NO122265B - - Google Patents
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- Publication number
- NO122265B NO122265B NO15160064A NO15160064A NO122265B NO 122265 B NO122265 B NO 122265B NO 15160064 A NO15160064 A NO 15160064A NO 15160064 A NO15160064 A NO 15160064A NO 122265 B NO122265 B NO 122265B
- Authority
- NO
- Norway
- Prior art keywords
- dione
- tetraol
- pregnadiene
- hours
- residue
- Prior art date
Links
- 150000003431 steroids Chemical class 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 25
- 241000187654 Nocardia Species 0.000 claims description 14
- -1 methylene, hydroxymethylene Chemical group 0.000 claims description 11
- 241000203720 Pimelobacter simplex Species 0.000 claims description 8
- QQCBOIWDENXRLP-BYZMTCBYSA-N (8s,9s,10r,13r,14s,17s)-17-ethyl-10,13-dimethyl-6,7,8,9,11,12,14,15,16,17-decahydro-3h-cyclopenta[a]phenanthrene Chemical class C1CC2=CCC=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](CC)[C@@]1(C)CC2 QQCBOIWDENXRLP-BYZMTCBYSA-N 0.000 claims description 7
- 229910052739 hydrogen Inorganic materials 0.000 claims description 7
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 7
- 239000001257 hydrogen Substances 0.000 claims description 6
- 241000894007 species Species 0.000 claims description 4
- 241001442589 Convoluta Species 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 125000002252 acyl group Chemical group 0.000 claims description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- 229910052736 halogen Chemical group 0.000 claims description 2
- 150000002367 halogens Chemical group 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
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- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
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- 239000012085 test solution Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- MUUHXGOJWVMBDY-UHFFFAOYSA-L tetrazolium blue Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC=CC=2)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 MUUHXGOJWVMBDY-UHFFFAOYSA-L 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 239000006150 trypticase soy agar Substances 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
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- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F24—HEATING; RANGES; VENTILATING
- F24C—DOMESTIC STOVES OR RANGES ; DETAILS OF DOMESTIC STOVES OR RANGES, OF GENERAL APPLICATION
- F24C7/00—Stoves or ranges heated by electric energy
- F24C7/04—Stoves or ranges heated by electric energy with heat radiated directly from the heating element
- F24C7/043—Stoves
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- H—ELECTRICITY
- H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
- H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
- H05B3/00—Ohmic-resistance heating
- H05B3/10—Heating elements characterised by the composition or nature of the materials or by the arrangement of the conductor
- H05B3/16—Heating elements characterised by the composition or nature of the materials or by the arrangement of the conductor the conductor being mounted on an insulating base
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- Combustion & Propulsion (AREA)
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- Resistance Heating (AREA)
Description
Fremgangsmåte for fremstilling av nye steroidforbindelser av 1,4-pregnadienrekken. Process for the production of new steroid compounds of the 1,4-pregnadiene series.
Foreliggende oppfinnelse vedrører en fremgangsmåte for fremstilling av nye steroidforbindelser av 1,4-pregnadienrekken. The present invention relates to a method for the production of new steroid compounds of the 1,4-pregnadiene series.
I den senere tid er en rekke av steroider av pregnen- og pregnadienrekken, f. eks. hydrocortison og l(2)-dehydro-hydrocortison blitt viktige terapeutiske midler og nyttige som mellomprodukter for fremstilling av andre terapeutisk nyttige steroider. De nye 1,4-pregnadiener som fåes ifølge foreliggende oppfinnelse, er brukbare som anti-inflammasjonsmidler ved behandling av arthritis, asthma, brannsår, bursitis o. 1., og også ved behandling av hudsykdommer og kollagen-sykdommer. Forbindelsene anvendes i forening med fyllstoffer, binde-midler osv. i tabletter, pulver, piller osv. De kan også anvendes parenteralt i en opp-løsning eller i en suspensjon. In recent times, a number of steroids of the pregnene and pregnadiene series, e.g. hydrocortisone and 1(2)-dehydro-hydrocortisone have become important therapeutic agents and useful as intermediates for the preparation of other therapeutically useful steroids. The new 1,4-pregnadienes obtained according to the present invention are useful as anti-inflammatory agents in the treatment of arthritis, asthma, burns, bursitis etc., and also in the treatment of skin diseases and collagen diseases. The compounds are used in combination with fillers, binders, etc. in tablets, powders, pills, etc. They can also be used parenterally in a solution or in a suspension.
I henhold til foreliggende oppfinnelse fremstilles de nye 1,4-pregnadiener, idet en 4-pregnenforbindelse av den alminnelige formel: According to the present invention, the new 1,4-pregnadienes are produced, a 4-pregnane compound of the general formula:
hvor X er hydrogen eller halogen, Y en methylen-, hydroxy-methylen- eller carbonyl-gruppe, Z en hydroxymethylen- eller lavere alkanoyloxymethylengruppe, R' er hydrogen eller en hydroxylgruppe og R er hydrogen eller en lavere alkanoylgruppe, påkjent måte utsettes for den fermentative, enzymatiske innvirkning av artene av slekten Nocardia eller arten Corynebacterium simplex, hvoretter det resulterende steroid kan hydrolyseres eller forestres ved hjelp av kjente fremgangsmåter. where X is hydrogen or halogen, Y a methylene, hydroxymethylene or carbonyl group, Z a hydroxymethylene or lower alkanoyloxymethylene group, R' is hydrogen or a hydroxyl group and R is hydrogen or a lower alkanoyl group, is exposed to the fermentative, enzymatic action of the species of the genus Nocardia or the species Corynebacterium simplex, after which the resulting steroid can be hydrolyzed or esterified using known methods.
De ved fremgangsmåten ifølge oppfinnelsen dannede 1,4-pregnadiener svarer til den generelle formel: The 1,4-pregnadienes formed by the method according to the invention correspond to the general formula:
hvor de enkelte symboler har den ovenfor angitte betydning. where the individual symbols have the meaning stated above.
Ved utførelse av fremgangsmåten ifølge oppfinnelsen kultiveres en sopp av slekten Nocardia, (Bergey, Manual, 6. Edition), som f. eks. corallina, ATCC 999 og ATCC 4273 (Lederle Culture nr. 13), asteroides, ATCC 3308, keratolyticus, ATCC 12484, transvalensis, ATCC 12485, erythropolis, ATCC 4277, convoluta, ATCC 4275, gardneri, ATCC 9604, leishmanii, ATCC 6855, opaca, ATCC 4276, blackwelii, ATCC 6846, caviae, ATCC 6848, cuniculi, ATCC 6864, globerula, ATCC 9356, polychromogenes, ATCC 3409, sylvodifera, ATCC 7372, farcinica, ATCC 3318, sylvodifera, ATCC 4919 eller andre arter av Nocardia eller fungus Corynebacterium simplex aerobisk i et passende næringsmedium med et 4-pregnenderivat. Under veksten av organismen under gunstige forhold elimineres to hydrogenatomer fra steroidforbindelse ring A, og det fåes herved en dobbeltbinding i 1(2)-stilling. Den nøy-aktige mekanisme for denne dehydrogene-ring er uklar, men er resultatet av enzymer som dannes av organismen under vekstfor-løpet. Et passende næringsmedium inneholder en oppløselig kilde for carbon, nitrogen og mineralstoffer. Carbonkilder omfatter sukkerarter, som f. eks. glucose, sucrose, maltose, dextrose, xylose og galac-tose, og også alkoholer som glycerol eller mannitol, maisstivelse, organiske syrer som citronsyre, eplesyre og eddiksyre og forskjellige naturprodukter, som inneholder carbohydrater, som f. eks. maisutlutningsvæske, sojabønnemel, bomullsfrømel og mange andre tilgjengelige materialer som er blitt anvendt tidligere som carbonkilde ved fermenteringsprosesser. Vanligvis kan en variasjon av ovennevnte anvendes i mediet med gode resultater. When carrying out the method according to the invention, a fungus of the genus Nocardia is cultivated (Bergey, Manual, 6th Edition), which e.g. corallina, ATCC 999 and ATCC 4273 (Lederle Culture No. 13), asteroides, ATCC 3308, keratolyticus, ATCC 12484, transvalensis, ATCC 12485, erythropolis, ATCC 4277, convoluta, ATCC 4275, gardneri, ATCC 9604, leishmanii, ATCC 6855, opaca, ATCC 4276, blackwelii, ATCC 6846, caviae, ATCC 6848, cuniculi, ATCC 6864, globerula, ATCC 9356, polychromogenes, ATCC 3409, sylvodifera, ATCC 7372, farcinica, ATCC 3318, sylvodifera, ATCC 4919 or other species of Nocardia or fungus Corynebacterium simplex aerobically in a suitable nutrient medium with a 4-pregnene derivative. During the growth of the organism under favorable conditions, two hydrogen atoms are eliminated from steroid compound ring A, and a double bond is thereby obtained in the 1(2) position. The exact mechanism for this dehydrogenation is unclear, but is the result of enzymes formed by the organism during the growth process. A suitable nutrient medium contains a soluble source of carbon, nitrogen and minerals. Carbon sources include sugars, such as glucose, sucrose, maltose, dextrose, xylose and galactose, and also alcohols such as glycerol or mannitol, corn starch, organic acids such as citric acid, malic acid and acetic acid and various natural products, which contain carbohydrates, such as e.g. corn leachate, soybean meal, cottonseed meal and many other available materials that have been used in the past as a carbon source in fermentation processes. Usually a variation of the above can be used in the medium with good results.
Egnede kilder for nitrogen omfatter enkelte av de ovennevnte stoffer, som f. eks. maisutlutningsvæske, sojabønnemel, bom-ullsfrømel og lignende, og forskjellige andre slags substanser som f. eks. kjøtteks-trakt, casein, gjær, enzymatisk nedbyggede proteiner og andre nedbygningsprodukter, innbefattet peptoner, aminosyrer og mange andre tilgjengelige proteinholdige materialer som har vist seg å være skikket til å understøtte veksten av sopper o. 1. Anorganiske kilder for nitrogen, omfattende urin-stoffer, ammoniumsalter, nitrater, og lignende kan anvendes i mediet som en kilde for assimilert nitrogen for å tilveiebringe et gunstig vekstmedium for organismen. Suitable sources for nitrogen include some of the above substances, such as e.g. corn leach liquid, soybean meal, cottonseed meal and the like, and various other kinds of substances such as e.g. meat extract, casein, yeast, enzymatically degraded proteins and other breakdown products, including peptones, amino acids and many other available proteinaceous materials which have been shown to be suitable for supporting the growth of fungi etc. 1. Inorganic sources of nitrogen, including urine substances, ammonium salts, nitrates, and the like can be used in the medium as a source of assimilated nitrogen to provide a favorable growth medium for the organism.
