AROMATIC DI-KETO DERIVATIVES, PROCESSES FOR THEIR PRODUCTION AND THEIR USE AS A PHARMACEUTICAL

Abstract: The present invention relates to new aromatic di-keto derivatives and to their pharmaceutically acceptable salts, esters, ethers and other obvious chemical equivalents. The derivatives are glucose-6-phosphate translocase inhibitors and can be used in the treatment of diabetes mellitus. The present invention further relates to a process for the production of the derivatives, to the use of the derivatives and their pharmaceutically acceptable salts, esters, ethers and other obvious chemical equivalents as pharmaceuticals, in particular to their use in the treatment of diabetes mellitus, and to pharmaceutical compositions comprising the derivatives, pharmaceutically acceptable salts, esters, ethers or other obvious chemical equivalents thereof.

Description: Aromatic di-keto derivatives, processes for their production and their use as a pharmaceutical The present invention relates to new aromatic di-keto derivatives and to their pharmaceutically acceptable salts, esters, ethers and other obvious chemical equivalents The derivatives are glucose-6-phosphate translocase inhibitors and can be used in the treatment of diabetes mellitus The present invention further relates to a process for the production of the derivatives, to the use of the derivatives and their pharmaceutically acceptable salts, esters, ethers and other obvious chemical equivalents as pharmaceuticals, in particular to their use in the treatment of diabetes mellitus, and to pharmaceutical compositions comprising the derivatives, pharmaceutically acceptable salts, esters, ethers or other obvious chemical equivalents thereof 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 catalysed 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 at least 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 phosphohydrolase, 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 EP-A-587 087 and EP-A-587 088. Kodaistatins A, B, C, and D described in PCT/EP 98/02247 are the first glucose-6-phosphate translocase inhibitors from microbial sources. The aromatic di-keto derivatives according to the present invention may be derived from a compound named mumbaistatin. Mumbaistatin is described in PCT/EP99/04127. It is a natural product obtainable by cultivation of the microorganism Streptomyces litmocidini, a sample of which has been deposited on July 4, 1997, with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession no. DSM 11641. The structural formula of mumbaistatin has now been determined and is given below: It has been found that certain derivatives of mumbaistatin have improved activity and are better tolerated in the mammalian body than mumbaistatin itself. Also, the separated diastereomers of mumbaistain have advantages over the mumbaistatin mixture of diastereomers. The present invention accordingly provides compounds of the general formula wherein R 4 , R 5 , R 6 and R 7 are independently H, OH, halogen, optionally substituted alkyl, aryl or acyl, X-alkyl or X-aryl, where X is O, NH, N-alkyl or S, K is a group of the formula II or III below: ?" *? & ■ C (CH-). CH, II ■ X,R, L is a group of the formula IV or V below: or K and L form, together with the respective carbon atoms to which they are bound, a group of the formula VI, Vll or VIII below: wherein Ri and R 3 are independently a cation, H, alkyl or aryl, R 2 is H, alkyl, aryl or acyl, X-i, X 2 , X3, X4, X5, Xe and X 7 are independently O, NH, N-alkyl or S, and the cyclus ring is, together with the C-atoms marked ,c' and ,d', an optionally substituted saturated, partly unsaturated or aromatic, carbocyclic or heterocyclic, simple or condensed ring system, with the exclusion of the compound where K is a group of the formula II and L is a group of the formula IV in which X-i, X 2 , X 3 , X , X 5 , ε and X 7 are O, R 1 ( R 2 and R 3 are H, R 4 is OH, R 5 , Re and R 7 are H and cyclus is 3, 8, di-hydroxy anthraquinone, and the compound where K and L form together a group of the formula VI in which Xi, X 2 , X 3 , X 4 , X 5 , Xe and X 7 are O, Ri is CH 3 , R 2 and R 3 are H, R 4 is OH, R 5 , R 6 and R 7 are H and cyclus is 3, 8, di-hydroxy anthraquinone and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. The present invention furthermore includes compounds of the general formula IX wherein M is a group of the formula X X 2 R 2 "(CH,), C H, ■ c II- -x.R . and, N is a group of the formula XI or M and N form, together with the C atom to which they are bound, a residue of the formula XII which is bonded through the C atom marked ,e', O is a group of the formula XIII |6 v Cyclus' and P is a group of the formula XIV or O and P form, together with the C atom to which they are bound, a residue of the formula XV which is bonded through the C atom marked ,f, and wherein R- \ to R 7 , X- \ to X 7 , cyclus, c and d are as defined in claim 1 , and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. The term 'alkyl' as used herein represents a straight or branched, optionally substituted Cι-C 6 -alkyl, preferably a C C -alkyl such as: methyl, ethyl, n- propyi, i-propyl, n-butyl or i-butyl, a straight or branched, optionally substituted, C 2 -C 6 -alkenyl, preferably C 2 -C -alkenyl such as allyl, a straight or branched, optionally substituted C 2 -C 6 -alkynyl, preferaby C 2 -C 4 -alkynyl such as allylene. The term 'aryl' as used herein represents an optionally substituted benzyl or phenyl. The term 'acyl' as used herein represents an optionally substituted aliphatic, aromatic or heterocyclic acyl, for example Cι-C 4 aliphatic acyl, such as acetyl or propionyl, aromatic acyl, such as, benzoyl or toluyl, and heterocyclic acyl which is derived from 5- or 6-membered rings with 1 -4 hetero atoms, such as, nicotinoyl, furyl, pyrrolyl, thienyl, thiazolyl and oxazolyl. Optionally substituted' as used herein means that the group in question is optionally substituted by one or more, preferably 1 , 2, 3 or 4, identical or different substituents selected from: hydroxyl, C -C alkyl, C C^lkenyl, C-i- C alkoxy, C-ι-C 4 alkoxycarbonyl, carbamoyl, carboxyl, trifluoromethyl, cyano, nitro, amino, Cι-C 4 alkylamino, diC-ι-C 4 alkylamino, amidino, aryloxy, arylamino and halogen. Halogen represents I, Br, Cl or FI, preferably Cl or Br. The term 'cation' represents an inorganic metal ion or an organic ammonium ion. Examples which may be mentioned are, in particular, pharmacologically acceptable alkali metal ions or alkaline earth metal ions, preferably sodium, potassium, calcium or magnesium ion, the ammonium ion and, from the organic ammonium ions, in particular, an optionally substituted alkylated ammonium ion, such as, for example, the t ethylammonium or diethanolammonium ion, as well as the morpholine, benzylammonium and procaine, L-arginine and L-lysine ion. The cyclus ring, which includes the carbon atoms marked 'c' and 'd' as used in the formulae may represent an optionally substituted, saturated, partly unsaturated or aromatic, carbocyclic or heterocyclic, simple or condensed ring system. A simple ring system means a monocyclic ring containing 3 to 6 ring atoms and a condensed ring system means a condensed dicyclic or tricyclic ring containing 6 to 14 ring atoms. The saturated carbocyclic ring system may represent a 3 to 14 membered ring system, preferably a simple 3 to 8 membered ring such as cyclo-C 3 -C 8 alkyl, more preferably cyclo-C 3 -C 6 alkyl, for example, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. It may also represent a bi- or tri-cyclic condensed ring system such as bicyclo[3.3.1]nonane and tetradecahydrophenanthrene. The partly unsaturated carbocyclic ring system differs from the saturated carbocyclic ring system in having one or two double or triple bonds. Thus it may represent a 3 to 14 membered ring system, preferably a 3 to 8 membered ring such as cyclo-C 3 -Csalkene, for example, cyclopentadiene or cyclooctatetraene, more preferably cyclo-C 3 -C 6 alkene, or cyclo-C 5 -C 8 alkyne. The aromatic carbocyclic simple or condensed ring system may represent a 5 to 14 membered monocyclic, dicyclic or tricyclic ring system such as phenyl, naphthyl, phenanthrene or anthraquinone. The heterocyclic ring system may be saturated, partly unsaturated or aromatic and may be a simple or condensed ring system as defined above. The heterocyclic ring system represents the carbocyclic ring system as defined above in which 1 , 2, 3 or 4 of the C atoms are replaced by identical or different heteroatoms selected from N, O and S. It may, for example, represent a 5- or 6-membered ring which has 1 to 4 hetero-atoms, selected from O, S and N, in particular N, optionally together with S or O as ring atoms. Some examples of heterocyclic ring systems are heteroalkyls such as pyrrolidine, piperidine, tetrahydrofuran, oxazolidine and thiazolidine, and heteroaryl residues such as pyridyl, pyrimidyl, furanyl, benzothiazoyl, benzofuranyl and indolyl. Preferably, the cyclus ring is a group of the formula XVI wherein R 8 is H, alkyl, aryl or acyl, R 9 is a cation, H, alkyl, aryl or acyl, R 1 0, ιι, R1 2 and R 1 3 are independently H, alkyl, -Xι 0 H or -X 1 0R, or R 10 and R 11 and/or Rι 2 and R 13 together are =Xιo, X 8 , X 9 and X 10 are independently O, NH or N-alkyl or S, R is alkyl, aryl or acyl, ' ' is an optional bond, and the cyclus is bound by the C-atoms marked 'c' and 'd'. More preferably the cyclus ring is a residue of the formula XVIA wherein X 8 and X 9 are independently H or O, R 8 and R 9 are independently H or alkyl, R 10 to R 13 are H, or R 10 and R 13 together and/or R 12 and R 3 together are = O, and the cyclus is bound by the C-atoms marked 'c' and 'd'. The cyclus part of the structure may be any one of a variety of different ring structures. It is advantageous, however, to have a substitution, preferably hydroxyl or