FI70594C - PROCEDURE FOR THE PRODUCTION OF PHARMACOLOGICAL PROPERTIES OF HYDROXIAMINKOLVAETEFOSPHONYRADERIVAT - Google Patents

Description

70594

The process prepares pharmacologically valuable hydroxyaminohydrocarbon phosphonic acid derivatives. - Förfarande för f ramstälining av pharmacologiskt värdefulla hydroxiaminkol-vätefosfonsyraderivat.

By dividing the separated patent application 772280 from Avdelad ur patent application 772280

The present invention relates to a process for the preparation of pharmacologically valuable hydroxyaminohydrohosphonic acid derivatives of the formula

OH O

Ί, I tl R1 - N - A'- P - OH (I ")

a I

OH

wherein R 1 'is acetyl and A' is trimethylene or 2-hydroxy-trimethylene; or R 1 'is formyl and A' is trimethylene or 3.

trans-1-propenylene and pharmaceutically acceptable salts thereof.

In general, the compound (I ") as defined above is prepared by culturing a microorganism belonging to the genus Streptomyces in a conventional manner. More specifically, the compound (I") is prepared by culturing a microorganism belonging to the genus Streptomyces, such as the following: Streptomyces rubellomurinus, Streptomyces rubellomurinus subsp. indigoferus, Streptomyces lavendulae and the like. Compound (I ") in which R 1 'is acetyl and A' is trimethylene (hereinafter referred to as FR-900098) is prepared by fermentation of Streptomyces rubellomurnius and its subspecies indigoferus; compounds (I") in which R 1 'is formyl and A 'is trimethylene (hereinafter referred to as FR-31705) and R 1' is formyl and A 'is trans-1-propenylene, 3 _______ - ΤΓ ^ 70594 2 is prepared by fermentation of Streptomyces lavendulae; and the compound (I ") in which R * 'is acetyl and A' is 2-hydroxytrimethylene is prepared by fermentation of Streptomyces rubel.lo-murinus subsp. indigoferus.

Fermentation of said microorganisms is performed in a nutrient medium containing assimilable carbon and nitrogen sources, preferably under aerobic conditions (e.g., shake culture, subsurface culture, etc.). Details of the method are given below.

Recommended carbon sources for nutritional sodium include glucose, fructose, glycerin and starch. Other useful sources include lactose, arabinose, xylose, dextrin, molasses and the like.

Recommended nitrogen sources include yeast extract, peptone, gluten flour, cottonseed meal, soybean meal, corn steep liquor, dry yeast, wheat germ, etc. Both inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium sulfate). i, etc.), urea, amino acid and the like.

Carbon and nitrogen sources, although used in combination, do not need to be used in pure form, as impurity substances containing trace amounts of growth factors and significant amounts of mineral nutrients are also suitable. If desired, mineral salts such as calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, magnesium salt, copper salt and the like may also be added to the medium. If necessary, especially when the culture medium foams a lot, an antifoam agent such as liquid paraffin, fatty oil, vegetable oil, mineral oil and silicones can be added.

3 70594

As with the recommended methods used to prepare other antibiotics in bulk, aerobic subsurface culture is used to produce large amounts of compound (I "). Shorter amounts are used in a shake culture or surface culture in a container or bottle. In addition, when growing in large containers, it is recommended to use vegetative form of the organism to avoid growth delay in the production of compound (I "). Thus, it is desirable to first prepare a vegetative inoculum of the organism by inoculating a relatively small amount of culture medium with the spores or mycelium of the organism and grow this and aseptically transfer the cultured vegetative inoculum into large containers. The medium in which the vegetative inoculum is prepared may be substantially similar to or different from the medium used to prepare compound (I ").

Mixing and aeration of the culture mixture can be performed in many different ways. Mixing can be accomplished with a propeller or similar mechanical stirrer, by rotating or shaking the fermentor, by means of various pumping devices, or by passing sterile air through the medium. Aeration can be performed by passing sterile air through the fermentation broth.

The fermentation is usually carried out at a temperature between about 20 and 40 ° C, preferably at 30 ° C for 50 to 100 hours.

Compound (I ") can be recovered from the culture medium in the usual manner commonly used in the recovery of other known antibiotics.

In general, most of the formed compound (I ") is grown in 1 solution and thus the compound (I") can be separated from the filtrate obtained by filtration or centrifugation of the solution by a conventional method such as concentration under reduced pressure, lyophilization, solvent extraction, adjusting pH, by treatment with a resin (e.g., anionic or cationic ion exchange resin, nonionic absorption resin), treatment with an adsorbent (e.g., activated carbon, silica, silica gel, cellulose, alumina), crystallization, recrystallization, and the like.

(I). Preparation of compound (I ") wherein R 1 'is acetyl and A' is -CH 2 -CH 2 -CH 2 -, i.e. 3- (N-acetyl-N-hydroxyamino) propylphosphonic acid (hereinafter referred to as FR-900098):

The antibiotic FR-900098 can be prepared by fermenting in a nutrient medium a strain of the genus Streptomyces that forms the antibiotic FR-900098, such as the strain Streptomyces rubelle-murinus and the strain Streptomyces rubellomurinus subsp. indigoferus strain.

(1) Micro-organisms:

The microorganisms that can be used in the preparation of the novel antibiotic FR-900098 are strains of Streptomyces rubellomurinus recently isolated in Japan, Mt. Hira,

Siga Prefecture, from a soil sample taken, and Streptomyces rubellomurinus subsp. indigoferus strains recently isolated from a soil sample at Koganei city,

Toki o, Japan.

A culture of the live Streptomyces rubellomurinus was deposited with the ATCC No. 31215 in the permanent culture repository of the American Type Culture Collection. The culture of the organism is further recorded under the number 3563 in Japanese Il 5 70594 5 in the collection of the Fermentation Research Institute, Agency of Industrial Science and Technology.

Live Streptomyces rubellomurinus subsp. the culture of the indigoferus organism is deposited under ATCC No. 31304 in the permanent culture stock of the American Type Culture Collection. The culture of the organism is further stored in the Japanese collection of the Fermentation Research Institute, Agency of Industrial Science and Technology.

The preparation of a new antibiotic is not limited to the use of a particular organism described herein, which is presented for illustrative purposes only. Ts. both artificial mutants and natural mutants can be used to make the antibiotic. Such an artificial mutant is made from the organism described herein in the usual manner, such as by X-ray irradiation, UV irradiation, N-methyl-N'-nitrosoguanidine, 2-aminopurine, and nitrogen mustard oils.

1) Microbiological properties 1) —1 Streptomyces rubellomurinus ATCC 31215:

Streptomyces rube1lomurinus ATCC 31215 has the following morphological, cultural and physiological characteristics: 1. Morphological characteristics

The morphology of the culture was observed microscopically for 10 to 14 days at 30 ° C on mycelium grown on the following media: sucrose nitrate, agar, glycerol-asparagine agar, yeast malt extract agar and oatmeal agar.

70594 6 (1) Method of branching of spore-forming hyphae:

Monopodial branching (2) Shape of spore-forming hyphae:

Straight or curved (rectiflex) (3) Number of spores: 10 to 50 spores (4) Appearance and size of spore surface:

Smooth, 0.4 to 0.8 x 1.1 to 1.6 microns (5) Occurrence of zoospores:

Not observed (6) Occurrence of spore colonies:

Not observed (7) Spore formation:

In the air system 2 · Vi 1 j elyomi na isities:

The strain had the following culture characteristics when grown for 10 to 14 days at 30 ° C on the following media.

