NO132241B - - Google Patents
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- Publication number
- NO132241B NO132241B NO3024/70A NO302470A NO132241B NO 132241 B NO132241 B NO 132241B NO 3024/70 A NO3024/70 A NO 3024/70A NO 302470 A NO302470 A NO 302470A NO 132241 B NO132241 B NO 132241B
- Authority
- NO
- Norway
- Prior art keywords
- antibiotic
- salt
- acid
- culture
- medium
- Prior art date
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- 150000003839 salts Chemical class 0.000 claims description 76
- ORQHMODRGXTBFU-LWNYNHHKSA-N (6r,7s)-3-(acetyloxymethyl)-7-[[(5r)-5-amino-5-carboxypentanoyl]amino]-7-methoxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid Chemical compound S1CC(COC(C)=O)=C(C(O)=O)N2C(=O)[C@@](OC)(NC(=O)CCC[C@@H](N)C(O)=O)[C@H]21 ORQHMODRGXTBFU-LWNYNHHKSA-N 0.000 claims description 70
- 238000000034 method Methods 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 13
- 241000187395 Streptomyces microflavus Species 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 45
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 23
- 230000003115 biocidal effect Effects 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000002609 medium Substances 0.000 description 18
- 238000004519 manufacturing process Methods 0.000 description 15
- 239000000203 mixture Substances 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000002253 acid Substances 0.000 description 12
- 229920001817 Agar Polymers 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 11
- 239000008272 agar Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 239000002054 inoculum Substances 0.000 description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 230000012010 growth Effects 0.000 description 8
- 239000012531 culture fluid Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 241000187747 Streptomyces Species 0.000 description 5
- 239000003463 adsorbent Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 239000013587 production medium Substances 0.000 description 5
- 239000000741 silica gel Substances 0.000 description 5
- 229910002027 silica gel Inorganic materials 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- HOKIDJSKDBPKTQ-GLXFQSAKSA-N Cephalosporin C Natural products S1CC(COC(=O)C)=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H](N)C(O)=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000589771 Ralstonia solanacearum Species 0.000 description 3
- 241000975692 Syphacia obvelata Species 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- -1 alkali metal salts Chemical class 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000000507 anthelmentic effect Effects 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- HOKIDJSKDBPKTQ-GLXFQSAKSA-M cephalosporin C(1-) Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H]([NH3+])C([O-])=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-M 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 230000037213 diet Effects 0.000 description 3
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000006916 nutrient agar Substances 0.000 description 3
- 238000004816 paper chromatography Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- OYIFNHCXNCRBQI-UHFFFAOYSA-N 2-aminoadipic acid Chemical compound OC(=O)C(N)CCCC(O)=O OYIFNHCXNCRBQI-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 241000244185 Ascaris lumbricoides Species 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000760148 Aspiculuris tetraptera Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- XPDXVDYUQZHFPV-UHFFFAOYSA-N Dansyl Chloride Chemical compound C1=CC=C2C(N(C)C)=CC=CC2=C1S(Cl)(=O)=O XPDXVDYUQZHFPV-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- 208000012766 Growth delay Diseases 0.000 description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000002814 agar dilution Methods 0.000 description 2
- 229910052783 alkali metal Inorganic materials 0.000 description 2
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 2
- 150000003863 ammonium salts Chemical class 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 description 2
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 244000309464 bull Species 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003610 charcoal Substances 0.000 description 2
- PAFZNILMFXTMIY-UHFFFAOYSA-N cyclohexylamine Chemical compound NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 description 2
- JQVDAXLFBXTEQA-UHFFFAOYSA-N dibutylamine Chemical compound CCCCNCCCC JQVDAXLFBXTEQA-UHFFFAOYSA-N 0.000 description 2
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 2
- DMBHHRLKUKUOEG-UHFFFAOYSA-N diphenylamine Chemical compound C=1C=CC=CC=1NC1=CC=CC=C1 DMBHHRLKUKUOEG-UHFFFAOYSA-N 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 235000012054 meals Nutrition 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
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- 235000019813 microcrystalline cellulose Nutrition 0.000 description 2
- 239000008108 microcrystalline cellulose Substances 0.000 description 2
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- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 150000007530 organic bases Chemical class 0.000 description 2
- 230000005789 organism growth Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 229940066779 peptones Drugs 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- DLYUQMMRRRQYAE-UHFFFAOYSA-N tetraphosphorus decaoxide Chemical compound O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 2
- 241001446247 uncultured actinomycete Species 0.000 description 2
- UQZSVIGPZAULMV-DKWTVANSSA-N (2s)-2,4-diamino-4-oxobutanoic acid;propane-1,2,3-triol Chemical compound OCC(O)CO.OC(=O)[C@@H](N)CC(N)=O UQZSVIGPZAULMV-DKWTVANSSA-N 0.000 description 1
- ICSNLGPSRYBMBD-UHFFFAOYSA-N 2-aminopyridine Chemical compound NC1=CC=CC=N1 ICSNLGPSRYBMBD-UHFFFAOYSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000244186 Ascaris Species 0.000 description 1
- 241000760149 Aspiculuris Species 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical compound C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 241000498255 Enterobius vermicularis Species 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000243780 Heligmosomoides polygyrus Species 0.000 description 1
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000191938 Micrococcus luteus Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
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- 241000607132 Salmonella enterica subsp. enterica serovar Gallinarum Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
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- 229920002472 Starch Polymers 0.000 description 1
- 241000187435 Streptomyces griseolus Species 0.000 description 1
- 241000187216 Streptomyces halstedii Species 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
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- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 229910052792 caesium Inorganic materials 0.000 description 1
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical compound [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
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- 229910052701 rubidium Inorganic materials 0.000 description 1
- IGLNJRXAVVLDKE-UHFFFAOYSA-N rubidium atom Chemical compound [Rb] IGLNJRXAVVLDKE-UHFFFAOYSA-N 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
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- 238000012807 shake-flask culturing Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
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- 229910052712 strontium Inorganic materials 0.000 description 1
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- 238000004809 thin layer chromatography Methods 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 229940086542 triethylamine Drugs 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- 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
- C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
- C12P35/08—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin disubstituted in the 7 position
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
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- Health & Medical Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Compounds Of Unknown Constitution (AREA)
- Cephalosporin Compounds (AREA)
Description
Foreliggende oppfinnelse angår en fremgangsmåte for fremstilling av et nytt antibiotikum A16884, og dets salter. Antibiotikum A16884 har formelen: The present invention relates to a method for the production of a new antibiotic A16884, and its salts. Antibiotic A16884 has the formula:
Antibiotikum A16884 er et nytt antibiotikum fremstilt ved fermentering av en hittil ubeskrevet, antibiotikum A16884-produserende stamme av Streptomyces lipmanii. Saltene av A16884 oppnås lett ved å omsette A16884 med en egnet syre eller base. Antibiotikum A16884 og dets salter oppviser antibakteriell og anthelmintisk aktivitet. Den antibakterielle aktivitet oppvises både overfor gram-positive og gram-negative organismer, såvel som overfor•plantepato-gene organismer. Antibiotic A16884 is a new antibiotic produced by fermentation of a previously undescribed antibiotic A16884-producing strain of Streptomyces lipmanii. The salts of A16884 are readily obtained by reacting A16884 with a suitable acid or base. Antibiotic A16884 and its salts exhibit antibacterial and anthelmintic activity. The antibacterial activity is shown both against gram-positive and gram-negative organisms, as well as against • plant pathogenic organisms.
