JPS6361951B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to C-15003PHOs and a method for producing the same. The present inventors searched for a method of converting a certain maytansinoid compound into another compound using a microorganism, and found that when a culture of a certain microorganism or its processed material is applied to a certain maytansinoid compound, the maytansinoid The inventors discovered that it was possible to introduce a hydroxyl group into the 15th position of a compound, and as a result of further research based on this knowledge, the present invention was completed. That is, the present invention provides (1) general formula () [In the formula, one of R ₁ and R ₂ represents a hydroxyl group and the other represents hydrogen, and R ₃ represents hydrogen or an alkanoyl having 5 or less carbon atoms. However, if R ₁ is a hydroxyl group and
When R ₂ is hydrogen, R ₃ is hydrogen or

Represents an alkanoyl having 5 or less carbon atoms other than [Formula]. When R ₁ is hydrogen and R ₂ is a hydroxyl group, R ₃ represents hydrogen or alkanoyl having 5 or less carbon atoms. ] Maytansinoid compounds () and (2) general formula () [In the formula, _R'3 represents hydrogen or alkanoyl having 5 or less carbon atoms. ] of the maytansinoid compound ()
A method for producing a maytansinoid compound (), which is characterized by contacting a culture or treated product of a microorganism belonging to the genus Streptomyces, the genus Chiainia, or the genus Streptosporangium, which has the ability to introduce a hydroxyl group at the 15th position. . In the above general formula, the alkanoyl having 5 or less carbon atoms represented by R ₃ or R′ ₃ is, for example,
Formyl (-CHO), acetyl (-COCH ₃ ), propionyl (-COCH ₂ CH ₂ ), butyryl (-
COCH ₂ CH ₂ CH ₃ ), isobutyryl

[Formula] Valeryl (-COCH ₂ CH ₂ CH ₂ CH ₃ ), Isovaleryl

Examples include [Formula]. In this specification, "C-15003PHO" or "PHO" is a general term for all compounds in which R ₁ is a hydroxyl group and R ₂ is hydrogen in the above general formula (), a mixture of two or more, or a mixture of two or more, or a mixture of two or more, or a mixture of two or more of them. It is referred to as a single compound. In addition, in the general formula (), R ₁ is a hydroxyl group, R ₂ is hydrogen,
A compound where R ₃ is hydrogen is called "C-15003PHO-O" or "PHO-O", and a compound where R' ₃ is -COCH ₃ is called "C-15003PHO-1" or simply "PHO-1".
, a compound in which R ₃ is -COCH ₂ CH ₃ is called "C-
15003PHO-2” or “PHO-2” and R ₃

The compound of [formula] is called "C-15003PHO-3" or "PHO-3", and R ₃ is

The compound of the formula will be referred to as "C-15003PHO-4" or "PHO-4", respectively. In addition, "C-15003epi-PHO" is a general term for all compounds in which R ₁ is hydrogen and R ₂ is a hydroxyl group in the above general formula (), a mixture of two or more, or a single compound of each. shall be taken as a thing. In addition, in the general formula (), a compound in which R ₁ is hydrogen, R ₂ is a hydroxyl group, and R ₃ is hydrogen is referred to as "C
−15003−epi−PHO−0” or “epi−PHO−
0'' and a compound where R ₃ is -COCH ₃ is called ``C-
15003epi-PHO-1” or “epi-PHO-1”
, a compound in which R ₃ is -COCH ₂ CH ₃ is called "C-
15003epi-PHO-2” or “epi-PHO-2”
and R ₃ is

The compound of [formula] is "C-15003epi-PHO-3" or "epi-PHO-3"
and R ₃ is

The compound of [formula] is "C-15003epi-PHO-4" or "epi-PHO-
4" respectively. Also, in the general formula (), the compound maytansinol "P-0" where R' ₃ is hydrogen and the compound maytansinol "P-0" where R' ₃ is -COCH ₃
The compound maytanacin is referred to as “P-1”, and R′ ₃ is −
The compound maytansinol propionate of COCH ₂ CH ₃ is called “P-2” and R′ ₃ is

