Abstract:
Racemization-free methods are disclosed for the synthesis of carfilzomib. Novel intermediates and methods of making carfilzomib employing fragment condensation using the novel intermediates are disclosed. Amorphous carfilzomib and methods of making same are disclosed.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. patent application Ser. No. 14/923,068 filed Oct. 26, 2015 and claims the benefit of U.S. Provisional Application No. 62/068,928 filed Oct. 27, 2014, the entireties of which are incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The presently disclosed subject matter relates to the synthesis of carfilzomib. 
     BACKGROUND 
     Carfilzomib, an epoxomicin derivative, is a selective proteasome inhibitor. Carfilzomib is used to treat patients with multiple myeloma who have already been treated with at least two other medications. 
     
       
                 
         
             
             
         
      
     
     SUMMARY OF THE INVENTION 
     Novel methods and intermediates are disclosed for the production of carfilzomib. Processes disclosed herein may be employed to produce amorphous form carfilzomib. 
     In one or more embodiments methods employ a fragment-based approach involving active esters. For example, active esters obtained from hydroxyl-benzotriazoles, hydroxy-aza-benzotriazoles, succinimide esters, substituted phenols, etc. may be employed. The methods are efficient and highly reproducible in large scale industrial processes. The methods employ fragment condensation using novel synthetic intermediates and provide racemization-free approaches. The disclosed methods produce carfilzomib in a reduced number of steps compared to existing methods in the literature. In accordance with an embodiment powder x-ray diffraction reveals the resulting carfilzomib active pharmaceutical ingredient (API) made according to a method disclosed herein is in amorphous form. 
     In one embodiment compounds having the formula I and pharmaceutically active esters and salts thereof are disclosed wherein Y is  1 -hydroxybenzotriazole, azabenzotriazole-succinimidyl, pentafluorophenyl, trichlorophenyl, nitrophenyl, pentachlorophenyl, 3,4-dehydro-4-oxo-1,2,3-benzotriazinyl (Dhbt), or fluorenylmethyl. 
     
       
                 
         
             
             
         
      
     
     Compounds of formula I may include active esters obtained from corresponding hydroxy compounds and substituted phenols. Compounds of formula I may be employed as intermediates in methods disclosed herein for making carfilzomib. 
     In further embodiments, methods of making compounds of formula I and derivatives thereof are disclosed. 
     In some embodiments active esters of formula I may be synthesized using reagents such as carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N′-dicyclohexylcarbodiimide (DCC), and N,N′-diisopropylcarbodiimide (DIPC). 
     In some embodiments, methods involve obtaining a free acid of a compound of formula I by various chemical methods and activating a resulting intermediate using a hydroxy and/or phenolic compound. Examples of such chemical methods include but are not limited to bis-silylation, using silylating agents, an organic base, and/or hydrolysis of esters by using alkali metal hydroxides including NaOH, KOH, LiOH and their corresponding carbonates, etc. 
     Compounds of formula I may be isolated using various organic solvents including but not limited to methanol, ethanol, acetonitrile, MDC, chloroform, ethylacetate, etc. 
     In other emboditments, compounds having the general structure IA, are disclosed 
                                
wherein Y, R 1  and R 2  are as defined in Table I for compounds having the formulas II, III, and IV.
 
     
       
         
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Compound 
                 R 1   
                 R 2   
               
               
                   
               
             
             
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 Chloro 
                 O—Y 
               
               
                 II 
                   
                   
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 Morpholino 
                 O—Y 
               
               
                 III 
                   
                   
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 Morpholino 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 IV 
               
               
                   
               
               
                 Y = 1-hydroxyberizotriazole, azabenzotriazole, succinimidyl, pentafluorophenyl, trichlorophenyl, nitrophenyl, pentachlorophenyl, 3,4-dehydro-4-oxo-1,2,3-benzotriazinyl (Dhbt), or fluoroenylmethyl. 
               
