GB1595234A - Thienamycin derivatives - Google Patents

Abstract

Antibiotic deacetyl 890A10 of the structure <IMAGE> and its pharmaceutically acceptable salts; it is produced from the compound 890A10 of the structure <IMAGE> by treatment with an enzyme which is capable of hydrolysing the N-acetyl group.

Description

(54) THIENAMYCIN DERIVATIVES (71) We, MERCK & CO. INC., a corporation duly organized and existing under the laws of the State of New Jersey, United States of America, of Bahway, New Jersey, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed to be particularly described in and by the following statement: The discovery of the remarkable antibiotic properties of penicillin stimulated great interest in this field which has resulted in the finding of many other valuable antibiotic substances such as other penicillins, streptomycin, bacitracin, tetracyclines, chloramphenicol and erythromycins. In general, the antibacterial activity of each of these antibiotics does not include certain clinically important pathogenic bacteria. For example, some are principally active against only gram-positive types of bacteria. Acquired resistance over the course of widespread use of existing antibiotics in the treatment of bacterial infection has caused a serious resistance problem to arise.

Accordingly, the deficiencies of the known antibiotics have stimulated further research to find other antibiotics which will be active against a wider range of pathogens as well as resistant strains of particular microorganisms.

This invention provides the new antibiotic desacetyl 890A1(), including its pharmaceutically acceptable salts. The invention encompasses the antibiotic in dilute forms, as crude concentrates and in pure forms. It also provides a composition comprising an antibacterially effective amount of the compound desacetyl 890A1() and a non-toxic pharmaceutically acceptable carrier, and a composition comprising an antibacterially effective amount of desacetyl 890A,(, and a carrier or diluent.

It has been found that desacetyl 890at() is highly effective in inhibiting the growth of various gram-negative and gram-positive microorganisms.

In accordance with the present invention, desacetyl 890Al() is produced by hydrolysing the N-acetyl group of 890at() using an amidohydrolase capable of hydrolysing the N-acetyl group. A convenient source of an amidohydrolase with this capability is amidohydrolaseproducing strains of the microorganism Protaminobacter rube. The particular enzyme produced by Protaminobacter ruber is N-acetyl-890Al() amidohydrolase, a member of the sub-group of enzymes designated E.C. 3.5.1 according to the recommended enzyme nomenclature of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry.

The microorganism capable of carrying out the deacetylation process was isolated from a soil sample and, based upon taxonomic studies, was identified as belonging to the species Protaminobacter ruber and has been designated MB-3528 in the culture collection of MERCK & CO., Inc., Rahway, New Jersey. A culture thereof has been placed on unrestricted permanent deposit with the culture collection of the Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, and has been assigned accession No. NRRL B-8143.

The morphological and cultural characteristics of Protaminobacter ruber NRRL B-8143 as well as carbon and nitrogen utilization and biochemical reactions are as follows: Morphology - Cells are rod-shaped with rounded ends, 0.9-1.2 x 2.4-4.6 microns, occurring singly or in pairs. Twenty-four and forty-eight hour cells stain gam-negative with a granular appearance. The granules, especially the polar granules, stain black with Sudan Black B. Cells are motile at 28"C., but motility is questionable at 370C.

Cultural Characteristics - Nutrient agar colonies are at first thin, punctiform, semitransparent and colorless; then becoming low convex, opaque, smooth, edge entire, somewhat dry in consistency and pigmented rose to rose-red.

Nutrient broth cultures are uniformly turbid with no pellicle.

Pigment production is not dependent on light or temperatures tested (28"C. and 37"C.).

Pigment is soluble in acetone but insoluble in water or chloroform.

Growth on nutrient agar and brain-heart infusion agar under aerobic conditions is somewhat slow but good at 28"C.; growth is moderate to good but slower at 370C.; there is no growth at 50"C.

Utilization of Carbon and Nitrogen Sources Using a basal salts medium with ammonium sulfate as nitrogen source, growth is good with arabinose, moderate with xylose, and poor with dextrose, fructose, mannose, rhamnose, lactose, maltose, sucrose, raffinose, cellulose, inositol and mannitol.