Mineralkravene for fermenteringen tilveiebringes vanligvis i råmaterialene, som ofte anvendes som kilder for carbon og nitrogen eller forekommer også i vannet som anvendes ved prosessen. Det er imid-lertid vanligvis tilrådelig å supplementere de mineraler som vanligvis er til stede, med tilsatte mengder for å få en maksimal vekst av Nocardia. Kationer og anioner som kan være ønskelige i de tilsatte mengder, omfatter natrium, calcium, kalium, magnesi-um, fosfat, sulfat, klorid, kobolt, mangan og forskjellige andre. Anvendelse av spor-elementer som f. eks. bor, kobber, kobolt, molybden og krom er ofte ønskelige. The mineral requirements for the fermentation are usually provided in the raw materials, which are often used as sources of carbon and nitrogen or also occur in the water used in the process. However, it is usually advisable to supplement the minerals normally present with added amounts to obtain maximum growth of Nocardia. Cations and anions which may be desirable in the amounts added include sodium, calcium, potassium, magnesium, phosphate, sulfate, chloride, cobalt, manganese and various others. Application of track elements such as e.g. boron, copper, cobalt, molybdenum and chromium are often desirable.
Veksten av Nocardia fungus eller av Corynebacterium simplex fungus finner sted under aerobiske forhold og luftingen i kolber kan f. eks. oppnåes ved omrøring på en reciproserende eller roterende rysteinnretning eller i flasker eller tanker, ved å tvinge stabil luft gjennom fermenterings-blandingen. Det er ønskelig at den stabile luft tvinges gjennom mediet i en mengde av fra y3 til 2 volumdeler luft pr. volum av medium pr. minutt. Omrøring i flasken eller The growth of Nocardia fungus or of Corynebacterium simplex fungus takes place under aerobic conditions and the aeration in flasks can e.g. achieved by agitation on a reciprocating or rotary shaking device or in bottles or tanks, by forcing stable air through the fermentation mixture. It is desirable that the stable air is forced through the medium in an amount of from y3 to 2 parts by volume of air per volume of medium per minute. Stirring in the bottle or
fermenteringsbeholderen bevirkes ved hjelp the fermentation vessel is effected by means of
av et mekanisk røreapparat. Nocardia fungus vil vokse eller gro ved temperaturer of a mechanical stirrer. Nocardia fungus will grow or thrive at temperatures
mellom 5 og 45° C, men det er å foretrekke å utføre prosessen under anvendelse av en temperatur fra 25° C til 37° C. Corynebacterium simplex vil vokse ved temperaturer mellom 10° C og 45° C, men fortrinsvis anvendes en temperatur fra 25° C til 38° C. between 5 and 45° C, but it is preferable to carry out the process using a temperature from 25° C to 37° C. Corynebacterium simplex will grow at temperatures between 10° C and 45° C, but preferably a temperature from 25°C to 38°C.
For å tilveiebringe et inokulat anvendes 1,0 ml av en vasket vegetativ cellesuspensjon av fungi av slekten Nocardia eller av Corynebacterium simplex for å inokulere 100 ml av en steril Trypticase-soja-dyrkningsvæske i en 500 ml Erlenmeyerkolbe. Trypticase-sojavæsken inneholder 3 pst. av tryptisk nedbrutte sojaproteiner som et dehydratisert pulver (Baltimore Bacte-riological Laboratories), glycerin — 5 pst., kjøttekstrakt (Armour) — 0,3 pst., og denne blanding steriliseres i 15 minutter ved en temperatur av 120° C ved et damptrykk av 1,1 kg/cm<2>. Etter sterilisering er pH i området 7,4 til 7,7. Den inokulerte eller podete kolbe inkuberes ved 37° C på en rysteinnretning i ca. 4—8 timer. Et slikt inokulat kan anvendes for å pode større charger av sterilt medium i flasker, og slike flaskekul-turer kan etter fermentering anvendes for å pode store charger av medium i fermen-teringstanker. Istedenfor den ovenfor be-skrevne Trypticase-sojavæske kan det anvendes andre medier, slik som det fremgår av følgende eksempler. To provide an inoculum, 1.0 ml of a washed vegetative cell suspension of fungi of the genus Nocardia or of Corynebacterium simplex is used to inoculate 100 ml of a sterile Trypticase-soy culture medium in a 500 ml Erlenmeyer flask. The trypticase soy liquid contains 3 percent tryptic-degraded soy proteins as a dehydrated powder (Baltimore Bacteriological Laboratories), glycerin — 5 percent, meat extract (Armour) — 0.3 percent, and this mixture is sterilized for 15 minutes at a temperature of 120° C at a vapor pressure of 1.1 kg/cm<2>. After sterilization, the pH is in the range 7.4 to 7.7. The inoculated or inoculated flask is incubated at 37° C on a shaker for approx. 4-8 hours. Such an inoculum can be used to inoculate larger loads of sterile medium in bottles, and such bottle cultures can, after fermentation, be used to inoculate large loads of medium in fermentation tanks. Instead of the Trypticase soy liquid described above, other media can be used, as can be seen from the following examples.
De ved fremgangsmåten ifølge oppfinnelsen anvendbare, i 4-stilling umettede forbindelser av pregnanrekken omfatter bl. a.: 4-pregnen-ll|3,16a,17a,21-tetraol-3,20-dion, 4-pregnen-9a,fluor-ll(3,16a,17a,21-tetraol- 3,20-dion og 4-pregnen-16a,17a,21-triol-3,ll,20-trion samt estere og lignende derivater av disse forbindelser. Ved anvendelsen av de ovennevnte steroidsubstrater ved fermenteringen, dannes de frie steroidforbindelser. Disse steroider tilsettes generelt til fermenteringen i oppløsning eller i findelt form. En fortrinsvis anvendt fremgangsmåte er å oppløse steroidet i ethanol eller et annet vann-blandbart oppløsningsmiddel, og tilsette det til fermenteringsmediet på et øn-sket trinn i fremgangsmåten. Skjønt steroidet kan utfelles fra oppløsningen når det tilsettes på denne måten, dispergeres det i mediet som en fin suspensjon og blir lett tilgjengelig for organismen for å bevirke oxydasjon. Mengden av steroid som tilsettes til dyrkningen kan variere ganske betrak-telig, men er generelt av størrelsesordenen 1/10 til 1 gram pr. liter av mediet. The 4-position unsaturated compounds of the pregnane series which can be used in the method according to the invention include a.: 4-pregnene-ll|3,16a,17a,21-tetraol-3,20-dione, 4-pregnene-9a,fluoro-ll(3,16a,17a,21-tetraol- 3,20-dione and 4-pregnene-16a,17a,21-triol-3,11,20-trione as well as esters and similar derivatives of these compounds. When the above-mentioned steroid substrates are used in the fermentation, free steroid compounds are formed. These steroids are generally added to the fermentation in solution or in finely divided form. A preferably used method is to dissolve the steroid in ethanol or another water-miscible solvent, and add it to the fermentation medium at a desired step in the method. Although the steroid may precipitate from the solution when added in this way, it is dispersed in the medium as a fine suspension and becomes readily available to the organism to effect oxidation. The amount of steroid added to the culture can vary quite considerably, but is generally of the order of 1/10 to 1 gram per liters of the medium.
Under dyrkningsprosessen kan det være ønskelig å tilsette anti-skumnings-midler, som f. eks. siliconer, glyceridolj er og lignende. Disse forbindelser tilsettes fra tid til annen og i de krevete mengder. During the cultivation process, it may be desirable to add anti-foaming agents, such as e.g. silicones, glyceride oil and the like. These compounds are added from time to time and in the required quantities.
Ved fremgangsmåten ifølge oppfinnelsen inkuberes vanligvis 10 ml's charger av podet medium i 100 ml's rysterør i en periode av ca. 16 til 40 timer ved en temperatur av ca. 32° C. Ved dette tidspunkt tilsettes 2 mg sterilt substrat (4-pregnenforbindelse) oppløst i 0,2 ml ethanol til hvert rør, og dyrkingen eller fermenteringen fortset-tes ved ca. 32° C. Dyrkingen får anledning til å fortsette i en periode som er tilstrekke-lig til å oppnå en maksimal omdannelse av 4-pregnen til 1,4-pregnadien. Denne tids-periode kan variere fra 1 til 72 timer eller lenger. In the method according to the invention, 10 ml chargers of inoculated medium are usually incubated in 100 ml shaking tubes for a period of approx. 16 to 40 hours at a temperature of approx. 32° C. At this point, 2 mg of sterile substrate (4-pregnene compound) dissolved in 0.2 ml of ethanol is added to each tube, and the cultivation or fermentation is continued at approx. 32° C. The cultivation is allowed to continue for a period which is sufficient to achieve a maximum conversion of the 4-pregnade into the 1,4-pregnade. This time period can vary from 1 to 72 hours or longer.
Ved avslutningen av dyrkingsprosessen utvinnes det ønskede 1,4-pregnadien ved hjelp av den følgende fremgangsmåte, som beskriver som eksempel en 10 ml fermentering. Dette er en generell fremgangsmåte, og kan utføres for dyrkinger av forskjellig størrelse. At the end of the cultivation process, the desired 1,4-pregnadiene is recovered by means of the following method, which describes as an example a 10 ml fermentation. This is a general procedure, and can be carried out for cultivations of different sizes.
Innholdet i dyrkingsrøret ekstraheres fire ganger med 4 volum methylenklorid. De fire ekstrakter forenes og den resulterende oppløsning vaskes derpå én gang med 2 pst.s vandig natriumbicarbonat mettet med natriumklorid, og vaskes derpå to ganger med mettet natriumkloridoppløsning. Den vaskete methylenkloridoppløsning kan tørkes over vannfritt magnesiumsulfat og filtreres. Filtratet konsentreres på dampbad ved atmosfærisk trykk til et lite volum og konsentratet overføres til en 10 ml's målekolbe og fylles med methylenklorid. Denne oppløsning anvendes for bestem-melse av steroidinnholdet, som beskrevet i det følgende. The contents of the culture tube are extracted four times with 4 volumes of methylene chloride. The four extracts are combined and the resulting solution is then washed once with 2% aqueous sodium bicarbonate saturated with sodium chloride, and then washed twice with saturated sodium chloride solution. The washed methylene chloride solution can be dried over anhydrous magnesium sulfate and filtered. The filtrate is concentrated on a steam bath at atmospheric pressure to a small volume and the concentrate is transferred to a 10 ml volumetric flask and filled with methylene chloride. This solution is used to determine the steroid content, as described below.
Ved dyrkingsprosesser i stor målestokk kan det rå produkt eller produktene utvinnes fra dyrkingsmaterialet ved hjelp av en enkel ekstraksjon med et oppløsningsmid-del, idet det anvendes et egnet oppløsnings-middel, som ikke er blandbart med vann, som f. eks. klorerte lavere hydrocarboner, alkoholer, estere, ketoner osv. Ytterligere rensning og utskillelse av steroidprodukter fra ekstraktene kan utføres ved hjelp av fremgangsmåter som er kjent for fagfolk på området. Utvinningen og rensningen av steroidblandingen krever ofte anvendelse av kromatografi. In the case of cultivation processes on a large scale, the raw product or products can be extracted from the cultivation material by means of a simple extraction with a solvent, using a suitable solvent which is not miscible with water, such as e.g. chlorinated lower hydrocarbons, alcohols, esters, ketones, etc. Further purification and separation of steroid products from the extracts can be accomplished by methods known to those skilled in the art. The recovery and purification of the steroid mixture often requires the use of chromatography.
