CS254050B1

CS254050B1 - Process for the preparation of acetyldigoxin

Info

Publication number: CS254050B1
Application number: CS383986A
Authority: CS
Inventors: Jan Fuska; Bohumil Proksa; Alzbeta Khandlova
Original assignee: Jan Fuska; Bohumil Proksa; Alzbeta Khandlova
Priority date: 1986-05-26
Filing date: 1986-05-26
Publication date: 1987-12-17

Abstract

Riešenie sa týká spósobu přípravy acetyldigoxinu tak, že sa do tekutého média obsahujúceho zdroj uhíka a dusíka naočkuje kultúra Penicillium vermiculatum Dang. CCM F-276 alebo Aspergillus niger CCM F- -330, potom sa přidá 10—60 mg lanatozidu C na 100 ml pódy a nechá sa kultivovat pri 26 až 30 °C po dobu 8 až 24 hodin. Filtrát vyfermentovanej půdy sa extrahuje chlorovanými uhlovodíkmi, s výhodou chloroformom, extrakt sa zahusti a získaný pevný produkt sa přečistí rekryštalizáciou zo zmesi rozpúšťadiel chloroform : benzén. Filtrát pódy možno extrahovat tiež estermi kyseliny octovej s výhodou octanom etylnatým.The solution relates to a method of preparing acetyldigoxin by inoculating a culture of Penicillium vermiculatum Dang into a liquid medium containing a source of carbon and nitrogen. CCM F-276 or Aspergillus niger CCM F-330, then 10-60 mg of lanatoside C per 100 ml of the soil is added and cultured at 26-30°C for 8-24 hours. The filtrate of the fermented soil is extracted with chlorinated hydrocarbons, preferably chloroform, the extract is concentrated and the obtained solid product is purified by recrystallization from a solvent mixture of chloroform:benzene. The soil filtrate can also be extracted with acetic acid esters, preferably with ethyl acetate.

Description

254050 . Vynález sa týká spůsobu přípravy acetyl- digoxinu selektívnou mikrobiálnou degluko- zidáciou lanatozidu C pomocou mikroorga-254050. The present invention relates to a process for the preparation of acetyl digoxin by selective microbial deglucosidation of lanatoside C by microorganisms

nizmov Penicillium vermiculátum Dang. CCM F-276 alebo Aspergillus niger CCM F-330. V predkladanom riešení sa uvádza spo-sob přípravy acetyldigoxinu, ktorého pod-stata spočívá v tom, že sa do živnej půdyobsahujúcej zdroj uhlíka, organického a an-organického dusíka, naočkovanej kultúrouP. vermiculátum přidá lanatozid C (100 až500 mg . L-1) rozpuštěný v polárnom roz-púšťadle, s výhodou v dimetylformamide akultivuje sa 8—24 hodin pri teplote 26—30stupňov Celzia za stálého miešania a pre-vzdušňovania. Po transformácii sa filtrátpůdy, ktorá obsahuje acetyldigoxin extra-huje chloroformom, získaný extrakt sa povysušení, za zníženého tlaku zbaví rozpúš-ťadla a surový produkt sa rekryštalizuje zozmesi rozpúšťadiel benzen : etanol (3 : 1).Na extrakciu látky z filtrátu možno takistopoužit ďalšie chlorované uhlovodíky s poč-tom uhlíkov 1—5 s výhodou dichlórmetánalebo estery kyseliny octovej s výhodou oc-tan etylnatý.Penicillium Vermiculum Dang. CCM F-276 or Aspergillus niger CCM F-330. The present invention provides a process for the preparation of acetyldigoxin, the principle of which is to inoculate a culture medium containing carbon, an organic nitrogen and an organic nitrogen. vermiculumate adds lanatoside C (100-500 mg. L-1) dissolved in a polar solvent, preferably in dimethylformamide, is cultured for 8-24 hours at 26-30 degrees Celsius with constant agitation and aeration. After transformation, the filtrate containing the acetyldigoxin is extracted with chloroform, the extract obtained is dried, the solvents are removed under reduced pressure, and the crude product is recrystallized from the benzene: ethanol (3: 1) solvent mixture. hydrocarbons having carbon numbers 1-5 are preferably dichloromethane or acetic acid esters, preferably ethyl acetate.