N-acetylethanolamine can be utilized as the sole carbon and nitrogen source.

No acid or gas is produced from dextrose or lactose in OF Basal Medium (Difco Laboratories, Detroit, Michigan) under aerobic or anaerobic conditions.

Biochemical Reactions The biochemical reactions are based on standard methods as described in Manual of Microbiological Methods, edited by the Society of American Bacteriologists, McGraw-Hill Book Co., New York, 1957.

Catalase - positive Oxidase - negative Starch not hydrolyzed Casein not hydrolyzed Gelatin not liquefied Litmus milk unchanged in consistency but becomes slightly alkaline after 7 days.

Indol - negative H2S - negative Nitrates not reduced Urease - positive Lysine and ornithine decarboxylase - negative.

890A,o is the term applied to the antibiotic having the formula

890A,,, its description and processes of production are set forth in the specification of our copending application No. 47324/77 (Serial No 1595233).

The novel antibiotic of the present invention, desacetyl 890at,), has the structural formula

and is prepared by enzymatic hydrolysis of 890A1(), using an amidohydrolase present in species of genus Protaminobacter.

The novel process of the present invention involves the cleavage of the N-acetyl group of the compound of the formula:

by intimately contacting the said compound with an amidohydrolase capable of hydrolysing the N-acetyl group. More specifically, the process of the present invention provides for the N-deacetylation of 890A,(, by intimately contacting said compound with the amidohydrolase, N-acetyl-890A1 amidohydrolase.

By an unexpected homology between N-acetylethanolamine and 89()A1(), extracts of microorganisms with the enzyme N-acetylethanolamine amidohydrolase arc in many cases able to hydrolyse 890at().

The compound 89()A1() is prepared by the fermentation of broth with the microorganism Streptomyces flavogriseus.

In the detailed description and examples that follow, the words "Dowex", "Whatman", "Eastman", "Chromagram", "Difco", "Hyflo", "Super-Cel", "Amberlitc" and "XAD" are trade marks. Parts are by weight unless otherwise indicated. Mesh sizes in the examples are U.S. standards.

Physical and Chemical Properties of Desacetyl 890ago Table 1 lists the antibacterial spectrum profile of desacetyl 890also TABLE I ASP&num; Organism, MB&num; (ATCC&num;) Inhib. Zone Diam., mm 1 Bacillus sp. 633 28 2 Proteus vulgaris 1012 5 3 Pseudomonas aeruginosa 979 0 4 Serratia marcescens 252 (990) 9 5 Staphylococcus aureus 108 (6538 P) 23 6 Bacillus subtilis 964 (6633) 31 7 Sarcina lutea 1101 21 8 Staphylococcus aureus 698 20 9 Streptococcus faecalis 753 0 10 Brucella bronchiseptica 965 (4617) 0 11 Vibrio percolans 1272 (8461) 27 12 Proteus vulgaris 838 (21100) 14 13 Escherichia coli 1418 12 14 Pseudomonas stutzeri 1231 (11607) 16 15 Klebsiella pneumoniae 1264 8 16 Aerobacter aerogenes 835 9 17 Erwinia atroseptica 1159 (4446) 11 18 Pseudomonas aeruginosa 2824 14H 19 Corynebacterium pseudodiph 261 (9742) 14 20 Escherichia coli 60 (9637) 10 21 Streptococcus faedum 2820 0 22 Streptococcus agalactiae 2875 23 23 Proteus vulgaris 2112 (episome) 15 24 Proteus mirabilis 3126 0 25 Vibrio percolans 1272 + 2 x 105 u/ml 10 penicillinase 26 V. percolans 1272 + Lactamase from Enterobacter MB2646 28 27 Micrococcus flavus 369 (10240) 17 Table 2 lists the 300 MHz nuclear magnetic resonance (NMR) spectra signal of deacetylated 890Alo determined relative to internal standard DSS (sodium 2,2-dimethyl-2silapentane-5-sulfonate) in D2O (24"C.).

