KR820001589B1 - Process for preparing demethyl maytansinoids - Google Patents

Abstract

The method of preparation for Demethyl maytansinoids was described. As reacting maytansinoid compsn. for the culture fluid of microbs. such as Bacillus, Streptomyces or Actinomyces, methoxy group was substituted for OH group and then the products were deacylated. Bacillus IFO 12108 was inoculated the medium containing of dextrin 2, peptone 0.5, yeast ext. 0.5, and meat ext. 0.5, and incubated at 30≰C for 16 hr. If ansamitocin P-4 were added and was reacted at 30≰C for 51 hr, ansamitocin P-4 was lost and produced PPM-4. X = Cl, H; R1 = H, acyl.

Description

Process for the preparation of the demethyl maytansinoid compound

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

MEANS TO SOLVE THE PROBLEM The present inventors searched for the method of converting a maytansinoid compound into another compound by a microorganism. When a culture of a microorganism or its processed substance is acted on a maytansinoid compound, the methoxy group of 20th place It can be converted to a hydroxyl group, it was found that the deacylation reaction of the resulting compound to obtain a compound having a hydroxyl group at the third position, and further studies based on these have completed the present invention.

The compound prepared by the method of the present invention is represented by general formula (I)

Wherein X represents C1 or H, and R ₁ represents H or an acyl group, respectively.

It is a demethyl maytansinoid compound (I) which is represented as (1) General formula (II)

Wherein X represents C ₁ or H, and R ₁ represents H or an acyl group, respectively.

Culture of microorganisms belonging to the genus Bacillus, Streptomyces or Actinomyces having the ability to convert methoxy groups of the 20th position of the compound (II) to hydroxyl groups in the maytansinoid compound (II) represented as The following general formula (III) is characterized by contacting water.

Wherein X and R ₁ represent the same meaning as described above.

The method of dimethyl maytansinoid compound (Ⅲ) represented by and (2) group the R ₁ R ₁ '(R _1' in the formula (II) stands acyl group, hydrogen or an acyl group having the group R ₁ Is a acyl group represented by the same acyl group), genus Bacillus, Streptomyces or the like having the ability to convert a methoxy group on the 20th position of the compound (IV) to a hydroxyl group in the maytansinoid compound (IV) Contacting a culture of a microorganism belonging to the genus Actinomyces or a treated product thereof to obtain a compound (V) wherein R ₁ is an acyl group represented by R ′ ₁ in General Formula (III), and then acylating the compound. General formula (Ⅳ)

(Wherein X has the same meaning as described above).

It is a manufacturing method of the demethyl maytansinoid compound (IV) shown by this.

-아미노산형 아실기 및 카르바모일형 아실기 등을 포함한다.In the above general formula, the acyl groups represented by R ₁ and R ₁ "include acyl groups derived from carboxylic acids or carbamic acid derivatives having a molecular weight of about 300 or less or acyl groups having 1 to 20 carbon atoms. Such acyl groups are, for example, saturated or unsaturated aliphatic acyl groups, saturated or unsaturated alicyclic acyl groups, aromatic acyl groups, saturated or unsaturated heterocyclic acyl groups, and N-acyl-

-An amino acid type acyl group, a carbamoyl type acyl group, and the like.

As these acyl groups, for example, the formula -CO-R ₂ (wherein R ₂ is a hydrogen atom or an alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl or bicyclic group which may have a substituent) ), And the cycloalkyl, cycloalkenyl, aryl and bicyclic groups may be bonded to the carbon atom of carbonyl via an alkylene chain.

-위치의 탄소원자에 결합하고 있어도 좋고, 또한 R₄는 수소원자 또는 치환기를 가지고 있어도 좋은 알킬, 시클로알킬, 시클로알킬알킬, 아릴 혹은 벤질를 나타내며, R₅는 수소원자, 알콕시, 보르닐옥시, 이소보르닐 옥시, 벤질옥시 또는 치환기를 가지고 있어도 좋은 알킬, 알케닐, 시클로알킬, 시크로알케닐, 아릴 혹은 이항환기를 나타내며, 이 시클로 알킬, 시클로알케닐, 아릴 및 이항환기는 알킬렌쇄를 통하여 질소원자의 카르보닐의 탄소원자에 결합하고 있어도 좋다)로서 표시되는 기, 또는 식

(식중 R₆및 R₇은 동일하거나 다르며, 수소원자 또는 이항환기를 나타내며, R₆과 R₇이 연결하여 인접한 질소원자와 함게 이항환기를 형성하고 있어도 좋다)로서 표시되는 기를 예거할 수 있다.(Wherein R ₃ represents a hydrogen atom or an alkyl, cycloalkyl, aryl, indolyl or imidazolyl which may have a substituent, which cycloalkyl, aryl, indolyl and imidazolyl may be represented by an alkylene chain)

R ₄ represents alkyl, cycloalkyl, cycloalkylalkyl, aryl or benzyl, which may be bonded to a carbon atom in the position, and R ₅ represents a hydrogen atom, alkoxy, bornyloxy, iso Bornyloxy, benzyloxy or alkyl, alkenyl, cycloalkyl, cycloalkenyl, aryl or dicyclic groups which may have substituents, and these cycloalkyl, cycloalkenyl, aryl and dicyclic groups are nitrogen atoms via an alkylene chain. May be bound to a carbon atom of carbonyl)

The group represented by (wherein R <_6> and R <_7> is the same or different and represents a hydrogen atom or a bicyclic ring group, and R <_6> and R <_7> may connect and may form the bicyclic ring group with the adjacent nitrogen atom may be mentioned.

In the group in which the acyl groups represented by R ₁ and R ′ ₁ described above are represented by the formula, -CO-R ₂ (wherein R ₂ has the same meaning as described above), examples of the alkyl group represented by R ₂ include, for example Alkyl groups having 1 to 18 carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, bentyl, isobentyl, 1-methylpropyl, hexyl, isohexyl, heptyl, 3 -Heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, leptadecyl, octadecyl, 1,2-dimethylpropyl, 1-ethylpropyl, 1,2,2-trimethylpropyl , 1-propylbutyl, 2-methylhexyl group) and the like, and among them, an alkyl group having 1 to 6 carbon atoms is preferable.

As the alkenyl group represented by R ₂ , for example, an alkenyl group having about 2 to 18 carbon atoms (eg, vinyl, aryl, 1-methyl-vinyl, 1-octenyl, 1-decenyl, 1,3-pentadienyl , An oleyl group), and an alkenyl group having about 2 to 4 carbon atoms is particularly preferable.

Examples of the alkenyl group represented by R ₂ include a cycloalkyl group having about 3 to 10 carbon atoms (e.g., cynopropyl, cynobutyl, cyclopentyl, cynonuclear, cynoloheptyl, cyclooctyl, norbornyl, argon). Danyl group) are exemplified, and these may be further condensed with a benzene ring (eg, 1- or 2-indanyl, benzocyclobutyl).

As a cycloalkenyl group represented by R <_2> , for example, a cycloalkenyl group having 3 to 10 carbon atoms (e.g., 1-cyclobutenyl, 1-2- or 3-cyclopentenyl, 1-, 2-, or 3) -Cyclohexenyl, 4-cyclohaptenyl, 4-cyclooctenyl, 1,4-cyclohexadienyl, 4-norbornenyl, 2,4,6-cyclohepta trienyl group), and the like.

As an aryl group represented by R <_2> , a phenyl group, (alpha)-, or (beta) -naphthyl group etc. are mentioned, for example, A phenyl group is preferable among these.

Examples of the binary ring group represented by R ₂ include N, O or / and S-containing 4, 5 or 6 membered binary ring groups. These may be either saturated or unsaturated vents, and the benzene ring may be conjugated.

As an N-containing 4, 5 or 6-membered bicyclic ring group, for example, azetidinyl, pyridyl, 1,2,3,4-tetrahydropyridyl, piperidyl, quinolyl, 1,2-dihydroqui Noryl, 3- or 4-isoquinoryl, 1,2-dihydroisoquinoryl, pyridyl, pyridylyl, indoryl group, and the like. Examples of the 0- or 5-membered rasulized bicyclic group include For example, a prill, a pisinyl, a dihydropyranyl, a bezopryl, a benzopyranyl group, etc. are mentioned, As a 5 or 6 membered former bicyclic group containing S, a thienyl benzothienyl etc. are mentioned, respectively, for example. Can be.

Said bicyclic ring group may contain 2 or more and 4 or more same or different hetero atoms, such as N, O, or S. As such a bicyclic group, for example, imidazolyl, pyrazolyl, pyrazinyl, pyrimidyl, Pyridazinyl, 2-imidazolyl, imidazolidinyl, benzoimidazoli, indazolyl, quinosalyl, quinazolinyl, cinnolinyl, 1,4-dioxanyl, 1,4-benzodioxanyl , 1,2- or 1,3-dithioranyl, 1,3-dithioanyl, isooxazolyl, oxazolyl, morpholinyl, benzoisoxazolyl, benzooxazolyl, isothiazolyl, thiazolyl, benzo Isothiazolyl, benzothiazolyl, benzothiadinyl, 1,2,4-, 1,2,5-or 1,3,4-oxadiazolyl, 1,2,3-, 1,2,4- , 1,2,5-or 1,3,4-thiadiazolyl, 1,2,4-, 1,2,5- or 1,3,4-triazolyl, 1,3,5-triadinyl , Benzotriazolyl, 1,2,3,4-tetrazolyl group and the like.

And among the above-described dicyclic groups, for example, azetidinyl, 1,2,3,4-tetrahydropyridyl, piperidyl, 1,2-dihydroquinolyl, 1,2-dihydroisoquinolyl, Pyrrolyl, pyrrolidinyl, pyrrolidinyl, pyrazolyl, 2-imidazolinyl, imidazolidinyl, indazolyl, morpholinyl, 1,2,3-, 1,2,5- or 1,34, -NH-containing bicyclic ring having a strong basicity such as triazolyl, benzotriazolyl, 1,2,3,4, tetrazolyl group has a suitable substituent described below on its N or an alkylene chain described below is bonded to the N-position It is generally desirable to do this.

Cyclic group (optionally substituted cycloalkyl, cycloalkenyl, aryl, and binary group) of the above-mentioned R ₂ may be bonded to the carbonyl carbon atom of the -CO-R ₂ by interposing an alkylene chain.

Accordingly, when the cyclic group and the alkylene chain described above are bonded, R ₂ represents an optionally substituted cycloalkylalkyl group, cycloalkenylalkyl group, aralkyl group or bicyclic-alkyl group.

As such an alkylene chain, for example, a linear or branched alkylene chain having 1 to 4 carbon atoms [eg, methylene, ethylene, methylmethylene (ethyllimen), propylene, butylene, 1-, 2- or 3-methylpropylene , 1- or 2-ethylethylene, propylmethylene, 1,1- or 1,2-dimethylethylene, isopropylmethylene].

Examples of such cycloalkylalkyl groups include 1-adamantylmethyl, cyclohexylmethyl, 3-cyclohexylpropyl, 2-cyclopentylmethyl, 2-cyclopentylethyl, 2-cyclopropylmethyl-cyclobutylethyl, cyclopentylmethyl, Cyclohexyl ethyl, 2-methyl-3- cyclohexyl propyl, etc. are mentioned.

Examples of cycloalkenyl alkyl groups include 1-, 2-, or 3-cyclopentenylmethyl, 1-, 2-, or 3-cyclohexenylmethyl, 4-cycloheptenyl-3-propyl, 1,4-cyclohexadi Ethyl methyl group etc. are mentioned.

-메틸벤질, 1-메틸-3-페닐출로필, 4-페닐부틸기 등을 들 수 있다.Examples of aralkyl groups include, for example, benzyl, phenethyl, 3-phenylpropyl,

-Methylbenzyl, 1-methyl-3-phenyl fluoropropyl, 4-phenylbutyl group, etc. are mentioned.

Examples of the bicyclic alkyl group include, for example, 3-indolylmethyl, 3- (3-indolyl) propyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 4- (2-thienyl) Propyl, 2-benzothiazolylmethyl, 2-benzooxazolylmethyl, 3-benzoisothiazolylmethyl, 3-benzoisooxazolylmethyl, furfuryl, 2-tenyl group, and the like.

When R ₂ is an N-containing bicyclic group whose N atom is a type bonded to the carbonyl carbon of the acyl group represented by —CO—R ₂ , the bicyclic ring is always bonded to the carbonyl group via the alkylene chain described above.

Examples of the bicyclic-alkyl group in which the alkylene chain is bonded to the N atom include 1-pyrrolylmethyl, 2-oxo-1-pyrrolidinylmethyl, 1-imidazolylmethyl, 3,5-dimethyl-1-pyrazolyl Methyl, 1-piperidylmethyl, 4-morpholinylmethyl, 1-tetrazolylmethyl, 2,5-dioxo-1-pyrrolidinylmethyl, 1,3-dioxo-2-isoindolylmethyl, 2-thioxo-4-oxo-3-thiazolidinylmethyl, 3,5-diiodine-4-oxo-1,4-dihydropyridine-1-methyl, 4-methyl-1-piperadinylmethyl, 1-indolylmethyl, etc. are mentioned.

The alkyl group, alkenyl group, cycloalkyl group, cycloalkenyl group, aryl- group and dicyclic group as R ₂ described above may optionally have a substituent. Examples of such substituents include alkoxy groups having 1 to 4 carbon atoms (e.g., methoxy, Ethoxy, propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy groups, alkanoyl groups having 2 to 4 carbon atoms (e.g. acetyl, propionyl, n- Butyryl, iso-butyryl group), alkanoyloxy group having 2 to 4 carbon atoms (e.g., acetyloxy, propionyloxy, n-butyryloxy, isobutyryloxy group), alkoxycarbonyl group having 2 to 4 carbon atoms (e.g., Methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, iso-propoxycarbonyl group), halogen atom (e.g. chlorine, fluorine, cancel, oxo), nitro group, cyano group, trifluoromethyl group, di -C _1-4 alkylamino (e.g., dimethylamino, diethylamino, dipropylamino, diisopropyl amino , Dibutylamino group), alkylthio group having 1 to 4 carbon atoms (e.g., methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio, tert-butyl tea Group), methylsulfonyl group, oxo group, thioxo group, alkanoylamide group having 1 to 4 carbon atoms (e.g., formamide, acetamide, propionylamide, butyrylamide, isobutylamide group), and the like. And when R ₂ is a cyclic group (cycloalkyl, cycloalkenyl, aryl and bicyclic group), an alkyl group having 1 to 4 carbon atoms (eg, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec -Butyl, tert-butyl group) are also illustrated as a substituent. These substituents are the same or different, and one or more and three may be present.

Examples of the alkyl group having 1 to 18 carbon atoms having a substituent represented by R ₂ include methoxymethyl, butoxymethyl, methylthiomethyl, methylthioethyl, ethylthiomethyl, isopropylthioethyl, butylthioethyl, isobutylthioethyl , Acetyloxymethyl, acetylthiooxyethyl, ethoxycarbonylmethyl, butoxycarbonylethyl, fluoromethyl, chloromethyl, chloroethyl, 3-chloropropyl, 4-chlorobutyl, 3,3,3-trichloro Propyl, trifluoromethyl, bromomethyl, 4-bromobutyl, 5-bromopentyl, iodinemethyl, 2-iodineethyl, 1,1-dimethyl-2,2-dichloroethyl, 2-chloro-1- Chloromethyl-1-methylethyl, cyanomethyl, methylsulfinylethyl, methylsulfonylmethyl and the like.

1-chlorovinyl etc. are mentioned as an example of a C2-C10 alkenyl group which has a substituent shown as R <_2> .

As an example of a C3-C10 cycloalkyl group which has a substituent represented as R <_2> , 2, 2- dimethyl cyclopropyl, 2-propyl cyclopropyl, 2-butyl cyclopropyl, 4-isobutyl cyclohexyl, 2-bromocyclo Propyl, 2-chlorocyclobutyl, 4-chlorocyclohexyl, 2-iodocyclohexyl, 2,2-difluorocyclobutyl, 3-methoxycyclohexyl, 2,2-dimethyl-3-acetylcyclobutyl, 4-acetylcyclohexyl, 2-cyanocyclohexyl, 2-cyanocyclobutyl, 4-cyanocyclohexyl, 4-dimethylaminocyclohexyl, etc. are mentioned.

Examples of the cycloalkenyl group having 3 to 10 carbon atoms having a substituent represented by R ₂ include 2-cyano-2-cyclohexenyl, 3,3-dimethyl-4-cyclobutenyl, 4-ethoxycarbonyl-1 -Cyclohexenyl, 4-butoxycarbonyl-1-cyclohexenyl, etc. are mentioned.

Examples of the aryl group having a substituent represented as R ₂ are 2-, 3- or 4-methylphenyl, 4-tert-butylphenyl, 2-, 3- or 4-chlorophenyl, 2-, 3- or 4-bro Morphenyl, 2-, 3- or 4- iodinephenyl, 2-, 3- or 4-fluorophenyl, 2- or methoxyphenyl, 4-butoxyphenyl, 4-methoxycarbonylphenyl, 3-acetyl Phenyl, 2-, 3- or 4-nitrophenyl, 3- or 4-cyanophenyl, 4-dimethylaminophenyl, 4-diethylaminophenyl, 4-acetoxyphenyl, 4-butyryloxyphenyl, 2, 4-2,5-, 2,6-, 3,4- or 3,5-dichlorophenyl, 3,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 3,4-methylenedioxy Phenyl, 3-trifluoromethylphenyl, 4-methylthiophenyl, 4-methylsulfonylphenyl, 4-acetamide phenyl, and the like.

Or not having a substituent represented as R ₂ or 4,5. 6 examples of the transposition of a ventilation source, for example 1-acetyl-2-azetidinyl, 1-methyl-2-pyrroline, 3-methoxy- 2-pril, 3-methyl-2-pril, 5-methyl-2-pril, 5-nitro-2-pril, 3-methyl-2-thienyl, 3-bromo-4,5-dimethyl-2 -Thienyl, 2-methyl-4-thiazolyl, 1,2-dimethyl-4-chloro-5-imidazolyl, 1-butyl-4-pyrazolyl, 2,4-dichloro-4-isothiazolyl, 5-methyl-1,2,3-thiadizolyl-4,3,5-dimethyl-4-isokisazolyl, 2-methyl-5-diisoisopropylamino-4-oxazolyl, 5-methyl-1 , 2,5-oxadiazolyl-3,4-methoxy-1,2,5-oxadiazolyl-2,5-methyl-1,2,3-oxadiazolyl-2,3-1,2, 3-thiadiazolyl-5,5-methyl-3,4-thiadiazolyl-2,5-methyl-1,2,3-thiadiazolyl, 4,1-methyl-1,2,3-triazolyl -4,2-ethyl-1,2,3,4-tetrazolyl-5,5-nitro-2-pyridine, 6-ethyl-4-pyridyl, 5-ethoxycarbonyl-3-pyridyl, 5 -Chloro-3-pyridyl, 1-butyryl-2-piperi , 2-oxo-5-pyranyl, 7-methoxy-3,4-dihydro-2H-2-pyranyl, 1-acetyl-2-pyrrolidinyl, 1-propyl-5-oxo-3-pyro Ridinyl, 3-methyl-2,4-dioxo-5-thiazolidinyl, 4-, 5-, 6- or 7-nitro-3-indolinyl, 5-fluoro-2-indolinyl, 2- Methyl-5-methoxy-3-indoryl, 1-methyl-2-indoryl, 5-chloro-2-benzothienyl, 3-methyl-2-benzopril, 1-methyl-2-benzoimidazolyl , 6-nitro-2-benzothiazolyl, 4-chloro-3-quinolyl, 6-methoxy-2-quinolyl, 2,4-dimethoxy-3-quinolyl, 2-methyl-1-oxo- 3-isocarbostyryl, 7-methyl-3-chmaryl, 4-methyl-quinazolyl, 3-propyl-2,4-dioxo-5-imidazolinyl-7-methoxycarbonyl-2- Oxo-1,2-dihydro-3-quinazolyl, 2-pril, 2-thienyl, 3-isooxazolyl, 4-imidazylyl, 1,2,5-thiadiazol-3-yl, 2 , 3- or 4-pyridyl, 2-pyridinyl, 2-pyrimidinyl, 2-S-triadinyl, 1,2-dithiooranyl, 3-indolyl, 2-benzothienyl, 2- Benzopril, 3-benzopyrazolyl, 2-benzo Imidazolyl, 2-benzooxazolyl, 3-benzoisoxazolyl, 3-benzoisothiazolyl, 2-benzothiazolyl, 2-benzo-1,4-oxadinyl, 3-quinolyl, 1-isoqui Teasing, etc. may be mentioned.

