DE3404470C2 - - Google Patents

Description

The invention relates to an anthracyclinone glucuronide, its Derivatives, a process for its preparation and a medicinal product. It has been found that anthracyclinone glucuronide, which is hereinafter referred to as "FCE 22724", is effective is to stop the growth of malignant tumors, such as P-388 leukemia, inhibit in experimental animals.

The invention relates to an anthracyclinone glucuronide Antitumor effect of the general formula A

The invention further relates to a method of manufacture an anthracyclinone glucuronide, wherein R is a sodium atom means that is characterized in that in itself known microorganisms of the strain Streptomyces peucetius var. vinaceus (DSM 2597) in aerobic conditions in an assimilable source of carbon and nitrogen and aqueous culture medium containing mineral salts be, the breeding for 3 to 7 days in egg  ner temperature of 25 to 37 ° C and a pH that at the beginning in the range of 6.5 to 7.0 and towards the end of breeding is in the range of 6.5 to 8.0, the mycelium the mycelium is removed from the culture medium extracted with a water-miscible organic solvent the extracts are concentrated under reduced pressure be, the crude anthracyclinone glucuronide of the general Formula A, wherein R represents a sodium atom, obtained is and for cleaning a concentrated aqueous solution the crude compound on a hydrophobic cross-linked, polymeric divinylbenzene resin is absorbed with an aqueous low alcohol is eluted, the pH of the eluate to release the anthracyclinone glucuronide of the general Formula A, wherein R represents a hydrogen atom, to 4.0 the eluate is extracted with methyl isobutyl ketone, the anthracyclinone glucuronide in pure form by adding precipitated n-hexane and it into the pure sodium salt by treatment with one equivalent of methanolic sodium hydroxide is converted again and then with diethyl ether is canceled.

It is preferred to use 10 vol% as the aqueous lower alcohol Use n-propanol in water.

The invention also relates to a pharmaceutical preparation, which is a therapeutically effective amount of an anthracyclinone glucuronide, mixed with a pharmaceutically acceptable Contains diluents and / or carriers.

Finally, the invention relates to the microorganism Streptomyces peucetius var. vinaceus DSM 2597. The microorganism Streptomyces peucetius var. Vinaceus is a mutant strain from Streptomyces peucetius var. aureus ATCC 31428.  

The microorganism

The one used in the method according to the invention Microorganism was induced by back mutation after the mutagenic treatment with UV rays from Streptomyces peucetius var. Aureus strain of 416 F.I. (ATCC 31428; DSM 1367; F.R.I. 4622) received as it by Cassinelli et al. (J. Antib., Vol. 35, No. 2, pages 176-183, 1982). The new one thus obtained Stamm was given the code number M 83 FCE by Farmitalia Carlo Erba collection of Microorganisms and at the German Collection for Microorganisms filed under the number DSM 2597.

The new microorganism differs from that Stock culture and from all others from Streptomyces peucetius mutants obtained mainly by that it forms a pigment of the substrate mycelium, which pink in young cultures on all solid media to red to wine-like and deep purple when aged becomes. This change in tint is also observed with the excess soluble pigment, which is on all solid media that normally used for the classification of Streptomycetes be forms. Such media include Bennet’s agar, Czapek's agar, asparagine-glucose agar, glycine-glycerol  Agar, potato glucose agar, asparagine-glycerol agar (Waksman S.A .: "The Actinomycetes", Volume II, 1961, William Wilkins & Co., Baltimore) and inorganic salt starch agar (T.G. Pridham, P. Anderson, C. Foley, L.A. Lindenfelser, C. A. Hesseltine and R. G. Benedict: A selection of media for maintenance and taxonomic studies of Streptomyces. Antibiotics Ann. 1956/1957, 947-953).

It is therefore assumed that the strain M 83 FCE is a Variant of Streptomyces peucetius which is the name Streptomyces peucetius var. Vinaceus becomes. The microorganism Streptomyces peucetius var. Vinaceus is also the subject of this Er finding.

