CH663415A5 - LUZOPEPTIN E (2). - Google Patents

Description

This invention relates to a novel cyclic depsipeptide antibiotic and a process for its manufacture and recovery.

GB-PS 2 050 384A describes an antibacterial antitu-mor complex called BBM-928 and its preparation by fermentation of a new strain of Actonomycetes called G455-101 (ATCC 31491). This document discloses that the BBM-928 complex consists of at least six components, designated BBM-928 A, B, C, D, E and F. Components A, B, C and D were fully characterized in the publication, while components E and F were characterized only by their Rf values in two silica gel thin-layer chromatography systems and by in vitro antimicrobial activities against certain aerobic bacteria were.

The BBM-928-producing microorganism strains G455-101 were later identified as new species of the genus Actinomadura and as Actinomadura luzonensis nov. sp. (see J. Antibiotics 33 [10], 1098-1102, 1980).

In J. Am. Chem. Soc. 103, 1241 (1981) describes the structures of BBM-928 A, B and C, also in Peptide Chemistry 1980, K. Okawa (Ed), Protein Research Foundation, Osaka, Japan, pp. 119-124 (1981) and J. Antibiotics 34 20 (2), 148-159 (1981).

The production, isolation, characterization and anti-tumor activity of the BBM-928 components are described in J. Antibiotics 33 (10), 1087-1097 (1980). Just as in GB patent application 2 050 384A, the 25 components E and F are characterized only by their Rf values in two silica gel thin layer chromatography systems.

A possible mechanism for the anti-tumor activity of BBM-928A is described in Biochemistry 19, 5527 (1980).

The BBM-928 antibiotics are referred to here as lucopeptin-30 antibiotics, based on the type which produces them. The terms lucopeptin A, B, C etc. used here are identical to those previously used in the literature BBM-928 A, B, C etc.

Explanations of the drawings:

35 Figure 1 shows the infrared absorption spectrum of luzopeptin E2, pelletized in KBr.

FIG. 2 shows the nuclear magnetic resonance spectrum of luzopeptin E2, dissolved in CDCb.

FIG. 3 shows the carbon 13 nuclear magnetic resonance spectrum of luzopeptin E2, dissolved in CDCh.

The invention relates to the antitumor antibiotic with the name luzopeptin E2, which is defined in claim 1, and a process for its preparation and isolation, as defined in claim 2, wherein it is obtained in a purified state, free of co-produced substances. The antibiotic is produced according to the invention by cultivating Actinomadura Luzonensis ATCC 31491 or a mutant thereof producing lucopeptin E2 in an aqueous medium with assimilable carbon and nitrogen sources under 50 submerged aerobic conditions, and then isolating the lucopeptin E2 from the culture medium. The lucopeptin E2 obtained is essentially free of co-produced substances.

Luzopeptin E2 shows both antimicrobial and 55 antitumor activities.

Luzopeptin E2 provided by the present invention has been found as a minor component of the lucopeptin (or BBM-928) antibiotic complex described 60 in GB patent application 2,050,384A. This application discloses the fermentation of Actinomadura luzonensis sp. nov for the preparation of a lucopeptin-antibiotic complex and the separation of such a complex into six bioactive components, which are now referred to as lucopeptins A, B, C, D, E and F. However, there is no disclosure of the antibiotic component luzopeptin E2, which is also obtained as a product in the fermentation of Actinomadura luzonensis.

The new antibiotic according to the invention is from the

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Actinomadura luzonensis fermentation broth has been isolated in substantially pure form and has been characterized by its physical, chemical and biological properties, as will be described in more detail below.

Further research has shown that luzopeptin E2 (see e.g. GB patent application 2 050 384A) actually does not contain only a single antibiotic component,

but represents a mixture of several such components, which includes lucopeptin E2.

Properties of Luzopeptin E ::

Luzopeptin E2 is a cyclic depsipeptide antibiotic that contains a quinoline nucleus as chromophores. Spectral and chemical analyzes show that lucopeptin E2 has the following structural formula:

HC CH,

3 \ / 3

C-OH

I.

»3?

