Patent Publication Number: US-2009240049-A1

Title: Purification of air sensitive steroids

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present invention claims the benefit of the following U.S. Provisional Patent Applications Nos. 61/069,994, filed Mar. 18, 2008, and 61/060,638, filed Jun. 11, 2008. The contents of these applications are incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a method for purifying air sensitive steroids from their corresponding oxidation impurities. 
     BACKGROUND OF THE INVENTION 
     Steroids are a wide class of compounds including many different chemical entities. They represent one of the milestones in chemistry applied to health care of humans. Their structures share the skeleton of formula I. 
     
       
         
         
             
             
         
       
     
     Table 1 lists several of the most commonly used Steroids. 
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Common name 
                 A 
                 B 
                 C 
                 D 
                 E 
                 G 
                 M 
                 Q 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Betamethasone 
                 Double 
                 F 
                 H 
                 OH 
                 beta CH 3   
                 OH 
                 OH 
               
               
                   
                 bond 
               
               
                 Betamethasone propionate 
                 Double 
                 F 
                 H 
                 OH 
                 beta CH 3   
                 EtCOO 
                 EtCOO 
               
               
                   
                 bond 
               
               
                 Betamethasone acetate 
                 Double 
                 F 
                 H 
                 OH 
                 beta CH 3   
                 OH 
                 MeCOO 
               
               
                   
                 bond 
               
               
                 Betamethasone 17 valerate 
                 Double 
                 F 
                 H 
                 OH 
                 beta CH 3   
                 nBuCOO 
                 OH 
               
               
                   
                 bond 
               
               
                 Beclomethasone 
                 Double 
                 Cl 
                 H 
                 OH 
                 beta CH 3   
                 EtCOO 
                 EtCOO 
               
               
                 dipropionate 
                 bond 
               
               
                 Diflorasone diacetate 
                 Double 
                 F 
                 F 
                 OH 
                 beta CH 3   
                 MeCOO 
                 MeCOO 
               
               
                   
                 bond 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Fludrocortisone acetate 
                 H 
                 H 
                 F 
                 H 
                 OH 
                 H 
                 OH 
                 MeCOO 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Difluprednate 
                 Double 
                 F 
                 F 
                 OH 
                 H 
                 nPrCOO 
                 MeCOO 
               
               
                   
                 bond 
               
               
                 Flumethasone pivalate 
                 Double 
                 F 
                 F 
                 OH 
                 alpha CH 3   
                 OH 
                 tBuCOO 
               
               
                   
                 bond 
               
               
                 Fluorometholone 
                 Double 
                 F 
                 CH 3   
                 OH 
                 H 
                 OH 
                 H 
               
               
                   
                 bond 
               
               
                 Fluorometholone 17 
                 Double 
                 F 
                 CH 3   
                 OH 
                 H 
                 MeCOO 
                 H 
               
               
                 acetate 
                 bond 
               
               
                 Deoxymethasone 
                 Double 
                 F 
                 H 
                 OH 
                 alpha CH 3   
                 H 
                 OH 
               
               
                   
                 bond 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Amcinonide 
                 Double 
                 F 
                 H 
                 OH 
                 Ciclopentanone ketal 
                 MeCOO 
               
               
                   
                 bond 
               
               
                 Desonide 
                 Double 
                 H 
                 H 
                 OH 
                 acetone ketal 
                 OH 
               
               
                   
                 bond 
               
               
                 Budesonide 
                 Double 
                 H 
                 H 
                 OH 
                 nPr-CHO ketal 
                 OH 
               
               
                   
                 bond 
               
               
                 Flunisolide 
                 Double 
                 H 
                 F 
                 OH 
                 Acetone ketal 
                 OH 
               
               
                   
                 bond 
               
               
                 Fluocinolone acetonide 
                 Double 
                 F 
                 F 
                 OH 
                 Acetone ketal 
                 OH 
               
               
                   
                 bond 
               
               
                 Fluocinonide 
                 Double 
                 F 
                 F 
                 OH 
                 Acetone ketal 
                 MeCOO 
               
               
                   
                 bond 
               
               
                 Triamcinolone acetonide 
                 Double 
                 F 
                 H 
                 OH 
                 Acetone ketal 
                 OH 
               
               
                   
                 bond 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 16 alpha hydroxy 
                 Double 
                 H 
                 H 
                 OH 
                 OH 
                 OH 
                 OH 
               
               
                 prednisolone 
                 bond 
               
               
                   
               
            
           
         
       
     
