HU177391B - Process for producing maytanzinol and 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 novel process for the preparation of maytanzinol, maytanacin and maytanzinol propionate. The compounds listed have anti-tumor activity.

Among these compounds, maytanacin and maytanzinol propionate are known to have potent antitumor activity (Kupchan et al., Journal of the American Chemical Society 97, 5294 (1975)). Maytanzinol, on the other hand, has only a weak antitumor activity (see above), but is a useful intermediate for the preparation of maytanacin and maytanzinol propionate and other derivatives.

Maytanzinol and maytanacin are described by Kupchan et al. was obtained from the bark of Putterlickia verrucosa (a plant of the maytenus genus) and yielded very low yields. More specifically, 1 kg of dried bark was obtained in an amount of 0.025 mg of the former and 0.36 mg of the latter. However, maytanzinol propionate was prepared by chemical propionation of maytanzinol.

In the course of the present invention, other soil samples were collected and analyzed.

Our investigations led to the discovery that some of the microorganisms so isolated are capable of collecting maytanzinol, maytanacin, or maytanzinol propionate in nutrient 3 (soil. These microorganisms also belong to the genus Nocardia. mutants derived from these microorganisms can be prepared by culturing the media in suitable medium containing suitable nutrients.

Further studies based on the above findings have led to the development of the present invention.

Thus, the present invention relates to a novel process for the preparation of maytanzinol, maytanacin or maytanzinol propionate in a culture medium containing assimilable carbon sources and digestible nitrogen sources of Nocardia C-15003 deposited under ATCC 31281 at a temperature of 20-35 ° C. Is cultured at an initial pH of 7.0, and the collected maytanzinol, maytanacin or maytanzinol propionate is isolated in a manner known per se.

In the prior art, the above compounds can be prepared from plants, but the number of suitable plants is very limited and preparation is extremely costly and time-consuming in all steps such as growth, drying and extraction, isolation and purification of the plant. However, the yield is extremely low.

In contrast, the process of the invention is readily and smoothly carried out by culturing the microorganism and is capable of producing a large amount of the compounds,

The process of the present invention is the first to produce these compounds as metabolites of microorganisms and as such is characterized as a very excellent method for the preparation of the compounds.

The process of the invention is used for the C-15003, ATCC No. 31281 deposited actinomycete strain which was isolated from soil and in ¹⁰ other samples also suitable for searching for the production of antibiotics of microorganisms.

The microbiological characteristics of strain C-15003 were examined using a procedure similar to that proposed by Schirling and Gottlib (Intemational Journal of ¹⁵ Systematic Bacteriology 16, 313-340 (1966)). The results of the observation at 28 ° C after 21 days are described below.

1. Morphological characteristics

The vegetative mycelium multiplies well on both agar and liquid media and branches. Many 25 fungal cells are 0.8-1.2μηι in diameter and in some cases may divide into short cells resembling or branching to rod-shaped bacteria. The strain exhibits good growth in various taxonomic media where aerial lecithium builds on the vegetative mycelium, although it often forms coremia-like bodies (50-200 x 200-1000 pm) on which additional aerial growth may occur. Many of the cgmicellium are elastic, and in some cases a straight or loose spiral-like configuration has been observed. Microscopic examination of aged cultures showed that only a few cases of conidia-like cells appear in chains, while cell suspensions obtained from the top of such cultures show, under microscopic observation, many elongated 40 ellipsoids (0.8-1.2 pm). x 4.8-6.8 µm) and ellipsoid (0.8 1.2 x 1.0-2.0 mm) shaped like arthrospores-like bodies.

Electron microscopy showed these bodies to have smooth surfaces. 45

2. Components of cells

The strain was cultured on modified ISP 1 medium by shaking at 28 ° C for 50 to 66-99 hours. At the end of the culture, the cells were harvested & rinsed. B. Becker et al. (Applied Microbiology 12, 421/1964 / and P. Lechevalier, Journal of Laboratory and Clinical Medicine 71, 934/1968 /) examined the diamino-pimelic acid and sugar content of finnish cells. that they were meso-shaped while we also found sites that corresponded to galactose and arabinose.

