CH622823A5 - Process for the preparation of antibiotics, namely mimosamycin and chlorcarcins A, B and C - Google Patents

Description

The present invention relates to a process for the preparation of new antibiotics, namely mimosamycin and chlorcarcine A, B and C.

In particular, the present invention relates to a process for the preparation of new antibiotics, namely mimosamycin, which has antibacterial properties against gram-positive bacteria and in particular against acid-resistant bacteria, and of three new antibiotics, namely the chlorocarbins A, B and C, and their acid addition salts which have anti-tumor effects. This method consists of cultivating Streptomyces lavendulae strain No. 314 (NRRL 11002), obtaining a complex of chlorcarcins and mimosamycin from a culture broth and isolating mimosamycin and the chlorcarcins A, B and C from this complex.

In particular, the present invention relates to a process for the preparation of mimosamycin, which consists in cultivating Streptomyces lavendulae strain No. 314 and obtaining mimosamycin from the culture broth, and to a process for the production of chlorocarcinols A, B and C and of Acid addition salts thereof, which consists in cultivating Streptomyces lavendulae strain No. 314, extracting a complex of the chlorocarbins from a culture broth and then the chlorcarcins from this complex of the chlorocarbins

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A, B and C isolated.

Isolation of approximately 2,000 different types of antibiotic substances has been disclosed, and it has become increasingly difficult to find a new antibiotic substance. Since the usual antibiotics are resistant to certain microorganisms, it is increasingly necessary to arrive at a new antibiotic substance. The development or research of new antibiotic substances also requires the appearance of new infectious diseases, which arise from the general use of antibiotics with a broad antibacterial spectrum, from steroid hormones, from antitumor agents or anti-immune substances.

Research has now been carried out for a new method for an important manufacturing method of antibiotic substances which are obtained by improving the breeding conditions with the aid of a known microorganism producing antibiotic substances. Based on this research, it was found that a Streptomyces lavendulae microorganism capable of producing the known antibiotic streptothricin also leads to new and valuable antibiotic substances, namely chlorcarcins A, B and C and mimosamycin, and it has also been found that Streptomyces lavendulae strain No. 314 are able to produce high effective units of these new antibiotic substances.

The aim of the present invention is therefore to provide a process for the preparation of said new antibiotics, namely mimosamycin and chlorocarcinols A, B and C, which have excellent biological properties.

The present invention further relates to a method for the fermentative production of these new antibiotics.

Streptomyces lavendulae strain no. 314 can be isolated from a soil sample that can be found in Kyoto and belongs to the genus Streptomyces. This strain is under number 3218 from Technical Research Institute of Microbial Indu-stry, Agency of Industriai Science & Technology, Ministry of International Trade and Industry, Japan, and also under number NRRL-11002 in the Northern Regional Research Laboratori Northern Central Region , Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois, USA. He was deposited here on August 10, 1976.

Observation of the aerial mycelium and spores of Streptomyces lavendulae strain No. 314 is carried out by cultivating the strain on media according to the International Streptomyces Project (ISP) (Shirling. EB & D. Gottlieb; International J. Systematic Bacteriol. 16,313-340,1966 ) carried out. This is done by placing a yeast-starch agar medium, an agar medium containing inorganic salts and starch and / or a basal medium containing maltose on agar plates using a carbon source pattern (Pridham-Gottlieb agar medium) for 1 to 2 weeks grows at 27 ° C. The colors of the mycelium with ripe spores, the vegetative mycelium and so on were determined according to the color scale described in "Descriptive Color Name Dictionary", Container's Corporation of America, 1950, and "Color Harmony Manual", Container Corporation of America, 1958.

The trunk no. 314 develops a wavy, wrinkled aerial mycelium, which has long branches with a through-hole 622823

has a diameter of approximately 0.6 to 1.0 n and shows some cylindrical spores. The spores have a size of 0.6 to 1.0 u * 0.8 to 2.0 (j,. According to the standard information in ISP, it can be assumed that the strain corresponding to the above-mentioned morphological properties belongs to the subgroup Rectiflexibles. However the aerial hyphae have eyelets or incomplete or elongated spirals with 1 to 2 turns, therefore strain No. 314 is morphologically defined as belonging to the subgroup Retinaculiaperti The no. 314 strain has special properties in that the aerial hyphae are morphologically ascribed to the subgroup Retinaculiaperti, the color of the aerial hyphae with ripe spores on various media is pink to lavender, the color of the vegetative mycelium on a synthetic one Medium is now and then blue to bluish brown and the production of melanin pigment posi tiv runs. When researching strains of the genus Streptomyces, which are described in "The Actinomycetes", S.A. Waksman, volume 2.1959 and "Bergey's Determinative Bacteriology", 8th edition, 1974, it can be assumed that this strain has a strong similarity to Streptomyces lavendulae. The strain No. 314 is inoculated in a medium customary for the preparation of an antibiotic substance and the culture medium is shaken at 27 ° C. for a growth period of 18 to 72 hours in order to obtain a culture filtrate which is highly effective against coliform bacilli. The filtrate thus obtained is then treated with activated carbon under alkaline conditions, eluted with acetone acidified with acid or over a weak cation exchange resin, e.g. B. Amberlite IRC-50 (trademark of Rohm & Haas Co., USA) adsorbed and subjected to desorption by means of 0.1 l hydrochloric acid in order to obtain a substance which is anti-acterial against coliform bacilli. The substance thus obtained is purified by chromatography on, for example, Amberlite CG-50 (trademark of Rohm & Haas Co.) and crystallized in the form of the corresponding Reinecke salt or in the form of the picrate, whereupon this substance is identified as streptothricin.

