DE1939045C - Method of making antibiotic 289-F - Google Patents

Description

used and breeding in the presence of fatigue.

Anthraquinone mono- or anthraquinone disulfone- The bacterial strains No. 613-15 (ATCC 21395)

acid or its metal salts and that and No. 1-125 (ATCC 21396) were taken from Str.

Separates antibiotic in the usual way. vertivillatus isolated. The bacterial strain No. 2-89

2. The method according to claim 1, characterized in 15 (ATCC 21397) was identified as a Streptomyces strain from that the anthraquinone mono- one taken from Takatsuki City, Osaka, Japan, or -disulfonic acid or its metal salts are isolated in soil samples. The bacterial strain number 2> · '-.' A concentration of 0.1 to 2.0 ° / ₀ (. Gew./Vol) (ATCC 21397) has a striking similarity / u applies. St. phaeoverticiilatus on. These bacterial strains

ao are able to use the antibiotic 289-F in KuIt ₁

to form liquid when in the medium, welc! ·.

contains a type of anthraquinone sulfonate, bred; · ι will. These bacterial strains differentiate between s! ■

The invention relates to a method for the production of Str. Phaeoverticiilatus with regard to. or of the antibiotic 289-F by cultivating one of the morphological properties, that is, this strain belonging to the Streptomyc family, phaeoverticiilatus, does not form secondary vertebrae, while Str. phai. Bakicriensiamms forms secondary vertebrae with spirals in a carbohydrate and stick- verticillatus,
nutrient medium containing substances under aerobic conditions. Those of St. phaeoverticiilatus different · -.-!

The antibiotic which can be produced according to the invention Cultivation conditions of the strains No. 613- '·. 289-F has an inhibitory effect on the growth of gram- 3 ° No. 1-125 and No. 2-89 are in the following TjIh.'v. positive and gram negative bacteria; furthermore suitable compiled; other properties are dage.p · ;; it is indistinguishable from one another to prevent honest ascites.
addiction carcinoma in mice. Based on these results, it is proposed that

The new antibiotic 289-F was first derived from the streptomycetes, which belong to the above bacteria in a culture broth of a bacterial strain, 35 strain category, to be referred to as Str. Phacoverticillaius which belongs to the group of Streptomyces phaeoverticil- var. Takatsukiensis,
latus, especially St. phaeov. var. takatsukiensis If one of these stems in an ordinary

(ATCC 21395, 21396 and 21397). Medium grows, one hardly observes the formation

Further investigations into the formation of 289-F in the culture broth.

of 289-F showed that in the culture broth to 4 ° However, if you have these microorganisms in one next another antibiotic, namely 289-FO, grows medium, which in addition to the usual and only then forms 289-F. 289-FO becomes anthraquinone mono- or Didiese due to the nutrient medium Strains, depending on the regulation of the fermentation time, contain sulfonic acid or metal salts thereof, so forms formed by fermentation. a large amount of 289-F in the culture broth.

Both 289-FO and 289-F are produced as pure crystals Get substances. Different physical consists, therefore, in the fact that one is called a bacterial strain kaiische and chemical properties of this sub-ATCC 21395, 21396 or 21397 used and the punches are given below; chemical growth in the presence of anthraquinone mono-structure is not yet known. It yielded or anthraquinone disulfonic acid or its metal itself but that both 289-FO and 289-F by salting 5 ° perform and the antibiotic in more common Treating with acetic anhydride the same acetylated way separates.

