halogenated hydrocarbons such as chloroform, ethers such as ethyl ether and dioxane, ketones such as acetone, aromatic hydrocarbons such as benzene and toluene, nitromethane, dimethylformamide and Aprotic polar solvents such as dimethyl sulfoxide, and the like can be used alone or in a mixture. If desired, the bases mentioned above may also be used as the solvent.

The microorganism strain used for the preparation of the substance 4181-2 may be any strain as long as it can produce the substance 4181-2. As an example, mention may be made of strain 4181 in the Streptomyces genus isolated by the present inventors. The strain 4181 is a microorganism belonging to the genus Streptomyces, which the present inventors separated from soil samples collected from Sichuan Province, Imei-Shan, People's Republic of China, 1-Himeshi, Yatabe-machi, Yatabe-machi, Ibaraki It was deposited with the accession number FERM BP-1010 dated April 2, 1986, under the Budapest Convention to the Institute of Microbiological Industry and Technology, Ministry of Commerce, Industry and Technology, 1-3. In addition, this strain was deposited on June 2, 1987, with the accession number KFCC-10368 to the Korean spawn association. The bacteriological properties of strain 4181 are as follows.

Microbiological properties

(a) form

Spore Basin: Simple Basin

Natural chain form: straight chain (Lectus flexibilis) (circular spore)

Number of spores: 10 spores or more per chain

Spore surface: soft

Spore Size: 0.8 ~ 1.0 × 1.1 ~ 1.5㎛

Single Mother: None

Spore sac: none

Implantation location of spores

Sustained cell: None

(b) Culture characteristics in various media are shown in Table 1.

TABLE 1

(c) physiological properties

1) Growth temperature range: Good growth at 26 to 30 ° C; Stop growth at 40 ° C and above

2) Liquefaction of gelatin (glucose-peptone gelatin medium): positive (after 3 weeks)

3) Milk coagulation: negative, milk peptonation: positive

4) Preparation of melanoid pigments: negative in tyrosine agar (ISP-7) and peptone yeast extract iron agar (ISP-6)

5) Preparation of hydrogen sulfide: negative

6) Hydrolysis of Starch (Starch Agar): Positive

7) reduction of nitrates: negative

8) Degradation of cellulose: positive

(d) Use of carbon sources (Friedham-Gottlieb agar, ISP-9)

The strain grows well using all of the following carbohydrates: L-arabinose, D-xylose, D-glucose, D-fructose, inositol, L-ramnose, raffinose, D-mannitol Sucrose, starch, D-mannose, maltose, D-sorbitol, cellulose and inulin.

(e) Cell composition

Analysis by Becker et al. (Appl. Microbiol. 12,421-423,1964) revealed that the hydrolyzate of the entire strain contained LL-diaminopimelic acid and a small amount of glycine. .

Strain 4181 forms a mycelium with many spore chains from substrate hyphae, the cell component diamino pimelic acid is LL-type, and the strain is streptomycin with the above biological properties without the flagella or spore sac. Clearly it is a strain belonging to the genus Seth. Thus, the strain was named Streptomyces species 4181.

Substance 4181-2 according to the present invention can be prepared by, for example, culturing the Streptomyces sp. 4181 or a mutant thereof in an appropriate medium.

The culturing method may be any method as long as it is commonly used for culturing microorganisms, but in general, it is preferable to cultivate the material 4181-2-producing strain by aerobic culturing such as shaking culture or floating culture according to liquid culture. The culture medium is one containing a nutrient source available to the strain. In this sense, any of synthetic, semi-synthetic, and natural media can be used. As the carbon source, glucose, sucrose, fructose, glycerol, textine, starch, molasses, corn steep liquor, organic acid and the like can be used alone or in combination. As a nitrogen source, Pharmamedia (Pharmamedia, trade name, a product of the US Traders Oil Company, containing 60% protein, filled with ash, fiber, etc.), peptone, meat extract, yeast extract, soy flour, casein, amino acid, Organic nitrogen content such as urea and the like, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate may be used alone or in combination. If desired, minerals such as sodium salts, potassium salts, magnesium salts, phosphate salts, heavy metal salts and the like may be incorporated into the medium.