Furthermore, using Streptomyces lavendulae IFM 1031, which is a streptothricin-producing strain, Streptomyces racemochromogenes IFM 1081, which appears to be identical to Streptomyces lavendulae and is capable of producing streptothricin, and for ultimate identification using the strain in question according to the present invention Comparisons were made with the breeding characteristics and the physiological characteristics. The results can be found in Tables 1, 2 and 3. Specifically, it was found that strain No. 314 is identical to Streptomyces lavendulae in terms of any characteristic properties that are currently used to identify the strain in the genus Streptomyces, although in in some respects, for example regarding the utilization of L-arabinose etc., slight differences have been found. Based on these findings, this strain was identified as Streptomyces lavendulae. Streptomyces racemochromogenes can also produce streptothricin, but this strain is obviously different due to the comparative results mentioned above compared to strain No. 314.

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Table I

Comparison of Streptomyces strain No. 314 with known strains

medium

Strain No. 314

S. lavendulae IFM 1031

S. racemochromogenes IFM 1081

Sucrose

VM

abundant, spread, abundant, spread

"Faint brown" to

Nitrate-

tend, "light olive"

tend, colorless to «faint

"Purple brown" (11 nl)

Agar

(1 Vi ie) *

brown »

(Czapek-

AT THE

abundant, «white» to abundant, «white» to abundant, «ivory»

Medium)

«Ivory» (2 db) with

«Ivory» (2 db) with

(2 de) to «gray»

Lavender shade

Lavender shade (5 ge to

(5 de)

(5 ge-4 ig)

4 ig)

SP

none up to «faint brown»

"Faint brown"

"Faint brown"

Glucose-

VM

plentifully, plentifully plentifully, plentifully

Asparagine

tend, colorless to «light tend,« grayish blue »

spreading "brown" to

Agar

olive »(l'Aie)

(10 pn)

"Blue" (10 pn)

AT THE

abundant, «grayish abundant,« white »late

abundant, «reddish

pink »(5 ec) with lavender

ter in «reddish gray»

gray »with lavender

color (5 ge-4 ig)

with lavender hue (5 ge) transition hue (5 ge)

SP

none up to «faint brown»

none up to «faint brown»

none up to «faint brown»

Glycerol

VM

abundant, spread abundant, spread dark olive (lVipn)

Asparagine

tend, colorless tend, dark olive

Agar

(l '/ 2 pn)

AT THE

moderate, «faint brownish moderate,« faint brownish moderate, «silver gray»

gray »(2 fe) to« silver gray (2 fe) to «silver

(3fe)

gray (3 fe)

gray (3 fe)

(ISP)

SP

none light brown

"Faint olive brown"

Tyrosine agar

VM

plentifully, plentifully plentifully, plentifully

tend, «faint brown» to tend, «faint brown» to broad, «mustard

"Mustard brown" (2 ni)

"Mustard brown" (2 ni)

brown »(2 ni)

AT THE

plentiful, «reddish abundant, reddish plentiful,« reddish

gray »(7 ge) with La

«Gray» (7 ge) with gray »(7 ge)

vendel shade (5 ge to

Lavender shade

4 ig)

5 ge-4 ig)

SP

none up to «faint brown»

none up to «faint brown»

none up to «faint brown»

Calcium hy-

VM

abundant, spread abundantly, spread abundantly, spread droxy-

succinate agar

tend, «faint brown»

tend, «bluish brown»

tend, «bluish brown»

to «dark olive» (1 ni)

(3pn)

(3pn)

AT THE

moderate, thin, powdery, «white» in «shell tint» (3 ba) none at all

SP

none up to «faint brown»

"Faint olive brown"

"Faint olive brown"

inorganic

VM

moderate, colorless moderate, colorless to

"Dark olive" (2 po)

Agar containing salts and starch

(ISP) nutrient agar

AT THE

SP VM

AM SP

abundant, spreading, «yellowish gray» (3 de)

none abundant, spreading, glittering surface, «camel» (3 ie) none «brown»

"Black olive" (2 pn)

abundant, spreading, «yellowish gray» (3 de)

none abundant, spreading, glittering surface, «camel» (3 ie) none «brown»

abundant, «gray» (3 de), «bluish gray» (13 ee)

«Faint brown» abundant, spreading, glittering surface, «camel» (3 ie) none

"Faint brown"

5 622823

Medium strain No. 314 S. lavendulae S. racemochromogenes

IFM 1031 IFM 1081

Yeast extract

VM

abundant, spread abundant, spread wrinkled, «faint brown»

Malt extract-

tend, strongly folded,

tend, colorless to

Agar

colorless to «faint brown»

"Faint brown"

AT THE

abundant, «grayish abundant,« grayish abundant, «reddish

pink »(5 ec) with lavas pink» (5 ec) with lavas gray »(5 ge)

del color (5 ge-4 ig)

del color (5 ge-4 ig)

(ISP)

SP

"Oak brown" (4 pi)

"Oak brown" (4 pi)

"Dark brown" (4 pi)

Oatmeal

VM

moderate, colorless moderate, «dusty blue»

"Bluish brown" (15 ni)

Agar

(15 ni)

AT THE

moderate, «grayish pink»

moderate, «rose wood» with moderate, «rose wood»

(5 ec) with lavender

Lavender shade

(3 ca)

color (5 ge-4 ig)

(5 ge-4 ig)

(ISP)

SP

none none up to «bluish brown»

none up to «faint brown»

Egg medium

VM

spreading, spreading, spreading

wrinkled, «chocolaté

wrinkled, chocolate

«Chocolaté» (4 pl)

brown »(4 pn)

brown »(5 pn)

AT THE

none none little to none

SP

"Faint brown" to

"Faint brown" to

"Chocolate"

"Chocolate brown"

"Chocolate brown"

(4 pl)

(4 pl)

VM: vegetative mycelium; AM: aerial mycelium; SP: soluble pigment * "Color Harmony manual code"

Table 2 Table 3

Comparison of the physiological properties of comparison of a carbon source utilizing pattern of