             
          
         
       
     
     The compounds of formula II, III, and IV and derivatives thereof, including stable active esters, are useful as intermediates in the synthesis of carfilzomib. 
     Formula II, III and IV active esters may be obtained from corresponding hydroxy compounds and phenols. 
     In further embodiments, methods of making compounds of formula II, III, and IV and derivatives thereof are disclosed. 
     In some embodiments active esters of formulas II, III, and IV may be synthesized using reagents such as CDI, EDC, DCC, and DIPC. 
     In some embodiments, methods involve obtaining a free acid of a compound of formula II, III, and IV by various chemical methods and activating a resulting intermediate using a hydroxy and/or phenolic compound. Examples of such chemical methods include but are not limited to bis-silylation, using silylating agents, an organic base, and/or hydrolysis of esters by using alkali metal hydroxides including NaOH, KOH, LiOH and their corresponding carbonates, etc. 
     Compounds of formula II, III, and IV may be isolated using various organic solvents including but not limited to methanol, ethanol, acetonitrile, MDC, chloroform, ethylacetate, etc. 
     Purification of compounds I, IL, III and IV may be effected using any suitable technique including but not limited to preparative RP-HPLC. 
     In other embodiments methods are disclosed for making carfilzomib by employing a compound of the formula I or derivatives thereof using a 2+2+1 fragment condensation. 
     In other embodiments methods are disclosed for making carfilzomib by employing a compound of the formula I (A) or derivatives thereof using a 3+2 fragment condensation. 
     In other embodiments methods are disclosed for making carfilzomib by employing a compound of the formula II or derivatives thereof using a 3+2 fragment condensation. 
     In other embodiments methods are disclosed for making carfilzomib by employing a compound of the formula III or derivatives thereof using a 3+2 fragment condensation. 
     In accordance with another embodiment, a method of making amorphous form carfilzomib is disclosed which employs the synthetic route: 
                                
wherein Y is 1-hydroxybenzotriazo, azabenzotriazolc, succinimidyl, pentafluorophenyl, trichlorophenyl, nitrophenyl, pentachlorophenyl 3,4-dehydro-4-oxo-1,2,3-benzotriazinyl (Dhbt), or fluorenylmethyl; X is HCl, trifluoroacetic acid (TFA), HCOOH, p-toluenesulfonic acid (TsOH), or methanesulfonic acid (MsOH); and R is H, methyl (Me), ethyl (Et), benzyl (Bzl), or isopropyl (iPr).
 
     In accordance with another embodiment, a method of making amorphous form carfilzomib is disclosed which employs the synthetic route: 
                                
wherein Y is 1-hydroxybenzotriazole, azabenzotriazole, succinimidyl, pentafluorophenyl, trichlorophenyl, nitrophenyl, pentachlorophenyl, 3,4-dehydro-4-oxo-1,2,3-benzotriazinyl (Dhbt), or fluorenylmethyl and X is HCl, TFA, HCOOH, TsOH, MsOH or isopropyl (IPr).
 
     Amorphous carfilzomib may be isolated using organic solvent extraction wherein the solvent may be MDC, chloroform, ethylacetate, diethylether, methyl-tert-butyl ether, diisopropyl ether, etc. 
     In another embodiment, amorphous form of carfilzomib is disclosed. X-ray powder diffraction data of amorphous carfilzomib made in accordance with a method disclosed herein is shown in  FIG. 1 . 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       For the purposes of illustration, there are forms shown in the drawings that are presently preferred, it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. 
         FIG. 1  is a graphical depiction of powder XRD data of a batch of carfilzomib made in accordance with at least one embodiment of the present disclosure. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following is a detailed description of the invention provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety. 
     Methods 
     The present invention describes in one aspect the synthesis of amorphous carfilzomib by employing active esters as novel intermediates. 
                                
wherein Y is 1-hydroxybenzotriazole, azabenzotriazole, succinimidyl, pentafluorophenyl, trichlorophenyl, nitrophenyl, pentachlorophenyl, 3,4-dehydro-4-oxo-1,2,3-benzotriazinyl (Dhbt), or fluorenylmethyl,
     X is HCl, TFA, HCOOH, TsOH, or MsOH, and   R is H, methyl (Me), ethyl (Et), benzyl (Bzl), or isopropyl (IPr).   

                                
wherein Y is 1-hydroxybenzotriazolelt, azabenzotriazole, succinimidyl, pentafluorophenyl, trichlorophenyl, nitrophenyl, pentachlorophenyl, 3,4-dehydro-4-oxo-1,2,3- benzotriazinyl (Dhbt), or fluorenylmethyl; and X is HCl, trifluoroacetic acid (TFA), HCOOH, p-toluenesulfonic acid (TsOH), or methanesulfonic acid (MsOH) or isopropyl (iPr).
 