Den fremgangsmåte som anvendes for å identifisere de steroider som er til stede i det ekstraherte dyrkingsmateriale, som foran nevnt, er papirstrimmelkromatogra-fien. Oppløsningsmiddelsystemet som anvendes, er vann-methanol-benzen frem-stillet ved rystning av ca. 50 pst. vann — 50 pst. metnanol med benzen i en skille-trakt, hvorpå man lar de to lag skille seg fra hverandre. En del av det nedre lag an-bringes i en åpen skål på bunnen av en stor glass-sylinder. Det øvre lag er oppløsnings-fasen og anvendes til å fylle den traufor-mete del inne i sylinderen. Det fremstilles en standard steroid-oppløsning ved å opp-løse 10 mg av hver av de følgende steroider i 10 ml methylenklorid: 4-pregnen-lip,17a,21-triol-3,20-dion, 4-pregnen-ll|3,21-diol-3,20-dion, 4-pregnen-17a,21-diol-3,20-dion, 4-pregnen-lla,17a,21-triol-3,20-dion, 4-pregnen-ll|3,16a,17a,21-tetraol-3,20-dion, 4-pregnen-lip,16a,17ni,21-tetraol-3,20- dion-16,21-diacetat, The method used to identify the steroids present in the extracted culture material, as mentioned above, is paper strip chromatography. The solvent system used is water-methanol-benzene prepared by shaking approx. 50 per cent water — 50 per cent methanol with benzene in a separatory funnel, after which the two layers are allowed to separate from each other. Part of the lower layer is placed in an open bowl at the bottom of a large glass cylinder. The upper layer is the dissolution phase and is used to fill the trough-shaped part inside the cylinder. A standard steroid solution is prepared by dissolving 10 mg of each of the following steroids in 10 ml of methylene chloride: 4-pregnene-lip,17a,21-triol-3,20-dione, 4-pregnene-11|3 ,21-diol-3,20-dione, 4-pregnene-17a,21-diol-3,20-dione, 4-pregnene-lla,17a,21-triol-3,20-dione, 4-pregnene-ll |3,16a,17a,21-tetraol-3,20-dione, 4-pregnen-lip,16a,17ni,21-tetraol-3,20- dione-16,21-diacetate,
4-pregnen-9a-fluor-ll|3,16a,17a,21-tetraol-3,20-dion, 4-pregnene-9a-fluoro-ll|3,16a,17a,21-tetraol-3,20-dione,
4-pregnen-9a-fluor-ll(5,16a,17a,21-tetraol-3,20-dion-16,21-diacetat. 4-pregnene-9a-fluoro-11(5,16a,17a,21-tetraol-3,20-dione-16,21-diacetate.
(Andre steroider kan anvendes i standard-oppløsningen når dette er ønskelig.) (Other steroids can be used in the standard solution when this is desired.)
I det minste en standard steroid-opp-løsning kromatograferes samtidig hver • gang man undersøker en ukjent oppløs-ning. Nøyaktig 0,025 ml av standard-ster-oidprøveoppløsningen påføres papirstrim-len ved startlinjen, 10 cm fra den øvre ende av strimlen, som foldes over kanten av trauet og neddykkes i oppløsningsmiddel-fasen i dette. Strimlen fremkalles derpå i 2—4 timer ved ca. 37° C. På lignende måte påføres 0,1 ml av den ukjente oppløsning på en annen strimmel, som derpå foldes inn i det samme trau og fremkalles samtidig med standard steroidstrimlen. Trauet muliggjør at det kan fremkalles mange strimler samtidig. Etter passende fremkal-ling av papirstrimlene, fjernes disse fra ap-paratet og tørkes med luft ved ca. 37° C. Etter tørkingen påsprøytes strimlene en alkalisk oppløsning av blått Tetrazolium, som fremkaller farve på de steder hvor det er til stede steroider. Farvefremkalte strim-t ler legges opp ved siden av i det minste en standard strimmel og det foretas en sam-menligning. De forskjellige steroider kan da identifiseres ved deres rekkefølge eller plasering på strimlene. At least one standard steroid solution is chromatographed at the same time • each time an unknown solution is examined. Exactly 0.025 ml of the standard steroid test solution is applied to the paper strip at the starting line, 10 cm from the upper end of the strip, which is folded over the edge of the trough and immersed in the solvent phase therein. The strip is then developed for 2-4 hours at approx. 37° C. In a similar manner, 0.1 ml of the unknown solution is applied to another strip, which is then folded into the same trough and developed at the same time as the standard steroid strip. The trough enables many strips to be developed simultaneously. After suitable development of the paper strips, these are removed from the apparatus and dried with air at approx. 37° C. After drying, the strips are sprayed with an alkaline solution of blue Tetrazolium, which induces color in the places where steroids are present. Color developed strips are placed next to at least one standard strip and a comparison is made. The different steroids can then be identified by their order or placement on the strips.
De ønskede, i 1- og 4-stilling umettede steroider er mer polare enn de tilsvarende i 4-stilling umettede steroider. Det skal vi-dere nevnes at når de ønskede i 1,4-stilling umettede steroidforbindelser er blitt iso-lert og bestemt, kan de selv anvendes i en standard-steroidoppløsning for å forbedre fremgangsmåten. The desired 1- and 4-position unsaturated steroids are more polar than the corresponding 4-position unsaturated steroids. It should also be mentioned that when the desired 1,4-position unsaturated steroid compounds have been isolated and determined, they can themselves be used in a standard steroid solution to improve the method.
I de følgende eksempler er det i detalj beskrevet dehydrogeneringen av i 4-stilling umettede forbindelser av pregnenrekken under dannelse av de tilsvarende i 1- og 4-stilling umettede steroider av pregnadienrekken. In the following examples, the dehydrogenation of 4-position unsaturated compounds of the pregnane series to form the corresponding 1- and 4-position unsaturated steroids of the pregnadiene series is described in detail.
Eksempel 1. Example 1.
l, 4- pregnadien- 9a- fluor- l lfi, l 6a, l 7a, 21 - l, 4- pregnadiene- 9a- fluoro- l lfi, l 6a, l 7a, 21 -
tetraol- 3, 20- dion. tetraol-3,20-dione.
En med gjærekstrakt tilberedt reagens-glass-skråkultur av Nocardia sp. (ATCC 12483) ble vasket med 7 ml steril saltopp-løsning, og den erholdte vegetativcellesus-pensjon ble anvendt for å inokulere 100 ml av en steril (som beskrevet ovenfor) Trypticase-sojabuljong i en 500 ml Erlenmeyerkolbe. Kolben ble inkubert i 7 timer ved 37° C. Dette inokulat ble derpå anvendt for å inokulere sterilt Trypticase-sojabuljongmedium i 100 ml's rysterør. 1 ml av inoku-latet ble anvendt for å inokulere hver 10 ml av et medium som inneholder melasse (Grandma's) 2 pst., kjøttekstrakt (Difco) 1 pst. og glucose 1 pst. i 100 ml rysterør. Rørene ble inkubert ved 32° C i 40 timer. Derpå ble det til hvert rør tilsatt 2 mg 4-pregnen-9a-fluor-lip,16a,17a,21-tetraol-3,20-dion oppløst i 0,2 ml ethanol. Inkube-ringen ble derpå fortsatt i ytterligere 24 timer. A test tube slant culture prepared with yeast extract of Nocardia sp. (ATCC 12483) was washed with 7 ml of sterile saline, and the resulting vegetative cell suspension was used to inoculate 100 ml of a sterile (as described above) Trypticase soy broth in a 500 ml Erlenmeyer flask. The flask was incubated for 7 hours at 37°C. This inoculum was then used to inoculate sterile Trypticase soy broth medium into 100 ml shake tubes. 1 ml of the inoculum was used to inoculate each 10 ml of a medium containing molasses (Grandma's) 2%, meat extract (Difco) 1% and glucose 1% in 100 ml shaker tubes. The tubes were incubated at 32°C for 40 hours. 2 mg of 4-pregnene-9a-fluoro-lip,16a,17a,21-tetraol-3,20-dione dissolved in 0.2 ml of ethanol was then added to each tube. Incubation was then continued for a further 24 hours.
10 ml av dyrkingsproduktet fra et av de ovennevnte rør ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene ble forenet og inndampet til tørrhet under vakuum. Residuet ble opptatt i 2 ml aceton, og en passende prøve ble analysert ved hjelp av den foran be-skrevne papirstrimmelteknikk. Papirstrim- 10 ml of the culture product from one of the above tubes was extracted four times with 40 ml of methylene chloride each time. The extracts were combined and evaporated to dryness under vacuum. The residue was taken up in 2 ml of acetone, and an appropriate sample was analyzed using the paper strip technique described above. paper strip
melkromatogrammet viste tilstedeværelsen av l,4-pregnadien-9a-fluor-ll(3,16a,17a,21-tetraol-3,20-dion. the flour chromatogram showed the presence of 1,4-pregnadiene-9a-fluoro-11(3,16a,17a,21-tetraol-3,20-dione.
Eksempel 2. Example 2.
l, 4- pregnadien- llfi, 16a, 17a, 21- tetraol-3, 20- dion. 1, 4- pregnadiene- 11fi, 16a, 17a, 21- tetraol-3, 20-dione.
En potetdekstrose-agarskråkultur av Nocardia gardneri (ATCC 9604) ble vasket med 5 ml av en steril saltoppløsning, og 1 ml's porsjoner av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml's porsjoner av det sterile medium nr. 13, regulert til pH7, i 100 ml's rysterør. Rørene ble rystet på en reciproserende rysteinnretning ved 32° C i 40 timer, og derpå ble der til hvert rør tilsatt 2 mg 4-pregnen-ll[3,16a,17a,21-tetraol-3,20-dion oppløst i 0,2 ml 70 pst.s vandig ethanol. Rørene ble høstet etter 6, 24 og 72 timers inkubering etter tilsetning av substrat. A potato dextrose agar slant culture of Nocardia gardneri (ATCC 9604) was washed with 5 ml of a sterile saline solution, and 1 ml portions of the resulting vegetative cell suspension were used to inoculate 10 ml portions of the sterile medium No. 13, adjusted to pH7, in 100 ml shaker tubes. The tubes were shaken on a reciprocating shaker at 32° C. for 40 hours, and then 2 mg of 4-pregnene-11[3,16a,17a,21-tetraol-3,20-dione dissolved in 0, 2 ml 70% aqueous ethanol. The tubes were harvested after 6, 24 and 72 hours of incubation after addition of substrate.
Den dyrkede mesk ble ekstrahert fire ganger med 40 ml methylenklorid. Ekstraktene fra hver mesk ble forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble opptatt i 2 ml pyridin, og 0,5 ml eddiksyreanhydrid ble tilsatt. Disse blandinger ble opphetet på et dampbad i 10—15 minutter, og derpå ble de konsentrert til tørre residuer under vakuum. Hvert residuum ble oppløst i 2 ml aceton, og passende volumer av de resulterende oppløsninger ble kromatografert. Kromatogrammene viste at substratet var blitt omdannet til l,4-pregnadien-ll|3,16a, 17a,21-tetraol-3,20-dion. The cultured mash was extracted four times with 40 ml of methylene chloride. The extracts from each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added. These mixtures were heated on a steam bath for 10-15 minutes and then concentrated to dry residues under vacuum. Each residue was dissolved in 2 ml of acetone, and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-11|3,16a,17a,21-tetraol-3,20-dione.