Acetyldigoxin je biela krystalická látka,u ktorej holi namerané nasledujúce hodno-ty: t. t. 203—206°C (rozklad), pre sumárnyvzorec C43H66O15 823,0, vypočítané: C 62,75pere. H 8,08 %, nájdené C 62,59 %, H 7,96 %.IC (KBr) v cm"1: 3 410, 2 966, 2 943, 1741,1720, 1 631, 1457, 1 347. Krystalická látkaposkytla pri tenkovrstevnej chromatografiina Silufole v sústavách benzén : etanol :: voda (30 : 10 : 0,3) jednu škvrnu s hod-notami Rf 0,41 a v sústave octan etylnatý :: metanol : voda (79 : 19 : 11) škvrnu s Rf0,85. Důkaz aglykonu sa robil po hydrolýzezískanej látky s kyselinou sírovou. Pri ten-kovrstevnej chromatografii na SilufoleR vsústave benzén : octan etylnatý (1:1) po-skytla látka jednu škvrnu s Rf 0,28 a v sú-stave octan etylnatý taktiež jednu škvrnu sRf 0,11, ktoré bolí identické so skvrnami zís-kanými rovnakým postupom zo standarduacetyldigoxinu. V pokusoch použité kmene P. vermicula-tum aj A. niger transformujú přidaný lana-tozid C velmi selektívne, bez dalších sprie-vodných metabolitov takmer kvantitativnéza 8—24 hodin. V nasledujúcich príkladoch sú uvedenésposoby transformácie lanatozidu C na a-cetyldigoxin. Příklad 1Acetyldigoxin is a white crystalline substance having the following values measured: mp 203-206 ° C (decomposition), for C 43 H 66 O 15 823.0, calculated: C, 62.75; H 8.08%, found C 62.59%, H 7.96% .IC (KBr) in cm -1 1: 410, 2966, 2943, 1741, 1720, 1,631, 1457, 1,347. in the case of Silufole thin film chromatography in benzene: ethanol: water systems (30: 10: 0.3), a spot with an Rf value of 0.41 and a spot of ethyl acetate: methanol: water (79: 19: 11) Proof of aglycone was carried out after the hydrolysis of the recovered substance with sulfuric acid.For thin layer chromatography on Silufol®, benzene: ethyl acetate (1: 1) gave one spot with an Rf of 0.28 and acetate as acetate. Also, one strain of sRf 0.11 which is identical to the spots obtained by the same procedure from standard acetyldigoxin was used in the experiments. almost quantitative 8-24 hours In the following examples, the transformation of lanatoside C to α-cetyl digest oxine Example 1

Do 25 varných baniek o objeme 500 mlpřipraví sa podá nasledujúceho zloženia:(g . L-1) glukóza 20, kvasničný extrakt 5,sójová múka 5, NaCl 5, KH2PO4 5, vodovod-ná voda 1 liter, pH 6,5-7,0. Póda sa sterili-zovala 15 minút pri 120 °C a po ochladení sanaočkovala 15 ml vegetatívneho inokula Pe-The following composition is prepared in 25 500 ml flasks: (g. L-1) glucose 20, yeast extract 5, soy flour 5, NaCl 5, KH 2 PO 4 5, tap water 1 liter, pH 6.5-7 , 0. The stage was sterilized at 120 ° C for 15 minutes and inoculated with 15 ml of the vegetative inoculum Pe-

nicillium vermiculátum. Kultivácia pri tep-lote 28 °C na rotačnej trepačke s 220 ot .. min-1. Po 24 hodinovej kultivácii sa dokaždej banky přidalo 30 mg lanatozidu Crozpuštěného v 0,3 ml dimetylformamidu.Celkom sa v pokuse použilo 720 mg lanato-zidu C. Transformácia sa ukončila v 20. ho-dině kultivácie, kedy bol přidaný substráttakmer kvantitativné transformovaný. My-célium použitého mikroorganizmu sa oddě-lilo filtráciou, získaný filtrát (1,900 ml, pH 6.1) , sa extrahoval trikrát za sebou chloro-formom (v pomere 2 : 1), za neustáléhomiešania po dobu 15—20 minút, pri labora-torně]' teplote. Získané extrakty sa spojili,bezvodým síranom sodným výsušili a zazníženého tlaku zahustili. Po zahuštění aochladení na +4°C sa vylúčil acetyldigo-xin, ktorý sa oddělil filtráciou a premyl di-etyléterom. Po přidaní dietyléteru, ktorý sazískal po premytí prvého podielu surovéhoproduktu k matečným lúhom, vypadla ďal-šia časť acetyldigoxinu. Získané surové pro-dukty sa spojili a rekryštalizovali zo zmesirozpúšťadiel chloroform : benzén (1 : 3).Celkom sa získalo 420 mg čistého acetyldi-goxinu, čo představuje výťažok 62 % vzhťa-dom na použitý substrát. Příklad 2nicillium vermiculátum. Cultivation at 28 ° C on a rotary shaker with 220 rpm. After 24 hours of cultivation, 30 mg of lanatoside dissolved in 0.3 ml of dimethylformamide was added to each flask. 720 mg of laatonide C was used in the assay. The transformation was terminated in the 20th hour of culture, when the added substrate was quantitatively transformed. The microorganism used was separated by filtration, the filtrate obtained (1.900 ml, pH 6.1), extracted three times in succession with chloroform (2: 1), stirring for 15-20 minutes, at room temperature. ] 'temperature. The extracts were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. After concentration and cooling to + 4 ° C, acetyldigoxine precipitated, which was collected by filtration and washed with diethyl ether. After the diethyl ether was added to the mother liquors after washing the first portion of the crude product, a further portion of the acetyldigoxin precipitated. The crude products obtained were combined and recrystallized from a solvent mixture of chloroform: benzene (1: 3). A total of 420 mg of pure acetyldioxine was obtained, representing a yield of 62% based on the substrate used. Example 2

