JPS635396B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the following general formula [] (In the formula, R represents -OH or an unterminated amino residue of bleomycins.) The present invention relates to N-methylbleomycin acid or N-methylbleomycins, non-toxic salts thereof, and a method for producing the same. N-methylbleomycin acid is represented by the following general formula [] when R is -OH in the above formula []. As a result of research, the inventors have found that N-methylbleomycins represented by the above formula [] and N-methylbleomycin acid represented by the above formula [] are bleomycin inactivating enzyme (hereinafter referred to as "inactivating enzyme"; Umezawa et al. : Journal of Antibiotics, Vol. 27, p. 419, 1974), and completed the present invention. Bleomycin is an antitumor antibiotic discovered by Umezawa et al. and is produced by the actinomycete Streptomyces verticillus (Umezawa et al.: Journal
of Antibiotics Volume 19A, Page 200, 1966
Year). A mixture containing bleomycin A ₂ and B ₂ as the main ingredients is currently used in cancer chemotherapy, especially in areas such as head and neck cancer, skin cancer, penile cancer, cervical cancer, esophageal cancer, lung cancer, and malignant lymphoma. It has shown excellent effects on epithelial cancer. However, conventional bleomycins have the following formula due to the action of an inactive enzyme: [In the formula, Z represents a bleomycin residue. ] It was revealed using organs such as rat liver that the partial structure (2,3-diaminopropionic acid amide) is inactivated by hydrolysis. It was found that bleomycin is relatively not inactivated in the skin and lungs, where bleomycin has a strong effect, but is easily inactivated in the stomach, where bleomycin is said to have no effect. Furthermore, it was found that this inactivating power was low for murine squamous cell carcinoma caused by 20-methylcholanthrene, but high for sarcoma caused by the same cause (Umezawa et al.: Journal of Antibiotics, Vol. 25, p. 409). , 1972; Umezawa et al.: Journal of Antibiotics vol. 27, p. 419.
(1974). Furthermore, the effect of inactivating bleomycin has been observed in squamous cell carcinomas that occur in the human head and neck, and this inactivation is particularly noticeable in poorly differentiated squamous cell carcinomas, for which bleomycin is said to be relatively ineffective. (Müller et al.: Kancer vol. 40, p. 2787, 1977). As can be seen from these findings, bleomycin cannot be sufficiently effective against cancers that have strong inactivated oxygen. This is one of the reasons why bleomycin is currently expected to be improved further. Therefore, the inventors believe that if a type of bleomycin derivative that is not inactivated can be found, it would be possible to more effectively treat squamous cell carcinoma in head and neck cancer, esophageal cancer, lung cancer, and other areas where conventional bleomycin is ineffective. We thought that it would be possible to treat adenocarcinoma, such as gastric cancer. As a result of various studies from this point of view, the present invention was completed by discovering that N-methylbleomycin acid and N-methylbleomycins are not affected by inactivating enzymes at all. The amino residue of the bleomycins R in the general formula [] of the present invention is, for example, (1) an alkylamino group (which may have a substituent on the alkyl group,
Substituents on alkyl groups include phenyl group, lower alkylthio group, lower alkoxy group, hydroxyl group, cyano group, guanidino group, carboxyl group, carbamoyl group, piperidino group, biperazino group, morpholino group, imidazolyl group, pyrrolidino group, amino group, lower alkoxycarbonyl group,

[Formula] etc., and the phenyl group, alkyl group, amino group, piperidino group, morpholino group, and pyrrolidino group in these substituents may further have a substituent, (2)
Anilino group (lower alkoxy group on the phenyl nucleus,
It may be substituted with a lower alkyl group, nitro group, cyano group, sulfonic acid group, sulfamoyl group, phenyl group, etc. ), (3) β-naphthylamino group, (4)
These include pyridylamino group, (5) 1,2,4-triazolyl amino group, (6) thiazolyl amino group, etc. More specifically, alkylamino groups include ethylamino group, propylamino group, etc. Examples of the alkylamino group include the following. (1) Phenyl-substituted lower alkylamino groups such as benzylamino group, aminomethylbenzylamino group, 2-phenylethylamino group, 1-phenylethylamino group, etc. (2) Lower alkylthio-substituted lower alkylamino groups such as methylthioethyl Amino group, methylthiopropylamino group, propylthiopropylamino group, etc., (3) lower alkoxy-substituted lower alkylamino group, such as methoxypropylamino group, ethoxypropylamino group, methoxyethylamino group, etc., (4) amino lower alkylamino group For example, 3-aminopropylamino group, 2-aminoethylamino group, 2-aminopropylamino group, etc., and these groups have an alkyl group, phenyl group, phenyl-substituted lower alkyl group, amino lower alkyl group (the amino group may be further substituted with the groups mentioned herein), cyclohexyl group, pyrrolidino lower alkyl group, piperidino lower alkyl group, morpholino lower alkyl group, cyclohexyl lower alkyl group, hydroxy lower alkyl group or lower alkoxy-lower alkyl group, examples of which include N,N-dimethylaminoethylamino group, N,N-diethylaminoethylamino group, N,N-dimethylaminopropylamino group. , N,N-dimethylaminopropylamino group, n-butylaminopropylamino group, octylaminopropylamino group, N,N-diphenylaminopropylamino group, benzylaminopropylamino group, 1-phenylethylaminopropylamino group group, 2-phenylethylaminopropylamino group, aminopropylaminopropylamino group, 6-aminohexylaminopropylamino group, N-aminopropyl-N-butylaminopropylamino group, N-aminopropyl-N-ethylamino group Propylamino group, N-aminopropyl-
