KR100343394B1 - Novel aklavinone derivative and preparation method thereof - Google Patents

Abstract

The present invention relates to a novel aclabinone derivative and a method for producing using a microorganism, and a transformant used for the preparation of the compound, and more particularly, to aclabinone C-11 hydroxy isolated from a strain of Streptomyces spp. Biosynthetic mutants were transformed with a vector containing a silase gene, followed by culturing the transformant to prepare 2,11-dihydroxyaclabinone, a novel aclabinone derivative of Formula 1 below. It can be used as a precursor for preparing new anthracycline antibiotics, and the anthracycline antibiotics obtained by the bioconversion method can be used as an effective antibiotic with low side effects due to low cytotoxicity.

Description

Novel aklavinone derivative and preparation method thereof

The present invention relates to a novel aclabinone derivative and a method for producing using a microorganism, and a transformant used for the preparation of the compound, and more particularly, to aclabinone C-11 hydroxy isolated from a strain of Streptomyces spp. The present invention relates to a novel aclabinone derivative prepared by transforming a biosynthetic mutant with a vector containing a silase gene, and then culturing the transformant, and a method for preparing the same.

Anthracycline-based antibiotics include doxorubicin, alaccinomycin, and daunorubicin, and are widely used as anticancer agents because they exhibit excellent cytotoxic effects. These anthracycline antibiotics can be broadly divided into aclacinomycin-based precursors of aclabinone and daunorubicin-based precursors of 11-hydroxyaclabinone, which are biosynthesized according to the type of precursor. The substance is known to be determined (Connors, NC et al ., J. of General Microbiology 136, 1887-1894 (1990)). Such alaccinomycin-based anticancer drugs, especially doxorubicin, a daunorubicin-based antibiotic, are severely toxic to the kidney and thus are limited in human dosage and duration of use, and therefore, to find new anthracycline antibiotics with fewer side effects. Efforts have been continued. Specifically, research has been conducted to synthesize derivatives capable of reducing oral acute and chronic kidney toxicity by modifying existing daunorubicin antibiotics, especially doxorubicin. As a result, epirubicin and aidarubicin (idarubicin) and the like were developed by organic synthesis. In addition, studies have been conducted to obtain new derivatives by modifying the aclocinomycin-based antibiotics. 11-hydroxy aclocinomycin using aclabinone C-11 hydroxylase derived from a strain of Streptomyces spp. A, 11-hydroxy aclocinomycin B, 11-hydroxy aclocinomycin X, 11-hydroxy aclocinomycin M, 11-hydroxy aclocinomycin G, 11-hydroxy aclocinomycin S, 11-hydroxy aclocinomycin Y and 11-hydroxy aclocinomycin T have been prepared (Korean Patent No. 1452943, Japanese Patent No. 2731367, US Pat. No. 5843738). Many researchers have been using recombinant genes to create novel anthracycline-based substances with reduced cytotoxic side effects (Jarmo Niemi et al . Microbiology 140, 1351-1358 (1994)).

On the other hand, research has been conducted to prepare a new anthracycline by a biotransformation method using anthracycline biosynthetic mutants using various anthracyclines as precursors (Osamu Johdo et al., J. of Antibiotics 49, 669-675 ( 1996), to date, when the anthracyclinone used as a precursor is a 2-hydroxyaclavinone having a hydroxy group at the C-2 position, 2-hydroxy only if a biosynthetic deficiency variant of the aclocinomycin family is used. Biotransformation with several glycosides such as siaclacinomycin A has only been reported (Toshikazu Oki et al ., J. of Antibiotics 34, 916-918 (1981)), yet glycosides in biosynthetic deficient strains of the Daunorubicin family (Glycoside) has not been reported to bioconvert (Akihiro Yoshimoto et al., J. of Antibiotics 33, 1158-1166 (1980)).

Accordingly, the present inventors have been trying to find a new aclabinone derivative (anthracyclinone) as a precursor that can be bioconverted by a daunorubicin-based biosynthetic deficiency mutant to make a new anthracycline antibiotic. When a transformant of Streptomyces sp. Transformed with a vector containing a hydroxylase gene is cultured, a new aclabinone derivative can be obtained from the culture medium. The present invention was completed by revealing that it is a substance 2,11-dihydroxyaclavinone.

It is an object of the present invention to provide a novel aclabinone derivative, a method for preparing the same, and a transformant used therein, and also to provide a method for producing an anthracycline antibiotic by bioconverting the aclabinone derivative.

