CS205127B2 - Process for preparing 3-/s/-1'-phenylethylamino-propylaminobleomycine - Google Patents

Description

BACKGROUND OF THE INVENTION The present invention relates to a process for the preparation of the novel 3 - [(SJ-lenylethylamino) propylaminobleomycin of formula I.

Ό-Χ X * F 2 YEAH O • \ ' ^ž X

and non-toxic salts thereof.

Bleomycins are carclostatic antibiotics discovered by Umezawa, one of the authors of the present invention, and his co-workers [Journal of Antibiotics, 19A, 200 (1966)]. They are water-soluble basic glycopeptides produced by the strain of actinomycetes Streptomyces verticilus, which readily form chelates with a single divalent copper atom. In normal culture, 16 components of bleomycin are produced, all of which have been isolated [see, for example, Umezawa et al., Journal of Antibiotics, 19A, 210 (1966)]. Of the known bleomycins, mixtures of copper free of bleomycins A 1, Az, As, B2 and demethyl-Az are commonly used to treat cancer in clinical practice (the term "bleomycin complex" is used hereinafter). The bleomycin complex has proved to be particularly useful in the treatment of epidermal carcinomas of the skin, carcinomas of the head and neck, cervical cancer, lung cancer and malignant lymphoma.

Concerning side effects, it has been reported. that patients undergo lung fibrosis and other side effects. Of the bleomycin-induced side effects, lung fibrosis is the most feared. It is easy to imagine that the carclnostatic efficacy of bleomycins would be even more evident in clinical application if the above-mentioned side effect were more or less suppressed.

Against this background, the inventors have been involved in the synthesis of various bleomycins for several years and have investigated in animal tests their carcinostatic activity and toxicity, including the effect on induction of lung fibrosis as the main test. As a result of their study, they have discovered 3 - [(S) -1'-phenylethylamino] propylaminobleomycin (in the following description this new bleomycin is designated by the symbol "NK 631", which includes both a copper containing form and a copper free form). novel bleomycin, obtained by reacting a reactive derivative of the carboxyl group of bleomycin acid with N - [(S) -1'-phenylethyl] -1,3-diaminopropane, in which the side effect causing lung fibrosis is significantly suppressed, its carcinostatic efficacy not being commercial bleomycin complex and other known bleomycins. Based on this discovery, the present invention has been realized. The purpose of the invention is to provide a process for the preparation of novel bleomycins of formula I, 3 - [(S) -1'-phen bleomycins of formula I, 3 - [(S) -1'-phenylethylamino] propylaminobleomycin, and non-toxic salts thereof. Further details of the invention will be apparent from the following description.

The excellent biological activity of the novel bleomycin of formula I results from the experimental tests below.

The biological activity of the NK 631 bleomycin was evaluated by comparing it to that of 3 - [(R, S) -1'-phenylethylamino] propylaminobleomycin (copper free form) monosulfate (hereinafter "RS-" for this bleomycin) form ”) and the efficacy of the bleomycin complex in the four following tests:

lj effect on induction of lung fibrosis

2) antlneoplastic activity

3) antimicrobial activity

4) toxicity.

For comparison, the RS form of bleomycins used was obtained by the fermentation method described in U.S. Patent No. 3,846,400.

1) Lung Toxicity in mice (effect on injection, once daily for ten fibrosis)

Twelve-year groups of 15-week-old male ICR mice were used for testing. Each pharmaceutical formulation tested was administered at a dose of 5 mg / kg. Doses were administered intraperitoneal on consecutive days. Mice were housed for an additional 5 weeks after drug administration. After the observation period, the mice were sacrificed and the extent of lung fibrosis was determined by autopsy. The incidence and degree of lung fibrosis induced in mice after administration of the novel bleomycin of Formula I, the commercial bleomycin complex, and the RS form of the antibiotic were compared. The results are shown in the table

AND.

Table I Incidence of fibrosis

Number of mice with pulmonary relative fibrosis (%) value

Grade of disease Total score - Relative evaluation of fibrosis grade lung value ^x : total number of samples

Monosulfate NK 631 (copper-free form) 4/12 (0.33) 0.36 6/36 (0,17) 0.25 RS form 7/12 (0,58) 0.63 12/36 (0.33) 0.49 Bleomycin complex 11/12 (0,92) 1.0 24/36 (0,67) 1.0

^x Assessment of lung fibrosis grade:

point - no fibrosis point - exudate accumulation in alveoli and fibrosis similar changes in alveolar septum points - fibrosis at several lung sites 4 points - scattered fibrosa of points - fibrosis on more than two-thirds of the entire lung area

As can be seen from the results, NK 631 monosulfate (copper free form) lung fibrosis is reduced to about 1/2 and 1/3, respectively, as compared to lung fibrosis caused by RS antibiotic and bleomycin complex. and about 1/2 or 1/4, respectively, as to the degree of the disease. This implies the benefit of NK 631 monosulfate for clinical use.

2. Antineoplastic activity - 1. Effect in vitro on HeLa S0 cell cultures

For each of the above bleomycin, the percent inhibition of cell growth after 72 hours of culture in the presence of test bleomycin was determined and the IDm value was calculated. NK 631 was found to have an ID 50 - 0.82 µg / ml, as opposed to a bleomycin complex having an IDm = 1.70 µg / ml, meaning that bleomycin NK 631 inhibits cell growth twice as strongly as bleomycin complex. The ID 50 for the RS form of the antibiotic is 0.80 µg / ml, which means that its inhibitory effect on cell growth is comparable to that of NK 631 monosulfate (copper-free forms).