N-methylaminopropylamino group, N-aminoethyl-N-methylaminopropylamino group, n
-Butylaminopropylaminopropylamino group, cyclohexylaminopropylaminopropylamino group, cyclohexylaminopropylamino group, 2-hydroxycyclohexylaminopropylamino group, 3-pyrrolidinopropylaminopropylamino group, 3-piperidinopropylaminopropyl Amino group, morpholinopropylaminopropylamino group, 3-hydroxypropylaminopropylamino group, 2-hydroxypropylaminoethylamino group, 3-methoxypropylaminopropylamino group, etc. (5) Guanidino lower alkylamino group such as guanidinobutylamino (6) Piperazino lower alkylamino group, such as piperazinoethylamino group,
Piperazinopropylamino group, 4-(3-aminopropyl)-piperazinopropylamino group, etc. (7)
Morpholino lower alkylamino group such as morpholinopropylamino group, morpholinoethylamino group, etc. (8) Pyrrolidino lower alkylamino group such as pyrrolidinopropylamino group (9) for example

[Formula] (10) Hydroxy lower alkylamino group such as hydroxyethylamino group, hydroxypropylamino group, etc. (11) Lower alkoxycarbonyl lower alkylamino group such as methoxycarbonylmethylamino group, methoxycarbonylpropylamino group, etc. The N-methylbleomycins represented by the general formula [ ] of the present invention can be obtained by reductive methylation of bleomycin acid, that is, by reacting it with formaldehyde in the presence of a reducing agent in an inert solvent, the partial structural formula of bleomycin acid [ ] amino group of

A methyl group is introduced into [formula] to obtain N-methylbleomycin acid represented by formula [], and then the general formula [] R'-H...[] (wherein R' is the terminal amino residue of bleomycins) Instead of reacting the amine represented by the conventional method for forming a peptide bond, for example, in the presence of a condensation reagent for peptide synthesis, or in the presence of a condensation reagent for peptide synthesis, N-methylbleomycin acid of the general formula [ ] can be used. It can be obtained by reacting with an amine of the general formula [] as an active ester used in the usual peptide synthesis. The inert solvent used for reductive methylation is preferably one that dissolves bleomycin acid, such as water, water-containing methanol, dimethylformamide, dimethyl sulfoxide, etc., and the reducing agent is sodium cyanoborohydride, etc. borohydride compounds are preferred. In addition, bleomycin acid is
Microorganisms belonging to the genus Fusarium as described in 19677, for example, Fusarium Roseum Foundation Fermentation Research Institute preservation number
It is produced by treating cultured bacterial cells such as IFO7189 with bleomycin _B2 . In the present invention, the terms N-methylbleomycin acid and N-methylbleomycins are
Unless otherwise specified, both copper-free bodies and copper-containing bodies are included. The method for producing N-methylbleomycins of the general formula [] will be explained in more detail. First, the bleomycin acid methylation step (first step) will be described. Bleomycin acid (preferably the decoppered form) is dissolved in aqueous methanol. This is maintained at a constant temperature of 0° to 30°C, and formaldehyde and sodium cyanoborohydride are added while stirring. After reacting for 1 to 24 hours, the pH of the reaction solution is lowered to 1.0 with hydrochloric acid to decompose the sodium cyanoborohydride and stop the reaction. The relative amount of formaldehyde may be in the range of about 0.5 to 3 moles per mole of bleomycin acid, but preferably 1.0 to 1.5 moles. The appropriate amount ratio of sodium cyanoborohydride is 0.6 to 2 moles per mole of bleomycin acid. Next, a method for extracting N-methylbleomycin acid from the reaction solution will be explained. After adjusting the Hz of the reaction solution to 6.0 with sodium hydroxide and distilling off methanol under reduced pressure, an adsorption resin such as Amberlite XAD-2 (Rohm & Co., Ltd.) was used for desalting.
(manufactured by Haas) is injected into a column packed with distilled water to adsorb the target substance. After washing away the salts with distilled water, add acidic water-containing methanol, e.g.
50N hydrochloric acid aqueous solution - methanol (1:4v/v)
elute with a wavelength of 290 millimicrons and collect the fractions that exhibit maximum absorption at a wavelength of around 290 millimicrons. Anion exchange resin, Dowex 44 (OH ^- type; manufactured by The Dow Chemical Company)
After neutralization, the mixture is concentrated under reduced pressure and freeze-dried to obtain a crude powder of N-methylbleomycin acid. Furthermore, the following operation is performed to increase the purity. When the above powder is dissolved in distilled water and basic copper carbonate is added and stirred, N-methylbleomycin acid is converted to a copper-containing compound. This was equilibrated in advance with PH4.5, 1/20 molar acetic acid-sodium acetate buffer.