7 70594

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70594 8 3. Physiological properties: (1) Growth temperature range (Bennett's agar slant 11a):

12-40 ° C, optimum: 27 ° C

(2) Liquefaction of gelatin (glucose-peptone-gelatin slant): negative (3) Hydrolysis of starch (starch-inorganic salts on agar): positive (4) Coagulation and peptonization of skimmed milk:

Coagulation: positive

Peptonization: weak (5) Production of melanoid pigment (tyrosine-agar11a, peptone-yeast iron agar and tryptone-yeast extract-1 solution): negative (iv) Cell wall: type I (contains LL-diaminopimelic acid) (7) Use of carbon sources (on Pridham-Gottlieb agar)

Carbon source Growth L-arabinose ++ D-xylose + Note: ++ = Very good D-glucose ++ + = Very S-fructose + ± = Uncertain

Sucrose ± - = Not applicable

Inositol L-rhamnose

Raffinose ± D-mannitol - D-mannose

Salicin 70594 9

From the literature, "Bergey's Manual of Determinative Bacteriology" Eighth Edition (1975), "The Actinomycetes" Vol. II (1961), by SA Waksman and "The International Streptomyces Project Reports", by EB Shirling and D. Gottlieb (cf. International Journal of Systematic Bacteriology Vol. 18, pages 69 and 279 (1968), Vol. 19, page 391 (1969) and Vol. 22, page 265 (1972), strains with the above-mentioned characteristics have been searched for). relatively analogous properties to strain ATCC No. 31215, have been found to be Streptomyces sindenensis, Streptomyces xanthocidicus and Streptomyces exfaliatus.

However, strain ATCC No 31215 differs from these analogous species in the following respects: (i) Streptomyces sindenensis:

The mature spore chains of Streptomyces sindenensis are usually short. The spores of the species are weak on starch-inorganic salts-agar. A little glycerol-asparagine agar11a is formed in the air mycelium of the species. One strain of the species can assimilate D-mannitol.

(ii) Streptomyces xanthocidicus:

The aerial mycelium of Streptomyces xanthocidicus is abundant on both glycerol-asparagine agar and yeast-malt extract agar. Some strains of the species produce melanoid pigments. One strain of the species can assimilate sucrose and raffinose relatively strongly.

10 70594 (iii) Streptomyces ex foii atus:

Streptomyces exf oi iatus forms an air mycelium on glycerol-asparagine agar. The spores of the species are very abundant on yeast maleate agar. One strain of the species can assimilate sucrose and raffinose relatively well. No rupture and sporulation of the species in the substrate mycelium is observed.

In view of the result of the above examination and the fact that strain ATCC 31215 is capable of producing the new antibiotic FR-900098, strain ATCC 31215 can be considered as a new species belonging to the genus Streptomyces, named Streptomyces rubellomurinus.

l) -2 Streptomyces rubellomurinus subsp. indigoferus ATCC 31304:

Streptomyces rubellomurinus subsp. indigoferus ATCC 31304 has the following morphological, cultural and physiological characteristics: 1. Morphological characteristics

Cultures grown for 10-14 days at 27 ° C on sucrose nitrate agar, glycerin-asparagine agar, yeast malt agar, oatmeal agar and inorganic salts on starch agar were observed microscopically.

(1) Morphology of sporophores: monopodial branching, rectiflex

Spore chains are usually long, more than 10 spores per chain.

(2) Spore surface: smooth l n 70594 (3) spore size: 0.4 to 0.9 x 1.0 to 1.6 microns (4) No hyphal cleavage or spore formation occurs in the substrate mycelium.

Spore colonies and zoospores are not detectable.

2. Cultivation characteristics:

The strain has the following culture properties when grown on the following media for 10 days below 27 ° C.

i2 70594

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13 3. Physiological properties: (1) Growth temperature range (on Bennett agar surfaces):

12-40 ° C, optimum: 27 ° C

(2) Liquefaction of gelatin (glucose-peptone gelatin skew): negative (3) Hydrolysis of starch (starch-inorganic salts on agar): strongly hydrolyzed (4) Coagulation and peptonization of skimmed milk (followed by peptonization: coagulation and subsequent coagulation) (tyrosine agar11a, peptone-yeast iron agar and tryptone-yeast extract solution): negative (6) Use of carbon sources (Pridham-Gott1ieb agar11a): H i i1ilource Growth L-Arab inosis ++

Cellulose D-fructose + D-galactose + D-glucose +

Glycerin +

Inositol - Sign + = positive

Lactose - = does not use D-maltose + D-mannitol D-mannose

Raffinose L-rhamnose --- 'Il 14 70594

Carbon Source Growth

Salicin Starch + Sign + = positive

Sucrose - = does not use D-xylose +

The above-mentioned microscopic examinations and culture studies show that the strain ATCC 31304 belongs to the genus Streptomyces. This organism was thus compared to published descriptions of Streptomyces species. Based on the above information, strain ATCC 31304 can be considered as closely resembling Streptomyces rubellomurinus ATCC 31215. However, this species was found to differ from strain ATCC 31303 in that its vegetative mycelium is indigo-colored on a medium containing yeast extract. As a result of these comparisons, strain ATCC 31304 is considered a subspecies of Streptomyces rubellomurinus and has been given the name Streptomyces rubellomurinus subsp. ind igoferus.

(2) Fermentation

To prepare the antibiotic FR-900098, the fermentation can be carried out by the conventional means described above, and the isolation of the antibiotic FR-900098 can also be carried out by the conventional means described above.

However, as previously mentioned, both antibiotic FR-33289 and FR-900098 are simultaneously formed in the culture broth when Streptomyces rubellomurinus subsp. indigoferus species.

Both of these antibiotics can be separated in the usual manner, such as chromatographically. The following is one example of a separation method.

15 70594

Culture filtrate (Step a) Acidified absorbent

absorbate

Elution i Eluate (Step b) Anion exchange resin

Eluate (Step c) Cellulose column chromatography

I I

FR-90098 FR-33289

Step a;

The filtrate is acidified in the usual manner, for example by adjusting the pH to 2.8, and the solution is passed through a column containing a suitable absorbent such as carbon. Elution is performed with an aqueous solvent (e.g. methanol, acetone, etc.).

Step b:

The eluate is passed through a column containing an anionic ion exchange resin (e.g. DEAE-Sephadex, Duolite A-6, etc.). The elution is carried out, for example, with aqueous sodium chloride (for example 0.3 N), aqueous ammonia (for example 0.2 M) and the like. These steps (steps a and b) are preferably repeated several times.

70594 BC

Step c:

The eluate is column chromatographed using cellulose and a suitable developer (e.g. aqueous propanol, etc.). Antibiotic FR-90098 can be separated by boiling with 75% aqueous propanol and antibiotic FR-33289 can be separated by boiling with 70% aqueous propanol.

The antibiotic FR-900098 formed in the culture medium or separated from the culture medium can be isolated in free form, i. FR-900098 per se and when a solution or concentrate containing the antibiotic FR-900098 is treated with an alkali metal or alkaline earth metal (e.g. sodium or potassium hydroxide or calcium carbonate, etc.) during the treatment steps, e.g. extraction, isolation or purification, the antibiotic FR-900098 can be isolated. as its early metal or alkaline earth metal salt.