FIG. 1 shows the predicted biosynthetic pathways of 2,11-dihydroxyaclabinone and 2,11-dihydroxyanthracycline,

2 shows the UV spectrum of 2,11-dihydroxyaclabinone,

Figure 3 shows the ¹ H-NMR spectrum and the structure of 2,11-dihydroxyaclabinone,

FIG. 4 shows the ¹³ C-NMR spectrum of 2,11-dihydroxyaclabinone,

5 shows the ESI-MS spectrum of 2,11-dihydroxyaclabinone,

FIG. 6 shows the UV spectrum of IDB-G4, a glycoside material of 2,11-dihydroxyaclabinone, produced from a doxorubicin producing deficiency mutant strain using 2,11-dihydroxyaclabinone as a precursor. ,

Figure 7 shows the ¹ H-NMR spectrum of the IDB-G4,

8 shows the FAB-MS spectrum of the IDB-G4.

In order to achieve the above object, the present invention provides a novel aclabinone derivative, 2,11-dihydroxyaklavinone (2,11-dihydroxyaklavinone) represented by the following formula (1).

Formula 1

In another aspect, the present invention is a Streptomyces gal transformed with a recombinant plasmid pMC213 containing a gene encoding aclavinone C-11 hydroxylase as a transformant for preparing the compound Relieus ( Streptomyces galilaeus ATCC 31671 / pMC213) is provided. As host cells, any other strain which does not have aclabinone C-11 hydroxylase activity and produces anthracycline may be used.

In addition, the present invention provides a method of producing 2,11-dihydroxyaclabinone by culturing the transformant to express aclabinone C-11 hydroxylase. Depending on the application, 2,11-dihydroxyaclabinone may be produced by separating and purifying the expression product from the culture and using the enzyme directly in biosynthesis of anthracycline antibiotics.

In another aspect, the present invention provides a method for producing an anthracycline antibiotic by a bioconversion method using a biosynthetic deficiency mutant using 2,11-dihydroxyaclabinone of the formula (1) as a precursor (aglycone). More specifically, the present invention provides a method for producing 2,11-dihydroxyanthracycline (2,11-dihydroxyanthracycline) by bioconverting the 2,11-dihydroxyaclabinone using a biosynthetic deficiency mutant strain. do. As a biosynthetic deficiency mutant strain used in the bioconversion process, a doxorubicin-producing mutant strain may be used, and in particular, it is preferable to use Streptomyces fusetius NU23 strain.

In another aspect, the present invention provides 2,11-dihydroxyanthracycline, which is an anthracycline-based antibiotic prepared by a bioconversion method using 2,11-dihydroxyaclabinone as a precursor.

Hereinafter, the present invention will be described in detail.

The aclobinone C-11 hydroxylase gene used in the present invention is involved in the mechanism of synthesizing ε-rhodomycinone and daunorubicin by acting on aclabinone (aklavinone). Encodes aclobinone C-11 hydroxylase and can be cloned from a strain of the genus Streptomyces (Korean Patent No. 14444, US Pat. No. 5,584,378). Preferably the gene of the present invention comprises an aclobinone C-11 hydroxylase gene isolated from Streptomyces fusettius (ATCC 27952), the base sequence of the gene and the aclabinone C-11 hydroxide The amino acid sequence of the loxylase is as described in Korean Patent No. 144443.

The aclabinone C-11 hydroxylase of the present invention is transformed from a host cell with a recombinant vector pMC213 encoded by the gene and containing the gene, and cultured the transformant, thereby culturing the transformants 2,11 Obtain dihydroxyaclavinones.

As a host cell, one which has no aclabinone C-11 hydroxylase activity and produces anthracycline is used. Specifically, Streptomyces lividans, Streptomyces fusetius , Streptomyces galileus , and the like can be used, and among them, Streptomyces fusetius, Streptomyces galileus, etc. are preferable. Do. More preferably, it is preferable to use Streptomyces galilaeus ( ATCC 31671), which is a mutant strain of Streptomyces galilaeus (ATCC 31133), which is an aclocinomycin producing strain. , 2-hydroxyaclabinone and a number of anthracyclinones (US Pat. No. 4373094, Akihiro Yoshimoto et al ., Jpn. J. of Antibiotics 44. 277-286 (1991)), Lack of aclabinone C-11 hydroxylase activity (Oki T. et al. , J. of Antibiotics 32, 801-819 (1979)).

As a method for transforming a host cell, a method known in the art may be used.For example, in the case of Streptomyces spp ., A method such as Hopwood et al. (Genetic Manipulations of Streptomyces : A labolatory manual.The John InnesFoundation , Norwich, United Kingdom, 1985).

The transformed host cell can express the gene by culturing in an appropriate culture condition. According to the application, an expression product enzyme (aclavinone C-11 hydroxylase) is isolated and purified from the host cell culture. Or may be directly involved in the biosynthesis of anthracycline antibiotics in cells. Specifically, in the present invention, a new anthracycline antibiotic which was not present in a wild-type host cell was produced from a microorganism which produced anthracycline antibiotic without showing the activity of aclabinone C-11 hydroxylase through the latter method. do.

