NO125678B - - Google Patents
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- NO125678B NO125678B NO3467/68A NO346768A NO125678B NO 125678 B NO125678 B NO 125678B NO 3467/68 A NO3467/68 A NO 3467/68A NO 346768 A NO346768 A NO 346768A NO 125678 B NO125678 B NO 125678B
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- Prior art keywords
- flavensomycin
- methanol
- benzene
- extract
- evaporating
- Prior art date
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- ASNAXPLWUAPUIY-ITNOJFFJSA-N [(e)-7,9,11,13,15,17,19,21,23-nonaoxooctatriacont-3-enyl] (e)-4-[(2-hydroxy-5-oxocyclopenten-1-yl)amino]-4-oxobut-2-enoate Chemical compound CCCCCCCCCCCCCCCC(=O)CC(=O)CC(=O)CC(=O)CC(=O)CC(=O)CC(=O)CC(=O)CC(=O)CC\C=C\CCOC(=O)\C=C\C(=O)NC1=C(O)CCC1=O ASNAXPLWUAPUIY-ITNOJFFJSA-N 0.000 claims description 24
- 239000003242 anti bacterial agent Substances 0.000 claims description 12
- 229940088710 antibiotic agent Drugs 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical class N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 229910052757 nitrogen Chemical class 0.000 claims description 3
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000012043 crude product Substances 0.000 claims description 2
- 230000002538 fungal effect Effects 0.000 claims description 2
- 229910052500 inorganic mineral Chemical class 0.000 claims description 2
- 239000011707 mineral Chemical class 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical class [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- 241000958313 Streptomyces cavourensis Species 0.000 claims 1
- 229910052799 carbon Inorganic materials 0.000 claims 1
- 239000012141 concentrate Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 claims 1
- 239000002917 insecticide Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 54
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 241000187747 Streptomyces Species 0.000 description 8
- 230000003115 biocidal effect Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000000287 crude extract Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- QGJOPFRUJISHPQ-UHFFFAOYSA-N Carbon disulfide Chemical compound S=C=S QGJOPFRUJISHPQ-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- QFFVPLLCYGOFPU-UHFFFAOYSA-N barium chromate Chemical compound [Ba+2].[O-][Cr]([O-])(=O)=O QFFVPLLCYGOFPU-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000012499 inoculation medium Substances 0.000 description 2
- 230000000749 insecticidal effect Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000013587 production medium Substances 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 241001446247 uncultured actinomycete Species 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000257226 Muscidae Species 0.000 description 1
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007665 chronic toxicity Effects 0.000 description 1
- 231100000160 chronic toxicity Toxicity 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- -1 petrol Chemical compound 0.000 description 1
- 235000019271 petrolatum Nutrition 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- QMYDVDBERNLWKB-UHFFFAOYSA-N propane-1,2-diol;hydrate Chemical compound O.CC(O)CO QMYDVDBERNLWKB-UHFFFAOYSA-N 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
- C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
- C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
- C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D241/24—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D241/26—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with nitrogen atoms directly attached to ring carbon atoms
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
- Plural Heterocyclic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
Fremgangsmåte til fremstilling av et antibiotisk middel. Method for the preparation of an antibiotic agent.
Det er kjent at antibiotiske midler som først bare ble brukt for terapeutiske formål, nu også brukes for andre formål med tilfredsstillende resultater. It is known that antibiotic agents, which were initially only used for therapeutic purposes, are now also used for other purposes with satisfactory results.
Det er funnet at en actinomycet når It has been found that an actinomycete reaches
den dyrkes i passende flytende, nærende medier er i stand til å gi et antibiotisk stoff som kan ekstraheres fra kulturmediet ved hjelp av passende metoder. it is grown in suitable liquid nutrient media is capable of yielding an antibiotic substance which can be extracted from the culture medium by suitable methods.
