JPS6213959B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a demethyl maytansinoid compound in which the 20th position of the maytansinoid compound is demethylated, and a method for producing the same. The present inventors searched for a method for converting maytansinoid compounds into other compounds using microorganisms, and found that when a culture of a certain microorganism or its processed product was applied to a maytansinoid compound, the methoxy group at position 20 was We learned that it can be converted to a hydroxyl group, and that a compound in which the 3-position is a hydroxyl group can be obtained by subjecting the obtained compound to a deacylation reaction.As a result of further research based on these findings, we completed the present invention. That is, the present invention provides (1) General formula () [In the formula, X represents Cl or H, and R represents a hydrocarbon residue which may have a substituent. ]
Demethylmaytansinoid compound represented by (), and (2) general formula () [In the formula, X represents Cl or H, and R represents a hydrocarbon residue which may have a substituent. ]
It is characterized by contacting a maytansinoid compound represented by () with a culture of a microorganism belonging to the genus Bacillus or the genus Streptomyces, or a treated product thereof, which has the ability to convert the methoxy group at position 20 of the compound into a hydroxyl group. This is a method for producing a demethylmaytansinoid compound () represented by the general formula (). In the above general formula, the hydrocarbon residue represented by R is a hydrocarbon residue having about 1 to 18 carbon atoms, such as an alkyl group, a cycloalkyl group, an aryl group,
Examples include aralkyl groups. As the alkyl group, for example, carbon number 1-
Alkyl groups of about 18 (e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec
-butyl, tert-butyl, pentyl, hexyl,
(heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 1-ethylpropyl, neopentyl, 1-ethylpentyl, 1- or 2-ethylhexyl group), among which carbon atoms 1-8
An alkyl group of about 100% is preferred. As a cycloalkyl group, for example, carbon number 3
-10 or so cycloalkyl groups (eg, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl group). Examples of the aryl group include a phenyl group and an α- or β-naphthyl group. Examples of the aralkyl group include the above-mentioned aryl group, particularly a group in which a phenyl group is substituted with an alkyl group having about 1 to 4 carbon atoms, such as benzyl, phenethyl, 1- or 3-phenylpropyl, 1-phenylethyl, 1-methyl- Examples include 3-phenylpropyl and 4-phenylbutyl groups. The above hydrocarbon residue may have a substituent, and such a substituent includes, for example, a carbon number of 1-
4 alkoxy groups (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy groups), phenoxy groups, benzyloxy groups, halogen atoms (e.g., fluorine, chlorine,
(bromine, iodine), cyano group, etc., and these substituents may be the same or different and may be substituted with one or more and up to three. Among the groups exemplified as the hydrocarbon residues, substituents for groups other than alkyl groups, that is, cyclic groups such as cycloalkyl groups, aryl groups, and aralkyl groups, include alkyl groups having 1 to 4 carbon atoms in addition to the above-mentioned substituents. groups (e.g., methyl, ethyl, propyl, isopropyl, butyl groups), halogenated alkyl groups having 1-4 carbon atoms (e.g., chloromethyl, bromomethyl, dichloromethyl, chlorodifluoromethyl, trifluoromethyl groups), etc. . Specific examples of the alkyl group having a substituent as R include 2-methoxyethyl, 2-ethoxyethyl, 2-butoxyethyl, 4-ethoxybutyl, chloromethyl, 1- or 2-chloroethyl, 2-bromoethyl, 2-fluoroethyl, 3-chloropropyl, 2,3-dichloropropyl, 2-chloroisopropyl, 1-chloromethyl-2-chloroethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, 1-
Examples of the cycloalkyl group having a substituent include 1-methylcyclobutyl, 1-methylcyclopentyl, 1-methylcyclopentyl, and 2-cyanopropyl.
Examples of the aralkyl group having a substituent such as -methylcyclohexyl include 2-, 3- or 4-chlorobenzyl, 4-bromobenzyl, 4-methylcyclohexyl, etc.
-methoxybenzyl, 2,5- or 3,4-dimethoxybenzyl, 3-chloro-4-methylbenzyl, etc. Examples of the aryl group having a substituent include 2-, 3- or 4-methylphenyl, 2-, 3- or 4-methoxyphenyl, 4
-ethoxyphenyl, 2-, 3- or 4-chlorophenyl, 4-fluorophenyl, 2,4-dichlorophenyl, 2-, 3- or 4-chloromethylphenyl, 4-trifluoromethylphenyl, Examples include 4-bromophenyl and 3-dimethylaminophenyl. The microorganism used in the method of the present invention may be a microorganism belonging to the genus Bacillus or Streptomyces, or a mutant thereof, which has the ability to convert the methoxy group at position 20 of the maytansinoid compound () into a hydroxyl group. Either is fine. Specific examples of microorganisms that can be used in the method of the present invention include Bacillus megaterium IFO 12108, Streptomyces flavotricini IFO 12770, and Streptomyces platensis.
platensis) IFO 12901. above
Microorganisms with IFO numbers are listed in the Institute for Fermentation Osaka List of Cultures, 6th edition, 1978.