Streptomyces strain No. 314 and known strains “° Streptomyces lavendulae strain No. 314 and known strains

Tribes

Physiological strain S. lavendulae S. racemo- carbon source strain S. lavendulae S.racemo-

Property No. 314 IFM 1031 chromogenes 45 No. 314 IFM 1031 chromogenes

IFM 1081 IFM 1081

nitrate

D-xylose *

-

±

-

reduction

L-arabinose *

+

-

-

(14 days)

+

+

+

so L-rhamnose *

-

-

-

liquefaction

D-glucose *

+

+

+

of gelatin

D-fructose *

±

±

-

(18 ° C,

Sucrose *

+

+

+

21 days)

+

+

+

Lactose

-

-

+

soluble pigment brown brown brown

55 maltose

+

+

+

Hydrolysis of cellulose

-

Raffinose * mannitol *

_

_

(21 days)

-

-

i-inositol *

-

-

-

Litmus milk

Sodium acetate

+

+

+

Coagulation

+ +

+ +

+ +

60 sodium citrate

+

+

+

Peptonization

+ +

+ +

+ +

Sodium succinate

+

+

+

pH

8.0

7.8

7.8

Blind test

-

-

-

Melanin

education

+

+

+

ss * carbon source described in ISP

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The chlorcarcinol complex compound and the mimosamy - evaporated to dryness, the concentrate in a little ethyl acetate, which are obtained according to the invention, have so far been dissolved, the solution in succession with an aqueous sodium

not yet from a culture broth of the above-mentioned, best hydrogen carbonate solution, sodium carbonate solution and known microorganisms, which are shaken and finally sodium hydroxide solution to Streptomyces lava.

dulae belong, have been isolated. 5 separates, whereupon the acidic substances in the aqueous phase

According to the invention, the chlorcarcin complex is obtained. The solvent phase is treated with ln-salt

binding and the mimosamycin extracted by cultivation of streptic acid, adjusted to a pH of 9 to 10 with an aqueous ammonia solution on myces lavendulae strain No. 314 and with chloroform

The breed can be extracted mainly for itself. This process is repeated several times by cultivation of methods known from microorganisms, whereupon the solvent phase is reduced, but usually pressure is dried to dryness to submerged cultures in order in this way to grip a crude basic liquid medium. As a component according to the invention which contains the chlorcarcin complex and mimicable medium, any nutrient containing samyein can be obtained. This component will be used over the medium, which the StammNr. 314 the Gat silica gel with a mixture of benzene and ethyl acetate of the Streptomyces tung can utilize. In particular, 15 column chromatography can be subjected, whereby one arrives at a synthetic, semisynthetic or natural media, which mainly consists of chlorocarcin A and which, as examples of medium components Koh- after developing with ethyl acetate, a fraction which contains sources of oxygen such as e.g. B. contains glucose, maltose, fructose, xylose, mainly mimosamycin. These fractions starch, glycerin, etc., as nitrogen sources, for example, are extracted several times by silica gel chromatography-meat extracts, peptone, gluten flour, cottonseed oil, soybean oil, chlorinated chlorine A and mimosamycin in the form of bean flour, corn steep liquor, dry yeast, yeast extract, one yellow syrups or yellow crystals. Ammonium sulfate, ammonium chloride, urea and others The chromatography column eluted previously can then be called organic or inorganic nitrogen sources. a mixture of ethyl acetate and acetone eluted, whereby one can also get to the medium carbonates, phosphates or to a fraction which mainly add the chloro-other metal salts. If there is an excess of 25 B and C during cultivation, whereupon the fraction should be foamed to dryness, it is customary and advantageous and the concentrate, namely a mixture of chlorocarbon, contains an anti-foaming agent , e.g. B. a vegetable B and C, again a silica gel chromatography under-shear oil, such as soybean oil, silicone oil, polyoxyalkylene-type, in order to add the chlorocarcin B and the chlorocar agent, mineral oils, etc., in this way. to get a C as a yellow powder.

The breeding temperature is generally 30

Ranges from approximately 27 to 30 ° C. As the volume of medi-mimosamycin increases, it is advantageous to use a seed culture. Raw mimosamycin is recrystallized from a mixture of ethylades and then this seed culture for inoculation with a acetate and ethyl ether to add to pure yellow Kri medium. The breeding period usually takes a long time to arrive.

about 18 hours to about 24 hours. 35 The ethyl alcohol obtained after extraction with l-hydrochloric acid

Depending on the production of the acetate layer according to the invention, the mixture is evaporated to dryness and the residue used in available antibiotics is dissolved in a suitable amount of methanol. The culture conditions are specified for this solution. enough water to add a water

To get the antibiotic content of 10% by volume enriched in culture broth in this way. Then the aqueous table substances are usually found within the solution extracted with n-hexane to remove the impurities

Mycelia and the culture fluid and are removed from those by lower polarity. The extract is thereupon

Centrifuging or filtering collected mycelia was concentrated to dryness and the residue was ethyl acetate and the filtrate was recovered. This is how they can be solved according to the invention. The solution thus obtained is washed successively with ln salt

according to available antibiotic substances by acid and in sodium hydroxide solution per se and thereon

Usually evaporated to dryness for the production of a natural product 45, taking measures used to obtain a residue and cleaning, for example, containing the raw neutral components,

the mimosamycin is also isolated and cleaned in the above in suitable solvents, the separability and the manner described from the components, the difference in a different speed

Procedure from a solution, the difference in the absorption 50 A. Physicochemical properties of mimosamycin possibility and in the affinity for different absorbents 1. Color and condition: yellow prism agents, the difference in the distribution between two 2nd melting point 227 to 231 ° C.

liquid phases, etc. These measures read 3. Elemental analysis:

if desired, alone or optionally below C = 61.51%, H = 4.79%, N = 5.87%

suitable combinations or repeat. A 55 4th molecular weight (mass spectrum): 233

suitable method is explained in more detail below. 5. Empirical formula: C12H11NO4

