IE45888B1 - Method for producing maytansinol and its derivatives - Google Patents

Abstract

Maytansinol, maytanacin and/or maytansinol propionate are prepared by culturing a microorganism belonging to the genus Nocardia, which is able to produce maytansinol, maytanacin and/or maytansinol propionate, with concentration of maytansinol, maytanacin and/or maytansinol propionate, and isolating it from the culture broth. These compounds are useful antitumour agents, i.e. useful cytostatics.

Description

The present invention relates to a method for preparing maytansinol, maytanacine and maytansinol propionate which are aiti-tumor agents.

Xt has been known tha% among the above compounds, maytanacine and maytansinol propionate have a strong anti-tumor activity [Kupehan et al.; Journal of the American Chemical Society 97. 529¾ (1975)]. Cu the other hand, maytansinol itself has only a weak anti-tumor activity (see the above reference) but it is a useful intermediate for the ready preparation of maytanacine and maytansinol propionate and various other derivatives.

Maytansinol and maytanacine have been obtained by the above Kupehan et al. from the hark of Putterlickia verrucosa (a plant belonging to the genus Maytenus)and thus the yield is extremely low, such as 0.025 mg of the former compound and 0.36 mg of the latter from 1 kg of the dried hark of the plant. As regards maytansinol propionate, it has been obtained by the chenioal propionation of maytansinol^ . We have collected various soil and other samples and investigated antibiotics produced hy microorganisms I which are isolated from these samples . As a result we have found that some of the microorganisms thus isolated can accumulate maytansinol, maytanacine or maytansinol propionate in the culture medium, these microorganisms belonging to the genus Nocardia, ami that these compounds can also be obtained by cultivating mutants derived from these microorganisms in a proper nutrient medium under appropriate conditions.

The present invention is based on these findings and our further studies.

Thus, the present invention relates toamethod for preparing maytansinol, maytanacine or maytansinol propionate, which comprises cultivating in a suitable medium a microorganism winch belongs to the genus Nocardia and is capable of producing maytansinol, maytanacine or maytansinol propionate, to accumulate maytansinol, maytanacine or maytansinol propionate in the culture broth; and recovering the accumulated maytansinol, maytanacine or maytansinol propionate.

According to the prior art, the above compounds are obtainable from plants, but the plants are limited to specific plants and great expense and a long period of time is required for the production ot each stage of growth, felling, drying and pulverizing of the plants and extraction, separation and purification. Further, the yield is extremely low.

On the contrast, the processes of the present invention can be conducted easily and smoothly hy cultivation of the microorganism, and a large amount Λ5888 produced and of the desired compounds can be/obtained when desired.

The present invention is the first example of obtaining these compounds as metabolites of microorganisms, and constitutes an acceptable method for the preparation thereof.

As an example of the microorganism usable in the present method, there may be mentioned an actinomycetes strain No. C-15005 which we isolated from soil and other samples on our screening for antibiotic-producing microorganisms.

The microbiological characteristics of Strain No. C-15003 were investigated by procedures analogous to those proposed by Schirling & Gottlieb [international Journal of Systematic Bacteriology 16, 313-340 (1966)]. The results of our observations at 28°C over 21 days are as follows. l) Morphological character The vegetative mycelium extends well and develops into branches, both on agar and in liquid media. Many of the hypae measure 0.8· to 1.2 Jim in diameter and, in certain instances, may divide into fragments resembling rod bacteria or branched short lengths of hypae. The strain gives good growth on various taxonomieal media, with an aerial mycelium being superimposed on the vegetative mycelium, although it frequently forms eoremia-like bodies 5888 (50-200 x 200 - lOOOum) on which further aerial growth takes place. Many of the aerial mycelia are sinuous or straight, a loosely spiral-like configuration being encountered on a few occasions.Microscopic examination of aged cultures reveals that only in a few eases do conidia-like cells occur in chains, while the cell suspensions obtained from the surfaces of such cultures, as microscopically examined, contain many elongated ellipsoidal (0.8-1.2 pm x 4,8-6.8 pm) and ellipsoidal (0,8-1.2 x 1.0-2.0 pm) bodies resembling arthrospores.

Electron-microscopic examination showed that these bodies had smooth surfaces. 2) The constitut.ents of cells The strain was shake-cultured in a modified ISP 15 No. 1 medium at 28°C for 66 to 90 hours, at the end of which time the cells were collected and rinsed. By the method of B. Becker et al. [Applied Microbiology 12, 421 (1964)] and tho method of M.P. Leehevalier.

