DD220046A1 - PROCESS FOR THE PREPARATION OF BETA-RHODOMYCIN II AND ITS AGLYKONS - Google Patents

Abstract

The invention relates to a process for the preparation of an anthracycline antibiotic and its aglycone, which are of potential use for the chemotherapy of tumor, virus and bacterial diseases in humans and livestock. In particular, the invention relates to a process for the preparation of the hitherto unavailable antibiotic beta-rhodomycin II, its salts and its hydrolytic degradation products using a novel microorganism obtained after interspecific recombination and as Streptomyces violaceus subsp. iremyceticus, strain ZIMET 43,687. The aim of the invention is to obtain an anthracycline antibiotic with potential cancerostatic properties and its hydrolytic degradation products as starting substrates for mutase synthesis and semisynthesis to expand the range of such drugs. The object is achieved by beta-rhodomycin II formed in media with suitable C, N sources and mineral salts by aerobic submerged fermentation of the new microorganism ZIMET 43 687, isolated by suitable methods from mycelium and culture filtrate and subsequently purified and optionally hydrolyzed.

Description

Field of application of the invention

The invention relates to a process for the preparation of an antibiotically active substance and its derivatives, which are of potential use in the chemotherapy of tumor, virus and bacterial diseases in humans and livestock. In particular, the invention relates to the recovery of a novel anthracycline antibiotic called beta-rhodomycin II, its salts, and its aglycone.

Characteristic of the known technical solution,

Anthracycline, one or more sugar residues containing glycosides of anthracyclinones, are antibiotically active compounds. The biologically inactive aglycone of anthracycline antibiotics (= anthracyciinones), which is derived from their carbon skeleton as derivatives'7,8,9,10-tetrahydro Depending on the number and position of the hydroxyl groups on the anthraquinone ring system, they can be subdivided into different groups with characteristic electron absorption spectra (Brockmann, H., 1963. Fortschr. Chem. Org. Naturstoffe , 21,121). More than 100 anthracyclines are known to date, the majority of which are mixtures of hard to separate individual components. ,

Two anthracycline antibiotics (doxorubicin - US patent 3590028; daunorubicin GB patent 1003383) are in clinical use because of their chemotherapeutic efficacy against acute leukemias and solid human tumors. A major problem in the cancer chemotherapeutic application of anthracycline antibiotics results from their general, hematological, digestive and cardiac toxicity, which restricts more widespread use in tumor chemotherapy and, for. B. in cardiac patients leads to contraindication. Since the cardiac toxicity of anthracyclines has proved to be particularly disturbing, new derivatives of already known compounds are constantly being prepared and investigated with regard to reduced toxicity. A disadvantage here is that chemical methods such as total and partial synthesis and biochemical methods for enzymatic conversion are available to obtain chemically rriodifizierter anthracycline antibiotics, but their application leads to an insufficient economy of product production, which does not allow large-scale extraction of these derivatives. In the hitherto known microbiological production process for anthracycline antibiotics preferably mixtures of chemically similar anthracyclines are obtained which consist of the mono-, di-, tri- or polysaccharides of different and under industrial conditions practically indivisible anthracyclinones. This entails standardization problems for the end product if, depending on the fermentation process, qualitative and quantitative shifts occur in the multicomponent mixture. Anthracyclines whose aglycones can be assigned to beta-rhodomycinone have hitherto been obtained with the help of the species Streptomyces purpurascens (Brockmann, Waehneldt and Niemeyer, 1969. Tetrahedron Lett., (6), 415; Brockmann, Scheffer and Stein, 1973. Tetrahedron Lett. , (38), 3699, Brockmann and Greve, 1975. Tetrahedron Lett., (11), 831), Streptomvces bobiliae (Biedermann and Bräuninger, 1972. Pharmacy, 27.782), Streptomyces griseoruber 4620 (Podojil, Blumauerovä, Prikrylova, Vanek, Gauze and Maksimova, 1980. Folia Microbio!, 25, 464), Streptomyces violaceus (Fleck, Strauss, Koch, Kramer and Prauser, 1974., Z. Allig. Mikrobiol., 14, 551), Streptomyces roseovilaceus A 529 (IFO 13081 Matsuzawa, Yoshimoto, Oki, Inui, Takeuchi and Umezawa, 1979. J. Antib., 32, 420) by fermentative route as well as by biosynthesis and glycosidation of beta-rhodomycinone using an aclacinomycin-deficient mutant (Oki, Yoshimoto and Matsuzawa, 1980. J. Antib. 33,1331) as mixtures or as minor components in very g Eringen quantities with unknown chemotherapeutic activity have been obtained. Detailed information on the physicochemical properties of beta-rhodomycins as well as their biological effects such as antimicrobial, antiviral, cytostatic and antitumor activity are only available from beta-rhodomycin I. This beta-rhodomycinone monoglycoside was obtained by fermentation as Haubtkomponente from a mutant of Streptomyces violaceus subsp. iremyceticus ZlMET 43678 (Him, Schlegel, Fleck, Tresselt, Gutsche, Sedmera and Vokoun, 1983. Pharmacy, in press) and isolated as a pure Einzelkomponehte. ,