After the cultivation has ended, the culture broth is filtered, 6. Ultraviolet absorption spectrum (according to FIG. 1):

to separate the mycelia from the filtrate. The filtrate becomes UV A.N? Ghano1 nm Q0g e). 230 (shoulder) (4.16), 317 (4.14), 396 (3.56)

by adding 10N sodium hydroxide to a pH value of UV A. ^ thano1 nm (log e): 277 (3.78), 370 (3.53)

set from 8.0. To achieve a better infrared absorption spectrum (according to FIG. 2), the filtrate can:

Ren extraction effect with a solvent beforehand on the IR vgjgï enr1: 1685,1655,1635,1585 half or a third of the volume. With the 8th NMR spectrum (CDCb) (according to FIG. 3):

5: 2.10 (3H, see), 3.69 (3H, see), 4.20 (3H, see), 7.12 (1H, see), 8, 28 (1H, s.)

basic, water-soluble antibiotics, e.g. B. Streptothricin, 9. Rotational dispersion spectrum (C = 4.37 • 10 ~ 7, MeOH):

which are generated simultaneously with strain no. 314, in 65 [0] 20 (nm): -1601 (500), -1373 (490), -1373 (480), -1144 (470),

Filtrate back while the chlorcarcin complex, -1144 (460), -1144 (450-390), -1373 (380), -1144 (370-350),

the mimosamycin etc. go into the solution phase. The -1373 (340), -1831 (330), -2288 (320), -2288 (310), -1831 (300),

Solvent phase is then applied under reduced pressure -1601 (290), -2059 (280), -2517 (270), -3661 (260), -5492 (250)

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10. Solubility:

Easily soluble in methanol, ethanol, chlorine, esters and acetone; sparingly soluble in ethyl ether and n-hexane; insoluble in water

11. Color reaction:

Honest reaction: positive (orange)

Ninhydrin and Dragendorff reaction: negative

To a certain extent, this substance has biological properties against gram-positive bacteria, but is not effective against gram-negative bacteria and fungi at concentrations of 50 (ig / ml).

The substance is particularly effective against acid-resistant bacteria and against strains which are highly resistant to streptomycin in human tubercle bacilli and against normal strains of tubercle bacilli. The minimum inhibitory concentrations for Mycobacterium tuber-culosis are determined after 4 weeks of incubation at 37 ° C in a Sauton medium. The results can be found in Table 4 below.

Table 4

Antimicrobial effects of mimosamycin against tubercle bacilli

Test organism

MIC (| ig / ml)

Mycobacterium tuberculosis

H37RV

1.56

Mycobacterium bovis No. 10

6.25

Mycobacterium tuberculosis SMR

1.56

Mycobacterium smegmatis

ATCC607

25.0

Remarks:

Culture medium, 37 ° C, 48 hours for smegmatis. Liquid Sauton medium, 37 ° C, 4 weeks for other three strains. The strains SMR and Matsudo are resistant to streptomycin at 1000 ng / ml and more.

Mice (18 to 20 g) tolerated intravenous injections of 50 mg / kg mimosamycin.

The above physicochemical and biological properties indicate that the substance is a new antibiotic substance, which is particularly effective against tubercle bacilli.

Mimosamycin can be used for therapeutic purposes as an antimicrobial or antituberculosis agent in the form of various pharmaceutical preparations, e.g. B. in the form of tablets, powders, oral suspensions, syrups, capsules, solutions or suspensions for injection, etc., are used. These preparations can be easily prepared by using the antibiotic in a known manner. The antibiotic can be administered orally or parenterally, and more preferably intramuscularly or intravenously. The dosage to be administered varies depending on the conditions, but generally depends on the extent of the disease, the age and body weight of the patient, etc. In general, a daily dosage in the range of between 200 mg and 2 g will be used in adults, which can be done in one batch or in the form of different dosage amounts.

Chlorcarcine A, B and C

Chlorcarcins A, B and C are basic, antibiotic substances and can form the corresponding acid addition salts. Typical examples of such salts are those with inorganic or organic acids, such as. As hydrochloric acid, sulfuric acid, phosphoric acid, stearic acid, propionic acid, tartaric acid, maleic acid, etc. As expected, these types of salts have the same antibiotic effect as the free antibiotic substances, although certain differences in the range of action and in the range of solubility can be found.

The physicochemical properties of Chlorcarcine A, B and C can be found below:

B. Physicochemical Properties of Chlorcarcin A

1. Color and condition: yellow syrup (free base)

2nd melting point: 140 to 144 ° C (HC1 salt, decomposition)

3. Elemental analysis (as HCl salt):

C = 45.74%; H = 4.73%; N = 6.94%; Cl = 16.14%

4. Molecular weight (mass spectrum): 535

5. Empirical formula: C24H26N309C1-2HCI- H2O

6. Specific rotation:

[aï? = -4 ° (C = 1.0, methanol)

7. Ultraviolet absorption spectrum (according to FIG. 4, in which the solid and dashed lines show the UV spectra in MeOH and O.ln-HCl-MeOH, respectively)

UV> Ähano1 nm (log e) = 268 (3.83)

^ Methanol nm (lQg e) = 238 (3.68)

^ ain-HCl-MeOH nm (log £) = 263 (3.85)

^ -mïnn HC 'MeOH nm (log e) = 234 (3.68)

8. Infrared absorption spectrum (according to Fig. 5):

IRv £ axCh cm-1: 1685,1665,1610

9. NMR spectrum (CDCb) (according to FIG. 6):

8: 1.23 (3H, see)

I, 95 (3H, s., J = 7 Hz)

2.26 (3H, d., J = 7 Hz)

4.05 (3H, s.)

6.70 (IN, s.)