Det nye aktive stoff som dannes ved The new active substance that is formed by
dyrking av actinomyceten er gitt navnet «flavensomycin». De fysikalske og kjemiske karakteristika for flavensomycin er forskjellige fra de hittil kjente antibioti-kas. De nye antibiotiske middel er utvun-net av kulturvæsker av en streptomyces som ble isolert fra jordbunnen i et gårds-bruk i Cavour, Piemonte. Denne streptomyces-art tilhører den kromogene gruppe og er gitt navnet Streptomyces Cavouren- cultivation of the actinomycete is given the name "flavensomycin". The physical and chemical characteristics of flavensomycin are different from the previously known antibiotics. The new antibiotics are extracted from culture fluids of a streptomyces that was isolated from the soil on a farm in Cavour, Piedmont. This streptomyces species belongs to the chromogenic group and has been given the name Streptomyces Cavouren-
sis, stamme nr. 829.8.52. Den er deponert i Institute of Microbiology, Rutgers Uni-versity, New Brunswick, New Jersey, USA i desember 1957, og registrert under nr. W. 3758. Dessuten er denne stamme sis, trunk no. 829.8.52. It has been deposited in the Institute of Microbiology, Rutgers University, New Brunswick, New Jersey, USA in December 1957, and registered under No. W. 3758. Moreover, this strain
registrert i Commonwealth Micological Institute, Kew, Surrey under nummer 70852, samt i National Collection of Indu-strial Bacteria, Teddington, Middlesex under nr. 8918. registered in the Commonwealth Mycological Institute, Kew, Surrey under number 70852, and in the National Collection of Industrial Bacteria, Teddington, Middlesex under number 8918.
Denne organismes vegetative mycelium dannes av lange, tynne forgrenede eller sammenbuntede hyfer. Luftmyceliet dannes av lange, rette eller bølgeformede hyfer som bærer avrundede konidier (sporer) som ikke er gruppert som busk-vekster. The vegetative mycelium of this organism is formed by long, thin branched or bundled hyphae. The aerial mycelium is formed by long, straight or wavy hyphae bearing rounded conidia (spores) which are not grouped as bushy growths.
Spiraler er ikke observert. Kulturkarakteristika for Streptomyces 829 angis i følgende tabell 1. Spirals are not observed. Culture characteristics of Streptomyces 829 are given in the following Table 1.
Denne streptomyces-art kan dyrkes ved hjelp av vanlige metoder, deriblant med stasjonære og neddykkede kulturer. Sistnevnte gir de beste kulturer. Kultur-mediene må inneholde kullhydrater, en passende kilde for nitrogen samt mineralsalter. Dyrkingen kan utføres ved temperaturer mellom 24 og 32° C og er avhengig av luftningen, omrøringen og det nærende mediums sammensetning. I praksis oppnåes i alminnelighet det maksimale ut-bytte fra det fjerde til det syvende dyrk-ningsdøgn. This Streptomyces species can be cultivated using standard methods, including stationary and submerged cultures. The latter gives the best cultures. The culture media must contain carbohydrates, a suitable source of nitrogen and mineral salts. Cultivation can be carried out at temperatures between 24 and 32° C and depends on the aeration, stirring and the composition of the nutrient medium. In practice, the maximum yield is generally achieved from the fourth to the seventh day of cultivation.
Under dyrkningen stiger pH verdien til 7,8—8, og den bør ikke overstige disse verdier. During cultivation, the pH value rises to 7.8-8, and it should not exceed these values.
Den kulturvæske man får tilbake efter dyrkningen av streptomyces-arten som gir flavensomycinet, inneholder forskjellige stoffer som rester av nærende elementer, «lysated» mycelium, foruten det aktive stoff som kan nå en konsentrasjon på 1600 enheter pr. ml eller høyere, avhengig av kulturmediet og dyrknings-betingelsene. The culture liquid that you get back after the cultivation of the streptomyces species that produces the flavensomycin contains various substances such as remnants of nutritious elements, "lysated" mycelium, in addition to the active substance which can reach a concentration of 1,600 units per ml or higher, depending on the culture medium and the cultivation conditions.
Flavensomycin kan ekstraheres fra kulturvæsken ved hjelp av forskjellige Flavensomycin can be extracted from the culture fluid using various
oppløsningsmidler som ikke. er blandbare med vann, som eter, kloroform, butanol, etylacetat, bensin, benzen, osv., ved en pH verdi mellom 7,5 og 8,5. , solvents that do not. are miscible with water, such as ether, chloroform, butanol, ethyl acetate, petrol, benzene, etc., at a pH value between 7.5 and 8.5. ,
Det erholdte ekstrakt inneholdende det antibiotiske middel som råprodukt inndampes i vakuum ved temperaturer mellom og inklusive 20 og 30° C. The obtained extract containing the antibiotic agent as raw product is evaporated in vacuum at temperatures between and including 20 and 30°C.