Object of the invention

The invention serves to produce a novel anthracycline antibiotic and its aglycone as well as the salts of the antibiotic to avoid the disadvantages of previously known methods and the range of potentially cancerostatic, virostatic and antimicrobial anthracyclines by a representative, which is a single component to and to use the hydrolytic degradation products as a starting substrate for the mutasynthesis and semisynthesis of new anthracycline antibiotics.

Explanation of the essence of the invention

The invention has for its object to provide a technologically simple method that allows to produce the anthracycline antibiotic beta-Rhodomycin II using a microorganism of readily available and inexpensive starting materials in good yields as a single component. According to the invention, the object is achieved by cultivating a new microorganism or its variants under aerobic and sterile culture conditions in a liquid nutrient medium with: corresponding carbon dioxide, nitrogen sources and mineral salts. The new microorganism used in this process was prepared by interspecific hybridization of genetically-labeled and secondary metabolically-blocked mutants of the violamycirc-former, Streptomyces violaceus. Stamm IMET JA 6844 (described in the GDR patent 100494) and the turimycin former, Streptomyces hygroscopicus, strain IMET

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Jena-GDR, deposited under the number ZIMET 43687. In terms of its morphological properties, the beta-rhodomycin II former ZIMET 43687 corresponds to the strain ZIMET 43615, which is described in the GDR patent 140672 and by Schlegel and Fleck 1980 (Z. AiIg. Mikrobiol., 20, 527-530) as Streptomyces violaceus sufasp. iremyceticus has been described. According to this, the beta-rhodomycin II-formner ZIMET 43687 belongs to the genus Streptomyces and corresponds to the diagnosis of Streptomyces violaceus (Rossi-Doria 189.1, Krassilnikov 1941, Waksmart 1953), as described after examination of the Neo-Type strain ISP 5082 (= INMI 1, RIA 656, ATCC 15888) in the "International Streptomyces Project" by Shiriing and Gottlieb (Int Jystyst Bact., 19, 49797/1969) The beta-rhodomycin II former ZlMET 43687 is produced by selection processes Population of the interspecific recombinant ZIMET 43615 and differs from the starting recombinant in that other anthracycline antibiotics are formed., While the initial recombinant ZlMET 43 615 the anthracycline antibiotic Iremycin ([10-aL-modosaminyl] -y-rhodomycinon; DD 140672), the new anthracycline antibiotic beta-rhodomycin II is synthesized by the mutant ZlMET 43687. The cultivation of the strain ZIMET 43687 and its variants takes place under aerobic conditions. Sporulated or lyophilically dried spore material or mycelial fragments are inoculated into suitable agar media and subsequently into liquid, previously sterilized nutrient media and the resulting mycelium is in a conventional manner at a temperature between 25 ° C and 37 ⁰ C, preferably 28 ° C, over a Period of 2 to 6 days, preferably 4 days, cultured at a Äquidität which is at the beginning of the fermentation process between pH 6.2 to pH 7.0 and at the end of the process between pH 7.5 and pH 8.3. The nutrient medium consists of carbon and nitrogen sources as well as inorganic salts. As _Λ .

