NL7908047A - ANTIBIOTIC, METHOD FOR PREPARING THE SAME, AND THIS ANTIBIOTIC CONTAINING USE COMPOSITION. - Google Patents

Description

Lx 5619

Title: Antibiotic, method of preparation thereof, and this antibiotic-containing use composition.

The invention relates to a new antibiotic, which will be designated SF-2052. The invention furthermore relates to a method for preparing this antibiotic by cultivating a strain of actinomycetes, in particular of the species Dactylosporangium or of the species Mikromono 3? o -r a in a culture media. Finally, the invention relates to a bacterial control composition comprising the aforementioned substance SF-2052 as the active ingredient.

Useful antibiotics can be prepared and separated from a culture fluid of various strains of micro-organisms. In an attempt to find new and useful antibiotics, which are active against gram-negative and gram-positive bacteria, and against various strains of bacteria, which are resistant to the known antibiotics, a study was carried out on the fermentation liquid of a new micro- organism called Dactylosporangium mat-suzakiense SF-2052. It has been found that a new antibiotic is obtained by cultivating this new microorganism in a culture medium containing nutrients which can be taken up under aerobic conditions. In addition, this new antibiotic could be separated from the culture liquid, which antibiotic has been given the designation SF-2052. The different properties of this substance have been determined. It has further been found that this new antibiotic can also be obtained by cultivating a strain of another new micro-organism, to which the name Mikro-25 monos po ra sp. SF-2098 is given.

The invention provides as a new antibiotic the aforementioned substance SF-2052, which can be used as a bactericidal agent. The invention further provides methods of preparing it from a strain of actinomycetes. All this will be explained in more detail in the description below.

According to the invention, as a new antibiotic, the aforementioned substance SF-2052 and a pharmaceutically acceptable acid addition salt thereof is provided, which substance is a water-soluble basic compound which has a bactericidal action, while the hydrogen chloride salt thereof is in the form of a colorless amorphous 7908047 * - 2 - powder, which is at least substantially stable under neutral to acidic conditions, but is unstable under basic conditions, has no further melting temperature, but gradually browns in the vicinity of 170 ° C, while at 209 · « Decomposition under foaming occurs at 210 ° C, which substance also has a positive reaction with ninhydrin, Lemieux reagent and Greig-Leiback reagent, but shows a negative reaction with Sakaguchi reagent and silver nitrate, which powder is finally soluble in water, is soluble in methanol, and is insoluble in ethyl acetate, chloroform, benzene and ethyl 10 ether.

The further properties of this salt are: (a) element composition: 35.69 #C, 7.04% H, 11.80 (b) no characteristic absorption peak at 220.370 nm in the UV absorption spectrum in a aqueous solution; (C) the IR absorption spectrum in a KBr tablet corresponds to the attached Figure 1; (d) the magnetic resonance absorption spectrum of hydrogen, determined in D2O, substantially corresponds to the attached FIG. 2; (E) the magnetic resonance absorption spectrum of carbon determined in substantially corresponds to the attached FIG. 3; O (f) the optical rotation in aqueous solution (c * 1) [α] ζ = + 87 J and 25 (g) a single spot is obtained at Rf = 0.46 by thin layer chromatography on cellulose with a mixture of n-propanol, pyridine, acetic acid and water (15: 10: 3 * 12) as developing solution, and at Rf = 0.09 by paper chromatography with a mixture of n-butanol, acetic acid and water (2: 1: 1) as developing liquid when applying the ascent method.

For example, the novel substance of the invention may be in the form of an acid addition salt of a pharmaceutically acceptable inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid, or a pharmaceutically acceptable organic acid such as acetic acid, citric acid, benzoic acid, ascorbic acid and malexneic acid.

The properties of the hydrochloric acid salt of SF-2052 are: 1 · Appearance: Colorless amorphous powder; 7908047 3 - 3 - 2. Melting Behavior: No particular melting temperature, slow discoloration to brown when heated to about 170 ° C, followed by decomposition at 209.210 ° C with foaming; 3 3 «Element composition C C 35 * 69 #, ï 7.0½ #, N 11.80 UV absorption: No characteristic absorption peak can be observed in the range 220-370 nm; IR Absorption: After tablet formation with KBr, the spectrum of Fig. 1 is obtained, with the main 10 absorption peaks occurring at 290, 350, 172, 16½, 151, 112 and 105 mra; 6. Molecular Weight: Since SF-2052 is unstable at basic center, the free base could not be obtained in stable form, so that direct determination of molecular weight by mass spectrometry was very difficult; it can be deduced from the magnetic resonance spectrum of carbon and any other available data that the free base should have a molecular weight of about 400 to 50; 7. Solubility: Easily soluble in water, soluble in methanol, and at least practically insoluble in organic solvents such as chloroform, ethyl acetate, hexane, benzene, ethyl ether and the like; 8. Stability: Relatively stable in acidic to neutral medium, but unstable in basic medium; 9 · Thin layer chromatography on cellulose: (a) Rf = 0.6 when developing with n-propanol / pyridine / acetic acid / water (15: 10: 3: 12) (b) Rf = 0.10 when developing with n-butanol / acetic acid / water (2: 1: 1); 10. Paper chromatography (ascending): (a) Rf '= 0, ^ 0 when developing with n-propanol / pyridine / acetic acid / water (15: 10: 3: 12) (b) Rf = 0.09 at development with n-butanol / acetic acid / water (2: 1: 1); 11. Color reactions: Positive for: ninhydrin, Greig-Leiback and Lemieux reagent 79 0 8 0 4 7, 1 - if -

Negative for: Sakaguchi reagent and silver nitrate;

12. Magnetic resonance: Hydrogen spectrum of SF-2052-HCl in D ^ O

at 100 MHz with external TMS standard, 5 is shown in Figure 2; the main signals are at 1.3½ (d), 2.00 (m), 3.16 (s), 3.51 (s), 5j33 (d), and 7 »99 Cs)» which number value - 6 represent the 10 parts, while Fig. 3 shows the carbon fiber drum with dioxane as the internal standard; 13. Optical rotation: £ cc] ^ = + 87 ° (cell, H2O).