TABLE 2 H 58 CH3 1.51 (6.2)b

3.14 3.43 3.07 (18.0,8.5) 3.43 (18.0,9.0) H6 3.88 (5.6,8.9) H5 4.36 H8 4.7-4.9(estimated) "shifts relative to calculated position of internal DSS bvalues in parenthesis are coupling constants.

The mobility of deacetylated 890A,o is determined on Dowex-1. Approximately 100 ssg of desacetyl 890A1() is applied to a column (0.7 x 15 cm.) of Dowex-1 x 2 (Cl-), 200-400 mesh, at an ionic strength of less than 0.01 M and is eluted with a solution containing 0.10 M NaCI + 0.011 M NH4Cl + 0.0001 M NH3 at pH 7.2 in deionized water. The main peak of desacetyl 890A() occurs between eluted volumes 65 ml. and 124 ml. with a maximum at eluted volume 109 ml.

Desacetyl 890A", has a maximum absorbance in the ultraviolet spectra at 296 nm.

The mobility of desacetyl 890A1() is determined on thin layer chromatography and electrophoresis.

Upon thin layer chromatography of desacetyl 890A,(, on Eastman Chromagram 6064 cellulose sheets at 230C. in 66% (v/v) ethanol, the antibiotic displays an Rf of 0.68 as measured by bioautography.

Upon electrophoresis of desacetyl 890A,() on Whatman 3MM chromatography paper using a buffer containing 35.2 g. KH2PO4 + 57.2 g. Na2HPO4 per liter, at 47 volts/cm. for 50 minutes, antibiotic desacetyl 890A,(, moved .65 mm. toward the positive electrode.

Under the same conditions, antibiotic 890A,(, moved 118 mm. toward the positive electrode. The mobilities are measured by bioautography, using chloramphenicol as a reference marker whose mobility is assumed to be zero.

Desacetyl 890A1(), the compound of this invention is a valuable antibiotic active against various gram-positive and gram-negative bacteria and, accordingly, finds utility in human and veterinary medicine. The compound of this invention can be used as an antibacterial drug for treating infections caused by gram-positive or gram-negative bacteria, for example against susceptible strains of Staphylococcus aureus, Proteus mirabilis, Escherichia coli.

Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The antibacterial material of the invention may further be utilized as an additive to animal feeding-stuffs for preserving foodstuffs and as a disinfectant. For example, it may be used in aqueous compositions in concentrations ranging from 0.1 to 100 parts of antibiotic per million parts of solution or preferably in concentrations ranging from 1 to 10 parts of antibiotic per million parts of solution in order to destroy and inhibit the growth of harmful bacteria on medical and dental equipment and as bactericides in industrial applications, for example in water-based paints and in the white water of paper mills to inhibit the growth of deleterious bacteria.

The antibiotic of this invention may be used in any one of a variety of pharmaceutical preparations as the sole active ingredient or in combination either with one or more other antibiotics or with one or more pharmacologically active substances. As an example of the former, an aminocyclitol antibiotic such as gentamicin may be coadministered in order to minimize any chance that resistant organisms will emerge. As an example of the latter, diphenoxylate and atropine may be combined in dosage forms intended for the therapy of gastroenteritis. The antibiotic may be used in capsule form or as tablets, powders or liquid solutions or as suspensions or elixirs. It may be administered orally, topically, intravenously or intramuscularly.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; lubricants, for example, magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example, potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to well known methods. Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product, for reconstitution with water or other suitable vehicles before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; nonaqueous vehicles, which may include edible oils, for example, almond oil or fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; preservatives, for example methyl or propyl phydroxybenzoates, or sorbic acid. Suppositories will contain conventional suppository bases, e.g. cocoa butter or other glyceride.

Compositions for injection may be presented in unit dose form in ampoules, or in multidose containers with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g.

sterile pyrogen-free water, before use.

The compositions may also be prepared in suitable forms for absorption through the mucous membranes of the nose and throat or bronchial tissues and may conveniently take the form of powder or liquid sprays or inhalants, lozenges or throat paints. For medication of the eyes or ears, the preparations may be presented as individual capsules, in liquid or semi-solid form, or may be used as drops. Topical applications may be formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.