Rensningen av råproduktet utføres ved kromatografi av den inndampede væs-ke gjennom en aluminiumoksydkolonne som er tilført en av de foran nevnte opp-løsningsmidler, fortrinsvis benzen. The purification of the crude product is carried out by chromatography of the evaporated liquid through an alumina column which has been supplied with one of the above-mentioned solvents, preferably benzene.
Rensning av det antibiotiske middel oppløst i benzen (rå-ekstrakt) kan utføres på to forskjellige måter: 1) Ved kromatografi av rå-ekstraktet i en aluminiumoksydkolonne. Man tilfører denne kolonne en benzenoppløsning, vasker den omhyggelig med benzen for å fjerne fett og fraksjoner som er inaktive og har gul til rød farve. Man vasker derpå med etylacetat, hvorved en annen inaktiv gulfarvet fraksjon elueres. Sluttelig vasker man med absolutt metanol, hvorved det antibiotiske middel elueres. 2) Rå-esktraktet felles med petroleter, bunnfallet oppløses i aceton, felles på-ny med petroleter, oppløses i benzen og kromatograferes i aluminiumoksyd. Kolonnen vaskes med benzen og etylacetat, hvorpå det antibiotiske middel elueres med absolutt metanol. Purification of the antibiotic agent dissolved in benzene (crude extract) can be carried out in two different ways: 1) By chromatography of the crude extract in an aluminum oxide column. A benzene solution is added to this column, it is washed carefully with benzene to remove fat and fractions that are inactive and have a yellow to red color. It is then washed with ethyl acetate, whereby another inactive yellow-coloured fraction is eluted. Finally, wash with absolute methanol, whereby the antibiotic is eluted. 2) The crude extract is combined with petroleum ether, the precipitate is dissolved in acetone, combined again with petroleum ether, dissolved in benzene and chromatographed in aluminum oxide. The column is washed with benzene and ethyl acetate, after which the antibiotic is eluted with absolute methanol.
Flavensomycin utvinnes fra metanol-oppløsningen ved å fordampe metanolet. Det er et krystallinsk sitrongult pulver. Flavensomycin is recovered from the methanol solution by evaporating the methanol. It is a crystalline lemon yellow powder.
I det følgende beskrives som eksempel en utførelsesform for fremgangsmåten ifølge oppfinnelsen. In the following, an embodiment of the method according to the invention is described as an example.
Eksempel: Streptomyces-stammen dyrkes i et agar-asparaginglycerolmedium ved 27° C. På dette medium oppnåes en meget god vekst av sporer. Produksjonsmediet kan podes direkte med sporer eller med et pod-ningsmedium bestående av en 24 timer gammel streptomyces-kultur som.er dyr-ket på et medium av samme sammensetning som produksjonsmediet, ved 27° C og under neddykkede betingelser. Example: The Streptomyces strain is cultivated in an agar-asparagine glycerol medium at 27° C. A very good growth of spores is achieved on this medium. The production medium can be inoculated directly with spores or with an inoculation medium consisting of a 24-hour-old streptomyces culture which has been grown on a medium of the same composition as the production medium, at 27° C. and under submerged conditions.
Til hver kolbe eller hvert dyrknings-apparat ble brukt følgende medium: -maltose 20 . g -pepton ................ 5 » -kornstøpvæske ........ 2.5 » -ledningsvann .......... 1 liter The following medium was used for each flask or culture apparatus: - maltose 20 . g -peptone ................ 5 » -grain pouring liquid ........ 2.5 » -tap water .......... 1 liter
Kolbens eller dyrkningsapparatenes innhold podes med sporer eller podnings-medium og dets pH verdi innstilles på 7 med natriumhydroksyd. The contents of the flask or culture apparatus are inoculated with spores or inoculation medium and its pH value is adjusted to 7 with sodium hydroxide.
