DE2261832C3 - 5-Dihydrocoriolin C and process for its preparation - Google Patents

Description

OH

2. A process for the preparation of 5-Dihydrocoriolin C from a filtrate obtained by cultivating the strain ATCC 20105 Coriolus consors in a medium appropriate for formation of 5-Dihydrocoriolin C medium at a temperature of 15- 30 ⁰ C under aerobic conditions, separating a portion of the mycelium formed, extracting the same with an organic solvent, concentrating the extract and filtering off the coriolin B which has separated out, characterized in that the mother liquor obtained is further concentrated, the 5-dihydrocoriolin C which has separated out is filtered off and, if desired, recrystallized or the 5 -Dihydrocoriolin C-containing filtrate in the usual way chromatographies.

The subject of the patent 21 06696 is a process for the production of Coriolin B by aerobic cultivation of microorganisms of the strain Coriolus consors ATCC 20305 at 15-30 ⁰ C and separation from the nutrient medium with the help of a solvent.

The process of the invention is now characterized in that one obtained above Concentrate the mother liquor even further, the precipitated 5-dihydrocoriolin C is filtered off and, if desired recrystallized or else the filtrate containing 5-dihydrocoriolin C is chromatographed in the usual way.

The 5-dihydrocoriolin C of the present invention is a new substance capable of being oxidized Form Coriolin C and 2 '»Dehydrocoriolin C with antibacterial and anti-tumor effects.

The IR absorption spectrum of the invention 5-dihydrocoriolin C (determined on a tablet made from 5-dihydrocoriolin C and potassium bromide) can be found in the attached drawing.

The 5-dihydrocoriolin C obtained according to the invention is in the crude and crystalline state obtained and has no antibacterial or anti-tumor effect itself, but show Coriolin C and 2'-Dehydrocoriolin C obtained by oxidation of 5-Dihydrocoriolin C with an oxidizing agent will have an antibacterial effect against Staphylococcus aureus and Bacillus subtilis and one life-prolonging effect in Ehrlich's ascites tumor in mice and mouse leukemia L-1210, as can be seen from the following test results (for Coriolin C see Umezawa et al .: Tetrahedron betters, No. 19 [1970], pp. 1637-1639; 2'-Dehydrocoriolin C is a new compound).

The results of a study of the antibacterial effects of Coriolin C and 2'-Dehydr < coriolin C according to the agar-agar dilution method are given in Table 1 below.

Table I.

Antibacterial effect of Coriolin C
and 2'-dehydrocoriolin C

Test organisms Coriolin C

St. aureus

FDA 209-P 1.56 *)

Terajima 1.56

Smith 1.56

M. flavus FDA-16 <0.78

B. anthracis <0.78

B. subtilis NRR B-558 <0.78

E. coli NIHJ 25.0

S. typhi-T63> 100.0
enteritis 12.5

Ps. Aeruginosa AS> 100.0
·) Minimum inhibitory concentration (iig / ml).

2-dehydrocaloriolin C.

1.56 *) 1.56
L, 56
1.56

<0.78
1.56

100.0

> 100.0

12.5

100.0

These two compounds inhibit the growth of gram positive microorganisms, and with both Substances can have almost the same effects.

The effects of Coriolin C and 2'-dehydro coriolin C on Ehrlich's ascites tumor at de Mouse, Mouse Leukemia L-1210, and Growth of Yoshida sarcoma cells in tissue cultures were examined and the following results were obtained:

The treatment of Ehrlich ascites tumors be Mice were given a daily dose of 0.19 to 12.5 mg / kg of each of the two compounds. carried out over 10 days. During aiii Control animals within 2 "* days after Ein vaccination of the tumor cells were dead, mice survived which were infected with 12.5, 6.25, 3.12, 1.56 and 0.678 mng / kj Coriolin C or 2'-dehydrocoriolin C was treated with administration in the abdominal cavity were 90%, 80%, 65%, 63% and 60% (when administering Coriolin C) more than 50 days and zi 80%, 75%, 60%, 60% and 50% (when 2'-dehydrocoriolin C is administered) for more than 50 days, and e; no ascitesretentin was observed.