In the present invention, as a preferred embodiment, the mutant Streptomyces galilaeus ( ATCC 31671) is transformed with a recombinant vector pMC213 containing an aclobinone C-11 hydroxylase gene and transformed to Streptomyces. The aclabinone C-11 hydroxylase gene is expressed in Galileos to produce a novel aclabinone derivative, 2,11-dihydroxyaclabinone. More specifically, by the enzyme, the C-11 position of 2-hydroxyaclavinone in Streptomyces galileus (ATCC 31671) is hydroxylated to produce 2,11-dihydroxyaclavinone.

The 2,11-dihydroxyaclabinone produced by the above method was extracted with chloroform of mycelia of cultured transformant, passed through a silica gel column, and the filtrate was methanol: water: acetic acid = 65: 35: 5. Separation and purification can be accomplished by performing high performance liquid chromatography (HPLC) steps using phosphorus developing solvents, the separated compounds being characterized by UV spectra, ¹ H-NMR, ¹³ C-NMR and ESI-MS spectra. As a result, it was confirmed that it is a 2,11-dihydroxyaclabinone, a novel anthracycline precursor having a hydroxy group introduced at the C-11 position of 2-hydroxyaclavinone (see FIG. 1 ).

Next, the isolated 2,11-dihydroxyaclabinone was added to the culture solution of Streptomyces fusetius NU23, a doxorubicin synthetic deficiency strain, and bioconverted to thereby convert a new anthracycline antibiotic, 2,11-dihydroxyanthra. Prepare cyclin.

It is expected that the anthracycline antibiotic prepared by the above method may have different properties from the previously discovered anthracycline antibiotics. In the present invention, the anthracycline antibiotic may be used as a precursor to the 2,11-dihydroxyaclavinone. Dihydroxyanthracycline, IDB-G4, was biosynthesized and confirmed to be cytotoxic to human cancer cells.

The antibiotic 2,11-dihydroxyanthracycline prepared by the present invention may be used in the form of a pharmaceutically acceptable salt, and salts include acid addition salts formed by pharmaceutically acceptable free acid. useful. Organic acids and inorganic acids may be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, tartaric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, Methanesulfonic acid, acetic acid, glycolic acid, lactic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid, aspartic acid, and the like can be used.

The present invention encompasses, in addition to non-toxic, inert, pharmaceutically suitable excipients, pharmaceutical compositions which contain one or more compounds according to the invention or which consist of one or more active compounds according to the invention and methods for preparing the compositions. The compounds of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.

In actual clinical administration, it can be administered in a variety of oral and parenteral formulations, which are formulated using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants.

The invention also includes formulations of dosage units. The formulations are present in individual dosage forms, such as tablets, coated tablets, capsules, pills, suppositories, and ampoules, wherein the amount of active compound in the drug corresponds to the fraction or multiple of the individual dosage. Dosage units may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. Individual dosages preferably contain an amount in which the active compound is administered at one time, which usually corresponds to a total, 1/2, 1/3 or 1/4 of the daily dose.

Pharmaceutically suitable excipients which are nontoxic and inert are solid, semisolid or liquid diluents, fillers and formulation aids of all types.

Preferred formulations include tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. In addition to liquid paraffin, various excipients may be included, such as wetting agents, sweetening agents, fragrances, preservatives and the like.

Tablets, coated tablets, capsules, pills, and granules are conventional excipients, such as (a) fillers and weighting agents (e.g. starch, lactose, sucrose, glucose, mannitol and silicic acid), (b ) Binders (e.g. carboxymethylcellulose, alginates, gelatin and polyvinylpyrrolidone), (c) hygroscopics (e.g. glycerol), (d) disintegrants (e.g. agar, calcium carbonate and sodium carbonate), (e) Dissolution retardants (e.g. paraffin), (f) absorption accelerators (e.g. quaternary ammonium compounds), (g) wetting agents (e.g. cetyl alcohol and glycerol monostearate), (h) adsorbents (e.g. kaolin and bentonite) And compounds (i) effective compounds other than lubricating agents (e.g. talc, calcium stearate, magnesium stearate, magnesium styrate talc and solid polyethylene glycols), or mixtures of the materials described in (a) to (i) above. It may contain these.

Tablets, coated tablets, capsules, pills, and granules may be provided with conventional coatings and sheaths, optionally containing milky agents, and also with compositions which preferentially release only the active compound or the active compound in specific areas of the intestine. It may be made and, if necessary, formulated in sustained release using a sealant composition such as a polymeric material or wax.

If desired, the active compounds can also be formulated in microcapsule form using one or more of these excipients.