2—2. Carcinostatic effect on mouse Ehrlich's carcinoma (solid tumor)

Male 6-week-old male ICRs were transplanted subcutaneously into the inguinal region of 2 x 10 ⁶ cells each. After 24 hours, each mouse was treated with the test preparation in the same manner as described above in Test 1. On the fifteenth day after subcutaneous transplantation, mouse tumors were removed and their weights compared to those of the untreated control group, and the percent inhibition was determined.

As shown in Table 2, the carcinostatic activity of NK 631 monosulfate (copper free form) is comparable to that of the RS form of the antibiotic and about 1.4 X higher than that of the bleomycin complex.

Table 2

Dose Percentage inhibition mg / kg X 10 monosulfate NK 631 RS form bleomycin (copper free) complex

2.7 84 78 67 0.9 68 60 55 0.3 39 51 49 0.1 24 51 24 0.03 19 Dec 13 21 0 0 0 0 ID50 0.35 0.31 0.49 mg / kg / day

2—3. Carcinostatic efficacy against ascitic hepatoma (ascitic type) in rats which was administered intraperitoneally once daily for ten consecutive days. Within 30 days after transplantation was Donryu rats were transplanto- monitored the weight of individual rats and intraperitoneally after 1 X 10® AH 66 platoon of dead and surviving animals. The result- cells. In 24 hours after transplantation was are shown in Table 3. the application of the tested preparation has begun, Table 3 Relative mean survival of animals (number of days) Dose NK 631 monosulfate RS form Bleomycin mg / kg X 10 (free of copper) antibiotics complex 3.12 296 298 241 1.56 269 257 257 0.78 216 196 145 0.39 163 149 116 0.19 116 121 110 0 100 ALIGN! 100 ALIGN! 100 ALIGN!

As shown in Table 3, the potency of NK 631 (copper free form) monosulfate against AH 66 ascitic hepatone is comparable to that of the RS form of the antibiotic and is higher than that of the bleomycin complex at all doses.

2—4. Inhibitory effect on epidermal carcinoma of the skin induced by 20-methylcholanthren

A solution of 20-methylcholanthrene (hereafter referred to as "20-MC") in acetone was applied twice weekly, for 18 weeks, locally to the trimmed back of mice of the ddy strain (male 10 weeks of age). Five weeks after the start of 20-MC administration, administration of the test preparation, which was administered intraperitoneally at a dose of 62.5 µg / mouse, was started twice a week for 15 weeks. During the first week after the administration of the test preparation, the skin area to which 20-MC was applied was pathologically examined for cancer.

As shown in Table 4, NK 631 monosulfate (copper free form) and RS-form antibiotic are somewhat less effective in inhibiting 20-MC-induced carcinogenesis than bleomycin complex.

Table 4

Mortality Incidence Percentage of carcinomas (%) inhibition

Monosulfate NK 631 (copper free) 5/11 (45.4) 47.8 RS form 1/12 5/11 (45.5) 47.8 Bleomycin complex 2/12 5/10 (50.0) 42.5 control 1/24 20/23 (86.9) 0

All of the above test results show that NK 631 monosulfate (a copper-free form) has a markedly higher antineoplastic activity compared to a commercial bleomycin complex.

3. Antimicrobial efficacy

Antimicrobial activity against Mycobacterium 607 and Bacillus subtilis PCI was determined by the disc method using bleomycin Az as a standard (1000 J / mg). The results are shown in Table 5.

Table 5

M. 607 B. Sub monosulfate NK 631 (copper free) 7 848

RS-form 7 535 bleomycin complex 1234

1550

1400

886

As can be seen from Table 5, NK 631 monosulfate (copper free form) and the RS form of the antibiotic exhibit a much higher antimicrobial power than the bleomycin complex.

4. Toxicity

4—1. Acute toxicity in rats (LD 50) by intraperitoneal administration

As shown in Table 6, the rat LD 50 values found for NK 631 monosulfate (copper free forms) are approximately as high as those found for the bleomycin complex and the RS form of the antibiotic.

205127 11 12 Table 6 LD 50 mg / kg Reliability limits (5% materiality threshold) monosulfate NK 631 (copper free) 155.0 133.6 - 179.8 rat RS form 150.6 129.0 - 170.6 bleomycin complex 168.0 130.0 - 217.0

4—2. Subacute and chronic toxicity (copper-free forms) in rats and dogs is comparable to the analogous toxicity of the bleomycin complex. However, special mention should be made that necrosis has been observed at the injection site in dogs when all doses of bleomycin complex were administered (90 injections at a dose of 1.2 mg / kg), but not in any the dogs received the same dose of NK 631 monosulfate (copper free forms). The lung toxicity of NK 631 monosulfate (copper free form) is also lower than the toxicity of the bleomycin complex.

The above results show that the toxicity of NK 631 monosulfate (copper free form) is comparable to the toxicity of the bleomycin complex, except for lung and necrosis toxicity, which are significantly lower for NK 631.

5. Summary

From the above test results, it can be concluded that bleomycin NK 631 is a novel compound having the following advantageous properties:

(a) extremely low lung toxicity, b antimicrobial and antineoplastic efficacy greater than commercial bleomycin complex, systemic toxicity comparable to that of commercial bleomycin complex, and dj low local injection site toxicity.

