Synthesis of dolastatin 3

• US 5,076,973 A
• Filed: 10/24/1988
• Issued: 12/31/1991
• Est. Priority Date: 10/24/1988
• Status: Expired due to Fees

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Holzapfel, Cedric W., Pettit, George R.
• Primary Examiner(s): Lee, Lester L.
• Assistant Examiner(s): Davenport, Avis M.
• Application Number:
  US07/261,311
• Time in Patent Office: 1,163 Days
• Field of Search: 514/9, 514/326, 514/11, 530/333, 530/331, 530/330, 530/317
• US Class Current: 530/333
• CPC Class Codes: C07K 7/56 the cyclisation not occurring through 2,4-diamino-butanoic acid

Abstract

Synthesis of dolastatin 3 is accomplished by one amino acid unit addition from L-Pro-OMe employing diethyl phosphorocyanidate-triethylamine for peptide bond formation and N-Boc protection (trifluoroacetic acid cleavage). Thereafter Boc-L-Leu-L-(gln)Thz-(gly)Thz-L-Val-L-Pro-OMe was obtained, successively crystallized from ethanol-diethyl ether, converted to the OPfp active ester, Boc cleavage and cyclization in dioxane containing t-butanol and 4-pyrrolidinopyridine to yield synthetic (-)-dolastatin 3.

Claims

1. A process for synthesizing a peptide having the structure cyclo comprising the steps of adding triethylamine and diethyl phosphorocyanidate to S-proline methyl ester and N-t-Boc-L-valine in 1,2-dimethoxyethane at 0°

C. to form a dipeptide;

holding said depeptide at 0°

C. for one hour;

allowing said dipeptide to warm to room temperature over a period of 4 hours;

diluting the warmed depeptide with ethyl acetate to form a diluted mixture;

washing said diluted mixture first with 5% HCl;

then with sodium hydrogen carbonate and finally with saturated brine and removing the solvent therefrom in vacuo to provide N-t-Boc-L-Val-L-Pro-OMe as a colorless syrup;

stirring said colorless syrup into a solution of dry dichloromethane at 0°

C.;

adding trifluoroacetic acid to said syrup mixture and holding said syrup mixture at 0°

C. for one hour;

warming said cold syrup-mixture to room temperature;

holding said syrup-mixture at room temperature for one hour;

diluting said warmed syrup mixture with dry carbon tetrachloride;

removing the solvents from said diluted syrup mixture in vacuo to provide a colorless syrup residue;

drying said colorless syrup residue under vacuum for twelve hours to provide a dipeptide trifluoroacetate;

mixing said dipeptide trifluoroacetate and N-t-Boc-(gly)Thz in dry dimethoxyethane to form a solution;

cooling said solution to 0°

C.;

adding triethylamine and DEPC to said cooled solution;

holding said cooled solution 0°

C. for one hour;

warming said cooled solution to room temperature over four hours to for a warmed solution;

diluting said warmed solution with ethyl acetate;

washing said diluted warmed solution with 5% HCl and sodium hydrogen carbonate;

further washing the washed diluted warmed solution with saturated brine;

drying said further washed warmed solution with sodium sulfate;

removing the solvent from said solution in vacuo to provide a residue of N-t-Boc-(gly)Thz-L-Val-L-Pro-OMe as a colorless syrup;

placing said residue of N-t-Boc-(gly)Thz-L-Val-L-Pro-OMe in dry dichloromethane at 0°

C.;

adding trifluoroacetic acid to said residue and said dry dichloromethane to form a mixture;

holding said mixture at 0°

C. for one hour;

warming said last formed mixture to room temperature for one hour;

diluting said room temperature mixture with carbon tetrachloride;

removing the solvents from said diluted mixture in vacuo to leave a tripeptide trifluoroacetate as a colorless syrup;

drying said colorless syrup under vacuum for 24 hours;

dissolving said dried syrup in dry dimethylformamide to form a tripeptide trifluoroacetate/dimethylformamide solution;

adding N-t-Boc-(gly)Thz to said tripeptide trifluoroacetate/dimethylformamide solution;

cooling said solution to 0°

C.;

adding triethylamine and DEPC to said cooled solution;

holding said solution at 0°

C. for one hour;

warming said cool solution to room temperature for six hours;

removing the solvents from said warmed solution in vacuo to provide a residue of protected tetrapeptide N-t-Boc-(gly)Thz-(gly)Thz-L-Val-L-Pro-OMe;

dissolving said protected tetrapeptide in trifluoroacetic acid at 0°

C.;

warming said solution to room temperature;

diluting said solution with carbon tetrachloride;

removing said solvents in vacuo to leave a tetrapeptide trifluoroacetate as a residue;

drying said residue under vacuum for 24 hours;

dissolving said dried residue in dimethylformamide to form a dimethylformamide solution;