[Journal of Laboratory and Clinical Medicine 71. 93¾ (1968)], the above whole cells were examined for diaminopimelic acid and sugar composition. The former was found to be of the meso-form, while spots were detected which corresponded to galactose and arabinose. 3) Characteristics on taxonomical media The strain showed comparatively good growth on 43888 various media, with the vegetative mycelium being colourless to pale yellow in the initial phases of culture to yellowish tan and light yellowish tan/in the later phases. The strain produces soluble pigments, yellow to yellowish tan, in various taxonomical media. The aerial mycelium is powdery and generally gives moderate growth, being white to yellow or light yellowish tan. The characteristics of the strain in various taxonomical media are set forth in Table 1 just below as follows: Table 1 Cultural characteristics of of Strain No. C-15OO3 on a taxonomical media (A) Sucrose nitrate agar: Growth (G): Luxuriant, Bright Melon Yellow (3 ia)* to Amber (3 le)*, ooremia-lilce bodies formed Aerial mycelium (AM): Scant, white Soluble pigment (SP): None or pale yellowish tan (B) Glycerol nitrate agar: G : Moderate, Light Ivory (2 ca)*, coremia-lilce bodies formed AM : Moderate, white SP : None (C) Glucose asparagine agar: G ; Moderate, Bright Marigold (3 pa)* to Bright Yellow (2 pa)*.

AM : Scant, white SP : Bright Yellow (2 pa)* «»888 (D) Glycerol asparagine agar: G : Moderate, Light Ivory (2 ca)*, coremia-like bodies formed AM : Scant, white 5 SP : None (E) Starch agar: G : Moderate, Light Ivory (2 ca)* to Light Wheat (2 ea)*, coremia-like bodies formed AM : Abundant, Light Ivory (2 ca)* SP : None (F) Nutrient agar: G : Moderate, Light Ivory (2 ca)* to Colonial Yellow (2 ga)*, coremia-like bodies formed AM : Scant, white 15 SP : None (G) Calcium malate agar: G : Moderate, Light Ivory (2 ca)* to Light Wheat (2ea)*, coremia-like bodies formed.

AM : Moderate, white to Light Ivory (2 ca)* SP : None (H) Yeast extract-malt extract agar: G : Moderate, Amber (5 lc)* to Bright Yellow (3 la)*, coremia-like bodies formed AM : Moderate, white to Light Ivory (2 ca)* SP : None (i) Oatmeal agar: G : Moderate, Light Ivory (2 ca)* to Colonial Yellow (2 ga)*, coremia-like bodies formed AM : Scant, white to light yellow SP : None (J) Peptone yeast extract iron agar: G : Moderate, Colonial Yellow (2 ga)* AM : None SP : Colonial Yellow (2 ga)* (K) Tyrosine agar: G : Moderate, Light Ivory (2 ca)* to Light Melon Yellow (5 ea)*, coremia-like bodies formed AM : Moderate, white to Light Ivory (2 ea)* SP : Camel (3 ie)* * The colour codes according to the Colour Harmony Manual, 4th ed. (Container Corporation of America, 1958) 4) Physiological character The physiological characters of the strain are shown in Table 2. Temperature range for growth: 12°C to 38°C. The temperature range in which good aerial growth occurs on agar (ISP No. 2) is 20 to 35°θ· Table 2 The physiological characteristics of Strain No.C-15003 Temperature range for growth: 12 to 38°C Temperature range for aerial growth: 20 to 35° C Liquefaction of gelatin: Positive Hydrolysis of starch: Positive Reduction of nitrates: Positive Peptonization of milk: Positive Coagulation of milk: Negative Decomposition of casein: Positive Production of melanoid pigments: Negative (peptone yeast extract iron agar), positive (tyrosine agar) Decomposition of tyrosine: Positive Decomposition of xanthine: Negative Decomposition of hypozanthine: Negative Tolerance to lysozyme: Positive Tolerance to sodium chloride: 2% ) Utilization of various carbon sources The utilization of various carbon sources was investigated using a medium described in Pridham and Gottlieb [Journal oi' Bacteriology 56. 107 (1948)] and a basal medium of the same composition plus 0.1% oi' yeast extract. The resulting spectrum is shown in Table 3.