For these reasons, bleomycin NK 631 is expected to be advantageous in clinical use.

The principle of the preparation of the novel bleomycin NK 631 of the above formula I by the process according to the invention is that the 3-aminopropyl ester or the N-monosubstituted 3-aminopropyl bleomycin ester of formula II

HO, 'CI'

NH.

NH.

C °, _CH n ^Ho ~.

CH ^{No. 1}

CH, ii sCH _p

H i

zN \

N '^ _C J <A>

h

O

II ^ c.

CH HH

• C, / CH

O r H

WA ⁰ / ^H

~ ^Η / o _{x r} O ~ 'C''' 9 ”H

OH

H

N

ϊ

H

T ^C \ ^{C X} OH \ CH OH \ oy \ ^c <\ / OC from Γ ^V H OH ° \ H

JC

OH (1D reacts in a solvent environment with NJ (S) -1'-phenylethyl] -1,3-diaminopropane at 0 to 80 ° C for 1 to 72 hours, to one equivalent of bleomycin 3-aminopropyl ester or its The N-monosubstituted derivative used is 1 to 10 equivalents of N - [(S) -1'-phenylethyl] -1,3-diaminopropane, and the N-monosubstituted 3-aminopropyl ester is selected from the group consisting of:

3-acetylaminopropyl ester, 3-succinylaminopropyl ester, 3-benzoylaminopropyl ester, 3-benzylaminopropyl ester, 3-p-toluenesulfonylaminopropyl ester, 3- (2,4-dinitrophenyl) aminopropyl ester, 3- (3,5-dimethyl-3-oxocyclohexen-1-yl) Amino-propyl ester, 3- (tert-butoxycarbonyl-aminopropyl) and 3- (salicylidene) imino-propyl ester Water, methanol, dimethylformamide, dimethylsulfoxide or a mixture thereof are used as solvents and the resulting product is converted to the corresponding non-toxic salt by treatment with hydrochloric acid, sulfuric acid or sulfuric acid or acetic acid. .

The starting material, N- [(S) -1'-phenylethyl] -1,3-diaminopropane, is a novel compound synthesized first by the inventors as follows.

To the phenylethylamine cooled to about 0 ° C, about equivalent amount of acrylonitrile is added and the reaction is completed by heating the mixture at 80 ° C to 100 ° C for 10-24 hours. The excess acrylonitrile was distilled off under reduced pressure and the remaining crude product was distilled off under reduced pressure. The obtained 3 - [(S) -1'-phenylethylamino] propionitrile is reduced in a conventional manner, for example with hydrogen in the presence of Raney nickel, to give the desired N- [(S) -1'-phenylethyl] -1,3-diaminopropane (in the following). of the description, this substance is referred to as the "amino compound." The physicochemical properties of that amino compound are summarized in Table 7.

Table 7

Physico-chemical properties of N- [(S) -l'-phenylethyl] -1,3-diaminopropane

Free base Dibydrochloride

1. Appearance at room temperature colorless liquid of white acicular crystals

2. Melting point

3. Boiling point

4. Ultraviolet absorption maxima (nm)

5. Molecular extinction coefficient, ε (256 nm)

6. Specific optical rotation [a] _D ²⁵

Dd ² «

Seventh _Rf value in thin layer chromatography, eluent: n-propanol-acetic-pyridine. acetic-water (15: 10: 3: 12, I / O), plate m. Avicel-SF ^R

8. R _m value in high-voltage electrophoresis, formic acid-acid system. acetic-water (25: 75: 900, V / V), Avicel-SF ^R plate,

800 V, 10 min., Rt = 1.0 _m alanine

9. IR absorption spectrum, cm ^-1 , KBr tablet up to 103 ° C at 0.267 kPa

241, 247, 252, 257, 263, 267 (methanol) -56.38 ° (undiluted, 1 = 1)

0.73

1.52

223.5-224 ° C

250, 256, 259, 266 (distilled water)

206 (distilled water) —20.6 ° (c = 1, distilled water)

0.73

1.52

10. Summary formula (molecular weight)

700, 760, 820, 910, 1025, 1080, 1130, 1200, 1304, 1350, 1370, 1450, 1595, 1605, 2850, 2940, 3300, 3375

C11H18N2 (178.28)

690, 750, 820, 910, 980

1020, 1065, 1075, 1150, 1205, 1385, 1460, 1500, 1515, 1590, 2500, 2850, 3000, 3500

C11H20N2Cl2 (251.20)

Detailed description of the procedure:

Bleomycin 3-aminopropyl ester used in the process is readily prepared in the form of the dihydrochloride (copper form) by thermal decomposition of bleomycin A2 to 3- (methylmercapto) propylaminobleomycin and reaction of the thus obtained bleomycin with halogenonitrile, haloacetic acid, haloacetic acid ester or an haloacetamide in an acidic medium (see U.S. Patent No. 3,886,133, incorporated herein by reference).