On the basis of the aforementioned properties of SF-2052-HCl, it can be assumed that its free base has the following structural formula: 15. NH-CH, m2 \ Γ

COCH.NHCH

The invention will be explained in more detail below with reference to a drawing; 1 shows a curve of the infrared absorption of a sample of the HCl salt of the new substance according to the invention, taken after tablet formation with KBr; FIG. 2 is a curve of the magnetic resonance spectrum of hydrogen of the HCl salt of this substance in DIO at 100 MHz; and Fig. 3 shows a corresponding absorption spectrum of carbon at 25 MHz.

The action of the novel substance of the invention in the sulfate form in suppressing the grits of various bacteria was determined by a conventional dilution method using cardiac infusion agar as incubator. Incubation was effected at a temperature of 37 ° C for 16 hours. The minimum suppression concentration follows from Table A below.

7908047

a

t - 5 -

Table A

Minimum under dr.

Micro-organism concentration (μκ / ml)

Bacillus subtillis PCI-219 1.56

Staphylococcus aureus 209P 3 »12 5 Sarcina lutea 0.78

Escherichia coli 12.5

Escherichia coli B 6.25

Escherichia coli K-12-R 5 * 12

Klebsiella pneumonia 3f12 10 Proteus vulgaris 1.56

Pseudomonas aeruginosa 100

Mycobacterium saegmatis 607 Km-B 100 ii ii 607 Sm-B 1.56

This table shows that the novel substance according to the invention has a powerful effect against both gram-negative and gram-positive bacteria ·

This new substance having the aforementioned properties does not coincide with any of the known substances, so that this substance is clearly new. The invention further provides a method for preparing this new substance, which method comprises the following steps;

Ca) cultivation of a substance producing actinomyces, especially of this substance. Dactylosporangium matsu-25 zakiense SF-2052, in an aqueous culture medium containing absorbable carbon and nitrogen sources, the cultivation being carried out under aerobic conditions for a time sufficient to generate the SF-2052 in the culture medium and accumulate; and (b) separating the SF-2052 from the culture.

The microorganism used according to the invention to generate SF-2052 is Dactylosporangium matsuzakiense SF-2052, which had been separated from a soil sample found in Japan in 1977 (Inagami Shrine, Matsuzaki). cho, Kamo-gun, Shizuoka Prefecture). The SF-2052 strain was separated by incubating the soil sample in an aqueous suspension on a separating culture medium consisting of 0.2% soluble starch, 0.04% ammonium sulfate, 0.02% potassium biphosphate, 0.02 % magnesium sulfate, 0.0% calcium carbonate, and 1.5% agar agar 7908047-6% (without pH adjustment) existed, which operation was performed at 28 ° C for 30 d. This strain has been deposited with the Japanese Fermentation Research Institute under number FEEM-P 4670, and also with the American Type Culture Collection in Washington D.C. (US) under number ATCC 31570 (1979, 09, 26).

This strain trembles the following microbiological properties. The observations were made on the data provided by E.B. Shirling and D. Gottlieb in Int. J. Syst. Bact. 16 (1966) 312 * 34θ described; I. Morphological Observations ^ 10 Carrier mycelia grow in a wave-like form with very numerous branches, and have a diameter of 0.3, 0.6 µm. Neither in the agar agar nor in the liquid was disruption of this mycelia usually observed.

Aerial mycelia were virtually unobserved, so it can be assumed that they are not actually formed. The carrier mycelium of the SF-2052 strain carries sporangia, individually or in clumps, on the surface of the agar agar. Sporangia are relatively liberally formed on starch agar, but sparingly on glycerol asparagine agar, tyrosine agar and oatmeal agar. The sporangia are finger-shaped, and have dimensions of 0.9… 1.4 µm at 4.0… 6.0 µm. Each sporangium usually contains a single row of three spores. Most spores are cylindrical or ovoid, while their surface is smooth. Each track has a size of 0.8-1.3 µm by 1.1..1.6 µm. For example, when the spores are suspended in an aqueous soil extract and remain therein for 15-3 min, they exhibit mobility.

II. Feature £ and several different breeding centers:

The cultivation properties of the SF-2032 strain follow from Table B. The color designations enclosed in square brackets correspond to the “Color Harmony Manual *” of Container Corp. of America. The incubation equipment was 28 ° C, while the observations took place after 21 d. .

