NO172856B - PROCEDURE FOR MANUFACTURING TUMOROUSING AGENTS - Google Patents
PROCEDURE FOR MANUFACTURING TUMOROUSING AGENTS Download PDFInfo
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- NO172856B NO172856B NO882722A NO882722A NO172856B NO 172856 B NO172856 B NO 172856B NO 882722 A NO882722 A NO 882722A NO 882722 A NO882722 A NO 882722A NO 172856 B NO172856 B NO 172856B
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- dichloromethane
- deoxy
- compound
- concentrate
- methanol
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- 238000000034 method Methods 0.000 title claims description 16
- 238000004519 manufacturing process Methods 0.000 title description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
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- 239000000203 mixture Substances 0.000 claims description 12
- PDQGEKGUTOTUNV-TZSSRYMLSA-N 4'-deoxy-4'-iododoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](I)[C@H](C)O1 PDQGEKGUTOTUNV-TZSSRYMLSA-N 0.000 claims description 10
- 241000187081 Streptomyces peucetius Species 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical class N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
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- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 10
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 150000001298 alcohols Chemical class 0.000 description 4
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- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 description 3
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- 229920001817 Agar Polymers 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
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- 239000005909 Kieselgur Substances 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
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- 239000001888 Peptone Substances 0.000 description 2
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- RIXHOXYAFWQBGU-RUELKSSGSA-N (7s,9s)-7-[(2r,4s,5s,6s)-4-amino-5-iodo-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione;hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](I)[C@H](C)O1 RIXHOXYAFWQBGU-RUELKSSGSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 1
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- 238000011735 C3H mouse Methods 0.000 description 1
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- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
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- 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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- 229910017052 cobalt Inorganic materials 0.000 description 1
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
Foreliggende oppfinnelse omhandler fremstilling av 4<1->deoksy-13(S)-dihydro-4<*->jododoksorubicin med formel (II) ;Mikroorganismen Streptomvces peucetius stamme DSM 2444 anvendes for stereoselektiv reduksjon av syrekloridet av den 13-keton funksjonelle gruppe av 4<1->deoksy-4•-jododoksorubicin (I) ;for å gi spesifikt 4<1->deoksy-13-(S)-dihydro-4<1->jododoksorubicin-HCl, som er en av de to mulige C-13 stereoisomere 4<1->deoksy-13-dihydro-4<1->jododoksorubiciner. Den nye forbindelse (II) heretter betegnet FCE 24883 er anvendelig som et tumorhemmende middel og oppviser på eksprimentelle svulster en aktivitet som kan sammenlignes med den til 4'-deoksy-4<1->;jododoksorubicin (I). Substratet for den mikrobielle stereo-sellektive reduksjon er en doksorubicin semi-syntetisk analog beskrevet i søkers US patent 4438105 (20. mars 1984). ;Mer spesielt omhandler foreliggende oppfinnelse en biosyn-tetisk fremgangsmåte hvorved en mutant av slekten Stre<p->tomvses peucetius betegnet stamme M 87 F.I. og deponert ved Deutsche Sammlung von Mikroorganismen hvor den er registrert under tilgangsnummer DSM 2444 og som er særpreget ved dets egenskap stereoselektivt å redusere den 13-keton funksjonelle gruppe av 4<1->deoksy-4<1->jododoksorubicin (I). Forbindelse FCE 24883 (II) som fremkommer, akummulerer i farmenter-ingsvæsken. FCE 24883 (II) kan gjenvinnes fra farmenterings-mediet og urene oppløsninger av dette kan konsentreres og ;renses. ;Oppfinnelsen gir således en fremgangsmåte for fremstilling av 