Synthesis of dolastatin 15

• US 5,554,725 A
• Filed: 09/14/1994
• Issued: 09/10/1996
• Est. Priority Date: 09/14/1994
• Status: Expired due to Term

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Pettit, George R.
• Primary Examiner(s): Scheiner, Toni R.
• Assistant Examiner(s): HUFF, SHEELA JITENDRA
• Application Number:
  US08/306,146
• Time in Patent Office: 727 Days
• Field of Search: 530/330, 530/329, 530/303
• US Class Current: 530/330
• CPC Class Codes: C07K 11/00 Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof

Abstract

The present invention relates to the synthesis of natural (-)- dolastatin and the elucidation of the absolute configuration of this important sea hare constituent. A segment synthetic strategy was utilized for obtaining the Dolabella auricularia (Indian Ocean sea hare) depsipeptide dolastatin 15. Reaction of protected (S)-Hiva-(S)-Phe (2c) with isopropenyl chloroformate followed by Meldrum'"'"'s ester, cyclization (2c→3a) of the product in toluene and finally methylation afforded the key (S)-dolapyrrolidine (Dpy) derivative (3b). Condensation of tripeptide (8) with the three unit Dpy segment (5b) followed by deprotection and coupling (diethyl phosphorocyanidate) led to dolastatin 15 in 11% overall yield. The powerful and selective activity of dolastatin 15 against the U.S. National Cancer Institute'"'"'s panel of human cell lines is reported.

Claims

1. A method of synthesizing dolastatin 15 comprising the steps of:

coupling tripeptide (8) and tripeptide (5b) to form peptide (9a);

hydrogenating said peptide (9a) to form peptide (9b);

coupling said peptide (9b) with Dov to form dolastatin 15; and

isolating said dolastatin 15.

2. The method according to claim 1, further including the step of:

purifying said dolastatin 15.

3. The method according to claim 1, wherein said step of coupling tripeptide (8) and tripeptide (5b) involves using DEPC to couple said tripeptide (8) and tripeptide (5b).

4. The method according to claim 3, further including the step of:

purifying said dolastatin 15.

5. The method according to claim 1, wherein said tripeptide (8) is produced by:

coupling Z-NMe-(S)-Val with (S)-Pro-OMe to form dipeptide (6);

hydrogenating said dipeptide (6) followed by coupling with Z-(S)-Val to form tripeptide (7); and

removing said methyl ester group from said tripeptide (7) to form said tripeptide (8).

6. The method according to claim 5, wherein said step of coupling tripeptide (8) and tripeptide (5b) involves using DEPC to couple said tripeptide (8) and tripeptide (5b).

7. The method according to claim 6, further including the step of:

purifying said dolastatin 15.

8. The method according to claim 1, wherein said tripeptide (5b) is produced by:

diazotating S-valine to form S-(Hiva);

coupling said S-(Hiva) with Phe-OMe to form (2a);

protecting said (2a) as silyl ether (2b);

saponifying said (2b) to form (2c);

cyclizing said (2c) to form (3a);

methylating said (3a) to form (3b);

removing the silyl group from said (3b) to form alcohol (4);

esterifying said alcohol (4) to form (5a) having a Boc-protecting group; and

removing said Boc-protecting group from said (5a) to form said (5b).

9. The method according to claim 8, wherein said step of coupling tripeptide (8) and tripeptide (5b) involves using DEPC to couple said tripeptide (8) and tripeptide (5b).

10. The method according to claim 9, further including the step of:

purifying said dolastatin 15.

11. The method according to claim 8, wherein said tripeptide (8) is produced by:

coupling Z-NMe-(S)-Val with (S)-Pro-OMe to form dipeptide (6);

hydrogenating said dipeptide (6) followed by coupling with Z-(S)-Val to form tripeptide (7); and

removing said methyl ester group from said tripeptide (7) to form said tripeptide (8).

12. The method according to claim 11, wherein said step of coupling tripeptide (8) and tripeptide (5b) involves using DEPC to couple said tripeptide (8) and tripeptide (5b).

13. The method according to claim 12, further including the step of:

purifying said dolastatin 15.

14. A method of synthesizing dolastatin 15 comprising the steps of:

a) coupling tripeptide (8) and peptide (5b) with diethylphosphorocyanidate (DEPC) to form peptide (9a),

b) deprotecting peptide (9a) by hydrogenolysis to form peptide (9b),

c) coupling peptide (9b) and Dov with DEPC to form dolastatin 15, and

d) isolating said dolastatin 15.

15. The method according to claim 14 in which said tripeptide (8) is produced by condensing N-Z (wherein Z is a carbobenzoxy protecting group), N-Me-(S)-Val and (S)-Pro-Me with DEPC to form dipeptide (6), cleaving the carbobenzoxy protecting group by hydrogenolysis to produce deprotected dipeptide (6), and coupling the deprotected peptide with mixed anhydride prepared from pivaloyl chloride and Z-(S)Val.

16. The method according to claim 14 in which said isolated dolastatin 15 is subsequently purified on a SEPHADEX LH-20 chromatography column.

17. The method according to claim 14 in which said depsipeptide comprises an amorphous powder having a melting point of about 175°

-175.5°

C., [α

]_D²⁴ -77°

(C, 0.2, CH₃ OH).

18. The method according to claim 14 in which said depsipeptide, when identified by TLC, ¹ H and ¹³ C-NMR is identical to natural (-)- dolastatin 15.

19. The method of claim 14 in which said peptide (5b) is produced by coupling S-Hiva and Phe-OMe hydrochloride with diethyl phosphorocyanidate to form (S)-Hiva-(S)-Phe-OMe (2a);

reacting said (S)-Hiva-(S)-Phe-OMe (2a) with t-butyldimethylsilyl chloride;

cleaving the methyl ester under mild alkaline conditions (2a→

2c);

deriving dolapyrolidone (3a) from (2c) and isopropenyl chloroformate in the presence of 4-methylamino-pyridine;

methylating dolapyrolidone (3a) with dimethylsulfate to produce silyl ether (3b);

cleaving the silyl ether (3b), using trifluoroacetic acid, to alcohol (4);

esterifying alcohol (4) with Boc-(S)-Pro using dicyclohexylcarbodiimide to produce depsipeptide (5a); and

converting depsipeptide (5a) with trifluoroacetic acid to form tripeptide (5b).