It is injected into a column packed with CM-Sephadex C-25 (Na ⁺ type; manufactured by Pharmacia Fine Chemicals) and adsorbed. When eluted by a linear gradient method in which the sodium concentration is gradually increased to 1.0 mol by continuously adding sodium chloride to the above buffer solution, N-methylbleomycin acid is eluted at around 0.35 mol. At this time, unreacted raw materials and by-products tend to elute more quickly, so they can be detected and separated and removed using an ultraviolet absorption monitor, such as Ubicord (manufactured by LKB). If impurities are found in the fraction of the target product, the above-mentioned chromatography may be performed again to ensure complete removal. The fraction of the target product obtained in this way was dewarmed by the Amberlite XAD-2 dewarming method used previously, and then freeze-dried to form a bluish-purple amorphous powder containing the copper-containing N-methylbleomycin acid. It can be obtained with Next, the step (second step) of converting the above intermediate into N-methylbleomycins will be explained. It is characterized in that the carboxyl group is activated with a condensation reagent for peptide synthesis and reacted with the amine of the general formula [] to lead to N-methylbleomycins. To explain one example specifically, N-methylbleomycin acid is dissolved in water, an organic solvent, or a mixture thereof. Dimethylformamide or dimethylsulfoxide is used as the organic solvent. A base catalyst such as N-methylmorpholine and a condensation reagent for peptide synthesis such as 6-chloro-1-parachlorobenzenesulfonyl oxide benztriazole (hereinafter referred to as "CCBT") are mixed at a constant temperature of 0° to 30°C with stirring.
That's what it means. ) is added first. Next, the general formula []
After adding this amine and reacting for 1 to 5 hours, acetic acid is added to stop the reaction, and a reaction solution containing N-methylbleomycin is obtained. When a copper-containing body is used as a raw material, a copper-containing body is obtained, and when a copper-free body is first used, a copper-free body is obtained, but the copper-containing body is preferable in the next isolation step. The relative ratio of the condensation reagent for peptide synthesis to the amine of the general formula [] is preferably 1 to 5 moles per mole of N-methylbleomycin acid. Next, a method for extracting the product from the reaction solution will be explained using the case of a copper-containing body as an example. The reaction solution was desalted using the Amberlite XAD-2 desalting method described above, and the resulting powder was dissolved in distilled water and purified by column chromatography using the CM-Sephadex C-25 used in the first step. do. The sodium concentration at which the target product is eluted differs depending on the type of N-methylbleomycin. For example, a type that does not have a basic group on the terminal amine elutes at around 0.50 mol, a type that has one basic group elutes at around 0.65 mol, and a type that has two basic groups elutes at around 0.80 mol. A fraction of the target substance showing an absorption maximum near the wavelength of 292 millimicrons eluted from the above column was collected, desalted, and purified to pH 6.0 using Dowex 44 (OH ^- type).
If the mixture is adjusted to the following conditions and freeze-dried, a copper-containing N-methylbleomycin compound is obtained as a blue-purple amorphous powder. The N-methylbleomycin (copper-containing compound) obtained in this way is processed using known methods such as EDTA.
A copper-free body can be obtained by removing copper using a method using (Japanese Patent Publication No. 52-31875). One example is Amberlite, which is made by dissolving a copper-containing body in distilled water and filling it with distilled water.
Inject into XAD-2 column and adsorb. When the column is washed with an aqueous solution consisting of 2% sodium chloride and 5% disodium ethylenediaminetetraacetic acid (hereinafter referred to as "EDTA.Na ₂ "), copper ions are carried away by EDTA:Na ₂ and N-methyl Bleomycins (decoppered) remain on the resin. The resin was washed with 2% sodium chloride to remove EDTA/Na ₂ and further washed with distilled water. Finally, it is eluted with acidic water-containing methanol, such as 1/50 N hydrochloric acid aqueous solution-methanol (1:4 v/v), and a fraction exhibiting an absorption maximum around a wavelength of 290 millimicrons is collected. After adjusting the pH to 6.0 using Dowex 44 (OH ^- type), concentrating under reduced pressure and freeze-drying, the copper-free hydrochloride of N-methylbleomycin is obtained as a white amorphous powder. If, for example, a sulfuric acid aqueous solution is used instead of the above-mentioned hydrochloric acid aqueous solution, the desired product can be obtained as a sulfate.
