DK143570B - METHOD OF PREPARING MAYTANSINOL MAYTANACIN AND OR MAYTANSINOL PROPIONATE - Google Patents

Description

(19) DANMARK (§)(19) DENMARK (§)

|p Π2) FREMLÆGGELSESSKRIFT (,,) 143570 B| p Π2) PUBLICATION WRITING (,,) 143570 B

DIREKTORATET FOR PATENT- OG VAREMÆRKEVÆSENETDIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Ansøgning nr. 4589/77 (51) lnt.Cl.e C 12 p 17/1$ (22) Indleveringsdag 14. okt. I977 (24) Løbedag 14. okt. 1977 (41) Aim. tilgængelig 1 . okt. 1978 (44) Fremlagt 7· sep. 1981 (86) International ansøgning nr.(21) Application No. 4589/77 (51) lnt.Cl.e C 12 p 17/1 $ (22) Filing date 14 Oct. I977 (24) Race day 14 Oct. 1977 (41) Aim. available 1. October 1978 (44) Posted 7 Sep 1981 (86) International application no.

(86) International indleveringsdag (85) Videreførelsesdag -(62) Stamansøgning nr. -(86) International filing day (85) Continuation day - (62) Master application no. -

(30) Prioritet 21. mar. 1977* 37167/77, JP(30) Priority Mar 21 1977 * 37167/77, JP

(71) Ansøger TAKEDA CHEMICAL INDUSTRIES LTD., Osaka, JP.(71) Applicant TAKEDA CHEMICAL INDUSTRIES LTD., Osaka, JP.

(72) Opfinder Elji Higashide, JP; Mitsuko Asal, JP: Seiichi Ta= nida, JP.(72) Inventor Elji Higashide, JP; Mitsuko Asal, JP: Seiichi Ta = nida, JP.

(74) Fuldmægtig Dansk Patent Kontor ApS.(74) Plenipotentiary Danish Patent Office ApS.

(54) Fremgangsmåde til fremstilling af raaytanelnol, maytanacin og/eller maytansinolpropionat.(54) Process for the preparation of raytanelnol, maytanacin and / or maytansinol propionate.

Den foreliggende opfindelse angår en fremgangsmåde til fremstilling af maytansinol, maytanacin og/eller maytansinolpropionat med formlenThe present invention relates to a process for the preparation of maytansinol, maytanacin and / or maytansinol propionate of the formula

Cl CH, O RCl CH, O R

I 3 II o c> CH3°\Α/ ' CV^pXNr/CH3I 3 II o c> CH3 ° \ Α / 'CV ^ pXNr / CH3

β ' CHβ 'CH

o Vo V

o λ.o λ.

m /0m / 0

I [OHIn [OH

q ch3 och3 Hq ch3 and3 H

2 145570 hvori R betyder H, CH^CO eller CH^CI^CO.Wherein R is H, CH 2 CO or CH 2 Cl 2 CO.

Det er kendt, at maytanacin og maytansinolpropionat har stærk antitumoraktivitet [Kupehan et al.: Journal of the American Chemical Society 9J_, 5294 (1975) ]. På den anden side har maytansinol selv kun svag antitumoraktivitet (jf. ovennævnte reference), men er et nyttigt mellemprodukt til enkel fremstilling af maytanacin og maytansinolpropionat samt forskellige andre derivater.It is known that maytanacin and maytansinol propionate have strong antitumor activity [Kupehan et al: Journal of the American Chemical Society 9J, 5294 (1975)]. On the other hand, maytansinol itself has only weak antitumor activity (cf. the above reference), but is a useful intermediate for simple preparation of maytanacin and maytansinol propionate as well as various other derivatives.

Maytansinol og maytanacin er opnået af Kupehan et al. fra barken af Putterlickia verrucosa (en plante, som tilhører slægten Maytenus), og herved er udbyttet ekstremt lavt, såsom 0,025 mg af den førstnævnte forbindelse og 0,36 mg af den sidstnævnte fra 1 kg tørret plan-tebark. Maytansinolpropionat er opnået ved kemisk propionering af maytansinol.Maytansinol and maytanacin have been obtained by Kupehan et al. from the bark of Putterlickia verrucosa (a plant belonging to the genus Maytenus), thereby yielding extremely low yields such as 0.025 mg of the former compound and 0.36 mg of the latter from 1 kg of dried plant bark. Maytansinol propionate is obtained by chemical propionation of maytansinol.

Ved undersøgelse af mikroorgansimer isoleret fra forskellige jordprøver har det vist sig, at stammen Nocardia nr. C-15003 (IFO 13726) er i stand til at producere maytansinol, maytanacin og/eller maytansinolpropionat i et egnet næringsmedium under passende betingelser.Upon examination of microorganisms isolated from various soil samples, it has been found that the strain Nocardia No. C-15003 (IFO 13726) is capable of producing maytansinol, maytanacin and / or maytansinol propionate in a suitable nutrient medium under appropriate conditions.

I overensstemmelse hermed er fremgangsmåden ifølge opfindelsen ejendommelig ved, at man dyrker en mikroorganisme, som hører til den art, hvortil stammen Nocardia nr. C-15003 (IFO 13726) hører, i et dyrkningsmedium, der indeholder assimilerbare carbonkilder og omsættelige nitrogenkilder, og udvinder maytansinol, maytanacin eller maytansinolpropionat eller flere af disse stoffer fra den opnåede dyrkningsblanding.Accordingly, the process of the invention is peculiar to the cultivation of a microorganism belonging to the species to which the strain Nocardia No. C-15003 (IFO 13726) belongs, in a culture medium containing assimilable carbon sources and transferable nitrogen sources, and extracting maytansinol, maytanacin or maytansinol propionate or more of these substances from the culture mixture obtained.

Ifølge den kendte teknik er de ovenstående forbindelser opnåelige fra planter, men disse planter er begrænset til specifikke arter, og der kræves store omkostninger og lang tid til produktionen på hvert trin af væksten, fældningen, tørringen og pulveriseringen af planterne og ekstraktionen, adskillelsen og rensningen af stofferne. Endvidere er udbyttet ekstremt lavt.According to the prior art, the above compounds are obtainable from plants, but these plants are limited to specific species and large costs and long time are required for the production at each stage of growth, precipitation, drying and pulverization of the plants and the extraction, separation and purification. of the substances. Furthermore, the yield is extremely low.

I modsætning hertil kan den omhandlede fremgangsmåde gennemføres let ved dyrkning af mikroorganismen, og der kan fremstilles en større 143570 3 mængde af de omhandlede forbindelser, som ikke tidligere har været fremstillet ved anvendelse af mikrobiologiske fremgangsmåder.In contrast, the process of the present invention can be readily accomplished by growing the microorganism, and a greater amount of the subject compounds can be prepared which have not previously been prepared using microbiological methods.

De mikrobiologiske karakteristika af stamme nr. C-15003 undersøgtes ved metoder, der er analoge med de, der foreslås af Schirling & Gottlieb (International Journal af Systematic Bacteriology 16, 313-34-0 (1966)). Resultaterne af observationer ved 28°C i 21 dage er som følger: 1) Morfologiske karakteristika:The microbiological characteristics of strain No. C-15003 were investigated by methods analogous to those proposed by Schirling & Gottlieb (International Journal of Systematic Bacteriology 16, 313-34-0 (1966)). The results of observations at 28 ° C for 21 days are as follows: 1) Morphological characteristics:

Det vegetative mycelium er veludviklet og forgrenet både på agar og i flydende medium. Mange af hyferne måler 0,8 til 1,2 u i diameter og kan under visse omstændigheder dele sig i fragmenter, der ligner stavformede bakterier, eller forgrenede,korte hyfestykker. Stammen giver god vækst på forskellige taksonomiske medier med luftmyceliet overlejret på det vegetative mycelium, skønt det ofte danner coremia-lignende legemer (50-200 x 200-1000 μ ), hvorpå videre vækst i luften finder sted. Mange af luftmycelierne er bugtede eller lige, og en løs spirallignende konfiguration træffes ved enkelte lejligheder.The vegetative mycelium is well developed and branched both on agar and in liquid medium. Many of the hyphae measure 0.8 to 1.2 microns in diameter and may, in some circumstances, divide into fragments similar to rod-shaped bacteria, or branched short hyphae. The stem gives good growth on various taxonomic media with the aerial mycelium superimposed on the vegetative mycelium, although it often forms coremia-like bodies (50-200 x 200-1000 μ), whereupon further growth in the air takes place. Many of the aerial mycelia are curved or straight, and a loose spiral-like configuration occurs on a few occasions.

Mikroskopisk undersøgelse af ældre kulturer viser, at de konidielig-nende celler kun i enkelte tilfælde optræder i kæder, mens de cellesuspensioner, der opnåedes fra overfladen af sådanne kulturer, ved mikroskopisk undersøgelse sås at indeholde mange forlængede ellipsoi-de (0,8-1,2 μ x 4,8-6,8 μ ) og ellipsoide (0,8-1,2 x 1,0-2,0 μ ) legemer, som lignede arthrosporer.Microscopic examination of older cultures shows that the conidial-like cells appear only in chains in some instances, while the cell suspensions obtained from the surface of such cultures were seen by microscopic examination to contain many elongated ellipsoids (0.8-1 , 2 μ x 4.8–6.8 μ) and ellipsoid (0.8–1.2 x 1.0–2.0 μ) bodies resembling arthrospores.

