CA1092999A - Method for producing maytansinol and its derivatives - Google Patents

Abstract

METHOD FOR PRODUCING MAYTANSINOL AND ITS DERIVATIVES

Abstract of the Disclosure Maytansinol, maytanacine or maytansinol propionate is (are) produced by cultivating a microorganism belonging to the genus Nocardia and being capable of producing maytansinol, maytanacine or maytansinol propionate, causing the microorganism to accumulate maytansinol, maytanacine or maytansinol propionate and recovering the same.
Those compounds are useful for anti-tumor agents.

Description

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The present invention relates to a method for preparing maytansinol, maytanacine and maytansinol pro-pionate which are anti-tumor agents.
It has been known that, among the above com-pounds, maytanacine and maytansinol propionate have strong anti-tumor activity [Kupchan et al.: Journal of the American Chemical Society 97, 5294 ~1975)]. On the other hand, maytansinol itself has only weak anti-tumor activity (See the above reference) but it is a useful inter-mediate for easy preparation of maytanacine and maytansinol propionate and other various derivatives.
Maytansinol and maytanacine have been obtainedby the above Kupchan et al. from bark of Putterlickia verruscosa ~Plant belonging to the genus Maytenus) and thus yield is extremely low, such as 0.025 mg of the former compound and 0.36 mg of the latter from 1 kg of dried bark of the plant. ~s to maytansinol propionate, it has been obtained by chemical propionation of maytan-sinol.

The present inventors have collected various soil and other samples and investigated antibiotics produced by microorganisms which are isolated from the samples, and these studies have reached findings that some of the microorganisms thus isolated can accumulate maytansinol, maytanacine or maytansinol propionate in the culture medium, these microorganisms belong to the genus Nocardia, and that these compounds can also be

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obtalned by cultivating mutants derived from these microorganisms in a proper nutrient medium under appro priate conditions.
Further studies on the basis of these findings have now reached completion of the present invention.
Thusg the present invention relates to a method for preparing maytansinol, maytanacine or maytansinol propionate, which comprises cultivating a microorganism, which belongs to the genus Nocardia and is capable of producing maytansinol, maytanacine or maytanslnol propionate 9 in a culture medium to accumulate maytansinol, maytanacine or maytansinol propionate in the cultured broth and recovering the same.
According to the prior art~ the above compounds are obtainable from plants, but the plants are limited to specific ones3 and great expense and a long period of time are required for the production at each stage of growth, felling~ d~ying and pulverizing of the plants and extractiong separation and purification. Further, the yleld is extremely low.
On the contraryg the processes of the present invention can be conducted easily and smoothly by cultivation of the microorganism, and a large amount of the objective compounds can be produced and obtained when desired.
me present invention is the first example of obtaining these compounds as metabolites of microorganismsg ~o~z99~

and can be said as an excellent method for preparation thereof.
As an example of the microorganism usable in the present methodg there may be men~ioned an actinomycete strain No. C-15003 which we isolated from soil and other samples in our screening for antibiotic-producing microorganisms.
me microbiological characters of Strain No. C-15003 w2re investigated by procedures analogous to those proposed by Schirling & Gottlieb [International Journal of Systematic Bacteriology 16~ 313-340 (1966)]. The results of observations at 28C over 21 days are as ~ollows.
1) Morphological characters The ve~etative mycelium extends well and develops into branches~ both on agar and in liquid medium. Many of the hyphae measure 0.8 to 1.2 ~m in diameter and, in certain instances9 may divide'into fragments resembling rod bacteria or branched short lengths of hypha~., m e strain gives good growth on various taxonomical media~ with aerial mycelium being superimposed on the vegetative mycelium3 although it frequently forms coremia like bodies (50 200 x 200 - lOOO~m) on which further aerial growth takes place. Many of the aerial mycelia are flexuous, straight or a loosely spiral like configuratlon being encountered on a few occasions.