Cultures 1978 sixth edition). The microorganisms listed in this list can be obtained from the Fermentation Research Institute. In general, Bacillus and Streptomyces are susceptible to changes in their properties, such as through X-ray irradiation, ultraviolet irradiation, radiation irradiation, and artificial mutagenesis methods (e.g., nitrosoguanidine, ethyleneimine, etc.). Can mutate easily. Even among such mutant strains, any strain having the ability to convert the methoxy group at position 20 of the maytansinoid compound () into a hydroxyl group can be used in the method of the present invention. The medium used for culturing the above-mentioned microorganisms in the method of the present invention may be either liquid or solid as long as it contains a nutrient source that the microorganisms can use, but it is more appropriate to use a liquid medium when processing a large amount. be. The medium contains a carbon source that can be assimilated by the above-mentioned microorganisms, a nitrogen source that can be digested, inorganic substances, micronutrients, and the like as appropriate. Examples of carbon sources include glucose, lactose, sucrose, maltose, dextrin,
Starch, glycerol, mannitol, sorbitol, etc., fats and oils (e.g., soybean oil, lard oil, chicken oil, etc.), and other nitrogen sources include meat extract, yeast extract, dried yeast, soybean flour, corn, etc.
steep liquor, peptone, cotton powder, blackstrap molasses,
Urea, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.) and others are used. Furthermore, salts containing sodium, potassium, calcium, magnesium, etc., metal salts such as iron, manganese, zinc, cobalt, nickel, etc., salts such as phosphoric acid, boric acid, etc., and salts of organic acids such as acetic acid, propionic acid, etc. are used as appropriate. . Other amino acids (e.g., glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline, etc.), peptides (e.g., dipeptides, tripeptides, etc.), vitamins (e.g., B ₁ , B ₂ , nicotinic acid, B ₁₂ ,C,E
etc.), nucleic acids (eg, purines, pyrimidines, derivatives thereof, etc.), and the like. Of course, inorganic or organic acids, alkalis, buffers, etc. may be added for the purpose of adjusting the pH of the medium, or appropriate amounts of oils and fats, surfactants, etc. may be added for the purpose of defoaming. The culturing method may be static culture, shaking culture, or aeration/agitation culture. It goes without saying that for large-scale treatment, it is desirable to use so-called deep aeration agitation culture. It goes without saying that culture conditions vary depending on the state and composition of the medium, the type of microorganism, the culture method, etc., but they are usually selected to have a temperature of 20℃ to 45℃ and an initial pH of around neutrality. It is better. In particular, the temperature during the middle stage of culture is 24℃ ~
Conditions of 37°C and initial pH of 6.5 to 8.5 are desirable.
The culture time may be about 6 to 100 hours, but especially 16 hours.
~60 hours is good. The "culture" used in the present invention refers to what is obtained by the above-mentioned culture. The "processed product" used in the present invention refers to the culture obtained as described above subjected to physicochemical treatment such as filtration, centrifugation, ultrasonication, French press treatment, alumina grinding, lytic enzyme treatment, surfactant or organic Refers to bacterial cells obtained by solvent treatment or crushed bacterial cells containing demethylase. Furthermore, demethylase obtained by purification by a known method, or bacterial cells or demethylase immobilized by a known method can also be used. The method of the present invention is carried out by bringing the raw material compound () into contact with the culture of the above-mentioned microorganism or its treated product. The concentration of the raw material compound in the reaction solution is 1 to 400.
