CS210673B2 - Manufacturing process of 3-(ls)p-1 - phenylethylamino prropylaminobleomycine - Google Patents

Abstract

PURPOSE:To prepare a carcinostatic bleomycin derivative (NK 631) by culturing bleomycin-producing fungi belonging to Streptomyces genus in a medium containing N-[(S)-1'-phenylethyl)-1,3-diaminopropane. CONSTITUTION:Bleomycin-producing fungi belonging to Streptomyces genus, e.g. Streptomyces verticillus NK-68-144 (FERM-P No.4108) is cultured in a nutrient medium contaning N-[(S)-1'-phenylethyl]-1,3-diaminopropane, which is a precursor of the title compound, under aerobic condition, and the title conpound (NK 631) is separated from the medium. The concentration of the precursor in the medium is usually 0.2-2mg/ml, and pref. 1mg/ml. The NK-631 has remarkably low tendency of lung fibrosis compared with conventional bleomycins, while keeping high carcinostatic activity.

Description

The present invention relates to a process for the preparation of a novel optically active bleomycin, 3 - [(S) -Γ-phenylethylamine] laminobleomycin of formula I

i \ X · / \ л z ^ = o

X * ο-χ lf

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21 * \ X / °

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5-δ р-г'-Е v

£ o-o-u,

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The principle of the preparation of 3 - [(S) -1'-phenylethylamino] propylaminobleomycin according to the invention is that the strain of Streptomyces verticllus FERM-P number 4108 (ATCC 31 307) or a mutant strain of Streptomyces verticilus

FERM-P No. 4108 (ATCC 31 307) obtained by irradiation with ultraviolet and / or X rays is inoculated into the culture medium and cultured in the presence of N- ((S) -1'-phenylethyl) -1,3-diaminopropane of formula II

210B73

or a salt thereof at 23 to 37 ° C for 5 to 10 days to selectively form 3 - [(S) -1'-phenylethylamino ·] propylaminobleo-. mycine, which is then recovered from the culture liquid.

N - [(S) -Γ-phenylethyl] -1,3-diaminopropane or a salt thereof is added to the culture liquid expediently at a concentration of 0.2 to 2 mg / ml medium.

The salt of N- [(S) -1'-phenylethyl] -1,3-diaminopropane is preferably hydrochloride, sulfate or acetate.

Bleomycin is an antineoplastic active anibiotic discovered by Umezawa and co-workers [Umezava et al .: Journal of Antibiotics, 19A, 200 (1966) ·]. It is a basic, water-soluble sugar peptide capable of forming a chelate with one divalent copper atom. It can be obtained by culturing a strain of bleomycin-producing microorganisms belonging to Streptomyces verticillus in a culture medium with aeration and stirring, and then isolating the product from the culture liquid by suitable methods, such as using ion-exchange resins. In a conventional culture method, 16 different types of bleomycin are produced and isolated. See, e.g., Umezawa et al., Journal of Antibiotics, 19A, 210 (1966).

It is also known that said bleomycins have the following general formula III

in which

R is different for each bleomycin species.

It is also disclosed in U.S. Pat. No. 3,846,400 that when a strain of bleomycin producing microorganisms is grown in a culture medium containing it. an amino compound, the amino compound incorporated into the bleomycin side chain molecule represented by the substituent R in formula III, to form a new bleomycin that differs from any known bleomycin and cannot be obtained by a conventional culture method.

Examples of some of these novel bleomycins are listed below:

Substituent R

H — N — CH2CH2CH2 —S — CH5

O

H II - N - CH 2 CH 2 CH 2 - S - CH 3

CH3

H + / - N - CH 2 CH 2 CH 2 - S \

CHs

H — N — CH2CH2CH2NH — CH2CH2CH2CH2NH2

NH

H || —N — CH2CH2CH2CH2 — NH — C — NH2 — NH— (CH2) - NHCH3 — NH— (CH2) ₃ NHC4H9

Name of bleomycin demethyl A2

3- (methylmercapto) p roylamino amine Olein

Ai

Also

As

B2

3- (mythylamino) -polyoylamino] Oleoylin

3- [bbtylamiήoOpooyylamine.cObeomycin

3- (pyrrolidino) pyrrolidin-olomyomyin

Of these bleomycins, mixtures of copper-grown bleomycins Αι, Α2, Α5, B2, demethyl-A2 m of some others (in another yoyis of the invention, this copper-gray mixture of bleomycins is referred to as "bleomycin comylex") are currently widely used on a large scale. in clinical practice for the treatment of cancer, particularly for the treatment of oyidermal carcinoma of the skin, carcinomas of the head and neck, cervical cancer, carcinoma of the cancer, malignant lymphoma and other cancerous diseases.