The N-monosubstituted 3-aminopropyl bleomycinate derivative having the protected amino group is obtained in high yield by dissolving the 3-aminopropyl bleomycinate 3-aminopropyl ester in water or in an organic solvent such as methanol or a mixture thereof and to a solution an equivalent amount or a slight excess of any of the known amino protecting agents, either in solid powder form or as a solution in an organic solvent such as methanol, is slowly added, with vigorous stirring, and the reaction is allowed to proceed at room temperature or with cooling ice, maintaining the pH of the reaction mixture continuously at a pH of 5.0 to 5.0

7.5 by adding an organic base such as trimethylamine, triethylamine, pyridine, 1,3-diazibicyclo [5.4.0] -7-undecene, 1,5-diazobicyclo [3.4.0] -5-nonene 1,4,4-diazabicyclo [2.2.2] octane, or N-methylmorpholine. Typical N-monosubstituted derivatives include, for example, 3-ace205127 tylaminopropyl bleomycinate monohydrochloride,

3-succinylaminopropyl bleomycin ester monohydrochloride 3-succinylaminopropyl bleomycin ester monohydrochloride 3-succinylaminopropyl bleomycin ester monohydrochloride } Aminopropyl bleomycinate, 3- (3,5-dimethyl-3-oxocyclohexen-1-yl) aminopropyl bleomycinate, 3-N-tert-butoxycarbonylaminopropyl bleomycinate monohydrochloride, or 3-N-salicylideniminopropyl ester monohydrochloride 3,886,133).

In the process, bleomycin NK 631 is formed by reacting a 3-aminopropyl or N-monosubstituted 3-aminopropyl bleomycinate ester with an amino compound. When N-monosubstituted 3-aminopropyl bleomycinate is used as the starting material, either pure isolated compounds or reaction mixtures, optionally concentrated, can be used in the process, obtained by reacting 3-aminopropyl bleomycinate with an amine-protecting agent.

Suitable solvents for the reaction are

3-aminopropyl ester or N-monosubstituted 3-aminopropyl bleomycinate with an amino compound may use water or organic solvents such as methanol, dimethylformamide or dimethyl sulfoxide. The reaction mixture is allowed to stand for 1 to 72 hours at 0 to 80 ° C under neutral or alkaline conditions; in this case, aminolysis occurs and bleomycin NK 631 is formed. At a higher pH, a longer reaction time at relatively low temperatures is required, whereas at a lower pH a short reaction time at higher temperatures is preferable. A suitable ratio of the reactants in the reaction mixture is 1 to 10 equivalents of amino compounds per equivalent of 3-aminopropyl bleomic acid ester or N-monosubstituted derivative thereof.

In order to isolate bleomycin NK 631 from the reaction mixture, the bleomycin component is first completely precipitated by adding 2-5 volumes of acetone per volume of reaction mixture. The precipitate formed is filtered off, washed thoroughly with acetone and dissolved in the smallest possible volume of distilled water. The aqueous solution obtained is quickly adjusted to pH 6.0 with hydrochloric acid or the like and applied to the upper end of a chromatographic column prepared from CM-Sephadex (§) C-25 (NH &^lt; ^{+ &gt};form; supplied by Pharmacia Fine Chemicals) slurry in 0.05 M aqueous ammonium chloride solution. The bleomycin components are adsorbed to the resin and eluted sequentially or continuously with an aqueous solution of ammonium chloride from 0.05 M to 1.0 M with bleomycin acid 3-aminopropyl ester or its N-monosubstituted derivative and bleomycin NK 631. Said substances are discharged from the column in the form of a blue-colored eluent (absorbing in the ultraviolet region at 292 nm) at ammonium chloride concentrations of 0.15 to 0.20, M and 0.35 to 0.45 M respectively. freed from salts by adsorption on an Amberlite® XAD-2 or Diaion ® IIIP 40 column, washing the column with water and eluting with bleomycin, NK 631 (a copper-containing form) is obtained as a blue-colored amorphous powder. with water acidified with hydrochloric acid or sulfuric acid to elute the column. The bleomycin acid or N-monosubstituted derivative thereof is preferably regenerated in this process. The method described above, which uses NK-Sephadex (phi) C-25 to isolate bleomycin NK 631, is presented as a typical illustrative example, and may be modified within certain limits without substantially affecting the results. For example, aqueous sodium chloride or ammonium sulfate may be used as eluent.

The above-described bleomycin NK 631 (copper-containing form) can be converted to a copper-free form using the known copper removal procedures described above in connection with process 1. If desired, the copper-free bleomycin NK 631 obtained can be converted to its non-toxic salts, such as its hydrochloride, sulfate or acetic acid salt.

The basic physicochemical properties of bleomycin NK 631 are shown in Tables 8 and 9.

The structural formula I of bleomycin NK 631 was confirmed as follows; NK 631 monosulfate (copper free form) was dissolved in heavy water and measured by its ¹³ C-NMR spectrum by the proton noise suppression method, using dioxane as internal standard. Signals belonging to all eleven ¹³ C atoms contained in the remainder

3 - [(S) -1'-phenylethylamino] propylamine in the side chain of bleomycin NK 631 was recorded at 19.4, 26.3, 37.0, 43.5, 58.9, 128.3 (two signals), 130.0 (two signals) and 136.3 ppm. The signals induced by the other carbon atoms correspond to those characteristic of the substances of the bleomycin group [see Naganawa et al., Journal of Antibiotics, 30, 388 (1977)].