Examples of the cycloalkyl alkyl group having a substituent represented by R ₂ include, for example, 3-acetyl-2,2-dimethyl-1-cyclobutylmethyl, 3-acetoxy-2,2-dimethyl-1-cyclobutylmethyl, 2- (3-chloro-1-cyclobutyl) ethyl, 2,3-dimethyl-1-cyclopentylmethyl, 2-isopropyl-1-cyclopentylmethyl, cis- or trans-4-acetamide-1-cyclo Hexylmethyl, cis- or trans-4-butyl-1-cyclohexylmethyl, cis- or trans-2- (4-acetamide-1-cyclohexyl) ethyl group and the like.

Examples of the cycloalkenyl alkyl group having a substituent represented by R ₂ include, for example, 2- (4-isopropyl-1-cyclohexenyl) ethyl and 1-ethyl-2- (4-isopropyl-1-cyclohex Cenyl) ethyl, 3-methoxy-4-methyl-3-cyclohexenylmethyl group, etc. are mentioned.

-메틸펜에틸 등을 들 수 있다.Examples of the aralkyl group having a substituent represented as R ₂ include 4-bromobenzyl, 2-, 3- or 4-chlorobenzyl, 2,5- or 3,4-dimethoxybenzyl, 4-ethoxybenzyl, 4 -Fluorobenzyl, 3- or 4-methoxybenzyl, 4-methoxyphenylethyl, 1- or 2-naphthylmethyl, 2-, 3- and 4-nitrobenzyl, 3-nitrophenethyl, benzyl, 2 -, 3- or 4-phenylpropyl, 2-, 3- or 4-methylbenzyl, 3,4,5-trimethoxybenzyl,

-Methyl phenethyl, etc. are mentioned.

As an example of the bicyclic-alkyl group which has a substituent represented by R <_2> , 5-ethyl-3- indorylmethyl, 5-fluoro-3- indorylmethyl, 5-methoxy-3- indorylmethyl, 5-methyl- 3-indorylmethyl, 1-methyl-5- tetrazolylmethyl, 2- (1-piperidinyl) ethyl, etc. are mentioned.

The acyl group represented by R ₁ or R ₁ ′ of the former

(Wherein R ₃ , R ₄ and R ₅ are consistent with the definitions above), the alkyl, cycloalkyl and aryl groups represented by R ₃ , R ₄ and R ₅ , respectively, may be groups of the examples mentioned in R ₂ . good. As a result, examples of the cycloalkyl group may be the group mentioned for R ₂ .

Alkenyl, cycloalkenyl, or transposition ventilation represented by R ₅ may be the same date referred to in R _5.

Examples of the alkoxy represented by R ₅ include an alkoxy group having about 1 to 7 carbon atoms (eg, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy , Pentoxy, hexyloxy, heptyloxy group) are mentioned, and the alkoxy group of 1-4 carbon atoms is especially preferable.

위의 탄소원자에 결합하고 있어도 좋다. 또 상기한 R₅로서의 시클로알킬, 시클로알케닐, 아릴 및 이항환기는 알킬렌쇄를 거쳐서 질소원자의 카르보닐의 탄소원자에 결합하고 있어도 좋다.Cycloalkyl, aryl, indolyl, and imidazolyl as R ₃ described above are substituted via an alkylene chain.

It may be bonded to the above carbon atom. The cycloalkyl, cycloalkenyl, aryl and bicyclic group as R ₅ described above may be bonded to the carbon atom of the carbon atom of the nitrogen atom via the alkylene chain.

Examples of such alkylene chain is Yeager to as alkylene chain in which R ₂ electricity.

Therefore, when passing through an alkylene chain, it will represent cycloalkyl, aralkyl, indorylalkyl, cycloalkenylalkyl, or bicyclic alkyl.

Examples of such cycloalkylalkyl, cycloalkenylalkyl, aralkyl and disulfide alkyl include the same groups represented by R ₂ described above.

As an example of each group which has a substituent represented by R <_3> , R <_4> and R <_5> , the same thing as the group which has a substituent as said R <_{2> is} mentioned.

Examples of indoryl groups having a substituent represented by R ₃ include 5-fluoro-2-indoryl-, 5-methoxy-2-indoryl, 1-methyl-2-indoryl, 5-methyl-2-indoryl Etc., and examples of the imidazolyl group having a substituent include 1-methyl-5-imidazolyl, 3-methyl-5-imidazolyl, 2-methyl-4-imidazolyl and the like. .

Examples of indolylalkyl having a substituent represented by R ₃ are, for example, 5-bromo-2-indoylmethyl, 5-bromo-2-indoylethyl, 5-chloro-2-indoylmethyl, 5 -Chloro-2-indoylethyl, 5-fluoro-2-indoylmethyl, 5-fluoro-2-indoylethyl, 5-methoxy-2-idorylmethyl, 5-methoxy-2-indo Ryl ethyl, 1-methyl-2- indoyl methyl, 1-methyl-2- indoyl ethyl, 5-methyl-2- indoyl ethyl, 5-methyl-2- indoyl ethyl, and the like.

Examples of imidazolyl alkyl having a substituent represented as R ₃ are, for example, 1-methyl-5-imidazolylmethyl, 1-methyl-5-imidazolylethyl, 3-methyl-5-imidazolyl Methyl, 3-methyl-5-imidazolylethyl, 2-methyl-4-imidazolylmethyl, 2-methyl-4-imidazolylmethyl, 2-ethyl-4-imidazolylmethyl, 1-methyl- 4-imidazolyl ethyl etc. are mentioned.

Examples of benzyl having a substituent represented as R ₄ include 4-bromobenzyl, 2-, 3- or 4-chlorobenzyl 4-fluorobenzyl, 4-methoxybenzyl, 4-ethoxybenzyl, 2-, 3 Or 4-methylbenzyl, 3,4-dimethoxybenzyl group, and the like.

The following formula described in detail above

-아미노아실기의 대표적인 예로서는, N-아세틸-N-메틸-그리실, N-벤조일-N-메틸-그리실, N-(4-클로로벤조일)-N-메틸-그리실, N-아세틸-N-벤질-아라닐, N-아세틸-N-메틸-로이실, N-아세틸-N-메틸-페닐아라닐, 2-(N-아세틸-N-메틸)-3-메톡시카르보닐프로피오닐, 2-(N-아세틸 -N-메틸)-3-메틸멜캅토프로피오닐, 2-(N-아세틸-N-메틸)-3-에틸멜캅토프로피오닐, N

-아세틸-N

N'-디메틸히스티디닐, N- 아세틸-N-메틸이소로이실,-N아세틸-N-메틸-로이실, N-아세틸-N-메틸-메티오닐, N-아세틸-N-메틸-페닐아라닐, N-아세틸-N-메틸-트리프로페닐, N-아세틸-N-4'-아세톡시-디로시닐, N-벤질-N-메틸-바릴, N-아세틸-N-메틸-페닐그리실, N-이소니코틴노일-N-메틸-

-아미노부티릴, N-아세틸-N-메틸-3-시아노아라닐, N-아세틸-N- 메틸-N-

-(2-티아조릴)그리실, N-아세틸-N-메틸(4'-디메틸아미노)-페닐아라닐 등을 들 수 있다.N-acyl- denoted by

Representative examples of the -aminoacyl group include N-acetyl-N-methyl-grisyl, N-benzoyl-N-methyl-grisyl, N- (4-chlorobenzoyl) -N-methyl-grisyl and N-acetyl- N-benzyl-alanyl, N-acetyl-N-methyl-loyl, N-acetyl-N-methyl-phenylalanyl, 2- (N-acetyl-N-methyl) -3-methoxycarbonylpropionyl , 2- (N-acetyl-N-methyl) -3-methylmelcaptopropionyl, 2- (N-acetyl-N-methyl) -3-ethylmelcaptopropionyl, N

-Acetyl-N

N'-dimethylhistidinyl, N-acetyl-N-methylisoroyl, -Nacetyl-N-methyl-roysil, N-acetyl-N-methyl-methionyl, N-acetyl-N-methyl-phenyl Aranyl, N-acetyl-N-methyl-tripropenyl, N-acetyl-N-4'-acetoxy-dirosinyl, N-benzyl-N-methyl-baryl, N-acetyl-N-methyl-phenyl Grisyl, N-isonicotinoyl-N-methyl-

Aminobutyryl, N-acetyl-N-methyl-3-cyanoalanyl, N-acetyl-N-methyl-N-

-(2-thiazolyl) grisyl, N-acetyl-N-methyl (4'-dimethylamino) -phenylalanyl, and the like.

The acyl group represented by the aforementioned R ₁ and R ₁ ′ is

(Wherein R ₆ and R ₇ have the same meanings as mentioned above), for the hydrocarbon residue represented by R ₆ and R ₇ , for example, an alkyl group, an alkenyl group, a cycloalkyl group, A cycloalkenyl group, a cycloalkyl group, a cycloalkenyl allyl group, an aryl group, an aralkyl group, a phenylcycloalkyl group, a cycloalkylphenyl group, a biphenyl group, etc. are mentioned.

As an example of said alkyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl, aryl, and aralkyl, the same thing as each group represented by said R <_2> is mentioned.

As the phenylcycloalkyl group, a phenyl group is substituted with the above-mentioned cycloalkyl group having 3 to 10 carbon atoms (especially 3 to 7), and examples thereof include 2-phenylcyclopropyl and 4-phenylcyclohexyl group. As the cycloalkylphenyl group, As what the said cycloalkyl group substituted by the phenyl group, 4-cyclopentylphenyl group 4-cyclohexylphenyl group etc. are mentioned, for example, As a biphenyl group, 4-biphenyl group etc. are mentioned, respectively.

Examples of the bicyclic group represented by R ₆ and R ₇ include N, O or / and S-containing 4,5 or 6-membered bicyclic groups, which may be either saturated or unsaturated vents, and benzene rings may be condensed. good.

As such a bicyclic group, for example, acetidinyl, pyrrolyl, prill, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidylyl, pyrazinyl And riazinyl, quinolyl, quinazolyl, quinosalyl, indoryl, benzoprayl, benzothienyl group and the like.

In addition, R ₆ and R ₇ may be linked to each other to form a binary ring group with adjacent N atoms. As such a bicyclic group, an azetidinyl, a pyrrolidinyl, a piperadinyl, a morpholinyl group, etc. are mentioned, for example.

으로서의 이항환기는 임의의 치환기를 가질수 있으며, 이러한 치환기로서는 예를들면 탄소수 1내지 4의 알콕시기, (예, 메톡시, 에톡시, 프로폭시, 이소프로폭시, 부톡시, 이소부톡시 sec-부톡시기), 탄수 1 내지 4의 알킬티오기(예, 메틸티오, 에틸티오, 프로필티오, 이소프로필티오, 부틸티오기), 페녹시기, 페닐티오기, 시클로헥실기, 할로겐원자(예, 불소, 염소, 취소, 옥소), 시아노기, 탄소수 2 내지 5의 알콕시카르보닐기(예, 메톡시카르보닐, 애톡시카르보닐, 프로폭시카르보닐, 이소프로폭시카르보닐, 부톡시카르보닐, 이소부톡시카르보닐, sec-부톡시카르보닐기), 벤질옥시카르보닐기, 니트로기, 아미노술포닐기, 디알킬아미노기(예, 디메틸아미노, 디에틸아미노, 디이소프로필아미노,디부틸아미노기)등을 들수 있으며, 이들의 치환기는 동일 또는 상이하게 1개 이상 3개까지 치환되어도 좋다.Hydrocarbon residue and bicyclic group represented by R ₆ and R ₇ described above, or

The dicyclic group as may have an arbitrary substituent, and examples of such substituent include an alkoxy group having 1 to 4 carbon atoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy sec-butoxy group). ), An alkylthio group having 1 to 4 carbon atoms (e.g., methylthio, ethylthio, propylthio, isopropylthio, butylthio group), phenoxy group, phenylthio group, cyclohexyl group, halogen atom (e.g., fluorine, chlorine) , Canceled, oxo), cyano group, alkoxycarbonyl group having 2 to 5 carbon atoms (e.g., methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl group), benzyloxycarbonyl group, nitro group, aminosulfonyl group, dialkylamino group (e.g., dimethylamino, diethylamino, diisopropylamino, dibutylamino group) and the like, and their substituents are the same. Or different It may be substituted by one or more 3 or more.

Substituents of alkyl groups, alkenyl groups, and other groups, i.e., hydrocarbon residues having a cyclic moiety and bicyclic groups in the groups exemplified as the above hydrocarbon residues, include alkyl groups having about 1 to 4 carbon atoms in addition to the substituents described above (R ₆ and R ₇ hydrocarbon residue may be substituted), for example methyl, ethyl, propyl, isopropyl, butyl trifluoromethyl, chloromethyl, 2-cyanoethyl, methoxymethyl, ethoxycarbonylmethyl, A dimethylaminomethyl group etc. are mentioned, As a substituent of the alkyl group as R <_6> and R <_7> , In addition to the group illustrated as a substituent of the above-mentioned hydrocarbon residue, Even if it has a bicyclic group like a bicyclic group as R <_6> and R <_7> Good).

-메틸벤질, 1,1-디메틸펜에틸, 4-메톡시펜에틸, 2,3-또는 4-피콜릴, 5-메틸-2-텐닐, 5-메틸플프릴, 3-피페라진오프로필, 2-모르포리노에틸, 4-메틸-1-피페라디닐프로필, 2-(1-메틸-2-피롤리디닐)에틸, 2-티아졸릴메틸, 2-메틸-4-옥사졸릴메틸, 5-클로로-1-메틸-3-인도릴에틸기 등을 들 수 있다.Specific examples of the hydrocarbon residue having a substituent as R ₆ and R ₇ include 2-methoxyethyl, 3-methoxy propyl, β-methoxyisopropyl, 3-isopropoxypropyl, 3-sec-butoxypropyl, 3 -Cyclohexyloxypropyl, 3-phenoxypropyl, 2-chloroethyl, 3-chloropropyl, 4-chlorobutyl, 2-propylthioethyl, 2-phenylthioethyl, 2-cyanoethyl, 5-cyanopentyl , 4-cyanocyclohexylmethyl, methoxycarbonylmethyl, ethoxycarbonylmethyl, 1- or 2-methoxycarbonylethyl, 1-methoxycarbonylisobutyl, 5-methoxycarbonylpentyl, dimethyl Aminopentyl, trifluoromethyl, 2-, 3- or 4-rollyl, xylyl, 2,4,5- or 2,4,6-trimethylphenyl, 2-, 3- or 4-chlorophenyl, 2, 5-dichlorophenyl, 2-, 3- or 4-fluorophenyl, 4-bromophenyl, 4-iodinephenyl, 2- or 3-trifluorophenyl, 2-, 3- or 4-nitrophenyl, 4 -Chloro-3-trifluoromethylphenyl, 2-methyl-4-nit Phenyl, 5-nitro-1-naphthyl, 8-chloro-1-naphthyl, 4-methoxycarbonylphenyl, 4-ethoxycarbonylphenyl, 4-aminosulfonylphenyl, 2-, 3- or 4 -Methoxyphenyl, 4-ethoxyphenyl, 2,5-dimethoxyphenyl, 1-methoxycarbonyl-2-phenethyl, 1-methoxycarbonyl, 1-phenylmethyl, 2-, 3- or 4 -Methylbenzyl, 2-, 3- or 4-chlorobenzyl, 2- or 3-fluorobenzyl, 3-iodine-benzyl, 2,4- or 3,4-dichlorobenzyl, 4-methoxybenzyl,

Methylbenzyl, 1,1-dimethylphenethyl, 4-methoxyphenethyl, 2,3- or 4-picolyl, 5-methyl-2-tenyl, 5-methylflpril, 3-piperazinopropyl, 2-morpholinoethyl, 4-methyl-1-piperadinylpropyl, 2- (1-methyl-2-pyrrolidinyl) ethyl, 2-thiazolylmethyl, 2-methyl-4-oxazolylmethyl, 5 And -chloro-1-methyl-3-indoleylethyl group.

로서 표시되는 이항환기가 치환기를 갖는 경우의 구체예로는, 2-,3-또는 4-메틸-1-피페리디닐, 4-메틸-1-피페라지닐, 2,6-디메틸모르포리노, 2-프로필-1-피페리디닐기등을 들 수 있다.Examples of the bicyclic group with or without substituents as R ₆ and R ₇ include 1-methyl-2-azetidinyl, 1-methyl-2-pyrrolyl, 5-methyl-2-pril,, 5-nitro-2 -Pril, 3-methyl-2-thienyl, 4,5-dichloro-2-thienyl, 2-methyl-4-thiazolyl, 1,1-methyl-4-imidazolyl, 2-dimethyl-4- Chloro-5-imidazolyl, 3,5-bis-methylthio-4-isothiazolyl, 3-methyl-5-isoxazole, 2-ylmeth-4-tiloxazolyl, 1-methyl-3-pyra Zolyl, 2-, 3- or 4-pyridyl, 4,5,6-trichloro-2-pyrimidyl, 3,5,6-trichloro-2-pyridinyl, 4,6-dichloro-2- S-triadinyl, 3- or 4-quinolyl, 2-quinazolyl, 2-quinoxaryl, 5-fluoro-1-methyl-3-indolyl, 2-benzopril, 2-benzothienyl group, etc. You can also

As a specific example in the case where the bicyclic ring represented as a substituent has a substituent, 2-, 3- or 4-methyl-1- piperidinyl, 4-methyl-1- piperazinyl, 2, 6-dimethyl morpholino And a 2-propyl-1-piperidinyl group.

로서 표시되는 기의 대표적인 예로서는 디메틸아미노 디에틸아미노, 디프로필아미노, 디이소프로필아미노, 디이소부틸아미노, 디벤질아미노, 디펜에틸아미노, 디페닐프로필아미노, N-메틸-N-벤젠아미노, N-에틸-N-부틸아미노, N-메틸-N-시클로펜틸아미노, N-메틸-N-시클로헥실아미노, -N-메틸프르프릴아미노기등을 들 수 있다.R ₆ and R ₇ are other than hydrogen atoms

Representative examples of the group represented as dimethylamino diethylamino, dipropylamino, diisopropylamino, diisobutylamino, dibenzylamino, diphenethylamino, diphenylpropylamino, N-methyl-N-benzeneamino, N -Ethyl-N-butylamino, N-methyl-N-cyclopentylamino, N-methyl-N-cyclohexylamino, -N-methylprprilamino group, etc. are mentioned.