Carbon sources can be starch, glucose, glycerin, mannitol, dextrin, sucrose, soybean oil and soybean meal. Nitrogen sources other than the above-mentioned nitrogenous substrates include dry yeast, meat peptone and casein. Good results can be achieved with the addition of mineral salts. The latter favor the course of the fermentation depending on the nutrient medium used. In complex media containing various flours, additions of calcium carbonate, sodium phosphate or potassium phosphate have proven to be beneficial. The fermentation of the image generator ZIMET 43687 can be carried out in steep-breasted bottles and round-bottomed flasks of various contents, in a glass fermenter and in V2 A steel tanks. The isolation of the beta-Rhodomycin II is carried out in such a way that the antibiotic from the culture filtrate with organic, water-immiscible or only slightly miscible, and from the Mycsl with organic, water-miscible and then water-immiscible or only slightly miscible solvents extracted, after extracting the extracts with a water-immiscible solvent ', dried over anhydrous sodium sulfate, the solvent was removed by distillation in vacuo, precipitated by adding petroleum ether with stirring, the solvent removed by filtration, washed with petroleum ether, then dried , then dissolved in lower alcohols, the solvent is removed by distillation in vacuo, the dried residue is treated with water and extracted at pH 8.0 with chloroform, the chloroform phase separated, dried over anhydrous sodium sulfate, brought to dryness in vacuo and on y the usual way is purified by column chromatography on silica gel.

Beta-Rhodomycin II crystallizes from chloroform / methanol in red-brown crystals, which dissolve well in chloroform, moderately in lower alcohols, and almost not in water and petroleum ether. The basic antibiotic has indicator properties and is red-yellow in acidic solutions, in alkaline blue-violet. Beta-Rhodomycin II forms in the reaction with the equivalent amount of methanolic hydrochloric acid in ether, a red Hydrochiorid of melting point 190/193 ° C. The hydrochloride is in contrast to the basic antibiotic well soluble in water and lower alcohols, but almost insoluble in chloroform and ether. Both the free base and the anthracycline hydrochloride represent according to their thin-layer chromatographic behavior on silica gel plates in different flow systems and at different pH values defined, chemically pure individual components.

Beta-Rhodornycin II forms a yellow pentaacetate C ₄₆ H ₅₈ Oi ₇ N ₂ (M ⁺ 910, mass spectrum) upon acetylation with acetic anhydride in pyridine. Upon hydrolysis with dilute acid in the heat, the anthracycline provides one mole of beta-rhodomycinone and two moles of rhodosamine. The identification of the aglycone as beta-rhodomycinone (Brockmann and Franck, 1955. Chem. Ber., 88, 1792) and that of the sugar component as L-rhodosamine (Brockmann, Spohler and Waehneldt, 1963. Chem. Ber., 96, 2925) was confirmed by comparative thin-layer chromatographic and various spectroscopic (MS, UV / VIS, IR and ¹ H NMR) studies. ,

The preparative electrochemical reduction of beta-rhodomycin II on a large-area mercury electrode results in reductive cleavage of the glycoside bond at the C-7 atom to the anthracycline antibiotic iremycin (Ihn, Schlegel, Fleck and Sedmera, 1980. J. Antibiotics, 33, 1457). , ,.

The structure of the antibiotic beta-rhodomycin II shown in Fig. 1 was determined by the chemical degradation and derivatization reactions as well as extensive spectroscopic studies on the intact antibiotic molecule. In our opinion, beta-rhodomycin II is identical to the rhodomycin A described in the literature (Brockmann and Patt, 1955. Chem. Ber., 88, 1455), although the empirical formula for rhodomycin A from elementary analysis is erroneous and physicochemical data only incomplete available. Important physicochemical parameters of beta-rhodomycin II are: '. ''; UV / VIS λ max (hydrochloride in methanol) nm: 235, 253, 294, 494

^IR max ^Cm " ^{1: 1595} i ^H - ^C0 :.

MS m / z (acetate): M ⁺ 910,476 (bisanhydroglycol triacetate), 434 (bisanhydroglycone diacetate), 392 (bisanhydroglycol diacetate)

Monoacetate), 350 (bisanhydroglycone, base peak) 200 (sugar acetate-OH), 158 (sugar-OH) ¹ H-NMR {pyridine-D _s ) ^:

1-H:. 7.86 Q, J ₁₂ = 7.5 Hz

2-H:: - 7.65T J ₁ ^ ₃ = 1.2 Hz

3-H: - 7,36Q '

7-H: '.. / _ _ _ 5,22u (X) 8a-H :). ^ ca. "2 ^ 3Ö - 2,60 (A) 8b-H :)., (B)"

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10-H:

CH ₃ VOnC ₂ H ₆ :

CH ₂ VOnC ₂ H ₅ :

2'-CH ₂ ; 2 "-CH ₂ : 3'-H;3" -H: 3'-N (CH ₃ J ₂ ; 3 "-N (CH ₃ );,: 4'-H;4" -H: 5'- H, 5 "-H: 5'-CH _3; 5" -CH _3:

Abbreviations: S = singlet, T = triplet, Q = quartet, Μ = multiple, u = signal unresolved.