μg/ml, more preferably 1-200 μg/ml, more suitably 50-200 μg/ml. A reaction temperature of 20 to 50°C and a pH of 5 to 10 are suitable, but a temperature of 24 to 40°C and a pH of 6 to 9 are particularly good. The reaction time is
10 minutes to 100 hours, more preferably 1 to 48 hours,
More preferably, it is 8 to 48 hours. Further, the reaction may be carried out under static conditions or under conditions of shaking, aeration or stirring, but it is better to carry out the reaction under shaking, aeration or stirring. A reaction accelerator, enzyme stabilizer, etc. may be added to the reaction solution if desired. Examples of the reaction promoter include coenzymes such as nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), and their precursors. body (e.g. adenine, adenosine, adenylic acid, nicotinamide, flavin, riboflavin, etc.), metal salts (e.g. magnesium chloride, manganese chloride, ferrous chloride, ferric chloride, zinc chloride, etc.), surfactants Example, Triton
100 (manufactured by Rohm and Haas), Brij-
58 (manufactured by Kao Atlas Co., Ltd.)], 3', 5'-cyclic adenylic acid, and the like. Examples of enzyme stabilizers include cysteine, 2-mercaptoethanol, dithiothreitol, sucrose, and glycerin. The novel substance thus obtained can be detected by thin layer chromatography (hereinafter abbreviated as TLC). The reaction solution was extracted with ethyl acetate, concentrated to 1/100 volume, and transferred to a silica gel glass plate (Merck & Co., Kieselgel, West Germany).
60F ₂₅₄ , 0.25 mm, 20 x 20 cm) as a carrier (solvent: chloroform: methanol = 9:
1) Measurement is performed using an absorption image detected by irradiation with ultraviolet rays of 253 Å. In order to collect the target product ( ) from the reaction solution, separation and purification methods normally used for collecting microbial metabolites can be used as appropriate, since this group of substances is weakly acidic and fat-soluble. For example, there are methods that utilize differences in solubility with impurities, methods that utilize differences in adsorption affinity of various adsorbents such as activated carbon, macroporous nonionic resins, silica gel, and alumina, and methods that use ion exchange resins to remove impurities. may be used alone, in combination, or repeatedly. When using the difference in solubility,
Suitable solvents for extraction from liquids include organic solvents that are immiscible with water, such as fatty acid esters such as ethyl acetate and amyl acetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform,
Ketones such as methyl isobutyl ketone are used. Extraction is carried out in a slightly acidic environment, preferably
It is carried out using ethyl acetate from a culture solution adjusted to pH 6. After washing the extract with water, it was concentrated under reduced pressure.
A non-polar solvent such as petroleum ether or hexane is added to collect the crude substance (i) containing the active ingredient. Since many spots other than the new substance demethylmaytansinoid compound (2) were observed in this on TLC, the following purification process was used in stages.
In other words, various types of adsorption chromatography are effective as commonly used purification methods, and commonly used carriers such as silica gel, alumina, macroporous nonionic adsorption resins, etc. can be used as adsorbents; Silica gel is most effectively used for the purification of (i), and new compounds can be developed by developing from non-polar solvents, such as petroleum ether and hexane, and by adding polar solvents such as ethyl acetate, acetone, ethanol, and methanol. Perform elution of demethylmaytansinoid compound (). For example, using silica gel (West German Merck, 0.05-0.2 mm) as a carrier, column chromatography was performed while increasing the mixing ratio of hexane and ethyl acetate, and the eluate was examined by TLC to determine whether demethyl maytansinoid was detected. The fractions containing compound () are collected, concentrated under reduced pressure, and petroleum ether or hexane is added to obtain crude substance (ii). Since this still contains other impurities, the next purification is performed. For example, purification is performed using a second silica gel column using a different solvent system. In this case, developing solvents include halogen-containing hydrocarbons such as dichloromethane and chloroform, as well as polar solvents such as alcohols such as ethanol and methanol, and ketos such as acetone and methyl ethyl ketone. Separate and collect the substance demethylmaytansinoid compound (). First,
The combination of solvent systems for the second silica gel column may be reversed, and other commonly used organic solvents may be appropriately combined. When using a macroporous adsorptive resin as a means of purifying the crude substance (ii), in order to elute the new substance demethylmaytansinoid compound (), it is necessary to combine lower alcohols, lower ketones, or esters with water. Use a mixture. Examples of lower alcohols include methanol, ethanol, propanol, and butanol; examples of lower ketones include acetone and methyl ethyl ketone; and examples of esters include ethyl acetate. As an example, the crude substance (ii) was dissolved in 50V/V% methanol water, passed through a Diaion HP-10 (Mitsubishi Kasei) column, and adsorbed at 50V/V.
After washing with % methanol water and eluting with 90V/V% methanol water, the new target substance demethylmaytansinoid compound () is eluted. The demethylmaytansinoid compound () thus obtained is concentrated under reduced pressure and isolated and collected as crystals from ethyl acetate, or concentrated under reduced pressure.