Derivative result. From this fact and from the expediency one applies the anthraquinone-

Investigation of the mode of formation, however, has to assume mono- or -disulphonic acid in a concentration that the chemical structures of the two assume 0.1 to 2.0% (w / v).
Substances are quite similar to each other and that 55 In the following the fermentation conditions for 289-FO in the first stage of fermentation are described as metabolic production of 289-F,
product formed by the microorganism and then achieved by the formation of 289-FO in the culture broth

further fermentation is converted into 289-F. its maximum after 65 hours of fermentation. After that

The anti of the invention for the formation decreases the amount of 289-FO gradually since spectrum antibiotic F-289 viable microorganisms were ^6o separated by this further continuous fermentation in 289-F of Streptomyces phaeoverticiilatus; is also converted. The fermentation time for the separation they were separated from a soil sample taken at Takatsuki City, Osaka-Fu, by 289-F must therefore at least more than 65 hours in Japan. Under- amount. For the production of 289-F one cultivates e.g. B. investigations of the characteristic properties of the Streptomyces phaeoverticiilatus var. Takatsukiensis microorganisms showed that they are to be considered as new variants ⁶ 5 (e.g. ATCC 21395) in a suitable aqueous medium which is in addition to the usual Streptomyces phaeoverticiilatus; they were therefore referred to as Streptomyces phaeoverticil nutrient medium or anthraquinone mono- or disullatus var. takatsukiensis and contain americic acid or its metal salts.

Table I.
Comparison between St. phaeoverticillatus and St. phaeoverticillatus var. Lakatsukiensis

medium

St. phaeoverticillaius var. Takatsukiensis No. 613-15 | No. 1-125

(ATCC 21395) (ATCC 21396)

No. 2-89
(ATCC 21397)

St. phaeoverticillatus

Starch agar

Glucose Czapek Agar

Sucrose czapek agar

Glucose asparagine agar

G: Moderately

LM: white to slightly yellow-brown

SM: white to pale yellow-brown

LP: Very faint yellow-brown

G: Good LM: Light brown

SM: brown to red-brown LP: brown to red-brown

G: Wrinkled well

LM: weak, white SM: light yellow brown to yellow brown LP: yellow brown

G: Weak

LM: Moderate, white to weak

brown-gray SM: white to ivory

LP: None

Morphological
properties

Mainly monoverticillus spirals

Mainly spirals G: growth; LM: air mycelium; SM: Suhstrat mycelium; LP: Soluble pigment.

G: Moderately

LM: weak, white to light brown-gray

SM: Colorless to pale yellow-brown

LP: Almost colorless

G: Good

LM: Moderate, white to light yellow-gray

SM: light yellow-brown to dull yellow

LP: dull yellow

G: Moderately

LM: weak to scanty SM: light yellow-brown

LP: light yellow brown

G: Moderately LM: Careless

SM: Colorless to very weak

Tan LP: None G: Moderately
LM: Weak, white

SM: colorless to white
LP: none

G: Good
LM: Brownish white

SM: brown
LP: brown

G: Good

LM: Weak, white

SM: white to brownish white

LP: Slightly red-brown

G: Weak
LM: Little to no

SM: Colorless to white
LP: None
Mainly spirals

G: Creamy creamy yellow LM: White to brownish white

SIw: Slightly yellow-brown to

olive-nut brown LP: weak, light loose

G: Creamy orangechamois LM: White to brownish white

SM: Chamois, grainy L ?: Orange chamois

G: beige to cinnamon colored

LM: white to brownish white SM: pink beige to chamois

LP: pink beige to chamois

G: cream, orange, chamois LM: white to brownish white

SM: Chamois, grainy LP: Orangechamois Biverticülus spiracles

CO W CD

5 6

In the context of the invention, the usual diameters of the silica gel can be 0.05 to 0.2 mm

Media for growing Streptomyces in stationary carried out using a mixture of

or aqueous culture can be used. Practically benzene and low molecular weight alkyl alcohols as

however, it becomes the aqueous culture method for the eluents.