The microorganism used in the method of the present invention is genus Bacillus, Streptomyces or Actinomyces, which has the ability to convert methoxy groups of the maytansinoid complex (II) to hydroxy groups with hydroxyl groups. Any of the microorganisms belonging to the genus and its mutant strains may be used.

Specific examples of microorganisms that can be used in the method of the present invention include, for example, Bacillus, megaterium (Bacillus megat-erium), IFO12108, Streptomyces. Streptomyces flavotricini IFO 12770, Streptomyces, Streptomces platensis, IFO12901, Streptomyces. Streptomyces libani IFO 13452 and Actinomyces. Nigrescens (Actinomyces nigrescens) IFO 12894 and the like.

The above-mentioned IFO numbered microorganisms are a list of fermentation laboratories. Orb. It is listed in Cultures 1972, 1975 and 1978. The microorganisms listed in this list can be obtained from this fermentation laboratory.

In general, genus Bacillus, Streptomyces and Actinomyces are susceptible to changes in their properties, such as X-ray irradiation, ultraviolet irradiation, irradiation, artificial modifiers (eg, nitro). It can be easily mutated as an artificial transformation means using guaniazine, etirenimine, etc.).

Even in such a mutant strain, any of those having the ability to convert methoxy group at position 20 of the maytansinoid compound (II) into hydroxide 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 liquid or solid as long as it contains a nutrient source that can be used by the microorganisms, but it is more appropriate 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 micronutrient, and the like. Examples of carbon sources include, for example, glucose, lactose, sucrose, maltose, dextrin, starch, glissoulol, mannitol, sorbitol, and other fats and oils (e.g., soybean oil, lard oil, chicken oil, etc.) and other nitrogen sources. Kiss, yeast extract, dry yeast, soy flour, coon stripe, lyca, peptone, cottonseed flour, molasses, urine, ammonium salts (e.g. ammonium sulfate, ammonium acetate, 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 vinegar acid and propionic acid are suitably used.

Other amino acids (e.g., gurutamic 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 and the like), nucleic acids (eg, purine, 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 of fat or oil or surface active agent may be added for the purpose of defoaming.

The medium may be stationary culture, or a means such as shaking culture or aeration stirring culture. Mass processing is preferably done by submerged aerobic culture.

It is natural that the conditions of the culture are not constant due to the condition of the medium, the composition, the type of microorganisms, the means of the culture, etc., but they usually select the initial pH at a temperature of 20 ° C. to 45 ° C. as the neutral vicinity. good. In other words, the temperature of the culture medium is preferably 24 to 37 ° C, and the initial pH is 6.5 to 8.5. The cultivation time is good as about 6 to 100 hours, but especially 16 to 60 hours is good.

The culture used in this invention means what is obtained as said culture. The treated product used in the present invention is a physicochemical treatment such as filtration, centrifugation, sonication, French press treatment, alumina chain, lytic enzyme treatment, surfactant or organic solvent treatment, etc. It refers to a cell lysate containing the obtained cell or demethylation enzyme.

Moreover, the demethylation enzyme obtained by refine | purifying by a well-known method, or the 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 above-described microorganisms or a treated product thereof.

The concentration of the starting compound in the reaction solution is suitably 1 to 200 µg / ml. Although reaction temperature is 20-50 degreeC and pH 5-10 is suitable, temperature 24-40 degreeC and pH 6-9 are especially preferable. The reaction time is 10 minutes to 100 hours, more preferably 1 to 48 hours. Moreover, although reaction is good also under the conditions of shaking, aeration, or stirring, even if it stops, shaking, aeration, or stirring is preferable.

In the reaction solution, a reaction accelerator, enzyme stabilizer, or the like may be added as desired. As this reaction promoter, for example, nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotitrin acid (NADP), prabin mononucleotides (FMN), and pravin adenine dinucleotides Coenzymes such as (FAD), their precursors (e.g. adenine, adenosine, adenylic acid, nicotinamide, pravin, riboprabin, etc.), metal salts (e.g. magnesium chloride, manganese chloride, ferrous chloride) , Ferric chloride, zinc chloride, etc.), surfactants (eg, Triton X-100 (manufactured by Rohm and Haas), Brij-58 (manufactured by Gua Atlas), etc.), 3'5 ' -Cyclic adenylic acid. As an enzyme stabilizer, cysteine, 2-mercaptoethanol, dithiositol, sucrose glycerin, etc. are mentioned, for example.

The detection of the novel substance (III) thus obtained can be measured by thin layer chromatography (hereinafter referred to as TLC). Extract the reaction mixture as ethyl acetate, and the solution concentrated to 1/100 capacity silica gel glass plate and a (Mel West Germany keusa, key easel gel 60F _254, 0.25mm, 20X20cm) as a support subjected to TLC solvent: chloroform: methanol: 9: 1), It is measured as an absorbed image detected by irradiating an ultraviolet ray of 2537 Å.

In order to collect the target substance (III) from the reaction solution, since the present substance group is slightly acidic and fat-soluble, it is possible to suitably use the method of separation and purification used for collecting conventional microbial metabolites. For example, means for utilizing the difference in solubility with impurities, means for using the difference in adsorption-extinguishing power of various adsorbents such as activated carbon, microporous nonionic resin, silica gel, alumina, ion exchange resin and impurities It is used alone or in combination as a means or repeatedly.

When a difference in solubility is used, a solvent suitable for extraction from the filtrate is an organic solvent which does not mix with water, for example, fatty acid esters such as ethyl acetate and amyl acetate, and halogenated hydrocarbons such as chloroform such as butanol and methyl isobutyl ketone Ketones, such as these, 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 pH6.

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. Since the TLC of this crude material has a large number of spots other than the novel substance demethyl maytansinoid compound (III), the following purification step is carried out stepwise. That is, various adsorption chromatography is effective as a purification method commonly used, and carriers commonly used as adsorbents, for example, silica gel, alumina, microporous nonionic desorbent resins, and the like, can be used. Silica gel is most effectively used for the purification from), starting with a nonpolar solvent such as petroleum ether, hexane, and then adding a polar solvent such as ethyl acetate, acetone, ethanol, methanol, etc. The methyl maytansinoid compound (III) is eluted. As an example, silica gel (West Germany Melk Co., Ltd. 0.05-0.2 mm) 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 demethyl maytansinoid. Fractions containing compound (III) are combined and concentrated under reduced pressure to give crude (ii) by addition of petroleum ether or hexane. Since this part still contains another impurity, the following purification is performed. For example, it refine | purifies with the 2nd silica gel column which changed the solvent.

In this case, the development solvent starts with halogen-containing hydrocarbons such as dichloromethane and chloroform, and the addition of polar solvents such as alcohols such as ethanol and methanol and ketones such as acetone and methyl ethyl ketone is added. Methyl maytansinoid compound (III) is isolate | separated and collected.

The combination of the solvent systems of the first and second silica gel columns may be reversed before and after, and other commonly used organic solvents are suitably combined.

When using microporous adsorbent resin as a means for purifying crude material (ii), to elute the novel substance demethylmaytansinoid compound (III), a lower alcohol, or a mixture of lower ketones, esters and water is used. .

As lower alcohols, methanol, ethanol, propanol, butanol, etc. are mentioned, for example, As lower ketones, acetone, methyl ethyl ketone ester, ethyl acetate etc. can be used, for example. As an example, the crude material (ii) is dissolved in 50V / V% methanol water, passed through a diamond ion HP-10 (Mitsubishi Kasei) column and adsorbed, and washed with 50V / V% methanol water and then 90V / V. Elution with% methanol water elutes the target substance, demethylmaytansinoid compound (III).

The demethyl maytansinoid compound (I) thus obtained is concentrated under reduced pressure, isolated as crystals with ethyl acetate or concentrated under reduced pressure, and then petroleum ether is added to obtain a powder.

In the demethyl maytansinoid compound (III), a demethyl maytansinoid compound represented by general formula (VI) by deacyl chemical reaction of compound (V), wherein R is a baby represented by R ₁ ′ ( VI).

That is, in the demethyl maytansinoid compound (III), the deketyl maytansinoid compound represented by general formula (VI) by deacylation reaction of the compound (V) in which R is an acyl group represented by R ₁ ′ ( VI). That is, demethylyl maytansinoid compound (VI) whose R <_1> is hydrogen in compound (III) can be obtained by deacylation reaction of the demethyl maytansinoid compound (V). In this case, since the position of the acyl group is above the beta of the carbonyl group, a commonly used reductive cleavage reaction is advantageously used, and the reaction can be carried out according to the conventional method. That is, at low temperature (e.g., -20 to 10 ° C), a complex metal hydrogen compound, for example, lithium aluminum hydride (LiA1H ₄ ) is used, and other functional groups such as carbonyl group, epoxy group, carbon-carbon double Compound (IV) can be obtained by carrying out reduction cleavage of a 3rd O-ester bond, without affecting a bond or the like.

Extraction and purification of compound (IV) can be performed in the same manner as in the above-described extraction and purification method.

As described above, demethyl maytansinoid compound (I) is a stereoisomer (eg, D-isomer, L-isomer) in the structure of acyl group R ₁ , each of its isomers and mixtures thereof. , Include. 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 known as separation means in the process of preparing the compound (II) as described later. For example, silica gel chromatography and its liquid chromatography may separate the respective isomers.

In general, the isomer relationship in the compound (I) in the method of the present invention is as many as the isomer in the compound (II).

In the case where a mixture of these isomers is used as the starting compound (II), the target (I) is obtained as a form of the mixture of isomers. In general, it is possible to separate into individual isomers by a separation method such as a method known per se, for example, silica gel chromatography.

Compound (I) of the present application can be used as antifungal, antiprotozal, antitumour, and its toxicity is low.

Compound (I) can also be used as a useful pharmaceutical synthetic intermediate. The bioactivity test was done about the compound obtained in the Example mentioned later.

Biological activity

A) antimicrobial activity

Tripticase. Using the agar medium (Baltimore Biologicals Limited, U. S. A.) as a test medium, the ability to inhibit the development of the following microorganisms was examined by the paper disk method. That is, paper 0.02 ml of 300 µg / ml solution of the demethylmaytansinoid compound (I) on the following microbial-containing platelet medium. It contained in the disc (Toyo Seisakusho, thin, diameter 8mm), and the growth inhibition ability was examined.

This results in the microorganism Escherichia. Cory, Proutus. Bulgarias, Proutus. Mirabiris, Schudomonas. Aelginosa, Staphyrocox. Aureus, Bacillus, Subchilles, Bacillus, Cereus. Crebra. New Chamonix, Serratia. Marsessen, Mycobacterium. Abium. There was no shout for.

On the other hand, using agar plate of assay batch [3.5 g of sodium diphosphate, 0.5 g of potassium phosphate-0.5 g, homo extract (made by Dipco Co.), 5 g of gurcose, 15 g of agar, 1000 ml pH 7.0 of distilled water] . Averanea (Hamigera avellanea) IFO 7721 was used as a test bacterium, and the antimicrobial activity of the test compound was examined by the paper disc method. That is, 0.02 ml of a 100 µg / ml solution of the demethyl maytansinoid compound (I) was papered on the microorganism-containing flat plate medium described above. Growth inhibition ability was examined by containing in a disk (Toyo Seisakusho, thin, diameter 8mm). As a result, the demethyl maytansinoid compound (I) showed growth inhibition.

The low support to compound (I) was shown in the 1st table | surface. Tetrahimena again. Tetraphy mena pyriformis W strain was used as the test microorganism, and assay batch [Triphytose. 20 g of Phenon (made by Dipco Co., Ltd.), 1 g of yeast extract, 2 g of ruggose, 1,000 ml of distilled water, and 1 molar phosphate buffer pH 7.0, 10 ml] were incubated at 28 ° C. for 44 hours to 48 hours, and then, by liquid dilution assay. The antibiotic's ability to inhibit microbial growth was examined.

As a result, the demethyl maytansinoid compound (I) showed growth inhibition.

Table 1 shows the ability to inhibit the growth of microorganisms with respect to compound (I).

TABLE 1

B) antitumor activity

In the therapeutic test with mice in which P-388 tumor cells were transplanted intraperitoneally, the demethylmaytansinoid compound (I) was clearly observed for a life-long effect in nine consecutive days once intraperitoneally.

C) Acute Toxicity

Acute toxicity by intravenous injection was measured using mice. As a result, demethylmaytansinoid compound (I) was found to have died of 1000 µg / kg.

As mentioned above, the demethyl maytansinoid compound (I) has a strong growth inhibitory ability against filamentous fungi and protozoa, and is also useful as a fungicide or an antiprotozoal agent.

In addition, the demethyl maytansinoid compound (I) is expected to be useful as an antitumor agent because of its life-saving effect on a mammal having a species (eg, a mouse).

If demethylmaytansinoid compound (I) is to be used as a fungicide and an antiprotozoal agent, for example, it can be advantageously used to test bacterial ecology such as soil, activity, sludge or animal body fluids. That is, when separating useful bacteria from the soil, or testing the action of bacteria other than circles or molds in the operation and interpretation of the active 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 solid medium, and 0.1 ml of an aqueous solution containing 10 to 100 µg / ml of 1% methanol of demethylmaytansinoid compound (I) per 1 ml of the medium is incubated. .

Compound (I) of the present invention can be used as an antitumor agent because it has a life-long effect on mixed-blooded mammals (eg, mice, rats, dogs, cats, humans) having tumors.

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 by subcutaneous, intraperitoneal, intravenous, intramuscular injection, etc., and the dosage is, for example, about 50 to about 800 µg / kg body weight / once Although the range is, considering the symptoms, objects, etc. can be appropriately selected. For example, about 500 µg to 10 mg of Compound (I) of the present invention was dissolved in a ratio of about 0.5 ml of alcohol (for example, methanol and ethanol), and then the physiological saline was added so that the total amount was 10 ml. You may prepare. If the dose is small, this solution can be prepared by diluting it with physiological saline.

Demethyl maytansinoid compound (I) was found to have a significantly increased solubility in water.

Hereinafter, in the present specification, the compound of R = COCHCH ₃ ) ₂ and X = CI in General Formula (I) is represented by "PDM-3", and the compound of R = CO (CH ₂ ) ₂ -CH ₃ and X = CI. Are referred to as "PDM-3 '", and compounds of R = COCH ₂ (CH ₃ ) ₂ and X = CI are referred to as "PDM-4", respectively.

In addition, in the starting compound (II) of the method of the present invention, a compound of R ₁ = -CO-CH (CH ₃ ) ₂ and X = CI is represented by "Ansamytocin P-3" and R ₁ = -CO (CH ₂ ) ₂ also the compound of X = CI to "My Ansari cytosine P-3 _{'", R 1 = -COCH 2 -CH (} CH 3) 2 also referred to each compound of X = CI to "My Ansari cytosine P-4" Shall be.

As a compound (II) used as a raw material compound in the manufacturing method of the compound of this invention, you may use well-known compounds, such as a maytansine-ansamytocin.

Maytansine is, for example, US Pat. 3,896,111.

Maytanasin, maytansinol, and propionate can be prepared by the methods described in the Journal of the American Chemical Society 97,5284 (1975) or by Nocardia sp. It is also obtained by incubating C 15003 (FERM-P No.3992: IFO-13726: ATCC-31281) in a culture medium and collecting it in culture. (See Japanese Patent Application Laid-Open No. 53-121998).

Ansamytocin P-3, ansamytocin P-3 ', and ansamytocin P-4 can be obtained by culturing the aforementioned Nocardia sp. C-15003. (See Japanese Patent Application Laid-Open No. 53-130698)

(FERM: Institute of Microbiological Technology, Korea Advanced Institute of Technology).

IFO: Fermentation Research Institute.

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

In addition, compound (II) used as a raw material compound in the method of the present invention is a carboxylic acid represented by maytansinol or dechloromaytansinol as R ₁ OH, wherein R ₁ has the same meaning as described above. Or it can manufacture also by acylating as a reactive derivative in the carboxyl group.

Maytansinol used in the preparation of the above-mentioned compound (II) is a known compound as a plant component [Kupchan et al, J. Amer. Chem. Soc. 97, 5294 (1975), which can be obtained by reductive cleavage of maytansine.

In addition, maytansinol is nocarudia sp. The following formula is produced by incubating C-15003 (FERM-P No. 3992, IFO-13726, ATCC-31281) in a medium.

Maytanasin, maytansinol, propionate or ansamytocin represented by [wherein R ₈ represents an acetyl group, propionyl group, iso-butylyl group, n-butylyl group or iso-bareryl group] Can also be produced by a reductive cleavage reaction using a metal hydride such as LiAlH ₄ . [Japanese Patent Application Laid-Open No. 53-130693, Nature Vol. 270, 721 (1977).

Dechloro maytansinol is obtained by reducing the compound whose X is CI in compound (II) as a metal hydrogen compound. As the metal hydrogen compound, for example, a metal complex, in particular lithium aluminum hydride (LiAlH 4), is preferable, and the amount of the metal hydrogen compound is usually about 1 to 25 mol, preferably about 4 to 1 mol of the raw material compound (II) (X = CI). About 10 moles.

The present reduction reaction is preferably carried out in the presence of a conventional solvent, and ethers such as diethyl ether and tetrahydrofuran are used as the solvent, and tetrahydrofuran is particularly preferable.

The reaction can usually be carried out at a temperature of about -70 ° C to about + 80 ° C, preferably about -40 ° C to + 20 ° C.

In this reaction, a compound in which only the acyl group at the third position of the conventional compound (II) (X = CI) is 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. This is purified by separation using silica gel chromatography, high performance liquid chromatography, or the like to obtain the desired dechloromaytansinol.

In the maytansinoid compound (II) which is a raw material compound of the present invention, a compound in which R ₁ is an acyl group, that is, the following formula

[Formula X and R ₁ 'represent the significance as described above] the compound represented by maytansinol or dechloro maytansinol

R ₁ '-OH (VIII)

It can be produced by reacting with a carboxylic acid represented by [wherein R ₁ 'has the same meaning as described above or a reactive derivative in the carboxyl group thereof].

As said acylation means, the method of acylating maytansinol or dechloro maytansinol as carboxylic acid (VIII) in presence of carbodiimide, for example is mentioned.

Although carboxylic acid (VIII) may use about 1-500 molar equivalents with respect to maytansinol or dechloro maytansinol, about 1-30 molar equivalents are also preferable in many cases.

Although carbodiimide may use about 1-700 molar equivalents with respect to maytansinol or dechloro maytansinol, for example, about 1-50 molar equivalents are often preferable. Carbodiimide which can be used should just have a carbodiimide bond (-NH-CO-NH-), and what can be converted into the urine bond (-NH-CO-NH) in this acylation reaction. For example, what is shown as following formula is mentioned.

R ₉ -N = C = NR ₁₀ (IX)

[R ₉ and R ₁₀ each represent an organic moiety capable of converting a carbodiimide bond to a urine bond during this acylation reaction,]

Examples of the organic residue represented by R ₉ and R ₁₀ include, for example, di-lower (C _1-6 ; the same as below), C _3-7 cycloalkyl group, di-lower alkylamino group or morpholino group with or without alkylamino group. A lower alkyl group having or without, or a phenyl group having or not having a lower alkyl group may be appropriately selected.

