Title of Invention

VANCORESMYCIN, A PROCESS FOR ITS PRODUCTION AND ITS USE AS A PHARMACEUTICAL

Abstract

ABSTRACT " VANCORESMYCIN AND A PROCESS FOR PRODUCING THE SAME" IN/PCT/2001/609/CHE This invention relates to a compound named Vancoresmycin, which is obtainable by cultivation of the microorganism HIL-006734 (DSM 12216), and to its pharmaceutically acceptable salts. The present invention further relates to a process for the production of Vancoresmycin, to the microorganism HIL-006734 (DSM 12216), to the use of Vancoresmycin and its pharmaceutically acceptable salts as pharmaceuticals, in particular to their use as antibiotics, and to pharmaceutical compositions comprising Vancoresmycin or a pharmaceutically acceptable salt thereof

Full Text

This invention relates to a compound named Vancoresmycin, which is obtainable by cultivation of the microorganism HIL-006734 (DSM 12216), and to its pharmaceutically acceptable salts and derivative?. The present invention further relates to a process for the production of Vancoresmycin, to the microorganism HIL-006734 (DSM 12216), to the use of Vancoresmycin and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular to their use as antibiotics, and to pharmaceutical compositions comprising Vancoresmycin or a pharmaceutically acceptable salt or derivative thereof. Methicillin resistant Staphylococcus aureus (MRSA) infections are known to be predominant in several infectious conditions like wounds and bums. Vancomycin and teicoplanin, belonging to the glycopeptide class, are the only two antibiotics clinically used for the treatment of MRSA infections. However, lue to the recent emergence of vancomycin- and teicoplanin-resistant strains, hese infections are reported to have become menacing and fatal. An htensive search for a structurally different class of compounds active against hese vancomycin and teicoplanin resistant strains has, therefore, been pitiated. For instance, methylsuifomycin I, a cyclic thiopeptide, has been escribed earlier [European Patent Publication No. 0818539 filed July 11, 1996] s an antibiotic active against vancomycin- and teicoplanin-resistant strains. has now been found that a novel compound named Vancoresmycin has ptibtotic activity. The present invention thus relates to Vancoresmycin, a impound of the fomnula: and to its pharmaceutically acceptable salts and derivatives, such as esters, ethers and obvious chemical equivalents, including all stereoisomeric forms and all tautomeric forms. Vancoresmycin has the molecular formula C71H126N2O21 (MW1343.80) and may be characterized by any one or more of its physico-chemical and spectral properties given below, such as its 1H NMR spectroscopic data and itsC NMR spectroscopic data, both provided in Table 2. Vancoresmycin may be described as a new antibiotic active against vancomycin and teicoplanin resistant strains. Vancoresmycin has a hitherto unreported new structure with a tetramic acid moiety bearing at the 3-position an acyl substituent with a highly oxygenated long alkyl chain substituted with an amino sugar. A chemical abstract literature search established Vancoresmycin to be a new compound. No other compound represented the structural features of Vancoresmycin. Vancoresmycin is obtainable by cultivation of a microorganism refen'ed to as culture no. HIL-006734 (henceforth referred to as HIL-006734). This microorganism used for the production of Vancoresmycin was isolated from a soil sample collected from National Park, Borivli, Mumbai, India. The microorganism HIL-006734 belongs to the order of Actinomycetales, genus Amycolatopsis, and has been deposited on 4 June 1998 with the German Collection of Microorganisms and Cell Cultures (DSM2 - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), Braunschweig, Germany and has been given the accession number DSM No. 12216. Thus, the present invention further provides a process for the production of the novel compound named Vancoresmycin from amycolatopsis species HIL-006734, its mutants and variants, under aerobic conditions in a nutrient medium containing one or more sources of carbon and one or more sources of nitrogen and optionally nutrient inorganic salts and/or trace elements, followed by isolation of the said compound and purification in a customary manner. Mutants and variants of the microorganism ST101170 may also be able