Fermentation process

The production takes place according to the usual, good in itself known methods and includes growing the microorganism in a previously sterilized, liquid Culture medium under aerobic conditions and temperature in the range from 25 to 37 ° C (preferably at 28 ° C) over a period of time varying from 3 to 7 days (preferably 5 days), and at a pH that initially in the range of 6.5 to 7.0 and towards the end of Fermentation process is in the range of 6.5 to 8.0. The culture medium contains carbon and nitrogen sources as well as mineral salts. Suitable carbon sources are starch, dextrin, glucose, glycerin, mannitol, Maltose, corn steep liquor, stillage or others at the Distillation products, soybean oil and soybean meal. Suitable nitrogen sources are those like the above carbon sources that contain nitrogen, Dry yeast, meat peptone or casein. Quality  Results are obtained when ammonium salts, such as ammonium nitrate, ammonium sulfates and diammonium phosphates, used. The mineral salts needed for production can be used according to the Vary medium. In media that have different substances, like different flours and fermentation residues included is the addition of calcium carbonate and sodium or potassium phosphates are useful. With media, which contain glucose or ammonium salts are higher Contents of mineral salts, such as potassium, sodium or Calcium salts, and small additions of iron, zinc, Copper, magnesium and manganese salts required. The Fermentation can be carried out in Erlenmeyer flasks or in laboratory or industrial fermentation devices with various Capacities are carried out.

Analytical procedures

Be samples of the fermentation broths and raw mixtures thin layer chromatography (TLC) with silica gel precoated panels using a mixture from chloroform-methanol-acetic acid-water (80/20/7/3, in vol.) as eluent, so finds FCE 22724 with an Rf mean of 0.22 as red colored main ingredient. A quantitative determination from FCE 22724, which is in the fermentation broths can be present using the following procedure be performed. Add to a sample of the broth 2 vol. Acetonitrile and the resulting mixture is Sonicated for 1 min at room temperature and then filtered. Take a sample of the filtrate's silica gel TLC analysis subjected using the above elution system a spectrophotometric determination of  FCE 22724 at 493 nm can be performed by using the corresponding, red-colored zone scraped off and with a Mixture of chloroform-ethanol (9/1, vol.) Eluted.

Isolation process

After fermentation is complete, FCE 22724 becomes mainly found in the mycelia, which from the fermentation liquids by filtration at pH 7.5 be separated with the help of diatomaceous earth. The Mycelium cake is mixed with a water-miscible organic solvents, such as acetonitrile, dioxane, Methanol and other low alcohol, extracted; prefers methanol is used. The mycelium extracts are collected and concentrated under reduced pressure and the aqueous concentrate contains FCE 22724 in the form an alkali salt. FCE 22724 can from the filter broth by extraction at pH 4 with one with water not miscible, organic solvents, such as methyl isobutyl ketone, Methyl propyl ketone and n-butanol, won will; Methyl isobutyl ketone is preferably used. FCE 22724 can be extracted from the organic extracts at pH 7 in water as sodium salt with aqueous sodium hydroxide or sodium bicarbonate are extracted again. From the filtered broths and aqueous concentrates can FCE 22724 on hydrophobic, polymeric, cross-linked Divinylbenzene resins such as Amberlite XAD 2 or XAD 4 and ER 180 (Rohm and Haas Co., Inc.); ER 180 is preferably used. That absorbed FCE 22724 is easily preferred with aqueous lower alcohols 10 vol% n-propanol in water, eluted.  

Cleaning process

Raw, aqueous concentrates, the pH of which by addition of sodium chloride (5%) was set to 7 passed through a column of ER 180 resin. After washing with water the elution takes place with 10 vol% n-propanol in water and the eluate is in fractions collected. From the selected fractions using Silica gel TLC are checked, obtained after the concentration in vacuum and freeze drying raw FCE 22724 in the form of its sodium salt. To pure FCE To obtain 22724, the pH of an aqueous Solution of the raw sodium salt adjusted to 4 and then is extracted with methyl isobutyl ketone. The organic Extract results in a concentration after vacuum low volume, precipitation with n-hexane and crystallization pure FCE 22724 from methyl isobutyl ketone as free Acid in the form of fine, red, crystalline needles. The Treatment of the alcoholic solution of pure FCE 22724 (I) with one equivalent of alcoholic sodium hydroxide and then concentration and precipitation with diethyl ether gives the corresponding, pure Sodium salt (II). The acid catalyzed esterification of FCE 22724 in methanol provides the corresponding Methyl ester (III).

