Abstract:
A method of preparing an anthracyclin such as epirubicin from a starting material comprising 13-dihydrodaunorubicine (13-daunorubicinol). The method comprises producing N-Trifluoroacetyl-13-daunorubicinol from 13-daunorubicinol by acylation. The N-Trifluoroacetyl-13-daunorubicinol is reacted with an aprotic solvent and an acylating agent to produce an intermediate sulfoxy salt which is treated with a strong base to produce 4′-keto-N-Trifluoroacetyldaunorubicin. The 4′-keto-N-Trifluoroacetyldaunorubicin is reacted with a reducing agent, such as borohydride of an alkaline metal, to produce N-Trifluoroacetyl-4′-epi-daunorubicin. The N-Trifluoroacetyl-4′-epi-daunorubicin is hydrolyzed in a basic solution to produce an intermediate compound. The intermediate compound is reacted with a halogenizing agent to produce a 14-Hal-derivative. The 14-Hal derivative is hydrolized in the presence of a formate of an alkaline metal to produce the desired final compound.

Description:
RELATED APPLICATIONS  
       [0001]     This Application claims the benefit of U.S. provisional Application No. 60/751,765, filed on Dec. 20, 2005, in accordance with 35 U.S.C. Section 119(e), and any other applicable laws. U.S. provisional Application No. 60/751,765 is hereby incorporated by reference in its entirety as if set forth fully herein. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The field of the invention generally relates to chemical methods used to produce anthracyclines, a compound which is useful as an anticancer chemotherapeutic drug. More specifically, the field of the invention relates to methods of producing anthracyclines in the form of Formula (1) (wherein An −  is an anion of any strong acid; for example, in one non-limiting case of 4′-epirubicin, An −  comprises Cl − ).  
                         
 
       BACKGROUND OF THE INVENTION  
       [0003]     Anthracyclines form one of the largest families of naturally occurring bioactive compounds. Several members of this family have shown to be clinically effective anti-neoplastic agents. These include, for example, daunorubicin, doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, aclarubicin, and carminomycin. For instance, these compounds have shown to be useful in treatment of breast carcinoma, acute lymphocytic and non-lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin&#39;s lymphoma, and other solid cancerous tumors.  
         [0004]     Anthracyclin antibiotics possess very high antineoplastic activity allowing for their effective application in the treatment of a wide spectrum of tumors. The starting material for the synthesis of the majority of anthracyclin antibiotics is Daunorubicin having the form shown in Formula (2). Epirubicin of Formula (1) differs from Daunorubicin which is produced by fermentation, by having a 14-oxymethyl group and equatorial orientation of the HO-4′-C.  
                         
 
         [0005]     Conversion of Daunorubicin to Epirubicin is achieved by the oxidation of 4′-hydroxyl fragment to ketone, accompanied by a loss of the optical center, with additional stereospecific reduction (in a needed conformation) and further transformation of the epi-daunorubicin to epirubicin via bromination of the 14-CH 3 —(CO)—fragment and hydrolysis of the resulting 14-CH 2 Br—fragment to—(CO)—CH 2 OH radical. This process is shown diagrammatically in Diagram 1, below.  
                         
                         
 
         [0006]     This synthetic pathway was developed by Farmitalia as described in U.S. Pat. No. 4,345,068 to Suarato et al. Other methods of epi-daunorubicin synthesis have been previously described, for example, in U.S. Pat. No. 5,945,518 to Bigatti et al. and U.S. Pat. No. 5,874,550 to van der Rijst et al. However, all existing methods of synthesizing epi-daunorubicin utilize the same starting material, namely Daunorubicin of Formula (2).  
       SUMMARY OF THE INVENTION  
       [0007]     The present invention is directed to an innovative method for producing an epirubicin compound using a new starting material for its synthesis. Specifically, the new starting material is 13-daunorubicinol (13-dihydrodaunorubicin as shown in Formula (3)). The key difference between daunorubicinol and daunorubicin is the presence of a hydroxyl group in position 13 of the anthracyclin nucleus as opposed to a 13-keto group.  
                         
 
         [0008]     The steps involved in the novel process of the present invention are as follows:  
         [0009]     (1) the first stage of the new process is the selective placement of the protective group on the amino-group of the antibiotic&#39;s glycoside part, as shown in Formula (4); the 13-OH and 4′-OH groups preferably remain unmodified.  
                         