As carbodiimide, dicyclohexyl carbodiimide is preferable practically, For example, diphenyl carbodiimide, di-0-tricarbodiimide, di-p-tricarbodiimide, di-tertiary Butylcarbodiimide, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide, 1-cyclohexyl-3- (4-diethylaminocyclohexyl) carbodiimide, 1-ethyl-3- (2-diethylaminopropyl) carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and the like.

The acylation reaction may be carried out in the presence of a suitable solvent, and examples of the solvent include esters (e.g. ethyl acetate), ethers (e.g. diethyl ether, dioxane, tetrahydrofuran, etc.) and halogenation. Hydrocarbons (e.g. methylene chloride, chloroform, etc.), nitriles (e.g. acetonitrile), aromatic hydrocarbons (e.g. benzene) nitromethane, pyridinedimethylformamide, dimethylsulfoxide, sulfolane or a suitable mixture thereof have.

The acylation reaction may be usually performed at an appropriate temperature within the range of the reflux temperature of the reaction system under ice cooling.

-, β-또는 r-피코린, 2,6-루티딘, 4-디메틸아미노피리딘, 4-(1-피를리디닐) 피리딘, 디메틸아닐린, 디에틸아닐린과 같은 방향족 3급아민], 알로겐화 알칼리 금속(예, 불화칼륨, 무수옥화리롬 등), 유기산염(예, 초산나트륨)등을 들 수 있으며, 산촉매로서는, 예를들면, 루이스산[예,무수염화아연, 무수염화알루미늄(AlCl₃), 무수염화제이철, 사염화티탄(TiCl₄), 사염화주석(SnCl₄), 오염화안티몬, 염화코발트, 염화제이동삼불화붕소에테레이드 등], 무기 강산(예, 황산 과염소산, 염화수소, 취화수소 등), 유기강산(예, 벤젠술폰산), p-톨루엔술폰산, 트리플루오로초산, 트리클로로초산 등), 산성 이온 교환수지(예, 폴리스틸렌술폰산)등을 들 수 있다.The acylation reaction proceeds more advantageously by using a catalyst capable of promoting acylation of maytansinol or dechloromaytansinol. As such a catalyst, a base catalyst and an acid catalyst are mentioned, for example. Such base catalysts include, for example, tertiary amines [eg, aliphatic tertiary amines such as triethylamine, pyridine,

-aromatic tertiary amines such as β- or r-phycoline, 2,6-lutidine, 4-dimethylaminopyridine, 4- (1-pyridinyl) pyridine, dimethylaniline, diethylaniline], allo Generated alkali metals (e.g., potassium fluoride, lithium anhydride, etc.), organic acid salts (e.g., sodium acetate), and the like, and acid catalysts include, for example, Lewis acids [e.g. zinc anhydride, aluminum anhydride (AlCl). ₃ ), anhydrous ferric chloride, titanium tetrachloride (TiCl ₄ ), tin tetrachloride (SnCl ₄ ), antimony chloride, cobalt chloride, chlorinated boron trifluoride etheride, etc.], inorganic strong acids (e.g. sulfuric acid perchloric acid, hydrogen chloride, embrittlement) Hydrogen, etc.), an organic strong acid (e.g., benzenesulfonic acid), p-toluenesulfonic acid, trifluoroacetic acid, trichloroacetic acid and the like, an acidic ion exchange resin (e.g. polystyrenesulfonic acid), and the like.

In the above formula (VIII), when carboxylic acid having an acyl group represented by the formula -CO-R ₂ (wherein R ₂ has the same meaning as described above) is generally used as the R 1 ′ in the reaction, Among the catalysts, 4-dimethylaminopyridine or 4- (1-pyrrolidinyl) pyridine and the like are preferable, and among the acyl groups, R ₁ in formula (VIII)

-아미노아실기를 갖는 카르본산을 반응에 사용할 경우에는 무수염화아연이 바람직한 촉매로서 사용된다.N-acyl- represented by [Formula and R ₃ , R ₄ and R ₅ have the same meaning as described above].

When carboxylic acid having an aminoacyl group is used for the reaction, anhydrous zinc chloride is used as the preferred catalyst.

The amount of the catalyst used may be about the amount of catalyst capable of promoting acylation of maytansinol or dechloromaytansinol by carboxylic acid (VIII), and usually about 0.001 to 10 molar equivalents, preferably (VIII). Preferably about 0.01 to 1 molar equivalent.

By the use of such a catalyst, the yield of maytansinoid compound (II) can be remarkably improved. The amount of carboxylic acid (VIII) can also be saved, for example about 1-10 molar equivalents for maytansinol or dechloromaytansinol.

를 갖는 카르본산에 이성체, 예를들면 D-이성체, L-이성체가 존재할 경우에는 (VⅢ)로서 개개의 이성체 또는 이들 임의의 비율의 혼합물을 사용할 수 있다. 본 반응에 의해 메이탄시놀 또는 데클로로메이탄시놀의 3위의 수산기에 광학 활성체인 아실기를 도입할 경우 그것에 상당하는 카르본산(VⅢ)의 광학활성체를 사용하면 바람직하다.In this reaction, carboxylic acid (VIII), for example, N-acyl-aminoacyl group

If isomers, such as D-isomers and L-isomers, are present in the carboxylic acid having a (VIII), individual isomers or mixtures of any of these ratios can be used. In the case where the acyl group, which is an optically active substance, is introduced into the hydroxyl group of maytansinol or dechloromaytansinol by this reaction, an optically active substance of carboxylic acid (VIII) corresponding thereto is preferably used.

Even when optically active carboxylic acid (VIII) is used, maytansinoid compound (II) is sometimes prepared as a mixture of D- and L-isomers.

As an acylation means which uses the reactive derivative in the carboxyl group of carboxylic acid (VIII), for example, acylated the 3rd position of maytansinol or dechloro maytansinol which acquires the acid anhydride of carboxylic acid (VIII). The method of acylating using the derivative which has a functional group which can be mentioned is mentioned.

As a solvent and a catalyst in an acylation means, the thing similar to the case of the acylation performed in presence of said carbodiimides is illustrated. The reaction temperature is usually about 20 to 100 ° C, more preferably about 20 to 40 ° C, and may be further heated to increase the reaction rate.

(식중, R₆및 R₇은 전기한 바와 같은 의미를 갖는다)로서 나타내는 화합물은 예를들면 메이탄시놀 또는 데클로로메이탄시놀을 카르바모일화반응시켜 제조할 수 있다.In the raw material compound (II) of the present invention, R ₁ ′ is

(Wherein R ₆ and R ₇ have the same meanings as mentioned above), for example, can be produced by carbamoylation of maytansinol or dechloromaytansinol.

(X)[식중, R₆및 R₇은 전기와 같은 의의, Z는 할로겐원자를 존나타냄]로서 표시되는 카르바민산 할라이드를 염기의 존재하에서 반응시키는 방법을 들 수 있다.As a means of carbamoylation, for example mayoltanol or dechloromaytansinol

And a method of reacting the carbamic acid halide represented by (X) (wherein R ₆ and R ₇ are the same as in the above, Z represents a halogen atom) in the presence of a base.

Examples of the halogen atom represented by Z in the formula (X) include oxygen, cancellation, and the like.

Examples of the base used in the reaction include alkali metals (e.g., lithium, sodium, etc.) or alkali metal alkyls (e.g., n-butyllithium, sec-butyllithium, phenyllithium, sodium naphthalene, sodium methanesulfinylmeside, etc.). Of these, n-butyllithium is preferred.

The reaction is carried out in a solvent and ethers (e.g., tetrahydrofuran, dimethyl ether, diethyl ether, etc.), aromatic hydrocarbons (e.g. toluene, xylene, etc.) or a suitable mixture of these solvents are used. The most widely used.

The reaction is carried out by dissolving maytansinol or techloromaytansinol in these solvents, and then adding the alkali metal (compound) described above to this, followed by adding a carbamic acid halide of formula (X).

The alkali metal (compound) is preferably used in an amount of about 3 to 10 molar equivalents to 1 mole of maytansinol or dechloro maytansinol, and more preferably 4 to 6 molar equivalents. Carbamic acid halides may be used in an amount of about 2 to 10 molar equivalents, preferably about 3 to 5.5 molar equivalents.

Although reaction is performed as temperature suitably selected in the range of -78 degreeC-+50 degreeC, it is good to perform normally as -30 degreeC-40 degreeC. Subsequently, the reaction solution is neutralized with a dilute aqueous solution if necessary, and then the product can be isolated using a method known per se, which is described later, as it is or after organically dissolving the solvent.

In addition, this reaction can also be performed using the other reactive derivative in the carboxyl group of carbamic acid instead of the carbamic acid halide mentioned above.

Other methods of carbamoylation include, for example, maytansinol or dechloromaytansinol,

R ₆ -N = C = O (XI)

There is a method of reacting the compound represented by (wherein R ₆ is as defined above). According to the present invention, a maytansinoid compound in which R ₇ is a hydrogen atom in formula (II) is obtained. The present reaction is preferably carried out in a solution state, and the solvent is not particularly limited as long as it is a solvent that does not have an active hydrogen atom capable of reacting with compound (XI) (eg, -OH, -NH- group hydrogen atom, etc.). Halogenated hydrocarbons (e.g., dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, etc.), aromatic hydrocarbons (e.g., diethyletherdimethoxyethane, dioxane, tetrahydrofuran, etc.) , Benzene, toluene, xylene, chlorobenzene and the like), nitriles (e.g. acetonitrile, propionitrile and the like), ethers (e.g. ethyl acetate and the like) and suitable mixtures thereof. It is preferable to perform reaction in these solvents which do not contain water as much as possible.

The amount of compound (XI) required for the reaction may be about equimolar amount relative to maytansinol or dechloromaytansinol, but is consumed by water in the solvent, and in some cases, compound (XI) itself is active. On the other hand, in consideration of work consumed by a reaction such as dimerization and the like, it is preferable to use it slightly in excess, and it is preferable to use about 2 to 20 times molar amount, preferably about 2 to 5 times molar amount. The reaction can be carried out in a temperature range of -20 ° C to 80 ° C, preferably 5 ° to 40 ° C. In the case of the active compound (XI) (typically having a nitro, polyhalogen substituent, etc.), the reaction can be carried out by simply mixing both reaction raw material compounds, but it is usually preferred to use a catalyst. As a catalyst, a basic substance, such as a tertiary amine (triethylamine, pyridine) and alkali metal alkoxide (potassium tert-butylate), is usually used as a catalyst used for carbamoylation of alcohols or phenols with isocyanate esters. , Sodium methyllaart), alkali metal acetates (e.g. lithium, sodium potassium, rubidium and cesium acetate) or metal salts (e.g. lead, bismuth, tin, cobalt, zinc, kidmium, manganese, titanium, iron, copper, etc.) Chlorides, organic carbonates, etc.), metal complexes or organometallic compounds (e.g., 2,4-pentadiene-metal complexes, fetocene, dibutyltin oxide, dioctyltin oxide, dibutyltin-bislauroaart Etc)

Among these, anhydrous zinc chloride is used as a catalyst which is especially suitable for the present reaction in view of the selectivity of the reaction, the reaction rate and the like.

The amount of the catalyst used is good as the amount of catalyst capable of promoting the reaction, and is usually sufficient in an amount of about 0.01 to about 10 body equivalents, preferably about 0.1 to about 3 molar equivalents, relative to compound (XI).

As the catalyst described above, for example, when the first copper chloride is used,

대신에 수소 원자를 갖는 화합물(II)로 전환시킬수 있다.(Wherein X and R ₆ are the same as described above), a compound may be produced. This compound (XII) is ninth place by treatment with acid

Instead, it can be converted to the compound (II) having a hydrogen atom.

Examples of the acid used in the reaction include a mineral acid (e.g., hydrogen chloride, hydrochloric acid, sulfuric acid, phosphoric acid, etc.), an organic strong acid (e.g., benzenesulfonate, toluenesulfonic acid, methanesulfonic acid, trifluoroacetic acid, trichloroacetic acid, and the like). Among them, trifluoroacetic acid is suitable. It is preferable to perform reaction as a solution state, As a solvent, the solvent similar to the case of carbamoylation by the compound (XI) mentioned above can be used. The reaction proceeds slowly, typically at temperatures of about -20 ° to about 40 ° C.

The present reaction can also be carried out by simply passing a carbamoylated reaction solution containing compound (XII) through a silica gel column.

The maytansinoid compound (II) of the present invention produced by each of the above-described methods can be isolated and extracted from the mixture using conventional means as appropriate, for example, by concentration, solvent extraction, chromatography, recrystallization, and the like. When compound (II) is prepared as a mixture of isomers (e.g., D-isomers, L-isomers), they may generally be separated into their respective isomers by means of known separation means such as silica gel chromatography. Maytansinoid compounds of the present invention include their individual isomers and mixtures thereof.

Hereinafter, the present invention will be described in more detail with reference to Examples and Examples, but the scope of the present invention is not limited thereto. In addition, percentage (%) represents weight / capacity percentage (W / V%) unless there is particular notice.

Reference Example 1

99.6 mg (0.176 mmol) of maytansinol is dissolved in 5 ml of dry dichloromethane. In this solution, 377.2 mg (1.76 mmol) of anhydrous acidic anhydride (capronic anhydride) and 43.5 mg (0.366 mmol) of p- Dimethylaminopyridine (DMAP) is added and the mixture is stirred at room temperature (about 23 ° C.) for 6 hours, then 30.5 mg (0.25 mmol) of DMAP is added and stirred at room temperature for 18 hours. 5 ml of 1 N hydrochloric acid is added to the reaction solution, the mixture is separated, the organic layer is taken, washed with 10% sodium bicarbonate (10 ml), and dried over anhydrous sodium sulfate. The solvent was then distilled off and the residue was subjected to silica gel (75 g) column chromatography, first eluting with ethyl acetate (about 250 mL), then ethyl acetate / ethyl saturated acetate = 2/1 (V / V) (about 900 mL) Let's do it. 16 g of the eluate was fractionated, and the fraction No. 13 to 30 are combined and the solvent is distilled off to give 43.0 mg of crude product. After dissolving this in ethyl acetate, ether was added to recover the precipitated white powdery solid to obtain 34.3 mg of maythasinol 3-hexanoate. Melting point 159-162 degreeC (decomposition).

Reference Example 2

In a solution of 23.5 mg of maytansinol dissolved in 1.0 ml of dichloromethane, acet-formic anhydride (cooling 2 ml of acetic anhydride to -5 ° to 0 ° C. at about 22 ° C., followed by stirring, 1 ml of 99 % Formic acid was added at a temperature of −5 ° to 0 ° C. over 10 minutes, followed by heating at 50 ° C. for 15 minutes, followed by quenching to 0 ° C.), 70.5 mg (about 10 mmol equivalent) and 11.7 mg 4-dimethylamino Pyridine is added and half-mixed at about room temperature (22 ° C.) at night. Subsequently, 10 drops of methanol was added to the reaction mixture, stirred and mixed at room temperature for 3 hours, and then dried under reduced pressure. The residue was then spotted on a silica gel formulation thin layer chromatograph and developed twice as ethyl acetate, followed by about 6.0 to 50 ml of steamer. The 8.0 cm silica gel is scraped off and eluted as 10% methanol dichloromethane. The molten metal is then distilled off under reduced pressure to yield 8.35 mg of maytansinol 3-formate as a colorless glassy material.

Reference Example 3

By the method of the reference example 1 or 2, the compound shown below is obtained.

(A) White sand-like substance maytansinol 3-octanoate is obtained from maytansinol and molar octanoic anhydride (caprylic anhydride). Melting Point 151-160 ° C (Decomposition)

(B) A white form of solid maytansinol 3-decanoate is obtained from maytansinol and decanoic acid (capric acid). Melting point 130-134 degreeC (decomposition).

(C) Maytansinol 3-heptanoate is obtained from maytansinol and heptanoic acid. Melting point 158-160 degreeC (decomposition).

(D) Maytansinol 3-tridecanoate is obtained from maytansinol and tridecanoic acid. Melting point 110-116 degreeC (decomposition).

(E) Maytansinol 3-hexadecanoate is obtained from maytansinol and hexadecanoic acid (palmitic acid) as a white powder. Melting point 105-116 degreeC (decomposition).

(F) Maytansinol 3-valerate is obtained from maytansinol and valeric anhydride. Melting point 165-168 degreeC.

Reference Example 4

Ethylcyclohexyl carbodiimide (DCC) (267 mg, 1.296 mmol) in which maytansinol (103.2 mg, 0.183 mmol) and cyclohexane carboxylic acid (140 mg, 1.094 mmol) were dissolved in 5 ml of dry dichloromethane. After stirring and stirring at room temperature until the non-condensate starts to precipitate, p-dimethylaminopyridine (50.8 mg, 0.416 mmol) of DMAFX is added and stirred and mixed at room temperature overnight. After the insolubles were filtered off, the filtrate was washed with 0.5 N hydrochloric acid (about 10 mL), then with saturated sodium bicarbonate (about 10 mL), dried over anhydrous sodium sulfate, and the solvent was distilled off. Subsequently, the obtained residue was subjected to silica gel curl (75 g) lump chromatography, eluted with ethyl acetate, and the eluate was collected by fractionation of 16 g each. Fraction No. 14 to 30 were combined and the elution was distilled off to obtain 59 mg of crude animal, which was dissolved in ethyl acetate and ether was added to give 24.3 mg of maytansinol 3-cyclohexanecarboxylate. Melting point 202 degrees-206 degreeC (decomposition).

Reference Example 5

The compound shown below is obtained by the method similar to the reference example 4.

(A) Maytansinol 3-cyclopropane carboxylate is obtained from maytansinol and cyclopropane carboxylic acid. Melting point 182 degrees-187 degreeC (mei).

(B) Maytansinol 3-phenylacetate is obtained from tancinol and phenylacetic acid. Melting | fusing point 180 degrees-182 degreeC (decomposition).

(C) Maytansinol 1-benzoate is obtained from maytansinol and benzoic acid. Melting point 174 ° -177 ° C. (decomposition).

(D) Maytansinol 3-p-chlorobenzoate is obtained from maytansinol and p-chloro benzoic acid. Melting point 178 ° to 183 ° C. (decomposition).

(E) Maytansinol 3- (2-furan) carboxylate is obtained from maytansinol and 2-furancarboxylic acid. Melting point 180 degrees-189 degreeC (decomposition).

(F) Maytansinol 3-phenylpropionate is obtained from maytansinol and phenylpropionic acid. Melting point 160 degrees-163 degreeC (decomposition).

(G) A white powder of maytansinol 3-nicotinate is obtained from maytansinol and nicotinic acid. Melting point 184 ° to 187 ° C. (decomposition).

(H) Maytansinol 3-picolinate is obtained from maytansinol and picolinic acid. Melting point 190 ° to 193 ° C. (decomposition).

(I) Maytansinol 3-isonicotinate of white crystals is obtained from maytansinol and isonicotinic acid. Melting point 185 ° to 187 ° C. (decomposition).

이 233,244,253 및 282인 유리상)(J) Maytansinol 3- (N-acetyl) -L-propynate as white crystals is obtained from maytansinol and N-acetyl-L-proline. Melting Point 195 ° ~ 198 ° C (UV Spectrum

Glass 233,244,253 and 282)

(K) Maytansinol 3- (2-thiophene) carboxylate of a glassy solid is obtained from maytansinol and 2-thiophene carboxylic acid. Melting | fusing point 161 degrees-163 degreeC (decomposition).