Table 3 The utilization of carbon sources by Strain No. C-15003 Source of earbon Growth Source mf carbon Growth D-Xylose + +)· * Raffinose + ±* L-Arabinose + + Melibiose + + D-Glucose ++ ++ i-Inositol D-Galactose + + D-Sorbitol D-Fructose +H- +l· D-Mannitol +( ++ L-Bhamnose + + Glycerol - + D-Mannose +++ ++ Soluble starch + + Sucrose ++ ++ Control Lactose - .* Maltose ± + Trehalose + ++ *Basal medium with 0.1$6 yeast extract added Note: ++: Luxuriant growth 4+: Good growth +: Growth +: Poor growth -: No growth 6) Other eharacteristics The cells were harvested hy the procedure previously described at (2) above and DNA was prepared by a procedure analogous to that of J. Marmur et al. [Journal of Molecular Biology, ^,208, 1961]. The G-C(GuanineCytosine) content of the DNA was found to be about 71 mole $, Gram-staining of the vegetative mycelium of this strain was positive.

The above characteristics of Strain No. C-15OO3 43888 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 of Determinative Bacteriology, 8th ed, 1974; and similar literature references.

Whilst this strain was thought to belong to Group 111 of the genus Nocardia, the failure to find any species having the characteristics so far described among the known strains' led us to conclude that this strain represented a novel species of microorganism.

The present Strain No. C-15OO3 has been deposited at the Fermentation Research institute, Agency of Industrial Science and Technology (FERM) under tho receipt number of 3992, at the Institute for Fermentation, Osaka, Japan (IFO) under the accession number of IFO 13726 and at the American Typo Culture Collection (ATCC), Maryland, U.S.A. under Lhe accession number of 312S1.

While Strain No. C-I5003 is a novel species of the genus Nocardia as just mentioned, it is liable , as are microorganisms generally, to undergo variations and mutations, whether spontaneously or under the influence of a mutagen. For example, the many variants of the strain which are obtainable for example by irradiation with X-rays, gamma rays or ultraviolet light, by monocell isolation, by culture on media containing various chemicals, or by any 43888 other mutagenic treatment, as well as the mutants spontaneously derived from the strain, should not in general he considered to represent any other distinct species hut, rather, any of such variants and mutants capable of elaborating maytansinol, maytanacine and maytansinol propionate may he invariably utilized for the purposes of this invention as the strain No. C-15OO3.

Thus, for example, subjecting Strain No. C-15OO3 to various mutagenic treatments yields mutants substantially lacking the ability to produce soluble pigments, mutants with sushtrate mycelia which are colourless, yellowish green, reddish tan or orange red, mutants whose hyphae are ready to fragment into bacillary elements or branched short hyphal fragments, and mutants with abundant white aerial mycelia or substantially without aerial mycelia.

The medium employed for the cultivation of a strain capable of producing maytansinol, maytanacine or maytansinol propionate (hereinafter, sometimes abbreviated as a producable strain) may be either a liquid or a solid medium provided it contains nutrients whioh the strain may utilize, although a liquid medium is preferred for high-production runs. The medium may comprise carbon and nitrogen sources which Strains No, C-15OO3 may assimilate and digest, inorganic matter, trace nutrients, etc. As examples of said carbon sources there may he mentioned glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol or sorbitol, fats or oils (e.g. soybean oil,lard oil or chicken oil ). The nitrogen sources may be for example meat extract, yeast extract, dried yeast, soybean meal, corn steep liquor, peptone, casein, cottonseed flour, spent molasses, urea or ammonium salts (e.g. ammonium sulphate, ammonium chloride, ammonium nitrate or ammonium acetate) and so on.

The medium may further contain for example salts of sodium, potassium, calcium or magnesium , salts of iron, manganese, zinc, cobalt or nickel, salts of phosphoric acid or boric acid and organic acid salts such as acetates or propionates. Further, the medium may contain, for example as additives various amino acids (e.g. glutamic acid, aspartic acid, alanine, glycine, lysine, methionine or proline), peptides (e.g. dipeptidcs or tripeptides), vitamins (e.g. Β^, B,,, nicotinic acid, B^o, C or E) or nucleic acids (e.g. purine, pyrimidine or derivatives thereof. For the purpose of adjusting the pll of the medium, there may be added for example an inorganic or organic acid, alkali or buffer.

