WO1989007612A1

WO1989007612A1 - Novel antibiotic compounds

Info

Publication number: WO1989007612A1
Application number: PCT/GB1989/000128
Authority: WO
Inventors: Medha Athalye; Anna Louisa Elson; Martin Leonard Gilpin
Original assignee: Beecham Group Plc
Priority date: 1988-02-10
Filing date: 1989-02-09
Publication date: 1989-08-24
Also published as: EP0368949A1

Abstract

Glycopeptide antibiotics MM 47761 and MM 49721 of formula (I), wherein R is chlorine or hydrogen are produced by Amycolatopsis orientalis strain NCIB 12608.

Description

Novel antibiotic compounds.

The present invention relates to a novel antibacterially active material obtainable from a microorganism, to a process for its production, and to its pharmaceutical use.

A large number of microorganisms have been isolated from nature and certain of those microorganisms have been found to produce various metabolites, which can be isolated and some of which have useful antibacterial activity. One such metabolite is a substance which has been designated MM 47761. It is believed to be a novel glycopeptide compound and it has been found to have useful antibacterial activity.

The present invention accordingly provides the novel substance MM 47761, and pharmaceutically acceptable derivatives thereof, such as pharmaceutically acceptable acid addition salts.

The present invention also provides a process for the production of the substance MM 47761 which comprises cultivating an MM 47761-producing microorganism and subsequently isolating MM 47761 or a derivative thereof from the culture.

The present invention furthermore provides a process for the preparation of the substance MM 47761 which comprises separating MM 47761 or a derivative thereof from a solution of MM 47761 in admixture with other antibacterially active substances and/or inactive substances by adsorption onto an affinity resin. The substance MM 47761 has the following characteristics:

(i) it may be obtained by the cultivation of a microorganism of the order Actinomycetales;

(ii) its retention time in high-performance liquid chromatography (h.p.l.c), using a C18 μ bondapak (Trade Mark) column packing (column size 3.9mm diameter × 300mm long), with an aqueous 0.1M NaH2PO₄ solvent system at pH 6.0 containing 10% acetonitrile at a flow rate of 2ml/min, is approximately 3.8 minutes as measured by u.v. absorption at 210 and 280 nm (packed h.p.l.c. column supplied by Waters Associates, U.S.A.); and

(iii) it shows antibacterial activity against Staphylococcus aureus V573.

MM 47761 may be obtained by the cultivation of an MM 47761 producing microorganism and the recovery of Mm 47761 or a derivative thereof from the culture.

The term 'cultivation' (and derivatives of that term) as used herein means the deliberate aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen, sulphur and mineral salts. Such aerobic growth may take place in a solid or semi-solid nutritive medium, or in a liquid medium in which the nutrients are dissolved or suspended. The cultivation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically defined. It has been found that suitable microorganisms for use in the cultivation process according to the invention include bacterial strains belonging to the order Actinomycetales that are capable of elaborating

MM 47761. It has further been found that an example of such a strain is Actinomycete sp. NCIB 12608 and also mutants thereof, which has been isolated from nature.

The term 'mutant' as used herein includes any mutant strain which arises spontaneously or through the effect of an external agent whether that agent is applied deliberately or otherwise. Suitable methods of producing mutant strains including those outlined by H.I. Adler in 'Techniques for the Development of Microorganisms' in 'Radiation and Radioisotopes for Industrial Microorganisms', Proceedings of a Symposium, Vienna, 1973, page 241, International Atomic Energy Authority, and these include:

(i) Ionizing radiation (e.g. X-rays and λ-rays), u.v. light, u.v. light plus a photosensitizing agents (e.g. 8-methoxypsoralen), nitrous acid, hydroxylamine, pyrimidine base analogues (e.g. 5-bromouracil), acridines, alkylating agents (e.g. mustard gas, ethyl-methane sulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and

(ii) Genetic techniques, including, for example, recombination, transformation, transduction, lysogenisation, lysogenic conversion, protoplast fusion and selective techniques for spontaneous mutants.

Actinomycete sp. NCIB 12608 is believed to be a previously unreported strain in the species amvcolatopsis orientalis and therefore also forms a part of the present invention, particularly in biologically pure form. It has been deposited at the

National Collections of Industrial and Marine Bacteria Ltd. (N.C.I.B), Aberdeen, Scotland under number 12608 on 1st December, 1987.

