Abstract:
Methods for producing amrubicin and structural analogs thereof. The present invention encompasses synthetic pathways for the production of amrubicin (Formula I) and structural analogs thereof. The synthetic pathways of the present invention preferably employ as a starting material an anthracycline compound having the generic Formula II: 
     
       
                 
         
             
             
         
       
     
     Compounds of Formula II may have any combination of the following identities for the indicated moieties: R 1 , R 2 , R 3 , R 4 , and R 8  may be H, OH, or alkoxy; R 5  may be H, alkyl, or alkoxycarbonyl; R 6  may be H or alkyl; R 7  may be OH or alkyl. In certain embodiments, ε-rhodomycinone or daunomycinone may be used as starting materials according to Formula II. The present invention employs a compound of Formula II as part of a semi-synthetic method that combines traditional chemical synthetic steps with biosynthetic steps to produce amrubicin, derivatives thereof, and structural analogs thereof. The methods of the present invention preferably include a glycosylation reaction whereby an algycon of amrubicin or structural analog thereof is glycosylated to produce the final product.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Application Ser. No. 61/173,440 filed on Apr. 28, 2009. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates generally to the novel process to produce a cytotoxic product, amrubicin, which is useful in cancer treatment. The process described herein is also useful to create novel 4-deoxy-9-amino anthracyclines for drug development purposes. 
         [0004]    2. Description of the Background 
         [0005]    Amrubicin (Formula I) is an anthracycline antibiotic currently under investigation in the United States for the treatment of small cell lung cancer and in market in Japan. Amrubicin (RN: 110267-81-7) provides some improved aspects in cancer chemotherapy such as reduced dose-limiting cardio toxicity and efficiency in lung cancer (see ref 1 and the references therein). Total chemical synthesis of amrubicin has been described in publication by Ishizumi ( J. Org. Chem.,  1987, 52, 4477) and respective patent (U.S. Pat. No. 4,673,668). Due to its large, multi-ring structure and numerous side groups, the synthesis of amrubicin is complicated and requires multiple purification steps. In addition, the prior art method for synthesis of amrubicin and its structural analogs employ large amounts of potassium cyanide and barium salts, both of which are highly toxic. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0006]    Furthermore, care should be taken during synthesis to maintain the stereochemistry at the multiple chiral centers of the molecule. If stereochemistry of the products is not maintained during synthesis, then costly and laborious separation of enantiomers would be required to obtain the biologically active amrubicin enantiomer. 
         [0007]    Semi-synthesis is a type of chemical synthesis that employs compounds produced by natural sources as starting materials. The naturally produced starting material is often complex itself and allows the final product to be synthesized in fewer steps than through traditional chemical synthesis methods. Additionally, biosynthetic steps, i.e. processes that utilize enzyme activities to accomplish chemical transformations, can be employed to achieve efficient synthesis of structurally complex organic compounds with very high enantiomeric purity. 
         [0008]    The present invention improves upon the prior art synthetic schemes for amrubicin by combining biosynthetic preparation of the starting material, and traditional chemical approaches to arrive at a safe and efficient method for the production of amrubicin and its structural analogs. 
       SUMMARY OF THE INVENTION 
       [0009]    The present invention encompasses synthetic pathways for the production of amrubicin (Formula I) and structural analogs thereof. The synthetic pathways of the present invention preferably employ as a starting material an anthracycline compound having the generic Formula II: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0010]    Compounds of Formula II may have any combination of the following identities for the indicated moieties: R 1 , R 2 , R 3 , R 4 , and R 8  may be —H, —OH, or alkoxy; R 5  may be —H, alkyl, or alkoxycarbonyl; R 6  may be —H, alkyl or acyl; R 7  is —OH. Compounds of Formula II can be chemically modified to yield aglycone of amrubicin (Formula III). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    or its structural analogs, which can be chemically glycosylated with respective sugar moiety to form amrubicin (or respective structural analogs thereof). Glycosylation of compound with formula III and its analogs has been described by Ishizumi ( J. Org. Chem.,  1987, 52, 4477), which is hereby incorporated by reference. 
         [0011]    In certain presently preferred embodiments, the starting anthracycline may be ε-rhodomycinone, having the Formula IV: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0012]    In other presently preferred embodiments, the starting anthracycline may be daunomycinone, having the Formula V: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0013]    In other presently preferred embodiments, the starting anthracycline may be 4-demethoxy-daunomycinone (idarubicinone) having the Formula VI 
         [0000]    
       