It is isolated and collected as a powder by adding petroleum ether. Furthermore, by subjecting the demethylmaytansinoid compound () to a deacylation reaction, the general formula () [In the formula, X has the same meaning as above. ] It is also possible to obtain a demethylmaytansinoid compound represented by: In this case, since the acyl group is located at the beta position of the carbonyl group, a commonly used reductive cleavage reaction can be advantageously used, and the reaction can be carried out according to conventional methods. That is, using a complex metal hydride compound [e.g., lithium aluminum hydride (LiAlH ₄ )] at low temperatures (e.g., -20 to 0°C), other functional groups, such as carbonyl groups, epoxy groups, and carbon-carbon double bonds, are used. Compound () can be obtained by reductive cleavage of the O-ester bond at the 3-position without affecting the above. Compound () can be collected and purified in the same manner as the collection and purification method described above. The demethylmaytansinoid compound () detailed above may contain stereoisomers (e.g., D-isomer, L-isomer) if they exist in the structure of R. It also includes mixtures thereof. In general, these isomer relationships already exist in the compound () which is the starting material of the present invention, and these isomers are
As will be detailed later, in the process of producing compound (), generally known separation means,
For example, each isomer may be separated by silica gel chromatography or high-performance liquid chromatography. Generally, in the method of the present invention, the isomerism in compound () is often the same as that in compound (). Furthermore, when a mixture of these isomers is used as the starting material compound (), the target compound () can be obtained in the form of a mixture of isomers. Furthermore, it can be separated into each isomer by a method generally known per se, for example, a separation means such as silica gel column chromatography. The compound () of the present application can be used as an antifungal agent, an antiprotozoal agent, and an antitumor agent. Also, toxicity is low. Compound () or () can also be used as a synthetic intermediate for useful pharmaceuticals. The biological activities of the compounds obtained in the Examples below are shown below. (A) Antimicrobial activity Tryptecase soy agar medium (Baltimore Biologicals Limited, U, S,
A) was used as a test medium, and its ability to inhibit the growth of the following microorganisms was tested using the paper disc method. That is, demethylmaytansinoid compound (
The growth inhibition ability was examined using a paper disk (Toyo Seisakusho, thin type, diameter 8 mm) containing 0.02 ml of a 300 μg/ml solution. the result,
It showed no activity against the following microorganisms. Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Staphylocotcus aureus, Bacillus subtilis, Bacillus cereus, Klebsiella niumoniae, Serratia marcescens, Mycobacterium avium. On the other hand, assay medium [disodium phosphate 3.5 g,
Using an agar plate containing 0.5 g of monopotassium phosphate, 5 g of yeast extract (manufactured by Difco), 10 g of glucose, 15 g of agar, 1000 ml of distilled water, pH 7.0], Hamigera avellanea IFO
7721 was used as a test bacterium and tested using the paper disk method. That is, 100μ of demethylmaytansinoid compound () on the above microorganism-containing plate medium.
The growth inhibiting ability was determined by placing 0.02 ml of the g/ml solution in a paper disk (Toyo Seisakusho, thin type, 8 mm in diameter). As a result, demethylmaytansinoid compound (2018) showed growth inhibition. In addition, a test microorganism of Tetraphymena pyriformis W strain was used, and the assay medium [tryptose/peptone (manufactured by Difco) 20 g, yeast extract 1 g, glucose 2 g, distilled water 1000 ml, 1 molar phosphate buffer
PH7.0, 10ml] at 28℃ for 44 hours or 48 hours.
After culturing for a period of time, the ability of the antibiotic to inhibit the growth of the microorganism was examined by liquid dilution assay. the result,
Demethylmaytansinoid compound () showed growth inhibition. (B) Antitumor activity In a therapeutic experiment using mice in which P-388 tumor cells were intraperitoneally implanted, demethylmaytansinoid compound () was clearly shown to have a survival effect when administered intraperitoneally once a day for 9 consecutive days. Ta. (C) Acute toxicity When acute toxicity was measured by intravenous injection using mice, no deaths were observed for the demethylmaytansinoid compound () at 1000 μg/Kg. As mentioned above, the demethylmaytansinoid compound (2) has a strong ability to inhibit the growth of filamentous fungi and protozoa, and is therefore useful as a fungicidal agent or an antiprotozoal agent. In addition, demethylmaytansinoid compounds () have shown a survival effect on tumor-bearing mammals (e.g., mice), so
It is also expected to be useful as an antitumor agent. The demethylmaytansinoid compound (2) can be advantageously used as a fungicidal agent and an antiprotozoal agent, for example, when examining the bacterial ecology of soil, activated sludge, animal body fluids, and the like. In other words, when separating useful bacteria from soil, or when testing the effects of protozoa or bacteria other than mold during the operation and analysis of activated sludge methods used in wastewater treatment, mold or bacteria living in the sample may be detected. It is possible to selectively develop bacterial ecology without allowing protozoa to develop. Specifically, a test sample is added to a liquid or solid medium, and 0.1 ml of a 1% methanol-containing aqueous solution of demethyl maytansinoid compound (10 to 100 μg/ml) is added per 1 ml of the medium, followed by culturing. Since the compound () of the present invention exhibits a survival effect on tumor-bearing mice, it can also be used as an anti-tumor agent in the treatment of tumor-bearing warm-blooded mammals (e.g. mice, rats, dogs, cats). can. The compound () of the present invention can be administered parenterally or orally as an antitumor agent. When administered parenterally, injection is preferably selected from subcutaneous, intraperitoneal, intravenous, and intramuscular injections, and the dosage is, for example, about 12.5 to about 1000 μg, preferably about 50 to about 800 μg.