In terms of side effects, however, bleomycin has some undesirable properties, for example, that it induces fibrosis of yolk. Fibrosa ylic is the most common side effect of bleomycin. It can be easily deduced that if this side effect were to be yo-pushed in any way, the carcinostatic efficacy of bleomycin could still be utilized in clinical yraxis.

For the above reasons, the inventors have been involved in the preparation of various bleomycins for several years and have investigated in animal tests their carcinostatic activity and toxicity, especially their effect on the induction of yolk fibrosis. As a result of their study, they found a new bleomycin, namely 3 - [(S) -1'-phenylethylamino) yroyylaminobleomycin of the above formula I (in another yoyis this new bleomycin is designated by the symbol "NK 631", which includes both copper and copper (I) form, which can be obtained by culturing a strain of bleomycin-producing microorganisms in the presence of N- [[S) -phenylethyl] -1,3-diammonoyroyl as a recursor, and which is not worse in carcinostatic effect than currently available bleomycin comlexes or any another known bleomycin in which the side effect of yolk fibrosis is significantly suppressed.

The foregoing is a feature of the invention. The purpose of the invention is to provide a formative approach to the production of the novel bleomycin NK 631. Further details of the invention will be apparent from the following yoyis.

One of the most important biochemical reactions that occur during the growth yeasts of microorganisms is isomerization, in which various oytically active substances are often converted to their motiodes or racemic compounds. Notwithstanding, it is also evident that the oytically active amino compound may undergo non-biochemical racemization during sterile sterilization or during the cultivation of microorganisms. Numerous studies have clarified that one oyticky active compound is clearly different from the other in ^scho, yoosti yronikání · wall microbial cells or cell membranes, and also to the substrate and syecificitou · affinity to enzymes that participate in the biochemical and metabolic yochodů.

It was naturally expected that blosynte210873 о

to a certain extent in a selective manner, and that the possibility that the amino compound from bleomycin will behave on the microbe upon receiving an optically active amino compound or that the resulting bleomycin will undergo racemization cannot be excluded.

The inventors analyzed the composition of bleomycin obtained by culturing a strain of bleomycin-producing microorganisms in a culture medium containing N- [(R, S) -1'-phenylethyl] -1,3-diaminopropane as a precursor, and found that the residue N- [ The β-phenylethyl] -1,3-diamoinopropane incorporated into the bleomycin molecule always has an almost constant R / S ratio of 4: 1. Although the reason why bleomycin is not an RS-form is unknown, the above experimental fact clearly shows that The method according to claim 1, wherein the optically active amino compound is introduced into the bleomycin molecule with significant selectivity by always incorporating the R-form over the S-form in the biosynthetic system of bleomycin-producing Streptomyces verticillus microorganisms.

For this reason, when cultivating in a medium containing N- ((S) -1'-phenylethyl] -1,3-diaminopropane as a precursor, it was expected that it would be extremely difficult to introduce the selectively said amino compound into the bleomycin molecule to obtain bleomycin NK 631 in high optical purity and high yield.

In spite of the above experimental fact, the inventors have unexpectedly found that when optically pure N- [(S) -r-phenylethyl] -1,3-diaminopropain is added to the culture system, it is not subject to racemization and is surprisingly built into the bleomycin molecule to the same extent of the R / S ratio as in the case where an amine compound in pure form is added to the culture medium.

Although at present no clear explanation of this phenomenon can be given, it is possible to accept the hypothesis that the R form of the amino compound acts competitively or antagonistically and as a result, the introduction of

The S-form of the amino compound is inhibited, and formation of the R-form of bleomycin takes place preferentially. Thus, a process has been found to provide in high yield a novel bleomycin NK 631 which is of great importance for the treatment of cancer. This disclosure is at the heart of the present invention. The invention makes it possible for the first time to successfully produce an optically active bleomycin by fermentation.

N - [(S) -1‘-Phenylethyl] -1,3-diaminopropane, used as a precursor in the process of the invention, is a novel compound that was first synthesized by the inventors. It can be prepared as follows:

Approximately equivalent amounts of acrylonitrile are added to the phenylethylamine pre-cooled to 0 ° C and the mixture is then heated to 85-100 ° C for 10-24 hours. After completion of the reaction, excess acrylonitrile was distilled off under reduced pressure and the remaining crude product was also 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 (hereinafter described) the name 'amino compound' is used for this substance.

The physico-chemical properties of said amino compound are shown in Table 1.