Elektronmikroskopiske undersøgelser viste, at disse legemer havde en glat overflade.Electron microscopic studies showed that these bodies had a smooth surface.

2) Cellebestanddelene:2) The cell components:

Stammen dyrkedes under omrystning i modificeret ISP nr. 1 medium ved 28°C i 66 til 90 timer, hvorefter cellerne opsamledes og skylledes. Yed metoden ifølge B. Becker et al. (Applied Microbiology 12, 421 (1964)) og metoden ifølge Μ, P. Lechevalier (Journal of Laboratory and Clinical Medicine £1, 934 (1968)) undersøgtes de ovennævnte he- 4 Μ357Ό le celler for diaminopimelinsyre og sukkersammensætning. Førstnævnte fandtes at være meso-formen, medens der påvistes pletter, som svarede til galactose og arabinose.The strain was grown under shaking in modified ISP # 1 medium at 28 ° C for 66 to 90 hours, after which the cells were collected and rinsed. The yed method of B. Becker et al. (Applied Microbiology 12, 421 (1964)) and the method of Μ, P. Lechevalier (Journal of Laboratory and Clinical Medicine £ 1, 934 (1968)), the above-mentioned whole 4Μ357Ό cells for diaminopimelic acid and sugar composition were examined. The former was found to be the meso-form, while stains corresponding to galactose and arabinose were detected.

3) Karakteristika på taksonomiske medier:3) Characteristics of taxonomic media:

Stammen viste forholdsvis god vækst på forskellige medier, idet det vegetative mycelium var farveløst til bleggult i den indledende dyrkningsfase og lyst gulbrunt til gulbrunt i senere faser. Stammen producerer opløselige pigmenter, som er gule til gulbrune,! forskellige taksonomiske medier. Luftmyceliet er pulveragtigt og giver generelt moderat vækst, idet det er hvidt til gult eller lyst gulbrunt. Stammens karakteristika i forskellige taksonomiske medier er vist i tabel 1.The stem showed relatively good growth on various media, with the vegetative mycelium being colorless to pale yellow in the initial cultivation phase and light yellow to yellow-brown in later stages. The strain produces soluble pigments which are yellow to yellowish brown! various taxonomic media. The aerial mycelium is powdery and generally produces moderate growth, being white to yellow or light yellowish brown. The characteristics of the strain in different taxonomic media are shown in Table 1.

Jabel 1Jabel 1

Dyrkningskarakteristika for stamme nr. C-15003 på taksonomiske medier: (A) Sucrose-nitrat-agar: Vækst (G): Overdådig, "Brite Melon Yellow" (3 ia)* til "Amber" (3 lc)#, coremia-lignende legemer dannet Luftmycelium (AM): Sparsomt, hvidtCultivation characteristics of strain No. C-15003 on taxonomic media: (A) Sucrose nitrate agar: Growth (G): Sumptuous, "Brite Melon Yellow" (3 ia) * to "Amber" (3 lc) #, coremia- similar bodies formed Air mycelium (AM): sparse, white

Opløseligt pigment (SP): Intet eller blegt gulbrunt (B) Glycerol-nitrat-agar: G : Moderat, "Lt Ivory" (2 ca)*, coremia-lignende legemer dannet AM : Moderat, hvidt SP : Intet (C) Glucose-asparagin-agar: G : Moderat, "Brite Marigold" (3 pa)4' til "Brite Yellow" (2 pa)* AM : Sparsomt, hvidt SP : "Brite Yellow" (2 pa)* (D) Glycerol-asparagin-agar: G : Moderat, "Lt Ivory" (2 ca)w, coremia-lignende legemer dannet AM : Sparsomt, hvidt SP : Intet (E) Stivelsesagar: G : Moderat, "Lt Ivory" (2 ca)1' til "Lt Wheat" (2 ea)*, coremia- lignende legemer dannet 143570 5 AM : Rigeligt, "Lt Ivory" (2 ca)1 SP : Intet (F) Næringsagar: G : Moderat, "Lt Ivory" (2 ca)1 til "Colonial Yellow" (2 ga)1, coremia-lignende legemer dannet AM : Sparsomt, hvidt SP : Intet (G) Calciummalatagar: G : Moderat, "Lt Ivory" (2 ca)1 til "Lt Wheat" (2 ea)1, coremia- lignende legemer dannet AM : Moderat, hvidt til "Lt Ivory" (2 ca)1 SP : Intet (H) Gærekstrakt-maltekstrakt-agar: G : Moderat, "Amber" (3 lc)1 til "Brite Yellow" (3 la)1, coremia- lignende legemer dannet AM : Moderat, hvidt til "Lt Ivory" (2 ca)1 SP : Intet (I) Havremelsagar: G : Moderat, "Lt Ivory" (2 ca)1 til "Colonial Yellow" (2 ga)1, coremia-lignende legemer dannet AM : Sparsomt, hvidt til lyst gult SP : Intet (I) Pepton-gærekstrakt-jern-agar: G : Moderat, "Colonial Yellow" (2 ga)1 AM : Intet SP : "Colonial Yellow" (2 ga)1 (K) Tyrosinagar: G : Moderat, "Lt Ivory" (2 ca)1 til "Lt Melon Yellow" (3 ea)1, coremia-lignende legemer dannet AM : Moderat, hvidt til "Lt Ivory" (2 ca)1 SP : "Camel" (3 ie)1Soluble pigment (SP): No or pale yellowish brown (B) Glycerol nitrate agar: G: Moderate, "Lt Ivory" (2 ca) *, coremia-like bodies formed AM: Moderate, white SP: None (C) Glucose -asparagine agar: G: Moderate, "Brite Marigold" (3 pa) 4 'to "Brite Yellow" (2 pa) * AM: Thrifty, white SP: "Brite Yellow" (2 pa) * (D) Glycerol- asparagine agar: G: Moderate, "Lt Ivory" (2 ca) w, coremia-like bodies formed AM: sparse, white SP: None (E) Starch agar: G: Moderate, "Lt Ivory" (2 ca) 1 ' to "Lt Wheat" (2 ea) *, coremia-like bodies formed 143570 5 AM: Abundant, "Lt Ivory" (2 ca) 1 SP: None (F) Nutrition Agar: G: Moderate, "Lt Ivory" (2 ca) ) 1 to "Colonial Yellow" (2 ga) 1, coremia-like bodies formed AM: sparse, white SP: None (G) Calcium malatagar: G: Moderate, "Lt Ivory" (2 ca) 1 to "Lt Wheat" ( 2 ea) 1, coremia-like bodies formed AM: Moderate, white to "Lt Ivory" (2 ca) 1 SP: None (H) Yeast extract malt extract agar: G: Moderate, "Amber" (3 lc) 1 to "Brite Yellow" (3) la) 1, coremia-like bodies formed AM: Moderate, white to "Lt Ivory" (2 ca) 1 SP: None (I) Oatmeal agar: G: Moderate, "Lt Ivory" (2 ca) 1 to "Colonial Yellow" (2 ga) 1, coremia-like bodies formed AM: sparse, white to light yellow SP: None (I) Pepton yeast extract-iron agar: G: Moderate, "Colonial Yellow" (2 ga) 1 AM: No SP : "Colonial Yellow" (2 ga) 1 (K) Tyrosine Agar: G: Moderate, "Lt Ivory" (2 ca) 1 to "Lt Melon Yellow" (3 ea) 1, coremia-like bodies formed AM: Moderate, white to "Lt Ivory" (2 ca) 1 SP: "Camel" (3 ie) 1

Farvekoderne er dem, der er angivet i "Color Harmony Manual", 4th ed. (Container Corporation of America, 1958).The color codes are those listed in the "Color Harmony Manual", 4th ed. (Container Corporation of America, 1958).

r 143570 b 4) Fysiologiske karakteristika:r 143570 b 4) Physiological characteristics:

Stammens fysiologiske karakteristika er vist i tabel 2. Temperaturområde for vækst: 12 til 38°C. Temperaturområdet, hvori der er god luftvækst på agar (ISP nr 2), er 20 til 35°C.The physiological characteristics of the strain are shown in Table 2. Temperature range for growth: 12 to 38 ° C. The temperature range in which there is good air growth on agar (ISP # 2) is 20 to 35 ° C.