Table 8

Physico-chemical properties of bleomycin NK 631 (forms containing copper)

Salt Dihydrochloride Monosulfate 1. Appearance blue amorphous powder blue amorphous powder 2. Solubility Ύ τ soluble in water, methanol, dimethylsulfoxide, dimethylformamide; sparingly soluble in dioxane; insoluble in ethanol, acetone, ether, benzene same as dihydrochloride 3. Melting point (decomposition) Noc: 2 ° C 205 to 207 205 to 207 4. Specific rotation (distilled water, c = 1,0) _r . 25 '] 436 —95.6 ° —93.7 ° 5. TL-chromatography V R _F value (a) 0,72 (b) 0,75 aj 0,72 bj 0,75 6. Electrophoresis ² / R _m value (alanine = 1) 0.80 0.80 Comment: 1 / a) on a thin layer of Silica G (G) (Trademark adsorbent for thin film ·

sillkagel, manufactured by Merck Inc, USA) in methanol-10% ammonium acetate-10% ammonia (10: 9: 1, v / v). (b) on a thin layer of Avicel SF (§) (a trademark of crystalline cellulose thin-layer chromatography adsorbent manufactured by FMC Corporation, USA) in the n-propanol-pyridine-acetic acid-water system (13: 10:: 3: 12, vol / vol.

2) On Avicel SF (§) in the formic acid - acetic acid - water system (25: 75: 900, v / v). 800 V; Fifteen minutes.

Table 9

Physico-chemical properties of bleomycin NK 631 (copper-free forms)

Salt Dihydrochloride Monosulfate Diacetate

1. Appearance pale yellow-white colored amorphous powder pale yellow-white colored amorphous powder pale yellow-white colored amorphous powder 2. Solubility Soluble in water, methanol, oc-acid

dimethyl, dimethylsulfoxide, dimethylformamide; sparingly soluble in dioxane; insoluble in ethanol, acetone, ether, benzene

3. Melting point

(dec.), ° C 195 to 197 196 to 198 188 to 190 4. Specific rotation, (distilled water, —2.1 ° —2.0 ° —2.0 ° -iniri 436 (c = 1,0) ₂₅ 5th chromatography ^TL-1 / R _t value a) 0.56 b) 0.70 a) 0.56 b) 0.70 (a) 0,56 (b) 0. 6. Electrophoresis ² / R _m value (alanine = 1) 0.94 0.94 0.94 Comment: 1 / a) Gel Gel G (R) ; methanol - 10% ammonium acetate - 10% ammonia (10: 9: : 1, v / v) (b) Avicel SF (§); n-propanol-pyridine - acetic acid - water (15: 10: 3: : 12, v / v) 2 / Avicel SF®; formic acid - acetic acid - water (25: 75: 900) v / v); 800 V; Fifteen minutes

In the Annex, Figures 1 and 2 show ultraviolet absorption curves and Figures 3 and 4 infrared absorption curves of bleomycin NK 631 of formula I.

Figure 1 shows the ultraviolet absorption curve of 3 - [(S) -1'-phenylethylamino-propylaminobleomycin dihydrochloride (copper containing forms); Figure 2 ultraviolet absorption curve of 3 - [(S) -1'-phenylethylamino] propylaminobleomycin monosulfate (copper free forms), to Figure 3 shows the infrared absorption curve of 3 - [(S) -1'-phenylethylamino iopropylaminobleomycin dihydrochloride (copper-containing forms) measured in a potassium bromide tablet, and Figure 4 shows the infrared absorption curve of 3- [(S) -1'-phenylethylamino iopropylaminobleomycin. (copper free forms) also measured in a potassium bromide tablet.

The process according to the invention is explained in detail in the following non-limiting examples.

Example 1

Synthesis of NK 631 dihydrochloride (forms containing copper)

To a solution of 9.0 g of 3-N-benzoylaminopropyl blcomycinate monohydrochloride (copper-containing forms) was added 9.16 g of the amino compound while cooling to 0 ° C. The reaction mixture was stirred at 0 ° C for 40 hours and then sufficient cold acetone was added to precipitate the reaction products. The precipitate is filtered off, washed with acetone and dissolved in 300 ml of distilled water. The solution was immediately acidified with 0.1 N hydrochloric acid to pH 6.0 and poured onto a column prepared from 1 liter of CM-Sephadex ®

C-25 (NHd ⁺ form) mixed in 0.05 M aqueous ammonium chloride solution The column with adhered bleomycins is washed with 10 liters of aqueous ammonium chloride solution, whose concentration is continuously increased from 0.05 M to 1.0 M. Unreacted

Bleomycin 3-N-benzoylaminopropyl ester is eluted from the column with a solution of about 0.2 M ammonium chloride and about 0.45 M ammonium chloride 631 ammonium chloride. Fractions containing these bleomycins exhibiting absorption in the UF region at 272 nm, are separately connected. The portion containing bleomycin NK 631 is poured onto an ion exchange resin column prepared from 1.3 liters of Amberlite (§) XAD-2. The column was washed thoroughly with water, and the product was eluted with 0.01 N hydrochloric acid in methanol / water (4: 1, v / v). A total of 2.5 liters of blue-colored fractions were collected which showed absorption in the UF region of the spectrum at 292 nm. Methanol is distilled off from the pooled fractions, the remaining aqueous bleomycin solution is adjusted to pH 6.0 with Dowex (β) 44 (OH ^- form) and then lyophilized. 8.2 g (yield 90%) of bleomycin dihydrochloride NK 631 (copper-containing forms) are obtained in the form of a blue-colored amorphous powder powder having an antimicrobial power of 8030 J / mg.

In a similar manner, 910 mg of unreacted 3-N-benzoylaminopropyl bleomycinic acid monohydrochloride (copper-containing forms) was obtained from the preceding fractions.