The free-form antibiotic FR-900098 can also be converted into its salt with a base such as an inorganic base (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) or an organic base (e.g., ethanolamine, trimethylamine, dicyclohexylamine, etc.).

The salt of the antibiotic FR-900098 can be easily converted to the free form by treatment with an acid such as a mineral acid (e.g. hydrochloric acid, etc.) in the usual manner.

(3) Antibiotic FR-900098:

The monosodium salt of the antibiotic FR-900098 obtained according to the above method has the following physical and chemical properties: li 17 70594 (a) Elemental analysis (%) s C 27.73; H 5.03; N 6.66 (other; phosphorus and oxygen)

(b) M.p .: 193-194 ° C

(c) Specific rotation capacity; [0 () 25 = o (C = 1.0 in water)

D

(d) Ultraviolet absorption spectrum; H 2 O or 0.1N HCl = absorption terminal

0, IN NaOH

λ max = 230 nm (shoulder) 1% (E 1 cm = 325) (e) Infrared absorption spectrum: v KBr = 3450, 3400, 3350, 3100, 2930, 2800, ma 2420, 2320, 1615, 1570, 1495, 1450, 1420, 1370, 1310, 1280, 1240, 1220, 1200, 1180, 1160, 1090, 1080, 1050, 1040, 990, 980, 925, 910, 885, 810, 780, 760, 740, 710 cm -1 (f) Solubility:

Freely soluble in water and methanol Slightly soluble in acetone and propanol

Insoluble in ethyl acetate, chloroform, benzene.

(g) Staining reaction:

Positive; all reactions with ferric chloride, potassium permanganate and iodine vapor. Negative; ninhydrin reaction and Molish reaction.

ie 70594 (h) Form and color of crystals: Colorless prisms (recrystallized from a mixture of methanol and acetone) (I) Thin layer chromatography:

Carrier; Eastman chromatogram Sheet Cellulose No. 13254 (trade name, manufactured by Eastman Kodak Co.)

Development solution Rf value 75% aqueous propanol solution 0.5 Saturated aqueous solution of n-butanol 0 70% aqueous acetonitrile solution 0.4

As a result of the analysis of the above-mentioned physical and chemical properties and other identification studies of the chemical structure, the structure of the antibiotic FR-900098 has been confirmed as follows:

OH O

I II

CH3CO NCH2CH2CH2P OH

OH

[3- (N-acetyl-N-hydroxyamino) propylphosphonic acid] (II). The compound (I ") wherein R 1 'is formyl and A' is -CH 2 -CH 2 -CH 2 - i.e. 3- (N-formyl-N-hydroxyamino) propylphosphonic acid (hereinafter referred to as FR-31705) and / or compound (I ") wherein R 1 'is formyl and A' is trans-1-propenylene, i.e. Preparation of 3- (N-formyl-N-hydroxyamino) -trans-1-propenylphosphonic acid (hereinafter referred to as FR-900136).

The antibiotic FR-31705 and / or the antibiotic FR-900136 can be prepared by fermentation in a nutrient medium of a strain of the genus Streptomyces FR 193155 which produces the antibiotic FR-31705 and / or the antibiotic FR-900136, for example a strain of Streptomyces lavendulae.

(1) Micro-organisms:

The microorganism that can be used to prepare the new antibiotics FR-31705 and / or FR-900136 is a strain of Streptomyces lavendulae recently isolated from a soil sample taken from Fukue city, Nagasaki Prefecture, Japan.

The culture of the living organism is recorded under ATCC No. 31279 in the American Type Culture Collection and under No. 3808 in the Japanese Collection Fermentation Research Institute, Agency of Industrial Science and Technology.

2) Microbiological properties 2) -1 Streptomyces lavendulae ATCC 31279

Streptomyces lavendulae ATCC 31279 has the following morphological, cultural and physiological characteristics: 1. Morphological characteristics:

The morphology of the culture was observed microscopically on mycelium grown on glycerol-asparagine agar, 11th housing agar 11a, oat flour agar 11a and inorganic salts-starch agar.

(1) Method of branching of spore-forming hyphae:

Monopodial branching (2) Shape of spore-forming hyphae:

Retinal ulcer: open loop, hook and sometimes rectus and spiral 70594 20 (3) Number of spores: 10 to 50 spores (4) Appearance and size of spore surface:

Smooth, 0.5 to 1.2 x 1.4 to 2.0 microns (5) Occurrence of zoospores:

Not observed (6) Occurrence of spore colonies:

Not observed (7) Spore formation:

Ilmar in humans (8) Substrate mycelium cleavage:

Not observed 2. Culture characteristics:

The strain has the following culture characteristics when grown for 10 to 14 days at 30 ° C on the following media.

il 70594 21 3 t

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c Φ CO Ή a) </) (/)

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ft ω ft ω ω: π P ^ ω S ω Se

j I

ft I

f | i: <T3 (0

- <P

0) c

C -M -H

p OI> 1: nj a £> cap M -ι-t Cu - -p -: m (0 -: ic P: (0 * -: io: ro S (0 Φ -: (0 (0 -pcp: ia pcppp cu φ cn

: (0 Φ -P> · Ρ φ -P -H y Φ Ή Ji -P

c -HC Φ C Φ C φ Φ - (OM c «κι ->, φ c -p. * ra. * -p. *. * cp co -p. * pca C φ (0 4 * E - * Φ - * - * Φ P φ (Ο -VP Φ Qj

• p -p P: ra P: ro p: ra: (0 C C Q, P: (0 2 C S

> CL Λ h ie Φ ci — i ι / i en -p: ra; (oh cn: (0 -h Ci: (0 ρ ρ Φ Φ φ φ Φ guu>: iii®i> ρ p • p -h ji a | aa - Οι: Φ -h: ro -r-ι .v c.: (0: (0 - -p PC φ C 'CCC> φ p cn C 4-)> C Φ (0: 0 - * (0: <0: C: (0 (0: ro: 0: i0 C. * Ρ -p ro: (0 P (0 C tn: 0 ^ 4) U 4L φ Ή JJ-H Φ-H UH (0- ^ Φ> | Φ · Η φ D (0 (0 Ρ Φ -Ρ: ί0 * —IC -Ή C ι — ι C · Η C Ε -Φ ft a I — IC --Η 5 Ε CD · ρ * (0 ςηΡίΛΦΦΡφΦΦΡφΕφί-ιαΦΦ' — icfiCccitfi Φ: Φ Φ (0 -ρ: (0 · γ-ι (Ο · ρ: ι0 -ρ φ φ φ S (0 · η φ ro φ φ: ro Φ> | > a> a |> ο ^> a> a hi δϋ ρ> a. * ϋ ρ> a Ρ

> i C CC

χ: φ φ φ

> 1 C CC

ι — I -Ρ - C - C - Ή - · Ρ (ΟίΟφίΟΦ- (ΟΦΦΦ c ggciocc (οεφε Φ CS Ε · Η Ε · Η φ ΕφΕφ, c φ ft ρ ra ρ ra c fid Ch O-HC r-ΗίθΕΦΕ - ^ (Or-1 (0 rl PO-Η -px: ra x; ra o C χ: -h χ; · ρ c KJiiB -hCHCHi: ® ΊΞ> Έ> - Φ

