Increased rate of hepatic glucose output is a general feature of diabetes mellitus. In particular, there is a strong correlation between fasting plasma glucose level in non- insulin dependent diabetes mellitus (NIDDM) and hepatic glucose output. The two pathways by which glucose is produced in the liver are gluconeogenesis and glycogenolysis. The terminal steps of both pathways is catalyse by the microsomal glucose-6-phosphatase, a key enzyme in the homeostatic regulation of blood glucose levels. The level of this enzyme has also been known to be elevated in both experimental and pathological conditions of diabetes. Interference with this enzyme system should, therefore, result in a reduced hepatic glucose production.

Hepatic glucose-6-phosphatase is a multicomponent system comprised of atleast three functional activities : a glucose-6-phosphate translocase (T1), a glucose-6-phosphate phosphohydrolase and a phosphate/pyrophosphate translocase (T2). The glucose-6- phosphate translocase facilitates transport of glucose-6-phosphate into the lumen of the endoplasmic reticulum (ER). The phoshohydrolase, with its active site situated on the lumenal surface of the ER, hydrolyses glucose-6-phosphate and releases glucose and phosphate into the lumen. While the efflux of phosphate is facilitated by the phosphate/pyrophosphate translocase, the exact mechanism of glucose efflux is still not clear.

The high degree of substrate specificity of glucose-6-phosphate translocase makes this a potential target for pharmacological intervention in the treatment of diabetes mellitus.

Thus, amongst physiologically occurring sugar phosphates. only glucose-6-phosphate

is transported by the translocase. In contrast, the phosphatase is non-specific and is known to hydrolyse a variety of organic phosphate esters.

A series of non-specific inhibitors of glucose-6-phosphatase has been described in the literature, e. g. phlorrhizin [J. Biol. Chem., 242,1955-1960 (1967)], 5,5'-dithio-bis-2- nitrobenzoic acid [Biochem. Biophys. Res. Commun., 48,694-699 (1972)], 2,2'- diisothiocyanatostilbene and 2-isothiocyanato-2'-acetoxystilbene [J. Biol. Chem., 255, 1113-1119 (1980)]. The first therapeutically utilizable inhibitors of the glucose-6- phosphatase system are proposed in European Patent Nos. 0587087 (Application No.

93114260.8) and 0587088 (Application No. 93114261.6). Kodaistatin A [Indian Patent Application No. 69/BOM/97 dt. Feb. 4,1997 ; EP Appl. No. 97106453.0 dt. April 18, 1997] and Kodaistatins B-D [Indian Patent Application No. 562/BOM/97 dt. Sept. 26, 1997] are the first glucose-6-phosphate translocase inhibitors from microbial sources.

According to one object of the invention, there is provided a compound with the moiecular formular C28H8011 (L 970885) obtainable from from an Actinomycete sp (culture number HIL-000337). L 970885 may be described as a glucose-6- phosphate translocase inhibitor.

L 970885 has a hitherto unreported new structure belonging to anthraquinone class of compounds. A chemical abstract literature search using search keys of the molecular formula established L 970885 to be a new compound. No other compound represented the structural features of L 970885.

The microorganism, culture number HIL-000337 used for the production of L970885 was isolated from a soil sample collecte from Rahne Fall, Madhya Pradesh, India.

The microorganism HIL-000337 belongs to the order of Actinomycetales and has been deposited with the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany with an accession number DSM No. 11992.

Therefore, another object of the present invention is to provide a process for the production of a new compound named L 970885 from culture number HIL-000337,

its mutants and variants. The said process comprises cultivation of culture H) L- 000337, its mutants and variants, under aerobic conditions in a nutrient medium containing sources of carbon and nitrogen, nutrient inorganic salts followed by isolation and purification of the said compound from the culture filtrate.

Further aspects of the invention include: pharmaceutical compositions containing the compound L 970885 or chemical derivatives thereof together with a pharmaceutically tolerated auxiliary and/or excipient, the use of the compound L 970885 or chemical derivatives thereof for the manufacture of a medicament for the treatment and/or prophylaxis of diabetes mellitus and the Actinomycete sp. culture number HIL-000337 (DSM 11992) and ist mutants and/or variants.