Chemical and physical properties

FCE 22724 as the free acid (I) is soluble in alkaline, aqueous media, lower alcohols and polar, organic solvents, somewhat soluble in acetone, ethyl acetate, diethyl ether, methylene dichloride and chloroform and hardly soluble in water, benzene and n-hexane. FCE 22724 as sodium salt (II) is soluble in water, lower alcohols and polar, organic solvents and somewhat soluble or insoluble in other organic solvents. FCE 22724 methyl ester (III) is soluble in lower alcohols, acetone, ethyl acetate, methylene dichloride and chloroform, somewhat soluble in diethyl ether, but insoluble in water and n-hexane. FCE 22724 has the following chemical and physical properties:
Melting point: 168 to 169 ° C (dec.)
UV and VIS absorption spectrum: in methanol: λ _max 233, 252, 288, 360, 474, 493 (525 Sch.) Nm
(E 490, 495, 144, 47, 184, 184, 100)
in 0.01 N methanolic sodium hydroxide: λ _max 233, 252, 290 (350 sh.), 540 and 580 nm
in M / 15 phosphate buffer pH 7: λ _max 232, 252, 288, 352, 474, 488 nm
(E 562, 526, 175, 55, 193, 187).
IR spectrum (KBr): peaks at the following frequencies:
3420, 2920, 1730, 1620, 1585, 1430, 1405, 1330, 1245, 1210, 1175, 1120, 1060, 940, 910, 870, 830 and 800 cm ^-1
Molecular formula: C₂₈H₂₈O₅, MW 604, 53, based on the elementary analysis and ¹³C-NMR spectrum ¹H-NMR spectrum (CDCl₃ + 1 drop of DMSO-d₆) at 200 MHz:
0.99 (t, J = 7.3 Hz, 3H, CH ₃CH₂); 1.40, 1.66 (two m, 2 H, CH₃ CH ₂); 1.9-2.3 (m, 2H, H-8 ); 3.56 (s, 3H, COO CH₃ ); 3.5-3.8 (m, 3H, H-2 ′, H-3 ′, H-4 ′); 3.94 (d, J = 9 Hz, 1H, H-5 ′); 4.14 (s, 1H, H-10 ); 4.85 (d, J = 7 Hz, 1H, H-1 ′); 5.14 (br.s, 1H, H-7 ); 7.65 (d, J = 4.4 Hz, 2H, H-1 , H-3 ); 8.00 (t, J = 4.4 Hz, 1H, H-2 ); 13.30 and 13.60 δ (two s, 2 H, OH-6 , OH-11 )
13 C-NMR spectrum (CDCl₃ / CD₃OH 1: 1, vol.) At 50 MHz:
6.08 (C-14); 32.38 (C-13); 3442 (C-8); 51.52 (C-10); 51.89 (COO CH ₃); 61.64 (C-4 ′); 71.22 (C-7); 71.56 (C-9); 72-89 (C-2 ′); 7514 (C-3 ′, 5 ′); 101.50 (C-1 ′); 122.03 (C-1); 124.44 (C-3); 133.09 (C-12a); 134.92 (C-6a); 135.81 (C-2); 138.16 (C-10a); 155.47 (C-11); 156.24 (C-6); 158.36 (C-4); 170.61 (C₅, - CO OCH₃); 171.52 (C₁₀, - CO OCH₃); 186.07 (C-5) and 187.13 δ (C-12).