     EXPERIMENTS AND EXAMPLES 
     Example 1( a ) 
     Morpholine Derivative-Dipeptide Acid (One Pot) 
     To a solution of morpholin-4-yl-acetic acid (100.0 mmol) in 250 ml dry tetrahydrofuran (THF), isobutyl chloroformate (100.0 mmol) and N-methylmorpholine (NMM) (110.0 mmol) were added and the mixture was stirred at 0° C. for 30 min. In another flask, homo-phenylalanine (100.0 mmol), trimethylsilyl chloride (TMS-Cl) (200 mmol) and diisopropylethylamine (DIPEA) (200 mmol) were refluxed in 300 mL of dry dichloromethane for 1 h. The mixture was cooled to 0° C. and added in one portion to a preformed mixed anhydride of formula 1a and the combined reaction mixture was stirred for 30-45 min. The reaction was concentrated under reduced pressure and the residue was dissolved in 10% Na 2 CO 3  and washed with ether. The aqueous phase was acidified with 1N HCl and extracted with 500 mL EtOAc/dichloromethane (MDC). The organic layer was washed with water (2×200 mL) and brine (1×200 mL) and dried over anhydrous Na 2 SO 4 . The Na 2 SO 4  was removed by filtration and the solvent evaporated under reduced pressure to give a dipeptide acid of the formula Ib. 
     
       
                 
         
             
             
         
      
       
                 
         
             
             
         
       
     
     To a 500 mL three-neck round bottom flask equipped with a magnetic stirrer and thermometer pocket arranged in a tub was charged a chloroacetamido derivative of H-hPhe-OH (10.0 g) of formula 3, THF (100 mL) and the reaction mixture was stirred at 20-30° C. KI (0.25 equiv.) and morpholine (1.2 equiv.) were added dropwise into the reaction mass at 5-15° C. After 15 minutes the temperature was increased to 20-30° C. The reaction was maintained for 12 to 16 h and upon confirmation of completion of the reaction, the solvent was distilled completely under vacuum at below 40° C. The residue was dissolved in MDC (300 mL) and washed with process water and brine solution. The organic layer was dried over anhydrous sodium sulphate (5 g) and distilled under vacuum at below 40° C. The compound Ib was precipitated with diethyl ether (150 mL) and filtered. 
     Example 2 
     Preparation of Chlroacetamido Derivative of Homophenylalanine Methyl Ester 
     A 500 mL three-neck round bottom flask equipped with a mechanical stirrer and thermometer pocket was arranged in a tub. A hydrochloride salt of H-hPhe-OMe (30 g) and MDC (200 mL) were charged into the flask and stirred under nitrogen atmosphere. Triethylamine (2 equiv.) was slowly added into the reaction mass at 0° C. to 10° C. and stirred for 10 min., followed by chloroacetyl chloride (1.1 equiv.) added drop wise at 0° C. to 10° C. with vigorous stirring. After 15 min. the temperature of the reaction mixture was raised to 30° C. and maintained 3 h. Upon completion of the reaction (confirmed by TLC), the reaction mass was diluted with MDC (200 mL), quenched with process water and washed with process water (2×200 mL) and brine (200 mL). The organic layer was dried over anhydrous sodium sulfate (10 g) and concentrated under vacuum at below 40° C. and precipitated by adding diethyl ether. 
     
       
                 
         
             
             
         
      
     
     Example 3( a ) 
     Morpholine Derivative-Dipeptide Ester 
     A 500 mL single neck flask equipped with a magnetic stirrer was arranged in a tub. Morpholine-4-yl-acetic acid (100 mmol) and DMF (200 mL) were charged into the flask and stirred at 20-30° C. DIPEA (100 mmol) was added slowly into the reaction mixture at 20-30° C., followed by addition of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU) (100 mmol) and hydroxybenzotriazole (HOBt) (100 mmol) and the reaction mixture was stirred at 20-30° C. In another flask, a homophenylalanine methyl ester hydrochloride (100 mmol) and DMF (150 mL) were charged and the solution cooled at 0-10° C. DIPEA (100 mmol) was slowly added into the solution at 0-10° C. and stirred for 5 min. The solution was carefully transferred to the earlier reaction mixture at 20-30° C. The reaction was maintained at 20-30° C. for 2 hours. MDC (500 mL) was charged, and the mixture was washed with process water (8×100 mL). The organic layer was washed with brine solution (2×100 mL) and the organic layer separated. The organic layer was dried over sodium sulphate (5 g) and distilled under vacuum at below 40° C. Diethyl ether (200 mL) was charged and the mixture cooled at 0-5° C. The separated solid was filtered and dried. 
     