Thus, by selecting the type of acid used in the above elution step, a non-toxic salt with any pharmaceutically acceptable acid can be obtained by extremely easy application of the known method. It is. In the carbon-13 NMR spectra (measured by the proton noise decoupling method in heavy water) of N-methylbleomycin acid and N-methylbleomycins produced by the method described above, there are The signal of the introduced methyl group was detected at a chemical shift of 32.7 ppm. Also, N-methylbleomycin acid or N-methylbleomycin acid
- When methylbleomycins were subjected to hydrolysis in 6N hydrochloric acid water at 105°C for 24 hours, it was confirmed that 3-amino-2-methylaminopropionic acid and methylamine were produced in the hydrolysis solution. At the same time, other degradation products common to bleomycins were detected except for 2,3-diaminopropionic acid. Furthermore, in N-methylbleomycins, amines introduced in the second step were detected. The above facts support that N-methylbleomycin acid and N-methylbleomycins produced by the method of the present invention have the chemical structures represented by the above formula [] and the above formula [], respectively. Examples of the amine of general formula [] used in the above reaction include the compounds shown below. Hydroxyamine, hydrazine, phenylhydrazine, semicarbazide, thiosemicarbazide,
dicyclohexylcarbodiimide, methylamine, ethylamine, n-propylamine, iso-
Propylamine, n-hexylamine, n-laurylamine, allylamine, dimethylamine, dipropylamine, cyclohexylamine, cyclopentylamine, aniline, aminopyridine, aminopyrimidine, aminothiazole, aminoimidazole, aminopyrazole, aminotriazole, naphthylamine, Examples include aminoquinoline, ethyleneimine, pyrrolidine, piperidine, morpholine, etc., and those with substituents include cyanomethylamine, β-hydroxyethylamine,
β-cyanoethylamine, β-bromoethylamine, 1,3-trimethylenediamine, 1,4-diaminobutane, 4-guanidinobutylamine, di-β-chloroethylamine, amino acid esters (e.g. glycine methyl ester, phenylglycine, methyl ester, phenylalanine methyl ester, lysine methyl ester, etc.), cyclohexylmethylamine, chloraniline, nitroaniline, anisidine, toluidine, cyanoaniline, aminoacetophenone, aminoacetanilide, biphenylamine, benzylamine, phenethylamine, sulfanilic acid, sulfur phanilamide, sulfadiazine, sulfathiazole,
Sulfadimethoxine, homosulfamine, furfurylamine, 4-(5-nitro-2-furyl)
Thiazolylamine 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, N-
(2-aminoethyl)-1,2-diaminoethane,
N-(3-aminopropyl)-1,3-diaminopropane, N-(3-aminopropyl)-1,4-diaminobutane, N-(3-aminopropyl)-1,
6-diaminohexane, N-(5-aminopentyl)-1,5-diaminopentane, N,N'-bis(2-aminoethyl)-diaminomethane, N,
N'-bis(2-aminoethyl)-1,2-diaminoethane, N,N'-bis(3-aminopropyl)
-1,4-diaminobutane, N,N'-bis(3
-aminopropyl)-1,6-diaminohexane,
N,N'-bis(5-aminopentyl)-1,5-
Diaminopentane, 2,2,3-trimethylpentamethylenediamine, 1,2-diaminopropane, N-methyl-1,3-diaminopropane, N
-butyl-1,3-diaminopropane, N,N-
Diethyl-1,2-diaminoethane, N,N-diethyl-1,2-diaminoethane, N,N-dimethyl-1,3-diaminopropane, N,N-diethyl-1,3-diaminopropane, 3- aminopropyl-trimethyl-ammonium bromide,
N-(3-dimethylaminopropyl)-1,3-diaminopropane, N-(3-methylaminopropyl)-1,2-diaminoethane, N-(3-methylaminopropyl)-1,4-diaminobutane , N
-(3-methylaminopropyl)-1,6-diaminohexane, N-(3-methylaminopropyl)-
1,8-diaminooctane, N-butyl-N'-
3-Aminopropyl-1,3-diaminopropane, N-(2-dimethylaminoethyl)-1,3-
Diaminopropane, N-(4-dimethylaminobutyl)-1,3-diaminopropane, N-(6-dimethylaminohexyl)-1,3-diaminopropane, N-(8-dimethylaminooctyl)-1,
3-diaminopropane, N-(3-hydroxypropyl)-1,3-diaminopropane, N-(2-
hydroxypropyl)-1,2-diaminoethane,
N-(2-hydroxyethyl)-1,3-diaminopropane, N-(3-methoxypropyl)-1,3
-diaminopropane, N-(3-octyloxypropyl)-1,3-diaminopropane, N-(3
-amino-1-methylpropyl)-1,3-diaminopropane, N-(3-amino-1-ethylpropyl)-1,3-diaminopropane, N,N-
Bis(3-aminopropyl)-methylamine, N,
N-bis(3-aminopropyl)-ethylamine,
