KR900005957B1 - Compound 4181-2 its derivatives - Google Patents

Abstract

Antileukemia 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 antitumor activity. Dose is 0.004-12mg per day for adult.

Description

Substance 4181-2 and its derivatives

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

2 shows the ultraviolet absorption spectrum (CHCl ₃ ) of compound 1,

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

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

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

6 shows the ¹ H-NMR spectrum in CDCl ₃ of compound 4.

The present invention relates to novel substance 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 the genus Streptomyces, the above-mentioned substance 4181-2 was produced and the substance was Gram- It has been found that not only has a broad spectrum of antibiotics, including positive and gram-negative bacteria and several types of fungi, but also excellent anti-tumor activity. In addition, the present inventors found that derivatives obtained by substituting acyl groups for both hydrogen atoms of two hydroxyl groups of the material 4181-2 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 obtainable by chemically modifying one or both of the 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 composition is provided wherein 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 by administering an effective amount of a compound of formula (II) to the mammal.

Unless stated otherwise to be cited herein by formulas (I) and (II), the term "acyl" refers to an acyl group having 2 to 5 carbon atoms, such as acetyl, propionyl, butyryl, valeryl, and the like, 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 (published March 10, 1986), compounds similar to general formula (I) according to the present invention are described. 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 Laid-Open No. 102897/1982, European Patent Application No. 54601 and The Journal of Antibiotics Vol.XXXV, No. In June, June, 1982, the above-mentioned serbinomycins A ₁ and A ₂ are described not by structural formula but by physical and chemical properties. However, the document only mentioned above that serbinomycin A ₁ and A ₂ and 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 the general formula (I) and (II), compounds in which substituent R of formula (I) or substituents R ₁ and R ₂ of formula (II) are hydrogen (ie, substance 4181-2) are prepared by culturing microorganisms. can do. As such, the microbial strain capable of producing Substance 4181-2 is incubated under appropriate conditions and the Substance 4181-2 generated from the mycelia cake or culture filtrate is collected.

The acyl derivatives of the material 4181-2 or alkyl derivatives can be synthesized by carrying out conventional acylation or alkylation reactions with the material 4181-2. For example, the acyl derivatives of substance 4181-2 may be prepared by treating substance 4181-2 with or without organic solvents and with organic bases or metal hydroxides such as ammonia, pyridine, triethylamine, and the like (eg, 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.) from about -50 deg. C to about 100 deg. C, preferably room temperature to the solvent used. It can be prepared by reacting with an acyl halide or acid anhydride at temperatures between isotropic points.

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.

Regarding the acylating agent, examples of alkyl halides having 1 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 the acid anhydride include fatty acid anhydrides having 2 to 5 carbon atoms, acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride and the like.

Regarding the alkylating agent, examples of the alkyl halide include 1 to 5 carbon atoms, and 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, iso amyl chloride, t-amyl chloride, 2-bromopentane, and the like. Examples of 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-butyl 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 acylation or alkyllase is used in an amount of at least about 2 moles, preferably about 2 to 10 moles per 4181-21 moles of substance, the reaction is mainly R ₁ and R ₂ in formula (II) and 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 and 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, Tsububa-gun, 305, Japan. It was deposited with the accession number FERM BP-1010 on April 2, 1986, under the Budapest Convention to the Ministry of Trade, Industry and Technology, Institute for Industrial Technology and Technology, located in 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 Characteristics]

(a) form

Spore Basin: Simple Basin

Natural chain form: fetters (Lectus flexibilis)

(Circular spore)

Number of spores: at least 10 spores in a chain

Spore surface: soft

Spore Size: 0.8 ~ 1.0 × 1.1 ~ 1.5㎛

Single Mother: None

Spore sac: none

Implantation 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 ~ 30 ℃; Stop growth at 40 ° C and above

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

3) milk solidification: negative

Peptonation of Milk: 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 strains are well described using all of the following carbohydrates. : L-arabinose, D-xylose, D-glucose, D-fructose, inositol, L-lamnose, raffinose, D-mannitol sucrose, starch, D-mannose, maltose Oz, 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 the substrate hyphae, the cell component diaminopimelic acid is LL-type, and the strain has the above biological characteristics, which does not have flagella or spore cysts. It is clear that the strain belongs to the genus Myse. Thus, the strain was named Streptomyces species 4141.

Substance 4181-2 according to the present invention can be prepared, for example, by culturing the Streptomyces spis 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 culture the material 4181-2-producing strain by aerobic culturing such as shaking cultivation or suspension cultivation according to liquid culture. The culture medium is used containing a nutrient source that the strain can use. In this sense, any of synthetic medium, semisynthetic medium and natural medium can be used. As the carbon source, glucose, sucrose, fructose, glycerol, dextrin, starch, molasses, corn steep liquor, organic acid and the like can be used alone or in combination. As a nitrogen atom, 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 necessary, minerals such as sodium salts, potassium salts, magnesium salts, phosphates, heavy metal salts and the like can 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 octadecanol, tetradecane, higher alcohols such as heptadecanol, various kinds of silicone compounds and the like may be added.

The pH of the culture medium is preferably maintained at slightly acidic or near neutral. Since 4181-2-producing strains are generally thought to grow at temperatures between about 20 ° C. and 37 ° C., the culturing is generally carried out at about 20 ° C. and 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.

Material 4181-2 accumulates in both the culture broth and mycelia cake. Therefore, the culture 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 that 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. Evaporation of the solvent underneath afforded 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, the purification may be repeated to add purification, or may be performed in combination with recrystallization from a solvent such as chloroform, methanol or acetone and / or other techniques. In this way, material 4181-2 of high purity can be separated.