(Oi — I E 1 C C (O H 10 H ι — 1C (ODfuOCC

E (0 (0 Φ D> -ρ> -ρ (O -ρ> 3> D Φ -P

£> Hca (o> (j >> 5 (o α (ο a c (o

• H ι — I · Ή jJ xJ t JJ Jj * f ~ (P

P - -r-ι ou LU li U - Φ pp Ci p O Φ (0ρ> 4 *:> ίφ> 1φ> 1ρχ; φ>, φ> -. * X; edoppcx: cx: do cx: cx: ft d

H £ 2 5> p>, p> | £ (Jd 3> 3> (0 (0 -P -H

mo a> ft ft ft ft —ι o -ft | ft ft.-ι ft ft> · η ω ω

I P

Φ Φ I I C (Λ

ι — I -ι — I - r-l · rH

p c -H C

P -r-l CD (0 (0

(0 - · —t (0 (0 I IP

(0 CD Ci CD Φ -H c

Ci (O (O Cl ρ P C -P

ρ p a ο ρ ο φ o a p ra cn: ra ra 3 cd ρ O (0 caraacDPcnraac> I CO I Φ Ρ (0 (0 (0 I Φ-H -r-l

• p (0 ·> - ι 1 (0 I CD ft O a> -P P

cn ι c cn σ-r-i ίο h jz i -p x: ra

0-r-1-d> | «C I <o 3 · Η C ICD

o in -π η ι -π OE <0 cn -r-ι -pra CC Cl φ p -pp ι -n Q -P ci (0 oa) jc (C mc (o ro opo ΟΌ u <; cjcpinp4 » rpo> pp j <(0p pp

_C (0D (0> i (0COP ι> -P (0D OP-p CuD

(0 CD ι-H: rD, r-l Öi: (OD> r (0 -P CD (Ο Η φ IJ φ (0 ic d c u it o (0 H k h os χον: ο σΣ ftp

Jj 1 »'---! --- I- s = r-l CM p LTl i'cC r- CC jCT> rH <22 70594 3. Physiological properties (1) Growth temperature range (Bennett's agar):

12-40 ° C, optimum: 26 ° C

(2) Liquefaction of gelatin (on glucose-peptone-gelatin slant): negative (3) Hydrolysis of starch (starch-inorganic salts-agar11a): positive (4) Coagulation and peptonization of skimmed milk:

Coagulation: negative

Peptonization: Peptonization is slow (5) Melanoid pigment production: (a) Positive on peptone-yeast-iron agar (b) Negative on tyrosine agar (6) Use of carbon sources (on Pridham-Gottlieb agar)

Carbon source Growth of L-arabinose

Cellulose - D-fractose D-glucose + D-galactose +

Inositol - Signs: + = good D-mannose ± ± = uncertain D-mannotol - = not use L-rhamnose

raffinose

Suk.roosi

Received isine + D-xylose at 23 7 0 5 9 4

The above microbiological properties indicate that strain ATCC 31279 belongs to the genus Streptomyces. As a result, the eighth edition of "Bergey's Manual of Determinavite Bacteriology" (1975), "The Actinomycetes", was searched in the literature.

Butter. II (1961), author S.A. Waksman and "The International Streptomyces Project Reports," by E.B. Shirling and D. Gottlieb [Vert. International Journal of Systematic Bacteriology Vol. 18, pages 69-189 and 279-392 (1968), Vol. 19, pages 391-512 (1969) and Vol. 22, pages 265-394 (1972)] strains having the above-mentioned properties, confirming that the microbiological properties of strain ATCC 31279 are identical to Streptomyces lavendulae.

This finding was also confirmed by comparing the microbiological properties of strain ATCC 31279 and the type culture, Streptomyces lavendulae IAM 0009.

Based on the above findings, the strain has been given the name Streptomyces lavendulae.

(2) Fermentation:

Fermentation to produce antibiotic FR-31705 and antibiotic FR-900136 can be carried out by the above-mentioned conventional methods, and also the isolation of these antibiotics can be carried out by the above-mentioned conventional methods, respectively.

As mentioned above, the culture medium contains both antibiotic FR-31705 and antibiotic FR-900136. Both of these antibiotics can be separated in the usual manner, such as chromatographically. The following is one example of a separation method.

24 70594

Culture filtrate (Step a ') Acidified absorbent

absorbate

The elution

Eluate (Step b ') Anion exchange resin

Eluate (Step c ') Cellulose column chromatography

I- I

FR-31705 FR-900136

Step a *;

The filtrate is acidified in the usual manner (for example, adjusted to pH 2.0) and the solution is passed through a column containing a suitable adsorbent such as carbon. Elution is performed as an aqueous solvent (e.g. methanol, acetone, etc.).

Step b1:

The eluate is passed through a column containing an anion exchange resin (e.g. DEAE-Sephadex, Duolite A-6, etc.). The elution is carried out, for example, with aqueous sodium chloride (for example 0.3 M) and aqueous ammonia (for example 0.2 M).

25 705 94

Step c ':

The eluate is column chromatographed using cellulose and a suitable developer solvent (e.g. aqueous propanol, etc.). For example, FR-31705 can be separated by development with 97% aqueous propanol and FR-900136 can be separated by development with 95% aqueous propanol.

Antibiotics FR-31705 and FR-900136 formed in or separated from the culture medium can be isolated in free form, i. FR-31705 per se. These antibiotics can further be isolated as their alkali or alkaline earth metal salts by treating the solution containing the antibiotics during treatment with alkali or alkaline earth metal (e.g. sodium or potassium hydroxide or calcium carbonate, etc.), e.g. extraction, isolation or purification.

Antibiotics obtained in their free form can also be converted into their salts with bases such as an inorganic base (e.g. sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) and an organic base (e.g. ethanolamine), trimethylamine, dicyclohexylamine, etc.).

Salts of these antibiotics can be readily converted to the free acid by treatment with a conventional method such as a mineral acid (e.g., hydrochloric acid, etc.).

3) Antibiotics FR-31705 and FR-900136: 3) -1 Antibiotic FR-31705

The monocalcium salt of the antibiotic FR-31705 obtained by the above method has the following physical and chemical properties: 26 70594 (a) Elemental analysis (%): C 21.62; H 4.07; N 6.36; K 17.99 (b) M.p .: 202-204 ° C (dec.) (C) Infrared absorption spectrum: new

Vmax = 2950, 2925, 2850, 2550, 2380, 1650, 1460, 1410, 1395, 1375, 1320, 1300, 1260, 1220, 1190, 1150, 1120, 940, 890, 810, 785, 700 cm-1 (d ) NMR spectrum δ (ppm) in D 2 O 1.25-2.3 (4H, m), 3.65 (2H, t, J = 6Hz), 8.00 (s)

8.35 (s) · * 1H

Based on the above physical and chemical properties and other identification studies of the chemical structure, the chemical structure of the antibiotic FR-31705 has been identified as follows:

OH O

I II

HCO-NCH2CH2CH2P-OH

OH

f 3- (N-Formyl-N-hydroxyamino) propyl phosphonic acid] 3) -2 Antibiotic FR-900136

The monopotassium salt of the antibiotic FR-900136 obtained by the above method has the following physical and chemical properties.