Suppositories include, in addition to the active compounds, conventional water-soluble or water-insoluble excipients, for example fats such as polyethylene glycol, cacao fats, higher esters (e.g. C ₁₄ -alcohols with C ₁₆ -fatty acids), witepsol, macrogol , Tween 61, laurin butter and glycerol gelatin or mixtures of these substances.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.

Ointments, pastes, creams and gels, in addition to active compounds, include conventional excipients, such as animal and vegetable fats, wax paraffins, starches, targacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide Or mixtures of these materials.

Powders and sprays may contain, in addition to the active compound, conventional excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these materials. Sprays may further contain customary sanding agents, for example chlorofluorohydrocarbons.

Liquids and emulsions, in addition to the active compounds, include conventional excipients such as solvents, solubilizing agents and emulsifiers, such as water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, Of 1,3-butylene glycol, dimethylformamide, oils, especially cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol, glycerol form alcohol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan Fatty acid esters, or mixtures of these materials.

Solutions and emulsions for parenteral administration can be formulated in sterile form that is isotonic with blood.

Suspending agents, in addition to the active compounds, include conventional excipients, for example liquid diluents (such as water, ethyl alcohol and propylene glycol, polyethylene glycol), and suspending agents (such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan) Esters), microcrystalline cellulose, aluminum metahydroxy, bentonite, agar and injectable esters such as tragacanth, ethyloleate or mixtures of these materials.

The formulations may also contain colorants, preservatives and additives that improve odor or taste, such as peppermint oil, eucalyptus oil and sweeteners (eg saccharin).

The therapeutically effective compound should be contained in the pharmaceutical formulation in an amount of about 0.1 to 99.5% by weight, preferably 0.5 to 95% by weight of the total mixture.

The formulation may contain, in addition to the compound according to the invention, other pharmaceutically effective compounds.

The formulations are prepared in a known manner, for example in a conventional manner in which the active compound is mixed with excipients.

The formulations can be administered to humans and animals as oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), subretinal alveolar, intratracheal or vaginal topical (powders, ointments, drops). Possible suitable formulations are injection solutions, oral and suspension solutions, and gels, infusions, emulsions, ointments or drops. In addition to ophthalmic and dermatological solutions, ie silver salts and other salts, eardrops, eye ointments, powders or solutions may be used for topical treatment.

In general, in medicinal and veterinary medicines, it is advantageous to administer the active compounds according to the invention in a total amount of about 0.5 to about 500 mg / kg body weight, suitably 5 to 300 mg / kg body weight every 24 hours. It has been demonstrated that it is advantageous to administer in several individual dosage forms as necessary to obtain the desired result. Individual dosage forms suitably contain from about 1 to about 100 mg / kg body weight, in particular from 3 to 80 mg / kg body weight, of the active compounds according to the invention. However, the dosage may need to be changed, and in particular, may be changed in consideration of the constitution specificity and weight of the subject to be treated, the type and depth of the disease, the nature of the formulation, the nature of the drug administration, and the duration or interval of administration.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 Cloning and Recombinant Plasmid Construction of Aclabinone C-11 Hydroxylase Gene

Cloning of the aclabinone C-11 hydroxylase gene was carried out as follows.

Chromosomal DNA of Streptomyces fusettius (ATCC 27952) was isolated and partially digested with restriction enzyme Sau3AI, followed by centrifugation at a 10-40% sugar solution gradient to pIJ702 (KCTC 1167). Was cloned into BgI II site. These strains were transformed with Streptomyces lividans 1326 to screen for strains resistant to platelets containing thiostrepton (50 μg / ml), and then again containing adriamycin (100 μg / ml). Final selection in media. Plasmids isolated from these resistant strains were digested with restriction enzymes such as BamHI, SacI, KpnI, and PstI, containing fragments of the same size by each restriction enzyme and pMC1, pMC2, pMC3 with fragments inserted in the same direction. , pMC4 was found. Computer analysis of these sequences revealed that in addition to the two resistance genes (drrA, drrB), there are open reading frames encoding two different proteins. Among them, a high copy number of a DNA fragment containing the gene for expressing a gene containing an open reading frame dnrF upstream of the resistance gene, ie, aclabinone C-11 hydroxylase gene, in a large amount. It was introduced into the vector pIJ702 to construct a high expression recombinant plasmid vector. To this end, the first clone pMC1 containing the resistance gene was partially digested with BamHI and a 2.2 kb DNA fragment recovered by agarose gel electrophoresis was inserted into a Streptomyces-only vector pIJ702 digested with BalII and named as recombinant plasmid pMC213. The transformants containing the same were used for the biotransformation experiments.

Example 2 Transformation and Culture of Streptomyces Strain

Strains of Streptomyces spp. Were transformed with the recombinant plasmid pMC213 containing the aclabinone C-11 hydroxylase gene obtained in Example 1, and the transformants were cultured.