3. Characteristics of taxonomic media

The strain showed relatively good growth on various media, where the vegetative mycelium changed from 65 colorless to pale yellow in the initial stages of culture and was between light yellow and light brown in subsequent phases. It has been found that the strain produces soluble pigments, which vary in color from yellow to yellow-brown in various taxonomic media. Aerial mycelium has a powdery and generally moderate growth and is white to light tan. The characteristics of the strain on various taxonomic media are summarized in Table 1 below.

Table 1

Culture characteristics of strain C-15003 on various taxonomic media (A) Sucrose nitrate agar:

Growth (G): Moderate, light melon between yellow (3 ia) ^x and amber (3 lc) ^x , coremia-like bodies.

Aerial mycelium (AM): low white Soluble pigment (SP): None or light tan.

(B) Glycerol nitrate agar:

G: Moderate, light ivory (2 ca) ^x coremia-like bodies.

AM: Moderate, white.

SP: None.

(C) Glucose Asparagine Agar:

G: Moderate, bright stork teal color between (3 pa) ^x and bright yellow (2 pa) ^x . AM: Little white. SP: Light yellow (2 pa) ^x .

(D) Glycerol Asparagine Agar:

G: Moderate, light ivory (2 ca) ^x coremia-like bodies.

AM: Little white.

SP: None.

(E) Starch agai:

G: Moderate, light ivory (2 ca) ^x to light wheat (2 ea) ^x , coremia-like bodies are formed.

AM: Light, light ivory (2 ca) ^x

SP: None.

(F) Nutrient Agar;

G: Moderate, light ivory (2 ca) ^x to colonial yellow (2 ga) ^x , coremia-like bodies are formed.

AM: Little white.

SP: None.

Continuation of Table 1 (G) Calcium malt agar:

G: Moderate, light ivory (2 ca) ^x to light wheat (2 ea) ^x , coremia-like bodies are formed.

SP: None.

(H) Yeast extract - malt extract agar: G: Moderate, amber (3 lx) ^x vivid yellow (3 la) ^x , coremia-like bodies are formed.

AM: Moderate white to light ivory (2 ca) ^x ,

SP: None.

(I) Oatmeal Agar:

G: Moderate, light ivory (2 ca) ^x colonial yellow (2 ga) ^x , coremia-like bodies are formed.

AM: Few, between white and light yellow.

SP: None.

(J) Pepton Yeast Extract Iron Agar:

G: Moderate, colonial yellow (2 ga) ^x AM: None

SP: Colonial Yellow (2 ga) ^x (K) Tyrosine Agar:

G: Moderate, light ivory (2 ca) ^x light melon to yellow (3 ea) ^x , coremia-like bodies.

AM: Moderate white to light ivory (2 ca) ^x .

SP: Camel colored (3 ie) ^x .

^X Colors were determined according to the 4th edition of the Color Harmony Manual (Container Corporation of America, 1958).

4. Physiological characteristics

The physiological characteristics of the strain are summarized in Table 2. During growth, the temperature was between 12 and 38 ° C. The temperature range within which good air growth is observed on agar (ISP NO. 2) is 20-35 ° C.

Table 2

Physiological Characteristics of Strain C-15003 Temperature range for growth: 12-38 ° C.

Temperature range suitable for air growth: 20-35 ° C.

Melting gelatine: positive.

Hydrolysis of starch: positive.

Nitrate reduction: positive.

Peptonization of milk: positive.

Milk coagulation: negative.

Continuation of Table 2

Casein breakdown: positive.

Production of melanoid pigments:

negative (peptone yeast extract iron agar) positive (tyrosine agar)

Tyrosine breakdown: positive. Xanthin resolution: negative.

Lysozyme tolerance: positive. Tolerance to sodium chloride: 2%.

5. Use of different coal sources

The use of various carbon sources ^ such. we tested using Pridham and Gottlieb (Journal of Bacteriology 56, 107 (1948)) and a basic medium of the same composition plus 0.1% yeast extract. The resulting spectrum is shown in Table 3.