Table 1

Physico-chemical properties

N- [(S) -1'-phenylethyl] -1,3-diaminopropane

Free base Dihydrochloride

1) Appearance

2) Melting point

3) Boiling point

4) Ultraviolet absorption maximum (nm) j Molar extinction coefficient ε (at 256 nm)

6) Specific rotation [ ' «jo ²⁵ colorless liquid (at room temperature) to 103 ° C (0.27 kPa)

241, 247, 252, 257, 263, 267 (methanol) white needle crystals

223.5-224 ° C

250, 256, 259, 266 (distilled water)

206 (in distilled water) —56.38 ⁰ (undiluted, 1-1) —20.6 ⁰ (c = 1, distilled water)

Free base Dihydrochloride

7) Thin Layer Chromatography (R _P ) n-propanol-pyridine-acetic acid-water (14: 10: 3: 12, v / v) Avicell-SF®

8} 'High Voltage Electrophoresis (R _1U ) Formic Acid - - Acetic Acid - -' Water (25: 75: 900, I / O)

Avicell-SF

800 V, 10 minutes Rm of alanine laid equal to 1.0

9) Infrared absorption spectrum (cm -1 ⁾ ,

KBr tablet)

10) Summary formula (molecular weight)

0.73

1.52

700, 760, 820, 910, 1025

1080, 1130, 1200, 1305, 1350

1370, 1450, 1495, 1605, 2850, 2940, 3300, 3375

C11H18N2 (178.28)

0.73

1.52

690, 740, 820, 910, 980

1020, 1065, 1075, 1150, 1205,

1385, 1460, 1500, 1515, 1590, 2500, 2850, 3000, 3500

C11H20 ‰ C12 (251.20)

In the practice of the process of the present invention, a culture medium containing mainly sugars and organic nitrogenous substances such as millet, porridge, glucose, starch, soybean meal, corn steep liquor and the like, and in addition containing small amounts of inorganic substances such as potassium phosphate, copper sulfate, zinc sulfate, table salt, nitrates and the like (this type of broth is described, for example, in U.S. Patent Nos. 3,681,491 and 3,846,400).

N- ['(S) -Γ-Phenylethyl] -1,3-diaminopropane, used as a precursor, can be added to the broth either before the cultivation is started or in portions throughout. cultivation, or · combine it in both ways. The amino compound added is generally first neutralized with hydrochloric acid and used as the hydrochloride. However, it is possible to add a free amino compound to the culture medium. In the latter case, the pH of the medium as a whole is adjusted and then the culture is started.

The concentration of N- [(S) -r-phenylethyl] -1,3-diaminopropane in the culture medium is usually in the range of 0.2 to 2 mg / ml, preferably 1 mg / ml. After sterilization, the broth is inoculated with bleomycin producing microorganisms belonging to the Streptomyces verticillus strain, for example Streptomyces verticillus strain NK 68-144, deposited with the Japanese Fermentation Research Institute, Agency of Industrial Science. and Technology, Japan) under the designation FERM-P No. 4108 and in the American Type Culture Collection under the designation ATCC 31307, or inoculated with a seed culture of bleomycin-producing microorganisms belonging to other Streptomyces strains verticillus, and the microorganisms are then cultured at 23-37 ° C, with aeration and stirring, usually for 5-10 days.

Cultivation is terminated when the maximum antimicrobial potency in the medium is reached (monitored by a bioassay using Mycobacteria 607 as the microorganism tested). Upon completion of the culture, the culture liquid is filtered and the filtrate is allowed to flow through a column of anion exchange resin carrying suitable reactive groups, such as carboxyl groups. The desired product is adsorbed onto the resin and then eluted with aqueous hydrochloric acid.

The eluate is neutralized, the dissolved crude bleomycin is adsorbed to activated carbon, the product is eluted with acetone / hydrochloric acid, the eluate is concentrated and then lyophilized. The crude product is taken up in methanol, the solution is chromatographed on. alumina using methanol as eluent, the eluate is concentrated and lyophilized. The product obtained is chromatographed on a Sephadex (g) G-25 column (a trademark for gel filtration material composed of cross-linked dextran microscopic beads manufactured and sold by Pharmacia Fine Chemicals Inc, Sweden) and then re-chromatographed on a CM column.

Sephadex®

C-25 (trademark for cation exchange type Sephadex, which consists of microscopic beads of carboxymethylated dextran polysaccharide and manufactured and sold by Pharmacia Fine Chemicals, Inc., Sweden), using an aqueous ammonium chloride solution as the eluent. An eluate is obtained containing the desired bleomycin NK 631 in the form containing copper. The aliquot containing the bleomycin is poured onto a Diaion (40) HP 40 ion exchange resin column, and the adsorbed column is washed successively with an aqueous solution of disodium ethylenediaminetetraacetic acid (abbreviated as "EDTA. Na 2") in the following description. sodium sulfate solution and water, and then the product is eluted with acetone / water. The eluate is concentrated and lyophilized; Bleomycin NK 831 monosulfate (copper free forms) is obtained as a light yellow-white amorphous powder.