The fermentation medium for cultivating NCIB 12608 suitably contains sources of assimilable carbon and assimilable nitrogen together with inorganic salts. Suitable sources of nitrogen include yeast extract, soyabean flour, meat extract, cottonseed, flour, malt, distillers dried solubles, amino acids, protein hydrolysates and ammonium and nitrate nitrogen. Suitable carbon sources include glucose, lactose, maltose, starch and glycerol. Suitably the culture medium also includes alkali metal ions (for example, sodium), halogen ions (for example, chloride), and alkaline earth metal ions (for example calcium and magnesium), as well as trace elements such as iron and cobalt.

The cultivation may suitably be effected at a temperature of about 20 to 35°C , advantageously 20 to 30°C, and the culture may suitably be harvested up to 7 days, advantageously about 3 to 5 days, after the initiation of fermentation in order to give an optimum yield of MM 47761.

The desired MM 47761 or a derivative thereof may then be isolated from the culture medium and worked up and purified using conventional techniques for glycopeptide compounds. All such isolation and purification procedures may conveniently be effected at cool to ambient temperature, for example at a temperature within the range of from 4 to 30°C, conveniently from

20 to 25°C.

The desired product is generally obtained predominantly from the culture filtrate, and it is therefore convenient for the first isolation step to involve removal of solid material from the fermentation broth by, for example, filtration or centrifugation, to give a clarified culture filtrate.

Further isolation of the desired MM 47761 from the clarified culture filtrate may conveniently be effected by adsorption onto an affinity resin such as D-alanyl-D-alanine-sepharose affinity resin.

The desired compound may readily be identified in a routine manner by testing for antibacterial activity and/or by monitoring the h.p.l.c. retention time.

Suitably, the separation procedure may include a high-performance liquid chromatography step, preferably as the last step. Elution may be effected using aqueous NaH₂PO₄/acetonitrile.

MM 47761 and its derivatives may be crystalline or non-crystalline and, if crystalline, may optionally be hydrated or solvated.

Culture NCIB 12608 has also been found to produce a further glycopeptide antibiotic which has been designated MM49721 and has the characteristics set out hereinbelow in the Examples. This compound, its pharmaceutically acceptable derivatives, processes for its production, and its pharmaceutical use, form a further aspect of the invention. Processes for the preparation of MM49721 in accordance with the invention are as set out above in relation to

MM47761.

MM 49721 and its derivatives may be crystalline or non-crystalline and, if crystalline, may optionally be hydrated or solvated.

MM 47761 and MM 49721 are believed to be the compounds of formula (I):

wherein R is chlorine (MM 47761) or hydrogen (MM 49721).

A further aspect of the invention provides a process for the preparation of the pseudoaglycone of MM 47761 or MM 49721, that is, a compound of formula (II):

wherein R is as defined above, which process comprises acid catalysed hydrolysis of MM 47761 or MM 49721. The hydrolysis is typically carried out at elevated temperature, for example in refluxing 5M hydrochloric acid for about 5 minutes.

The compounds according to the invention are suitably provided in substantially pure form, for example at least 50% pure, suitable at least 60% pure, advantageously at least 75% pure, preferably at least 85% pure, more preferably at least 95% pure, especially at least 98% pure, all percentages being calculated as weight/weight. An impure or less pure form of a compound according to the invention may, for example, be used in the preparation of a more pure form of the same compound or of a related compound (for example a corresponding derivative) suitable for pharmaceutical use.

The compounds of the invention have antibacterial properties and are useful for the treatment of bacterial infections in animals, especially mammals, including humans, in particular humans and domesticated animals (including farm animals). The compounds may be used for the treatment of infections caused by a wide range of organisms including, for example, those mentioned herein. The present invention provides a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable derivative thereof together with a pharmaceutically acceptable carrier or excipient.

The present invention also provides a method of treating bacterial infections in animals, especially in humans and in domesticated mammals, which comprises administering a compound of the invention or a pharmaceutically acceptable derivative thereof, or a composition according to the invention, to a patient in need thereof.

The compounds and compositions according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics.

The compounds and compositions according to the invention may be formulated for administration by any route, for example oral, topical or parenteral. The compositions may, for example, be made up in the form of tablets, capsules, powders, granules, lozenges, creams, syrups, or liquid preparations, for example solutions or suspensions, which may be formulated for oral use or in sterile form for parenteral administration by injection or infusion.

Tablets and capsules for oral administration may be in unit dosage form, and may contain conventional excipients including, for example, binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; and pharmaceutically acceptable wetting agents, for example sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain conventional additives, including, for example, suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia;. non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters (for example glycerine), propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavouring and colour agents.

Compositions according to the invention intended for topical administration may, for example, be in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, impregnated dressings, and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.