Obtainment of 289-F preferred. 5 If methanol is used as a low molecular weight alkyl

289-F can be used, for example, with good active alcohol, if a degree of solvent can be used under aerobic conditions and stirring in a mixture of benzene-methanol (volume ratio 9: 1 over a period of 3 to 5 days at a temperature of up to 7: 3); is preferably a benzene 25 to 35 ° C, preferably 25 to 30 ⁰ C, by fermentation of methanol mixture in a volume ratio of 8: turns ίο be prepared. 2 For example, the silica gel column

The general procedures for growing others (Mallincrodt silica, 0.147 mm) with the benzene

Streptomyces strains can also be prepared for culturing methanol mixture (volume ratio 8: 2),

of the bacterial strains according to the invention apply. and then that is previously in the same solution

The nutrient medium preferably contains the following constituents, dissolved crude product at the top of the column: carbon, e.g. B. in the form of i ₅ and eluted with the same solvent. Starch, glucose, maltose or mixtures thereof; Various absorption nitrogen, e.g. B. in the form of meat extract, Peplon, tion bands from pale yellow to purple. The soybean meal, corn water, peanut meal, flax- respective fractions are collected in an automatic seed meal, ammonium salts, nitrates, yeast and fraction collector and on their mixtures; inorganic salts, e.g. Example in the form ao their color reaction with 2 ° / ₀ sodium nickel (in of potassium salts, sodium salts, phosphates or methanol) was investigated. The fraction with the 289-F sulfates, which dissociate into ions in the nutrient medium. This results in a bluish to purple color and the different metal fractions can optionally also be mixed with the 289-FO a gray-blue colored ion, such as B. Magnesium, manganese, zinc, cobalt reaction.

or iron, in small quantities or in traces to the »5 The individual fractions are then at lower

Growing medium can be added. Temperature under reduced pressure to dryness

For technical production, the preferred hangs evaporated. A crude crystalline is obtained here

Composition of the respective nutrient medium of or amorphous powder (289-F). In the case of the alumina

the Streptomyces strain used in each case, such as column chromatography, is preferably used

This also applies to the production of other antibiotics 30 with a water-saturated mixture of low molecular weight

using other strains of Streptomyces alkyl alcohol and low molecular weight alkyl acetate than

the case is. Eluent on. Examples of useful low-

For example, the following fermentation molecular alcohols are methanol or ethanol,

conditions for the production of 289-F. As ethyl acetate are z. B. methyl acetate, ethyl

35 acetate, propyl acetate or butyl acetate.

Nutrient medium _D j _e two solvents are used in the following

Meat extract 0.5% / ₀ proportions applied.

Sodium chloride 0.5 ° / ₀ Benzene: ROH = 7: 1 to 4: 1, preferably 7: 1st

Calcium carbonate 0.6 ^υ / ₀

Soybean oil 0.1% 40 The working method of the alumina column chromatography

Peptone 0.5% graph is the same as silica gel chromatography.

dried yeast 0.3 ° / “If you get 289-F in the column chromatography in

Glucose 4.0% / ₀ amorphous form, it is converted from acetonitrile

Sodium anthraquinone-2,7-disulfonate 0.5 / ₀ ° crystallized to give orange-yellow crystals is obtained.

45 These crystals are then made again from methanol

After inoculation of Streptomyces phaeo recrystallized, whereby one orange-colored nadelverticillatus var. takatsukiensis is cultivated in shaped crystals from pure 289-F. The self-aqueous medium at a temperature of 28 ° C shafts of 289-F are described in more detail below carried out under aerobic conditions for 90 hours. wrote. An attempt was made because the 289-F is unstable Separating 289-F from nutrient broth 50 will produce a stable derivative of this compound is carried out as follows. In this connection it was found that dii

The culture broth is filtered off, and the acetylated compound filtrate is very stable and easy to add is used by studying biological activity with a cation exchange resin passed through filled column. The eluate will be with can. Acetylation di extracted from an organic solvent, and the 55 chemotherapeutic properties of this anti Solvent extract is evaporated, further improving biotics and reducing their toxicity 289-F is obtained as a crude product. According to another set.

usable procedure is the broth after the The new antibiotic 289-F has the following)

.Filtration directly with an organic solvent, physical and chemical properties to medium at a weakly alkaline pH value extra- 60 biological effects on:
hiert without a treatment with a cation.

exchange resin takes place; the solvent extract ^{becomes w Kns talie}

then, as in the procedure described above, a 289-F orange-yellow needles (first made from acetonitr steamed. and then recrystallized from methanol).