Bleomycin monosulfate NK 631 (copper-free forms) obtained by the process of the invention is hydrolysed with strong acid for indication and determination of optical purity, the hydrolysate is separated by high-voltage paper electrophoresis and two-dimensional paper chromatography and the substances are detected with ninhydrin; this demonstrates the presence of N- (l‘-phenylethyl) -1,3-diaminopropane.

The amine released by hydrolysis is isolated and purified, its specific rotation and ultraviolet absorbance are measured, and the optical values are determined for the optical purity of bleomycin NK 631. Specifically, the optical purity is determined as follows:

About 50 mg of bleomycin NK 631 monosulfate (copper free form) was heated to reflux (110-115 degrees Celsius) for 16 hours with 6 N hydrochloric acid. The resulting N - [(S-1'-phenylethyl) -1,3-diaminopropane is isolated from the mixture of decomposition products, purified and then its optical rotation (at λ = 365 nm) at 25 ° C determined using The absorbance at λ = 256 nm is then determined and the whole ultraviolet curve is recorded. The curve in the range 280 to 310 nm is extrapolated to λ = 256 nm and this value is used as the baseline at 256 nm.

The optical purity x value can be expressed by the following equations:

S ^X ~ S + R (1), x = 0.5 + 5.735 X (2) in which

S = left-handed optical rotation, R = right-handed optical rotation, a = observed optical rotation and OD = observed absorbance.

Bleomycin NK monosulfate (copper free form) prepared according to the process of the invention yields N- [(S) -1'-phenylethyl] -1,3-diaminopropane, which typically has a specific rotation of [?]] After acid acid hydrolysis. ²⁵ = -64.18 ° (c = 0.38, water). The optical purity of this bleomycin monosulfate NK 631 (copper free form), calculated according to the above equations, is 98.5%.

Tables 2 and 3 show the basic physical and chemical properties of bleomycin NK 631.

The structure of bleomycin NK 631 represented by formula I was confirmed by dissolving bleomycin NK 631 monosulfate (copper free form) in heavy water and measuring its ¹³ C-NMR spectrum by the proton noise suppression method, using dioxane as an internal standard.

The following signals were indicated:

19.4, 26.3, 37.0, 43.5, 58.9, 128.3 (two signals), 130.0 (two), 130.2 and 136.3 ppm attributable to eleven ¹⁵ C atoms present in the amino compound residue in the side chain of the bleomycin molecule NK 631, namely the 3 - [(3) -1'-phenylethylamino] propylamine residue. All other observed carbon atom signals corresponded to the characteristic signals of the remaining bleomycin molecule as described in the literature [see Naganawa et al., Journal of Antibiotics, Volume 30, p. 388 (1977)].

Table 2

Physical and chemical properties of bleomycin

NK 631 (molds containing copper)

Dihydrochloride Monosulfate

1. Appearance

2. Solubility

3. Melting point (decomposition) ° C

4. Specific rotation [ja] ^ 25 (distilled water, c = 1,0)

5. T-layer chromatography (Rf) (see note 1)

6. Electrophoresis (R _m ) (R _{m of} alanine laid · equal to ¹ 1.0;

(see note 2) blue colored amorphous powder soluble in water, methanol, acetic acid, dimethylsulfoxide and dimethylformamide; sparingly soluble in dioxane; insoluble in. ethanol, acetone, ether. and benzene,

205-207-95.6 °

(a) 0,72

(b) 0,75

0.80 blue amorphous powder same as dihydrochloride

205-207-93.7 °

(a) 0,72

(b) 0,75

0.80

Note 1:

a) on a Silica gel G ® adsorbent (trademark for silica gel for thin-layer chromatography, manufactured by Merck Inc, USA), in the system methanol-10% ammonium acetate - 10% aqueous ammonia (10: 9: 1, V / IN).

(b) on Avicel SF (§) (thin film chromatography adsorbent brand name, consisting of talc-cellulose rat manufactured by FMC Corporation, USA) in the n-propanol-pyridine-acetic acid-water system (15: 10: 12: 12).

Note 2:

on Avicdl SF (§) adsorbent in formic acid - acetic acid - water (25: 75: 900, v / v), 800 V, 15 minutes.

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Table 3

Physical and chemical properties of bleomycin NK 631 (copper free forms)

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1 and 2 show ultraviolet absorption curves, and 3 and 4 infrared absorption curves of bleomycin NK 631 of formula 1. FIG. 1 shows the ultraviolet absorption curve of the dihydrochloride

3 - [(S) - [phi] phenylethylamino] propplaminobleomycin of formula I (copper-containing forms), Fig. 2 shows an ultraviolet absorption curve of the corresponding moypsulfate (copper-free forms); Figure 3 shows the infrared absorption curve of 3 - [(S) -1'-phenylethylamino] propylamino-aminomycin (copper-containing forms) measured in a potassium bromide tablet, and Figure 4 shows the infrared absorption curve of the corresponding monosulfate (copper-free forms) measured the same way.