adding N-t-Boc-L-Leu to said dimethylformamide solution;

cooling the resulting solution to 0°

C.;

adding triethylamine and DEPC to said cooled solution;

holding said cooled solution at 0°

C. for one hour;

warming said cooled solution to room temperature for four hours;

removing the solvents from said room temperature solution in vacuo to leave a residue;

purifying said residue on a silica column in 7% methanol and ethyl acetate to produce a protected pentapeptide N-t-Boc-L-Leu-L-(gln)Thz-(gly)Thz-L-Val-L-Pro-OMe;

placing the protected pentapeptide in dioxane and water;

treating the dioxane/water solution with sodium hydroxide at room temperature for three hours;

diluting the treated solution with diethyl ether to form a diluted treated solution having an aqueous phase;

collecting the aqueous phase of said diluted treated solution;

adjusting the pH of said aqueous phase to 2 with all cold 2N HCl;

extracting said pH adjusted aqueous phase with chloroform to create a plurality of extracts;

combining said extracts;

drying said combined extracts with anhydrous sodium sulfate;

removing the solvent from said dried extracts in vacuo leaving an N-Boc-pentapeptide;

precipitating N-Boc-pentapeptide from ethanol with diethyl ether;

placing the N-Boc-pentapeptide precipitate in dry dimethoxyethane and dimethylformamide to form a solution;

treating said solution with pentafluorophenol and N,N¹ -dicyclohexyl-carbodiimide at -20°

C. in a nitrogen atmosphere;

warming said treated solution to room temperature;

holding said warmed solution at room temperature for 12 hours;

filtering said warmed solution to remove dicyclohexylurea therefrom;

removing the solvents from said filtered warmed solution in vacuo to leave a pentapeptide pentafluorophenyl ester;

treating said pentapeptide pentafluorophenyl ester with trifluoroacetic acid at room temperature to form a reaction mixture;

diluting said reaction mixture with carbon tetrachloride;

removing the solvents from said diluted reaction mixture in vacuo to leave a pentapeptide trifluoroacetate ester as the residue;

drying said residue under vacuum;

dissolving said dried residue in dioxane to produce a solution of 4-pyrrolidinopyridine in t-butanol and dioxane at 95°

C.;

filtering said solution through celite to create a residue; and

removing the solvent from said residue in vacuo to produce Cyclo-[L-Val-L-Pro-L-Leu-L(gln)Thz-(gly)Thz] as an amorphous white powder.

2. A process for synthesizing dolastatin 3 having the structure cyclo, according to claim 1, comprising the first step of adding triethylamine and diethyl phosphorocyanidate to S-proline methyl ester and N-t-Boc-L-valine in 1,2-dimethoxy at 0°

C. to form a dipeptide.

3. A process for synthesizing a dolastatin 3 according to claim 2, including the further steps of holding said dipeptide at 0°

C. for one hour, allowing the dipeptide to warm to room temperature over 4 hours, and thereafter diluting the warmed dipeptide with ethyl acetate to form a diluted mixture.

4. A process for synthesizing a dolastatin 3 according to claim 3, including the further steps of washing said diluted mixture with 5% HCl followed by sodium hydrogen carbonate followed with saturated brine and removing the solvent therefrom in vacuo to provide N-t-Boc-L-Val-L-Pro-OMe as a colorless syrup.

5. A process for synthesizing a dolastatin 3 according to claim 4, including the further steps of stirring said colorless syrup into a solution of dry dichloromethane at 0°

C. and addition trifluoroacetic acid thereto and holding said syrup mixture at 0°

C. for one hour.

6. A process for synthesizing a dolastatin 3 according to claim 5, including the further steps of raising said cold syrup-mixture to room temperature, holding said syrup-mixture at room temperature for one hour;

diluting the warmed syrup mixture with dry carbon tetrachloride;

removing the solvents from said last formed mixtures in vacuo to provide a colorless syrup residue.

7. A process for synthesizing a dolastatin 3 according to claim 6, including the further step of drying said colorless syrup residue under vacuum for twelve hours to leave a dipeptide trifluoroacetate.

8. A process for synthesizing a dolastatin 3 according to claim 7, including the further steps of mixing said dipeptide trifluoroacetate and N-t-Boc-(gly)Thz in dry dimethoxyethane to form a solution, and cooling the solution to 0°

C.

9. A process for synthesizing a dolastatin 3 according to claim 8, including the further steps of adding triethylamine and DEPC to the solution;

holding the solution 0°

C. for one hour; and

thereafter warming the solution to room temperature over four hours to form a warmed solution.

10. A process for synthesizing a dolastatin 3 according to claim 9, including the further step of diluting said warmed solution with ethyl acetate, washing said diluted warmed solution with 5% HCl and sodium hydrogen carbonate, and thereafter further washing the washed diluted warmed solution with saturated brine.