The RoI product containing 289-F then becomes 65

further on silica gel or alumina column chromato- w i> melting points

graphically cleaned. 289-F 211.5 to 213.0 ⁰ C (decomposition immediately

Silica gel column chromatography (more preferably after melting).

(3) Elemental Analysis 289-F C 67.04 H 7.12 N 4.15

(4) UV spectrum

The UV spectrum of compound 289-F in methanol is shown in FIG. 2 shown. The absorption maxima and absorption coefficients are given in the following table:

ιημ (El *) 289 F 244 (E 807), 270 (shoulder), 434 (E 210)

(5) 1R spectrum

im (KBr) of compound 289-F

(8) Paper chromatography of 289-F

Rf

0.14
0.71

solvent

Acetonitrile butyl acetate dibutyl ether (3: 1)

Other requirements

Filter paper, developed according to the ascending method at 2O ⁰ C

The Rf values were obtained from a yellow spot or by staining with nickel acetate.

289-F 3450, 1620, 1610, 1585

(6) Color reactions of 289-F

(9) Thin layer chromatography of 289-F Other requirements Rf solvent on Silicage!
on silica gel 0.81
0.06 Ethanol / 14 ° / _O iges
Ammonia water
(4: 1)
Ethanol / pyridine
(4: 1)

_a "f one

through

(10) Optical rotation

must "KupIe, ni., a. at ^

lischer solution.

(7) Solubility of 289-F Soluble in: methanol, ethanol, acetone

Ethyl acetate, benzene, chloroform, acetonitrile, aqueous acidic solution.

Slightly soluble in: Aqueous alkaline as well neutral solution.

Very slightly soluble in: water.

289-F

+ 456.7 °

conditions

l, 5 ° / _o in chloroform at 20 ⁰ C

(11) Acute Toxicities

289-F
Iyomicin B ₁

LD intravenous

12.5 to 4.4 to 5.5

mg / kg intra peritoneal

12.5 to 12.5 to 15.5

Experimental animal

Mouse mouse

(12) Antibiotic Activity

Staphylococcus aureus 209-P · ·

MHTococcus flavus

Sar.ma lutea: ATCC 9341 ....

Bacillus subtilis PCI-219

Eschcrichia coli B

Pseudomonas aeruginosa

Proteus vulgaris

Streptococcus pyogenes TI Streptococcus pyogenes S-8 .. ■ Diplococcus pneumoniae DP-I Lactobacillus fermenti 36 .. ■■ _ Mycobacterium tuberculosis W Candida albicans YU-1200 .. · Trichophyton interdigitale. · ·

Aspergillus niger

Xantosnonas oryzae

Shigella dysenteriae

Salmonella typhosa 901W ··· Minimum inhibitory concentration μg / ml 289-FO ») 1 289-F **) | Acetyl 289-F **)

100 100 100 3.12
0.39
0.39
3.12

0.78

1.56
0.39
3.12

100 100 100 100

100

0.39 0.04 0.09 0.78 3.12 12.5

1.56 0.02 0.09 0.01 3.12 0.78 50.0

0.39 6.25 6.25

100 100

100 100

100

0.6 0.78 0.78 1.56 25.0

1.56 0.39 0.39 0.39 0.78 25.0

12.5 25.0

1.56

·) Determined from the » » * · ♦> Determined from the ll 205684/3 «

ίο

(13) Antitumor activities for Ehrlich ascites carcinoma

tumor Minimal concentration
for full
Inhibition µg / mouse
289-F Honest ascites
Carcinoma
3 days after administration
7 days after administration 50
12th

From the table above there is an interesting sustained inhibitory effect on carcinoma, with an inhibition of the growth of tumor cells even when a lower dose is administered occurs at a later date. The above

Results were obtained according to the following procedure:

48 hours after intraperitoneal administration of the diluted at various concentrations Antibiotic was administered intraperitoneally to 10 million cancer cells causing ascites inoculated. Then an ascites sample was taken after each administration and the sample smeared and stained with dye,

ίο The stained samples were compared to a control sample compared, and the concentration required for complete inhibition was determined, in which the number of cells decreased to 0 and 5% of the comparison cells.