Eksempel 3. Example 3.
1, 4- pregnadien- ll [3,2 6a, l 7a, 21 - tetraol-3, 20- dion. 1, 4-pregnadiene-ll[3,2 6a,l 7a,21-tetraol-3,20-dione.
En potetdekstrose-agar-skråkultur av Nocardia leishmanii (ATCC 6855) ble vasket med 5 ml av en steril saltoppløsning, og 1 ml's porsjoner av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml's porsjoner av det sterile medium nr. 13, regulert til pH 7, i 100 ml rysterør. Rørene ble rystet på en resiprose-rende rysteinnretning ved 32° C i 40 timer, og på dette tidspunkt ble det til hvert rør tilsatt 2 mg av substratet, 4-pregnen-lip, 16a,17a,21-tetraol-3,20-dion, oppløst i 0,2 ml 70 pst.s vandig ethanol. Rørene ble høstet etter 6, 24 og 72 timers inkubering etter tilsetning av substrat. A potato dextrose agar slant culture of Nocardia leishmanii (ATCC 6855) was washed with 5 ml of a sterile saline solution, and 1 ml portions of the resulting vegetative cell suspension were used to inoculate 10 ml portions of the sterile medium No. 13, adjusted to pH 7, in 100 ml shaker tube. The tubes were shaken on a reciprocating shaker at 32° C. for 40 hours, at which time 2 mg of the substrate, 4-pregnen-lip, 16a,17a,21-tetraol-3,20- dione, dissolved in 0.2 ml of 70% aqueous ethanol. The tubes were harvested after 6, 24 and 72 hours of incubation after addition of substrate.
De dyrkete meskprodukter tale hver ekstrahert fire ganger med 40 ml methylenklorid. Ekstraktene av hver mesk tale forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble opptatt i 2 ml pyridin, og der ble tilsatt 0,5 ml eddiksyreanhydrid. Disse blandinger ble opphetet på et dampbad i 10—15 minutter, og derpå konsentrert til tørre residuer under vakuum. Hvert residuum ble oppløst i 2 ml aceton, og passende volumer av de resulterende oppløsninger ble kromatografert. Kromatogrammene viste at substratet var blitt omdannet til 1,4-pregnadien-lip, 16a,17a,21-tetraol-3,20-dion. The cultured mash products were each extracted four times with 40 ml of methylene chloride. The extracts of each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added thereto. These mixtures were heated on a steam bath for 10-15 minutes, and then concentrated to dry residues under vacuum. Each residue was dissolved in 2 ml of acetone, and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-lip, 16a,17a,21-tetraol-3,20-dione.
Eksempel 4. Example 4.
1, 4- pregnadien- 11^, 16a, l 7a, 21 - tetraol-3, 20- dion. 1, 4- pregnadiene- 11^, 16a, 1 7a, 21 - tetraol-3, 20-dione.
En potetdekstrose-agarskråkultur av Nocardia caviae (ATCC 6848) ble vasket med 5 ml av en steril saltoppløsning, og 1 ml's porsjoner av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml's porsjoner av det sterile medium nr. 13, regulert til pH 7, i 100 ml's rysterør. Rørene ble rystet på en reciproserende rysteinnretning ved 32° C i 40 timer, og derpå ble det til hvert rør tilsatt 2 mg av substrat, 4-pregnen-ll(3,16a,17a, 21-tetraol-3,20-dion, oppløst i 0,2 ml 70 pst.s vandig ethanol. Rørene ble høstet etter 6, 24 og 72 timers inkubering etter tilsetning av substrat. A potato dextrose agar slant culture of Nocardia caviae (ATCC 6848) was washed with 5 ml of a sterile saline solution, and 1 ml portions of the resulting vegetative cell suspension were used to inoculate 10 ml portions of the sterile medium No. 13, adjusted to pH 7 , in 100 ml shaker tubes. The tubes were shaken on a reciprocating shaker at 32° C. for 40 hours, and then to each tube was added 2 mg of substrate, 4-pregnene-11(3,16a,17a,21-tetraol-3,20-dione, dissolved in 0.2 ml of 70% aqueous ethanol The tubes were harvested after 6, 24 and 72 hours of incubation after addition of substrate.
Den dyrkete mesk ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene av hver mesk ble forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble opptatt i 2 ml pyridin, og det ble tilsatt 0,5 ml eddiksyreanhydrid. Disse blandinger ble opphetet på et dampbad i 10—15 minutter, og derpå konsentrert til tørre residuer under vakuum. Hvert residuum ble oppløst i 2 ml aceton, og passende volumer av de resulterende oppløsninger ble kromatografert. Kromatogrammene viste at substratet var blitt omdannet til 1,4-pregnadien-lip,16a,17a,21-tetraol-3,20-dion. The cultured mash was extracted four times with 40 ml of methylene chloride each time. The extracts of each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added. These mixtures were heated on a steam bath for 10-15 minutes, and then concentrated to dry residues under vacuum. Each residue was dissolved in 2 ml of acetone, and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-lip,16a,17a,21-tetraol-3,20-dione.
Eksempel 5. Example 5.
1, 4- pregnadien- ll$, 16a, 17 a, 21- tetraol-3, 20- dion. 1, 4- pregnadiene-ll$, 16a, 17a, 21- tetraol-3, 20-dione.
En potetdekstrose-agarskråkultur av Nocardia convoluta (ATCC 4275) ble vasket med 7 ml av en steril saltoppløsning og 1 ml av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml av det sterile medium nr. 13, regulert til pH 7, før autoklavatehandling (pH etter autoklavbehandling var 6,75) i et 100 ml's rysterør. Røret ble rystet på en reciproserende rysteinnretning i 16 timer ved 32° C. En ml's porsjoner av den resulterende vekst ble anvendt for å inokulere fem 10 ml's porsjoner av det sterile medium nr. 13 i 100 ml's rysterør. Disse rør ble inkubert på den reciproserende rysteinnretning i 16 timer ved 32° C, og deretter ble tilsatt 2 mg av substrat, 4-pregnen-lip, 16a,17a,21-tetraol-3,20-dion, oppløst i 0,2 ml 70 pst.s vandig ethanol, til hvert rør. Rørene ble høstet etter på hinannen føl-gende inkuberingsperioder av y2, 2, 7, 24 og 72 timer. A potato dextrose agar slant culture of Nocardia convoluta (ATCC 4275) was washed with 7 ml of a sterile saline solution and 1 ml of the resulting vegetative cell suspension was used to inoculate 10 ml of the sterile medium No. 13, adjusted to pH 7, before autoclaving. (pH after autoclave treatment was 6.75) in a 100 ml shaking tube. The tube was shaken on a reciprocating shaker for 16 hours at 32°C. One ml portions of the resulting growth were used to inoculate five 10 ml portions of Sterile Medium No. 13 in 100 ml shaking tubes. These tubes were incubated on the reciprocating shaker for 16 hours at 32°C, and then 2 mg of substrate, 4-pregnen-lip, 16a,17a,21-tetraol-3,20-dione, dissolved in 0.2 ml 70 percent aqueous ethanol, to each tube. The tubes were harvested after successive incubation periods of y2, 2, 7, 24 and 72 hours.
Den dyrkete mesk ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene av hver mesk ble forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble opptatt i 2 ml pyridin, og der ble tilsatt 0,5 ml eddiksyreanhydrid. Disse blandinger ble opphetet på et dampbad i 10—15 minutter og derpå konsentrert til tørre residuer under vakuum. Hvert residuum ble oppløst i 2 ml aceton, og passende volumer av de resulterende oppløsninger ble kromatografert. Kromatogrammene viste at substratet var blitt omdannet til 1,4-pregnadien-lip,16a,17a,21-tetraol-3,20-dion. The cultured mash was extracted four times with 40 ml of methylene chloride each time. The extracts of each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added thereto. These mixtures were heated on a steam bath for 10-15 minutes and then concentrated to dry residues under vacuum. Each residue was dissolved in 2 ml of acetone, and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-lip,16a,17a,21-tetraol-3,20-dione.
Eksempel 6. Example 6.
l, 4- pregnadien- llfi, l 6a, l 7a, 21- tetraol-3, 20- dion. l, 4- pregnadiene- llfi, l 6a, l 7a, 21- tetraol-3, 20-dione.
En gjæreekstrakt-agar-skråkultur av Nocardia corallina (ATCC 999) ble vasket med 7 ml av en steril saltoppløsning. Den resulterende suspensjon ble tilsatt til 100 ml av et Trypticase-sojabuljongmedium uten glycerol i en 500 ml Erlenmeyerkolbe. Blandingen ble inkubert under rystning ved 37° C i 8 timer. 1 ml av dette inokulat ble anvendt for å inokulere hver av 15 Erlenmeyerkolber, idet hver kolbe inneholdt 100 ml av det sterile medium nr. 13. Kolbene ble inkubert under rystning ved 32° C i 40 timer. 20 mg 4-pregnen-ll[3,16a, 17a,21-tetraol-3,20-dion oppløst i 2 ml ethanol ble tilsatt hver flaske. Kolbene ble derpå inkubert under rystning ved 32° C i 8<1>/2 time. Innholdene av kolbene ble forenet og man fikk en mesk med pH 7,3. Det forente produkt etter høstning ble ekstrahert på vanlig måte, og man fikk 352 mg rå krystaller. Krystallisering fra aceton-petrol-ether ga 145 mg, smeltepunkt 216— 218°C. Rekrystallisering fra aceton-petrolether økte smeltepunktet til 229—231° C. Det infrarøde spektrum var identisk med spektret av en autentisk prøve av 1,4-pregnadien-ll(3,16a,17a,21-tetraol-3,20-dion. A yeast extract agar slant culture of Nocardia corallina (ATCC 999) was washed with 7 ml of a sterile saline solution. The resulting suspension was added to 100 ml of Trypticase soy broth medium without glycerol in a 500 ml Erlenmeyer flask. The mixture was incubated with shaking at 37°C for 8 hours. 1 ml of this inoculum was used to inoculate each of 15 Erlenmeyer flasks, each flask containing 100 ml of the sterile medium No. 13. The flasks were incubated with shaking at 32° C. for 40 hours. 20 mg of 4-pregnene-11[3,16a,17a,21-tetraol-3,20-dione dissolved in 2 ml of ethanol was added to each bottle. The flasks were then incubated with shaking at 32° C. for 8<1>/2 hours. The contents of the flasks were combined and a mash with a pH of 7.3 was obtained. The combined product after harvest was extracted in the usual manner to give 352 mg of crude crystals. Crystallization from acetone-petroleum-ether gave 145 mg, melting point 216-218°C. Recrystallization from acetone-petroleum ether increased the melting point to 229-231° C. The infrared spectrum was identical to the spectrum of an authentic sample of 1,4-pregnadiene-11(3,16a,17a,21-tetraol-3,20-dione.