Protoplasts of Streptomyces lividans 1326 were prepared by slightly modifying the method of Hopwood et al. (Genetic Manipulation of Streptomyces , The John Innes Foundation, Norwich, England, 1985) and transformed by the following method.

Streptomyces strains were prepared using R2YE medium [103g of sugar, 0.25g of K ₂ SO ₄ , 10g of MgCl _{2 ·} 6H ₂ O, 0.1g of cassanoic acid, 5.0g of yeast extract, TES (N-tris [hydroxymethyl] methyl-2-aminoethane sulfonic acid) 5.73 g was dissolved in 790 ml of distilled water and sterilized, and 5 ml of 1M NaOH, 10 ml of 0.5% KH ₂ PO ₄ , 20 ml of 1M CaCl ₂ , 15 ml of 20% proline, trace element solution Used by adding 2 ml. The trace component solution used was ZnCl ₂ 40 mg, FeCl ₃ 6H ₂ O 200 mg, CuCl ₂ 2H ₂ O 10 mg, MnCl ₂ 4H ₂ O 10 mg, Na ₂ B ₄ O ₇ 10H ₂ O 10 mg, (NH ₄ ) Was prepared by dissolving 10 mg of ₆ Mo ₇ O ₂₄ 4H ₂ O in 1 L of distilled water. After inoculating in 10 ml and incubating for 2 to 3 days at 28 ° C., 100 ml of R2YE medium containing 0.5% glycine in a 500 ml flask was used. It was incubated at 28 ℃ for 1 ~ 2 days before the second metabolite production was re-inoculated.

Centrifuge the cells for 30 minutes at 3000 rmp and add 20 ml of P buffer containing Lysozyme (1 mg / ml) (Hopwood et al , Genetic Manipulation of Streptomyces.A Laborative Manual, John Innes Foundation, Norwich, UK) After 15-60 minutes at, the degree of protoplast formation was confirmed under the microscope. The cells without protoplasts were removed by filtration with a sterile cotton filter. The filtered cells were washed with P buffer, dissolved in P buffer, and then aliquoted at about ^{8-9 for} protoplasts and stored at -80 ° C. During transformation, the stored protoplasts were rapidly dissolved at 37 ° C. and 20 μl recombinant plasmid immediately after addition of 1 ml of T buffer (Hopwood et al., Genetic Manipulation of Streptomyces.A Laborative Manual, John Innes Foundation, Norwich, UK). DNA (1 μg) was mixed, and after 1 minute, washed with 5 ml of P buffer and dissolved in 1 ml of P buffer. After dispensing 330 μl each, 2.5 ml of R2YE medium added with 0.6% agar to each sample was mixed well, and then inoculated in R2YE regeneration medium and incubated. The culture conditions were different according to each strain used as a host cell, specifically as follows.

(1) Culture of Streptomyces galileus (ATCC 31671)

After inoculating and incubating the R2YE regeneration medium, Streptomyces galileus 31671 strains were YS solid medium [3 g yeast extract, 10 g soluble starch, 15 g agar, containing chistrepton (25 μg / ml), pH 7.2], the transformants were grown by incubation at 28 ℃ for 80 hours. The transformants were pre-incubated at 28 ° C. for 2 days in YS medium containing yeast streptone (25 μg / ml) (3 g of yeast extract, 10 g of soluble starch, pH 7.2), followed by intermediate culture in YS medium. SGM medium [15g soluble starch, glucose 10g, soy flour 30g, yeast extract 1g, NaCl 1g, MgSO ₄ 1g, KH ₂ PO ₄ 1g, CuSO ₄ 0.007 g, FeSO ₄ 0.001 g, MnCl ₂ 0.008 g, ZnSO ₄ 0.002 g] fermentation culture at 28 ℃ 7-10 days to analyze the metabolite.

(2) Culture of Streptomyces Lividans

Actinordin producing strain Streptomyces lividans ( KCTC 9059) was used as a transformation host for major cloning operations.

Streptomyces lividans inoculated in the R2YE regeneration medium was incubated at 30 ° C. for 18 to 24 hours, and then 3 ml of 0.6% soft R2YE agar medium containing chistrepton (25 μg / ml) was spread over each plate. . The transformants were grown by incubating at 28 ° C. for 3-5 days and analyzed.

Example 3 Extraction, Separation and Analysis of 2,11-Dihydroxyaclavinone

(1) Extraction and separation of 2,11-dihydroxyaclavinone

After centrifuging the Streptomyces galileus bacteria cultured in Example 2 (1) at 7000 rpm for 30 minutes, the mycelium was repeatedly extracted twice with acetone, concentrated and reextracted with chloroform. It was adsorbed on HP20 resin, eluted with acetone, concentrated and reextracted with chloroform. Silica gel column chromatography using chloroform: methanol = 20: 1 as the developing solvent was carried out to obtain a fraction, which was subjected to Sephadex LH20 gel filtration using methanol as a solvent. Preparative HPLC (YMC ODS-A column (10 × 290 mm)) was then carried out to obtain pure 2,11-dihydroxyaclabinone, with methanol: water: acetic acid = 65: 35: 5 as a developing solvent. Was used.