V pokuse sa použila půda rovnakého zlo-ženia ako v pokuse 1. Po 24 hodinovej kul-tivácii sa do půdy přidalo 60 mg lanatoziduC na 100 ml půdy. Transformácia sa ukon-čila v 24 hodině. Číry filtrát (1,800 ml, pH 6.2) sa trikrát za sebou extrahoval octanometylnatým (v pomere 2:1). Spojené extrak-ty po vysušení bezvodým síranom sodnýmsa zahustili a po ochladení na —(-4 °C sa vy-lúčil acetyldigoxin. K matečným lúhom sapřidal dvojnásobný objem dietyléteru, čímsa získal další podiel acetyldigoxinu. Suro-vé produkty sa rekryštalizovali zo zmesi oc-tan etylnatý : benzén (3 : 1). Celkom sa zís-kalo 510 mg čistého acetyldigoxinu, čo před-stavuje 63,7 % výťažok, vzhladom k použi-tému substrátu. Příklad 3In the experiment, soil of the same composition was used as in Experiment 1. After 24 hours of cultivation, 60 mg of lanatoside C per 100 ml of soil was added to the soil. The transformation was terminated at 24 hours. The clear filtrate (1.800 mL, pH 6.2) was extracted three times in succession with octanomethyl (2: 1). The combined extracts were dried over anhydrous sodium sulphate and, after cooling to - (4 ° C, acetyldigoxin was precipitated. Two volumes of diethyl ether was added to the mother liquors to give an additional amount of acetyldigoxin. Ethyl ethyl: benzene (3: 1) A total of 510 mg of pure acetyldigoxin was obtained, representing a yield of 63.7% relative to the substrate used.

Postup biotransformácie lanatozidu C bolrovnaký ako je uvedený v příklade 1, alevytvořený acetyldigoxin sa extrahoval di-chlórmetánom. Výťažok vzhfadom k použi-tému substrátu bol 57,2 °/o. Příklad 4The biotransformation procedure of lanatoside C was as described in Example 1, but the acetyldigoxin formed was extracted with dichloromethane. The yield of substrate used was 57.2 °. Example 4

Postup biotransformácie bol rovnaký ako je uvedený v příklade 1, ale ako mikroorga- nizmus sa použil kmen Aspergillus niger CCM F-330. Výťažok acetyldigoxinu vzhfa- dom k použitému substrátu bol 48,7 %.The biotransformation procedure was as described in Example 1, but the Aspergillus niger strain CCM F-330 was used as a microorganism. The yield of acetyldigoxin relative to the substrate used was 48.7%.

Claims

254050 PREDMET

A process for the preparation of acetyldigoxin characterized in that a Peni-cillium vermiculatum Dang culture is inoculated into a liquid medium containing a carbon source, an organic nitrogen and an organic nitrogen. CCM F-276 or Aspergillus niger CCM F-330, 10 to 60 mg of lanatoside C dissolved in a polar solvent per 100 ml of water are added, cultured at 26 to 30 ° C with stirring and aeration for 12-24 h The filtrate is extracted with organic solvents, the extract is concentrated after drying and the residue is crystallized.

2. The process according to claim 1, wherein dimethyl polar amide is used as the polar solvent to dissolve lanatoside C.

3. A process according to claim 1, wherein the filtrate after cultivation is extracted with chlorinated hydrocarbons having a carbon number of 1 to 4, preferably chloroform, or acetic acid esters with alcohols having carbon numbers of 1 to 3, preferably ethyl acetate.

4. Process according to claim 1, characterized in that the residue after evaporation of the extractant is recrystallized from chloroform-benzene or ethyl acetate-benzene.