Når dyrkningen er avsluttet, heves pH verdien til 7,5—8,5 og man arbeider ved en temperatur på 27—28° C. Myceliet tas ut og det antibiotiske middel ekstraheres fra dyrkningsmediet med tre porsjo-ner benzen i forholdet benzen og dyrk-ningsmedium på 3 : 1. Herunder passer man omhyggelig at pH verdien ligger mellom 7,5 og 7,8. Under disse betingelser oppløses flavensomycin i benzen. When cultivation is finished, the pH value is raised to 7.5-8.5 and one works at a temperature of 27-28° C. The mycelium is taken out and the antibiotic agent is extracted from the cultivation medium with three portions of benzene in the ratio of benzene to culture -ning medium of 3 : 1. Here you carefully ensure that the pH value is between 7.5 and 7.8. Under these conditions, flavensomycin dissolves in benzene.
Ekstraktets volum minskes ved inn-dampning i vakuum innen et temperatur-område mellom 20 og 40° C. Selv de siste spor av vann fjernes ved denne operasjon. The volume of the extract is reduced by evaporation in a vacuum within a temperature range between 20 and 40° C. Even the last traces of water are removed by this operation.
Produktet renses ved kromatografi av det inndampede ekstrakt i en aluminiumoksydkolonne som er tilført benzen. The product is purified by chromatography of the evaporated extract in an alumina column to which benzene has been added.
1) Det antibiotiske middel absorberes sammen med noen fraksjoner med gul til rød farve mens aktive fraksjoner og røde til fiolette pigmenter ikke absorberes. Kolonnen vaskes flere ganger med benzen og derpå med etylacetat, hvorved der elueres en gul, inaktiv fraksjon. Sluttelig vaskes der med absolutt metanol hvorved den aktive 1) The antibiotic agent is absorbed together with some fractions with a yellow to red color, while active fractions and red to violet pigments are not absorbed. The column is washed several times with benzene and then with ethyl acetate, whereby a yellow, inactive fraction is eluted. Finally, it is washed with absolute methanol, whereby the active
fraksjon (flavensomycin) tas ut. Noen rødfarvede inaktive fraksjoner som kan elueres med aceton og vann blir tilbake i kolonnen. fraction (flavensomycin) is taken out. Some red-colored inactive fractions that can be eluted with acetone and water remain in the column.
2) Det konsentrerte benzenekstrakt av det antibiotiske middel felles med petroleter. Bunnfallet som utgjør den aktive bestanddel oppløses i aceton og felles påny med petroleter. De fettaktige pig-menterte fraksjoner med gul til rød farve som ikke adsorberes i alumini-umsoksydkolonnen og således krever 2) The concentrated benzene extract of the antibiotic agent is combined with petroleum ether. The precipitate which constitutes the active ingredient is dissolved in acetone and redissolved with petroleum ether. The fatty pigmented fractions with a yellow to red color which are not adsorbed in the alumina column and thus require
en meget langvarig vaskning av kolonnen med benzen, fjernes nesten full-stendig ved hjelp av denne metode. Bunnfallet oppløses i benzen og kromatograferes i en aluminiumoksydkolonne. Efter vasking med benzen og etylacetat elueres den aktive fraksjon med absolutt metanol. a very long washing of the column with benzene, is almost completely removed by means of this method. The precipitate is dissolved in benzene and chromatographed in an alumina column. After washing with benzene and ethyl acetate, the active fraction is eluted with absolute methanol.
Flavensomycin utvinnes fra metanol-oppløsningen ved å fordampe metanolen. Man får et sitrongult, krystallinsk pulver uten lukt og ikke hygroskopisk. Flavensomycin is recovered from the methanol solution by evaporating the methanol. You get a lemon-yellow, crystalline powder that is odorless and not hygroscopic.
Det nye antibiotiske middel viser føl-gende kjemiske og fysikalske egenskaper: Flavensomycinets klare, citrongule farve forandres ikke i alkaliske eller i sure media. Produktet inneholder 2,11 % nitrogen, og inneholder ikke svovel eller halo-gen. The new antibiotic shows the following chemical and physical properties: Flavensomycin's clear, lemon-yellow color does not change in alkaline or acidic media. The product contains 2.11% nitrogen, and does not contain sulfur or halogen.
