JPH0463880B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a new method for producing an aminopropylaminobleomycin derivative represented by the general formula () below, which has antitumor activity and is expected to be an anticancer agent. [Prior art] Some of the aminopropylaminobleomycin derivatives represented by the general formula [] below are disclosed in Japanese Patent Application Laid-open No. 1983-
No. 116497, etc., and is produced by a method in which bleomycins represented by the general formula [] below are reductively condensed with a carbonyl compound represented by the general formula [] below in the presence of sodium cyanoborohydride. [Problems to be solved by the invention] However, according to the above method, about 5 to 7% of by-products of unknown structure are produced, and on the other hand, it is relatively difficult to remove these by-products, making the purification process complicated. In addition, there is a drawback that hydrogen gas under cyanide is generated during the reaction, which worsens the working environment. [Means for solving the problem] Therefore, the present inventors have developed a method that generates fewer by-products and
In addition, the following general formula [] [BX] -NH- (CH ₂ ) ₃ -A-Y-B [] [In the formula, [BX] is the following formula, which does not worsen the working environment. represents a residue obtained by removing the hydroxyl group from the carboxyl group of bleomycin acid (in the case of a copper-containing substance, chelate copper is omitted), Y represents a lower alkylene group, and A represents the formula

【formula】 【formula】

[expression] or

[Formula] represents a group (where R ₁ is a hydrogen atom, a lower alkyl group or a benzyl group, R ₂ is a lower alkyl group or a benzyl group, and R is a lower alkyl group), and B is

A group represented by [Formula] (where R ₅ and R ₆ are each a hydrogen atom or a lower group optionally substituted with one or more phenyl, naphthyl, furyl, thiophene, norbornene, or cycloalkyl groups) Alkyl group (phenyl group further includes halogen atom, lower alkyl group, lower alkoxy group, cyano group, halogenomethyl group,
May be substituted with one or more substituents selected from cycloalkyl groups and benzyloxy groups (hydrogen on the benzene ring may be substituted with a halogen atom, lower alkoxy group, or benzyloxy group) . )). However, at least one of R ₅ and R ₆ is a group other than a hydrogen atom. ]
As a result of various studies on new methods for producing the aminopropylaminobleomycin derivative represented by
General formula [] [BX]-NH-( _CH2 ) ₃ -A-Y- _NH2 [] [In the formula, [BX], Y and A are the same as above. ] Aminopropylbleomycins represented by the general formula [] R ₃ -CO-R ₄ [] [wherein R ₃ and R ₄ are each a hydrogen atom, a cycloalkyl group, a cycloalkyl group, or one or more phenyl groups] Lower alkyl groups that may be substituted (phenyl groups, which may be further substituted with halogen atoms), halogen atoms, lower alkyl groups, lower alkoxy groups, cycloalkyloxy groups, benzyloxy groups (halogen atoms, lower alkoxy hydrogen of the benzene ring may be substituted with a benzyloxy group), a phenyl group, a naphthyl group, a thiophene group, or a furyl group, which may be substituted with one or more types of cyano group, halogenomethyl group. shows. ] When a carbonyl compound represented by the formula is reductively condensed with a boron cyanide compound in a solvent while an inert gas is passed through the solvent, the formation of structurally unknown by-products can be greatly suppressed and the generation thereof. It was also discovered that hydrogen cyanide gas can be removed. The present invention has been completed based on the above findings. Examples of the lower alkyl group in the general formulas [], [], [] include methyl group, ethyl group,
_C1 to _C4 alkyl groups such as propyl group, isopropyl group, n-butyl group, isobutyl group, halogens include fluorine, chlorine, bromine, etc., lower alkyl groups include ethylene group, trimethylene group, etc. The C ₂ - _{C 6} alkylene groups such as C, tetramethylene and hexamethylene groups are cycloalkyl groups such as cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecanyl and cycloundecanyl groups. Examples of lower alkoxy groups include _C5 to _C12 cycloalkyl groups such as methoxy group, ethoxy group,
Examples of C ₁ -C ₄ alkoxy such as propoxy group and butoxy group and halogenomethyl group include trifluoromethyl group and trichloromethyl group. In A above

For example, [formula] is

【formula】 【formula】 【formula】

【formula】 etc., but

For example, [formula] is

[In the formula, R ₁ ″ represents lower alkyl, and X represents a halogen atom.]