Also, in addition to a carrier, the compositions of the present invention may include other ingredients such as stabilizers, binders antioxidants, preservatives, lubricators, suspending agents, viscosity agents or flavoring agents.

In veterinary medicine, such as in the treatment of cows, sheep and pigs, the compositions may, for example, be formulated as intramammary preparations in either long-acting or quick-release bases. They can also be used for treating chickens.

The dosage to be administered depends to a large extent upon the condition of the subject being treated, the weight of the host, the type of infection and the route and frequency of administration, the parenteral route being preferred for generalized infections and the oral route for intestinal infections.

In the treatment of bacterial infections in man, the compound of this invention is administered orally or parenterally, in accordance with conventional procedures for antibiotic administration, in an amount of from 2 to 600 mg./kg./day and preferably 5 to l()() mg./kg./day in preferably divided dosage, e.g. three to four times a day. It may be administered in dosage units containing, for example, 25, 250, 330, 400 or 1000 mg. of active ingredient with suitable physiologically acceptable carriers or excipients. The dosage units are in the form of liquid preparations such as solutions or suspensions or as solids in tablets or capsules. It will, of course, be understood that the optimum dose in any given instance will depend upon the type and severity of infection to be treated, and that smaller doses will be involved in pediatric use, all of such adjustments being within the skill of the practitioner in the field.

The examples, which follow, illustrate the methods by which the products of this invention may be obtained. The examples are illustrative.

EXAMPLE 1 Method of Isolation of N-Acetyl-890A", amidohydrolase-producing Organisms A 1% (w/v) suspension of fertile lawn soil is prepared by suspending 1 g. of lawn soil in 100 ml. sterile phosphate-buffer-saline solution of the following composition: Phosphate-Buffer-Saline Solution NaCI 8.8 g.

1M Phosphate Buffer, pH 7.5* 10 ml.

Distilled H2O 1000 ml.

*IM Phosphate Buffer, pH 7.5 16 ml. 1M KH2PO4 is mixed with 84 ml. 1M K2HPO4.

The pH of the phosphate buffer is adjusted to 7.5 by adding small quantities of either IM KH2PO4 or 1M K2HPO4.

Aliquot portions of this 1% stock soil suspension are used to prepare 10x, 100x and 1,000x dilutions.

One-ml. portions of the stock suspensions or 1-ml. portions of the 10x, 100x, and 1,000x dilutions are added to 2-ml. portions of sterile 1.0% agar solutions at 48"C. The mixtures are quickly poured over the surface of sterile petri dishes of 85 mm. diameter containing 20 ml. of Medium A. Medium A has the following composition: Medium A KH2PO4 3.0 g.

K2HPO4 7.0 g.

MgSO4 0.1 g.

Distilled H,O 1000 ml.

N-Acetylethanol amine solution 8.5 ml.

*N-acetylethanolamine Solution N-acetylethanolamine is diluted 10x in H2O and membrane sterilized. This solution is added after autoclaving.

For solid media: Add 20 g. agar The petri dishes are incubated for 18 days at 28"C. Well-isolated colonies are picked and streaked on Medium B. Medium B has the following composition: Medium B Tomato Paste 40 g.

Ground Oatmeal 15 g.

Distilled H2O 1000 ml.

pH adjusted to 6 using NaOH For solid media: add 20 g. agar Individual clones are selected and grown for two days at 28"C. on slants of Medium B.

A portion of the growth of the slants is used to inoculate a 250-ml. Erlenmeyer flask containing 50 ml. of Medium A; a 250-ml. Erlenmeyer flask containing 50 ml.

supplemented Medium B (supplemented after autoclaving with 0.4 ml. of a membranesterilized solution of N-acetylethanolamine diluted 10x with water); and a 250-ml.

Erlenmeyer flask containing 50 ml. Medium C. Medium C has the following composition: Medium C Dextrose 20 g.

Pharmamedia 8 g.

Corn Steep Liquor (wet basis) 5 g.

