KR820001342B1 - Process for rpeparing demethyl maytansinoids compounds - Google Patents

Abstract

Title compds. (I; X = Cl, H; R = substituted hydrocarbon group), having antiprotozoal and antitumor activities, were prepd. by introducing OH into the 20 position of a maytansinoid compd. (II) employing a member of the genus streptomyces. Thus, streptomyces pratensis was cultivated in a nutrient medium at 28≰C for 24 hr. The cultivated liq. 5l and maytansinol-3-isopropyl carbonate 20 mg were reacted at 28≰C for 48 hr to give demethyl maytansinol-3-isopropyl carbonate.

Description

Preparation of Dimethyl Maitan Synoid Compound

The present invention relates to a method for preparing a dimethyl maytansinoid compound in which the 20th position of the maytansinoid compound is dimethylated.

The present inventors have searched for a method for converting a maytansinoid compound into another compound by a microorganism, and when a culture or a processed product of a microorganism is applied to the maytansinoid compound, the methoxy group at position 20 is a hydroxyl group. It was found that the compound was converted into the amine, and the obtained compound was added to the deacylation reaction to obtain a compound having a hydroxyl group at the third position.

That is, the present invention is of general formula (II)

Wherein X represents Cl or H and R represents a hydrocarbon residue which may have a substituent, respectively.

A maytansinoid compound (II) represented as a contact with a culture of a microorganism belonging to the genus Bacillus or Streptomyces or having a capability of converting a methoxy group at the 20th position of the compound into a hydroxyl group or a treated product thereof Formula (I)

It is a manufacturing method of the dimethyl maytansinoid compound (I) represented as a.

In the general formula, the hydrocarbon residue represented by R includes a hydrocarbon residue having about 1 to 18 carbon atoms, for example, an alkyl group, a cycloalkyl group, an aryl group, an araryl group, and the like.

As said alkyl group, For example, a C1-C18 alkyl group (for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertt- butyl, pentyl, hexyl, heptyl, octyl, nonyl, Decyl, undecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 1-ethylpropyl, neopentyl, 1-ethylpentyl, 1- or 2-ethylhexyl group), and in the air about 1-8 The alkyl group of is preferable.

As a cycloalkyl group, a C3-C10 cycloalkyl group (for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooxyl, adamantyl group) is mentioned, for example.

As said aryl group, a phenyl group, (alpha)-, or (beta) -naphthyl group is mentioned, for example.

As the aralkyl group, a group in which the aforementioned aryl group, in particular, a phenyl group is converted to an alkyl group having 1 to 4 carbon atoms, for example benzyl, phenethyl, 1- or 3-phenylpropyl, 1-phenylethyl, 1-methyl- 3-phenylpropyl, 4-phenylbutyl group, etc. are mentioned.

The above hydrocarbon residue may have a substituent, and examples of such substituent include an alkoxy group having 1 to 4 carbon atoms (eg, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec- Butoxy group), phenoxy group, benzyloxy group, halogen atom (e.g., fluorine, chlorine, cancellation, oxo), cyano group and the like, and these substituents may be substituted one or three or the same or differently. .

As substituents of cyclic groups such as cycloalkyl group, aryl group, aralkyl group and the like other than the alkyl group in the groups exemplified as the above hydrocarbon residues, other alkyl groups having 1 to 4 carbon atoms (e.g., methyl, ethyl, propyl, Isopropyl, butyl groups), halogenated alkyl groups having 1 to 4 carbon atoms (e.g., chloromethyl, bromomethyl, dichloromethyl, chlorodifluoromethyl, trifluoromethyl group) and the like.

As a specific example of the alkyl group which has a substituent as R, For example, 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-trifluoromethyl, 1- or 2-cyanopropyl, and the like, and examples of the cycloalkyl group having a substituent include 1-methylcyclobutyl, 1-methylcyclopentyl, 1-methylcyclohexyl, and the like. As the aralkyl having this substituent, for example, 2-, 3- or 4-chlorobenzyl, 4-bromobenzyl, 4-methoxybenzyl, 2,5- or 3,4-dimethoxybenzyl, 3-chloro 4-methylbenzyl and the like, and 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- Luo the like phenyl, 2,4-dichlorophenyl, 2-, 3- or 4-methylphenyl, 4-fluoro-trimethylphenyl, 4-bromophenyl, 3-dimethylaminophenyl respectively Yeager.

The microorganism used in the method of the present invention may be any of microorganisms belonging to the genus Bacillus or Streptomyces, which has the ability to convert the methoxy group at the 20th position of the maytansinoid compound (II) into a hydroxyl group, and the mutant strains thereof.