By reacting the halide represented by the general formula [] [In the formula, [BX], Y, R ₁ ″, and Z are the same as above.D
is a tert-butyloxycarbonyl group using 6N hydrochloric acid under ice cooling using a known method. By removing the group, a compound of the general formula [] can be obtained.
Examples of compounds obtained by this method include
One example is A5MMHH. Next, when R ₁ and R ₂ are different, the compound of the general formula [] is reductively condensed with the compound of the above formula [] to form the general formula [] [In the formula, Y is the same as above], and this compound is reacted with a compound of the general formula [] to form the general formula [] [In the formula, Y, R ₁ ″, R ₂ , Z, and D are the same as above.] The protecting group is then removed by the above method to obtain a compound of the general formula [ ]. Examples of compounds obtained by this method include A5MMHH. Among the carbonyl compounds represented by the general formula [], 3,4-di(p-chlorobenzyloxy)benzaldehyde, 3,4-di(3',4'-dichlorobenzyloxy)benzaldehyde, 3,
4-(p-methoxybenzyloxy)benzaldehyde, 4-(4'-benzyloxybenzyloxy)benzaldehyde), 4-cyclooctylmethyloxybenzaldehyde is a new compound and is produced by the method described below. [In the formula, R ₇ is p-methoxybenzyl, 3,4-dichlorobenzyl, p-chlorobenzyl, p-benzyloxybenzyl, cyclooctylmethyl, X is halogen, and n is 0 or 1] That is, the formula Compounds represented by [] and formula []
In a solvent such as acetone, a compound represented by
The reaction may be carried out at room temperature to the boiling point of the solvent. After the reaction is completed, the solvent is distilled off, and water and an organic solvent such as chloroform or ether are added to the residue to obtain aldebide in the organic layer. If necessary, a pure product can be obtained by recrystallization or by purifying the crude product by column chromatography using silica gel or adsorption resin. Furthermore, cyclotridecanecarbaldehyde is a new compound and can be produced by the method described below. That is, when titanium tetrachloride is added to a mixture of zinc dust, anhydrous tetrahydrofuran, and dibromomethane, and cyclotridecanone is added and reacted, methylene cyclotridecane is obtained. This is then purified by epoxidation with metachloroperbenzoic acid.
Reaction of this with boron trifluoride ether complex in anhydrous dichloromethane yields cyclotridecane carboxaldehyde. [Example] Next, the manufacturing method of the present invention will be explained with reference to Examples. Example 1 (A) 3-[N-Mekyl-N-(3-aminopropyl)amino]propylaminobleomycin 3
Dissolve 0.500 g (0.310 mmol) of copper-containing hydrochloride (hereinafter abbreviated as BLM APMP) in 40 ml of methanol, and dissolve 3,4-bis(benzyloxy).
Benzaldehyde (hereinafter abbreviated as BBBA)
0.296 g (0.93 mmol) is added, followed by 39 ml (0.62 mmol) of sodium borohydrocyanide dissolved in 5 ml of methanol. After raising the temperature to 40°C, nitrogen gas is bubbled into the reaction solution at a rate of 20 to 25 ml/min, and the reaction is allowed to proceed with stirring for 48 hours. After the reaction, cool to room temperature and stop supplying nitrogen gas. CM pre-filled with methanol
- Inject the reaction solution into a Toyopearl 650M (manufactured by Toyo Soda Co., Ltd.) column (50 ml volume) to adsorb the target substance. After adsorption, pour 150ml of methanol into the
Removes cyanide, boron compounds and BBBA. Elution is then carried out by a sequential concentration gradient method in which sodium chloride is added stepwise to 50% (v/v) methanol water to increase the sodium concentration by 0.05 mol to 0.30 mol. (Flow 50ml of the eluate at each concentration.) Run 300ml of the eluate and collect 150ml of the blue fraction that elutes at a sodium concentration of around 0.20M. According to high performance liquid chromatography quantitative method, this fraction contains 3-
{N-methyl-N-[3′-(bis(3″,4″-bis(benzyloxy)benzyl)amino)propyl]amino}propylaminobleomycin 3
Contains 0.499 g (0.225 mmol) of copper-containing hydrochloride (hereinafter abbreviated as BB-BLM). Yield 72.6% (from BLM APMP copper-containing body). This fraction is injected into a Diaion HP 20SSZ (manufactured by Mitsubishi Chemical Corporation) column (40 ml volume) packed with 50% (v/v) methanol water to adsorb the target product. Next, purification is carried out by eluting methanol water containing 0.1% (v/v) acetic acid by a sequential concentration gradient method in which the methanol concentration is increased from 50% to 90% in 10% increments. (Flow 60ml of the eluate at each concentration.) Pour 300ml of the eluate,
80ml of blue fraction with high performance liquid chromatography purity of 99.5% eluting at methanol concentration of around 80%
Collect. Among these are copper-containing bodies of BB-BLM.
Contains 0.449g (0.203mmol). Purification yield 90.2%. (B) The fraction obtained in (A) with a methanol concentration of 10%
Amberlite XAD-2 (manufactured by Rohm and Haas) column (50 ml volume) concentrated to
inject into and absorb. Resin with sodium chloride 5