27,70594 (a) Elemental analysis (%) C 21.32; H 3.26; N 6.00; H 2 O 1.49 (b) M.p .; 178-180 ° C (dec.) (C) Infrared absorption spectrum; nujoii

Vmax = 2960, 2930, 2870, 2600, 2350, 1660, 1530, 1460, 1440, 1400, 1380, 1365, 1290, 1250, 1180, 1125, 1070, 1010, 980, 960, 950, 890, 830, 780, 700 cm-1 (d) NMR spectrum δ (ppm) in D 2 O 4.30 (2H, m), 6.01 (1H, m), 6.38 (1H, m), 8.02 (s) j

8.32 (s) * 1H

Based on the above physical and chemical properties and other identification studies of the chemical structure, the chemical structure of the antibiotic FR-900136 has been identified as follows:

OH O

I II

HCO-n-CH 2 CH = CHP oh

OH

[3- (N-formyl-N-hydroxyamino) -trans-1-propenylphosphonic acid] (III). The compound (I ") wherein R 1 'is acetyl and cl

A 'is OH

-ch2-ch-ch2-, ______ - ι; 28 70594 so. Preparation of 3- (N-acetyl-N-hydroxyamino) -2-hydroxypropylphosphonic acid (hereinafter referred to as FR-33289):

The antibiotic FR-33289 can be prepared by fermentation in a food medium of a strain of the genus Streptomyces that produces the antibiotic FR-33289, such as Streptomyces rubellomurinus subsp. indigoferus strain.

(1) Micro-organisms:

An example of a recommended microorganism that can be used in the preparation of the new antibiotic FR-33289 is Streptomyces rubellomurinus subsp. indigoferus ATCC 31304.

The microbiological properties of strain ATCC 31304 have been described above, with reference where appropriate.

It should also be noted that Streptomyces rubellomurinus subsp. ind igoferus ATCC 31304 can simultaneously produce both antibiotic FR-900098 and antibiotic FR-33289 in the culture medium, as mentioned above.

(2) Fermentation:

Fermentation to produce the antibiotic FR-33289 can be carried out by the above-mentioned normal means, and also the antibiotic isolation can be carried out by the above-mentioned conventional means.

As mentioned above, the culture medium contains both antibiotic FR-900098 and antibiotic FR-33289. Both of these antibiotics can be distinguished.

The recommended separation methods are the same as mentioned above.

29 70594 (3) Antibiotic FR-33289:

The monosodium salt of the antibiotic FR-33289 obtained by the above method has the following physical and chemical properties: (a) Infrared absorption spectrum:

KBR

Vmax = 3300, 2900, 2400, 1740, 1620, 1420, 1240, 1140, 1040, 900 cm -1 (b) NMR spectrum: δ (ppm) in δ 2 O 1.88 (2H, d, d. J = Δ S 18 Hz), 2.16 (3H, s), 3.66 - 3.9 (2H, m), 4.30 (1H, m) (c) Staining reaction:

Positive: reaction with ferric chloride, potassium permanganate and iodine vapor.

(a) Thin layer chromatography:

Carrier; Eastman chromatogram Sheet Cellulose No. 13254 (trade name, manufactured by Eastman Kodak Co.)

Development solution Rf 60% aqueous propanol 0.6

Based on the above chemical properties and other identification studies of the chemical structure, the chemical structure of the antibiotic FR-33289 has been identified as follows:

OH OH O

l I II

CH 3 CO-N-CH2CHCH2-P-OH

OH

30 70594 [3- (N-Acetyl-N-hydroxyamino) -2-hydroxypropylphosphonic acid

Biological properties of hydroxyamino hydrocarbon phosphonic acid derivatives

Antimicrobial activity:

The target compounds, hydroxyaminohydrocarbon phosphonic acid derivatives (I), their phosphono group esters and their salts, have been found to have potent antibacterial activity against pathogenic microorganisms such as gram-positive and negative bacteria, including Bacillus, Salecella, Sareina, Escher, Against the genera Pseudomonas, Shigella and Enterobacter. The target compounds of this invention are thus useful in the treatment of infectious diseases caused by such pathogenic bacteria in humans and animals. The biological properties of some typical target compounds (I) are illustrated below. 1

II

Monosodium salt of 3- (N-acetyl-N-hydroxyamino) -propylphosphonic acid:

Minimum Preventive Concentration (M.I.C.) The M.I.C. test was performed using a standard serial dilution method on nutrient agar incubated for 20 hours at 37 ° C. The M.I.C. value is expressed as the lowest concentration (microgram / ml) of the monosodium salt of 3- (N-acetyl-N-hydroxyamino) propylphosphonic acid that inhibits the growth of the microorganism. The results are as follows: „70594

Test organism M.I.C (pg / ml)

Staphylococcus aureus FDA209P JC-1> 1000

Bacillus subtilis ATCC6633 125

Sareina lutea PCI 1001 8

Escherichia coli NIHJ JC-2 63

Escherichia coli 1341-29 32

Klebsiella pneumoniae NCTC 418 500

Proteus vulgaris IAM 1025 125

Proteus mirabilis 1> 1000

Proteus morganii 30> 1000

Proteus rettgeri 15 63

Pseudomonas aeruginosa IAM 1095 250

Salmonella typhi T-287 2

Shigella flexneri IaEW8 8

Serratia narcescens 5 250

Citrobacter freundii 20,500

Enterobacter aerogenes 10 32

Enterobacter cloacae 25 63

Protective effect in mice experimentally induced infections: The in vivo activity of the monosodium salt of 3- (N-acetyl-N-hydroxyamino) propylphosphonic acid against Escherichia coli was tested using ICR strain male mice weighing 20-25 g. Two groups, each consisting of four mice, were fasted for 24 h before testing.

Each mouse was injected intraperitoneally with a suspension of the pathogenic bacterium, Scherichia coli strain No. 1341-29, in 2.5% aqueous Mucin (0.5 ml).

(Challenge dose: 1 x 106 live cells / mouse). One group was used to measure the protective effect and the other _._-ti 32 70594 as a control.

One hour after infection, each mouse in the experimental group was injected with 3- (N-acetyl-N-hydroxyamino) propylphosphonic acid monosodium salt (4 mg) in water (0.5 ml). Mice in the control group were not treated with antibiotics.

Animals from both groups that died and survived were counted for one week.

All mice in the experimental group survived, while all mice in the control group died.

Acute toxicity: A solution of 3- (N-acetyl-N-hydroxyamino) propylphosphonic acid monosodium salt in water (0.5 ml) was injected intravenously into five mice (dose: 5 g / kg). All results were normal 10 days after administration.

Hypolipidemic Activity: The target compound (I) of the present invention also has hypolipidemic activity, such as hypocholesterolenic activity, and is useful as a therapeutic agent in the treatment of hyperlipemia.

For example, 3- (N-hydroxyamino) propylphosphonic acid, one of the target compounds, has hypocholesterolemic activity, i.e., almost the same as the commercially available "Clofibrate", in a test in which Wistar rats were fed a high-fat cholesterol diet.