Tabel 2Table 2

Fysiologiske karakteristika for stamme nr. C-15003:Physiological characteristics of strain C-15003:

Temperaturområde for vækst: 12 til 38°CTemperature range for growth: 12 to 38 ° C

Temperaturområde for luftvækst: 20 til 35°CTemperature range for air growth: 20 to 35 ° C

Gelatinesmeltning: PositivGelatin melting: Positive

Hydrolyse af stivelse: PositivHydrolysis of starch: Positive

Reduktion af nitrater: PositivNitrate reduction: Positive

Peptonisering af mælk: PositivPeptonization of milk: Positive

Koagulation af mælk: NegativMilk coagulation: Negative

Dekomposition af kasein: PositivDecomposition of casein: Positive

Produktion af melanoide pigmenter: Negativ (pepton-gærekstrakt- jern-agar), positiv (tyrosin-agar)Production of Melanoid Pigments: Negative (Peptone Yeast Extract Iron Agar), Positive (Tyrosine Agar)

Dekomposition af tyrosin: PositivDecomposition of tyrosine: Positive

Dekomposition af xanthin: NegativDecomposition of xanthine: Negative

Dekomposition af hypoxanthin: NegativDecomposition of hypoxanthine: Negative

Tolerance for lysozym: PositivLysozyme tolerance: Positive

Tolerance for natriumchlorid: 2% 5) Udnyttelse af forskellige carbonkilder:Sodium chloride tolerance: 2% 5) Utilization of various carbon sources:

Udnyttelse af forskellige carbonkilder undersøgtes under anvendelse af et medium beskrevet i Pridham and Gottlieb (Journal of Bacteriology ^6, IO7 (1948)) og et basalmedium af samme sammensætning plus 0,1% gærekstrakt. Det resulterende spektrum er vist i tabel 3.Utilization of various carbon sources was investigated using a medium described in Pridham and Gottlieb (Journal of Bacteriology ^ 6, IO7 (1948)) and a basal medium of the same composition plus 0.1% yeast extract. The resulting spectrum is shown in Table 3.

Tabel 3Table 3

Udnyttelsen af carbonkilder af stamme nr. C-I5OO3 Carbonkilde Vækst Oarbonkilde Vækst D-Xylose + ++* Raffinose ± 7Exploitation of carbon sources of strain No. C-I5OO3 Carbon source Growth Oxygen source Growth D-Xylose + ++ * Raffinose ± 7

Carborkilde Vækst Carbonkilde Vækst L-Arabinose + + Melibiose + + D-Glucose ++ ++ i-Inositol - - D-Galactose + + D-Sorbitol -- D-Fructose +++ ++ D-Mannitol ++ ++ L-Rhamnose + + Glycerol - ± D-Mannose +++ ++ Opløselig stivelse + +Carbon Source Growth Carbon Source Growth L-Arabinose + + Melibiosis + + D-Glucose ++ ++ i-Inositol - - D-Galactose + + D-Sorbitol - D-Fructose +++ ++ D-Mannitol ++ ++ L -Rhamnose ++ Glycerol - ± D-Mannose +++ ++ Soluble Starch ++

Sucrose ++ ++ Kontrol - -Sucrose ++ ++ Control - -

Lactose - -*Lactose - - *

Maltose ± +Maltose ± +

Trehalose + ++ * Basalmedium med 0,1% gøerekstrakt tilsatTrehalose + ++ * Basal medium with 0.1% yeast extract added

Mote: +++: Overdådig vækst ++: God vækst + : Vækst ±: Ringe vækst Ingen vækst 6) Andre karakteristika:Fashion: +++: Sumptuous growth ++: Good growth +: Growth ±: Low growth No growth 6) Other characteristics:

Cellerne udvandtes ved den ovenfor i 2) beskrevne metode, og DNA'et fremstilledes analogt med metoden ifølge J. Marmur et al. (Journal of Molecular Biology, 208, (1961)), G-C(guanin“cytosin)-indholr-det i DNA'et fandtes at være ca, 71 mol-%.The cells were purified by the method described above in 2) and the DNA was prepared analogously to the method of J. Marmur et al. (Journal of Molecular Biology, 208, (1961)), the G-C (guanine "cytosine) content of the DNA was found to be about 71 mole%.

Gramfarvning af det vegetative mycelium af denne stamme var positiv.Gram staining of the vegetative mycelium of this strain was positive.

De ovennævnte karakteristika af stamme nr. C-15003 sammenlignedes med beskrivelserne i S. A. Waksman's "The Actinomycetes Vol. 2" (The Williams and Wilkins Co., (1961)); R. E. Buchanan and N. E. Gibbons, "Bergey's Manual og Determinative Bacteriology, 8th ed., 197^", og anden litteratur.The above characteristics of strain No. C-15003 were compared with the descriptions in S. A. Waksman's "The Actinomycetes Vol. 2" (The Williams and Wilkins Co., (1961)); R. E. Buchanan and N. E. Gibbons, "Bergey's Manual and Determinative Bacteriology, 8th ed., 197 ^," and other literature.

Da stammen formodedes at tilhøre gruppe III i slægten Nocardia, og daman ikke fandt arter med de hidtil beskrevne karakteristika blandt de kendte stammer, konkluderede man, at denne stamme repræsenterer en hidtil ukendt mikroorganismeart.As the strain was thought to belong to group III of the genus Nocardia, and daman did not find species with the characteristics described so far among the known strains, it was concluded that this strain represents a novel microorganism species.

143570 8143570 8

Den foreliggende stamme nr. C-15003 er deponeret hos Fermentation Research Institute, Agency of Industrial Science and Technology (FERM) under indleveringsnummeret 3992, hos Institute for Fermentation, Osaka (IFO) under accessionsnummeret IFO 13726 og hos The American Type Culture Collection (ATCC), Maryland, U.S.A. under accessionsnummeret 31281.The present strain No. C-15003 is deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology (FERM) under filing number 3992, with the Institute for Fermentation, Osaka (IFO) under accession number IFO 13726 and with The American Type Culture Collection (ATCC ), Maryland, USA under the accession number 31281.

Stamme nr. C-15003 tilhører en ny art af slægten Nocardia, som netop nævnt, og den er tilbøjelig til, ligesom mikroorganismer generelt, at undergå variationer og mutationer enten spontant eller under indflydelse af et mutagen. De mange varianter af stammen, som er opnåelige ved bestråling med røntgenstråler, gammastråler, ultraviolet lys og lignende, ved isolering af enkelte celler ved dyrkning på medier indeholdende forskellige kemikalier, eller ved enhver anden mutagen behandling, såvel som de mutanter, der spontant afledes fra stammen, og hvis egenskaber i det væsentlige ikke afviger fra de givne karakteristika, og som er i stand til at frembringe maytansinol, maytanacin og maytansinolpropionat, kan udnyttes ved den omhandlede fremgangsmåde ligesom stamme nr. C-15003. Hvis stamme nr. C-15003 eksempelvis underkastes forskellige mutagene behandlinger, opnås mutanter, som i det væsentlige mangler evnen til at producere opløselige pigmenter, mutanter med substratmycelier, der er farveløse, gulgrønne, rødbrune eller orangerøde, mutanter, hvis hyfer er rede til at fragmentere i bacillære elementer eller forgrenede korte hyfefragmenter, og mutanter med rigeligt, hvidt luftmycelium eller i det væsentlige uden luftmycelium.Strain No. C-15003 belongs to a new species of the genus Nocardia, just mentioned, and it, like microorganisms in general, tends to undergo variations and mutations either spontaneously or under the influence of a mutagen. The many variants of the strain obtainable by radiation with X-rays, gamma rays, ultraviolet light and the like, by isolating single cells by culturing on media containing various chemicals, or by any other mutagenic treatment, as well as the mutants that spontaneously derive from strain, and whose properties do not differ substantially from the given characteristics and are capable of producing maytansinol, maytanacin and maytansinol propionate, can be utilized by the process of the present invention as is the case of strain C-15003. For example, if strain C-15003 is subjected to various mutagenic treatments, mutants which are substantially lacking in the ability to produce soluble pigments, mutants with substrate mycelia that are colorless, yellow-green, red-brown or orange-red, are obtained, mutants whose hyphae are ready to fragments in bacillary elements or branched short hyphal fragments, and mutants with abundant white aerial mycelium or substantially without aerial mycelium.