79 0 8.0-4 r "4« - 7 -

Table B

Air Soluble

Kweekmidden_ Growth and. color mycelium pigment

Sucrose nitrate agar normal growth, amber kl. to pale orange f3 lc] none none

Glucose Asparagine Agar Regular Growth, Red-Orange | f4pc] None None

Glycerol asparagine agar slight thin growth, pale melon yellow none none 13 ea] 5 Starch agar normal to good growth, red-orange none none (> P °]

Oatmeal agar normal growth, orange [4 lc-5 nc] none none

Yeast extract-malt extract- normal growth, agar coiled, none no amber- to light brown [3 lc-4 ng]

Tyrosine agar good growth, some brownish gray-orange to pink light brown l ^ l · lc-4 ng]

Food agar very low growth, none none pale orange 10 Bennet agar normal to good growth, red orange none none [k pc-5 pc]

Calcium maleate agar low to normal growth, light orange none none [3 ga-4 ga] III. _PhysiolOggic_features_: (1) Growth temperature: The SF-2052 strain grows within an area

. O

of 15..H-0 C on starch agar, where o * 15 the optimum temperature 25 · «3? C be; (2) Liquefaction of gelatin: No liquefaction occurred when incubation at 20 ° C for 21 d; (3) Starch hydrolysis: Positive (when incubated at 28 ° C for 21 d); 7908047 - 8 - a (½) Nitrate reduction: Negative (when incubating at 28 ° C for 21 d) j

(5) Curdling of buttermilk: Negative (when incubated at 28 ° C

for 21 d); -

5 (6) Peptization of buttermilk: Negative (when incubated at 28 ° C

for 21 d); (7) Melanoid pigment formation: Negative.

IV. Use of cabbage sources: An estimate was made when incubating at 28 ° C in the middle with the following composition:

Yeast Extract (Difco) 5 g

Calcium carbonate 1 g

Agar (Difco) 15 g

Distilled water 1000 ml 15 Pe results thereof are listed in Table C.

Table C

Carbon source Growth D-glucose +++ D-xylose +++ 20 D-fructose ++ L-arabinose +++ D-mannitol +++ X-inositol. +

Rhamnose +++ 25 Sucrose +++

Eaffinose +

No addition + V. Composition of the cell wall;

The cell wall and whole cell composition in the SF-2052 strain was examined in the manner described by T. Yamaguchi in J. Bact. 89 (1985) ^^ •• ^ 53> by B. Becker et al. In Appl. Microbiol. 12 (196½) ** 21..423 »by M.P. Lechevalier in J.Lab. Clin. Med. 71 (1968) 93 ^. 9½½.

From the results thus obtained it appears that this strain has 35 cell walls of. type II, since these walls contain hydroxydiamine-pimelic acid and glycine. Furthermore, the whole cell sugar pattern is of type A or D, since the whole cell hydrolyzate contains xylose and galactose along with a trace of arabinose.

7908047 - 9 -

In view of the aforementioned properties of this strain, it is believed to belong to the genus Dactylospo-r a n g i a of the order Actinomycetales. Two species are already known of the genus Dactylosporangia, namely 5 D. auranti & c njn and D. thailandense, see J. E. Thiemann et al., Arch. Mikrobiol. 58 (1967) k2 .. $ 2 and "Bergey's Manual of Determinative Bacteriology" 8th ed. (197 ^) · A comparison of the present strain with the known species is made in Table D. __, y / Λ t /

X

r / / y t *> / / / / ..

/ / 7210 8 0 4 7 - .10 - Λ

CM

ΙΛ 93 • η «a Ο 4) ο φ φ ο (Μ 'ϋ Ö d -4- Ο φ tO · 0

Ό · Η · / - »CO

ö Φ Φ 60 · Η ® Ι> Μ Β Ο, r "j Ή 4) id VO Ρ Η h Ö U Ο d ΛΑ,. Λ. · Η • Η b0 Cd Φ d Ή Β · Η * Η * Η * Η CM Τ 'Xl φ V, Ο 60 Ρ 3 Ρ © Φ Φ * ^ Φ Λ Φ Ο Η Φ Μ «U · Η · Η · Η Ο -μ Ö> Ο · Π Λ jd +> -Ρ +> COJ > Ο .61 d 'brf'Ö d ΧΪ · Η Β · Η Xl ΙΛΙ • 3 φ φ φ φ Ο Φ Ο 10 bO Q Φ Φ Q © 3> OhO Φ Ο Ο Φ Ο Ο · τ)

