DE3403125A1 - ANTIBIOTIC LUZOPEPTIN E (DOWN ARROW) 2 (DOWN ARROW), METHOD FOR THE PRODUCTION THEREOF AND PHARMACEUTICAL AGENT CONTAINING THIS COMPOUND - Google Patents

Description

• m *

• · * a

M / 25 004; kJ.

The invention relates to a new cyclic depsipeptide, which is an antibiotic, and a method its manufacture and extraction. The invention also relates to pharmaceutical agents which contain this new antibiotic included, and the use of this antibiotic as an antimicrobial agent and as an anti-tumor center 1.

GB application 2,050,384A describes an antibacterial one Antitumor complex designated BBM-928 and its production by fermentation of a new strain of Actinomycates. This strain is designated G455-101 (ATCC 31491). These The reference also mentions that the BBM-928 complex Consists of at least six components, labeled BBM-928 A, B, C, D, E and F. The parts A, B, C and D are fully characterized in this document, while components E and F only by their Rf values in two silica gel TLC (thin layer chromatography) systems and by determining their in vitro antimicrobial effectiveness against certain aerobic bacteria are characterized.

The BBM-928 producing organism was later found to be strain G455-101 a new species of the genus Actinomadura. This strain was later named Actinomadura luzonensis nov.sp. denotes [see J. Antibiotics 53 (10): 1098-1102 (1980)].

Both in J.Am.Chem.Soc. 103 ; 1241 (1981) and in Peptide Chemistry 1980, K. Okawa (editor), Protein

. :::::::: ··: · 3A03125 Μ / 25 004 ^

Research Foundation, Osaka, Japan, pp. 119-124 (1981) and J. Antibiotics £ 4 (2): 148-159 (1981) are the Structures of BBM-928 A, B and C described.

The manufacture, isolation, characterization and anti-tumor efficacy of the BBM-928 ingredients are in J. Antibiotics 52 (10) ι 1087-1097 (1980) disclosed. As in GB application 2,050,384A are the ingredients E and P merely by their Rf values in two SiIikagel TLC systems characterized.

In Biochemistry V% \ 5537 (1980) a mechanism for the anti-tumor efficacy of BBM-928A is suggested.

The BBM-928 antibiotics are currently referred to as lucopeptin antibiotics from the species that produce them. In the following, the designation luzopeptin A ₉ B ₈ C, etc. used here is identical to the designation BBM-928A, B ₉ C ₉ etc. previously used in the literature.

FIG. 1 shows the IR spectrum of lucopeptin E ₂ , recorded in KBr-PiIIe ₈

Fig. 2 shows the proton NMR spectrum of lucopeptin E ₀₉ dissolved in CDCl3.

Fig. 3 shows the -T ~ NMR spectrum of lucopeptin E ₂₉ dissolved in CDCl ,.
30th

The invention relates to a new antitumor antibiotic which is designated _{lucopeptin E 2.} The invention also relates to a process for its production and isolation in purified feed

Μ / 25 004

stood and free of substances produced together with it. The antibiotic is obtained by culturing a lucopeptin E ₂ -producing strain of Actinomadura luzonensis, preferably Actinomadura luzonensis ATCC 31491, or a mutant thereof in an aqueous medium containing assimilable carbon and nitrogen sources under submerged aerobic conditions. Cultivation is carried out until a sufficient amount of lucopeptin Eg has been produced by said organism in said culture medium. The lucopeptin E _{2 is then obtained} from the culture medium in purified form so that it is essentially free of the substances produced together with it.

Luzopeptin E ₂ exhibits both antimicrobial and antitumor properties.

It has been found that the lucopeptin E ₂ provided according to the invention is a minor component of the antibiotic lucopeptin (or BBM-928) complex disclosed in GB application 2,050,384A. This application describes the fermentation of Actinomadura luzonensis sp.nov. for the production of an antibiotic lucopeptin complex and the separation of this complex into six bioactive components, which are now referred to as lucopeptins A, B, C, D, E and F. However, this publication does not disclose the antibiotic component lucopeptin E ₂ , which - as has now been found - is co-produced during the fermentation of Actinomadura luzonensis.

Μ / 25 004 y £> ·

The novel antibiotic according to the invention was obtained from the fermentation broth of Actinomadura luzonensis in im essentially pure form and isolated by its physical, chemical and biological properties characterized. These properties are described in more detail below.

Studies j, which were carried out after the isolation and characterization of lucupeptin E ₂ , have shown that lucopeptin E (see for example GB application 2 050 384A) is not a single, antibiotic component, but rather a mixture of components including lucopeptin E ₂ represents.