The excellent biological activity of bleomycin NK 631 according to the invention results from the experimental results below.

The biological activity of bleomycin NK 631 was compared with that of a commercialobleomycin complex and that of 3 - [(RSpr-phenylethylamino) propylaminoblepmycin monosulfate (copper-free form) (hereinafter referred to as "RS-form") in the following four tests:

1] the effect on inducing lung fibrosis is antineoplastic activity

3) anti-microbial activity

4) toxicity

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

1. Mouse lung toxicity (fibrosis induction)

Twelve-year groups of 15-week-old male ICR mice were used for testing. Test substances were administered at a constant dose of 5 mg / kg in the form of intrareritic injections once daily for 10 consecutive days. Mice were housed and observed for 5 weeks after drug administration. The animals were sacrificed and the extent of lung fibrosis was determined by autopsy.

The incidence and degree of lung fibrosis induced in the group of mice treated with bleomycin NK 631 mpnpsulfate (copper free form) was compared with the incidence and degree of lung fibrosis in the groups of mice receiving the RS form of the antibiotic and the blnomycius complex. The results are shown in Table 4.

Table 4

Number of mice with lung fibrosis (%) Occurrence Relative value Degree of disease Overall assessment of lung fibrosis grade: total number of samples (see note) Relative value mpypsuieát bleomycin NK 631 (copper-free form) 4/12 (0.33) 0.36 .. 6/36 - (0.17) - 0.25 · RS form 7/12 (0.59) 0.63 12/36. (0.33) -. . . 0.49 blnpmyciyový 11/12 (0.91) 1.0 24/36 (0.67) 1.0.

complex

Note:

Scale used to assess lung fibrosis grade:

point - no fibrosis point - accumulation of exudate in the alveoli and fibrosis similar changes in alveolar septum points - fibrosis at several points points - scattered fibrosis of points - fibrosis at more than two-thirds - whole area

As shown in Table 4, Bleomycin monosulfate NK 631 (a copper-free form) reduces the incidence and extent of lung fibrosis by about half compared to the RS antibiotic form, and by about a quarter and the extent by about a quarter compared to the bleomycin complex. The results found clearly demonstrate the benefit of bleomycin monosulfate NK 631 for clinical use.

2. Antineoplastic activity

2—1. Effect in vitro on HeLa cultures. S3 cells

A value was determined for each of the above bleomycin. ID50 inhibition of HeLa S3 proliferation. cells after 72 hours of culture in the presence of test bleomycin. For. bleomycin complex was found to be ID 50 = 1.70 µg / ml and for bleomycin NK 631 ID 50 = 0.82 µg / ml. These data indicate that bleomycin NK 631 inhibits the proliferation of HeLa S3 cells twice as much as the bleomycin complex. For the RS form of the antibiotic, an LD50 of 0.180 µg / ml was found, indicating that the inhibitory effect of this bleomycin is comparable to that of bleomycin monosulfate NK 631 (copper free forms).

2—2. Karciniost-atiCic effect in mice Ehrlich's carcinoma (solid tumor)

Male 6-week-old male ICRs were transplanted subcutaneously into the inguinal region of 2X10-8 cells. Twenty-four hours after transplantation, 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 weighed, and the weights compared to those of the control group, untreated and the% inhibition was found.

As shown in Table 5, the carcinostatic activity of bleomycin monosulfate NK 631 (copper free form) is comparable to that of the RS antibiotic form and about 1.4 times higher than that of bleomycin complex.

Table 5

Dose Grade. inhibition (%)

(mg / kg X10) Monosulfate NK 631 (copper-free form) RS-form antibiotic Bleomycin 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 (mg / kg / day) 0.35 0.31 0.49

2—3. Carcinostatic efficacy against ascitic hepatoma in rats (ascitic type)

Donry strain rats were transplanted into the abdominal cavity with 1X10-6 AH 66 cells. Twenty-four hours after transplantation, the test preparation was administered intraperitoneally and continued once daily for ten consecutive days. The body weight of rats and the number of surviving animals were monitored 30 days after transplantation. The results are shown in Table 6.

Table 6

Relative average survival (number of days)

Dose (mg / kg X10) Monosulfate NK 631 (copper-free form) RS-form antibiotic Bleomycin 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 10O 100 ALIGN! 100 ALIGN!