Treatment of murine leukemia L-121C by daily administration of 6 to 100 mg / kg Coriolin C or 2'-dehydrocoriolin C over 10 days resulted in mice that were 6, 12.5, 25, 50 and respectively 100 mg / kg Coriolin C or 2'-Dehydrocoriolin C administered had a life-prolonging effect of 119%, 131%, 140%, 150% and 175%, respectively Coriolin C and of 115%, 120%, 125%, 135% and 160% for 2'-dehydrocoriolin C (based on the Survival time of the control animals [100%]).

In the investigation of growth inhibition in tissue cultures of Yoshida sarcoma cells Rats underwent a 60% reproduction inhibition with 0.75 μg / mg with coriolin C or 2-dehydrocoriolin C received.

Studies of the acute toxicity of coriolin C and 2-dehydrocoriolin C in mice showed that the DL ₅₀ when administered into the abdominal cavity is above 50 mg / kg and when administered subcutaneously is above 90 mg / kg. Further, in the case of continuous administration to the abdominal cavity for 10 days, all of the mice tested survived for more than 50 days even when the total dose reached 100 mg / kg, and the toxicity was found to be low.

From the above, it follows that Coriolin C and 2'-Dehydrocoriolin C are excellent antibacterial agents and have anti-tumor activity.

Between the properties of 5-dihydrocoriolin C.

OH

OH

O — CO-CH- (CH ₂ I ₅ -CH,

OH
Coriolin C

Ο — CO — C— (CH ₂ J ₅ -CH, O

2'-dehydrocoriolin C.

O-CO-CH- (CH ₂ J ₅ -CH ₃ OH

and the advantageous pharmacological properties of the coriolins accessible therefrom by oxidation

40

45

there is a clear causal relationship, such as the structural comparison of the corresponding formulas shows, with the keto group in 5-position presumably a decisive role for the pharmacological Effect.

Coriolin C can be produced directly by fermentation, but the yield is so low that that such a process can hardly be carried out industrially. In contrast, 5-Dihydrocorio-Hn C obtained according to the invention in high yield, and it can be smoothly by certain oxidizing agents can be converted into Coriolin C. The Production of Coriolin C by Oxidation of 5-Dihydrocoriolin C is therefore more advantageous and economical than the direct fermentative route.

The 5-dihydrocoriolin C obtained according to the invention has the following properties:

5-Dihydrocoriolin C is obtained in the form of colorless crystals which melt at 183 ° C and are soluble in methanol, ethanol, ethyl acetate, acetone, chloroform and benzene, but are slightly or not soluble in water and petroleum ether. 5-Dihydrocorion C shows the following IR absorption spectrum (measured on KBr tablets). The main absorptions are at the wave numbers (cm " ¹ ) 3490, 3360, 2950, 1741, 1649, 1460, 1419, 1389, 1370, 1340, 1315, 1270, 1184, 1139, 1109, 1081, 1032, 1003, 983, 965 , 949, 921, 891, 868, 841, S09, 788, 781, 751, 716 and 672. The spectrum is shown in the attached drawing.

5-Dihydrocoriolin C has a molecular weight of 424 (determined by mass spectrometry) and shows at the elemental analysis showed the following values: C 65.07% and H 8.55%; it does not contain nitrogen.

On the basis of the above values of the elemental analysis and the molecular weight determined by mass spectrometry, the empirical formula C ₂₃ H ₃₆ O ₇ (molecular weight: 424) was found.

The above structural formula of 5-dihydrocoriolin C was obtained based on the analysis of the NMR spectrum as well as its behavior towards reagents determined.