                 
         
             
             
         
       
     
         [0014]    The present invention preferably employs a compound of Formula II as biosynthetically prepared starting material of a semi-synthetic method that combines traditional chemical synthetic steps with biosynthetic starting material to produce amrubicin, derivatives thereof, and structural analogs thereof. The enantiomeric purity of the product is achieved in part by biosynthetic production of the starting compound (Formula II). The particular chemical moieties that are chosen for groups R 1  through R 7  will determine whether the product is amrubicin aglycon or a structural analog thereof. 
         [0015]    While synthesis of starting compounds with formula II as defined above is known in the art, biochemical synthesis of respective antracycline analogs where R 7  is —NH 2  (as in aglycon of amrubicine) or modification of biochemically synthesized compounds with formula II to R 7  is —NH 2  is not known by the state of art. 
         [0016]    Antracycline compounds analogous to formula II with R 7  is —NH 2  have been only synthesized by total chemical synthesis which is a difficult multi-step process. 
         [0017]    The goal of the invention was therefore to provide methods for chemical modification of compounds with formula II where R 7  is —OH to analogues where R 7  is —NH 2 , while maintaining the configuration of all chiral carbon atoms in the molecule. 
         [0018]    The goal is achieved through subjecting compounds with formula II to a Ritter reaction with a suitable nitrile to form a cyclic intermediate, which is then hydrolyzed in acidic conditions to yield respective derivates with R 7 =—NH 2 . 
         [0019]    The starting compound according to Formula II is preferably subjected to chemical reactions modifying the substituent R 7  from —OH to —NH 2  to achieve synthesis of the aglycons of amrubicin or structural analogs thereof while maintaining appropriate chirality. Finally, the aglycons of amrubicin or structural analogs thereof may be converted into amrubicin or structural analogs thereof through a chemical glycosylation. 
         [0020]    The present invention provides many benefits over the prior art. Generally, the reactions of the present invention are performed under mild conditions, resulting in reduced production of dangerous wastes. The enantiomeric purity of the aglycon molecule is achieved by the biosynthesis of the starting compound. Therefore, none of the following chemical tools are required—asymmetric catalysis, asymmetric synthesis, nor separation of enantiomers of the product. 
         [0021]    In contrast to total chemical synthesis of amrubicin and its structural analogs in the prior art where large amounts of potassium cyanide and barium salts are consumed, no such toxic or dangerous reagents are required here during the synthesis. The present invention further avoids photochemical reactions and therefore avoids using special apparatuses employed for such reactions. 
         [0022]    The present invention also provides synthetic methods for production of amrubicin aglycone and its structural analogs that require less time and resources, and is therefore considerably more cost-effective and environmentally friendly. Smaller number of chemical steps, smaller amounts of dangerous wastes, and no requirement for the use of expensive equipment make the commercial preparation of amrubicin aglycone and therefore also amrubicin and its structural analogs more efficient and less expensive. All these factors increase the availability of this potent drug for cancer patients. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0023]    It is to be understood that the description of the present invention has been simplified to illustrate elements that are relevant for a clear understanding of the invention, while eliminating, for purposes of clarity, other elements that may be well known. 