Eksempel 7. Example 7.
l, 4- pregnadien- llfi, 16a, 17a, 21- tetraol-3, 20- dion. 1, 4- pregnadiene- 11fi, 16a, 17a, 21- tetraol-3, 20-dione.
En potetdekstrose-agar-skråkultur av Nocardia globerula (ATCC 9356) ble vasket med 5 ml av en steril saltoppløsning, og 1 ml porsjoner av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml's porsjoner av det sterile medium nr. 13, regulert til pH 7, i 100 ml rysterør. Rørene ble rystet på en reciproserende rysteinnretning ved 32° C i 40 timer, på hvilket tidspunkt det ble tilsatt 2 mg substrat, 4-pregnen-ll|3,16a,17a,21-tetraol-3,20-dion. Rørene ble høstet etter 6, 24 og 72 timers inkubering etter tilsetning av substrat. A potato dextrose agar slant culture of Nocardia globerula (ATCC 9356) was washed with 5 ml of a sterile saline solution, and 1 ml portions of the resulting vegetative cell suspension were used to inoculate 10 ml portions of the sterile medium No. 13, adjusted to pH 7, in 100 ml shaker tube. The tubes were shaken on a reciprocating shaker at 32°C for 40 hours, at which time 2 mg of substrate, 4-pregnene-11|3,16a,17a,21-tetraol-3,20-dione, was added. The tubes were harvested after 6, 24 and 72 hours of incubation after addition of substrate.
Den fermenterte mesk ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene av hver mesk ble forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble opptatt i 2 ml pyridin og det ble tilsatt 0,5 ml eddiksyreanhydrid. Disse blandinger ble opphetet på dampbad i 10—15 minutter og derpå konsentrert til tørre residuer under vakuum. Hvert residuum ble oppløst i 2 ml aceton og passende volumer av de resulterende oppløsninger ble kroma-tografért. Kromatogrammene viste at substratet var blitt omdannet til 1,4-pregnadien-ll(3,16a,17a,21-tetraol-3,20-dion. The fermented mash was extracted four times with 40 ml of methylene chloride each time. The extracts of each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine and 0.5 ml of acetic anhydride was added. These mixtures were heated on a steam bath for 10-15 minutes and then concentrated to dry residues under vacuum. Each residue was dissolved in 2 ml of acetone and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-11(3,16a,17a,21-tetraol-3,20-dione.
Eksempel 8. Example 8.
1, 4- pregnadien- ll (3,2 6a, l 7a, 21 - tetraol-3, 20- dion. 1, 4- pregnadiene-ll (3,2 6a, l 7a, 21 - tetraol-3, 20-dione.
En gjærekstrakt-agarskråkultur av Nocardia corallina (ATCC 999) ble vasket med 7 ml av en steril saltoppløsning, og den resulterende suspensjon ble anvendt for å inokulere 100 ml av det sterile medium nr. 13 i en 500 ml Erlenmeyerkolbe. Blandingen ble inkubert under rystning i 7y2 time ved 37° C. 1 ml's porsjoner av denne kultur ble anvendt for å inokulere 10 ml's porsjoner av det sterile medium i A yeast extract agar slant culture of Nocardia corallina (ATCC 999) was washed with 7 ml of a sterile saline solution, and the resulting suspension was used to inoculate 100 ml of the sterile medium No. 13 in a 500 ml Erlenmeyer flask. The mixture was incubated with shaking for 7y2 hours at 37° C. 1 ml portions of this culture were used to inoculate 10 ml portions of the sterile medium in
100 ml's rør. Rørene ble inkubert ved 32° C i 40 timer, og derpå ble 2 mg 4-pregnen-llfi,16a,17a,21-tetraol-3,20-dion, oppløst i 0,2 ml ethanol, tilsatt til hvert rør. Rørene ble derpå inkubert i 24 timer. Innholdet av et rør (10 ml) ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene ble forenet og konsentrert til et tørt residuum under redusert trykk. Residuet ble opptatt i 2 ml pyridin, og 0,5 ml eddiksyreanhydrid ble tilsatt. Blandingen ble opphetet på dampbad, i 10—15 minutter og derpå konsentrert til et tørt residuum under vakuum. Residuet ble oppløst i 2 ml aceton, og et passende volum av den resulterende oppløsning ble kromatografert. Kromatogrammet viste at substratet var blitt omdannet til l,4-pregnadien-ll|3,16a, 17d,21-tetraol-3,20-dion. 100 ml tubes. The tubes were incubated at 32° C. for 40 hours, and then 2 mg of 4-pregnene-llfi,16a,17a,21-tetraol-3,20-dione, dissolved in 0.2 ml of ethanol, was added to each tube. The tubes were then incubated for 24 hours. The contents of one tube (10 ml) were extracted four times with 40 ml of methylene chloride each time. The extracts were combined and concentrated to a dry residue under reduced pressure. The residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added. The mixture was heated on a steam bath for 10-15 minutes and then concentrated to a dry residue under vacuum. The residue was dissolved in 2 ml of acetone, and an appropriate volume of the resulting solution was chromatographed. The chromatogram showed that the substrate had been converted to 1,4-pregnadiene-11|3,16a,17d,21-tetraol-3,20-dione.
Eksempel 9. Example 9.
l, 4- pregnadien- ll$, 16a, 17a, 21- tetraol-3, 20- dion. l, 4- pregnadiene- ll$, 16a, 17a, 21- tetraol-3, 20- dione.
Et dyrkingsforsøk som beskrevet i eksempel 6 ble utført under anvendelse av Nocardia sp. (ATCC 12 483) istedenfor Nocardia corallina. Produktet, når det ble prøvet på et papirstrimmelkromatogram, ga l,4-pregnadien-lip,16a,17a,21-tetraol-3,20-dion, som var identisk med produktet i henhold til eksempel 6. A cultivation experiment as described in Example 6 was carried out using Nocardia sp. (ATCC 12 483) instead of Nocardia corallina. The product, when tested on a paper strip chromatogram, gave 1,4-pregnadiene-lip,16a,17a,21-tetraol-3,20-dione, which was identical to the product of Example 6.
Eksempel 10. Example 10.
1, 4- pregnadien- l 1 (3,2 6a, l 7a, 21 - tetraol-3, 20- dion. 1, 4- pregnadiene-l 1 (3,2 6a, l 7a, 21 - tetraol-3, 20-dione.
En potetdekstrose-agar-skråkultur av Nocardia polychromogenes (ATCC 3409) ble vasket med 5 ml av en steril saltopp-løsning, og 1 ml's porsjoner av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml's porsjoner av det sterile medium nr. 13, regulert til pH 7, i 100 ml's rysterør. Rørene ble rystet på en reciproserende rysteinnretning ved 32° C i 40 timer, på hvilket tidspunkt 2 mg av substrat, 4-pregnen-ll|3,16a,17a,21-tetraol-3,20-dion, oppløst i 0,2 ml 70 pst.s vandig ethanol, ble tilsatt til hvert rør. Rørene ble høstet etter 6, 24 og 72 timers inkubering etter tilsetning av substratet. A potato dextrose agar slant culture of Nocardia polychromogenes (ATCC 3409) was washed with 5 ml of a sterile saline solution, and 1 ml portions of the resulting vegetative cell suspension were used to inoculate 10 ml portions of the sterile medium No. 13, regulated to pH 7, in 100 ml shaker tubes. The tubes were shaken on a reciprocating shaker at 32°C for 40 hours, at which time 2 mg of substrate, 4-pregnene-ll|3,16a,17a,21-tetraol-3,20-dione, dissolved in 0.2 ml of 70% aqueous ethanol was added to each tube. The tubes were harvested after 6, 24 and 72 hours of incubation after addition of the substrate.
Den dyrkete mesk ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene av hver mesk ble forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble optatt i 2 ml pyridin, og 0,5 ml eddiksyreanhydrid ble tilsatt. Disse blandinger ble opphetet på et dampbad i 10—15 minutter og derpå konsentrert til tørre residuer under vakuum. Hvert residuum ble oppløst i 2 ml aceton, og passende volumer av de resulterende oppløsninger ble kromatografert. Kromatogrammene viste at substratet var blitt omdannet til l,4-pregnadien-ll|3,16a, 17a,21 -tetraol-3,20 -dion. The cultured mash was extracted four times with 40 ml of methylene chloride each time. The extracts of each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added. These mixtures were heated on a steam bath for 10-15 minutes and then concentrated to dry residues under vacuum. Each residue was dissolved in 2 ml of acetone, and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-11|3,16a,17a,21-tetraol-3,20-dione.
Eksempel 11. Example 11.
1, 4- pregnadien- 9a- fluor- llfi, l 6a, l 7a, 21 - 1, 4- pregnadiene- 9a- fluoro- llfi, l 6a, l 7a, 21 -
tetraol- 3, 20- dion. tetraol-3,20-dione.
En gjærekstrakt-agar-skråkultur av Nocardia corallina (ATCC 999) ble vasket med 7 ml av den sterile saltoppløsning, og den resulterende suspensjon ble vasket for å inokulere 100 ml av medium nr. 13 i en 500 ml Erlenmeyerkolbe. Blandingen ble inkubert under rystning ved 37° C i iy2 time. 1 ml's porsjoner av denne kultur ble derpå anvendt for å inokulere 10 ml's porsjoner av det sterile medium nr. 13 i 100 ml's dyrkningsrør. De inokulerte rør ble inkubert i 40 timer ved 32° C. Derpå ble tilsatt til hvert rør 2 mg 4-pregnen-9a-fluor-ll|3,16a,17a,21-tetraol-3,20-dion, oppløst i 0,2 ml ethanol. Rørene ble inkubert i 72 timer. Deretter ble innholdet av et rør ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene ble forenet og konsentrert til et tørt residuum under vakuum. Residuet ble opptatt i 2 ml pyridin, og der ble tilsatt 0,5 ml eddiksyreanhydrid. Denne blanding ble opphetet på et dampbad i 10—15 minutter og derpå konsentrert til et tørt residuum under vakuum. Residuet ble opløst i 2 ml aceton, og et passende volum av den resulterende oppløs-ning ble kromatografert. Kromatogrammet viste vesentlige mengder 1,4-pregnadien-9a-fluor-ll(3,16a,17a,21-tetraol-3,20-dion-16,21-diacetat. Således viste det seg at 1,4-pregnadien-9a-fluor-ll(3,16a,17a,21-tetraol-3,20-dion var blitt dannet i betydelige mengder under dyrkingen. A yeast extract agar slant culture of Nocardia corallina (ATCC 999) was washed with 7 ml of the sterile saline solution and the resulting suspension was washed to inoculate 100 ml of medium No. 13 in a 500 ml Erlenmeyer flask. The mixture was incubated with shaking at 37°C for 1 and 2 hours. 1 ml portions of this culture were then used to inoculate 10 ml portions of sterile medium No. 13 in 100 ml culture tubes. The inoculated tubes were incubated for 40 hours at 32° C. Then 2 mg of 4-pregnene-9a-fluoro-11|3,16a,17a,21-tetraol-3,20-dione, dissolved in 0 .2 ml of ethanol. The tubes were incubated for 72 hours. Then the contents of a tube were extracted four times with 40 ml of methylene chloride each time. The extracts were combined and concentrated to a dry residue under vacuum. The residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added thereto. This mixture was heated on a steam bath for 10-15 minutes and then concentrated to a dry residue under vacuum. The residue was dissolved in 2 ml of acetone, and an appropriate volume of the resulting solution was chromatographed. The chromatogram showed significant amounts of 1,4-pregnadiene-9a-fluoro-11(3,16a,17a,21-tetraol-3,20-dione-16,21-diacetate. Thus, it turned out that 1,4-pregnadiene-9a -fluoro-11(3,16a,17a,21-tetraol-3,20-dione had been formed in significant amounts during cultivation.