alkoxyl, on the cyclus. The cyclus is preferably an aromatic ring structure of the formula XVIB below: c wherein R 9 is H or Cι-C 4 -alkyl, and X 9 is O Preferred compounds of the present invention have the general formula XVIII below: R 7 XVIII wherein Ri to R 7 , Xi to X 7 , cyclus and c and d are as defined above, with the exclusion of the compound where X to X 7 are O, Ri, R 2 and R 3 are H, R 4 is OH, R 5 , R 6 and R 7 are H and cyclus is 3, 8, di-hydroxy anthraquinone, and its pharmaceutically acceptable salts, esters and ethers and other obvious equivalents, in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. Preferably the carbon marked with an asterisk has an S configuration, in which case the exclusion mentioned above is not applicable. Suitably, Ri, R 2 and R 3 are Cι-C 6 -alkyl, preferably d-C 4 alkyl, such as methyl. Conveniently, any one or more of Xi to X are O. An example of a compound of the formula XVIII above is given below: A further example of a compound of the formula XVIIIB above is given below: XVIIIB The alkylated mumbaisatin derivatives of the formula XVIIIA and formula XVIIIB are obtained by dissolving mumbaistatin in a solvent, preferably an organic solvent such as alkanol, for example methanol, and reacting with an alkylating agent such as diazoalkane, for example diazomethane, diazoethane, or diarylmethyldiazomethane such as diphenyldiazomethane. The alkyl substituent in the above compounds of the formula XVIIIA and formula XVIIIIB is preferably a C C -alkyl. When the Cι-C 4 -alkyl is methyl, for example, the methylated mumbaistatin derivatives may be obtained by reacting mumbaistain in solution with a methylating agent such as diazomethane. The mumbaistain has ideally previously been treated with acid, preferably low molecular organic acid, for example formic acid, acetic acid or thfluoroacetic acid. The reaction product is subsequently isolated, preferably by chromatography. Isolation of the compounds according to the present invention from the reaction medium can be effected by methods which are in themselves known and which depend on the solubility of the resulting compounds. A further example of a compound of the formula XVIII is the diastereomer given below: XVIIIC wherein the C atom marked with an asterisk has the S configuration. A diastereomer of the formula XVIIID according to the present invention is shown below: XVIIID wherein the carbon atoms marked 'a' and 'b' in form of a half-ketal or ketal have independently the S or R configuration. A further example of the compound of the formula XVIII is given below: XVIIIE Some of the preferred compounds of the formula I exemplified above may be generalised as hydroxy-diketo-dicarbonic acid derivatives. The invention further relates to compounds of the general formula XIX below: wherein Ri to R 7 , Xi to X 7 , cyclus and c and d are as defined above, with the exception of the compound where Ri is methyl, R is -OH, Xi -X 7 are O and the cyclus is 3, 8, di-hydroxy anthraquinone, and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. Preferably, Ri is Cι-C 6 -alkyl, such as methyl. R is suitably hydroxy or Cι-C 6 -alkoxy, such as methoxy. An example of a compound of the formula XIX is given below: XIXA A further example of a compound of the formula XIX is given below: XIXB A yet further example of a compound of the formula XIX is given below: XIXC Another example of a compound of the formula XIX is the diastereomer below: XIXD wherein the C atom marked with an asterisk * has an 'S' configuration and the C atoms marked respectively with 'a' and 'b' both have either an S or R configuration. One process for the preparation of a compound of the formula XIXA, XIXB or XIXIC comprises dissolving mumbaistatin in a solvent, preferably an organic solvent, for example an alkanol such as methanol, and reacting with a methylating agent such as diazomethane. Mumbaistain has ideally previously been treated with acid such as trifluoroacetic acid. The reaction product is isolated, preferably by chromatography. Mumbaistatin is of limited stability in solution at a pH of around 6 to 9. At acid pH mumbaistatin rapidly undergoes a complex conversion, for example to the compound of the formula XIXD above. Because the acid form of mumbaistain is reacted with diazomethane to produce the methylated compounds of the formula XVIIIA, XVIIIB, XIXA, XIXB and XIXC above, special precautions need to be taken to ensure that native, defined methylation products are obtained. It has been found that the required methylation products are obtained under cold conditions such as at temperatures of -1°C to 3°C, preferably 0° C, and/or when the process is carried out without prolonged reaction times. It has surprisingly been possible to crystallize at least one of the methylation products by using a mixture of water and acetonitrile. This enabled determination of the structure of the compounds by X-radiation spectrometry. Table 