Monoammoniumsaltet av antibiotikum A16884 er et hvitt, amorft stoff, som spaltes ved ca. 180°C, er lett løselig i vann, løselig i dimetylsulfoksyd, ubetydelig løselig i lavere alkanoler og uløselig i acetonitril, er amfotert, har fire titrerbare grupper av pK^ = 3,9; pK'a2 =5,3,<*> pK'a3 =9,2 og pK'a4 = 10,5 når tit- ;rert i 66% dimetylformanid ved en begynnelses-pH på 5,8, har en til-synelatende molekylvekt på ca. 435 bestmt fra titreringsdata, har ;25 o o ;en spesifikk optisk dreining [ a] — på +140,9 (C = 1 vekt/vol.-% i vann), har i mineraloljesalve følgende klart adskillbare bånd i dets infrarøde absorpsjonsspektrum ved følgende bølgelengder i ^a: 3,18 (bredt bånd), 5,66, 6,26, 6,57, 6,89, 7,15, 7,28, 7,40, 7,73, 8,00, 8,14, 8,79, 9,24, 9,65, 9,79 og 10,40, og gir positive forsøk med ninhydrin-, Pan Deutscher-, Benedict-, Molisch-, jod- og dansylklorid-reagenser, og negative forsøk med Fehling-, ferriklorid, biuret- og Sakaguchi-reagenser. Fremgangsmåten karakteriseres ved dyrking av Streptomyces lipmanii NRRL 3584 i et kulturmedium inneholdende assi-limilerbare forråd av karbon, nitrogen og uorganiske salter under neddykkede, aerobe betingelser inntil en vesentlig mengde A16884 ;er fremstilt av nevnte organisme i nevnte kulturmedium. ;Antibiotikum A16884 kan brukes.som sådant eller som ;et salt, for eksempel et syreaddisjonssalt eller et salt med et kation. I tilfellet av et salt med et kation kan saltet enten være et mono- ellér di-salt. Det er ofte foretrukket å fremstille saltene direkte i renseprosessen slik at antibiotikumet når det er separert, er i saltform. Antibiotikum A16884 har vært separert på denne måte og av den grunn karakteriseres det heretter som monoammoniumsaltet. ;Monoammoniumsaltet av antibiotikum A16884 ér et ;hvitt, amorft, fast stoff som spaltes ved ca. 180°C, er lett løse- ;lig i vann, løselig i dimetylsulfoksyd (DMSO), svakt løselig i ;lavere alkanoler og tilnærmet uløselig i acetonitril og andre organiske løsningsmidler. Ovennevnte verdi av den spesifikke op- ;tiske dreining av monoammoniumsaltet av antibiotikum A16884 er basert på saltet tørket ved romtemperatur i vakuum over vannfritt kalsiumklorid i ca. 15 timer. ;Elektrometrisk titrering av monoammoniumsaltet av antibiotikum A16884 i 66% dimetylformanid-vannløsning ved en begynnelses-pH på 6,6 viser nærvær av firetitrerbare grupper: pK^ = 3,5, pK'a2 = 5,2, pK'a3 =9,2 og pK'a4 = 10,3. Ved lik titrering av en senere prøve, med unntagelse av en begynnelses-pH på 5,8, var, som angitt ovenfor, de respektive verdier: pK'a^ = 3,9, pK'a2 = 5,3, pK'a3 = 9,2 og pK'a^ = 10,5. Når monoammoniumsaltet av antibiotikum A16884 omdannes til syreformen, forsvinner pK'a ved 9,2 . ;Elementæranalyse av monoammoniumsaltet av A16884 tørket i vakuum ved 80°C over fosforpentoksyd ga følgende verdier : ;Analyser viste et metoksylinnhold på 6,64% og et acetylinnhold på 9,23% og en Van Slyke-undersøkelse på aminonitrogen viste 5,09%. ;Det infrarøde absorpsjonsspektrum for monoammoniumsaltet av antibiotikum A16884 i en mineraloljesalve er vist i figur 1 på den medfølgende tegning. De klart adskillbare bånd i det infrarøde spektrum over området 2,0 til 15,0 ^a er som ovenfor angitt. ;Det ultrafiolette absorpsjonsspektrum for monoammoniumsaltet av antibiotikum A16884 i vandig løsning viser absorp-1 °/ 1 % sjonsmaksima ved 242 mia (Et- = 126) og ved 625 mu (Er- = 158) . ;J /i cm . / 1 cm Sirkulær dikroisme ble også målt i vandig løsning og viste en positiv Cotton-effekt ved 263 ima og en negativ Cotton-effekt ved 236 ima • ;Papirkromatografi av monoammoniumsaltet av antibiotikum Al688*1- på Whatman nr. 1-papir ga en R^-verdi på 0,79 i et losningsmiddelsystem av propanol, acetonitril og vann i et vo-lumforhold 1:1:1. Bioautografier ble oppnådd ved å plassere pa-pirkromatografen på agarplater Inokulert med folsomme organismer, slik som Salmonella gallinarum, som prbveorganismer. The monoammonium salt of antibiotic A16884 is a white, amorphous substance, which decomposes at approx. 180°C, is slightly soluble in water, soluble in dimethylsulfoxide, slightly soluble in lower alkanols and insoluble in acetonitrile, is amphoteric, has four titratable groups of pK^ = 3.9; pK'a2 =5.3,<*> pK'a3 =9.2 and pK'a4 =10.5 when titrated in 66% dimethylformanide at an initial pH of 5.8, has an apparent molecular weight of approx. 435 determined from titration data, ;25 o o ;has a specific optical rotation [ a] — of +140.9 (C = 1 wt/vol.-% in water), has in mineral oil ointment the following clearly distinguishable bands in its infrared absorption spectrum at the following wavelengths in ^a: 3.18 (broadband), 5.66, 6.26, 6.57, 6.89, 7.15, 7.28, 7.40, 7.73, 8.00, 8 .14, 8.79, 9.24, 9.65, 9.79 and 10.40, giving positive tests with ninhydrin, Pan Deutscher, Benedict, Molisch, iodine and dansyl chloride reagents, and negative experiments with Fehling, ferric chloride, biuret and Sakaguchi reagents. The method is characterized by growing Streptomyces lipmanii NRRL 3584 in a culture medium containing assimilable stores of carbon, nitrogen and inorganic salts under submerged, aerobic conditions until a significant amount of A16884 is produced by said organism in said culture medium. Antibiotic A16884 can be used as such or as a salt, for example an acid addition salt or a salt with a cation. In the case of a salt with a cation, the salt can be either a mono or di salt. It is often preferred to prepare the salts directly in the purification process so that the antibiotic, when separated, is in salt form. Antibiotic A16884 has been separated in this way and for that reason it is henceforth characterized as the monoammonium salt. The monoammonium salt of antibiotic A16884 is a white, amorphous, solid which decomposes at approx. 180°C, is easily soluble in water, soluble in dimethylsulfoxide (DMSO), slightly soluble in lower alkanols and almost insoluble in acetonitrile and other organic solvents. The above value of the specific optical rotation of the monoammonium salt of antibiotic A16884 is based on the salt dried at room temperature in vacuum over anhydrous calcium chloride for approx. 15 hours. ;Electrometric titration of the monoammonium salt of antibiotic A16884 in 66% dimethylformanide aqueous solution at an initial pH of 6.6 shows the presence of four titratable groups: pK^ = 3.5, pK'a2 = 5.2, pK'a3 =9, 2 and pK'a4 = 10.3. On equal titration of a later sample, with the exception of an initial pH of 5.8, the respective values were, as indicated above: pK'a^ = 3.9, pK'a2 = 5.3, pK'a3 = 9.2 and pK'a^ = 10.5. When the monoammonium salt of antibiotic A16884 is converted to the acid form, the pK'a disappears at 9.2. ;Elementary analysis of the monoammonium salt of A16884 dried in vacuum at 80°C over phosphorus pentoxide gave the following values : ;Analyses showed a methoxy content of 6.64% and an acetyl content of 9.23% and a Van Slyke examination on amino nitrogen showed 5.09% . ;The infrared absorption spectrum of the monoammonium salt of antibiotic A16884 in a mineral oil ointment is shown in Figure 1 of the accompanying drawing. The clearly distinguishable bands in the infrared spectrum over the range 2.0 to 15.0 µa are as indicated above. ;The ultraviolet absorption spectrum for the monoammonium salt of antibiotic A16884 in aqueous solution shows absorption maxima at 1 °/1% at 242 mia (Et- = 126) and at 625 mu (Er- = 158). ;J /in cm . / 1 cm Circular dichroism was also measured in aqueous solution and showed a positive Cotton effect at 263 ima and a negative Cotton effect at 236 ima • ;Paper chromatography of the monoammonium salt of antibiotic Al688*1- on Whatman No. 1 paper gave a R^ value of 0.79 in a solvent system of propanol, acetonitrile and water in a volume ratio of 1:1:1. Bioautographs were obtained by placing the paper chromatograph on agar plates inoculated with susceptible organisms, such as Salmonella gallinarum, as test organisms.
NMR-spektret til A16884 i DgO viste fblgende karakteristika: 5,16 ppm. (1H, enkel), 4,86, 4,68 ppm. (2H, AB firedoblet, J = 12,5 Hz), 3,9 - 3,7 ppm. (1H, mangedoblet), 3.67» 3,29 ppm. (2H, AB firedoblet, J = 18 Hz), 3»53PPm. (3H, enkel), 2,6 - 2,3 ppm. (2H, mangedoblet), 2,10 ppm. (3H, enkel), 2,1 - 1,6 ppm. (4H, mangedoblet). The NMR spectrum of A16884 in DgO showed the following characteristics: 5.16 ppm. (1H, single), 4.86, 4.68 ppm. (2H, AB quadrupled, J = 12.5 Hz), 3.9 - 3.7 ppm. (1H, multiplied), 3.67» 3.29 ppm. (2H, AB quadrupled, J = 18 Hz), 3»53PPm. (3H, simple), 2.6 - 2.3 ppm. (2H, multiplied), 2.10 ppm. (3H, simple), 2.1 - 1.6 ppm. (4H, multiplied).
Papirkromatografi av monoammoniumsaltet ble også utfort 1 andre losningsmiddelsysterner med fblgende resultater: Paper chromatography of the monoammonium salt was also carried out in other solvent systems with the following results:
Når monoammoniumsaltet av A16884 ble underkastet tynnskikts-kromatografi på silikagelplater i 70 % vandig acetonitril, under anvendelse av en ninhydrin-dusj som en detektor, hadde det en Rf-verdi på ca. 0,4?, på celluloseplater i 70 % vandig acetonitril, under anvendelse av den samme fremgangsmåte for påvisning, hadde det en Rf-verdi på 0,45. When the monoammonium salt of A16884 was subjected to thin layer chromatography on silica gel plates in 70% aqueous acetonitrile, using a ninhydrin shower as a detector, it had an Rf value of approx. 0.4?, on cellulose plates in 70% aqueous acetonitrile, using the same method of detection, had an Rf value of 0.45.
Aminosyreanalyser av et surt hydrolysat av antibiotikum A16884, utfort ved Spackman-Moore-Steln-teknikk, viste to ninhydrin-reaksjonstopper, hvorav én ble eluert identisk med glycin (0,758 yumol/mg) og den annen ble eluert akkurat like for glycin og ble identifisert som alfa-aminoadipinsyre (2,39yumol/ mg). Ved lik analyse av en senere prove var de observerte verdi- Amino acid analyzes of an acid hydrolyzate of antibiotic A16884, carried out by the Spackman-Moore-Steln technique, showed two ninhydrin reaction peaks, one of which eluted identically to glycine (0.758 yumol/mg) and the other eluted exactly the same for glycine and was identified as alpha-aminoadipic acid (2.39 yumol/ mg). In the same analysis of a later sample, the observed values were
er henholdsvis 0,49 jmol/ mg og 1,2 ^imol/mg. are respectively 0.49 jmol/mg and 1.2 ^imol/mg.
En rekke kvalitative kjemiske undersbkelser har vært utfort med antibiotikum A16884. Antibiotikum A16884 gir en positiv prove med ninhydrin-, Pan Deutscher-, Benedict-, Molsoh-, jod- og dansylklorid-reagenser, men ikke med Fehling-,- ferriklorid, biuret- og Sakaguchi-reagenser. A number of qualitative chemical investigations have been carried out with antibiotic A16884. Antibiotic A16884 gives a positive sample with ninhydrin, Pan Deutscher, Benedict, Molsoh, iodine and dansyl chloride reagents, but not with Fehling, ferric chloride, biuret and Sakaguchi reagents.