         [0024]    The present invention provides methods for the synthesis of amrubicin and structural analogs thereof. The synthesis preferably begins with an anthracycline starting compound that has been produced through fermentation or semi-synthetic processes. The compound has the formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0025]    The compound shown in Formula II may include any combination of the following identities for the indicated moieties: R 1 , R 2 , R 3 , R 4 , and R 8  may be H, OH, or alkoxy; R 5  may be H, alkyl, or alkoxycarbonyl; R 6  may be —H, alkyl or acyl; R 7  is —OH. Compounds according to Formula II may be synthesized biochemically according to well-known processes. See, e.g., U.S. Pat. No. 5,986,077, which is hereby incorporated by reference in its entirety. Daunomycinone, where R 1  is —H, R 2  is —OCH 3 , wherein R 3  is —OH, wherein R 4  is —OH, wherein R 5  is —H, wherein R 6  is —COCH 3 , wherein R 7  is —OH, and R 8  is —OH (Formula V) can be further modified to 4-demethoxydaunomycinone (idarubicinone), where R 1  is —H, R 2  is —OCH 3 , wherein R 3  is —OH, wherein R 4  is —OH, wherein R 5  is —H, wherein R 6  is —COCH 3 , wherein R 7  is —OH, and R 8  is —OH (Formula VI), as described in WO 01/87814 and US 2006/0047108. 
         [0026]    Idarubicinone may be directly aminated to aglycone of amrubicin by employing methods of the present invention. 
         [0027]    While the synthetic steps taken from the compound having Formula II to amrubicin or the structural analog of amrubicin will depend on the specific character of each of the moieties, one of skill in the art will recognize that multiple pathways are available within the scope of the present invention. In general, the starting compound of Formula II is modified through a series of chemical reactions to generate aglycons of amrubicin and structural analogs thereof. 
         [0028]    The method of choice for amination of antracyclines with Formula II, preferably antracyclines with formula II where R 7 =—OH and R 8 =—OH, in the present invention is Ritter reaction of said compounds with suitable nitriles to get cyclic intermediate with formula VII. 
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         [0029]    Or a non-cyclic amide intermediate with the formula VIIa 
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         [0000]    , where R 1 -R 6  are as defined above for formula II and R 9  is alkyl, halogenated alkyl, hydrogen or aryl group depending on the used nitrile, followed by hydrolysis of said intermediates with formula VII in acidic conditions. 
         [0030]    Hydrolysis of compounds with formula VII is preferably carried out with aqueous solution of a strong acid, preferably strong inorganic acid, most preferably sulfuric acid. Organic solvents, for example tetrahydrofurane, can be added to hydrolysis reaction mixture to improve solubility of anthracyclines and also to facilitate separation of reaction products by extraction. 
         [0031]    Other chemical methods like Mitsunobu reaction, or tosylation with the subsequent Gabriel reaction or other chemical amination methods can theoretically also be used for replacement of hydroxyl group with amino group, but most of them require protection of other hydroxyl groups and other functional groups in the molecule. Also, formation of cyclic intermediate with formula VII in the Ritter reaction helps to maintain the enantiomeric purity of the aglycon. 
         [0032]    The aglycon product is then preferably glycosylated by 6-bromotetrahydro-2H-pyran-3,4-diyl diacetate or other protected derivatives of 2-deoxy-D-ribose diacetyl-ribosylbromide and then deprotected, providing amrubicin (ref. Ishizumi ( J. Org. Chem.,  1987, 52, 4477)). 
       EXAMPLES 
       [0033]    To more clearly demonstrate the present invention, examples are provided herein below. These are not meant to be limiting, but are instead exemplary embodiments of the general approaches within the scope of the present invention. The yields in the below examples are not optimized. Optimization would involve evaluating multiple parameters, including reactor design and recovery approaches according to processes well known to those of skill in the art. While such efforts would improve the yield, that optimization does not impact the fundamental elements of the disclosed inventive process. 
       Example 1 
       [0034]    In the present example, the R 7  in 4-dehydroxy-ε-rhodomycinone (R 1 ═R 2 ═H, R 3 ═R 4 ═R 8 ═OH, R 7 ═OH, R 6 ═C 2 H 5 , R 5 ═COOCH 3 ) is transformed into —NH 2  by Ritter amination. 