If foams remarkably occur during fermentation, For example, Vegetable oils, such as a soybean oil and linseed oil; Antifoaming agents such as higher alcohols such as octadecanol, tetradecanol, heptadecanol, various kinds of silicone compounds and the like may be added.

The pH of the culture medium is preferably maintained near weak acidity or neutrality. Since 4181-2-producing strains are generally believed to grow at temperatures between about 20 ° C. and 37 ° C., the culture is generally carried out at about 20-37 ° C., preferably at about 27-30 ° C. In the case of liquid culture, material 4181-2 accumulates after 2 to 5 days of culture.

Of course, the optimal point of the culture conditions should be selected from the conditions depending on the producer of the particular substance 4181-2 used, its properties, and other conditions. Separation of the prepared material 4181-2 in the culture may be performed by a conventional method for separating microbial metabolites. For example, solvent extraction, pH adjustment, crystallization, and other techniques may be used alone or in appropriate combinations in the proper order.

Substance 4181-2 accumulates in both the culture and mycelia cakes. Therefore, the culture solution is first centrifuged or filtered and the mycelia cake is treated with a solvent such as acetone, methanol or a mixture thereof to extract substance 4181-2. On the other hand, the supernatant or filtrate is treated with a solvent which is not mixed with water, such as ethyl acetate, chloroform, butanol, or mixtures thereof, to extract substance 4181-2 into the organic layer and dehydrated by adding sodium sulfate to the organic layer, and then The solvent is evaporated to prepare a crude extract containing material 4181-2. If necessary, effective extraction can be carried out by adjusting the pH with hydrochloric acid or sulfuric acid or by adding brine.

For purification, conventional purification methods for lipophilic low molecular weight materials can be used. As described above, various adsorption chromatography methods using an adsorbent such as silica gel, alumina, macroporous nonionic adsorbent resin, and the like may be used. Especially preferred is silica gel chromatography using chloroform-methanol as eluent. If necessary, further purification may be performed by repeating the above chromatography process or by recrystallization from a solvent such as chloroform, methanol or acetone and / or in combination with other techniques. In this way, material 4181-2 of high purity can be separated.

Antitumor compositions containing the substance 4181-2 or a derivative thereof as an active ingredient in accordance with the present invention can be administered in various dosage forms for the treatment of humans and mammals. These dosage forms can be oral administration, injections, suppositories, and the like, each of which can be prepared by established pharmaceutical methods. In the preparation of oral solid preparations, the substance 4181-2 or derivatives thereof may be formulated with or without addition of excipients or binders, disintegrants, lubricants, colorants, flavors, flavors, and the like, and tablets. , Coated tablets, granules, powders, capsules and the like are processed according to known methods. For the preparation of parenteral preparations, material 4181-2 or derivatives thereof may be administered subcutaneously, intramuscularly, intravenously or by other routes in preparation with pH adjusting agents, buffers, stabilizers, tonicity agents, local anesthetics and the like. For suppositories, substance 4181-2 or derivatives thereof are formulated with suppositories according to known methods by formulating with or without a surfactant with an appropriate suppository base.

Examples of excipients used in the preparation of tablets, capsules, granules or powders include lactose, sucrose, starch, talc, magnesium, stearate, crystalline cellulose, methyl cellulose, carboxy methyl cellulose, glycerol, sodium alginate , Gum arabic and the like can be mentioned. Examples of such binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, gum arabic, shellac, sucrose and the like. Examples of such lubricants include magnesium stearate, talc and the like. Colorants, flavors, disintegrating agents and other auxiliaries may be used in common in the known art. Tablets are coated by established pharmaceutical methods.

Suppository bases used in suppository preparations include, among others, oily bases such as cacao butter, polyethylene glycol, lanolin, fatty acid triglycerides, Witsol (trademark of Witepsol Dynamite Nobel, mono, di or triglycerides).