N,N-bis(3-aminopropyl)-n-butylamine, 3-aminopropyl-dimethylsulfonium bromide, 3-aminopropyl-dimethylsulfonium chloride, 3-acetamidopropyl-dimethylsulfonium bromide, 3-amino-3 -Carboxypropyldimethylsulfonium chloride, 3-amino-3-carbamoylpropyl-dimethylsulfonium chloride, 4
-(aminobutyl)-guanidine, 3-amidinopropylamine, N-(3-aminopropyl)-pyrrolidine, N-(3-aminopropyl)-piperidine, N-(2-aminoethyl)-piperazine, N-
(3-aminopropyl)-piperazine, N-(3-
aminopropyl)morpholine, N-(3-aminopropyl)-N'-methylpiperazine, N-(3-
aminopropyl)-N'-ethylpiperazine, N,
N′-bis(3-aminopropyl)-piperazine,
N-(3-pyrrolidino-propyl)-1,3-diaminopropane, N-(3-piperidino-propyl)
-1,3-diaminopropane, N-(3-morpholinopropyl)-1,3-diaminopropane, N
-benzyl-1,3-diaminopropane, N,N
-dibenzyl-1,3-diaminopropane, N-
(1-phenylethyl)-1,3-diaminopropane, N-(p-methylbenzyl)-1,3-diaminopropane, m-xylene diamine, p-xylene diamine, N-cyclohexyl-1,3-diaminopropane, N-(3-cyclohexylaminopropyl)-1,3-diaminopropane, N-(2
-hydroxycyclohexyl)-1,3-diaminopropane, N-(2-phenylethyl)-1,3
-Diaminopropane, N-(2-p-tolylethyl)-1,3-diaminopropane, N-benzyl-N'-(3-aminopropyl)-1,3-diaminopropane, N-(1-phenylethyl)- N′−
(3-aminopropyl)-1,3-diaminopropane, N-(2-furfuryl)-1,3-diaminopropane, N-(5-methyl-2-furfuryl)-
1,3-diaminopropane, N,N-dimethyl-
1,3-diaminopropane, 4-piperidylmethylamine, 2-(4-imidazolyl)-ethylamine, N,N-dimethyl-N'-acetyl-1,3
-diaminopropane, N,N-dimethyl-N'-
Propionyl-1,3-diaminopropane, 1-
carboxy-4-dimethylaminobutylamine,
1-carbamoyl-4-dimethylaminobutylamine, N,N-dimethyl-N'-benzoyl-1,
3-diaminopropane, N-[2-(β-pyridyl)-ethyl]-1,3-diaminopropane, N-
(2-methoxyethyl)-1,3-diaminopropane, N-(1-methyl-3-methoxypropyl)-
Examples include 1,3-diaminopropane. In addition to the CCBT, N-ethyl-
5-phenyl isoxazolium-3'-sulfonate (NEPIS), N-tertiary butyl-5-methylisoxazolium perchlorate, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, di-p-nitrophenyl sulfite,
Dicyclohexylcarbodiimide (DCC), 1-
Ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-cyclohexyl-3-(2-
Morpholinoethyl) carbodiimide and the like are used. Examples of the N-methylbleomycins of the present invention obtained as described above include the following compounds. N-methylbleomycins R′=−NH−(CH ₂ ) ₂ CH ₃ −NH−(CH ₂ ) ₅ CH ₃ −
NH−(CH ₂ ) ₂ −NH ₂ −NH−(CH ₂ ) ₃ −NH ₂ −NH−(CH ₂ ) ₃ −NH−CH ₃ −NH−(CH ₂ ) ₃ −
NH− (CH ₂ ) ₃ −CH ₃ −NH− (CH ₂ ) ₃ −NH−
( _CH2 ) ₆ − _NH2 −NH−(CH ₂ ) ₃ −NH−(CH ₂ ) ₃ −NH ₂ −NH−(CH ₂ ) ₃ −NH−(CH ₂ ) ₃ −OH−NH−
(CH ₂ ) ₃ −NH− (CH ₂ ) ₃ −OCH ₃ −NH−(CH ₂ ) ₃ −NH−(CH ₂ ) ₂ −OCH ₃ −NH−(CH ₂ ) ₂ −OH−NH CH ₂ CN Next, Tables 1 to 3 show the main physical and chemical properties investigated for representative examples of N-methylbleomycin acid and N-methylbleomycins. Note that the compound numbers in the table indicate the following compounds. Compound (1) N-methylbleomycin acid compound (2) N-methylbleomycin A ₂ Compound (3) N-methylbleomycin B ₂ Compound (4) N-methylbleomycin A ₂ ′ ^-b (R=-NH-
( _CH2 ) ₃ - _NH2 ) Compound (5) 3-[(S)-1'-phenylethylamino]propylamino-N-methylbleomycin Compound (6) 3-(3-n-methylaminopropylamino)
Propylamino-N-methylbleomycin (R
=-NH( _CH2 ) ₃ -NH-( _CH2 ) _3- NH-n-
_{C4H9 )}

【table】

【table】

【table】

[Table] Various biological properties investigated using representative examples of the intermediate N-methylbleomycin acid and N-methylbleomycins produced by the method of the present invention are explained below. 1 Resistance test to inactivated enzyme (1) Extraction method of inactivated enzyme The liver of a female Donrieux rat was crushed by adding 1/15 mol of twice its weight in a phosphate buffer solution of PH7.2 to make a tissue emulsion. . Centrifugation was performed at 105,000×G for 60 minutes, and the supernatant was dialyzed. This high molecular fraction was used as an extract of the inactivated enzyme. (2) Measuring method for inactivation reaction Add 1 ml of the above extract to 1 ml of the substrate solution (800 mcg
It also contains N-methylbleomycin acid or N-methylbleomycins. ) and 37℃
Reactions were carried out for 15, 30, 60, and 120 minutes.