         [0035]    Step 1: Ritter reaction. Trifluoroacetic acid (40 ml) and acetonitrile (20 ml) were mixed and 1 g (0.0024 mol) of 4-dehydroxy-ε-rhodomycinone was added to the solution. The mixture was stirred at 60° C. for 3 h and subsequently concentrated on rotary evaporator to obtain a viscous residue. The residue was dissolved in chloroform (80 ml) and washed with water (100 ml). The water phase was extracted twice with chloroform (both 20 ml), and the combined chloroform extracts washed with saturated aqueous sodium bicarbonate solution (100 ml), water (100 ml) and saturated sodium chloride solution (80 ml). The organic phase was dried over sodium sulfate, and the solvent removed in vacuo. The dry residue was purified by silica gel chromatography (eluent: methanol/chloroform 1:40) and 948 mg of the cyclic Ritter reaction product was obtained as orange crystalline solid. A yield of 90% was achieved. 
         [0036]    Step 2: Hydrolysis of the Ritter reaction product. 100 mg of Ritter reaction product from example 1 was dissolved in mixture of 1 ml of conc. sulfuric acid, 2 ml of water and 9 ml of tetrahydrofurane. The reaction mixture was heated under reflux for 5 days. The reaction mixture was extracted 2 times with 10 ml diethyl ether to remove side products. After that, reaction mixture was neutralized with 0.5 M sodium hydroxide to pH ˜5, and then with saturated sodium bicarbonate solution to pH 7.8 and extracted with chloroform (2 times 20 ml). Combined chloroform fractions were washed with water (30 ml) and saturated sodium chloride solution (20 ml). The organic phase was dried over sodium sulfate and solvent was evaporated and remaining solid was dried in vacuo. The yield was 39 mg of pure 9-amino-4-dehydroxy-ε-rhodomycinone. 
       Example 2 
       [0037]    In daunomycinone (R 1 ═H, R 2 ═OCH 3 , R 3 ═R 4 ═R 8 ═OH, R 5 ═H, R 6 ═COCH 3 , R 7 ═OH; Formula IV above), the R 7  is transformed into —NH 2  by Ritter amination. 
         [0038]    Step 1: Ritter reaction. Trifluoroacetic acid (100 ml) and acetonitrile (40 ml) were mixed and 2 g (0.005 mol) of daunomycinone was added to the solution. The mixture was stirred at 60° C. for 3 h and subsequently concentrated in vacuo. The resultant solid was dissolved in chloroform (100 ml) and washed with water (100 ml). The water phase was extracted twice with chloroform (150 and 120 ml), and the combined chloroform extracts washed with saturated aqueous sodium bicarbonate solution (100 ml). The organic phase was dried over sodium sulfate, and the solvent removed in vacuo. The dry residue was purified by silica gel chromatography (eluent: 2.5% methanol/chloroform) and 900 mg of the Ritter intermediate (imidazoline cycle) was obtained. 
         [0039]    Step 2a. Ritter intermediate hydrolysis. Experiment 1. 450 mg of the Ritter intermediate of example 2 was dissolved in a mixture of tetrahydrofurane (33.75 ml), concentrated sulfuric acid (3.75 ml) and water (7.5 ml) and heated at 60° C. for 30 h. The reaction mixture was extracted with ether (2×200 ml) and ethyl acetate (4×100 ml) and the pH was taken to 3.5 with 0.5 M sodium hydroxide solution. The solution was extracted with chloroform (200 ml). Saturated aqueous sodium bicarbonate solution was added to obtain the pH of 7.5, followed by extraction with chloroform (200 ml). The final chloroform extract was washed with water (200 ml) and brine (400 ml), which were subsequently back-extracted with chloroform (200 ml). The chloroform phases were combined and dried over sodium sulfate. The solution was filtered, the solvent removed under reduced pressure and the product dried in vacuo. The dry residue (200 mg) was dissolved in chloroform (5 ml), followed by the addition of 3M methanolic hydrochloric acid (3 equivalents) and ether (5 ml) and left at −20° C. overnight. The solution was filtered, dried in vacuo and 145 mg of the 9-deoxy-9-amino-daunomycinone hydrochloride obtained. 
         [0040]    Step 2b. Ritter intermediate hydrolysis. Experiment 2. 450 mg of the Ritter intermediate of example 2 was dissolved in 3 N sulfuric acid (45 ml) and heated at 60° C. for 30 h. The after-treatments were carried out in the same manner as in experiment 1 2. 200 mg of the obtained 9-deoxy-9-amino-daunomycinone was dissolved in chloroform (5 ml), followed by the addition of 3M methanolic hydrochloric acid (3 equivalents) and ether (5 ml) and left at −20° C. overnight. The solution was filtered, dried in vacuo and 115 mg of the 9-deoxy-9-amino-daunomycinone hydrochloride obtained. 