Reference Example 6

15.0 g of the antibiotic ansamytoxin mixture (ansamitocin P-2 = 12%; ansamitocin P-3 = 71%, ansamitocin P-4 = 17%) were dissolved in 800 ml of dry tetrahydrofuran (THF) and dried nitrogen. Cool to −50 ° C. using a dry ice-ethanol bath under airflow. Then, 13.0 g of lithium aluminum hydride (LAH) was added all at once, stirred and mixed at -50 ° C to 22 ° C for 2 hours, cooled to -28 ° C again, and 3g of lithium aluminum hydride (LAH) was added, After stirring for 1 hour and 20 minutes at -28 ° C to 22 ° C, the mixture was again cooled to -50 ° C, and then carefully added dropwise 750 ml of 2N hydrochloric acid over 30 minutes, and the reaction solution was washed with 2.6 L, 1.6 L and 0.8 L. extracted three times with ethyl acetate, dried (MgSO _4, 250g), washed (twice each 100㎖) as a saturated aqueous solution of sodium chloride. the extract combined. 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 developed and eluted as ethyl acetate / water = 98.5: 1.5 (V / V), and the eluate was collected by fractionation of 400 g each. Fraction No. 35 to 52 were combined, the solvent was distilled off, and dried in vacuo to yield 7.25 g of maytansinol. 1.55 g of an almost equimolar mixture of maytansinol and dechloromaytansinol at 53 to 68 and fraction No. 0.78 g of dechloromaytansinol is obtained in 69 to 86. As a result, white powder of 0.71 g of chloromaytansinol is obtained from recrystallization of chloroform-hexane. Melting point 174 to 179 (decomposition).

Reference Example 7

In a solution of 100.0 mg (0.189 mmol) of dechloromethanesinol in 15 ml of dry dichloromethane, 69 mg (0.476 mmol) of N-acetyl-N-methyl-L-alanine, 117 mg (0.568 mmol) of DCC and anhydrous 39 mg (0.287 mmol) of zinc chloride were added sequentially, and the mixture was stirred at room temperature (about 23 ° C.) for 30 minutes, and again 55 mg (0.379 mmol) of N-acetyl-N-methyl-L-alanine (DCC 98 mg (0.476 mmol)) And 31 mg (0.228 mmol) of anhydrous zinc chloride are added, followed by stirring and mixing at room temperature for 2 hours. The insolubles were filtered off, the filtrate was washed with water, dried and concentrated to dryness. The residue was dissolved in 30 ml of ethyl acetate, the insolubles were filtered off, and the filtrate was concentrated to dryness and the residue was washed with about 5 ml of ethyl acetate. The solution was dissolved and attached to silica gel column chromatography (2 mm (outer diameter) x 500 mm), followed by elution with ethyl acetate / ethyl saturated ethyl acetate = 2/1 (V / V), followed by elution with ethyl saturated acetate, and the eluate was 15 g each. Fraction. When fractions No. 55 to 103 are combined and the solvent is distilled off, 53 mg of crude dechloromaytansine is obtained, which is dissolved in ethyl acetate and cooled by addition of ether to give 24 mg of L-dechloromaytansine as colorless crystals. Melting Point 184 ~ 186 ℃ (Decomposition)

Fraction No. of the chromatography described above. When 168-221 are mixed and the solvent is distilled off, 65 mg of D-dechloromaytansine is obtained. After dissolving this in chloroform, the precipitated crystals were filtered by adding ether to give 21 mg of colorless microcrystals of D-decolomytansine. Melting point 175-178 degreeC (decomposition).

Reference Example 8

By the method similar to the reference example 7, the compound shown below is obtained.

(A) Dechloromaytansinol 3-isobutylate is obtained as white prismatic crystals from dechloromaytansinol and isobutyl anhydride. Melting point 250-252 degreeC (decomposition).

(B) Dechloromaytansinol 3-nicotinate is obtained from dechloromaytansinol and nicotinic acid. Melting point 170-173 degreeC (decomposition).

(C) Chlorotantansinol 3-cyclohexanecarboxylate is obtained from dechloromaytansinol and cyclohexanecarboxylic acid. Melting point 217-220 degreeC (decomposition).

(D) Dechloromaytansinol 3-phenylacetate is obtained from dechloromaytansinol and phenylacetic acid. Melting point 165-170 degreeC (decomposition).

Reference Example 9

In a solution of 150.0 mg (0.265 mmol) of maytansinol in 30 mL of dry dichloromethane, 124.0 mg (0.663 mmol) of N-acetyl-N-methyl-L-leucine and dicyclohexylcarbodiimide (DCC) 174.2 After adding mg (0.846 mmol) and stirring at room temperature for a while, 46 mg (0.338 mmol) of anhydrous zinc chloride are added, stirring at room temperature for 30 minutes, and again adding 46 mg of anhydrous zinc chloride and stirring at the same temperature for about 45 minutes. . Subsequently, 104.3 mg (0.558 mmol) of N-acetyl-N-methyl-L-leucine, 141 mg (0.686 mmol) of DCC and 46 mg of anhydrous zinc chloride were added again, followed by stirring at the same temperature for 2.5 hours. The reaction solution was washed with water, the organic layer was dried over Na ₂ SO ₄ , the solvent was distilled off under reduced pressure, and the residue was added to silica gel (75 g) column chromatography, and the resultant was eluted with ethyl acetate (about 600 mL), followed by saturated ethyl acetate. As an elution, the eluate was separated and collected in 17 g portions. Fraction No. 14 to 34 were combined, and the residue (100 mg) obtained by distilling off the solvent was added again to silica gel (35 g) column chromatography (solvent: chloroform / methanol = 60/1 (V / V), and the eluate was separated by 25 g. Combine 16-30 to dissolve the solvent, dissolve the residue in ethyl acetate and cool, filter the precipitated crystals to obtain 89 mg of Compound A. Combine fractions No. 35 to 56 of the first chromatograph and distill the solvent Water was purified by silica gel (40 g) chromatography (solvent: chloroform / methanol = 60/1 (V / V) (about 200 mL), and 40/1, 1 L: 25 g each). The residue was dissolved in ethyl acetate, ether was added, and the precipitated precipitate was filtered off to give 52 mg of Compound B. Compounds A and B are both targets and from the following physicochemical data, these compounds were each Maytansinol 3- (N-acetyl-N-methyl) -L-leucine ester and maytansinol Judging from 3- (N-acetyl-N-methyl) -D-leucine ester.

Compound A: melting | fusing point 172-175 degreeC (decomposition).

Compound B: melting | fusing point 157-159 degreeC (decomposition).

Reference Example 10

The following compounds are manufactured by the same method as in Reference Example 9.

(A) Isomers differing only in the stereo configuration of the maytansinol 3- (N-acetyl-N-benzyl) alanine esters from the mayansinol and N-acetyl-N-benzyl-D-alanine with the substituents above 2 ' Get as. Melting point 174 to 177 ° C (decomposition), 163 to 166 ° C (decomposition),

(B) Maytansinol and maytansinol 3- (N-acetyl-N-methyl) phenylalanine esters in maytansinol and N-acetyl-N-methyl-L-phenylalanine as isomers differing only in the stereo configuration of the substituent on 2 ' Get Melting point 189-193 degreeC (decomposition), 212-214 degreeC (decomposition).

: 234,244,254,282,290], 및 메이탄시놀 3-(N-tert-부틸옥시카르보닐-N-메틸-D-알라닌) 에스테르[UV-스펙트럼

: 234,241(sh),253.5,282,290]을 얻는다.(C) Maytansinol 3- (N-tert-butyloxycarbonyl-N-methyl-L-alanine) ester in maytansinol and N-tert-butoxygaryl-N-methyl-L-alanine [UV-spectrum

: 234,244,254,282,290], and maytansinol 3- (N-tert-butyloxycarbonyl-N-methyl-D-alanine) ester [UV-spectrum

: 234,241 (sh), 253.5,282,290].

(D) Maytansinol 3- (N-acetyl) zalcosine esters from maytansinol and N-acetylzalcosine are obtained as a glassy substance.

ppm : 0.87(3H,s), 1.28(3H,d,J=5Hz), 1.68(3H,S), 2.14(3H,s), 2.19(1H,dd,J=3Hz 및 14Hz), 2.55(1H,dd,J=11Hz 및 14Hz), 2.76(1H,d,J=9Hz), 3.07(2H,s), 3. 13(3H,s), 3. 18(3H,s) 3.35(3H,s), 3. 47(1H,d,J=9Hz), 3.52(1H,d,J=13Hz),3.98(3H,s), 4.18(1H,m), 4.92(1H,dd,J=3Hz 및 11Hz),5.74(1H,dd,J=9Hz 및 15Hz), 6.18(1H,d,J=11Hz), 6.44(1H,dd,J=11Hz 및 15Hz), 6.53(1H,s), 6.82(2H,s)NMR Spectrum (in CDCI ₃ )

ppm: 0.87 (3H, s), 1.28 (3H, d, J = 5 Hz), 1.68 (3H, S), 2.14 (3H, s), 2.19 (1H, dd, J = 3 Hz and 14 Hz), 2.55 (1H) , dd, J = 11Hz and 14Hz), 2.76 (1H, d, J = 9Hz), 3.07 (2H, s), 3.13 (3H, s), 3.18 (3H, s) 3.35 (3H, s ), 3. 47 (1H, d, J = 9 Hz), 3.52 (1H, d, J = 13 Hz), 3.98 (3H, s), 4.18 (1H, m), 4.92 (1H, dd, J = 3 Hz and 11 Hz), 5.74 (1H, dd, J = 9 Hz and 15 Hz), 6.18 (1H, d, J = 11 Hz), 6.44 (1H, dd, J = 11 Hz and 15 Hz), 6.53 (1H, s), 6.82 (2H , s)

(E) Maytansinol 3- (N-acetyl) -glycine ester is obtained from maytansinol and N-acetylglycine. Melting point 189-192 ° C. (decomposition),

Reference Example 11

Maytansinol (300 mg, 0.5315 mmol) and N-acetyl-N-methyl-L-alanine (1.585 g, 10.62 mmol) were dissolved in 80 mL dichloromethane, and 3.285 g of dicyclohexylcarbodiimide was added to this solution. 72.5 mg (0.532 mmol) of anhydrous zinc chloride is added, stirred and mixed at about 20 ° C. for 6 hours, and left to stand at the same temperature for 11 hours. Then, N-acetyl-N-methyl-L-alanine (530 mg) dicyclohexylcarbodiimide (1095 mg) and anhydrous zinc chloride (150 mg) were added, and after 2 hours, the reaction mixture was filtered, and the filtrate was about 150 Wash with ml water and dry over anhydrous sodium sulfate. The insolubles were filtered off and the filtrate was attached to silica gel (60 g) column chromatography, eluted with chloroform / methanol = 40/1 (V / V), and fractionated 25 g after excluding the current fraction. Fraction N0.14 to 25 was concentrated down, and chromatographed using silica gel (65 g), followed by developmental elution as ethyl acetate / ethyl saturated ethyl acetate = 2/1 (V / V), excluding the current. 16 g each and fraction fraction No. Obtain 149.3 mg (Compound A) at 2560. The second fractions of the chromatographic fractions Nos. 23, 24 and 61 to 100 were combined and concentrated to dryness to obtain 20.5 mg of the material, which was added to the thin layer chromatography of silica gel preparation ((Kieselgel) 60F ₂₅₄ ArT 5717, Merck) for 10%. As isopropanol-chloroform of 6.3 mg of Compound A is obtained. The fractions No. 101 to 105 of the second chromatography were combined and concentrated to obtain 320 mg of the product, which was then chromatographed again with a column filled with 75 g of silica gel [solvent: chloroform / methanol = 40/1 (V / V)]. 95.7 mg of Compound A is obtained (Compound B).

The total yield of compound A is 155.6 mg and the yield of compound B is 95.7 mg.

Compound A is a natural maytansine (L-type) by comparing the following various data on this with those of the natural maytansine described in Journal of Organic Chemistry, 42, No. 14, 2349-2357 (1977). )

When Compound A is dissolved in a mixture of acetic acid ether and left in a refrigerator, crystals are obtained, which are recrystallized once in ethyl acetate-ether and then twice in dichloromethane-ether to obtain crystals of Compound A. Colorless tablet, melting point 191-195 ° C. (decomposition).

Compound B is considered D- maytansine again by identifying several of the following data relating to this with the data of maytansine and identifying the isomer of maytansine.

The chloroform solution of compound B was treated with diethyl ether to recrystallize. The crystal thus obtained was recrystallized twice using the same solvent system to obtain a crystal of a compound having a melting point of 155 to 178 DEG C (slow decomposition).

Reference Example 12

By the method similar to the reference example 11, the compound shown below can be obtained.

(A) Maytansinol and N-methyl-N-propionyl-L-alanine in natural form (type L) maytanfurin colorless needle, melting point 185-189 ° C. (slight degradation) and D-maytanpurin Colorless needles and melting points of 192 to 197 ° C (decomposition) are obtained.

(B) From maytansinol and N-isobutyryl-N-methyl-L-alanine, natural form (L type) maytanbutin [colorless needle melting point 185-187 degreeC (decomposition)] is obtained.

(C) Maytansinol and N-isobareryl-N-methyl-L-alanine obtain natural form (type L) maytanvarin and D-maytanbarin.

NMR spectrum of native maytanbarin (in CDCl ₃ ) 8: 0.79 (3H, s), 0.91 (3H, d, J = 6 Hz), 0.95 (3H, d, J = 6 Hz), 1.27 (3H, d, J = 6 Hz), 1.30 (3H, d, J = 7 Hz), 1.64 (3H, s), 2.13 (2H, d, J = 7 Hz), 2.15 (1H, dd, J = 14 Hz and 3HZ), 2.60 (1H , dd, J = 14Hz and 11Hz), 2.83 (3H, s), 3.00 (1H, d, J = 9Hz), 3.07 (1H, d, J = 13Hz), 3.17 (3H, s), 3.34 (3H, s), 3.47 (1H, d, J = 9 Hz), 3.59 (1H, br), 3.65 (1H, d, J = 13 Hz), 3.95 (3H, s), 4.27 (1H, m), 4.74 (1H, dd, J = 12 Hz and 3 Hz), 5.35 (1H, q, J = 7 Hz), 5.64 (1H, dd, J = 15 hZ and 9 Hz), 6.28 (1H6.69 (1H, br, s), 6.39 (1H, dd, J = 15 Hz and 11 Hz), 6.67 (1H, d, J = 2 Hz), d, J = 11 Hz), 6.69 (1H, d, J = 1 Hz), 0.7-2.0 (3H).

Mass spectrum (m / e): 733,672,485,470,450,170

NMR spectrum of D-maytanbarin (in CDCl ₃ ) 8: 0.89 (3H, s), 0.93 (3H, d, J = 6 Hz), 0.96 (3H, d, J = 6 Hz), 1.26 (3H, d, J = 4 Hz), 1.49 (3H, d, J = 7 Hz), 1.69 (3H, s), 2.66 (1H, dd, J = 15 Hz and 12 Hz), 3.02 (3H, s), 3.12 (3H, s), 3.18 (d, J = 13Hz), 3.32 (3H, s), 3.42 (1H, d, J = 9Hz), 3.50 (1H, d, J = 13Hz), 3.96 (3H, s), 4.29 (1H, m ), 4.92 (1H, dd, J = 11 Hz and 3 Hz), 5.00 (1H, q, J = 7 Hz), 5.05 (1H, br), 5.78 (1H, dd, J = 15 Hz) and 9 Hz, 6.17 (1H) , d, J = 11Hz), 6.24 (1H, s) 6.43 (1H, dd, J = 15Hz and 11Hz), 6.77 (1H, d, J = 1.5Hz), 6.83 (1H, d, J = 1.5Hz) , 0.8-2.5 (7H).

Mass spectrum (m / e): 733, 672, 485, 470, 450, 170

Reference Example 13

(I) 53.5 g (0.52 mol) of N-methyl-L-alanine is suspended in 600 ml of methanol and 76 g of dry hydrogen chloride gas is dissolved with ice-cooling stirring. As the reaction proceeds, the suspension of the raw material is reduced and a uniform solution is stirred overnight at room temperature. 85 g (0.8 mole) of ortho formic acid methyl e-sxpr was added to the reaction solution, and the mixture was left at room temperature for 24 hours, and then a small amount of insoluble matter was filtered off and the filtrate was concentrated under reduced pressure to obtain a solid of N-methyl-L-alanine methyl ester hydrochloride. Lose.

NMR spectrum (in DMSO-d ₆ ) 8: 1.50 (3H, d, J = 7H), 2.60 (after 3H, m heavy water addition, s), 3.75 (3H, s), 4.12 (1H, m, after heavy water addition , q, J = 7 Hz), 9.83 (2H, br)

(II) 33.7 g (0.22 mol) of N-methyl-L-alanine methyl ester hydrochloride was dissolved in 300 ml of chloroform, 65 ml of anhydrous acetic acid and 110 ml of triethylamine were added and left at room temperature for 24 hours. After decomposition, neutralize by adding sodium bicarbonate. The chloroform layer was separated, the water layer was extracted with ethyl acetate (120 mL × 5), the chloroform layer and the ethyl acetate layer were combined and concentrated under reduced pressure. The brown oil obtained was dissolved in chloroform, washed with an aqueous solution of sodium bicarbonate, and concentrated under reduced pressure. 31.8 g of N-acetyl-N-methyl-L-alanine methyl ester is obtained.

NMR spectrum (in CDCl ₃ ) 8: 1.38 (3H, d, J = 7 Hz), 2.12 (3H, s) 2.97 (3H, s), 3.70 (3H, s), 5.23 (1H, q, J = 7 Hz)

The obtained ester is dissolved in 100 ml of methanol and 170 ml of 1 N sodium hydroxide aqueous solution, and the mixture is left at room temperature for 2 hours. The methanol is removed under reduced pressure, and the alkaline aqueous solution is extracted with chloroform. The pH of the aqueous layer was adjusted to 1 with hydrochloride while ice-cooling, followed by extraction with ethyl acetate (140 mL x 5), which was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a white solid. Recrystallization with ethyl acetate hexane gave 8.1 g of colorless acicular crystal of N-acetyl-N-methyl-L-alanine.

[α] ²⁵ _D -58.5 ° (c = 1, DMF)

-74.3 ° (c = 1, H ₂ O)

Melting Point 121 ~ 122 ℃

(III) The following compound is manufactured by the method similar to the above.

(A) N-methyl-N-propionyl-L-alanine (colorless columnar crystals) is obtained from N-methyl-L-alanine methyl ester hydrochloride and propionic anhydride. Melting Point 108 ~ 110 ℃

(B) N-isobutyl-N-methyl-L-alanine (colorless columnar crystals) is obtained from N-methyl-L-alanine methyl ester hydrochloride and isobutyl chloride. Melting Point 117 ~ 118 ℃

(C) N-methyl-N-isovaleryl-L-alanine (colorless needle crystal) is obtained from N-methyl-L-alanine methyl ester hydrochloride and isovaleryl chloride. Melting Point 88 ~ 89 ℃

Reference Example 14

(I) Preparation of β-methoxycarbonylethyl isocyanate

26.4 g of succinate monomethyl ester is dissolved in 600 ml of dry toluene, followed by 55 g of diphenylphosphoryl azide and 22 g of triethylamine. After standing at room temperature for 3 hours, the mixture was washed with iced water, dried, concentrated to about 1/3 the solvent and refluxed for 2 hours. After distilling off the solvent completely, distillation under reduced pressure afforded 13.6 g of the title compound.