Suitable amounts of oils, fats or surfactants, for example may also be added as antifoaming agents.

The cultivation may be conducted under stationary shaking submerged aerobic or other suitable cultural conditions. For high production runs, submerged aerobic culture is preferred. While the conditions of culture depend upon the condition and composition of the medium, the strain, the cultural method and other factors, it is normally preferred to carry out incubation at 20 to 35 °C with an initial pH of substantially 7.0. A temperature of from 23 to 30°C is particularly desirable in an intermediate stage of cultivation, with an initial pH of 6.5 to 7.5. While the incubation time is also variable according to the same factors as mentioned above, it is advisable to continue the incubation until the titer of the desired antibiotic product roaches a maximum. In the case of shaking culture or submerged aerobic culture in a liquid medium, the time required normally ranges from about 48 to 144 hours.

The potency of the antibiotic was assayed with Tetrahymena pyriformis W as an assay organism. Thus, the above microorganism was grown on a test medium [20 g of Proteosepeptone (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 for from 4'4 to 48 hours and the potency of the antibiotic was determined by the serial dilution method with a monitoring of the turbidity of growth and by a thin-layer chromatographic (briefly, TIC) assay to be described hereinafter.

Maytanacine, maytansinol propionate or maytansinol is produced and accumulated in the resulting cultured broth, both ex l.raceJ lulariy und inlraoeIlularJy, None ol these substances shows distinct antibiotic activity, and thus they have been detected by 'HjC which is set forth in parallel to detection by activity to the Tetrahyniena strain. Thus, the fermentation broth is separated into cells and filtrate by filtration or centrifuging and the filtrate is extracted with the same volume of ethyl acetate. The same amount of 70$ acetonewater is added to the cells as the filtrate and, after an hour's 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 coiucntratcd to l/iOO hy volume and subjected to thin-layer chromatography on a silica gel-glass plate (Merck, West Germany Kieselgel 60 F 0.25 mm, x 20) (solvent system: chloroform-methanol = 9:1), The potency was determined on the basis of the Intensity of spots detected by irradiation with ultraviolet light at 2557 ’.A.

Maytanacine, maytansinol propionate and maytansinol which are thus produced in the culture broth are lipophyllc and neutral, and they can he conveniently recovered by the separation and purification procedures which are normally employed for the harvesting of such microbial metabolites. For example, there may he employed a procedure which utilizes the difference in solubility between the antibiotic and an impurity, means which utilize the fractionating adsorptive affinity of various adsorbents such as activated carbon, macroporous non-ionic resins, silica gel or alumina, a procedure of removing the impurities by means of ion-exchange resins, and so on, as applied singly or in a suitable combination or as applied in repetition.

Since, as aforesaid, maytanaeine, maytansinol propionate or maythnsinol occurs in both the filtrate and cells, the antibiotics are separated and purfied by means of such an adsorbent, if one is employed, either directly or after a solvent extraction in the ease of the filtrate, or after a solvent extraction in the case of microbial cells. The solvent extraction may be performed by any of the following and other methods e.g. (l) solvent extraction from the culture broth prior to separation of cells or (2) solvent extraction of the cells and the filtrate obtained by filtration, centrifuging or a like process.

To extract the filtrate and cells independently, the following procedure may be advantageously used.

The solvents suitable for the extraction of the filtrate are water-immiscible organic solvents such as fatty acid esters, e.g. ethyl acetate or amyl acetate; alcohols, e.g. butanol; halogenated hydrocarbons, 45838 e.g. chloroform; or ketones, e.g. methyl isobutyl ketone.