Although the range is μg/Kg body weight/one dose, symptoms,
It can be determined as appropriate in consideration of the target animal, etc. The injection solution can be prepared using a conventional method, for example, about 100 μg to about 10 mg of the compound () of the present invention, preferably about 500 μg to about 10 mg.
It may be prepared by dissolving μg to about 10 mg in alcohol (eg, methanol, ethanol) in a ratio of about 0.5 ml, and adding physiological saline to make a total volume of 10 ml. When the dose is small, this solution can be prepared by diluting it with physiological saline. Demethylmaytansinoid compounds () have been found to have significantly increased solubility in water. The maytansinoid compound (), which is the raw material compound used in the method of the present invention, has the formula maytansinol or dechloromaytansinol represented by [wherein, It can be produced by reacting a halogenocarbonic ester represented by ] in the presence of a base. Regarding the above formula (), examples of the halogen atom represented by Y include a chlorine atom and a bromine atom. The reaction is usually carried out in the presence of a solvent, such as ethers (e.g. dimethyl ether, diethyl ether, dioxane, tetrahydrofuran), hydrocarbons (e.g. petroleum ether, hexane, benzene, toluene, xylene).
and mixed solvents thereof, among which tetrahydrofuran is particularly advantageously used. Examples of the base used in the reaction include alkali metals (e.g., lithium, sodium), alkali metal hydrides (e.g., sodium hydride), organometallic compounds of alkali metals [e.g., butyllithium, sec-butyllithium, tert- Examples include butyl lithium, naphthalene sodium, deimucyl sodium (CH ₃ SOCH ₂ Na)], and phenylmagnesium bromide. The amount of the base to be used is usually about 3 to 20 mol, preferably about 4 to 10 mol, per 1 mol of the compound (). Halogeno carbonate () is compound ()1
It is usually used in an amount of about 3 to 20 mol, preferably about 5 to 10 mol, based on the molar amount. Although the order of the reaction is not particularly limited, it is usually convenient to dissolve the compound () in a solvent, add a base solution thereto, and then add the halogenocarbonate () and react. The reaction is usually carried out at a temperature of about -78°C to +50°C, preferably about -30°C to +40°C. The maytansinoid compound (2), which is the raw material compound of the present invention produced by the above method, can be separated and purified from the reaction mixture as appropriate by known separation and purification means such as concentration, solvent extraction, chromatography, recrystallization, etc. It can be used to isolate and collect. Maytansinol used in the production of the above raw material compound () is a compound known as a plant component [Kupchan et al. J. Amer. Chem. Soc., 97 , 5294
(1975)] and can also be obtained by the reductive cleavage reaction of maytansines. Maytansinol is also Nocaldeia Dp.No.
C-15003 (FERM-P No.3992, IFO 13726,
Formula for producing and accumulating ATCC-31281) by culturing it in a medium [In the formula, R' is an acetyl group, a propionyl group, an iso-
butyryl group, n-butyryl group or iso-valeryl group], maytansinol propionate or ansamitocins, by subjecting them to a reductive cleavage reaction using a metal hydride such as _LiAlH4. [Unexamined Japanese Patent Publication No. 53-130693,
See Nature vol.270, p.721 (1977)]. Dechloromaytansinol can be obtained by reducing the compound () with a metal hydride.
