CA1276400C

CA1276400C - Process for producing (amido)n-substituted bleomycins

Info

Publication number: CA1276400C
Application number: CA000453841A
Authority: CA
Inventors: Akio Fujii; Yasuhiko Muraoka; Tokuji Nakatani; Takeo Fukuoka; Katsutoshi Takahashi; Hamao Umezawa
Original assignee: Nippon Kayaku Co Ltd
Current assignee: Nippon Kayaku Co Ltd
Priority date: 1983-05-14
Filing date: 1984-05-08
Publication date: 1990-11-13
Anticipated expiration: 2007-11-13
Also published as: AT380886B; JPS59210100A; YU43845B; CS346884A2; ES8506308A1; CS241086B2; DK237184D0; KR840009304A; YU83784A; ATA157084A; KR910001725B1; JPH03879B2; DK237184A; HUT34223A; HU189110B; ES532369A0

Abstract

ABSTRACT OF THE DISCLOSURE
A process for producing (amido)N-substituted bleomycins represented by the general formula wherein BM is a residue of bleomycin skelton, X is a C1 to C10 alkyl group which may be substituted by a C1 to C4 alkyl-amino, di(C1 to C6 alkyl) amino or N-[(C1 to C4 alkyl)amino-C1 to C4 alkyl]N-C1 to C4 alkylamino group, wherein the alkyl substituent may have a phenyl substituent, which phenyl may be substituted with a halogen atom; R1 is (1) a cycloalkyl group having 6 to 10 carbon atoms, (2) a C1 to C4 alkyl group having a phenyl substituent (the phenyl may further have a halogen substituent), or (3) a phenyl group which may have one or more substituents selected from halogen atoms, cyano and benzyloxy, and R2 is a halogen atom or -CH2-R1, wherein R1 is as defined above, which comprises allowing an (amindo)N-substituted derivative represented by the general formula

Description

This invention relates to a process for producing (amido)N-substituted bleomycins. More particularly, the present invention relates to a proces~ for producing (amido)N-substituted bleomycins of low toxici-ty represented by the gene~al formula [I]
CONH2 N~I2 Cl~ 2 Nll CEI R~
CH CH2 CO N~l IH3 IIH2 N ` ~ ~ CO-NH--(C~I2) 3-N- ~CH2) 3 N R2 BM- ~
S H [I]

wherein BM is a residue of bleomycin skelton, X is a Cl to C10 alkyl group which may be substituted by a Cl to C4 alkyl-amlno, di~Cl to C6 alkyl) amino or N-[(Cl to C~ alkyl)amino-Cl to C4 alkyl~N-Cl -to C~ alkylamino group, wherein the alkyl substi-tuents may have a phenyl substituent, which phenyl may be substituted with a halogen atom; Rl is (1) a cycloalkyl group having 6 to 10 carbon atoms, (2) a Cl to C4 alkyl group having a phenyl substituent (the phenyl may further have a halogen substituent), or (3) a phenyl group which may have one or more substituents selected from halogen a~oms, cyano and benzyloxy, and R2 is a halogen atom or -CH2-Rl~ wherein Rl is as defined above.

Bleomycin is a family of carcinos-tatic antib.iotic substances discovered in 1966 by Umezawa, one of the present inventors, and collaborators [Vmezawa et al., Journal of Antibiotics, 19A, p. 200 (196~)]. It is produced by Streptomyces verticillus, an actinomyceke, and 1 is a basic water-soluble glycopeptide capable of readily chelating one atom of divalent copper. In ordinary culture, 16 members of the blemocin family are produced and are each isolated [e.g., Umezawa et al., Journal of Antibiotics, l9A, p. 210 (1966)]. Of these bleomycins, Al, A2, A5, B2 and demethyl-A2 are currently being widely used in the form o~ copper-free mixture (herein-after referred to as bleomycin complex) in clinical fields of cancer therapy; more particularly, they are successfully used in the treatment of squamous ce~l carcinoma as major target, skin cancer, head and neck cancer, lung cancer, and malignant lymphoma.
Various belomycins are also disclosed in U.S.
Patent 3,922,262 and U.S. Patent Re 30,451.
These bleomycins are generally produced in copper-containing form in the ordinary fermentation.
The copper-free form is obtained by removing the copper from the copper-containing form. The term "bleomycin", as herein employed, includes both the copper-containing form and the copper-free form, unless specifically indicated.
The above bleomycins are represented by the general formula (II) Cl ONH 2 Nl H 2 ,________~_________________________________~
~ ~ O
CIH3 H!1 ' ~SJ~3 CH3 N C CH O CH CH2 ~
CH N \CH3 HO \CH3 \CH2 S H
CH H
H O ~ N
/ C o C\ H~l ` N~ H
¦ HOCH2 OH H H
H~ l C~ ,/f H , (II) OH / f o- ~CH20H

C I C

0'~ ~ NH2 .

