DD298427A5 - PROCESS FOR PREPARING 3-METHYL-5,6 (7), 8-TRI-HYDROXY-2-AZA-ANTHRACETHONE (9,10) - Google Patents

Abstract

The invention relates to a process for obtaining a new aza-anthraquinone which has the structure of a * in detail. The object associated with the aim of producing these novel metabolites as tolypocladin in an effective manner is achieved by using in the biological process stage a cyclosporin A-forming fungal microorganism, preferably a strain of the species Tolypocladium inflatum or Sesquicilliopsis rosariensis, under sterile conditions is cultivated in aerobic Submersfermentation. The said metabolite is subsequently extracted from the mycelium or mycelial residues obtained here with hot water or with polar organic solvents at acid p H values and, after concentration of the solvent, recovered in almost pure form. For fine cleaning, an ordinary chromatographic process may optionally be connected. Tolypocladin has been shown to inhibit cancer cell culture in selected in vitro test systems. {Novel aza-anthraquinone; * Name Tolypocladin; fungal production organisms Tolypocladium inflatum and Sesquicilliopsis rosariensis; Cyclosporin A generator; aerobic submerged fermentation}

Description

For this 3 pages drawings

Field of application of the invention

The invention relates to a microbial process for the preparation of a novel 2-aza-anthraquinone-based metabolite containing a strong chromophore with the p-quinone moiety present.

The field of application of the invention lies in microbiological-pharmaceutical research and industry.

-2- 298 Characteristic of the known technical solutions

Various fungi form, under certain culture conditions and depending on the nutrient broth composition, chinoid colored substances belonging to the structural type of the o- and p-quinones, naphthoquinones, anthraquinones and other polyketido (Turner, B., Fungal Metabolites, Vol.11, Academic Press, Vol. 1983). In addition to carbocyclic structures, heterocyclic quinones were also found, which, like the carbocyclic representatives, are distinguished by chromogenic properties (Thomson, R.H., Naturally Occurring Quinones, Academic Press, New York, 1971). A number of naturally occurring quinoidal compounds possess beneficial biological properties due to their reducibility by respiratory chain enzymes of living cells or DNA intercalation, which make them suitable for use as antineoplastic agents. p-quinonoid compounds of the anthracycline type are e.g. B. in the therapy of cancers in use. Due to various side effects (cardiotoxicity, alopecia, nausea, immunosuppression, etc.), the search for more effective derivatives and new Kanzerostatika continues to be of interest. By chemosynthetic modification of natural basic structures and by partial syntheses, starting from natural basic bodies, effect-optimized remedies can be produced.

Isoquinoline structures have hitherto been found to be a component of secondary metabolites of the alkaloid type (K.Mothes et al., Biochemistry of Alkolites, VEB Deutscher Verlag der Wissenschaften, Berlin, 1986). However, 2-aza-anthraciiinones (benzo-isoquinolinediones) from fungi or prokaryotic microorganisms have not been described so far.

Object of the invention

The invention aims to effectively produce the new fungal metabolite 3-methyl-5,6 (7), 8-trihydroxy-2-aza-anthraquinone- (9,10), in order to directly or potentially exclude the range of potential carcinostatic agents after chemical modification usable active ingredients to enrich a new basic type.

Darleguno of the essence of the invention The invention has for its object to describe a technologically simple microbiological process that the

fermentative production of a new fungal metabolite from easily accessible and cheap feedstocks into good ones

Yields possible. According to the invention this object is achieved in its basic feature by a fungal cyclosporin Α-forming microorganism

is cultured aerobically at a temperature between 2O ⁰ C and 37 ⁰ C. The new metabolite 3-methyl-5,6 (7), 8-trihydroxy-2-aza-anthraquinone- (3-methyl) -5,6 (7), 8-trihydroxy-2-aza-anthraquinone- (3), which is present during culture c and / or residues or auxiliary products present after cyclosporin A removal, is 9,10), to which the name Tolypocladin is given below, then extracted under acidic conditions with water or with polar organic solvents.