4'-deoksy-13(S)-dihydro-4<1->jododoksorubicin av formel (II), hvilken fremgangsmåte omfatter å dyrke Streptomyces peucetius stamme M 87 F.I. (DSM 2444) i nærvær av 4<1->deoksy-4<1->jododoksorubicin og gjennvinne det resulterende 4<1->deoksy-13(S)-dihydro-4'-jododoksorubicin som sådan som angitt i krav l's karakteriserende del. ;Detaljert beskrivelse av oppfinnelsen ;Mikroorganismen Stre<p>tom<y>ces peucetius underart aureus ATCC 31428 har blitt mutert ved å bruke nitrosoguanidin for å gi en laboratorie mikroorganisme betegnet Streptomyces peucetius stamme M 87 F.I. som selektivt omdanner forbindelse (I) til forbindelse (II). S. peucetius stamme M 87 F.I. har blitt gitt tilgangsnummeret DSM 2444 av Deutsche Sammlung von Mikroorganism, Vest-Tyskland hvor den har blitt deponert. ;Morfologien av mutantstammen M 87 F.I. kan ikke sjelnes fra den til den opprinnelige S. peucetius ATCC 31428, men begge kulturer er klart adskillbare i sine kulturelle og bio-kjemiske særtrekk. Faktisk danner mutantstammen M 87 F.I. på agar ikke,det strågule til sitrongule oppløselige pigment som særpreger dets opphav S. peucetius ATCC 31428. ;Videre kan mutantstammen M 87 F.I. selektivt transformere forbindelsen (I) til forbindelsen (II) mens opphavet S. peucetius ATCC 31428 ikke er selektiv i dette henseende. Denne egenskap til mutanten M 87 F.I. gjør den meget anvendelig som beskrevet heri. ;Transformerings-prosessen ;Den stereosélektive biotransformasjon ifølge foreliggende oppfinnelse kan utføres i en voksende kultur av S. peucetius stamme M 87 F.I. ved å tilsette forbindelse (I) som substrat til kulturen under inkuberingsperioden. ;Forbindelse I som hydrokloridet kan tilsettes etter oppløs-ning i sterilt destilert vann. Det foretrukne men ikke begrensende konsentrasjonsområdet av forbindelse (I) i kulturen er ca. 50-200 jig per liter. Kulturen dyrkes i et nær-ingsmedium, inneholdende karbonkilder f.eks. et assimiler-bart karbohydrat samt en nitrogenkilde f.eks. en assimiler-bar nitrogenforbindelse eller proteinholdig materiale. Foretrukne karbonkilder innebefatter glukose, sukleose, glyse-rol, stivelse, kornstivelse, dekstrin, sirup og lignende. Foretrukne nitrogenkilder innebefatter silosaft, gjærekstrakt, tørrgjær, soyabønnemel, bomullskornmel, kornmel, kasein, fiskemel, fast gjæringsmateriale, animalsk pepton, kjøtt ekstrakt, ammoniumsalter o.l. Kombinasjoner av disse og karbon kilder kan bli brukt fordelaktig. Spormetaller f.eks. sink, magnesium, mangan, kobolt, jern o.l. behøver ikke nødvendigvis tilsettes fermenteringsmediene siden springvann og ikke rensede bestandeler blir brukt som kompo-nenter av mediet før sterilisering. ;Biotransformasjonsprosessen kan foregå fra ca. 72 timer til 8 dager. Inkuberingstemperaturen under biotransformasjonsprosessen kan ligge fra ca. 25°C til ca. 37°C, hvor 29°C er foretrukket. Inneholdet av transformasjonskamrene aeres ved omrystning ved ca. 250 r.p.m. eller ved omrøring med sterilisert luft for å lette vekst av mikroorganismene, og dette øker effektiviteten av transformasjonsprosessen. ;Analysemetoder ;Utviklingen av den mikrobielle transformasjonsreaskjon over-våkes ved å trekk ut prøver av farmateringene ved forskjel-lige tidsintervaller og ekstraherer ved pH 8,0 med en 9:1 diklormetan:metanolblanding. Når en prøve av det organgiske ekstrakt underkastes tynnsjiktskromatografi (TLC) ved å bruke som eluent en blanding av kloroform:metanol:eddik-syre:vann 80:20:7:3 (per volum), finnes at forbindelsen FCE 24883 (II) foreligger ved Rf middelverdi 0,50 mens 4'-deoksy-4'-jododoksorubicin (I) finnes ved Rf 0,60. En kvantitativ vurdering av de to antrasykliner kan utføres etter TLC ved å bruke ovenfor nevnte elueringssysterner ved å skrape av og eluere med metanol de tilsvarende rødfargete soner og endelig spektrofotometrisk bestemmelse ved 496 nm. ;Isoleringsprosedyre ;Hele fermateringsmediet hvori forbindelse (I) har blitt underkastet omdannelse til FCE 24883 (II), filtreres ved hjelp av diatomholdig jord. Den røde myceliale kake ekstra-heres med et organisk oppløsningsmiddel som er blandbart med vann så som metanol og andre lavere alkoholer, dioksan, acetonitril, aceton, og fortrinnsvis anvendes aceton. De myceliale ekstrakter samles opp, konsentreres under redusert trykk