60 63 Ο UIO h 63 d ft d ft bO H

O ft

• Η O

& O +> d Φ Λ · Η £ d • · §

Λ * H

• Η Ο Φ

• Η ® d O

Φ O <d ε o d _ 60 3 (-1 (3

OH to Φ · Η · B

Β φ bO φ vs Xl CO

Η Ο -Η Φ .¾ d g d +> + »u xi xi 3 3 d © to

d Μ Φ · Φ Ο d 't-ι «Η * H Β -Ρ I

(Ö OO ΪΟΦΦΦΤ * S +> CM

d Φ H 60 63 I -Η Η Η * Φ B ΙΛ

p Β> + »+» + »+> · Η> O

do d μ d h Λ S. • 'i * ri J r? ® <y

3 +> © Φ +> © Φ O 60 (0 OH & i .O I

• φ · Η> Φ · Η Φ Φ -Η Φ O O Φ ft

0 60 3 Ο 63 3 Μ Η Η Ö ft ft 3 · A W

Η Φ • d © ·} Ή fl Q d S ·· * O + »d

H 'Ο · Η B

φ Xl ► 60 _o d

% d d I

b Φ Φ -p ^ t * xi d • rl φ Φ (Ö φ d A d ö odd h o id o® £> + i r bO Odd

ο · Η Φ ®> B

1 φ φ φ Ό JO Φ O

CM O vs d H 2 §

D d d Jci Β Φ, Λ P

Ο «> B d d O'h'h'hKX B · Η

CM d 30 d Φ Φ Φ * Φ d O

I φ o 63 I 60 Η Η H Xl © H

ft d I +> -Ρ-Ρ + »O Xl B

CQOOd d Λ d d Bddd rPdO

30Φ ΦΟΦ Φ bo bo to Φ ω Φ I

ΦΟΦ φ Η © Φ ΦΦΦΦ 60 *> + »60 d«> 63 H 60 60 d d d 60 3 „2

Η Λ B

Xl O d

I Φ O -P

X O Xl X

d. I> © CM

• rl ft 60 I * -P #

60 O -rl d Φ d T- CO

d ft φ B d Φ * -rl d d a d d ω η φ bo φ BPBdtOlO-P 60 Μ ft.HbOd © Bd d ft H ft v CO 60 B B d S Η Η Ο <O * ddddl-ΡΕΗΗΦ d.

Φ I d B to 10 * Φ 60 Φ .Ü ft Φ d 60 60 Oo8 ft ω · Η Γ ΛΙ ΙΛ ft CO oBd Hi ft ti ® H ft »# * BO ft I Ή ft i» s B Β H +> d J3 O 0} Φ Xl ΟΦ Xl> -P ft X>

Up -P Φ X d OBd ·· bh dBo do B to p ft φ d dbo d H l> Β HB dxll> © Φ> d> ft η © M © to bCft S ft ft H d H © d • HO bOSO 60ft Ο Φ-ΡΦΟ · Η ft d ft do oBsd x • Η Η · Η Ή · Η Η Η Β ® H d φ da υ φ a -pd φ! » d d * h φ

OB dH O dd B O d -P U <H Q

dtOOBd Ο Φ 60 d Φ Η B B ft

CD Β> O Cfl> BBC3> 3 W «O

7908047 - 11 -

The SF-2052 strain does not coincide with any of the known species of the genus Dactylosporangia, so it can be considered a new species, which is referred to as Dactylosporangium matsuzakiense SF-2052.

5 A new micro-organism of an in

Choshi, Chiba prefecture, Japan, to separate collected soil sample, which micro-organism has been called SF-2098 strain. This strain appears to belong to the order of the Actinomycetales, and to the genus Hikromonospora. This strain is registered in Japan under number 10 FEBM-P 5073, and under number with the American Type Culture Collection. It has been found that this SF-2098 strain can produce the aforementioned substance SF-2052 according to the invention.

The invention provides a method of preparing SF-2052. which includes the following steps: (a) Growing a strain of Mi kr om o n o s po r a sp. SF-2098 (registered under FEEM-P 5073 and ATCC) in an aqueous liquid culture medium containing absorbable carbon and nitrogen sources under aerobic conditions for a time sufficient to propagate the SF-2052 through the culture medium bring and accumulate; and (b) Separating the SF-2052 from the culture.

The SF-2098 strain has the following microbiological properties: 25 I · Morphological observation:

The SF-2098 strain does not form air mycelium on various agar culture media, which are generally used for culturing actinomycete s strains. Microscopic examination has shown that the carrier mycelium grows with branches and that a trace is formed at various points thereof. When observed with an electron microscope, the spores were found to be approximately spherical, while the diameter was approximately 1.0 to 1.3 µm. The surface of the spores showed blunt protuberances.

II. Culture behavior on different culture media: The development of SF-2098 cultures incubated on different culture media for 15 days at 28 ° C was observed, with the results reported in Table E. The color indications in square brackets are based on the Color Harmony Manual from Container Corp. of America.

7908047 *

- 12 -Table E

Soluble _ Culture medium Growth and color pigment

Sucrose nitrate agar normal to good growth, no arc, orange [4 la-4 na]

Glucose asparagine agar very weak growth, no colorless 5 Glycerol asparagine weak growth, yellow agar brown [3 ie], later different in olive green [1 »5 ge]

Starch agar normal growth, orange brown [3 lc], later changing to dark olive green [1,5 pn]

Yeast extract-malt extract- normal to good growth, agar light olive green none [1.5 11..1.5

Oatmeal agar normal growth, orange-brown [3 lc], later changing to olive green [1.5 ig]

Tyrosine agar 'weak growth, orange to no yellow-brown [3 lc. * 3 ie] 10 Food agar weak growth, weak yellow- no brown [3 gc]

Bennet agar normal growth, moist, no orange-brown [3 lc]

Calcium malate agar weak growth, light olive- no green [1.5 g] lil. Physiological Properties ^ (1) Growth Temperature Range: The SF-2098 strain grows in a range of 15 · 5 ^ 5 ° C; (2) Gelatin Flowing Positive (incubated at 20 ° C for the 21 d); (3) Starch hydrolyses Positive (incubated at 28 ° C for 14 d); 20 (* 0 Effect on buttermilk: Positive to peptization and curdling (incubated at 28 ° C for 21 d); (5) Melanoid pigment formation: Negative; 7908047 - 13 - (6) Nitrate reduction: Negative (incubated at 28 ° C for 14 d) 5 (?) NaCl tolerance (estimated according to Intern. J. Syst. Bacteriol.

21, (1971) 240) $ 5 good growth at .0..1.5 weak growth at 3 · «4 and no growth at more than 5% * IV. Consumption of carbon sources ^ The estimate was made by incubating at 28 ° C in a center with the following composition:

Yeast Extract (Difco) 0.5%

Calcium carbonate 0.1%

Bacto agar (Difco) 1.5%

Distilled water, rest 15 The results are shown in table Έ.