The extraction is generally carried out at a pH near neutral and preferably, the culture fluid previously adjusted to pH 7 is extracted with ethyl acetate. The extract is washed with ether and concentrated under reduced pressure. Then, 'a non-polar solvent such as petroleum, ether or hexane is added to the concentrate and the crude product (f) containing the active compound is recovered. Because, on TLC, a number of spots are detected other than maytanacine, maytansinol .propionate or maytansinol, the product (I) is sequentially subjected to the following purification procedures. Thus, a routine purification procedure, particularly adsorption chromatography is useful and, for this purpose , one of the common adsorbents such as silica gel, alumina or a maeroporoas non-ionic adsorbent resin, may be employed. For purification from the crude product (l), silica gel is most useful. Development may be carried out, for example, starting with petroleum ether or hexane, and elution is performed by the addition of a polar solvent sueli as ethyl aeetate, acetone, ethanol or methanol. In a typical process, using silica gel (Merck, West Germany, 0.05 - 0.2 mm) as a carrier, column chromatography is carried out with a serial increase in the hexane to ethyl acetate ratio. The eluate is sampled and investigated by TW and the fractions containing effective ingredients are pooled and concentrated under reduced pressure, Then, petroleum ether or hexane is added to the concentrate, whereby the crude product (ll) is obtained. Since this product still contains impurities, it is further purified as follows. For example, the product (ll) may be purified by means of a second silica gel column using a different solvent system, The developing system for this purpose may consist in 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 methyl ethyl ketone, or the like. In this way, maytanaoine, maytansinol propionate or maytansinol is isolated. The order of solvent systems for the first and second silica gel columns may he reversed and, in addition, ordinary organic solvents may be used in conjunction with the above systems if necessary.

Where a macroporous adsorbent resin is used as a purification means for the crude product (II) elution of maytanaoine, maytansinol propionate or maytansinol is accomplished with a mixture of water with a lower alcohol, a lower ketone or an ester. The lower alcohol may for example he methanol, ethanol, propanol or butanol and the lower ketone may for example he acetone or methyl ethyl ketone. The ester may for example he ethyl acetate.

In a typical procedure, the crude product (II) is dissolved in 600 methanol-water and adsorbed on a column of Diaion (Trade Mark) HP-10 (Mitsubishi Kasei K.K.). The column is washed with 700 mel.hanol-water and, then, elution is carried out with 900 methanoi-water. In this way, maytanacine, maytansinol propionate or maytansinol is eluted from the column.

In either of the processes described above, the fractions containing the defined components aro pooled and concentrated under reduced pressure. To the dry product are added 5 to 8 volumes of ethyl acetate and the mixture is allowed to stand, whereupon crystals of maytanacine, maytansinol propionate or maytansinol separate, respectively, when tlie crystals contain maytanacine and maytansinol propionate; they are then separated from each other by means of an adsorbent such as those mentioned hereinbefore. Thus, using silica gel or a macroporous non-ionic adsorbent resin and the above solvents, the desired compounds may be fractionally eluted. When, for example, silica gel is employed, development is carried out with hexane, ethyl acetate, or eblorol'orm-methanol, whereby maytansinol propionate and maytanacine emerge in that order. After detection by TLC, the fractions arc respectively concentrated under reduced pressure and ethyl acetate is added to the concentrates, in this manner, the respective compounds can bo obtained 5888 as crystals. When a macroporous non-ionic adsorbent resin is employed, gradient elution with a varying ratio of alcohol, ketone or ester to water may he utilized. 60$ methanol-water and 9555 methanol-water, with 555 sodium chloride added, maytanaeine and maytansinol propionate emerge In the order mentioned. After sampling and detection by TLC, each group of active fractions is concentrated under reduced pressure and crystallized from ethyl acetate. The isolated crystals include ethyl acetate as a solvent of crystallization and, after drying over phosphorus pentoxide at 70°C for 8 hours, show the following physical and .chemical properties (Table 4), Table 4 Maytanaeine Maytansinol propionate ^31^41^^2^9 Maytansinol α23Η3701Ν2Ο8C30 h39cin2o9 Melting point(°C) 235-236° 188-190° 172.5-174° Specific [a]22 (c=o °-121°±10° [a]22°„i27°±10° (C=O.35,CHC13) [α]β2°-313ο±10ο rotation .25,CHCl (0=0.22,CHCIg) c 59.62 59.93 59.28 Analysis Found if 6.93 6.82 6.38 (55) N 4.28 4.32 5.02 Cl 5.74 5.57 6.15 Analysis c 59-85 59 -9'4 59.52 Calcd. H 6.48 6.65 6.60 (SS) N 4.61 4.51 4.96 Cl 5.84 5.71 6.27 Maytanacine ^30^39^^2^9 Maytansinol propionate C31H4lC1N2°9 Maytansinol C28H37C1N2°8 Ultraviolet absorption spectrum nm(e) 233(30330) 240(sh.28240) 252(27850) 280(5680) 288(5660) 233(30240) 240(sh.28400) 252(27650) 280(5740) 288(5710), 232(32750) 244(sh.30850) , 252(31650) 281(5750) 288(5700) Infrared absorption spectrum (cm-ϊ) 1740,1730,1670 .1580 1740,1730,1670, 1580 1715,1670,1580 Mas& spectrum (m/e) 545,485,470,450 559,485,470,450 503,485,470, 450 Acid,neutral or · . basic lipophyl and neutral substance lipophyl and neutral substance lipophyl and neutral substance Color reactions Dragendorff reaction: positive Beilstein reaction: positive Dragendorff reaction: positive Beilstein reaction: positive Dragendorff reaction: positive Beilstein reaction: positive The above-mentioned, data of elemental analysis, specific rotation, UV spectra, III spectra and mass spectra, are in good agreement with the data, of maytanacine, maytansinol propionate and maytansinol which are given in the lite.raLurc (sec e.g. Kupchun ot ai, The Journal of the Ameiican Chemical Society 97, 5294(1975)).