1: Crystal data and structure refinement for trimethyl-mumbaistatin (formula XVIIIA). Identification code sh608 Empirical formula C 33 H 27 N Oιι Formula weight 613.56 Temperature 293(2) K Wavelength 0.71073 A Crystal system Monoclinic Space group P2(1) Unit cell dimensions a = 12.907(4) A α = 90°. b = 11.253(5) A β = 96.56(2) c = 20.003(6) A y = 90°. Volume 2886.2(17) A3 Density (calculated) 1.412 Mg/m 3 Absorption coefficient 0.107 mm "1 F(000) 1280 Crystal size 0.04 x 0.1 x θ.2 mm 3 Theta range for data collection 2.08 to 20.83°. Index ranges -12<=h<=12, -11 <=k<=11 , -19<=l<=19 Reflections collected 9796 Independent reflections 5833 [R(int) = 0.0447] Completeness to theta = 20.83° 98.7 % Absorption correction maximum: 0.862, minimum: 0.632 Refinement method Full-matrix least-squares on F^ Data / restraints / parameters 5833 / 1 / 822 Goodness-of-fit on F^ 1.064 Final R indices [l>2sigma(l)] R1 = 0.0510, wR2 = 0.0966 R indices (all data) R1 = 0.0981 , wR2 = 0.1171 Absolute structure parameter 1(2) Extinction coefficient 0.0035(4) Largest diff. peak and hole 0.194 and -0.174 e.A' 3 Table 2. Chemical shift of tetramethyl-mumbaistatin lactone di-spiroketone (Formula a) A and B correspond to both diastereomer forms (ratio A:B approx. 1.2 :1.0). Table 3 Comparison of the aromatic protons of formula XVIIIA and formula XVIIIB. Table 4 Chemical shift of Mumbaistatin lactone-di-spiroketal-monomethylester (formula XIXA) in DMSO at 300K. a) For these nuclei no signal was observed in the 13 C-Spectrum. Where two sets of signals were observed (ratio approx. 1.1 :1.0) they corresponded to the two diastereomer forms. Both values are separated by a comma in the case where the diastereomers show different chemical shifts (the first value corresponds to the main component). The invention also relates to a compound of the general formula XX wherein Ri to R 7 , Xi to X 7, cyclus and c and d are as defined above, and its pharmaceutically acceptable salts, esters and ethers and other chemical equivalents, in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. Preferably one or more of Xi to X 7 are O. The invention furthermore relates to compounds of the general formula XXI : wherein Ri to R 7 , Xi to X 7 , cyclus and c and d are as defined above, and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents, in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. Preferably one or more of Xi to X are O. The invention additionally relates to a compound of the general formula XXII wherein Ri to R 7 , Xi to X 7 , cyclus and c and d are as defined above, and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents, in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. Preferably one or more of Xi to X 7 are O. An example of a compound of the formula XXII is given below: XXIIA A process for the preparation of a compound of the formula XXIIA comprises dissolving mumbaistatin in a solvent, preferably an organic solvent such as alkanol, and reacting with an amide source such as an ammonia solution. The process is carried out under cold conditions, preferably at a temperature of -1°C to 3° C, more preferably at 0°C. The reaction product is subsequently isolated. The invention furthermore relates to compounds of the general formula XXIV XXIV wherein Ri to R 7 , i to X 7 , cyclus and c and d are as defined above, and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents, in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. Preferably, one or more of Xi to X 7 are O. The compounds according to the present invention are tautomers in which open and closed forms exist in equilibrium. The closed structures of the formula XIX to XXIV above can be converted to the open structure of the formula XVIII by reaction with a suitable base. Suitable bases which can be used for the reaction are inorganic or organic bases. Thus, tertiary amines and alkali metal carbonates, such as sodium carbonate, sodium bicarbonate, potassium bicarbonatere, potassium carbonate, lithium carbonate may be used. An Example of tautomers according to the present invention in equilibrium are compounds of the formula XXIIIA and XVIIIF shown below: XXI 11 A XVIIIF The compounds according to the invention may be converted into pharmaceutically acceptable salts and obvious chemical equivalents, like esters and ethers, which are all covered by the present invention. The invention also covers all salts and obvious chemical equivalents of the present compounds 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 the present aromatic di-keto derivatives and their salts, esters, ethers and other obvious chemical equivalents in all their stereoisomeric forms and tautomeric forms. The salts of the derivatives (e.g. Na, K, ammonium salts) 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 the present compounds with suitable sodium or potassium bases. Esters may be prepared, for example, by reacting the present compounds 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. 