og kombineres med de filtrerte fermenteringsvæsker, justert til pH 8,0 og derpå ekstrahert med et organisk oppløsningsmiddel som ikke er blandbart med vann så som n-butanol, kloroform, diklormetan eller fortrinnsvis en diklormetan:metanol 9:1 blanding. De organiske ekstrakter inneholder FCE 24883 (II) sammen med forbindelser (I) og enkelte små nedbrytningsprodukter. ;Opprenskningsprosedyre ;Det organiske ekstrakt konsentreres under redusert trykk til tørrhet og residier oppløst i diklormetan kromatograferes på en kolonne av silikagel bufret ved pH 7 med en gradient av diklormetan:metanol:vannblanding. Forbindelse I elueres først med en 95:5:0,25 blanding fulgt av FCE 24883 (II) med en 90:10:0,5 blanding. Fra de sammenslåtte fraksjoner etter vask med vann, konsentrasjon til et lite volum i nærvær av en propanol tilsetning av en ekvivalent saltsyre og av et overskudd av en heksan, erholdes en utfelling av ren FCE 24883 (II) som hydrokloridet. ;Kjemiske og fysiske egenskaper ;FCE 24883 (II) som fri base er oppløselig i polare organiske oppløsningsmidler og vandige alkoholer, mens dets hydroklorid er oppløselig i vann og lavere alkoholer, men har begrenset løsbarhet i organiske oppløsningsmidler. Hydro-klorider til FCE 24883 har følgende fysiokjemiske egenskaper : ;Smeltepunkt: 200°C (dec) ;Spesifikk rotasjon: [cx]23" + 188°(c 0,05,CH3OH) ;U.V. og VIS absorpsjonsspektrum: X^^g 232, 254, 290 og 480 nm (E<1>%cm = 492, 370, 127, 163). I. R. spektrum (KBr): topper seg ved de følgende frekvenser: 3400, 2970, 2920, 1610, 1580, 1472, 1440, 1410, 1380, 1355, 1320, 1280, 1235, 1210, 1110, 1080, 1060, 1030, 1010, 985, 965, 940, 920, 900, 890, 870, 860, 830, 810, 785, 755, 730, 710, 540, 480, 450 og 415 can"1. ;1H-NMR spektrum (DMS0d6, 200 MHz, 22°C): 14,03 (bs, 2H, 0H-6, OH-11), 7,6-7,9 (m, 3H, H-2,H-3), 5,26 (m.lH,H-l'), 4,96 (d, J=5,2 Hz, 1H, OH-13), 4,92 (m, lH,H-7), 4,55 (m, 1H,H-4'), 4,51 (t, J=6,7 Hz, 1H, OH-14), 4,20 (s, 1H, OH-9), 3,97 (s, 3H, 4-OCH3), 3,76 (ddd, J=3,5, 6,7, 11,0 Hz, 1H, CH (H) - OH), 3,60 (dq, J=1,0, 6,0 Hz, H-5<1>), 3,48 (ddd. J=7,2, 6,7, II, 0 HZ, 1H, CH(H)-OH), 3,37 (ddd, J=5,2, 3,5, 7,2 Hz, 1H, ;H-13), 3,02 (m, 1H, H-3•), 2,81 (m, 2H, CH2-10), 2,15 (dd. J=2,0, 15,3 Hz, 1H, H- 8e). 1,97 (dd, J=6,0, 15,3 Hz, 1H, H^ 8ax), 1,7-1,9 (m, 2H, CH2-2') og 1,14S (d, J=6,0 Hz, 3H, CH3-5<«>) ;Molekylformel: C27<H>30NIO10 • HC1 ;m/z i FD ekvivalent til fri base: 656(MH); ;655(M); og 416(M<+>) tilsvarende aglysonet. ;En selektiv høytrykksvæske kromatografi (HPLC) metode til-later å adskille (to topper med tilbakeholdelsestider på 18,8 og 19,3 min.) de to C-13 stereoisomere alkoholer som er tilstede i en prøve av syntetisk 41-deoksy-13-dihydro-41-jododoksorubicin fremstilt ved en NaBH4 reduksjon av I. ;Ved å bruke samme HPLC-metode opptrer FCE 24883 (II) som en enkelt topp med en tilbakeholdelsestid på 19,3 minutter, tilsvarende den til den saktere bevegelige bestandel av det syntetiske 13-dehydroderivat. ;HPLC metode ;Kolonne: 2 RP Spherisorb S30DS2 (C18 3p, Phase Seperation England) 150 x 4,5 mm tilkoblet i serie. ;Temperatur: 45°C ;Mobil fase A: 0,05 M vandig KH2P04, justert til pH 3,0 med ;1 M H3PO4/CH3OH = 80/20 (per volum) ;Mobil fase B: CH30H ;Eluering: Isokratisk i 30 minutter (42%a + 58%B) Strømningsgrad: 0,6 ml/min. ;Påvisning: 254 nm. ;Strukturbestrømmelse ;Sur hydrolyse av II (0,2 N vandig HC1, 80°C, 30 minutter) gir en rød utfelling av det tilsvarende aglyson (III), mens sukkerbestandelen, nemlig 3-amino-2,3,4,6-tetradeoksy-4-jod-L-lyksoheksoheksose (IV) som er tilstede i den vandige fase, ikke har blitt identifisert etter sammenligning med en autentisk prøve erholdt ved sur hydrolyse av forbindelse I. ;Den absolutte (S)-konfigurasjon ved C-13 av III har blitt bestemt ved direkte sammenligning (1H-NMR og masse spektrum, TLC) av dets 9,13-0-isopropyliden-14-0-t-butyldifenylsilyl derivat med tilsvarende derivat av autentiske prøver av 13-(S)-dihydroadriamycinion erholdt som beskrevet av S. Penco et al. i Gazzetta Chimica Italiana, 115, 195, 1985. ;Biologisk aktivitet ;Den cytotoksiske aktivitet av FCE 24883 (II) ble undersøkt "in vitro" på Hela og P 388 cellers kolonidannelse sammen-lignet med forbindelse I og doksorubisin. Som angitt i tabell I var forbindelse II