11. A process for synthesizing a dolastatin 3 according to claim 10 including the further steps of drying said further washed solution with sodium sulfate and removing the solvent therefrom in vacuo to provide a residue of N-t-Boc-(gly)Thz-L-Val-L-Pro-OMe as a colorless syrup.

12. A process for synthesizing a dolastatin 3 according to claim 11 including the further steps of placing said residue of N-t-Boc-(gly)Thz-L-Val-L-Pro-OMe in dry dichloromethane at 0°

C., adding trifluoroacetic acid thereto to form a mixture and holding said mixture at 0°

C. for one hour.

13. A process for synthesizing a dolastatin 3 according to claim 12, including the further steps of warming said last formed mixture to room temperature for one hour, diluting the room temperature mixture with carbon tetrachloride, removing the solvents from the diluted mixture in vacuo to leave the tripeptide trifluoroacetate as a colorless syrup, drying the colorless syrup under vacuum for 24 hours, and dissolving the dried syrup in dry dimethylformamide to form a solution.

14. A process for synthesizing a dolastatin 3 according to claim 13 including the further steps of adding N-t-Boc-L-(gln)Thz to the tripeptide trifluoroacetate/dimethylformamide solution, cooling the solution to 0°

C. adding triethylamine and DEPC to the cooled solution, holding said solution at 0°

C. for one hour, warming the cool solution to room temperature for six hours, and removing the solvents from said warmed solution in vacuo leaving as the residue the protected tetrapeptide N-t-Boc-L-(gln)Thz-(gly)Thz-L-Val-L-Pro-OMe.

15. A process for synthesizing a dolastatin 3 according to claim 14 including the further steps of dissolving said protected tetrapeptide in trifluoroacetic acid at 0°

C., warming said solution to room temperature, diluting said solution with carbon tetrachloride, removing said solvents in vacuo to leave a tetrapeptide trifluoroacetate as a residue, drying said residue under vacuum for 24 hours, and dissolving the dried residue in dimethylformamide to form a solution.

16. A process for synthesizing a dolastatin 3 according to claim 15 including the further steps of adding N-t-Boc-L-Leu to said last formed solution, cooling the resulting solution to 0°

C., adding triethylamine and DEPC to said cooled solution, holding said cooled solution at 0°

C. for one hour;

warming said cooled solution to room temperature for four hours;

removing the solvents from said room temperature solution in vacuo to leave residue;

purifying said residue on a silica column in 7% methanel and ethyl acetate to produce the protected pentapeptide N-t-Boc-L-Leu-L-(gln)Thz-(gly)Thz-L-Val-L-Pro-OMe.

17. A process for synthesizing a dolastatin 3 according to claim 16, including the further steps of placing the protected pentapeptide in dioxane and water, treating the solution formed with sodium hydroxide at room temperature for three hours, and diluting the treated solution with diethyl ether.

18. A process for synthesizing a dolastatin 3 according to a claim 17, further steps of collecting including the aqueous phase of the diluted treated solution adjusting the pH of said aqueous phase to 2 with all cold 2N HCl, extracting said pH adjusted aqueous phase with chloroform to create a plurality of extracts;

combining said extract, drying said combined extracts with anhydrous sodium sulfate;

removing the solvent from said dried extracts in vacuo leaving the N-Boc-pentapeptide; and

precipitating the N-Boc-pentapeptide from ethanol with diethyl ether.

19. A process for synthesizing a dolastatin 3 according to claim 18, including the further steps of placing the N-Boc-pentapeptide precipitate in dry dimethoxyethane and dimethylformamide to form a solution, treating said solution with pentafluorophenol and N.N¹ -dicyclohexylcarbodiimide at -20°

C. in a nitrogen atmosphere;

warming said treated solution to room temperature, holding said warming solution at room temperature for 12 hours;

filtering said warmed solution to remove dicyclohexylurea therefrom;

removing the solvents from said filtered warmed solution in vacuo to leave a pentapeptide pentafluorophenyl ester; and

treating said ester with trifluoroacetic acid at room temperature to form a reaction mixture.

20. A process for synthesizing a dolastatin 3 according to claim 19, including the further steps of diluting said reaction mixture with carbon tetrachloride;

removing the solvents from said diluted reaction mixture in vacuo to leave pentapeptide trifluoroacetate ester as the residue;

drying said residue under vacuum, dissolving said dried residue in dioxane to produce a solution of 4-pyrrolidinopyridine in t-butanol and dioxane at 95°

C.;

holding said solution and filtering said solution through celite to create a residue.

21. A process for synthesizing a dolastatin 3 according to claim 20 including the further steps of removing the solvent from said residue in vacuo to leave as an amorphous white powder Cyclo-[L-Val-L-Pro-L-Leu-L(gln)Thz-(gln)Thz].