As a specific example of the microorganism which can be used by the method of this invention, it is Bacillus, for example. Megathelium IFO 12108, Streptomyces. Prabotryciny IFO 12770, Streptomyces. Pratensis IFO 12901 and the like. The above-mentioned IFO numbered microorganisms are listed by the Foundation. off. It is listed in the Institute for fermentation osaka list of cultures 1978 Sixth edition. The microorganisms described in this list can be obtained from the publication laboratory.

In general, Bacillus spp. And Streptomyces spp. Are easily changed in their properties, for example, X-ray irradiation, UV irradiation, irradiation, artificial modifiers (eg, nitrosoguanine, ethyleneamine, etc.) It can be easily transitioned by a transition means or the like.

Even in such a mutant strain, any of those having the ability to convert a maytansinoid compound (II) to a methoxy-based hydroxyl group at position 20 can be used in the method of the present invention.

The medium used for culturing the microorganisms in the method of the present invention may be liquid or solid as long as it contains a nutrient source that the microorganism can use, but it is more suitable to use a liquid medium when processing a large amount. The medium is suitably formulated with a carbon source capable of assimilation of the microorganisms, a digestible nitrogen source, an inorganic substance, a trace element, and the like. As the carbon source, for example, glucose, lactose, sucrose, maltose, dextrin, starch glycerol, mannitol, sorbitol, and other fats and oils (e.g., soybean oil, laard oil, chicken oil, etc.), etc. Dry Yeast, Soybean Meal, Coon. Stiff. Lacca, peptone, cottonseed meal, waste molasses, urine, ammonium salts (eg ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.) are used. Salts containing sodium, potassium, calcium and magnesium, metal salts such as iron, manganese, zinc, cobalt and nickel, salts such as phosphoric acid and boric acid, and salts of organic acids such as acetic acid and propionic acid are appropriately used.

Other amino acids (e.g. glutamic acid, aparaginic acid, alanine, glycine, lysine, methionine, florin, etc.), peptides (e.g. dipeptides, tripeptides, etc.), vitamins (e.g., B ₁ , B ₂ nicotinic acid, B ₁₂ , C , E, etc.), nucleic acids (for example, prin, pyrimidine and derivatives thereof) and the like. Of course, an inorganic or organic acid, an alkali, a buffer, or the like may be added for the purpose of adjusting the pH of the medium, or an appropriate amount such as an oil or fat or a surfactant may be added for the purpose of defoaming.

The means for culturing may be political culture, or a means such as shaking culture or aeration stirring culture may be used. It goes without saying that it is preferable to carry out so-called deep aeration agitation culture for mass processing. Naturally, the conditions of the culture are not constant depending on the condition of the medium, the composition, the type of microorganisms, the means of the culture, and the like. In particular, the medium temperature is preferably 24 ° C.-37 ° C., and the initial pH is 6.5-8.5. Incubation time is good as about 6-100 hours, but especially at 16-60 hours.

The culture used in this invention means what was obtained by said culture.

The treated product used in the present invention is a physicochemical treatment, for example, filtration, centrifugation, sonication, French press treatment, alumina grinding, lytic enzyme treatment, surfactant treatment or organic solvent treatment. The cell lysate containing the cell or demethylation enzyme obtained as such. Moreover, the demethylation enzyme obtained by purification by a well-known method or the microbial cell or demethylation enzyme immobilized by a well-known method can also be used.

The method of the present invention is carried out by bringing a raw material compound (II) into contact with a culture of the microorganisms described above or a treated product thereof. The concentration of the starting compound in the reaction solution is 1-400 µg / ml, more preferably 1-200 µg / ml, and 50-200 µg / ml is more suitable. Although reaction temperature is suitable for 20-50 degreeC and pH 5-10, temperature 24-40 degreeC and pH 6-9 are especially preferable. The reaction time is 10 minutes-100 hours, more preferably 1-48 hours, again preferably 8-43 hours. Although the reaction may be under conditions of shaking, aeration or agitation even under stationary conditions, shaking, aeration or agitation is preferred. In the reaction solution, a reaction accelerator, an enzyme stabilizer, or the like may be added as desired. As the reaction promoter, for example, coenzymes such as nicotinamide adenine nucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and precursors thereof (examples) , Adenine, adenosine, adenylic acid, nicotinamide, flavin, riboflavin, etc., metal salts (e.g. magnesium chloride, manganese chloride, ferrous chloride, ferric chloride, zinc chloride, etc.), surfactants, Triton X- 3 ', 5'- cyclic adenylic acid, etc. are mentioned, such as 100 (made by Rohm and Haas) and Brij-58 (made by Kao Atlas). As a fermentation stabilizer, cysteine, 2-mercaptoethanol, dithiositol, sucrose, glycerin, etc. are mentioned, for example.