like aktiv som 4'-deoksy-4'-jododoksorubicin (I) og doksorubicin. ;Den "in vivo" antitumorale aktivitet ble for FCE 24883 (II) undersøkt mot disseminert Gross leukemi. C3H mus ble inji-sert intravenøst med 2*IO<6> celler/mus og behandlet med forbindelser under studium 24 timer etter tumorinjektsjonen. The present invention relates to the production of 4<1->deoxy-13(S)-dihydro-4<*->iododoxorubicin with formula (II); The microorganism Streptomvces peucetius strain DSM 2444 is used for the stereoselective reduction of the acid chloride of the 13-ketone functional group of 4<1->deoxy-4•-iododoxorubicin (I) ; to give specifically 4<1->deoxy-13-(S)-dihydro-4<1->iododoxorubicin-HCl, which is one of the two possible C-13 stereoisomers 4<1->deoxy-13-dihydro-4<1->iododoxorubicins. The new compound (II) hereinafter referred to as FCE 24883 is useful as a tumor suppressor and exhibits on experimental tumors an activity comparable to that of 4'-deoxy-4<1>;iododoxorubicin (I). The substrate for the microbial stereo-selective reduction is a doxorubicin semi-synthetic analog described in applicant's US patent 4438105 (March 20, 1984). More particularly, the present invention relates to a biosynthetic method by which a mutant of the genus Streptomvses peucetius designated strain M 87 F.I. and deposited at the Deutsche Sammlung von Mikroorganismen where it is registered under accession number DSM 2444 and which is characterized by its ability to stereoselectively reduce the 13-ketone functional group of 4<1->deoxy-4<1->iododoxorubicin (I). Compound FCE 24883 (II) which appears accumulates in the fermentation liquid. FCE 24883 (II) can be recovered from the fermentation medium and impure solutions thereof can be concentrated and purified. The invention thus provides a method for the production of 4'-deoxy-13(S)-dihydro-4<1->iododoxorubicin of formula (II), which method comprises cultivating Streptomyces peucetius strain M 87 F.I. (DSM 2444) in the presence of 4<1->deoxy-4<1->iododoxorubicin and recover the resulting 4<1->deoxy-13(S)-dihydro-4'-iododoxorubicin as such as set forth in claim 1's characterizing share. ;Detailed description of the invention ;The microorganism Stre<p>tom<y>ces peucetius subspecies aureus ATCC 31428 has been mutated using nitrosoguanidine to give a laboratory microorganism designated Streptomyces peucetius strain M 87 F.I. which selectively converts compound (I) to compound (II). S. peucetius strain M 87 F.I. has been given the accession number DSM 2444 by the Deutsche Sammlung von Mikroorganism, West Germany where it has been deposited. ;The morphology of the mutant strain M 87 F.I. cannot be separated from that of the original S. peucetius ATCC 31428, but both cultures are clearly distinguishable in their cultural and biochemical characteristics. Indeed, the mutant strain M 87 forms F.I. on agar not the straw yellow to lemon yellow soluble pigment that characterizes its origin S. peucetius ATCC 31428. Furthermore, the mutant strain M 87 F.I. selectively transform the compound (I) into the compound (II) while the parent S. peucetius ATCC 31428 is not selective in this respect. This characteristic of the mutant M 87 F.I. makes it very useful as described here. ;The transformation process ;The stereoselective biotransformation according to the present invention can be carried out in a growing culture of S. peucetius strain M 87 F.I. by adding compound (I) as substrate to the culture during the incubation period. Compound I to which the hydrochloride can be added after dissolution in sterile distilled water. The preferred but not limiting concentration range of compound (I) in the culture is approx. 50-200 jig per litre. The culture is grown in a nutrient medium, containing carbon sources, e.g. an assimilable carbohydrate and a nitrogen source, e.g. an assimilable nitrogenous compound or proteinaceous material. Preferred carbon sources include glucose, sucleose, glycerol, starch, corn starch, dextrin, syrup and the like. Preferred nitrogen sources include silage juice, yeast extract, dry yeast, soybean meal, cottonseed meal, corn meal, casein, fish meal, solid fermentation material, animal peptone, meat extract, ammonium salts and the like. Combinations of these and carbon sources can be used advantageously. Trace metals e.g. zinc, magnesium, manganese, cobalt, iron etc. does not necessarily need to be added to the fermentation media since tap water and non-purified ingredients are used as components of the medium before sterilization. ;The biotransformation process can take place from approx. 