Table F

Carbon source Growth

No additive + D-glucose +++ 20 D-fructose "++ D-xylose +++

Glycerol + L-arabinose + D-mannitol + 25 I-innositol +

Sucrose +++ α-melibiosis +

Baffinose +

Shaanose + 30 V. Chemical composition of the cell wall:

The cell wall composition of the SF-2098 strain was examined as described by B. Becker et al. In Appl. Microbiol.

13 (1965) 236. From the results thus obtained it appeared that the cell vand contained hydroxydiaminopimelic acid.

In view of the aforementioned properties of the strain examined, taking into account in particular the fact that no air mycelium is formed on agar media, that furthermore this strain is a mesophilic actinomyces, which has a single trace on the wire 7908047 V - + - - + + carries germycelium, and that the cell wall contains hydroxydiaminopimeline acid, it can be concluded that this strain belongs to the species Mikro-monospora. Therefore, this strain is now referred to as Mikromonospora sp. SF-2098. In the fermentation of the SF-2052 according to the invention, using either Dactyl o sporangium matsuzakiense SF-2052 or Mikromonospora sp. SF-2098, the strain producing SF-2052 can be grown for its intended purpose in a known manner in a culture medium containing food sources which can be taken up by common microorganisms. For this purpose use can be made of known nutrients which are generally used for the cultivation of known strains of actinomycetes. Examples thereof are glucose, glycerine, sucrose, starch, dextrin, maltose syrup, molasses, and soybean oil as a carbon source, soybean meal, wheat germ, meat extract, peptone, yeast extract, dried yeast, corn oil, cottonseed meal, ammonium sulfate and sodium nitrate as a nitrogen source. If desired, inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, cobalt chloride, phosphates and the like can be added. Furthermore, organic or inorganic substances, which promote the growth of the SF-2052 or SF-2098 strain used and the formation of the SF-2052 product, may be included in the culture medium. As to the culture methods to be used, it may be noted that cultivation in a liquid, and in particular in the immersed state, is generally preferred, as is the case with the known antibiotics. In the present case, cultivation is carried out under aerobic conditions. The SF-2052 strain can be incubated at a temperature of 25 · 37 ° C, and usually at 28 ... 32 ° C. Thereby, the formation of the SF-2052 substance 30 in the culture mass reaches a maximum at the end of an incubation period of 2.10 d, both in shaking culture and in culture in a tray.

The SF-2098 strain is incubated at a temperature of 25 - 0 ° C, and

O

usually at about 30 ° C. The culture medium is preferably kept neutral or weakly basic. When growing in a liquid of this die, the formation of SF-2052 in the culture mass usually reaches a maximum within 3 * 10 d.

For example, to calibrate the SF-2052 formed in this manner, a calibration medium containing 0.5% polypeptone, 0.3% beef extract, and 1.5% agar (pH 7 * 0) may be used, as 7908047 - 15 - Calibrator Baci-llus subtillis PCI-219 jug. are used. At a concentration of 50..10 µg / ml of the SF-2052, the relationship between the logarithm of the concentration and the centerline of the suppression region is represented by a line, which corresponds to a range of 21.2..12, 8 mm with the usual determination with a paper disc.

The SF-2052 substance of the invention is a water-soluble basic antibiotic, while when produced by cultivation of the S3P-20 $ 2 or SF-2098 strain, it is essentially in the liquid phase of the culture mass is present and can be separated therefrom using the physicochemical properties of this substance. Separation and purification can be done by chromatography using an absorption plastic such as Amberlite XAD-2 (consisting of a microporous copolymer of styrene and divinylbenzene supplied by Rohm & Haas CoO and Diaion HP-20 (consisting of a microporous copolymer of styrene and divinylbenzene, supplied by Mitsubishi Kasei Co.) or a gel filtration agent such as Sephadex G-10 (consisting of dextran with epichlorohydrin cross-connections supplied by 20 Pharmacia Co.) and Sephadex LH-20 (a gel consisting of a derivative of dextran sulfate, also supplied by Pharmacia Co.), or a cation exchange resin such as Amberlite IRC-50 or Amberlite CG-50 (a weakly acidic exchange resin consisting of a copolymer of methacrylic acid and divinylbenzene supplied by Rohm & Haas Co.) and 25 CM-Sephadex (a gel filtration agent consisting of a cross-linked dextran gel substituted with carboxy-methyl supplied by Pharmacia Co.). Separating the SF-2052 from the culture mass can be done as follows. This mass is stripped of the solids by filtration or centrifugation, after which the residue is treated with Diaion HP-20 to adsorb the active substance, which is then washed out of the resin by development with a 50% aqueous acetone solution. (without pH adjustment) and with a 50 # * s aqueous acetone solution (pH = 2.0, adjusted using hydrochloric acid) as the detergent, after which the washed liquid is concentrated under reduced pressure to give a crude powdery product is obtained, which consists of SF-2052 hydrochloride.

The crude product is then purified by column chromatography using CM-Sephadex (H + form), 7908047-16 * CM-Sephadex (Na + form) and / or activated charcoal with a suitable composition * in which a highly purified product is obtained * consisting of SF-2052 hydrochloride. Since the free base of SF-2052 is very unstable under basic conditions, it is difficult to obtain the pure product in the form of a free base. It is important to always carry out the separation and purification of SF-2052 under neutral or acid conditions *, where it is recommended that a weakly acidic solution of SF-2052 be subjected to a freeze-drying process as a last step * in which SF-2052 is in the hydro -10 chloride or sulfate form if a colorless amorphous powder is obtained.