After protein removal from 0.3 ml of the reaction solution, residual activity against Mycobacterium smegmatis ATCC607 was measured. As a control, a similar test was conducted using non-N-methylated bleomycin acid and bleomycins. The results are shown in FIGS. 1 to 6. In the figure, 1 is the result for the compound of the present invention, and 2 is the result for the control compound. As is clear from FIGS. 1 to 6, conventional bleomycin acid and bleomycins are highly susceptible to inactivation enzymes and are inactivated over time, whereas the present invention's N-methylbleomycin It turns out that acids and bleomycins are very insensitive to inactivating enzymes. 2 DNA strand cleavage activity in a cell-free system The mechanism of anticancer action of bleomycins is thought to be DNA strand cleavage carried out by the cooperation of bleomycin (decopper-free form), divalent iron, and molecular oxygen. . Therefore, it was confirmed that N-methylbleomycins also have similar activity. (1) Method Divalent iron and N-methylbleomycin (decoppered form) were mixed in advance and dissolved together with tritium-labeled DNA in a 1/10 mol phosphate buffer with a pH of 7.4. The reaction was carried out for 5 minutes at 37°C in the presence of oxygen. 25% trichloroacetic acid was added to the reaction solution, and the resulting precipitate was centrifuged. The radioactivity of this supernatant (acid-soluble fraction) was measured and the acid solubilization rate was calculated. This was used as an indicator of the degree of DNA cleavage. A similar test was performed using bleomycin A ₂ and B ₂ as controls. The results are shown in Table 4. 3 Growth inhibitory effect on cultured He La S ₃ cells (1) Method: He La S ₃ cells were inoculated into a plastic sheared culture medium (MEM supplemented with 10% calf serum), and two days later, N-methylbleomycin acid or N-methyl was added. Bleomycins were added.

[Table] After continuing the culture for an additional 3 days, the number of cells was measured. The growth inhibition rate is calculated using the following formula: Inhibition rate (%) = (1-A-C/B-C) x 100 [In the formula, A is the final cell count on the third day after adding the test sample, and B is the number of cells without adding the test sample. The final cell number in the control, C, represents the cell number at the time of addition of the test sample. ]
Calculated using The ID ₅₀ value (concentration for 50% inhibition) was determined from the graph of sample concentration and inhibition rate. As a control, bleomycin acid and bleomycins were similarly tested. The results are shown in Table 5. 4 Mycobacterium smegmatis
Antibacterial activity against ATCC607 (1) Method Using the above-mentioned test bacteria, it was measured by the agar plate cylinder method. However, the standard material bleomycin A ₂
(decoppered product) was set to 1000mcg titer/mg. The results are shown in Table 6.

【table】

[Table] As is clear from the results in Table 6, N-methylbleomycins also exhibit excellent antibacterial activity. As can be seen from the experimental results shown above, N-
Methylbleomycin acid and N-methylbleomycins are not inactivated by inactivating enzymes.
In addition, N-methylbleomycins retain DAN cleaving activity similar to bleomycins, and
It has strong growth inhibitory activity on He La S ₃ cells and also has excellent antibacterial activity. These facts support the fact that this compound would be useful as an antitumor agent, bactericidal agent, etc. Next, the present invention will be specifically explained using examples. Example 1 Synthesis of N-methylbleomycin acid (copper-containing form) 1.0 g of bleomycin acid (copper-free form) was dissolved in 48 ml of water-containing 85% methanol, and the mixture was stirred at 30°C.
Add 30 mg formaldehyde as an aqueous solution,
This was followed by the addition of 33 mg of sodium cyanoborohydride. After reacting for 12 hours, the pH of the reaction solution was lowered to 1.0 with a 1N aqueous hydrochloric acid solution, and the reaction was stopped by standing for 10 minutes. After neutralizing with a 1N aqueous sodium hydroxide solution, methanol was distilled off under reduced pressure, and distilled water was added to the residual liquid to make 10 ml. In order to desalt this, it was injected into an Amberlite XAD-2 column (300 ml volume) filled with distilled water and adsorbed. After washing with distilled water, it was eluted with a 1/50 N hydrochloric acid aqueous solution-methanol (1:4 v/v), and a fraction showing an absorption maximum around a wavelength of 290 millimicrons was collected. After neutralizing with anion exchange resin Dowex 44 (OH ^- type), it was concentrated under reduced pressure and freeze-dried. This was dissolved in 10 ml of distilled water, 113 mg of basic copper carbonate was added thereto, and the mixture was stirred at room temperature for 2 hours. Excess basic copper carbonate was removed by filtration, and the blue-purple solution was equilibrated with 1/20 molar, PH4.5 acetic acid-sodium acetate buffer solution.
It was injected into a −25 (Na ⁺ form) column (200 ml volume) and adsorbed. By the linear gradient method, the sodium concentration was gradually increased to 1.0 molar by continuously adding sodium chloride to the above buffer solution.
The eluate was run. A blue-purple fraction eluting at around 0.35 mol was collected, and after performing the above chromatography again to increase purity, it was desalted using the Amberlite XAD-2 desalting method used earlier. After adjusting the pH to 6.0 using Dowex 44 (OH ^- ), it was concentrated under reduced pressure and lyophilized to give 250 mg of N.
- A blue-purple amorphous powder of methylbleomycin acid (copper-containing substance) was obtained. Yield 24%. The ultraviolet absorption curve of this product measured using distilled water is shown in FIG. 7, and the infrared absorption curve measured using the potassium bromide tablet method is shown in FIG. 11. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water) 292 (150) 246 (151) Other physical and chemical properties are as shown in Table 1. Example 2 Synthesis of N-methylbleomycin acid (copper-containing form) 1.0 g of bleomycin acid (copper-free form) was dissolved in 50 ml of water-containing 90% methanol, and the mixture was stirred at 0°C.