1 wherein R is a terminal amine residue of bleomycin; and the chelated copper is omitted in the case of copper-containing form.
3 b/eo~ ycin5 The above LlG~myci..G exhibit excellent effects, however, cause side effects such as plumonary toxicity depending upon the way they are applied. Accordingly, their application is now restricted. Hence, development ~7~

1 of bleomycins causing less side effects has been desired.
In an effort to satisfy this desire, based on an extensive study, the present inventors had previously invented the (ami~o)N-subs~ituted bleomycins represented by the general formula [I] mentioned above.
As a result of further studies, the present inventors have ound that the (amido)N-substituted bleo-mycins represented by the general formula [I] can be produced in a large amount by reductively condensing an (amido)N-substituted derivative of 3-[N-methyl-N-(3-aminopropyl)amino]propylaminobleomycin represented by the general formula [III]

CH2 ~CH/ `CH2/ \CO-NH-X CIH3 IM N ~ Co-NH-(cH2)3-N-(cH2)3 NH2 wherein BM and X each have the same definition as above, with an aldehyde represented by the general formula [IV]

RlCHO [IV]

wherein R1 has the same definition as above. Based on this finindg, the present invention has been completed.
The residue of bleomycin skelton represented by 1 BM in the general formula [I] of the present invention is the portion enclosed by dotted lines in the general formula [II~ of bleomycins and includes both the copper-containing form and the copper-free form.
Of the aldehydes of the general formula [IV]
used in the present invention, cycloalkyl aldehydes include, for example, cycloalkyl aldehydes of 5 to 13 carbon atoms and, specifically, cyclopentyl aldehyde, cyclooctyl aldehyde, cyclododecanoyl aldehyde, cyclo-undecanoyl aldehyde, etc. Phenyl-substituted saturated aliphatic aldehydes whose phenyl substituent per se may have substituents include saturated aliphatic aldehydes of l to 5 carbon atoms having a phenyl substituent such as phenylacetaldehyde, phenylethylaldehyde and halogen-substituted phenylacetaldehydes (e.g. chlorophenyl-~3 acetaldehyde). Benzaldehydes include, for example, h a lo b e rl ~ a ld ~h yole s benzaldehyde, halogcnobc~aldchyd06 le.g. p-chlorobenzalde-hyde, m-bromobenzald~hyde), cyanobenzaldehyde, and benzyloxybenzaldehydes (e.g. m,p-dibenzyloxybenzaldehyde).
The compound represented by the general formula [III~ as a starting material of the present invention can be produced by hydrolyzing 3-[N-methyl-N-(3-amino~
propyl)amino]propylaminobleomycin (hereinafter referred to as bleomycin APMP) by the use of a bleomycin-inactivating enzyme (hereinafter referred to as in-activating enzyme; Umezawa et al., Journal of Anti-biotics, vol. 27, p. 419, 1974) to obtain 3-[N-methyl-N-(3-aminopropyl)amino]propylaminodeamidobleomycin ~7~

l (hereinafter referred to as deamidobleomycin ~PMP) repre-sented by the formula CONH2 ll H2 C~I2 ~ ~ NH~ CEI / ~COOH

N 11_ CONH--(CH2) 3--N--(CH2) 3 NH2 BM l~ ~ S'~`H

and then condensing the deamidobleomycin APMP with an amine represented by the general formula [V]

NH2 X [V]

wherein X is same as in the general formula [I].
As X, there can be mentioned, for example, alkyl groups of 6 to lO carbon atoms and alkyl groups of 2 to 6 carbon atoms having a primary amino substituent.
Particularly preferred are n-octyl, phenylethylamino-propyl, 3-[N-methyl-N-[3-(2-~p-chlorophenyl)ethyl)-aminopropyl]amino]propyl and di-n-butylaminopropyl.
The compound of the general formula [III] can be produced, for example, as follows.
Bovine liver is homogenized with a phosphate buffer solution. The resultant homogenate is sub~ected to centrifugation at 8,000 rpm. The supernatant is dialyzed against a phosphate buffer solution to obtain 7~

1 a crude enzyme solution. To the crude enzyme solution is added the copper-rree form of bleomycin APMP dissolved in a phosphate buffer solution. The mixture is subjected to reaction at 37C for 5 to 48 hours. The reaction mixture is freed of proteins by an appropriate method.
[For example, trichloroacetic acid (hereinafter referred to as TCA) is added to a concentration of 5~, to pre-cipitate proteins. The precipitate is removed by centri-fugation. The precipitate is washed three times with 5~ ~rCA. The supernatant and all washings are combined.]
After neutralization, copper acetate is added to the protein-free reaction mixture so that the copper acetate is in excess relative to bleomycin, whereby the intended product is converted into a copper-chelate. For desalt-ing, the copper-chelate solution is poured into a column packed with an adsorptive resin, Diaion HP-40 with distilled water, to effect the adsoption of the intended product. After washing off the salt with distilled water, elution is conducted with a mixture of 1/50 N aqueous hydrochloric acid solution and methanol (1O4 v~v) to collect fractions having an absorption maximum at a wave-length of around 290 m~. After neutralization with an anion exchange resin, Dowex 44 lOH type, manufactured by Dow Chemical Co.), the combined fraction is concent-rated under reduced pressure and lyophilized.
The powder obtained above is dissolved indistilled water. The resulting solution is poured into a column packed with CM-Sephadex C-25 (Na type, 1 manufactured by Pharmacia Fine Chemicals Co.) previously equilibrated with a 1/20 M acetic acid-sodium acetate buffer solution of pH 4.5, to effect adsorption.
Elution is conducted by the linear concentration gradient method in which sodium chloride is continuously added to the above mentioned buffer solution to gradually increase the sodium concentration to 1.0 M, to collect the bluish-violet fractions ~f the intended product. The combined fraction is freed of the salt according to the same desalting method by Diaion HP-40 as employed previously and then lyophilized, whereby the copper-containing form o~ deamidobleomycin APMP is obtained as a blue amorphous powder.
The thus obtained deamidobleomycin APMP is then condensed with an amine represented by the general formula [V]
NH2 X [V]