The compound of [formula] is "C-15003P- 3" or "P-3" and R' ₃ is -
The compound of COCH ₂ CH ₂ CH ₃ is called “C-15003P-3′”
or “P-3′” and R′ ₃ is

The compound of the formula will be referred to as "C-15003P-4" or "P-4", respectively. P-0, P-1, P-2, P-3, P-3', P
-4, for example, Nocardia sp.
No. C-15003 strain [FERM-P No. 3992 to the Institute of Microbial Technology, Agency of Industrial Science and Technology, IFO-13726 to the Fermentation Research Institute, The American Type Culture Collection (The
American Type Culture Collection) ATCC
-31281, respectively. ] can be obtained by culturing it in a medium and collecting and purifying it from the culture [JP-A-53-1211998;
−130693 Publication, Nature
vol. 270, p. 721 (1977), Tetrahedron vol. 35, p. 1079 (1979)]. P-0 can also be obtained by subjecting P-3, P-3' and/or P-4 to a deacylation reaction [JP-A-53-130693, Nature
vo1.270, P.721 (1977), Tetrahedron Volume 35,
See page 1079 (1979)]. Furthermore, compounds of the general formula () in which R′ ₃ is a substituent other than hydrogen, P-0 has a general formula derived from the corresponding carboxylic acid. [In the formula, R ₄ represents an alkanoyl having 5 or less carbon atoms. ] or the general formula R ₄ X ( ) [wherein R ₄ has the same meaning as above. X represents halogen. ] It can be produced by reacting an acid halide represented by: Examples of the alkanoyl having 5 or less carbon atoms represented by R ₄ include the same as R' ₃ above. Examples of the halogen represented by X in the above general formula () include chlorine, bromine, and iodine. It may be preferable to carry out the above reaction in the presence of a base. Examples of the base include triethylamine, tributylamine, pyridine, 4-dimethylaminopyridine, α-, β
Examples include tertiary amines such as -, γ-picoline, 2,6-lutidine, dimethylaniline, diethylaniline, and N-methylmorpholine. In addition, in the above reaction, a suitable solvent may be used, such as esters such as ethyl acetate, ethers such as diethyl ether, dioxane, and tetrahydrofuran, and halogenated hydrocarbons such as methylene chloride and chloroform. , nitriles such as acetonitrile, aromatic hydrocarbons such as benzene, nitromethane, dimethylformamide, dimethyl sulfoxide, sulfolane, etc., and mixtures thereof may also be used. Further, the above base may be used as a solvent, or a mixture of the base and the above solvent may be used as a solvent. The temperature at which the above reaction is carried out is not particularly limited, but -20°C to 40°C is suitable. The thus obtained compound of general formula () in which R′ ₃ is a substituent of hydrogen or more can be obtained by appropriately using commonly used separation and purification means such as extraction with a solvent, chromatography, recrystallization, etc. It can be purified by The microorganism used in the method of the present invention belongs to the genus Streptomyces, Chainia, or Streptosporangium, and has the ability to introduce a hydroxyl group into the 15-position of the maytansinoid compound (). Any microorganism or mutant strain thereof may be used. Examples of microorganisms that can be used in the method of the present invention include Streptomyces
Streptomyces sclerotialus
IFO 12246, Streptomyces castneus IFO13670, Streptomyces flavochromogenes
myces flavochromogenes) IFO13443, Streptomyces olivaceiscle−roticus
IFO13484, Streptomyces flaviscleroticus
IFO13357, Chainia nigra
nigra) IFO13362 and Streptosporangium roseum (Streptosporangium roseum)
roseum) IFO3776. above
IFO112246, IFO13670, IFO13443, IFO13484,
IFO13357, IFO13362 and IFO3776 are listed in the List of Cultures 1978 by the Fermentation Research Institute.
, 6th edition (Institute for Fermentation,
Osaka List of Cultures 1978 sixth edition), and both can be obtained from the Fermentation Research Institute. In general, Streptomyces, Chiainia, and Streptosporanporagium are susceptible to changes in their properties, such as X-ray irradiation, ultraviolet irradiation, radiation irradiation, and artificial mutagens (e.g., nitrosoguanidine, ethyleneimine, etc.). ) can be easily mutated using artificial mutation means. Even in such mutant strains, maytansinoid compounds () 15