The detection of the novel substance (I) thus obtained can be measured by thin layer chromatography (hereinafter abbreviated as TLC). The reaction solution was extracted as ethyl acetate and the concentrated solution to 100/1 volume was attached to TLC with a silica gel glass plate (West Germany Melk Co., Ltd., Kigel Gel 60F254, 0.25 mm, 20-20 cm) as a carrier (solvent: chloroform: methanol = 9: 1), It measures with the absorbed phase which irradiates 2537 Hz of ultraviolet-rays and detects.

In order to collect the target substance (I) from the reaction solution, since the present substance group is slightly acidic and fat-soluble, the separation and purification method usually used for collecting microbial metabolites can be suitably used. For example, any of means for using the difference in solubility with impurities, means for using the difference in adsorption affinity of various adsorbents such as activated carbon, macroporous nonionic resin, silica gel and alumina, and means for removing impurities by ion exchange resin Each of them is used alone, in combination, or repeatedly. When a difference in solubility is used, suitable solvents for extraction from the filtrate are organic solvents which do not mix with water, for example fatty acid esters such as ethyl acetate and amyl acetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform and methyl isobutyl Ketones, such as ketones, are used. Extraction is performed in the vicinity of weakly acidic acid, and is preferably performed using ethyl acetate in a culture filtrate adjusted to pH 6. The extract is washed with water, concentrated under reduced pressure, and a crude substance (i) containing an active ingredient is collected by adding a nonpolar solvent such as petroleum ether or hexane. In this, many spots other than the novel substance demethyl maytansinoid compound (I) are seen on TLC, and the following purification process is used step by step. That is, various adsorption chromatography is effective as a commonly used purification method, and as the adsorbent, carriers generally used, for example, silica gel, alumina, macroporous nonionic adsorption resin, and the like can be used. Purification from) is the most effective use of silica gel, starting with a nonpolar solvent such as petroleum ether, hexane and adding a new polar solvent such as ethyl acetate, acetone, ethanol and methanol. The maytansinoid compound (I) is eluted. As an example, silica gel (West Germany Melk 0.05-0.2mm) is used as a carrier, column chromatography is performed while increasing the mixing ratio of hexane and ethyl acetate sequentially, and the eluate is irradiated with TLC to give a demethyl maytansinoid compound ( Collect the fructose containing I), concentrate under reduced pressure and add petroleum ether or hexane to obtain crude (ii). Since this part still contains another impurity, the following purification is performed.

For example, it refine | purifies by the 2nd silica gel column which changed the solvent system. In this case, the development solvent starts with halogen-containing hydrocarbons such as dichloromethane and chloroform, and a new substance is added by adding a polar solvent such as alcohols such as ethanol and methanol and ketones such as acetone and methyl ethyl ketone. Methyl maytansinoid compound (I) is isolate | separated and collected. In the combination of the solvent systems of the first and second silica gel columns, the front and rear surfaces may be reversed, and other commonly used organic solvents may be appropriately combined.

When the macroporous adsorbent resin is used as the refining means of the crude substance (ii), to dissolve the novel substance tetametantansinoid compound (I), a lower alcohol, or a mixture of lower ketones, esters and water is used. use. As lower alcohols, for example, methanol, ethanol, propanol, butanol and the like are lower ketones. For example, acetone, methyl ethyl ketone, esters and ethyl acetate can be used. As an example, the crude material (ii) was dissolved in 50V / V% methanol water, passed through a DIION HP-10 (Mitsubishi Chemical Co., Ltd.) column for adsorption, and 90V / V% after washing with 50V / V% methanol water. Elution with methanol water elutes the novel substance Temethylmaytansinoid Compound (I) as a target.

The demethyl maytansinoid compound (I) thus obtained is concentrated under reduced pressure and isolated from ethyl acetate as crystals or concentrated under reduced pressure, and isolated by powdering with petroleum ether.

Again, deacylated reaction of demethyl maytansinoid compound (I) was carried out to general formula (III).

Wherein X has the same meaning as electricity.

A demethyl maytansinoid compound represented as can be obtained. In this case, since the acyl group is located at the beta position of the carbonyl group, a commonly used reductive cleavage reaction is advantageously used, and the reaction can be carried out in accordance with the conventional method.

That is, a complex metal hydrogen compound (eg, lithium aluminum hydride (LiA ₁ H ₄ )) is used at low temperature (eg, -20-0 ° C), and other functional groups such as carbonyl group, epoxy group, carbon-carbon doublet Compound (III) can be obtained by reducing cleavage of the 0-ester bond of the third position without affecting the bonding or the like. Extraction and purification of compound (III) can be carried out in the same manner as in the aforementioned extraction and purification method.