~1~9Z999 Microscopic examination of aged cultures reveals that only in few cases the conidia like cells occur in chains, while the cell suspensions obtained from the surfaces o~ such culturesg as microscopically examined~ contained many elongated ellipsoidal (0.8-1.2 ~m x 4~8-6.8 ~m) and ellipsoidal (0.8-1.2 x l.C-2.0 ~m) bodies resembling arthrospores.
Electron-microscopic examinations showed that these bodies had smooth surfaces.
2) The constituents of cells The strain was shake-cultured in modified ISP
No. 1 medium at 28C for 66 to 90 hours, at the end of which time ~he cells were collected and rinsed. By the method of B. Becker et al. [Applied Microbiology 12, l~21 (1964)] and the method of MoP ~ Lechevalier. `
[Journal of Laboratory and Clinical Medicine 71, 934 (1968)], the above whole cells were examined for diaminopimelic acid and sugar compoSition. The former was found to be the meso--~orma while spots were detected, which corresponded to galactose and arabinose.

3) Characteristics on taxonomical media The strain showed comparatively good growth on various media~ with the vegetative mycelium being colorless to pale yellow in initial phases of culture and light yellowish tan to yellowish tan in later phases.
The strain produces soluble pigments~ yellow to yellowi~h .

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tan9 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.
Table 1 Cultural characteristics of Strain No. C-15003 on taxonomical media (A) Sucrose~nit~atê agaro Growth (G): Luxuriantg Brite Melon Yellow (3 ia)*
to Amber (3 lc~, coremia like bodies formed Aerial mycelium (AM): Scant, white Soluble pigment (SP) None or pale yellowish tan (B) Glycerol.nitrate agàro G Moderate, Lt Ivory (2 ca)*, coremia like bodies formed AM: Moderateg white SP: None (C) Glucose~ asparàgin~ a~ar:
G Moderate 3 Brite Marigold (3 pa)~ toBrite Yellow (2 pa~*
AM: Scant, white SP: ~rite Yellow (2 pa)*
(D) Glycerol;aspar~gine ag~ro G : Moderate, Lt Ivory (2 ca)*~ coremia like bodies ~ormed .

AM: Scantg white SP: None (~) Starch agarO
G : Moderateg Lt Ivory (2 ca)~ to Lt Wheat (2 ea)*g coremia like bodies formed AM: Abundant, Lt Ivory (2 ca)*
SP: None (F) Nutrient agar:
G : Moderate, Lt Ivory (2 ca)* to Colonial Yellow (2 ga)*, coremia like bodies formed AM: Scant, white SP: None (G) Calcium malate agar:
G : Moderate~ Lt Ivory ~2 ca)* to Lt wheat (2 ea)*, coremia like bodies formed.
AM: Moderateg white to Lt Ivory (2 ca)*
SPo None (H) Yeast extract-malt extract agar.
G : Moderate~ Amber (3 lc)* to Brite Yellow (3 la)*g coremia lik~ bodies formed ~M: Moderate, white to Lt Ivory (2 ca)*
SP: None (I) Oatmeal agar:
G : Moderate, Lt Ivory (2 ca)* to Colonial Yellow (2 ~a)*, coremia like bodies formed AM: Scant, white to light yellow SP: None (J) Peptone yeas~ extract iron ag~r G : Moderate~ Colonial Yellow (2 ga)*
AM: None SP: Colonial Yellow (2 ga)*
(K) Tyrosine agar-G : Moderate9 Lt Ivory ~2 ca)* to Lt Melon Yellow (3 ea)*, coremia like bodies formed AM: Moderate, white to Lt Ivory (2 ca)*
SP: Camel (3 ie)*
* The color codes aceording to Color Harmony Manual, 4th ed. (Container Corporation of America, 1958)

4) Physiological characters The physiological characters of the strain are shown in Table 2. Temperature range for growth: 12C to 38C.
me temperature ran~e in which good aerial growth occurs on agar (ISP NoO 2) is 20 to 35Co Table 2 The physiological characters of Strain No. C-15003 Temperature range for growth: . 12 to 38C
Temperature range fo~ aerial growth: 20 to 35C
~iquefaction of gelatin: Positive Hydrolysis of starch: Positive Reduction of nitrates: Positive Peptonization of milk: Positive Coagulation of milk: Negative Z~9 Decomposition of casein: Positive Production of melanoid pigments: Ne~ative (peptone yeast extract iron agar), positive ~tyrosine agar) Decomposition of tyrosine: ~ositive Decomposition of ~nthine: Negative Decomposition of hypoxanthine. Ne~ative Tolerance to lysozyme: Positive Tolerance to sodium chloride: 2%