Distilled H2O 1000 ml.

pH adjusted to 7 with NaOH or HCI N-acetylethanol amine solution8 8.5 ml.

*N-acetylethanolamine Solution N-acetylethanolamine is diluted 10x in H2O and membrane sterilized. This solution is added after autoclaving.

The flasks are shaken at 28"C. on a 220 rpm (2" throw) shaker for 4 days. A 30-ml.

portion from each flask is centrifuged for 15 minutes at 8,000 rpm. The supernatant portion is removed, leaving only enough to form a thick suspension of cells and media solids. Half of the suspension is subjected to ultrasonic disruption using a Branson Instrument Model LS-75 Sonifier with a 1/2 inch probe. The input power is set at position No. 4, and four successive 15-second cycles of irradiation are used, while chilling the suspension in ice water during and between disruption. To test for the presence of N-acetyl-890A lo amidohydrolase activity, a 10-pl. portion of the sonicate is mixed with 25 Fl. of an 890A1() solution containing 500 Fg. per ml. Controls containing antibiotic and buffer alone; and sonicated cells and buffer without antibiotic are also carried out. After incubation overnight at 280C., 10 Fl.

quantities are applied to Schleicher and Schuell No. 2043-B paper. Using 0.03 M potassium phosphate buffer pH 7.1, a voltage gradient of 50 volts/cm. is applied for 30 minutes. The paper is placed on a Staphylococcus aureus ATCC 6538P plate and incubated at 370C. for 18 hours.

The assay plates are prepared as follows: an overnight growth of the assay organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% yeast extract is diluted with nutrient broth, plus 0.2% yeast extract to a suspension having 60% transmittance at a wavelength of 660 nm. This suspension is added to Difco nutrient agar supplemented with 2.0 g./l. Difco yeast extract at 47"C. to 48"C., to make a composition containing 33.2 ml. of the suspension per liter of agar. Forty ml. of this suspension is poured into 22.5 cm x 22.5 cm. petri plates, and these plates are chilled and held at 4"C. until used (5 day maximum).

The unchanged bioactive 890A,(, spot migrated 8 cm. toward the positive pole. A new bioactive spot due to desacetyl 890A,(, is detected and migrated 4 cm. toward the positive pole. Control incubation mixtures of antibiotic plus buffer, and cell sonicate plus buffer produce no bi6active material migrating 4 cm. toward the positive pole.

EXAMPLE 2 Deacetylation of 890A zo A portion of the growth on a slant of Protaminobacter ruber MB-3528 is used to inoculate a 250-ml. Erlenmeyer flask containing 50 ml. of Medium C. Medium C has the following composition: Medium C Dextrose 20 g.

Pharmamedia 8 g.

Corn Steep Liquor (wet basis) 5 g.

Distilled H2O 1000 ml.

pH adjusted to 7 with NaOH or HCI N-acetylethanolamine solution 8.5 ml.

*N-acetylethanolamine Solution N-acetylethanolamine is diluted 10x in H2O and membrane sterilized. This solution is added after autoclaving.

The flask is shaken at 28"C. on a 220 rpm (2-inch throw) shaker for four days. A 25-ml.

portion from the flask is centrifuged for 15 minutes at 8,000 rpm. The supernatant is removed and the cells on the surface of the media solids scraped off into 0.5 ml. of 0.05M potassium phosphate buffer, pH 7.4. The resulting suspension is subjected to ultrasonic disruption using a Branson Instrument Model LS-75 Sonifier with a 1/2-inch probe at setting 4 for four 15-second intervals, while chilling the suspension in ice water during and between disruption. A 10-l. portion of the sonicate is mixed with 25 ul. of an 890A1() solution containing 500 Ecg. per ml. Controls containing antibiotic and buffer alone; and sonicated cells and buffer without antibiotic are also run. After incubation overnight at 280C., 10-l.

quantities are applied to Schleicher & Schuell No. 2043-B paper. Using 0.03M potassium phosphate buffer, pH 7.1, a voltage gradient of 50 volts/cm. is applied for 30 minutes. The paper is placed on a Staphyloccoccus aureus ATCC 6538P assay plate and incubated at 37"C. for 18 hours.