72 hours to 8 days. The incubation temperature during the biotransformation process can range from approx. 25°C to approx. 37°C, where 29°C is preferred. The contents of the transformation chambers are aerated by shaking at approx. 250 r.p.m. or by stirring with sterilized air to facilitate the growth of the microorganisms, and this increases the efficiency of the transformation process. ;Analysis methods ;The development of the microbial transformation reaction is monitored by withdrawing samples of the formulations at different time intervals and extracting at pH 8.0 with a 9:1 dichloromethane:methanol mixture. When a sample of the organic extract is subjected to thin layer chromatography (TLC) using as eluent a mixture of chloroform:methanol:acetic acid:water 80:20:7:3 (by volume), it is found that the compound FCE 24883 (II) is present at Rf mean value 0.50 while 4'-deoxy-4'-iododoxorubicin (I) is found at Rf 0.60. A quantitative assessment of the two anthracyclines can be carried out after TLC using the above-mentioned elution systems by scraping off and eluting with methanol the corresponding red colored zones and final spectrophotometric determination at 496 nm. ;Isolation Procedure ;The entire fermentation medium in which compound (I) has been subjected to conversion to FCE 24883 (II) is filtered using diatomaceous earth. The red mycelial cake is extracted with an organic solvent which is miscible with water such as methanol and other lower alcohols, dioxane, acetonitrile, acetone, and preferably acetone is used. The mycelial extracts are collected, concentrated under reduced pressure and combined with the filtered fermentation liquids, adjusted to pH 8.0 and then extracted with an organic solvent immiscible with water such as n-butanol, chloroform, dichloromethane or preferably a dichloromethane: methanol 9:1 mixt. The organic extracts contain FCE 24883 (II) together with compounds (I) and some small degradation products. ;Purification procedure ;The organic extract is concentrated under reduced pressure to dryness and residues dissolved in dichloromethane are chromatographed on a column of silica gel buffered at pH 7 with a gradient of dichloromethane:methanol:water mixture. Compound I is first eluted with a 95:5:0.25 mixture followed by FCE 24883 (II) with a 90:10:0.5 mixture. From the combined fractions after washing with water, concentration to a small volume in the presence of a propanol, addition of an equivalent of hydrochloric acid and of an excess of a hexane, a precipitate of pure FCE 24883 (II) is obtained as the hydrochloride. ;Chemical and physical properties ;FCE 24883 (II) as a free base is soluble in polar organic solvents and aqueous alcohols, while its hydrochloride is soluble in water and lower alcohols but has limited solubility in organic solvents. Hydrochlorides of FCE 24883 have the following physiochemical properties : ;Melting point: 200°C (dec) ;Specific rotation: [cx]23" + 188°(c 0.05,CH3OH) ;U.V. and VIS absorption spectrum: X^^g 232, 254, 290 and 480 nm (E<1>%cm = 492, 370, 127, 163). IR spectrum (KBr): peaks at the following frequencies: 3400, 2970, 2920, 1610, 1580, 1472, 1440, 1410, 1380, 1355, 1320, 1280, 1235, 1210, 1110, 1080, 1060, 1030, 1010, 985, 965, 940, 920, 900, 890, 870, 860, 830, 75, 85, 81 730, 710, 540, 480, 450 and 415 can"1. ;1H-NMR spectrum (DMS0d6, 200 MHz, 22°C): 14.03 (bs, 2H, 0H-6, OH-11), 7.6-7.9 (m, 3H, H-2,H -3), 5.26 (m.1H,H-1'), 4.96 (d, J=5.2 Hz, 1H, OH-13), 4.92 (m, 1H,H-7) , 4.55 (m, 1H,H-4'), 4.51 (t, J=6.7 Hz, 1H, OH-14), 4.20 (s, 1H, OH-9), 3, 97 (s, 3H, 4-OCH3), 3.76 (ddd, J=3.5, 6.7, 11.0 Hz, 1H, CH (H) - OH), 3.60 (dq, J= 1.0, 6.0 Hz, H-5<1>), 3.48 (ddd. J=7.2, 6.7, II, 0 HZ, 1H, CH(H)-OH), 3, 37 (ddd, J=5.2, 3.5, 7.2 Hz, 1H, ;H-13), 3.02 (m, 1H, H-3•), 2.81 (m, 2H, CH2 -10), 2.15 (dd. J=2.0, 15.3 Hz, 1H, H- 8e). 