Structure determination

The structure assigned to FCE 22724 and which corresponds to 4-O- ( β -D-glucuronyl) - ε -rhodomycinone (I) in Figure 1 is determined as follows: An aqueous, acidic hydrolysis gives ε -Rhodomycinone (IV) as insoluble, purple-colored aglycon and D-glucuronic acid (V), both of which were identified by direct comparison with authentic samples. Incubation of the FCE-22724 sodium salt with different glucuronidases enzymatically confirms the presence of a part of the molecule of D-glucuronic acid (V) which is bound to ε- rhodomycinone (IV). The position and β configuration of the glycosidic bond in FCE 22724 was assigned based on UV, VIS, IR, 1 H and 13 C NMR spectra. ε - Rhodomycinone (IV) and its 7-O-glucosides are known components of various anthracycline complexes, which are characterized by different species of the genus Streptomyces, such as pyrromycin (Chem. Ber. 92, 1904, 1959), cinerubin A and B (US PS 38 64 480), musettamycin and marcellomycin from bohemic acid complex (US Pat. No. 40 39 736) can be obtained. FCE 22724 (I) is the first biosynthetic, anthracycline-like anti-tumor compound that contains a β- D-glucuronyl group in its molecule, which is bound to the phenolic hydroxyl group in the 4-position of the ε - rhodomycinone.

The following examples illustrate the invention.

Example 1

A culture of Streptomyces peucetius var. Vinaceus, Strain M 83 FCE, is 14 days at 28 ° C on agar slant plates of the medium SA (glucose 3%; brewer's dry yeast 1.2%; NaCl 0.1%; KH₂PO₄ 0.5%; CaCO₃ 0.1%; MgSO₄ 0.005%; FeSO₄ · 7 H₂O 0.0005%; ZnSO₄ · 7 H₂O 0.0005%; CuSO₄ · 5 H₂O 0.0005%; Agar 2%; Tap water up to 100 ml; pH 6.7;  sterilization is carried out by heating in an autoclave grown at 115 ° C for 20 min).

The mycelium fragments scraped off from the agar culture are collected in 3 ml of sterile, distilled water and homogenized with a vortex homogenizer. The suspension thus obtained is used to inoculate 300 ml Erlenmeyer flasks containing 60 ml of the following liquid growth medium: dry brewer's yeast 0.3%; Peptone 0.5%; Ca (NO₃) ₂ · 4 H₂O 0.05%; Tap water up to 100 ml; sterilization is carried out by heating in an autoclave at 120 ° C. for 20 minutes; after sterilization, the pH is between 6.8 and 7.0. The inoculated flasks are shaken for 2 days at a temperature of 28 ° C on a rotary shaker, which is operated at 250 min ^-1 and describes a circle of 7 cm in diameter. 1.5 ml of the culture grown as described above is used to inoculate 300 ml alder flasks containing 50 ml of the following production medium: glucose 6%; dry brewery yeast 2.5%; Soybean meal 1%; NaCl 2%; KH₂PO₄ 0.1%; ZnSO₄ · 7 H₂O 0.001%; CuSO₄ · 5 H₂O 0.001%; Tap water up to 100 ml; pH 6.7; sterilization is carried out by heating in an autoclave at 115 ° C for 20 min. The flasks are incubated for 7 days at 28 ° C under the same conditions as described above for the vaccination phase. The maximum concentration of FCE 22724 was reached between the 6th and 7th day of fermentation with a production of 50 mcg / ml.

Example 2

The entire fermentation process was carried out according to Example 1 performed, the only difference in use of the following production medium: Glycerin 6%; dry brewer's yeast 2.5%; Soybean flour 1%; NaCl 2%; KH₂PO₄ 0.1%; CaCO₃ 0.2%; MgSO₄ 0.01%; FeSO₄ · 7 H₂O 0.001%; ZnSO₄ · 7 H₂O 0.001%; CuSO₄ · 5 H₂O 0.001%; Tap water up to 100 ml; pH 6.7; the sterilization was done by heating in an autoclave 115 ° C for 20 min.

The maximum concentration of FCE 22724 was between the 6th and 7th day of fermentation with a production reached 100 mcg / ml.