Monoammoniumsaltet av antibiotikum A16884 er stabilt ved. pH 3-9 ved 5°C i 8 dogn, relativt stabilt ved pH 3-9 ved 25°C i 4 dogn og ustabilt ved varierende pH-verdier ved 100° innen 5 minutter. Biologisk aktivitet tapes langsomt ved pH 3-9 ved en temperatur på 37°C, idet halvparten tapes etter 4 dogn. The monoammonium salt of antibiotic A16884 is stable at pH 3-9 at 5°C for 8 days, relatively stable at pH 3-9 at 25°C for 4 days and unstable at varying pH values at 100° within 5 minutes. Biological activity is lost slowly at pH 3-9 at a temperature of 37°C, with half being lost after 4 days.
Antibiotikum A16884 har en hemmende virkning på vekst av både gram-positive og gram-negative bakterier. Nivåene Antibiotic A16884 has an inhibitory effect on the growth of both gram-positive and gram-negative bacteria. The levels
ved hvilke delvis renset monoammoniumsalt av antibiotikum A16884 viser hemmende virkning mot vekst av illustrerende organismer er angitt i tabell I. De hemmende nivåer ble bestemt ved agarfortynningsprbven eller ved kulturvæskefortynningsprbven (angitt i at which the partially purified monoammonium salt of antibiotic A16884 shows inhibitory action against the growth of illustrative organisms are listed in Table I. The inhibitory levels were determined by the agar dilution test or by the culture fluid dilution test (listed in
tabellen ved bokstavene henholdsvis "a.d." og "b.d.". the table by the letters respectively "a.d." and "b.d.".
I agarfortynningsprbven utstrykes prbveorganismen på en serie agarplater inneholdende forskjellige konsentrasjoner av monoammoniumsaltet av antibiotikum A16884 for å bestemme de minste konsentrasjoner i mcg/ml (mikrogram pr. milliliter) i agar-substratet som hemmer veksten av organismen over et tidsrom på 48 timer (72 timer i tilfellet med de plantepatogene organismer) . In the agar dilution test, the test organism is plated on a series of agar plates containing different concentrations of the monoammonium salt of antibiotic A16884 to determine the minimum concentrations in mcg/ml (micrograms per milliliter) in the agar substrate that inhibit the growth of the organism over a period of 48 hours (72 hours in the case of the plant pathogenic organisms) .
I kulturvæskefortynningsprbven ble en rekke ror inneholdende næringskultur med varierende konsentrasjoner av monoammoniumsaltet av antibiotikum A16884 innokulert med prbveorga-nismene for å bestemme den minste konsentrasjon av monoammoniumsaltet av A16884 i mcg/ml i substratet som hindrer organisme-veksten i et tidsrom av tyve timer. In the culture liquid dilution test, a number of tubes containing nutrient culture with varying concentrations of the monoammonium salt of antibiotic A16884 were inoculated with the test organisms to determine the minimum concentration of the monoammonium salt of A16884 in mcg/ml in the substrate which prevents the growth of the organism for a period of twenty hours.
Ingen binding av hesteserum ble iakttatt i noen av de ovennevnte prøver. No binding of horse serum was observed in any of the above samples.
Som det vil sees av foregående tabell, oppviser antibiotikum A16884 som monoammoniumsalt aktivitet mot gram-posi- As will be seen from the preceding table, antibiotic A16884 as a monoammonium salt exhibits activity against gram-positive
tive og gram-negative bakterier. tive and gram-negative bacteria.
Mer høyverdig renset monoammoniumsalt av antibioti- More high quality purified monoammonium salt of antibiotics
kum A16884 ble videre undersøkt for antibakteriell aktivitet i et forsøk hvori ble anvendt kulturvæskefortynningsteknikken beskrevet ovenfor. Resultatene uttrykt i det minste antall mikrogram pr. milliliter som kreves for oppnåelse av hemming, er angitt i tabell II nedenfor. kum A16884 was further examined for antibacterial activity in an experiment using the culture fluid dilution technique described above. The results expressed in the minimum number of micrograms per milliliters required to achieve inhibition are listed in Table II below.
Antibiotikum A16884 og dets salter oppviser også Antibiotic A16884 and its salts also exhibit
in vivo aktivitet mot et antall av ovennevnte organismer og er følgelig brukbare for å kontrollere infeksjoner foranlediget av slike organismer i vertdyr. Delvis renset antibiotikum A16884 som monoammoniumsaltet oppviser en ED^0 på 23 mg/kg i mus infisert med Proteus PR6 og en ed^q på 33,8 mg/kg i mus infisert med Shigella SH3 . Mer høyrenset A16884 som ammoniumsaltet oppviser en ED,-Q på 3,64 mg/kg i mus infisert med Escherichia coli EC14 og E°50 på 23 mg/kg mus i infisert med Salmonella typhosa SA12 og in vivo activity against a number of the above organisms and are consequently useful for controlling infections caused by such organisms in host animals. Partially purified antibiotic A16884 as the monoammonium salt exhibits an ED^0 of 23 mg/kg in mice infected with Proteus PR6 and an ed^q of 33.8 mg/kg in mice infected with Shigella SH3. More highly purified A16884 as the ammonium salt exhibits an ED,-Q of 3.64 mg/kg in mice infected with Escherichia coli EC14 and an E°50 of 23 mg/kg in mice infected with Salmonella typhosa SA12 and
en ED^0 93,4 mg/kg i mus infisert med Klebsiella Kl. Administrering ble foretatt under huden. an ED^0 93.4 mg/kg in mice infected with Klebsiella Kl Administration was carried out under the skin.
Som ovenfor nevnt, oppviser antibiotikum A16884 og dets salter anthelmintisk aktivitet i tillegg til bakteriell aktivitet. Følgelig kan antibiotikum A16884 eller saltet derav ad-ministreres til varmblodige dyr for å kontrollere forskjellige innvortes parasitter, spesielt mage- og innvollsormer som Ascaris lumbricoides var. suum, Nematospiroides dubius, Aspiculuris tetraptera, Syphacia obvelata og lignende. Administreringen foretas fortrinnsvis oralt, for eksempel ved innlemmelse av antibiotikum A16884 eller et salt i dyreføde, ved administrering av tabletter, medisin etc. inneholdende A16884 eller et salt, eller på andre måter. Generelt er doser fra 1 til 500 milligram pr. kg eller mer legemsvekt effektive i enkeltdose administrering. Hvis antibiotikum A16884 eller et-salt derav tilføres som As mentioned above, antibiotic A16884 and its salts exhibit anthelmintic activity in addition to bacterial activity. Accordingly, antibiotic A16884 or its salt can be administered to warm-blooded animals to control various internal parasites, especially stomach and intestinal worms such as Ascaris lumbricoides var. suum, Nematospiroides dubius, Aspiculuris tetraptera, Syphacia obvelata and the like. The administration is preferably carried out orally, for example by incorporating antibiotic A16884 or a salt in animal feed, by administering tablets, medicine etc. containing A16884 or a salt, or in other ways. In general, doses are from 1 to 500 milligrams per kg or more body weight effective in single dose administration. If antibiotic A16884 or a salt thereof is added as
en bestanddel av regulær føde, gir konsentrasjoner på fra 0,001 a component of regular food, gives concentrations of from 0.001
til 0,05% eller mer gode resultater. Et foretrukket konsentrasjons-område for antibiotikum A16884 eller et salt derav i føde er fra 0,01 til 0,05%. to 0.05% or more good results. A preferred concentration range for antibiotic A16884 or a salt thereof in food is from 0.01 to 0.05%.
Den anthelmintiske aktivitet av antibiotikum A16884 The anthelmintic activity of antibiotic A16884
er illustrert ved følgende forsøk. is illustrated by the following experiment.
I et første forsøk ble antibiotikum A16884-monoammoniumsalt administrert i en enkel dose til hver av to mus infisert med Aspiculuris tetraptern og Syphacia obvelata (nålormer). Dosen var 500 milligram antibiotikum A16884-monoammoniumsalt pr. individuelt dyrs kroppsvekt administrert som en suspensjon av fysiologisk salt-lake inneholdende 0,125% metylcellulose som suspenderingsmidde1. In a first experiment, the antibiotic A16884 monoammonium salt was administered in a single dose to each of two mice infected with Aspiculuris tetraptern and Syphacia obvelata (needleworms). The dose was 500 milligrams of antibiotic A16884-monoammonium salt per individual animal body weight administered as a suspension of physiological saline containing 0.125% methylcellulose as suspending agent1.
En kontrollgruppe mus infisert med Aspiculuris tetraptera og Syphacia obvelata ble anvendt ved forsøket. Begge grupper ble holdt under normale laboratoriebetingelser i 48 timer etter dosering av den be-handlede gruppe. Alle mus ble så slaktet og undersøkt for bestemmelse av nærvær av og antall av nålormer, hvilket er angitt i den følgende tabell: A control group of mice infected with Aspiculuris tetraptera and Syphacia obvelata was used in the experiment. Both groups were kept under normal laboratory conditions for 48 hours after dosing of the treated group. All mice were then slaughtered and examined to determine the presence and number of pinworms, as indicated in the following table:
I et annet forsøk ble antibiotikum A16884-monoammoniumsalt blandet med standard museføde for oppnåelse av et flertall be-handlede føder inneholdende antibiotikum A16884-monoammoniumsalt i - konsentrasjoner på 0,005, 0,01 og 0,05 vektprosent. Føden ble brukt som dietter for separate grupper av mus, fem mus pr. gruppe. In another experiment, antibiotic A16884 monoammonium salt was mixed with standard mouse chow to obtain a plurality of treated diets containing antibiotic A16884 monoammonium salt in concentrations of 0.005, 0.01 and 0.05 percent by weight. The food was used as diets for separate groups of mice, five mice per group.
Ca. 24 timer etter inntak av føden ble musene infisert med Ascaris lumbrigoides var. suum. Alle grupper ble matet med deres respektive føde og holdt under normale laboratoriebetingelser for et tidsrom av ti døgn, hvoretter alle mus ble tatt av fra mating. Den ellevte dag ble alle mus slaktet og lungene undersøkt for bestemmelse av nærvær av og i tilfelle nærvær antall lesjoner av Ascaris lumbricoides var. suum. About. 24 hours after consuming the food, the mice were infected with Ascaris lumbrigoides var. hum. All groups were fed their respective food and kept under normal laboratory conditions for a period of ten days, after which all mice were removed from feeding. On the eleventh day, all mice were sacrificed and the lungs examined to determine the presence and, if present, the number of Ascaris lumbricoides lesions. hum.