Compositions according to the invention may be formulated as suppositories, which may contain conventional suppository bases, for example cocoa-butter or other glycerides.

Compositions according to the invention intended for parenteral administration may conveniently be in fluid unit dosage forms, which may be prepared utilizing the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, may be either suspended or dissolved in the vehicle. In preparing solutions, the compound may be dissolved in water for injection and filter-sterilised before being filled into a suitable vial or ampoule, which is then sealed. Advantageously, conventional additives including, for example, localanaesthetics, preservatives, and buffering agents can be dissolved in the vehicle. In order to enhance the stability of the solution, the composition may be frozen after being filled into the vial, and the water removed under vacuum; the resulting dry lyophilized powder may then be sealed in the vial and a accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions may be prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilisation cannot be accomplished by filtration. The compound may instead be sterilised by exposure to ethylene oxide before being suspended in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in such suspensions in order to facilitate uniform distribution of the compound.

A compound or composition according to the invention may suitable be administered to the patient in an antibacterially effective amount.

A composition according to the invention may suitably contain from 0.1% by weight, preferably from 10 to 60% by weight, of a compound according io the invention (based on the total weight of the composition), depending on the method of administration.

The compounds according to the invention may suitably be administered to the patient at a daily dosage of from 1.0 to 50 mg/kg of body weight. For an adult human (of approximately 70 kg body weight), from 50 to 3000 mg, for example about 1500 mg, of a compound according to the invention may be administered daily. Suitably, the dosage for adult humans is from 5 to 20 mg/kg per day. Higher or lower dosages may, however, be used in accordance with normal clinical practice.

When the compositions according to the invention are presented in unit dosage form, each unit dose may suitably comprise from 25 to 1000 mg, preferable from 50 to 500 mg, of a compound according to the invention.

The following Examples illustrate the present invention. Production and Isolation of MM 47761

Example 1

a) Fermentation

Culture NCIB 12608 was grown for 7 days at 26°C on a solid agar slant in a McCartney bottle. The agar medium had the following composition:

Constituent Amount (g/l)

Yeast extract 4.0

Malt extract 10.0 Dextrose 4.0

Agar 20.0

Deionised water To 1 litre

[The constituents were all 'Bacto' products (Bacto is a Trade Mark) as supplied by Difco Laboratories, P.O. Box 14B, Central Avenue, East Molesey, Surrey]. The medium was adjusted to pH7.3 before sterilisation.

A spore suspension was prepared by adding 10ml of sterilised water containing 0.005% Triton X 100 to a McCartney bottle agar culture of NCIB 12608, followed by sonication of the suspension for 1 minute. 1ml of this suspension was added to a flask of seed stage medium [Triton X 100 was obtained from B.D.H Chemicals Ltd., Poole, Dorset].

The seed stage fermentation was carried out in a 500ml conical flask containing 100ml of seed stage medium. The seed stage medium contained: Constituent Amount (g/l)

Soya bean flour 10

Glycerol 20

Maltose 2

CoCl₂·6H₂O 0.005

Stock trace elements 10ml

Deionised water to 1 litre

The stock trace element solution contained:

Constituent Amount (g/1)

CaCl₂.2H₂O 10

MgCl₂.6H₂O 10 NaCl 10

FeCl₃ 3

ZnCl₂ 0.5

CuCl₂.2H₂O 0.5

MnSO₄.4H₂O 0.5

The medium was adjusted to pH7.3 before sterilisation at 117°C for 15 minutes.

[The soya bean flour was Arkasoy 50 supplied by the British Arkady Co. Ltd., Old Trafford, Manchester]. Incubation was carried out for 72 hours at 26°C and 240r.p.m. on a gyratory shaker.

5ml of the seed culture was used to inoculate each of 30; 500ml conical flasks containing 100ml of fermentation medium. The fermentation medium was identical to the seed stage medium. The fermentation was carried out for 96 hours at 26°C and 240r.p.m. on a gyratory shaker. The harvested broth was clarified by centrifugation. b) Isolation of MM 47761

The glycopeptide, MM 47761 was isolated from the clarified broth by adsorption onto D-alanyl-D-alaninesepharose affinity resin.

The affinity adsorbent was prepared from D-alanyl-D-alanine immobilised on Activated CH-Sepharose 4B [6-Aminohexanoic acid-activated-sepharose-4B was obtained from Sigma Chemical Co., Poole, Dorset].