5-Dihydrocoriolin C is also formed by basic diomycetes, but differs in terms of physical and chemical properties, molecular formula and structural formula from the already known products Coriolin, Coriolin B, Corio-Hn C, 5-Ketocoriolin B or hirustic acid C. It is new. To produce the filtrate containing 5-dihydrocoriolin C used as starting material, a microorganism which is capable of forming 5-dihydrocoriolin C, namely Coriolus consors (Berk) Imaz ATCC no Kyushu (Japan) widely used and detailed in "Zoku Genshoku Nippon Kinrui Zukan" (colored Japanese "bacterial picture book", cont.) By Rokuya I ma ζ eki and Jiro Hongo, pages 141 and 142, Hoikusha Publishing Co., Japan, edition from 1968 is grown in a suitable medium under aerobic conditions. Although it is possible to use a solid cultivation method for culturing, a liquid culture method is preferred for mass production, typically the culture at 15 to 30 ° C and especially at 25 is carried out until 28 ⁰ C.

As a culture medium for the formation of 5-dihydrocoriolin C, a carbon source, a A medium containing nitrogen source, inorganic salts, product ins promoters, etc. can be used.

Commercially available sugars, oils and fats, etc. can be used as the carbon source such as glucose, glycerin, starch, dextrin, maltose, lactose, sucrose, oil and fat or Molasses in the pure or raw state.

Soybean powder, meat extract, peptone, dried brewer's yeast, yeast extract, Grain softeners, casein, cottonseed oil, fish meal, nitrates, ammonium salts, urea or amino acids such as glutamic acid or the like can be used.

Inorganic salts are sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate or phosphates or very small amounts of salts of heavy metals such as copper, manganese, or iron Zinc into consideration. Furthermore, the addition, in particular partial addition, of acetic acid or malonic acid increases and mevalonic acid, the formation of 5-dihydrocoriolin C during cultivation.

5 J 6

An example of a suitable culture medium for 5-ketocoriolin B is included. However, since Coriolin B in

the preparation of the ethyl acetate containing 5-dihydrocoriolin C is the least soluble, it becomes through

Filtrate is indicated below: Concentration of the liquid extract excreted first

,.,. .. and can be removed by filtration.

Composition I (inoculation medium) _s Experience has shown that 5-dihydrocoriolin C is now used

Glucose 5.0% from the mother liquor obtained by concentration

Peptone 0.2% and let the concentrated liquid stand in shape

Dried brewer's yeast 0.3% excreted from needle-like crystals, as 5-di-

Potassium dihydrogen phosphate 0.3% hydrocoriolin C is easier to crystallize than 5-keto

Magnesium sulfate 0.1% ι ο corolin B.

However, if containing polyoxyethylene groups, a lot of impurities in the extract

surfactant contained 0.01% is the excretion of 5-dihydrocoriolin C crystals difficult, and the 5-dihydro-

Composition II (production _{medium) coriolin c is therefore from 5} . _K etocoriolin B usual-

Glucose 5.0% 15 instruct by chromatographic methods

Peptone 0.2% using silica gel as a carrier medium and one

Dried brewer's yeast 0.3% solvent mixture of methanol-chloroform,

Potassium dihydrogen phosphate 0.3% chloroform or benzene separately as eluent.

Magnesium sulfate 0.1% During the elution, 5-ketocoriolin B is eluted first

20 containing polyoxyethylene groups and then 5-dihydrocorioline C. Their separation

surfactant 0.01% is satisfactorily possible because the two

Calcium carbonate *) 1.6% bonds due to two hydroxyl groups in the

*) Separately sterilized and clearly differentiated from the medium at the beginning of the culture

added The by evaporation of the 5-dihydroconoline C

25 containing eluate to dryness obtained crude powder

An incubated inoculation liquid is combined in a small amount of an organic

Settlement I is then dissolved with shaking in the solvent and the 5-dihydrocoriolin C

Composition II designated medium through some of it in the form of needle-like crystals

Cultivated for days at 15 to 30 ° C. In the case of the addition of solvents in which 5-dihydro-

Large-scale cultivation using 30 coriolin C is sparingly soluble, for example n-hexane

in a fermentation tank, the inoculation and / or water is excreted.