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

Cultivation of culture No. HIL-000337 may be carried out at temperatures between 25-30°C and pH between 6.0 and 8.0. Preferably culture No. HIL-000337 is cultivated at 27°C ( 1 °C) and pH 7.0.

The fermentation is preferably carried out for 40 to 70 hours when an optimal yield of L 970885 of the present invention is obtained. It is particularly preferred to carry out the fermentation for 60-70 hours under submerged conditions in shake flasks and 43-48 hours in laboratory fermenters. If desired, Desmophene (Polypropylene oxide) may be used as an antifoam agent in the fermenters. The progress of fermentation and formation of L 970885 can be detected by measuring the inhibition

of glucose-6-phospate activity in untreated and triton X-1009 disrupted rat liver microsomes in microtitre plates at room temperature using a colorimetric assay as described in Methods in Enzymology, 174,58-67 (1989) with some modifications. In the resulting culture broth, L 970885 is present only in the culture filtrate. Thus, it can be recovered by extraction of the culture filtrate 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),"Amberlite XAD (Rohm and Hass Industries U. S. A.) activated charcoal or ion exchange chromatography at pH 5-8. The preferred method is adsorption over"Diaion HP-20"followed by desorption of the compound using eluants such as water, methanol, acetone or acetonitrile 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: normal phase chromatography (using alumina or silica gel as stationary phase and eluents such as ethyl acetate, chloroform, methanol or combinations thereof), reverse phase chromatography (using reverse phase silica gel like dimethyl- octadecylsilyisilica gel, also called RP-18 or dimethyloctylsilylsilica 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 or acetone, or combinations of these solvents), gel permeation chromatography using resins such as"Sephadex LH-20@" (Pharmacia Chemical Industries, Sweden), TSKgel Toyopearl HW-40F (TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol, chloroform or 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 and chloroform. These techniques may be used repeatedly or a combination of the different techniques may be used. The preferred method is chromatography over Sephadex LH-20 followed by preparative HPLC over reverse phase modified silica gel (RP-18).

L 970885 can be administered orally, intramuscularly or intravenously.

Pharmaceuticais which contain L 970885 or in combinations as the active substance is subject of the present invention also. They can be prepared by mixing the active compound with one or more pharmacologically tolerated auxiliaries and/or excipients such as, for example, fillers, emulsifiers, lubricants, masking flavours, colorants or buffer substances, and converted into a suitable pharmaceutical form such as, for example, tablets, coated tables, capsules, granules, powders, mulsions, 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 suspension 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.

The dosage of the active compound L 970885 to be administered and the frequency of administration depend on the potency of the active compound and additionally on the nature and severity of the disease to be treated and on the sex, age, weight and individual responsiveness of the mammal to be treated. On an average, the daily dose of the compound L 970885 in a human patient of about 75 kg weight is at least 1 mg to at most 500 mg, preferably from about 10 mg to 250 mg, it being possible, according to requirement, in several doses per day, and also, where appropriate, to be lower or higher.

The compound L 970885 may be converted into its pharmaceutically acceptable salts and derivatives like esters and ethers. Salts like sodium may be prepared by treating the active compound L 970885 with the corresponding bases. Esters may be prepared by reacting the compound L 970885 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 literature [Advanced Organic Synthesis, 4th Edition, J. March, John Wiley & Sons, 1992].

Ethers may be prepared from L 970885 by reaction with alkylating agents under basic conditions. Other methods of preparation of ethers are given [Advanced Organic Synthesis, 4th Edition, J. March, John Wiley & Sons, 1992].

The following examples are illustrative of the present invention but not limitative of the scope thereof : Example 1 Isolation of the culture HIL-000337 from soil (a) Composition of nutrient isolation medium: Corn starch : 10.0 g Casein: 1.0 g Peptone: 1.0 g Yeast extract: 1.0 g K2HP04: 0.5 g Agar powder: 13.0 g Demineralized water: 1.0 litre pH: 7.5 (b) Soil plating and isolation: 10 g of soif collecte from Rahne Fall, Madhya Pradesh, India was added to 90 ml of sterilized demineralized water in 250 ml Erlenmeyer flask which was then shaken for 2 hours on a rotary shaker (220 rpm). The above soil suspension was then serially diluted in steps of 10 upto 10-5. From the last dilution, 1 ml of suspension was placed at the centre of a sterile glass petri plate (15 cms diameter) in which was then poured approximately 50 ml of the above isolation medium supplemented with 75 sig/ml of cycloheximide, 5, ug/ml of ampicillin and 25 lg/ml of framycetin. The medium was cooled to 45°C before pouring and the plate swirled thoroughly. The mixture of soil suspension and medium was allowed to settle and incubated at 30°C ( 1°C) for 7 days. The petri plate was periodically observed and the culture No.