To prove the novelty of the antibiotic 289-F according to the invention are in the following table the properties of similar well-known antibiotics are listed. (The specified data donors the main differences compared to the invention

ao appropriate substances again.)

R. Melting point LD 50 UV absorption Elcmentar- mole IR-off I. tion maxima analysis kular sorption I. ⁰ C
_ mg / kg λ (E) found Weight cm " ¹ According to the invention established connection 289-F 211.5 to 213.0 12.5 to 20 244 (807) N 4.51 1620 (p. V.) H 7.12 676.78 1610 12.5 to 20 270 C 67.04 (i. p.) (Shoulder) Well-known antibiotics (Comparison) Iyomycin B, > 270 4.4 to 5.5 234 to 244 (500) N 3.89 1100 1740 (The Journal of Mouse, i. v. 270 (330) Antibiotics Α., 12.5 to 20 9 [3], pp. 117 to 123 Mouse, i. p. 430 (96) [1964]) Iomycin B ₄ > 270 100 243 to 244 (360) N 8.94 (J. A. Α., 9 [3], (Mouse, i. P.) 265 (250) H 5.81 Pp. 117 to 123 [1964]) 430 (59) C 55.30 Pluramycin A 177 12.5 to 25 345 (672) N 3.66 1744 (J. A. A., 9 [2], (gets dark) (Mouse, i. P.) 265 to 270 H 6.30 Pp. 75 to 81 [1956]) (Crystals (Shoulder) C 66 ^ 83 from ÄtOH) 200 to 215 (gets dark) (Crystals from ÄtOAc) Rubiflavine not 15th 395 N 3.45 412 (Antimicrobial crystalline (Mouse, i. P.) (Shoulder) H 7.50 Tl * Agents and C 68.50 Chemotherapy 1964, Pp. 68 to 74) Hedamycin 243 to 245 0.3 245 (800) N 3.76 743 1660 (Anti. Ag. & Chem., (Decomposition) ^ Mouse, «p.) 260 to 265 H 6.89 1630 1966, pp. 606 to 619) (Shoulder) C 65.65 430 Anthracidin (Anti. Ag. & Chem., 1963, pp. 63 to 67) A. 114 to 116 233 (784) N 3.91 452 358 (465) H 8.11 . 111 to 112.5 233 (74η C 66.49
N 3.69 397 350 (390) H 7.90 C 63.64

11 12

It is believed that the molecular weight of Example 2

of the antibiotic produced according to the invention ". . . ,. . . . _{lt u} · λ i, w tri..m '

289-F is about 600 to 800. Example 1 was repeated, except that sodium

So 289-F is a new and useful compound. anthraquinone-ß-sulfonate instead of sodium anthra-die The invention is now based on the following 5 quinone-2,7-disulfonate was used. Examples further explained. The sterilized nutrient solution (151) was placed in a

glass fermentation device with a holder

_R 's The 1 1 capacity of 301 was introduced and 1.5 1 strepto-

myces phaeoverticillatus var. takatsukiensis No. 2-89

150 liters of a nutrient solution of the following ₁₀ (atCC 21397) were inoculated and cultured at 28 to 30 ° C. Composition made. The nutrient solution was prepared in the same way as in the

Glucose 4% game 4 processed, taking 4.5 g of raw antibiotic

Peptone 0 ' ⁵ % ²⁸⁹ " ^cases have been reached.