5) Utilization of various carbon sources The utilization of various carbon sources was in~estigated using a medium described in Pridham and Gottlleb [Journal of Bacteriology 56, 107 (1948)]
and a basal medium of the same composition plus 0.1%
of yeast extract. The resultant spectrum is shown in Table 3.
Table 3 me utili2ation of carbon sources by Strain No.C-15003 Source of carbon Growth Sources of carbon Growth D-Xylose + ++* Raffinose + +*
L= Arabinose + + Melibiose + +
D= Glucose ~ +~ i-Inositol - -D-Galactose + + D~Sorbitol - -D-Fructose ~+ ++ D-Mannitol +~ ~+
L-Rhamnose + + 51ycerol _ +
D-l~annose ~+ ++ Soluble starch +
Sucrose ~+ +~ Control " : .. ~

g9 Lactose - - *
Maltose +
Trehalose -~ ~

* Basal medium with 0.1% yeast extract added Note: +~: Luxuriant growth ~: Good growth Growth +~ Poor growth -~ ~o growth

6) Other characteristics me cells were harvested by the proceaure previously described in 2) and DNA was p~epared by a procedure analogous to that of ~. Marmur et al. ~Journal of Molecular Biology, 3,2083 1961]. The G-C(Guanine-Cytosine) content of the DNA was found to be about 71 mole %.
Gram-staining of the vegetative mycelium of this strain was positive.
me above characteristics of Strain No. C-15003 were compared with the descriptions in S. A. Waksman's '~he Actinomycetes Vol. 2"[The Williams and Wilkins Co., 1961]; R. E. Buchanan and N. E. Glbbons,'~ergey's Manual of Determinative Bacteriology, 8th ed, 1974 ";
and other literatures.
Whilst this strain was thought to belong to Group m of the genus Nocardia~ the failure to find any species having the characters so far described among , ~ ~

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the known strains led us to conclude that this straln represented a novel species of microorganism.
The present Strain NoO C--15003 has been deposited at Fermentation Research Instltute, Agency of Industrial Science and Technology (FERM) under the receipt number of 3992 and at Institute for Fermentation~ Osaka (IFO) under the accession number of IFO 13726 and at The American Type Culture Collection (ATCC), Marylandg U.S.A.
under the accession number of 31281.
~ hile Strain No. C-15003 is a novel species of the genus Nocardia as just mentioned3 it is liable3 as are microorganisms generally, to undergo variations and mutations, whether spontaneously or under the influence of a mutagen. For exampleg the many variants of the strain which are obtainable by irradiation with X-raysg ~amma raysg ultraviolet light9 etc., by monocell isolationg by culture on media containing various chemicals, or by any other mutagenic treatment, as well as the mutants spGntaneously derived from the strain, should not be substantially considered io represent any other distinct species butg rather~ any of such vàriants and mutants capable of elaborating maytansinol~ maytanacine and maytansinol propionate may be invariablY utilized for the purposes of this invention as the strain NoO C-15003 By way of example~ subjecting Stra~n No. C-15003 to various mutag~enic treatments yields mutants substantially l~Zgg~

lacking the ability to produce soluble pigments~ mutants with substrate mycelia which are colorlessg yellowish green3 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 strain capable of producing maytansinol~ maytanacine or maytansinol propionate (hereinafter~ sometimes abbreviated as l'producible strain") may be whichever of a liquid and a solid medium only if it contains nutrients ~hich the strain may utilize9 although a liquid medium is preferred for high-production runs.
The medium may comprise cArbon and nitrogen sources ~hich Strain No. C-15003 may assimilate and digest, inorganic matter~ trace nutrients3 etcO As examples of said carbon sources may be mentioned glucose, lactoseg sucrose, maltose, dextring starchg glycerol, manni~ol, sorbitol~ etc., fats and oils (eOg. soybean oila lard oil, chicken oil, etc.) and so forth. The nitrogen sources may for example be meat extract~ yeast extractj dried yeast3 soybean mealg corn steep liquor~ peptone, casein, cottonseed flour, spent molasses, ure~a ammonium salts (e.g. ammonium sulfate, ammonium chlorideg ammonium nitrate, ammonium acetate, etc.) and so fQrth.