As the metal hydride compound, for example, a metal complex compound, especially lithium aluminum hydride (LiAlH ₄ ) is preferable, and the amount used is usually about 1-25 mol, preferably about 4-10 mol, per 1 mol of the compound (). . This reduction reaction is usually conveniently carried out in the presence of a solvent, such as ethers (eg diethyl ether, tetrahydrofuran, etc.), with tetrahydrofuran being particularly preferred. The reaction can be carried out usually at a temperature of about -70°C to +80°C, preferably about -40°C to +20°C. In this reaction, a compound in which only the acyl group at the 3-position of compound () is removed, ie, maytansinol, is often produced as a by-product. After the reduction reaction,
Excess reducing agent is removed by adding water, acetic acid, ethyl acetate, etc., and the reaction solution is then made acidic and extracted with a suitable solvent (eg, ethyl acetate) to obtain a crude product. Dechloromaytansinol can be obtained by separating and purifying this product using silica gel column chromatography or high performance liquid chromatography. The present invention will be explained in more detail below using reference examples and examples, but the scope of the present invention is not limited thereto. Reference Example 1 Antibiotic ansamitocin mixture (Ansamitocin P-2, 12%; Ansamitocin P-3, 71%; Ansamitocin P-
4.17%) was dissolved in 800 ml of dry THF (tetrahydrofuran) and cooled to -50°C using a dry ice-ethanol bath under a stream of dry nitrogen. Next, 13.0g of lithium aluminum hydride (LAH) was added at once, and the temperature was then heated from -50℃ to -22℃.
Stir at ℃ for 2 hours, cool again to -28℃,
g of LAH was added, stirred at -28℃ to -22℃ for 1 hour and 20 minutes, cooled again to -50℃, and then carefully added 750ml of 2N hydrochloric acid dropwise over the next 30 minutes to cool the reaction solution.
Extract three times using 2.6, 1.6, and 0.8 portions of ethyl acetate, and combine the extracts, wash with saturated saline (twice to 100 ml), and dry (MgSO ₄ , 250 g).
The solvent was distilled off under reduced pressure, and the residue (13.6 g) was subjected to silica gel (1.2 Kg) column chromatography, developed and eluted with ethyl acetate/water = 98.5:1.5 (V/V),
Fractionate and collect 400 g of the eluate. Fraction No.35−
52 were combined, the solvent was distilled off, and then vacuum dried to obtain 7.25 g of maytansinol. Fraction No. 53 was obtained in the same manner.
-68 to 1.55 g of an approximately equimolar mixture of maytansinol and dechloromaytansinol and fraction No. 69
0.78 g of dechloromaytansinol is obtained from -86. By reprecipitating this product from chloroform-hexane, 0.71 g of dechloromaytansinol is obtained as a white powder. Melting point: 174-179℃ (decomposition) NMR-spectrum (in CDCl ₃ ) δppm: 0.86
(3H, s), 1.27 (3H, d, J=ca 6Hz),
1.65 (3H, s), 2.63 (1H, d, J=9Hz),
3.07 (1H, d, J=13Hz), 3.23 (3H, s),
3.35 (3H, s), 3.42 (1H, d, J=13Hz),
3.75 (1H, d, J=9Hz), 3.81 (3H, s),
4.37 (1H, m), 5.51 (1H, dd, J=9Hz and 15
Hz), 6.10 (1H, d, J = 11Hz), 6.41 (1H,
dd, J = 11Hz and 15Hz), 6.56 (1H, d, J = 2
Hz), 6.60 (1H, s), 6.70 (1H, mostly s), 6.97 (1H, mostly s), Other MS-spectrum (m/e): 469, Other UV-spectrum (λ ^MeOH _nax ) nm: 231.5 , 241
.Five,
250.5, 277.5, 286 Reference Example 2 Process for producing maytansinol 3-isopropyl carbonate. 57 mg of maytansinol is dissolved in 2.0 ml of dry tetrahydrofuran and treated with 5 molar equivalents of 15% n-butyllithium (in n-hexane) at -20°C in a stream of nitrogen. Add 61 mg of isopropyl chloroformate to this solution, stir for 15 minutes, and bring the reaction solution to 0°C.
0.5 ml of saturated saline and 20 ml of tetrahydrofuran are added to separate the organic layer. After drying the organic layer, the solvent was distilled off, and the residue was separated and purified using silica gel column chromatography to obtain 5 mg.