HIL-000337 was isolated from amongst the growing microorganisms.

Example 2 Maintenance of the culture HIL-000337 Culture No. HIL-000337 was maintained on the following medium : Malt extract: 10.0 g Yeast extract: 4.0 g Glucose: 4.0 g Agar powder: 13.0 g Demineralized water: 1.0 litre pH: 7.0 After dissolving the above mentioned ingredients throughly by heating, it was distributed in test tubes and then sterilized at 121°C for 20 minutes. The test tubes were then cooled and allowed to solidify in a slanting position. The agar slants were streaked with the growth of the culture No. HIL-000337 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.

Example 3 Fermentation of culture HIL-000337 in shake flasks Composition of seed medium: Glucose: 15.0 g Soyabean meal: 15.0 g Corn steep liquor: 5.0 g NaCI: 5.0 g CaCO3: 2.0 g Demineralized water: 1.0 litre pH : 7.0

The above seed medium was distributed in 80 mt amounts in 500 ml Erlenmeyer flasks and autoclave at 121° C for 20 minutes. The flasks were cooled to room temperature and each flask was then inoculated with a loopful 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 OC) to give seed culture.

Composition of the production medium Glucose: 20.0 g Soyabean meal: 10.0 g CaCO3: 0.2 g Cobalt chloride: 0.001 g Demineralized water: 1.0 litre pH: 7.0 The production medium was distributed in 60 ml amounts in 500 ml Erlenmeyer flasks and autoclave at 121°C for 20 minutes. The flasks were cooled to room temperature and then inoculated with the above mentioned seed culture (1% v/v).

The fermentation was carried out on a rotary shaker at 240 rpm and at a temperature of 27°C ( 1 OC) for 66 hours.

The production of L 970885 was monitored by measuring the inhibition of glucose-6- phosphate translocase. After harvesting, the culture broth was centrifuged and L 970885 was isolated from the culture filtrate and purified as described in Example 5.

Example 4 Fermentation of the culture No. HIL-000337 in fermenters Stage 1: Preparation of seed culture in shake flasks The seed medium of Example 3 was distributed in 150 ml amounts in 1 L Erlenmeyer flasks and autoclave for 20 minutes. The seed culture was grown in these flasks as described in Example 3.

Stage 2: Preparation of seed culture in fermenter 20 litres of the seed medium, as described in Example 3, in a 30 litre fermenter was sterilized in situ for 45 minutes at 121°C, cooled to 27° + 1 °C and seeded with 1.4 litres of the seed culture mentioned above.

The fermentation was run with the following parameters : Temperature: 27°C (+ 0. 5°C) Agitation 200 rpm Aeration 20 1 pm Harvest time: 24 hrs.

Stage 3: Large scale fermentation 250 litres of the production medium, as described in Example 3, in a 390 litre fermenter along with 50 ml of desmophene (Polypropylene oxide) as antifoam agent was sterilized in situ for 45 minutes at 121°C, cooled to 27° 1°C and seeded with 20 litres of the seed culture from Stage 2.

The fermentation was run with the following parameters : Temperature: 27°C (+ 0. 5°C) Agitation 100 rpm Aeration 120 1 pm Harvest time: 45 hrs.

The production of the compound was monitored by measuring the inhibition of glucose-6-phosphate translocase. When fermentation was discontinued, the pH of the culture broth was 6.0-7.0. The culture broth was centrifuged after harvesting and the glucose-6-phosphate translocase inhibitor L 970885 was isolated from the culture filtrate as described below in Example 5.

Example 5: Isolation and Purification of L 970885 from HIL 000337 Approximately 300 litres of culture broth was harvested and separated from the mycelium by centrifugation. L970885 was found to be present in culture filtrate. The culture filtrate (285 litres) was passed through a column of Diaion HP-20 (7.5 litres, 2.5 % v/v). The column was washed thorougly with demineralized water (25 litres) and then eluted with gradient of methanol in water. Fractions were collecte in 10 litre size. The active eluates (4 x 10 litres), obtained with 50% methanol in water were combined, concentrated under reduced pressure of 10-100 mm of Hg at 35 °C and lyophillized to yield the crude active material (160 g) showing an IC50 of 29 jg/m !.