to synthesize the compound according to the present invention. Such mutants may be produced in a known manner by physical means, for example irradiation such as with ultraviolet- or X-rays, or chemical mutagens, such as ethylmethylansulfonate (EMS), 2-hydroxy-4-methoxy-ben2ophenone (MOB) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The screening for suitable mutants and variants which can produce the compound according to the invention can be confirmed by detennination of the biological activity of the compounds accumulated in the culture broth, for example by testing the antibacterial action. The nutrient medium preferably contains sources of carbon, nitrogen and nutrient inorganic salts. The carbon sources are, for example, starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose or galactose, preferably starch. The sources of nitrogen are, for example, soyabean meal, peanut meal, yeast extract, beef extract, peptone, malt extract, corn steep liquor, gelatin or casamion acids, preferably peptone and yeast extract. The nutrient inorganic salts are, for example sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, calcium chloride, calcium carbonate, potassium nitrate, ammonium sulphate or magnesium sulphate, preferably calcium carbonate, sodium chloride and magnesium sulphate. The cultivation of HIL-006734 may be carried out at temperatures between 25-35°C and pH between 6.0 and 8.0. Preferably HIL-006734 is cultivated at 30°C (±1°C) and pH7.0. The cultivation of mL-006734 is preferably carried out for 60-96 hours when an optimal yield of the antibiotic of the invention is obtained. It is particularly preferred to carry out the cultivation by fermentation for 68-96 hours under submerged conditions for example in shake flasks as well as in laboratory fermenters. The progress of fermentation and formation of the Vancoresmycin can be detected by High Pressure Liquid Chromatography (HPLC) and by measuring the bioactivity of the culture broth against Staphylococci and Enterococci species by the known microbial agar plate diffusion assay method. The preferred culture is Staphylococcus aureus 3066, which is a resistant strain to methicillin, a p-lactam antibiotic reported in the literature and Entrococcus faecium ( E. faecium VR-1), which is resistant to vancomycin. In the resulting culture broth Vancoresmycin is present in the culture filtrate as well as in mycelium and can be isolated using known separation techniques. Thus, it can be recovered from the culture filtrate by extraction with a water immiscible solvent such as ethyl acetate, dichloromethane, chloroform or butanol at pH 5-8 or by hydrophobic interaction chromatography using polymeric resins such as "Diaion HP-20®" (Mitsubishi Chemical Industries Limited, Japan), "Amberiite XAD®" (Rohm and Hass Industries U.S.A.), activated charcoal or ion exchange chromatography at pH 5-8. The preferred method is extraction with ethyl acetate. The active material can also be recovered from mycelium by extraction with a water miscible solvent such as methanol, acetone, acetonitrile, n-propanol or iso-propanol or a water immiscible solvent such as ethyl acetate, dichloromethane, chlorofonn or butanol at pH 5-8 and the preferred method is the extraction with ethyl acetate. Concentration and lyophilization of the extracts gives the active crude material. The antibiotic Vancoresmycin of the present invention may, for example, be recovered from the crude material as follows : By fractionation using any of the following techniques: normal phase chromatography (using alumina or silica gel as stationary phase and eluents such as petroleum ether, ethyl acetate, methylene chloride, acetone, chloroform,' methanol or combinations thereof and additions of amines such as NEta), reverse phase chromatography (using reverse phase silica gel like dimethyloctadecylsilylsiiica gel, also called RP-18 or dimethyloctylsilyl silica gel also called RP-8 as stationary phase and eluents such as water, buffers viz. phosphate, acetate, citrate (pH 2-8) and organic solvents such as methanol, acetonitrile, acetone, tetrahydrofuran or combinations of these solvents), gel permeation chromatography using resins such as ®Sephadex LH-20 (Pharmacia Chemical Industries, Sweden), TSKgelToyopeari HW (TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol, chloroform, acetone, ethyl