by the use of a known method for forming an acid amide linkage such as the method described in U.S. Patent 4,195,018 and, if necessary, further subjected to copper removal, to obtain a reaction mixture containing the compound of the general formllla [III].
In the above condensation reaction, there is used a condensing agent such as cyclohe~ylcarbodiimide, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide, l-cyclo-hexyl-3-(2-morpholinoethyl)carbodiimide, diisopropyl-carbodiimide, diphenylphosphor azidate (DPPA), ~ 6~

1 die~hylphosphoro cyanidate (DEPC), 6-chloro-1-p-chloro-benzenesulfonyloxybenztriazole ~CCBT) or the like.
Together with the above condensing agent, condensaticn additives such as p-nitrophenol, o,p-dinitrophenol, pentachlorophenol, 2,4,5-trichlorophenol, pentafluorophenol, N-hydroxys~ccinimide, N-hydroxy-5-norbornene-2,3-dicarboxyimide, l-hydroxybenztriazole or the like can be used.
The condensation is carried out in a solvent such as water, dimethylformaldehyde, acetonitrile or a mixture thereof. The reaction temperature is -20 to 50C and preferably -10 to 30C. The reaction time is not particularly restricted but 1 to 70 hours is sufficient.
The compound of the general formula [III] is isolated from the reaction mixture as follows. To the above obtained reaction mixture is added an organic solvent such as acetone, ether ox the like to precipitate the intended product. Then, the precipitate is dissolved in distilled water. The solution is adjusted to pH 6 and, for desalting, poured into a column packed with an adsorp-tive resin such as Amberlite XAD-2 (manufactured by Rohm and ~aas Co.) with distilled water, to effect the adsorp-tion of the intended product. After the salt has been washed off with distilled water, elution is conducted with an acidic, water-containing methanol, for example, a mixture of 1/50 N aqueous hydrochloric acid solution and methanol (1:4 v/v), to collect fractions showing an absorption maximum at a wave length of around 290 m~.

1 After neutralization with an anion exchange resin, Dowex 44 ~OH type, manufactuxed by ~ow Chemical Co.), the combined frac-tion is condensed and lyophilized to obtain a crude powder of the compound of the general formula LIII].
There is a case wherein the above desalting step can be omitted. In this case, the above precipitate is dissolved in distilled water and subjected to the fol-lowing step.
The above obtained powder is dissolved in distilled water. The solution is poured into a column packed with CM-Sephadex C-25 (Na type, manufactured by Pharmacia Fine Chemicals Co~) previously equilibrated with 1/20 M acetic acid-sodium acetate buffer solution of pH 4.5, to effect adsorption. Elution is conducted by the linear concentration gradient method in which sodium chloride is continuously added to the above buffer solution to gradually increase the sodium concentration to 1.0 M~ Because the unreacted materials and the by-products have a property of being eluted earlier, they can be separated and removed by the use of an ultraviolet absorption monitor. If fractions of the intended product are found to contain impurities~ the above chroma~ography is repeated for complete removal of the impurities.
The above chromatography may be replaced by a chromatography using an adsorptive resin such as Amberlite XAD-Z. In this chromatography, an aqueous solution of crude product is poured into a column packed with a resin, for example, with 1% aqueous ammonium acetate solution, 7~ 3 1 to effect adsorption of the intended product. Elution is conducted by a linear concentration gradient method in which methanol is continuously added to the above buffer solution to gradually increase the methanol concentration. Because it occurs at this time that the unreacted materials are eluted earlier and the by-products later, they can be separated and removed by the use of an ultraviolet absorption monitor. If fractions of the intended product are found to contain impurities, the above chromatography is repeated for complete removal of the impurities.
The above two purification procedures may be used individually or in combination.
The fraction of the intended product thus obtained is freed of the salt by the desalting method using an adsorptive resin such as Amberlite XAD-2 as described above and then lyophilized to obtain the copper containing form of the compound of the general formula [III] as a blue amorphous powder.
This copper-containing form can be convereted into a copper-free form by a known method such as the method using EDTA described in U.S. Patent 3,929,993.
An example of the conversion into a copper-free form is described below. The copper-containing form is dissolved in distilled water. The solution is poured into a column packed with Amberlite XAD-2 with distilled water to effect adsorption. The resin in the column is washed with an aqueous solution containing sodium chloride ~.%~7~
l and 5% of disodium salt of ethylenediaminetetracetic acid (hereinafter referred to as EDTA.2Na), whereby copper ion is carried away by EDTA.2Na and the copper-free form remains on the resin. The resin is washed with sodium chloride to remove EDTA.2Na and is further washed with distilled water. Subsequently, elution is conducted by the use of an acidic, water-containing methanol, for example, a mixture of l/50 N aqueous hydrochloric acid solution and methanol (1:4 v/v) to collect fractions showing an absorption maximum at a wave length of around 290 m~. The combined fraction is adjusted to pH 6.0 by the use of Dowex 44 (OH type, manufactured by Dow Chemical Co.) and then concentrated under reduced pressure and lyophilized to obtain a hydrochloride o the copper-free form of the compound represented by the general formula [III] as a white amorphous powder.
If, in the above, an aqueous sulfuric acid is used in place of an aqueous hydrochloric acid solution, a sulfate is obtained. Thus, by appropriately selecting the kind of an acid in the above elution step, a salt with any desired acid can be obtained.
In order to carry out the present invention, the compound of the general formula [III] thus obtained is reductively condensed with an aldehyde represented by the general formula [IV].
As the reducing agent used in this condensa-tion, there can be mentioned boron hydride compounds ~e.g.
sodium cyanoborohydride, sodium borohydride, lithium 6~6~