Nivået av antibiotikum Al6884-monoammoniumsalt i dietten og det gjennomsnittlige antall lungelesjoner pr. dyr i hver gruppe er angitt i den følgende tabell: The level of antibiotic Al6884-monoammonium salt in the diet and the average number of lung lesions per animals in each group are indicated in the following table:
Antibiotikum A16884, som monoammoniumsaltet, ble undersøkt for kontrollen av plantepatogene bakterieorganismer. Ved dette forsøk ble antibiotikum A16884-monoammoniumsaltet anvendt i en vandig dusj ved en konsentrasjon på 400 deler pr. million ferdig sammensetning. 30 døgn gamle tomatplanter ble brukt ved vurderingen, 2 planter i hver potte. Plantene i en potte ble behandlet med den ovenfor beskrevne løsning, fikk tørke i luft og ble så inokulert med et medium inneholdende en aktiv kultur av Pseudomonas solanacearum. Plantene i den annen potte ble dusjet med en vandig dusj løsning identisk med den ovenfor beskrevne behandlings-løsning, men uten antibiotikum, som blindprøve. Plantene som tjente som blindprøve, ble deretter likeledes inokulert. Alle plantene ble holdt i 24 timer i et fuktig rom, deretter fjernet fra rommet og holdt i syv døgn under gode dyrkningsforhold. Ved slutten av denne periode ble alle plantene undersøkt for bestemmelse av nærvær, og i tilfelle, graden av infeksjon. Plantene behandlet med antibiotikum A16884-monoammoniumsalt var fullstendig fri for sykdoms-symptomer foranlediget av Pseudomonas solanacearum, mens blind-prøveplantene derimot oppviste utstrakte symptomer som kunne til-skrives Pseudomonas solanacearum. Antibiotic A16884, as the monoammonium salt, was investigated for the control of plant pathogenic bacterial organisms. In this experiment, the antibiotic A16884 monoammonium salt was used in an aqueous shower at a concentration of 400 parts per million finished composition. 30-day-old tomato plants were used in the assessment, 2 plants in each pot. The plants in a pot were treated with the solution described above, allowed to dry in air and then inoculated with a medium containing an active culture of Pseudomonas solanacearum. The plants in the second pot were showered with an aqueous shower solution identical to the treatment solution described above, but without antibiotics, as a blank test. The plants that served as blanks were then likewise inoculated. All plants were kept for 24 hours in a humid room, then removed from the room and kept for seven days under good growing conditions. At the end of this period, all plants were examined to determine the presence and, if applicable, the degree of infection. The plants treated with antibiotic A16884-monoammonium salt were completely free of disease symptoms caused by Pseudomonas solanacearum, while the blind test plants, on the other hand, showed extensive symptoms that could be attributed to Pseudomonas solanacearum.
Sammenligninqsforsøk Comparison test
Cephalosporin C og A16884 ble samtidig undersøkt in vitro mot en rekke arter og in vivo mot Escherichia coli. Begge undersøkelsesmetoder var av vanlig type. De oppnådde data ved in vivo-forsøkene er som følger: Cephalosporin C and A16884 were simultaneously tested in vitro against a number of species and in vivo against Escherichia coli. Both survey methods were of the usual type. The data obtained in the in vivo experiments are as follows:
Det samme aktivitetsmønster ble oppnådd ved in vitro-undersøkelsen: The same pattern of activity was obtained in the in vitro study:
Antibiotikum A16884 ble videre funnet å være bedre enn Cephalosporin C mot to andre organismer: Antibiotic A16884 was further found to be superior to Cephalosporin C against two other organisms:
Fremstilling og isolering av Cephalosporin C er beskrevet i US-patenter 3.093.638, 3.184.454 og 3.467.654. Videre er det også en omfattende beskrivelse av dette i kapittel 1 av "Cephalosporins and Penicillins" av Edwin H. Flynn (Academic Press, N.Y., 1972) . Preparation and isolation of Cephalosporin C is described in US patents 3,093,638, 3,184,454 and 3,467,654. Furthermore, there is also a comprehensive description of this in Chapter 1 of "Cephalosporins and Penicillins" by Edwin H. Flynn (Academic Press, N.Y., 1972).
Antibiotikum A16884 fremstilles ved å dyrke en Antibiotic A16884 is prepared by growing a
nylig funnet og hittil ubeskrevet organismestamme isolert fra jordprøver fra Syd-Amerika. recently discovered and hitherto undescribed strain of organism isolated from soil samples from South America.
Organismen ble isolert fra ovennevnte jordprøver ved The organism was isolated from the above soil samples at
å suspendere p®6sjoner av jordprøvene i sterilt, destillert vann og ved å stryke ut suspensjonene på næringsagar. De sådde nærings-agarplater ble inkubert ved 25-35°C i flere døgn. Ved slutten av inkubasjonstiden ble kolonier av den antibiotikum A16884-produser-ende organisme ved hjelp av en steril platinaøse overført til agar-skråkulturer. Agar-skråkulturene ble så inkubert for tilveiebring-else av passende mengder inokulat for fremstilling av antibiotikum A16884. to suspend portions of the soil samples in sterile, distilled water and by spreading the suspensions on nutrient agar. The seeded nutrient agar plates were incubated at 25-35°C for several days. At the end of the incubation period, colonies of the antibiotic A16884-producing organism were transferred to agar slants using a sterile platinum spoon. The agar slants were then incubated to provide appropriate amounts of inoculum for the production of antibiotic A16884.
Actinomyceten brukt i henhold til oppfinnelsen for fremstilling av antibiotikum A16884 har blitt betegnet som en stamme av Streptomyces lipmanii Waksman og Curtis. The actinomycete used according to the invention for the preparation of antibiotic A16884 has been designated as a strain of Streptomyces lipmanii Waksman and Curtis.
Den nye organisme som er istand til å produsere antibiotikum A16884 har blitt plassert som permanent depositum uten re-striksjoner med hensyn til tilgjengelighet i kultursamlingen i Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture (tidligere Northern Regional Research Laboratories), Peoria, Illinois 61604, og er til-gjengelig for almenheten under kulturnummeret NRRL 3584. The new organism capable of producing antibiotic A16884 has been placed in permanent deposit without restriction as to availability in the culture collection of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture (formerly Northern Regional Research Laboratories), Peoria, Illinois 61604, and is available to the public under culture number NRRL 3584.
Streptomyces lipmanii NRRL 3584's karakteristika er Streptomyces lipmanii NRRL 3584's characteristics are
gitt i de følgende tabeller. Fremgangsmåten anbefalt av internatio-nal Streptomyces Project (Shirling m.fl.: "Methods for Characteri-zation of Streptomyces Species", Intern. Bull. Systematic Bacteriol. 16: 313-340 (1966)) for karakteriseringen av Streptomyces-arter har vært brukt sammen med supplerende forsøk. Fargebéteghelser ble fastsatt i henhold til ISCC-NBS-metoden beskrevet av Kelly m.fl. i "The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names" (U.S. Department of Commerce Circ. 553, Washington, D.C., 1955) . Tallene i parenteser refererer seg til Tresner og Backus fargeserier (Tresner m.fl. "System of Color Wheels for Streptomyces Taxonomy", Appl. Microbiol. 11: 335-338 (1963)) og fargetabellbetegnelser er understreket. Maerz og Pauls fargebeteg-nelser (Maerz m.fl.: "Dictionary of Color (McGraw-Hill Book Co., Inc., New York, 1950) er angitt i parenteser. Kulturer ble dyrket ved 30°C i 14 døgn hvis ikke annet er angitt. given in the following tables. The procedure recommended by the International Streptomyces Project (Shirling et al.: "Methods for Characterization of Streptomyces Species", Intern. Bull. Systematic Bacteriol. 16: 313-340 (1966)) for the characterization of Streptomyces species has been used in conjunction with supplementary trials. Color defects were determined according to the ISCC-NBS method described by Kelly et al. in "The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names" (U.S. Department of Commerce Circ. 553, Washington, D.C., 1955). The numbers in parentheses refer to Tresner and Backus color series (Tresner et al. "System of Color Wheels for Streptomyces Taxonomy", Appl. Microbiol. 11: 335-338 (1963)) and color table designations are underlined. Maerz and Paul's color designations (Maerz et al.: "Dictionary of Color (McGraw-Hill Book Co., Inc., New York, 1950)) are indicated in parentheses. Cultures were grown at 30°C for 14 days if not otherwise is indicated.
Karakteristika for den anvendte kultur er meget like de som nylig er publisert (Shirling E.B. og D. Gottlieb, 1968. Co-operative description of type cultures of Streptomyces. II. Species description from first study. Intern. Bull. Systematic Bacteriol. 18: 69-189) for S. lipmanii. Den adskiller seg fra S. lipmanii ved utnyttelsen av visse karbonkilder og også ved farven på glycerol-asparagin agar. Arter som ligner på S. lipmanii omfatter S. fellus, S. flavovireus, S. griseolus og S. halstedii. Characteristics of the culture used are very similar to those recently published (Shirling E.B. and D. Gottlieb, 1968. Co-operative description of type cultures of Streptomyces. II. Species description from first study. Intern. Bull. Systematic Bacteriol. 18: 69-189) for S. lipmanii. It differs from S. lipmanii by the utilization of certain carbon sources and also by the color of glycerol-asparagine agar. Species similar to S. lipmanii include S. fellus, S. flavovireus, S. griseolus and S. halstedii.
Som ovenfor nevnt fremstilles antibiotikum A16884 ved dyrkning av NRRL 3584. Kulturmediet anvendt ved fremstilling av antibiotikum A16884 ved dyrkning av den ovenfor identifiserte organisme kan være hvilket som helst av flere media, idet, som det vil fremgå av de ovenfor beskrevne utnyttelsesforsøk, organismen er istand til å utnytte forskjellige energikilder. Av produksjonsøko-nomiske grunner, maksimalt utbytte av antibiotikum og lettheten ved å isolere antibiotikumet foretrekkes imidlertid visse relativt enkle næringskilder. Media som er brukbare ved fremstilling av antibiotikumet omfatter for eksempel.en assimilerbar karbonkilde som glukose stivelse glycerin, melasse, dekstrin og lignende. As mentioned above, antibiotic A16884 is produced by cultivation of NRRL 3584. The culture medium used in the production of antibiotic A16884 by cultivation of the above-identified organism can be any of several media, since, as will be apparent from the above-described utilization trials, the organism is to utilize different energy sources. However, for reasons of economic production, maximum yield of antibiotic and the ease of isolating the antibiotic, certain relatively simple food sources are preferred. Media which are usable in the preparation of the antibiotic include, for example, an assimilable carbon source such as glucose, starch, glycerin, molasses, dextrin and the like.