       
                 
         
             
             
         
      
     
     Example 3( b ) 
     To a 500 mL three-neck round bottom flask equipped with a magnetic stirrer, and thermometer pocket arranged in a tub was charged a chloroacetamido derivative of H-hPhe-OMe (10.0 g), THF (100 mL) and the reaction mixture was stirred at 20-30° C. KI (0.25 equiv.) and morpholine (1.2 equiv.) were added dropwise into the reaction mass at 5-15° C. After 15 minutes the temperature was increased to 20-30° C. The reaction was maintained for 12 to 16 h and upon confirmation of completion of the reaction; the solvent was distilled completely under vacuum at below 40° C. The residue was dissolved in MDC (300 mL) and washed with process water and brine solution. The organic layer was dried over anhydrous sodium sulfate (5 g) and distilled under vacuum at below 40° C. The compound Ia was precipitated with diethyl ether (150 mL) and filtered. 
     
       
                 
         
             
             
         
      
     
     Example 4 
     Hydrolysis of Morpholine Derivative-Dipeptide Ester 
     To a 500 mL three-neck round bottom flask equipped with a mechanical stirrer and thermometer pocket arranged in a tub was charged a morpholine derivative dipeptide ester of Formula Ia (100 mmol). Methanol (240 mL) was added and the reaction mixture was stirred at 0-10° C. LiOH (3 equiv.) in process water (80 mL) was added into the reaction mass at 0-10° C. The reaction mixture was maintained for 3-4 h at 0-10° C. The reaction mixture was evaporated to dryness under vacuum and the residue was dissolved with water (100 mL) and washed with ether (100 mL). The aqueous layer was acidified using 3N HCl solution (40 mL). The aqueous layer was extracted with ethylacetate, dried with anhydrous sodium sulphate and evaporated. 
     
       
                 
         
             
             
         
      
     
     Example 5 
     Preparation of Dipeptide Active Ester 
     To a solution of morpholine-h-phenylalanine-dipeptide (Formula Ib) (100.0 mmol) in 300 ml dry THF, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl) (150 mmol), an N-hydroxy compound/substituted phenol of Formula 6 (100 mmol) wherein Y is 1-hydroxybenzotriazole (OBt) and 4-dimethylaminopyridine (DMAP) (0.1 equiv.) were added, and the reaction mixture was stirred for 2 h at 0° C. After completion of the reaction, confirmed by TLC, the reaction mixture was evaporated to dryness and the residue was dissolved in MDC and washed with process water and brine. The organic layer was dried over anhydrous Na 2 SO 4  and the solvent was evaporated under reduced pressure to give a dipeptide ester of the Formula I. 
     
       
                 
         
             
             
         
      
     
     Example 6 
     Preparation of Boc-hPhe-Leu-OH 
     To a solution of Boc-hPhe-OH (100.0 mmol, 23.12 g) in 250 ml dry THF, isobutyl chloroformate (100.0 mmol) and NMM (110.0 mmol) were added and the mixture was stirred at 0° C. for 30 min. In another flask, H-Leu-OH (100.0 mmol), TMS-Cl (200 mmol), and DIPEA (200 mmol) were refluxed in 300 mL of dry dichloromethane for 1 h. The mixture was cooled to 0° C. and added in one portion to a preformed mixed anhydride of Formula 5 and the combined reaction mixture was stirred for 30-45 min. The reaction was concentrated under reduced pressure and the residue was dissolved in 10% Na 2 CO 3  and washed with ether. The aqueous phase was acidified with citric acid and extracted with 500 mL EtOAc/MDC. The organic layer was washed with water (2×200 mL) and brine (1×200 mL) and dried over anhydrous Na 2 SO 4 . The solvent was evaporated under reduced pressure to give a dipeptide acid of Formula V. 
     