The following examples are further illustrative but by no means limitative of the invention, wherein part(s) is (or are) based on weight unless otherwise specified and the relationship between part(s) and part(s) by volume corresponds to that between gram(s) and milliliter(s), and is based on weight/volume unless otherwise specified.

Example 1 Maytansinol-, maytanacine- and maytansinol propionate-producible Nocardia No. C-15OO3 (IFO 13726; FERM 3992; ATCC 31281) as grown on a medium (yeast extractmalt extract agar) was used to inoeulate a 200 parts by volume fermenter containing AO parts by volume of a seed culture medium (2$ glucose, 3$ soluble starch, 1$ raw soybean meal, 1$ corn steep liquor, 0.5$ Polypepton, 0.3$ NaCl and 0.5$ CaCO^, pH 7.0). The inoculated medium was incubated at 28°C for 48 hours to obtain an inoculum.

A 0.5 part hy volume portion of the inoculum thus obtained was transferred to a 200 parts hy volume fermenter containing 40 parts hy volume of a fermentation medium composed of 5$ dextrin, 3$ corn steep liquor, 0.1$ Polypepton and 0.5$ CaCO^ (pH 7.0), and cultivated at 28°C for 90 hours to give an inoculum (seed culture).

As determined hy the serial dilution method using Tetrahyniena pyriformis W as an assay organism and maytansinol propionate as the standard product, the above culture was found to have a titer of 25 ug/ml.

Example 2 A 10 parts hy volume portion of the inoculum (seed) obtained in Example 1 was transferred to a 2000 parts by volume fermenter containing 500 parts by volume of a seed culture medium (same as above), and incubated at 28°C for' 48 hours. A 500 parts hy volume portion of the resulting culture was transferred to a 50,000 parts by volume tank of stainless steel containing 30,000 parts by volume of a seed culture medium and cultivated at 28°C under aeration (30,000 parts by volume/min.), agitation [280 r.p.m. (1/2DT)] and internal pressure (l kg/cm ) to yield a seed culture. This culture was used to seed a 200,000 parts hy volume tank of stainless steel containing 100,000 parts hy volume of a fermentation medium similar to that used in Example 1 at an inoculation rate of 10$. The inoculated medium was incubated at 28°C under aeration (100,000 parts by volume/min.), agitation [200 r.p.m. (l/2 DT) ] and internal pressure (lkg/em2) for 90 hours.

As determined by the same procedure as that described in Example 1, the culture obtained above was found to have a titer of 25 ug/ml.

To 90,000 parts by volume of the culture obtained above were added 2000 parts of Ilyflo-Supercel (Trade Mark, Johns-Manville Products, U.S.A.) and, after thorough mixing, the mixture was filtered on a pressure filter to obtain 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 hy volume of ethyl acetate. This procedure was repeated onee again. The ethyl acetate layers were pooled, washed twice with 30,000 parts by volume portions of water, dried by the addition of 500 parts of anhydrous sodium sulphate and concentrated under reduced pressure to 200 parts byvolume. Petroleum ether was added to the concentrate and the resulting precipitate was recovered by filtration (53 parts). This crude product (I) was stirred with 100 parts by volume of ethyl acetate and. the insolubles were filtered off. The filtrate was stirred with 10 parts of silica gel (Merck, West Germany, 0.05-0.2 mm) and the ethyl acetate was removed under reduced pressure. The residue was applied to the top of a silica gel column (400 parts by volume). Elution was carried out with 500 parts by volume of hexane, 500 parts by volume of hexaneethyl acetate (3:1), 500 parts by volume of hexane-ethyl acetate (l:l), 500 parts by volume of hexane-ethyl acetate (1:3), 500 parts by volume of ethyl acetate and 1000 parts by volume of ethyl acetate-methanol (50:l), and 1000 parts by volume of ethyl acetate-methanol (25:1), with the eluate being collected in 100 parts by volume fractions.