1 borohydride, potassium borohydride), bis(triphenylphos-phine)tetrahydroborato copper, etc. Catalytic reduction may be conducted by the use of a catalyst such as palladium carbon. Use of 2 moles or less and preferably 1 to 1.5 moles of the aldehyde represented by the general formula [IV] relative to 1 mole of the compound of the general formula [III] results in formation of compounds of the general formula [I] wherein R2 is substantially hydrogen. Use of 3 moles or more results in formation of a compound of the general formula [I] wherein R2 ls -CH2 ~1 The desirable addition amount of the aldehyde is 1.2 moles for obtaining a comp~und of the general formula [I] wherein R2 is hydrogen and 5 to 15 moles, preferably about 10 moles for obtaining a compound of the general formula [I] wherein R2 is -CH2-Rl. When the aldehyde of the general formula [IV] happens to be a compound difficultly soluble ln methanol such as m,p-dibenzyloxybenzaldehyde, its amount may be reduced on the condition that the reaction time be prolonged.
There is no particular restriction to the solvent used in the above condensation reaction, unless the solvent takes part in the reaction. As the solvent, there are used polar solvents such as water, methanol, ethanol, dimethylformamide, dimethylsulfoxide, tetra-hydrofuran and acetonitrile, mixtures thereof and so forth.
The reaction temperature is -5 to 80C and ordinarily 0 to 50C. More particularly, it is desirable that 0 to 25C be used for obtaining a compound wherein R2 1 is hydrogen and 35 to 50C for obtaining a compound wherein R2 is -CH2-Rl by the use of an aldehyde of large steric hindrance or of low solubility. An appropriate reaction time is 3 to 70 hours. When an aldehyde of large steric hindrance or of low solubility is used, a longer reaction time ~e.g. 150 hours) is more effective.
From the reaction mixture thus obtained, the compound of the general formula [I] is isolated as follows.
When a boron hydride compound has been used, the reaction mixture is adjusted to pH 1 with hydrochloric acid and stirred at room tempe~rature for 5 to 10 minutes to decompose the reducing agent in excess. Thereafter, the reaction mixture is neutralized, freed of methanol by distillation under reduced pressure, freed of aldehyde in excess and ketone by ether or butanol extraction, and subjected to the following desalting step. That is, the reaction mixture thus obtained is poured into a column packed with an adsorptive resin such as Amberlite XAD-2 (manufactured by Rohm and Haas Co.) with distilled water to effect the adsorption of the intended product.
Afier the salt has been washed off with distilled water, elution is conducted with an acidic, water-containing methanol, for example, a mixture of 1/50 N aqueous hydro-chloric acid solution and methanol (1:4 v/v~ to collect fractions of a blue bleomycin derivative. The combined fraction is, if necessary, neutrali~ed with an anion exchange resin, Dowex 44 (OH type, manufactured by Dow Chemical Co.), concentrated under reduced pressure and 1 lyophilized to obtain a blue crude powder of the intended product.
In order to make a further purification, the following step is co~ducted.
The above powder is dissolved in distilled water. The solution is poured into a column packed with CM-Sephadex C-25 INa type, manufactured by Pharmacia Fine Chemicals Co.) previously equlibrated with a 1/20 M
acetic acid-sodium acetate buffer solution of p~ 4.5, to effect adsorption. Elution is conducted by a linear concentration gradient method in which sodium chloride is continuously added to the above buffer solution to gradually increase the sodium concentration to 1.0 M.
If fractions of the intended product are found to contain impurities, the above chromatography may be followed by a chromatography using an adsorptive resin such as Amberlite XAD-2. The combined fraction is poured into a column packed with a resin, for example, with 4% aqueous ammonium acetat~ solution to effect the adsorption of the intended product. Elution is conducted by a linear concentration gradient method in which methanol is continuously added to the above buffer solution to gradually increase the methanol concentration. In this case, the unreacted materials are eluted earliest, the compound in which R2 is hydrogen is eluted next and the compound in which Rl and R2 are same is eluted last.
The separation of these fractions is effected satis-factorily by the use of an ultraviolet absorption monitor.