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. Other obvious chemical equivalents include reduction or oxidation products and addition products such as hydrates. For example, the anthraquinone group of mumbaistatin may be reduced with a reducing agent to hydroquinone. The resultant product is an effective inhibitor of glucose-6-phosphate translocase with an IC 50 of = ~ 5 nM. Glucose-6-phosphate translocase activity has been shown in several biochemical test systems for mumbaistatin. The yield of mumbaistatin from the culture filtrate of Streptomyces litmocidini is extremely low, however, which has hindered further development of the compound. Moreover, until now it has not been possible to ascertain the structural formula of mumbaistatin due of numerous factors including the compounds inability to crystalize and instability in solution. A process has now been found, however, which enables the isolation of mumbaistatin from an extract in relatively high yield. The present invention accordingly provides a process for the isolation of mumbaistatin comprising extracting a culture filtrate including mumbaistain by ion exchange chromatography at a pH of 5-8, preferably 6 or 7. Although the use of ion exchange is generally mentioned in PCT/EP99/04127, it is clear that the use of ion exchange for the purpose of improving yield was not recognised. This is seen from the examples in the above patent application PCT/EP99/04127 where ion exchangers are not used for the isolation of mumbaistatin and where from 730 litres of culture filtrate merely 70 mg of pure mumbaistain is obtained. The process of the present invention allows the isolation and enrichment of mumbaistatin and mumbaistain-related compounds by means of an ion exchange process whereby yields of at least more than 50 %, more usually > 70 % are obtained. Mumbaistain obtained according to the present process has an improved IC 50 of = ~ 5 nM in comparison to the mumbaistain obtained in PCT/EP99/04127. In the process for the isolation of mumbaistatin according to the present invention various ion exchangers may be used. Examples are QAE-, DEAE- and THAE-anion exchangers. Preferably, substituted or unsubstituted amino groups are carried on the chosen matrix. More preferably, DEAE-anion exchangers are used such as DEAE- ® Sepharose Fast Flow or ® Fractogel EMD DEAE. The anion exchangers may be used in a known manner. An organic solvent content of 5 to 85 % in a buffer system may be used. It is preferable, however, that the organic solvent used has a high content of buffer system, preferably therefore, an organic solvent content of 10 to 40 % in the aqueous buffer solution is used. Examples of suitable organic solvents are water-miscibie organic solvents such as lower alcohols, acetone, acetonitrile, glycol, dioxane, dimethyl sulfoxide, formamide and the like. Preferred solvents are methanol, ethanol, isopropanol and acetone. With the process described, > 99% pure mumbaistatin can be obtained and the compound can be enriched in a yield of more than 70 %. The resultant enriched mumbaistatin may be purified in a simple manner by, for example, molecular sieve- and/or reverse-phase-chromatography. The compounds according to the invention inhibit rat liver microsomal glucose-6- phosphate translocase. The compounds 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. The compounds according to the present invention and their pharmaceutically acceptable salts, esters, ethers and other obvious chemical equivalents 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 aromatic di-keto derivatives and their pharmaceutically acceptable salts, esters, ethers and others obvious chemical equivalents for use as pharmaceuticals and to the use of the derivatives and their pharmaceutically acceptable salts, esters, ethers and other obvious chemical equivalents 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 which contain an effective amount of the derivatives and/or one or more pharmaceutically acceptable salts, esters, ethers and/or obvious chemical equivalents thereof together with a pharmaceutically acceptable carrier. The compounds according to the invention can be administered orally, intramuscularly, intravenously or by other modes of administration. Pharmaceutical compositions which contain the present compounds or a pharmaceutically acceptable salt or obvious chemical equivalent 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 flavours, 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 which may be mentioned are starch, 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. Pharmaceutical compositions comprising one or more of the present compounds or a pharmaceutically acceptable salt or obvious chemical equivalent 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. On an average, the daily dose of a compound according to the present invention