Mediet, som anvendes til dyrkningen af den maytansinol-, maytanacin-eller maytansinolpropionatproducerende stamme (i det følgende undertiden forkortet som "den producerende stamme"), kan være et hvilket som helst flydende og fast medium, hvis det blot indeholder næringsstoffer, som stammen kan udnytte, skønt et flydende medium er foretrukket til større produktioner. Mediet skal indeholde carbon- og nitrogenkilder, som stamme nr. C-15003 kan assimilere og omsætte, uorganiske stoffer, mikronæringsstoffer m.m.. Som eksempler på carbon-kilder kan nævnes glucose, lactose, sucrose, maltose, dextrin, stivelse, glycerol, mannitol og sorbitol, fedtstoffer og olier (f.eks. sojabønneolie, svinefedt og kyllingefedt). ETitrogenkilderne kan f.eks. være kødekstrakt, gærekstrakt, tørgær, sojabønnemel, majsstøbevand, pepton, kasein, bomuldsfrømel, "spent molasses", urinstof, ammonium- 9 w$s;ro salte (f.eks. ammoniumsulfat, ammoniumchlorid, ammoniumnitrat og ammoniumacetat). Mediet kan endvidere indeholde salte af natrium, kalium, calcium, magnesium og lignende, salte af jern, mangan, zink, cohalt, nikkel og lignende, salte af phosphorsyre og horsyre og salte af organiske syrer, såsom acetater og propionater. Endvidere kan mediet indeholde forskellige aminosyrer (f.eks. glutaminsyre, asparaginsyre, alanin, glycin, lysin, methionin og prolin), peptider (f.eks. dipeptider og tripeptider), vitaminer (f.eks. B^, nicotinsyre, B·^, C og E), haser fra nucleinsyrer (f.eks. purin, pyrimidin og derivater deraf). Til indstilling af me-r-diets pH kan der f.eks. tilsættes en uorganisk eller organisk syre, et alkali eller en puffer. Egnede mængder olier, fedtstoffer, overfladeaktive stoffer og lignende kan også tilsættes som antiskum-midler.The medium used for the cultivation of the maytansinol, maytanacin or maytansinol propionate strain (hereinafter sometimes abbreviated as "the producing strain") may be any liquid and solid medium if it merely contains nutrients which the strain may contain. exploit, although a liquid medium is preferred for larger productions. The medium must contain carbon and nitrogen sources which strain C-15003 can assimilate and react with, inorganic substances, micronutrients, etc. Examples of carbon sources include glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol and sorbitol, fats and oils (eg soybean oil, lard and chicken fat). The ETrogen sources may e.g. be it meat extract, yeast extract, dry yeast, soybean meal, corn molding water, peptone, casein, cotton seed meal, spent molasses, urea, ammonium 9 w $ s; ro salts (eg ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium acetate). The medium may further contain salts of sodium, potassium, calcium, magnesium and the like, salts of iron, manganese, zinc, cohal, nickel and the like, salts of phosphoric and horsic acid and salts of organic acids such as acetates and propionates. Furthermore, the medium may contain various amino acids (e.g., glutamic acid, aspartic acid, alanine, glycine, lysine, methionine and proline), peptides (e.g., dipeptides and tripeptides), vitamins (e.g., β, nicotinic acid, β · , C and E), rabbits from nucleic acids (e.g., purine, pyrimidine and derivatives thereof). For example, to adjust the pH of the diet, e.g. an inorganic or organic acid, an alkali or a buffer is added. Suitable amounts of oils, fats, surfactants and the like can also be added as antifoam agents.

Dyrkningen kan gennemføres stationært, under omrystning, suhmers aeroht og under enhver anden dyrkningsbetingelse. Til større produktioner foretrækkes naturligvis suhmers aeroh dyrkning. Skønt dyrknirøgs--hetingelserne naturligvis afhænger af tilstanden og sammensætningen af mediet, stammen, dyrkningsmetoden og andre faktorer foretrækkes det normalt at gennemføre inkubation ved 20 til 35°C med et indledende pH på ca. 7,0. Særligt ønskelig er en temperatur fra 23 til 30°CCultivation can be carried out stationary, under shaking, suherm aeroht and under any other cultivation condition. Of course, for larger productions, the aeroh cultivation of suhm is preferred. Of course, although the animal smoke fumes depend on the state and composition of the medium, strain, culture method and other factors, it is usually preferred to incubate at 20 to 35 ° C with an initial pH of approx. 7.0. Particularly desirable is a temperature of 23 to 30 ° C

1 et mellemliggende dyrkningstrin med et indledende pH på 6,5 til 7,5· Da inkubationstiden også er variabel afhængig af de samme faktorer som nævnt ovenfor, er det tilrådeligt at fortsætte inkubationen, indtil titeren af det ønskede antibiotiske produkt bliver maksimal. X tilfælde af en rystekultur eller aerob suhmers kultur i flydende medium, er den nødvendige tid normalt fra ca. 48 til 144 timer.In an intermediate culture step with an initial pH of 6.5 to 7.5 · Since the incubation time is also variable depending on the same factors mentioned above, it is advisable to continue the incubation until the titre of the desired antibiotic product is maximized. X case of a culture of shaking or aerobic sucker culture in liquid medium, the required time is usually from approx. 48 to 144 hours.

Styrken af antibiotikumet prøvedes med Tetrahymena pyriformis W som prøveorganisme. Således dyrkedes ovennævnte mikroorganisme på et testmedium (20 g proteose-pepton (Difco), 1 g gærekstrakt (Difco), 2 g glucose, 1000 ml destilleret vand og 10 ml 1 M phosphatpuffer (pH 7,0)) ved 28°C i 44 til 48 timer, og styrken af antibiotikumet bestemtes ved seriefortyndingsmetoden med en kontrol af vækstens turbiditet og ved en tyndtlagskromatografisk (forkortet TLC) prøvning, som skal beskrives i det følgende.The strength of the antibiotic was tested with Tetrahymena pyriformis W as the test organism. Thus, the above microorganism was grown on a test medium (20 g of proteose peptone (Difco), 1 g of yeast extract (Difco), 2 g of glucose, 1000 ml of distilled water and 10 ml of 1 M phosphate buffer (pH 7.0)) at 28 ° C. 44 to 48 hours, and the potency of the antibiotic was determined by the serial dilution method with a control of growth turbidity and by a thin layer chromatographic (abbreviated TLC) test to be described below.

Maytanacin, maytansinolpropionat eller maytansinol produceres og akkumuleres i den resulterende kulturvæske, både extracellulært og 143570 10 intr acellulært.Maytanacin, maytansinol propionate or maytansinol are produced and accumulated in the resulting culture fluid, both extracellularly and intracellularly.

Ingen af disse stoffer viser tydelig antibiotisk aktivitet, og de bestemtes således ved TIC, som udførtes parallelt med påvisning ved aktivitet på Tetrahymena-stammen. Således adskilles fermentationsnæringsvæsken i celler og filtrat ved filtrering eller centrifugering, og filtratet ekstraberes med det samme volumen ethylacetat. Til cellerne sættes den samme mængde 70%'s acetone-vand som filtratet,og efter 1 times omrøring ved 20°C filtreres suspensionen. Acetonet fjernes fra filtratet, og det resulterende vandige filtrat ekstra-beres med ethylacetat. Hver af ekstrakterne koncentreres til 1/100 volumen og underkastes tyndtlagskromatografi på en silicagel-glas-plade (kiselgel 60 ^,25 mm, 20 x 20) (opløsningsmiddelsystem: cbloroform-metbanol = 9:1)· Styrken bestemtes på basis af intensiteten af pletterne, der påvistes ved bestråling med ultraviolet lys ved 2537 Å.None of these substances show clear antibiotic activity, and thus were determined by TIC, which was performed in parallel with detection by activity on the Tetrahymena strain. Thus, the fermentation broth is separated into cells and filtrate by filtration or centrifugation and the filtrate is extracted with the same volume of ethyl acetate. To the cells is added the same amount of 70% acetone water as the filtrate and after 1 hour stirring at 20 ° C the suspension is filtered. The acetone is removed from the filtrate and the resulting aqueous filtrate is extracted with ethyl acetate. Each of the extracts is concentrated to 1/100 volume and subjected to thin layer chromatography on a silica gel glass plate (silica gel 60 µ, 25 mm, 20 x 20) (solvent system: cbloroform-metbanol = 9: 1) · The strength was determined based on the intensity of the spots detected by ultraviolet light irradiation at 2537 Å.

Maytanacin, maytansinolpropionat eller maytansinol, som produceres i kulturvæsken, er lipofile og neutrale. De kan bekvemt udvindes ved de adskillelses- og rensningsmetoder, der normalt anvendes til udvinding af mikrobielle metaboliter. F.eks. kan der anvendes en metode, som udnytter forskellen i opløselighed mellem antibiotikumet og urenheden, metoder, som udnytter den fraktionerende adsorptive affinitet af forskellige adsorptionsmidler, såsom aktivt kul, makroporøse ikke-ioniske harpikser, silicagel og alumina, og en metode til fjernelse af urenhederne ved hjælp af ionbytterharpikser anvendt enkeltvis eller i en egnet kombination eller anvendt gentagne gange.Maytanacin, maytansinol propionate or maytansinol produced in the culture fluid are lipophilic and neutral. They can be conveniently recovered by the separation and purification methods normally used for the extraction of microbial metabolites. Eg. For example, a method utilizing the difference in solubility between the antibiotic and the impurity may be employed, methods utilizing the fractional adsorptive affinity of various adsorbents such as activated carbon, macroporous non-ionic resins, silica gel and alumina, and a method of removing the impurities by using ion exchange resins used singly or in a suitable combination or used repeatedly.

Som tidligere anført findes maytanacin, maytansinolpropionat eller maytansinol både extracellulært og intracellulært i den udfermenterede kulturvæske. Til oparbejdning af denne kan den opdeles i et filtrat og en cellemasse på gængs måde, hvorpå filtratet underkastes opløsningsmiddelekstraktion med et med vand ikke-blandbart organisk opløsningsmiddel. Cellerne i cellemassen nedbrydes og underkastes opløsningsmiddelekstraktion med et med vand ikke-blandbart organisk opløsningsmiddel. Derpå behandles de isolerede organiske faser med et adsorptionsmiddel til fjernelse af urenheder. Eventuelt kan den udfermenterede kulturvæske underkastes opløsningsmiddelekstraktion inden isolering af cellerne.As previously stated, maytanacin, maytansinol propionate or maytansinol are found both extracellularly and intracellularly in the fermented culture fluid. For working it up, it can be divided into a filtrate and a cell mass in the usual way, undergoing the filtrate with solvent extraction with a water-immiscible organic solvent. The cells in the cell mass are degraded and subjected to solvent extraction with a water-immiscible organic solvent. Then, the isolated organic phases are treated with an adsorbent to remove impurities. Optionally, the fermented culture fluid may be subjected to solvent extraction before isolating the cells.