       
                 
         
             
             
         
      
     
     Example 7 
     Synthesis of H-hPhe-Leu-OH. Salt 
     To the protected dipeptide of Formula V, was added 80% Trifluoroacetic acid (TFA) in dichloromethane and the reaction mixture was stirred at room temperature for 2 h. Upon confirmation of completion of the reaction monitored by TLC, the mixture was concentrated and the product was precipitated by adding cold ether. The solid was filtered, washed with ether and dried using vacuum. In the scheme below X is TFA. 
     
       
                 
         
             
             
         
      
     
     Example 8 
     Synthesis of Morpholine Intermediate 
     To a solution of morpholine-4-yl-acetic acid (100.0 mmol) in 250 ml dry THF, isobutyl chloroformate (100.0 mmol) and NMM (110.0 mmol) were added and the mixture was stirred at 0° C. for 30 min. TFA.H-hPhe-Leu-OH (100.0 mmol), TMS-Cl (200 mmol), and DIPEA (300 mmol) were refluxed in 300 mL of dry dichloromethane for 1 h. The mixture was cooled to 0° C. and added in one portion to the preformed mixed anhydride of Formula 1a and the combined reaction mixture was stirred for 30-45 min. The reaction was concentrated under reduced pressure and the residue was dissolved in 10% Na 2 CO 3  and washed with ether. The aqueous phase was acidified with citric acid and extracted with 500 mL EtOAc/MDC. The organic layer was washed with water (2×200 mL) and brine (1×200 mL) and dried over anhydrous Na 2 SO 4 . The Na 2 SO 4  was removed by filtration and the solvent evaporated under reduced pressure to give the tripeptide acid of Formula IIIa. 
     
       
                 
         
             
             
         
      
     
     Example 9 
     Preparation of Tripeptide Active Ester 
     To a solution of morpholine-hPhe-Leu-OH tripeptide (100.0 mmol) in 300 ml dry THF, EDC.HCl (150 mmol), an N-hydroxy compound/substituted phenol of Formula 6 (100 mmol), and DMAP (0.1 equiv.) were added and the reaction mixture was stirred for 2 h at 0° C. After completion of the reaction, confirmed by TLC, the reaction mixture was evaporated to dryness and the residue was dissolved in MDC and washed with process water and brine. The organic layer was dried over anhydrous Na 2 SO 4  and the solvent was evaporated under reduced pressure to give the tripeptide ester of the Formula III. 
     
       
                 
         
             
             
         
      
     
     Example 10 
     Preparation of Dipeptide with Epoxyketone 
     A 500 mL single neck flask equipped with a magnetic stirrer was arranged in a tub. Boc-Phe-OH (100 mmol) and DMF (200 mL) were charged into the flask and stirred at 20-30° C. DIPEA (100 mmol) was slowly added into the reaction mixture at 20-30° C., followed by HBTU (100 mmol) and HOBt (100 mmol) which were added and stirred at 20-30° C. In another flask H-Leu-epoxy ketone salt (in which X is Boc)(100 mmol) and MDC were charged and cooled to 0° C. to 5° C. and trifluoroacetic acid (150 mL) slowly added and stirred for 120 min and distilled under vacuum completely. DMF (150 mL) was charged and the solution cooled at 0-10° C. DIPEA (100 mmol) was slowly added into the solution at 0-10° C. and stirred for 5 min. The solution was carefully transferred to the earlier reaction mixture at 20-30° C. The reaction was maintained for 2 h at 20-30° C. MDC (500 mL) was charged into the reaction mixture, and the mixture was washed with process water (8×100 mL). The organic layer was washed with brine solution (2×100 mL) and the organic layer separated. The organic layer was dried over sodium sulphate (5 g) and distilled under vacuum at below 40° C. Diethyl ether (200 mL) was charged in the vessel and the mixture cooled at 0° C. to 5° C. The separated solid was filtered and dried. 
     
       
                 
         
             
             
         
      
     
     Example 11 
     Preparation of H-Phe-Leu-Epoxy Ketone Salt 
     To the protected dipeptide epoxyketone derivative of Formula VII was added 80% TFA in dichloromethane and the reaction mixture was stirred at −10-0° C. for 2 h. Upon confirmation of completion of the deprotection, monitored by TLC, the mixture was concentrated and the product of Formula VIII in which X is TFA was precipitated by adding cold ether. The solid was filtered and washed with ether and dried in vacuum. 
     
