US3799841A - Microbiological reduction of 11-hydroxy-15-keto prostaglandin intermediates - Google Patents

Microbiological reduction of 11-hydroxy-15-keto prostaglandin intermediates Download PDF

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Publication number
US3799841A
US3799841A US00308480A US30848072A US3799841A US 3799841 A US3799841 A US 3799841A US 00308480 A US00308480 A US 00308480A US 30848072 A US30848072 A US 30848072A US 3799841 A US3799841 A US 3799841A
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hydroxy
dihydroxy
nrrl
cis
oxoprosta
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US00308480A
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English (en)
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W Marsheck
M Miyano
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GD Searle LLC
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GD Searle LLC
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Priority to US00308480A priority Critical patent/US3799841A/en
Priority to DE2357815A priority patent/DE2357815A1/de
Priority to GB5392573A priority patent/GB1423273A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C405/00—Compounds containing a five-membered ring having two side-chains in ortho position to each other, and having oxygen atoms directly attached to the ring in ortho position to one of the side-chains, one side-chain containing, not directly attached to the ring, a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, and the other side-chain having oxygen atoms attached in gamma-position to the ring, e.g. prostaglandins ; Analogues or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P31/00—Preparation of compounds containing a five-membered ring having two side-chains in ortho position to each other, and having at least one oxygen atom directly bound to the ring in ortho position to one of the side-chains, one side-chain containing, not directly bound to the ring, a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, and the other side-chain having at least one oxygen atom bound in gamma-position to the ring, e.g. prostaglandins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/85—Flavobacterium