The clarified broth (2400ml) prepared as described in a) was stirred for 1 hour with D-alanyl-D-alaninesepharose affinity resin (30ml wet volume) . The mixture was filtered onto a glass scinter funnel and the filtrate discarded. The affinity resin was washed with distilled water (100ml) and filtered as before. The resin was washed twice more with distilled water. The glycopeptide was eluted from the affinity resin by stirring with 0.1M ammonia containing 50% acetonitrile (50ml) for 15min. The mixture was filtered and the filtrate evaporated under reduced pressure to dryness, to yield a cream solid.

The solid was re-suspended in water (50ml) and the glycopeptide adsorbed once more onto D-alanyl-D-alanine-sepharose affinity resin (30ml wet volume). The resin was washed with water and the glycopeptide eluted with 0.1M ammonia containing 50% acetonitrile as previously described. The eluant was evaporated to dryness to yield 195mg of substantially pure MM 47761.

The positive ion Fast Atom Bombardment mass spectrum gave a peak at 1560 ± 1 believed to indicate a molecular weight of 1559 ± 1. Acid hydrolysis of MM 47761 gave aspartic acid and N-methyl leucine. Example 2 :

a) Fermentation.

Culture NCIB 12608 was grown for 7 days at 28°C on solid agar in a petri dish. The agar medium had the following composition:

Constituent Amount (g/l)

Yeast Extract* 5.0

Malt Extract* 10.0

Glycerol 10.0

Neutralised Soya Peptone* 5.0 Agar* 18.0

Deionised water To 1 litre

[*These constituents were supplied by Oxoid Limited, Basingstoke, Hampshire, England]. The medium was adjusted to pH 6.5 before sterilisation.

An 8mm diameter agar plug was used to inoculate each 500ml conical flask, fitted with cotton gauze caps, containing 100ml of seed stage medium, having the following composition:

Constituent Amount ( g/l)

Soyabean flour 10.0

Glycerol 20.0

Maltose 2.0

CoCl₂.6H₂O 0.005

Stock trace elements 10ml

Deionised water To 1 litre The stock trace element solution contained:

Constituent Amount (g/l)

CaCl₂·2H₂O 10.0

MgCl₂.6H₂O 10.0

NaCl 10.0

FeCl₃ 3.0

ZnCl₂ 0.5

CuCl₂.2H₂O 0.5

MnSO₄.4H₂O 0.5

[Soya bean flour was Arkasoy '50', supplied by Arkady A.D.M., Manchester, England.]

The medium was adjusted to pH 7.0 before sterilisation in an autoclave at 121°C for 15 minutes. The flasks were then incubated on a gyratory shaking table at 240r.p.m. for 48 hours at 28°C.

15 litres of seed stage medium, with the same composition as above, but with the addition of 0.1% antifoaming agent, polypropylene glycol P2000 was sterilised in a 20L, fully baffled fermenter for 1 hour at 121°C. 200ml of the flask seed stage were used as inoculum and the fermentation was incubated at 26°C for 48 hours. The fermenter was stirred by an agitator, fitted with three, vaned-disc impellers, at 200r.p.m. and supplied with sterile air at 0.5 volumes per volume per minute. An overpressure of air of 0.5 bar was maintained throughout.

For the final fermentation, 300L of medium with the same composition as the 20L seed stage were sterilised in a 450L fully baffled fermenter for 1 hour at 121°C. 8L of vegetative inoculum from the 20L fermenter were used as inoculum and the fermentation was incubated at

26°C until harvest at 72 hours. The fermenter was stirred by an agitator fitted with three vaned-disc impellers at 50r.p.m. and supplied with sterile air at

0.5 volumes per volume per minute. An overpressure of air of 0.5 bar was maintained throughout.

b) Isolation

i) Clarified broth (310L) from example 2a was applied to a 12L Amberlite IRA 458 anion-exchange column, (Cl- form) at a flow rate of lLmin^-1. [Resin supplied by Rohm and Haas Ltd., Lenning House, 2 Mason' s Avenue, Croydon, Surrey.]

The column was washed with 20L of deionised water. The percolate and water wash were combined, adjusted to pH 7.0 and loaded onto a 6.9L column of Diaion HP20 at a flow rate of 600ml.min^-1. [Resin supplied by Mitsubishi Chemical Industries, Tokyo, Japan.] The column was washed with 10L of deionised water and the percolate and water wash discarded. The active material was eluted from the column with propan-1-ol/water, 40/60. 1 litre fractions were collected. Fractions with antibiotic activity (6-24) were bulked and evaporated in vacuo to 2.1L. The HP20 column was further eluted with propan-1-ol/water, 50/50 containing 0.1M ammonia. 1 litre fractions were collected. Fractions 25-45 were bulked and evaporated in vacuo to 1.6L. The combined concentrates were centrifuged and the supernatant filtered through GF/D glass fibre filters [Filters supplied by Whatman, Springfield Mill, Maidstone, Kent, England].

