Title:  Mumbaistatin, a process for it's production and its use as a pharmaceutical

United States Patent:  6,297,043

Assignee:  Aventis Pharma Deutschland GmbH (Frankfurt am Main, DE)

Filed:  June 23, 1999

Foreign Application Priority Data:  Jun 24, 1998[EP] (98111636)

The present invention relates to a compound named Mumbaistatin, its pharmaceutically acceptable salts and derivatives, a process for its production, and its use as a pharmaceutical. Mumbaistatin is obtainable by cultivation of the microorganism HIL-008003 (DSM 11641). Mumbaistatin is a glucose-6-phosphate translocase inhibitor and can be used in the treatment of diabetes mellitus. The present invention further relates to a process for the production of Mumbaistatin, to the microorganism HIL-008003 (DSM 11641), to the use of Mumbaistatin and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular to their use in the treatment of diabetes mellitus, and to pharmaceutical compositions comprising Mumbaistatin or a pharmaceutically acceptable salt or derivative thereof.

Description of the Invention

The present invention relates to a compound named Mumbaistatin which is obtainable by cultivation of the microorganism HIL-008003 (DSM 11641), and to its pharmaceutically acceptable salts and derivatives. Mumbaistatin is a glucose-6-phosphate translocase inhibitor and can be used in the treatment of diabetes mellitus. The present invention further relates to a process for the production of Mumbaistatin, to the microorganism HIL-008003 (DSM 11641), to the use of Mumbaistatin and its pharmaceutically acceptable salts and derivatives as pharmaceuticals, in particular for their use in the treatment of diabetes mellitus, and to pharmaceutical compositions comprising Mumbaistatin or a pharmaceutically acceptable salt or derivative thereof.

Mumbaistatin has a hitherto unreported novel structure belonging to quinone class of compounds. A chemical abstract literature search using search keys of the molecular formula established Mumbaistatin to be a novel compound. No other compound represented the structural features of Mumbaistatin.

Mumbaistatin is obtainable by cultivation of a microorganism referred to as culture no. HIL-008003 or also as culture no. Y-9645974 (henceforth referred to as HIL-008003). This microorganism used for the production of Mumbaistatin was isolated from a soil sample collected from the Hiranyakeshi riverbed near Amboli, Maharashtra, India. The microorganism HIL-008003 has been identified as Streptomyces litmocidini. The microorganism was deposited on Jul. 4, 1997, with the German Collection of Microorganisms and Cell Cultures (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), Mascheroder Weg 1b, D-38124, Braunschweig, Germany and given the accession number DSM 11641.

Thus, the present invention further provides a process for the production of the novel compound named Mumbaistatin and its pharmaceutically acceptable salts and derivatives from Streptomyces species HIL-008003, its mutants and variants. The said process comprises cultivation of culture no. HIL-008003, its mutants or 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.

The nutrient medium preferably contains sources of carbon, nitrogen and nutrient inorganic salts and optionally trace elements. The carbon sources are, for example, starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose or galactose, preferably glucose. The sources of nitrogen are, for example, soybean meal, peanut meal, yeast extract, beef extract, peptone, tryptone, malt extract, corn steep liquor, gelatin or casamino acids, preferably soybean meal and corn steep liquor. The nutrient inorganic salts and trace elements are, for example, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, cobalt chloride, calcium chloride, calcium carbonate, potassium nitrate, ammonium sulfate or magnesium sulfate, preferably cobalt chloride and calcium carbonate.

Cultivation of culture no. HIL-008003 is usually carried out at temperatures between 25-30.degree. C. and pH between 6.0 and 8.0. Preferably culture no. HIL-008003 is cultivated at 27.degree. C. (.+-.1.degree. C.) and pH 7.0.

The fermentation of HIL-008003 is preferably carried out for about 40 to 70 hours in order to obtain an optimal yield of Mumbaistatin. It is particularly preferred to carry out the fermentation for about 40 to 48 hours under submerged conditions, for example, in shake flasks as well as in laboratory fermenters. If desired, DESMOPHEN.RTM. (polypropylene oxide) may be used as an antifoam agent in the fermenters. The progress of fermentation and formation of Mumbaistatin can be detected by measuring the inhibition of glucose-6-phospate translocase activity in untreated and TRITON X-100.RTM. disrupted rat liver microsomes in microtiter plates at room temperature using a calorimetric assay as described in Methods in Enzymology 174, 58-67 (1989) with some modifications. In the resulting culture broth, Mumbaistatin is present primarily in the culture filtrate and can thus be recovered by extraction of the culture filtrate with a water immiscible solvent such as, for example, ethyl acetate, dichloromethane, chloroform or butanol at pH 5-8, or by hydrophobic interaction chromatography using polymeric resins such as DIAION.RTM. HP-20 (Mitsubishi Chemical Industries Limited, Japan), AMBERLITE.RTM. XAD (Rohm and Haas Industries, U.S.A.), or activated charcoal, or by ion exchange chromatography at pH 5-8. The preferred method is adsorption over DIAION.RTM. HP-20 followed by desorption of the compound using eluants such as water, methanol, acetone, acetonitrile, n-propanol, isopropanol or combinations thereof. Concentration and lyophilization of the active eluates gives the crude compound.