Example 1 a d 2

Synthesis of NK 631 dihydrochloride (forms containing copper)

To a solution of 950 mg of 3-Np-toluene sulfonylaminopropyl bleomycinate monohydrochloride (copper-containing forms) in 3 mL of methanol was added with stirring at 0 ° C 980 mg of the amino compound and stirred at 0 ° C for 48 hours. Dilute 15 ml of cold acetone with stirring to precipitate bleomycin fractions The precipitate is filtered, washed with acetone and dissolved in 30 ml of distilled water, acidified immediately with 0.1 N hydrochloric acid to pH 6.0 and purified as described in step And an example

5. 833 mg (85% yield) of bleomycin dihydrochloride NK 631 (copper-containing forms) are obtained in the form of a blue-colored amorphous powder which exhibits an antimicrobial power of 8,010 J / mg.

Example 3

Synthesis of NK 631 dihydrochloride ((forms containing copper)

To a solution of 850 mg of bleomycinic acid 3-acetylaminopropyl ester (copper form) in 5 ml of dimethylformamide was added, with stirring, at 0 ° C, 950 mg of the amino compound was added and the mixture was stirred at 0 ° C for 45 hours. The reaction mixture was diluted with 20 ml of cold acetone with stirring to precipitate the bleomycin component. The precipitate was filtered off, washed with acetone and dissolved in 30 ml of distilled water. The solution was immediately acidified with 0.1 N hydrochloric acid to pH 6.0 and treated in the same manner as described in Step A of Example 5. 570 mg (63% yield) of bleomycin dihydrochloride NK 631 (copper containing forms) were obtained as of a colored amorphous powder having an antimicrobial power of 8,010 J / mg.

Example 4

Synthesis of NK 631 dihydrochloride (forms containing copper)

2BS127

To a solution of 900 mg of 3-N- (2,4-dlnitrophenyl) aminopropyl bleomycinate monohydrochloride (copper-containing forms) in 3 ml of dimethylsulfoxide was added 793 mg of the amine compound while stirring at 0 ° C and stirred for 24 hours at 0 ° C. Noc: 2 ° C. The reaction mixture is then diluted with stirring with 15 ml of cold acetone, the precipitated bleomycin fractions are filtered off and washed with acetone. The crude product was dissolved in 30 ml of distilled water, the solution was immediately acidified with 0.1 N hydrochloric acid to pH 6.0 and then treated as described in Step A of Example 1. 690 mg (78 percent yield) of bleomycin dihydrochloride NK 631 were obtained. (copper-containing forms) in the form of a blue-colored amorphous powder having an antimicrobial cardinality of 8,010 J / mg. Example 5

Synthesis of copper-containing NK 631 dihydrochloride and copper-free NK 631 dihydrochloride

To a solution of 1.0 g of 3-aminopropyl bleomycinate dihydrochloride (copper containing forms) in 10 ml of methanol was added dropwise 105 mg of benzoyl chloride dropwise over 30 minutes while maintaining the pH of the mixture by adding 1,4-diazabicyclo [2.2.2] ] octane in the range 6.5 to 7.5. The solution was stirred for an additional 30 minutes and then concentrated to a volume of 2 mL. The concentrated solution was cooled to 0 ° C, 1.1 g of the amino compound was added with stirring, and the mixture was stirred at 0 ° C for 72 hours. The reaction mixture is diluted with 6 rolls of cold acetone while stirring, the precipitated bleomycin fractions are filtered off and washed with acetone. The crude product is dissolved in 30 ml of distilled water, the solution is immediately acidified with 0.1 N hydrochloric acid to pH 6.0 and poured onto a column prepared from 100 ml of CM-Septhadex® (in NHd ⁺ form) mixed in 0.05 M aq. ammonium chloride solution. The adsorbed bleomycin components were eluted from the column in the same manner as described in step A of Example 5. 779 mg (73% yield) of bleomycin dihydrochloride NK 631 (copper containing forms) were obtained as a blue amorphous powder having an antimicrobial power of 8,000 j / mg.

In addition to this product, 240 µg of unreacted 3-N-benzoylaminopropyl bleomycinate monohydrochloride is obtained.

Step B: A solution of 700 mg of Bleomycin NK 631 dihydrochloride (copper form) obtained in Step A above, in 20 ml of distilled water is poured onto a column prepared from 65 ml of Diaion ® HP-40 ion exchanger mixed in distilled water. The column containing the adsorbed bleomycin is washed successively with 200 ml of a 5% aqueous solution of EDTA.Na2, then with 250 ml of a 5% aqueous solution of sodium chloride and then with 100 ml of distilled water. Then, the product was eluted from the column with a mixture of methanol with 0.0025 N aqueous hydrochloric acid [1: 1, v / v) and the fractions showing ultraviolet absorption at 290 nm (total 98 mL) were collected. Methanol was distilled off from the pooled fractions under reduced pressure, and the remaining aqueous solution was treated with Dowex® 44

OH form to pH 6.0 and then lyophilized. 658 mg (98% yield) of Bleomycin NK 631 dihydrochloride (copper free form) were obtained as a light yellow-white amorphous powder having an antimicrobial power of 7 834 J / mg.