Stoffets absorpsjonsspektrum i det ul-trafiolette område viser bare ett maksi-mum, nemlig ved 251. The substance's absorption spectrum in the ultraviolet region shows only one maximum, namely at 251.
Dets absorpsjonsspektrum i det infra-røde område viser hovedmaksima ved 2,90 3,26 — 3,45 — 5,86 — 5,94 — 6,17 — 6,51 — 6,92 — 8,02 — 9,05 — 10,04 — 10,32 — 10,91 — 13,02 — 13,13 mikroner ( se vedføyede tegning). Its absorption spectrum in the infrared region shows the main maxima at 2.90 3.26 — 3.45 — 5.86 — 5.94 — 6.17 — 6.51 — 6.92 — 8.02 — 9.05 — 10.04 — 10.32 — 10.91 — 13.02 — 13.13 microns (see attached drawing).
De angitte første seks maksima tilsva-rer følgende funksjonelle grupper: OH; CH, CO, konjugert CO, konjugert dobbeltbinding, fenyl. Nærværet av sistnevnte gruppe og av konjugert CO er ikke sikker. The indicated first six maxima correspond to the following functional groups: OH; CH, CO, conjugated CO, conjugated double bond, phenyl. The presence of the latter group and of conjugated CO is not certain.
Absorpsjonsspektret i infrarødt for flavensomycin i kaliumbromid ved konsentrasjon på 3 % (nederste kurve B) er i vedføyede tegning vist i sammenligning med dette absorpsjonsspektrum for en ka-liumbromidskive (øverste kurve A) bestemt med et Beckmann IR/2 spektrofoto-meter. The infrared absorption spectrum for flavensomycin in potassium bromide at a concentration of 3% (bottom curve B) is shown in the attached drawing in comparison with this absorption spectrum for a potassium bromide disk (top curve A) determined with a Beckmann IR/2 spectrophotometer.
Det nye antibiotiske middel som frem-stilles ved hjelp av foreliggende oppfin-nelse er meget lett oppløselig i noen opp-løsningsmidler som metanol, etanol propylenglykol og pyridin. Det er lett opp-løselig i noen alkoholer som normalt pro-panol, normal butanol og amylalkohol. Det er også oppløselig i iseddik og i eddiksy-rens metyl-, etyl-, propyl- og butylestere. Det er også oppløselig i aceton, kloroform, dioksan og benzen, samt vann. The new antibiotic agent produced by means of the present invention is very easily soluble in some solvents such as methanol, ethanol, propylene glycol and pyridine. It is easily soluble in some alcohols such as normal propanol, normal butanol and amyl alcohol. It is also soluble in glacial acetic acid and in acetic acid's methyl, ethyl, propyl and butyl esters. It is also soluble in acetone, chloroform, dioxane and benzene, as well as water.
Det nye antibiotiske middel er uopp-løselig i glycerol, kullstofftetraklorid, kullstoffdisulfid, eter, heksan, benzen og vaselinolje. The new antibiotic agent is insoluble in glycerol, carbon tetrachloride, carbon disulphide, ether, hexane, benzene and petroleum jelly.
Vandige oppløsninger av flavensomycin kan fåes ved å blande oppløsninger av stoffet i et oppløsningsmiddel med vann, f. eks. oppløsninger på opp til 0,2 g pr. ml i etanol. Oppløsningene skummer. Aqueous solutions of flavensomycin can be obtained by mixing solutions of the substance in a solvent with water, e.g. solutions of up to 0.2 g per ml in ethanol. The solutions foam.
Flavensomycin gir farvereaksjoner med Milisch's, Fehling's og Ehrlich's reagenser. Det gir ikke farvereaksjoner med Seliwanoffs, Tollens', Millon's, Lieber-mann's, Sakaguchi's reagenser og heller ikke med biuret. Med FeCl3 gir det et bunnfall uten farvereaksjon. Flavensomycin gives color reactions with Milisch's, Fehling's and Ehrlich's reagents. It does not give color reactions with Seliwanoff's, Tollens', Millon's, Liebermann's, Sakaguchi's reagents nor with biuret. With FeCl3 it gives a precipitate without a color reaction.