In its further embodiment, the method in the microbiological process stage is characterized as follows: Cultivation is carried out in sterile submerged fermentation, whereby for cultivation mycelial areas of surface cultures of Cyclosporin Α -forming fungal strains of the species Tolypocladlum inflatum, preferably of strain T. inflatum Wb 6-5, or

the species Sesqulcllliopsls rosarlensls, preferably from strain S. rosarlensls F 605, are used. Biologically pure

Samples of the two above-mentioned fungal strains are in connection with earlier patent applications of Establishment of origin in the depository of micro-organisms of the GDR, Central Institute of Microbiology and

experimental therapy of the AdW, Beutenbergstr.11, Jena, DDR - 6900 under the registration marks

IMET43899 for strain Tolyprucladlum inflatum Wb 6-5 as well

IMET 43887 for strain Sesnuicilliopis rosariensis F 605

deposited lands.

The fermentation medium consists of carbon and nitrogen sources as well as inorganic salts. It is preferred

used for culturing a liquid Näht medium, which is the only nitrogen source ammonium salts, preferably

Ammonium sulfate and / or Diammoniumhydrogenphosphat contains. The cultivation of the method to be used according to the method Strains are carried out in steep-breasted bottles and round-bottomed flasks of various volumes, in glass fermentors with stirrer or in V2A-steel tanks. The biomass of T.lnflatum strains obtained in the culture stages is aerobic in the main culture

and sterile conditions at 25 ⁰ C to 32 ⁰ C, preferably to 27 ⁰ C, in an ammonium salt nutrient medium above

Labeling at an acidity between pH 4.0 and pH 7.0 fermented for a period of 3 days to 8 days. In contrast, the biomass of S. rosarlensis strains available after the cultivation stages becomes aerobic in the main crop

and sterile conditions at 25 ° C to 35 ⁰ C in an ammonium salt nutrient medium above labeling also in a

Acidity between pH 4.0 and pH 7.0 fermented for the duration of 5 days to 11 days. For the further embodiment of the overall process is now also characteristic that in its processing stage the Extraction of Tolypokladins in several, sometimes complementary variants can be made, namely under acidic Conditions either

a) by extraction of the fresh mycelium, preferably the fresh wet mycelium,

b) by extraction of mycelial residues, as they are present after separation of the cyclosporir, -A, or

c) by extraction of such chromatographic support materials which have been used as preferably aluminum oxide for the purification of crude cyclosporin A.

The extraction in each of these variants is pH 1.0 to pH 4.0, preferably at pH 1.0 to pH 2.0,

with oxalic acid hot water, acetone or methanol (oxalic acid addition in each case 2%) or with hydrochloric acid methanol or dimethylformamide

executed. By reextraction of the aqueous extract or the concentrated residue of the extraction with organic

Solvents such as CHCl ₃ and / or n-butanol and subsequent concentration of the extract, the above-mentioned new metabolite

together with low levels of similar compounds (congeners).

By chromatography on oxalic acid silica gel and on organophilic dextran gels, tolypocladin in pure form

be won, d. H. the mentioned small amounts of by-products with lower polarity, which in changing

Quantity can be formed depending on the given fermentation and the according to their electron spectra and their

pH behaviors similar to those of 3-methyl-5,6 (7), 8-trihydroxy-2-aza-anthraquinone (9,10) are attributed to these

Completely separated. The chemical identity of the new metabolite is evidenced by physicochemical parameters shown in Table 1. In the Fig. 1 indicated structure of the new compound 3-methyl-5,6 (7), 8-trihydroxy-2-aza-anthraquinone (9,10) is further by the NMR spectroscopic results (Varian XL-400,400MHz or 100MHz for ¹ H and ¹³ C NMR spectra, respectively)

secured.

The 400 MHz proton spectrum shown in Figure 2 demonstrates the presence of three aromatic uncoupled protons

(6.7647 ppm, 7.8681 ppm, 9.2183 ppm), an N-CH ₃ group (2.7227 ppm), and the OH groups (13.27 ppm, 12.0 ppm;

The 100 MHz ¹³ C spectrum (Figure 3) shows the presence of two carbonyl groups, 11 aromatic sp ² -hybridized Carbon atoms and bonded to the heterologous aromatic ring methyl group. With the given physicochemical data, the structure of the new compound as shown in Fig. 1 becomes sufficient

determined. (To describe the exact localization of the OH group and the proton at positions 6 and 7 is not yet possible with the structural aides used).