When SF-2052 is to be separated from the culture mass of the SF-2098 strain, it makes sense and effective to adjust this mass to a pH of 2.0 by adding hydrochloric acid, and then filter this mass, for example with using a diatomaceous earth filter to remove the mycelium cake and other solids therefrom, and the filtrate is passed through a column of Amberlite IRC-50 (Na + form) to adsorb the active substance. Finally, the resin column is washed with 0.4 N 20 hydrochloric acid. The resulting liquid is neutralized with aqueous sodium hydroxide, and then desalted with activated carbon. The resulting solution is further purified by column chromatography with CM-Sephadex, activated carbon and / or Sephadex LH-20 to obtain a highly purified product consisting of SF-2052 hydrochloride.

To determine the acute toxicity of SF-2052 sulfate, this substance was injected intravenously into different groups of mice in an isotonic aqueous solution of sodium chloride.

SF-2052 sulfate was found to exhibit an LD ^ Q value of 350 mg / 30 kg in mice, and can therefore be considered non-toxic. The invention further provides a bactericidal agent comprising as an active agent an effective amount of at least one of the pharmaceutically acceptable acid addition salts of SF-2052, together with a pharmaceutically acceptable liquid or solid carrier. The amount of this active substance can thereby be 5 · 50% by weight.

The bactericidal agent of the invention can be used as an aqueous solution containing 0.01 to 0.1% by weight of SF-2052 acid addition salt, which solution is suitable for sterilization of surgical equipment and instruments. , as well as other appliances and areas to be kept sterile. This agent can further be used in an oral form such as tablets, capsules, powders, solutions and suspensions, by an amount of a pharmaceutically acceptable acid addition salt of SF-2052 with a conventional pharmaceutically acceptable solid carrier such as by mixing starch, sucrose, talc or calcium carbonate, or by dissolving or suspending this substance in a pharmaceutically acceptable liquid such as ethanol and water. The amount ratio between the active substance according to the invention and the carrier material depends on the nature of the prepared agent to be taken in the mouth, and is usually about 1: 1: 1: 100.

This bactericidal agent can also be used as an injectable solution or suspension, in which case the active substance is dissolved or suspended in an amount of 0.1..10 wt.% In a physiological saline solution or the like carrier, for example Ringer's solution. , with or without addition of a suitable spreading agent. Such a solution or suspension can be used for intravenous, intramuscular or intraperitoneal injection.

It will be clear that the amount of the active substance according to the invention used depends on the nature of the composition used for administration, furthermore on the manner of administration, and on the disease to be treated. Obviously, account should be taken of age, body weight, gender, diet, time of administration, route of administration, degree of excretion, use of other drugs, susceptibilities and severity of the disease. Usually about 0.5, 100 mg / kg of the active compound per day is given to an adult. Optimal amounts can be determined in the usual way, taking into account past experience.

The invention will be explained in more detail below by means of a number of non-limiting examples.

Example I

(1) Incubation of the SF-2052 strain

A stock culture of Dactylosporangium matsu-zakiense SF-2052 (registered as FERM-P A-670 and ATCC No.

31570) was used, while sterilized 7908047 f.

- 18 - seed culture medium was used which contained 2.0% glucose, 0.5% peptone, 0.2 # meat extract, 0.3% yeast extract, 0.2% soybean meal and 0.1% calcium carbonate (pH 7.0 before sterilizing).

From this stock culture, 5 to 6 loop fills were seeded 5 to 20 inl of the aforementioned seed culture medium in a 100 ml Erlenmeyer flask and incubated at 28 ° C for 6 d to yield a first seed culture. This first seed culture was formed in 10 flasks. Total 200 ml of the first seed culture was seeded in 20 ml portions in 10 500 ml Erlenmeyer flasks, each containing 80 ml of the aforementioned seed culture medium, and incubated at 28 ° C for 72 h.

In this manner, a second seed culture was obtained, which was seeded in 5 ml portions into 150 500 ml Erlenmeyer flasks, each containing 80 ml of a preparation culture medium. The latter contained 4.0% glucose, 1.0% peptone, 0.4 # meat extract, 0.6% yeast extract, 0.4% soybean meal and 0.2% calcium carbonate, i.e. each constituent in twice the amount than in the aforementioned seed culture medium. Culturing was done at 28 ° C for 6 d with shaking as usual.

Subsequently, the culture mass was subjected to a continuous centrifugation operation using a cooled centrifuge, whereby 10 1 of filtrate was obtained in about 2 h.

(2) Separating_the_SF-2052 £

The aforementioned filtrate (10 1) was passed through a column containing 750 ml of the aforementioned Diaion HP-20, in order to adsorb the active substance. This resin column was then washed with 3 L of water, rinsed with 1.8 L of a 50 # aqueous acetone solution (pH not adjusted), then with 1.8 L of 50% FS aqueous acetone solution (pH 2.0) with HCl). In this manner, the S 2 -2052 was washed from the resin column. The washings were collected in 1.8 L aliquots.

The active ingredients (together 3 * 6 L) containing the SF-2052 were compacted under reduced pressure by evaporation of the acetone. The concentrate was then left to stand, then filtered to remove the precipitate formed. The filtrate was passed through a 300 ml column of the aforementioned CM-Sephadex C-25 (H + form) to adsorb the active substance.