in a patient of about 75 mg weight is at least 0.001 mg to at most 100 mg, preferably at most 10.0 mg. Apart from use as pharmaceutically active ingredients and as intermediates in the production of derivatives, the present compounds and their salts and obvious chemical equivalents 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. The following examples are illustrative of the present invention, but not limitative of the scope thereof. Abbreviations: MeOH methanol; DMSO dimethylsulfoxide; TFA trifluoroacetic acid Example 1 Maintenance of the culture Streptomyces litmocidini , DSM 11641. Culture DSM 11641 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 Streptomyces litmocidini , DSM 11641 , 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 2 Fermentation of culture Streptomyces litmocidini , DSM 11641 in fermenters. Stage 1 : Preparation of seed culture 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 CaC0 3 2.0 g Demineralized water 1.0 litre pH 7.0 The above seed medium was distributed in 160 ml amounts in 1 L Erlenmeyer flasks and autoclaved 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 1 and shaken on a rotary shaker for 72 hours at 240 rpm at 27°C (± 1°C) to give seed culture. Composition of the production medium Glucose 20.0 g Soyabean meal 10.0 g CaC0 3 0.2 g Cobalt chloride 0.001 g Demineralized water 1.0 litre PH 7.0 Stage 2 : Preparation of seed culture in fermenter 80 litres of the seed medium, as described above, in a 100 litre Marubishi fermenter was sterilised in situ for 45 minutes at 121 °C, cooled to 27°C ± 1 °C and seeded with 4.5 litres of the seed culture mentioned above. The fermentation was run with the following parameters : Temperature 27°C (± 0.5°C) Agitation 80 rpm Aeration 50 Ipm Harvest time 24 hours Stage 3 : Large scale fermentation 700 litres of the production medium, as descπbed above, in a 1000 litre Marubishi fermenter along with 150 ml of ® Desmophen (polypropylene oxide) as antifoam agent was sterilised in situ for 45 minutes at 121 °C, cooled to 27°C ± 1 °C and seeded with 75 litres of the seed culture from Stage 2. The fermentation was run with the following parameters : Temperature 27°C (± 0.5°C) Agitation 50 rpm Aeration 450 Ipm Harvest time 40-44 hours 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 Mumbaistatin was isolated from the culture filtrate as described below in Example 3. Example 3 Isolation of Mumbaistatin by anion exchange Approximately 200 litres of culture broth was harvested and separated from mycelium (12 kg) by centrifugation. The desired compound Mumbaistatin was found to be present primarily in the culture filtrate. The culture filtrate ( 180 litres with 120 mg mumbaistatin ) was passed over a column filled with adsorption resin ® MCI GEL CHP20P (20 cm diameter x 45 cm height, content 14 litres). The column was eluted with a gradient process of from 120 litres 0.1% phosphate buffer, pH 6.3 to 120 litres 45% isopropanol in water. The column through-flow was 18 litres/hour. The largest amount of mumbaistatin (102 mg in 12 litres) was present in the salt-free fraction which was eluted with a step gradient of 25 to 28 % isopropanol in water. The resultant active eluate was passed through DEAE- ® Sepharose Fast Flow filled column (3 litres) which had been equilibrated to pH 7.0 with phosphate buffer. Mumbaistatin was eluted in a gradient process with 20 % isopropanol in 0.1 % sodium phosphate buffer, pH 7.0 as A-buffer and 20 % isopropanol in 0.1 % phosphate buffer and 0.25 % NaCI as B-buffer. Using a flow rate of 50 ml /min., 100 fractions were collected in which fractions 72 to 74 contained 81 mg of highly enriched mumbaistatin and fraction 75 a further 18 mg which was less pure. The fractions were pooled and concentrated in vacuum. The material was further purified by passing through a ® Nucleosil 100-10 CisAB column (2.1 cm x 25 cm) and eluted at pH 6.3 with a step gradient of 5 - 35 % acetonitrite in 0.05 % ammonium acetate buffer. Freeze drying of the pure fractions resulted in a total of 86 mg (73 + 13 mg ) pure mumbaistatin ammonium salt. The sodium salt of mumbaistain was prepared by dissolving 40 mg of the ammonium salt in 10 ml water (pH 6.4) and increasing the flow of the solution with sodium chloride to 12 mS/cm 2 . The resultant aqueous solution was then passed over a ® MCI GEL CHP20P column ( 1cm wide x 9cm high ). The elution results with a water/40% acetonitrile in water gradient, the column flow was 5 ml per minute and the fraction sizes were 10 ml. In fractions 16 to 19 the sodium salt was found and the purifed solution had a pH of 8.5. From these fractions resulted 32 mg mumbaistatin sodium salt after freeze-drying with a purity of 99%, measured by HPLC. UV maxima, dissolved in methanol: 219 nm, ε = 33 000; 257 nm, ε = 19 500; 285 nm, ε = 19 000; 414 nm, ε = 5 100. Inhibition of glucose-6-phosphate translocase from rat liver microsomes was with an IC 50 of = 5 nM. Inhibition of microsomal glucose-6-phosphatase in 10 μM solution: activity was not demonstrable. Example 4 Mumbaistatin methylation products 18 mg mumbaistatin obtained according to Example 3 was dissolved in 50 ml water, cooled to 0°C and maintained at a pH of 2.8 with cold thfluoroacetic acid (TFA). Directly thereafter the resultant mixture was passed over a column ( 1 cm x 8 cm) filled with 6.2 ml ® MCI GEL, CHP20P, (75 - 150 urn), and eluted using a gradient of 0.01% TFA to 30 % acetonitrile in 0.01 % TFA. The flow rate was 2.5 ml /min. The eluates were cooled and the mumbaistatin-containing fractions directly frozen to - 40° C and lyophilised. The freeze-dried product ( 15 mg ) was dissolved in methanol and methylated with diazomethane. After concentration in vacuum the reaction mixture, a mixture of more than ten methylation products was separated by passing over ® LiChrosorb RP18, 10u, column with dimensions 1 cm x 25 cm (width x length). Acetonitrile in water, 5 to 55 %, was used as the solution. The fractions were pooled cold and maintained under cold conditions during further processing. The fractions were concentrated in vacuum. Fraction 19 was mumbaistatin-mono-methylether-dimethyl ester corresponding to formula XVIIIA having a molecular weight of 590. The characteristic NMR data for the compound are shown in Table 3 above. Inhibition of glucose-6- translocase by a 3 μM solution: 42 %. A compound corresponding to formula XIXB was obtained from fraction 34 after concentration in vacuum under cold conditions. Crystallographic data for the compound are provided in Table 1 above. There exist the diastereomers S,R,R and S,S,S for the compound which are shown above. Inhibition of glucose-6-phosphate translocase: IC5o = > 100 μM. Fraction 26 contained a compound which, after storage, was the mumbaistatin tetramethyl derivative corresponding to formula XIXC. The relevant 1 H and 13 C-NMR data for this compound are provided in Table 2 above. Example 5 Mumbaistatin hemiketal-amide (Formula XXIIIA) A 1 ml concentrated aqueous ammonia solution was added dropwise under an argon atmosphere at 0°C with stirring to a solution of 10 ml mumbaistatin in 1 ml methanol. The mixture was stirred at this temperature for 2 hours and subsequently the solution was removed in vacuum. 10 mg mumbaistatin-amide was obtained in the form of a beige powder. The molecular weight (548, M + H + ) was determined by electron spray mass spectrometry corresponding to the chemical formula C 28 H 2 ιNOn. 1 H-NMR (500 MHz, DMSO-d6): δ = 7.8 (d, 1 H), 7.75 (t, 1 H), 7.35 (m, 1 H), 7.25 (s, 1H), 6.85 (t, 1 H), 6.55 (d, 1H), 3.85 (m, 1H), 2.2-2.35 (m), 2.05 (m, 1H), 1.8 (m, 1H), 1.2-1.4 (m) ppm. Mumbaistatin amide of the formula XXIIIA inhibites glucose-6-phosphate translocase with an IC 50 = ~ 1 μm Example 6 Manufacture of mumbaistatin lactone diketal mono-methyl-esters 10 mg mumbaistatin obtained from Example 3 was dissolved in 1 ml absolute methanol, reacted with 0.1 % strength aqueous TFA and allowed to stand at room temperature for 5 hours. The reaction product was purified by preparative chromatography as described in Example 3 and after freeze-drying the active fractions contained 7 mg of mumbaistain lactone mono methyl ester (formula XIXA). The molecular weight of the compound was 544 Da (ESI-MS). 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Claims: Claims A compound of the formula wherein R 4 , R 5 , R 6 and R are independently H, OH, halogen, optionally substituted alkyl, aryl or acyl, X-alkyl or X-aryl, where X is O, NH, N-alkyl or S, K is a group of the formula II or III below: L is a group of the formula IV or V below: or K and L form, together with the respective carbon atoms to which they are bound, a group of the formula VI, Vll or VIII below: wherein Ri and R 3 are independently a cation, H, alkyl or aryl, R 2 is H, alkyl, aryl or acyl, Xi, X2, X3, X4, X5, XΘ and X are independently O, NH, N-alkyl or S, and the cyclus ring is, together with the C-atoms marked ,c' and ,d', an optionally substituted saturated, partly unsaturated or aromatic, carbocyclic or heterocyclic, simple or condensed ring system, with the exclusion of the compound where K is a group of the formula II and L is a group of the formula IV in which Xi, X 2 , X 3 , X 4 , X 5 , X 6 and X 7 are O, Ri, R and R 3 are H, R 4 is OH, R 5 , R 6 and R 7 are H and cyclus is 3, 8, di-hydroxy anthraquinone, and the compound where K and L form together a group of the formula VI in which Xi, X 2 , X 3 , X 4 , X 5 , X 6 and X 7 are O, Ri is -CH 3 , R 2 and R 3 are H, R is OH, R 5 , R 6 and R 7 are H and cyclus is 3, 8, di-hydroxy anthraquinone, and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. A compound as defined in claim 1 of the formula IX wherein M is a group of the formula X and, N is a group of the formula XI or M and N form, together with the C atom to which they are bound, a residue of the formula XII which is bonded through the C atom marked ,e', O is a group of the formula XIII and P is a group of the formula XIV -X 5 R 2 XIV or O and P form, together with the C atom to which they are bound, a residue of the formula XV: which is bonded through the C atom marked ,f, and wherein Ri to R 7 , Xi to X 7 , cyclus, c and d are as defined in claim 1 , and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. A compound as defined in claim 1 of the formula XVIII below XVIII wherein Ri to R 7 , Xi to X 7 , cyclus, c and d, are as defined in claim 1 and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. A compound of the formula I as defined in claim 1 or of the formula XVIII as defined in claim 3 wherein Ri , R 2 and R 3 are alkyl. 