maytansinol 3-isopropyl carbonate is obtained. Silica gel thin layer chromatography (Merck HPTLC): Rf = 0.44 (developing solvent, chloroform:methanol = 95:5), MS
- Spectrum (m/e), 650 (M ⁺ ), 589 (M ⁺ -
61). Reference Example 3 In the same manner as in Reference Example 2, the compound shown below is obtained. (A) Maytansinol 3-n-octyl carbonate is obtained from maytansinol and n-octyl chloroformate. Silica gel thin layer chromatography (Merck HPTLC): Rf=
0.61 (Developing solvent chloroform: methanol =
95:5), MS-spectrum (m/e), 659
(M ⁺ −61). (B) Maytansinol 3-phenyl carbonate is obtained from maytansinol and phenyl chloroformate. Silica gel thin layer chromatography (HPPTLC manufactured by Merck) Rf = 0.45 (developing solvent chloroform:methanol = 95:5), MS
- Spectrum (m/e), 623 (M ⁺ -61). (C) Dechloromaytansinol 3-benzyl carbonate is obtained from dechloromaytansinol and benzyl chloroformate. Silica gel thin layer chromatography (Merck HPTLC)
Rf=0.54 (developing solvent chloroform:methanol=95:5), MS-spectrum (m/e), 603
(M ⁺ −61). Example 1 Bacillus megaterium IFO 12108 was inoculated into a medium (PH7.5) containing 2% dextrin, 0.5% peptone, 0.5% yeast extract and 0.5% meat extract,
Incubate with shaking at 28℃ for 16 hours. In this culture solution 5
When 100 mg of dechloromaytansinol 3-benzyl carbonate was added and reacted by shaking at 28°C for 48 hours, dechloromaytansinol 3
- Benzyl carbonate completely disappeared, and thin layer chromatography (TLC) showed that demethyldechloromaytansinol 3-benzyl carbonate was produced. Example 2 Ethyl acetate was added to the culture solution 5 obtained in Example 1.
2.5 was added, stirred and extracted, and suctioned through a nuttie containing 15 g of Hyflo Super Cell (manufactured by Johnsmanville Products, USA), and this operation was repeated twice. Combine the ethyl acetate layers and add 1 part water.
The mixture is washed twice with water, dried by adding 30 g of anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude substance (i). The obtained crude substance (i) was dissolved in a small amount of chloroform and mixed with silica gel (manufactured by Merck, Germany) prepared in advance.
Pour 10 g of the column (0.05-0.2 mm) into the top of a column (diameter 1.2 cm), pour in 300 ml of chloroform, and elute with chloroform/methanol (20:1). Fractionate the eluate into 20 ml portions and transfer each fraction to a silica gel glass plate (manufactured by Merck, Germany).
Kieselgel 60F ₂₅₄ 0.25mm, 20×20) from the bottom edge
Spot it at a position of 2.5 cm and develop it about 17 cm using the developing solvent chloroform:methanol (9:1). After development, examine the absorption image under ultraviolet light (2537Å) Rf0.44
Collect fractions that are absorbed nearby and concentrate under reduced pressure.
63 mg of crude material (ii) is obtained. Dissolve the crude substance (ii) in a small amount of chloroform and apply it on six silica gel glass plates in a straight line at a position 2.5 cm from the bottom edge of each plate.After developing with chloroform:methanol (9:1), scrape off the absorption image of Rf0.44. Extract twice with ethyl acetate containing a small amount of water, wash the extracted ethyl acetate with water, dry with sodium sulfate, concentrate under reduced pressure, add petroleum ether, and dry the precipitate. 48 mg of white powder of demethyldechloromaytansinol 3-benzyl carbonate is obtained. Silica gel thin layer chromatography (manufactured by Merck): Rf = 0.44 (developing solvent chloroform:methanol = 9:1), MS-spectrum (m/e),
589 (M ⁺ −61). Example 3 Streptomyces flavotricini IFO 12770
, dextrin 1%, glucose 1%, glycerol 1%, peptone 0.5%, yeast extract 0.5%, meat extract 0.5%, salt 0.3% and calcium carbonate 0.5
% in a medium (PH7.2) and culture with shaking at 28°C for 24 hours. When 50 mg of maytansinol 3-phenyl carbonate was added to this culture solution 5 and reacted by shaking at 28°C for 48 hours, maytansinol 3-phenyl carbonate decreased and demethylmaytansinol 3 -TLC shows that phenyl carbonate is formed. Example 4 The culture solution obtained in Example 3 was purified in the same manner as in Example 2, subjected to thin layer chromatography in the same manner as in Example 2, and a fraction around Rf0.38 was collected, and demethylmaytansinol was obtained. 24 mg of crude material (ii) of 3-phenyl carbonate is obtained. Demethylmaytansinol 3-phenyl carbonate was purified in the same manner as in Example 2.