1 If fraction of the intended product are ~ound to contain impurities, the above chromatography is repeated for complete removal of the impurities.
In this step, the column of CM-Sephadex C~25, can be replaced by the column of CM-Sepharose CL-6B
(manufactured by Pharmacia Fine Chemicals Co.) or CM
Toyopearl 650 (manufactured by TOYO SODA MFG CO.; or the like packed and washed with methanol. In this case, the desalting step after reaction may be omitted. The -~eaction mixture is directly poured into a column to effect the adsorption of the intended product. After the reagents, etc. have been washed off with methanol, elution is conducted with an aqueous methanol containing 0.1 M sodium chloride. The combined fraction is subjected to a chromatography using Amberlite XAD-2.
The combined fraction of the intended product thus obtained is desalted by the desalting method using Diaion HP-40 as employed previously and then lyophilized to obtain the copper-containing form of an (amido)N-substituted bleomycin of the general formula [I] as ablue amorphous powder.
The copper-containing form of an (amido)N-substituted belomycin thus obtained can be subjected to copper removal by the above described method to obtain a desired salt of a copper-free form as a white amorphous powder.
The typical examples of the compound of the present invention are shown in Table 1 and thelr physico-chemical properties in Table 2.

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1 The biological properties as determined on the typical compounds of the present invention are described below.
1. Antimicrobial activities against Mycobacterium Smegmatis ATCC 607 and 3acillus subtilis~
The antimicrobial activity was tested against the captioned test organisms by the method of agar plate-cylinder. The activity was recorded on the assumption that the activity of standard sample of bleomycin A2 (copper-free form) is 1,000 mcg poteney/mg.
2. Growth inhibitory activity against cultured HeLa S3 cells.
HeLa S3 cells were inoculated into a medium (MEM with 10% Calf serum) placed in a plastic petri dish.
Two days thereafter, a bleomycin was added to the dish.
After 3 days of breedlng, the number of cells were counted. The percentage growth inhibition was calculated using the following equation:
Percentage inhibition ~%) = 100 x (B-A)/(B-C) where A is the final number of cells after 3 days from the addition of test sample, B is the final r.umber of cells in the control without addition of the test sample, and C is the number of cells at the time of addition of the test sample. ID50 (concentration of 50~ inhibition) was estimated from the graph prepared by plotting the concentration of test sample against the percentage inhibition.
The results obtained in 1 and 2 are shown in Table 3.

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,Q ~ a a - . .. - - -~ Z ~ o o c~ - -1 3. Pulmonary toxicity (pulmonary fibrosis) in mice.
ICR strain mice Imale, 15 weeks old) in groups of 9 members were used. Each test pxeparation was admini-stered by intraperitoneal injection once a day for 10 consecutive days at a dose rate of 5 mg/kg. After comple-tion of the administration, the mice were bred for 5 weeks under observation, then slaughtered and autopsïed to examine the incidence and grade of the pulmonary fibrosis.
The evaluation was made by comparing the number of admini stered mice suffering from pulmonary fibrosis and the grade of the disease. The results are shown in Table 4.
The grade was numerically rated as follows:
Number of points 0: No fibrosis.
1: Accumulation of exudate in alveolus and fibrosis-like change in alveolar septum.
2: Fibrosis in several areas.
4: Scattered fibrosis.
6: Fibrosis in more than two-thirds of the total area.
The "ratio" in Table 4 was calculated by comparison with "Bleomycin Complex".

Table 4 _.
Incidence Grade Compound Number of mice Total score of No. with pulmonary pulmonary fibrosis/ Ratio fibrosis total number of t%) samples (%) _ .
1 . 2/9 (20) 2/27 (7) 0.07 3 0~1 (0) 0/3 ~0) 0 6 0/9 (0) 0/27 (o) 0 7 0/9 (0) 0~27 (0) 0 8 0/9 (0) 0/27 (o) 0 9 0/9 (0) 0/27 (0) 0 0/9 (0) 0/27 (o) 0 . _ _ _ .

1 As is obvious from the above results, the compounds of the present invention have a high growth-inhibitory activity against cultured HeLa S3 c~lls as well as a distinguished antimicrobial activity, and are very low in pulmonary toxicity, positively suggesting their usefulness in clinical fields.
The present invention will be explained in more detail below by way of Examples, however r is in no way restricted to them.

