Synthesis of dolastatin 10

• US 4,978,744 A
• Filed: 01/27/1989
• Issued: 12/18/1990
• Est. Priority Date: 01/27/1989
• 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., Singh, Sheo B.
• Primary Examiner(s): LEE, LESTER
• Application Number:
  US07/304,572
• Time in Patent Office: 690 Days
• Field of Search: 530/330, 514/17
• US Class Current: 530/330
• CPC Class Codes: C07K 5/0205 containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof

Abstract

A complicated but extremely important scheme of synthesis has been developed for synthesizing, dolaisoleuine, dolaproine, dolaphenine and dolavaline from readily available starting materials such as Z-(S,S)isoleucine, S-phenylalaline, S-phenylalaninol, S-prolinol, S-mandelate, and S-valine. The requisite amino acids have been combined using several peptide coupling procedures to create pharmaceutically pure dolastatin 10.

Claims

1. The method of synthesizing dolastatin 10 comprising the steps of forming a solution of Z-(S,S)-isoleucine and methyl iodide in tetrahydrofuran;

cooling said solution to about 0°

C.;

stirring said cooled solution under argon;

adding sodium hydride to said stirred cooled solution to form a suspension;

stirring said suspension at room temperature;

adding ethyl acetate and ice water to said stirred suspension to form an aqueous layer;

washing aqueous layer with ether;

acidifying said washed aqueous layer with acid at 0°

C.;

extracting said acidified aqueous layer with ethyl acetate; and

washing said extract;

removing said solvent from said extract to provide N-Z-N-methyl- (S,S)- isoleucine;

dissolving said N-Z-N-methyl-(S,S)- isoleucine in tetrahydrofuran at 0°

C. under argon;

adding borane-tetrahydrofuran complex to said solution with stirring;

extracting the product from said solution with ethyl acetate;

evaporating the solvent from extracted product to form an oil;

filtering said oil through a column of silica gel;

eluting said column with hexane-acetone (7;

3) to form N-Z-N-methyl-(S,S)-isoleucinol;

admixing said N-Z-N-methyl-(S,S)-isoleucinol with triethylamine in dimethylsulfoxide at room temperature;

cooling the mixed solution to 0°

C.;

adding sulfur trioxide-pyridine complex to said solution under argon;

extracting the aldehyde from said solution with ether;

washing said ethereal solution successively with citric acid, water, and sodium bicarbonate to yield chromotographically pure N-Z-N-methyl- (S,S)-isoleucinal;

preparing a solution of lithium diisopropylamide from n-butyllithium and diisopropylamine in tetrahydrofuran at -78°

C.;

warming said solution to -20°

C.;

recooling said solution to -78°

C.;

adding tert-butyl acetate to said recooled solution;

stirring said solution while rewarming said solution to -20°

C.;

recooling said solution to -78°

C. and adding said chromatographically pure N-Z-N-methyl-(S,S)-isoleucinal thereto;

treating the resulting mixture with ice water;

extracting the mixture with ether;

drying said extract;

solvent evaporating said dry extract to yield a viscous oil;

chromatographing and eluting said viscous oil with hexane-acetone (47;

3) to yield tert-butyl(3S,4S, 5S)-3-hydroxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate and tert-butyl (3R,4S,5S)-3-hydroxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate;

adding boron trifluoride etherate to a cooled and stirred solution of tert-butyl (3R,4S,5S)-3-hydroxy-4-(N-Z-N-methyl) -amino-5-methyl-heptanoate in dichloromethane under argon;

thereafter adding an anhydrous solution of diazomethane in dichloromethane to said solution;

filtering the polymethylene side products from said solution to form a filtrate;

concentrating said filtrate;

chromatographing said concentrated filtrate on a silica gel column;

eluting said column with hexane-acetone (97;

3) to yield tert-butyl (3R,4S,5S)-3-methoxy-4-(N-Z-N-methyl) -amino-5-methyl-heptanoate;

admixing trifluoroacetic anhydride in dichloromethane into a cooled -78°

C. solution of dimethylsulfoxide in dichloromethane with stirring;

adding N-Boc-S-prolinol in dichloromethane to said cool stirred mixture and continue stirring for about an hour;

adding triethylamine to said solution;

warming said solution to -20°

C.;

extracting said solution with ether to form an ethereal solution;

washing and concentrating said ethereal solution to form an oil;

dissolving said oil in acetone;

filtering said oil-acetone solution through a silica-gel column;

eluting said column with hexane-acetone (93;

7) to yield N-Boc-S-prolinal;

adding 2-S-Propionyloxy-1,1,2-triphenylethanol to a solution of lithium diisopropylamide in tetrahydrofuran at -78°

C. under argon to form a reaction mixture;

permitting said reaction mixture to warm to room temperature over two hours to form a yellow solution;

cooling the yellow solution to - 95°

C.;

adding freshly synthesized magnesium bromide to said cooled yellow solution;

thereafter adding a solution of Boc-S-prolinal in tetrahydrofuran to said yellow solution and allowing the reaction therebetween to proceed for two hours at -95°