Cultivation carried out in two stages. For the conversion of 5 - dihydrocoriolin C into According to the usual procedure, the coriolin C or 2'-dehydrocoriolin C is oxidized Cultivation in an inoculation medium in one dation are different methods of the shake flask first inoculation liquid obtained initially 35 Addition: (1) the oxidation with chromic acid in pyridine, in a previously provided with inoculation medium (2) the oxidation with manganese dioxide in neutral Transferred to fermentation tank and subjected to cultivation solution or (3) oxidation with a dicyclohexyl. Then the second inoculating liquid is carbodiimide solution in dimethyl sulfoxide. Advantageous However, procedures (1) and (2) are halted in another fermentation tank Production medium inoculated and the 40 turns.

Subjected to cultivation; the second stage can

however, they can also be omitted. play explained.

In this cultivation Coriolin, Coriolin B,

B-ketocoriolin B and 5-dihydrocoriolin C formed; example 1

since the coriolin is mainly in the liquid part 45

is present, while Coriolin B, 5-Ketocoriolin B and a) starting material
5-dihydrocoriolin C are mainly present in the mycelium, the coriolin B, 5-ketocoriolin B 25 g wood chips (corresponding to approx. 0.3 to 0.85 mm and 5-dihydrocoriolin C containing mycelium of the clear mesh size) from Magnolia hypoleuca was Culture fluid is separated off according to generally known methods, such as filtration or centrifugation, in a 1 liter Erlenmeyer flask. If able, and with 170 ml of a 2% glucose, this mycelium fraction is subjected to an extraction with organic liquid solvents containing 0.5% dried brewer's yeast, such as methanol, acetone, ethyl acetate, culture medium (hereinafter referred to as "GY medium" be-butyl acetate or methyl isobutyl ketone) Then the bottle was added, the 5-dihydrocoriolin C is sterilized in the organic autoclave at 120 ° C for 20 minutes and then transferred to the solvent layer to form 5-dihydrocoriolin C when a with the extraction Water-miscible capable microorganisms (ATCC 20305) inoculated from an organic solvent, e.g. acetone, Me-agar slant culture, which had been cultivated at 25 to 27 ° C for 10 to 14 days using ethanol or ethanol as an extractant. The result is, the extract liquid is pressurized under reduced animal culture as a wood chip inoculum and the concentrated liquid is then used.

a renewed extraction with a non-hydro- This then became 500 ml of sterilized GY-phile organic solvent such as medium added. 100 ml each of the microorganism ethyl acetate or methyl isobutyl ketone, men suspension were placed in 5 Erlenmeyer flasks from whereby water-soluble impurities are removed 65 5 1 content with 1 1 medium of the following combination. Settlement I brought and 72 hours on a rotating-In the extract liquid are next to the desired shaking cultivator with 190 rpm at 27 ° C th 5-dihydrocoriolin C in addition to Coriolin B and cultivated.

Composition I (inoculation medium)

Glucose 5.0%

Peptone 0.2%

Dried brewer's yeast 0.3%

Potassium dihydrogen phosphate 0.3%

Magnesium sulfate 0.1%

A surfactant containing polyoxyethylene groups
Mean 0.01% ίο

The entire amount of this first seed liquid was placed in a 200 liter stainless fermentation tank Steel introduced with 1201 of a medium with the same composition as above and κ 72 hours with stirring (200 rpm) and with an aeration rate of 120 l / minute at 27'C cultivated.

Thereafter, 12 1 of the resulting second inoculum fluid brought in a 200-1 fermentation tank made of stainless steel zo-free 1201 medium of the following composition II and 168 hours as cultured under the same culture conditions above.

Composition 11 (production medium)

Glucose 5.0%

Peptone 0.2%

Dried brewer's yeast 0.3%

Potassium dihydrogen phosphate 0.3%

Magnesium sulfate 0.1% o

A surfactant containing polyoxyethylene groups

Mean 0.01%

Calcium carbonate 1.6%

·) Separately sterilized and added to the medium at the beginning of the culti- ^ added fourth.