     Archiv for Pharmaci og Chemi, Scientific Edition 8, 1980, 187-206 and the Journal of Organic Chemistry, 1963, 28, 2001, describe the oxidation of the alcohol at the 21 carbon (“21-OH”) to the corresponding aldehyde (“21-CHO”). As reported by these references, the α-ketolic side chain of steroids is easily oxidized by air to give the corresponding 21-dehydro derivative (steroid-glyoxal), which undergoes further oxidative degradation. This oxidation takes place mostly under neutral or alkaline conditions and is catalyzed by traces of metals, such as iron. 
     Thus, it is difficult to control and/or avoid the formation of the 21-aldehyde impurity, which will eventually contaminate the steroid. 
     Impurities in steroids are undesirable and, in extreme cases, might even be toxic to a patient being treated with a dosage form containing the steroid. Because of the toxicity of the 21-aldehyde impurities of steroids, their established limit in the API is often stated to be less than 0.10%, see, for example, the 2008 U.S. Pharmacopoeia wherein the limit for 21-dehydro budesonide in Budesonide is defined as 0.07% (ref. USP 31, Vol. 2, p. 1565-6). 
     As known by those skilled in the art, the management of process impurities is greatly enhanced by understanding their chemical structures and synthetic pathways and by identifying the parameters that influence the amount of impurities in the final product. 
     Thus, methods that would allow for easy purification of steroids from their 21-aldehyde impurities as well as stabilize the steroids against further oxidation are needed. The present invention addresses this need, providing steroids free of 21-aldehyde oxidation products, and means for preparation thereof. 
     SUMMARY OF THE INVENTION 
     In one embodiment, the present invention provides a method for purifying air-sensitive steroids of the following formula I 
     
       
         
         
             
             
         
       
     
     from the 21-aldehyde steroid impurity, in either hydrated or non-hydrated form, of the following formula, 
     
       
         
         
             
             
         
       
     
     comprising combining an air-sensitive steroid with an amino acid or an analogue thereof of formula II: 
     
       
         
         
             
             
         
       
     
     to obtain a mixture; and 
     recovering the air sensitive steroid from the mixture to obtain a purified air sensitive steroid, 
     wherein A and B are each H or together represent a double bond;
         C is H, F, Cl, or OH;   D is H, CH 3 , Cl, or F;   E is H, OH or a carbonyl;   G is H, OH, CH 3  or an oxygen atom that together with M forms a ketal or acetal with a C 1 -C 6  linear or branched or cyclic carbonyl;   M is H, OH, an OH esterified with a C 1 -C 6  linear or branched mono or bicarboxylic acid or with benzoic acid, or an oxygen atom that together with G forms a ketal or acetal with a C 1 -C 6  linear or branched or cyclic carbonyl;   Q is OH or an OH esterified with a C 1 -C 6  linear or branched carboxylic acid;   A 1  is SH or OH;   B 1  is NHR 2  or NHCOR 4;      R 1  is H, COOH or COR 5;      R 2 , R 3  are each independently H, CH 3  or C 2 H 5 ;   R 4  is a linear or branched C 1 -C 4  alkyl;   R 5  is a peptide of the following formula:       

     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein X and Y are each independently substituents of an amino-acid; N 1  and 
                 N 2  are each independently NH or N 1  forms together with X and/or N 2  forms together with Y an amino acid substituent; and 
                 m=0 or 1 or 2; and 
               
             
             n=0 or 1. 
           
         
       
    
     In another embodiment, the present invention provides an adduct of formula III, 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             A 2 =S or O; 
             B 2 ═NR 2  or NCOR 4 ; 
             A and B are each H or together represent a double bond; 
             C is H, F, Cl, or OH; 
             D is H, CH 3 , Cl, or F; 
             E is H, OH or a carbonyl; 
             G is H, OH, CH 3  or an oxygen atom that together with M forms a ketal or acetal with a C 1 -C 6  linear or branched or cyclic carbonyl; 
             M is H, OH, an OH esterified with a C 1 -C 6  linear or branched mono or bicarboxylic acid or with benzoic acid, or an oxygen atom that together with G forms a ketal or acetal with a C 1 -C 6  linear or branched or cyclic carbonyl; 
             R 1  is H, COOH or COR 5;    
             R 2 , R 3  are each independently H, CH 3  or C 2 H 5 ; 
             R 4  is a linear or branched C 1 -C 4  alkyl; 
             R 5  is a peptide of the following formula: 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein X and Y are each independently substituents of an amino-acid; N 1  and 
                 N 2  are each independently NH or N 1  forms together with X and/or N 2  forms together with Y an amino acid substituent; and 
                 m=0 or 1 or 2; and 
               
             
             n=0 or 1. 
           