The demethylmaytansinoid compound (I) described in detail above, when stereoisomers (eg, D-isomers, L-isomers) are present in the structure of R, includes each of these isomers and even mixtures thereof.

In general, these isomers are already present in the compound (II) which is a starting material of the present invention, and these isomers are generally self-producing in the process of preparing the compound (II), as described in detail later. In some cases, it is separated into individual isomers by known separation means, for example, silica gel chromatography or high performance liquid chromatography.

In general, in the method of the present invention, the heterogeneity in the compound (I) is often the same as that in the compound (II).

In addition, when the mixture of these isomers is used as compound (II) which is a starting material, the target object (I) is obtained as a form of the mixture of isomers. In general, it can be separated into individual isomers by a separation means such as a method known per se, for example, silica gel column chromatography.

Compound (I) of the present invention can be used as a fungicide, an antiprotozoal agent or an antitumor agent. In addition, the toxicity is low.

Compound (I) or (III) can also be used as a synthetic intermediate of a useful medicine.

The biological activity with respect to the compound obtained by the Example mentioned later is shown below.

A) antimicrobial activity

Trypticase. The soy agar medium (Baltimore Biologicals Limited.U.S.A.) Was used as a test medium, and paper was developed for the microorganisms shown below. Checked by the disk method. That is, 0.02 ml of a 300 µg / ml solution of the tetymethanane synoid compound (I) on the following microbial-containing platelet medium was paper. The growth hypotension was examined by embedding in a disk (Toyo Seisakusho, thin, 8 mm in diameter). As a result, the following microorganisms did not show activity.

Esheriah. collie. Proteus, Bulgari, Proteus, Miraphyris, Pseudomonas, Aelginosa, Staphylococcus, Aureus, Bacillus, Zvetiris, Bacillus. Cereus, Crevcela. Pneumoniae, Serratia, Marsessen, Mycobacterium. Abium.

On the other hand, using agar plate of assay medium [3.5 g of disodium phosphate, 0.5 g of 1 potassium phosphate, 5 g of yeast Ekis (made by Diffco Corporation), 10 g of glucose, 15 g of agar, 1000 ml of distilled water, pH 7.0], was used. Paper with Abelanea IFO 7721 as test bacteria. It checked by the disk method. That is, 0.02 ml of a solution of 1000 µg / ml of the demethyl maytansinoid compound (I) was papered on the microorganism-containing plate medium described above. The growth hypotension was examined by embedding it in a disc (Toyosei Corporation, thin, 8 mm in diameter). As a result, the demethyl maytansinoid compound (I) showed growth inhibition.

In addition, tetrahimena. Pyriformis W strain was used as a test microorganism, and assay medium (triptoose, peptone (20 g of Diff Co., yeast 1 g, glucose 2 g, distilled water 1000 ml, 1, 1 mol phosphate buffer pH 7.0, 10 ml) was used. The culture was inhibited for 44 hours to 48 hours at 28 ° C., and the antimicrobial growth inhibition ability of the antibiotic was determined by liquid dilution assay, and as a result, the demethylmaytansinoid compound (I) showed growth inhibition.

B) antitumor activity

In the therapeutic experiment with mice in which P-388 tumor cells were transplanted intraperitoneally, the demethylmaytansinoid compound (I) clearly showed a life-saving effect in continuous administration once a day for 9 days.

C) Acute Toxicity

Mice were used to measure acute toxicity by intravenous injection. As a result, no demethylmaytansinoid compound (I) died at 1000 µg / kg.

As described above, the demethylmaytansinoid compound (I) has a strong growth inhibitory ability against filamentous fungi and protozoa, and thus is also useful as a fungicide or an antiprotozoal agent. In addition, the demethylmaytansinoid compound (I) is expected to be useful as an antitumor agent because of its life-saving effect on a mammal (eg, a mouse) having a tumor.

If demethyl maytansinoid compound (I) is used as an antiseptic and an antiprotozoal agent, for example, it can be advantageously used when examining bacterial ecology such as soil, activated sludge or animal body fluid. In other words, when separating useful bacteria from the soil, or when testing the action of bacteria other than protozoa or fungi in the operation and interpretation of the activated sludge method used for wastewater treatment, it does not develop live mold or protozoa in the sample. It is possible to selectively develop bacterial ecology.