10. Solubility:

Easily soluble in ethyl ether, esters, chloroform, acetone, alcohols, 0.1 HCl; sparingly soluble in n-hexane and O.ln-NaOH; insoluble in water

II. Color reaction:

Dragendorff reaction: positive

Ninhydrin, FeCb and anthrone reaction: negative

C. Physicochemical Properties of Chlorcarcin B (Free Base)

1. Color and condition: yellow powder

2. Melting point: 78 to 81 ° C

3. Elemental analysis:

C = 60.89%; H = 6.20%; N = 6.71%; Cl = 5.20%

4. Molecular weight (mass spectrum): 553

5. Empirical formula: C29Hî206N3C1 * 5 / 4H2O

6. Ultraviolet absorption spectrum (see FIG. 7), in which the solid and dashed lines represent the UV spectra in MeOH and O.ln-HCl-MeOH, respectively)

UV ^ hano1 nm (log e): 268.5 (4.25), 370 (3.38)

? Äthano1 nm (log e): 234 (3.99), 338 (3.34)

> ^ anx "HCI" Me0H nm (log e): 262 (4.18), 380 (3.34)

^ ojn-HCi-MeOH nm (iog e). 23l (3.95), 354 (3.32)

7. Infrared absorption spectrum (according to Fig. 8):

IRv £ gFJ cm-1: 1715.1683.1655.1612

8. NMR spectrum (CDCb) (according to FIG. 9):

8: 1.28 (3H, m.), 1.92 (3H, s.),

2.04 (3H, see), 2.27 (3H, see),

2.50 (3H, see), 4.04 (3H, see),

4.08 (3H, s.), 4.42 (1H, s.),

6.84 (1H, d., J = 8 Hz)

9. Circular dichroism spectrum:

(C = 8.63-10 "5, methanol): Ae (nm):

-2.45 (360) (negative maximum)

-0.352 (320) (positive maximum)

-13.0 (2.78) (negative maximum)

5

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15

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50

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65

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10. Solubility:

Easily soluble in lower alcohols, chloroform, esters, acetone, benzene and ethyl ether; sparingly soluble in n-hexane; insoluble in water

11. Color reaction:

Dragendorff and Meyer reactions: positive Ninhydrin and Ehrlich reactions: negative

D. Physicochemical Properties of Chlorcarcin C

1. Color and condition: yellow powder

2. Melting point: 79 to 84 ° C '3. Elemental analysis:

C = 60.88%; H = 6.11%; N = 6.54%; Cl = 6.71% '4. Molecular weight (mass spectrum): 567

5. Empirical formula: C3oH3406N3C1 «3 / 2H2O

6. Ultraviolet absorption spectrum (according to FIG. 10, in which the solid and dashed lines represent the UV spectra in MeOH or O.ln-HCl-MeOH)

UV> Ähano1 nm (log e): 268 (4.26), 368 (3.34)

^ Methanol nm (lQg 8): 231 (3.91)

^ n-HCi-MeOH nm (log e): 262.5 (4.22), 380 (3.40)

^ Mn-HCi-MeOH nm (log e); 228.5 (3.87), 351 (3.36)

7. Infrared absorption spectrum (according to Fig. 11):

IRv £ Sp cm-1: 1718.1683.1655.1618

8. NMR spectrum (Fig. 12):

8: 1.42 (3H, s.), 2.01 (3H, s.),

2.15 (3H, see), 2.35 (3H, see),

2.59 (3H, s.), 3.59 (3H, s.),

4.05 (3H, see), 4.07 (3H, see),

6.71 (lH, d., J = 8Hz)

9. Circular dichroism spectrum:

(C = 9.38xl0 "5, methanol); Ae (nm):

-3.88 (360) (negative maximum)

-2.26 (310) (positive maximum)

-24.5 (273) (negative maximum)

10. Solubility:

Easily soluble in lower alcohols, chloroform, esters, acetone, benzene and ethyl ether; sparingly soluble in n-hexane; insoluble in water

11. Color reaction:

Dragendorff and Meyer reactions: positive Ninhydrin and Ehrlich reactions: negative

Chlorcarcins A, B and C have an antibacterial and anti-tumor effect and can also be used as medicines. The antimicrobial spectra of Chlorcarcin A, Chlorcarcin B and Chlorcarcin C can be found in Table 5, in which the antibiotics mainly show an antibacterial effect against gram-positive bacteria, but have a low antibacterial effect against most Gram-negative bacteria. Chlorcarcins A, B and C have cytostatic effects against L 1210 leukemia in mice, causing 100% cell killing in a suspension culture of 0.05 (ig / ml, 10 jig / ml and 10 ng / ml, respectively).

Table 5

Antimicrobial spectra of chlorcarcins A, B and C

Test organism Chlorcarcin Chlorcarcin Chlorcarcin

ABC

Staphylococcus aureus 209 P.

0.1

12.5

12.5

Staphylococcus

aureus smith

0.02

12.5

12.5

Staphylococcus

albus

0.1

50

50

Staphylococcus

citreus

0.1

25th

25th

Test organism

Chlorcarcin

. Chlorcarcin

Chlorine

A

B

C.