For Tolypocladin, in selected in vitro test systems, an inhibitory effect on cancer cell cultures, e.g. B. on L 1210 cells have been found. embodiments example 1

1.1. 1 to ² cm ² large agar pieces from single colonies of surface cultures of the strain Tolypocladlum inflatum Wb 6/5 are used to inoculate a stock culture medium of the following composition (g / l) in 500 ml Erlenmeyer flasks (80 ml content, 25 ° C, rotary shaker, 150 rpm, 4 days Culture time): (NH ₄ I ₂ SO ₄ 7; (NH ₃ ) ₈ HPO ₄ 2; MgSO ₄ .7H ₂ O 0.5; ZnSO ₄ 0.008; Glucose 100; CaCO ₃ ; pH 5.3 to pH 5.7 ,

The second pre-breeding is carried out on the same medium in 5 l bottles with 4 ml of ml contents (72 hours, 25 ° C, Rotary shaker, 150 rpm). For the scale up to 2Ol and the main culture in 500 l fermentors is a Medium of the following composition used (g / l): glucose 80; (NH ₄ I ₂ HPO ₄ 2; (NH ₄ I ₂ SO ₄ 5; MgSO ₄ .7H ₂ O 0.5; ZnSO ₄ 7H ₂ O 0.008; CaCO ₃ 5.1; Distilled water ad 1 i; pH 5.3 to 5.9; Sterilization for 30 minutes, 120 ° C. The main culture is in 500-I-V2A Tankfermentoren for a period of 7 days at a temperature of 25 ⁰ C to 27 ⁰ C.

(aeration rate: 0.25: 1 [v / v]; stirrer speed: 300 rpm). The foam control is done with sterile corn oil.

1.2. The mycelium from 450I broth is harvested by filtration and rotary cells in 400I water (pH 2.0) res'jspendiert and heated to 9O ⁰ C for 30 minutes. Subsequently, the extracted biomass is separated by rotary cell filtration and discarded. The acidic, aqueous, red solution is extracted twice with 0.1 volume of butyl acetate. The concentrated extract with organic solvents is taken up in 2 liters of chloroform and washed twice with 1 liter of 0.1 M Na ₂ CO ₃ solution in H ₂ O. The metabolite goes into the aqueous solution as a violet sodium salt. The aqueous phase is filtered and adjusted to pH 4 with oxalic acid. Then the aqueous phase is extracted ₃ times with CHCl ₃ in the ratio 1: 1 (aqueous phase: CHCl ₃ ). The CHCI ₃ extract is concentrated to 100 ml. The metabolite precipitates as red-brown amorphous powder in at least 90% purity (yield 2 g / 450 l fermentation solution). Further purification is achieved in that the powder 2 times with water (100ml) and 2 times with petroleum ether (3O ⁰ C to 5O ⁰ C, 100ml) is washed and dried. For an optionally desirable fine purification of the new metabolite 3-methyl-5,6 (7), 8-trihydroxy-2-azaanthraquinone- (9,10), one of the following chromatographic sub-processes is finally used:

Chromatography on silica gel (0.063-0.2 mm) suspended in acetone / CHCl ₃ (2: 1, v / v) with 1% oxalic acid. First colored congeners appear, followed by 3-methyl-5,6 (7), 8-trihydroxy-2-azaanthraquinone (9,10). The fractions containing the metabolites are concentrated and the oxalic acid is removed by partitioning between CHCl ₃ and water phase.

Chromatography on organophilic dextran gels with 0.05% oxalic acid and methanol. In this case, higher molecular weight brown, iron complex-forming compounds may optionally be separated as a forerun.