1.97 (dd, J=6.0, 15.3 Hz, 1H, H^ 8ax), 1.7-1.9 (m, 2H, CH2-2') and 1.14S (d, J= 6.0 Hz, 3H, CH3-5<«>) ;Molecular formula: C27<H>30NIO10 • HC1 ;m/z in FD equivalent to free base: 656(MH); ;655(M); and 416(M<+>) corresponding to the aglyzone. ;A selective high pressure liquid chromatography (HPLC) method allows to separate (two peaks with retention times of 18.8 and 19.3 min.) the two C-13 stereoisomeric alcohols present in a sample of synthetic 41-deoxy-13 -dihydro-41-iododoxorubicin prepared by a NaBH4 reduction of I. ;Using the same HPLC method, FCE 24883 (II) appears as a single peak with a retention time of 19.3 min, corresponding to that of the slower moving component of the synthetic 13-dehydroderivative. ;HPLC method ;Column: 2 RP Spherisorb S30DS2 (C18 3p, Phase Seperation England) 150 x 4.5 mm connected in series. ;Temperature: 45°C ;Mobile phase A: 0.05 M aqueous KH2P04, adjusted to pH 3.0 with ;1 M H3PO4/CH3OH = 80/20 (by volume) ;Mobile phase B: CH3OH ;Elution: Isocratic in 30 minutes (42%a + 58%B) Flow rate: 0.6 ml/min. ;Detection: 254 nm. ;Structure analysis ;Acid hydrolysis of II (0.2 N aqueous HCl, 80°C, 30 minutes) gives a red precipitate of the corresponding aglyzone (III), while the sugar component, namely 3-amino-2,3,4,6- tetradeoxy-4-iodo-L-lyxohexohexose (IV) present in the aqueous phase has not been identified after comparison with an authentic sample obtained by acid hydrolysis of compound I. ;The absolute (S) configuration at C-13 of III has been determined by direct comparison (1H-NMR and mass spectrum, TLC) of its 9,13-0-isopropylidene-14-0-t-butyldiphenylsilyl derivative with the corresponding derivative of authentic samples of 13-(S)-dihydroadriamycin ion obtained as described by S. Penco et al. in Gazzetta Chimica Italiana, 115, 195, 1985. ;Biological activity ;The cytotoxic activity of FCE 24883 (II) was investigated "in vitro" on Hela and P 388 cell colony formation in comparison with compound I and doxorubicin. As indicated in Table I, compound II was as active as 4'-deoxy-4'-iododoxorubicin (I) and doxorubicin. ;The "in vivo" antitumoral activity was investigated for FCE 24883 (II) against disseminated Gross leukaemia. C3H mice were injected intravenously with 2*10<6> cells/mouse and treated with compounds under study 24 hours after the tumor injection.
Tabell II viser resultatene av begge eksprimenter. Ved den optimale dose ble FCE 24883 (II) funnet å være mere virksom enn doksorubisin og like virksom som 4'-deoksy-4<1->jododoksorubicin (I) med en liten toksisitet oppvist ved de aktive doser. Antitumor-aktiviteten av forbindelse II vurdert som midlere overlevelsestid av behandlete i forhold til kontrollmus kan sammenlignes med den til I og doksorubisin. Table II shows the results of both experiments. At the optimal dose, FCE 24883 (II) was found to be more active than doxorubicin and as active as 4'-deoxy-4<1->iododoxorubicin (I) with little toxicity shown at the active doses. The antitumor activity of compound II assessed as mean survival time of treated versus control mice is comparable to that of I and doxorubicin.
Beskrivelse av foretrukne utførelsesformer Description of preferred embodiments
De følgende ikke-begrensende eksempler er gitt for å beskrive i større detalj fremgangsmåten ifølge foreliggende oppfinnelse. The following non-limiting examples are given to describe in greater detail the method according to the present invention.
Eksempel 1 Example 1
En kultur av Stre<p>tomyces peucetius stamme M 87 F.I (DSM 2444) har blitt dyrket i 14 dager ved 28"C på agar skrå-plater i det følgende opprettholdelsesmedium (medium SA): glukose 3%, tørrgjær 1,2%, NaCl 0,1%, KH2P04 0,05%, CaC03 0,1%, MgS04 0,005%, FeS04.7H20 0,0005%, ZnS04.7H20 0,0005%, CuS04.5H20 0,0005%, agar 2%, springvann opp til 100 ml, pH 6,7 hvor sterilisering utføres ved oppvarming i en autoklav til 115°C i 20 minutter. A culture of Stre<p>tomyces peucetius strain M 87 F.I (DSM 2444) has been grown for 14 days at 28"C on agar slants in the following maintenance medium (medium SA): glucose 3%, dry yeast 1.2% , NaCl 0.1%, KH2P04 0.05%, CaC03 0.1%, MgS04 0.005%, FeS04.7H20 0.0005%, ZnS04.7H20 0.0005%, CuS04.5H20 0.0005%, agar 2% , tap water up to 100 ml, pH 6.7 where sterilization is carried out by heating in an autoclave to 115°C for 20 minutes.