Streptococcus

hemolyticus

Coock

6.25

6.25

6.25

Streptococcus

hemolyticus

090R

50

100

100

Streptococcus

salivarius

6.25

50

50

Streptococcus

faecalis

25th

> 100

> 100

Bacillus

substilis PCI 219

0.2

25th

25th

Bacillus

cereus

50

50

50

Corynebacterium

diphtheriae

0.003

0.05

0.05

Corynebacterium

xerosis

0.003

0.39

0.39

Lactobacillus

arabinosus

12.5

50

50

Nocardia

asteoides

25th

50

50

Mycobacterium

smegmatis

ATCC607

100

> 100

> 100

Mycobacterium

phlei

100

> 100

> 100

Mycobacterium

avium

100

> 100

> 100

Escherichia

coli

> 100

> 100

> 100

Shigella

dysenteriae

> 100

> 100

> 100

Salmonella

typhimurium

> 100

> 100

> 100

Klebsiella

pneumoniae

100

50

50

Serratia

marcescens

> 100

> 100

> 100

Pseudomonas

aeruginosa

> 100

> 100

> 100

Brucella

abortus

> 100

> 100

> 100

Candida

albicans

> 100

> 100

> 100

Saccharomyces

cerevisiae

> 100

> 100

> 100

Tolura rubra

> 100

> 100

> 100

Penicillium

glaucum

> 100

> 100

> 100

Aspergillus

-

Niger

> 100

> 100

> 100

Aspergillus

oryzae

> 100

> 100

> 100

Mucormucedo

> 100

> 100

> 100

Trichophyton

mentagrophytes

100

> 100

> 100

Medium and breeding conditions:

1% glucose nutrient agar (3% glycerol nutrient agar for acid-resistant bacteria, blood agar for Streptococcus hae-molyticus and Brucella abortus) 37 ° C, 24 or 48 hours.

Sabouraud dextrose agar for fungi, 27 ° C, 48 hours (72 hours for Trichophyton mentagrophytes).

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Chlorcarcins A, B and C are relatively little toxic and tolerable in mice with an average body weight of 18 to 20 g when administered intravenously at 25 mg / kg (A) and 250 mg / kg (B and C).

Chlorcarcin A was tested for anti-tumor effects by intraperitoneal administration using an Ehrlich carcinoma. In all mice, prolonged lifespan of 30 days or more was observed when 80 (ig / mouse was administered for 1 week, while prolonged lifespan was observed in 60% of mice with daily doses of only 60 (ig / mouse for 1 week could.

test

The antibacterial effects during cultivation and extraction measures were tested using Bacillus subtilis PCI 219 according to the agar plate method. The streptothricin test was carried out using Escherichia coli strain Fi because strain no. 314 can produce this antibiotic in a large amount. The coexistence of streptothricin can easily be determined by determining an antibacterial effect against E. coli, because the chlorcarcin complex, namely the fraction extracted into a solvent, has no effect against E. coli. The mimosamycin was similarly tested using Mycobacterium smegmatis ATCC 607 as a test organism.

As mentioned above, Chlorcarcins A, B and C have an anti-tumor effect and can be used for the treatment of neoplasms in warm-blooded animals. It can also be assumed that they can be used as antineoplasmic agents. The estimated effective dosage for chlorcarcin A should be within 10 mg to 1 g daily in adults. For chlorine carcins B and C, daily doses of 100 mg to 10 g can be expected. The chlorcarcins can be administered in the form of similar pharmaceutical preparations as is the case for mimosamycin, with parenteral and in certain cases advantageously being administered orally. Usually you will work intramuscularly or intravenously.

A particular embodiment for the production of said antibiotic substances is described in the following examples.

example 1

Shake culture in flask A. Seed culture

A suspension of a freeze-dried preparation of Streptomyces lavendulae strain No. 314 in physiological saline was prepared and inoculated on an agar culture with the following composition.

Glucose 10 g L-asparagine 5 g KH2PO4 5 g

Agar 15 g

Tap water 1000 ml pH 6.8-7.0

The cultivation was carried out at 27 ° C for 1 week, whereby a seed culture with abundant growth and some spores was obtained.

B. culture medium

100 shake flasks, each with a volume of 500 ml, which contained 100 ml of the culture medium defined above, were inoculated with spores collected from the above-mentioned seed culture medium in amounts of two eyes per flask.

622823

glucose

1.0 g

Strength

10.0 g

Polypeptone (available from Wako Pure

10.0 g

Chemical Industries, Ltd., Japan)

Meat extract (dto.)

5.0 g

NaCl

3.0 g

Silicone KM 72

(Shin-Etsu Chemical Co., Ltd., Japan)

10 ml

tap water

1000 ml pH 7.0

The cultivation was carried out for 40 hours at 27 ° C. using a shaker (125 strokes per minute, 8 cm vibration).

After the cultivation was complete, the flask contents were combined and the mycelia isolated using a continuous centrifugal separator. The floating part thus obtained (10 liters) was adjusted to a pH of 8.0 and then extracted three times with a third volume of chloroform.

The extracts were combined, filtered through filter paper and then concentrated to dryness under reduced pressure to give 2.8 g of a dark brown, solid substance. A solution of this substance in 50 ml of ethyl acetate was shaken successively with a 5% aqueous sodium hydrogen carbonate solution, an in sodium carbonate solution and finally an in sodium hydroxide solution in respective portions of 25 ml to remove the acidic substances. The organic layer was extracted five times with 25 ml of 1N hydrochloric acid each. The extracts were combined with the aqueous layer, whereupon the mixture was adjusted to pH 9-10 by adding aqueous ammonia and extracted five times with the same volume of chloroform. The extracts were combined and evaporated to dryness. The residue was dissolved in 50 ml of a 10% aqueous acetic acid solution and the pH of this solution was increased to 9-10 by adding aqueous ammonia, followed by extraction five times with equal amounts of chloroform. The extract was concentrated under reduced pressure to obtain 200 mg of a crude extract containing the chlorcarcin complex and mimosamycin.

The crude extract thus obtained was introduced into a glass column which had a diameter of 1.0 cm and a length of 50 cm and was charged with silica gel [70 to 230 meshes (mses), available from Merck & Co., USA] . This column was developed with mixtures of benzene and acetate in a mixing ratio of 2: 1.1: 1 and 1: 2, whereby fractions were obtained which predominantly contained chlorocarcin A and mimosamycin. Each fraction was then chromatographed in a silica gel column (230 mesh, available from Merck & Co.), whereby 5.2 mg of chlorocarcinol A as a yellow powder and 2.8 mg of mimosamycin as yellow crystals were obtained.