The crude material can be further purified by using any of the following techniques: by normal phase chromatography using alumina or silica gel as stationary phase and eluants such as ethyl acetate, chloroform, methanol or combinations thereof; by reverse phase chromatography using reverse phase silica gel like dimethyloctadecylsilylsilica gel, also called RP-18, or dimethyloctylsilylsilica gel, also called RP-8, as stationary phase and eluants such as water, buffers such as phosphate, acetate, citrate (pH 2-8), and organic solvents such as methanol, acetonitrile, acetone, tetrahydrofuran or combinations of these solvents; by gel permeation chromatography using resins such as SEPHADEX.RTM. LH-20 (Pharmacia Chemical Industries, Sweden), TSKgel TOYOPEARL.RTM. HW-40F (TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol, chloroform, acetone, ethyl acetate or combinations of these solvents or SEPHADEX.RTM. G-0 and G-25 in water; or by counter-current chromatography using a biphasic eluant system made up of two or more solvents such as water, methanol, ethanol, isopropanol, 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 TOYOPEARL.RTM. followed by reverse phase modified silica gel (RP-18).

The compound Mumbaistatin 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 invention also covers all salts and derivatives of Mumbaistatin which themselves are not suitable for use as pharmaceuticals but which can be used as intermediates in the preparation of pharmaceutically acceptable salts and derivatives. The invention covers Mumbaistatin and all its salts and derivatives in all their stereoisomeric forms and tautomeric forms. 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 Mumbaistatin with suitable sodium or potassium bases.

Esters may be prepared, for example, by reacting Mumbaistatin with carboxylic acids in the presence of reagents such as dicyclohexylcarbodiimide (DCC), or by treating the compound with acylating agents such as acid chlorides. Other methods of preparation of esters are given in the literature, for example in J. March, Advanced Organic Synthesis, 4th Edition, John Wiley & Sons, 1992.

The esters of Mumbaistatin covered by the present invention include intramolecular esters, i.e. lactones. A compound specifically mentioned as being a subject of the present invention is the compound which has been named L970860 and the pharmaceutically acceptable salts and derivatives thereof, in all their stereoisomeric and tautomeric forms. Compound L970860 is a lactone obtainable by treatment of Mumbaistatin with trifluoroacetic acid. It has the molecular formula C₂₈ H₁₈ O_11.

Ethers may be prepared, for example, from Mumbaistatin by reaction with alkylating agents under basic conditions. Other methods of preparation of ethers are given in the literature, for example in Advanced Organic Synthesis, 4th Edition, J. March, John Wiley & Sons, 1992.

Mumbaistatin potently inhibits rat liver microsomal glucose-6-phosphate translocase. The results obtained in pharmacological tests are given below. Mumbaistatin and its pharmaceutically acceptable salts and derivatives are therefore useful as pharmaceutically active ingredients, in particular in the treatment of diabetes mellitus, and more generally in the treatment or prophylaxis of conditions which are caused by or associated with an elevated activity of glucose-6-phosphate translocase, or of conditions in which it is intended to reduce glucose-6-phosphate translocase activity. Mumbaistatin 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 enteral or parenteral administration. Accordingly, the present invention also relates to Mumbaistatin and its pharmaceutically acceptable salts and derivatives for use as pharmaceuticals and to the use of Mumbaistatin and its pharmaceutically acceptable salts and derivatives for the production of medicaments for reducing glucose-6-phosphate translocase activity, in particular for the production of medicaments for the treatment of diabetes mellitus. The present invention further relates to pharmaceutical compositions that contain an effective amount of Mumbaistatin and/or one or more pharmaceutically acceptable salts and/or derivatives thereof together with a pharmaceutically acceptable carrier.

Mumbaistatin can be administered orally, intramuscularly, intravenously or by other modes of administration. Pharmaceutical compositions, which contain Mumbaistatin or a pharmaceutically acceptable salt or derivative thereof singly or in combinations, can be prepared according to standard techniques by mixing the compound(s) with one or more pharmacologically acceptable excipients and/or auxiliaries such as, for example, fillers, emulsifiers, lubricants, masking flavors, colorants or buffer substances, and converting the mixture into a suitable pharmaceutical form such as, for example, tablets, coated tablets, capsules or a suspension or solution suitable for enteral or parenteral administration.

Examples of auxiliaries and/or excipients that may be utilized are starch, tragacanth, lactose, talc, agar-agar, polyglycols, ethanol and water. Suspensions or solutions in water are suitable and preferred for parenteral administration. It is also possible to administer the active substances as such, without vehicles or diluents, in a suitable form, for example, in capsules. Pharmaceutical compositions comprising Mumbaistatin or a pharmaceutically acceptable salt or derivative thereof may also contain other pharmaceutically active ingredients.

As 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.

Apart from use as pharmaceutically active ingredients and as intermediates in the production of derivatives, Mumbaistatin and its salts and derivatives can also be employed as auxiliaries for diagnostic purposes, for example in in vitro diagnoses, and for research purposes in biochemical investigations in which an inhibition of glucose-6-phosphate translocase is desired.

Claim 1 of 13 Claims

We claim:

1. Mumbaistatin, comprising a compound of molecular formula C₂₈ H₂₀O₁₂, further defined by an ¹H NMR spectrum as shown in FIG. 9, and a ¹³ C NMR spectrum as shown in FIG. 10, or a pharmaceutically acceptable salt, ester, or ether of said compound.

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