Both fresh fermentation solutions of the beta-rhodomycin H-generator, 2IMET 43687, and the beta-rhodomycin II isolated from them possess antibiotic activity. Beta-Rhodomycin II preferentially inhibits the growth of Gram-positive bacteria and mycobacteria. In contrast, beta-rhodomycin II does not exhibit growth-inhibiting activity against representatives of gram-negative bacteria, yeasts or filamentous fungi. In determining the antibacterial efficacy of beta-rhodomycin II compared to the corresponding efficacy of the anthracycline antibiotic doxorubicin clinically used as a cancer chemotherapeutic agent and the known anthracycline Antibiotics iremycin and beta-rhodomycin I gave the following differences in activity under standardized conditions against the spore-forming agent Bacillus subtilis SG 119: anthracycline antibiotics activity (%)

5.49 p UCH ₃ -CH ₂ = 7,0 HZ J5-H, 5-CH ₃ = 6'5 HZ 1.35T Js-hu-ch, - 6/5 Hz ca.2,15M Σ J about 8 Hz 5,77 u (Signal superposition from 1'-H and 1 - "- H) about 2.0-2.60 M 3,26 m 3,40 M ' 2.58 s 2,71 p 4,22 approx. S (u) ι " 4,37 approx. S (u) 4.26 approx. Q 4,59 approx. Q 1,55 D 1,62D

Iremycin 100 Beta-Rhodomycin II 766 Beta-Rhodomycin I 322 doxorubicin 738

Interestingly, the significant increase in antimicrobial activity of the beta-rhodomycin derivative according to the invention, which is evidently caused by the second rhodosaminyl residue bound to the aglycone, leads to the corresponding activity of the known beta-rhodomycin I.

Compared to the cytostatic and cancerostatic anthracycline antibiotics daunorubicin and doxorubicin, beta-rhodomycin Il hydrochloride shows qualitatively comparable effects on the interaction with DNA in vitro and DNA metabolism in the cell, which i.a. be made responsible for the cytostatic effect of anthracycline antibiotics in microorganisms and mammalian cells and for the antitumor activity in animal experiments. Beta-Rhodomycin II also selectively suppresses the replication of DNA viruses in Gram-negative bacteria (e.g., temperate lambda phage in Escherichia coli C 600) at concentrations that do not simultaneously affect the metabolism and proliferation of host cells.

Beta-Rhodomycin II may be considered as a hitherto unavailable anthracycline antibiotic whose biological activity enables its potential application as a chemotherapeutic agent against tumor, virus and bacterial diseases in humans and livestock.

embodiments

The following examples serve to illustrate the invention without limiting it thereto. '.

  1. Two 500 ml breast bottles each contain 80 ml of the following pre-breeding medium:

Glucose '1.5%

Soybean meal 1.5%

Na chloride. '0.5%. ,

Ca carbonate .0.1%

primary K-phosphate 0.03%,

in tap water

Sterilization: 35min. at 1 ° C. Acidity after sterilization is at pH 6.3. Each steep-vial is inoculated with a spore or mycelial suspension of beta rhodomycin II builder ZIMET 43687 which is made up with sterile, isotonic saline from a 10 to 14 day culture of the beta-rhodomycin U-builder grown in a test tube on the following culture medium becomes: . , ''

Oatmeal 1.0%

Oatmeal, ground 1.0%

Agar-agar 2.0%

in tap water -

Sterilization: 35min. at 120 ° C, natural. '. ,.

The inoculated pre-cultivation cultures are 48h. incubated at 28 ° C on a shaker with a frequency of 180 U / min. 8 ml of a pre-culture so incubated are used to inoculate ÖOOVnl-Steiibrustflaschen, each containing 80ml of the following production medium:.