(2) Physicochemical Properties of 2,11-dihydroxyaclabinone

The metabolite isolated in (1) was identified by TLC and HPLC. In the case of TLC analysis (Silica gel 60 F-254 plate, Merck), the developing solvent was used by mixing 15: 1 chloroform: methanol. HPLC (ODS A-320 Column, YMC Co., Ltd., Japan, was used as a developing solvent using acetonitrile: 20 mM phosphoric acid solution (Acetonitrile: 20 mM phosphoric acid) as 42:58.

As a result, 2,11-dihydroxyaclabinone, a novel aclabinone derivative isolated in (1), had a melting point of 244 to 246 ° C as a red purple powder, and was dissolved in methanol and chloroform. In the UV spectrum (Milton Roy, solvent used, methanol), the aclabinone showed UV absorption at 230, 281, and 483 nm, whereas for 2,11-dihydroxyaclabinone, the hydroxy group was at position 11 carbon. Due to the introduction, a bathochromic effect showing absorption maximum at 230, 258, 281, and 491 nm was shown (see FIG. 2 ). In addition, the ¹ H-NMR spectrum (CDCl ₃ solvent, Varian UNITY-300 spectrometer, 300 MHz) showed the same pattern as 2-hydroxyaclavinone, but as shown in Table 1 , the 7.65 ppm singlet corresponding to H-11 was lost. It can be seen that the position of the hydroxylation is the carbon position 11 (see FIG. 3 ), and also 154.3 ppm of carbon 11 in the ¹³ C-NMR spectrum (CDCl ₃ solvent, Virian UNITY-300 spectrometer, 75.4 MHz) It was confirmed that the carbon at position 11 was hydroxyated from the low-length shift to (see FIG. 4 ). Molecular ion peaks were also observed at m / z 443 (MH-) in ESI-MS (Platform (VG, UK)), and the molecular weight of the calculated 2,11-dihydroxyaclabinone plus 32 mass units was added to the aclabinone. It was confirmed that a molecular weight of 444, which is a corresponding value, was obtained (see FIG. 5 ).

NMR Chemical Arrangement of 2,11-Dihydroxyaclabinone Proton chemical shift (ppm) carbon chemical shift (ppm) One H 6.95 d One CH 104.6 3 H 6.97 d 2 C 155.3 7 H 5.09 br d 3 CH 111.9 8 H 2.10 br d 4 C 166.5 8 Ha 2.02 d 4a C 109.3 10 H 4.10 s 5 C 188.3 12 Ha 1.65 m 5a C 112.0 12 Ha 1.43 m 6 C 154.3 OCH ₃ 3.57 s 6a C 134.3 7 CH 61.6 8 CH ₂ 34.5 9 C 71.4 10 CH 52.2 10a C 137.1 11 C 154.3 11a C 115.4 12 C 185.5 12a C 130.5 13 CH ₂ 32.4 14 CH ₂ 5.6 15 C 171.3 16 OCH ₃ 51.17

Example 4 Bioconversion of 2,11-dihydroxyaclabinone

Using 2,11-dihydroxyaclabinone isolated in Example 3 as a precursor, a new backbone anthracycline was prepared by the method of biotransformation in Streptomyces fusettius NU23 strain. Streptomyces fusettius NU23 used is a biosynthetic deficient variant of the doxorubicin producing strain Streptomyces peucetius supsp.caesius (ATCC 27952) , which exhibits a doxorubicin synthetic deficiency.

Specifically, the bioconversion of 2,11-dihydroxyaclabinone was carried out by the following method.

Streptomyces fusettius NU23 strain used as a biotransformation host is composed of NDYE solid medium (45 g malt per sugar, 4.3 g NaNO ₃ , 0.23 g K ₂ HPO ₄ , 5.2 g HEPES buffer, 0.12 g MgSO ₄ , and other minor component solutions 2 ㎖, agar 15 g, pH 7.4) and incubated at 28 ℃ for 4 days, inoculated in NDYE liquid medium and incubated for 5 days at a shaking speed of 250 rpm at 28 ℃ 2, so that the final concentration is 30 mg per liter of culture medium , 11-dihydroxyaclabinone was added. Thereafter, the culture was further incubated for 4 days under the same conditions to bioconvert 2,11-dihydroxyaclabinone.