The assay plates are prepared as follows: an overnight growth of the assay organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% yeast extract is diluted with nutrient broth, plus 0.2% yeast extract to a suspension having 60% transmittance at a wavelength of 660 nm. This suspension is added to Difco nutrient agar supplemented with 2.0 g./l. Difco yeast extract at 47"C. to 48"C., to make a composition containing 33.2 ml. of the suspension per liter of agar. Forty ml. of this suspension is poured into 22.5 cm. x 22.5 cm. petri plates, and these plates are chilled and held at 40C. until used (5-day maximum).

In addition to the unchanged bioactive 890A,(, spot migrating 8 cm. toward the positive pole, a new bioactive spot due to desacetyl 890A,(, is found migrating 4 cm. toward the positive pole. Control incubation mixtures of antibiotic plus buffer, and of cell sonicate plus buffer, produce no bioactive material migrating 4 cm. toward the positive pole.

EXAMPLE 3 Preparation of Antibiotic 890Aso Two frozen vials each containing 2 ml. of MA-4638 inoculum are slowly thawed, and the contents aseptically transferred to two seed flasks each containing 500 ml. of C Medium.

The seed flask, which is a 2-liter, triple-baffled shake flask equipped with a side arm. is stoppered with cotton.

C Medium Autolysed Yeast (Ardamine*) 10.0 g.

Glucose 10.0 g.

MgSO4 7H2O 0.05 g.

Phosphate Buffer** 2.0 ml.

Distilled Water 1000 ml.

pH adjusted to 6.5 with NaOH before sterilization *Ardamine: Yeast Products, Inc.

**Phosphate buffer solution: KH2PO4 91.0 g.

Na2HPO4 95.0 g.

Distilled Water 1000 ml.

The inoculated seed flasks are shaken for 24-30 hours at 280C t 1"C. on a 210 rpm gyrotory shaker, 2-inch throw.

The growth from the seed flasks are used to inoculate two 14-liter glass fermentors containing 10 liters of production medium (D medium plus 0.15% soybean oil, v/v).

D Medium Dextrin (CPC Modified Starch) 40.0 g.

Distiller's Solubles 7.0 g.

Yeast Extract 5.0 g.

CoCl2 6H2O 50.0 mg.

Distilled Water 1000 ml.

pH adjusted to 7.3 with NaOH before sterilization The fermentors are operated at 24"C. using an agitation rate of 640 rpm (about Kd 5.5), and an air flow of 0.5 VVM, for 72 hours. Defoamer, Hodag-MF (Hodag Chemical Corp.) is used as required, but not to exceed 0.1%.

Contents of the two fermentors are pooled after the fermentation run, about 20 liters.

200-ml. portions of the batch are centrifuged in a Servall RC-2B centrifuge at 9000 rpm for 20 minutes. The supernatant is filtered through a 1-cm. bed of Hyflo Super-Cel in a 13-inch Lapp funnel with a cloth filter. The filtrate has a bioactiyity of 20 units/ml.

The filtered broth, (18 liters) is applied to a column (8.2 x 29 cm.) of Dowex-lx2(Cl-) 50-100 mesh, at 150 ml./min. The column is then washed with 1 liter of deionized water followed by 15 liters of 0.15 M NaCI + 0.01M Tris-HCl buffer, pH 7.0 + 25 FM neutral EDTA in 50% MeOH at 150 ml./min.

The antibiotic 890A,(, is then eluted with 3.2% NaCI + 0.02 M Tris-HCl buffer pH 7.0 + 251lM neutral EDTA in 80% MEOH at 100 ml./min. Fractions are collected as follows: one fraction of 1 liter, four fractions of 500 ml. each, and twelve fractions of 1 liter each are collected . Bioactivity and absorbance at 220 nm is measured on each fraction, and those fractions leaving bioactivity/A22(, ratios of greater than 0.6 units/A22(, unit, and which also contained more than 10% of the total recovered activity per fraction are combined for further purification. Thus, fractions 4 to 8 are combined, containing a total of 22% of the applied bioactivity.