Table 3

By various tribes carbon sources utilization C-15003 carbon sources Growth carbon sources Growth D-xylose ♦ raffizóz + ± ^x L-arabinose 4 4 melibiose 4 4 D-glucose 44 44 i-inositol - - - D-galactose + 4 D-sorbitol - - D-fructose 4 + 4 44 D-mannitol 44 44 Lramnóz 4+ glycerol ± D-mannose 444 4t soluble starch ++ Sucrose Lactose Maltose Trehalose 44 44 __X ± + 4 44 control

^x Basic medium with 0.1% yeast extract.

Comment:

♦ **: excellent growth «·: good growth +: growth ±: bad growth

-: no growth

6. Other Features

Cells were harvested by the method described previously under point 2 and DNA preparation was performed by J. Marmur et al. (Journal of Molecular Biology 3: 208, 1961) The DNA (deoxynucleic acid) G-C (guanincytosine) content was found to be about 7 'mol%.

The Gram water test of the vegetative mycelium of the strain gave a positive result.

The above characteristics of strain C-15003 were compared in S. A. Waksman's "The Actinomycetes Vol. 2" (The Williams and Wilkins Co. 1961) and R. E. Buchanan and N. E. Gibbons, "Bérgey's Manual of Determinative Bacteriology", 8.

edition 1974 ”and other literature.

After the strain is Nocardia genus III. group, the fact that we did not find among the known strains that had the same properties, we had to conclude that the strain we were investigating represented a new specie.

Strain C-15003 was deposited under number 3992 in the Fermentation Research Institute, Ferromation Collection of the Agency for Industrial Science and Technology (FERM), while the strain was deposited under IFO 13726 in The Institute for Fermentation, Osaka (IFO) and The The American Type Culture Collection (ATCC), Maryland, USA, was assigned the number 31281.

While strain C-15003 is a new species of the Nocardia genus, as mentioned, it, like other microorganisms, tends to produce different variants and mutants, either spontaneously or under the influence of mutagens. For example, many variants of the strain can be obtained by exposure to X-rays or by gamma rays, ultraviolet rays, single-cell isolation, culture in 21 media containing various chemicals, or any other mutagenic treatment. Mutants produced spontaneously from the strain are also not considered to be a separate species; on the contrary, any such variant or mutant capable of producing maytanzinol, maytanacin and maytanzinol propionate may be used for the purposes of the invention. For example, strain C-15003 is subjected to various mutagenic treatments to produce mutants that lack essentially soluble pigment production in which mycelia are colorless, yellowish-green, reddish-tan or orange-red, fungal cells of other mutants are capable of bacillus-like cells, others have little or no aerial mycelium.

A culture medium for the production of maytanzinol, maytanacin or maytanzinol propionate (sometimes referred to as a "productive" strain) may be liquid or solid media if it contains nutrients that the strain can utilize, although it is advantageous for use in large-scale production.

The medium used for the cultivation of such antibiotic C-15003 producing strains may be any liquid or solid medium, provided that it contains nutrients which the strain is capable of utilizing, although in the case of large-scale production liquid media are preferred. The media may contain nitrogen and carbon spinning, which can be assimilated and digested by microorganism C-15003, inorganic materials, trace amounts of nutrients and other substances. Examples of carbon sources include glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol, sorbitol, fats and oils (e.g., soybean oil, lard, chicken fat). Examples of nitro gene sources include: meat extract, yeast extract, dried yeast, soybean flour, corn stripping liquid, peptone, linseed meal, malt waste, urea, ammonium salts (e.g., ammonium sulfate, ammonium nitrate, ammonium nitrate). The medium may additionally contain sodium, potassium, calcium, magnesium and other salts such as iron, manganese, zinc, cobalt, nickel salts, phosphoric acid, boric acid salts and organic acid salts such as acetates and propionate. The medium may also include various amino acids (e.g., glutamic acid, alanine, glycine, lysine, methionine, proline), peptides (e.g. dipeptides, tripepti5 triglycerides), vitamins (e.g., Bi, B ₂ vitamins, nicotinic acid, Bi _2, vitamins C and E) , nucleic acids (such as purine, pyrimidine and their derivatives). An inorganic or organic acid, alkaline, buffers and the like may be added to adjust the pH of the medium. Suitable amounts of oils, fats, surfactants, and other suitable materials may be used as antifoam agents.