Boiling Point 8: 64 to 66 ° C

(II) Preparation of 5-dimethylaminopentyl isocyanate

23.5 g of 6-N, N-dimethylaminocarboxylic acid methyl ester and 10.2 g of hydrazine are dissolved in 136 ml of ethanol and left to reflux for several days. Excess hydroxyl ethanol liquid is added to this, and a white precipitate is collected by filtration. The material was extracted with heating to 300 ml containing 50% ethanol, and after cooling, some of the impurities were removed and concentrated to dryness to give 23.3 g of a white powder. This whole powder is suspended in 136 ml of water and treated with 12.3 g of sodium nitrite under cooling. The reaction solution was adjusted to pH 10.5 with 5 N caustic soda and extracted three times with 150 mL of benzene. The benzene layer was combined and left to reflux for 1 hour after washing with water. The solvent is carefully distilled off and the residue is distilled under reduced pressure to give 6.1 g of the title compound.

Boiling Point 14: 110 ° to 115 ° C.

Reference Example 15

56 mg (0.099 mmol) of maytansinol was dissolved in 10 ml of dry dichloromethane, followed by 24 mg (0.202 mmol) of phenyl isocyanate, followed by 30 mg (0.221 mmol) of anhydrous chloride at room temperature (18-23 ° C.). Zinc is added and stirred at the same temperature for 3 hours. The reaction solution was washed with water (Na ₂ SO ₄ ), concentrated and the residue was subjected to silica gel chromat [solvent: ethyl acetate / ethyl acetate 3/1 at 4/1 (V / V)], and the eluate was 17 g. Gather fractionally. 58 mg of maytansinol 3-N-phenylcarbamate is obtained by combining fractions No. 9 and 17 and distilling off the solvent.

Melting point 187-189 ° C. (recrystallized from ethyl acetate-hexane)

Reference Example 16

To a solution of 54 mg (0.0956 mmol) of maytansinol in 10 ml of dichloromethane, 50 mg (0.977 mmol) of methyl isocyanate and 30 mg of cuprous chloride were added thereto, stirred at room temperature for 4 hours, and the reaction solution was filtered and concentrated. The residue was evaporated with chloroform / methanol 40/1 (V / V) on a silica gel column (diameter 25 mm x length 45 mm), and the eluate was collected by dividing by 25 g each. The fractions No. 34 to 44 are collected and concentrated to dryness to yield 44 mg of white glassy material. This material is reprecipitated with chloroform-hexane to give 28 mg of maytansinol 3- (N-methyl) carbamate 9- (2,4-dimethyl) altophanate as a white powder.

Melting point 149-151 degreeC (decomposition).

10 mg of mesitasinol 3- (N-methyl) carbamate 9- (2,4-dimethyl) allophanate is dissolved in 0.2 ml of dichloromethane and two drops of trifluoro acetic acid are added thereto. After stirring for 5 minutes at room temperature, dichloromethanol was added, washed with sodium bicarbonate water, the solvent was distilled off, and the residue was chromatographed using 12 g of silica gel to obtain 5.2 mg of maytansinol 3- (N-methyl) carbamate. Get

Reference Example 17

The compound shown below is manufactured by the method similar to the reference examples 14-16.

(A) Maytansinol 3- (N-methyl) carbamate obtained from maytansinol and methyl isocyanate. Melting Point 196 ~ 200 ℃ (Decomposition)

(B) Obtained maytansinol 3- (N-butyl) carbamate from maytansinol and butyl isocyanate. Melting point 162-165 deg.

(C) Maytansinol 3- (N-octadecyl) carbamate obtained from maytansinol and octadecyl isocyanate. Melting point 105-109 degreeC.

(D) Maytansinol 3- (N-cyclohexyl) carbamate obtained from maytansinol and cyclohexyl isocyanate. Melting point 175-178 degreeC.

(E) Maytansinol 3- (N-α-naphthyl) carbamate obtained from maytansinol and α-naphthyl isocyanate. 172-175 degreeC.

(F) Maytansinol 3- (N-p-ethoxyphenyl) carbamate obtained from maytansinol and p-ethoxyphenylisocyanate. Melting point 221-223 degreeC.

Dichloromaytansinol 3- (N-phenyl) carbamate is obtained as a colorless glassy substance from (G) dichloromaytansinol and phenylisocyanate.

NMR spectrum (CDCl ₃ ) δ ppm: 0.87 (3H, s), 1.26 (3H, d, J = 6 Hz), 1.70 (3H, s), 2.03 (3H, s), 2.23 (1H, dd, J = 2.5 Hz and 14 Hz), 2.69 (1H, dd, J = 11 Hz and 14 Hz), 2.87 (1H, d, J = 9 Hz), 3.23 (3H, s), 3.30 (3H, s), 3.42 (1H, d, J = 9 Hz), 3.49 (1H, d, J = 14 Hz), 3.85 (3H, s), 4.30 (1H, m), 4.78 (1H, dd, J = 2.5 Hz and 11 Hz), 5.37 (1H, dd, J) = 9 Hz and 15 Hz), 6.10 (1H, d, J = 10.5 Hz), 6.39 (1H, s), 6.43 (1H, dd, J = 10.5 Hz and 15 Hz), 6.57 to 7.56 (about 9H, m)

(H) Maytansinol 3-isopropylcarbamate obtained from maytansinol and isopropylisoanate.

Scalpel spectrum (m / e), 588 (M ⁺ -61)

(I) Maytansinol 3- (Nm-pyridyl) carbamate is obtained from maytansinol and pyridyl-3-isocyanate. Scalpel spectrum (m / e), 625 (M ⁺ -61).

(J) Maytansinol 3- (N-5-dimethylaminopentyl) carbamate is obtained from maytansinol and 5-dimethylaminopentylisocyanate. Scalpel spectrum (m / e), 659 (M ⁺ -61).

(K) Maytansinol 3- (N-β-methoxycarbonylethyl) carbamate is obtained from maytansinol and β-methoxycarbonylethyl isocyanate. Mess spectrum (m / e), 632 (M ⁺ -61).

(L) Obtained maytansinol 3-N, N-dimethylcarbamate from maytansinol and N, N-dimethylcarbamoylchloride. Rf = 0.39 (using chloroform: methanol = 95: 5 as the developing solvent), mass spectrum (m / e), 574 (M ⁺ -61).

Reference Example 18

Cardier sp. Of Maytansinol, Maytanasin and Maytansinol Propionate Produced in East Extract-Maltose Extract Slope Agar Media. No. C-15003 (IFO 13726, FERM-P No.3992; ATCC-31281) was treated with 2% glucose in a 200 ml Erlenmeyer flask, 3% soluble starch, 1% raw soy flour, 1% coonsticker, and 0.5% polypeptone. Inoculate in 40 ml seed medium containing 0.3% NaCl, 0.5% CaCO ₃ and PH 7.0, and incubate on a 28 ° C, 48 hour rotary shaker to obtain seed culture solution.

0.5 ml of the obtained seed culture solution was transplanted into a 40 ml main medium containing 5% of dextrin, 3% of Coon Stiff liquor, 0.1% of polypeptone, 0.5% of CaCO ₃ and _P H 7.0 in a 200 ml Erlenmeyer flask, and C, incubated on a rotary shaker for 90 hours. This culture broth has a production titer of 25 g / ml when tetrahymenapiripolmis W is used as an assay and maytansinol propionate is used as a standard and assayed by a liquid dilution method.

Reference Example 19

용 사카구치 플라스크에 이식하고 28˚C 48시간 왕복 진탕기 위에서 배양했다. 이 배양액 500㎖를 종배양 30

를 함유한 50

용 스텐레스 스틸 탱크에 이식하고 28˚C, 통기 30

분, 교반 280회전/분(1/2DT) 내압 1kg/cm²의 조건으로 48시간 배양에서 종배양액을 얻었다. 얻어진 종배양액을 이식률 10%로 참고예 18에 표시한 것과 마찬가지의 주배지 100

를 함유한 200

들이 스텐레스 스틸 탱크에 이식하고, 28˚C, 통기 100

/분, 교반 200회전/분(1/2DT), 내압 1kg/cm²의 조건으로 90시간 배양했다. 얻어진 배양액은 참고예 18의 검정법으로 25㎍/㎖의 생산역가를 나타냈다.10 ml of the seed culture solution obtained in Reference Example 18 containing 2,500 mL of the seed culture.

It was transplanted into a sakaguchi flask and incubated on a 28 ° C 48 hour reciprocating shaker. Species culture of 500 ml of this culture

50 containing

Implanted in stainless steel tank for 28 ° C, vented 30

The seed culture liquid was obtained by incubating for 48 hours on the conditions of 1 kg / cm <^2> of internal pressures of 280 revolutions / minute (1 / 2DT), and internal pressure for 1 minute. Main culture 100 similar to that shown in Reference Example 18 using the obtained seed culture solution at a transplantation rate of 10%.

Containing 200

Implanted into a stainless steel tank, 28 ° C, aeration 100

It culture | cultivated for 90 hours on the conditions of / min, stirring 200 revolutions / min (1 / 2DT), and internal pressure 1 kg / cm <^2> . The obtained culture had a production titer of 25 µg / ml by the assay of Reference Example 18.

90 kg of the obtained culture solution is added 2 kg of Hypro Superset (Johnson Manville Prodact) and mixed well. The mixture is filtered through a pressure filter to give 85 L of filtrate and 32 L of wet cells. 30 L of ethyl acetate was added to 85 L of the filtrate, followed by stirring extraction. This operation was repeated twice. The ethyl acetate layer was combined, washed twice with 30 liters of water, 500 g of anhydrous sodium sulfate was added, the mixture was dried under reduced pressure to 200 ml, and the precipitate precipitated by adding petroleum ether was collected by filtration (53 g). 100 ml of ethyl acetate was added to the obtained crude substance I, mixed, and the insolubles were filtered off. 10 g of silica gel (0.05 to 0.2 mm) was added to the filtrate, and the mixture was added and mixed with ethyl acetate, followed by distillation under reduced pressure, and a silica gel column (400) prepared in advance. ML of n-hexane, 500 mL of n-hexane ethyl acetate (3: 1), 500 mL of n-hexane ethyl acetate (1: 1), 500 mL of n-hexane ethyl acetate (1: 3) , 500 ml of ethyl acetate, 1 L of ethyl acetate (50: 1), and 1 L of ethyl acetate (25: 1) were applied, and the eluate was fractionated every 100 ml. 1 ml of each fraction was concentrated and firmly added, and 0.1 ml of ethyl acetate was added and spotted at a position of 2.5 cm from the bottom of a silica gel glass plate (West Germany Merck Kieselgel 60F ₂₅₄ 0.25 mm 20 × 20). It develops about 17 cm with methanol (19: 1). After the development, the absorption phase was irradiated under ultraviolet light (2537 Hz), and fraction No. 1 having absorption near Rf 0.58 to 0.63. No 38 to 40, each having an absorption from 25 to 30 and Rf 0.25 to 0.30, are collected and concentrated under reduced pressure to about 20 ml. 150 ml of petroleum ether is added to each concentrate to obtain 5 g of crude II and 2 g of crude maytansinol.

0.5 g of the obtained crude substance II was dissolved in 10 ml of ethyl vinegar, 4 g of silica gel (0.05 to 0.2 mm of West German Merk Co., Ltd.) was added thereto, mixed well, and ethyl acetate was distilled off under reduced pressure. It is placed on the top of 300 ml of the prepared silica gel column and washed with 500 ml of chloroform, and then eluted with 500 ml of chloroform methanol (50: 1), 500 ml of chloroform methanol (20: 1) and 500 ml of chloroform methanol (10: 1). . The eluate is partitioned into 25 ml portions, 05 ml of each fraction is concentrated under reduced pressure, 0.05 ml of ethyl acetate is added, and this sample is subjected to thin layer chromatography (chloroform / methanol 9: 1 is used as a developing solvent). Rf 0.48 to 0.50 and fraction No. exhibiting an absorption phase at 2357 kPa. 4. When 0-41 is concentrated under reduced pressure and 0.5 ml of ethyl acetate is added and left to stand, 50 mg of crystals of maytanacin and maytansinol propionate are obtained.

50 mg of the obtained maytanacin and maytansinol propionate mixed crystals are dissolved in 5 ml of methanol, and 100 mg of salt and 5 ml of water are added thereto to dissolve. 200 ml of Diaion HP-10 (Mc. Bishigasei) was charged to a 1.8 cm diameter column and adjusted by washing with 600 ml of 50% methanol containing 5% salt. The sample solution prepared above was washed with 300 ml of 60% methanol containing 5% salt, and then eluted between 1.5 L of 60% methanol containing 5% salt and 1.5 L of 95% methanol. The eluate is separated by 15 ml and each fraction is detected by thin layer chromatography. Maytanasine is detected in fractions 130-135 and maytacinol propionate is detected in fractions 138-142. Gather and concentrate each, add 30 ml of water and 50 ml of ethyl acetate to dissolve it, place it in a separatory funnel, shake, separate the water layer, wash twice with 30 ml of water, and dry the ethyl acetate layer with herbs. Each crystal is precipitated. This crystal is collected by the filter cake and dried. Maytanasin 13 mg, maytansinol pyridionate 25 mg.

0.2 g of crude maytansinol obtained as described above is dissolved in 3 ml of ethyl acetate, 0.5 g of silica gel (0.05 to 0.2 mm of West Germany Merk Co., Ltd.) is added and mixed well, and ethyl acetate is distilled off under reduced pressure. It was placed on the top of 80 ml of the prepared silica gel column, washed with 150 ml of chloroform, and then chloroform. 150 ml of methanol (50: 1), chloroform. 150 ml of methanol (20: 1), chloroform. Elution with 300 ml of methanol (10: 1). The eluate was fractionated in 10 ml portions, 95 ml of each fraction was concentrated under reduced pressure, 0.05 ml of ethyl acetate was added, and this sample was subjected to thin layer chromatography. [Use of chloroform / methanol (9: 1) as a developing solvent] Concentrated oils No. 50 to 52, which represent an absorbing phase, were dried under reduced pressure in 2357 cc of Rf 0.33 to 0.38, and 0.5 ml of ethyl acetate was added thereto to leave 20 mg of maytansinol. The crystal of is obtained.

The physical and chemical properties of the obtained maytansinol, maytansinine and maytansinol propionate are shown in the following table.

TABLE 2

Reference Example 20

Nocardea No. cultivated in yeast extract maltose extract agar medium C-15003 (IFO 13726, FERM-P No. 3992; ATCC-31281) in 200 ml Erlenmeyer flasks containing 2% glucose, 3% soluble starch 1% soy flour, 1% coonsticker, 0.5% polypeptone, 40 ml of seed medium containing 0.3% NaCl, 0.5% CaCO ₃ , _P H 7.0 and incubated for 48 hours at 28 ° C. on a rotary shaker to obtain a seed culture solution.

0.5 ml of the resulting culture broth was transplanted into a 40 ml main medium containing 5% dextrin, 3% coonsticker, 3% polypeptone, 0.5% CaCO ₃ , _P H 7.0 in a 200 ml Erlenmeyer flask and at 28 ° C. Incubated on a rotary shaker for 90 hours. The culture solution was found to exhibit a production titer of 25 µg / ml when detrahimena pyriformis W was assayed and assayed by liquid dilution using ansa-mytocin P-3 as a standard.

Reference Example 21

10 ml of the seed culture solution obtained in Reference Example 20 was transplanted into a 2 L sakaguchi flask containing 500 ml of seed medium and incubated on a reciprocating agitator at 28 ° C. for 48 hours. 500 ml of this culture solution was transplanted into a 50 liter stainless steel tank containing 30 ml of seed medium, and subjected to 28 ° C., aeration 30 l / min, agitation 280 revolutions / min (1/2 DT), and internal pressure of 1 kg / cm ² . After transplanting for 48 hours, 200% containing 100 liters of the main medium as shown in Reference Example 20 was transplanted into a 10% stainless steel tank at 28 ° C., aeration 100 lL, stirring 200 revolutions (1 / 2DL), and internal pressure 1 kg. Incubated for 90 hours under the condition of / cm 2. When the resultant culture solution was used as the determination method of Reference Example 20 and each other, a production titer of 25 L / mL was shown.

Reference Example 22

To 95 liters of the culture solution obtained in Reference Example 21, 2 kg of a hyperprocell (Jones Manville Product Co., Ltd.) is added and mixed well. The mixture is filtered through a pressure filter to give 85 liters of the filtrate and 32 kg of wet cells. 30 L of ethyl acetate was added to 82 L of the filtrate, followed by stirring extraction. This operation was repeated twice. The ethyl acetate layers were combined, washed twice with 30 ml of water, and 500 g of anhydrous sodium sulfate was added, followed by drying under reduced pressure to 200 ml, petroleum ether was added, and the precipitate precipitate was collected by filtration (53 g). 100 ml of ethyl acetate was added to the obtained crude substance I, mixed and the insolubles were filtered off, and 10 g of silicagen (West Germany Merk Co., 0.05-0.2 mm) was added to the filtrate, followed by mixing. Place on top of (400 ml) n-nucleic acid 500 ml, n-nucleic acid. 500 ml of ethyl acetate (3: 1), n-nucleic acid. 500 ml of ethyl acetate (1: 1), n-nucleic acid. 500 ml of ethyl acetate (1: 3), 500 ml of ethyl acetate, and 1 liter of ethyl acetate (50: 1) were poured, and the eluate was fractionated every 100 ml. 1 ml of each fraction was concentrated to dryness, 0.1 ml of ethyl acetate was added, and spotted at a position of 2.5 cm from the bottom of the silica gel glass plate (West Germany Merck Kieselgel 60F ₂₅₄ 0.25 mm 20 x 20), acetic acid as a developing solvent. Develop about 17 cm using ethyl / methanol (19: 1). After the development, the absorbed phase was irradiated under ultraviolet light (2537 kPa), and the oil fractions No. 23 to 28 with absorption near Rf 0.6 to 0.65 were collected and concentrated under reduced pressure to about 20 ml. 150 ml of petroleum ethyl is added to the concentrate to obtain 15 g of crude substance II.

Reference Example 23

50 L of 70% acetone water was added to 32 kg of the cells obtained in Reference Example 22, followed by stirring extraction for 3 hours and filtering with a pressure filter.

The extraction was repeated with 50 L of 70% acetone water again, the filtrates obtained by filtration were similarly combined, and acetone was distilled off by concentration under reduced pressure. The resulting aqueous solution was poured into a 5 liter column of Diaion HP-10 (Mitsubishi Chemical), washed with 20 liter water and 50% methanol water, and eluted with 15 liter 90% methanol water. The eluate is concentrated under reduced pressure to 3 liters, 3 liters of water and 3 liters of ethyl acetate are added, shaken and mixed, and this operation is repeated twice. The combined layers of ethyl acetate were washed with water, anhydrous sodium sulfate, dried, concentrated under reduced pressure to 200 ml, and the precipitate precipitated by addition of petroleum ether was collected by filtration (28 g). The crude product obtained was purified by silica gel column in the same manner as in Example 20 to obtain 8.0 g of crude material II.