Den foretrukne karbonkilde er glukose. Ytterligere anvendbare media omfatter en assimilerbar nitrogenkilde som soyabønnemel, faststoffer som maisstøp, gjær, bomullsfrømel, kjøttekstrakt, peptoner (kjøtt eller soyabønner), kasein, aminosyreblandinger og lignende. Foretrukne nitrogenkilder er peptoner, soyabønnemel, aminosyreblandinger og lignende. Blant uorganiske næringssalter som er istand til å gi natrium-, kalium-, ammonium-, kalsium-, fosfat-, sulfat-, klorid-, karbonat- og lignende ioner. The preferred carbon source is glucose. Additional useful media include an assimilable nitrogen source such as soybean meal, solids such as corn meal, yeast, cottonseed meal, meat extract, peptones (meat or soybean), casein, amino acid mixtures, and the like. Preferred nitrogen sources are peptones, soybean meal, amino acid mixtures and the like. Among inorganic nutrient salts capable of giving sodium, potassium, ammonium, calcium, phosphate, sulphate, chloride, carbonate and similar ions.
Små mengder elementer nødvendige for optimal vekst Small amounts of elements necessary for optimal growth
og utvikling av organismen brukt for fremstilling av antibioti- and development of the organism used for the production of antibiotics
kum A16884 kan også innlemmes i kulturmediet. Slike sporelemen- kum A16884 can also be incorporated into the culture medium. Such trace element-
ter opptrer vanligvis som forurensninger i de andre bestanddel- usually act as contaminants in the other constituents
ene i mediet i mengder tilstrekkelig til å oppfylle vekstkrave- in the medium in quantities sufficient to fulfill growth requirements
ne til actinomyceten anvendt i foreliggende oppfinnelse. ne to the actinomycete used in the present invention.
Den opprinnelige pH i kulturmediet kan variere. Det er imidlertid funnet ønskelig at mediets opprinnelige pH ligger mellom 6,5 og 7,2. Som iakttatt ved andre actinomyceter, øker pH i mediet gradvis under organismens vekstperiode for fremstillingen av antibiotikumet og kan oppnå et nivå på fra 6,7 til 7,5 og derover, idet den endelige pH i det minste er avhengig av mediets opprinnelige pH, tilstedeværende puffere i mediet og tidsrommet for organismens vekst. The initial pH of the culture medium can vary. However, it has been found desirable that the medium's original pH is between 6.5 and 7.2. As observed with other actinomycetes, the pH of the medium increases gradually during the organism's growth period for the production of the antibiotic and can reach a level of from 6.7 to 7.5 and above, the final pH being at least dependent on the initial pH of the medium, present buffers in the medium and the time period for the organism's growth.
Neddykket, aerobe dyrkningsbetingelser i sterile be-holdere velges for fremstilling av antibiotikum A16884. Mediet i den sterile beholder kan inokuleres med en sporesuspensjon, men på grunn av vekstforsinkelsen ved anvendelse av en sporesuspensjon som inokulat, foretrekkes en vegetativ form for dyrkingen. Ved på denne måte å unngå vekstforsinkelsen, oppnåes en mer effektiv utnyttelse av dyrkningsutstyret. Det er følgelig ønskelig først å fremstille et vegetativt inokulat av organismen ved å inokulere en relativt liten mengde av kulturmediet med organismens sporeform og når et ungt, aktivt, vegetativt inokulat er oppnådd, overføres det vegetative inokulat aseptisk til den store beholder. Mediet hvori det vegetative inokulat er fremstilt, kan enten være det samme eller forskjellig fra det som anvendes for fremstilling av antibiotikum A16884 i stor målestokk. Submerged, aerobic cultivation conditions in sterile containers are chosen for the production of antibiotic A16884. The medium in the sterile container can be inoculated with a spore suspension, but due to the growth delay when using a spore suspension as inoculum, a vegetative form of cultivation is preferred. By avoiding the growth delay in this way, a more efficient utilization of the cultivation equipment is achieved. It is therefore desirable to first prepare a vegetative inoculum of the organism by inoculating a relatively small amount of the culture medium with the organism's spore form and when a young, active, vegetative inoculum is obtained, the vegetative inoculum is aseptically transferred to the large container. The medium in which the vegetative inoculum is produced can either be the same or different from that used for the production of antibiotic A16884 on a large scale.
Organismen som produserer antibiotikum A16884, kan vokse over et bredt temperaturområde, mellom 25 - 37°C. Optimal produksjon av A16884 synes å opptre ved temperaturer på 26 - 30°C. Generelt opptrer maksimal produksjon av antibiotikumet innen ca. 36-72 timer etterinokulering av kulturmediet. The organism that produces antibiotic A16884 can grow over a wide temperature range, between 25 - 37°C. Optimal production of A16884 appears to occur at temperatures of 26 - 30°C. In general, maximum production of the antibiotic occurs within approx. 36-72 hours after inoculation of the culture medium.
Som vanlig ved aerobe, neddykkete dyrkningsprosesser, blåses steril luft gjennom kulturmediet. For effektiv vekst av . organismen og antibiotikum Al6884-produksjon er volumet av anvendt • luft i beholderen for fremstilling av A16884 fra 0,2 til 0,4 volumdel luft pr. minutt pr. volumdel kultur. Det foretrukne volum er 0,40 volumdel luft pr. minutt pr. volumdel kulturmedium. As usual with aerobic, submerged cultivation processes, sterile air is blown through the culture medium. For efficient growth of . the organism and antibiotic Al6884 production is the volume of air used • in the container for the production of A16884 from 0.2 to 0.4 volume part of air per minute per volume part culture. The preferred volume is 0.40 parts by volume of air per minute per volume part culture medium.
Konsentrasjonen av antibiotikumaktivitet i kulturmediet kan lett følges under dyrkningsperioden ved å undersøke prøver av kulturmediet med henblikk på dets hemmende aktivitet The concentration of antibiotic activity in the culture medium can be easily monitored during the culture period by examining samples of the culture medium for its inhibitory activity
overfor veksten av organismer som man vet vil bli hemmet ved nær- against the growth of organisms which are known to be inhibited by near-
vær av antibiotikum A16884. Organismene Sarcina lutea og Salmonella gallinarium er funnet å kunne brukes for dette formål. Undersøkelsen av prøvene kan utføres ved hjelp av den kjente turbidometriske eller kopp-platemetoden. be of antibiotic A16884. The organisms Sarcina lutea and Salmonella gallinarium have been found to be useful for this purpose. The examination of the samples can be carried out using the known turbidometric or cup-plate method.
Generelt opptrer maksimal produksjon av A16884 innen ett til tre døgn etter inokulering av kulturmediet i neddykket, aerob dyrkning eller rystekolbedyrkningsmetoder. In general, maximum production of A16884 occurs within one to three days after inoculation of the culture medium in the submerged, aerobic culture, or shake flask culture methods.
Antibiotika-aktiviteten frembragt under fermenteringen av A16884 opptrer i antibiotikakulturvæsken. Følgelig er isolerings-teknikken anvendt ved fremstillingen av A16884 utformet for å oppnå maksimal utvinning av antibiotikumet fra kulturvæsken. Således fjer-nes for eksempel myceliet og uløste faststoffer fra fermenterings-kulturvæsken på vanlig måte, som for eksempel filtrering eller sentrifugering, og antibiotikumet A16884 kan utvinnes fra den filtrerte eller sentrifugerte kulturvæske ved anvendelse av ekstraksjons- eller adsorpsjonsteknikk. The antibiotic activity produced during the fermentation of A16884 appears in the antibiotic culture fluid. Consequently, the isolation technique used in the preparation of A16884 is designed to achieve maximum recovery of the antibiotic from the culture fluid. Thus, for example, the mycelium and undissolved solids are removed from the fermentation culture liquid in the usual way, such as filtration or centrifugation, and the antibiotic A16884 can be recovered from the filtered or centrifuged culture liquid using extraction or adsorption techniques.
For utvinning av A16884 ved adsorpsjon kan anvendes forskjellige adsorpsjonsmidler og ionevekslerharpikser, for eksempel kull, silikagel, aluminiumoksyd og ionevekslerharpikser. " Antibiotikum A16884 oppnådd fra fermentering kan enten være i amfoter form eller saltform avhengig av fermenteringsbetingelsene. Uavhengig av hvilken form kan det adsorberes på ett av ovennevnte eller lignende adsorpsjonsmidler fra løsning i et passende løsningsmiddel. Det adsorberte antibiotikum A16884 eller salt kan så elueres fra adsorpsjonsmidlet ved passende elueringsteknikk, slik som ved vasking av adsorpsjonsmidlet hvorpå A16884 eller dets salt er adsorbert, med et løsningsmiddel. Hvor eluering utføres ved vasking med et løs-ningsmiddel, f.eks. ammoniumformiat eller natriumacetat, resulterer prosessen i eluering av antibiotikum A16884 som henholdsvis ammonium-eller natriumsalt. Slike salter kan lett tilbakedannes til antibiotikum A16884 på vanlig måte. I den foregåendeutvinningsprosess kan også mikrokrystallinsk cellulose brukes som adsorpsjonsmiddel. For the extraction of A16884 by adsorption, different adsorbents and ion exchange resins can be used, for example coal, silica gel, aluminum oxide and ion exchange resins. " Antibiotic A16884 obtained from fermentation can be either in amphoteric form or salt form depending on the fermentation conditions. Regardless of which form, it can be adsorbed onto one of the above or similar adsorbents from solution in a suitable solvent. The adsorbed antibiotic A16884 or salt can then be eluted from the adsorbent by suitable elution technique, such as by washing the adsorbent onto which A16884 or its salt is adsorbed, with a solvent. Where elution is carried out by washing with a solvent, eg, ammonium formate or sodium acetate, the process results in elution of antibiotic A16884 as respectively ammonium or sodium salt. Such salts can easily be regenerated to antibiotic A16884 in the usual way. In the preceding extraction process, microcrystalline cellulose can also be used as an adsorbent.