The cultivation can be performed by any stationary, shaking, immersing aerobic or other method. Large-scale; Of course, submerged aerobic cultures are the most suitable for production. While the culture conditions will of course depend on the composition of the medium, the strain, the method used and other factors, the incubation is carried out at 20 to 35 ° C) at an initial pH of about 7.0. It is particularly desirable for the temperature to be between 23 and 30 ° C during the intermediate stages of culture and for the initial pH to be between 6.5 and 7.5. Although incubation time is also a _; Depending on the above factors, it is advisable to incubate until the desired antibiotic titer is at its maximum. In the case of shaking cultures or aerobic immersion cultures, this time is usually between 48 and 144 hours.

) The antibiotic potential was tested with Tetrahymena pyriformis W. Thus, the above microorganism was assayed in a test medium (20 g of protease 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) at 28 ° C. , Cultured for 44-48 hours, and the potency of the antibiotic was determined by serial dilution, monitoring for changes in turbidity, assaying for effects on peritoneal tumor cells, and thin-layer chromatography as described below.

Maytanacin, maytanzinol or maytanzinol propionate is produced and harvested on the resulting medium, both extracellularly and intracellularly. None of the substances exhibited antibiotic activity and were detected by thin layer chromatography in parallel with the activity against the Tetrahymena strain. Thus, the fermentation broth was separated on cells and filtrate by filtration or centrifugation and the filtrate was extracted with an equal volume of ethyl acetate. To the cells was added the same amount of 70% acetone / water mixture as for their hairs and after stirring for 1 hour at 20 ° C the suspension was filtered. The acetone was removed from the filtrate and the resulting aqueous filtrate was extracted with ethyl acetate. Each extract was concentrated to 1/100 volume and analyzed by TLC on a silica gel plate (Merek, Germany, Kieselgel 60 F 254, 0.25 mm, 20 x 20). Chloroform: methanol (9: 1) was used as the solvent system. The potential was determined by the intensity of the spots as determined by irradiation at 2537 A with ultraviolet light.

The media thus obtained maytanacin, maytanzinol propionate or maytanzinol are lipophilic and inert materials and can be readily recovered by commonly used isolation and purification procedures which are usually used to harvest these types of organisms. For example, a method that utilizes the difference in solubility of antibiotics and impurities, i.e., one that utilizes adsorptive affinity for various adsorbents (such as activated carbon, macroporous, non-ionic resins, silica gel, alumina), may be used. In addition, impurities can be removed by ion exchange resins and other methods, which may be used alone or in combination, optionally repeated.

Since these compounds are present in the filtrate as well as in the cells, the isolation and purification of antibiotics by such adsorbents, if any, can be carried out either directly or after filtration by solvent extraction or by microbiological cells by solvent extraction. Solvent extraction can be carried out by any of the following methods:

(1) Solvent extraction of fermentation broth before separation of cells; (2) Solvent extraction of cells and filtrate obtained by filtration, centrifugation or other methods. Preferably, the following procedure is used for the independent extraction of filtrate and cells.