Flavensomycin er temmelig stabilt når det oppbevares i vakuum som et tørt pulver eller som oppløsninger i organiske oppløsningsmidler. Flavensomycin is fairly stable when stored in vacuum as a dry powder or as solutions in organic solvents.
I vanlige oppløsninger, særlig med lave konsentrasjoner er det tilbøyelig til delvis å tape sin aktivitet. Det er observert at det antibiotiske middel blir raskere inaktivt ved en pH verdi på 8—10 enn ved en pH verdi på 6—4. In ordinary solutions, especially at low concentrations, it tends to partially lose its activity. It has been observed that the antibiotic becomes inactive faster at a pH value of 8-10 than at a pH value of 6-4.
En vandig oppløsning av flavensomycin med en konsentrasjon på 100 mikrogram pr. ml, pH verdi 7, taper ved 18° C omkring 40 °/o av sin aktivitet efter tre døgn. En vandig oppløsning på 1 mg pr. ml med pH verdi likeledes på 7, er efter oppbevaring ved 4° C uforandret efter syv døgn. Samme oppløsnings aktivitet minskes hundrede ganger efter seks timers oppbevaring ved 70° C og efter tredve mi-nutters oppbevaring ved 100° C. An aqueous solution of flavensomycin with a concentration of 100 micrograms per ml, pH value 7, loses at 18° C around 40 °/o of its activity after three days. An aqueous solution of 1 mg per ml with a pH value likewise of 7, after storage at 4° C is unchanged after seven days. The activity of the same solution is reduced a hundredfold after six hours of storage at 70°C and after thirty minutes of storage at 100°C.
Den beste stabilitet i vandige oppløs-ninger har man ved pH verdi på 6,3—7. The best stability in aqueous solutions is achieved at a pH value of 6.3-7.
Lyset synes å ha liten innflytelse på The light seems to have little influence on
inaktivering av flavensomycin. inactivation of flavensomycin.
Flavensomycin har en vesentlig virkning mot fungusarter og er særlig aktivt overfor gjærarter av saccaromyces-typen samt på trådformede fungi av peni-cillium-familien. Det er ikke aktivt overfor Schizomycetes ved konsentrasjoner som er mindre enn 100 mikrogram pr. ml. Flavensomycin has a significant effect against fungal species and is particularly active against yeast species of the saccaromyces type as well as filamentous fungi of the penicillium family. It is not active against Schizomycetes at concentrations less than 100 micrograms per ml.
I nedenstående tabell angis det antibiotiske spektrum for flavensomycin på basis av følgende bestemmelser: fortyn-ning i agar, vandige oppløsninger av det antibiotiske middel fremstillet av 10 %'s etanoloppløsninger: The table below shows the antibiotic spectrum for flavensomycin on the basis of the following determinations: dilution in agar, aqueous solutions of the antibiotic prepared from 10% ethanol solutions:
Foruten sin sterke fungiside virkning viser flavensomycin også en bemerkelses-verdig insektisid virkning. I nedenstående tabell III vises denne insektiside virkning basert på lokal påføring på husfluer: In addition to its strong fungicidal effect, flavensomycin also shows a remarkable insecticidal effect. Table III below shows this insecticidal effect based on local application to houseflies:
Flavensomycinets giftighet er bestemt ved forsøk på hvite mus under anvendelse av intraperitonal injeksjon. Man fikk føl-gende resultater: LD50 = 1 mg/kg, LDo = 350 mikrogram/kg. Ved subkutan injeksjon ble funnet LD50 = 2 mg/kg, LDo = 1 mg/kg. Flavensomycin's toxicity has been determined by experiments on white mice using intraperitoneal injection. The following results were obtained: LD50 = 1 mg/kg, LDo = 350 micrograms/kg. By subcutaneous injection, LD50 = 2 mg/kg, LDo = 1 mg/kg was found.