Examples Step A
Eight hundred grams of fresh bovine liver was homogenized with 1.6 Q of a 0.05 M phosphate buffer solution l of pH 7.2. The homogenate was subjected to centrifugation at 8,000 rpm for 30 minutes. The supernatant was dialyzed against a 0.05 M phosphate buffer solution by using cellulose membrane to obtain a crude enzyme solution.
To 32.7 g of bleomycin APMP was added 1.5 Q of the crude enzyme solution. They were subjected to reaction at 37C
for 24 hours. To the reaction mixture was added 150 ml of 55~ trichloroacetic acid (hereinafter referred to as TCA) to precipitate proteins. The precipitate was removed by centrifugation and washed three times with 5% TCA.
The supernatant and all washings were combined. After neutralization with 4 M NaOH, the combined solution was mixed with 5 g of copper acetate (1.2 equivalents relative to bleomycin) to convert the intended product into a copper-chelate. For desalting, the copper-chelate was poured into a column of 7 liter in volume packed with an ad~orptive resin, Diaion HP 4- (manufactured by Mitsubishi Chemical Co.) with water to effect the adsorption of the intended product. The salt was washed off with 14 liters of distilled water. Elution was conducted with a mixture of 1/50 N aqueous hydrochloric acid solution and methanol (1:4 v/v) to collect fractions showing an absorption maximum at a wave length around 290 m~. The combined fraction was neutralized with Dowex 44 (OH type, manu-factured by Dow Chemical Co.) and then concentrated underreduced pressure.
The concentrate obtained was poured into a column of 3 liter in volume packed with CM Sepadex C-25 7~

1 (Na type, manufactured by Pharmacia Fine ~hemicals Co.) previously equilibrated with 1/20 M acetic acid-sodium acetate buffer solution of pH 4O5~ to effect adsorption.
Elution was conducted by a linear concentration gradient method in which sodium chloride was continuously added to the above buffer solution to gradually increase the sodium concentration to 1.0 M. Blue fractions which were eluted at a sodium concentration of around 0.6 M were collected and combined. The combined fraction was sub-jected to desalting by the desalting method by DiaionHP 40 as previously employed and then lyophilized to obtain 21.7 g of the copper-containing form of deamido-bleomycin APMP as a blue amorphous powder. The yield was 66%.
I5 The blue powder showed ultraviolet absorption maxima (E 1%/1 cm) at 241 m~ (127) and 292 m~ (104), as measured in distilled water.
The powder showed infrared absorption maxima (in wave number, cm 1) as measured by the KBr-tablet 20 method at 3350, 1725, 1650, 1560, 1460, 1380, 1200, 1140, 1100, 1060, 1020, 990, 880 and 770.
Other physicochemical properties are shown in Table 5.

Step B
In 100 ml of dimethylformamide were dissolved 5 g of the copper-containing form of deamidobleomycin APMP
obtained in Step A as well as 8.3 g (10 equivalents l relative to bleomycin in moles) of 3-[tS~ phenylethyl-amino]propylamine hydrochloride (hereinafter referred to as PEP amine hydrochloride). To the resulting solution being cooled at 0C with stirring were added 13.4 g (30 equivalents relative to bleomycin in mole) of 1-hydroxybenztriazole and 3.63 ml of N-methylmorpholine and subsequently 6.8 g (lO equivalents relati~e to bleomycin in moles) of dicyclohexylcarbodiimide. The mixture was subjected to reaction at room temperature ~or 6 hours with stirring.
Acetone (10 times volume of the reaction mixture) was added to the reaction mixture to precipitate the intended product. After thorough washing with acetone, the precipitate was dissolved in distilled water. The solution was poured into a column, 500 ml in volume, packed with CM Sephadex C-25 (Nat type, manufactured by Pharmacia Fine Chemicals Co.) previously e~uilibrated with l/20 M acetic acid-sodium acetate buffer solution of pH

4.5, to effect adsorption. Elution was conducted by a linear concentration gradient method in which s~odium chloride was continuously added to the above buffer solution to gradually increase the sodium concentration to l.0 M. Blue fractions which were eluted at a sodium concentration of around 0.7 M were collected and combined. The combined fraction was subjected to desalt-ing by the desalting method by Diaion HP-40 (500 ml used) as employed previously and then lyophilized to obtain 3.3 g of the copper-containing form of 3-(~-methylaminopropyl~-~:7~

1 aminopropylamino-(amido)N-[3-((S) l'-phenylethylamino)-propyl]-bleomycin (hereinafter referred to as dAPMP-PEP) as a blue amorphous powder. The yield was 66%. This product showed ultraviolet absorption maximum (E 1%/1 cm) as measured in distilled water, at 241 m~ (108) and 292 m~
(87). The product showed infrared absorption maxima (in wave number, cm 1) as measured by the Ksr-t~blet method, at 3350, 1725, 1645, 1560, 1460, 1380, 1250, 1140, 1100, 1060, 1015l 990, 880, 765 and 700.
Other physicochemical properties are shown in Table 5.
In the above procedure of Step B, PEP amine hydrochloride and N-methylmorpholine were replaced by n-octylamine, whereby there was obtained 3-(N-methyl-aminopropyl)aminopropylamino-(amido)N-(n-octyl)-bleomycin (hereinafter referred, to as dAPMP-OCT).
This product showed ultraviolet absorption maxima (E 1~/1 cm) as measured in distilled water, at 241 m~ (118) and 292 m~ (95).
The product showed infrared absorption maxima lin wave number cm 1) as measured by the KBr-tablet method, at 3350, 2940, 1720, 1640, 1555, 1460, 1370, 1250, 1115, 1100, 1060, 1010, 995, 930, 880 -~nd 760.
Other physicochemical properties are shown in Table 5.