11 f«me11 f «me

Til ekstraktion af filtratet og cellerne uafhængigt af hinanden kan følgende fremgangsmåde med fordel benyttes.For the extraction of the filtrate and the cells independently, the following procedure can be advantageously used.

Egnede opløsningsmidler til ekstraktion af filtratet er med vand ikke-blandbare organiske opløsningsmidler, såsom fedtsyreestere, f.eks. ethylacetat og amylacetat, alkoholer, f.eks. butanol, halogenerede carbonhydrider, f.eks. chloroform, og ketoner, f.eks. methyl i s obu tylketon. Ekstraktionen udføres ved et pH nær neutralpunktet, og dyrkningsvæsken, der forinden fortrinsvis er indstillet til pH 7, ekstraheres f.eks. med ethylacetat. Ekstrakten vaskes med vand og koncentreres under reduceret tryk. Derefter sættes et upolært opløsningsmiddel, såsom petroleumsether eller hexan, til koncentratet , og råproduktet I, som indeholder den aktive forbindelse, udvindes.Suitable solvents for extracting the filtrate are water-immiscible organic solvents such as fatty acid esters, e.g. ethyl acetate and amyl acetate, alcohols, e.g. butanol, halogenated hydrocarbons, e.g. chloroform, and ketones, e.g. methyl i s obu tylketone. The extraction is carried out at a pH near the neutral point, and the culture liquid, which is previously preferably adjusted to pH 7, is extracted e.g. with ethyl acetate. The extract is washed with water and concentrated under reduced pressure. Then, an unpolar solvent such as petroleum ether or hexane is added to the concentrate and the crude product I containing the active compound is recovered.

Da der ved TLC påvises et antal pletter foruden maytanacin, maytan-sinolpropionat eller maytansinol, underkastes produktet I successivt de følgende rensningsmetoder. Således er en rutinerensningsmetode, især adsorptionskromatografi, nyttig, og til dette formål kan anvendes et af de almindelige adsorbenser, såsom silicagel, alumina eller enmakroporøs ikke-ionisk adsorberende harpiks. Til rensning af råproduktet I er silicagel det nyttigste, Eluering kan f.eks. begynde med petroleumsether og hexan og gennemføres ved tilsætning af et polært opløsningsmiddel, såsom ethylacetat, acetone, ethanol eller methanol. Ved en typisk fremgangsmåde,under anvendelse af silicagel (0,05-0,2 mm) som bærer, udføres søjlekromatografi med en gradvis forøgelse af hexan til ethylacetatforholdet. Eluatet opsamles og undersøges ved TLC og fraktionerne, der indeholder effektive ingredienser, samles og koncentreres under reduceret tryk. Derefter sættes petroleumsether eller hexan til koncentratet, hvorved råproduktet II opnås. Da dette produkt stadig indeholder urenheder, renses det yderligere som følger. F.eks. kan produktet II renses ved hjælp af en anden silica-gelsøjle under anvendelse af et andet opløsningsmiddelsystem. Elu-eringssystemet til dette formål kan bestå af et halogeneret carbon-hydrid, såsom dichlormethan eller chloroform, med tilsætning af et polært opløsningsmiddel, såsom en alkohol, f.eks. ethanol eller methanol, en keton, f.eks. acetone eller metbylethylketon. På denne måde isoleres maytanacin, maytansinolpropionat eller maytansinol. Rækkefølgen af opløsningsmiddelsystemerne til den første og anden silicagelsøjle kan være omvendt, og desuden kan almindelige organiske opløsningsmidler anvendes i forbindelse med de ovenstående systemer.Since TLC detects a number of stains besides maytanacin, maytanicinol propionate or maytansinol, product I is successively subjected to the following purification methods. Thus, a routine purification method, especially adsorption chromatography, is useful and for this purpose one of the common adsorbents such as silica gel, alumina or single macroporous nonionic adsorbent resin may be used. For purification of the crude product I, silica gel is the most useful. begin with petroleum ether and hexane and is carried out by the addition of a polar solvent such as ethyl acetate, acetone, ethanol or methanol. In a typical process, using silica gel (0.05-0.2 mm) as the carrier, column chromatography is performed with a gradual increase of hexane to the ethyl acetate ratio. The eluate is collected and examined by TLC and the fractions containing effective ingredients are collected and concentrated under reduced pressure. Then petroleum ether or hexane is added to the concentrate to give the crude product II. As this product still contains impurities, it is further purified as follows. Eg. For example, the product II can be purified by another silica gel column using a different solvent system. The elution system for this purpose may consist of a halogenated hydrocarbon such as dichloromethane or chloroform, with the addition of a polar solvent such as an alcohol, e.g. ethanol or methanol, a ketone, e.g. acetone or methbylethyl ketone. In this way, maytanacin, maytansinol propionate or maytansinol are isolated. The order of the solvent systems of the first and second silica gel columns may be reversed, and in addition ordinary organic solvents may be used in connection with the above systems.

143570 12143570 12

Hvis en makroporøs adsorberende harpiks anvendes som rensningsmiddel for råproduktet II, udføres eluering af maytanacin, maytansi-nolpropionat eller maytansinol med en blanding af vand og en lavere alkohol, en lavere keton eller en ester. Den lavere alkohol kan f.eks. være methanol, ethanol, propanol eller butanol, og den lavere keton kan f.eks. være acetone eller methylethylketon. Esteren kan f.eks, være ethylacetat. Ved en typisk fremgangsmåde opløses råproduktet II i 60%'s methanol-vand og adsorberes på en søjle af "Diaion HP-10", som er en non-ionisk makroporøs ionbytterharpiks. Søjlen vaskes med 70%'s methanol-vand, hvorefter eluering udføres med 90%'s methanol-vand. På denne måde elueres maytanacin, maytansinolpropionat eller maytansinol fra søjlen.If a macroporous adsorbent resin is used as the purifier of the crude product II, elution of maytanacin, maytansanol propionate or maytansinol with a mixture of water and a lower alcohol, a lower ketone or an ester is performed. The lower alcohol may e.g. be methanol, ethanol, propanol or butanol, and the lower ketone may e.g. be acetone or methyl ethyl ketone. For example, the ester may be ethyl acetate. In a typical process, the crude product II is dissolved in 60% methanol-water and adsorbed on a column of "Diaion HP-10" which is a nonionic macroporous ion exchange resin. The column is washed with 70% methanol-water, followed by elution with 90% methanol-water. In this way, maytanacin, maytansinol propionate or maytansinol are eluted from the column.

Ved de ovenfor beskrevne fremgangsmåder samles fraktionerne, der indeholder de ønskede forbindelser, og koncentreres under reduceret tryk. Til det tørre produkt sættes 5 til 8 volumen ethylacetat ,og blandingen henstår, hvorved der udskilles krystaller af maytanacin, maytansinolpropionat eller maytansinol, eller hvis krystallerne indeholder maytanacin og maytansinolpropionat, adskilles de derefter fra hinanden ved hjælp af et adsorbens, såsom et af de ovennævnte. Under·anvendelse af silicagel eller en makroporøs ikke-ionisk adsor-verende harpiks og de ovenstående opløsningsmidler kan de ønskede forbindelser således elueres fraktioneret. Hvis f.eks. silicagel anvendes, udføres elueringen med hexan, ethylacetat eller chloroform-methanol, hvorved maytansinolpropionat og maytanacin fremkommer i den nævnte rækkefølge. Efter påvisning med TLC koncentreres fraktionerne under reduceret tryk,og ethylacetat sættes til koncentraterne. På denne måde kan de respektive forbindelser opnås som krystaller. Hvis der anvendes en makroporøs ikke-ionisk adsorberende harpiks, kan der benyttes gradienteluering med et varierende forhold af alkohol, keton eller ester til vand. Ved gradientelueringsmeto-den, under anvendelse af 60%'s methanol-vand og 95%'s methanol-vand, med 5% natriumchlorid tilsat, fremkommer maytanacin og maytansinolpropionat i den nævnte rækkefølge. Efter opsamling og påvisning med TLC koncentreres hver gruppe af aktive fraktioner under reduceret tryk og krystalliseres fra ethylacetat. De isolerede krystaller indeholder ethylacetat som krystallisationsopløsningsmiddel og viser efter tørring over phosphorpentoxid ved 70°C i 8 timer de følgende fysiske og kemiske egenskaber. (Tabel h).In the methods described above, the fractions containing the desired compounds are collected and concentrated under reduced pressure. To the dry product is added 5 to 8 volumes of ethyl acetate and the mixture is left to separate crystals of maytanacin, maytansinol propionate or maytansinol, or if the crystals contain maytanacin and maytansinol propionate, they are then separated by an adsorbent such as one of the above. . Thus, using silica gel or a macroporous nonionic adsorbent resin and the above solvents, the desired compounds can be eluted fractionally. For example, If silica gel is used, the elution is carried out with hexane, ethyl acetate or chloroform-methanol to give maytansinol propionate and maytanacin in the order mentioned. After detection by TLC, the fractions are concentrated under reduced pressure and ethyl acetate is added to the concentrates. In this way, the respective compounds can be obtained as crystals. If a macroporous nonionic adsorbent resin is used, gradient elution with a varying ratio of alcohol, ketone or ester to water can be used. By the gradient elution method, using 60% methanol-water and 95% methanol-water, with 5% sodium chloride added, maytanacin and maytansinol propionate appear in the above order. After collection and detection by TLC, each group of active fractions is concentrated under reduced pressure and crystallized from ethyl acetate. The isolated crystals contain ethyl acetate as the crystallization solvent and after drying over phosphorus pentoxide at 70 ° C for 8 hours show the following physical and chemical properties. (Table h).