Definitions

  • the present invention is concerned with a process for stereoselectively reducing the 15-keto function of ll-hydroXy-9,l-dioxoprosta-8(12),13-dienoic acid to the hydroxy stereoisomers such that the resulting 11,15 hydroXy groups are in the cis stereochemical relationship to one another.
  • This conversion is achieved by the fermentative action of Flavobacterium sp. NRRL B-5641.
  • S dihydroxy- 9-oxoprostanoic acids.
  • the latter pair shows biological hypotensive activity distinct from dihydro PGE as indicated in U.S. Pat. 3,696,144.
  • hypotensive activity For the determination of hypotensive activity the following assay is used:
  • Male Charles River rats weighing 100-350 g. are anesthetized by intraperitoneal injection of 1.5 g./kg. of urethane, whereupon cardiovascular reflexes are blocked by subcutaneous injection of 3 mg. of atropine sulfate dissolved in 0.3 ml. of aqueous 0.85% sodium chloride and sensitization is induced by subcutaneous injection of 5 mg. of pentolinium tartrate dissolved in 1 ml. of aqueous 0.85% sodium chloride.
  • the trachea is intubated and both femoral veins and a carotid artery are cannulated, the latter being connected to a calibrated transducer, amplifier and recorder. After surgery, 5 mg./kg.
  • heparin sodium is introduced via one of the venus cannulae as a 2% solution in aqueous 0.85% sodium chloride and rectal temperature is adjusted to 35 C. by means of a regulator and external heat source.
  • a high and a low dose with a ratio of high dose to low dose of 2:1, of the test compound and a high and a low dose, also in the ratio of 2: l, of the standard prostaglandin B are individually administered intravenously and in a random pattern and the blood pressure of each animal is permitted to return to normal between successive injections.
  • the relative potency of the test compound compared to the standard prostaglandin E is determined by a 4 point bioassay using a randomized block design. The results are analyzed by NRRL cultures can be obtained at A.R.S. Culture Collectron, 1815 North University Street, Peoria, 111. 61604.
  • Scheme I illustrates the conversions achieved by the process of the present invention and the usefulness of the products produced thereby as intermediates for other compounds.
  • Chemical reduction as described in US. Pat. 3,696,144typically with sodium borohydride, afiords a mixture of racemic cis-11,15-dihydroxy and trans-11,15-dihydroxy compounds, Scheme Scheme 1 ll 00 H 1 TV ⁇ / ⁇ / W 11%) 0 l Microbiological reduction COzH on W H6 H6 H?
  • the preparation of the fermentation substrate, ll-hydroxy 9,15 dioxoprosta 8(12),13 dienoic acid, is described in US. Pat. 3,687,811.
  • the 11-hydroxy-9,15-dioxoprosta-8(12),13-dienoic acid is subjected to the fermentative action of Flavo'bacterium sp. NRRL B-5641 and the reduction product is isolated.
  • the resulting dl-cis- 1 1,15-dihydroxy-9-oxoprosta-8(12),13-dienoic acid may be resolved with common resolving agents and the 8(12), 13 carbon to carbon double bonds are reduced by catalytic hydrogenation. If resolution is not effected before hydrogenation, the diastereomers resulting from hydrogenation may be separated by column chromatography.
  • Fermentation is ordinarily carried out in the medium wherein the organism is cultured. However, it is likewise possible to separate the bacterial cells from the culture medium by centrifugation or other means and use the resultant cellular matter to implement the fermentation. Moreover, the cells can be ruptured ultrasonically or otherwise to facilitate access to enzymes present, which can be isolated by filtration or extracted with a solvent such as acetone or water and substituted for the organism or cells thereof.
  • a solvent such as acetone or water
  • a nutrient medium is required for culture of the organism, which is to say one containing assirnilable nitrogen and carbon; and an adequate supply of sterile air should be maintained therein; for example, by exposing a large surface of the medium to the air or preferably passing it through the medium in quantities sufiicient to support submerged growth.
  • Suitable nitrogen sources are thus normally employed for the purpose, including soy bean meal, corn-steep liquor, meat extract, protein (optionally digested), peptone, yeast extract, distillers solubles, casein hydrolysate, cottonseed meal, nitrate and/or ammonium compounds. All of the foregoing materials excepting sometimes the last two serve also as carbon sources.
  • Other carbon-containing substances satisfactory and conventionally used as nutrients are the carbohydrates, for example, glycerol, glucose, fructose, sucrose, lactose, maltose, inositol, dextrin, starch and whey.
  • Phosphate, magnesium, and/or ferrous ions likewise may be incorporated in the culture medium as growthpromoting factors, if desired; buffers may be added to assure that growth is initiated at a suitable pH; and Wetting agents may be employed to improve contact between the prostaglandin and the fermenting agent.
  • An antifoaming agent is usually beneficial. Where isolated cells or enzymes are used to induce fermentation rather than the intact and growing organism, nutrients need not, of course, be present; but in either event the medium is customarily preponderantly aqueous.
  • the medium consists of 4 parts beef extract, 4 parts of commercial meat peptone, 1 part of yeast extract, 10 parts of dextrose, 2.5 parts of sodium chloride and 6,000 parts of distilled water.
  • the medium is sterilized by heating at approximately to 15 pounds per square inch pressure for about 20 minutes; then is cooled to room temperature and inoculated with a culture of Flavobacterium sp. NRRL B-5641.
  • the resulting fermentation mixtures are incubated with moderate aeration and agitation for about 29 hours at room temperature. At the end of that time there is added a solution of 1.1 parts of 11-hydroxy-9,l5-dioxoprosta-8(l2), 13-dienoic acid in 10 parts of acetone. That fermentation reaction is continued for about 65 hours, at the endof which time the pH of each mixture is adjusted to approximately 4.0 by the addition of citric acid.
  • the crude product is isolated from each mixture by extraction with methylene chloride.
  • the compounds of this invention are obtained as racemic mixtures. Resolution of these racemates is conveniently achieved by standard methods utilizing optically active amines such as the d or 1 enantiomers of brucine, morphine, quinine, quinidine, strychnine, menthylamine, cinchonine, cinchonidine and a-phenylethylamine. In that manner, cis-11,15 dihydroxy 9 oxoprosta-8(12),13- dienoic acid, for example, is resolved to afford its individual d (11a,15(S); [@15 +233. M.P. 71) and 1 (1113,15(SQ, [ah- 23.0, M.P. 71) enantiomers.
  • optically active amines such as the d or 1 enantiomers of brucine, morphine, quinine, quinidine, strychnine, menthylamine, cinchonine, cinchonidine and a-phenyle
  • EXAMPLE 1 A medium consisting of 4 parts of beef extract, 4 parts of commercial dextrose, 2.5 parts of sodium chloride and 6,000 parts of distilled water is divided into ten 600-part portions. Each portion is sterilized by heating at approximately 120 at 15 pounds per square inch pressure for about 20 minutes, then is cooled to room temperature and inoculated with a culture of Flavorbacterium sp. NRRL B-5641. The resulting fermentation mixtures are incubated with moderate aeration and agitation for about 29 hours at room temperature. At the end of that time there is added to each of the mixtures a solution of .08 part of 11-hydroxy-9,l5-dioxoprosta 8(12),13 dienoic acid in 1 part of acetone.
  • That fermentation reaction is continued for about 65 hours, at the end of which time the pH of each mixture is adjusted to aproximately 4.0 by the addition of citric acid.
  • the crude product is isolated from each mixture by extraction with methylene chloride.
  • the oily residue is dissolved in a solution of benzene-ethyl acetate-acetic acid (69.5300.5) v./v. and placed on a silica gel chromatographic column.
  • the d1- cis 11,15 dihydroxy-9,l5-oxoprosta-8(12),13 dienoic acids are eluted with a solvent system consisting of benzene-ethyl acetate-acetic acid (59.5400.5) v./v.
  • a process for the stereoselective reduction of 11-hydroxy-lS-keto prostaglandins to cis 11,15 dihydroxy prostaglandins which comprises subjecting the 11-hydmxy-lS-keto prostaglandins to the fermentative action 5 of 'Flavobacterium sp. NRRL B-5641 and isolating the cis-11,15-dihydroxy prostaglandins.
  • a process for the conversion of dl- 11-hydroxy-9,15-dioxoprosta-8(12),13-dienoic acid to dlcis-l1,l5-dihydroxy-9-oxoprosta 8(12),13 dienoic acid which comprises subjecting the d1-11-hydroxy-9,15-dioxoprosta-8(12),l3-dienoic acid to the fermentative action of Flavobacterium sp. NRRL B-5641 and isolating the d1-cis-11,15 dihydroxy-9-oxoprosta-8(12),13-dienoic acid.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
US00308480A 1972-11-21 1972-11-21 Microbiological reduction of 11-hydroxy-15-keto prostaglandin intermediates Expired - Lifetime US3799841A (en)