 
         [0010]     (2) the second stage of the process involves oxidation of the 13-OH and 4′-OH groups to the corresponding ketones by treating the N-Trifluoroacetyl-13-daunorubicinol of Formula (4), with dimethylsulfoxide activated by different acylating agents (AcX), to produce the compound as shown in Formula (5):  
         [0011]     AcX=PySO 3 , SOCl 2 , PHal 3 , POHal 3 ; Hal=Cl, Br;  
         [0012]     Ac=AlkCO; OC—(CH 2 ) n —CO, n=0-4; AlkSO 2 ; ArCO; ArSO 2 ,  
         [0013]     Alk=alkyl of halogenalkyl radical  
         [0014]     Ar=phenyl or substituted phenyl radical  
         [0015]     X=Cl, Br, I, OAc.  
                         
 
         [0016]     (3) during the third stage, the 4′-keto group of the 4′-keto-N-Trifluoroacetyl-daunorubicin of Formula (5), is reduced to the equatorial 4′-OH group, without modification of the 13-keto group. This is accomplished by reacting the 4′-keto-N-Trifluoroacetyl-daunorubicin with a reducing agent, such as a derivative of a borohydride of an alkaline metal MHBL 3 , where M=Li, Na, K; L=AlkO, AlkCOO, ArCOO (Alk=Me, Et, n-Pr, All, —(CH 2 ) n , n=0-4; Ar=Ph or subst. Ph=Ph-Alk, resulting in the N-Trifluoroacetyl-4′-epi-daunorubicin as shown in Formula (6).  
                         
 
         [0017]     (4) hydrolyzing N-Trifluoroacetyl-4′-epi-daunorubicin in a basic solution to produce a derivative as shown in Formula (7).  
                         
 
         [0018]     (5) halogenization of the 4′-epi-daunorubicin of Formula (7) at the C14 position is accomplished by reaction with the complex halogenides described by Formula (8):  
                         
 
 where R 1  through R 6  are defined as H or a hydrocarbon radical of 1 to 4 carbon chains (C 1 -C 4 ); Hal is Cl, Br, I. 
 
         [0019]     (6) the attained 14-Hal-derivative,as shown in Formula (9) (where Hal is Cl, Br, or I; and An −  is an anion of a strong acid), is hydrolyzed by well-known methods in the presence of a formate of an alkaline metal with a final result of a product of Formula (1).  
                         
 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0020]     the method of preparing an epirubicin compound using daunorubicinol as the starting material according to the present invention comprises the following steps.  
         [0021]     I. Synthesis of N-trifluoroacetyl-13-daunorubicinol  
         [0022]     N-TFA-13-daunorubicinol is produced from 13-daunorubicinol by acylation of the latter by trifluoroacetic anhydride in dry, aprotonic, immiscible-with-water solvents, preferably in dichloromethane, with further soft hydrolysis of the resulting amidoester in a two-phase system of aqueous base—organic solution of the amidoester to N-TFA-Daunorubicinol (this is shown in Diagram 2, below).  
                         
 
         [0023]     II. Synthesis of 4′-keto-N-trifluoroacetyldaunorubicin  
         [0024]     4′-keto-N-TNF-daunorubicin is derived by interacting N-TFA-13-daunorubicinol with dimethylsulfoxide, activated by various acetylating agents (AcX). N-TFA-13-daunorubicinol is converted to its sulfoxy salt (4), which further splits to 4′-keto-N-TFA-daunorubicin (as shown in Formula (10), among other products.  
                         
 
         [0025]     In certain conditions, the yield of the target ketone may exceed 85% (see Diagram 3).  
                         
 
         [0026]     Diagram 3 Legend:  
         [0027]     AcX=Py SO 3 , SOCl 2 , PHal 3 , POHal 3 ; Hal=Cl, Br;  
         [0028]     Ac=AlkCO, OC—(CH 2 ) n —CO, n=0÷4, AlkSO 2 , ArCO, ArSO 2 ,  
         [0029]     Alk=alkyl or halogenalkyl radical,  
         [0030]     Ar=phenyl or substituted phenyl radical.  
         [0031]     X=Cl, Br, J, OAc.  
                         