The amount of substance 4181-2 or its derivatives in the pharmaceutical composition according to the present invention will vary depending on the condition of the patient, the particular dosage form and other factors, but will generally be from about 0.04 to 4 mg per dosage, about 0.004 to 2 mg per injection And about 0.04 to 4 mg per suppository. The daily dosage also varies according to the patient's symptoms and other factors but generally ranges from about 0.004 to 12 mg for adults.

The preparation of the compounds according to the invention is shown in the following examples. In these examples, the compounds of the present invention may be prepared by bioanalysis and bioautomography using Staphylococcus aureus 209p, Salcina lutea ATCC 9341, etc. Examine the cytotoxic effect on the culture of pharyngeal cancer (KB cell) -derived cells.

Example 1

Preparation of Substance 4181-2 (Compound 1)

Fill a 500 ml conical flask with 100 ml of medium containing 0.1% glucose, 0.1% polypeptone, 0.3% meat extract, 0.5% yeast extract, 2.4% soluble starch and 0.3% calcium carbonate (pH 7.0) After sterilization, Streptomyces SPICE 4181 (FERM BP-1010) is inoculated with platinum and shaken with a rotary shaker at 27 ° C. for 48 hours (180 r.pm amplitude 10 cm). The 500 ml conical flask is then charged with 100 ml of medium containing 4.0% glycerol, 1.0% Pharmamedia, 0.2% sodium chloride, 0.2% magnesium sulfate 0.3% calcium carbonate (pH 7.0). After sterilization, 5% of the inoculum was added thereto, and incubated for 96 hours at 27 ° C. on a rotary shaker. After the incubation, the culture (7.4ι, pH 7.4) is centrifuged and filtered and the mycelia cake is extracted three times in succession with methanol (about 1ι each). The extract is concentrated under reduced pressure and the residue is stirred three times with ethyl acetate (0.3ι each). The pH of the culture filtrate is adjusted to 3 with dilute hydrochloric acid and extracted three times with ethyl acetate (0.8ι each).

The extracts are combined, combined with the mycelial extract obtained above, washed with water, then dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure to give a brown oil (6.42 g). This oil is dissolved in ethyl acetate (0.3ι) and joined n-hexane (0.5ι). The resulting precipitate is filtered and evaporated to dryness to give a reddish brown crude powder (801 mg) containing material 4181-2.

The powder is dissolved in chloroform (about 10 ml) and adsorbed onto a silica gel chromatography column (Merck, Kigel spheres, 5.9 x 19 cm). The column is washed with chloroform (1.4ι) and then eluted with 3.9ι chloroform-methanol (20: 1). Fractions containing material 4181-2 are collected to distill off the solvent. The residue is recrystallized from chloroform-acetone and dried to give a red orange powder (83 mg).

Melting Point> 260 ° C (Decomposition)

The ¹ H-NMR spectrum (DMSO-d ₆ ), ultraviolet absorption spectrum (CDCI ₃ ) and infrared absorption spectrum (KBr) of compound 1 are shown in FIGS. 1, 2 and 3, respectively.

Example 2

Synthesis of Diacetyl Derivative of Compound 4181-2 (Compound 2)

To a mixture of chloroform (15 ml) and pyridine (15 ml) is added dropwise acetic anhydride (7.5 ml) while cooling with ice the solution of substance 4181-2 (29 mg) and the mixture is stirred at room temperature overnight. The reaction mixture is concentrated and the residue is dissolved in chloroform and subjected to silica gel column chromatography (Merck, Kigel sphere, 5.9 × 19 cm). Then eluted with chloroform-methanol (50: 1) and the eluate was concentrated to dryness to afford the title compound (28 mg, yield 83%).

Melting point 208-212 ° C (decomposition)

¹ H-NMR spectrum (CDCI ₃ ) of compound 2 is shown in FIG. 4.