Example 5 Isolation and Analysis of Bioconversion Substances of 2,11-Dihydroxyaclabinone

(1) Isolation of 2,11-dihydroxyaclavinone bioconversion material

After centrifugation of the bioconversion terminated culture medium at 7000 rpm for 30 minutes, the cells were extracted with methanol and acetone, the culture filtrate was extracted with a solvent mixed with chloroform and methanol 9: 1, HP20 adsorption resin, and concentrated. Chromatography was carried out with chloroform and methanol as a developing solvent from 20: 1 to 2: 1 to recover the IDB-G fraction.

Four glycosides such as IDB-G1, G2, G3, and G4 were obtained by performing HPLC (YMC ODS-A column) on glycosides (IDB-G) identified by thin layer chromatography (TLC). It was confirmed that there is.

To analyze the bioconversion material, oxalic acid was added to the culture medium incubated for 7-8 days at 28 ° C. under aerobic conditions (shaking culture 250-300 rpm) in a production medium, at a concentration of 30 mg / ml. Hydrolysis at 55 ° C. for 45 minutes and adjusted to pH 8.5 using 10N NaOH. An equal amount of a chloroform: methanol (9: 1) mixed solution was added to the reaction mixture, mixed well, the metabolite was extracted with a solvent layer, centrifuged at 3,000 rpm for 30 minutes, the solvent layer was recovered, and concentrated under reduced pressure. It was dissolved in methanol and used for TLC or HPLC analysis. For TLC analysis (Silica gel 60 F-254 plate, Merck), the developing solvent for biosynthetic intermediates in the form of glycosides is 80: 20: 2 in chloroform: methanol: formic acid. As a mixed solvent, a developing solvent of aglycone, a sugar-free intermediate, was used in which hexane: chloroform: methanol was mixed at 5: 5: 1. HPLC (ODS-120T C-18 column, Tosoh Co., Ltd.) was used as a developing solvent in which SDS was added to the final 10 mM concentration in a mixed solvent of acetonitrile: 20 mM phosphate solution (42:58).

IDB-G4, considered dual final metabolite, was isolated by preparative HPLC.

(2) Physicochemical Properties of 2,11-Dihydroxyanthracycline IDB-G4

The antibiotic 2,11-dihydroxyanthracycline IDB-G4 isolated in (1) is a red powder, and doxorubicin shows UV absorption maximum at 230, 254 and 495 nm in the UV spectrum measured by dissolving in methanol. In the case of 11-dihydroxyanthracycline IDB-G4, the absorption peak was exhibited at 230, 281, and 491 nm due to the introduction of a hydroxyl group at the carbon position 2 (see FIG. 6 ). In addition, the ¹ H-NMR spectrum (CDCl ₃ solvent, Varian UNITY-300 spectrometer, 300 MHz) shows that in the case of doxorubicin, the proton located at carbon 2 appears as a triplet at 7.5-7.9 ppm and is located at carbon 1 and 3 Proton appears as a doublet at 7.6 ppm, whereas in the case of IDB-G4 the triplet located at carbon 2 is lost and the protons of carbons 1 and 3 are changed from 6.5-7.1 ppm to a doublet, indicating that the carbon 2 position is It can be seen that it is oxylated (see FIG. 7 ). In addition, under the conditions of chloroform: methanol: formic acid = 80: 20: 2, TB development solvent of doxorubicin, IDB-G4 showed lower R _f value than doxorubicin due to the introduction of hydroxy group, In hexane: chloroform: methanol = 5: 5: 1, it showed a lower R _f value than doxorubicinone, indicating that IDB-G4 was a glycoside. Molecular ion peaks were also observed in FAB-MS (JEOL HX-110) at m / z 560 (M + H +), corresponding to the addition of doxorubicin by 16 mass units by hydroxylation (see FIG. 8 ). Therefore, it can be seen from the above results that IDB-G4 is 2-hydroxy doxorubicin, a 2,11-dihydroxyanthracycline, which is an anthracycline having a structure similar to doxorubicin. .

Experimental Example 1 Anticancer Activity of the Anthracycline Compound

Anticancer activity was measured by the following method using a human cancer cell line panel reduced for the anticancer agent 2,11-dihydroxyanthracycline IDB-G4 obtained in Example 5.