CH-jO

OH

0 = C-NH-CH -CO-N-CH —CON— CH

2 2 èo u

CONH-CH-CO-N ^^ i I I

CH2 hn

0

1

CO I

T CH — N — CO-CH -N-CO-CH -NH-C ili 2 I 2 II ho-k: ch3 ch3 0

H3C ch3

1

0

1

CH,

NH I

N-CO-CH-NH-CO

Luzopeptin E2 is a white solid with a melting point of 201-203 ° C, a molecular weight of 1315.3732 and a molecular formula C60H78N14O20. The elementary analysis provides the following proportions in% by weight:

Carbon 53.19

Hydrogen 5.40

Nitrogen 12.92

Oxygen (difference) 28.49

The infrared absorption spectrum of lucopeptin E2, pelletized in KBr, is shown in Figure 1 of the drawings. Characteristic infrared absorption bands are present at the following frequencies, expressed in cm-1: 3470.3370, 2980, 2983, 2850.1740, 1645, 1520, 1418, 1350, 1285, 1230, 1195, 1150, 1128, 1105, 1065 , 1025, 905, 885, 795, 785, and 750.

The proton magnetic resonance and carbon B nuclear magnetic resonance spectra of luzopeptin E2 were determined using a Bruker model WM-360 spectrometer,

working at 360 MHz or 90 MHz. The PMR and CMR spectra are shown in Figures 2 and 3 of the drawings.

In a solvent system consisting of xylene / methyl ethyl ketone / methanol (5: 5: 1 v / v), luzopeptin E2 shows an Rf value of 0.33, determined by silica gel thin layer chromatography.

The key structural feature that distinguishes lucopeptin E2 from known lucopeptin components is the presence of a hexahydropyridazine residue instead of a tetrahydropyridazine group.

Production of Luzopeptin E2:

Luzopeptin E2 can be produced by culturing Actinomadura luzonensis ATCC 31491 or a mutant thereof producing lucopeptin E2 under submerged aerobic conditions in an aqueous nutrient medium.

The microorganisms for producing lucopeptin E2 and the fermentation conditions therefor are described in GB patent application 2,050,384A. While the preferred microorganism is Actinomadura luzonensis strain G455-101 (ATCC 31491), any lucopeptin E2-producing strain or mutant of the preferred microorganism can be used. The mutants mentioned

25th can be made from such microorganisms by conventional means, e.g. X-ray, ultraviolet, nitrogen gas treatment, phage exposure, and the like. Methods using the named strains or mutant producing lucopeptin E2 are also intended to be encompassed in the present invention.

The producing microorganism is grown in conventional nutrient media which contain an assimilable carbon source, such as starch, glucose, dextrin, 35 maltose, lactose, sucrose, fructose, mannose. Molasses and glycerin. The nutrient medium should also contain an assimilable nitrogen source such as protein, protein hydrolyzates, polypeptides, amino acids, corn water, casein and urea. It should also contain inorganic salts as nutrients, which provide inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride and nitrate ions.

Although not essential for the production and production of luzo-45 peptin E2, a sulfur-containing compound, selected from the following group, is preferably added to the nutrient solution: D, L-methionine, S-methyl-cysteine, S-ethyl-L-cysteine , D, L-ethionine, D-methionine, L-methionine, acetylmethionine, 2-aminoethanethiol, 2-hydroxy-4- (methyl-50 thio) butyric acid, D, L-0-methyl-serine, L-cysteine and L -Me-thionyl-glycine. The preferred sulfur-containing compounds are D, L-methionine and S-methyl-cysteine. Such sulfur-containing compounds can be added, for example, in a concentration of 0.05-0.5% by weight, based on the fermentation medium. The most preferred concentrations for D, L-methionine and S-cysteine are 0.15 and 0.1 wt%, respectively. It has been found that the above-mentioned addition of the sulfur-containing compound increases the production of lucopeptin E2 so that it can be tested and isolated more easily.

In the production of lucopeptin E: any temperature can be used which leads to a satisfactory growth of the producing microorganism. Temperatures in the range of 20-45 ° C are generally used, with the range of 27-35 ° C being preferred. The maximum production of lucopeptin E2 is generally obtained after 72-96 h.