Reference Example 24

1.5 g of crude substance II obtained in Reference Example 22 was dissolved in 10 ml of ethyl vinegar acetate, 4 g of silica gel (0.05 to 0.2 mm of West Germany Merk Co.) was added thereto, followed by sufficient mixing. The ethyl acetate was distilled off under reduced pressure. It is placed on top of 300 ml of the prepared silica gel column and washed with 500 ml of chloroform, followed by 500 ml of chloroform / methanol (50: 1), 500 ml of chloroform / methanol (20: 1), and 500 ml of chloroform / methanol (10: 1). Elutes. The eluate is fractionated by 25 ml, 0.5 ml of each fraction is concentrated under reduced pressure, 0.05 ml of ethyl acetate is added, and the sample is subjected to thin layer chromatography on silica gel. (Electrosolvent chloroform / methanol 9: 1) Fraction No. showing an absorption phase at 2537 Pa as Rf 0.50 to 0.60. When 39,40 were concentrated under reduced pressure and dried, and 2 ml of ethyl acetate was added and left to stand, 150 mg of a mixture of ansamytocin P-3, P-3 'and P-4 was obtained.

150 mg of the mixture of the above obtained ansamytocin P-3, P-3 'and P-4 is dissolved in 15 ml of methanol, and 30 mg of salt and 15 ml of water are added thereto to dissolve. 200 ml of Diaion HP-10 (Mitsubishi Chemical) was charged into a 1.8 cm diameter column and adjusted using 600 ml of 5% methanol water containing 5% saline. After using the sample solution prepared above, the solution was gradient eluted between 60% containing 5% saline, 1.5 L of methanol and 1.5 L of 95% methanol. The eluate was fractionated by 15 ml and each fraction was detected by thin layer chromatography on silica gel. Ansamytocin P-3 is eluted at fractions 145-153, and ansamytocin P-3 'and P-4 are eluted at fractions 167-180. Gather and concentrate each, add 50 ml of water and 100 ml of ethyl acetate, dissolve it, add to a separatory funnel, shake, separate the water layer, wash twice with 50 ml of water, and dry the ethyl acetate layer with forget-me-not and leave it to stand. Each crystal precipitates. The crystals are collected by filtration and dried.

Ansamytocin P-3 70mg, Ansamytocin P-3 ', P-4 18mg, Ansamytocin P-4 15mg

18 mg of ansamytocin P-3 'and P-4 mixed crystals were dissolved in 0.3 ml of ethyl acetate, and a straight line was placed at a position of 2.5 cm from the bottom of three silica gel glass plates (West Germany Merck's Kigel gel 60F ₂₅₄ 0.25 mm 20 x 20). Apply onto phase and develop with ethyl acetate (19: 1). After about 18 cm of development, each of the absorption phases of Rf 0.68 (Ansamytocin P-4) and Rf 0.65 (Ansamytocin P-3 ') was scraped and extracted twice with ethyl acetate containing a small amount of water. The extracted ethyl acetate is washed with water, concentrated under reduced pressure with anhydrous sodium sulfate, and left to stand. Crystals of anosamytocin P-4 10 mg at an Rf value of 0.68 and ansamytocin P-3 '3 mg at an Rf value of 0.65 are obtained.

The physicochemical properties of ansamytocin P-3, ansamytocin P-3 'and ansamytocin P-4 obtained by the above are shown in the following table.

TABLE 3

Reference Example 25

15 mg of the mixture of ansamytocin P-3, P-3 'and P-4 obtained in Reference Example 24 is dissolved in tetrahydrofuran and cooled to -5 ° C. Add 12 mg of lithium aluminum hydride and leave for 2 hours.

0.5 ml of 1% H ₂ SO ₄ water was added to the reaction solution, followed by extraction with 2 ml of ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to preparative TLC (solvent: acetic acid: methanol = 19; 1) using silica gel in the same manner as shown in Reference Example 7. The mixture is collected, extracted with ethyl acetate containing a small amount of water, washed with water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to precipitate crystals. The crystals were filtered off and dried to obtain 10 mg of ketansinol. Melting point 174 ° C.

Elemental analysis

Found: C 59.65, H 6.58, N 5.02, Cl 6.51 C ₂₈ H ₃₇ ClN ₂ O ₈

Calculated Value: C 59.52, H 6.60, N 4.96, Cl 6.27

IR: 1715,1670,1580 (cm ^-1 )

UV (nm): 232 (32750), 244 (sh30850), 252 (31650), 281 (5750), 288 (5700)

The properties of this product correspond to the maytansinol obtained in 19 in the above reference.

Reference Example 26

100 mg of maytansinol was dissolved in 0.7 ml of pyridine and ice-cooled, 0.35 ml of acetic anhydride was added thereto, and the reaction solution was then poured into 5 ml of cold water after being left at room temperature (23-25 ° C.) for 18 hours. The mixed and precipitated precipitate was collected by filtration and dried to obtain 85 mg of a powdery substance, which was then dissolved in 1 ml of a mixture of chloroform methanol 10/1 (V / V), added with 0.2 g of silica gel, mixed well, and prepared in advance. Place on top of column (80 mL) and elute with elution with unsaturated ethyl acetate. The eluate was partitioned into 10 ml portions, the fractions from NO.37 to 44 were collected, concentrated under reduced pressure, a small amount (about 0.5 ml) of ethyl acetate was added to the residue, the precipitated crystals were dissolved again, and the mixture was left at room temperature. The precipitated crystals were collected by filtration to obtain 36.0 mg of maytanacin. It was melting point 231 degreeC, and it considered that it is the same compound as maytanacin obtained by the reference sample 19 by comparing the mass spectrum, IR spectrum, UV spectrum, and thin layer chromatography.

Example 1

Bacillus megaterium IFO 12108 is inoculated in medium (pH7.5) containing 2% dextrin, 0.5% peptone, 0.5% yeast extract and 0.5% meat extract, and incubated at 30 ° C for 16 hours. When 110 mg of ansamytocin P-4 was added to 2.75 liters of this solution and shaken for 30 hours at 30 ° C., the anzamytocin P-4 was completely lost and PDM-4 was produced. TLC).

Example 2

1.3 liters of ethyl acetate was added to 2.75 liters of the culture solution obtained in Example 1, followed by extraction with stirring. The resultant was filtered by suction using expanded brass of 30 g of Hyprosupercell (Johnson-Manville, Products, Inc.), and the operation was repeated twice. Combine the ethyl acetate layer and wash twice with 800 ml of 20% hydrochloric acid at a prescribed concentration, then 400 ml of 0.5% sodium bicarbonate water, wash twice with 400 ml of water, add 10 g of anhydrous sodium sulfate, and concentrate under reduced pressure to 2 ml. The precipitates added by filtration are collected by filtration (126 mg). The crude material (I) of the obtained PDM-4 was dissolved in a small amount of chloroform and poured into the top of a column (1 cm in diameter) filled with 5 g of a silica gel (West Germany Merk Co. 0.05-0.2 mm) prepared in advance, and 100 ml of chloroform and chloroform / ethanol (40 : 1) 100 ml and 200 ml of chloroform / methanol (20: 1) were flowed to fractionate the eluate by 10 ml. Each fraction is spotted at a position of 2.5 cm from the bottom of a silica gel glass plate (West Germany Mergel Kiesel Gel 60F ₂₅₄ 0.25 mm 20 × 20), and is about 17 with a developing solvent and ethyl vinegar / methanol (19: 1). It develops by cm. After the development, the absorption phase was irradiated under ultraviolet light (2537Å), and the oil fraction No. Collect 13-17 and concentrate under reduced pressure to about 1ml. 30 ml of petroleum ether is added to the concentrate to obtain 79 mg of crude (II) of PDM-4.

Example 3

79 mg of crude material (II) of PDM-4 obtained in Example 2 was dissolved in a small amount of chloroform, using four sheets of silica gel glass plates (West Germany Merk Co., Kiegel Gel F ₂₅₄ 2 mm 20 × 20), each of which was 2.5 cm Was applied linearly at, and developed with ethyl acetate / methanol (19: 1), and then an absorbent phase having an Rf value of 0.6 was collected and extracted twice with ethyl acetate containing a small amount of water. After washing with water and concentrated under reduced pressure with anhydrous sodium sulfate, petroleum ether was added to obtain 68 mg of white powder of PDM-4.

Example 4

에 220mg의 메이탄시놀을 첨가하고, 30℃에서 30시간 진탕하여 반응시키면, 메이탄시놀은 감소하고, 데메틸메이탄시놀이 생성되어 있음을 TLC로 확인할 수 있다.Culture medium of the same bacteria obtained by the same method as in Example 1 5. 5

When 220 mg of maytansinol was added to the mixture, and the mixture was shaken at 30 ° C. for 30 hours to react, maytansinol decreased and it was confirmed by TLC that demethyl maytansinol was formed.

Example 5

The culture solution obtained in Example 4 was purified in the same manner as in Example 2 and subjected to thin layer chromatography in the same manner as in Example 2, where the fraction near Rf value of 25 was collected to obtain 61 mg of crude substance of dimethyl maytansinol. . The purification was carried out in the same manner as in Example 3 below, to obtain 33 mg of white powder of demethylmaytansinol.

Example 6

에 500mg의 메이탄시놀, 프로피오네이트를 첨가하고, 30℃로 28시간 진탕해서 반응시키면, 메이탄시놀, 프로피오네이트는 완전히 소실하고, 데메틸메이탄시놀이 생성되어 있음을 TLC로 확인할 수 있다.Culture medium of the same bacteria obtained in the same manner as in Example 1

500 mg of maytansinol and propionate were added to the mixture, and the mixture was shaken at 30 ° C. for 28 hours to react completely. TLC showed that maytansinol and propionate disappeared completely and demethylmaytanol was formed. You can check it.

Example 7

를 식초산 에틸 12

를 써서 실시예 2와 같은 방법으로 정제하여, 실시예 2와 같은 박층 크로마토그래피를 해서, Rf치 0. 58부근의 프랙숀을 모아서 감압 농축 건조하여 식초산에틸 5㎖를 가하여 방치하면, 데메틸메이탄시놀프로피오네이트의 결정 235mg이 얻어진다.Culture medium obtained in Example 6 12.5

Ethyl vinegar 12

Purification was carried out in the same manner as in Example 2, and the thin layer chromatography was carried out in the same manner as in Example 2. The fractions near Rf value of 58 were collected, concentrated to dryness under reduced pressure, and 5 ml of ethyl vinegar was added and left to stand. 235 mg of crystals of maytansinol propionate are obtained.

Example 8

에 10mg의 메이탄시놀, 메이타나신, 메이탄시놀. 프로피오네이트, 안사마이토신 P-3 및 안사마이트신 P-4의 혼합물을 첨가하고 30℃로 24시간 진탕해서 반응시키면, 상기의 혼합물의 데메틸체가 생성되어 있음을 TLC로 확인된다.Culture solution of the same bacteria obtained in the same manner as in Example 1

In 10mg maytansinol, maytansine, maytansinol. When a mixture of propionate, ansamytocin P-3, and ansamycin P-4 was added and shaken at 30 ° C. for 24 hours for reaction, it was confirmed by TLC that demethyl bodies of the mixture were produced.

Developing solvent Ethyl acetate. Methanol (19: 1) develops dimethyl maytansinol at Rf values from 0.25 to 0.3, demethylmaytanasine at Rf values from 54 to 50 deg. Methylmaytansinol. The propionate has an absorption phase of PDM-3 at Rf value 0.61 and PDM-4 at Rf value 0.64. Further, when developed with a developing solvent, chloroform / methanol (9: 1), demethyl maytansinol at Rf value 0.30 and demethyl maytansinol at Rf value 0.40. Propionate has an absorption phase of PDM-3 at Rf value 0.42 and PDM-4 at Rf value 0.44.

Example 9

Streptomyces, Flavotrysini IPO12770, dextrin 1%, glucose 1%, glycerol 1%, peptone 0.5%, yeast extract 0.5%, meat extract 0.5%, salt 0.3% and calcium carbonate 0 Inoculate in medium containing 5% pH 7.2 and incubate for 48 hours at 28 ° C. 20 mg of anasamytocin P-3 is added to 2 liters of this culture, followed by shaking for 48 hours at 28 캜. Purification in the same manner as in Examples 2 and 3 yields 12 mg of white powder of PDM-3.

Example 10

에 20mg의 메이타나신을 첨가해서, 28℃에서 48시간 진탕해서 반응시킨다. 실시예 2, 3과 같은 방법으로 정제하고 소량의 식초산에틸를 가해서 방치하면 데메틸메이타나신의 결정 6mg가 얻어진다.Culture medium of the same bacteria obtained in the same manner as in Example 9

20 mg of maytanacin is added to the mixture, and the mixture is shaken at 28 ° C. for 48 hours for reaction. Purification was carried out in the same manner as in Examples 2 and 3, and a small amount of ethyl vinegar was added and left to obtain 6 mg of crystals of demethylmethanacin.

Example 11

에 10mg의 메이탄 시놀. 프로피오네이트를 첨가해서 28℃로 48시간 진탕해서 반응시키면 메이탄시놀, 프로피오네이트는 완전히 소실하고 데메틸메이탄시놀. 프로피오네이트가 생성되어 있음이 TLC로 확인된다.Culture solution 1 obtained by culturing Actino micros and nigresense IFO12894 in the same manner as in Example 9.

In 10mg maytansinol. When propionate was added and shaken at 28 ° C. for 48 hours to react, maytansinol and propionate completely disappeared and demethyl maytansinol. TLC confirms that propionate has been produced.

Example 12

에 10mg의 메이탄시놀 프로피오네이트를 첨가하고 28℃에서 48시간 진탕해서 반응시키면 메이탄시놀 프로피오네이트는 완전히 소실하고, 데메틸메이탄시놀 프로피오네이트가 생성되어 있음을 TLC로 확인할 수 있다.Culture medium 1 obtained by culturing Streptomyces and Fapthenis IFO12901 in the same manner as in Example 9

10 mg of maytansinol propionate was added to the mixture, and the mixture was shaken at 28 ° C. for 48 hours to completely dissolve the maytansinol propionate and to form demethylmaytansinol propionate. You can check it.

Example 13

80 mg of the crystals of demethylmaytansinol and propionate obtained in Example 7 were dissolved in 30 ml of tetrahydrofuran and cooled to -5 ° C. 80 mg of lithium aluminum hydride is added thereto. The reaction solution is transferred to an ice bath and stirred for 30 minutes. 10 ml of ethyl vinegar and 10 ml of 200% regular hydrochloric acid are added, and then 50 ml of ethyl vinegar is added and extracted. The ethyl vinegar layer was washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel TLC, followed by 17 cm development with ethyl acetate / methanol (19: 1), followed by an Rf value of about 0.25 to about 40. The absorbent phase is scraped off, extracted with ethyl vinegar containing a small amount of water, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 41 mg of dimethyl maytansinol.

Its physicochemical properties were consistent with the singular phase of demethyl maytansinol obtained in Example 5.

Example 14

50 mg of PDM-3 powder obtained in Example 9 was dissolved in 20 mg of tetrahydrofuran, cooled to -5 ° C and 50 mg of lithium aluminum hydride was added. When this is processed and purified in the same manner as in Example 13, 27 mg of white powder of demethyl maytansinol is obtained. The physical and chemical properties of this material are consistent with those of demethylmaytansinol obtained in Example 5.

Example 15

When 50 mg of PDM-4 powder obtained in Example 3 was treated and purified in the same manner as in Example 13, 23 mg of demethylmaytansinol was obtained. Its physical and chemical properties were consistent with those of demethylmaytansinol obtained in Example 5.

Example 16

50 mg of the crystal of demethylmaytanasin obtained in Example 10 was treated and purified in the same manner as in Example 13 to obtain 29 mg of demethyl maytansinol. The water chemical properties of this material were consistent with those of demitylmethane tancinol obtained in Example 5.

Example 17

30 mg of a mixture of demethylmethanacin, demethylmaytansinol, propionate, PDM-3, and PDM-4 obtained in Example 8 was dissolved in 20 ml of tetrahydrofuran, cooled to -5 ° C, and lithium aluminum hydride. 30 mg of lead is added. When this was treated and purified in the same manner as in Example 13, 12 mg of demethyl maytansinol was obtained. The physical and chemical properties of the foreign matter were consistent with those of the demethyl maytansinol obtained in Example 5.

Example 18

에 10mg의 안사마이토신 P-3을 첨가하고 28℃에서 48시간 진탕해서 반응시키면, 안사마이토신 P-3은 완전히 소실하고 PDM-3이 생성되어 있음을 TLC로 확인할 수 있다.Streptomyces. Culture amount 1 obtained by culturing Ribani IFO13452 in the same manner as in Example 9

When 10 mg of ansamytocin P-3 was added to the mixture and shaken at 28 ° C. for 48 hours for reaction, it was confirmed by TLC that ansamytocin P-3 disappeared completely and PDM-3 was produced.

It shows in the following table of the compound obtained by said Example.

TABLE 4

Compared with the known compound group in the physicochemical properties, antimicrobial synthesis and anti-swollen activity described above, the same group as the antibiotic was not recognized. Thus, each is compared with the corresponding antibiotic ansamytocin. The data of the mass spectrum of ansamytocin P-3 is expressed as follows.

In PDM-3, there are 14 units of units each, and 559, 471, 456, and 436 are recognized, and PDM-3 is a compound in which one methyl group of ansamytocin is converted to hydrogen. In comparison with the nuclear magnetic resonance spectra, the methyl group signal is recognized as δ 3. 18, 3.38, and 4. 00 in ansamytocin P-3, but the signal of δ 4. 00 is lost in PMD-3. It can be easily measured that the time was converted to a hydroxyl group, and it can be seen that PDM-3 is a novel compound. Thousands of PDM-4, Demethylmaytansinol propionate, Demethylmaytanasin and Demethyl maytansinol are also mentioned. From the above data, the measurement structural formulas of demethyl maytansinol, demethyl maytanasin, demethyl maytansinol propionate, PDM-3, and PDM-4 are shown in FIG.

Example 19

Basilis megaterium IFO 12108 is inoculated in a medium containing 2% dextrin, 0.5% peptone, 0.5% yeast extract and 0.5% yukupgi, and incubated for 16 hours at 28 ° C. When 1.5 mg of this culture solution was added with 30 mg of maytansinol 3-picolinate and reacted by shaking at 28 ° C. for 29 hours, maytansinol 3-picolinate disappeared completely and demethylmaytansinol 3 The formation of picolinate can be seen by thin layer chromatography (TLC).

Example 20

750 ml of ethyl acetate was added to 1.5 l of the culture broth obtained in Example 19, followed by extraction with stirring, suction filtration of 15 g of Hyprosupercell (Johnson Manville Prodact, USA), and suction filtration, and this operation was repeated twice. The combined ethyl acetate layers were washed twice with 300 ml of water, dried with 10 g of anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude (I). The crude substance (I) was dissolved in a small amount of chloroform and pre-healed silica gel ( West Germany, Merck, 0.05 to 0.2 mm) was poured into the top of a column (5 cm diameter), and 150 ml of chloroform and chloroformmethanol (20: 1) were flowed to fractionate the eluate by 10 ml. Each fraction was spotted at a position of 2.5 cm from the lower end of a silica gel glass plate (West Germany, Merck, Kitatgel 60F ₂₅₄ , 0.25 mm, 20 × 20), and was diluted with developing solvent chloroform methanol (9: 1). It develops 17 cm. After the development, the absorbed phase is irradiated under ultraviolet light (2537 kPa) to collect fractions Nos. 12 to 16 with absorption near Rf 0.40, and are compressed under reduced pressure to obtain 18 mg of crude substance (II). Crude substance (II) was dissolved in a small amount of chloroform, coated with a silica glass plate of 4 sheets in a straight line at a position of 2.5 cm from each lower end, and developed with ethyl acetate in water saturation, followed by an absorption phase of Rf 0.30. The resulting mixture was extracted twice with ethyl acetate containing a small amount of water, and the resulting extracted ethyl vinegar was dried under reduced pressure with anhydrous sodium sulfate and washed with petroleum ether. 6 mg of white powder of dimethyl maytansinol 3-picolinate was added. Is obtained.