         
       
    
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides a method for purifying steroids, especially air-sensitive steroids, from their corresponding oxidation impurity, a 21-aldehyde derivative. 
     During synthesis of these steroidal products, especially industrial synthesis, the following two scenarios occur: 
     1. Oxygen is present, even if an inert atmosphere is used during process operations for manufacturing the steroidal product, because of its solubility in water and in the organic solvents commonly used in the manufacturing processes. Common industrial process operations, wherein the steroid is exposed to air, are, for instance: filtration of the solid after crystallization, discharge from a Buchner funnel or from a centrifuge, loading into the vacuum oven, collection of the dried product and packaging, and storage in a closed container; in addition, if the product is micronized, the package is reopened and subsequent loading, discharging, and packaging may again occur in the presence of air. Moreover, micronization enlarges the surface area of the solid, thereby increasing possible exposure of the steroid in particles to air and to oxidation. 
     2. Traces of metals such as Chromium, Nickel, Molybdenum and Iron may be present in the raw materials or in the equipment that is used to prepare the steroidal product. These metals catalyze the oxidation of the steroid (see example 16). 
     Thus, the 21-aldehyde impurity that is difficult to purify from by conventional methods (for example, see examples 2 and 9), can be formed at any stage of the preparation, packaging and storage of the steroid. 
     Due to structure similarity between the aldehyde impurity and the steroid, there is a need for repetitive purification steps. However, this decreases the yield and can lead to the exposure of the product to oxygen, thereby possibly partially re-forming the 21-aldhyde impurity. 
     As is known in the field of steroids and their production, the term “air sensitive” in reference to steroids refers to steroids with an α-ketolic side chain (Q=OH in Table 1) or esters prepared from intermediates with an α-ketolic side chain, thereof of the following formula 
     
       
         
         
             
             
         
       
     
     wherein A and B are each H or together represent a double bond;
         C is H, F, Cl or OH;   D is H, CH 3 , Cl or F;   E is H, OH or a carbonyl;   G is H, OH, CH 3  or an oxygen atom that together with M forms a ketal or acetal with a C 1 -C 6  linear or branched or cyclic carbonyl;   M is H, OH, an OH esterified with a C 1 -C 6  linear or branched mono or bicarboxylic acid or with benzoic acid or an oxygen atom that together with G forms a ketal or acetal with a C 1 -C 6  linear or branched or cyclic carbonyl; and   Q is OH or an OH esterified with a C 1 -C 6  linear or branched carboxylic acid,
 
that, when exposed to even trace amounts of air, the 21-OH group (Q=OH) is oxidized, providing the 21-aldehyde steroid analogue. Examples of such steroids are listed in Table 1.
       

     As known in the art, the term an “amino acid substituent” is the side-chain of an amino acid. 
     As used herein, the term “room temperature” refers to a temperature of about 20° C. to about 30° C., more preferably about 20° C. to about 25° C. 
     As used herein, the term “percent” or “%” refers to the percent area as measured by HPLC, unless otherwise indicated. 
     As used herein, dipolar aprotic solvent refers to a solvent lacking acidic hydrogens and containing at least one polarized bonds between carbon and a heteroatom, typically a multiple bond between carbon and either oxygen or nitrogen. 
     As used herein, apolar solvent refers to solvents having a low dielectric constant and not miscible with water. 
     In one embodiment, the present invention provides a method of purifying air-sensitive steroids of the following formula I 
     
       
         
         
             
             
         
       
     
     from the 21-aldehyde steroid impurity, in either hydrated or non-hydrated form, of the following formula, 
     
       
         
         
             
             
         
       
     
     comprising combining an air-sensitive steroid with an amino acid or an analogue thereof of formula II: 
     
       
         
         
             
             
         
       
     
     to obtain a mixture; and 
     recovering the air sensitive steroid from the mixture to obtain a purified air sensitive steroid, 
     wherein A and B are H or together represent a double bond;
         C is H, F, Cl, or OH;   D is H, CH 3 , Cl, or F;   E is H, OH a carbonyl;   G is H, OH, CH 3  or an oxygen atom that together with M forms a ketal or acetal with a C 1 -C 6  linear or branched or cyclic carbonyl;   M is H, OH, an OH esterified with a C 1 -C 6  linear or branched mono or bicarboxylic acid or with benzoic acid or an oxygen atom that together with G forms ketal or acetal with C 1 -C 6  linear or branched or cyclic carbonyl compounds;   Q is OH or an OH esterified with a C 1 -C 6  linear or branched carboxylic acid;   A 1  is SH or OH;   B 1  is NHR 2  or NHCOR 4;      R 1  is H, COOH, COR 5;      R 2 , R 3  are each independently H, CH 3 , C 2 H 5 ;   R 4  is a C 1 -C 4  linear or branched alkyl;   R 5  is a peptide of the following formula:       

     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein X and Y are each independently substituents of an amino acid; N 1  and 
                 N 2  are each independently NH or N 1  forms together with either X and/or N 2  forms together with Y an amino acid substituent; and 
                 m=0,1 or 2; and 
               
             
             n=0 or 1. 
           