Example 6

Synthes of NK 631 monosulfate (copper-containing forms) and NK 631 diacetate (copper-free forms)

Step A: To a solution of 1.0 g dihydrochloride

Bleomycin 3-aminopropyl ester (copper form) in 10 ml of methanol is added dropwise with vigorous stirring at room temperature over 30 minutes with 104 mg of benzoyl chloride while maintaining the pH of the solution at 5.0 to 7.5 by adding N-methylmorpholine. . The reaction mixture is stirred for an additional 30 minutes, then diluted with 50 ml of acetone and the precipitated bleomycin fractions are filtered off and washed with acetone. The precipitate was dissolved in 3 mL of methanol, 579 mg of the amino compound was added with stirring at 0 ° C, and the mixture was stirred at 0 ° C for 42 hours. The reaction mixture is diluted with 10 ml of cold acetone with stirring, the precipitated reaction product is filtered off, washed with acetone and dissolved in 30 ml of distilled water. The aqueous solution was immediately acidified with 0.1 N hydrochloric acid to pH 6.0 and then treated using a CM-Sephadex ® column in the same manner as described in Step A of Example 5 above. from 65 ml of Diaion (R) HP-40 ion exchanger mixed in distilled water and adsorbed bleomycins are eluted after washing the column with water with methanol / 0.01 N aqueous sulfuric acid (1: 1, v / v), collecting fractions showing ultraviolet absorption at 292 nm, total Ϊ30 ml). Methanol was distilled off from the pooled fractions, the remaining aqueous bleomycin solution was adjusted to pH 6.0 with Dowex® 44 (OH-form) and then lyophilized. 813 mg (75% yield) of bleomycin monosulfate NK 631 (copper-containing forms) are obtained in the form of a blue-colored amorphous powder which exhibits an antimicrobial power of 7 819 j / mg.

Stage 800: 800 mg of Bleomycin NK 631 monosulfate (copper-containing forms) obtained in Stage A above, was deprived of copper in the same manner as described in Stage B in Example 9 except 5% was used in the column elution phase aqueous ammonium acetate solution and a mixture of methanol with 0.01 N aqueous acetic acid (1: 1, v / v) instead of a 5% aqueous sodium chloride solution and a mixture of methanol with 0.0025 N aqueous hydrochloric acid (1: 1, v / v) obj). In this way 748 mg (96% yield) of bleomycin diacetate NK 631 (copper free forms) are obtained in the form of a pale yellow-white colored amorphous powder having the following spectral and antimicrobial characteristics: Ultraviolet absorption maximum:

Λmax ^nm E 0.1 0.1N / HCl

290 105

Antimicrobial cardinality: 7,620 j / mg. Example 7

Synthesis of copper-containing NK 631 dihydrochloride and copper-free NK 631 dihydrochloride

Step A: To a solution of 1.0 g dihydrochloride

Of 3-aminopropyl bleomycinate in 3 ml methanol at room temperature with stirring dropwise over 60 minutes 193 mg of S-tert-butoxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine while maintaining the pH of the mixture by adding 14% triethylamine solution in % methanol at 6.0 to 7.4. The reaction mixture was stirred for an additional 2 hours, the resulting solution of 3-N-tert-butoxycarbonylaminopropyl bleomycinate was cooled to 0 ° C, 1.2 g of the amino compound was added thereto, and the mixture was stirred for 70 hours. The reaction mixture is diluted with 10 ml of cold acetone, the precipitated bleomycin fractions are filtered off and washed with acetone. The crude product was dissolved in 30 ml of distilled water, the aqueous solution was immediately acidified with 0.1 N hydrochloric acid to pH 6.0 and then purified as described in Example A, Step A

5. 597 mg (56% yield) of NK 631 dihydrochloride (copper-containing forms) are obtained as a blue amorphous powder having an antimicrobial strength of 8,010 J / mg.

Step B: To a solution of 500 mg of Bleomycin NK 631 dihydrochloride (copper containing form) obtained as described in Step A above, in 50 ml of methanol, a 1 mm internal diameter tube submerged below the surface of the solution was introduced under vigorous stirring, until dissolved in the solution in a large excess (about 1 hour). After the hydrogen sulphide has ceased, the solution is allowed to stand at room temperature for 30 minutes, the precipitated copper sulphide is filtered off and washed with 50 ml of methanol saturated with hydrogen sulphide. The filtrate and washings were combined and the methanol and hydrogen sulfide removed by distillation under reduced pressure. The residue is dissolved in 50 ml of methanol, 100 ml of ethyl ether are added, the precipitate formed is filtered off, washed with ether and dried. 394 mg (82% yield) of Bleomycin NK 631 dihydrochloride (copper free forms) are obtained in the form of a pale yellow-white amorphous powder having an antimicrobial power of 7 820 J / mg.

Example 8

Synthesis of copper-containing NK 631 dihydrochloride and copper-free NK 631 dihydrochloride.

Step A: To a solution of 1.0 g of 3-aminopropyl bleomycinate dihydrochloride (copper-containing form) in 2 ml of methanol, 98 mg of salicylaldehyde is added under vigorous stirring at room temperature, maintaining the pH of the mixture at 7.0 by addition of triethylamine. up to 7.4; 3-N-salicylideniminoiminopropyl bleomycinate is formed in solution. After stirring for 1 hour, the mixture was cooled to 0 ° C and 600 mg of the amino compound was added with stirring. The reaction mixture is stirred for 30 hours, then diluted with 10 ml of cold acetone and the precipitated bleomycin fractions are filtered off and washed with acetone. The crude product was dissolved in 30 ml of distilled water, the aqueous solution was acidified to pH 6.0 with 0.1 N hydrochloric acid and then purified as described in Step A of Example 5. 501 mg (47% yield) of bleomycin NK hydrochloride were obtained. 631 (copper containing forms), which exhibits an antimicrobial cardinality of 8,013 J / mg.