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The medium may further contain salts of sodiumy potassium9 calcium, magnesiuma etc. 9 salts of iron, manganese, zinc~ cobaltg nickel~ etc. 9 salts of phosphoric acid3 boric acid~ etc and organic acid salts such as acetates and propionates. Further, the medium may containg as added, various ami~o acids (e.g.
glutamic acidg aspartic acida alanine, glycine3 lysine, methionine3 proline3 etc. )3 peptides (e.g. dipeptides, tripeptidesa etc.)~ vitamins (e.g. Bl/ B23 nicotinic acid3 B12~ C, E, etc.), nucleic acids (e.g. pur~ne~
pyrimidine and derivatives thereof and so forth. For the purpose of adjusting the pH of the medium~ there may be added an inorganic or organic acid, alkali~
buffer or the like. Suitable amounts of oils, fats, surfactants, etc. may also be added as antifoams.
The cultivation may be conducted by any of the stationary a shake, submerged aerobic and other cultural conditions. For hi~h production runs 3 submerged aerobic culture is of course preferred. ~hile the conditions of culture, of course, depends upon the condition and composition of medium, the strain~ cultural method and other factors, it is normally preferred to carry out incubation at 20 'o 35C with an initial pH of about

7.0 or thereabouts. Particularly desirable is a temperature from 23 to 30C in an intermediate stage of cultivation, with an initial pH of 6.5 to 7.5. While 3~

the incubation time also is variable according to the same factors as mentioned aboveg it is advisable to continue the incubation until the titer of the desired antibiotic product becomes max-Lmal~ In the case of shake culture or aerobic submerged culture in liquid medium9 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 or~anism. Thusg the above microorganism was grown on a test medium 20 g of Protesse-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 28C
for 44 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~ TLC) assay to be described hereinaf~er.
Maytanacine 9 maytansinol propionate or maytansinol is produced and accumulated in the resultant cultured broth, both extracellularly and intracellularly.
None of these substances shows distinc~ antibiotic activity, and thus they have been detected by TLC which is set forth in parallel to detection by activity to the Tetrahymlena strain. Thusg the fermentation broth is separated into cells and filtrate by ~lltration ~2999 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 an hour's stirring at 20C9 the suspension is filtered. The acetone is removed from the filtrate and the resultant aqueous filtrate is extracted with ethyl acetate. Each of the extracts is concentrated to 1/100 by volume and sub~ected to thin-layer chromatography on a silica gel-gl~ss plate (Merck, West Germany~ ~ieselgel* 60 F 254~ 0.25 mm, 20 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 2537 Ao Maytanacine 3 maytansinol propionate or maytansinol which ls thus produced in the cultured broth, are lipophyl and neutral, they can be conveniently recovered by separation and purification procedures which are normally emplo~ed for the harvest of such microbial metabolites. For example~ there may be employed a procedure which utilizes the difference in solubility between the antibiotic and impurity9 means which utilizes the fractionating adsorptive affinity of various adsorbents such as activated carbong macroporous nonionic resins, silica gel, alumlna, etc., a procedure of removing the impuritles by means of ion exchange *TTademark , 2~

resins9 and so forth, as applied singly or in a suitable combination or as applied in repetition.
Since, as aforesaidg maytanacine, maytansinol propionate or maytansinol occur in both the filtrate and cellsg the antibiotics are separated and ~urlfied by means of such an adsorbentg if one is employed, either directly or after a solvent extraction in the case of the filtrate a 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. (1) solvent extraction from the culture broth prior to separation of cells and (2) solvent extraction of the cells and the filtrate obtained by filtration3 centrifugation or other process. To extract the filtrate and cells independently, the follow-ing procedure may be taken advantageously.
The solvents suitable for extraction of the filtrate are water-immiscible organic sol~ents such as fatty ac~d esters 3 e.g. ethyl acetate and amyl acetate; alcohols, e.g. butanol3 halogenated hydrocarbons, e.g. chloroform; and ketonesg e.g. methyl isobutyl ketone. The extraction is carried out at a pH near neutral and, preferablyg the culture fluid previously adjusted to pH 7 is extracted with ethyl acetate.
The extract is washed with water and concentrated under reduced pressure. Then, a nonpolar solvent such as ~z~