Luzopeptin E ₂ is an antibiotic, cyclic depsipeptide that contains a quinoline nucleus as a chromophore. From spectral and chemical analyzes, luzopeptin E _{2 was assigned} the following structural formula?

M / 25

Μ / 25 004 X ^ S

Luzopeptin E ₂ is a white solid with a melting point of 201 to 203 ° C "It has a molecular weight of 1315» 3732 and the following empirical formula% ⁰ OO ^ Te ¹¹ 14 ° 20 * ^Elem ®ntar analyzes showed the following average percentages by weight:

Carbon 53.19

Hydrogen 5 * 40
Nitrogen 12.92

Oxygen 28.49
(by difference)

The XR spectrum of lucopeptin S ₂ , recorded in KBr-PiIIe ₉ is shown in FIG. 1. Characteristic IR absorption bands are found at the following frequencies, expressed in cm " ¹ 2 3470, 3370, 2980, 2983, 2850 , 1740, 1645 ₉ 1520, 1418, 1350, 1285, 1230, 1195, 1150, 1128 ₉ 1105, 1065, 1025, 905, 885, 795, 785 and 750.

The ¹ H and ^ C-WMR spectrum of luzopeptin E ₂ was recorded with a Bruker spectrometer WM-360 at 36Ο MHz and 90 MHz. The PMR and CMR spectra are shown in Figures 2 and 3,

In a solvent of lg @ @ _p miseh consisting of xylene-methyl ethyl ketone-M®thanol (5sfKi Y / V) ₉ Luzopeptin Ep has in © © n Rf value of 0.33 as determined by silica gel Dünnschichtchromatographi ©.

The main structural difference from lucopeptin Eg g © g © nüb @ r d @ n "b © kaBat © a, Lusopeptia ingredients lies in the aw © g © ah © it © iner hexahydropyridazine unit instead of © in © r tetrahydropyridazine group. 35

M / 25 004 ^

Luzopeptin E ₂ can be produced by cultivating a lucopeptin E _2- producing strain of Actinomadura luzonensis, preferably a strain of Actinomadura luzonensis with the properties of ATCC 31491 or a mutant thereof, under submerged, aerodynamic conditions in an aqueous nutrient medium.

The organisms producing lucopeptin E ₂ and the corresponding fermentation conditions are described in GB application 2,050,384A.

Actinomadura luzonensis strain G455-1O1 (ATCC

31491) a. However, any lucopeptin E ₂ -producing strain or a mutant of the preferred organism which can be obtained from such an organism by conventional methods, for example irradiation with X-rays or UV rays, treatment with nitrogen mustard, exposure to phages and the like, can be obtained, use according to the invention.

The producing organism is left in a usual Grow nutrient medium that contains an assimilable source of carbon. One such source of carbon is for example starch, glucose, dextrin, maltose, lactose, sucrose, fructose, mannose, Molasses, glycerin and the like. The nutrient medium should also include an assimilable source of nitrogen, for example Protein, protein hydrolyzate, polypeptides, amino acids, corn steep liquor, casein, urea and the like., as well as inorganic nutrient salts that provide inorganic anions and cations, such as potassium, Sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, nitrate and the like.

M / 25 004 ff AO.

Although it is not necessary for the production and recovery of lucopeptin Ep, it is preferred to add a sulfur-containing compound to the nutrient medium. This sulfur-containing compound is selected from D, L-methionine, S-methyl-cystelene, S-ethyl-L-cysteiny D, L-ethionine ₉ DM © th ± onin ₉ L-methionine, acetyl methionine, 2-amino @ thanthiol , 2-Hydroxy-4- (methylthio) -butyric acid, D _s LO-methyl-serine, L-cysteine and L-methionyl glycine · The preferred sulfur-containing compounds are D _s ! «Methionine, and S-methylcysteine. These sulfur-containing compounds can be added to the fermentation medium, for example, in a concentration of about 0.05 to about 0.5% (G®w _o / vol.), DI® the most preferred concentrations for DyL-Methionln and S-methyl-cysteine are at Og159o and & _$ Λ% _Ο It has not been found that the addition of the above-mentioned sulfur-containing compound increases the production of lucopeptin Eg, so that it can be produced and isolated more quickly. "

In the production of Luz © p © PTIN Eg you can at any temperature arbelt © n _o di © Is conducive to satisfactory growth of the producing organisms for ©. One can at temperature © N ₉ is between about 20 ° and about 45 ⁰ C li © © n g ₀ D r © particularly preferred Temperaturb © © r i is a maximum production of Luzopeptin E ₂ 27-35 ⁰ receives G. one generally after about 72 to 96 hours «