2. 3- (N-Formyl-N-hydroxyamino) propylphosphonic acid monoammonium salt: 33 70594

Minimum Preventive Concentration (M.I.C.): The M.I.C. assay was performed using a standard serial dilution method (inoculum: 10 cells / ml) on nutrient agar incubated for 20 hours at 37 ° C. The M.I.C. value is expressed as the lowest concentration (μg / ml) of 3- (N-formyl-N-hydroxyamino) propylphosphic acid that inhibits the growth of microorganisms. The results are as follows:

Test organism M.I.C (pg / ml)

Staphylococcus aureus FDA209P JC-1 800

Bacillus subtilis ATCC6633 6.25

Sareina lutea PCI 1001 0.1

Escherichia coli NIHJ JC-2 200

Escherichia coli 1341-18 (R) 12.5

Klebsiella pneumoniae NCTC 418 100

Proteus vulgaris IAM 1025 3.13

Proteus mirabilis 1432-75 6.25

Proteus morganii 1433-2 800

Proteus rettgeri 1434-3 1.56

Proteus inconstans 1436-21 3.13

Pseudomonas aeruginosa IAM 1095 0.78

Salmonella enteritidis 1891 0.39

Salmonella typhi 0-901 0.39

Salmonella paratyphi A-1015 12.5

Salmonella typhimurium 1406 25

Shigella flexneri IaEW8 12.5

Shigella sonnei I EW33 100

Serratia marcescens 1421-4 100

Citrobacter freundii 1381-3 3.13

Enterobacter aerogenes 1402-10 6.25

Enterobacter cloacae 1401-4 6.25 34 70594

Protective effect in experimental infections in mice: (a) Test compound: 3- (N-formyl-N-hydroxyamino) propylphosphonic acid monammonium salt (b) Experimental animal: ICR strain male mice 4 weeks old and weighing 24 ± 1 g . Each experimental group comprised 8 animals.

(c) Test method:

Each experimental animal was injected intraperitoneally with a certain amount of pathogenic bacteria in 5% aqueous Mucin solution (0.5 ml).

Thereafter, each test animal was administered the above test compound in water (0.25 ml) subcutaneously three times with emotions 0, 1, and 3, or orally once an hour 1 after infection with pathogenic bacteria.

Survival or death of all experimental animals was monitored for one week and ED50 values were calculated by the probit method. The results are given in the following table.

il 70594 35

Per inoculated per mouse - ED5Q (mg / mouse)

Pathogenic live oral subcutaneous bacterial cell count when administered

Pseudomonas aeruginosa 1.2 x 10δ 0.228 0.280 1101-76 ___________ F s c h e r i c h a coli 1341-67_6.9 x 107_0.167_2.559

Proteus mirabilis 1432-75_8.0 x 107__0,236_4,331

Acute toxicity (a) Test compound: 3- (N-formyl-N-hydroxyamino) propyl phosphonic acid monosodium salt.

(b) Experimental animal: male and female mice of the ICR strain, age 6 weeks.

(c) Observation times:

One week (d) Calculation method:

Litchfield and Wilcoxon method LD5O (mg / kg) orally subcutaneously

Animal Sex i_given_given

Mice in the dog 11,000 8,050 ________ female _____ 11,000__8,270__

Rat___ male___ 11,000___8,000__ 70594 36 3. 3- (N-Formyl-N-hydroxyamino) -trans-1-propenylphosphonic acid monopotassium salt:

Minimum Preventive Concentration (M.I.C.) The M.I.C assay was performed using a standard serial dilution method (inoculum: 10 cells / ml) on nutrient agar incubated for 18 hours at 37 ° C. The M.I.C value is expressed as the monopotassium salt of 3- (N-formyl-N-hydroxyamino) -trans-1-propenylphosphonic acid at the lowest concentration (pg / ml) which inhibits the growth of microorganisms. The results are as follows:

Test organism M.I.C (pg / ml)

Staphylococcus aureus FDA209P JC-1 100

Bacillus subtilis ATCC6633 6.25

Sareina lutea PCI 1001 0.2

Escherichia coli 1341-18 (R +) 25

Klebsiella pneumoniae NCTC 418 100

Proteus vulgaris IAM 1025 1.56

Proteus mirabilis 1432-75 0.39

Proteus morganii 1433-2 100

Proteus rettgeri 1434-3 6.25

Proteus inconstans 1436-21 25

Pseudomonas aeruginosa IAM 1095 1.56

Salmonella enteritidis 1891 6.25

Salmonella typhi 0-901 0.78

Salmonella paratyphi A-1015 25

Salmonella typhimurium 1406 12.5

Shigella flexneri IaEW8 50

Shigella sonnei I EW33 25

Serratia marcescens 1421-4 100

Citrobacter freundii 1381-3 12.5

Enterobacter aerogenes 1402-10 50

Enterobacter cloacae 1401-4 12.5

II

70594 37

Pharmaceutical Composition Containing Hydroxyaminohydrocarbon Phosphonic Acid Derivatives The target compounds (I) of this invention, the hydroxyaminohydrocarbon phosphonic acid derivatives of this invention in its phosphono group, may be formulated for administration in any suitable manner, analogously to known antibiotics, in admixture with a non-toxic carrier.

The pharmaceutically acceptable salt of the compound (I) may be a salt with an inorganic or organic base such as sodium salt, potassium salt, calcium salt, ammonium salt, ethanolamine salt, triethylamine salt, dicyclohexylamine salt and the like, and a salt with an inorganic or organic acid , fumarate, tartrate, p-toluenesulfonate and the like, and a further salt with an amino acid such as arginine salt, aspartic acid salt, glutamic acid salt, and the like.

The antimicrobial composition may thus be used as a pharmaceutical preparation, for example in solid, semi-solid or liquid form, containing the active target compound in admixture with a pharmaceutical organic or inorganic carrier or excipient suitable for external, enteral or parenteral administration. The active ingredient may be admixed with, for example, conventional non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. Useful carriers include water, glucose, lactose, acacia, gelatin, mannitol, starch paste, 70594 38 magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, in the form of a liquid or other carriers suitable for the preparation of preparations, and in addition, excipients, stabilizers, thickeners and coloring agents and perfumes may be used. Antimicrobial compositions may also contain preservatives or bacteriostatic agents to keep the active ingredient of the desired preparations stable infect a process or condition.

When this mixture is administered to humans, it is best by intravenous, intramuscular or oral administration. Although the dosage or therapeutically effective amount of a target compound of this invention will vary and depend on the age and condition of each individual patient being treated, a daily dose of about 2 to 100 mg of active ingredient per kilogram of human or animal body weight is usually used to treat diseases. The average single dose is usually about 50 mg, 100 mg, 250 mg and 500 mg.

Example of fermentation (1) A culture medium (100 ml) containing 2% starch, 1% cottonseed meal and 1% dried yeast was poured into five 500 ml Sakaguchi flasks and sterilized for 20 minutes at 120 ° C. Looped Streptomyces rubellomu- Rinus FERM No. (ATCC No. 31215) in slant culture of the microorganism was inoculated into each medium and grown for 2 days at 30 ° C. The resulting culture was inoculated into a medium (20 L) containing 5% soluble starch, 0.5% cottonseed meal, 2.5% gluten meal, 0.5% dry yeast,

II

70594 39 1% MgSO 4 · 4H 2 O, 1% KH 2 PO 4 and 0.7% Na 2 HPO 4. I2H2O in a 30 liter fermentor pre-sterilized for 20 minutes at 120 ° C and grown for 3 days at 30 ° C.