petroleum ether or hexane is added to the concentrate and the crude product I containing the active compound is recoveredO Because, on TLCg a number of spots are detected ot;her than maytanac~ne3 maytansinol propionate or maytansinol 9 the product I
is sequentially sub~ected to the following purification procedures. Thus, a routine purification procedure, particularly adsorption chromatography is useful and, for this purpose~ one of those common adsorbents such as silica gel, alumina, macroporous nonionic adsorbent resin, etc. may be employed. For purificatlon from the crude product I, silica gel is most useful. And development may be carried out, for example starting ~ith petroleum ether and hexane and elution is performed by the addition of a polar sol~ent such as ethyl acetate9 acetone, ethanol or methanol. In a typical process, using silica gel (Merck, West Germany9 0.05-O.2 mm) as a carrier, column chromatography is carried out t~ith a serial increase in the hexane to ethyl acetate ratio. The eluate is sampled and investigated by TLC and the fractions containing e~fective ingredients are pooled and concentrated under reduced pressure. ~hen, petroleum ether or hexane is added to the concentrate, whereby the crude product ~ is obtained. S:ince this product still contains impurities, it is further purified as follows. For example, the lO~Zg~9 product ~ may be purified by means of a second silica gel column using a different solvent system.
The developing systemfOr this purpose may conslst in a halogenated hydrocarbon such as dichloromethane or chloroform9with the addition of polar solvent such as an alcohol, e.g. ethanol or methanol, a ketone 3 e.g.
acetone or methyl ethyl ketone, or the like. In this way, maytanacir.e, maytansinol propionate or maytansinol is isolated. The order of solvent systems for the first and second silica gel columns may be reversed and, in addition, ordinary organic solvents may be used in con~unction with the above systems if necessary.
~ lhere a macroporous adsorbent resin is used as purification means for crude product ~ elution of maytanacineg maytansinol propionate or maytansinol ls accomplished with a mi~ture of water with a lower alcohol, a lower ketone or an ester. The lower alcohol may for example be methanol, ethanol, propanol or butanol and the lower ketone may ~or example be acetone or methyl ethyl ketone. The ester may for example be ethyl acetate. In a typical procedure, the crude product ~ is dissolYed in 60% methanol-water and adsorbed on a column of Diaion*HP-10 (Mitsubishi Kasei K~K.). The column is washed with 70% methanol-water and~ then, elution is carried ou~
with 90% methanol-water. In this way, maytanacine, *Trademark :~o~9~
maytansinol propionate or maytansinol is eluted from the column.
In either of the processes described above3 the fractions containing the object components are pooled and concentrated under reduced pressure To the dry product is added 5 to 8 volumes of ethyl acetate and the mixture is allowed to standg whereupon crystals of maytanacine, maytansinol propionate or maytansinol separate3 respectively when the crystals contain maytanacine and maytansinol propionateg they are then separated from each other by means of an adsorbent such as those mentioned hereinbefore. Thus~ using silica gel or a macroporous nonionic adsorbent resin and the above solvents~ the desired compounds may be fractionally eluted. When, for exampleg silica gel is employed~
development is carried out with hexaneg ethyl acetate, or chloroform-methanol~ whereby maytansinol propionate and maytanacine emerge in that order. After detection by TLC~ the fractions are respectively concentrated under reduced pressure and ethyl acetate is added to the concentrates. In this manner9 t.he re.spective compounds can be obtained as crystals. When a macroporous nonionic adsorbent resin.is employed3 gradient elution with a varying ratio of alcohol~
ketone or ester to water may be utilized. For example, by the gradient elution method involving the use of 60Z methanol-water and 95% methanol-water, with 5%
sodium chloride added, maytanacine and maytansinol propionate emerge in the order mentioned. After sampling and detection by TLC 3 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 and3 after drying over phosphorus pentoxide at 70C for 8 hoursg show the followin~
physical and chemical properties. (Table 4) Table 4 Maytanacine Maytansinol Maytansinol propionate C30H39ClN209 C31H41ClN2 9C28H37ClN208 . .