Example 3

The culture of strain M 83 FCE was carried out according to example 1. The mycelium fragments of the three oblique cultures were collected and collected in 10 ml of sterile, distilled water. After carrying out the homogenization according to Example 1, the suspension was used to inoculate 2 l round-bottom flasks with baffles which contained 500 ml of the seed medium described in Example 1. The flasks were incubated at a temperature of 28 ° C. on a rotary shaker, which is operated at 120 min ^-1 and describes a circle of 7 cm in diameter. The entire inoculation material was used to inoculate an 80 l stainless steel fermentation vessel containing 50 l of the inoculation medium described in Example 1 and sterilized by steam pressure at 120 ° C. for 30 min. The inoculation phase takes place at 28 ° C. with stirring at 230 min ^-1 and an air flow of 0.5 1/1 medium / min. After 30 hours, this inoculum was used to inoculate 500 liters of 800 liter stainless fermentation tanks containing the production medium described in Example 2. It was sterilized under a steam pressure at 120 ° C for 30 min. The fermentation took place at 28 ° C. with stirring at 250 min ^-1 and aeration with an air flow of 0.7 l / l medium / min. The maximum concentration of FCE 22724 was reached between the 6th and 7th day of fermentation with a production of 70 mcg / ml.

Example 4

The whole broth (3 l) from the fermentation, obtained according to Example 1, was at a pH of 7 below Filtered using 3% diatomaceous earth as a filter aid. The wet filter cake was washed with methanol (3 l) extracted. After filtering, two additional Extractions (with 3 l or 2 l methanol) carried out, to ensure that all of the red pigments are recovered were. The combined extracts are reduced Pressure concentrated to 300 ml. The pH the filtered broth obtained as described above was adjusted to 4 with sulfuric acid and then extracted with methyl isobutyl ketone (3 l). After two the organic extracts became additional extractions combined, concentrated and again with 3% aqueous Extracted sodium bicarbonate. To the watery one Layer with the aqueous concentrate of the Mycelium extract was combined, sodium chloride was added (5%) and the whole was on a column (3.2 × 27 cm), which was filled with resin Er 180® (200 ml). First was washed with water that was discarded  and then eluted with 10 vol% n-propanol in water. The selected fractions by TLC were found under reduced pressure concentrated and then freeze-dried, taking one obtained crude FCE 22724 as sodium salt (1 g).

Example 5

The pH of an aqueous solution (200 ml) of crude FCE 22724 as sodium salt (1 g), obtained according to example 4, is adjusted to 4 with sulfuric acid and then it is extracted with methyl isobutyl ketone. The organic Extracts are concentrated under reduced pressure and almost pure FCE 22724 (0.3 g) was added n-hexane precipitated. Crystallization from methyl isobutyl ketone gave pure red needles from FCE 22724 (0.25 g) as free acid; Mp 168 ° C (dec.).

Example 6

A methanolic solution (20 ml) of FCE 22724 in Form of free acid (0.12 g), obtained according to example 5, was made with an equivalent (0.2 mmol) of methanolic Treated sodium hydroxide. After concentration and Precipitation with diethyl ether gives pure FCE 22724 in the form of its sodium salt (0.11 g); Mp 190 up to 191 ° C (dec.).

Example 7

A solution of FCE 22724 (0.1 g) in anhydrous methanol (100 ml) is treated with 1 N methanolic hydrochloric acid (1 ml). After 2 h at room temperature the reaction mixture is diluted with ethyl acetate (100 ml) and concentrated to 50 ml under reduced pressure. After washing with aqueous sodium hydrogen carbonate and water, the organic phase is dried over anhydrous sodium sulfate and concentrated to a small volume. The addition of n-hexane gives the pure methyl ester of FCE 22724 (0.09 g) as a red, microcrystalline powder; Mp 150-152 ° C; UV and VIS spectrum: λ 223, 252, 288, 474, 493 (525 Sch.) Nm (E 505, 500, 147, 48, 178, 178, 96). Its molecular formula C₂₉H₃₀O₁₅, which is based on elemental analysis, was confirmed by field desorption mass spectrometry (m / e 618) and the assigned structure (III) was confirmed by IR and NMR spectroscopy:
1 NMR spectrum (CDCl₃) at 200 MHz: 1.12 (t, J = 7.2 Hz, 3 H, CH ₃CH₂); 1.12, 1.52 (two m, 2 H, CH ₂CH₃); 2.0-2.4 (m, 2H, H-8 ); 3.69 (s, 3H, C₁₀-COO CH ₃); 3.82 (s, 3H, C₅, -COO CH ₃); 3.8-4.0 (m, 3H, H-3 ′, H-4 ′, H-2 ′); 4.20 (s, 1H, H-10 ); 4.28 (d, J = 8 Hz, 1H, H-5 ′); 5.06 (br.s, 1H, H-1 ′), 5.19 (br.s, 1H, H-7 ); 7.2-7.6 (m, 3H, H-1 , H-2 , H-3 ); 13.21, 13.47 δ (two s, 2 H, OH-6 , OH-11 ).