The original column was then further developed with a mixture of ethyl acetate and acetone (mixing ratio 1: 1), whereby fractions (73 mg) were obtained which contained the chlorocarbins B and C. The fraction thus obtained was repeatedly chromatographed on a silica gel column (230 mesh, available from Merck & Co.) using acetone as the developing solvent, whereby 28 mg of chlorocarcin B and 37.3 mg of chlorocarcin C were obtained.

Example 2

Cultivation in fermentation vessels

A. The seed culture was prepared in the same manner as in Example 1, except that the cultivation was carried out for 24 hours.

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622823

10th

B. 4 fermentation vessels with a volume of 20 liters each, each containing 15 liters of the culture medium mentioned below, were sterilized under pressure in a conventional manner.

Glucose 5.0 g

Thickness 5.0 g

Polypeptone (available from Wako Pure Chemical Industries, Ltd., Japan) 10.0 g

Meat extract (dto.) 5.0 g

NaCl 3.0 g

Tap water 1000 ml pH 7.0-7.2

10th

The cultivation was carried out for 18 hours under the following conditions:

Seed culture 1%

Cultivation temperature 27 ° C

Stirring speed 550 rpm

Flow rate of 1 volume medium 20

aseptic air per minute

Antifoam (silicone KM 72)

if necessary admitted

25th

After the above-mentioned period of time, the maximum titers of the chlorcarcin complex and mimosamycin produced were obtained, and the titers decreased rapidly. The pH of the broth dropped below 6.0 and then rose again to approximately 6.8. The mycelium volume increased rapidly and the glucose was practically exhausted at that point. At the maximum titer of chlorocarcin, a dilution method using Bacillus subtilis PCI 219 as the test organism gave 512 dilution units, while an agar plate diffusion method gave an inhibition zone of approximately 40 mm. The maximum generation of streptothricin was reached after about 30 hours. Separate mycelia from the culture broth (approx. 60 liters) using a continuously operating centrifugation device. The culture liquid was concentrated to a third of the volume.

The concentrate was adjusted to a pH of 8.0 by adding in sodium hydroxide solution and extracted three times with equal volumes of chloroform. The extracts were combined and concentrated under reduced pressure to give 16.4 g of a mixture of crude chlorocinyl complex and mimosamycin. The mixture was dissolved in 10 ml of ethyl acetate and the solution thus obtained was washed with 50 ml of a 5% strength aqueous sodium bicarbonate solution, then with an in-sodium carbonate solution and then four times with an in-sodium hydroxide solution with stirring. The sodium hydroxide layer contained acidic, ineffective components, especially stearic acid. The ethyl acetate layer was shaken five times with 60 ml of a 1N hydrochloric acid solution, after which basic substances were extracted into the acid. The acidic extracts were combined, adjusted to pH 9-10 by adding aqueous ammonia, extracted five times with equal volumes of chloroform, and the organic layer was separated and concentrated under reduced pressure to give crude, basic substances. These substances were dissolved in 60 ml of a 10% aqueous acetic acid solution and the solution was then adjusted to a pH of 9 to 10 by adding aqueous ammonia and then extracted five times with the same volume of chloroform. The extracts were combined and concentrated under reduced pressure to give approximately 0.98 g of crude product containing chlorcarcin complex and mimosamycin. .

A total of approximately 0.2 to 1.5 g of crude product was obtained from the four fermentation vessels.

The cultivation was repeated 140 times using four fermentation vessels, using a total of 840 liters of culture broth. In this way, 8.67 g of crude product was obtained.

The crude product dissolved in a little benzene was introduced into a glass column which had a diameter of 5 cm and was loaded with silica gel (70 to 230 meshes, available from Merck & Co.) soaked in benzene. The column was developed with a mixture of benzene and ethyl acetate (mixing ratio 2: 1). After the impurities were eluted, the fraction containing mainly chlorcarcin A and the fraction mainly containing mimosamycin were successively eluted with the same developer solvent. These fractions were concentrated and chromatographed on silica gel (230 mesh, Merck & Co.) to give 42 mg of mimosamycin and 85 mg of chlorocarcin A.

The original column was eluted with a 1: 1 mixture of ethyl acetate and acetone to give a mixture containing chlorocarbins B and C. This mixture was repeatedly chromatographed using silica gel (230 mesh, Merck & Co.) and treated with the same solvent to give 215 mg of chlorocarcin B and 52 mg of chlorocarcin C in the pure state.

G

6 sheets of drawings

Claims (4)