acetate or their combinations or ®Sephadex G-10 and G-25 in water; or by counter-current chromatography using a biphasic eluent system made up of two or more solvents such as water, methanol, ethanol, Iso-propanol, n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride, chloroform, ethyl acetate, petroleum ether, benzene and toluene. These techniques may be used repeatedly or a combination of the different techniques may be used. The preferred method is chromatography over reverse phase silica gel (RP-18). The compound Vancoresmycin may be converted into pharmaceutically acceptable salts and derivatives, like esters and ethers and other obvious chemical equivalents, which are all covered by the present invention. The salts and derivatives can be prepared by standard procedures known to one skilled in the art. Salts like sodium and potassium salts, for example, may be prepared by treating Vancoresmycin with suitable sodium or potassium bases. Esters and ethers may be prepared by the methods given in the literature, for example, in Advanced Organic Synthesis, 4" Edition, J. March, John Wiley & Sons., 1992. The amino group of the sugar moiety can be alkylated or acetylated e. g. with acid chlorides by standard procedures known to one skilled in the art. Chemical equivalents may be stable complexes with metal ions e.g. transition metals like La*, Sm*, Eu*, Gd*, which are typical for tetramic acid derivatives and may be prepared by the methods given in the literature (K.Tanaka et. al., Chem. Phamn. Bull. 1979, 27, 1901. K. Matsuo, Chem. Phann. Bull. 1980, 28. 2494). The double bonds of the alkyl side chain may be reduced by the methods given in the literature, for example in P. N. Rylander, "Hydrogenation Methods", Academic Press, New York (1985). Chpt. 2 or may be hydrohalogenated by methods described by H.O. House in "Modern Synthetic Reactions". W.A. Benjymin, Inc.. New York (1972), pp 446-452. Hydroxylated derivatives may be produced by reaction of the double bonds with reagents like OSO4 as described in the literature e.g. in Chem. Rev. 1980, 80, 187. Derivatives may also be fomied by conversion of the double bonds into epoxides by oxidation e.g. with MCPBA like described in Advanced Organic Synthesis, 4"* Edition. J. March. John Wiley & Sons.. 1992. v'ancoresmycin has antibacterial activity. Minimum inhibitory concentrations of /ancoresmycin against a wide range of bacterial are given in Table 3 below, /ancoresmycin and its pharmaceutically acceptable salts and derivatives can be administered to animals, preferably to mammals, and in particular to humans as pharmaceuticals on their own, in mixtures with one another and in the form of pharmaceutical compositions which permit parenteral administration. Accordingly the present invention also relates to Vancoresmycin and its pharmaceutically acceptable salts and derivatives for use as pharmaceuticals and to the use of Vancoresmycin and its pharmaceutically acceptable salts and derivatives for the production of medicaments having antibacterial activity. The present invention further relates to phannaceutical compositions which contain an effective amount of Vancoresmycin and/or one or more phannaceuticaHy acceptable salts and/or derivatives thereof, together with a pharmaceuticaHy acceptable carrier. Vancoresmycin can be administered orally, intramuscularly, intravenously or by other modes of administration. Pharmaceutical compositions which contain Vancoresmycin or a phannaceutically acceptable salt or derivative thereof with other pharmaceutically active substances can be prepared by mixing the active compounds with one or more pharmacologically tolerated auxiliaries and/or excipients such as, for example, fillers, emulslfiers, lubricants, masking flavours, colorants or buffer substances, and converting the mixture into a suitable pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules, powders, emulsions, suspensions or solutions suitable for parenteral administration. Examples of auxiliaries and/or excipients which may be mentioned are tragacanth, lactose, talc, agar-agar, polyglycols, ethanol and water. Suitable and preferred for parenteral administration are suspensions or solutions In water. It is also possible to administer the active substances as such, without vehicles or diluents, in a suitable form, for example, in capsules. As is