5. A process for the preparation of a compound of the formula XVIII as defined in claim 4, comprising reacting a compound of the formula XVIII wherein Ri, R 2 and/or R 3 are H with an alkylating agent, and isolating the reaction product. 6. A compound as defined in claim 1 or claim 2 of the formula XIX below wherein Ri, R 4 to R 7 , Xi to X 7 , cyclus, c and d are as defined in claim 1 , and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. 7. A compound of the formula XIX as defined in claim 6, wherein Ri is alkyl and R is OH or alkoxy. 8. A process for the preparation of a compound as defined in claim 6, comprising reacting a solution of the compound of the formula XIX wherein R is H and R is OH with an alkylating agent and isolating the reaction product. 9. A process for the preparation of a compound as defined in claim 7, comprising reacting a solution of the compound of the formula XIX wherein Ri is H and R is OH in an alkyl alcohol with an acid and isolating the reaction product. 10. A compound of the formula XX below as defined in claim 1 or claim 2 wherein Ri to R 7 , Xi to X , cyclus, c and d are as defined in claim 1 above, and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. 11. A compound of the formula XXI below as defined in claim 1 wherein Ri to R 7 , Xi to X , cyclus, c and d are as defined in claim 1 , and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. 12. A compound as defined in claim 1 of the formula XXII below wherein X to X 7 , Ri to R 7 , cyclus, c and d are as defined in claim 1 , and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. 13. A compound of the formula XXII as defined in claim 12, wherein Ri to R 7 are H, X 3 is NH and R 4 is OH. 14. A compound as defined in claim 1 or claim 2 of the formula wherein Ri to R 7 , Xi to X 7 and cyclus are as defined in claim 1 , and its pharmaceutically acceptable salts, esters and ethers and other obvious chemical equivalents in all their stereoisomeric and tautomeric forms and mixtures thereof in any ratio. 15. A process for the preparation of a compound of the formula XVIII, XXII or XXIV as claimed in claims 3, 12 or 14, respectively, wherein -X 3 R 3 is -NH 2 , comprising reacting a compound of the formula XVIII, XXII or XXIV wherein X 3 R 3 is OH with an amide source. 16. A process according to claim 15 wherein the compound of the formula XVIII is mumbaistatin. 17. A compound as defined in any one of claims 1-4, 6, 7 and 10-14, respectively, wherein any one or more of Xi to X 7 are O. 18. A compound as defined in any one of claims 3, 4, 6, 7, 10-14, 17, respectively, wherein the carbon marked with an asterisk has an S configuration and the exclusion in claim 1 does not apply. 19. A compound as defined in any one of the preceding claims wherein cyclus is a group selected from: optionally substituted phenyl, benzyl, naphthyl, phenanthrene or anthraquinone. 20. A compound as defined in claim 19 wherein the cyclus is optionally substituted by one or more of OH, Cι-C -alkyl, -OCι-C 3 -alkyl, amino, nitro, halogen, -NH-CrC -alkyl, carboxy and cyano. 21 . A compound as defined in claim 19 or 20 wherein the cyclus is 3, 8- dihydroxyanthraquinone. 22. A compound as defined in any one of the preceding claims or a pharmaceutically acceptable salt thereof for use as a pharmaceutical. 23. A pharmaceutical composition, comprising an effective amount oτ a compouπα as defined in any one of claims 1 to 4, 6, 7, 10 to 14, 17 to 22 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 24. A compound as defined in any one of claims 1 to 4, 6, 7, 10 to 14, 17 to 22 or a pharmaceutically acceptable salt thereof for use as an inhibitor of glucose-6-phosphate translocase. 25. A compound as defined in any one of claims 1 to 4, 6, 7, 10 to 14, 17 to 22 or a pharmaceutically acceptable salt thereof for use in the treatment of diabetes mellitus.

Inventors: KURZ MICHAEL, PAULUS ERICH, VERTESY LASZLO, KURZ, MICHAEL, PAULUS, ERICH, VERTESY, LASZLO
Applicants: AVENTIS PHARMA GMBH, AVENTIS PHARMA DEUTSCHLAND GMBH
Application year: 2000
Main classification: C07C63
IPC codes: A61K31, A61P3, A61P43, C07C51, C07C63, C07C65, C07C66, C07C67, C07C69, C07C231, C07C235, C07D307, C07D493