The crude material was again passed through a Diaion HP-20 (2.5 litres) column.

The column was washed thorougly with demineralized water (10 litres) and then eluted with 50% methanol in water. The fractions were collecte in one litre size.

The active eluates were combined and concentrated under reduced pressure of 10- 100mm of Hg at 35 °C and lyophillized to obtain enriched material (40 g) having an IC5oof 20 rg/ml.

The above material was purified by passing through Sephadex LH-20 (2 litres) packed in glass column using demineralized water as the mobile phase. The flow rate was maintained at 1 ml/minute. The fractions were collecte in 25 ml. size. The active fractions were pooled, concentrated under reduced pressure of 10-100mm of Hg at 35 °C and lyophillized to obtain further enriched material (1.4 g) material having an ICso of 18, ug/ml.

The above material was further purified by passing through another Sephadex LH- 20 (800 ml) packed in glass column using demineralized water as the mobile phase.

The flow rate was maintained at 0.8 ml/minute. The fractions were collecte in 15 ml size. The active fractions were combined, concentrated under reduced pressure of 10-100 mm of Hg at 35 °C and lyophillized to obtain semipure material (200 mg) having an ICso of 15ag/ml.

The semipure material, thus obtained, was finally purified by preparative HPLC using LichroCART RP-18 column (250 x 10 mm). The mobile phase was 25% acetonitrile in 0. 1% trifluoracetic acid at a flow rate of 6 ml/min and detection at 210 nm. The pure compound, thus obtained, was 15 mg with an IC50 of 18 p. M.

The purity of the compound L 970885 was checked by HPLC (High Pressure Liquid Chromatography) on a LiChrocart (250 mm x 4 mm) RP Select B (5, u) column using a gradient of 0.1 % aqueous orthophospharic acid (pH 2.5) to CH3CN in 20 min at a flow rate of 1 ml/min and UV detection at 280 nm L 970885 inhibits potently the activity of rat liver microsomal glucose-6-phosphate translocase with an ICso of about 18 s1M.

The physico-chemical and spectral properties of L 970885 are summarized in Table 1.

Table 1 <BR> <BR> <BR> <BR> <BR> <BR> L 970885<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> Nature : Reddish brown solid Solubility: MeOH, DMSO and water Melting point: > 360° C [α]D: -490° (c 0.02, Water) HPLC RT: 10.63 min Fig. 1 of the accompanying drawings ESI-MS (Electrospray lonization: 529 (M-H)- Mass) Molecularformula: C28H18O11 UV: Fig. 2 of the accompanying drawings IR (KBr) cm~1: Fig. 3 of the accompanying drawings H NMR (300 MHz, DMSO-d6): Fig. 4 of the accopmanying drawings 13C NMR (300 MHz, DMSO-d6): Fig. 5 of the accompanying drawings BUDAPEST TREATY INTERNATIONALTHE RECOGNITION Or @E DEPOSIT OF MICROORGANIS@ : S FOR THE PURPOSES OF PATENT PItOCEnURE INTERNATIONAL FORM Hoechst Marion Roussel Deutschland GmbH 65926 Frankfurt/Main RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page 1. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the DEPOSITOR: Accession number given by the ° INTERNATIONAL DEPOSITARY AUTHORITY HIL 000337 DSM 11992 n. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I. above was accompanied by: () a scientific description (X) a proposed taxonomic designation (Mark with a cross where applicable). III. RECEIPT AND ACCEPTANCE This International Depositary Authority accepts the microorganism identified under 1. above, which was received by it on 19 9 8-0 2-10 (Date of the original deposit)'. IV. RECEIPT OF REQUEST FOR CONVERSION The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion). V. INTERNATIONAL DEPOSITARY AUTHORITY Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature (s) of person (s) having the power to represent the MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized official (s): Address: Mascheroder Weg lb D-38124 Braunschweig 40 Date: 1998-02-12 'Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired.

Form DSMZ-BP/4 (sole page) 0196