Common methods are used in the fermentation process

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used. For example, small quantities are usually produced in shake bottles or in surface cultures. Large quantities are preferably produced under submerged aerobic conditions in sterile tanks. In tank fermentation, a vegetative inoculum is first produced in a nutrient broth, in which the culture broth is inoculated with a spore of the microorganism in order to obtain a young, active culture which is then aseptically transferred into the medium of the fermentation tank. The tanks and bottles can be ventilated by pressing sterile air through or onto the surface of the fermentation medium and mechanically stirring in the tank. If necessary, anti-foaming agents such as silicone oil, soybean oil or bacon oil can be added.

The course of the fermentation can be followed by testing the fermentation medium from time to time using a microorganism sensitive to lucopeptin E2, such as Bacillus sub-tilis ATCC 6633. Lucopeptin E2 can also be in the UV range 360 nm or by its Rf value of 0.37 in silica gel thin layer chromatography system xylon-methyl-ethyl-ketone / methanol (9: 9: 1 v / v).

Isolation of luzopeptin E2:

Luzopeptin Ea can be isolated from the fermentation broth by conventional methods; for example by liquid extraction and chromatography methods, as described in detail in Example 1. Such processes allow the lucopeptin E2 component to be obtained in a purified form which is essentially free of co-produced products.

Biological activity:

The in vitro minimum inhibitory concentrations (MIC) of lucopeptin E2 were determined by an agar dilution series. In general, Mueller-Hinton agar was used for gram-positive and gram-negative bacteria, except for acid-resistant bacteria, for which the No. 1001 medium (3% glycerol, 0.3% sodium L-glutamate, 0.2% peptone, 0 , 31% Na2HPÛ4, 0.1% KH2PO4, 0.005% ammonium citrate, 0.001% MgSÜ4 and 1.5% agar) was used. The results are shown in Table 1 in comparison with the activity of lucopeptin A.

Table 1

In vitro antimicrobial activity for lucopeptin E2

MIC in µg / 'ml

Test organism lucopeptin lucopeptin

E2 A

Staphylococcus aureus 209P

0.4

0.4

Staphylococcus aureus Smith

0.4

0.8

Staphylococcus pyogenes S-23

<0.05

0.2

Sarcina lutea PCI 1001

0.1

0.2

Micrococcus flavus D12

0.2

0.2

Corynebacterium xerosis 53K-I

0.8

0.8

Bacillus subtilis PCI 219

0.2

0.8

Bacillus megaterium D-2

0.2

0.2

Bacillus anthracis A9540

0.1

0.2

Escherichia coli NIHJ

> 100

> 100

Klebsiella pneumoniae D-l 1

> 100

> 100

Proteus vulgaris A9436

> 100

> 100

Pseudomonas aeruginosa A9930

> 100

> 100

Mycobacterium smegmatis 607

0.8

0.4

Mycobacterium phlei D88

1.6

0.4

The antitumor activity of luzopeptin E2 against leukemia P388 was determined by known methods in BDFi mice (Can. Chem. Rep. 50,479, 1966 and Can. Chem. Rep. 3,1, [1972]). The results of this test are shown in Table 2 below, along with the results for lucopeptin A.

Table 2

Effect of luzopeptin E2 on P-388 lymphoblastic leukemia

Compound dose (mg / kg / day) tumor inhibition

MST (% T / C)

Lucopeptin E2 64 toxic

32 144

16 167

8 122

4 122

2 100

1 100 lucopeptin A 64 156

32 161

16 144

8 144

4 128

2 111

Treatment: once a day for 9 injections

Host: BDFi female mice

Evaluation- MST = mean survival time% T / C = MST

treatment: treated / MST control x 100

Criterion: T / C> 125 is more pronounced

Antitumor effect considered toxicity: <4/6 surviving, 5 day 5

In a second test, the antitumor activity of luzopeptin E2 against leukemia P388 in BdFi mice was determined by the method as described in J. Antibiotics 33, 1087-1097 (1980). As shown in Table 3, lucopeptins A and E2 have almost equivalent antitumor activity. The intraperitoneal toxicity, which is determined after multiple dosing (qd 1 -> - 9) of lucopeptin A and E2, is shown in Table 3 on the last line. The toxicity of luzopeptin E2 is almost Vi that of lucopeptin A.