Example 3

Synthesis of Monomethyl Derivative of Compound 4181-2 (Compound 3)

Dissolve material 4181-2 (100 mg) in 100 ml of a mixture of chloroform and methanol (10: 1), followed by silver oxide (45.2 mg), followed by methyl iodide (27.6 mg). The mixture is stirred overnight and the reaction mixture is filtered. The filtrate is concentrated, dissolved in chloroform again, and eluted with chloroform in silica gel column chromatography (Merck, Kigel sphere, 5.9 x 19 cm). Concentrate the eluate to dryness to afford the title compound (20 mg, yield 19.5%).

Melting Point> 300 ℃ (Decomposition)

¹ H-NMR spectrum (CDCI ₃ ) of compound 3 is shown in FIG. 5.

Example 4

Synthesis of Monomethyl Monoacetyl Derivative of Compound 4181-2 (Compound 4)

Methyl ether (40 mg) obtained in Example 3 is dissolved in chloroform (20 ml) -pyridine (20 ml), cooled with ice and 10 ml of acetic anhydride are added dropwise. The reaction mixture was treated in the same manner as in Example 2 to give the title compound (35 mg, yield 81.6%).

Melting Point 220 ~ 225 ℃ (Decomposition)

¹ H-NMR spectrum (CDCl ₃ ) of compound 4 is shown in FIG. 6.

Substance 4181-2 and acyl and / or alkyl derivatives thereof represented by general formula (I) or (II) not only have a broad antimicrobial spectrum including gram-positive bacteria, gram-negative bacteria and fungi, but also have excellent antimicrobial properties. Have tumor activity. Tests to determine the antibacterial, antifungal and antitumor activity of the compounds were performed as follows.

Pharmacology

1. Antibacterial Spectrum

Antibacterial and antifungal spectra of substance 4181-2 were investigated. The minimum inhibitory concentration (MIC) is determined by two-fold serial dilution using nutrient agar media for gram-positive and gram-negative bacteria and savoragar agar media for fungi. The antimicrobial spectrum of substance 4181-2 is shown in Table 2.

TABLE 2

2. Antitumor Activity

Antitumor activity of Substance 4181-2 and its derivatives was assessed by increased survival. 1 × 10 ⁶ leukemia p 388 cells are injected intraperitoneally of BDF _l mice. Each group consists of six birds. The next day, each of compounds 1-4 is suspended in 0.2% Tween 80 solution in physiological saline and administered intraperitoneally and the survival time is determined in days. The results are shown in Table 3. The percent increase in survival is calculated by the following equation.

In the above formula, D ₀ represents the average survival (days) of the untreated group and D represents the average survival (days) of the treated group.

TABLE 3

3. Acute Toxicity Study

Male fly rats (weight 25g) of ddy strain are used. Each of Compounds 1-4 was suspended in 0.2% Tween 80 solution in physiological saline and administered intraperitoneally. The LD ₅₀ value (mg / kg) is determined by the up-and-down method. The results are shown in Table 4.

TABLE 4

Examples of preparations using the compounds of the present invention are shown below.

The tablets are prepared using the above ingredients in the aforementioned proportions.

The granules are prepared by using the above components in the above-mentioned blending ratios.

The fine particles are prepared by using the above components in the above-mentioned blending ratio.

The capsules are prepared by using the above ingredients in the above-mentioned blending ratios.

Injectables are prepared by using the above ingredients in the above mentioned combination ratios.

The suppositories are prepared using the above ingredients in the above mentioned combination ratios.

Claims (4)

An anti-tumor agent containing a compound of the following general formula which is an active ingredient and a pharmaceutically acceptable carrier.

Wherein R ₁ and R ₂ are the same or different and each represents a hydrogen atom, an alkyl group or an acyl group. The antitumor agent according to claim 1, wherein R ₁ and R ₂ are each hydrogen atoms. A microorganism strain belonging to Streptomyces that can produce substance 4181-2 is cultured in a medium, and the substance 4181-2 is recovered from the culture medium, and the recovered substance 4181-2 as described above is converted into an alkylating agent or an acylating agent according to the purpose. Process for the preparation of a compound of the general formula, characterized in that the reaction.

Wherein R ₁ and R ₂ are as defined in claim 1. The method of claim 3, wherein the microbial strain is Streptomyces Species 4181 (FERM BP-1010 KFCC-10368).

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