point(C)235-236 188-190 172.5-174 Specific [~]D2 -121+10 [~]D2-127+10 [~]22_313O+loo rotation (C=0.25,CHC13) (C=0.35,CHC13) (C=0022~CHCl3) C59.62 59.93 59.28 Found H6.93 6.82 6.38 (%) N4.28 4.32 5.02 Cl5-74 5.57 6.15 _ Analysis C59.85 59.94 59.52 Calcd. H6.48 6.65 6.60 (%) N4.61 4.51 4.96 Cl5.84 5.71 6.27 99~

-Maytanacine Maytansinol Maytansinol propionate C30H39ClN209 C31H41 1 2 9 C28H37clN208 . I . _ .
Ultraviolet 233(30330) 1 233(30240) 232(32750) spectrum 240(sh.28240) 240(sh.28400) 244(sh.30~50) nm() 252(27850) 252(27650) 25~(31650) 280(5680) 2~0~5740) 281(5750) 288(5660j 288(5710) 288t5700) ....
Infrared 1740gl73091670 1740,173~il670, 1715,167091580 absorption 80 1580 spectrum 15 Mas~
s(pectrum 545,48534703450 5599485~470,450 503,485,4709 Acidgneu- lipophyl and Iipophyl and lipophyl and tral or neutral sub- neutral sub- neutral sub basic stance stance stance Dragendorff Dragendorff Dragendorff Color reaction reaction: reac-~ion:
reactions posi~ive pos~tive positive Beilstein Beilstein Beilstein reaction: reaction: reaction:
positive posi-tive positive Above mentioned data of elemental analysls~
specific rotation9 UV spectra3 IR spectra9 mass spectra9 etc. are in good agreement with the data of maytanacine, maytansinol propionate and maytansinol which are given in the literature Kupchan et al. (The Journal : . . .: . :.,. : . : . .

of American Chemical Society 979 5294 (1975) .
The following examples are further illustrative but by no means limitative of the invention, wherein "part(s)" is based on weight unless otherwise noted and the relationship between "part(s)" and "part(s) by volume" corresponds to that between "gram(s)"
and ''milliliter(s)''g and 'i%i' is based on "weight/volume"
unless otherwise noted.
Example 1 /~laytansinol-, maytanacine- and maytansinol propionate-producible Nocardia No. C-15003 (IF0 13726;
FERM 3992 ~ ATCC 31281) as grown on a medium (yeast extract-malt extract agar) was used to inoculate a 200 parts by volume fermenter containing 40 parts by volume of a seed culture medium (2% glucose9 3%
soluble starch, l$ raw soybean meal a 1% corn steep liquora 0.5% Polypepton*, 0.3% NaCl, 0.5% CaC03, pH 7.0). The inoculated medium was incubated at 28C
~or 48 hours to obtain an inoculum. A 0.5 part by volume portion of the inoculum thus obtained was transferred to a 200 parts by volume fermenter containing 40 parts by volume of a fermentation medium composed of 5% dextrin3 3% corn steep l~quor, 0.1% Polypepton and 0 5% CaC03 (pH 7 ) a ~and cultivated at 28C for 90 hours to give inoculum (seed culture?.
As determined by the serial d~lution method *Trademark ;~:. , ... .:,. -., . : , ... .. , .: . : .