Anything that has the ability to introduce a hydroxyl group into the position can be used in the method of the present invention. The medium used for culturing the above-mentioned microorganisms in the method of the present invention may be either liquid or solid as long as it contains a nutrient source that can be used by the strain, but it is more appropriate to use a liquid medium when processing a large amount. be. The culture medium contains carbon sources that can be assimilated by the above microorganisms,
Digestible nitrogen sources, inorganic substances, micronutrients, etc. are appropriately blended. Carbon sources include, for example, glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol, sorbitol, etc., oils and fats (eg, soybean oil, lard oil, chicken oil, etc.), and nitrogen sources include, for example. Meat extract, yeast extract, dried yeast, soy flour, corn steep, liquor, peptone, cotton powder, molasses, urea, ammonium salts (e.g. ammonium sulfate,
Ammonium chloride, ammonium nitrate, ammonium acetate, etc.) and others are used. Further, salts containing sodium, potassium, calcium, magnesium, etc., metal salts of iron, manganese, zinc, cobalt, nickel, etc., salts of phosphoric acid, boric acid, etc., and salts of organic acids such as acetic acid, propionic acid, etc. are used as appropriate. In addition, amino acids (e.g., glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline, etc.), peptides (e.g.,
dipeptides, tripeptides, etc.), vitamins (e.g.
B ₁ , B ₂ , nicotinic acid, B ₁₂ , C, E, etc.), nucleic acids (e.g., purines, pyrimidines and their derivatives, etc.)
etc. may be included. Of course, inorganic or organic acids, alkalis, buffers, etc. may be added for the purpose of adjusting the pH of the medium, or appropriate amounts of oils and fats, surfactants, etc. may be added for the purpose of defoaming. The culturing method may be static culture, shaking culture, or aeration/agitation culture. It goes without saying that for large-scale treatment, it is desirable to use so-called deep aeration agitation culture. It goes without saying that culture conditions vary depending on the condition and composition of the medium, the type of strain, the culture method, etc., but these are usually
It is best to select the initial pH to be around neutral at a temperature of 20°C to 45°C. In particular, the temperature during the middle stage of cultivation is between 24°C and 37°C.
℃, and the initial pH is preferably 6.5 to 8.5. Cultivation time may be about 6 to 100 hours, but especially 16 to 100 hours.
Good in 48 hours. The "culture" used in the present invention refers to what is obtained by the above-mentioned culture. The "processed product" used in the present invention refers to the culture obtained as described above subjected to physicochemical treatment such as filtration, centrifugation, ultrasonication, French press treatment, alumina grinding, lytic enzyme treatment, surfactant or organic It is called bacterial cells obtained by solvent treatment or crushed bacterial cells containing hydroxylase. Furthermore, hydroxylase obtained by purification by a known method, or bacterial cells or hydroxylase immobilized by a known method can also be used. The method of the present invention is carried out by bringing the raw material compound () into contact with the culture of the above-mentioned microorganism or its treated product. The concentration of the raw material compound in the reaction solution is 1~
500 μg/ml is appropriate. Reaction temperature is 20~50℃,
A pH of 5 to 10 is appropriate, especially at a temperature of 24 to 40℃,
PH6-9 is good. Reaction time is 1-200 hours,
More preferably, 24 to 72 hours is appropriate. Further, the reaction may be carried out under static conditions or under conditions of shaking, aeration or stirring, but it is better to carry out the reaction under shaking, aeration or stirring. The substance thus obtained can be detected by thin layer chromatography (hereinafter abbreviated as TLC). Extract the reaction solution with ethyl acetate and dilute to 1/10
The liquid concentrated to 0 volume was poured onto a silica gel glass plate (West German Merck & Co., Kieselgel 60F ₂₅₄ , 0.25