The combined filtrates (3.7L) were applied to a 0.825L CM Sephadex C25 cation-exchange column (Na⁺ form), previously equilibrated in 0.005M NaH₂PO₄ pH 6.6, at a flow rate of 20ml.min^-1. [CM Sephadex supplied by

Pharmacia Ltd., Uppsala, Sweden.] The percolate was discarded and the column was washed with 500ml³ deionised water. Antibiotically active material was eluted using an exponential gradient of 0.05M NaH₂PO₄ pH 7.0 to 0.2M Na₂HPO₄ pH 9.2 (1 litre mixing volume), at a flow rate of 20ml.min^-1. 1 minute fractions were collected. Fractions with antibiotic activity (91-240) were bulked to give 2.6L at pH 7.8 containing approximately 60g of substantially pure MM 47761.

ii) 3.7L of solution, obtained essentially as described in Example 2b(i), were loaded onto a 0.7L column of HP20. The column was washed with 1.9L of deionised water and eluted with 2.5L of propan-1-ol/water, 50/50 containing 0.1M ammonia. The eluate was evaporated in vacuo to 725ml. The combined percolate and water wash which still contained some antibiotic activity was loaded onto a second HP20 column of 1.4L. The column was treated as above and the evaporated eluates from both columns were combined.

NaH₂PO₄ was added to the concentrated solution (1.4L) to give a molarity of 0.005M and the pH adjusted to 6.7 with 5M NaOH.

The buffered solution was loaded onto a 0.8L CM Sephadex C25 column previously equilibrated in 0.005M NaH₂PO₄ pH 6.6 at a flow rate of 30ml.min^-1. The column was washed with 1L of 0.05M NaH₂PO₄ pH 7.0 and eluted using an exponential gradient of 0.05M NaH₂PO₄ pH 7.0 to 0.2M Na₂HPO₄ pH 9.2 (1L mixing volume). After 500ml of eluate had passed, 20ml fractions were collected. Those fractions containing MM 47761 (41-180) were combined and adjusted to pH 7.0 with 5M

HCl.

The solution (2.8L) was loaded onto a 1.3L column of HP20. The column was washed with 3.4L of deionised water and eluted with 2.5L propan-1-ol/water, 50/50 containing 0.1M ammonia. The eluate was evaporated in vacuo to 0.9L and the pH adjusted to 7.0 using 1M HCl.

The solution was freeze-dried to yield 39.0g of substantially pure MM 47761.

Example 3

Isolation of MM 47761 by preparative HPLC

The MM 47761 obtained from large scale fermentations of NCIB 12608, as described in Example 2, may contain residual amounts of MM 49721 and may be further purified using preparative HPLC.

100mg of MM 47761 containing approximately 5% MM 49721 was dissolved in 1ml of 0.1M NaH₂PO₄, pH 6.0. Six aliquots of 100 or 150μl (totalling 750μl) were loaded onto a Dynamax 150-A preparative HPLC column, 10mm × 300mm, containing 12μm particles of C₁₈ reverse phase silica. [Rainin Instrument Co., Woburn, MA, USA]. The column was eluted with 0.1M NaH₂PO₄ pH 6.0 containing 7% acetonitrile at a flow rate of 6.0ml.min^-1. The eluant was monitored at 210nm on a Waters Lambda-Max model 481 spectrophotometer, using a semi-preparative flow cell, (2.1mm path length).

From the commencement of elution of MM 47761 (4 minutes) 1 minute fractions were collected. The fractions were assayed on a Waters analytical HPLC column, as described in the Detection Methods section.

Fractions 1 from each run, containing MM 47761, free of MM 49721, were combined. Acetonitrile was removed by evaporation in vacuo and the product was desalted by treatment with D-alanyl-D-alanine Sepharose affinity resin as described in previous examples. The affinity eluate was evaporated in vacuo and freeze-dried to yield 50mg of MM 47761 containing less than 1% MM 49721.

Example 4

Isolation of MM 49721

The product obtained from large scale fermentations of culture NCIB 12608, as described in Example 2, may contain MM 49721.

The MM 49721 can be isolated using CM Sephadex C25 cation-exchange column chromatography.