         
       
    
     Typically, G and M, N 1  and X, N 2  and Y are connected to each other directly or via at least one atom, thus forming together a ketal or acetal (G and M) or an amino acid substituent (N 1  and X, N 2  and Y), respectively. 
     Typically, the air-sensitive steroid is a steroid, such as any one of those listed in Table 1, which is contaminated with its corresponding 21-aldehyde impurity. 
     Preferably, the air-sensitive steroid is Flunisolide, 16-alpha-hydroxyprednisolone, Budesonide or Deoxymethasone. More preferably the steroid is 16-alpha-hydroxyprednisolone or Budesonide, most preferably the steroid is Budesonide. 
     Preferably, A 1  in formula II is SH. 
     Preferably, B 1  is NHR 2 , more preferably NH 2 . 
     Preferably, R 1  is COOH or COR 5 , more preferably, COOH. 
     Preferably, R 2  and R 3  are H or CH 3 , more preferably H. 
     Preferably, R 4  is CH 3    
     Preferably, X and Y are substituents of the following amino acids: Alanine, Asparagine, Aspartate, Arginine, Cysteine, Glutamate, Glutamine, Glycine, Histidine, Isoleicine, Leucine, Lysine, Methionine, Phenylalanine, Serine, Threonine, Tryptophane, Tyrosine and Valine and Proline. 
     Preferably, the substituent is a substituent of Glycine or Alanine, more preferably of Glycine. 
     The aldehyde can exist in the carbonylic form or the hydrated form, depending on the conditions, for example, the carbonylic form is more predominant under anhydrous conditions. 
     The amino acid or analogue thereof of formula II can be either optically or not optically active, i.e. not optically active is without a chiral center or a racemic mixure. Also commercial salts, such as hydrochloride salts, can be used as a source for the amino acid and analogues thereof, by reacting them with a base. 
     The amino acid or analogue thereof of formula II reacts with the aldehyde impurity (hydrated or in the carbonylic form) generating an adduct of formula III, 
     
       
         
         
             
             
         
       
     
     wherein 
     A 2 =S or O; 
     B 2 ═NR 2  or NCOR 4 ; and 
     R 1 , R 2 , R 3 , R 4 , A, B, C, D, E, M and G are as described before, which can be easily removed by extractions and/or by crystallization, with minor loss of yield. 
     Further, the amino acid or analogue thereof of formula II forms complexes with the metals thus assisting in avoiding the catalysis of the oxidation reaction that is performed by the free form of the metal. 
     Preferably, A 2  in formula III is S. 
     The adduct of formula III wherein the steroid is Budesonide and the amino acid or analogue thereof of formula II is L-cysteine, prepared as a pure sample by conventional methods from the corresponding 21-aldehyde, is characterized by data selected from the group consisting of  1 H NMR (DMSO, 400.13 MHz, DMSO, 303° K): diastereoisomer 1: δ 7.301(d,1H), 6.158(dd, J 10.1,1.9,1H), 5.916(dd, J 1.9,1.2 1H), 2.518(m,1H), 2.287(dd,J 13.5,3.2 1H), 1.968(m,1H), 1.111(m,1H), 2.012(m, 1H), 1.027(dd, J 11.3, 3.4, 1H), 4.285(m, 1H), 1.864(dd, J 13.7, 3.7, 1H), 1.768(dd, J 13.7. 2.5, 1H), 1.569(m, 1H), 1.779(m, 1H), 1.601(m, 1H), 5.023(d, J 7.5, 1H), 0.865(s,3H), 1.375(s,3H), 4.872(d, J 4.0, 1H), 5.149(t, J 5.3, 1H), 1.595(m, 2H), 1.356(m, 2H), 0.875(t, J 7.4, 3H), 5.241(s, 1H), 2.974(d, J 5.6, 2H), 4.409 (t, J 5.6, 1H), 12.746(br s, 1H); diastereoisomer 2: 7.313(d, J 10.1 1H), 6.163(dd, J 10.1, 1.9, 1H), 5.917(dd, J 1.9,1.1,1H), 2.526(m, 1H), 2.294(ddd, J 13.5, 4.7, 1.7,1H), 2.001(m, 1H), 1.023(m, 1H), 2.089(m, 1H), 0.963(m, 1H), 4.134(m, 1H), 1.851(dd, J 13.5, 3.4 1H), 1.760(dd, J 13.5. 2.4, 1H), 1.554(m, 1H), 1.630(m, 1H), 1.519(m, 1H), 4.737(d, J 4.5, 1H), 0.815(s, 3H), 1.381(s, 3H), 4.929(d, J 4.1, 1H), 4.809(t, J 4.6, 1H), 1.546(m, 2H), 1.364(m, 2H), 0.874(t, J 7.4, 3H), 5.266(s, 1H), 3.020(dd, J 10.2, 4.2, 1H), 2.957(dd, J 10.2, 6.6, 1H), 4.424(dd, J 6.6, 4.2, 1H), 12.835(br, s, 1H); and LC/MS: at m/z 532 the molecular ion is present. 
     Preferably, the amino acid or analogue thereof of Formula II is chosen from the list consisting of L-cysteine, cysteamine, penicillamine, cysteyl-glycine (Cys-Gly), homocysteine and L-serine, as depicted in the following table. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Compound 
                 Structure 
               
               
                   
                   
               
             
            
               
                   
                 L-cysteine 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 cysteamine 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 DL-penicillamine 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 Cys-Gly 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 DL-homocysteine 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
               
                   
                 L-serine 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
                   
               
            