Specifically, the test sample is added to a liquid or a solid medium, and 0.1 ml of a 10-100 µg / ml 1% methanol-containing aqueous solution of demethylmaytansinoid compound (I) per 1 ml of the medium is added and cultured.

Since the compound (I) of the present invention has a life-long effect on, for example, a mouse having a tumor, it may be used as an anti-tumor agent in the treatment of tumor-bearing warm blood mammals (eg, mice, rats, dogs, cats). It may be.

When the compound (I) of the present invention is administered as an antitumor agent, it can be carried out parenterally or orally. In the case of parenteral administration, it is preferable to use by injection, for example, it is appropriately selected from subcutaneous, intraperitoneal, intravenous, intramuscular injection and the like, and the dosage is, for example, about 12.5 to about 1000 µg. Is in the range of about 50 to about 800 μg / kg body weight per dose, but may be appropriately determined in consideration of symptoms, animals, and the like. The injection solution is prepared by dissolving about 100 μg to about 10 mg of the usual means, for example, Compound (I) of the present invention, preferably about 500 μg to about 100 mg in the ratio of about 0.5 ml of alcohol (eg, methanol, ethanol). In addition, it may be prepared by adding physiological saline to a total amount of 10 ml. If the dose is small, this solution can be prepared by diluting it with physiological saline.

It is recognized that the demethyl maytansinoid compound (I) is remarkably increased in solubility in water.

The maytansinoid compound (II) which is a raw material compound used in the method of the present invention is, for example, a formula

Wherein X is of the same meaning as the periodical formula of maytansinol or dechloro maytansinol

It can be produced by reacting the halogenocarbonate ester represented by [wherein Y represents a halogen atom and R has the same significance as the foregoing] in the presence of a base.

Regarding the formula (V), examples of the halogen atom represented by Y include a chlorine atom, a cancel atom and the like.

The reaction is carried out in the presence of a common solvent. Examples of such a solvent include ethers (e.g., dimethyl ether, diethyl ether, dioxane, tetrahydrofuran), hydrocarbons (e.g., petroleum ether, hexane, benzene, toluene , Xylene), mixed solvents thereof, and the like, and tetrahydrofuran is particularly suitable for use.

Examples of the base used for the reaction include alkali metals (e.g. lithium, sodium), alkali metal hydrides (e.g. sodium hydride), and organometallic compounds of alkali metals [e.g. butyllithium, sec-butyllithium, tert -butyl lithium, naphthalene sodium, dimsil sodium (CH ₃ SOCH ₂ Na)] and the like can be mentioned phenyl magnesium bromide. The use amount of base is about 3-20 mol normally with respect to 5 mol of compounds (IV), Preferably it is about 4-10 mol.

The halogenocarbonate ester (V) is usually used in an amount of about 3 to 20 moles, preferably about 4 to 10 moles, based on 1 mole of the compound (IV).

The order of the reaction is not particularly limited, but it is best to dissolve the common compound (IV) in a solvent, add a base solution thereof, and then add a halogenolanate ester (V) to react.

The reaction is usually carried out under temperature conditions of about -78 ° C to + 50 ° C, preferably about -30 ° C to + 40 ° C.

The maytansinoid compound (II), which is a raw material compound of the present invention, prepared according to the above-described method, may be suitably used in a reaction mixture by using known separation and purification means such as concentration, solvent extraction chromatography, recrystallization, and the like. I can isolate it.

Maytansinoids used in the preparation of the above-mentioned compound (II) are known compounds as plant components [Kupchan et al. J. Amer. Chem. Soc., 97, 5294 (1975)], and can also be obtained by the reductive series of maytansine oil.

In addition, maytansinol is nocardia SP. No, C-15003 (FERM-P No, 3992, IFO 13726, ATCC-31281)

Wherein, R 'represents an acetyl group, propionyl group, isc-butyryl group, n-butyryl group, or isc-vareryl group] maytanacin, maytansinol, propionate or ansamytocins Can also be prepared by a reductive reheating reaction using a metal hydride, for example LiAlH ₄ [Publication No. 53-130693, Nature Vol. 270. p, 721 (1977).

Dechloromethanesinol is obtained by reducing compound (VI) as a metal hydrogen compound. As the metal hydrogen compound, for example, a metal complex compound, particularly lithium aluminum hydride (LiAlH ₄ ), is preferably used. The amount of the metal hydrogen compound is usually about 1 to 25 moles, preferably about 4 to 10 moles per mole of the compound (VI). .

It is preferable to perform this reduction reaction normally in the presence of a solvent, As such a solvent, ethers (for example, diethyl ether, tetrahydrofuran etc.) are used, Especially tetrahydrofuran is preferable.