         [0041]    Step 2c. Ritter intermediate hydrolysis, experiment 3. 700 mg of the Ritter intermediate of example 2 was treated as in experiment 1 using a mixture of 525 ml of tetrahydrofurane, 116.7 ml of concentrated sulfuric acid and 58.3 ml of water and heating at 60° C. for 7 h. 600 mg of the 9-deoxy-9-amino-daunomycinone was obtained and dissolved in chloroform (10 ml). After the addition of 3 M methanolic hydrochloric acid (2 equivalents) and ether (10 ml), the solution was filtered, dried in vacuo and 400 mg of the 9-deoxy-9-amino-daunomycinone hydrochloride was obtained 
       Example 3 
       [0042]    For preparation of aglycon of amrubicine, daunomycinone (R 1 ═H, R 2 ═OCH 3 , R 3 ═R 4 ═R 8 ═OH, R 5 ═H, R 6 ═COCH 3 , R 7 ═OH; Formula V above), first a demethoxylation of R 2 ═OCH 3  into R 2 ═OH is performed as known in the state of the art, followed by dehydroxylation of R 2 ═OH into R 2 ═H by methods described in (WO 01/87814 and US 2006/0047108). The resulting 4-demethoxy-daunomycinone (idamycinone, formula VI) is then converted to aglycon of amrubicine as follows. 
         [0043]    Step 1: Ritter reaction. Trifluoroacetic acid (32 ml) and acetonitrile (32 ml) were mixed and warmed to 55° C. and 456 mg (1.24 mmol) of 4-demethoxydaunomycinone was added to the solution. The mixture was stirred at 60° C. for 45 min and subsequently concentrated on rotary evaporator to obtain a viscous residue. The residue was dissolved in chloroform (75 ml) and washed with 1.6% sodium bicarbonate solution (75 ml) and two times with water (both 75 ml). The organic phase was dried over sodium sulfate, and the solvent removed in vacuo. The dry residue was purified by silica gel chromatography (eluent: first methanol/chloroform 1:50, then methanol:chloroform 1:10). 212 mg of the cyclic Ritter reaction product was obtained as orange crystalline solid. A yield of 44% was achieved. 
         [0044]    Step 2. Ritter intermediate hydrolysis. 10 mg of the cyclic Ritter reaction product from Step 1 was dissolved in 3N sulfuric acid and heated at 80° C. for 3 days. The mixture was neutralized by saturated NaHCO 3  solution and the product was extracted by 3×10 mL dichloromethane. The extracts were combined and evaporated in vacuo. The crude product was purified by preparative TLC. The molecular weight of product (366,09732) was consistent with the HRMS calculated for aglycone of amrubicine (366,09831 in negative mode). 
         [0045]    Glycosylation may be achieved through using chemical coupling (R 1 ═R 2 ═H, R 3 ═R 4 ═R 8 ═OH, R 7 ═NH 2 , R 5 ═H, R 6 ═COCH 3 ) with 6-bromotetrahydro-2H-pyran-3,4-diyl diacetate or other protected derivatives of 2-deoxy-D-ribose. 
         [0046]    In addition to the starting compounds described above in Examples 1 and 2, the present invention also contemplates reaction schemes that employ aklavinone as a starting compound. In general, the starting compound will determine the structure of the final product. As such, the use of different starting compounds within the context of the present invention will generate sets of novel compounds that would, in turn, serve as leads of new biologically active compounds. 
         [0047]    Nothing in the above description is meant to limit the present invention to any specific materials, geometry, or orientation of atoms. Many part/orientation substitutions are contemplated within the scope of the present invention and will be apparent to those skilled in the art. The embodiments described herein were presented by way of example only and should not be used to limit the scope of the invention. 
         [0048]    Although the invention has been described in terms of particular embodiments in an application, one of ordinary skill in the art, in light of the teachings herein, can generate additional embodiments and modifications without departing from the spirit of, or exceeding the scope of, the claimed invention. Accordingly, it is understood that the drawings and the descriptions herein are proffered only to facilitate comprehension of the invention and should not be construed to limit the scope thereof.