           
         
       
     
     The preferred amino acid or an analogue thereof of Formula II is L-cysteine. 
     Preferably, the purification comprises combining the air-sensitive steroid, an amino acid or an analogue thereof of formula II and a solvent selected from the group consisting of an alcohol, an ester, a ketone, a haloalkane, a dipolar aprotic solvent, a nitrile, an ether, and mixtures thereof, and mixtures thereof with water, to obtain a solution also comprising the adduct of formula III, from which the air sensitive steroid is recovered to obtain such purified steroid. 
     The amino acid or analogue thereof of formula II is present in the solution in sufficient excess to remove the 21-aldehyde impurity. Preferably, the mole ratio of the amino acid or analogue thereof of formula II to aldehyde is from about 2:1 to about 10:1, more preferably it is from about 3:1 to about 5:1, most preferably it is about 4:1, respectively. 
     Preferably, the alcohol is a linear or branched C 1 -C 5  alcohol, more preferably a C 1 -C 4  alcohol, even more preferably methanol, ethanol, isopropanol or isobutanol, most preferably methanol. 
     Preferably, the ester is a C 2 -C 5  ester, more preferably a C 3 -C 5  ester, most preferably ethyl acetate. 
     Preferably, the ketone is C 2 -C 5  ketone, more preferably acetone, butanone or isopropyl methyl ketone, most preferably acetone. 
     Preferably, the haloalkane is a C 1 -C 5  haloalkane, more preferably a C 1 -C 2  haloalkane, most preferably dichloromethane. 
     Preferably, the dipolar aprotic solvent is a C 2 -C 5  dipolar aprotic solvent, more preferably dimethylsulfoxide (DMSO), dimethylformamide (DMF) or dimethylacetamide (DMA), most preferably dimethylformamide. 
     Preferably the nitrile is a C 1 -C 5  nitrile, more preferably C 1 -C 3  nitrile, most preferably acetonitrile. 
     Preferably, the ether is a C 4 -C 6  ether, more preferably tetrahydrofuran, 2-methyltetrahydrofuran or dioxane, most preferably tetrahydrofuran. 
     Preferably, mixtures with water comprise an alcohol, a haloalkane and water, a dipolar aprotic solvent and water, an ether and water, a ketone and water, an alcohol and water, more preferably, the mixture is a mixture of methanol, dichloromethane and water, DMF and water, THF and water, dioxane and water, methanol and water or acetone and water, most preferably, the mixture is a mixture of methanol, dichloromethane and water. 
     Optionally, the solution can contain an acid, which can catalyze the formation of the adduct in the reaction of the aldehyde with the amino acid or analogue thereof of formula II. Preferably, the acid is an organic acid, more preferably, acetic acid, formic acid or paratoluene sulfonic acid (PTSA), most preferably, acetic acid. 
     Preferably, the mole ratio of catalyst:amino acid or analogue thereof of formula II is about 1:10 to about 5:1, more preferably 4:1, even more preferably 2:1. 
     Combining the air sensitive steroid and the solvent can provide a solution or a suspension, depending on the solvent. 
     To aid in dissolution, the suspension can be heated. Preferably, the dissolution is performed at a temperature of about room temperature to about 70° C., more preferably at about 40° C. to about 60° C., most preferably at about 60° C. depending on the solvent. Preferably, the dissolution is performed for about 30 minutes to about 2 hours, more preferably for about one hour. 
     The completion of the reaction can be determined by HPLC, by monitoring the increase in the amount of the adduct or the disappearance of the aldehyde. 
     Preferably, the concentration of the air sensitive steroid in the solvent is from about 10 g/l to about 200 g/l, more preferably it is about 100 g/l. 
     Prior to recovering the steroid, the reaction mixture can be concentrated. Preferably, the reaction mixture is a solution. 
     The recovery of the purified steroid may be done by separating the air sensitive steroid from the adduct of formula III. Preferably, the recovery can be done either by precipitation of the purified steroid from the reaction solution, followed by separation of it from the remaining soluble adduct to obtain a purified steroid, or by extracting the obtained adduct from the reaction solution. 
     Preferably, the precipitation is done by combining the reaction solution with an anti-solvent to obtain a second mixture, optionally followed by either cooling or by concentrating and cooling the obtained second mixture. 
     Preferably, the anti-solvent is water. 
     Preferably, concentration is done at a temperature of about 40° C. to about 80° C., more preferably of about 60° C. to about 70° C., most preferably at about 70° C. 
     Preferably, cooling is done to a temperature of about room temperature to about 0° C., more preferably, to about 0° C. 
     The precipitated purified steroid can then be filtered, i.e., separated from remaining solution containing the adduct of formula III, followed by washing the filtered purified steroid. 
     Preferably, the extraction is performed by combining the reaction solution with an acidic or basic aqueous solution; separating the obtained phases, wherein the organic phase contains the purified steroid and the aqueous phase contains the adduct and/or its decomposition products and the base or acid; washing the organic phase with water, and isolating the purified steroid from the organic phase. Preferably, the isolation can be done by either removing the solvent or by crystallization. 
     Preferably, the basic aqueous solution is an aqueous solution of sodium carbonate or sodium hydrogen carbonate, more preferably of sodium hydrogen carbonate. 
     Preferably, the pH of the basic aqueous solution is greater than 7, more preferably it is greater than 7 to about 8. 
     Preferably, the pH of the acidic aqueous solution is less than 7, more preferably it is less than 7 to about 5, most preferably less than 7 to about 6. Preferably, the acid is HCl. 
     Preferably, the crystallization is done by combining the organic phase with an anti-solvent, thus providing a suspension comprising the crystalline steroid. The precipitated steroid can then be filtered from the suspension. 
     Preferably, the anti-solvent is water or a C 4 -C 8  apolar solvent, more preferably the apolar solvent is a C 5 -C 8  alkane or a C 4 -C 6  ether. Preferably the C 5 -C 8  alkane is hexane or heptane. Preferably, the C 4 -C 6  ether is diisopropyl ether or diethyl ether or methyl tertbutyl ether. Most preferably the anti-solvent is water. 
     Preferably, the levels of the 21-aldehyde impurity in the purified steroid obtained by the purification process of the invention is less than or equal to about 0.15%, more preferably less than or equal to about 0.10%, most preferably less than or equal to about 0.01%, for example, between about 0.005% and about 0.10%, between about 0.008% and about 0.07%, between about 0.009% and about 0.05%. 
     Having described the invention with reference to certain preferred embodiments, other embodiments will become apparent to one skilled in the art from consideration of the specification. The invention is further defined by reference to the following examples describing in detail the purification of the air sensitive steroid using an amino acid or analogue thereof. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the invention 
     EXAMPLES 
     Analytical Methods Used 
     HPLC 
     The amount of air-sensitive steroid and/or its 21 aldehyde impurity were determined using HPLC. Common HPLC methods for these steroids and their 21-aldehyde impurities may be found in various monographs see for example for Budesonide and its 21-aldehyde impurity a HPLC method is reported in USP31-NF26, page 1565 (detection limit 0.02%), for Fluocinolone acetonide a HPLC method in European Pharmacopoeia (EP) 6.0, page 1915, and for Methylprednisolone a HPLC method in European Pharmacopoeia (EP) 6.0, page 2393. 
     Mass Spectrometry 
     