622823 PATENT CLAIMS 1. Process for the preparation of the new antibiotic substance mimosamycin in the form of neutral, yellow prisms, this substance having the following physical values: melting point 227 to 231 ° C .; Composition 61.51% carbon, 4.79% hydrogen and 5.87% nitrogen; Molecular weight 233 according to mass spectrum; empirical formula C12H11NO4; 1, UV / MjPh nm (log e): 230 (shoulder) (4.16), 317 (4.14), 396 (3.56), UV> nm (log e): 277 (3rd , 78), 370 (3.53); 2, IR v {£! £ cm-1: 1685.1655,1635.1585; NMR spectrum (CDCb) according to FIG. 3.8: 2.10 (3H, s.), 3.69 (3H, s.) 4.20 (3H, s.), 7.12 (1H, s. ), 8.28 (1H, s.); Rotational dispersion spectrum (C = 4.37 • 10-7, MeOH) of [0] 20 (nm): -1601 (500), -1373 (490), -1373 (480), -1144 (470), - 1144 (460), -1144 (450-390), -1373 (380), -1144 (370-350), - 1373 (340), -1831 (330), -2288 (320), -2288 (310) , -1831 (300), -1601 (290), -2059 (280), -2517 (270), -3661 (260), -5492 (250); wherein the substance is also readily soluble in methanol, ethanol, chloroform, an ester and acetone, sparingly soluble in ethyl ether and n-hexane and insoluble in water and is positive for Ehrlich's reagent and negative for ninhydrin and Dragendorff reagent, as well as of Acid addition salts thereof, characterized in that Streptomyces lavendulae strain No. 314 (NRRL11002) is cultivated, a complex of chlorocycles and mimosamycin is obtained from the culture broth and mimosamycin as such or in the form of an acid addition salt is isolated therefrom from this complex. 2. Process for the preparation of the new antibiotic substance chlorcarcin A in the form of a basic, yellow syrup, this substance having the following physical data: melting point 140 to 144 ° C. (with decomposition as the chlorohydrate salt); Composition (as chlorohydrate salt): 45.74% carbon, 4.73% hydrogen, 6.94% nitrogen and 16.14% chlorine; Molecular weight 535 according to mass spectrum; empirical formula Cî ^ ôNaOsCl- 2HC1 »H2O; specific rotation [a] ff = -4 ° (C = 1.0, methanol); 4, UV nm (log e): 268 (3.83), UV XMeOH nm (log e). 238 (3.68), UVXfeHCl-MeOH nm (log e): 263, (3.85), UV Âmn0H "° 'In" HC1 nm (log e): 234 (3.68); 5, IR v ^ SF 'cm-1: 1685, 1665, 1610; NMR spectrum (CDCb) according to FIG. 6.5: 1.23 (3H, s.), 1.95 (3H, s, J = 7 Hz), 2.26 (3H, s., J = 7 Hz ), 4.05 (3H, s.), 6.70 (1H, s.), This substance also being readily soluble in ethyl ether, an ester, chloroform, acetone, an alcohol and 0, ln-acidic acid, in n- Hexane and a 0.1 N sodium hydroxide solution is sparingly soluble and insoluble in water and reacts positively to Dragendorff reagent and negatively to ninhydrin, FeCb and anthrone reagent, and acid addition salts thereof, characterized in that Streptomyces lavendulae strain no 314 (NRRL 11002), obtains a complex of chlorcarcins and mimosamycin from the culture broth, and then isolates chlorcarcin A as such or in the form of an acid addition salt from this complex. 3. Process for the preparation of the new antibiotic substance chlorcarcin B in the form of a basic, yellow powder, this substance having the following physical data: melting point 78 to 81 ° C .; Composition 60.89% carbon, 6.20% hydrogen, 6.71% nitrogen and 5.20% chlorine; Molecular weight 553 according to mass spectrum; empirical formula C29H32O6N3CI-5/4 ^ 0; Ultraviolet absorption spectrum according to FIG. 7, UV nm (log e): 268.5 (4.25), 370 (3.38), UV / ^ eOH nm ^ e): 234 (3.99), 338 (3.34 ), UV ^ HC | -Me0H nm (log e): 262 (4.18), 380 (3.34), UV ^ J "-HC1-MeOH nm (log e): 231 (3.95), 354 (3.32); Infrared absorption spectrum according to FIG. 8, IR v & ajF1 cm-1: 1715.1683.1655.1612; NMR spectrum (CDCb) according to FIG. 9 Ô: 1.28 (3H, m.), 1, 92 (3H, s.), 2.04 (3H, s.), 2.27 (3H, s.), 2.50 (3H, s.), 4.04 (3H, s.), 4, 08 (3H, see), 4.42 (1H, see), 6.84 (1H, i.e., J = 8Hz); Circular dichroism spectrum (C = 8.63-10 "5, methanol), As (nm): -2.45 (360) negative maximum): -0.352 (320) (positive maximum); -13.0 (2.78) (negative maximum); this substance is readily soluble in an alcohol, chloroform, an ester, acetone, benzene and ethyl ether, is sparingly soluble in n-hexane and is insoluble in water and is positive for Dragendorff and Meyer reagents and for ninhydrin and Ehrlich reagent reacts negatively, as well as of acid addition salts thereof, characterized in that Streptomyces lavendulae strain No. 314 (NRRL 11002) is cultivated, a complex of chlorocarcines and mimosamycin is obtained from the culture broth and then chlorocarcin B as such or in the form of an acid addition salt thereof isolated from this complex. 4. A process for the preparation of the new antibiotic substance chlorcarcin C in the form of a basic, yellow powder, this substance having the following physical data: melting point 79 to 84 ° C .; Composition 60.88% carbon, 6.11% hydrogen, 6.54% nitrogen and 6.71% chlorine; Molecular weight 567 according to mass spectrum; empirical formula C3oH340eN3Cl'3 / 2H20; 10, UVAJ # | PH nm (log e): 268 (4.26), 368 (3.34), UV ^ oh nm (log e): 231 (3.91), UV ^ Wn- fra-MeOH nm (log e): 262.5 (4.22), 380 (3.40), UV ^ nn-HC | -Me0H nm (log e): 228.5 (3.87), 351 ( 3.36); 11, IRv £ aj cm-1: 1718.1683.1655.1618; NMR spectrum according to FIG. 12.5: 1.42 (3H, s.), 2.01 (3H, s.), 2.15 (3H, s.), 2.35 (3H, s.), 2.59 (3H, s.), 3.59 (3H, s.), 4.05 (3H, s.), 4.07 (3H, s.), 6.71 (1H, d., J = 8 Hz); Circular dichroism spectrum (C = 9.38-10-5, methanol), As (nm): -3.88 (360) (negative maximum): -2.26 (310) (positive maximum); -24.5 (273) (negative maximum); this substance being readily soluble in an alcohol, chloroform, an ester, acetone, benzene and ethyl ether, sparingly soluble in n-hexane and insoluble in water, and acid addition salts thereof, characterized in that Streptomyces lavendulae strain No. 314 (NRRL 11002), from the culture broth a complex of chlorocarcines and mimosamycin is obtained and then chlorocarcinol as such or in the form of an acid addition salt thereof is isolated from this complex.