Eksempel 12. Example 12.
1, 4- pregnadien- 9a- fluor- l 1 (3,2 6a, l 7a, 21 - 1, 4- pregnadiene- 9a- fluoro- l 1 (3,2 6a, l 7a, 21 -
tetraol- 3, 20- dion- 16, 21 - diacetat. tetraol-3,20-dione-16,21-diacetate.
7 ml av en steril saltoppløsning ble anvendt for å vaske en gjærekstrakt-agar-skråkultur av Nocardia corallina (ATCC 999), og den resulterende suspensjon ble 7 ml of a sterile saline solution was used to wash a yeast extract agar slant culture of Nocardia corallina (ATCC 999), and the resulting suspension was
anvendt for å inokulere 100 ml av det sterile medium nr. 13 i en 500 ml Erlenmeyerkolbe. Blandingen ble inkubert i 8 timer ved 37° C, og 1 ml's porsjoner av denne vekst ble anvendt for å inokulere 100 ml's porsjoner av det sterile medium i 32 500 ml's kolber. Disse kolber ble inkubert ved 32° C i 40 timer, og derpå ble 25 mg 4-pregnen-9a-fluor-ll(3,16«,17a,21-tetraol-3,20-dion-16,21-diacetat oppløst i 2,5 ml aceton, tilsatt til hver kolbe. Kolbene ble inkubert i 53 timer. Innholdet av flaskene ble forenet og et like volum av aceton ble tilsatt. Oppløsningen ble filtrert gjennom diatomé jord og acetonet ble avdampet under redusert trykk. Den vandige fase ble mettet med salt og ekstrahert fem ganger med 500 ml n-butanol. Butanolekstrakten ble inndampet under redusert trykk til tørrhet. Residuet ble oppslemmet med om-trentlig 1 liter kokende aceton for å fjerne det organiske residuum fra de anorganiske salter og derpå filtrert gjennom diatoméjord. Acetonet ble inndampet og residuet ble oppløst i pyridin og acetylert med eddiksyreanhydrid (romtemperatur over natten). Methanol ble tilsatt og oppløsningen ble inndampet under redusert trykk til tørr-het (vekt 1,17 g). Ved fordelingskromato-grafi under anvendelse av oppløsningsmid-delsystemet: 3 deler eddiksyreethylester, 2 deler petrolether (kokeområde 90 til 100° C), 3 deler methanol og 2 deler vann, ble oppnådd 0,50 g av det ønskede råprodukt, l,4-pregnadien-9a-fluor-ll(3,16a,17a,21-tetraol-16,21-diacetat. Krystallisering av residuet fra aceton-petrolether ga 341 mg substans, smeltepunkt 153—233° C under forutgående fuktning. Det vide smelte-punktområde er sannsynligvis et resultat av solvatisering. used to inoculate 100 ml of the sterile medium No. 13 into a 500 ml Erlenmeyer flask. The mixture was incubated for 8 hours at 37°C, and 1 ml portions of this growth were used to inoculate 100 ml portions of the sterile medium in 32,500 ml flasks. These flasks were incubated at 32° C. for 40 hours, and then 25 mg of 4-pregnene-9a-fluoro-11(3,16,17a,21-tetraol-3,20-dione-16,21-diacetate was dissolved in 2.5 mL of acetone, added to each flask. The flasks were incubated for 53 hours. The contents of the flasks were combined and an equal volume of acetone was added. The solution was filtered through diatomaceous earth and the acetone was evaporated under reduced pressure. The aqueous phase was saturated with salt and extracted five times with 500 ml of n-butanol. The butanol extract was evaporated under reduced pressure to dryness. The residue was slurried with approximately 1 liter of boiling acetone to remove the organic residue from the inorganic salts and then filtered through diatomaceous earth .The acetone was evaporated and the residue was dissolved in pyridine and acetylated with acetic anhydride (room temperature overnight). Methanol was added and the solution was evaporated under reduced pressure to dryness (wt. 1.17 g). By partition chromatography using solvent -subsystem: 3 de ler acetic acid ethyl ester, 2 parts petroleum ether (boiling range 90 to 100° C), 3 parts methanol and 2 parts water, 0.50 g of the desired crude product, 1,4-pregnadiene-9a-fluoro-ll(3,16a, 17α,21-tetraol-16,21-diacetate. Crystallization of the residue from acetone-petroleum ether gave 341 mg of substance, melting point 153-233° C. under previous moistening. The wide melting point range is probably a result of solvation.
Eksempel 13. Example 13.
1, 4- pregnadien- llfi, l 6a, l 7a, 21 - tetraol-3, 20- dion. 1, 4- pregnadiene- 11fi, 16a, 17a, 21- tetraol-3, 20-dione.
En potetdekstrose-agar-skråkultur av Nocardia asteroides (ATCC 3308) ble vasket med 5 ml av en steril saltoppløsning, og 1 ml's porsjoner av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml's porsjoner av det sterile medium nr. 13, regulert til pH 7, i 100 ml rysterør. Rørene ble rystet på en reciproserende rysteinnretning ved 32° C i 40 timer, og derpå ble det til hvert rør tilsatt 2 mg av substratet, 4-pregnen-ll(3,16a,17a, A potato dextrose agar slant culture of Nocardia asteroides (ATCC 3308) was washed with 5 ml of a sterile saline solution, and 1 ml portions of the resulting vegetative cell suspension were used to inoculate 10 ml portions of the sterile medium No. 13, adjusted to pH 7, in 100 ml shaker tube. The tubes were shaken on a reciprocating shaker at 32°C for 40 hours, and then 2 mg of the substrate, 4-pregnene-ll(3,16a,17a,
21-tetraol-3,20-dion oppløst i 0,2 ml 70 pst.s vandig ethanol. Rørene ble høstet etter 6, 24 og 72 timers inkubering etter tilsetning av substrat. 21-tetraol-3,20-dione dissolved in 0.2 ml of 70% aqueous ethanol. The tubes were harvested after 6, 24 and 72 hours of incubation after addition of substrate.
Den dyrkete mesk ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene av hver mesk ble forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble optatt i 2 ml pyridin, og 0,5 ml eddiksyreanhydrid ble tilsatt. Disse blandinger ble opphetet på et dampbad i 10—15 minutter og derpå konsentrert til tørre residuer under våkum. Hvert residuum ble oppløst i 2 ml aceton, og passende volum av de resulterende oppløsninger ble kromatografert. Kromatogrammene viste at substratet var blitt omdannet til l,4-pregnadien-ll|3, 16a,17a,21-tetraol-3,20-dion. The cultured mash was extracted four times with 40 ml of methylene chloride each time. The extracts of each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added. These mixtures were heated on a steam bath for 10-15 minutes and then concentrated to dry residues under vacuum. Each residue was dissolved in 2 ml of acetone, and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-11|3,16a,17a,21-tetraol-3,20-dione.
Eksempel 14. Example 14.
l, 4- pregnadien- llfi, 16a, 17a, 21- tetraol-3, 20- dion. 1, 4- pregnadiene- 11fi, 16a, 17a, 21- tetraol-3, 20-dione.
En potetdekstrose-agar-skråkultur av Nocardia cuniculi (ATCC 6864) ble vasket med 7 ml av en steril saltoppløsning, og 1 ml av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml av det sterile medium nr. 13, regulert til pH 7, for autoklavbehandling (pH etter autoklavbehandling var 6,75) i et 100 ml rysterør. Røret ble rystet på en reciproserende rysteinnretning i 16 timer ved 32° C. 1 ml's porsjoner av den resulterende vekst ble anvendt for å inokulere fem 10 ml's porsjoner av det sterile medium nr. 13 i 100 ml's rysterør. Disse rør ble inkubert på den reciproserende rysteinnretning i 16 timer ved 32° C, og derpå ble tilsatt til hvert rør 2 mg av substrat, 4-pregnen-ll(3,16a,17a,21-tetraol-3,2-dion, oppløst i 0,2 ml 70 pst.s vandig ethanol. Rørene ble høstet etter på-følgende inkuberingsperioder av y2, 2, 7, 24 og 72 timer. A potato dextrose agar slant culture of Nocardia cuniculi (ATCC 6864) was washed with 7 ml of a sterile saline solution, and 1 ml of the resulting vegetative cell suspension was used to inoculate 10 ml of the sterile medium No. 13, adjusted to pH 7 , for autoclaving (pH after autoclaving was 6.75) in a 100 ml shaking tube. The tube was shaken on a reciprocating shaker for 16 hours at 32°C. 1 ml portions of the resulting growth were used to inoculate five 10 ml portions of sterile medium No. 13 in 100 ml shaker tubes. These tubes were incubated on the reciprocating shaker for 16 hours at 32°C, and then 2 mg of substrate, 4-pregnene-11(3,16a,17a,21-tetraol-3,2-dione, dissolved in 0.2 ml of 70% aqueous ethanol The tubes were harvested after successive incubation periods of y2, 2, 7, 24 and 72 hours.
Den dyrkete mesk ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene av hver mesk ble forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble opptatt i 2 ml pyridin, og det ble tilsatt 0,5 ml eddiksyreanhydrid. Disse blandinger ble opphetet på et dampbad i 10—15 minutter og derpå konsentrert til tørre residuer under vakuum. Hvert residuum ble oppløst i 2 ml aceton, og passende volum av de resulterende oppløsninger ble kromatografert. Kromatogrammene viste at substratet var blitt omdannet til 1,4-pregnadien-11 (3,16a, 17a,21-tetraol-3,20-dion. The cultured mash was extracted four times with 40 ml of methylene chloride each time. The extracts of each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added. These mixtures were heated on a steam bath for 10-15 minutes and then concentrated to dry residues under vacuum. Each residue was dissolved in 2 ml of acetone, and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-11 (3,16a, 17a,21-tetraol-3,20-dione.
Eksempel 15. Example 15.
l, 4- pregnadien- llfi, l 6a, l 7a, 21 - tetraol-3, 20- dion. l, 4- pregnadiene- llfi, l 6a, l 7a, 21 - tetraol-3, 20-dione.