13 U357013 U3570

Maytanacin Maytansinol- Maytansinol propionat _C50H39C1N2Q9_C31H41C1N2°9 C28H37G1N2°8Maytanacin Maytansinol- Maytansinol propionate _C50H39C1N2Q9_C31H41C1N2 ° 9 C28H37G1N2 ° 8

Smeltepunkt (°C) 235-236° 188-190° 172,5-174°Melting point (° C) 235-236 ° 188-190 ° 172.5-174 °

Specifik [a]22 -121°±10° [α]22 -127O±10° [ a]^2°-313°±10° drejning (c =0,25, CHClj) (c =0,35,OHClj) (c =0,22,01101^) 0 59,62 59,93 59,28Specific [a] 22 -121 ° ± 10 ° [α] 22 -127O ± 10 ° [a] 2 ° -313 ° ± 10 ° rotation (c = 0.25, CHCl₂) (c = 0.35, OHCl₂ ) (c = 0.22.01101 ^) 0 59.62 59.93 59.28

Analyse, H 6,93 6,82 6,38 fundet {%) N 4,28 4,32 5,02 01 5,74- 5,57 6,15 0 59,85 59,94 59,52Analysis, H 6.93 6.82 6.38 found (%) N 4.28 4.32 5.02 01 5.74-5.57 6.15 0 59.85 59.94 59.52

Analyse, H 6,48 6,65 6,60 beregnet N 4,61 4-,51 4,96 (%) Cl 5,84 5,71 6,27Analysis, H 6.48 6.65 6.60 Calculated N 4.61 4-, 51 4.96 (%) Cl 5.84 5.71 6.27

Ultraviolet 233(30330) 233(30240) 232(32750) absorpti- 240(sh.28240) 240(sb.28400) 244(sh.30850) onsspektrum 252(27850) 252(27650)' 252(31650) nm(e) 280(5680) 280(5740) 281(5750) 288(5660) 288(5710) 288(5700)Ultraviolet 233 (30330) 233 (30240) 232 (32750) absorption 240 (sh.28240) 240 (sb.28400) 244 (sh.30850) on spectrum 252 (27850) 252 (27650) 252 (31650) nm (e 280 (5680) 280 (5740) 281 (5750) 288 (5660) 288 (5710) 288 (5700)

Infrarødt absorpti- 1740,1730,1670, 1740,1730,1670, 1715,1670,1580 onsspektrum 1580 1580 (cm-1)Infrared Absorption 1740,1730,1670, 1740,1730,1670, 1715,1670,1580 Spectrum 1580 1580 (cm -1)

Masse- spektrum 545,485,470,450 559^5^0,450 505,485,470, (m/e) 450Mass Spectrum 545,485,470,450 559 5 5 0.450 505,485,470, (m / e) 450

Surt, neu- lipofilt og ru*,, «u- lipofilt og neu- lipofilt og neutralt eller trait stof , _ , trait stof trait stof basisk 1A3 5 70 14Acid, neu-lipophilic and rough *, «non-lipophilic and neu-lipophilic and neutral or trait substance, trait substance trait substance basic

Maytanacin Maytansinol- Maytansinol propionat C30HJ9CUr209 03iH4101H2°9 C28H37011I208Maytanacin Maytansinol- Maytansinol propionate C30HJ9CUr209 03iH4101H2 ° 9 C28H37011I208

Farvereak- Dragendorffre ak- Dragendorffreak- Dragendorffreak- tioner tion: positiv tion: positiv tion: positivColor reaction- Dragendorffre action- Dragendorffreak- Dragendorffraction tion: positive tion: positive tion: positive

Beilsteinreakti- Beilsteinreakti- Beilsteinreaktion: positiv on: positiv on: positivBeilstein reaction- Beilstein reaction- Beilstein reaction: positive on: positive on: positive

Ovennævnte data for elementaranalyse, specifik drejning, UV-spektre, IE-spektre, massespektre m.m. er i god overensstemmelse med de data for maytanacin, maytansinolpropionat og maytansinol, der er givet i litteraturen, Eupchan et al. (The Journal of American Chemical Society 9Z, 5294- (1975)).The above data for elemental analysis, specific rotation, UV spectra, IE spectra, mass spectra and more. are in good agreement with the data for maytanacin, maytansinol propionate and maytansinol given in the literature, Eupchan et al. (The Journal of the American Chemical Society 9Z, 5294- (1975)).

De følgende eksempler skal yderligere belyse opfindelsen. Dele er vægtdele, medmindre andet er angivet, forholdet mellem dele og volumendele svarer til forholdet mellem g og ml, og % er baseret på vægt/volumen, medmindre andet er angivet.The following examples will further illustrate the invention. Parts are parts by weight, unless otherwise stated, the ratio of parts to volume parts is the ratio of g to ml, and% is based on weight / volume unless otherwise stated.

Eksempel 1.Example 1.

A.A.

Maytansinol-, maytanacin- og maytansinolpropionat-producerende No-cardia nr. C-15003 (IFO 13726; FEEM 3992; ATCC 31281) dyrket på et medium (gærekstrakt-maltekstrakt-agar) anvendtes til inokulation af en 200 volumendele fermentor indeholdende 40 volumendele af et podningskulturmedium (2% glucose, 3% opløselig stivelse, 1% råt sojabønnemel, 1% majsstøbevand, 0,5% polypepton, 0,3% EaCl, 0,5% CaCO^, pH 7,0). Det inokulerede medium inkuberedes ved 28°C i 48 timer, hvorved der opnåedes et inokulum. 0,5 volumendele af det således opnåede inokulum overførtes til en 200 volumendeles fermentor indeholdende 40 volumendele af et fermentationsmedium sammensat af ·5% dextrin, 3% majsstøbevand, 0,1% polypepton og 0,5% CaCO^ (pH 7,0) og dyrkedes ved 28°C i 90 timer, hvilket gav et inokulum (podningskultur) .Maytansinol, maytanacin and maytansinol propionate producing No-cardia No. C-15003 (IFO 13726; FEEM 3992; ATCC 31281) grown on a medium (yeast extract malt extract agar) was used to inoculate a 200 volume part fermentor containing 40 parts by volume. a seed culture medium (2% glucose, 3% soluble starch, 1% raw soybean meal, 1% corn cast water, 0.5% polypeptone, 0.3% EaCl, 0.5% CaCO 3, pH 7.0). The inoculated medium was incubated at 28 ° C for 48 hours to give an inoculum. 0.5 parts by volume of the inoculum thus obtained was transferred to a 200 parts by volume fermentor containing 40 parts by volume of a fermentation medium composed of · 5% dextrin, 3% maize cast water, 0.1% polypeptone and 0.5% CaCO 2 (pH 7.0) and grown at 28 ° C for 90 hours to give an inoculum (seed culture).

J.5 H3ST0J.5 H3ST0

Ved seriefortyndingsmetoden under anvendelse af Tetrahymena pyrifor-mis W som prøveorganisme og maytansinolpropionat som standardprodukt fandtes den ovenstående kultur at have en titer på 25 ug/ml.By the serial dilution method using Tetrahymena pyriformis W as a test organism and maytansinol propionate as the standard product, the above culture was found to have a titer of 25 µg / ml.

B.B.

10 volumendele af det under A opnåede inokulum (podningskultur) overførtes til en 2.000 volumendeles fermentor indeholdende 500 vo-lumendele af et podningskulturmedium (det samme som ovenfor) og inkuberedes ved 28°C i 48 timer. 5°0 volumendele af den resulterende kultur overførtes til en 50.000 volumendeles rustfri ståltank indeholdende 30.000 volumendele podningskulturmedium og dyrkedes ved 28°C under gennemluftning (30.000 volumendele/minut), omrøring (280 om-10 parts by volume of the inoculum obtained (A) obtained (graft culture) was transferred to a 2,000 volume part fermentor containing 500 volumes of graft culture medium (same as above) and incubated at 28 ° C for 48 hours. 5 ° 0 parts by volume of the resulting culture was transferred to a 50,000 part by volume stainless steel tank containing 30,000 parts by volume of seed culture medium and grown at 28 ° C under aeration (30,000 parts / minute), stirring (280 rpm).