The reaction can usually be carried out 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 3rd position of compound (VI) is normally released, that is, maytansinol is often produced by-product. After the reduction reaction, the excess reducing agent is removed by addition of water, acetic acid or ethyl acetate, and then the reaction solution is acidified and then extracted with a suitable solvent (e.g. ethyl acetate) to give a crude product. Dechloro maytansinol is obtained by separating and refining this using silica gel chromatography or the like.

(FERM: Institute of Microbiology and Industrial Technology, KAIST

IFO: Fermentation Research Institute

ATCC: The American Type Culture Collection, U.S.A.)

Hereinafter, the present invention will be described in detail with reference to examples and examples, but the scope of the present invention is not limited thereto.

Reference Example 1

15.0 g of the antibiotic ansamytocin mixture (Ansamytocin P-2.12%: copper P-3.71%: copper P-4.17%) is dissolved in 800 ml of dry THF (tetrahydrofuran) and dried ice-ethanol bath under a dry nitrogen stream. Cool to -50 ° C using. Subsequently, 13.0 g of lithium aluminum hydride (LAH) was added all at once, then stirred at -50 ° C to 22 ° C for 2 hours, cooled again to -28 ° C, 3 g of LAH was added, and 1 at -28 ° C to 22 ° C. After stirring for 20 minutes, the mixture was cooled to −50 ° C., and then carefully added dropwise to 750 ml of 2N hydrochloric acid for 30 minutes, and the reaction solution was extracted three times using 2.6 L, 1.6 L, and 0.8 L of ethyl acetate. The combined extracts were washed with saturated brine (twice per 100 ml) and dried (MgSO ₄ , 250 g). The solvent was distilled off under reduced pressure, and the residue (13.6 g) was added to silica gel (1.2 kg) column chromatography, and the mixture was eluted with ethyl acetate / water = 98.5: 1.5 (V / V), and the eluate was collected by fractionation of 400 g. Fraction No. 35-52 were combined, and the solvent was distilled off and dried in vacuo to obtain 7.25 g of maytansinol. 1.55 g of a nearly equivalent molar mixture of maytansinol and dechloromaytansinol at 53-68 and fraction no. 0.78 g of dechloromaytansinol is obtained in 69-86. 0.71 g of dechloromaytansinol is obtained as a white powder by reprecipitation in chloroform-hexane. Melting Point: 174 ~ 179 ℃ (Decomposition)

NMR-Spectle (ppm in CDCl ₃ : 0.86 (3H, s), 1.27 (3H, d, J = Ca 6 Hz), 1.65 (3H, s), 2.63 (1H, d, J = 9 Hz), 3.07 (1H , d, J = 13 Hz), 3.23 (3H, s), 3.35 (3H, s), 3.42 (1H, d, J = 13 Hz), 3.75 (1H, d, J = 9 Hz), 3.81 (3H, s) , 4.37 (1H, m), 5.51 (1H, dd, J = 9 Hz and 15 Hz), 6.10 (1H, d, J = 11 Hz), 6.41 (1H, dd, J = 11 Hz and 15 Hz), 6.56 (1H, d , J = 2 Hz), 6.70 (1H, s), 6.70 (1H, most s), 6.97 (1H, most s), others.

MS-Spectle (m / e): 469, others.

nm : 231.5, 241.5, 250.5, 277.5, 286.UV-Spectle

nm: 231.5, 241.5, 250.5, 277.5, 286.

Reference Example 2

Preparation of maytansinol 3-isopropylcarbonate.

57 mg of maytansinol is dissolved in 2.1 ml of dry tetrahydrofuran and treated with 5 molar equivalents of 15% n-butyllithium (n-hexane solution) at -20 ° C in nitrogen flow. 61 mg of isopropyl chloro formic acid was added to the solution, and the mixture was left to stir for 15 minutes. The reaction solution was returned to 0 ° C, and 0.5 ml of saturated saline and 20 ml of tetrahydrofuran were added to separate the organic layer. When the organic layer is dried, the solvent is distilled off, and the residue is purified by silica gel column chromatography to obtain 5 mg of maytansinol 3-isopropyl carbonate.

Silica gel thin layer chromatography (HPTLC: Rf = 0.44 (developing solvent, chloroform: methanol = 95: 5) manufactured by Melk Co., Ltd.), MS-Spectle (m / e), 650 (M ⁺ ), 589 (M ⁺ -6).

Reference Example 3

As a method similar to the reference example 2, the compound shown below is obtained.

(A) Maytansinol 3-n-octylcarbonate is obtained from maytansinol and chloro formic acid n-octyl. Silica gel thin layer chromatography (HPTLC manufactured by Melk Co., Ltd.): Rf = 0.61 (field solvent chloroform: methanol = 95: 5), MS-specule (m / e), 659 (M ⁺ -61).