Kronisk giftighet ved intraperitonal injeksjon: LDo = 250 mikrogram/kg pr. dag i IO dager. Giftighet ved oral innfør-ing: LD50 = 25 mg/kg, akutt (i metanol og vann); i propylenglykol-vann: LD50 = 19 mg/kg. Chronic toxicity by intraperitoneal injection: LDo = 250 micrograms/kg per day for IO days. Toxicity by oral administration: LD50 = 25 mg/kg, acute (in methanol and water); in propylene glycol water: LD50 = 19 mg/kg.
Claims (3)
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US66600467A | 1967-09-07 | 1967-09-07 | |
US71897668A | 1968-03-21 | 1968-03-21 |
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NO125678B true NO125678B (en) | 1972-10-16 |
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BE (1) | BE720233A (en) |
CA (1) | CA939345A (en) |
CH (1) | CH534168A (en) |
DK (1) | DK145892C (en) |
ES (1) | ES357877A1 (en) |
FI (1) | FI50975C (en) |
FR (1) | FR8490M (en) |
GB (2) | GB1214409A (en) |
IL (1) | IL30634A0 (en) |
NL (1) | NL6812003A (en) |
NO (1) | NO125678B (en) |
OA (1) | OA02886A (en) |
SE (1) | SE355811B (en) |
YU (2) | YU205268A (en) |
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DK2773633T3 (en) | 2011-11-02 | 2017-11-13 | Boehringer Ingelheim Int | Heterocyclic compounds, medicaments containing the compounds, their use and processes for their preparation. |
US8859559B2 (en) | 2011-12-20 | 2014-10-14 | Boehringer Ingelheim International Gmbh | Substituted pyrazines and their use in the treatment of disease |
EP2861577B1 (en) | 2012-05-25 | 2017-01-04 | Boehringer Ingelheim International GmbH | Tertiary amines, medicaments containing said amines, use thereof and processes for the preparation thereof |
EP2897940B1 (en) | 2012-09-24 | 2019-05-01 | Boehringer Ingelheim International GmbH | Heterocyclic compounds, medicaments containing said compounds, use thereof and processes for the preparation thereof |
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1968
- 1968-08-22 NL NL6812003A patent/NL6812003A/xx unknown
- 1968-08-26 IL IL30634A patent/IL30634A0/en unknown
- 1968-08-28 FI FI682412A patent/FI50975C/en active
- 1968-08-30 BE BE720233D patent/BE720233A/xx unknown
- 1968-08-31 CA CA028,994A patent/CA939345A/en not_active Expired
- 1968-09-02 YU YU02052/68A patent/YU205268A/en unknown
- 1968-09-03 GB GB53830/69A patent/GB1214409A/en not_active Expired
- 1968-09-03 GB GB41777/68A patent/GB1214408A/en not_active Expired
- 1968-09-04 OA OA53362A patent/OA02886A/en unknown
- 1968-09-05 SE SE11956/68A patent/SE355811B/xx unknown
- 1968-09-05 ES ES357877A patent/ES357877A1/en not_active Expired
- 1968-09-06 CH CH1340268A patent/CH534168A/en not_active IP Right Cessation
- 1968-09-06 AT AT872468A patent/AT289123B/en not_active IP Right Cessation
- 1968-09-06 DK DK429268A patent/DK145892C/en active
- 1968-09-06 NO NO3467/68A patent/NO125678B/no unknown
- 1968-12-06 FR FR176992A patent/FR8490M/fr not_active Expired
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1969
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YU34082B (en) | 1978-12-31 |
BE720233A (en) | 1969-02-28 |
YU205268A (en) | 1978-06-30 |
NL6812003A (en) | 1969-03-11 |
SE355811B (en) | 1973-05-07 |
DK145892B (en) | 1983-04-05 |
ES357877A1 (en) | 1970-05-01 |
GB1214409A (en) | 1970-12-02 |
CH534168A (en) | 1973-02-28 |
FI50975C (en) | 1976-09-10 |
FI50975B (en) | 1976-05-31 |
GB1214408A (en) | 1970-12-02 |
AT289123B (en) | 1971-04-13 |
IL30634A0 (en) | 1968-10-24 |
OA02886A (en) | 1970-12-15 |
DK145892C (en) | 1983-09-12 |
CA939345A (en) | 1974-01-01 |
FR8490M (en) | 1973-07-27 |
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