The pooled fractions are concentrated under reduced pressure to 190 ml., the precipitated salt is washed with deionized water, and the wash is added to the supernatant, bringing the final volume to 220 ml. The concentrate is adjusted to pH 6.5 with HC1 and is applied on a column (6.0 x 59 cm.) of Amberlite XAD-2 which has been previously washed with 8 liters of 60% aqueous acetone followed by 16 liters of deionized water. After application is completed, the column is rinsed with 5 x 10 ml. portions of deionized water, and the antibiotics are eluted with deionized water at 35 ml./min. Frac

Claims (23)

**WARNING** start of CLMS field may overlap end of DESC **. further purification. Thus, fractions 4 to 8 are combined, containing a total of 22% of the applied bioactivity. The pooled fractions are concentrated under reduced pressure to 190 ml., the precipitated salt is washed with deionized water, and the wash is added to the supernatant, bringing the final volume to 220 ml. The concentrate is adjusted to pH 6.5 with HC1 and is applied on a column (6.0 x 59 cm.) of Amberlite XAD-2 which has been previously washed with 8 liters of 60% aqueous acetone followed by 16 liters of deionized water. After application is completed, the column is rinsed with 5 x 10 ml. portions of deionized water, and the antibiotics are eluted with deionized water at 35 ml./min. Fractions are collected as follows: one fraction of 500 ml. followed by seven fractions of 250 ml. each, followed by four fractions of 500 ml. Bioactivity and HAEA304 values are measured on fractions from 3 to 12. Fractions 4 to 9 have HAEA3)4/A22f) ratios greater than 0.01, and are combined for further purification. Fraction 3, which contains about one-fifth of the applied salt, is also added to this pool. The combined pool contains 469 HAEA304 units. The pooled fractions are diluted to 4.1 liters and are applied to a column (2.15 x 42 cm.) of Dowex-lx4(Cl-) minus 400 mesh at 2 ml./min. The column is washed with 100 ml. of 50% methanol, and the antibiotic is eluted with 0.25 M NaCl + 0.01 M NH4Cl + 0.0001 M NH3 in 80% MeOH at approximately 2 ml./min. Fractions of from 8 to 12 ml. are collected. The bioactivity and the absorbance at 220 nm, 260 nm, and 300 nm are measured for every fifth fraction, and HAEA3(,(, is measured on the peak bioactive fractions. The 890A1() bioactivity appears in fractions 85 to 150, with a maximum at fraction 120. Fractions with HAEA300/A3(,(, values greater than 0.05 are combined for further purification. Thus, fractions 105 to 133 are combined, containing a total of 130 HAEA30o units. The pooled fractions 105 to 133 are diluted to 1900 ml. with deionized water, and the pH is adjusted to 7.6. The sample is applied to a column (2.15 x 42 cm.) of Dowex-lx2(Cl-), minus 400 mesh, which has been previously washed with 3 liters of 3% NaCI in 50% methanol followed by 50 ml. of 50% methanol. After the entire sample has been applied, the column is washed with 100 ml. of 30% methanol, and is eluted at 2 ml./min. with 0.26 M NaCl + 0.005 M NH4Cl + 0.0002 M NH3 in 30% methanol. Fractions of 10 to 12 ml. are collected. The main peak of antibiotic 890Alo appears in fractions 230 to 290, with a maximum at fraction 260. Those fractions with an A()(JA25(, ratio greater than 1.10 and an A3)1)/A22) ratio greater than 0.83 are combined for further purification. Thus, fractions 240 to 275 are combined, containing 91.6 HAEA(,(, units. The combined fractions 240 to 275 are concentrated under reduced pressure to 10 ml., and the concentrate is separated from the precipitated salt by pipetting. The salt is washed with 2 ml. deionized water, and the wash is added to the concentrate. The combined 12 ml. of concentrate and wash is applied to a column (2.15 x 76 cm.) of Bio-Gel P-2 (200-400 mesh), which has been previously washed with 30 ml. of saturated NaCI in deionized water, followed by 1500 ml. of deionized water and 50 ml. of 0.05 mM NH3 in deionized water. After application is complete, the column is rinsed with 3 x 1 ml. rinses of 0.05 mM NH3 in deionized water, and the antibiotic is eluted with 0.05 mM NH3 in deionized water at 0.73 ml./min. Fractions of 3.65 ml. each are collected. The main peak of antibiotic 890A1() appears in fractions 29 to 50, with a maximum at fraction 36. Those fractions having an A(,(,/A25(, ratio greater than 1.9, and also having an AXo(,/A22(, ratio greater than 1.45, are combined for lyophilization and NMR analysis. Thus, fractions 36 to 45 are combined. They contain 50.8 A(,(, units, of which 45.1 is hydroxylamine-extinguishable. The combined fractions 36 to 45 are concentrated under reduced pressure to I ml., and 5 ml. D2O is added. The sample is again concentrated to 0.835 ml., and 0.825 ml. of this is transferred to a glass vial and freeze-dried and lyophilized. The lyophilized sample contains 48.2 AX units and has 4.6 mg of solids. WHAT WE CLAIM IS:

1. The compound desacetyl 890A1(), which has the formula

and its pharmaceutically acceptable salts.

2. A process for producing desacetyl 890A,(,

that comprises intimately contacting the compound 890A10, which has the formula

with an enzyme capable of hydrolysing the N-acetyl group.

3. A process as claimed in Claim 2 in which the enzyme is an amidohydrolase.

4. A process as claimed in Claim 3 in which the amidohydrolase is an Nacetylethanolamine amidohydrolase.

5. A process as claimed in Claim 3 in which the amidohydrolase is an N-acetyl 890A,o amidohydrolase.

6. A process for producing desacetyl 890A,o comprising obtaining microorganisms with N-acetyl-890A,o amidohydrolase by selecting those microorganisms capable of using N-acetyl-ethanolamine for growth, testing these microorganisms for the presence of N-acetyl-890Alo amidohydrolase, and using the microorganisms with N-acetyl-890A amidohydrolase in a process as claimed in Claim 2 by intimately contacting them with the compound 890A,(,.

7. A process as claimed in Claim 3 carried out with an amidohydrolase produced by an amidohydrolase-producing strain of the microorganism Protaminobacter ruber.

8. A process for producing desacetyl 890A,(, that comprises intimately contacting 890A,o with the enzyme N-acetyl-890A,(, amidohydrolase produced by an amidohydrolaseproducing strain of the microorganism Protaminobacter rube.

9. A composition comprising an antibacterially effect amount of the compound desacetyl 890A,(, according to Claim 1 and a non-toxic pharmaceutically acceptable carrier.

10. A composition comprising an antibacterially effective amount of the compound claimed in Claim 1 and a carrier or diluent.

11. A composition as claimed in Claim 10 in the form of a disinfectant containing 0.1 to 1.00 ppm of the said compound.

12. A composition as claimed in Claim 9 also containing another antibiotic or pharmacologically active substance.

13. A composition as claimed in Claim 12, in which the other antibiotic or pharmacologically active substance is gentamycin, diphenoxylate or atropine.

14. A composition as claimed in Claim 9, 12 or 13 in the form of a capsule, tablet, powder, liquid solution, suspension, elixir, syrup or emulsion.

15. A composition as claimed in Claim 9, 12 or 13 in the form of a suppository.

16. A composition as claimed in Claim 9, 12 or 13 in injectable form.

17. A composition as claimed in Claim 9, 12 or 13 in the form of a throat spray, an inhalant, a lozenge or a throat paint.

18. A composition as claimed in Claim 9, 12 or 13 in topically administrable form.

19. A composition as claimed in Claim 18 in the form of an ointment, cream, lotion our paint.

20. A composition as claimed in Claim 10 in the form of a preparation suitable for administration in veterinary medicine.

21. A composition as claimed in Claim 20 in the form of an intramammary preparation.

22. A process as claimed in Claim 2 substantially as hereinbefore described in Example

23. A compound as claimed in Claim 1 when prepared by a process as claimed in any one of Claims 2 to 8 and 22.