         
         Instrument: Finnigan LCQ (ion-trap) 
         Method: ESI, positive ion mode 
         Conditions: Source Voltage (kV) 3.50, Source Current (μA) 1.85, Capillary Voltage (V) 17.86, Capillary Temp (° C.): 190.00. Mobile phase 10 mM ammonium fornate in 70% aqueous methanol. 
       
    
     Example 1 
     Purification of Flunisolide With cysteine 
     20 g Flunisolide (6α-fluoro-11β,6α, 17, 21-tetrahydroxy-pregna-1,4-dien-3,20-dione 16,17-acetonide) (0.046 mol) containing 0.15% 21-dehydro impurity (aldehyde) were dissolved in 225 ml acetone at 40° C. 27 mg L-cysteine (0.22 mmol) were added and the solution was left stirring for 1 hour. The mixture was then poured into water (500 ml), concentrated under vacuum, cooled to room temperature, filtered and washed with water. The crystallized product contained 0.09% dehydro derivative (the 21-aldehyde). 
     Comparative Example 2 
     Purification of Flunisolide Without cysteine 
     20 g Flunisolide (6α-fluoro-11β,16α, 17, 21-tetrahydroxy-pregna-1,4-dien-3,20-dione 16,17-acetonide) (0.046 mol) containing 0.15% 21-dehydro impurity (aldehyde) were dissolved in 225 ml acetone at 40° C. and the solution was left stirring for 1 hour. The mixture was then poured into water (500 ml), concentrated under vacuum, cooled to room temperature, filtered and washed with water. The crystallized product contained 0.15% dehydro derivative (the 21-aldehyde): no purification occurred. 
     Example 3 
     Purification of Flunisolide With cysteine 
     25 g Flunisolide (6α-fluoro-11β,16α, 17, 21-tetrahydroxy-pregna-1,4-dien-3,20-dione 16,17-acetonide) (0.057 mol) containing 0.15% 21-dehydro impurity (aldehyde) were dissolved in 350 ml methanol at 40° C. 40 mg L-cysteine (0.33 mmol) were added and the solution was allowed to stir at 40° C. for 1 hour. Water was added (585 ml) and crystallization occurred; the mixture was cooled to 0° C., the solid was filtered and washed. The crystallized product contained 0.07% dehydro derivative (the 21-aldehyde). 
     Example 4 
     Purification of 16-alpha-hydroxyprednisolone With L-cysteine 
     20 g of 16-alpha-hydroxyprednisolone (0.053 mol) were dissolved at 40° C. in a mixture of 80 ml methanol, 40 ml dichlorometane and 40 ml water. This solution was found to contain 0.11% (HPLC area) of 21-dehydro compound (aldehyde). Then 30 mg L-cysteine (0.25 mmol) were added and the solution was allowed to stir at 40° C. for 1 hr. The treated solution was found to contain a reduced amount of 21-dehydroderivative (0.03% HPLC area). After concentration under vacuum and water addition, the product was filtered and washed. The crystallized product contained &lt;0.01% 21-dehydro derivative (the 21-aldehyde). 
     Example 5 
     Purification of Budesonide With L-cysteine 
     20 g Budesonide (0.046 mol) containing 0.20% 21-dehydro derivative (HPLC area) and 50 mg L-cysteine (0.41 mmol) were dissolved in 250 ml methanol at 60° C. After stirring 1 hour at room temperature the product was crystallized by addition of 420 ml water. The mixture was then cooled to 0° C., the solid was filtered, washed with water and analyzed by HPLC: the 21-dehydro derivative (the 21-aldehyde) was not detected. 
     Example 6 
     Purification of Deoxymethasone With N-acetyl-cysteine 
     15 g of Deoxymethasone (9-Fluoro-11-beta, 21-dihydroxy-16-alpha-methylpregna-1,4-diene-3,20-dione) (0.040 mol) containing 0.61% 21-dehydro derivative (HPLC area) were dissolved in 150 ml dichloromethane and treated with 75 mg N-acetyl-cysteine (0.46 mmol). After stirring at room temperature for 30 minutes, the solution was washed with sodium hydrogen carbonate 2% in water (100 ml), then with water (50 ml). The organic phase was concentrated under vacuum, added of 75 ml methanol, concentrated at 70° C. to a reduced volume, cooled and finally treated with 150 ml water to crystallize the product. The solid was filtered, dried and analyzed by HPLC: the content of 21-dehydro derivative (the 21-aldehyde) was 0.28%. 
     Example 7 
     Purification of Deoxymethasone With D,L-cysteine 
     15 g of Deoxymethasone (9-Fluoro-11-beta, 21-dihydroxy-16-alpha-methylpregna-1,4-diene-3,20-dione) (0.040 mol) containing 0.61% 21-dehydro derivative (HPLC area) were dissolved in 150 ml dichloromethane and treated with 102 mg D,L-cysteine (0.84 mmol). After stirring at room temperature for 30 minutes, the solution was washed water (100 ml), then with aqueous sodium hydrogen carbonate (100 ml), then with water (150 ml). The organic phase was concentrated under vacuum, added of 75 ml methanol, concentrated at 70° C. to a reduced volume, cooled and finally treated with 150 ml water to crystallize the product. The solid was filtered, dried and analyzed by HPLC: the content of 21-dehydro derivative (the 21-aldehyde) was 0.06%. 
     Example 8 
     Purification of Deoxymethasone With D,L-cysteine 