With the Fem © ntationsv © rfahr © H one can use the usual Approach methods “This is how, for example, small Quantities in bulk bottles or through surface culture doors h © r. Larger sizes are produced by

M / 25 004 ff 41.

preferably works in sterile tanks under submerged, aerobic conditions. For tank fermentation one puts first prepare a vegetative inoculum in a nutrient broth by culturing the nutrient culture with spores from the The organism is inoculated to provide a young, active seed culture which can then be aseptically introduced into the Medium of the fermentation tank transferred. The ventilation in the tanks and bottles is achieved by blowing sterile air through or onto the surface of the Fermentation medium passes, stirring in the tanks with the help of a mechanical rotary stirrer. Antifoam agents such as silicone oil, soybean oil, and lard oil can be added if necessary.

The course of the fermentation can be followed by examining the fermentation medium from time to time with an organism that _{responds to lucopeptin E 2} , for example Bacillus subtilis ATCC 6633. Luzopeptin E _{2 can} also be determined in the UV spectrum at 360 nm or by its Rf value at 0.37 in the silica gel TLC system xylene-methyl ethyl ketone-methano1 (9: 9: 1 v / v).

Customary methods are used to isolate luzopeptin E ₂ from the fermentation broth. These include, for example, solvent extraction processes and chromatographic processes, which are described in more detail in Example 1 below. These processes make it possible to obtain the lucopeptin E ₂ component in a purified form that is essentially free of the products produced together with it.

M / 25 004 Iß / M

The in vitro minimum inhibitory concentrations (MIC) of lucopeptin E ₂ were determined by the serial agar dilution method. Mueller-Hinton agar has generally been used for gram-positive and gram-negative bacteria. In the case of acid-resistant bacteria, however, medium No. 1001 (3% glycerine, 0.3% sodium L-glutamate, 0.2% peptone, 0.31% Na ₉ HPOz ₁₈ 0.1% KH ₉ POz ₁ , 0.005% Ammo -10 sodium citrate, 0.001% MgSO ₄ and 1.5% agar) are used. The results obtained in comparison to the effectiveness of luzopeptin A are summarized in Table 1.

Table _1_

in vitro antimicrobial © activity of lucopeptin E _P

-— _— .. «. έ—

Test organism MIC in / µg / ml

Luzopeptin E ₂ Luzopeptin A

Staphylococcus aureus 209P Staphylococcus aureus Smith Streptococcus pyogenes S-23 Sarcina lutea PCI 1001 Micrococcus flavus D12 Corynebacteriuro xerosis 53K-I Bacillus subtilis PCI Bacillus megaterium D-2 Bacillus anthracis Ä9540 Escherichia coli NIHJ Klebsiella pneuxnoniae D-Il Proteus vulgaris A9436 Pseudomonas aeruginosa A9930 Mycobacterium smegmatis Mycobacteritun phlei D88

0 .4 0 .4 0 .4 0 .8th <0 .05 0 .2 0 .1 0 .2 0 .2 0 .2 0 .8th 0 .8th 0 .2 0 .8th 0 .2 0 .2 0 .1 0 .2 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 0. ,8th 0. .4 1. , 6 0. .4

M / 25 004 yffö.

The antitumor activity of lucopeptin E ₂ against leukemia P388 was determined with the aid of BDP ₁ mice according to methods described earlier (Can.Chem.Rep. 5JD: 479, 1966 and Can.Chem.Rep. J5: 1, 1972). The results of this test, as well as the results of the test for lucopeptin A, are shown in Table 2 below.

Table 2

Effect of lucopeptin E ₂ on P-388 lymphocytic leukemia ^

Compound dose (mg / kg / day) tumor inhibition \ MST (% T / C)

Luzopeptin E ₂ 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 nine injections
Host: BDF ₁ -Mouse (female)
Evaluation: MST = mean survival time

% T / C = MST treated / MST control χ 100

Criterion: T / C * 125 was considered a significant antitumor _K effect

Toxicity: <4/6 survivors, day 5

M / 25 004 / Υ

In a second test, the antitumor activity of lucopeptin E ₂ against leukemia P388 was determined on BDF ₁ mice in accordance with the method described in J. Antibiotics ££: 1087-1097 (1980). As a comparison compound, luzopeptin A was tested. As can be seen from Table 3, luzopeptin A and E _{2 show} almost equivalent antitumor activity. The toxicity determined intraperitoneally after multiple doses (qd 1 -> 9) of luzopeptin A and E ₂ is listed in the last line of Table 3. The toxicity of luzopeptin Ep is only half that of luzopsptin A.