Table 5 . . ~
Thin layer * Electro-*
compound chromatography 1 phoresis 2 of Cu-contalning of Cu-contain-form, Rf ing form, ~m (Rm of alanine=
_ _ _ . _ Deamidobleomycin APMP 0.28 ¦ 0.93 dAPMP-PEP 0.23 1.17 dAPMP-OCT 0.37 1.00 *1 Silica Gel 60F 254 Silanized (Merck Co.);
measured in methanol-6% ammonium acetate solution mixture (60-40 v/v) *2 Avicel SF ~FMC Co.); ormic acid-acetic acid~
water ~27:75:900 v/v); 800 V, 15 minutes 1 In the procedure of Step B, when PEP amine hydrochloride is replaced by 3-(di-n-butylamino)propyl-amine or N-{3-[N-methyl-N- E 3-(2-(p-chloroph~nyl~ethyl-amino)propyl]amino}propylamine, there are obtained corresponding deamidobleomycin APMP derivatives.

Step C
One gram of the copper-containing form of dAPMP-PEP obtained in Step B was dissolved in 100 ml of methanol. Thereto was added 700 mg (about 4 equivalents relative to bleomycin) of m,p dibenzyloxybenzaldehyde.
Then, there was added 46 mg ~about 1.3 equivalents relative to bleomycin) of cyanoborohydride. The mixture was s~jectad to reaction at 40C for 48 hours. The reaction Q~

1 mixture was poured into a column of 100 ml capacity packed with CM-Toyopearl 650 (Na type, manufactured by TOYO SODA MFG) previously washed with methanol, to effect the adsorption of the intended product. The column was washed with 200 ml of methanol. Elution was conducted by a linear concentration gradient method in which 500 ml of a methanol-water solution (7:3) containing 0.2 M sodium chloride was continuously added to 500 ml of a methanol-water solution (7:3). 130 ml of Blue fractions which were eluted at a salt concentration of about 0.12 M was poured into a column of 160 ml in volume packed with Amberlite XAD-2 to effect adsorption. Elution was conducted by a linear concentration gradient method in which 1 liter of methanol was continuously added to 1 liter of 0.05 M acetic acid-sodium acetate buffer solution of pH 4.5 go increase the methanol concentration linearly. Blue fractions which were eluted at a methanol concentration of around 90% were poured into a column of 100 ml capacity packed with Amberlite XAD-2 to effect the adsorption of the intended product. After washing with 200 ml of water, elution was conducted with a mixture of 1150 N hydrochloric acid solution and methanol (1:4 v/v) to collect blue fractions. The fractions were combined, neutralized with Dowex 44 (OH type, manufactured by Dow Chemical Co.) and concentrated to dryness to obtain 670 mg of a blue powder. This powder was dissolved in 50 ml of water. The solution was poured into a column, 100 ml in volume, packed with Amberlite XAD-2 to effect ~ ~7~i~0~

1 adsorption. The column was washed with 300 ml of 5%
aqueous EDTA-2NA solution, 100 ml of 2% aqueous sodium chloride solution and 200 ml of water in this order.
Then, elution was conducted with a mixture o~ 1/50 N
aqueous hydrochloric acid solution and methanol (1:4 v/v) to collect fractions showing an absorption maximum at a wave length of around 290 nm. They were adjusted to pH
6.0 by the use of Dowex 44 (OH type) and lyophili.zed to obtain 640 mg of the copper-free form hydrochloride of dDD-PEP as a white amorphous powder. The yield was 64~.
This product showed an ultraviolet absorption maximum (E 1%/1 cm) as measured in distilled water, at 285 nm (74.4). The product showed infrared absoxption maxima (in wave number, cm 1) as measured by the KBr-tablet method, at 3400, 2900, 1710, 1650, 1545, 1505, 1450, 1380, 1260, 1135, 1060, 1010, 800, 730 and 690.
Other physicochemical properties are shown in Table 1.
In Step C, when dAPMP-OCT was used as a material bleomycin and a procedure similar to the above was adopted, there was obtained dDD-OCT.
In Step C, when (1) there were used, as a material deamidobleomycin APMP derivatives obtained in Step B and corresponding to respective intended products, (2) there were used, as an aldehyde, cyclooctyl aldehyde, p-chlorobenzaldehyde and p-cyanobenzaldehyde corresponding to respective intended products, (3) these aldehydes were ~7~

1 used in an amount of 1.2 moles relative to 1 mole of deamidobleomycin APMP derivative, ~4) and reaction was conducted according to the above procedure, intended products shown in Table 1 were obtained, respectively.

Claims

1. A process for producing an (amido) N-substituted bleomycin represented by the general formula wherein BM is a residue of bleomycin skelton, X is a C1 to C10 alkyl group, which may be substituted by a C1 to C4 alkyl-amino, di(C1 to C6 alkyl) amino or N-[(C1 to C4 alkyl) amino-C1 to C4 alkyl] N-C1 to C4 alkylamino group, wherein the alkyl substitute may have a phenyl substituent, which phenyl may be substituted with a halogen atom; R1 is (1) a cycloalkyl group having 6 to 10 carbon atoms, (2) a C1 to C4 alkyl group having a phenyl substituent (the phenyl may further have a halogen substituent), or (3) a phenyl group which may have one or more substituents selected form halogen atoms, cyano and benzyloxy, and R2 is a halogen atom or -CH2-R1, wherein R1 is as defined above which comprises allowing an (amido) N-substituted derivative of 3-[N-methyl-N-(3-aminopropyl), amino] propylaminobleomycin represented by the general formula wherein BM has the same definition as above and X is an alkyl group which may have substituents, to condense reductively with an aldehyde represented by the general formula wherein R1 has the same definition as above.