~2g~9 using Tetrahymena pyriformis W as an assay organism and maytansinol propionate as the standard product, the above culture was found to have a titer of 25 ~g/ml.
Example 2 A lO parts by 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 28C for 48 hours. A 500 parts by volume portion of the resultant culture was transferred to a 50000 parts by volume tank of stainless steel containing ~0000 parts by volume of seed culture medium and cultivated at 28C under aeration (30000 parts by volume~min.), agitation [280 r.p.m. (1/2DT)]
and internal pressure (1 k~/cm2) to obtain a seed culture. This culture was used to seed a 200000 parts by volume tank of stainless steel containing 100000 parts by volume of a fermentation medium similar to the one used in Example 1 at an inoculation rate of 10%. The inoculated medium was incubated at 28C under aeration (100000 parts by volume/min.), agitation [200 r.p.m. (1/2 DT)] and internal pressure (1 kg/cm2) 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 ~g/ml.
To 900C)0 parts by volume of the culture obtained above was added 2000 parts of Hyflo-Super-Cel* (Johns -Manville, U. S. A.) and, after thorough mixing, the mixture was filtered on a pressure filter to obtain 85000 parts by volume of filtrate and 32000 parts of moist cells. The filtrate (85000 parts by volume) was stirred and extracted with 30000 parts by volume of ethyl acetate. This procedure was repeated once again. The ethyl acetate layers were pooled, washed t~ice with 30000 parts by volume portions 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 resultant 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 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 and 1000 parts by volume of ethyl acetate-*Trademark ~ 23 methanol (50:1)3 and 1000 parts by volume of ethyl acetate-methanol (25~ with the eluate being collected in 100 parts by volume fractions.
One part by volume portion of each fraction was concentrated to drynessg and ~.1 part by volume o~
ethyl acetate was added to the concentrate. The mixture was spotteed at 2.5 cm from the bottom edge of a silica gel-glass plate (Merck, West Germany, 60 F 2549 0.25 mm, 20 x 20) and developed for about 17 cm with a solvent systemofethyl acetate-metXanol (19:1). After developmentg de~ection was carried out with ultraviolet light t2537A).
The active fractions No. 25-30 of Rf 0.58-O.~3 and the fractlons No.38-40 of Xf 0.25-0.30 were collected and Goncentrated under reduced pressure to about 20 parts ~y vo-ume, respectively. To these con-centrates were added~ each 150 parts by volume of petroleum ether to obtain 5 parts of a crude product and 2 parts of crude màytansinol.
In 10 parts by volume of ethyl acetate was dissolved 0.5 part of the crude product ~ obtained abo~e and the solution was stirred well with 4 parts of silio~ gel (Merck~ West Germany~ 0.05-0.2 mm3. The ethyl acetate was removed under reduced pressure. The residue was applied to the top of a column of 300 parts by volume silica gel and the colu~n was first washed with 500 parts ~ 24 -.. : .: ,., lO~Z999 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 parts by volume o~ chloroform-methanol (10:1). 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 acetateg and the mixture as a sample was subjected to thin layer chromatography (developing system: chloroform-methanol=
9:1) .
The fraction Nos. 40 and 41 absorbing at 2537 A
in the ~one of Rf 0.48-0.50 were collected and concentrated to dryness under reduced pressure. To the residue was added 0.~ part by volume of ethyl acetate and the mixture was allowed to stand~ whereupon 0.05 part 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 methanolg fallowed by addition of 0.1 part of sodium chloride and 5 parts bX volume of water.
A column was packed with 200 parts b~ volume of Diaion*
HP-10 (Mitsubishi Kasei K.K.) and washed with 600 parts by volume of 50% methanol-water containing 5~ of NaCl.
The sample solution prepared above was passed through *Trademark -. : ..... ... ..

10~9~9 the column~ and gradient elution was carried out using 1500 parts by volume of 60% methanol-water containing 5%
NaCl and 1500 parts by volume of g5~ methanol-water.
The eluate was collected in 15 parts by volume fractions and each fraction was investigated by thin layer chromatography. The fractions 130 tQ 135 contained maytanacine, and the fractions 138-142 contained maytansinol propionate~
Each group of fractions was concentrated and dissolved by the addition of 30 ml of water and 50 ml of ethyl acetate. The solution was shaken in a separatory ~unnel and the water layer was separated andg after washing twice with 30ml-portions of water~ the ethyl acetate layer was dried over anhydrous sodium sulfateg concentrated and allowed to stand. In the above manner9 crystals were obtained from each ~roup of fractions. The crystals were collected by filtration and driedO Maytanacine 0.013 part. Maytansinol propionate 0O025 part.
In 3 parts by volume of ethyl acetate was dissolved 0.2 part of the crude maytansinol obtained above and the solution was stirred well with 0.5 part of silica gel (Merckg 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 by volume silica gel and the column was first washed with 150 parts by volume of chloroform and then eluted with 150 parts by volume of chloro~orm-methanol (50:1), 150 parts 3zg~9 by volume of chloroform-methanol (20:1) and 300 par~s by volume of chloroform-methanol ~10:1). The eluate was collected in 10 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: chloroform-methanol=