Table 5
Antitumor activity of lucopeptin A versus

P388 Leukemia _

Dose ⁴ "(ip) antitum or _T activity (% T / C% in MST) in mg / kg / day luzopepfin A lucopeptin E ₀

143 181 149 129 117

0.001 0.034

⁺ Dosage schedule qd 1 - ^ 9

From the above data üb © r di © antimicrobial activity and di © efficacy against tumors in mice is ersichtlichs that Luzopeptin E ₂ is useful as an antibiotic and as an anti-tumor agent for inhibiting malignant tumors _s wi © P388 leukemia in Säugetie including humans _ren »The invention therefore also relates to pharmaceuticals

0.1 89 0.03 174 0.01 168 0.003 141 0 _s 001 121 0.0003 0.001 MED ⁺ (mg / kg / day) O ₉ 019 LD ₅₀ ⁺ (mg / kg / day)

M / 25 004 / A ^

Agents containing an effective antimicrobial or tumor-inhibiting amount of lucopeptin E ₂ together with an inert, pharmaceutically acceptable carrier or diluent. These agents can also contain other active, antimicrobial, or anti-tumor agents, and can be formulated in any form suitable for the desired mode of administration.

These agents can, for example, be solid agents for oral administration, e.g. tablets, capsules, Pills, powders and granules. These agents can also be in liquid form for oral administration, for example as solutions, suspensions, syrups or elixirs. You can also use preparations for the represent parenteral administration, e.g. sterile solutions, suspensions or emulsions. You can also are in the form of solid, sterile means that are quickly in sterile water, physiological before use Saline or other sterile injectable medium can be dissolved.

The invention also relates to the use of lucopeptin E ₂ for the therapeutic treatment of a mammal (including humans) suffering from a microbial infection or a malignant tumor (eg P388 leukemia). For this purpose, an effective antimicrobial or tumor-inhibiting dose of lucopeptin E ₂ or a pharmaceutical agent containing this compound is administered to the mammal or the host.

The following examples serve to illustrate the invention in more detail without, however, restricting it. Pharmamedia is a trademark of Buckeye Oilseed Products Co., Fort Worth, Texas, for cottonseed meal.

M / 25 004 y // fC,

Nutrisoy is © in trademark of Archer Daniels Midland Co., Decatur, Illinois, for a soybean meal that is finely ground. Fermo 30 is a trademark of Yeast Products Inc., Clifton, New Jersey, for a yeast powder. Skellysolve B is a trademark of Skelly Oil Co., Kansas City, Missouri, contains a mineral oil solvent, the isomeric hexenes, and has a boiling point of 60-68 ⁰ C. Nylon Copol 8 is a tube-shaped piece of nylon that is used as a column instead of glass. "The nylon is resistant to certain solvents. The column can thus be cut after the end of the chromatography in order to obtain the fractions.

Example 1

Production of lucopeptin E ₂

A. Fermentation
20th

A well-grown, vegetative preparation of Actinomadura luzonensis strain G455-101 holds one Frozen (-12 ⁰ C) in sucrose ₉ wherein the final concentration is 20%. This frozen vegetative preparation is used to inoculate a vegetative medium containing 4% Cerelose _9, 1% Pharmamedia (cottonseed meal), 1% Nutrisoy (soybean meal powder) and 0.3% CaCO. The seed culture is incubated for 48 h at 35 ⁰ C on a rotary shaker (210 rev / min). 7 ml of the culture are then transferred to a 500 ml Erlenmeyer flask containing 100 ml of a fermentation medium consisting of 3% Cerelose, 2% Pharmamedia, 1% Ferao 30 (yeast powder), 0.5% CaCO ^ and 0.15 # DL-methionine is composed, adjusting the pH to 7.0 before sterilization.

• «· ft I

M / 25 004

The production of lucopeptin E ₉ reaches between 72

- Gm

and 96 h at 35 C incubation temperature a maximum.

B. Determination

Luzopeptin Eg can be isolated from the fermentation mixture by diluting the entire broth with an equal volume of a pH 8, 0.5 M Tris-HCl buffer and subsequent extraction with twice the broth volume of methylene chloride. After brief centrifugation to break the emulsion, the solvent layer is spotted on Analtech silica gel plates and developed in a xylene-methyl ethyl ketone-methanol (9: 9: 1) system. Luzopeptin E ₂ appears at R _f 0.37 between BBM-928A (R _f 0.57) and BBM-928B (R _f 0.27). All three components can be determined by UV at 360 nm or by bioautography using an agar adjusted to pH 8.0 and inoculated with Bacillus subtilis ATCC 6633.