This column was washed with successive amounts of 1.0 L NaCl, 0.1 N, 0.2 N, 0.4 N, respectively. 0.6 N. The coil

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Liquid was collected in 18 ml portions, washing out the active substance in 26 × 92 portions.

- The active ingredients were passed through a column of 200 ml of chromatographically activated carbon to adsorb the active substance, which column was washed with 900 ml of water, followed by rinsing with 1.2 l of 50% acetone aqueous solution (pH not adjusted) and then with 600 ml of 50% aqueous acetone solution (pH 2.0 adjusted with HCl) The rinsing liquid was collected in 1800 ml portions, after which the active parts (total 1800 ml) containing the washed SF-2052 , were combined and the resulting solution was compacted under reduced pressure. This compacted solution was freeze-dried to yield 170 mg of a raw white powdery product (purity about 20 #), which product was SF-2052 hydrochloride.

15 (3) Purification

The aforementioned crude product (170 mg) was dissolved in 5 ml of water, after which the resulting solution was passed through a column of 180 ml CM-Sephadex C-25 (Na + form), which column was soaked with 0.1 N NaCl. This column was then rinsed with 2 L 0.5 N NaCl followed by development with 1.0 N NaCl. The rinsing fluid was collected in 18 ml aliquots, and the 8 ·· 20 aliquots containing the SF-2052 were reassembled.

The active ingredients (together 220 ml) were passed through a column of 40 ml of chromatographically activated carbon to adsorb the active substance. This column was washed with 250 ml of water, followed by rinsing with 200 ml of 50 # aqueous acetone solution (pH not adjusted), then with 200 ml 5 ° aqueous acetone solution (pH 2.0) to remove the active substance from the column to rinse. The rinsing liquid was collected in 400 ml portions, after which the active ingredients (total 400 ml) were compacted under reduced pressure. The resulting solution was frozen to dryness to yield 26 mg of SF-2052-HCl in the form of a colorless powder, which was pure.

(4) Formation of SF-2052-sulfate-35 The aforementioned colorless powder was dissolved in 10 ml of water, after which the solution was passed through a column of 10 ml of strongly basic anion exchange resin, namely Amberlite IHA-4QQ (sulfate form). The flow-through liquid was compacted and freeze-dried, yielding 30 mg of the SF-2052 sulfate in the form of 7908047-20 - * a colorless powder. This product had a decomposition temperature above 220 ° C. .

Example II

The filtrate 5 (10 L) obtained in the step (1) of Example I was passed through an 800 ml column of Diaion HP-20 to adsorb the active substance. This column was washed with 1 L of water, then rinsed with 50% aqueous acetone solution (pH * 0, 0) while collecting the rinsing liquid in 500 ml aliquots. Portions 2 ·· 6 contain the rinsed active substance, which 10 portions were combined and stripped of the & c $ ton under reduced pressure ·

The remaining liquid was allowed to stand for some time, after which the precipitate formed was filtered off. The filtrate was passed through a 250 ml column of Amberlite 1BG-50 (Na + form), in order to adsorb the active substance. This column was then washed with 2 L of water, then rinsed with 0.1 N HCl. The rinsing liquid was collected in 18 ml portions, the active substance being rinsed in the portions 2 ^ 0..336. These were reunited and passed through a 150 ml Amberlite IB-5 (0H ~ -20 form) column to be neutralized.

The solution (pH 5 »**) leaving this column was fed into a column of 80 ml of chromatographically activated carbon to adsorb the active substance, which column was washed with kOO ml of water, then rinsed with 3 ° 0 ml of 5% aqueous acetone solution (pH not adjusted), then with 300 ml of 50% aqueous acetone solution (pH 2, 1/2 adjusted with HCl) to desorb the active substance from the resin column.

The active solution thus obtained (total 600 ml) was concentrated under reduced pressure, and then freeze-dried, yielding 260 mg of SF-2052-HCl in the form of a slightly yellow colored raw powder (purity 16 #).

Example III

(1) Incubation of the SF-2098 strain;

A stock culture of Mikromonospora sp. SF-2098 35 (registered as FERM-P 5073 and ATCC No.) was used, as well as a sterilized seed culture broth containing 2.0% glucose, 0.5% peptone, 0.2% meat extract, 0.3% yeast extract, 0 , 2% soybean meal, and 0.14 calcium carbonate (pH, not adjusted).

7908047> * - 21 -

From this stock culture, 3 or 4 loop fills were seeded to 20 ml of the aforementioned inoculum in a 100 ml Erlenmeyer flask, followed by incubation at 28 ° C for 4 d, yielding a seed culture. This was done in 20 flasks.

The resulting culture (total 40 ml) was seeded in 4 ml aliquots into 100 500 ml Erlenmeyer flasks, each containing 80 ml of a sterilized culture broth. This product contained b% maltose syrup, 0.3% soybean oil, 0.5% soluble vegetable protein, 1.0% cottonseed meal, 0.001 # ferrous sulfate, 0.0001% cobalt-10 chloride and 0.2% calcium carbonate (pH 7 .0 for sterilizing).

Incubation was done at 28 ° C for 2 d, as usual with shaking.

After incubation, the culture mass was adjusted to pH 2.0 by addition of 6 N HCl, then filtered with diatomaceous earth to yield 7.0 L of filtrate.

(2) Separating_the_SF-2052ï_

The aforementioned filtrate (71) was passed through a 220 ml column of Amberlite IBC-50 (Na + form) to adsorb the active substance.