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Application Number Priority Date Filing Date Title
US00308480A US3799841A (en) 1972-11-21 1972-11-21 Microbiological reduction of 11-hydroxy-15-keto prostaglandin intermediates
DE2357815A DE2357815A1 (de) 1972-11-21 1973-11-20 Mikrobiologische reduktion von 11-hydroxy-15-ketoprostaglandin-zwischenprodukten
GB5392573A GB1423273A (en) 1972-11-21 1973-11-21 Microbiological reduction of 11-hydroxy-15-keto prostaglandin intermediates

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3868306A (en) * 1973-11-12 1975-02-25 Wisconsin Alumni Res Found Method for preparing 3(s or r)-hydroxy-1-iodo-1-trans-octene
US3880712A (en) * 1973-01-15 1975-04-29 Ici Ltd Reduction process
US4247635A (en) * 1978-12-08 1981-01-27 Schering Aktiengesellschaft Microbiological reduction of 15-ketoprostaglandin intermediates

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3880712A (en) * 1973-01-15 1975-04-29 Ici Ltd Reduction process
US3868306A (en) * 1973-11-12 1975-02-25 Wisconsin Alumni Res Found Method for preparing 3(s or r)-hydroxy-1-iodo-1-trans-octene
US4247635A (en) * 1978-12-08 1981-01-27 Schering Aktiengesellschaft Microbiological reduction of 15-ketoprostaglandin intermediates

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DE2357815A1 (de) 1974-05-30
GB1423273A (en) 1976-02-04

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