C. Isolation

All the broth (10 L) from the fermentation of the Actinomadura luzonensis strain G455-101, grown in the presence of DL-methionine (1.5 g / l), is made up with sodium carbonate to pH 8 and stirred for 2 hours with 4 liters of n-butanol in the presence of diatomaceous earth as a filter aid. The resulting The mixture is filtered, the organic phase is separated off and concentrated under reduced pressure to a oily residue. The oily residue is then rubbed in with Skellysolve B (trademark of Skelly Oil Co. for a petroleum ether solvent that is isomeric

Μ / 25 004 ^ / ff.

Comprises hexanes and has a boiling point of 60 to 68 ⁰ C) and diethyl ether, filtered, dried and obtained 4 g of an enriched lucopeptin complex. The enriched lucopeptin complex is chromatographed on a dry silica gel column which was prepared according to Loθν and Goodman (Progress in Separation and Purification, Volume 3, page 73, 1970). Dry columnar silica gel (Merck; 280 g) is deactivated by adding 40 ml of distilled water, shaking vigorously for 3 hours and then equilibrating for a further 3 hours with 18 ml of a mixture of xylene methyl ethyl ketone (1: 1 V / V). The silica gel is then used to pack the column using a 160 (gage) nylon Copol 8 (4 × 100 cm) column with the aid of glass wool.

The lucopeptin complex (4 g) is dissolved in 30 ml of xylene methyl ethyl ketone (1: 1 V / V) *, filtered and coated with silica gel by removing the solvent under reduced pressure, and the coated silica gel is applied to the dry column. The column is developed with methyl ethyl ketone-xylene-methanol (5: 5: 1 V / V) until the eluent runs almost over the column bed. The column is then examined with a portable UV lamp at 360 nm, the yellow-fluorescent zones are cut out and the silica gel sections obtained are eluted with chloroform-methanol (1: 1 V / V). After analysis by thin layer chromatography (TLC) for SiO ₂ (methyl ethyl ketone-xylene-methanol, 5s5: 1 V / V), the solids containing luzopeptin E ₂ (494 mg) are combined. The fraction enriched with lucopeptin E ₂ (494 mg) is dissolved in methylene chloride, coated with silica gel and chromatographed on a 2 × 80 cm silica gel column (packed as a suspension in toluene) with a toluene

Methanol gradients from 0 to 10%. The fractions are collected and analyzed by TLC on silica gel (methyl ethyl ketone-xylene-methanol, 5: 5: 1 V / V). Elution with 7% methanol gives lucopeptin A and impurities. Then lucopeptin A and lucopeptin E ₂ (fractions 71 to 75) and pure lucopeptin E ₂ (fractions 76 to 80) are eluted. Crystallization of the pure luzopeptin Eg from methylene chloride-toluene gives 22 mg of crystalline luzopeptin E ₂ -

- blank page -

Claims (6)

M / 25 004 5 1. Claims antitumor antibiotic lucopeptin Ep of the formula OH u r ra CH. S / ^J η C-OH Γ 3 ^H 3 ^C , I. O-C-NH-CH ₂ -CO-N-CH ₂ -CO-N-CH L NH j N-CO-CH-NH-O L CH — N — CO-CH, -N-CO-CH, -NH ~ C - II ² I ² Z / HO-C CH ₃ CH ₃ O H ₃ C ^S CH ₃ 2. A method for producing the antitumor antibiotic lucopeptin E "according to claim 1, characterized , that one Actinomadura luzonensis ATCC 31491 or a Luzopeptin E "producing mutants thereof in an aqueous, assimilable carbon and nitrogen source containing nutrient medium cultivated under submerged, aerobic conditions until in this culture medium a sufficient amount of lucopeptin E "has been produced by the said organism, and Μ / 25 004 ² that luzopeptin E ₂ 'is isolated from the culture medium essentially free of substances produced together with it. 3. The method according to claim 2, characterized in that a sulfur-containing compound is selected is under D, L-methionine, S-methyl-cysteine, S-Ethy1-L-cysteine, D, L-ethionine, D-methionine, D-methionine, L-methionine, acetyl-methionine, 2-aminoethanethiol, 2-hydroxy-4- (methylthio) -butyric acid, D, L-O-methyl-serine, L-Cyst © in and L-methionylglycine, in the Gives the permentation stage to the nutrient medium, 4. The method according to claim 2, characterized in that D, L-methionine od @ r S-methyl-cysteine in the Fermentation stage gives to the nutrient medium. 5. A pharmaceutical agent containing lucopeptin E ₂ according to claim 1, optionally together with customary carriers and / or diluents. 6. Use of luzopeptin E ₂ according to claim 1 for combating microbial infections or malignant tumors in mammals including humans.