 
         [0032]     R 7 , R 8 , R 9 =alkyl or cycloalkyl;  
         [0033]     R 8 , R 9 =—(CH 2 ) n — where n=3 to 6. cyclic of polycyclic highly basic amine, for example DBU, quinuclidine.  
         [0034]     Aprotic solvent=non-aqueous, aprotic solvent, for example DMSO, DMAA, HMPA, DCM, and other halogenalkanes, aromatic hydrocarbons, and mixtures thereof.  
         [0035]     Reaction is conducted at temperatures from −80° C. to 0° C.; more optimally at −70±5° C. Increasing the reaction temperature significantly increases the amount of side products and impurities.  
         [0036]     III. Synthesis of 4′-epi-N-trifluoroacetyldaunorubicin  
         [0037]     4′-epi-N-trifluoroacetyldaunorubicin is synthesized by way of stereospecific reduction of the 4′-keto-N-TFA-daunorubicin in equatorial conformation with sodium borohydride (L=H).  
         [0038]     This reaction (see Diagram 4) increases yield of the desired epimer to more than 90%. However, utilization of this reducing agent also leads to a reduction of the 13-keto-group in the aglycon fragment of the molecule with formation of N-TFA-daunorubicinol.  
                         
 
         [0039]     Alternatively, the reducing agent may be sodium borohydride with L≠H; in particular, L=AlkO (Alk=Me, Et, n-Pr, All); AcO (Ac═CR 3 CO, R═H, Hal). Utilization of this borohydride decreases its reducing power, thus improving both regio-and stereoselectivity of the reaction.  
         [0040]     The reaction is conducted in non-reducible solvents, such as alcohols, ethers, hydrocarbons and halogenated hydrocarbons and mixtures thereof, preferably in methanol. The reaction is conducted at temperatures from −35° C. to 10° C., and more preferably at −20±5° C.  
         [0041]     The transformation of 4′-epi-N-TFA-daunorubicin to 4′-epi-daunorubicin by removal of the trifluoroacetyl protection group from 4′-epi-N-TFA derivatives of anthracyclins is attained by treatment with an aqueous base having a pH=10-13, at a temperature from 0° C. to 40° C., preferably 20±5° C.  
         [0042]     IV. Modification of 14-CH3 radical to 14-CH2OH in an aglycone fragment of 4′-epi-daunorubicin  
         [0043]     Halogenization of the 4′-epi-daunorubicin product (Formula (6)) shown in Diagram (4) is accomplished by utilization of complex halogenides as halogenizing agents. This approach decreases the number of synthesizing stages, and increases the yield and purity of the final product.  
                         