Suitable solvents for extracting the filtrate are water immiscible organic solvents such as fatty acid esters such as ethyl acetate, amyl acetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform and ketones such as methyl isobutyl ketone. The extraction is carried out at a pH close to neutral and preferably the liquid fermentation broth previously adjusted to pH 7 is extracted with ethyl acetate. The extract was washed with water and concentrated under reduced pressure. A non-polar solvent such as petroleum ether or hexane is then added to the concentrate and the crude product I containing the active ingredient is recovered. Since TLC shows several spots besides maytanacin, maytanzinol propionate and maytanzinol, product I is subjected to the following purification steps in several steps. Thus, adsorption chromatography can be used as a standard purification process, and one of the usual adsorbents such as silica gel, alumina, macroporous non-ionic adsorbent resin can be used as the adsorbent. The use of silica gel is most preferred for the purification of crude product I. For example, the elution can be accomplished by starting with petroleum ether and hexane and eluting with a polar solvent such as ethyl acetate, acetone, ethanol or methanol. According to a conventional procedure, using silica gel (Merek, USG, 0.05-0.2 mm) as a carrier, column chromatography is carried out by gradually increasing the hexane: ethyl acetate ratio. The eluate was sampled and examined by thin layer chromatography, and the fractions containing the active compounds were collected and concentrated under reduced pressure. Thereafter, petroleum ether or hexane was added to the concentrate to give II. of crude product. Since this product still contains impurities, it is further purified as follows. II. For example, crude product # 1 can be purified on a silica gel column using a variety of solvent systems. The development system used 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 such as methanol or ethanol, a ketone such as acetone or methylethylketone or the like. This method separates maytanacin, maytanzinol propionate and maytanzinol. The order of solvents applied to the first and second silica gel columns is reversible and other conventional organic solvents may be used in combination with the above systems, if necessary.

If a macroporous adsorbent resin is used as a means of purification, the method described in FIG. In the purification of crude product no., elution of maytanacin, maytanzinol propionate and maytanzinol is carried out in a mixture of water and a lower alcohol, lower ketone or ester. The lower alcohol may be, for example, methanol, ethanol, propanol or butanol and the lower ketone may be, for example, acetone or methylethylketone. As the ester, for example, ethyl acetate may be used. According to a typical procedure, the method described in FIG. crude product was dissolved in a 60% methanol-water mixture and a Diaion HP-10 (Mitsubishi Kasel

K.K.) column. The column was washed with 70% methanol / water and eluted with 90% methanol / water. This procedure elutes maytanacin, maytanzinol propionate or maytanzinol from the column.

In any of the methods described above, the fractions containing the active compounds were collected and concentrated under reduced pressure. To the dry product is added 5 to 8 volumes of ethyl acetate and the mixture is allowed to stand and the maytanacin, maytanzinol propionate or maytanzinol are separated. These compounds are then separated by means of an adsorbent. Any of the above-mentioned adsorbents can be used for this purpose. Thus, the desired compounds can be fractionally eluted using silica gel or a macroporous non-ionic adsorbent resin and working with the above solvents. For example, when silica gel is used, development is carried out with hexane, ethyl acetate, or chloroform-methanol, and maytanzinol propionate and maytanacin appear in the order given herein. For TLC detection, each fraction was concentrated individually under reduced pressure (177391) and ethyl acetate was added to the concentrates. In this way, the corresponding compounds are obtained in crystalline form. When using a macroporous non-ionic resin, mixtures of alcohol, ketone or ester and water of different compositions are used to perform the gradient elution technique. For example, using gradient elution techniques, 60% methanol-water, 95% methanol-water, and 5% sodium chloride, maytanacin and maytanzinol propionate are precipitated in the order indicated. After sampling and detection by TLC, each group of active fractions was concentrated under reduced pressure and crystallized from ethyl acetate. The isolated crystals contain ethyl acetate as the crystallization solvent. After drying over phosphorus pentoxide at 70 ° C for 8 hours, they have the following physical and chemical properties.