(B) Maytansinol 3-phenyl carbonate is obtained from maytansinol and phenyl formate. Silica gel thin layer chromatography (HPTLC manufactured by Melk Co., Ltd.): Rf = 0.45 (developing solvent: methanol = 95: 5), MS-specule (m / e), 623 (M ⁺ -61).

(C) Dechloromaytansinol 3-benzyl carbonate is obtained from dechloromaytansinol and chlor formic acid benzyl. Silica gel thin layer chromatography (HPTLC manufactured by Melk Co., Ltd.): Rf = 0.54 (developing solvent chloroform: methanol = 95: 5), MS-spectle (m / e), 603 (M ⁺ -61).

Example 1

Bacillus megaterium IFO 12108 is inoculated in a medium (pH 7.5) containing 2% dextrin, 0.5% Kapton, 0.5% yeast Ekis and 0.5% Yueki's, and incubated at 28 ° C for 16 hours. When 5 mg of this culture solution was added with 100 mg of dechloromaytansinol 3-benzyl carbonate, the reaction was carried out by shaking at 28 ° C. for 48 hours, and the dechloromaytansinol 3-benzyl carbonate was completely lost. It is confirmed in thin layer chromatography (TLC) that methyldechloromaytansinol 3-benzylcarbonate is being produced.

Example 2

2.5 L of ethyl acetate was added to 5 L of the culture solution obtained in Example 1, followed by extraction with stirring. The resultant was filtered by suction with a filter coated with 15 g of Hyprosupercell (manufactured by Jonesmanville, USA), and this operation was repeated twice.

The ethyl acetate layers were combined, washed twice with 1 liter of water, dried with 30 g of anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain crude (i). The crude material (i) obtained was dissolved in a small amount of chloroform, poured into a column (1.2 cm in diameter) filled with 10 g of a silica gel (West Germany, 0.05-0.2 mm manufactured by Melk Co., Ltd.) prepared in advance, and 300 ml of chloroform was poured, followed by chloroform: methanol ( Elution is carried out as 20: 1). 20 ml of the eluate was fractionated and fractionated, and each fraction was spotted at a position of 2.5 cm from the bottom of a silica gel glass plate (West Germany, Kigel gel 60F254 0.25 mm, 20 × 20) manufactured by Melk, and developed solvent chloroform: methanol (9: 1) Expand about 17 cm). After the development, the absorbed phase is irradiated under ultraviolet light (2537Å), the absorbed fractions are collected near Rf 0.44, concentrated under reduced pressure, and 63 mg of crude substance (ii) is obtained. Crude material (ii) was dissolved in a small amount of chloroform, and each of the silica gel glass plates was applied in a straight line at a position of 2.5 cm from the lower end, and developed as chloroform: methanol (9: 1), followed by absorption of Rf 0.44. The mixture was scraped off and extracted twice with ethyl acetate containing a small amount of water. The obtained extracted ethyl acetate was concentrated under reduced pressure after washing with sodium sulfate, washed with petroleum ether, and the precipitated precipitate was filtered off and dried. 48 mg of white powder of demethyl dechloro maytansinol 3-benzyl carbonate are obtained.

Silica gel thin layer chromatography (manufactured by Melk Corporation): Rf = 0.44 (open solvent chloroform: methanol = 9: 1), MS-spectle (m / e), 589 (M ⁺ -61).

Example 3

Contains Streptomyces prabotorishini = IFO 12770, 1% dextrin, 1% glucose, 1% glycerol, 0.5% peptone, 0.5% yeast, 0.5% yukeskie, 0.3% salt, and calcium carbonate Inoculate in one medium (pH 7.2) and incubate at 28 ° C for 24 hours. When 50 mg of maytansinol 3-phenyl carbonate is added to 5 liters of this culture, and reacted by shaking at 28 ° C. for 48 hours, maytansinol 3-phenyl carbonate is decreased and demethyl maytansinol is reacted. It is confirmed as TLC that 3-phenyl carbonate is produced.

Example 4

The culture solution obtained in Example 3 was purified in the same manner as in Example 2, the thin layer chromatography was carried out in the same manner as in Example 2, and the fraction near Rf 0.38 was collected to prepare a crude substance of dimethylmaytansinol 3-phenylcarbonate. (ii) 24 mg is obtained.

The purification was carried out in the same manner as in Example 2 below to obtain 13 mg of demethyl maytansinol 3-phenylcarbonate.