Glucose 3.0%:

Soybean flour 1.0% ·

Na chloride -. 0.5%

Ca-carbonate 0.3%. ",

in tap water

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2. The procedure is as in Example 1 with the difference that the production medium has the following composition:

Glucose 3.0%

Soya meal 3.0% ·. ',,

Na chloride '' 0.3%

Ca carbonate 0.3%,

in tap water sterilization: 35min. at 115 ^° C. The acidity after sterilization is at pH 6.2. , :

3. A culture of the beta-Rhodomycin II-formner from a solid medium, as described in Example 1, is used to inoculate 80 ml of the liquid pre-breeding medium mentioned in Example 1, which is contained in a SOQ ml steep-breast bottle. It incubates 48h. at 28 ° C on a shaker with a shaking frequency of 180U / min. 12 ml of the culture broth thus obtained are used to inoculate 400 ml of the same pre-growing medium, which is contained in a 2-liter glass cups. You incubate another 24 hours. at 28 ° C at a shaking frequency of 180 U / min, before the thus obtained 400ml culture broth for inoculation of 20I of the production medium described in Example 1 serve. The latter is contained in a 32 l glass fermenter. During the 96-hour fermentation, which is carried out at a stirring speed of 400 rpm and with an air supply of 15 l / min, foaming can be controlled by adding small amounts of sunflower oil or other silicone-containing antifoaming agents. 200 l of culture solution thus obtained are adjusted to pH 4.0 with hydrochloric acid and, after separation of the mycelium, 19.0 kg of mycelium and 150 l of potassium fluoride are obtained. receive. Thereafter, the 1501 culture filtrate are adjusted to pH 7) with sodium hydroxide solution and stirred with 100 l of butanol. The butanol extract thus obtained is then acidified to the color change with hydrochloric acid and in vacuo to V25 of the initial volume

concentrated. , .

From 19.0 kg of mycelium with 7Ol methanol, a first and second extract are prepared, with 300 ml of hydrochloric acid (diluted 1: 1 diluted with water) before the second extraction to the methanol. The resulting methanol extracts are filtered, adjusted to pH 6.0 with sodium hydroxide solution, combined and concentrated in vacuo. The aqueous methanolic extract-co-concentrate of the mycelium is exhaustively extracted at pH 7 to pH 9 with butanol, wherein 51 of the mycelial extract concentrate 51 water and 51 butanol are used. After separating the butanol phase containing the pigments, the latter is re-acidified with hydrochloric acid, followed by concentration, combined with the concentrate of the butanol extract of the culture filtrate, precipitated with petroleum ether, washed and concentrated in vacuo. Dried sulfuric acid. The yield of crude product is

  50g. , .. '.'. i; - ._

4. 10 g of crude product are taken up in 50 ml of methanol, treated with 11 water, adjusted to pH 7.9 with NaHCO ₃ and extracted exhaustively (3 times) with chloroform. The chloroform phase is separated, washed with water, dried over anhydrous sodium sulfate, filtered off and concentrated under reduced pressure at 20 ⁰ C to dryness. This gives 2.4 g of anthracycline crude product. To separate the beta-rhodomycin II, the crude product is taken up in chloroform and 2mai on silica gel (NaHCO ₃ -buffered - 0.05-43.2 mm) with CHCl ₃ -CHCl ₃ / acetone = 80:20 CHCVAcetoniMethanol = 80:20: 6 and then on silica gel (0.05-0.2) with CHCl ₃ : methanol: H ₂ O = 70: 20: 2 / chromatographed and isolated from chloroform / petroleum ether in pure form. The yield is 205 mg beta-rhodomycin IL This corresponds to a yield of 5.1 g of pure beta-rhodomycin II per m ³ fermentation liquid.

5. 100 mg Beta-Rhodomycin II are dissolved in 10 ml of 0.1 N hydrochloric acid and heated to 95 ° C for 70 minutes. The thereby precipitating beta-rhodomycinone is filtered off, washed several times with water and recrystallized from glacial acetic acid. Yield: 40mg red needles of melting point 223-226 ° C.

Claims (1)

-1 _T 255 894 5 Claim:; Process for the preparation of beta-rhodornycin II and its aglycone, characterized in that a new microorganism, Streptomyces violaceus subsp. iremyceticus, strain ZIMET 43687, under aerobic conditions in liquid nutrient media containing a carbon and nitrogen source and mineral salts, cultivated at a temperature between 25 ⁰ C and 37 ⁰ C for a period of 2 days to 6 days and the anthracycline formed Antibiotic beta-rhodomycin II extracted from both the mycelium and from the culture filtrate at an acidity between pH 3 and pH 9 using conventional solvents, subsequently concentrated by methods known per se, precipitated and chromatographically purified or with inorganic or organic acids in its salts converted or converted into its Aglykön by acid hydrolysis. '*' See 1 page illustration