A one part by volume portion of each fraction was concentrated to dryness, and 0.1 part by volume of ethyl acetate was added to the concentrate. The mixture was spotted at 2.5 cm from the bottom edge of a silica gel-glass plate (Merck, West German, 60 F254’ 0>25 mm> x 20) and developed for about 17 cm with a solvent system of ethyl acetate-methanol (19:l). After development, 0 detection was carried out with ultraviolet light (2537A).

The active fractions Nos. 25-30 of Rf 0.58-0.63 and the fractions Nos. 38-40 of Rf 0.25-0.30 were collected and concentrated under reduced pressure to ahout 20 parts by volume, respectively. To these concentrates were added 150 parts by volume portions of petroleum ether to obtain 5 parts of a crude product (XI) and 2 parts of crude maytansinol.

In 10 parts by volume of ethyl acetate was dissolved 0.5 part of the crude product (ll) obtained above and the solution was thoroughly stirred with 4 parts of silica gel (Merck, West Germany, 0.05-0.2 mm). The ethyl acetate was removed under reduced pressure. The residue was applied to the top of 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:l) and 500 parts by volume of chloroform-methanol (lO:l). The eluate was collected in 25 parts by volume fractions.

A 0.5 part by volume portion of each fraction was concentrated under reduced pressure. To the concentrate was added 0.05 part by volume of ethyl acetate, and the mixture as a sample was subjected to thin layer chromatography (developing system: ehloroform-methanol= 9:1). o Fractions Nos. 40 and 41 absorbing at 2537 A in the zone of Rf 0.48-0.50 were collected and concentrated to dryness under reduced pressure. To the residue was added 0.5 part hy volume of ethyl acetate and the mixture was allowed to stand, whereupon 0.05 part of mixed crystals of maytanacine and maytansinol propionate were obtained. 0.05 part of the above mixed crystals of maytanacine and maytansinol propionate was dissolved in 5 parts by volume of methanol, followed hy the addition of 0.1 part of sodium chloride and 5 parts by volume of water.

A column was packed with 200 parts hy volume of Diaion (Trade Mark) HP-10 (Mitsubishi Kasei K.K.) and washed with 600 parts hy volume of 500 methanoi-water containing 50 of NaCl. The sample solution prepared above was passed through the column, and gradient elution was carried out using 1500 parts hy volume of 600 methanoiwater containing 50 NaCl and 1500 parts by volume of 950 methanoi-water. The elute was collected in 15 parts hy volume fractions and each fraction was investigated hy thin layer chromatography. The fractions 150 to 135 contained maytanacine, and the fractions 138-142 contained maytansinol propionate.

Each group of fractions was concentrated and dissolved hy the addition of 30 ml of water and 50 ml of ethyl acetate. The solution was shaken in a separation funnel and the water layer was separated and, after washing twice with 30 ml-portions of water, the ethyl acetate layer was dried over anhydrous sodium sulphate, concentrated and allowed to stand. In the above manner, crystals were obtained from each group of fractions.

The crystals were collected hy filtration and dried.

Maytanacine: 0.013 part. Maytansinol propionate: 0.025 part.

In 3 parts hy volume of ethyl acetare was dissolved 0.2 part of the crude maytansinol obtained above and the resulting solution was thoroughly stirred with 0.5 part of silica gel (Merck, West Germany, 0.05-0.2 mm). The ethyl acetate was removed under reduced pressure. The residue was applied to the top of a column of 80 parts hy volume of silica gel and the column was first washed with 150 parts by volume of chloro form and then eluted with 150 parts by volume of chlorol'orm-mcthanol (50:1), 150 parts by volume of chloroform-methanol (20:1) and 300 parts by volume of chloroform-methanol (10:1). The eluate was collected in 10 parts hy volume fractions.