Add 34 mg formaldehyde as an aqueous solution,
Subsequently, 72 mg of sodium cyanoborohydride was added. After reacting for 24 hours, the reaction solution was treated according to the method of Example 1 to obtain 198 mg of a blue-purple amorphous powder of N-methylbleomycin acid (copper-containing substance). Yield 19%. This product showed the same physical and chemical properties as those obtained in Example 1. Example 3 Synthesis of N-methylbleomycin acid (copper-containing form) 1.0 g of bleomycin acid (copper-free form) was dissolved in 40 ml of water-containing 95% methanol, and the mixture was stirred at 23°C.
Add 25 mg of formaldehyde as an aqueous solution,
This was followed by the addition of 62 mg of sodium cyanoborohydride. After reacting for 6 hours, the reaction solution was treated according to the method of Example 1 to obtain 235 mg of a blue-purple amorphous powder of N-methylbleomycin acid (copper-containing substance). Yield 22%. The physical and chemical properties of this product were the same as those of Example 1. Synthesis A-Step of N-methylbleomycin _A2 N-methylbleomycin acid (copper-containing substance) 400 mg
was dissolved in 20 ml of dimethylformacide and mixed with 116 mg of N-methylmorpholine and 298 with stirring at 23°C.
mg of CCBT was added. After stirring for 5 minutes,
166 mg of 3-aminopropyldimethylsulfonium chloride (hydrochloride) was added. After reacting for 3 hours, 5.3 ml of glacial acetic acid was added to stop the reaction. The target product was precipitated by mixing 300 ml of acetone with the reaction solution, and the precipitate was dissolved in 10 ml of distilled water and then treated in the same manner as in Example 1. That is, after desalting using Amberlite XAD-2 desalting method, CM
-The product was subjected to column chromatography using Sephadex C-25, and fractions of approximately 0.65 mol were collected. This was desalted and freeze-dried to obtain 369 mg of a blue-purple amorphous powder of N-methylbleomycin A ₂ (copper-containing compound)-hydrochloride. Yield 82%. The ultraviolet absorption curve of this product measured using distilled water is shown in FIG. 8, and the infrared absorption curve measured using the potassium bromide tablet method is shown in FIG. 12. The maximum ultraviolet absorption [millimicron (E1% 1cm distilled water)] is as follows. 292 (122) 244 (148) Other physical and chemical properties are as shown in Table 1. B-Step Dissolve 300 mg of the copper-containing substance obtained in A-Step in 10 ml of distilled water, and add Amberlite XAD to remove copper.
-2 column (200 ml volume) and adsorbed.
600ml of an aqueous solution consisting of 2% sodium chloride and 5% EDTA・_Na2 , 400ml of a 2% sodium chloride aqueous solution
After washing the column with 250 ml of distilled water and 250 ml of distilled water,
1/50N hydrochloric acid aqueous solution - methanol (1:4v/
eluted with v), and fractions showing maximum absorption around a wavelength of 290 millimicrons were collected. Dowex 44
After adjusting the pH to 6.0 using (OH ^- form), it was concentrated under reduced pressure and lyophilized to obtain 265 mg of white amorphous powder of N-methylbleomycin A ₂ (decoppered form) hydrochloride. Ta. Yield 92%. The ultraviolet absorption curve of this product is shown in Figure 9, and the infrared absorption curve measured by the potassium bromide tablet method is shown in Figure 13. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water)
291 (93) Other physicochemical properties are shown in Table 2. Example 5 Synthesis of N-methylbleomycin B ₂ A-Step N-methylbleomycin acid (copper-containing compound) 400 mg
was dissolved in 20 ml of dimethyl sulfoxide, and 116 mg of N-methylmorpholine and 298
mg of CCBT was added. After stirring for 5 minutes,
Add 176 mg of agmatine (hydrochloride) and add 3 more
Time reacted. The reaction solution was treated according to the method of Example 4, A-Step to obtain 345 mg of a blue-purple amorphous powder of N-methylbleomycin B ₂ (copper-containing compound) hydrochloride. Yield 76%. The maximum ultraviolet absorption values of this product were as follows. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water) 292 (123) 244 (153) Other physical and chemical properties are shown in Table 1. B-Step 335 mg of the copper-containing body obtained in A-Step was added to Example 4B-
The copper was removed by the method described in Step 1 to obtain 280 mg of white amorphous powder of N-methylbleomycin B ₂ (decoppered form) hydrochloride. Yield 86%. The ultraviolet absorption curve measured with distilled water of this product is the 10th
FIG. 14 shows an infrared absorption curve measured by the potassium bromide tablet method. Ultraviolet absorption maximum: millimicrons (E1% 1cm distilled water) 290 (101) Other physical and chemical properties are shown in Table 2. Example 6 Synthesis of N-methylbleomycin A _2' -b A-Step N-methylbleomycin acid (copper-containing compound) 400 mg
was dissolved in 20 ml of dimethylformamide and mixed with 29 mg of N-methylmorpholine while stirring at 0°C.
mg of CCBT was added. After stirring for 5 minutes, 64
Add 5 more mg of 1,3-diaminopropane
Time reacted. The reaction solution was treated according to the method of Example 4, A-Step to obtain 402 mg of N-methylbleomycin A ₂ '-b.