Anti-tumor compositions containing substance 4181-2 or a derivative thereof as an active ingredient in accordance with the present invention may 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, substance 4181-2 or a derivative thereof can 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 formulated with a pH adjuster, buffer, stabilizer, isotonic agent, local anesthetic and the like and administered subcutaneously, intramuscularly, intravenously or by other routes. For suppository preparation, Material 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, methylcellulose, carboxymethyl 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 cacaoter, polyethyleneglycol, lanolin, fatty acid triglycerides, Witsol (trademark of Witepsol Dynamite Nobel, mono, di or triglycerides) and the like.

The amount of substance 4181-2 or derivatives thereof in the pharmaceutical composition according to the present invention varies depending on the condition of the patient, the particular dosage form and other factors, but is generally about 0.04 to 4 mg per unit oral dosage, about 0.004 to injection 2 mg, and about 0.04-4 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 bioanalytical and bioautomated photography using Staphylococcus aureus 209p, Salcina lutea ATCC9341, or the like as a test microorganism or in the human nasopharynx. Examine and confirm the cytotoxic effect on the culture of cancer (KB cells) derived cells.

Example 1

Preparation of Substance 4181-2 (Compound 1).

의 염화나트륨, 0.2%의 황산마그네슘 및 0.3%의 탄산칼슘(pH 7.0)을 함유하는 배지 100ml을 채운다. 멸균 후에, 상기 접종 배양액 5㎎를 가입하고, 회전 진탕기에서 27℃로 96시간 동안 진탕 배양한다. 배양이 끝난 후에, 배양액(7.4l, pH 7.4)를 원심분리 및 여과하고 균사 케이크를 메탄올(약 1l씩) 3회 연속 추출한다. 추출액을 감압하에 농축하고 잔류물을 교반하며 에틸 아세테이트(0.3l씩)로 3회 추출한다. 배양여액의 pH를 묽은 염산으로 3으로 조절하고 에틸 아세테이트 (0.8l씩) 로 3회 추출한다. 추출액을 취합하고, 상기에서 수득한 균사 추출액과 합쳐서 물로 세척한 후에, 무수 황산나트륨으로 탈수시키고 감압하에 농축하여 갈색 유상물(6.42g)을 수득한다. 이 유상물을 에틸 아세테이트(0.3l)에 용해시키고 n-헥산(0.5l)을 가입한다. 생성된 침전물을 여과하고 건조할때까지 증발시켜 물질 4181-2를 함유하는 적갈색 조분말(801㎎)을 수득한다.100 mg 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) in a 500 mg conical flask After filling and sterilizing, Streptomyses SPICE 4181 (FERM BP-1010) is inoculated with a platinum equivalent and shake incubated for 48 hours at 27 ° C. with a rotary shaker (180 r.pm amplitude 10 cm). Then, in a 500 ml conical flask, 4.0% glycerol, 1.0% Pharmamedia, 0.2

100 ml of a medium containing sodium chloride, 0.2% magnesium sulfate and 0.3% calcium carbonate (pH 7.0) is charged. After sterilization, 5 mg of the inoculation culture was added and shaken for 96 hours at 27 ° C. on a rotary shaker. After incubation, the culture solution (7.4 l, pH 7.4) is centrifuged and filtered and the mycelia cake is extracted three times in succession with methanol (about 1 l). The extract is concentrated under reduced pressure and the residue is stirred three times with ethyl acetate (0.3 l each). The pH of the culture filtrate is adjusted to 3 with dilute hydrochloric acid and extracted three times with ethyl acetate (0.8 l 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 (0.3 l) and joined n-hexane (0.5 l). 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 mg) and adsorbed onto a silica gel chromatography column (Merck, Kigel spheres, 5.9 x 19 cm). The column is washed with chloroform (1.4 l) and then eluted with 3.9 l of 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)

¹ H-NMR spectrum (DMSO-d ₆ ), ultraviolet absorption spectrum (CHCl 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 on acetic anhydride (7.5 ml) and the mixture is stirred overnight at room temperature. 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 (20 mg, 83% yield).

Welding 208 ~ 212 ℃ (decomposition)

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

Example 3

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

Dissolve substance 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 (CDCl 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 mg) -pyridine (20 ml), cooled with ice and 10 ml of acetic anhydride is 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%).

Welding 220 ~ 225 ℃ (decomposition)

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

Substance 4181-2 and its acyl and / or alkyl derivatives represented by formula (I) or (II) not only have a broad spectrum of 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.

[Pharmacological examination]

1. Antibacterial Spectrum

Antibacterial and antifungal spectra of substance 4181-2 were investigated. Gram-positive bacteria, nutrient-negative agar medium for gram-negative bacteria, and savoraoud agar medium for fungi are determined, and the minimum inhibitory concentration (MIC) is determined by two-fold serial dilution. 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 ₁ birds. 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 is determined in days. The results are shown in Table 3. The percent increase in survival is calculated by the following equation.

Where D ₀ represents the mean survival time (days) of the untreated group and D represents the mean survival time (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 is suspended in 0.2% Tween 80 solution in physiological saline and administered intraperitoneally to birds. 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.

[Example 1]

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

[Example 2]

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

[Example 3]

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

[Example 4]

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

[Example 5]

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

[Example 6]

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

Claims (2)

Compounds of the general formula.

Wherein R is hydrogen or an acyl group. The compound of claim 1, wherein R is a hydrogen atom.

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