The sample is subjected to TLC using a carrier (solvent: chloroform: methanol = 9:1) and measured by the absorption image detected by irradiation with ultraviolet rays of 2537 Å. To collect the target product from the reaction solution, separation and purification methods normally used for collecting microbial metabolites can be used as appropriate, since this group of substances is neutral fat-soluble. For example, there are methods that utilize differences in solubility with impurities, methods that utilize differences in adsorption affinity of various adsorbents such as activated carbon, macroporous nonionic resins, silica gel, and alumina, and methods that remove impurities using ion exchange resins. may be used alone, in combination, or repeatedly. When utilizing the difference in solubility, suitable solvents for extraction from liquids include organic solvents that are immiscible with water, such as fatty acid esters such as ethyl acetate and aluminum acetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform, etc. Ketones such as methyl isobutyl ketone are used. Extraction is performed near neutrality, preferably using ethyl acetate from a culture solution adjusted to pH 7. After washing the extract with water, it is concentrated under reduced pressure, and a non-polar solvent such as petroleum ether or hexane is added to collect the crude substance (i) containing the active ingredient. Some of this includes TLC
Since many spots other than the target substance PHO and target substance epi-PHO are observed above, the following purification steps are used in stages. In other words, various types of adsorption chromatography are effective as commonly used purification methods, and commonly used carriers such as silica gel, alumina, macroporous nonionic adsorption resins, etc. can be used as adsorbents; Silica gel is most effectively used for the purification of (i), and the target substance can be obtained by developing from non-polar solvents such as petroleum ether and hexane, and by adding polar solvents such as ethyl acetate, acetone, ethanol, and methanol. PHO and epi
- Perform elution of PHO. One example is using silica gel (West German Merck & Co., Ltd., 0.05-0.2 mm) as a carrier,
Perform column chromatography while gradually increasing the mixing ratio of hexane and ethyl acetate, and collect the eluate.
The fractions containing PHO and epi-PHO are collected by TLC analysis, concentrated under reduced pressure, and petroleum ether or hexane is added to obtain crude substance (ii). If this still contains many other impurities, the following purification is performed. For example, purification is performed using a second silica gel column using a different solvent system. In this case, the developing solvent may be a halogen-containing hydrocarbon such as dichloromethane or chloroform, or a polar solvent such as alcohols such as ethanol or methanol, or ketones such as acetone or methyl ethyl ketone. substance PHO, epi−
Separate and collect PHO. The combination of solvent systems for the first and second silica gel columns can be reversed or used alone, and other commonly used organic solvents may be appropriately combined. In addition to PHO, epi-PHO was present in the obtained crude substance (iii).
If it contains, the following purification step is further used. That is, although the above-mentioned various adsorption chromatography and partition chromatography are effective, it is advantageous to perform separation using a reversed-phase partition gel for purification of the crude substance (iii). Alcohols and ketones that mix with water are used as elution solvents. One example is a high-performance liquid chromatograph PrepLC/ _system500 (U.S., Waters Inc., PrepPAK-500/C ₁₈ ), etc.
Preparative fractionation is carried out using the Waters Co., Ltd.). When aqueous methanol is used as a solvent, epi-PHO,
Since it is eluted in the order of PHO, after detection by reverse phase TLC (Merck & Co., West Germany), epi-PHO, PHO
Concentrate the fraction under reduced pressure. The resulting concentrates are each extracted with ethyl acetate, concentrated under reduced pressure, and a small amount of methanol is added and allowed to stand, yielding colorless crystals. PHO-0 obtained in Example 5, PHO-1 obtained in Example 7, and PHO-1 obtained in Example 9, which will be described later.
PHO-2, PHO-3 obtained in Example 3, PHO-4 obtained in Example 11, PHO-4 obtained in Example 5
epi-PHO-0, epi-PHO obtained in Example 7
Table 1 shows the physicochemical properties of epi-PHO-2 obtained in Example 9, epi-PHO-3 obtained in Example 3, and epi-PHO-4 obtained in Example 11. and shown in Table 2.