Andre salter av antibiotikum A16884 enn ammonium- og alkalimetallsaltene fremstilles fortrinnsvis ved vanlig omsetning av antibiotikum A16884 i ikke-modifisert, amfoter form med den respektive syre eller base. Ved fremstilling av syreaddisjonssalter om-settes således antibiotikum A1S884 med en uorganisk eller organisk syre. Representative syrer omfatter korhydrogensyre, bromhydrogen-syre, jodhydrogensyre, sovelsyre, fosforsyre, eddiksyre, benzosyre, sulfaminsyre, vinsyre, citronsyre, maleinsyre, ravsyre, askorbinsyre og glykolsyre. Salts of antibiotic A16884 other than the ammonium and alkali metal salts are preferably prepared by ordinary reaction of antibiotic A16884 in unmodified, amphoteric form with the respective acid or base. When producing acid addition salts, antibiotic A1S884 is thus reacted with an inorganic or organic acid. Representative acids include corric acid, hydrobromic acid, hydroiodic acid, solubilic acid, phosphoric acid, acetic acid, benzoic acid, sulfamic acid, tartaric acid, citric acid, maleic acid, succinic acid, ascorbic acid and glycolic acid.
Antibiotikum A16884 danner også salter med kationer ved omsetning av A16884 i ikke-modifisert, amfoter form med uorganiske baser eller salter. Eksempler på slike salter er ammonium- og sub-stituerte ammoniumsalter, alkalimetallsalter som natrium, kalium, litium, cesium, rubidium, jordalkalimetallsalter som kalsium, stron-tium og barium, og salter med andre metaller som aluminium, kobber, sink, magnesium og sølv. Med hensyn til organiske baser er identi-teten av basen ikke kritisk, skjønt en base som i vann har en numer-isk pH på 3,0 eller derover foretrekkes. Representative organiske baser omfatter benzylamin, metylamin, dietylamin, trietylamin, pro-kain, diisopropylamin, etanolamin, cykloheksylamin, dicykloheksyl-amin, difenylamin, di-n-butylamin, kinolin og pyridylamin. Antibiotic A16884 also forms salts with cations upon reaction of A16884 in unmodified, amphoteric form with inorganic bases or salts. Examples of such salts are ammonium and substituted ammonium salts, alkali metal salts such as sodium, potassium, lithium, cesium, rubidium, alkaline earth metal salts such as calcium, strontium and barium, and salts with other metals such as aluminium, copper, zinc, magnesium and silver . With regard to organic bases, the identity of the base is not critical, although a base which in water has a numerical pH of 3.0 or above is preferred. Representative organic bases include benzylamine, methylamine, diethylamine, triethylamine, procaine, diisopropylamine, ethanolamine, cyclohexylamine, dicyclohexylamine, diphenylamine, di-n-butylamine, quinoline and pyridylamine.
Saltene av antibiotikum A16884 som er farmasøytisk akseptable, er generelt foretrukket. Imidlertid er alle salter brukbare som mellomprodukter ved fremstillingen, separasjonen og rens-ingen av antibiotikum A16884. For terapeutiske formål er enten ka-tioniske eller anioniske, farmasøytisk akseptable salter generelt ekvivalente med antibiotikum A16884, skjønt spesielle salter under tiden foretrekkes på grunn av en gunstig egenskap, slik som løselig-het, jevnført med den saltdannende halvdel. The pharmaceutically acceptable salts of antibiotic A16884 are generally preferred. However, all salts are useful as intermediates in the preparation, separation and purification of antibiotic A16884. For therapeutic purposes, either cationic or anionic pharmaceutically acceptable salts are generally equivalent to antibiotic A16884, although particular salts are currently preferred because of a favorable property, such as solubility, associated with the salt-forming moiety.
For mer fullstendig å illustrere utøvelsen av oppfinnelsen, gjengis i det følgende en del illustrerende eksempler. In order to more fully illustrate the practice of the invention, a number of illustrative examples are reproduced below.
Eksempel 1 Example 1
RYSTEKOLBEFREMSTILLING AV ANTIBIOTIKUM A16884 SHAKE FLASK PREPARATION OF ANTIBIOTICS A16884
En sporekultur av Streptomyces lipmanii NRRL 3584 ble fremstilt ved dyrkning av organismen på en næringsagar-skråkultur som hadde følgende sammensetning: A spore culture of Streptomyces lipmanii NRRL 3584 was prepared by culturing the organism on a nutrient agar slant having the following composition:
pH i mediet ble regulert til 7,0 ved tilsetning av nåtriumhydroksyd The pH of the medium was adjusted to 7.0 by adding sodium hydroxide
Agarskråkulturen ble inokulert med sporer av Streptomyces lipmanii NRRL 3584 og inkubert i 6 døgn ved 30°C. Agarskråkulturen ble så dekket med sterilt, destillert vann og forsiktig skrapet for å fjerne sporene og cellene som en vandig suspensjon. En milliliter av den resulterende suspensjon ble brukt til inokulering av hver av 100 ml porsjoner av et vegetativt medium med følgende sammensetning: The agar slant culture was inoculated with spores of Streptomyces lipmanii NRRL 3584 and incubated for 6 days at 30°C. The agar slant was then covered with sterile distilled water and gently scraped to remove the spores and cells as an aqueous suspension. One milliliter of the resulting suspension was used to inoculate each of 100 ml portions of a vegetative medium with the following composition:
pH i det vegetative medium ble regulert til 6,7 ved tilsetning av natriumhydroksyd. The pH in the vegetative medium was adjusted to 6.7 by adding sodium hydroxide.
Det vegetative inokulat ble rystet i 36 timer ved 30°C på en frem- og tilbakegående rysteinnretning med et ca. 5 cm slag ved 108 slag/min. Det således fremstilte inokulat ble brukt til fremstilling av A16884 som følger. The vegetative inoculum was shaken for 36 hours at 30°C on a reciprocating shaker with an approx. 5 cm stroke at 108 strokes/min. The inoculum thus prepared was used for the preparation of A16884 as follows.
Et produksjonsmedium med følgende sammensetning ble fremstilt: A production medium with the following composition was prepared:
100 milliliters porsjoner av produksjonsmediet ble anbragt i 500 milliliters Erlenmeyer-kolber som var sterilisert ved 120°C i 30 minutter. Etter avkjøling ble hver kolbe inokulert med et 5% vegetativt inokulat. Kolben ble rystet i 72 timer ved 30°C på en roterende rysteinnretning ved 250 rpm. Under fermenteringen ble mediet luftet med steril luft ved en hastighet av 0,4 vol./vol./min. Iso-leringen ble utført i det vesentlige som gjengitt nedenfor i eks. 8. 100 milliliter portions of the production medium were placed in 500 milliliter Erlenmeyer flasks which had been sterilized at 120°C for 30 minutes. After cooling, each flask was inoculated with a 5% vegetative inoculum. The flask was shaken for 72 hours at 30°C on a rotary shaker at 250 rpm. During the fermentation, the medium was aerated with sterile air at a rate of 0.4 vol./vol./min. The isolation was carried out essentially as reproduced below in ex. 8.
Eksempel 2 Example 2
Antibiotikum A16884 ble fremstilt i henhold til fremgangsmåten i eksempel 1, men under anvendelse av et produksjons- Antibiotic A16884 was prepared according to the procedure of Example 1, but using a manufacturing
medium med følgende sammensetning: medium with the following composition:
og under anvendlese av, istedenfor en roterende rysteinnretning, en frem- og tilbakegående rysteinnretning som arbeidet ved 108 slag pr. minutt. and using, instead of a rotary shaking device, a reciprocating shaking device which worked at 108 strokes per minute.
Eksempel 3 Example 3
Antibiotikum A16884 ble fremstilt som i fremgangsmåten ifblge eksempel 1 med unntagelse av., at det ble brukt et produksjonsmedium med fblgende sammensetning: Antibiotic A16884 was prepared as in the method according to example 1 with the exception that a production medium with the following composition was used:
Eksempel 4 Example 4
Antibiotikum A16884 ble fremstilt som i fremgangsmåten ifblge eksempel 1 med unntagelse av at det ble brukt et produksjonsmedium med fblgende sammensetning: Antibiotic A16884 was prepared as in the procedure according to example 1 with the exception that a production medium with the following composition was used:
Eksempel 5 Example 5
Antibiotikum A16884 ble fremstilt som i fremgangsmåten ifblge eksempel 1 med unntagelse av at det ble brukt et produksjonsmedium med fblgende sammensetning: Antibiotic A16884 was prepared as in the procedure according to example 1 with the exception that a production medium with the following composition was used:
Eksempel 6 Example 6
En annen sporekultur av Streptomyces lipmanii NRRL 3584 ble fremstilt ved å dyrke organismen på en nærlngsagar-skråkultur. Skråkulturen hadde i dette tilfelle fblgende sammensetning: Another spore culture of Streptomyces lipmanii NRRL 3584 was prepared by growing the organism on a near-agar slant. In this case, the oblique culture had the following composition:
pH i mediet ble regulert ved tilsetning av natriumhydroksyd til 7,0. The pH of the medium was adjusted by adding sodium hydroxide to 7.0.
Agar-skråkulturen ble inokulert med sporer av Streptomyces lipmanii NRRL 3584 og inkubert i 7 dogn ved 30°C, Agar-skråkulturen ble så skrapet for å fjerne sporer som ble tilsatt 2,0 ml sterilt kjbttserum. Til et sterilt lyofilt ror ble så overfort 0,1 ml av den resulterende serumsporesuspensjon. Den ble frysetbrket i form av pellets. The agar slant was inoculated with spores of Streptomyces lipmanii NRRL 3584 and incubated for 7 days at 30°C. The agar slant was then scraped to remove spores to which 2.0 ml of sterile bovine serum was added. To a sterile lyophilic tube was then transferred 0.1 ml of the resulting serum spore suspension. It was freeze-dried in the form of pellets.