customary, the galenic formulation and the method of administration as well as the dosage range which are suitable in a specific case depend on the species to be treated and on the state of the respective condition or disease, and can be optimized using methods known in the art. On average, the daily dose of active compound in a patient of about 75kg weight is at least 0.001 mg to at most 10mg, preferably at most 10mg. The following are illustrative examples of the present invention but not limitative of the scope thereof: b) Soil plating and isolation 10 g of soil collected from National Park, Borivli, Mumbai, India were added to 90 ml of sterilized water in a 250 ml Erienmeyer flask which was shaken for 2 hours on a rotary shaker (220 rpm). The above soil suspension was serially diluted in steps of 10 up to 10*. From the last dilution, 1 ml of suspension was placed at the centre of a sterile glass petri plate(15 cms diameter) to which was poured approximately 50 ml of the above isolation medium supplemented with 25 pg/mi of amphotericin B as antifungal agent and cooled to 45°C and the plate swirled thoroughly. The mixture of soil suspension and medium was allowed to settle and incubated at 28°C (±1°C) for 7 days. The petri plate was periodically observed and HIL-006734 (culture no. Y-9439786) was isolated from amongst the growing microorganisms. EXAMPLE 2 Maintenance of the culture no. HIL-006734 Composition of maintenance medium After dissolving the ingredients thoroughly by heating, the resultant solution was distributed in test tubes and sterilized at 121°C for 20 mins. The test tubes were cooled and allowed to solidify in a slanting position. The agar slants were streaked with the growth of HIL-006734 by a wire loop and incubated at 28°C (±1°C) until a good growth was observed. The well grown cultures were stored in the refrigerator at +8°C. The above medium was distributed in 100 ml amounts in 300 ml Erienmeyer flasks and autoclaved at 121'C for 20 minutes. The flasks were cooled to room temperature and inoculated with the above mentioned agar slant. The incubation was earned out for five days on a rotary shaker at 180 rpm and 28'C. 1.5 ml of this culture was mixed with 1.5 ml glycerol (99 %) and stored at -20°C. EXAMPLE 3 Fennentation of the culture no. HIL-006734 in shake flasks The above medium was distributed in 100 ml amounts in 500 ml Erienmeyer flasks and autoclaved for 20 mins. The flasks were cooled to room temperature and each flask was inoculated with a ioopful of the above mentioned well grown culture of Example 2 and shaken on a rotary shaker for 72 hours at 240 rpm at 27°C (±1°C) to give seed culture. The production medium was distributed in 100 ml amounts in 500 ml Erienmeyer flasks and autoclaved at 121°C for 20 mins. The flasks were cooled to room temperature and inoculated with the above mentioned seed culture(2% v/v).The fermentation was carried out on a rotary shaker at 240 rpm and 27°C (±1°C) for 48 hours. The production of the antibiotic was determined by testing the bloactivity against S. aureus 3066 and Ent. faecium VR-1 using the well diffusion method in a known manner. EXAMPLE 4 Cultivation of the culture no. HIL-006734 in fennenters Preparation of seed culture in shake flasks The seed medium of Example 3 was distributed in 150 ml amounts in 1000 ml Erienmeyer flasks and autoclaved at 121 °C for 20 mins. The seed culture was grown in these flasks as described in Example 3. 20 litres of the production medium in 22 litre fermenter (in two fermenters) and 9 litres of the production medium in 12 litre fermenter (in two fermenters) along with 1ml(/10 litre fermenter) of ®Desmophen as antifoaming agent was sterilized in situ for 40 mins. at 121°C, cooled to 27°C {±°C) and seeded with 1.5 litre(/22 lit. fermenter) or 0.75 litre (/12 lit. fermenter) of the seed culture mentioned above. The production of the antibiotic was determined by testing the bioactivity against S. aureus 3066 and Ent. faecium VR-1 and HPLC analysis. The final pH of the culture broth was 7.0-7.5. The culture broth was harvested and centrifuged and the antibiotic was isolated and purified from the culture filtrate and the mycelium by the method described in Example 5 or 6. EXAMPLE 5 Isolation and purification of Vancoresmvcin cm) in methanol repeatedly and the active fractions were pooled and concentrated. This