Table 4

Maytanacin C3 0H39CIN2 O9 Maytanzinol propionate C ₃ H ₄₁ ClN ₂ O ₉ Maytanzinol C ₂ sH _{3 7} C1N ₂ O ₈ mp (° C) 235-236 188-190 172.5 to 174 specific rotation [α] D ² ° -121 ° ± 10 ° [Α] -127 ° + 10 ° fa] ^ ² ° -313 ° + 10 ' ability (C = 0.25, CHCl 3) (C = 0.35, CHCl 3) (C = 0.22, CHCl ₃ ) Analysis C 59.62 59.63 59.28 calculated (%) H 6.93 6.82 6.38 N 4.28 4.32 5.02 cl 5.74 5.57 6.15 Analysis c 59.85 59.94 59.52 found (%) H 6.48 6.65 6.60 N 4.61 4.51 4.96 cl 5.84 5.71 6.27 ultraviolet absorption 233 (30330) 233 (30240) 232 (32750) spectrum (e) 240 (including 28240) 240 (including 28400) 244 (including 30850) 252 (27850) 252 (27650) 252 (31650) 280 (5680) 280 (5740) 281 (5750) 288 (5660) 288 (5710) 288 (5700) infrared absorption 1740, 1730, 1740, 1730, 1715, 1670, spectrum (cm ' ¹ ) 1670, 1580 1670, 1580 1580 Nuclear Magnetic Resonance Spectrum (m / e) 545, 485, 559, 485, 503, 485 470, 450 470, 450 470, 450 you are acidic, neutral lipophilic and lipophilic and lipophilic and alkaline neutral neutral neutral material material material color reactions Dragendorff- Dragendorff- Dragendorff- -reaction; -reaction: -reaction: positive positive positive Beilstein- Beilstein- Beilstein- -reaction: -reaction: -reaction: positive positive positive

The above chemical analysis, specific rotation, ultraviolet, infrared and mass spectral data are in good agreement with maytanacin, maytanzinol and maytanzinol propionate according to Kupchan et al. (See Journal and American Chemical

Society 97, 5294 (1975). 65

The following examples further illustrate the present invention without limiting it. In the examples, the relationship between "parts by weight" and "parts by volume" is the same as the expression "grams" and "milliliters". % Values, unless otherwise stated, refer to mixed%.

Example 1

A yeast extract-malt extract grown on culture medium containing Nocardia C-15003 (IFO 13726, FERM 3992, ATCC 31281) producing maytanzinol, maytanacin and maytanzinol propionate was inoculated with 200 volumes of inoculum medium. Inoculum medium: 2% glucose, 3% soluble starch, 1% raw soybean flour, 1% corn stripping liquid, 0.5% polypeptone, 0.3% sodium chloride, 0.5% calcium carbonate, pH 7 , 0). The inoculated medium is incubated at 28 ° C for 48 hours to produce an inoculum. 0.5 volumes of the inoculum thus obtained are added to a 200-volume fermenter containing 40 volumes of medium. The medium is composed of 5% dextrin, 3% corn dressing fluid, 0.1% polypeptone, 0.5% calcium carbonate, pH 7.0. The medium is cultured at 28 ° C for 90 hours to produce an inoculum (seed culture).

Determined by serial dilution using Tetrahymena pyriformis W as the test microorganism and C-15003 P-3 as the comparator, the titre of the above culture is 25 µg / ml.

Example 2 Volume of inoculum obtained in Example 1 was transferred to a 2000 volume volume of 500 volumes of inoculum culture medium (identical to Example 1) and incubated for 48 hours at 28 ° C. 500 volumes of the resulting culture were transferred to a 50,000 volume stainless steel container containing 30,000 volumes of inoculum culture medium and cultured at 28 ° C under aeration (280 rpm 1/2 DT /) and kg / cm ² internal pressure. The resulting inoculum culture was added to a 200,000 volume stainless steel fermenter containing 100,000 volumes of a culture medium similar to that used in Example 1 at a 10% inoculation rate. The inoculated medium is incubated at 28 ° C with aeration (100,000 volumes / min), agitation (200 rpm), and 1 kg / cm ² internal pressure for 90 hours. As determined by the method described in Example 1, the resulting culture titer was 25 pg / ml.