As shown in Table 6, the efficacy of Bleomycin NK 631 monosulfate (medium free form) against Aβ 63 ascitic hepatoma is comparable to that of the RS form of the antibiotic and is higher than the efficacy of bleomycin complex at all doses.

2—4. Anticarcinogenic effect against 20-methylcholanthren-induced epidermal skin cancer in mice

A solution of 20-methylcholanthrene (hereafter referred to as 20-Mc) in acetone was twice weekly, for 18 weeks, topically applied to the shaved back of male 10-week-old ddy mice. Six weeks after the start of 20-MC administration, the test preparation was administered intraperitoneally at a dose of 62.5 µg / mouse and continued twice weekly for 15 weeks. One week after the administration of the test preparation, the skin area to which 20-MC was applied was pathologically examined for carcinogenesis.

As shown in Table 7, Bleomycin monosulfate NK 631 (a copper-free form) and the RS-form of the antibiotic inhibit slightly 20-MC-induced carcinogenesis than the bleomycin complex.

Table 7 Degree of Inhibition (%) Mortality Degree of carcinogenesis (in ° / o) Monosulfate NK 631 (copper-free form) 1/12 5/11 (45.4) 47.8 RS form 1/12 5/11 (45.4) 47.8 bleomycin complex 2/12 5/10 (50.0) 42.5 control 1/24 20/23 (86.9) 0

All the above tests show that bleomycin monosulfate NK 631 (a copper-free form) has a markedly higher antineoplastic activity compared to the commercially available bleomycin complex.

3. The antimicrobial activity of Mycobacterium 607 and Bacillus subtiles PCI was determined by the disc method using bleomycin Аг as a standard (1000 J / mg potency). The results are shown in Table 8.

Antimicrobial activity against microorgaTable 8

Mycobacterium 607

Bacillus subtilis PCI

Monosulfate NK 631 (copper-free form) 7848 1550 RS form antibiotics 7535 1400 Bleomycin complex 1234 886

As shown in Table 8, bleomycin monosulfate NK 631 (copper free form) and the RS form of the antibiotic exhibit much higher antimicrobial activity than the bleomycin complex.

4. Toxicity

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

As shown in Table 9, the LD 50 values for rats found for bleomycin monosulfate NK 631 (copper free forms) are approximately the same as those found for the bleomycin complex and the RS form of the antibiotic.

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Table 9

LD 50 (mg / kg) Reliability limits (5% materiality level)

NK 631 monosulfate (copper free form) 155.0

133.6-179.8 rat

RS form.

antibiotics 150.6

129.0-170.6 bleomycin complex 168.0

130.0 to 217.0

4- ^ 2. Subacute and chronic toxicity in rats and dogs

The subacute and chronic toxicity of bleomycin monosulfate NK 631 (copper free forms) in rats and dogs is comparable to analogous toxicities of the bleomycin complex. However, it should also be noted that bleomycin monosulfate NK 631 (copper free form) does not induce injection site necrosis in dogs, whereas bleomycin complex causes injection site necrosis in all dogs in the high dose group (1.2 mg / kg, 90 times), although the dose level was the same for both bleomycin species. Bleomycin NK 631 also exhibits lower lung toxicity than bleomycin complex.

The above results show that ^; that the toxicity of bleomycin NK 631 monosulfate (copper-free forms) is comparable to the toxicity of bleomycin complex, with the exception of lung toxicity and necrosis induction, which are substantially lower for bleomycin NK 631.

5. Summary

From the above test results it can be concluded that bleomycin. NK 631 is a new compound having the following advantages:

a) extremely low lung toxicity,

(b) antimicrobial and antineoplastic activity greater than commercially available bleomycin complex;

(c) general toxicity comparable to that of commercially available bleomycin. a

d) lower local toxicity at the injection site than commercially available bleomycin complex.

For these reasons, it is expected that the novel bleomycin NK 631 will be advantageous in clinical use.

The process according to the invention is explained in more detail in the following examples, which are not intended to limit the scope of the invention in any way.

Examples

Example 1

One liter of broth (pH 7.0) containing 2% glucose, 0,75% peptone, 0,75% meat extract and 0,3% sodium chloride is placed in a 5-liter Erlenmeyer flask, the soil is sterilized, seeded. Streptomyces verticillus NK-68-144 microorganisms (FERM-P number · 4108, ATCC number · 31 387) and the microorganisms are cultured at 28 ° C for 48 hours with stirring and shaking; hatching is obtained. culture liquid.

At the same time, to the broth containing 7.17 percent millet slurry, 0.5% glucose, 3.5% soybean meal, 0.75% corn steep liquor, 0.3% chloride. solution, 0.1% dipotassium hydrogenphosphate, 0.05% zinc sulfate, 0.01 ° / o copper sulfate · 0.2% "of sodium nitrate and 0.03 ^Ό / ο · surfactant brand Pronal ST-1 ( () (Non-ionic surfactant and antifoam produced by Toho · Kakagu) add N- [· (S) -Γ-phenylethyl] -1,3-diaminopropane hydrochloride at 1 mg / ml soil and adjust the pH of the mixture to 6.5.