Table 3

Antitumor activity of luzopeptin A on P388 leukemia

Dose * (ip) antitumor activity (% T / C% in MST)

in mg / kg / day lucopeptin A lucopeptin E2

0.1

0.03

89

143

0.01

174

181

0.003

168

149

0.001

141

129

0.0003

121

117

MED * (mg / kg /

0.001

0.001

Day)

LD5o * (mg / kg /

0.019

0.034

Day)

* Dosage schedule: qd l - »- 9

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As shown by the antimicrobial and mouse, tumor data, luzopeptin E2 is useful as an antibiotic and also as an anti-tumor agent for inhibiting malignant tumors of warm-blooded animals, such as P388 leukemia. To prepare pharmaceutical compositions, an effective antimicrobial or anti-tumor amount of luzopeptin E2 is combined with an inert pharmaceutically acceptable carrier or diluent. Such compositions can also contain other active antimicrobial or antitumor substances and can be converted into any convenient form as required for the desired route of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups or elixirs and preparations for parenteral administration such as sterile solutions, suspensions or Emulsions. They can also be made into sterile solid compositions which can be dissolved in sterile water, physiological saline or some other sterile injectable media immediately before use.

The following example serves to explain the invention. "Pharmamedia" is a trademark of Buckeye Oilseed Products Co., Fort Worth, Texas, for cottonseed flour. "Nutrisoy" is a trademark of Archer Daniels Midland Co., Decatur, Illinois, for soybean grains, ground in flour. "Fermo 30" is a trademark of Yeast Products Inc., Clifton, New Jersey, for yeast powder. "Skelly-solve B" is a trademark of Skelly Oil Co., Kansas City, Missouri, for a petroleum solvent that contains isomeric hexanes and has a boiling point of 60-68 ° C. "Nylon Copol 8" is a tube-like piece made of nylon, which is used for columns instead of glass. The nylon can withstand certain solvents and when the chromatography is complete the column can be cut to obtain the fractions.

example

Production of Luzopeptin E2:

A. Fermentation

A well grown vegetative preparation of Actinomadura luzonensis strain G455-101 was stored frozen (- 12 ° C) in sucrose with a final concentration of 20%. This frozen vegetative preparation was used to inoculate a vegetative medium containing 3% Cerelose, 1% Pharmamedia (cottonseed flour), 1% Nutrisoy (soybean meal powder) and 0.3% CaCCh. The inoculum was incubated at 35 ° C for 48 h in a rotary shaker (210 revolutions / min) and 7 ml of the culture was transferred to a 500 ml Erlenmeyer flask containing 100 ml of the fermentation medium consisting of 3% cerelose, 2% Pharmamedia, 1% Ferme 30 (yeast powder), 0.5% CaCCh and 0.15% DL-methionine, and the pH was adjusted to 7.0 before sterilization. The production of luzopeptin E2 reached a maximum between 72 and 96 h at an incubation temperature of 35 ° C.

B. Evidence

Luzopeptin E2 can be isolated from the fermentation mixture by diluting the entire broth with an equal volume of 0.5 molar Tris-HCl buffer, pH 8, followed by extraction with two broth volumes of methylene chloride. After briefly centrifuging to break the emulsion, the solvent layer was spotted on Analtech silica gel plates and developed in a xylene / methyl ethyl ketone / methanol (9: 9: 1) system. Luzopeptin E2 appears at an Rf of 0.37 between BBM-928A (Rf 0.57) and BBM-928B (Rf 0.27). All three components can be detected by UV reading at 360 nm or by bioautography, using agar adjusted to pH 8.0 and inoculated with Bacillus stubti-lis ATCC 6633.