Step B: 450 mg of Bleomycin NK 631 dihydrochloride (copper form) obtained as described in Step A above was dissolved in 25 ml of 0.5 N aqueous hydrochloric acid and 25 ml of a 0.2% chloroform solution of dithiazone (diphenylthiocarbazone) was added. . The mixture is shaken rapidly and mixed and then allowed to stand, separating into two layers. The lower layer (chloroform fraction) was separated and 25 ml of fresh chloroform solution of dithizone was added to the upper aqueous layer; The mixture was shaken again and the chloroform layer was separated. The above procedure, i.e. shaking, separating into layers and adding fresh chloroform solution of dithizone, is repeated eight times. Subsequently, the upper aqueous layer was washed with chloroform, separated, the aqueous solution was adjusted to pH 6.0 with Dowex (44) (in the OH 'form) and the water was distilled off to dryness under reduced pressure. 415 mg (96% yield) of Bleomycin dihydrochloride NK 631 (copper free forms) are obtained in the form of a pale yellow-white colorless amorphous powder having an antimicrobial power of 7 845 J / mg.

Example 1 9

Synthesis of NK 631 dihydrochloride (forms containing copper)

To a solution of 1.0 g of bleomycinic 3-aminopropyl ester (copper form) dihydrochloride in 2 ml of methanol was added 1.0 g of the amine compound while cooling to 0 ° C with stirring, and the mixture was stirred for a further 72 hours. The reaction mixture is diluted with 6 ml of cold acetone with stirring and the precipitated bleomycin fractions are filtered off. The crude product was purified as described in Step A of Example 5 to give 128 mg (12% yield) of Bleomycin dihydrochloride NK 631 (copper containing forms) as a colored amorphous powder having an antimicrobial power of 8,015 J / mg.

Example 10

Synthesis of N [(S) -l'-phenylethyl] -1,3-diaminopropane ("amino compound")

To 50 g of (S) -1-phenylethylamine was added 33 g of acrylonitrile while cooling to 0 ° C with stirring. The flask containing the reaction mixture was fitted with a reflux condenser equipped with a chloro-calcium cap and the mixture was heated to 93 ° C with stirring for 18 hours. After heating, the excess acrylonitrile was distilled off under reduced pressure and the remaining crude product was distilled off under reduced pressure; The fraction boiling at 142-145 ° C at 0.93 kPa was collected. 53.5 g of 3 - [(5'-phenylethylamino) propionitrile are obtained.

The nitrile obtained is charged into a hydrogenation autoclave, 5 g of Raney nickel W-7 and 50 ml of ethanol containing 15% ammonia are added and the mixture is hydrogenated, under stirring at 1000 rpm, at 50 to 58 ° C, with hydrogen under partial pressure. 10 to 4 MPa for 1.5 hours. After completion of the hydrogenation, the reaction mixture was distilled under reduced pressure and the fraction boiling at 95-103 ° C at 0.27 kPa was collected. 48.3 g of N- [(S) -1'-phenylethyl] -1,3-diaminopropane are obtained (yield 66% of theory, calculated on the starting (S) -1-phenylethylamine).

Claims (5)

OBJECT OF INVENTION Process for the preparation of 3 - [(S) -1'-phenylethylamino] propylaminobleomycin of the formula T X ^ u-> u O OF X £ and nontoxic salts thereof, characterized in that the 3-aminopropyl ester or the N-monosub30-substituted 3-aminopropyl bleomycinate ester of the formula II O II WC In _z Ή-Ch ' CH NH, i * CK NK IPCH o II AND NO. ch ₃ 'ch' ^ch 'c' ⁿ 'chi II t sCH4 ,. O ^ CH'CH, HO, cn; Λο OH / CHO C ^{H 1} H XI C ^Z 1 ON 'C ^ i H ^ O. r H IN H V ΌΗ CH 2 OH ° C 44} o * 'nr, The INI is reacted in a solvent medium with N- [(S) -4'-phenylethyl] -1,3-diaminopropane at 0 to 80 ° C for 1 to 72 hours, and the product obtained is optionally converted by treatment with acid into an appropriate non-toxic salt. 2. The method of claim 1, wherein from 1 to 10 equivalents of N- [(S) -1'-phenylethyl] -1,3-diaminopropane is used per equivalent of 3-aminopropyl bleomycinate or N-monosubstituted derivative thereof. 3. The process of claim 1 wherein the N-monosubstituted 3-aminopropyl ester is selected from the group consisting of 3-acetylaminopropyl ester, 3-succinylaminopropyl ester, 3-benzoylaminopropyl ester, 3-benzylaminopropyl ester, 3-p-toluenesulfonylaminopropyl ester, 3- (2,4-dinitropynyl) aminopropyl ester, 3- (3,5-dimethyl-3-oxocyclohexen-1-yl) aminopropyl ester, 3- (tert-butyl) butoxycarbonyl amino aminopropyl ester and 3- (salicylidene) iminopropyl ester. 4. A process according to claim 1 wherein the solvent is water, methanol, dimethylformamide, dimethylsulfoxide or a mixture thereof. 5. A process according to any one of Claims 1 to 4 wherein the resulting product is converted to the corresponding non-toxic salt by treatment with hydrochloric acid, sulfuric acid or acetic acid.