Example 21

37 mg of maytansinol 3- (N-phenyl) carbamate was added to 2 liters of the same culture medium obtained by culturing in the same manner as in Example 19, followed by shaking at 228 ° C for 48 hours for reaction. It can be seen by TLC that the (N-phenyl) carbamate is completely lost and demethylmaytansinol 3- (N-phenylcarbamate is produced.

Example 22

The culture broth obtained in Example 21 was purified in the same manner as in Example 20, and the same fraction as in Example 20 was subjected to depleted chromatography to defraction of Rf 0.40 to collect fractions of methyl methyltansinol 3- (N-phenyl). 27 mg of crude substance (II) of carbamate is obtained. Purification was carried out in the same manner as in Example 20, to obtain 12 mg of white powder of demethylmaytansinol 3- (N-phenyl) carbamate.

UV spectrum (λ _max MeOH) _nm : 233, 252, 280, 288

Mass spectrum (m / e): 608, 471, 456, 436

Example 23

To 2 liters of the same culture medium obtained by culturing in the same manner as in Example 19, 37 mg of dechloromaytansinol 3-phenylacetate was added and the mixture was shaken at 28 ° C. for 48 hours to react with dechloromethane synol 3-phenylacetate. Disappears and TLC shows that demethyldechloromaytansinol 3-phenylacetate was produced.

Example 24

The culture solution obtained in Example 23 was purified in the same manner as in Example 20, and the thin layer chromatography was carried out in the same manner as in Example 20, and the fractions near Rf 0.43 were collected to prepare dimethyl dechloromaytansinol 3-phenylacetate. 30 mg of substance (II) is obtained. Hereinafter, the purification was carried out in the same manner as in Example 20 to obtain 14 mg of white powder of demethyldechloromaytansinol 3-phenylacetate.

UV spectrum (λ _max MeOH) _nm : 230, 240, 277, 285

Mass spectrum (m / e): 634 (M), 573, 437, 422

Example 25

18 mg of maytansinol 3-hexanoate is added to 1 liter of the same culture medium obtained by culturing in the same manner as in Example 19, and shaken at 30 ° C. for 48 hours for reaction. The ate disappears completely and TLC shows that demethylmaytansinol 3-hexanoate is produced.

Example 26

The culture solution obtained in Example 25 was purified in the same manner as in Example 20, and the thin layer chromatography was carried out in the same manner as in Example 20 to collect fractions near Rf 0.48, thereby preparing crude methyl methethanol 3-hexanoate. 9 mg of substance (II) is obtained. The purification was carried out in the same manner as in Example 20 below to obtain 4 mg of white powder of demethylmaytansinol 3-hexanoate.

UV spectrum (λ _max MeOH) _nm : 233, 240, 252, 280, 289

Mass spectrum (m / e): 587, 471, 456, 436

Example 27

19.5 mg of maytansinol 3-p-chlorobenzoate was added to 1 liter of the same culture medium obtained by culturing in the same manner as in Example 19, and the mixture was reacted by shaking at 30 ° C for 27 hours to react with maytansinol 3-p. It can be seen from TLC that -chlorobenzoate completely disappears and demethylmaytansinol 3-p-chlorobenzoate is produced.

Example 28

The culture solution obtained in Example 27 was purified in the same manner as in Example 20, and thin layer chromatography was carried out in the same manner as in Example 20 to collect fractions near Rf 0.43, thereby preparing crude methyl dimethyltansinol 3-p-chlorobenzoate. 12 mg of substance (II) is obtained. The purification was carried out in the same manner as in Example 20 below to obtain 6 mg of white powder of demethylmaytansinol 3-p-chlorobenzoate.

UV spectrum (λ _max MeOH) _nm : 240, 252, 280, 289

Mass spectrum (m / e): 627, 471, 456, 436

Example 29

15.9 mg of maytansinol 3-cyclohexanecarboxylate was added to 750 ml of the same culture medium obtained by culturing Streptomyces flabothricini IFO 12770 in the same manner as in Example 9. It can be seen by TLC that the maytansinol 3-cyclohexanecarboxylate is completely lost when reacted, and that demethylmaytansinol 3-cyclohexanecarboxylate is produced.

Example 30

The culture solution obtained in Example 29 was purified in the same manner as in Example 20, and the thin layer chromatography was carried out in the same manner as in Example 20 to collect fractions near Rf 0.45, and then dimethylmaytansinol 3-cyclohexanecarboxylate phenyl acetate. 9 mg of crude (II) is obtained. Purification was carried out in the same manner as in Example 20, to obtain 4 mg of white powder of demethylmaytansinol 3-cyclohexanecarboxylate.

UV spectrum (λ _max MeOH) _nm : 233, 240, 252, 280, 289

Mass spectrum (m / e): 599, 471, 456, 436

Example 31

40 mg of maytansinol 3-phenylacetate was added to 4 liters of the same culture medium obtained by culturing Streptomyces pratensis IFO 12901 in the same manner as in Example 9. TLC shows that phenol 3-phenylacetate is produced.

Example 32

The culture medium obtained in Example 31 was purified in the same manner as in Example 20, and subjected to thin layer chromatography in the same manner as in Example 20, where the fraction near Rf 0.43 was collected to obtain a crude substance of dimethylmaytansinol 3-phenyl acetate. (II) 22 mg are obtained. The purification was carried out in the same manner as in Example 20 below to obtain 8 mg of white powder of demethylmaytansinol 3-phenylacetate.

UV spectrum (λ _max MeOH) _nm : 233, 240, 252, 280, 289.

Mass spectrum (m / e): 607, 471, 456, 436

Example 33

To 2.8 liters of the same culture medium obtained by culturing Bacillus megaterium IFO 12108 in the same manner as in Example 19, 140 mg of dechloromaytansinol 3-isobutylate was added and shaken at 28 ° C. for 50 hours for reaction. It can be seen from TLC that dechloromaytansinol 3-isobutylate disappears and demethyl dechloromaytansinol 3-isobutylate is produced.

Example 34

The culture solution obtained in Example 33 was purified in the same manner as in Example 20, and the thin layer chromatography was carried out in the same manner as in Example 20, and the fractions around Rf 0.42 were collected to form demethyldechloromaytansinol 3-isobutylate. Obtain 113 mg of crude substance (II). The crude substance (II) is washed with a small amount of ether, and then dissolved in ethyl acetate and left to obtain 76 mg of crystals of demethyldechloromaytansinol 3-isobutylate.

Melting Point: 213 ~ 215 ℃

Specific luminance: -116.6 ° (c = 0.47, CHCl ₃ )

Elemental Analysis (%)

Found: C 63.27 H 7.33 N 4.61

Calculation: C ₃₁ H ₄₂ N ₂ O ₉

C 63.46 H 7.22 N 4.78

UV spectrum (λ _max MeOH) _nm (ε): 230 (27200), 240 (29700), 248 (29700), 277 (4100), 285 (3700)

Mes Spectrum (m / e): 525, 437, 422

Example 35

When the strain of Streptomyces pratensis IFO 12901 was incubated in the same manner as in Example 9, after adding 440 mg of L-maytansine to 4.4 L of the same culture medium, the mixture was shaken at 28 ° C. for 48 hours, It can be seen by thin layer chromatography (TLC) that tancin disappears completely and L-demethylmaytansine is produced.

Example 36

To 4.4 L of the reaction solution obtained in Example 35, 2.2 L of ethyl vinegar was added, stirred and extracted, suction filtered with 40 L of hyprosupercell (Jonesmanville Prodact, Inc.), and suction filtration, and this operation was repeated twice.

The ethyl vinegar layer was combined and washed twice with 1.2 L of hydrochloric acid at 20 ° C., followed by 1 L of 0.5% brine, washed twice with 1 L of water, 20 g of anhydrous sodium sulfate was added, and concentrated under reduced pressure to 2 mg. ML is added to precipitate and precipitate the precipitate (252 mg). The obtained crude substance (I) of L-demethyl maytansine was dissolved in a small amount of chloroform, poured into a top of a column (1.2 cm) filled with 10 g of silica gel (West Germany, Merck Co., Ltd. 0.05-0.2 mm) prepared in advance, and then glow-form 100 ml, 200 ml of chloroform methanol (20: 1) and 200 ml of chloroform methanol (10: 1) were flowed to fractionate the eluate by 2 ml. Each fraction was spotted at a position of 2.5 cm from the bottom of a silica gel glass plate (West Germany, Merck, Kiesel Gel 60F ₂₅₄ , 0.25 mm, 20 × 20), and developed with a developing solvent, ethyl vinegar / methanol (19: 1). It develops about 17 cm. Fraction No. 2 having a thick water near the Rf value of 923 by irradiating the absorbing phase under ultraviolet ray (2537A) after development. Collect 13217 and concentrate under reduced pressure to about 1 ml. 30 ml of petroleum ether is added to the concentrate to obtain 183 mg of crude substance (II) of L-demethylmaytansine.

Example 37

183 mg of crude substance (II) of L-demethylmaytansine obtained in Example 36 was dissolved in a small amount of chloroform to prepare 6 sheets of silica gel glass plates (West Germany, Merck, Kieselgel F ₂₅₄ , 2 mm, 20 × 20). After application, apply a straight line at 2.5cm from each lower end, develop with ethyl acetate / methanol (19: 1), scrape off the absorbed phase with an Rf value of 0.23, and extract twice with ethyl vinegar containing a small amount of water. After washing with water, the obtained extracted ethyl acetate was concentrated under reduced pressure with anhydrous sodium sulfate, and added with nucleic acid to leave 147 mg of demethyl maytansine crystal.

Example 38

260 mg of D-maytansine was added to 2.6 liters of the same bacterial culture medium obtained by the same method as Example 9, and then shaken and reacted at 28 ° C. for 31 hours, thereby losing D- maytansine. It can be seen from TLC that D-demethylmaytansine is produced.

Example 39

The reaction solution obtained in Example 38 was purified in the same manner as in Example 36. A thin layer chromatography similar to Example 36 was carried out to collect an oil having an Rf value of near 0.08 to obtain 106 mg of crude D-demethylmaytansine. . The purification was carried out in the same manner as in Example 37 below, to obtain 63 mg of white powder of D-demethylmaytansine.

Example 40

Actinomyces nigresense IFO 12894 was added to 2.2 liters of the same culture medium obtained in the same manner as in Example 9, and 217 mg of maytanfurin (D, L) was added thereto. (D, L) is lost and it turns out that demethyl maytanfurin (D, L) is produced | generated.

Example 41

The reaction solution obtained in Example 40 was purified in the same manner as in Example 36 to obtain 153 mg of crude material of dimethylmethanepurine (D, L).

In the same manner as in Example 37 below, five silica gel glass plates were applied in a straight line at 2.5 cm from each lower end, and then developed with ethyl acetate / methanol (19: 1), followed by scratching the absorption phases having an Rf of 0.40 and 0.28, respectively. The resulting mixture was extracted twice with ethyl acetate containing a small amount of water. The extracted ethyl acetate was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. When petroleum ether was added, 47 mg of white powder of L-demethylmethanepurine was obtained in the fraction of Rf 0.40. In the division of Rf 0.28, 38 mg of white powder of D-demethylmedanfurin is obtained.

Example 42

315 mg of maytanbutin (D, L) of 3.5 liters of the same culture medium obtained by culturing Actinomyces nigresense IFO 12894 in the same manner as in Example 9 was added and shaken at 28 ° C. for 48 hours to react. It can be seen by TLC that tanbutin (D, L) is lost and demethylmaytanbutin is produced.

Example 43

The culture solution obtained in Example 42 was purified in the same manner as in Example 36 to obtain 189 mg of crude material of dimethylmethylbutan (D, L).

TLC was carried out in the same manner as in Example 37, followed by development with ethyl acetate / methanol (19: 1), and the absorbing phases of Rf 0.47 and Rf 0.31 were scraped, respectively, and treated in the same manner as in Example 37. -85 mg of white powder of dimethylmethylbutanine was obtained by 78 mg of white powder of D- dimethylmethylbutanine in the division of Rf 0.31, respectively.

Example 44

530 mg of maytanvaline (D, L) was added to 5.3 liters of the same culture of Actinomyces nigresense IFO 12894 obtained in the same manner as in Example 9, and the mixture was shaken at 28 ° C for 48 hours to incubate. It can be seen from TLC that (D, L) is lost and demaytanvaline (D, L) is produced.

Example 45

The culture solution obtained in Example 44 was purified in the same manner as in Example 36 to obtain 223 mg of crude material of demaytanvaline (D, L).

In the same manner as in Example 37 below, the compound was subjected to flaptic TLC, developed with ethyl acetate / methanol (19: 1), and then the absorbed phases of Rf 0.55 and Rf 0.40 were scraped off and treated in the same manner as in Example 37 to obtain Rf 0.55. In the division, 95 mg of white powder of L-demethyl maytanvaline and 88 mg of white powder of D-demethylmethane valine in the division of Rf 0.40 are obtained, respectively.

Example 46

40 mg of L-maytansine was added to 4 liters of the same culture obtained by culturing Streptomyces flabotrysini IFO 12770 in the same manner as in Example 9, and reacted by shaking at 28 ° C for 48 hours. Purification in the same manner as in Examples 36 and 37 gave 5 mg of white powder of L-demethylmaytansine.

Example 47

40 mg of D-maytansine is added to 4 liters of the same culture medium obtained in the same manner as in Example 9 with Streptomicros Flavotrycin IFO 12770, followed by shaking at 28 ° C for 48 hours.

Purification in the same manner as in Examples 36 and 37, adding a small amount of ethyl acetate, and leaving it to obtain 6 mg of D-demethylmaytansine crystals.

[Example 48]

10 mg of L- maytansine was added to 1 liter of the same culture medium obtained by culturing Actinomyces nigresense IFO 13452 in the same manner as in Example 9, and the mixture was shaken at 28 ° C. for 48 hours to react with L- maytansine. It can be seen by TLC that it is decreasing and L-demethylmaytansine is produced.

Example 49

10 mg of L- maytansine was added to 1 liter of the same culture medium obtained by culturing Streptomyces ribini IFO 13452 in the same manner as in Example 9, and the mixture was shaken at 28 ° C. for 48 hours to react with L- maytansine. It can be seen by TLC that it is decreasing and L-demethylmaytansine is produced.

The physical properties of the compound obtained in the above Example are shown in the following table.

TABLE 5

Example 50

By the same method as Examples 1-8, 19-28, 33-34 using Bacillus megaterium IFO 1210, the compound shown below is obtained. Rf values of the starting compound, the product, and the product [electric solvent, CHCI ₃ : MeOH = 9: 1, plate: silica gel glass plate (Merck 60F ₂₅₄ thickness 0.25 mm)] are shown.

TABLE 6

Example 51

The compound shown below was obtained by the same method as Example 9, 29, 30, 46 using Streptomyces flabotriscine IFO 12770. Rf values of the starting compound, the product and the product (developing solvent: CHCl ₃ : MeOH = 9: 1, plate: silica gel glass plate (Merck 60F ₂₅₄ thickness 0.25 mm)) are shown in the following table.

TABLE 7

Example 52

The compound shown below is obtained by the same method as Example 12, 31, 32, 35-39 using Streptomyces pratensise IFO 12901. Rf values of the starting compound, the product and the product [developing solvent: CHCl ₃ : MeOH = 9: 1, plate: silica gel] The glass plate (Merck 60F ₂₅₄ thickness 0.25 mm) is shown in the following table.

TABLE 8

Example 53

50 mg of the crystal of demethyldechloromaytansinol 3-isobutylate obtained in Example 34 was dissolved in 20 mg of tetrahydrofuran, cooled to -5 ° C, and 50 mg of lithium aluminum hydride was added. The resultant was treated in the same manner as in Example 13 and subjected to flaptic TLC, followed by 17 cm of expansion with ethyl acetate / methanol (19: 1) to scrape off the Rf values of 0.20 to 0.25, and contained a small amount of water. After extracting with ethyl acetate, washing with water, drying with anhydrous sodium sulfate, and then concentrating under reduced pressure, 43 mg of powder of dimethyl dechloromaytansinol is obtained. This is dissolved in a small amount of ethyl acetate and left to crystallize. Dry after filtration 33 mg.

Melting point 198-201 degreeC (decomposition).

Elemental Analysis (%)

Found: C 62.48; H 7.25; N 5.19; O 24.89

Calculation: C ₂₇ H ₃₆ N ₂ O ₈

C 62.77; H 7.02; N 5.42; O 24.77

Mass spectrum (m / e): 455, 437

Spectrum (λ _max MeOH) nm: 230, 240, 248, 277, 285

Example 54

50 mg of the crystal of L-demethylmaytansine obtained in Example 37 was dissolved in 20 ml of tetrahydrofuran, cooled to -5 ° C and 50 ml of lithium aluminum hydride was added to react. The reaction solution is transferred to a water bath and stirred for 30 minutes. 10 ml of ethyl acetate and 10 ml of 200% hydrochloric acid are added, and then 50 ml of ethyl acetate is added and extracted. The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to flaptic TLC using silica gel, which was developed 17 cm with ethyl acetate / methanol (19: 1), and the absorbed phase near 0.25 of the Rf value was collected. Then, the mixture was 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 41 mg of demethyl maytansinol.

Its physical and chemical properties are consistent with those of demethylmaytansinol obtained in Example 5 above.

Melting point 195 ° C.

Elemental analysis

Found: C 58.67; H 6.54; N 4.83; CI 6.19 (%)

Calculation: for C ₂₇ H ₃₅ CIN ₂ O ₈

C 58.85; H 6.40; N 5.08; Cl 6.43 (%)

Mass spectrum: m / e 489, 471

UV Restraint Spectrum (nm): 232, 242, 251, 280, 288

Example 55

35 mg of the powder of D-demethylmaytansine obtained in Example 39 was dissolved in 14 mg of tetrahydrofuran, cooled to -5 ° C and 35 mg of lithium aluminum hydride was added. When this was processed and purified in the same manner as in Example 54, 19 mg of white powder of dimethyl maytansinol was obtained. Its physical and chemical properties are consistent with the growth of demethylmaytansinol obtained in Example 54. Melting point 196 ° C.

Example 56

30 mg of the powder of demethyl maytansinol 3- (N-phenyl) carbamate obtained in Example 22 was dissolved 14 mg of tetrahydrofuran, cooled to −5 ° C. or lower, and 30 mg of lithium aluminum hydride. Add. When this was processed and purified in the same manner as in Example 54, 17 mg of white powder of demethylmaytansinol was obtained. The physical and chemical properties of this material are consistent with those of demethylmaytansinol obtained in Example 54. Melting point 194 캜.

Claims (1)

General formula

In the maytansinoid compound represented by [wherein X represents Cl or H, and R ₁ represents H or an acyl group, respectively], Bacillus genus and streptoto having a capability of converting a methoxy group of the compound into a hydroxyl group A general formula characterized by contacting a culture of the microorganism belonging to the genus Micros or Actinomyces or a processed product thereof

A process for producing a demethyl maytansinoid compound represented by [wherein X and R ₁ represent the same meaning as in the foregoing].