To the resulting 95,000 volumes of culture, 2,000 parts by weight of Hyflo-supercel ® (Johnes and Manville Products, USA) were added and, after thorough mixing, the mixture was filtered through a pressure filter. 85,000 volumes of filtrate and 32,000 parts by weight of wet cells are obtained. The 85,000 volumes of filtrate were stirred and extracted with 30,000 volumes of ethyl acetate. The procedure is repeated once more. The ethyl acetate layers were collected, washed twice with 30,000 volumes of water, dried with 500 parts by weight of anhydrous sodium sulfate, and concentrated under reduced pressure to 200 volumes. Petroleum ether was added to concentrate '14 and the resulting precipitate (53 parts by weight) was collected by filtration. This crude product I is mixed with 100 volumes of ethyl acetate and the insolubles are filtered off. The filtrate was mixed with 10 parts by weight of silica gel (Merek, Germany, 0.05-0.2 mm) and the ethyl acetate was removed under reduced pressure. The residue was applied to the top of a 400 volume silica gel column. Elution with 500 volumes of hexane, 500 volumes of 3: 1 hexane-ethyl acetate mixture, 500 volumes of 1: 1 hexane-ethyl acetate mixture, 500 volumes of 1: 3 hexane-ethyl acetate mixture, 500 volumes of ethyl acetate and 1000 volumes of 50: 1 ethyl acetate methanol, collecting the eluates in fractions of 100 volumes.

One volume of each fraction was evaporated to dryness and 0.1 volume of ethyl acetate was added to the concentrate. The resulting mixture was dropped to 2.5 cm from the bottom corner of a silica gel glass plate (Merek, Germany, 60F _2S4 , 0.25 mm, 20 x 20) and run to about 17 cm with a 19: 1 ethyl acetate-methanol solvent system. live. After development, detection is performed with ultraviolet light (wavelength: 2537 A).

The 25-30 fractions with an Rf of 0.58-0.63 and the 38-40 fractions with an Rf of 0.25-0.30 were collected and concentrated under reduced pressure to about 20 volumes. To the concentrates obtained were added 150-150 volumes of petroleum ether to give 5 parts by weight of II. of crude product and 2 parts by weight of crude maytanzinol.

Dissolve the reaction mixture in II. 1.5 parts by weight of crude product. then the solution is well mixed with 4 parts by weight of silica gel (Merek, Germany, 0.05-0.2 mm). The ethyl acetate was removed under reduced pressure. The residue was applied to the top of a 300 volume silica gel column and the column was washed first with 500 volumes of chloroform and then eluted with 500 volumes of 20: 1 chloroform: methanol and 10: 1 chloroform: methanol. The eluates were collected in fractions of 25 volumes.

0.5 volumes of each fraction were concentrated under reduced pressure. To the resulting concentrate was added 0.05 volumes of ethyl acetate and the sample was analyzed by silica gel thin-layer chromatography (developing system: chloroform: methanol = 9: 1).

Fractions 40 and 41 absorbing at 2537 Λ in the Rf 0.48-0.50 zone are collected and evaporated to dryness under reduced pressure. To the residue was added 0.5 volumes of ethyl acetate and the mixture was allowed to stand at room temperature to give 0.05 parts by weight of mixed maytanacin, maytanzinol propionate crystals.

The thus obtained mixed crystals of maytanacin and maytanzinol propionate were dissolved in 0.05 parts by weight of methanol and then

0.1 parts by weight of sodium chloride and 5 volumes of water are added. One column was filled with 200 volumes of Diaion HP-10 (Mitsubishi Kaséi

KK) and 600 volumes of 50% methanol / water containing 5% sodium chloride. A sample of the above solution was passed through the column, eluted with gradient ₅ and technology, 1500 vol (containing 5% sodium chloride) with 60% methanol-water mixture solution of methanol-water mixture was 95% Ή 1500 vol.

The eluate was collected in fractions of 15 volumes and all fractions were analyzed by TLC. Fractions 13E-135 contain maytanacin and fractions 138-142 contain maytanzinol propionate. 15

All fractions were concentrated and dissolved in water (30 mL) and ethyl acetate (50 mL). The solution is shaken in a separatory funnel and the aqueous layer is separated and after washing twice with 30 ml of water, the ethyl acetate layer is dried over anhydrous sodium sulfate, concentrated and allowed to stand. By the above procedure, crystals were obtained from each fraction. The crystals were collected by filtration and dried. The amount of maytanacin is 0.013 g, while that of maytanzinol propionate is 0.013 g.

volume of ethyl acetate

0.2 parts by weight of crude maytanzinol obtained in the above step and the solution is well mixed with 30 0.5 parts by weight of silica gel (Merek, 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 volumes of silica gel and the column was washed first with 150 volumes of chloroform, then with 150 volumes of 50: 1 chloroform: methanol and 150 volumes of 20: 1 chloroform: methanol and finally 300 volumes of 10: 1. eluted with chloroform-methanol. The eluate was collected in 10 volumes of fractions.