【table】

【table】

【table】

[Table] As a result of comparing the above-mentioned physicochemical properties with known maytansinoid compounds, PHO-3 was found to be found in The Journal of Organic Chemistry, Vol. 42,
De-acetylmaytanbutacine described on page 2349 (1977)
It was in good agreement with those of Also, by the usual method
When PHO-3 was acetylated, the physicochemical properties matched well with the physicochemical properties of maytanbutacine described in the above literature. But other PHO and epi−
No compounds matching PHO were observed. First, let's compare epi-PHO-3 with PHO-3.
Since the same fragment is observed in the mass spectrum, epi-PHO-3 is PHO-3 as well as PHO-3.
It is easily presumed that this is a compound in which a hydroxyl group has been introduced into -3. Furthermore, when comparing the nuclear magnetic resonance spectra, hydrogen at position 15 is observed as a singlet at δ5.37 in PHO-3, but it is observed as a singlet at δ5.11 in epi-PHO-3. It is easily estimated that it is a stereoisomer at position 15 of , and epi-PHO-3 is found to be a new compound. Furthermore, the results of X-ray analysis
The absolute configuration of C15 in epi-PHO-3 was found to be the S configuration. Other PHOs
and epi-PHO are compared with PHO-3 and epi-PHO-3, respectively, and it is found that their structural formulas are shown in FIGS. 1 and 2.

【table】 \
_CH3

[Developing solvent: water-saturated ethyl acetate; plate: silica gel glass plate (manufactured by Merck & Co.)]

Reference Example 4 Epi-PHO-3 (33 mg) obtained in Example 3 was dissolved in 1 ml of pyridine, 4 drops of acetic anhydride was added, and the mixture was left at room temperature overnight. Thereafter, the same treatment as in Reference Example 3 was performed to obtain crystals of 15-acetyl epi-PHO-3 (23 mg). Mass spectrum m/e 692 (M ⁺ ) m/e 631, 571, 556, 536, 483, 468,
448 Rf=0.39 [Developing solvent: water-saturated ethyl acetate; plate: silica gel glass plate (manufactured by Merck & Co.)].

Claims (1)

[Claims] 1. General formula [In the formula, one of R ₁ and R ₂ represents a hydroxyl group and the other represents hydrogen, and R ₃ represents hydrogen or an alkanoyl having 5 or less carbon atoms. However, if R ₁ is a hydroxyl group and
When R ₂ is hydrogen, R ₃ represents hydrogen or an alkanoyl other than [Formula] having 5 or less carbon atoms. When R ₁ is hydrogen and R ₂ is a hydroxyl group, R ₃ represents hydrogen or alkanoyl having 5 or less carbon atoms. ] maytansinoid compound. 2 General formula [In the formula, _R'3 represents hydrogen or alkanoyl having 5 or less carbon atoms. ] maytansinoid compound is contacted with a culture of a microorganism belonging to the genus Streptomyces, genus Chiainia, or genus Streptosporangium, or a treated product thereof, which has the ability to introduce a hydroxyl group into the 15th position of the maytansinoid compound. A general formula characterized by [In the formula, one of R ₁ and R ₂ represents a hydroxyl group and the other represents hydrogen, and R ₃ represents hydrogen or an alkanoyl having 5 or less carbon atoms. However, if R ₁ is a hydroxyl group and
When R ₂ is hydrogen, R ₃ represents hydrogen or an alkanoyl other than [Formula] having 5 or less carbon atoms. When R ₁ is hydrogen and R ₂ is a hydroxyl group, R ₃ represents hydrogen or alkanoyl having 5 or less carbon atoms. ] A method for producing a maytansinoid compound represented by