After the completion of the cultivation, the obtained culture liquid was filtered to give 5.5 kg of moist Mycelium were obtained. This was transferred to a container and 9 l of acetone were added. To The solution was filtered through stirring and the mycelium was washed further with 31% acetone and the Wash liquid and the filtrate combined and concentrated. The concentrated liquid obtained was mixed with 5 l of ethyl acetate, stirred and extracted.

The solvent layer was separated and concentrated to 350 ml under reduced pressure and left to stand, with crystals of Coriolin B separating out. The crystals were filtered off and with washed a small amount of benzene.

b) According to the invention, the washing liquid and the filtrate were now combined and further to 150 ml concentrated, leaving a 5-dihydrocoriolin C precipitate was deposited. This was filtered off, washed with benzene and dried, yielding 8.59 g raw crystals were obtained.

The crude crystals were recrystallized from an 80% methanol aqueous solution, whereby 5.19 g of pure 5-dihydrocoriolin C (m.p .: 183 ° C) were obtained became.

Example 2
a) starting material

10 ml each of the second inoculation liquid obtained in the same way as in Example 1 was poured into 150 Shake flasks with a capacity of 500 ml with 125 ml of the medium according to the composition II and kept on a reciprocating shaker for 7 days at 27 ° C Cultivated 130 floats per minute. 15.5 l of the culture liquid obtained was filtered, whereby 550 g of wet mycelium was obtained.

This moist mycelium was mixed with 2 liters of acetone, stirred, extracted, filtered and washed with 1 liter of acetone washed. The washing liquid and the filtrate were combined and under reduced pressure constricted. 600 ml of the concentrated liquid was mixed with 1.21 ethyl acetate and extracted. the The resulting solvent layer was separated, concentrated to about 50 ml under reduced pressure and allowed to stand, whereby crystals of Coriolin B were precipitated. The crystals were filtered off and washed with benzene.

b) According to the invention, the filtrate and the washing liquid were now combined and reduced Pressure was reduced to dryness, yielding 5.6 g of a containing the desired 5-dihydrocoriolin C. oily material were obtained.

This oily material was dissolved in a small amount of chloroform and over one of 250 ml Silica gel (suspended in chloroform) packed column. Thereafter, a 1% methanol-chloroform mixed solvent was added passed through the column in an approximately equal volume of the column, whereby 5-octocoriolin B was eluted first. Subsequently, by further passing through the solvent mixture in about twice The amount corresponding to the column volume elutes the desired 5-dihydrocoriolin C through the column. The 5-dihydrocoriolin C fraction was collected, concentrated to dryness under reduced pressure and recrystallized from an aqueous 80% methanol solution, whereby 2.34 g of pure 5-dihydrocoriolin C (F .: 183 C).

The further processing of 5-dihydrocoriolin C can be seen from the following examples.

Example A.

3 g of 5-dihydrocoriolin C were in 30 ml of pyridine dissolved and with a suspension of a chromic acid-pyridine complex in pyridine, which by adding of 2.5 g of chromic acid to 40 ml of pyridine, mixed with stirring and added for 6 days 3 to 5 ° C brought to reaction.

After the reaction, 350 ml of ethyl acetate were added and the impurities deposited filtered off. The filtrate was washed gradually with a total of 500 ml of water, the upper one Layer separated and concentrated to dryness under reduced pressure, yielding 2.53 g of crude product were obtained.

The crude product was dissolved in 10 ml of ethanol and in one with 100 ml of a dextran derivative that is used for gel filtration in organic solvents (»Sephadex LH 20«), filled column chromatographed. The column was eluted with a total of 85 ml of ethanol. The last 25 ml of the eluted Liquid was collected and concentrated to dryness under reduced pressure. The narrowed one The residue was dissolved in 5 ml of chloroform and chromatographed in a column filled with 50 ml of silica gel. The column was eluted with 100 ml of a 1% M ethanol-chloroform solvent mixture, whereby an eluted fraction of 2'-dehydrocoriolin C was obtained.

709 623/203

The column was then filled with 100 ml of a 2% methanol-chloroform solvent mixture eluted, thereby obtaining a Coriolin C eluted fraction would.