dry yeast 0.3% B e i s ρ i e 1 3

Meat extract 0.5 ^u / ₀ »5 ^v

Sodium chloride ". 0. ^{5% 2} '° R ß ^emäß Example 1 produced crude anti-

Calcium carbonate °> ⁶ % biotikum 289-F were column chromatographed

Sodium anthraquinone-2,7-disulfonate 0.5% purified with activated clay. The column was cleaned

by ethyl acetate-methanol saturated with water

This nutrient solution was ^{eluted for 30 minutes at 120 °} C. * (7: 1) and the eluate was sterilized in the same way as in a 300-1 fermentation device. Treated on example 4. In the process, 350 mg of crude were finally cooled to 28 ° C. and obtained with 151 crystalline antibiotic 289-F. The self-Streptomyces phaeoverticiUatus var. Takatsukiensis shafts of the product were inoculated as in Example 1. No. 613-15 (ATCC 21305). The nutrient solution

was 90 hours at 27 to 28 ⁰ C with stirring »5 B eis ρ ie 1 4

and air supply grown aseptically. The fermentation broth _. . . . ,. . _L , _L. . _, t

was mixed with filter aid and filtered. According to Example 1 was repeated, but with the

adjusting the pH to 8.2 to 8.4 with bacterial strain AlCCiLJVo instead of the aqueous sodium hydroxide, the filtrate with terier strain ATCC 21395 was used. Extracted by ¹ L of its volume in chloroform three times. 3 Treatment of 1501 culture solution according to Example 1 E) If the chloroform layer was washed with water, 7.2 g of crude crystalline 289-F were obtained, dried with sodium sulfate and reduced in pressure

Pressure evaporated to dryness. After adding an example

Petroleum ether to the above residue were about _. . . . ,. ,,., -. ,,, O ₁

Flea product 35 containing 40 g of antibiotic 289-F. Example 1 was repeated, but with the exception that the bacteria precipitated. This crude product (5 g) was used in 30 ml of the strain ATCC 21397 instead of the bacteria-benzene-methanol (4: 1) and used on a silica strain ATCC 21 395. By Begel's column (2 8 χ 40 cm), which previously acted on 1501 culture solution in accordance with Example 1 with benzene-methanol (4: 1) had been charged. 6.4 g of crude crystalline 289-F were obtained. When fluing with the same solvent, 40

yellow to purple-red colored frac- applicability of those prepared according to the invention

eluted. The antibiotic 289-F containing compound 289-F for combating tumors

The tinging fractions were determined by paper chromato- combating sarcoma 180

graphic test and by color reaction with

2 ° / ieem methanolic nickel acetate determined and 45 subcutaneous solid tumors of the sarcoma 180 at The after evaporation of the solution dd mice were removed about 7 days after the transmittel at reduced pressure below 40 ° C. A sham marriage of this tumor Any remaining residue, in a small amount, was found in the area of the right armpit of each mouse Dissolved ethyl acetate. After adding petroleum ether, implanted using a trocar. The treatment crude crystals were precipitated from this solution. 50 with 289-F took place 24 hours after the onset The crystals were then planted with petroleum ether of the tumor. Here were at seven on washed and dried. 917 mg of 1 mg 289-F per k * were used on the following days. obtained crude crystalline antibiotic 289-F. Administered intravenously according to body weight, recrystallization of the crude crystals from aceto 40 days after implantation of the tumor.

nitrile, yellowish-orange sand-like crystals were found nonexistent in four of ten treated mice. Further recrystallization from Me- Tumor was found, while in the case of dehanol, orange or yellowish-orange needles in other mice initially inhibited the tumor with a melting point of 211.5 to 213, ^{0 0} C growth and then a strong regression de (decomposition after melting) obtained. The tumor was observed.

other properties were the same as already described 60 untreated comparison mice perished. ^{20 to 30 days after} planting the tumor.

1 sheet of drawings

Claims (1)

can type CuKure Collection, RockvUle. Md., Under patent claims: the numbers ATCC 21395, 21396 and 21397 deposited 1. Process for the preparation of the antibiotic Details of these microorganisms are in fol-289-F described by breeding one belonging to the Strepto- 5 family. Bacteria belonging to myces phaeoverticiilatus - Among the strains producing 289-F are suitable Strain in a carbohydrate and nitrogen from Str. Phaeoverticiilatus NRRL 3021 and from the nutrient medium containing aerobic conditions, above-mentioned soil sample separated Streptomyces characterized in that one as species, which as a variant of Str. phaeoverticil bacterial staram ATCC 21395, 21396 or 21397 io latus are to be construed, especially for the purpose of