Wash with 300 ml of an aqueous solution of 2% EDTA/2Na, wash with 100 ml of 2% sodium chloride, then 150 ml of distilled water, and finally add 1/50 N hydrochloric acid aqueous solution.
Elute with methanol (1:4v/v) wavelength 290n
Collect the fraction showing the absorption maximum near m. Dowex 44 (OH type; manufactured by The Dow Chemical Company)
After adjusting the pH to 6.0, 0.415 g (0.193 m
mol) as a colorless amorphous powder. Total yield
62.3% (from BLM APMP copper-containing body). This product matches the standard product in terms of ultraviolet absorption spectrum, infrared absorption spectrum, retention value in thin layer chromatography on a copper-containing body, and retention value in high performance liquid chromatography. Example 2 BLM APMP copper-containing substance 0.500g (0.310mmol)
was dissolved in 40 ml of methanol, 0.11 g (0.60 mmol) of cycloundecanecarboxaldehyde was added, and then sodium cyanoborohydride 13
mg (0.21 mmol) dissolved in 5 ml of methanol is added. Nitrogen gas is bubbled into the reaction solution at a rate of 35 to 40 ml/min, and the reaction is allowed to proceed at room temperature with stirring for 30 hours. After the reaction, the same operation as in Example 1 is performed. CM-Toyopearl 650M treatment allows 3-{N
-Methyl-N-[3'-(cycloundecanylmethylamino)propyl]amino}propylaminobleomycin trihydrochloride (hereinafter abbreviated as MCU-BLM) containing 0.469 g (0.264 mmol) of copper-containing substance Get a picture. Yield 85.2% (from BLM APMP copper-containing body).
Then, by Diaion HP 20SS purification,
A fraction containing 0.449 g (0.252 mmol) of copper-containing MCU-BLM is obtained. Purification yield 95.7%. This fraction was decoppered, concentrated under reduced pressure, and then lyophilized to yield 0.411 g (0.240 mmol) of decoppered MCU-BLM.
is obtained as a colorless amorphous powder. Total yield 77.4%
(From BLM APMP copper-containing body). This product matches the standard product in terms of ultraviolet absorption spectrum, infrared absorption spectrum, retention value in thin layer chromatography on a copper-containing body, and retention value in high performance liquid chromatography. Example 3 3-{N,N-dimethyl-N-[3'-(N,N-
Dissolve 0.553 g (0.310 mmol) of the copper-containing substance of dimethyl-N-(3″-aminopropylamino)propyl]amino}propylaminobleomycin trihydrochloride dichloride (hereinafter abbreviated as BLM PP4M) in 40 ml of methanol. 0.099g of benzaldehyde
(0.93 mmol) and then 39 mg (0.62 mmol) of sodium cyanoborohydride dissolved in 5 ml of methanol are added, and the same operation as in Example 2 is carried out. By CM-Toyo Pearl 650M treatment, 3-
{N,N-dimethyl-N-[3′-N,N-dimethyl-N-(3″-(dibenzylamino)propyl)amino)propyl]amino}propylaminobleomycin trihydrochloride dichloride (hereinafter referred to as PPDBZ) A fraction containing 0.493 g (0.251 mmol) of copper-containing body of -BLM is obtained. Yield 81.0% (from BLM PP4M copper-containing body). Then, by Diaion HP 20SS purification,
Copper content of PPDBZ-BLM 0.394g (0.201mmol)
Obtain a fraction containing Purification yield 79.9%. This fraction was decoppered, concentrated under reduced pressure, and lyophilized to give 0.363 g (0.191 m
mol) as a colorless amorphous powder. Total yield
61.6% (from BLM PP4M copper-containing body). This product matches the standard product in terms of ultraviolet absorption spectrum, infrared absorption spectrum, retention value in thin layer chromatography on a copper-containing body, and retention value in high performance liquid chromatography. Example 4 In Example 3, 3 instead of BLM PP4M
-[4′-(3″-aminopropyl)piperazily-1-
0.529 g of copper-containing substance of propylaminobleomycin tetrahydrochloride (hereinafter abbreviated as BLM PY)
(0.310 mmol), and perform the same operation using helium gas instead of nitrogen gas. By CM-Toyopearl 650M treatment, 3-
Copper-containing compound of {4′-[3″-(dibenzylamino)propyl]piperidyl}propylaminobleomycin tetrahydrochloride (hereinafter abbreviated as PYDBZ-BLM)
A fraction containing 0.468 g (0.248 mmol) is obtained. yield
80.0% (from BLM PY copper-containing body). Then, by Diaion HP 20SS purification,
Copper content of PYDBZ-BLM 0.422g (0.244mmol)
Obtain a fraction containing Purification yield 90.2%. This fraction was decoppered, concentrated under reduced pressure, and lyophilized to give 0.386 g (0.212 m
mol) as a colorless amorphous powder. Total yield 68.4% (from BLM PY copper-containing body). This product matches the standard product in terms of ultraviolet absorption spectrum, infrared absorption spectrum, retention value in thin layer chromatography on a copper-containing body, and retention value in high performance liquid chromatography. [Effects of the Invention] Table 1 shows the production rates of the structurally unknown substance and the compound represented by the general formula [] and the hydrogen cyanide concentration on the reaction liquid surface produced by the methods of Examples 1 to 4. In addition, the respective production rates and hydrogen cyanide concentrations obtained by methods that do not pass inert gas are shown as control examples.