- Fine purification of the sodium salt by gel chromatography sn hydrophilic dextran gels (type G-10, Molselekt, Sephadex, etc.). For this purpose, the metabolite (25 mg) is taken up in the minimum amount of Na ₂ CO ₃ solution, applied to a column 40 cm χ 1 cm and eluted with water. The violet fractions containing the metabolites are acidified to ρ H 1.0 with HCl and extracted several times with CHCl ₃ . After concentration of the solvent, the metabolite is recovered in pure form.

Example 2

The biological process step is carried out as in Example 1. Subsequently, the grown mycelium is harvested by separation and isolated from this moist mycelium in a conventional manner, the active ingredient cyclosporin A. From the resulting mycelial residues, a crude solution containing the metabolite is obtained by extraction with 2% oxalic acid solution in acetone or methanol. After concentration to dryness, the metabolite is taken up with 0.1 N Na ₂ CO ₃ and fat contents are removed by distribution between the CHCl ₃ and water phases. Further workup of the remaining in the basic aqueous phase V abolite is carried out as in Example

Example 3

Dio biological process stage and the separation of the grown mycelium are carried out as in Example 1. From the mycelium in a known manner the active substance cyclosporin A is extracted and separated by column chromatography (support material alumina). The metabolite tolypocladin is extracted either from oxalic acid (2%) acetone or methanol, from hydrochloric acid methanol (1N HCl) or from dimethylformamide (1% HCl conc.) From the strongly colored parts of the support material. and from the residue of corresponding concentrated extracts, the target substance is finally obtained analogously to the methodology described in Examples 1 and 2.

Table 1: Physicochemical and spectral properties of 3-methyl-5,6 (7), 8-trihydroxy-2-aza-anthrachirion (9,10)

- Properties:

red amorphous solid

soluble - 0.1 M Na ₂ CO ₃ ZH ₂ O; good with color change to violet - acetone, methanol, CHCl ₃ ; moderate

insoluble in hexane or petroleum ether melting point: ⁰ C (uncorrected)

Chromatographic properties: Rf 0.25 (silufole, oxalic acid impregnated, AcBtOn-CHCl ₃ , 2: 1, v / v)

- EI mass spectrum (Jeol JMS-D100): 'Molecular Formula (MS):

C ₁₄ H ₉ NO ₆ (M ⁺ ): m / z 271.0475; 271.0481 calc. For C ₁₄ H ₉ NO ₆ Characteristic MS fragment peaks:

m / z 243 (M ⁺ -CO); 228 (M ⁺ -CO-CH _3); 215 (M ⁺ -2CO); 201 (M ⁺ -C ₃ H ₂ O ₂ ); 197 (M ⁺ 2CO-H ₂ O); 169 (M ⁺ -H ₂ O-3CO); 145 (M ⁺ -C ₃ H ₂ O ₄ ).

- IR spectrum:

\ _m " (in KBr; cm" ¹ ): 630,645,690,705,740,755,760,790,805,810,850,925,950,1050,1120,1140,1170,1 210,1300,1360, 1405,1450,1455,1500.1 515,1535,1550,1575,1610, (chelated Quinone), 2320.3000.3400, 3710.1810.

- UV / VIS spectrum:

a) free acid A _m , _x (in MeOH, nm):

535

500 (ε = 3.5 × 10 ^e cm ² / mole)

460

330, <250 strong absorption

b) sodium salt X _n ,,,, (in MeOH, nm):

550.525 (both ε = 7.5cm ² / mole)

Λ20

290

c) Zn ⁺⁺ complex> _mix (inMe0H, nm):

550,390,335,268, <300 strong absorption

d) Cu ⁺⁺ complex A _mix (inMe0H, nm):

580.530, <300 strong absorption

e) Ni ⁺⁺ complex A _m "(inMe0H, nm):

595,560,510,400,270, <250 strong absorption

f) Mn ⁺⁺ complex A _mlx (inMe0H, nni):

550,390,330,268, <245 strong absorption

g) Ca ⁺⁺ complex ^, "(inMeOH.nm):

605,575,530,380,280, <245 strong absorption

h) Mg ⁺⁺ complex Ä _m , _x (inMeOH, nm):