C. Isolation

The whole beer (101) from the fermentation of Actinomadura luzonensis, strain G455-101, grown in the presence of DL-methionine (1.5 g / 1), was adjusted to pH 8 with sodium carbonate and for 2 h with 4 1 n-buta-nol stirred in the presence of diatomaceous earth as a filter aid. The resulting mixture was filtered and the organic phase was separated and concentrated to an oily residue under reduced pressure. Trituration of the oily residue with Skellysolve B (trademark of Skelly Oil Co. for petroleum solvents, containing isomeric hexanes with a boiling point of 60-68 ° C) and diethyl ether gave 4 g of an enriched lucopeptin complex after filtering and drying. The enriched lucopeptin complex was chromatographed on a dry silica gel column, manufactured according to Loev and Goodman (Progress in Separation and Purification, Vol. 3, p. 73, 1970). Dry column silica gel (Merck; 280 g) was reactivated by adding 40 ml of distilled water and shaking vigorously for 3 h, followed by equilibration for a further 3 h with 18 ml of a mixture of xylene / methyl ethyl ketone (1: 1 , Volume). The silica gel was then used to pack the column using a caliber 160 nylon Copol 8 (4x 100 cm) column and glass wool.

The lucopeptin complex (4 g) was filtered in 30 ml of xylene / methyl ethyl ketone (1: 1, volume), coated on silica gel with removal of the solvent under reduced pressure, and placed on a dry column. The column was developed with methyl ethyl ketone / xylene / methanol (5: 5: 1, volume) until the eluent had just flowed through the bed of the column. While observing the column with a portable UV lamp at 360 nm, the yellow fluorescent zones were cut out of the column, and the silica gel contained in these zones was eluted with chloroform / methanol (1: 1), volume), and after thin layer chromatography (TLC ) Analysis for SiO:

(Methyl ethyl ketone / xylene; 5: 5: 1, volume) the solids containing lucopeptin E2 were combined (494 mg). The enriched lucopeptin Ej fraction (494 mg) was dissolved in methylene chloride, applied to silica gel and chromatographed on a 2 x 80 cm silica gel column, which was packed in toluene with a slurry and eluted with a toluene / methanol gradient (0-10%) . The fractions were collected and analyzed by TLC on silica gel (methyl ethyl ketone / xylene / methanol: 5: 5: 1, volume). Elution with 7% methanol gave lucopeptin A and contaminants, after which lucopeptin A and lucopeptin E: (fractions 71-75) and pure lucopeptin E: (fractions 76-80) were eluted. The crystallization of pure luzo-

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Claims (4)

663 415 1 CO I CH9 ^ NH I k ^ N-KXKM-OTK »^ NvVj ^ S CH — N — C0-CHo-N-C0-CH ^ -NH-C - L I 1 2 1 2 II HO ho-k: ch3 ch3 o H3c ch3 2. A process for the preparation of the antitumor antibiotic luzopeptin E2 of formula I according to claim 1, characterized in that the microorganism Actinomadura luzonensis ATCC 31491, or a lucopeptin Ez-pro-reducing mutant thereof, in an aqueous nutrient medium, the assimilable carbon - Contains and sources of nitrogen, is cultivated under submerged aerobic conditions until a substantial proportion of the lucopeptin E2 has been generated by the microorganism in the culture medium, and the lucopeptin E2 is isolated from the culture medium so that it is essentially free of substances which are simultaneously created with it. 2nd PATENT CLAIMS 1. The antitumor antibiotic lucopeptin E2 of the formula 3. The method according to claim 2, characterized in that a sulfur-containing compound selected from the group consisting of D, L-methionine, S-methyl-cysteine, S-ethyl-L-cysteine, D, L-ethionine, D-methionine , L-methionine, acetyl-methionine, 2-aminoethanethiol, 2-hydroxy-4- (methyl-thio) -butyric acid, D, L-0-methyl-serine, L-cysteine and L-methionyl-glycine in the fermentation step is added to the nutrient solution. "3 ^ 3 ch3 H ^: Ç" "O = C-NH-ch2- <X> N-CH2-CO-N-ÇH, D | io CO-NH-CH-CO-W ^ I ^ «H ^ k2 a) 0 I 4. The method according to claim 2, characterized in that D, L-methionine or S-methyl-cysteine is added to the nutrient solution in the fermentation step.