was further chromatographed on 'oyopearf TSK HW 40F column (6 cm x 35 cm) in methanol. The active fractions were pooled and concentrated under reduced pressure to get 3 g of semipure material. The final purification was done by preparative HPLC using the following conditions: The semi-pure material was finally purified by preparative HPLC on a 25 mm X 250 mm Hibar-Lichrospher RP-18 (10 p using 67.5:32.5 Methanol: Phosphate buffer (0.01M, pH 6.5) as the eluant at a flow rate of 23 ml/min and detection at 220 nm. The active eluates were pooled and concentrated under reduced pressure to remove the solvent and then desalted on ®Diaion HP-20 (50 ml) column, eluted with acetonitrile:water (80:20) and concentrated under reduced pressure and lyophilized to get 70 mg of pure compound. EXAMPLE 6 Isolation and purification of Vancoresmvcin The culture broth (200 litres) was harvested and centrifuged to separate the mycelium and the culture filtrate. The mycelium was exhaustively extracted with methanol (30 - 40 L) and the extract concentrated 1:10 to obtain a colorless precipitation which was filtered off to obtain 30 - 50 g of crude material. This material was further purified by HPLC: The active fractions were eluted after 125 min. The pooled fractions were concentrated under reduced pressure and freeze dried. The Vancoresmycin-containing fractions eluted after 32 min. The pooled fractions were concentrated under reduced pressure and freeze dried. The yield of the two purification columns'was "50 %. The physico-chemical and spectral properties of Vancoresmycin are given in Tables 1 and 2 and the minimum inhibitory concentrations (MIC) against various bacteria are listed in Table 3. Figures 1 and 2 are referred to jn Table 1. Figure 1 shows the detection of by High Pressure Liquid Chromatography, and Figure 2 shows the ultraviolet absorption spectrum of in methanol. TABLE 1 Liquid Chromatography) [Column : LiChrocart ™ (250 mm x 4 mm) RP Select B (5|J; Eluant: Gradient of 0.1 % aqueous orthophospharic acid (pH 2.5) to CH3CN in 20 min; Flow rate : 1 ml/min.; Detection : 220 nm] Fig. 1 of the accompanying drawings lonisation Mass) HR-FAB-MS (High 1343.89142 (M+H)* resolution Fast Atom [Calcd forC71H122N202i : Bombardment Mass) 1343.889915 (M+H)*] We Claim: 1. Vancoresmycin, a compound of the formula: and Its pharmaceuticaify acceptable salts. Vancoresmycin, a compound of the molecular formula C71H126N2O21, obtainable by cultivation of the microorganism Amycolatopsis species HIL-006734 (DSM 12216) or one of its mutants under aerobic conditions in a nutrient medium containing sources of carbon and nitrogen, followed by isolation and purification in a customary manner, and its pharmaceutically acceptable salts, A process for the production of Vancoresmycin or a salt thereof as claimed in claim 1 or claim 2, comprising cultivation of the microorganism Amycolatopsis species HIL-006734 (DSM 12216) or one of its mutants under aerobic conditions in a nutrient medium containing sources of carbon and nitrogen, followed by isolation and purification in a customary manner. 4. The process as claimed in claim 3, further comprising reacting Vancoresmycin with a suitable agent to form a pharmaceutically acceptable salt. 5. Amycolatopsis species HIL-006734 (DSM 12216) or one of its mutants. 6. ' Vancoresrnycin or a pharmaceutically acceptable salt thereof as claimed in claim 1 or claim 2 for use as a pharmaceutical, 7. A pharmaceutical composition, comprising an effective amount of Vancoresmycin or a pharmaceutically acceptable salt thereof as claimed in claim 1 or claim 2, and a pharmaceutically acceptable carrier. 8. Vancoresmycin or a pharmaceutically acceptable salt thereof as claimed in claim 1 or claim 2, for use as an antibiotic.

Documents:

in-pct-2001-0609-che abstract.pdf

in-pct-2001-0609-che claims-duplicate.pdf

in-pct-2001-0609-che claims.pdf

in-pct-2001-0609-che correspondence-others.pdf

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in-pct-2001-0609-che description (complete).pdf

in-pct-2001-0609-che drawings.pdf

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in-pct-2001-0609-che form-13.pdf

in-pct-2001-0609-che form-19.pdf

in-pct-2001-0609-che form-26.pdf

in-pct-2001-0609-che form-3.pdf

in-pct-2001-0609-che form-5.pdf

in-pct-2001-0609-che others.pdf

in-pct-2001-0609-che pct.pdf