Twenty liters of said nutrient broth are introduced into a 39 liter fermenter and sterilized. The broth is inoculated with the above-described culture medium at 10%. Cultivation is performed with aeration and stirring at 400 rpm for 7 days at 28 ° C. Upon completion of the fermentation, the culture liquid, after addition of 5% diatomaceous earth, is filtered through a brand filter (trade name for diatomaceous earth Daikalite new filtering agent manufactured and sold by Toa Kasei Co., Japan) to yield 10 liters of antimicrobial filtrate. cardinality 326 J / ml (3.26 g cardinality).

The filtrate is combined with the wash water and the solution is passed through a column made of 630 ml Amberlite (§) IRC-50 · (H-form), adsorbing the desired product on the column on 210673. The column was washed with water and the product was eluted with 0 L of 0.5 N hydrochloric acid. The eluate was neutralized, the insoluble material was filtered off and the filtrate was passed through a column made of 50 ml of activated carbon. The column was washed with water and the product was eluted with 750 ml of a 0.02 N hydrochloric acid / acetone (1: 1) mixture. Elution ^Whether zneutrahzuje La-wexem ® Settle name anion-exchange resin composed of a copolymer of epichlorohydrin with ammonia, manufactured and sold by Dow Chemical Co., USA), the liquid was concentrated and the concentrated solution is lyophilised. The product obtained is dissolved in three times water, the solution is added dropwise to 9 volumes of methanol, and the insoluble matter formed is filtered off. The methanolic solution is passed through a column made of 25 ml of alumina and the product is eluted with 90% methanol. The eluate is neutralized, concentrated and then lyophilized. The residue was dissolved in 10 ml of water, the product was adsorbed on a column prepared from 100 ml ^G-25 and PA ^to the interpenetrated network of water; ©) Sephadex collecting blue-colored fraction.

The blue fractions were combined, the solution was passed through a column made up of 100 ml of CM-Ssphadex (g) C-25, and the product was eluted with an aqueous ammonium chloride solution ranging from 0.05 to 1 M. To the pooled bleomycin NK containing fractions 631 was added 1 g EDTA-Naz. The pH of the solution was adjusted to 0.5 with 12 N hydrochloric acid and then neutralized with 5 N sodium hydroxide solution. The product is adsorbed on a column made of 50 ml of Diainone © HP-40 (trade name for an absorbent resin consisting of styrene) a divinylbenzene copolymer manufactured and sold by Mitsubishi Chemical Co., Japan.

The column was washed with 5% EDTA solution. Naa (twice the column volume), then 5% aqueous sodium sulfate solution (1.5 times the column volume), water and the product eluted with 50% aqueous acetone (3 times the column volume), the eluate is concentrated and the concentrated solution is lyophilized . 100 mg of the final product, bleomycin monosulfate NK 631 (copper-free forms) having a cardinality of 8.30 mg are obtained. The yield of this product is 25.4% calculated on the content in the filtered culture liquid. Analysis for ^uses meto ^dy with CM-Sephadex ® b ^y l ly zen content 98.7% bleomycin NK · 631. Its optical purity was 98.5% · determined · · the method specified in the description.

The product obtained has the following spectral and antimicrobial characteristics:

Ultraviolet absorption spectrum:

% maximum, at 290 nm, E = 106 at 0.1 cm

N hydrochloric acid

Antimicrobial cardinality: 8077 j / mg.

Example 2

A 200 liter fermentation tank was charged with 120 liters of nutrient broth having the same composition as described in Example 1, and 120 g of N- [(S) -T-phenylethyl] -1,3 were added to the solution. - ^d iaminopropanu pH was adjusted to 6.5 and the mixture was sterilized. The culture medium is then inoculated at 10% with a seed culture medium of Streptomyces verticillus NK 68-14.4 (FERM-P No. 4108, ATCC 31 307 J) prepared by the same procedure and under the same conditions as described in Example 1. 1. Microorganisms were cultured under aeration and stirring at 350 rpm for 7 days at 28 ° C.

At the end of the cultivation, the filtrate (107 liters, 671 J / ml, 72 gm) was treated in the same manner as in Example 1. After extraction and purification, 4.7 g of Bleomycin NK 631 monosulfate (copper-free forms) were obtained. ) (38.9 g-mobutnostij. the yield is 47% calculated on the content of bleomycin in filtered fermentation liquid. Analysis methodology using a ^CM-dy Sephactexem was © o to ^l · ^ns · that the product contained 99.6% The obtained bleomycin NK 631 has an optical purity of 99.1% as determined by the method described above.