A blue-purple amorphous powder of (copper-containing substance) hydrochloride was obtained. Yield 92%. The maximum ultraviolet absorption values of this product were as follows. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water) 292 (122) 244 (148) Other physical and chemical properties are shown in Table 3. B-Step Example 4,
The copper was removed by the B-step method to obtain 333 mg of white amorphous powder of N-methylbleomycin A ₂ '-b (decoppered form) hydrochloride. Yield 89%. The maximum ultraviolet absorption values of this product were as follows. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water) 291 (101) Example 7 Synthesis of 3-[(S)-1'-phenylethylamino]propylamino-N-methylbleomycin A-Step N-methyl Bleomycin acid (copper-containing substance) 400mg
was dissolved in 20 ml of dimethylformamide and mixed with 29 mg of N-methylmorpholine while stirring at 27°C.
mg of CCBT was added. After stirring for 5 minutes,
154 mg of N-[(S)-1'-phenylethyl]-1,
3-Diaminopropane was added and the reaction was further continued for 3 hours. The reaction solution was treated according to the method of Example 4, A-Step to obtain 304 mg of 3-[(S)-1'-phenylethylamino]propylacino-N-methylbleomycin (copper-containing compound) hydrochloride. A blue-violet amorphous powder was obtained. Yield 65%. The maximum ultraviolet absorption values of this product were as follows. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water) 292 (114) 243 (140) B-step 290 mg of the copper-containing body obtained in A-step was added to Example 4,
After removing copper using the B-step method, 257 mg of 3-
A white amorphous powder of [(S)-1'-phenylethylamino]propylamino-N-methylbleomycin (decoppered form) hydrochloride was obtained. Yield 92%. The maximum ultraviolet absorption values of this product were as follows. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water) 290 (101) Example 8 3-(3-n-butylaminopropylamino)
Synthesis of propylamino-N-methylbleomycin A-Step N-methylbleomycin acid (copper-containing substance) 400mg
was dissolved in 20 ml of dimethylformamide and mixed with 29 mg of N-methylmorpholine while stirring at 0°C.
mg of CCBT was added. After stirring for 5 minutes,
Add 162 mg of N-3-aminopropyl-N'-n-butyl-1,3-diaminopropane and add 1
Time reacted. The reaction solution was treated according to the method of Example 4, A-Step (however, the elution concentration from CM-Sephadex C-25 was around 0.80 mol), and 246 mg of 3-(3-
A blue-purple amorphous powder of n-butylaminopropylaminopropylamino-N-methylbleomycin (decoppered form) hydrochloride was obtained. Yield 51%. The maximum ultraviolet absorption values of this product were as follows. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water) 292 (102) 243 (125) B-step 237 mg of the copper-containing body obtained in A-step was added to Example 4,
Decopper was removed using the B-step method to obtain 194 mg of 3-(3
A white amorphous powder of -n-butylaminopropylamino)propylamino-N-methylbleomycin (decoppered form) hydrochloride was obtained. Yield 85%. The maximum ultraviolet absorption values of this product were as follows. Maximum ultraviolet absorption: Millimicrons (E1% 1cm distilled water) 290 (99)

[Brief explanation of the drawing]

Figures 1 to 6 show the residual activity of the compounds of the present invention and the corresponding conventional bleomycins against inactivating enzymes, with the vertical axis showing the residual activity (%) and the horizontal axis showing the reaction time. 1 in Figure 1 is N-
Methylbleomycin A ₂ , 2 is bleomycin
A ₂ , 1 in Figure 2 is N-methylbleomycin B ₂ ,
2 is bleomycin B ₂ and the third 1 is 3-[(S)-
1'-Phenylethylamino]propylamino-N
-methylbleomycin, 2 is 3-[(S)-1'-
Phenylethylamino]propylaminobleomycin, 1 in Figure 4 is 3-(3-n-butylaminopropylamino)propylamino-N-methylbleomycin, 2 is 3-(3-n-butylaminopropylamino)propyl Aminobleomycin, 1 in Figure 5 is N-methylbleomycin
A ₂ '-b, 2 indicates bleomycin A ₂ '-b, 1 in FIG. 6 indicates N-methylbleomycin acid, and 2 indicates the residual activity of bleomycin acid. Figures 7 to 10 are the ultraviolet absorption curves of the compounds of the present invention, Figure 7 is N-methylbleomycin acid (copper-containing body), and Figure 8 is N-
Methylbleomycin A ₂ (copper-containing form), hydrochloride, Figure 9 shows N-methylbleomycin A ₂ (copper-free form).
Hydrochloride, Figure 10 is N-methylbleomycin
This is an ultraviolet absorption curve of B ₂ (decoppered product) hydrochloride.
Figures 11 to 14 are infrared absorption curves of the compounds of the present invention measured by the potash bromide tablet method. ₂ (copper-containing form)/hydrochloride; Figure 13 shows N-methylbleomycin A ₂ (copper-free form)/hydrochloride; Fig. 14 shows infrared absorption of N-methylbleomycin B ₂ (copper-free form)/hydrochloride. Show a curve.

Claims (1)

[Claims] 1. General formula (In the formula, R represents -OH or an unterminated amino residue of bleomycins.) N-methylbleomycin acid or N-methylbleomycins and non-toxic salts thereof. 2 By reductive methylation of bleomycin acid,
General formula below N-methylbleomycin acid represented by
The general formula is then reacted with an amine by conventional methods of peptide bond formation. (In the formula, R ' represents an unterminated amino residue of bleomycins.) A method for producing N-methylbleomycins and non-toxic salts thereof.