9:~). 0 me fraction Nos. 50 to 52 ~bsorbing at 2537 A
in the zone of Rf 0.33 to ~.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 crystals of maytansinol were obtained.
Example 3 With stirring, 32000 parts of the cells obtained in Example 2 were extracted ~Jith 50000 parts by volume o~
70% acetone-waterfor 3 hours andg ~heng filtered on a pressure filter. me extraction with 50000 parts by volume of 70% acetone-water and subsequent filtration was repeated once again. The filtrates were pooled and the acetone was remo~ed by concentration under reduced pressure. The resultant aqueous system T~as passed through a column of 5000 parts by volume Diaion*HP-10 (Mitsubishi l~asei K.K.). me column was washed with *Trademark , : . .; :...... . .

~o9z~99 20000parts by volume of waker and 50% aqueous methanol, followed by elution with 15000 parts by volume of 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 combined3 washed with water, dried by the addition of anhydrous sodium sulfate and concentrated under reduced pressure to 200 parts by volume. Following the addition of petroleum etherg the precipi-tate 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 ~ and 0O5 part of crude maytansinol.
Example 4 1000 Parts by volume of the culture of Exa~ple 2 was inoculated into a 2000Q0 parts by volume tank of stainless steel containing 100000 part~ by volume of a seed culture medium and the lnocu~ated medium was incubated at 28C under aeration (100000 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 2000000 parts by volume tank of stainless steel containing 1000000 parts by volume of a fermentation medium as same as that used in Example 1 at a transplantation rate of .... . . .

~ - ; ::: :

~L~9;~g~9 10%. Cultivation was carr~ed out at 28C under aera-tion (1000000 parts by volume/min.),agitation [120 r.p.m.
(1/3 DT)] and internal pressure (1 kg/cm2) for 90 hours.
The resultant culture was found to have a titer of 20 ~g/ml as assayed by the assay procedure described in Example 1.
To 90~00 parts by volume of the above-obtained culture was adde~ ~00000 parts by volume of acetone and, after an hour's stirring, 20Q00 parts o~ Hyflo-Super-Cel*
(Johns -- Manvilleg U.S~A.)9 was addedO The mixture was further stirred and filtered on a pressure filter machine.
To 1700000 parts by volume of the resultant filtrate was added 500000 parts by volum~ of w ter and9 ln a Podb~lniak*
(Podbielniak~ Inc., U.S.A.)g the mixture was extracted wlth 1000~00 parts by volume of ethyl acetateO The ethyl acetate layer was washed with water, dried by the addition of anhydrous sodium sulfate and concentrated under reduced pressure. To the concentrate was added petroleum ether and the resultant precipitate was recovered by filtration and dried. By the above procedure was obtained 680 parts of crude product I. Thereafterg as in Examples 2 and 3 9 this crude product was purified to obtain 1.1 parts of maytanacine,2,2 parts of maytansinol propionate, and 0.1 part of maytansinol.
*Trademark . .: ~ .. .. , ,., .. :

.. ~ . .: : . .

Claims (6)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for preparing a product selected from maytansinol, maytanacine, maytansinol propionate, and mixtures thereof, which comprises cultivating in a suitable culture medium containing assimilable carbon sources and digestible nitrogen sources, a microorganism belonging to the genus Nocardia and capable of producing maytansinol, maytanacine or maytansinol propionate until maytansinol, maytanacine or maytansinol propionate is sub-stantially accumulated therein, and recovering said product.

2. A method as claimed in claim 1, wherein the product recovered is maytansinol.

3. A method as claimed in claim 1, wherein the product recovered is maytanacine.

4. A method as claimed in claim 1, wherein the product recovered is maytansinol propionate.

5. A method as claimed in claim 2, 3 or 4 wherein the microorganism is Nocardia No. C-15003 (ATCC 31281; IFO 13726; FERM 3992).

6. A method as claimed in claim 2, 3 or 4, wherein the microorganism is a maytansinol, maytanacine or maytansinol propionate producing mutant of Nocardia No. C-15003 (ATCC 31281; IFO 13726; FERM 3992).