590,550,510,380,280, <245 strong absorption

i) Fe ⁺⁺ complex Ä _mex (inMe0H, nm):

630,480, <370 strong absorption

j) Fe ⁺⁺⁺ complex X _m "(inMe0H, nm):

650, <440 strong absorption

triacetate Production by reaction of 142 mg of methyl-trihydroxy-isoquinoline-dione in 3 ml of pyridine and 3 ml of acetic anhydride for 4 hours at 60 ^C C. Purification by column chromatography (silica 0.063 to 0.2 mm; CHCl ₃ acetone 10: 1) Yield 122 mg; Melting point 212 to 215 ⁰ C (yellow crystals) mass spectrometry

M ⁺ : m / z 397

M ⁺ -CH ₂ CO: m / z 355.0692

M ⁺ -2CH ₂ CO: m / z 313.0570

M ⁺ -3CH ₂ CO: m / z 271.0501

Characteristic fragment peaks: m / z 243.0542; m / z 165.1620; m / z 125.1319; m / z 111.1140; m / z 97.1000; m / z 71.0856;

m / z 57.0598; m / z 41.9829; m / z 41.0103 UVX _mlx (nm): 240.335

IR K _n , * (cm ^-1 ): 630, 880, 880, 895, 995, 995, 1020, 1090, 1115, 1160, 1180, 1270, 1315, 1350, 1400 .. 1420,

1575,1655,1665,1755,1760.

Claims (11)

A process for the preparation of 3-methyl-5,6 (7), 8-trihydroxy-2-aza-anthraquinone- (9,10) (Tolypocladin) by microbial route, characterized in that - a fungal cyclosporin Α-forming microorganism aerobically cultured at a temperature between 20 ⁰ C and 37 ⁰ C and - extracted the metabolite Tolypocladin from the obtained mycelium and / or from existing after the cyclosporin Α separation residues or Hilfspropu tiu under acidic conditions with water or with polar organic solvents. 2. The method according to claim 1, characterized in that one uses for culturing a fungal microorganism of the species Tolypocladium inflatum, 3. The method according to claim 2, characterized in that one uses for culturing the fungal microorganism Tolvpocladium Inflatum Wb 6-5. 4. The method according to claim 1 to 3, characterized in that cultivating the selected Tolypocladium inflatum strain under sterile conditions at 25 ° C to 27 ⁰ C in a liquid ammonium salt nutrient medium for a period of 3 days to 8 days. 5. The method according to claim!, Characterized in that one uses for culturing a fungal microorganism of the species Sesquicilliopsis rosariensis. 6. The method according to claim 4, characterized in that one uses for culturing the fungal microorganism Sesquicilliopsis rosariensis F 605. 7. The method according to claim 1,5 and 6, characterized in cultivating the selected Sesquicilliopsis rcsariensis strain under sterile conditions at 25 ⁰ C to 35 ⁰ C in a liquid ammonium salt nutrient medium for a period of 5 days to 11 days , 8. The method according to claim 1 to 7, characterized in that the metabolite Tolypocladin from the obtained wetland mycelium with oxalic acid hot water,
with oxalic acetone,
with oxalic or hydrochloric methanol or
with hydrochloric acid dimethylformamide
extracted and then optionally finely purified on chromatographir.chom ways. 9. The method according to claim 1 to 7, characterized in that the metabolite Tolypocladin present after the Cyclosporin A Abtrennurig present mycelial residues with oxalic acid hot water,
with oxalic acetone,
with oxalic or hydrochloric methanol or
with hydrochloric acid dimethylformamide
extracted and then optionally finely purified by chromatography. 10. The method according to claim 1 to 7, characterized in that the metabolite Tolypocladin used for cyclosporin Α-cleaning alumina with oxalic acid hot water,
with oxalic acetone,
with oxalic or hydrochloric methanol or
with hydrochloric acid dimethylformamide
extracted and then optionally finely purified by chromatography. 11. The method according to claim 1 and 8 to 10, characterized in that one carries out the extraction in each case at pH 1.0 to pH 4.0.