En potetdekstrose-agar-skråkultur av Nocardia sylvodifera (ATCC 4919) ble vasket med 5 ml av en steril saltoppløsning, og 1 ml's porsjoner av den resulterende vegetative cellesuspensjon ble anvendt for å inokulere 10 ml's porsjoner av det sterile medium nr. 13, regulert til pH 7, i 100 ml's rysterør. Rørene ble rystet på en reciproserende rysteinnretning ved 32° C i 40 timer, og derpå ble tilsatt til hvert rør 2 mg av substrat, 4-pregnen-ll|3,16a,17a,21-tetraol-3,20-dion, oppløst i 0,2 ml 70 pst.s vandig ethanol. Rørene ble høstet etter 6, 24 og 72 timers inkubering etter tilsetning av substrat. A potato dextrose agar slant culture of Nocardia sylvodifera (ATCC 4919) was washed with 5 ml of a sterile saline solution, and 1 ml portions of the resulting vegetative cell suspension were used to inoculate 10 ml portions of the sterile medium No. 13, adjusted to pH 7, in 100 ml shaker tubes. The tubes were shaken on a reciprocating shaker at 32° C. for 40 hours, and then to each tube was added 2 mg of substrate, 4-pregnene-11|3,16a,17a,21-tetraol-3,20-dione, dissolved in 0.2 ml of 70% aqueous ethanol. The tubes were harvested after 6, 24 and 72 hours of incubation after addition of substrate.
Den dyrkete mesk ble ekstrahert fire ganger med 40 ml methylenklorid hver gang. Ekstraktene av hver mesk ble forenet og konsentrert til et tørt residuum under redusert trykk. Hvert residuum ble opptatt i 2 ml pyridin, og 0,5 ml eddiksyreanhydrid ble tilsatt. Disse blandinger ble opphetet The cultured mash was extracted four times with 40 ml of methylene chloride each time. The extracts of each mash were combined and concentrated to a dry residue under reduced pressure. Each residue was taken up in 2 ml of pyridine, and 0.5 ml of acetic anhydride was added. These mixtures were heated
på et dampbad i 10—15 minutter, og derpå in a steam bath for 10-15 minutes, and then
konsentrert til tørre residuer under vakuum. Hvert residuum ble oppløst i 2 ml aceton, og passende volumer av de resulterende oppløsninger ble kromatografert. Kromatogrammene viste at substratet var blitt omdannet til l,4-pregnadien-ll|3,16a, 17a,21-tetraol-3,20-dion. concentrated to dry residue under vacuum. Each residue was dissolved in 2 ml of acetone, and appropriate volumes of the resulting solutions were chromatographed. The chromatograms showed that the substrate had been converted to 1,4-pregnadiene-11|3,16a,17a,21-tetraol-3,20-dione.
Eksempel 16. Example 16.
l, 4- pregnadien- llfi, l 6a, l 7a, 21 - tetraol-3, 20- dion. l, 4- pregnadiene- llfi, l 6a, l 7a, 21 - tetraol-3, 20-dione.
En gjærekstrakt-agar-skråkultur av Nocardia corallina (ATCC 999) ble vasket med en steril saltoppløsning. Den resulterende vegetative cellesuspensjon ble inn-ført i en 500 ml Erlenmeyerkolbe som inneholdt 100 ml av det sterile medium nr. 13. Denne kolbe ble anbrakt på en reciproserende rysteinretning ved 37° C i 7y2 time. 1 ml av den resulterende vekst ble inokulert i fem 100 ml's rysterør, inneholdende 10 ml av det sterile medium nr. 13. Rørene ble inkubert på en reciproserende rysteinnretning ved 32° C i 40 timer, og derpå ble 2 mg 4-pregnen-ll|3,16a,17a,21-tetraol-3,20-dion-16,21-diacetat oppløst i 0,2 ml ethanol tilsatt til hvert rør. Rørene ble høstet etter 2, 7y2, 24, 48 og 72 timer. Papirstrim-melforsøk på den ekstraherte dyrknings-væske viste at en vesentlig mengde av ut-gangsmaterialet var blitt omdannet til 1,4-pregnadien-ll|3,16a,17a,21-tetraol-3,20-dion. A yeast extract agar slant culture of Nocardia corallina (ATCC 999) was washed with a sterile saline solution. The resulting vegetative cell suspension was introduced into a 500 ml Erlenmeyer flask containing 100 ml of the sterile medium No. 13. This flask was placed on a reciprocating shaker at 37°C for 7y2 hours. 1 ml of the resulting growth was inoculated into five 100 ml shaker tubes containing 10 ml of the sterile medium No. 13. The tubes were incubated on a reciprocating shaker at 32°C for 40 hours, and then 2 mg of 4-pregnene-II |3,16a,17a,21-tetraol-3,20-dione-16,21-diacetate dissolved in 0.2 ml ethanol added to each tube. The tubes were harvested after 2, 7y2, 24, 48 and 72 hours. Paper strip tests on the extracted culture liquid showed that a significant amount of the starting material had been converted to 1,4-pregnadiene-11|3,16a,17a,21-tetraol-3,20-dione.
Eksempel 17. Example 17.
1, 4- pregnadien- ll p,2 6a, l 7a, 21 - tetraol-3, 20- dion. 1, 4- pregnadiene-ll p,2 6a, l 7a, 21 - tetraol-3, 20-dione.
En Trypticase-sojaagar-skråkultur ble vasket med 5 ml sterilt vann, og den resulterende cellesuspensjon av Corynebacterium simplex ble anvendt for å inokulere 100 ml sterilt Trypticase-soja-bulj ongmedium i en 500 ml Erlenmeyerkolbe. Denne blandingen ble inkubert under rystning ved 37° C i 8 timer. Fem og tyve 500 ml Erlenmeyerkolber, hver inneholdende 100 ml av det sterile Trypticase-sojabulj ongmedium uten glycerol, ble hver inokulert med 1 ml av det 8 timer gamle inokulat. Disse kolber ble inkubert ved 32° C i 40 timer. Derpå ble tilsatt til hver kolbe 40 mg 4-pregnen-ll(3,16a,17a,21-tetraol-3,20-dion oppløst i 4 ml ethanol, og dyrkingen ble fortsatt i 8 timer ved 32° C. Innholdet av alle 25 kolber ble forenet, og man fikk en mesk av pH 8,1. A Trypticase-soy agar slant culture was washed with 5 ml of sterile water, and the resulting cell suspension of Corynebacterium simplex was used to inoculate 100 ml of sterile Trypticase-soy broth culture medium in a 500 ml Erlenmeyer flask. This mixture was incubated with shaking at 37°C for 8 hours. Twenty-five 500 ml Erlenmeyer flasks, each containing 100 ml of the sterile Trypticase soy broth culture medium without glycerol, were each inoculated with 1 ml of the 8 hour old inoculum. These flasks were incubated at 32°C for 40 hours. 40 mg of 4-pregnene-11(3,16a,17a,21-tetraol-3,20-dione dissolved in 4 ml of ethanol was then added to each flask, and cultivation was continued for 8 hours at 32° C. The content of all 25 flasks were combined, and a mash of pH 8.1 was obtained.
Den forente mesk ble ekstrahert én gang med 3 liter methylenklorid og tre ganger med 2-liters porsjoner av methylenklorid. Den forente ekstrakt ble vasket én gang med mettet saltoppløsning og inndampet til tørrhet under redusert trykk. Dette ga 509 mg av et oljeaktig residuum, som ble oppløst i 1,5 ml av den stasjonære fase fra systemet, 3 deler ethylacetat, 2 deler petrolether (90—100° C), 3 deler methanol og 2 deler vann, og blandet med 3 g diatoméjord. Denne impregnerte diatoméjord ble derpå innført på toppen av en 1,5 x 35 cm glasskolonne som inneholdt 25 g diatoméjord, impregnert med 12,5 ml av den stasjonære fase fra det ovennevnte system. Den ønskede forbindelse ble derpå eluert med den mobile fase fra det ovennevnte system, så at man fikk 207 mg av et fast råprodukt. Dette ble krystallisert fra aceton-petrolether (60—70° C), så at man fikk 56 mg, smeltepunkt 195—200° C (Kofler blokk). Omkrystallisering fra det samme oppløsningsmiddelpar økte smeltepunktet til 202—205° C (blokk); smeltepunkt 229— 231° C (kapillarmetode). Ultrafiolett spektrum: 1maks<1>= 241 m|A (e = 14500)- The combined mash was extracted once with 3 liters of methylene chloride and three times with 2 liter portions of methylene chloride. The combined extract was washed once with saturated saline and evaporated to dryness under reduced pressure. This gave 509 mg of an oily residue, which was dissolved in 1.5 ml of the stationary phase from the system, 3 parts ethyl acetate, 2 parts petroleum ether (90-100° C), 3 parts methanol and 2 parts water, and mixed with 3 g diatomaceous earth. This impregnated diatomaceous earth was then introduced on top of a 1.5 x 35 cm glass column containing 25 g of diatomaceous earth, impregnated with 12.5 ml of the stationary phase from the above system. The desired compound was then eluted with the mobile phase from the above system, so that 207 mg of a solid crude product was obtained. This was crystallized from acetone-petroleum ether (60-70° C), so that 56 mg was obtained, melting point 195-200° C (Kofler block). Recrystallization from the same solvent pair increased the melting point to 202-205°C (block); melting point 229— 231° C (capillary method). Ultraviolet spectrum: 1max<1>= 241 m|A (e = 14500)-
De fysikalske og kjemiske egenskaper av forbindelsen tilsvarte egenskapene for 1,4-pregnadien-ll(3,16a,17a,21-tetraol-3,20-dion. The physical and chemical properties of the compound corresponded to those of 1,4-pregnadiene-11(3,16a,17a,21-tetraol-3,20-dione.
Eksempel 18. Example 18.
1, 4- pregnadien- ll p,2 6a, l 7a, 21 - tetraol-3, 20- dion. 1, 4- pregnadiene-ll p,2 6a, l 7a, 21 - tetraol-3, 20-dione.
Den forente væske fra en dyrkning som beskrevet i eksempel 17 ble ekstrahert med syv 2-liter porsjoner methylenklorid, og de forente ekstrakter inndampet til tørrhet under redusert trykk. Dette ga 1,585 g av en rå halv-fast substans, som ble kromatografert. Fraksjonene som inneholdt den ønskede forbindelse, ble forenet og inndampet til tørrhet så at man fikk 601 mg krystallinsk residuum. Krystallisering fra aceton-petrolether ga 278 mg, smeltepunkt 229—231° C. Omkrystallisering fra det samme opløsningsmiddelpar hevet smeltepunktet til 231—232° C. The combined liquid from a culture as described in Example 17 was extracted with seven 2-liter portions of methylene chloride, and the combined extracts evaporated to dryness under reduced pressure. This gave 1.585 g of a crude semi-solid, which was chromatographed. The fractions containing the desired compound were combined and evaporated to dryness to give 601 mg of crystalline residue. Crystallization from acetone-petroleum ether gave 278 mg, melting point 229-231° C. Recrystallization from the same solvent pair raised the melting point to 231-232° C.
Analyse: C21<H>28<O>0 (376,44) Analysis: C21<H>28<O>0 (376.44)
Beregnet: ..... 67,00 % C, 7,50 % H Funnet: ...... 66,82 % C, 7,27 % H. Calculated: ..... 67.00% C, 7.50% H Found: ...... 66.82% C, 7.27% H.
Produktet var identisk med en prøve av l,4-pregnadien-lip,16a,17a,21-tetraol-3,20-dion erholdt i henhold til eksempel 17. The product was identical to a sample of 1,4-pregnadiene-lip,16a,17a,21-tetraol-3,20-dione obtained according to Example 17.