When the culture was completed, diatomaceous earth (400 g) was added to the culture medium, and the mixture was filtered. The filtrate (20 L) was concentrated to 1 liter under reduced pressure. Methanol (4 L) was added to the concentrate, and the mixture was stirred to form a precipitate. The precipitate was collected by filtration and the filtrate was concentrated to 1 liter. The obtained concentrate was passed through a column containing activated carbon. The solution passed through the column was adjusted to pH 2.0 with cation exchange resin 11a, Duolite 020 (trade name, manufactured by Diamond Shamrock Chemical Co.) (H + type; 500 ml) and passed through Duolite A6 (trade name, manufactured by Diamond Shamrock Chemical Co.) (0H “ type; 500 ml) through the column. It was then eluted with 0.1 N aqueous sodium hydroxide solution (1500 ml). The eluate was adjusted to pH 2.0 with Duolite C-20 (H + type) resin and then passed through an activated carbon column. The title compound was eluted with 70% aqueous acetone (1 L). Fractions containing the target compound were collected and concentrated under reduced pressure. The residue thus obtained was adjusted to pH 6.5 with 6 N aqueous sodium hydroxide solution and column chromatographed on cellulose (300 ml) using eluent (80% aqueous propanol). The fractions containing the title compound were collected and dried under reduced pressure to give a white powder (600 mg). The powder was dissolved by heating in a small amount of methanol. A small volume of acetone was then added to the solution. The mixture was allowed to stand overnight at 40 ° C / to form crystals which were filtered and dried. There was thus obtained as a colorless crystals the monosodium salt of the antibiotic FR-900098 (300 mg).

(2) An aqueous medium containing 2% potato starch, 70594 40 1% gluten powder, 1% dry yeast and 1% corn steep liquor was adjusted to pH 7.0 with 6 N aqueous sodium hydroxide 1 solution. The medium was then poured into 100 ml of six 500 ml Erlenmeyer flasks and the flasks were sterilized for 20 minutes at 120 ° C. Each medium was inoculated with a looped Streptomyces lavendulae ATCC 31279 oblique surface culture and the organism was grown on a rotary shaker for 3 days at 30 ° C. Aqueous medium (20 L) containing 3% methyl oleate, 1% cottonseed meal, 1% wheat germ, 0.5% dry yeast, 05% corn steep liquor, 1% potassium bisphosphate, and 1% secondary sodium phosphate was separately poured into a 30 liter fermentor. , and sterilization was performed for 30 minutes at 120 ° C. To this medium was added the whole of the culture medium obtained above, and then the organism was grown for 3 days at 30 ° C. During the growth, the fermentation was carried out by stirring the culture solution with propeller equipment at a speed of 250 rpm. by passing sterile air 20 l / solution / min through the culture medium. and maintaining an internal pressure of 0.5 (kg / cm 2) in the fermentor.

When the culture was completed, the culture solution was filtered through diatomaceous earth (2 kg). The filtrate (15 L) was adjusted to pH 2.0 with 1 N hydrochloric acid and passed through a column containing activated carbon (5 L). The column was washed with water, then eluted with 70% aqueous acetone (4 L). The eluate was concentrated to 1 liter, adjusted to pH 2 with 6 N hydrochloric acid, and then passed through a column containing anion exchange resin Duolite A-6 (OH-type) (trade name, Diamond Shamrock Chemical Co.) (500 ml). The column was washed with water, then eluted with 0.1 N aqueous sodium hydroxide solution (1.5 L). The eluate was passed through a column containing cation exchange resin Duolite C-20 (H + type) (trade name, Diamond Shamrock Chemical Co.).

I! 70594 41

The column was washed with water, then eluted with 60% aqueous acetone (500 mL). The eluate was concentrated under reduced pressure to an oily residue which was subjected to column chromatography on cellulose (300 ml) (developing solvent: 80% aqueous acetonitrile). The fractions containing the title compound were collected and concentrated under reduced pressure to give an oily residue, which was adjusted to pH 7.0 with 1 N aqueous potassium hydroxide solution, and column chromatographed on cellulose (300 ml) (developing solvent: 60% aqueous propanol). The fractions containing the title compound were collected and concentrated under reduced pressure to an oily residue which was dissolved in ethanol (200 ml). Acetone (20 ml) and diethyl ether (200 ml) were added to the solution to give a precipitate which was separated by filtration and dried to a crude powder (150 mg). The powder was dissolved in water (150 ml) and the solution was adjusted to pH 7 and passed through a column containing activated carbon (100 ml). The column-passed solution was concentrated under reduced pressure to an oily residue, which was column chromatographed on cellulose (200 ml) (developing solvent: 65% aqueous propanol). The fractions containing the title compound were collected and concentrated under reduced pressure to an oily residue which was dissolved in methanol (1 ml). The solution was heated to 50 ° C and acetone (10 mL) was added. The mixture was allowed to stand overnight at 4 ° C to form crystals which were collected by filtration and dried. There was thus obtained the monopotassium salt (5 mg) of the antibiotic FR-31705 as needles.

(3) An aqueous medium containing 1% potassium starch, 1% glycerol, 1% flaxseed flour and 1% dry yeast was poured into ten 500 ml Erlenmeyer flasks and sterilized for 20 minutes at 120 ° C. One medium of inoculated Streptomyces lavendulae ATCC 31279 oblique surface culture was inoculated into each medium and the organism was grown in 70594 42 rotary strainers for 3 days at 30 ° C.

Separately, 80 liters of aqueous medium containing 3% methyl oleate, 1% cottonseed meal, 1% wheat germ, 0.5% corn steep liquor, 0.5% dry yeast, 1% potassium bisphosphate and 1% secondary sodium phosphate were poured into a 100 liter fermentor and sterilized. minutes at 120 ° C. This medium was inoculated in its entirety with the culture medium obtained above, and the organism was grown for 72 hours at 30 ° C. During the growth, the fermentation was carried out by stirring the medium with propeller equipment at a speed of 130 rpm, passing sterile air at 80 liters / solution / minute and maintaining the internal pressure of the fermenter at 1 (kg / cm 2).

After the culture was completed, the culture solution was filtered through diatomaceous earth (5 kg). The filtrate (70 L) was adjusted to pH 2 with 6 N hydrochloric acid and then treated with activated carbon (20 L). Elution was performed with 20% aqueous acetone. Acetone was removed from the eluate under reduced pressure. The obtained aqueous solution was adjusted to pH 2 with a cation exchange resin Duolite C-20 (H + type), and then the solution was passed through a column containing anion exchange resin Duolite A-6 (OH ~ type). Elution was performed with 0.2 N aqueous ammonia. The eluate was adjusted to pH 6 and lyophilized to a crude powder (100 g) which was dissolved in water (3 L). The solution was adjusted to pH 2 with 6 N hydrochloric acid and treated with activated carbon (6 L).

Elution was performed with 0.03 N aqueous ammonia. The eluate containing the target compound was concentrated to 1 liter under reduced pressure. The concentrate was adjusted to pH 2, further concentrated in vacuo and column chromatographed on cellulose (1 L). The column was washed with acetone (1 L). Elution was then performed with 97% aqueous acetone,

II

70594 43 to give an eluate containing the antibiotic FR-31705. The same procedure was performed with 95% aqueous acetone to give an eluate containing the antibiotic FR-900136. The eluate containing the antibiotic FR-900136 was neutralized with 1 N aqueous sodium hydroxide solution and concentrated under reduced pressure to a residue which was triturated with a mixture of acetone and diethyl ether. There was thus obtained the monosodium salt (50 mg) of the antibiotic FR-900136 as a powder.