2. A process according to Claim 1, wherein the aldehyde is (1) a cycloalkylaldehyde of 5 to 13 carbon atoms, (2) a saturated aliphatic aldehyde of 1 to 5 carbon atoms having a phenyl or halogenophenyl substituent, or (3) non-substituted benzaldehyde or benzaldehyde having one or more substituents selected from the group consisting of halogen atoms, cyano and benzyloxy.

3. A process according to Claim 1, wherein the X
is (1) an alkyl group of 6 to 10 carbon atoms or (2) an amino(lower)alkyl group wherein the amino has one or two alkyl (1 to 5 carbon atoms) substituents and the alkyls on the amino may have a non-substituted loweralkylamino, phenyl(lower)alkylamino or halogenophenyl(lower)alkyl-amino substituent.

4. A process according to Claim 3, wherein the X is octyl, phenylethylaminopropyl, N-methyl-N-[3-di(chloro-phenylethyl)aminopropyl]aminopropyl or dibutylaminopropyl.

5. A process according to Claim 1, wherein the X is octyl, phenylethylaminopropyl, N-methyl-N-[3-di-(chlorophenyl)aminopropyl]aminopropyl or dibutylamino-propyl, the aldehyde is cyclooctylaldehyde, halogeno-phenylacetaldehyde or dibenzyloxybenzaldehyde, and the R1 is cyclooctylmethyl, halogenophenylethyl or dibenzyloxy-phenylmethyl.

6. A process for producing 3-{N-methyl-N-[3-(2-(p-chlorophenyl)ethylamino)propyl]amino}propylamino-(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycin, 3-{N-methyl-N-[3-(p-cyanobenzylamino)propylamino}-propylamino-(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycin or 3-{N-methyl-N-[3-(cyclooctylmethylamino)-propyl]amino}propylamino-(amido)N-[3-((S)-1'-phenylethyl-amino)propyl]-bleomycin, which comprises allowing 3-(N-methylaminopropylamino)propyl-(amido)N-[3-((S)-1'-phenylethylamino)propyl]-bleomycin to condense reductively with p-chlorobenzaldehyde, p-cyanobenzaldehyde or cyclo-octylaldehyde, respectively.

7. A process for producing 3-{N-methyl-N-[3-(cyclooctylmethylamino)propyl]amino}propylamino-(amido)-N-[octyl]-bleomycin, 3-{N-methyl-N-[3-(2-(p-chloro-phenyl)ethylamino)propyl]amino}propylamino-(amido)N-[octyl]-bleomycin, 3-{N-methyl-N-[3-(p-cyanobenzylamino)-propyl]amino}propylamino-(amido)N-[octyl]-bleomycin or 3-{N-methyl-N-[3-bis(m,p-dibenzyloxybenzyl)aminopropyl]-amino}-propylamino-(amido)N-[n-octyl]-bleomycin, which comprises allowing 3-(N-methylaminopropyl)-aminopropyl-amino-(amido)N-(n-octyl)-bleomycin to condense reductively with cyclooctylaldehyde, p-chlorophenylacetaldehyde, p-cyanophenylbenzaldehyde or m,p-dibenzyloxybenzaldehyde, respectively.

8. A process for producing 3-{N methyl-N-[3-(2-(p-chlorophenyl)ethylamino)propyl]amino}-propylamino-(amido)N-[3-(di-n-butylamino)propyl]-bleomycin or 3-{N-methyl-N-[3-(p-cyanobenzylamino)propyl]amino}-propylamino-(amido)N-[3-(di-n-butylamino)propyl]-bleomycin which comprises allowing 3-(N-methylaminopropyl)aminopropyl-amino-(amido)N-[3-(di-n-butylamino)propyl]-bleomycin to condense reductively with p-chlorophenylacetaldehyde or p-cyanobenzaldehyde, respectively.

9. A process for producing 3-{N-methyl-N-[3-(2-(p-chlorophenyl)ethylamino)propyl]amino}propylamino-(amido)N-{3-[N-methyl-N [3-(2-(p-chlorophenyl)ethylamino)-propyl]amino]propyl}-bleomycin, which comprises allowing 3-(N-methylaminopropyl)aminopropylamino-(amido)N-{3-[N methyl-N-[3-(2-(p-chlorophenyl)ethylamino)propyl]-amino]propyl}-bleomycin to condense reductively with p-chlorophenylacetaldehyde.

10. A process for producing 3-[-N-methyl-N-[3-bis(m,p,-dibenzyloxybenzyl)aminopropyl]-amino}-propylamino-(amido)N-[n-octyl]-bieomycin which comprises allowing 3-(N-methylamino-propyl)-aminopropylamino-(amido)N-(n-octyl)bleomycin to condense yreductively with m,p-dibenzyloxybenzaldehyde.