De såldes oppnådde pellets ble brukt til å inokulere et vegetativt medium med fblgende sammensetning: The pellets thus obtained were used to inoculate a vegetative medium with the following composition:
pH i mediet var 6,7 og forble uforandret.. The pH in the medium was 6.7 and remained unchanged.
Eksempel 7 Example 7
FREMSTILLING AV ANTIBIOTIKUM A16884 I FORSOKSANLEGG MANUFACTURE OF ANTIBIOTIC A16884 IN PILOT FACILITIES
Til et 40 liters fermenteringskar av rustfritt stål ble tilsatt 24 liter av et medium med fblgende sammensetning: 24 liters of a medium with the following composition were added to a 40 liter stainless steel fermentation vessel:
Kaldt springvann til fortynning til 25 liter Cold tap water to dilute to 25 litres
Den opprinnelige pH var 6,5 og ble ikke regulert. Mediet ble sterilisert i 30 minutter ved 120°C, avkjblt og så inokulert med et 5 % inokulat fremstilt som i eksempel 6. Fermenteringen ble utfort ved 30°C i 66 timer, luftet med steril luft med en hastighet av 0,35 vol./vol./min. og omrbrt av en mekanisk rbre-innretning som gjorde 420 omdreininger pr. minutt. Den endelige pH var 7,5» The initial pH was 6.5 and was not regulated. The medium was sterilized for 30 minutes at 120°C, cooled and then inoculated with a 5% inoculum prepared as in Example 6. The fermentation was carried out at 30°C for 66 hours, aerated with sterile air at a rate of 0.35 vol. /vol./min. and rotated by a mechanical rbre device that made 420 revolutions per minute. The final pH was 7.5"
A16884 ble utvunnet fra kulturvæsken og isolert som A16884 was recovered from the culture fluid and isolated as
angitt i eksempel 8. given in Example 8.
Eksempel 8 Example 8
ISOLERING AV UBEARBEIDET ANTIBIOTIKUM A16884 SOM M0N0AMM0NIUM-SALTET ISOLATION OF CRUDE ANTIBIOTIC A16884 AS ITS M0N0AMM0NIUM SALT
Ca. 6o liter kulturvæske oppnådd som angitt i ek- About. 6o liters of culture fluid obtained as indicated in ek-
sempel 7, ble filtrert ved hjelp av "Hyflo Super-cel" (en di-atomé jord). Filtratet ble så fort over i en kolonne på 9,6 x 150 sample 7, was filtered using "Hyflo Super-cel" (a diatomic earth). The filtrate was then quickly transferred to a 9.6 x 150 column
cm pakket med aktivkull ("Pittsburgh Cal. 12 x 40"). Kolonnen ble vasket med vann inntil effluenten var fargelbs og den aktive del adsorbert på kullet ble fjernet ved å fore 50 fa vandig ace- cm packed with activated carbon ("Pittsburgh Cal. 12 x 40"). The column was washed with water until the effluent was colorless and the active part adsorbed on the charcoal was removed by passing 50 fa aqueous ace-
ton gjennom kolonnen. Fraksjonene inneholdende den aktive del ble slått sammen, konsentrert i vakuum for å fjerne aceton, og tilfort en 5,9 x 104 cm kolonne pakket med "IRA-68"-harpiks (kom-mersiell anionvekslerharpiks som var blitt vasket med maursyre for omdannelse av harpiksen til formiatcyklusen). Kolonnen ble vasket med vann inntil effluenten var klar og fargelbs, og den aktive del ble fjernet ved vasking med 0,1 M ammoniumformiatlbs- ton through the column. The fractions containing the active moiety were pooled, concentrated in vacuo to remove acetone, and applied to a 5.9 x 10 4 cm column packed with "IRA-68" resin (commercial anion exchange resin that had been washed with formic acid to convert the resin of the formate cycle). The column was washed with water until the effluent was clear and colorless, and the active fraction was removed by washing with 0.1 M ammonium formate.
ning. De aktive fraksjoner ble slått sammen og fort over i en i en 4,3 x 72 cm kolonne inneholdende aktivkull ("Pittsburgh 12 x 40"). Kolonnen ble vasket med en mengde vann lik seks ganger ko-lonnens volum og den aktive del ble eluert med 30 % vandig acetonitril. De aktive fraksjoner ble slått sammen, konsentrert i nothing. The active fractions were combined and transferred to a 4.3 x 72 cm column containing activated carbon ("Pittsburgh 12 x 40"). The column was washed with an amount of water equal to six times the volume of the column and the active part was eluted with 30% aqueous acetonitrile. The active factions were merged, concentrated in
vakuum for å fjerne acetonitril, og frysetbrket. Utbyttet var 25 vacuum to remove acetonitrile, and frozen. The yield was 25
til 30 gram fast stoff. to 30 grams of solids.
Det frysetbrkete preparat ble lost 1 et minimum av A minimum of the freeze-dried preparation was removed
vann og tilfort en 7,2 x 60 cm kolonne pakket med mikrokrystallinsk celluloseprodukt ("Avicel") suspendert i 70 % vandig acetonitril, og som var vasket med acetonitril for tilsetningen av den aktive prove. Etter tilsetning av prbven ble kolonnen vas- water and added to a 7.2 x 60 cm column packed with microcrystalline cellulose product ("Avicel") suspended in 70% aqueous acetonitrile, which had been washed with acetonitrile for the addition of the active sample. After addition of the sample, the column was washed
ket med ett kolonnevolum acetonitril og den aktive del eluert ket with one column volume of acetonitrile and the active part eluted
med metanol. De aktive fraksjoner ble slått sammen og konsen- with methanol. The active factions were merged and concen-
trert til ca. 200 ml og den aktive del felt ved tilsetning av 10 volumdeler aceton og tbrket i vakuum. Utbyttet var 9-12 gram. three to approx. 200 ml and the active part precipitated by adding 10 parts by volume of acetone and dried in vacuo. The yield was 9-12 grams.
20 gram material oppnådd som ovenfor beskrevet, ble 20 grams of material obtained as described above was
lost i et minimum av vann og tilfort en silikagelkolonne (7,2 x dissolved in a minimum of water and added to a silica gel column (7.2 x
60 cm). Silikagelen (Davison Chemical type "950") var på forhånd vasket med vann og deretter med metanol og suspendert i 70 % acetonitril for pakking av kolonnen. Etter tilforing av proven ble kolonnen vasket med ett kolonnevolum acetonitril og den aktive del eluert med 70 % acetonitril. De mest aktive fraksjoner ble slått sammen, konsentrert i vakuum til torrhet og lbst i metanol og deretter felt med 10 volumdeler aceton. Bunnfallet ble filtrert, vasket med aceton og tbrket 1 vakuum. Utbyttet var 8 gram. Mindre aktive fraksjoner ga ytterligere 6 gram. 60 cm). The silica gel (Davison Chemical type "950") was pre-washed with water and then with methanol and suspended in 70% acetonitrile for packing the column. After feeding the sample, the column was washed with one column volume of acetonitrile and the active part eluted with 70% acetonitrile. The most active fractions were pooled, concentrated in vacuo to dryness and dissolved in methanol and then precipitated with 10 parts by volume of acetone. The precipitate was filtered, washed with acetone and dried under vacuum. The yield was 8 grams. Less active fractions gave an additional 6 grams.
Eksempel 9 Example 9
RENSING AV A16884- MONOAMMONIUMSALT PURIFICATION OF A16884- MONOAMMONIUM SALT
Ett gram av det frysetbrkete produkt fremstilt som beskrevet i eksempel 8, ble lost i 4 ml vann og tilfort en 2 x éo cm kolonne pakket med 175 ml silikagel type <W>950<M> i 80 % vandig acetonitril. Kolonnen ble eluert med acetonitril/vann (4:1). Elueringen ble etterfulgt av provetaking og papirkromatografi. Som resultat av elueringen ble oppnådd et flertall fraksjoner. Fraksjonene inneholdende antibiotikum A16884 som monoammoniumsalt ble slått sammen, konsentrert til torrhet, lost i et lite volum dimetylsulfoksyd, deretter i atskillige milliliter etanol og den aktive del ble felt ved tilsetning av overskudd av eter. Bunnfallet ble sentrifugert og tbrket i vakuum. Utbyttet av antibiotikum Al6884-monoammoniumsalt var 91 mg. One gram of the freeze-dried product prepared as described in example 8 was dissolved in 4 ml of water and added to a 2 x 10 cm column packed with 175 ml of silica gel type <W>950<M> in 80% aqueous acetonitrile. The column was eluted with acetonitrile/water (4:1). The elution was followed by sampling and paper chromatography. As a result of the elution, a majority of fractions were obtained. The fractions containing antibiotic A16884 as monoammonium salt were pooled, concentrated to dryness, dissolved in a small volume of dimethylsulfoxide, then in several milliliters of ethanol and the active portion precipitated by addition of excess ether. The precipitate was centrifuged and dried in vacuo. The yield of antibiotic Al6884 monoammonium salt was 91 mg.
Eksempel 10 Example 10
FREMSTILLING AV ANTIBIOTIKUM Al6884 I SYREFORM PREPARATION OF ANTIBIOTIC Al6884 IN ACID FORM
200 mg monoammoniumsalt av A16884 ble lost i 30 ml vann og 6 ml "Dowex 50 x 12" (H+)-harpiks ble tilsatt. Blandin-gen ble omrbrt 1 40 minutter, filtrert, harpiksen vasket med vann på filtret og filtratene slått sammen. Det samlete filtrat hadde en pH på 2,7. Filtratet ble konsentrert i vakuum til ca. 200 mg of the monoammonium salt of A16884 was dissolved in 30 ml of water and 6 ml of "Dowex 50 x 12" (H+) resin was added. The mixture was stirred for 140 minutes, filtered, the resin washed with water on the filter and the filtrates combined. The collected filtrate had a pH of 2.7. The filtrate was concentrated in vacuo to approx.