4.18g of solid prepared essentially as in Example 2, containing 7% MM 49721, was dissolved in 300ml of 0.05M NaH₂PO₄ pH 6.0 and loaded onto a 0.5L CM Sephadex C25 cation-exchange column (Na⁺ form), previously equilibrated in the same buffer. The column was washed with 0.05M NaH₂PO₄ pH 6.0 (200ml) and then eluted with an exponential gradient of 0.05M NaH₂PO₄ pH 6.0 to 0.2M Na₂HPO₄ pH 10.0 (500ml mixing volume) at a flow rate of 6ml.min^-1. 20ml fractions were collected. Fractions found to be antibacterially active were further monitored by HPLC. Fractions 72-83 contained MM 49721, fractions 92-114 contained MM 47761. Fractions containing MM 49721 were bulked, adjusted to pH 6.5 and treated with D-alanyl-D-alanine Sepharose affinity resin as described in previous examples. After elution from the affinity resin the product was freeze dried to yield 143 mg of substantially pure MM

49721.

The positive ion Fast Atom Bombardment mass spectrum gave a peak at 1526±1 believed to indicate a molecular weight of 1525±1.

Detection Methods

Fermentation samples were monitored for antibiotic activity by bioassay on Staphylococcus aureus V573, using the conventional hole in plate method.

MM 47761

Preparations of MM 47761 were assayed on a Waters high performance liquid chromatography column (3.9 × 300mm) containing μ-bondapak C18 reverse phase material. [Waters Associates 34, Maple Street, Milford, Mass. U.S.A]. The glycopeptide was eluted from the column with 0.1M NaH₂PO₄ pH 6.0 containing 10% acetonitrile, at a flow rate of 2ml/min. The eluant was monitored at 210 and 280nm by a Hewlet-Packard 1040A diode array hplc monitor [Hewlet-Packard, Corvallis Division, 100 N.E. Circle Boulevard, Corvallis, Oregon, U.S.A]. Under these conditions MM 47761 had a retention time of 3.8 minutes (cf vancomycin R_T = 11.0 minutes, ristocetin R_T = 5.8 minutes under the same conditions).

Using the same system, but 0.1M Na phosphate buffer pH 7.5 containing 10% acetonitrile, and 0.005M sodium heptane sulphonate ion-pairing reagent at a flow rate of 2ml/min., MM 47761 had a retention time of 6.0 minutes.

MM 49721

Using the hplc system described for MM47761, and 0.1M NaH₂PO₄ pH 6.0 containing 10% acetonitrile as solvent (flow rate 2ml/mm), MM49721 had a retention time of 5.8 min (cf. MM47761 R_T = 4.6 min; vancomycin R_T = 11.4 min.) When the solvent used was 0.1M NaH₂PO₄ pH 7.5 containing 10% acetonitrile, and 0.005M sodium heptane sulphonate ion-pairing reagent, at a flow rate of 2ml/min; MM49721 had a retention time of 7.4 min.

(cf .MM47761 R_T = 7.4 min; vancomycin R_T = 13.4 min.)

Physical and spectral properties of MM 47761 after drying over P₂O₅: FAB-MS

(glycerol/thiogycerol/trifluoracetic acid) MH⁺ 1560±1 Molecular Weight: 1559 Molecular Formula: C₇₂H₈₆N₉O₂₈Cl UV (H₂O) λ_max 280nm (ε 6403) [α]_D ²⁰ -79° (c = 0.77% in H₂O) IR (KBr) 1653, 1587, 1501 cm^{-1 1}H NMR in DMSO ds at 373°k. Tetramethylailane as internal standard. δ_H 8.36 (1H, d, J 6.1 Hz), 7.98 (1H, d, J 6.0 Hz), 7.66 (1H, dd, J 7.7 and ca. 1.0 Hz), 7.55 (1H, dd, J 8.3 and ca. 1.0 Hz), 7.41 (1H, br. s), 7.33 (1H, br. d), 7.26 (1H, d, J 8.4 Hz), 7.15 (1H, br. s), 7.06 (1H, dd, J 8.4 and ca. 1.0 Hz), 6.82 (1H, dd, J 8.4 and 2.1 Hz), 6.73 (1H, d, J 8.4 Hz), 6.40 (2H, s), ca. 5.7 (1H, br), 5.69 (1H, br. s), 5.54 (1H, d, J 7.3 Hz), 5.43 (1H, br. S), 5.26 (1H, s), 5.18 (1H, s), 5.16 (1H, d, J

3.2 Hz), 4.73 (2H, m) , 4.54 (2H, m), 4.37 (1H, br.), 4.24 (1H, br.), 4.11 (1H, br. m), 3.78 (1H, m), 3.72

(1H, m), 3.68 (1H, dq, J 9.2 and 6.0 Hz), 3.54 (3H, overlapping), 3.35 (2H, overlapping), 3.25 (1H, dd, J