     15 g of Deoxymethasone (9-Fluoro-11-beta, 21-dihydroxy-16-alpha-methylpregna-1,4-diene-3,20-dione) (0.040 mol) containing 0.19% 21-dehydro derivative (HPLC area) were dissolved in 60 ml methanol and treated with 100 mg D,L-cysteine (0.83 mmol). After stirring at 45° C. for 30 minutes, the solvent was distilled off to small volume. The residue was cooled to room temperature, dissolved in dichloromethane (150 ml), washed with aqueous sodium hydrogen carbonate (150 ml), then with water (150 ml). The organic phase was concentrated under vacuum, was added of 75 ml methanol, concentrated at 70° C. to a reduced volume, cooled and finally treated with 150 ml water to crystallize the product. The solid was filtered, dried and analyzed by HPLC: the content of 21-dehydro derivative (the 21-aldehyde) was 0.02%. 
     General Procedure: Examples 9-15 
     Purification of Budesonide With and Without cysteine-analogues 
     1 g Budesonide (“Budesonide start”) was mixed with 0.05 g of the corresponding 21-aldehyde in 12.5 ml methanol resulting in Budesonide having 0.36% of the aldehyde impurity; the cysteine-analogue was added in a quantity of 4 moles/mole aldehyde. After 1 h at 60° C., 21 ml water were added, the mixture was cooled to 0° C., the product filtered, washed with water, dried and analyzed by HPLC. 
     Comparative Example 9 
     Purification of Budesonide Without L-cysteine 
     1 g Budesonide (2.32 mmol) containing 0.36% 21-dehydro derivative (HPLC area) was suspended in 12.5 ml methanol at 60° C. After heating the suspension turned into a solution. After 1 hour the product was crystallized by addition of 21 ml water. The mixture was then cooled to 0° C., the solid was filtered, washed with water and analyzed by HPLC: the 21-dehydro derivative was 0.29%. 
     Example 10 
     Purification of Budesonide With L-cysteine 
     1 g Budesonide (2.32 mmol) containing 0.36% 21-dehydro derivative (HPLC area) and 6 mg L-cysteine (0.049 mmol) were suspended in 12.5 ml methanol at 60° C. After heating the suspension turned into a solution. After 1 hour the product was crystallized by addition of 21 ml water. The mixture was then cooled to 0° C., the solid was filtered, washed with water and analyzed by HPLC: the 21-dehydro derivative was 0.07%. 
     Example 11 
     Purification of Budesonide With DL-penicillamine 
     1 g Budesonide (2.32 mmol) containing 0.36% 21-dehydro derivative (HPLC area) and 8 mg DL-penicillamine (0.054 mmol) were suspended in 12.5 ml methanol at 60° C. After heating the suspension turned into a solution. After 1 hour the product was crystallized by addition of 21 ml water. The mixture was then cooled to 0° C., the solid was filtered, washed with water and analyzed by HPLC: the 21-dehydro derivative was 0.04%. 
     Example 12 
     Purification of Budesonide With cys-gly 
     1 g Budesonide (2.32 mmol) containing 0.36% 21-dehydro derivative (HPLC area) and 12 mg cys-gly (0.057 mmol) were suspended in 12.5 ml methanol at 60° C. After heating the suspension turned into a solution. After 1 hour the product was crystallized by addition of 21 ml water. The mixture was then cooled to 0° C., the solid was filtered, washed with water and analyzed by HPLC: the 21-dehydro derivative was not detected. 
     Example 13 
     Purification of Budesonide With DL-homocysteine 
     1 g Budesonide (2.32 mmol) containing 0.36% 21-dehydro derivative (HPLC area) and 7.7 mg DL-homocysteine (0.054 mmol) were suspended in 12.5 ml methanol at 60° C. After heating the suspension turned into a solution. After 1 hour the product was crystallized by addition of 21 ml water. The mixture was then cooled to 0° C., the solid was filtered, washed with water and analyzed by HPLC: the 21-dehydro derivative was 0.03%. 
     Example 14 
     Purification of Budesonide With L-serine 
     1 g Budesonide (2.32 mmol) containing 0.36% 21-dehydro derivative (HPLC area) and 7 mg L-serine (0.067 mmol) were suspended in 12.5 ml methanol at 60° C. After heating the suspension turned into a solution. After 1 hour the product was crystallized by addition of 21 ml water. The mixture was then cooled to 0° C., the solid was filtered, washed with water and analyzed by HPLC: the 21-dehydro derivative was 0.08%. 
     Example 15 
     Purification of Budesonide With cysteamine and Acetic Acid 
     1 g Budesonide (2.32 mmol) containing 0.36% 21-dehydro derivative (HPLC area), 4.3 mg cysteamine (0.053 mmol) and 1.1 mg acetic acid (0.017 mmol) were suspended in 12.5 ml methanol at 60° C. After heating the suspension turned into a solution. After 1 hour the product was crystallized by addition of 21 ml water. The mixture was then cooled to 0° C., the solid was filtered, washed with water and analyzed by HPLC: the 21-dehydro derivative was 0.12%. 
     Example 16 
     Stabiliy of Budesonide 
     Budenoside from three production lots prepared in stainless steel reactors was compared to a lot of Budenoside produced in such a reactor and purified with cysteine according to example 5. All samples were stored in double plastic containers. The samples that were not treated with cysteine were kept at a temperature of 25±2° C. and relative humidity of 60%. The sample treated with cysteine was stored at a temperature that varied from about 0° C. to about 40° C. with no humidity control. As is shown in the table below the amount of the 21-aldehyde impurity in the Budenoside purified according to example 5 is less than 0.07% even after 6 years of storage. The aldehyde content of all samples was measured by HPLC. 
     