Silica gel thin layer chromatography (manufactured by Melk Corporation): Rf = 0.038 [developing solvent chloroform: methanol = (9: 1)], MS-spectle (m / e), 609 (M ⁺ -61).

Example 5

Streptomyces pratensis IFO 12901 is inoculated into the same medium as in Example 3, and cultured with shaking at 28 ° C. for 24 hours. To 5 liters of this culture solution, 50 mg of maytansinol 3-isopropyl carbonate was added, and when reacted by shaking at 28 ° C. for 48 hours, the maytansinol 3-isopropyl carbonate was lost and demethyl mate It is confirmed by TLC that synol 3-isopropyl carbonate is being produced.

Example 6

The culture solution obtained in Example 5 was purified in the same manner as in Example 2, and the thin layer chromatography was carried out in the same manner as in Example 2, and the fractions near Rf 0.43 were collected to prepare dimethylmaytansinol 3-isopropyl carbonate. 18 mg of substance (ii) is obtained. The purification was carried out in the same manner as in Example 2 below to obtain 14 mg of demethyl maytansinol 3-isopropyl carbonate.

Silica gel thin layer chromatography (Melco): Rf = 0.43 [developing solvent chloroform: methanol = (9: 1)]

MS-Spectle (m / e), 575 (M ⁺ -61).

Example 7

Using Bacillus mecaterium IFO 12108, the compounds shown below were obtained according to the same method as in Examples 1 and 2.

Rf values of starting material compounds, the product and the product _{[eluent: CHCl 3: MeOH = 9:} 1, plate: Silica gel glass plate (Melk Co. 60F254 thickness 0.25mm)] refers to.

Example 8

Using Streptomyces prabotriscine IFO 12770, the compounds shown below were obtained in the same manner as in Examples 3 and 4. Rf values of starting material compounds, the product and the product _{[eluent: CHCl 3: MeOH = 9:} 1, plate: Silica gel glass plate (Melk Co. 60F254 thickness 0.25mm)] refers to.

Example 9

Using Streptomyces Frantensis IFO 12901, the compounds shown below were obtained in the same manner as in Examples 5 and 6. Rf values of starting material compounds, the product and the product _{[eluent: CHCl 3: MeOH = 9:} 1 Plate: Silica gel glass plate (West Germany Melk Co. 60F254 thickness 0.25mm)] refers to.

Example 10

10 mg of the powder of demethyldechloromaytansinol 3-benzyl carbonate obtained in Example 2 is dissolved in 4 ml of tetrahydrofuran, cooled to -5 ° C and 10 mg of lithium alumina hydride is added. The reaction solution is transferred to jade and stirred for 30 minutes. 2 ml of ethyl acetate and 1.2 ml of 1/200 hydrochloric acid were added, followed by extraction with 10 ml of ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to pure paralytic TLC using silica gel. Methanol (19: 1) was used to develop 17 cm, the absorbent phase near Rf 0.20-0.25 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. 6.2 mg of a powder of tancinol is obtained.

When dissolved in a small amount of ethyl acetate and left to stand, crystals precipitate. The crystals are dried after filtration. The yield is 4.8 mg.

Melting Point: 198-201 ℃ (Decomposition)

Found: 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-Spectle (m / e) 455,437

nm : 230, 240, 248, 2 77, 285UV-Spectle

nm: 230, 240, 248, 2 77, 285

Example 11

It is dissolved in 4 ml of tetrahydrofuran powder of 10 mg of demethyl maytansinol 3-isopropyl carbonate obtained in Example 6, cooled to -5 DEG C, and 10 mg of lithium aluminum hydride is added. This was processed in the same manner as in Example 9, pure paralytic TLC using silica gel was carried out, and ethyl acetate. 17 cm development as methanol (19: 1) was carried out, and the absorption phase near 0.25-0.32 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. mg is obtained. Melting Point 196 ℃

Elemental Analysis Value (%)

Found: C 58.62 H 6.59 N 4.82 Cl 6.21

Calculation: C ₂₇ H ₃₅ CIN ₂ O ₈

C 58.85 H 6.40 N 5.08 Cl 6.43

MS-Spectle (m / e) 489,471

nm : 232, 242, 251, 280, 288UV-Spectle

nm: 232, 242, 251, 280, 288

Claims (1)

General formula

In the formula, the maytansinoid compound represented by [X represents Cl or H, and R represents a hydrocarbon residue which may have a substituent, respectively] is used to convert the methoxy group of the compound into a hydroxyl group. A general formula characterized by contacting a culture of a microorganism belonging to the genus Bacillus or Streptomyces or a processed product thereof

A process for producing a demethylmaytansinoid compound represented by the formula [X and R represent the same meaning as the foregoing].