Sporene av den således dyrkete kultur samles opp og suspen-deres i 3 ml sterilt destilert vann og suspensjonen inokuleres i 300 ml Erlenmeyer kolber inneholdende 60 ml av det følgende flytende vekst-medium: tørrgjær 0,3%, pepton 0,5%, Ca(N03)2.4H20 0,05%, springvann opp til 100 ml. Sterilisering ved oppvarming i autoklav til 120°C i 20 minutter. pH av dette medium etter sterilisering er mellom 6,8 og 7,0. De inokulerte flasker rystes i to dager ved en temperatur på 28°C på en rotasjonsrister som drives ved 250 r.p.m. og beskriver en sirkel på 7 cm i diameter. 1,5 ml av kulturen, dyrket som beskrevet ovenfor, inokuleres i 300 ml Erlenmeyer kolber inneholdende 50 ml av det følgende biotransformerende medium: gjærekstrakt 1,5%, KH2P04 0,25%, glukose 1,5%, springvann opp til 100 ml, pH 6,9 sterilisering ved oppvarming i autoklav til 115°C i 20 minutter. Glukoseoppløsningen steriliseres separat og tilsettes hver sterilisert flaske ved passende konsentrasjon. The spores of the thus grown culture are collected and suspended in 3 ml of sterile distilled water and the suspension is inoculated into 300 ml Erlenmeyer flasks containing 60 ml of the following liquid growth medium: dry yeast 0.3%, peptone 0.5%, Ca (N03)2.4H20 0.05%, tap water up to 100 ml. Sterilization by heating in an autoclave to 120°C for 20 minutes. The pH of this medium after sterilization is between 6.8 and 7.0. The inoculated bottles are shaken for two days at a temperature of 28°C on a rotary shaker operated at 250 r.p.m. and describes a circle of 7 cm in diameter. 1.5 ml of the culture, grown as described above, is inoculated into 300 ml Erlenmeyer flasks containing 50 ml of the following biotransformation medium: yeast extract 1.5%, KH2PO4 0.25%, glucose 1.5%, tap water up to 100 ml , pH 6.9 sterilization by heating in an autoclave to 115°C for 20 minutes. The glucose solution is sterilized separately and added to each sterilized bottle at the appropriate concentration.
Flaskene inkuberes derpå ved 28°C under betingelser beskrevet for vekstfasen i 24 timer. Ved denne tid ble 1,0 ml av en oppløsning av forbindelse I i sterilisert destilert, vann ved en konsentrasjon på 5 mg/ml, tilsatt hver flaske. De rystede flasker inkuberes i ytterligere 2 dager for å erholde en 70% omdannelse av forbindelse I til forbindelse The flasks are then incubated at 28°C under conditions described for the growth phase for 24 hours. At this time, 1.0 ml of a solution of compound I in sterilized distilled water at a concentration of 5 mg/ml was added to each bottle. The shaken bottles are incubated for an additional 2 days to obtain a 70% conversion of compound I to compound
II. II.
Eksempel 2 Example 2
En kultur av S. peucetius stamme M 87 F.I. dyrkes på fast medium som beskrevet i eksempel 1. Sporene av tre skråkultu-rer slås sammen og samles opp i 10 ml sterilt destilert vann hvor suspensjonen således erholdt inokuleres i en 2 liter rundbunnet flaske med hinderplater inneholdende 500 ml av vekstmediet beskrevet for eksempel 1. Flasken inkuberes i 48 timer på en rotasjonsryster som drives ved 120 r.p.m. og beskriver en sirkel med 7 cm i diameter ved en temperatur på 28°C. Hele veksten inokuleres i en 10 1 fermentator av rust-fritt stål inneholdende 7,5 1 av biotransformasjonsmediet beskrevet i eksempel 1, og sterilisert med damp ved 120°C i 3 0 minutter hvor glukoseoppløsningen blir sterilisert separat og tilsettes ved passende konsentrasjon til den sterili-serte fermentator. Kulturen tillates å vokse ved 28°C under omrøring ved 230 r.p.m. og aereres med en luftstrøm på 0,7 1/1 medium/min. Etter 48 timer tilsettes substratforbind-elsen ved konsentrasjonen beskrevet for eksempel 1 og kulturen inkuberes i 3 ytterligere dager for å erholde en 60% omdannelse av forbindelse I til forbindelse II. A culture of S. peucetius strain M 87 F.I. grown on solid medium as described in example 1. The spores of three slant cultures are combined and collected in 10 ml of sterile distilled water, where the suspension thus obtained is inoculated into a 2 liter round-bottomed bottle with barrier plates containing 500 ml of the growth medium described in example 1. The bottle is incubated for 48 hours on a rotary shaker operated at 120 r.p.m. and describes a circle with a diameter of 7 cm at a temperature of 28°C. The entire growth is inoculated into a 10 1 stainless steel fermenter containing 7.5 1 of the biotransformation medium described in Example 1, and sterilized with steam at 120°C for 30 minutes where the glucose solution is sterilized separately and added at an appropriate concentration to the sterile -certe fermenter. The culture is allowed to grow at 28°C with agitation at 230 r.p.m. and aerated with an air flow of 0.7 1/1 medium/min. After 48 hours, the substrate compound is added at the concentration described for example 1 and the culture is incubated for 3 further days to obtain a 60% conversion of compound I to compound II.