       
         
           
               
               
               
               
               
            
               
                   
               
               
                   
                   
                   
                   
                 Budenoside purified 
               
               
                   
                 Budenoside 
                 Budenoside 
                 Budenoside 
                 according to example 5 
               
               
                 Time 
                 HPLC Area % 
                 HPLC Area % 
                 HPLC Area % 
                 HPLC Area % 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (Years) 
                 21 Aldehyde 
                 Budenoside 
                 21 Aldehyde 
                 Budenoside 
                 21 Aldehyde 
                 Budenoside 
                 21 Aldehyde 
                 Budenoside 
               
               
                   
               
               
                 0 
                 nd 
                 99.0 
                 nd 
                 99.4 
                 nd 
                 99.2 
                 nd 
                 99.76 
               
               
                 1 
                 nd 
                 98.8 
                 nd 
                 98.9 
                 nd 
                 99.0 
               
               
                 2 
                 0.1 
                 98.7 
                 0.6 
                 98.5 
                 &lt;0.1 
                 99.1 
               
               
                 5 
                 0.2 
                 98.4 
                 0.9 
                 97.8 
                 &lt;0.2 
                 98.2 
               
               
                 6 
                   
                   
                   
                   
                   
                   
                 0.02 
                 99.61 
               
               
                   
               
               
                 nd = not detected