9.3 and 9.3 Hz), 3.03 (1H, dd, J 7.0 and 6.5 Hz), 2.96 (1H, d, J 9.4 Hz), 2.61 (1H, dd, J 15.7 and 3.6 Hz), 2.34 (3H, s), 2.21 (1H, dd, J 15.7 and 7.1 Hz), 1.98 (1H, br.), 1.80 (1H, m), 1.72 (1H, br. dd), 1.55 (1H, m), 1.44 (1H, m), 1.21 (3H, S), 1.21 (3H, d, J 6.0 Hz), 1.10 (3H, d, J 6.1 Hz), 0.94 (3H, d, J 6.6 Hz), 0.92 (3H, d, J 6.6 Hz) ppm.

Acid Hydrolysis (6MHCl/110°/18hrs/N₂/sealed tube) gives aspartic acid and N-methyl leucine. Physical and spectroscopic properties of MM 49721 after drying over P₂O₅:

FAB-MS (Glycerol/thiogycerol/trifluoracetic acid)

MH⁺ 1526±1

Molecular Weight: 1525

Molecular Formula: C₇₂H₈₇N₉O₂₈

UV (H₂0) λ_max 280nm (ε 5844) IR (KBr) 1653, 1587, 1500 cm^-1

Example 5

Preparation of MT 49729 (MM 47761 pseudoaglycone)

A stirred solution of MM 47761 (l.OOg) in 5M hydrochloric acid (20ml) was heated to reflux for 5 mins. The cooled mixture was then diluted with propanol and the solvents removed under reduced pressure.

The resulting solid was then dissolved in water (200ml) and the pH of the solution adjusted to 6.0 by addition of saturated sodium hydrogen carbonate solution. D-Ala-D-Ala sepharose affinity resin (40-50ml settled resin) was added and the suspension stirred for 30mins. The resin was collected by filtration and washed with distilled water (100ml). The glycopeptide was removed from the resin by suspension of the resin in 50% acetonitrile/50% 0.2M ammonium hydroxide solution (100ml).

After stirring for 5 mins the resin was again collected by filtration and the eluent evaporated to dryness under reduced pressure. This elution step was repeated a further four times and the combined eluents provided 0.70g (87%) of MT 49729. FAB mass spectroscopy indicated a MW of 1251.

Reference Example

Preparation of affinity adsorbent

The N-hydroxysuccinimide ester of 6-aminohexanoic acid Sepharose 4B (60g) was placed on a glass scinter and washed with ImM hydrochloric acid solution (2L) under suction. The wet cake was then added to a solution of D-alanyl-D-alanine (1.5g) in 0.1M sodium bicarbonate solution (60ml) and occasionally shaken over the next hour. The suspension was filtered under suction and the residue suspended in 0.1M tris

(hydroxymethyl)aminomethane (TRIS) (100ml) for 1 hour and then refiltered through a glass sinter. The cake was washed successively with 0.1M sodium bicarbonate solution, 0.05M TRIS (containing 0.5M sodium chloride), 0.05M formate buffer at pH 4.0 (containing 0.5M sodium chloride) and finally distilled water. The affinity resin was then stored at 4°C in aqueous suspension.

CLASSIFICATION OF NCIB 12608

Methods used

The methods followed were those recommended by the International Streptomyces Project for the characterization of Streptomyces species [Shirling, E.B. and Gottlieb, D. "Methods for the characterization of Streptomyces species" Int. J. Syst. Bacteriol.,16: 313-340 (1966)] and those recommended for the characterization of Amycolata and Amycolatopsis species [Lechevalier, M.P.,

Prauser, H., Labeda, D.A. and Ruan, J.-S. "Two new genera of nocardioform actinomycetes: Amyolata gen, nov. and Amycolatopsis gen, nov." Int. J. Syst. Bacteriol. 36: 29-37 (1986)].

The isomers of diaminopimelic acid (DAP) and the carbohydrates in hydrolysates of whole cells were established by Thin Layer Chromatography (TLC) using the methods described by Komagata and Suzuki [Komagata, K. and Suzuki, K.-I. "Lipid and Cell-Wall Analysis in Bacterial Systematics" Methods in Microbiology, 19: 161-207 (1987)]. Mycolic acids were determined by the methods described by Minnikin et al. [Minnikin, D. E., Hutchinson, I.G. and Caldicott, A.B. "Thin Layer Chromatography of Methanolysates of Mycolic Acid Containing Bacteria" J. Chromatogr. 188: 221-233 (1980)]. RESULTS.