(4) Four 500 ml Erlenmeyer flasks containing 100 ml of an aqueous medium containing 1% potato starch, 1% glycerol, 1% cottonseed meal and 1% dry yeast were sterilized for 10 minutes at 120 ° C. Each flask was inoculated with a looped Streptomyces rubellomurinus subsp. indigoferus ATCC 31304 oblique culture, after which the organism was grown on a rotary shaker for 3 days at 30 ° C. Aqueous medium (70 L) containing 2% soluble starch, 0.25% corn steep liquor, 0.25% dry yeast, 0.5% cottonseed meal, 0.5% wheat germ, 0.5% potassium dihydrogen phosphate was separately poured into a 100 liter fermentor. , 0.5% Na 2 HPO 4 · 12H 2 O and 0.000125% CO 2 Cl 2 * 12H 2 O, and sterilization was performed for 30 minutes at 120 ° C. The whole of the culture medium obtained above was added to the medium, and the organism was grown for 3 days at 30 ° C. During the growth period, the fermentation was carried out by stirring the culture medium with a propeller at 300 rpm, passing sterile air (70 liters / solution / minute) through the culture medium and maintaining the internal pressure of the fermentor at 0.5 kg / cm 2).

When the culture was completed, the culture solution was adjusted to pH 2.8 with 6 N hydrochloric acid. The resulting precipitate was filtered off. The filtrate was passed through a column containing activated carbon (10 L). Elution was then performed with 70% aqueous 70594 44 acetone (20 L). The eluate was concentrated under reduced pressure to a residue, to which was added water to give an aqueous solution (15 L). The aqueous solution was passed through a column of DEAE-Sephadex, (H + type) (8 L) (trade name, manufactured by Pharmacia A.B.) pretreated with 1/100 M phosphate spray solution (pH 6.0). Elution was performed with 0.3 M aqueous sodium chloride solution (10 L). The eluate was adjusted to pH 3.3 with 6 N hydrochloric acid and then passed through a column containing activated carbon (2 L). Water was added to the passed solution to give a total volume of 30 L. The resulting aqueous solution was adjusted to pH 2.8 with 6 N hydrochloric acid, and then activated carbon with 70% aqueous acetone was passed. The active fractions were collected, adjusted to pH 6.0 with 6 N aqueous sodium hydroxide solution and concentrated to 100 ml under reduced pressure. The concentrate was column chromatographed on cellulose (1 L). The column was developed with 75% aqueous propanol (2 liters) to give fraction (A), and then the column was developed with 70% aqueous propanol (2 liters) to give fraction (B).

Fraction (A) was concentrated to 40 ml under reduced pressure, and the obtained concentrate was passed through a column of Sephadex G-15 (1 L), manufactured by Pharmacia A.B., and then subjected to column chromatography on cellulose. The column was developed with 80% aqueous propanol. The active fractions were collected and concentrated under reduced pressure to a residue which was lyophilized to give the monosodium salt of the antibiotic FR-900098 (300 mg) as a white powder.

The fraction (B) obtained above was concentrated to 60 ml under reduced pressure, and the obtained concentrate was passed through a Sephadex G-15 (1 L) column and then subjected to column chromatography on cellulose. The column was developed with 75% aqueous propanol.

ii 70594 45

The active fractions were collected and evaporated to dryness to give the monosodium salt of the antibiotic FR-33289 as a white powder (600 mg).

Examples of the antimicrobial mixture (i) Preparation for injection (1) The vials were charged with the required amount of sterile antibiotic, 3- (N-acetyl-N-hydroxyamino) propylphosphonic acid monosodium salt, containing 500 mg of active ingredient. The bottles were hermetically sealed to prevent bacterial entry. When the vials are used, 2 ml of sterile distilled water for injections is added and the vial is used.

In substantially the same manner as in Example (1), the antibiotic injection preparation illustrated in the following Examples (2) to (4) was prepared.

(2) The monoammonium salt of 3- (N-formyl-N-hydroxyamino) propylphosphonic acid (250 mg) was used as the active ingredient of the injection.

(3) The monopotassium salt of 3- (N-formyl-N-hydroxyamino) trans-1-propenylphosphonic acid (250 mg) was used as the active ingredient of the injection.

(4) The monosodium salt of 3- (N-acetyl-N-hydroxyamino) -2-hydroxypropylphosphonic acid (500 mg) was used as the active ingredient of the injection.

(5) The monopotassium salt of 3- (N-formyl-N-hydroxyamino) -2-hydroxypropylphosphonic acid (250 mg) was used as the active ingredient of the injection.

_ ΤΓ ^ 70594 46 (ii) Tablet preparation (1) A suitable tablet formulation consists of the following mixture.

3- (N-Formyl-N-hydroxyamino) propylphosphonic acid monosodium salt 200 mg

Mannitol 400 mg Starch 50 mg

Magnesium stearate 10 mg (iii) Capsule preparation Monopotassium salt of 3- (N-formyl-N-hydroxyamino) propylphosphonic acid 300 mg

Magnesium stearate 15 mg These ingredients were mixed and then placed in a hard gelatin capsule as usual.

(iv) Oily suspension preparation 3- (N-formyl-N-hydroxyamino) propylphosphonic acid monosodium salt 200 mg lanetta wax SX (trade name) 50 mg

Vaseline 100 mg

Brilliant blue FCF 25 mg These ingredients were mixed with liquid paraffin to give a total of 3 g of infusion preparation.

Claims (5)

A process for the preparation of pharmacologically valuable hydroxyaminohydrocarbon phosphonic acid derivatives, the formula of which is OH 0 -I, I II R 1 - N - A '- P - OH (I') a I OH where R 1 or R 2 'is formyl and A' trimethylene or trans-1-propenylene and their pharmaceutically acceptable salts, characterized in that phosphonic acid derivatives of formula (I ") producing Streptomyces rubellomurinus ATCC 31215, Streptomyces rubellomurinus subsp. Indigoferus ATCC 31304 or Streptomyces lavendulae ATCC 31279 is grown in an aqueous nutrient medium under aerobic conditions until the medium becomes substantially antibiotically active and the thus produced acidic derivative of formula (I ") is recovered from the culture solution. A method according to claim 1, characterized in that a 3- (N-acetyl-N-hydroxyamino) propyl phosphonic acid producing strain belonging to the Streptomyces genus, Streptomyces rubellomurinus ATCC 31215 or Streptomyces rubellomurinus subsp. Indigoferus ATCC 31304 organism is grown in an aqueous nutrient substrate under aerobic conditions until said substrate becomes substantially antibiotically active and a 3- (N-acetyl-N-hydroxyamino) propylphosphonic acid antibiotic is obtained. Method according to claim 1, characterized in that the Streptomyces lavendulae ATCC 31279 organism is grown in an aqueous nutrient under aerobic conditions until said substrate becomes substantially antibiotically active and a 3- (N-formyl-N-hydroxyamino) propyl is obtained. -fosfonsyraantibiot.