Eksempel 19. Example 19.
1, 4- pregnadien- l lfi, 16a, l 7a, 21 - tetraol-3, 20- dion- l 6, 21 - diacetat. 1, 4- pregnadiene-l lfi, 16a, l 7a, 21 - tetraol-3, 20-dione- l 6, 21 - diacetate.
En blanding av 100 mg 1,4-pregnadien-lip,16a,17a,21-tetraol-3,20-dion i 10 ml pyridin inneholdende 2 ml eddiksyreanhydrid lot man henstå natten over ved romtemperatur, hvorpå oppløsningen ble inndampet til tørrhet under redusert trykk. Det faste residuum ble krystallisert fra ethyl-åcetatpetroleumether (90—100° C), og man fikk 105 mg (86 pst.) 1,4-pregnadien-lip, 16a,17a,21-tetraol-3,20-dion-16,21-diacetat, smeltepunkt 147—150° C. Omkrystallisering fra det samme oppløsningsmiddelpar økte smeltepunktet til 161—163° C. A mixture of 100 mg of 1,4-pregnadiene-lip,16a,17a,21-tetraol-3,20-dione in 10 ml of pyridine containing 2 ml of acetic anhydride was allowed to stand overnight at room temperature, after which the solution was evaporated to dryness under reduced Print. The solid residue was crystallized from ethyl acetate petroleum ether (90-100° C), and 105 mg (86 percent) of 1,4-pregnadiene-lip, 16a,17a,21-tetraol-3,20-dione-16 were obtained ,21-diacetate, melting point 147—150° C. Recrystallization from the same solvent pair increased the melting point to 161—163° C.
Eksempel 20. Example 20.
l, 4- pregnadien- 9a- fluor- llfi, 16a, 17a, 21-tetraol- 16, 21- diacetat. 1, 4- pregnadiene- 9a- fluoro- llfi, 16a, 17a, 21-tetraol- 16, 21- diacetate.
En agar-skråkultur ble vasket med 5 An agar slant culture was washed with 5
ml av en steril saltoppløsning, og den resulterende sporesuspensjon av Corynebacterium simplex ble tilsatt til 100 ml av det sterile Trypticase-sojabuljongmediet i en 500 ml Erlenmeyerkolbe. Blandingen ble inkubert ved 32° C i 8 timer. 1 ml av denne kultur ble anvendt for å inokulere hver av lO kolber, og hver kolbe inneholdt 100 ml av det sterile Trypticase-sojabuljongmediet. ml of a sterile saline solution, and the resulting spore suspension of Corynebacterium simplex was added to 100 ml of the sterile Trypticase soy broth medium in a 500 ml Erlenmeyer flask. The mixture was incubated at 32°C for 8 hours. 1 ml of this culture was used to inoculate each of 10 flasks, and each flask contained 100 ml of the sterile Trypticase soy broth medium.
De ti kolbene ble inkubert under rystning The ten flasks were incubated under shaking
ved 32° C i 16' timer. 20 mg 4-pregnen-9a-fluor-lip,16a,17a,21-tetraol-3,20-dion-16,21-diacetat oppløst i 2 ml ethanol ble tilsatt til hver kolbe, og kolbeinnholdene ble forenet. Den forenete oppløsning ble ekstrahert flere ganger med et stort volum methylenklorid, vasket med mettet salt-oppløsning og inndampet under redusert trykk. Residuet ble oppløst i methanol, be-handlet med' aktivt kull, filtrert gjennom diatoméjord og påny inndampet så at man fikk 277 mg av en olje og acetylert natten over. Papirstrimmelkromatografi viste om-trentlig like mengder av substrat og et mer polert produkt (l,4-pregnadien-9a-fluor-ll|3,16cc,17a,21-tetraol-3,20-dion-16,21-di- at 32° C. for 16' hours. 20 mg of 4-pregnene-9α-fluoro-lip,16α,17α,21-tetraol-3,20-dione-16,21-diacetate dissolved in 2 mL of ethanol was added to each flask, and the flask contents were combined. The combined solution was extracted several times with a large volume of methylene chloride, washed with brine and evaporated under reduced pressure. The residue was dissolved in methanol, treated with activated carbon, filtered through diatomaceous earth and re-evaporated to give 277 mg of an oil and acetylated overnight. Paper strip chromatography showed roughly equal amounts of substrate and a more polished product (1,4-pregnadiene-9a-fluoro-11|3,16cc,17a,21-tetraol-3,20-dione-16,21-di-
acetat) sammen med meget små mengder av to mindre polare produkter. Fordelings-kromatografi av 0,25 g av residuet (diato-méjordkolonne; system: 2 deler ethylace- acetate) along with very small amounts of two less polar products. Partition chromatography of 0.25 g of the residue (diatomaceous earth column; system: 2 parts ethylace-
tat, 3 deler petrolether (90—100° C), 3 de- tat, 3 parts petroleum ether (90—100° C), 3 de-
ler methanol og 2 deler vann) atskilte de mindre polare produkter og substratet. Det ønskede mest polare produkt forble på kolonnen og ble eluert med 500 ml methanol. Residuet (90 mg) fra den inndam- ler methanol and 2 parts water) separated the less polar products and the substrate. The desired most polar product remained on the column and was eluted with 500 ml of methanol. The residue (90 mg) from the
pete methanol ble påny fordelt på diatomé- pete methanol was redistributed on diatom
jord'. (system: 3 deler ethylacetat, 2 deler petrolether (90^-100° C), 3 deler methanol' earth'. (system: 3 parts ethyl acetate, 2 parts petroleum ether (90^-100° C), 3 parts methanol'
og 2 deler vann), og den delen som inne- and 2 parts water), and the part containing
holdt, det ønskede produkt (bestemt ved ultrafiolett absorpsj.onsspektrum)- ble inndampet under redusert trykk og man fikk 18 mg av et fast stoff. Krystallisering. fra kept, the desired product (determined by ultraviolet absorption spectrum) was evaporated under reduced pressure to give 18 mg of a solid. Crystallization. from
aceton-petrolether ga 13 mg fargeløse nå- acetone-petroleum ether gave 13 mg of colorless now-
ler av l,4-pregnadien-9a-fluor-lip,16a,17a, 21-tetraol-3,20-dion-16,21rdiacetat, smeltepunkt (Kofler blokk) ca. 150—240° C med betydelig, tap av oppløsningsmiddel ved 150° C. Omkrystallisering fra aceton-petrolether endret ikke smeltepunktet. clay of 1,4-pregnadiene-9a-fluoro-lip,16a,17a,21-tetraol-3,20-dione-16,21rdiacetate, melting point (Kofler block) approx. 150—240° C with considerable loss of solvent at 150° C. Recrystallization from acetone-petroleum ether did not change the melting point.
2,4-bis-dinitrof enylhydrazonet av den ovennevnte forbindelse ble fremstilt og dets ultrafiolette absorpsj onsspektrum bestemt: The 2,4-bis-dinitroph enylhydrazone of the above compound was prepared and its ultraviolet absorption spectrum determined:
CHCL CHCL
maks = 258>300 (infleksjon), 312 (inflek- max = 258>300 (inflection), 312 (inflection
sjon.). og 400 •m(x. tion.). and 400 •m(x.
Eksempel 21. Example 21.
1, 4- pregnadien- 9a- fluor- l 1$, 16a, 17a, 21-tetraol- 3, 20- dion. 1, 4- pregnadiene- 9a- fluoro- l 1$, 16a, 17a, 21-tetraol- 3, 20-dione.
En oppløsning av 100 mg 1,4-pregnadien-9a-fluor-lip,16a,17a,21-tetraol-3,20-dion-16,21-diacetat ble oppløst i 10 ml methanol og avkjølt til 0° C. Etter spyling-med nitrogen- ble det tilsatt eni oppløsning av 35 mg' kaliumhydroxyd i 2 ml- methanol til A solution of 100 mg of 1,4-pregnadiene-9a-fluoro-lip,16a,17a,21-tetraol-3,20-dione-16,21-diacetate was dissolved in 10 ml of methanol and cooled to 0° C. After flushing - with nitrogen - a solution of 35 mg of potassium hydroxide in 2 ml of methanol was added to
den steroide oppløsning. Etter henstand'the steroid solution. After grace'
ved romtemperatur i 1 time ble- oppløsnin- at room temperature for 1 hour, the solution
gen nøytralisert, iseddiksyren inndampet under nitrogenatmosfære til et hvitt fåst stoff. Vann ble tilsatt, og etter avkjøling ble- produktet filtrert og vasket med' vann så at man fikk 52 mg l,4-pregnadien-9a-fluor-np,16a-,l'7a,21-tetraol-3!,20-dion- av smeltepunkt 246—249° C: Tre omkrystallr-seringer fra acetonpetroleumether ga129 mg tetrol, smeltepunkt 260—262,5 <0> C. gen neutralized, the glacial acetic acid evaporated under a nitrogen atmosphere to a white solid. Water was added, and after cooling the product was filtered and washed with water so that 52 mg of 1,4-pregnadiene-9a-fluoro-np,16a-,1'7a,21-tetraol-3!,20- was obtained dione- of melting point 246—249° C: Three recrystallizations from acetone petroleum ether gave 129 mg of tetrol, melting point 260—262.5 <0> C.
Analyse: C21H|7O0F Analysis: C21H|7O0F
Claims (2)
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR943219A FR1371950A (en) | 1963-07-30 | 1963-07-30 | Method and device for mounting strong bare electric wires on an electrically insulating support and heating apparatus resulting therefrom |
Publications (1)
Publication Number | Publication Date |
---|---|
NO122265B true NO122265B (en) | 1971-06-07 |
Family
ID=8809530
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NO15160064A NO122265B (en) | 1963-07-30 | 1964-01-15 |
Country Status (5)
Country | Link |
---|---|
BE (1) | BE642342A (en) |
DK (1) | DK111454B (en) |
FR (1) | FR1371950A (en) |
GB (1) | GB1036777A (en) |
NO (1) | NO122265B (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2515914A3 (en) * | 1981-10-30 | 1983-05-06 | Seb Sa | Support for electric resistances in air heating appts. - uses wire coil supported at intervals on wire struts carried on plastics support ring |
DE3935054A1 (en) * | 1989-10-20 | 1991-04-25 | M U T Elektronische Und Elektr | Electrical air-heater elements for integral duct - uses narrow=profile resistors for good heat transfer and separable connections for versatile use |
-
1963
- 1963-07-30 FR FR943219A patent/FR1371950A/en not_active Expired
-
1964
- 1964-01-10 GB GB127464A patent/GB1036777A/en not_active Expired
- 1964-01-10 BE BE642342A patent/BE642342A/xx unknown
- 1964-01-15 NO NO15160064A patent/NO122265B/no unknown
- 1964-02-10 DK DK64364A patent/DK111454B/en unknown
Also Published As
Publication number | Publication date |
---|---|
FR1371950A (en) | 1964-09-11 |
DK111454B (en) | 1968-08-26 |
GB1036777A (en) | 1966-07-20 |
BE642342A (en) | 1964-05-04 |
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