OISLAND

drejninger/minut (1/2 DT)) og indvendigt tryk (1 kg/cm ), hvorved der opnåedes en podningskultur. Denne kultur anvendtes til podning af en 200.000 volumendeles rustfri ståltank indeholdende 100.000 volumendele af et lignende fermentationsmedium, som det i eksempel 1 anvendte i en inokulationsmængde på 10%. Det inokulerede medium inkuberedes ved 28°C under gennemluftning (lOO.OOO volumendele/minut), om- p røring (200 omdrejninger/minut (l/2 DT)) og indvendigt tryk (l kg/cm ) i 90 timer. Bestemt ved samme metode som under A fandtes den ovenfor opnåede kultur at have en titer på 25 yg/ml.revolutions / minute (1/2 DT)) and internal pressure (1 kg / cm) to obtain a seed culture. This culture was used to seed a 200,000 part by volume stainless steel tank containing 100,000 parts by volume of a similar fermentation medium as that used in Example 1 in an inoculation amount of 10%. The inoculated medium was incubated at 28 ° C under aeration (100.OOO volumes / minute), stirred (200 rpm (1/2 DT)) and internal pressure (1 kg / cm) for 90 hours. Determined by the same method as under A, the culture obtained above was found to have a titer of 25 µg / ml.

Til 90.000 volumendele af den ovenfor opnåede kultur sattes 2.000 dele "Hyflo-supercel", og efter grundig omrøring filtreredes blandingen på et trykfilter, hvorved der opnåedes 85.000 volumendele filtrat og 32.000 dele fugtige celler. Filtratet (85.000 volumendele) omrørtes og ekstraheredes med 30.000 volumendele ethylacetat. Denne fremgangsmåde gentoges endnu en gang. Ethylacetatlagene samledes, vaskedes to gange med 30·000 volumendele vand, tørredes ved tilsætning af 500 dele vandfrit natriumsulfat og koncentreredes under reduceret tryk til 200 volumendele. Petroleumsether sattes til koncentratet, og det resulterende bundfald udvandtes ved filtrering (53 dele). Dette råprodukt I omrørtes med 100 volumendele ethylacetat, og de uopløselige bestanddele filtreredes fra. Filtratet omrørtes med 10 dele silicagel (0,05-0,2 mm) og ethylacetatet fjernedes under reduceret tryk. Remanensen anbragtes på en silicagelsøjle (400 volumendele). Eluering udførtes med 500 volumendele hexan, 500 volumendele hexan-ethylacetat (3:1)? 500 volumendele hexan-ethylacetat (1:1), 500 volumendele hexan-ethylacetat (1:3), 500 volumendele ethylacetat 1C 143570 lb og 1000 volumendele ethylacetat-methanol (50:1), og 1000 volumendele ethylacetat-methanol (25:1), idet eluatet opsamledes i 100 volumendeles fraktioner.To 90,000 parts by volume of the culture obtained above, 2,000 parts of "Hyflo supercell" were added and after thorough stirring the mixture was filtered on a pressure filter to give 85,000 parts by volume of filtrate and 32,000 parts of moist cells. The filtrate (85,000 parts by volume) was stirred and extracted with 30,000 parts by volume of ethyl acetate. This procedure was repeated again. The ethyl acetate layers were collected, washed twice with 30 · 000 parts by volume of water, dried by the addition of 500 parts of anhydrous sodium sulfate and concentrated under reduced pressure to 200 parts by volume. Petroleum ether was added to the concentrate and the resulting precipitate was diluted by filtration (53 parts). This crude product I was stirred with 100 parts by volume of ethyl acetate and the insoluble ingredients were filtered off. The filtrate was stirred with 10 parts of silica gel (0.05-0.2 mm) and the ethyl acetate removed under reduced pressure. The residue was placed on a silica gel column (400 parts by volume). Elution was carried out with 500 parts by volume of hexane, 500 parts by volume of hexane-ethyl acetate (3: 1). 500 parts by volume of hexane-ethyl acetate (1: 1), 500 parts by volume of hexane-ethyl acetate (1: 3), 500 parts by volume of ethyl acetate 1C 143570 lb and 1000 parts by volume of ethyl acetate-methanol (50: 1), and 1000 parts by volume of ethyl acetate-methanol (25: 1) ), collecting the eluate in 100 volume fraction fractions.

1 volumendel af hver fraktion koncentreredes til tørhed, og 0,1 volumendel ethylacetat sattes til koncentratet. Blandingen anbragtes i pletter 2,5 cm fra den nederste kant af en silieagel-glasplade (60 ^254’ °>25 mm, 20 x 20) og elueredes ca. 17 cm med et opløsningsmiddelsystem af ethylacetat-methanol (19:1). Efter eluering gennemførtes påvisning med ultraviolet lys (2557 Å).One part by volume of each fraction was concentrated to dryness and 0.1 part by volume of ethyl acetate was added to the concentrate. The mixture was placed in spots 2.5 cm from the bottom edge of a silica gel glass plate (60 254 °> 25 mm, 20 x 20) and eluted approximately 17 cm with a solvent system of ethyl acetate-methanol (19: 1). After elution, ultraviolet light detection (2557 Å) was performed.

De aktive fraktioner nr. 25-30 med Rf 0,58-0,63 og fraktionerne nr. 58-40 med Rf 0,25-0,30 opsamledes og koncentreredes under reduceret tryk til hver ca. 20 volumendele. Til hvert af disse koncentrater sattes 150 volumendele petroleumether, hvorved der opnåedes 5 dele af et råprodukt II og 2 dele råt maytansinol.Active fractions Nos. 25-30 with Rf 0.58-0.63 and fractions Nos. 58-40 with Rf 0.25-0.30 were collected and concentrated under reduced pressure to about 20 volumes. To each of these concentrates was added 150 parts by volume of petroleum ether to give 5 parts of a crude product II and 2 parts of raw maytansinol.

I 10 volumendele ethylacetat opløstes 0,5 del af det ovenfor opnåede råprodukt II, og opløsningen omrørtes omhyggeligt med 4 dele si-licagel (0,05-0,2 mm). Ethylacetatet fjernedes under reduceret tryk. Remanensen anbragtes på en søjl-e af 300 volumendele silicagel, og søjlen vaskedes først med 500 volumendele chloroform og elueredes derefter med 500 volumendele chloroform-methanol (50:1), 500 volumendele chloroform-methanol (20:1) og 500 volumendele chloroform-methanol (10:1). Eluatet opsamledes i 25 volumendeles fraktioner.In 10 parts by volume of ethyl acetate, 0.5 part of the crude product II obtained above was dissolved and the solution was stirred carefully with 4 parts silica gel (0.05-0.2 mm). The ethyl acetate was removed under reduced pressure. The residue was placed on a column of 300 parts by volume of silica gel, and the column was first washed with 500 parts by volume of chloroform and then eluted with 500 parts by volume of chloroform-methanol (50: 1), 500 parts by volume of chloroform-methanol (20: 1) and 500 by volume. methanol (10: 1). The eluate was collected in 25 volume fractions.

0,5 volumendele af hver fraktion koncentreredes under reduceret tryk. Til koncentratet sattes 0,05 volumendel ethylacetat, og blandingen underkastedes tyndtlagskromatografi (elueringssystem: chloroform-methanol = 9:1), hvorved der opnåedes en prøveopløsning.0.5 parts by volume of each fraction was concentrated under reduced pressure. To the concentrate was added 0.05 volume portion of ethyl acetate and the mixture was subjected to thin layer chromatography (elution system: chloroform-methanol = 9: 1) to give a sample solution.

Fraktionerne nr. 40 og 41, der absorberede ved 2537 Å i Rf-området 0,48-0,50, opsamledes og koncentreredes til tørhed under reduceret tryk. Til remanensen sattes 0,5 volumendel ethylacetat, og blandingen henstod, hvorved der opnåedes 0,05 del blandede krystaller af maytanacin og maytansinolpropionat.Fractions Nos. 40 and 41, absorbed at 2537 Å in the Rf range 0.48-0.50, were collected and concentrated to dryness under reduced pressure. To the residue was added 0.5 part by volume of ethyl acetate and the mixture was allowed to give 0.05 part of mixed crystals of maytanacin and maytansinol propionate.

0,05 del af de ovennævnte blandede krystaller af maytanacin og maytansinolpropionat opløstes i 5 volumendele methanol, hvorefter der tilsattes 0,1 del natriumchlorid og 5 volumendele vand. En søjle pakkedes med 200 volumendele "Diaion KP-10" og vaskedes med 600 volumen- 17 143570 dele 50%'s methanol-vand indeholdende 5% NaCl. Den ovenfor fremstillede prøveopløsning sendtes gennem søjlen, og der udførtes gradient-eluering under anvendelse af 1500 volumendele 60%'s methanol-vand indeholdende 5% NaCl og 1500 volumendele 95%'s methanol-vand. Elua-tet opsamledes i 15 volumendeles fraktioner, og hver fraktion undersøgtes ved tyndtlagskromatografi. Fraktionerne 130 til 135 indeholdt maytanacin, og fraktionerne 138 til 142 indeholdt maytansinol-propionat.0.05 part of the above-mentioned mixed crystals of maytanacin and maytansinol propionate were dissolved in 5 parts by volume of methanol, after which 0.1 part of sodium chloride and 5 parts by volume of water were added. A column was packed with 200 parts by volume "Diaion KP-10" and washed with 600 volumes by volume of 50% methanol-water containing 5% NaCl. The above prepared sample solution was passed through the column and gradient elution was performed using 1500 parts by volume of 60% methanol-water containing 5% NaCl and 1500 parts by volume of 95% methanol-water. The eluent was collected in 15 volume fractions and each fraction was examined by thin layer chromatography. Fractions 130 to 135 contained maytanacin and fractions 138 to 142 contained maytansinol propionate.

