Preparation and diabetic use of Gibberellins

Abstract

The present invention relates to application of compounds of formula (1) (Gibberellins) and their derivatives for the preparation of a pharmaceutical composition or medicaments for the treatment of diabetes, its complications and associated conditions, including obesity, micro and macro vascular diseases, nephropathy, neuropathy, eye diseases, diabetic ulcerations and the like. The method results the normalization of serum glucose level and other physiological conditions.

1 - 29 . (canceled)  
   
   
       30 . A process for the preparation of Gibberellins including Gibberellin A 3 , including the steps of: 
 (a) incubating a Gibberellin-producing strain of microorganism in a fermentation broth;    (b) adjusting the pH of the fermentation broth to pH 6.5 to 7.0 and filtering to obtain a filter cake of microorganism mycelium, and a filtrate;    (c) washing the filter cake with water and combining the washing with the filtrate to form an aqueous solution;    (d) concentrating the aqueous solution;    (e) mixing the aqueous solution with an organic solvent at a temperature of 5 to 10° C. and adjusting the pH of the mixture to less than 2.0;    (f) allowing the mixture to separate into an aqueous phase and a first organic phase and removing the first organic phase;    (g) re-extracting the aqueous phase from step (f) with organic solvent to obtain a second organic phase;    (h) combining the first and second organic phases and concentrating to form a concentrated organic solution;    (i) heating the concentrated organic solution at 60-70° C. for 3 to 4 hours with stirring, until the precipitation of solid matter ceases;    (j) cooling the concentrated organic solution to room temperature and filtering to obtain a precipitate;    (k) washing the precipitate in cold organic solvent and drying to obtain an off-white solid containing about 80% Gibberellin A 3 , about 4% Gibberellin A 4  and about 4% Gibberellin A 7 .    
   
   
       31 . The process of  claim 30 , comprising the further steps of: 
 (l) dissolving the off-white solid in a mixture of 32.6% methanol, 2.2% water and 65.2% acetone to obtain a Gibberellin solution;    (m) diluting the Gibberellin solution with a 10:1 mixture of organic solvent and water;    (n) filtering the diluted Gibberellin solution and concentrating the filtrate by vacuum evaporation;    (o) heating the concentrate to a temperature of 60 to 80° C. for 2 to 3 hours with stirring, cooling to room temperature and filtering to obtain a solid crystalline precipitate;    (p) washing the precipitate with cold organic solvent and drying to obtain Gibberellin A 3  crystals.    
   
   
       32 . A process according to  claim 30  wherein the Gibberellin-producing strain of microorganism is  Gibberella fujikuroi.    
   
   
       33 . A process according to  claim 30 , wherein the concentration of the solutions in steps (d) and (h) is achieved using vacuum evaporation.  
   
   
       34 . A process according to  claim 30  wherein the organic solvent is ethyl acetate.  
   
   
       35 . A process according to  claim 31  wherein the organic solvent is ethyl acetate.  
   
   
       36 . A process according to  claim 31  comprising the further steps of: 
 (q) dissolving the Gibberellin A 3  in methanol;    (r) adding the Gibberellin solution to an equimolar aqueous solution of NaHCO 3 ;    (s) evaporating the mixed solutions to dryness to obtain a solid residue;    (t) dissolving the residue in water and freeze drying to obtain Gibberellin A 3  sodium salt.    
   
   
       37 . A process according to  claim 36 , comprising the further steps of dissolving the Gibberellin A 3  sodium salt in water, passing the solution through a column loaded with a zinc ion-exchange resin, washing the column with water, collecting and combining the effluent and washings and removing the water to obtain Gibberellin A 3  zinc salt.  
   
   
       38 . A process according to  claim 31  comprising the further steps of: 
 (q) dissolving the Gibberellin A 3  in a 50:1 ratio mixture of acetone to water;    (r) mixing the Gibberellin A 3  solution with equimolar amounts of triethylamine and ethyl chloroformate, and a one tenth molar amount of N-methyl morpholine, and stirring at −15° C. for 20 minutes;    (s) diluting the resultant mixture with anhydrous ethanol and stirring at room temperature;    (t) evaporating the diluted mixture to dryness and partitioning the residue between ethyl acetate and water in a 6:1 ratio; separating the ethyl acetate layer, washing with 2% HCl, followed by water,    (u) followed by 5% NaHCO 3 , followed by water, and evaporating under reduced pressure to dryness to give Gibberellin A 3  ethyl ester.