Example 8

A solution of FCE 22724 (0.1 g) in dioxane (2 ml) and 1% aqueous sulfuric acid (10 ml) is heated at 100 ° C for 2 h. The reaction mixture containing a red colored precipitate is diluted with water (20 ml) and extracted with ethyl acetate. The extract is washed with 3% sodium hydrogen carbonate and water and dried over anhydrous sodium sulfate and concentrated to a small volume under reduced pressure. The addition of an excess of n-hexane gives a red crystalline compound (0.045 g; mp. 210 ° C.), which was identified as e- rhodomycinone (IV) after comparison with an authentic sample. On the other hand, the acidic component present in the water-soluble fraction of the above hydrolyzate was identified by silica gel TLC as D-glucuronic acid (V) after comparison with an authentic sample.

Example 9

A solution of FCE 22724 (0.05 g) in M / 15 phosphate buffer at pH 7 (50 ml) is treated with an aqueous solution (50 ml) of β- D-glucuronidase (50 g) and at 37 ° C. for 1 h incubated. Ε -Rhodomycinone (IV; 0.022 g) is isolated from the ethyl acetate extract, while the presence of D-glucuronic acid (V) was confirmed again in the aqueous phase.

Biological activity

FCE 22724 shows cytotoxic activity in HeLa Cell cloning activity in vitro; it is 40 µg / ml as medium concentration, which is 50% of the growing cells inhibited (ID₅₀).

FCE 22724 also shows anti-tumor activity in vivo at P 388 leukemia in mice as shown in Table 1 going on.  

Table 1

in vivo effect of FCE 22724 on P-388 murine leukemia)

Claims (4)

1. The microorganism Streptomyces peucetius var. Vina ceus DSM 2597. 2. Anthracyclinone glucuronide with anti-tumor activity of the general formula A wherein R represents a hydrogen atom, a methyl group or a sodium atom. 3. Process for the preparation of an anthracyclinone glucuronide according to claim 2, wherein R represents a sodium atom, characterized in that known per se Wise microorganisms of the strain Streptomyces peuceutius var. vinaceus (DSM 2597) in aerobic conditions in an assimilable source of carbon and nitrogen and aqueous culture medium containing mineral salts be, the breeding for 3 to 7 days in egg  ner temperature of 25 to 37 ° C and a pH that at the beginning in the range of 6.5 to 7.0 and towards the end of breeding is in the range of 6.5 to 8.0, the mycelium the mycelium is removed from the culture medium extracted with a water-miscible organic solvent the extracts are concentrated under reduced pressure be, the crude anthracyclinone glucuronide of the general Formula A, wherein R represents a sodium atom, obtained is and for cleaning a concentrated aqueous solution the crude compound on a hydrophobic cross-linked, polymeric divinylbenzene resin is absorbed with an aqueous low alcohol is eluted, the pH of the eluate to release the anthracyclinone glucuronide of the general Formula A, wherein R represents a hydrogen atom, to 4.0 the eluate is extracted with methyl isobutyl ketone, the anthracyclinone glucuronate in pure form by adding precipitated n-hexane and it into the pure sodium salt by treatment with one equivalent of methanolic sodium hydroxide is converted again and then with diethyl ether is canceled. 4. Pharmaceutical preparation containing a therapeutic effective amount of a compound of claim 2 mixed with a pharmaceutically acceptable diluent and / or carrier.