       
                 
         
             
             
         
      
     
     Example 12 
     Synthesis of Carfilzomib 
     To a solution of tripeptide active ester of Formula III (100.0 mmol) in 250 mL organic solvent, a dipeptide epoxyketone derivative of Formula VIII (100 mmol) and equimolar organic base were added. The reaction was stirred at 0-25° C. for 2 h, concentrated under reduced pressure and the residue was dissolved in MDC washed with process water and brine. The organic layer was dried over anhydrous Na 2 SO 4  and the solvent was evaporated under reduced pressure to obtain the title compound. 
     Example 13 
     Preparation of Boc-Leu-Phe-OH 
     To a solution of Boc-Leu-OH (100.0 mmol) in 250 ml dry THF, isobutyl chloroformate (100.0 mmol) and NMM (110.0 mmol) were added and the mixture was stirred at 0° C. for 30 min. In another flask, H-Phe-OH (100.0 mmol), TMS-Cl (200 mmol), and DDPEA (200 mmol) were refluxed in 300 mL of dry dichloromethane for 1 h. The mixture was cooled to 0° C. and added in one portion to a preformed mixed anhydride of Formula 9a and the combined reaction mixture was stirred for 30-45 min. The reaction was concentrated under reduced pressure and the residue was dissolved in 10% Na 2 CO 3  and washed with ether. The aqueous phase was acidified with citric acid and extracted with 500 mL EtOAc/MDC. The organic layer was washed with water (2×200 mL) and brine (1×200 mL) and dried over anhydrous Na 2 SO 4 . The solvent was evaporated under reduced pressure to give the dipeptide acid of Formula IX. 
     
       
                 
         
             
             
         
      
     
     Example 14 
     Preparation of H-Leu-Phe-OH.Salt 
     To the protected Boc-Leu-Phe-OH dipeptide of Formula IX was added 80% TFA in dichloromethane and the reaction mixture was stirred at room temperature for 2 h. Upon confirming completion of the deprotection (monitored by TLC), the mixture was concentrated and the product was precipitated by adding cold diethyl ether. The solid of Formula X (in which X is TFA) was filtered, washed with ether and dried using vacuum. 
     
       
                 
         
             
             
         
      
     
     Example 15 
     Synthesis of Morpholine Derivative of Tetrapeptide 
     To a solution of morpholine derived dipeptide of Formula Ib (100.0 mmol) in 250 ml dry THF, isobutyl chloroformate (100.0 mmol) and NMM (110.0 mmol) were added and the mixture was stirred at 0° C. for 30 min. In the other flask, TFA.H-Leu-Phe-OH (100.0 mmol), TMS-Cl (200 mmol), and DIPEA (300 mmol) were refluxed in 300 mL of dry dichloromethane for 1 h. The mixture was cooled to 0° C. and added in one portion to the preformed mixed anhydride of Formula Ic and the combined reaction mixture was stirred for 1 h. The reaction was concentrated under reduced pressure and the residue was dissolved in 10% Na 2 CO 3  and washed with ether. The aqueous phase was acidified with 1N HCl and extracted with 500 mL MDC. The organic layer was washed with water (2×200 mL) and brine (1×200 mL) and dried over anhydrous Na 2 SO 4 . The Na 2 SO 4  was removed by filtration and the solvent evaporated under reduced pressure to give the tetrapeptide acid of Formula XI. 
     
       
                 
         
             
             
         
      
     
     Example 16 
     Synthesis of Boc-Leu-Phe-OMe 
     A 500 mL single neck flask equipped with a magnetic stirrer was arranged in a tub. Boc-Leu-OH of the formula 9 (100 mmol) and DMF (200 mL) were charged into the flask and stirred at 20-30° C. DIPEA (100 mmol) was slowly added into the reaction mixture at 20-30° C., followed by HBTU (100 mmol) and HOBt (100 mmol) which were added and stirred at 20-30° C. In another flask H-Phe-OMe salt of the formula 10 (100 mmol) and DMF (150 mL) were charged and the solution cooled at 0-10° C. DIPEA (100 mmol) was slowly added into the solution at 0-10° C. and stirred for 5 min. The solution was carefully transferred to the earlier reaction mixture at 20-30° C. The reaction was maintained for 2 h at 20-30° C. MDC (500 mL) was charged into the reaction mixture, and the mixture was washed with process water (8×100 mL). The organic layer was washed with brine solution (2×100 mL) and the organic layer separated. The organic layer was dried over sodium sulphate (5 g) and distilled under vacuum at below 40° C. Diethyl ether (200 mL) was charged in the vessel and the mixture cooled at 0° C. to 5° C. The separated solid product of Formula XII in which R is CH3 was filtered and dried. 
     