1 ml, 4 ml metanol ble tilsatt og syren ble felt ved tilsetning av 40 ml aceton. Bunnfallet ble fjernet ved sentrifugering og tbrket 1 vakuum. Utbyttet var 35 mg antibiotikum A16884 i syreform. Den oppviste fblgende pKa: 3»5» 5»2 og 10,3 ved titrering i 66 % dimetylformamid. ved en begynnelses-pH på 4,5»1 ml, 4 ml of methanol was added and the acid was precipitated by the addition of 40 ml of acetone. The precipitate was removed by centrifugation and vacuum applied. The yield was 35 mg of antibiotic A16884 in acid form. It showed the following pKa: 3»5»5»2 and 10.3 when titrated in 66% dimethylformamide. at an initial pH of 4.5"
Eksempel 11 Example 11
FREMSTILLING AV DINATRIUMSALT AV Al6884 PREPARATION OF DINATIUM SALT OF Al6884
180 mg Al6884-monoammoniumsalt ble lost i ca. 2 ml vann og pH regulert til 10 med IN NaOH. Lbsningen ble konsentrert i vakuum til lite volum, 4 ml metanol ble tilsatt og di-na tr iumsal tet felt ved tilsetning av 40 ml aceton'. Saltet ble fjernet ved sentrifugering og tbrket i vakuum. Det oppviste fol- 180 mg of Al6884 monoammonium salt was dissolved in approx. 2 ml of water and pH adjusted to 10 with IN NaOH. The solution was concentrated in vacuo to a small volume, 4 ml of methanol was added and the disodium salt was precipitated by the addition of 40 ml of acetone. The salt was removed by centrifugation and dried in vacuo. The demonstrated fol-
gende verdier av pKa: 3,9» 5»2 og 10,5 ved titrering i 66 % dimetylformamid ved. en begynnelses-pH på 10,4. Atomabsorpsjons- changing values of pKa: 3.9, 5,2 and 10.5 when titrated in 66% dimethylformamide at an initial pH of 10.4. atomic absorption
analyse viste 6 % natrium. analysis showed 6% sodium.
Eksempel 12 Example 12
FREMSTILLING AV ANTIBIOTIKUM A16884- HYDROKLORID PREPARATION OF ANTIBIOTIC A16884- HYDROCHLORIDE
200 mg monoammoniumsalt av A16884 ble lbst i 2 ml 200 mg of the monoammonium salt of A16884 was dissolved in 2 ml
vann og pH regulert til 2,0 med IN HC1. Reaksjonsblandingen ble fortynnet med 5 ml metanol og 50 ml aceton ble tilsatt for å water and pH adjusted to 2.0 with IN HC1. The reaction mixture was diluted with 5 ml of methanol and 50 ml of acetone was added to
felle ut det forbnskete antibiotikum Al6884-hyd.roklorid. Det ble separert ved sentrifugering, vasket med aceton og tbrket i va- precipitate the confounded antibiotic Al6884 hydrochloride. It was separated by centrifugation, washed with acetone and dried in water
kuum. Analyse viste 5>74 % klor og elektrometrisk titrering i 66 kuum. Analysis showed 5>74% chlorine and electrometric titration in 66
% dimetylformamid ved en begynnelses-pH på 5»0 viste titrerbare grupper ved 3,9» 5»2 og 10,4. % dimethylformamide at an initial pH of 5.0 showed titratable groups at 3.9, 5.2 and 10.4.
Eksempel 13 Example 13
ISOLERING AV RÅTT ANTIBIOTIKUM A16884 SOM MONONATRIUMSALT ISOLATION OF CRUDE ANTIBIOTIC A16884 AS MONONODIUM SALT
Ca. 6o liter kulturvæske oppnådd som angitt i ek- About. 6o liters of culture fluid obtained as indicated in ek-
sempel 7, ble filtrert ved hjelp av "Hyflo-Super-cel"-filtre-ringshjelpemiddel. Filtratet ble fort over i en 9»6 x 150 cm ko- sample 7, was filtered using "Hyflo-Super-cel" filter aid. The filtrate was quickly transferred to a 9"6 x 150 cm co-
lonne pakket med aktivkull ("Pittsburgh Ca. 12 x 40"). Kolonnen ble vasket med vann og den absorberte aktive del ble fjernet ved å fore 50 % vandig aceton gjennom kolonnen. Fraksjonene inneholdende den aktive del ble slått sammen, konsentrert i vakuum for å fjerne aceton og tilfort en 5>9 x 104 cm kolonne pakket med "IRA-68" (acetat-cyklus). Kolonnen ble vasket med vann inn- bag packed with activated charcoal ("Pittsburgh Ca. 12 x 40"). The column was washed with water and the absorbed active moiety was removed by passing 50% aqueous acetone through the column. The fractions containing the active moiety were pooled, concentrated in vacuo to remove acetone and applied to a 5>9 x 10 4 cm column packed with "IRA-68" (acetate cycle). The column was washed with water in
til effluenten var klar og fargelbs, og den aktive del ble fjer- until the effluent was clear and colourless, and the active part was removed
net ved vasking med 0,1 M natriumacetat. De aktive fraksjoner ble slått sammen og fort over i en 4,3 x 72 cm kolonne pakket med "Pittsburgh 12 x 4o"-kull. Kolonnen ble vasket med seks gan- net by washing with 0.1 M sodium acetate. The active fractions were combined and passed into a 4.3 x 72 cm column packed with "Pittsburgh 12 x 40" charcoal. The column was washed six times with
ger kolonnevolumet av vann og den aktive del eluert med 30 % vandig aceton. De aktive fraksjoner ble slått sammen, konsentrert gives the column volume of water and the active part eluted with 30% aqueous acetone. The active factions were merged, concentrated
i vakuum for å fjerne aceton og frysetbrket. Utbyttet var 20 - in vacuum to remove acetone and the frozen fraction. The yield was 20 -
30 g. Analyse viste 2,5 % natrium. 30 g. Analysis showed 2.5% sodium.
Claims (1)
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Publication Number | Publication Date |
---|---|
NO132241B true NO132241B (en) | 1975-06-30 |
NO132241C NO132241C (en) | 1975-10-08 |
Family
ID=25301838
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NO3024/70A NO132241C (en) | 1969-08-06 | 1970-08-05 |
Country Status (19)
Country | Link |
---|---|
JP (1) | JPS5218276B1 (en) |
AT (1) | AT300195B (en) |
BE (1) | BE754424A (en) |
BR (1) | BR6915133D0 (en) |
CA (1) | CA947680A (en) |
CH (1) | CH547855A (en) |
CS (1) | CS172332B2 (en) |
DE (1) | DE2039184C3 (en) |
DK (1) | DK125483B (en) |
ES (1) | ES382489A1 (en) |
FI (1) | FI45989C (en) |
FR (1) | FR2068468B1 (en) |
GB (1) | GB1312129A (en) |
IL (1) | IL35034A (en) |
NL (1) | NL156752B (en) |
NO (1) | NO132241C (en) |
OA (1) | OA03652A (en) |
SE (1) | SE362897B (en) |
ZA (1) | ZA705284B (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2221035C2 (en) * | 1972-04-28 | 1982-03-25 | Merck & Co., Inc., 07065 Rahway, N.J. | Process for the preparation of substituted 6-iminoopenicillins and 7-iminocephalosporins |
CA1090728A (en) * | 1976-02-04 | 1980-12-02 | Eli Lilly And Company | Antibiotic a-7413 mixture comprising factors a,b,c and d and a process for producing it |
JPS5538137A (en) * | 1978-09-07 | 1980-03-17 | Ichirou Minamimoto | Production of mesh shoe upper |
-
0
- BE BE754424D patent/BE754424A/en not_active IP Right Cessation
-
1969
- 1969-12-15 BR BR215133/69A patent/BR6915133D0/en unknown
-
1970
- 1970-07-30 ZA ZA705284*A patent/ZA705284B/en unknown
- 1970-07-31 CA CA089,709A patent/CA947680A/en not_active Expired
- 1970-07-31 IL IL35034A patent/IL35034A/en unknown
- 1970-08-04 NL NL7011547.A patent/NL156752B/en not_active IP Right Cessation
- 1970-08-04 GB GB3749070A patent/GB1312129A/en not_active Expired
- 1970-08-04 FI FI702155A patent/FI45989C/en active
- 1970-08-05 ES ES382489A patent/ES382489A1/en not_active Expired
- 1970-08-05 NO NO3024/70A patent/NO132241C/no unknown
- 1970-08-05 SE SE10714/70A patent/SE362897B/xx unknown
- 1970-08-05 DK DK403870AA patent/DK125483B/en unknown
- 1970-08-06 DE DE2039184A patent/DE2039184C3/en not_active Expired
- 1970-08-06 JP JP45068968A patent/JPS5218276B1/ja active Pending
- 1970-08-06 CS CS5503A patent/CS172332B2/cs unknown
- 1970-08-06 FR FR707029024A patent/FR2068468B1/fr not_active Expired
- 1970-08-06 CH CH1186070A patent/CH547855A/en not_active IP Right Cessation
- 1970-08-06 AT AT717870A patent/AT300195B/en not_active IP Right Cessation
- 1970-08-06 OA OA54000A patent/OA03652A/en unknown
Also Published As
Publication number | Publication date |
---|---|
SE362897B (en) | 1973-12-27 |
IL35034A (en) | 1974-05-16 |
ES382489A1 (en) | 1972-12-01 |
GB1312129A (en) | 1973-04-04 |
AT300195B (en) | 1972-07-10 |
BE754424A (en) | 1971-02-05 |
DE2039184C3 (en) | 1978-11-23 |
OA03652A (en) | 1971-12-24 |
CA947680A (en) | 1974-05-21 |
FI45989C (en) | 1972-11-10 |
JPS5218276B1 (en) | 1977-05-20 |
DE2039184A1 (en) | 1971-02-25 |
NL156752B (en) | 1978-05-16 |
BR6915133D0 (en) | 1973-04-12 |
IL35034A0 (en) | 1970-09-17 |
FI45989B (en) | 1972-07-31 |
CS172332B2 (en) | 1976-12-29 |
DK125483B (en) | 1973-02-26 |
NO132241C (en) | 1975-10-08 |
DE2039184B2 (en) | 1978-03-23 |
CH547855A (en) | 1974-04-11 |
FR2068468B1 (en) | 1974-06-14 |
FR2068468A1 (en) | 1971-08-27 |
ZA705284B (en) | 1972-03-29 |
NL7011547A (en) | 1971-02-09 |
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