Mouse and human-derived cancer cell lines were cultured in a 37 ° C., 5% CO ₂ incubator with RPMI 1640 medium supplemented with 10% Fetal Calf Serum. Cancer cell lines used were human-derived leukemia cell lines such as P388, P388 / ADM, and L1210, and human-derived cell lines such as gastric cancer cell lines SNU-1, SNU-1 / ADM, colon cancer cell line SNU-C4, and leukemia cell line HL60. Cytotoxicity of -dihydroxyanthracycline IDB-G4 was measured by the sulforhodamine B method. Cells in logarithmic growth phase were treated with trypsin-EDTA to make single cell suspensions, diluted to 5-8 × 10 ⁴ / ml concentrations, and 100 μl aliquots in 96 well plates and incubated for 24 hours. 100 μl was added to the medium by diluting step by step. After 48 hours of incubation, 50 μl of cold 50% trichloroacetic acid solution was added to the culture solution to fix the viability of the cells, and the cells were fixed at 4 ° C. for 1 hour. The fixed cells were washed 5 times with water, stained for 30 minutes with 100 µl of 0.4% sulforhodamine B (dissolved in 1% acetic acid), washed several times with 1% acetic acid solution, dried, and then washed with 10 mM Tris solution (pH). 10.5) 200 μl was added and the absorbance at 540 nm was measured in an ELISA reader to determine the cytotoxicity from the comparison with the control group.

The results of the experiment are shown in Table 2 .

In Vitro Cytotoxicity of IDB-G4 Against Cancer Cell Lines Cell line Doxorubicin IDB-G4 LeukemiaP388 0.02 0.80 Leukemia (Multi-drug resistance) P388 / ADM 1.00 10.20 Leukemia L1210 0.02 0.30 Leukemia (Multi-drug resistance) L1210 / MDR 1.50 25.00 Stomach CancerSNU-1 0.20 1.30 Stomach Cancer (Multi-drug Resistance) SNU-1 / ADM 10.00 25.00 Colon cancerSNU-C4 1.20 12.50 LeukemiaHL60 0.20 1.40 Leukemia (Multi-drug resistance) HL60 / ADM 10.40 25.00 Unit IC ₅₀ : μM

In vitro cytotoxicity of the mouse-derived leukemia cell lines, P388 and L1210, as a control drug as doxorubicin, was examined in both normal cancer cell lines and multidrug-resistant cell lines. Anthracycline showed 10-20 times lower cytotoxicity than doxorubicin. This result shows that the 2-hydroxyanthracycline-based material exhibits lower cytotoxicity than the 2-anhydroanthracycline-based material. The result is that Akihiro et al. (Japanese Patent No. 125826/81) have 2-hydroxyaclacinomycin. It is consistent with the report that the cytotoxicity of A shows 4 to 8 times lower toxicity than that of aclocinomycin A.

Secondly, cytotoxicity was measured using human-derived gastric cancer cell lines SNU-1 and SNU-1 / ADM (multi-drug resistant cell line), leukemia cell line HL60, and colon cancer cell lines SNU-C4 and SNU-C4 / ADM. As a result, attenuation of cytotoxicity was observed in the same trend as that of mouse-derived cell lines.

Experimental Example 2 Acute Toxicity in Mice

On the other hand, the following experiment was conducted to determine the acute toxicity of 2,11-dihydroxyanthracycline of the present invention.

Acute toxicity test was performed using 6 weeks old SPF SD mice. Two animals per group were dissolved in physiological saline containing 0.1% Tween 80 and 2,11-dihydroxyanthracycline, respectively, followed by single intravenous administration at a dose of 800 mg / kg / 0.5 ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal organs, thoracic organs, and kidneys. As a result, all animals treated with test substance showed no clinical symptoms and no dead animals, and no toxic changes were observed in weight change, blood test, blood biochemical test, autopsy findings. As a result, all of the tested compounds did not show toxic changes up to 800 mg / kg in mice, and the minimum intravenous dose was 800 mg / kg or more.

The novel aclabinone derivatives of the present invention, 2,11-dihydroxyaclabinone, can be used for the preparation of various novel anthracycline antibiotics through bioconversion processes, and the anthracycline system prepared using the compound as a precursor. Antibiotic 2,11-dihydroxyanthracycline is less toxic to cells and can be used as a safer and more effective anticancer agent than conventional anticancer agents.

Claims (7)

2,11-dihydroxyaklavinone of the formula (1). Formula 1 Streptomyces transformed with recombinant plasmid pMC213 containing a gene encoding aclabinone C-11 hydroxylase, and producing 2,11-dihydroxyaclabinone using 2-hydroxyaclabinone Ses galileus ( Streptomyces galilaeus ATCC 31671 / pMC213 , Accession No .: KCTC 0662BP). The method of producing a compound of claim 1, which is prepared by culturing the transformant of claim 2. A method for producing an anthracycline antibiotic by a bioconversion method using a biosynthetic deficient variant using the 2,11-dihydroxyaclabinone of claim 1 as an aglycone. The method for producing an anthracycline antibiotic according to claim 4, wherein the biosynthetic deficiency mutant is Streptomyces fusetius NU23. 5. The method of claim 4, wherein the anthracycline antibiotic is 2,11-dihydroxyanthracycline. 6. 2,11-dihydroxyanthracene prepared by the method of claim 4 clean.