       
                 
         
             
             
         
      
     
     Example 17 
     Synthesis of H-Leu-Phe-OR. Salt 
     To the protected Boc-Leu-Phe-OR dipeptide of Formula XII in which R is CH3 was added 80% TFA in dichloromethane and the reaction mixture was stirred at room temperature for 2 h. Upon confirming completion of the deprotection (monitored by TLC), the mixture was concentrated and the product was precipitated by adding cold diethyl ether. The solid of Formula XIII in which X is TFA and R is CH3 was filtered, washed with ether and dried using vacuum. 
     
       
                 
         
             
             
         
      
     
     Example 18 
     Synthesis of Tetrapeptide Active Ester 
     To a solution of a morpholine-hPhe-Leu-Phe-OH tetra-peptide of Formula XI (100.0 mmol) in 300 mL dry THF, EDC.HCl (150 mmol), an N-hydroxy compound/substituted phenol of the Formula 6 (100 mmol) and DMAP (0.1 equiv.) were added and the reaction mixture was stirred for 2 h at 0-25° C. After completion of the reaction, confirmed by TLC, evaporated the reaction mixture to dryness and the residue was dissolved in MDC and washed with process water and brine. The organic layer was dried over anhydrous Na 2 SO 4  and this organic layer used in Example 19. 
     
       
                 
         
             
             
         
      
     
     Example 19 
     Synthesis of Carfilzomib 
     The solution of tetrapeptide active ester of the Formula IV (100.0 mmol) from the above example was cooled at 0° C. to 5° C. H-Leu-epoxy ketone derivative of Formula 8 (100 mmol) and MDC were charged in a vessel and cooled to 0° C. to 5° C., and to this mixture trifluoroacetic acid (150 mL) was added slowly and stirred for 120 min and distilled under vacuum completely, and an equimolar quantity of diisopropylethylamine was added. This solution was slowly added to the above solution containing the tetrapeptide active ester of Formula IV. The reaction was stirred at 0-25° C. for 2 h, concentrated under reduced pressure and the residue was dissolved in MDC and washed with process water and brine. The organic layer was dried over anhydrous Na 2 SO 4  and the solvent was evaporated under reduced pressure to compound carfilzomib. 
     Example 20 
     Purification of Carfilzomib 
     Carfilzomib crude was dissolved in dimethylformamide, a polar aprotic solvent. Examples of suitable polar aprotic solvents include but are not limited to dimethylformamide, dimethylsulfoxide, or acetonitrile. To the solution was added 10% to 20% of citric acid solution. The resulting solution was stirred for 5 to 6 hours then filtered off to obtain the pure carfilzomib. This purification method provides highly pure carfilzomib in which the diasteromeric impurity is controlled at 5 to 10 percent. This method improves yield and reduces the time cycle in scale up purifications. 
     The purified carfilzomib is further purified by preparative HPLC to obtain the drug substance of carfilzomib with amorphous form. The eluted fractions from preparative HPLC were collected and made pH basic, preferably 8-10, most preferably 8-9. The resulting reaction mass was distilled off in such a way to remain 40% of reaction mass, under vacuum at below 30° C. and stirred for 60 to 90 minutes. Filtered off the reaction mass and again the wet solids were dissolved in MDC and treated with brine solution and sodium sulphate. The resulting reaction mass was subjected to distillation and strip out with diethyl ether. Finally the compound was dried under vacuum to obtain the drug substance of carfilzomib with amorphous form. The XRD profile of the compound is shown in  FIG. 1 . 
     Although the compositions and methods of the present disclosure have been described with reference to exemplary embodiments thereof, the present disclosure is not limited thereby. Indeed, the exemplary embodiments are implementations of the disclosed compositions and methods are provided for illustrative and non-limitative purposes. Changes, modifications, enhancements and/or refinements to the disclosed systems and methods may be made without departing from the spirit or scope of the present disclosure. Accordingly, such changes, modifications, enhancements and/or refinements are encompassed within the scope of the present invention.