A 0.5 part hy volume portion of each fraction was concentrated under reduced pressure. To the concentrate was added 0.05 part by volume of ethyl acetate, and the mixture as a sample was subjected to thin layer chromatography (developing system: chloroform-methanol=9:l). 43888 Fractions Nos. 50 to 52 absorbing at 2537 A in the zone of Rf 0.33 to 0.38 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 stand, whereupon 0.020 part of crystals of maytansinol was obtained.

Example 3 With stirring, 32,000 parts of the cells obtained iuExample 2 were extracted with 50,000 parts by volume of 70^ acetone-water for 3 hours and then filtered on a pressure filter. The extraction with 50,000 parts by volume of 70$ acetone-water and subsequent filtration was repeated once again. The filtrates were pooled and the acetone was removed by concentration under reduced pressure. The resulting aqueous system was passed through a column of 5,000 parts by volume of Diaion (Trade Mark) HP-10 (Mitsubishi Kasei K.K.). The column was washed with 2Q000 parts by volume of water and 50$ aqueous methanol, followed by elution with 15,000 parts by volume 90$ methanol-water. The eluate was concentrated under reduced pressure to 3000 parts by volume and shaken with 3000 parts by volume of water and 3000 parts by volume of ethyl acetate. The above procedure was repeated once •again. The ethyl acetate layers were combined, washed with water, dried by the addition of anhydrous sodium sulphate and concentrated under reduced pressure to 200 parts by volume. Following the addition of petroleum ether, the precipitate was recovered by filtration (280 parts).

The above-obtained crude product (I) was purified by means of a column of silica gel similarly to Example 1 to recover 1.0 part of crude product (ll) and 0.5 part of crude maytansinol.

Example 4 1000 parts by volume of the culture of Example 2 was inoculated into a 200,000 parts by volume tank of stainless steel 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 volume/ min.) and agitation (200 r.p.m.) for 48 hours to prepare a seed culture. This seed culture was transferred to a 2,000,000 parts by volume tank of.stainless steel containing 1,000,000 parts by volume of a fermentation medium similar to that used in Example 1 at a transplantation rate of 10$. Cultivation was carried out at 28°C under aeration (1,000,000 parts by volume/min.) agitation [120 r.p.m. (1/3 DT)] and internal pressure (l kg/cm ) for 90 hours.

The resulting culture was found to have a titer of 20 pg/ml as assayed by the assay procedure described in Example 1.

To 900,000 parts by volume of the above-obtained culture were added 900,000 parts by volume of acetone and, after 4S888 an hour's stirring, 20,000 parts of Hyflo-Supercel (Trade Mark, Johns - Manville, USA) were -added. The mixture was further stirred and filtered on a pressure filter machine.

To 1,700,000 parts hy volume of the resulting filtrate were added 500,000 parts hy volume of water and, in a Podhielniak (Trade Mark) extraction (Podhielniak, Inc., USA), the mixture was extracted with 1,000,000 parts hy volume of ethyl acetate. The ethyl acetate layer was washed with water, dried hy the addition of anhydrous sodium sulphate and concentrated under reduced pressure.

Petroleum ether was added to the concentrate and the resulting precipitate was recovered hy filtration and dried. 680 parts of a crude product (l) hy the above procedure were obtained thereafter, as in Examples 2 and 3, this crude product was purified to yield 1.1 parts of maytanaoine, 2.2 parts of maytansinol propionate and 0.1 part of maytansinol.

Claims (7)

CLAIMS:

1. A method for preparing maytansinol, maytanacine or maytansinol propionate which comprises cultivating a microorganism belonging to the genus Nocardia and capable of producing maytansinol, maytanacine or g maytansinol propionate in a culture medium containing assimilable carbon sources and digestible nitrogen sources until maytansinol, maytanacine or maytansinol propionate is substantially accumulated therein; and recovering the latter. 10

2. A method as claimed in Claim 1, wherein the product obtained is maytansinol.

3. A method as claimed in Claim I, wherein the product obtained is maytanacine.

4. A method as claimed in Claim 1, wherein the product 15 obtained is maytansinol propionate.

5. A method as claimed in Claim 1, wherein the microorganism is Nocardia No. C-15OO3 (ATCC 31281; IFO 13726; FERM 3992).

6. A method according to Claim 1 substantially as 20 herein described with reference to any of the specific examples.

7. Maytansinol, maytanacine or maytansinol propionate when produced by a process as claimed in any of Claims 1 to 6.