1. Cultural Characteristics:

Culture NCIB 12608 grew well on all the growth media recommended by the ISP, forming well developed, cream to pale brown substrate mycelium and abundant white aerial mycelium. No soluble pigments were detected on any of the media used. The cultural characterisitcs of NCIB 12608 on various media are summarised in Table 1.

2. Chemical Characteristics:

Hydrolysed whole-cells of the culture NCIB 12608 contained the meso isomer of diaminopimelic acid. Arabinose and Galactose were the major sugars present while ribose was detected in minor quantities. Mycolic acids were not present. These results indicate that culture NCIB 12608 has a cell-wall type IV with type A sugar pattern (Lechevalier et al, 1986). However, lack of mycolic acids places this culture firmly outside the genus Nocardia sensu stricto.

3. Physiological characterisitcs:

Key physiological characterisitcs of culture NCIB 12608 and Amycolatopsis orientalis ATCC 19795, listed in Table 2, show that these two cultures share many characteristics. Identification of NCIB 12608:

Based on key chemical, physiological and morphological features, culture NCIB 12608 is identified as a strain of Amycolatopsis orientalis. Although NCIB 12608 differs from the type strain of Amycolatopsis orientalis (ATCC 19795) in some tests, the differences are relatively minor and thus, culture NCIB 12608 is identified as a new strain of the species Amycolatopsis orientalis.

Table 1:

Growth characteristics of NCIB 12608 after 14 days at 28°C

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

MEDIUM GROWTH AERIAL MYCELIUM SUBSTRATE SOLUBLE Formation Colour MYCELIUM PIGMENT - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

ISP 2 Good Good White Cream None

ISP 3 Good Fair White White/ None cream

ISP 4 Good Good White Cream None

ISP 5 Good Good White Cream None

ISP 6 Fair Poor White White/ None cream

ISP 7 Good Good White Cream/ None white

Nutrient Good Fair White Cream None Agarr

Bennetts Good Good White Cream None Agar

Czapek- Good Fair White Cream/ NoneDox Agar white - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Physiological characterisitics of NCIB 12608 after 14 days at 28°C

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - NCIB Amycolatopsis

CHARACTERISTICS 12608 orientalis ATCC 19795 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Decomposition of:

Adenine - -

Casein + +

Hypoxanthine - +

Tyrosine + +

Xanthine + + Production of;

Nitrate reductase +/- +

Amylase + -

Urease + +

Melanin - -

Esculinase + + Decarboxylation of:

Benzoate - -

Citrate - +

Mucate - + -

Malate + Growth in the presence of:

Lysozyme (500 u./ml) + -

Salicylate + -

NaCl (5%, w/v) + +

Rifampicin (50 ug/ml) - + Growth at:

28 C + +

37 C + -

45 C - - Utilization of carbohydrates as sole carbon sources:

Adonitol + +

Arabinose + +

Dextrin +/- +

Erythritol + +

Galactose + +

Inositol + +

Lactose + +

Maltose - +

Mannitol + +

Melibiose - +

Raffinose + -

Rhamnose - +

Salicin + +

Sorbitol - -

Sucrose + +

Trehalose + +

Xylose + + (Control : no sugar) - -

Key: +/- = variable result; ND = Not determined ^•

Claims

1. A compound of formula (I), or a pharmaceutically acceptable derivative thereof:

wherein R is chlorine or hydrogen.

2. A process for the production of a compound of formula (I) as defined in claim 1 , which process comprises cultivating Amycolatopsis orientalis NCIB 12608 or a mutant thereof and subsequently isolating the compound of formula (I) or a derivative thereof from the culture.

3. Amycolatopsis orientalis NCIB 12608 or a mutant thereof, in biologically pure form.

4. A process for the preparation of a compound of formula (II):

wherein R is as defined in claim 1, which process comprises acid catalysed hydrolysis of a compound of formula (I) as defined in claim 1.

5. A pharmaceutical composition comprising a compound according to claim 1 together with a pharmaceutically acceptable carrier or excipient.

6. A method of treating bacterial infections in animals including humans, which comprises administering thereto an effective non-toxic amount of a compound according to claim 1 or a composition according to claim 5.

7. A compound according to claim 1, for use in therapy.

8. A compound according to claim 1, for use in the treatment of bacterial infections in animals includinghumans.

9. Use of a compound according to claim 1 in the manufacture of a medicament for use in the treatment of bacterial infections in animals including humans.