Eksempel 3 Example 3
Kulturen/mediet (5 1) fra en fermentering erholdt i henhold til eksempel 1 ble filtrert ved å bruke 2% diatomholdig jord som filter. Den våte filterkake ble ekstrahert med aceton (3 1). Etter filtrering ble 2 ytterligere ekstraksjoner med aceton utført for å sikre en fullstendig gjenvinning av de røde pigmenter. De sammenslåtte acetonekstrakter ble konsentrert under redusert trykk og konsentratet (1 1) ble slått sammen med det filtrerte medium og utstrakt ekstrakthert ved pH 8 med en diklormetan:metanol 9:1 blanding. Det organiske ekstrakt inneholdende forbindelsene I og II med enkelte nedbrytningsprodukter ble konsentrert under redusert trykk til tørrhet. Residiet oppløst i diklormetan ble kromatografert på en kolonne av silikagel bufret ved pH 7 (m/15 fosfat buffere) med en gradient av diklormetan:metanol:vann blanding. Etter enkelte nedbrytningsprodukter ble forbindelse I eluert med en 95:5:0,25 blanding fulgt av FCE 24883 (II) med en 90:10:0,5 blanding. The culture/medium (5 L) from a fermentation obtained according to Example 1 was filtered using 2% diatomaceous earth as a filter. The wet filter cake was extracted with acetone (3 L). After filtration, 2 further extractions with acetone were carried out to ensure a complete recovery of the red pigments. The combined acetone extracts were concentrated under reduced pressure and the concentrate (1 L) was combined with the filtered medium and extensively extracted at pH 8 with a dichloromethane:methanol 9:1 mixture. The organic extract containing compounds I and II with some decomposition products was concentrated under reduced pressure to dryness. The residue dissolved in dichloromethane was chromatographed on a column of silica gel buffered at pH 7 (w/15 phosphate buffers) with a gradient of dichloromethane:methanol:water mixture. After some degradation products, compound I was eluted with a 95:5:0.25 mixture followed by FCE 24883 (II) with a 90:10:0.5 mixture.
Fra de sammenslåtte fraksjoner etter vask med vannkonsen-trering til et lite volum i nærvær av n-propanol, tilsetning av en ekvivalent mengde saltsyre og et overskudd av n-heksan, ble ren FCE 24883 (II 0,30g, 60%) erholdt som hydrokloridet (smeltepunkt 200°C). Ved å følge samme fremgangsmåte ble også ikke transformert forbindelse I (0,13 g, 26%) erholdt som hydroklorid. From the combined fractions after washing with water concentration to a small volume in the presence of n-propanol, addition of an equivalent amount of hydrochloric acid and an excess of n-hexane, pure FCE 24883 (II 0.30g, 60%) was obtained as the hydrochloride (melting point 200°C). By following the same procedure, untransformed compound I (0.13 g, 26%) was also obtained as hydrochloride.
Eksempel 4 Example 4
En prøve på FCE 24833 (I) (200 mg) ble oppløst i 0,2 N vandig saltsyre (15 ml) og oppvarmet i 30 min. til 100 °C. En krystalinsk rød utfelling (0,12 g) av aglyson (III) ble samlet opp ved filtrering, vasket med vann og tørket. Masse-spektrum: m/e 416 (M<+>). Aglysonet (III) ble identifisert som 13-(S)-dihydroadriamycinon ved sammenligning med en autentisk prøve. A sample of FCE 24833 (I) (200 mg) was dissolved in 0.2 N aqueous hydrochloric acid (15 ml) and heated for 30 min. to 100 °C. A crystalline red precipitate (0.12 g) of aglysone (III) was collected by filtration, washed with water and dried. Mass spectrum: m/e 416 (M<+>). The aglyzone (III) was identified as 13-(S)-dihydroadriamycinone by comparison with an authentic sample.
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NO172856B true NO172856B (en) | 1993-06-07 |
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NO172856C (en) | 1993-09-15 |
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