Get 13mg. Silica gel thin layer chromatography (manufactured by Merck & Co.): Rf = 0.38 [developing solvent chloroform:methanol (9:1)] MS-spectrum (m/e), 609 (M ⁺ -61). Example 5 Streptomyces platensis IFO 12901 is inoculated into the same medium as in Example 3, and cultured with shaking at 28°C for 24 hours. When 50 mg of maytansinol 3-isopropyl carbonate is added to this culture solution 5 and reacted by shaking at 28°C for 48 hours, maytansinol 3-isopropyl carbonate disappears and demethylmaytansinol 3-isopropyl TLC that carbonate is produced
It is recognized in Example 6 The culture solution obtained in Example 5 was purified in the same manner as in Example 2, and the same thin layer chromatography as in Example 2 was performed to collect the fractions around Rf0.43. 18 mg of crude substance (ii) of Nor 3-isopropyl carbonate is obtained. Thereafter, purification was carried out in the same manner as in Example 2 to obtain 14 mg of demethylmaytansinol 3-isopropyl carbonate. Silica gel thin layer chromatography (manufactured by Merck & Co.): Rf = 0.43 [developing solvent chloroform:methanol (9:1)] MS-spectrum (m/e), 575 (M ⁺ -61). Example 7 Using Bacillus megaterium IFO 12108,
By the same method as in Examples 1 and 2, the following compounds are obtained. raw materials, products and products
Rf value [Developing solvent: CHCl ₃ :MeOH=9:1, plate: silica gel glass plate (Merckk 60
F ₂₅₄ thickness 0.25mm)].

[Table] Example 8 Streptomyces flavotrichini IFO 12770
Using the same method as in Examples 3 and 4, the following compounds are obtained. Rf values of raw material compounds, products, and products [Developing solvent: CHCl ₃ :MeOH=
9:1, plate: silica gel glass plate (Merck 60 F ₂₅₄ thickness 0.25 mm)].

[Table] Example 9 Using Streptomyces platensis IFO 12901, the following compound was obtained in the same manner as in Examples 5 and 6. Rf values of raw material compounds, products, and products [Developing solvent: CHCl ₃ :MeOH=9:
1. Plate: Silica gel glass plate (Merck 60 F ₂₅₄ thickness 0.25 mm)].

[Table] Example 10 10 mg of powder of demethyldechloromaytansinol 3-benzyl carbonate obtained in Example 2
was dissolved in 4 ml of tetrahydrofuran, cooled to -5°C, and 10 mg of lithium aluminum hydride was added. Transfer the reaction solution to a water bath and stir for 30 minutes. After adding 2 ml of ethyl acetate and 1.2 ml of 1/200N hydrochloric acid, further add 10 ml of ethyl acetate for extraction.
The ethyl acetate layer was washed with water, anhydrous sodium sulfate was added, dried, and concentrated under reduced pressure. Performed preparative TLC using silica gel, developed with ethyl acetate/methanol (19:1) for 17 cm, and obtained an absorption image around Rf 0.20 to 0.25. Scrape off, extract with ethyl acetate containing a small amount of water, wash with water, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain 6.2 mg of demethyldechloromaytansinol powder. When dissolved in a small amount of ethyl acetate and left to stand, crystals will precipitate. The crystals are filtered and then dried. Yield 4.8mg. Melting point 198-201℃ (decomposition) Elemental analysis (%) Actual value C 62.68; H 7.21; N 5.16; O 24.92 Theoretical value C ₂₇ H ₃₆ N ₂ O ₈ C 62.77; H 7.02; N 5.42; O 24.77 MS-spectrum (m/e) 455, 437 UV-spectrum (λ ^MeOH _nax ) nm: 230, 240,
248, 277, 285 Example 11 10 mg of demethylmaytansinol 3-isopropyl carbonate powder obtained in Example 6 was dissolved in 4 ml of tetrahydrofuran and cooled to -5°C.
Add 10 mg of lithium aluminum hydride. This was treated in the same manner as in Example 9, subjected to preparative TLC using silica gel, and developed with ethyl acetate/methanol (19:1) for 17 cm.
The absorption image around ~0.32 was scraped off, extracted with ethyl acetate containing a small amount of water, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain demethylmaytansinol.
6.8mg obtained. Melting point 196℃ Elemental analysis value (%) Actual value C 58.62; H 6.59; N 4.82; Cl 6.21 Calculated value C ₂₇ H ₃₅ ClN ₂ O ₈ C 58.85; H 6.40; /e) 489, 471 UV-spectrum (λ ^MeOH _nax ) nm: 232, 242,
251, 280, 288

Claims (1)

[Claims] 1. General formula [In the formula, X represents Cl or H, and R represents a hydrocarbon residue which may have a substituent. ]
Demethylmaytansinoid compound represented by 2 General formula [In the formula, X represents Cl or H, and R represents a hydrocarbon residue which may have a substituent. ]
A general formula characterized by contacting a maytansinoid compound represented by the above with a culture of a microorganism belonging to the genus Bacillus or the genus Streptomyces, or a treated product thereof, which has the ability to convert the methoxy group at position 20 of the compound into a hydroxyl group. [In the formula, X and R represent the same meanings as above. ] A method for producing a demethylmaytansinoid compound represented by