[Table] * Calculated from the values measured by liquid chromatography of the reaction solution.
As is clear from this table, in the control example, about 4 to 8% of substances with unknown structure are produced, whereas in the example of the method of the present invention, the production is about 0.5 to 1%, which is about 1/5.
The yield of the target compound of general formula [] has been improved by about 4 to 6%. In addition, the hydrogen cyanide concentration at the reaction liquid surface is 300~
It has dropped dramatically from 360ppm to 12-15ppm. As is clear from the above, the method of the present invention reduces side reaction products of unknown structure by approximately 1/5 to 1/8 compared to the conventional method.
The yield of the target product can also be improved. Furthermore, since the concentration of hydrogen cyanide is greatly reduced, the working environment is greatly improved.

Claims (1)

[Claims] 1 General formula [BX]-NH-(CH ₂ ) ₃ -A-Y-NH ₂ [In the formula, [BX] represents a residue obtained by removing the hydroxyl group from the carboxyl group of bleomycin acid, and A is a group represented by the formula [Formula] [Formula] [Formula] or [Formula] (where R ₁ is a hydrogen atom, a lower alkyl group or a benzyl group, R ₂ is a lower alkyl group or a benzyl group, Y and R are (lower alkylene group). Aminopropylaminobleomycin represented by the general formula R ₃ −CO−R ₄ [wherein R ₃ and R ₄ are each substituted with one or more hydrogen atoms, cycloalkyl groups, cycloalkyl groups, or phenyl groups] a lower alkyl group (the phenyl group may be further substituted with a halogen atom), a halogen atom, a lower alkyl group, a lower alkoxy group, a cycloalkyloxy group, a benzyloxy group (a halogen atom, a lower alkoxy group, hydrogen of the benzene ring may be substituted with a benzyloxy group), a phenyl group, a naphthyl group, a thiophene group, or a furyl group that may be substituted with one or more types of cyano group, halogenomethyl group. . ] [BX]-NH-(CH ₂ ) ₃ -A-Y-B [In the formula, [BX], Y and A are the same as above. B is a group represented by the formula (where R ₅ and R ₆ are each a hydrogen atom or a phenyl group, a naphthyl group, a furyl group, a thiophene group, a norbornene group,
A lower alkyl group that may be substituted with one or more cycloalkyl groups (the phenyl group is further substituted with a halogen atom, a lower alkyl group, a lower alkoxy group, a cyano group, a halogenomethyl group, a cycloalkyloxy group, a benzyloxy group (a halogen atom) The hydrogen on the benzene ring may be substituted with a lower alkoxy group, a benzyloxy group), and one or more substituents selected from the following. However, at least one of R ₅ and R ₆ is a group other than a hydrogen atom. ] A method for producing an aminopropylaminobleomycin derivative and a salt thereof.