0.5 to 0.5 volumes of each fraction was concentrated under reduced pressure. Ethyl acetate (0.05 volumes) was added to the concentrate and the mixture was analyzed by thin layer chromatography using chloroform / methanol 9: 1 as developing system.

Fractions 50-52 (Rf = 0.33-0.38) absorbing at 2537 A were collected and evaporated to dryness under reduced pressure. To the residue was added 0.5 volumes of ethyl acetate and the mixture was allowed to stand. 0.020 parts by weight of maytanzinol crystals are obtained.

Example 3

000 parts by weight of the cell obtained in Example 2 were extracted with 50,000 volumes of 70% acetone / water with stirring for 3 hours and then filtered through a pressure filter. The extraction was repeated once more with 50,000 volumes of 70% acetone / water. The filtrates were collected and the acetone was removed by concentration under reduced pressure. The resulting aqueous system was passed through a 65

5000 volumes on a column containing Diaion HP-10 (Mitsubishi Kaséi K.K.). The column was washed with 20,000 volumes of water and 50% methanol and then eluted with 15,000 volumes of 90% methanolic water. The eluate was concentrated under reduced pressure to 3,000 volumes and extracted with 3,000 volumes of water and 3,000 volumes of ethyl acetate. The above procedure is repeated once more. The ethyl acetate layer was combined, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 200 volumes. Light petroleum was added to the concentrate and the resulting precipitate (280 parts by weight) was collected by filtration. The crude product I obtained above is purified by chromatography on a silica gel column in a manner similar to Example 1. 1.0 parts by weight crude II. and 0.5 parts by weight of crude maytanzinol.

Example 4

1000 volumes of the culture obtained in Example 2 were inoculated in a stainless steel 200,000 volumes vessel containing 100,000 volumes of inoculum culture medium and the inoculated medium at 28 ° C, aeration (100,000 volumes / min) and mixing ( 200 rpm) for 48 hours to prepare the inoculum culture. This inoculum culture was transferred to a 200,000-volume stainless steel container containing 100,000 volumes of the same medium as described in Example 1. The transmission rate is 10%. Cultivation was performed at 28 ° C with aeration (1,000,000 volumes / min), agitation (120 rpm), and internal pressure (1 kg / cm ² ) for 90 hours. The titer of the resulting culture was found to be 20 µg / ml by the procedure described in Example 1. To 900,000 volumes of the above culture was added 900,000 volumes of acetone and, after stirring for one hour, 20,000 parts by weight of Hyflo-Supercel (Johnes and Mann, USA). The mixture is further stirred and suctioned. To the resulting filtrate was added 500,000 volumes of water to 170,000 volumes and the mixture was extracted with 1,000,000 volumes of ethyl acetate in a Podbielniak (Podbielniak, Inc. USA). The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. To the concentrate was added petroleum ether and the resulting precipitate was collected by filtration and dried. The above procedure gives 680 parts by weight of crude product I. Then, as in Examples 2 and 3, this crude product is purified when 1.1 parts by weight of maytanacin,

2.2 parts by weight of maytanzinol propionate and 0.1 parts by weight of maytanzinol are obtained.

Claims (1)

Claim: Process for the preparation of maytanzinol, maytanacin and maytanzinol propionate, characterized in that The microorganism Nocardia C-15003 deposited under ATCC 31281 is assimilated in a medium containing 18 active carbon sources and digestible nitrogen sources at a temperature of 20-35 ° C and an initial pH of 7.0, followed by accumulation of maytanzinol, maytanac and / or maytansin in itself isn »not separated.