Both fractions were separately concentrated to dryness under reduced pressure, yielding 267 g powdery 2'-dehydrocoriolin C (melting point: 51 to 55 ° C) and 851 mg of crude crystals of Coriolin C were obtained.

The crude crystals of Coriolin C were recrystallized from η-hexane to give 722 mg of Coriolin C (F .: 73 to 75 "C).

The Coriolin C obtained in the present example is a known substance of those already mentioned Structural formula

OH

-0-CO-CH- (CH ₁ I ₅ -CH ₃

OH

The 2'-dehydrocoriolin C, on the other hand, is a new compound with the following properties: Colorless powder; F .: 51-55 ° C; [«]? - 19.0 "(c = 1%. Chloroform); soluble in methanol, ethanol, ethyl acetate. Acetone, chloroform, benzene and carbon tetrachloride, but little or not soluble in water, η-hexane and petroleum ether. The elemental analysis of 2'- Dehydrocoriolin C gives 65.69% C and 7.67% H, which, taking into account the molecular weight determined by mass spectrometry, _{gives a sum formula of C 23} H ₃₂ O _7. As a result of the analysis of the NMR spectrum it was found that the 2'- Dehydrocoriolin C has the structural formula already mentioned

OH

J 1 — r> - cn —r—

owns.

O-CO-C- (CH ₂ Ij-CH ₃ O

Example B.

1 g of 5-dihydrocoriolin C was dissolved in 100 ml of anhydrous Benzene dissolved and mixed with 8 g of manganese dioxide with stirring and stored at room temperature for 32 h long reacted. The reaction mixture was filtered and the precipitate washed with anhydrous Benzene washed. The filtrate and the washing liquid were combined and under reduced pressure concentrated to dryness to give 610 mg of solid Malerial.

This solid material was dissolved in 3 ml of ethanol and filled with 25 ml of "Sephadex LH 20" Column chromatographies. The column was eluted with a total of 25 ml of ethanol. The last 9 ml of the

ίο eluted liquid fractions were collected and concentrated to dryness under reduced pressure.

The concentrated residue was dissolved in 1.2 ml of chloroform and filled into a 15 ml silica gel Column chromatographies. The column was filled with 30 ml of a 1% methanol-chloroform solvent mixture eluted, whereby an eluted fraction of 2'-dehydrocoriolin C was obtained. After that was obtained by eluting the columns with 30 ml of a 2% methanol-chloroform mixed solvent an eluted fraction of Coriolin C was obtained. Both fractions were taken under reduced pressure Concentrated to dryness, yielding 62 mg of powdered 2'-dehydrocoriolin C (F .: 51-55 ° C) and 180mg crude crystals of Coriolin C were obtained.

The latter were recrystallized from η-hexane, yielding 132 mg of Coriolin-C crystals with a Melting point of 73-75 ° C were obtained.

Examples

200 mg of 5-dihydrocoriolin C were dissolved in a solvent mixture of 1.2 ml of anhydrous dimethyl sulfoxide, 0.7 ml of benzene, 0.077 ml of pyridine and 0.038 ml of trifluoroacetic acid and dissolved with 300 mg Dicyclohexylcarbodiimide mixed with stirring. The solution was left to stand at room temperature for 1 hour and then for a further 15 hours at 3-4 ° C for reaction brought.

After the reaction had ended, 12 ml of ether and 130 mg of oxalic acid (as a 10% solution in Methanol) was added and after 30 minutes 12 ml of water were added. The precipitates obtained were filtered off and washed with ether.

The ethereal layer was separated from the filtrate and washed with a 5% aqueous sodium bicarbonate solution and then washed with water and concentrated under reduced pressure. The concentrate was separated by chromatography on silica gel in the same manner as in Example A, whereby 42 mg Coriolin C crystals (F .: 73-75 ° C) and 13 mg of powdery 2'-dehydrocoriolin C (F .: 51-55 C) were obtained.

1 sheet of drawings

Claims (1)

Patent claims: I. 5-Dihydrocoriolin C of Formula
OH HO γν - * -0-CO-CH- (CH ₂ ) S-CH,