This column was then washed with 1 l of water, followed by rinsing with 0.4 N HCl. The rinsing liquid was collected in 250 ml aliquots, with the active substance rinsed out in 2.5 aliquots. These were combined and the pH adjusted to 4..6 by adding 0.2 N NaOH and the liquid was passed through a 150 ml column of activated carbon to adsorb the active substance. This column was washed with 500 ml of water, then rinsed with 400 ml of 50% aqueous acetone solution (pH not adjusted), then with 400 ml of 50% aqueous acetone solution (pH 2.2), to remove the active desorb the cabbage column.

The active parts of the rinsing liquid (total 800 ml) were combined with each other and the acetone was removed under reduced pressure.

The solution thus obtained was passed through a column of 200 ml CM-Sephadex 0-25 to adsorb the active substance.

This column was washed with 1 L 0.3 N NaCl, then 35 with 1 L 0.5 N NaCl, followed by rinsing with 0.5 N NaCl. The rinsing liquid was collected in 20 ml aliquots, leaving the '102 aliquots. .172 containing the SF-2052 were combined. The active solution was passed through a column of 50 ml of chromatographically activated carbon to adsorb the active substance. This column was washed 7908047 ff - 22 - with 200 ml water, then rinsed with 200 ml 50 - # * s aqueous acetone solution (pH not adjusted), then with 200 ml 50% * b aqueous acetone solution (pH 2.0 adjusted) with HCl) rinsing out the active substance in good yield · The active parts (total 800 ml) were concentrated under reduced pressure and then frozen to yield 12 * mg of a raw white powder (purity 20 #) , which powder contained SF-2052-HCl.

120 mg of this crude powder was dissolved in ml of 50% aqueous methanol solution, which solution was passed through a column of 330 ml of Sephadex LH-20, which had been well washed with 90 ml of methanol solution. This column was developed with 90 aqueous methanol solution, collecting the rinsing liquid in 12 ml portions. The active substance was rinsed in portions 12..16. The combined active portions (total 60 ml) were concentrated under reduced pressure and frozen to afford 20 mg of the pure SF-2052-HCl as a colorless powder.

7908047

Claims (7)

1. Novel substance) named SF-2052, and a pharmaceutically acceptable acid addition salt thereof, which substance is a water-soluble basic compound exhibiting a bactericidal action, SF-2052-HCl being in the form of a colorless and amorphous powder, which is at least substantially stable in neutral to acid medium, but is unstable in basic medium, which substance further has no particular melting temperature, but discolours at or near 170 ° to brown, and decomposes under foaming at 209 · 210 ° C, which substance further reacts positively with ninhydrin, Lemieux reagent and Greig-Leiback-10 reagent, but negatively with Sakaguchi reagent and silver nitrate, and which substance is finally soluble in water, fairly soluble in methanol, and insoluble in ethyl acetate, chloroform, benzene and ethyl ether, characterized by: (a) a composition of 35 * 69% C, 7 * 04% H, 11.80% N; (B) the absence of a typical absorption peak in the 220-370 nm region of the UV absorption spectrum in an aqueous solution; (c) an IR absorption spectrum in a KBr tablet corresponding to the spectrum plotted in Figure 1; (D) an absorption spectrum for magnetic resonance of hydrogen, determined in D 2 O, which substantially corresponds to the spectrum shown in Figure 2; Ce) a magnetic resonance absorption spectrum of carbon, determined in D 2 O, which substantially corresponds to the spectrum shown in Figure 3; O (f) an optical rotation in aqueous solution (c = 1) [oe] ^ '= +87; and (g) a single spot at Rf = 0.46 by thin layer chromatography on cellulose with a mixture of n-propanol, pyridine, acetic acid and water (15: 10: 3: 12) as developing solution, and at Rf = 0.09 in paper chromatography with a mixture of n-butanol, acetic acid and water (2: 1: 1) as developing liquid by using the incremental method. 2. A substance according to claim 1, in the form of SF-2052 hydrochloride or sulfate. The method of forming the substance according to claim 1, characterized by: 7908047 r-2k - (a) culturing an SF-2052 producing strain of actinomyces, in particular Dactylosporangium matsuzakiense SF-2052, registered as FERM-P 4670 and ATCC 31 570, in an aqueous liquid culture medium containing absorbable carbon-5 and nitrogen sources, the cultivation taking place under aerobic conditions for a time sufficient to produce the SF-2052 in the culture medium and to accumulate; and (b) separating the SF-2052 · 10 k · Method from the culture according to claim 3 *, characterized in that the cultivation takes place at a temperature of 25 ° 37 ° C for 2..10 d. 5. A method according to claim 3 or 4, characterized in that the SF-2052 is separated from the culture mass in the hydrochloride form. A method of producing the substance according to claim 1, characterized by: (a) culturing a strain of Mikromonospora spo SF-2098, registered as FERM-P 5073 and ATCC, in an aqueous liquid culture medium, which is absorbable contains carbon and nitrogen sources, the cultivation being carried out under aerobic conditions for a time sufficient to generate the SF-2052 in the culture medium and to accumulate; and (b) separating the SF-2052 from the culture. The method according to claim 6, characterized in that the cultivation takes place at a temperature of 25 · · kO ° C for a time of 3 · 10 d. 8. A method according to claim 6 or 7 *, characterized in that the SF-2052 is separated from the culture mass in the hydrochloride form. · 9. A bacterial control composition comprising, as an active ingredient, an effective amount of at least one of the pharmaceutically acceptable acid addition salts of SF-2052, together with a pharmaceutically acceptable carrier for this ingredient. 7908047