 
         [0044]     Solvents utilized in this reaction are amides, simple ethers and mixtures thereof, preferably dimethylformamide and tetrahydrofuran.  
         [0045]     This reaction is conducted at a temperature of 20-60° C. for 2-20 hours; preferably at 50° C. for 3 hours. The attained 14-halogen derivative (Formula (9) is hydrolyzed in an aqueous acetone solution in the presence of salts of carboxylic acids, preferably sodium formate, pH=2.5-5.5. This results in the final product of Formula (1).  
       EXAMPLE 1  
       [0046]     (a) 5 grams of 13-dihydrodaunorubicine of Formula (3) is suspended in 200 ml of dichloromethane (DCM) and chilled to 0° C. While intensely stirring the suspension, drops of trifluoroacetic anhydride in DCM (8 ml:15 ml) are slowly added over a period of 1 hour.  
         [0047]     (b) The resulting mixture is kept at 0° C. for another 0.5 hours and is then poured into 250 ml of distilled water and mixed with further separation of the organic layer.  
         [0048]     (c) 200 ml of saturated solution of sodium bicarbonate is added to the resulting organic layer, and the mixture is left at room temperature, being intensely stirred, for 24 hours, in order to undergo hydrolysis resulting in a solution of 3′-N, 4′,13-di-O-tri-trifluoroacetyldaunorubicinol.  
         [0049]     (d) After completion of hydrolysis (controlled according to HPLC), the organic layer is separated and subjected to evaporation under reduced pressure conditions until fully dry.  
         [0050]     (e) After evaporation, 5 grams of N-trifluoroacetyl-13-daunorubicinol is produced with a purity of about 90% (this is confirmed by HPLC).  
         [0051]     (f) The N-trifluoroacetyl-13-daunorubicinol from step (e) of Example 1 is utilized in the next synthetic step in Example 2 without additional purification.  
       EXAMPLE 2  
       [0052]     (a) 8 ml of DMSO is dissolved in 100 ml of DCM and chilled down to −60° C. while being stirred. After that, 2 ml of oxalylchloride in 5 ml of DCM is added to the solution, which is then incubated at −60° C. for 40 minutes to produce a reaction mixture.  
         [0053]     (b) 5 grams of N-trifluoroacetyl-13-daunorubicinol is dissolved in 50 ml of DCM and is added to the reaction mixture over a 20-minute period, while maintaining the temperature in a −60±5° C. range. The reaction mixture is then incubated for 1 hour.  
         [0054]     (c) 10 ml of triethylamine is added to the reaction mixture at a temperature ≦−60° C. The total time of contact between the reaction mixture and the triethylamine is 10 minutes.  
         [0055]     (d) A solution of 5 ml of acetic acid in 10 ml of DCM is added to the reaction mixture and stirred for 2 minutes.  
         [0056]     (e) The reaction mixture is then poured into 300 ml of distilled water. This is stirred and an organic layer is separated. This step is repeated 3 times.  
         [0057]     (f) The organic layer is evaporated in a rotary evaporator under reduced pressure conditions.  
         [0058]     (g) After evaporation, 4.7 gram of 4′keto-N-trifluoroacetyldaunorubicin is produced with a purity of about 85% (this is confirmed by HPLC).  
         [0059]     (h) The 4′keto-N-trifluoroacetyldaunorubicin from step (g) of Example 2 is utilized in the next synthetic step in Example 3 without additional purification.  
       EXAMPLE 3  
       [0060]     (a) 4.7 grams of 4′keto-N-trifluoroacetyldaunorubicin is dissolved in 180 ml of tetrahydrofuran and, while stirring, 2.1 grams of sodium triacetylborohydride is added over a 40-minute period. While being agitated, the reaction mixture is incubated for 1 hour at a temperature range of 20±2° C.  
         [0061]     (b) The reaction mass is then transferred into a mixture of 150 ml of DCM+300 ml of distilled water+2 ml of 1M hydrochloric acid, and stirred. An organic layer is separated and then washed twice with 300 ml aliquots of distilled water.  
         [0062]     (c) After evaporation, 4.6 g of 4′epi-N-trifluoroacetyldaunorubicin is produced with a purity of about 79% (this is confirmed by HPLC).  
         [0063]     (d) The produced crude product then undergoes purification in a preparative chromatograph. After evaporation of the eluate, 3.0 grams of 4′epi-N-trifluoroacetyldaunorubicin is produced with a purity of about 95% (this is confirmed by HPLC).  
       EXAMPLE 4  
       [0064]     3.0 grams of 4′epi-N-trifluoroacetyldaunorubicin is suspended in 200 ml of distilled water at a temperature 30° C., and 10 ml of 1.0N NaOH solution is then added. The mixture is incubated for 30 minutes, and then neutralized to pH 7 with a solution of hydrochloric acid and is then purified using preparative chromatography. After evaporation of eluate, 2.1 grams of 4′epi-daunorubicin hydrochloride is produced with a purity of about 96% (Confirmed by HPLC).  
       EXAMPLE 5  
       [0065]     (a) 2.1 grams of 4′epi-daunorubicin hydrochloride is dissolved in 70 ml of dimethylformamide, and 2.8 grams of hydrogen dibromobromate bis (dimethylformamide) is added to the mixture. The mixture is then incubated at 40° C. for 2 hours.  
         [0066]     (b) The reaction mixture is poured into 350 ml of acetonitrile. The precipitated sediment is filtered and washed with acetonitrile; and the solvent is removed.  
         [0067]     (c) The solid sediment is dissolved in a mixture of 80 ml of acetone+80 ml of 0.25 M aqueous solution of hydrogen bromide+8 grams of sodium formate. The reaction mixture is incubated for 30 hours at 35° C.  
         [0068]     (d) The reaction mixture undergoes preparative chromatography wherein epirubicin-containing fraction is separated.  
         [0069]     (e) Eluate is evaporated, and the residue is crystallized by adding acetone.  
         [0070]     (f) The yield of this process is 1.3 g of epirubucin hydrochloride of 99.8% purity (this is confirmed by HPLC).