Abstract:
A staurosporine derivative with anti-cancer activity and anti-bacterial activity is produced by acidifying a solution containing staurosporine to isomerize staurosporine to its desired derivative, whereby the derivative is recovered from the acidified solution.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a process for producing UCN-01 from a solution containing UCN-02 in an efficient and simple manner without complicated steps. 
     2. Brief Description of the Background Art 
     UCN-01 is a well-known compound with anti-cancer activity and anti-bacterial activity, having the structure represented by the following formula (II).                           
     The following processes for producing UCN-01 are known: 
     (1) Japanese Published Unexamined Patent Application 87/220196 provides a fermentation method comprising culturing a microorganism of genus Streptomyces with an ability to generate UCN-01 in a culture medium and harvesting the generated UCN-01; 
     (2) WO 89/07105 provides a method comprising three-step chemical synthesis of UCN-01 from staurosporine represented by the following formula (III).                           
     Staurosporine is a potent inhibitor of Protein Kinase C and can be readily obtained from Streptomyces sp. Staurosporine is also commercially available, for instance, from Fermentek Corp. of Jerusalem, Israel. 
     (3) Japanese Published Unexamined Patent Application 94/9645 provides a method comprising oxidizing staurosporine in a solution comprising dimethyl sulfoxide and an aqueous alkali solution to a racemic mixture of UCN-02 represented by the following formula (I):                           
     and a steric isomer thereof, UCN-01 represented by the formula II above, and recovering UCN-01 by column chromatography using a carrier such as a synthetic adsorbent (for example, Diaion HP-20SS manufactured by Mitsubishi Chemical Industries, Co., Ltd.) and silica gel. 
     However, these methods are disadvantageous in that the culture titer by the fermentation of method (1) is low; the yield of UCN-01 resulting from the chemical synthesis of method (2) is low because 3 steps are required; and the recovery of UCN-01 is low in the column chromatography purification step of method (3) because UCN-02 must be removed as an impurity. Thus, known procedures are very complicated and are not applicable to large scale commercial synthesis. 
     SUMMARY OF THE INVENTION 
     The present invention relates to a process for producing UCN-01 in an efficient and simple manner. 
     More specifically, the present invention provides a process for producing UCN-01, comprising acidifying a solution containing UCN-02 so as to isomerize UCN-02 to UCN-01, and recovering UCN-01 from the resulting acidified solution. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The process for producing UCN-01 according to the present invention firstly requires only a solution containing UCN-02. Using such a solution, in conformity with the present invention, highly purified UCN-01 can be produced at an excellent efficiency in a simple manner. 
     Any solution containing UCN-02 may be used as the solution containing UCN-02 in accordance with the present invention. Suitable solutions containing UCN-02 include, for example, a solution obtained by oxidizing staurosporine in a solution comprising dimethyl sulfoxide and an aqueous alkali solution as discussed in aforementioned Japanese Published Unexamined Patent Application 94/9645. 
     Solutions containing UCN-02 may contain UCN-01. Therefore, when the solution containing both of UCN-01 and UCN-02, which is obtained by the process for producing UCN-01, is treated by the process of the present invention, the solution containing UCN-01, which consists of both of UCN-01 changed from UCN-02 and UCN-01 existing in the original solution, is obtained, and UCN-01 can be obtained in high yield without separation of UCN-02 from the solutions, 
     The reaction to isomerize UCN-02 to UCN-01 can be carried out by adding water so that the concentration of water in the solution is preferably 50 v/v % or more, more preferably 90 v/v % or more, and adjusting the solution to preferably pH 5 or less, more preferably pH 1 to 3, further more preferably pH 2 to 2.5. 
     The concentration of UCN-02 in the reaction mixture is preferably 25 g/L or less, more preferably 5 to 10 g/L. When UCN-01 is contained in the reaction solution, the combined concentration of UCN-01 and UCN-02 in a reaction mixture is adjusted to the aforementioned concentration. 
     Acids to adjust pH include, among others, acetic acid, hydrochloric acid, sulfuric acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid and xylenesulfonic acid. Further, hydrochloric acid or sulfuric acid is preferably used, and hydrochloric acid is more preferably used. 
     The reaction temperature is 0 to 50° C., preferably 20 to 30° C.; and the reaction time is 1 to 24 hours. 
     When the reaction is complete, the reaction mixture is adjusted to pH 7 or more, whereby UCN-01 is precipitated from the reaction mixture. The precipitate of UCN-01 is separated from the reaction mixture in a conventional manner such as a filtration. 
    
    
     The invention is now described in detail in the following examples. 
     EXAMPLES 1-6 
     A solution containing UCN-01 and UCN-02 was prepared by dissolving staurosporine (1.0 g; 2.1 mmol) in 100 ml of dimethyl sulfoxide, followed by addition of 10 ml of 0.3 mol/L sodium hydroxide, and then stirring the resulting mixture at room temperature for 8 hours. The UCN-02/UCN-01 ratio in the solution was 14.2%. 
     Water (33 ml) was added to the solution to dilute the solution; and the resulting dilution was passed through a column (25 ml volume) packed with Diaion SP-207 manufactured by Mitsubishi Chemical Industries, Co., Ltd. 
     After the solution was passed through the column, SP-207 was treated sequentially with 25 ml of a 70% dimethyl sulfoxide solution, 75 ml of water, and 125 ml of an aqueous 0.02 mol/L hydrochloric acid solution, followed by elution of UCN-01 and UCN-02 with an aqueous 60% acetone solution containing 0.005 mol/L hydrochloric acid. 
     The resulting elution was concentrated by two-fold to remove acetone. Then, by using (1) 1 mol/L hydrochloric acid, (2) 1 mol/L sulfuric acid, (3) 1 mol/L trifluoroacetic acid, (4) 1 mol/L methanesulfonic acid, (5) 1 mol/L p-toluenesulfonic acid, or (6) 1 mol/L xylenesulfonic acid, the resulting solution was adjusted to pH 2.2 and agitated overnight at room temperature, to isomerize UCN-02 to UCN-01. The UCN-02/UCN-01 ratio in each of these solutions are shown in Table 1. 
     By adjusting these solutions to pH 8.5 by using aqueous 1 mol/L sodium hydroxide and subsequently filtering the resulting solution, UCN-01 was recovered. The recovered weight and recovery are also shown in Table 1. 
     
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                 UCN-02/ 
                 Recovered 
                   
               
               
                   
                 UCN-01 
                 weight 
                 Recovery 
               
               
                 Acid (1 mol/L) 
                 (%) 
                 (g) 
                 (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Hydrochloric acid 
                 3.7 
                 0.8 
                 84 
               
               
                 2. 
                 Sulfuric acid 
                 4.0 
                 0.7 
                 73 
               
               
                 3. 
                 Trifluoroacetic acid 
                 5.0 
                 0.7 
                 74 
               
               
                 4. 
                 Methanesulfonic acid 
                 4.7 
                 0.8 
                 77 
               
               
                 5. 
                 p-Toluenesulfonic acid 
                 4.0 
                 0.7 
                 74 
               
               
                 6. 
                 Xylenesulfonic acid 
                 3.5 
                 0.8 
                 75 
               
               
                   
               
             
          
         
       
     
     Example 7 
     UCN-01 prepared under the conditions in Example 1 was dissolved in 200 ml of chloroform, and the solution was subsequently concentrated to 7 ml. UCN-01 was crystallized from the solution, and 0.7 g of UCN-01 crystal (purity 99%) was recovered. 
     The content of UCN-02 in the recovered UCN-01 crystal was 0.2%.