The product has the following spectral and antimicrobial characteristics:

Ultraviolet absorption maximum at 290%

nm, E = 107, in · 0,1 N chloro1 cm hydrogen chloride.

Anti-microbial cardinality: 8027 j / mg.

Example 3

Synthesis of N- [(S-r-phenylethyl) -1,3-diamlnopropane

To 50 g of (SJ-1-phenylethylamine cooled to 0 ° C) was added 33 g of acrylonitrile, the reaction flask was fitted with a reflux condenser equipped with a chloro-calcium cap, and the mixture was heated to 93 ° C for 18 hours with stirring. .

After completion of the reaction, the excess acrylonitrile was distilled off under reduced pressure and the remaining crude product was subjected to fractional distillation also under reduced pressure;

The fraction boiling at 142-145 ° C at 0.933 kPa was collected. 53.5 g of 3- [(S) -1'-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, with 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 liquid reaction mixture was distilled under reduced pressure and the fraction boiling at 95-103 ° C at 0.266 kPa was collected. 48.3 g of N- ((S) -1'-phenylethyl] -1,3-diaminopropane are obtained (yield 66% of theory, calculated on the starting (S1-1-phenylethylamine)).

Example 4

N - [(S) -1'-phenylethyl] -1,3-diaminopropane hydrochloride is added to the broth containing the same ingredients as used in Example 1 in an amount of 0.2 mg / ml soil and the pH of the soil is adjusted to 6, 5. Twenty liters of culture are placed in a 30 liter culture vessel and sterilized, then seeded at 10% with Streptomyces verticillus NK 71-125 seed culture fluid (which is a mutation strain obtained from Streptomyces verticillus FERM-P No. 4108, ATCC 31 307 irradiation with ultraviolet rays), prepared under the same conditions as in Example 1. Cultivation is carried out for 5 days at 37 ° C with aeration and stirring at 360 rpm. At the end of the culture, the antimicrobial potency of NK 631 in the culture medium is 376 J / ml.

Example 5

To the broth containing the same ingredients as used in Example 1, N- [(SJ-T-phenylethyl) 1,3-diaminopropane hydrochloride was added in an amount of 2 mg / ml soil and the pH of the soil was adjusted to 6.5. is introduced into a 30 liter culture vessel and sterilized, then seeded at 10% with Streptomyces verticillus NK 71-054 seed culture fluid (a mutant strain obtained from Streptomyces verticullus FERM-P # 4108, ATTC 31 307 by double irradiation). ultraviolet rays and one irradiation (X-rays) prepared from the same conditions as in Example 1. Cultivation is performed with aeration and stirring at 400 rpm at 23 ° C. After the cultivation, the antimicrobial potency of NK 631 in the culture medium is 571 µl. / mg.

Example 6

N - [(S) -1'-phenylethyl] -1,3-diaminopropane hydrochloride was added to the broth containing the same ingredients as used in Example 1 in an amount of 0.5 mg / ml and the pH of the soil was adjusted to 6.5. Twenty liters of culture are placed in a 1 liter culture vessel and sterilized, then seeded at 10% with Streptomyces verticillus NK 71-058 seed liquid (a mutant strain derived from Streptomyces verticillus FERM-P # 4108, ATCC).

307 by double irradiation with ultraviolet rays and double irradiation with X rays prepared under the same conditions as in Example 1. Cultivation was carried out for 7 days at 30 ° C with stirring at 400 rpm. At the end of the culture, the antimicrobial potency of NK 631 in the culture medium is 820 j / ml.

Claims (3)

SUBJECT OF THE INVENTION Process for the production of 3- [(S) -1'-phenylethyl] propylaminobleomycin, characterized in that a strain of Streptomyces verticillus FERM-P number 4108 (ATCC 31 307) or a mutation strain obtained from Streptomyces verticullus FERM-P No. 4108 (ATCC 31307) inoculated with ultraviolet and / or X rays into the culture medium and cultured in the presence of N- [(S) -1'-phenylethyl] -1,3-diaminopropane of formula II Or its salts at 23 to 37 ° C for 5 to 10 days to selectively form 3- [ ^: (S) -1-phenylethylamino] propylaminobleomycin, which is then isolated from the culture liquid. 2. The method of claim 1, wherein N - [(S) -r-phenylethyl] -1,3-diaminopropane or a salt thereof is added to the culture liquid at a concentration of 0.2 to 2 mg / ml medium. 3. A process according to claim 1, wherein the salt of N- [(S) -T-phenylethyl] -1,3-diaminopropane is hydrochloride, sulfate or octane.