Hver gruppe fraktioner koncentreredes og opløstes ved tilsætning af 30 ml vand og 50 ml ethylacetat. Opløsningen omrystedes i en skilletragt, og det vandige lag skiltes fra, og efter to ganges vask med 30 ml portioner vand tørredes ethylacetatlaget over vandfrit natriumsulfat, koncentreredes og henstod derefter. På ovennævnte måde opnåedes krystaller fra hver gruppe fraktioner. Krystallerne opsamledes ved filtrering og tørredes. Maytanacin 0,013 del. Maytansinol-propionat 0,025 del.Each group of fractions was concentrated and dissolved by adding 30 ml of water and 50 ml of ethyl acetate. The solution was shaken in a separatory funnel and the aqueous layer was separated and, after washing twice with 30 ml portions of water, the ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated and then allowed to stand. In the above manner, crystals were obtained from each group of fractions. The crystals were collected by filtration and dried. Maytanacin 0.013 Part. Maytansinol propionate 0.025 part.

I 3 volumendele ethylacetat opløstes 0,2 del af det ovenfor opnåede rå maytansinol, og opløsningen omrørtes godt med 0,5 del silicagel (0,05-0,2 mm). Ethylacetatet fjernedes under reduceret tryk. Remanensen anbragtes på en søjle af 80 volumendele silicagel, og søjlen vaskedes først med 150 volumendele chloroform og elueredes derefter med 150 volumendele chloroform-methanol (50:1), 150 volumendele chloroform-methanol (20:1) og 3volumendele chloroform-methanol (10:1). Eluatet opsamledes i 10 volumendeles fraktioner.In 3 parts by volume of ethyl acetate, 0.2 parts of the crude maytansinol obtained above was dissolved and the solution was well stirred with 0.5 part silica gel (0.05-0.2 mm). The ethyl acetate was removed under reduced pressure. The residue was placed on a column of 80 parts by volume of silica gel, and the column was first washed with 150 parts by volume of chloroform and then eluted with 150 parts by volume of chloroform-methanol (50: 1), 150 parts by volume of chloroform-methanol (20: 1) and 3 volumes of chloroform-methanol (10). : 1). The eluate was collected in 10 volume fractions.

0,5 volumendel af hver fraktion koncentreredes under reduceret tryk. Til koncentratet sattes 0,05 volumendel ethylacetat, og blandingen underkastedes tyndtlagskromatografi (elueringssystem: chloroform-methanol = 9:1).0.5 part by volume of each fraction was concentrated under reduced pressure. To the concentrate was added 0.05 volume portion of ethyl acetate and subjected to thin layer chromatography (elution system: chloroform-methanol = 9: 1).

Fraktionerne nr. 50 til 52, der absorberede ved 2537 Å i Rf-området 0,33 til 0,38, opsamledes og koncentreredes til tørhed under reduceret tryk. Til remanensen sattes 0,5 volumendel ethylacetat,og blandingen henstod, hvorefter der opnåedes 0,020 del krystaller af maytansinol.Fractions Nos. 50 to 52, absorbed at 2537 Å in the Rf range 0.33 to 0.38, were collected and concentrated to dryness under reduced pressure. To the residue was added 0.5 volume portion of ethyl acetate and the mixture was allowed to afford 0.020 part crystals of maytansinol.

C.C.

32.000 dele af de under B opnåede celler ekstraheredes under om- 18 «3570 røring med 50*000 volumendele 70%'s acetone-vand i 3 timer og filtreredes derefter på et trykfilter. Ekstraktionen med 50.000 volumendele 70%'s acetone-vand og den efterfølgende filtrering gentoges endnu en gang. Filtraterne samledes, og acetonet fjernedes ved koncentration under reduceret tryk. Det resulterende vandige system sendtes gennem en søjle af 5*000 volumendele "Diaion BP-10". Søj'len vaskedes med 20.000 volumendele vand og 50%'s vandig methanol, hvorefter der elueredes med 15*000 volumendele 90%'s methanol-vand. Eluatet koncentreredes under reduceret tryk til 3*000 volumendele og omrystedes med 3,000 volumendele vand og 3*000 volumendele ethylacetat. Denne fremgangsmåde gentoges endnu en gang. Ethylacetatlagene samledes, vaskedes med vand, tørredes ved tilsætning af vandfrit natriumsulfat og koncentreredes under reduceret tryk til 200 volumendele. Efter tilsætning af petroleumsether udvandtes bundfaldet ved filtrering (280 dele). Det ovenfor opnåede råprodukt I rensedes ved hj'ælp af en si-licagelsøj'le på lignende måde som under A, hvorved der opnåedes 1,0 del af råproduktet II og 0,5 del råt maytansinol.32,000 parts of the cells obtained under B were extracted with stirring with 50 * 000 parts by volume of 70% acetone water for 3 hours and then filtered on a pressure filter. The 50,000 volume extraction 70% acetone water extraction and subsequent filtration was repeated again. The filtrates were collected and the acetone was removed by concentration under reduced pressure. The resulting aqueous system was passed through a column of 5 * 000 volume parts "Diaion BP-10". The column was washed with 20,000 parts by volume of water and 50% aqueous methanol, then eluted with 15 * 000 parts by volume of 90% methanol-water. The eluate was concentrated under reduced pressure to 3 * 000 parts by volume and shaken with 3,000 parts by volume of water and 3 * 000 parts by volume of ethyl acetate. This procedure was repeated again. The ethyl acetate layers were collected, washed with water, dried by adding anhydrous sodium sulfate and concentrated under reduced pressure to 200 parts by volume. After addition of petroleum ether, the precipitate was diluted by filtration (280 parts). The crude product I obtained above was purified using a silica gel column in a similar manner as under A to give 1.0 part of crude product II and 0.5 part of raw maytansinol.

Eksempel 2 1000 volumendele af kulturen fra eksempel IB inokuleredes i en 200.000 volumendeles rustfri ståltank indeholdende 100.000 volumendele af et podningskulturmedium, og det inokulerede medium inkuberedes ved 28°C under gennemluftning (100.000 volumendele/minut) og omrøring (200 om-drejninger/minut) i 48 timer til fremstilling af en podningskultur. Denne podningskultur overførtes til en 2.000.000 volumendeles rustfri ståltank indeholdende 1.000.000 volumendele af det samme fermentationsmedium, som det i eksempel 1A anvendte, i en maaigde på 10%. Dyrkningen gennemførtes ved 28°C under gennemluftning (1,000.000 volumendele minut), omrøring (120 omdrejninger/minut (1/3 Dl)) og indvendigt tryk (l kg/cm ) i 90 timer. Den resulterende kultur fandtes at have en titer på 20 pg/ml, prøvet ved den i eksempel 1A beskrevne prøvningsmetode. Til 9OO.OOO volumendele af den ovenfor opnåede kultur sattes 900.000 volumendele acetone og efter 1 times omrøring 20.000 dele "Hyflo-Supercel". Blandingen omrørtes yderligere og filtreredes på en trykfiltreringsmaskine. Til 1.700.000 volumendele af det resulterende filtrat sattes 500.000 volumendele vand, og blandingen ekstraheredes i en "Podbielniak" med 1.000.000 volumendele ethylacetat. Ethylacetatlaget vaskedes med vand, tørredes ved tilsætning af vandfrit natriumsulfat og koncentreredes under reduceret tryk.Example 2 1000 parts by volume of the culture of Example 1B were inoculated into a 200,000 part by volume stainless steel tank containing 100,000 parts by volume of a seed culture medium and the inoculated medium was incubated at 28 ° C under aeration (100,000 parts by minute / minute) and stirred (200 rpm). for 48 hours to prepare a seed culture. This seed culture was transferred to a 2,000,000 volume part stainless steel tank containing 1,000,000 volume parts of the same fermentation medium used in Example 1A for a maximum of 10%. The cultivation was carried out at 28 ° C under aeration (1,000,000 parts by volume), stirring (120 rpm (1/3 Dl)) and internal pressure (1 kg / cm) for 90 hours. The resulting culture was found to have a titer of 20 µg / ml, tested by the test method described in Example 1A. To 900,000 parts by volume of the culture obtained above were added 900,000 parts by volume of acetone and after 1 hour stirring 20,000 parts of "Hyflo-Supercel". The mixture was further stirred and filtered on a pressure filtration machine. To 1,700,000 parts by volume of the resulting filtrate were added 500,000 volumes of water and the mixture was extracted into a "Podbielniak" with 1,000,000 volumes of ethyl acetate. The ethyl acetate layer was washed with water, dried by the addition of anhydrous sodium sulfate and concentrated under reduced pressure.