C. under methanol-liquid nitrogen;

terminating said reaction with saturated aqueous ammonium chloride;

warming said mixture to 0°

C.;

adding ether to said mixture to form an ethereal solution;

washing, drying and solvent evaporating said ethereal solution to provide a glassy solid;

dissolving said glassy solid to form a solution;

adsorbing said solution on silica gel;

chromatographing said adsorbed silica gel; and

eluting said chromatographed silica gel to provide N-Boc-2S-3'"'"'-[2"ethoy-(1"hydroxy-1",1",2"-triphenyl) 2'"'"'S-methyl-3'"'"'R-hydroxy- propionate]-pyrrolidine;

methylating said N-Boc-2S-3, propionate-pyrrolidine in dichloromethane with boron trifluoride etherate and diazomethane in anhydrous dichloromethane;

eluting said methylated pyrrolidine with hexane-acetone (9;

1) to provide N-Boc-2S-3'"'"'-[2"-ethoxy(1"-hydroxy-1",1",2"-triphenyl) 2'"'"'S-methyl-3'"'"'R-methoxy-propionate]-pyrrolidine;

adding potassium tert-butoxide to a solution of said (2'"'"'S,3'"'"'R) ester in tetrahydrofuran with stirring until the solution turns bright yellow;

warming the bright yellow solution to -15°

C. and stopping the epimerization thereof by the addition of sufficient saturated aqueous citric acid thereto to dilute said solution;

extracting said solution with dichloromethane to form a combined extract;

washing said extract and removing the solvent therefrom to provide a clear oil;

chromatographing said yellow oil on silica gel;

eluting said silica gel to provide N-Boc-2S-3'"'"'-[2"ethoxy(1"hydroxy- 1",1", 2"-triphenyl) 2'"'"'S- methyl-3'"'"'R-methoxy-propionate]-pyrrolidine;

forming a solution of N-Boc-S-phenylalaninol in anhydrous dimethylsulfoxide-triethylam;

ine;

oxidizing said solution with sulfur trioxide pyridine complex;

adding water to said oxidized solution to precipitate N-Boc-S-phenylalaninal;

forming a solution of N-Boc-S-phenylalaninal and 2-aminoethanethiol in anhydrous benzene;

stirring said solution under argon for four hours;

removing the solvents and excess reagents from said stirred solution by filtration through a silica gel pad to form a residue;

crystallizing said residue to form 2-(N-Boc-1'"'"'S-amino-2'"'"'-phenyl-ethyl)-2-(R,S)-thiazolidine [N-Boc-2(3),4(5)-tetrahydro-dolaphenine];

forming a suspension of battery grade manganese dioxide in anhydrous benzene;

adding 1'"'"'-ethyl (N-Boc-1'"'"'S-amino-2'"'"'-phenyl)-2(R,S)-thiazolidine [N-Boc-2(3),4(5)-tetrahydro-dolaphenine] in benzene to said suspension to form a mixture;

stirring said mixture under argon at 55°

C. for 24 hours;

adding additional manganese dioxide with continued stirring;

filtering said stirred solution through silica gel;

washing said silica gel sequentially with benzene, ethyl acetate, chloroform and chloroform acetone (1;

1) to form a combined eluant;

evaporating said combined eluant to form a residue;

chromatographing said residue on a silica gel column;

eluting said column with ethyl acetate-hexane; and

crystallizing from said eluant granules consisting of 2-(1'"'"'S-Boc-amino-2'"'"'-phenyl-ethyl)-2-thiazole[N-Boc-S-dolaphenine].

2. The method of claim 1 in which said N-Z-N-methyl-(S,S)-isoleucine is prepared by the steps of forming a solution of Z-(S,S)-isoleucine and methyl iodide in tetrahydrofuran;

cooling said solution to about 0°

C.;

stirring said cooled solution under argon;

adding sodium hydride to said stirred cooled solution to form a suspension;

stirring said suspension at room temperature;

adding ethyl acetate and ice water to said stirred suspension to form an aqueous layer;

washing said aqueous layer with ether;

acidifying said washed aqueous layer with acid at 0°

C.;

extracting said acidified aqueous layer with ethyl acetate; and

washing said extract; and

removing said solvent from said extract to provide N-Z-N-methyl-(S,S)-isoleucine.

3. The method according to claim 2 including the further steps of dissolving said N-Z,N-methyl-(S,S)-isoleucine in tetrahydrofuran at 0°

C. under argon;

adding borane-tetrahydrofuran complex to said solution with stirring;

extracting the product from said solution with ethyl acetate;

evaporating the solvent from extracted product to form an oil;

filtering said oil through a column of silica gel; and

eluting said column with hexane-acetone (7;

3) to form N-Z-N-methyl-(S,S)-isoleucinol.

4. The method of claim 1 in which said N,N-dimethyl-S-valine is prepared by the steps of forming a mixture of S-valine in water with formaldehyde and a catalyst;

hydrogenating said mixture;

heating said hydrogenated mixture at reflux;

removing said catalyst from said heated hydrogenated mixture by filtration to form a filtrate;

concentrating said filtrate in vacuo to create a residue;

adding water and ethanol to said residue to form a solution;

evaporating said solution to dryness;

repeating the steps of adding water and ethanol and evaporating said solution to dryness until all paraformaldehyde is removed therefrom and an amorphous powder remains;

admixing ethanol-acetone with said powder; and

crystallizing colorless needles therefrom, said needles consisting of N,N-dimethyl-S-valine.

5. The method according to claim 1 comprising the further steps of:

adding 5% Pd/C to a mixture of N-Z-dolaisoleuine tert-butyl ester in ethyl acetate-methanol (3;

1);

hydrogenating said mixture at ambient temperature and pressure;

filtering the hydrogenated mixture to remove said Pd/C therefrom and create a filtrate;

evaporating said filtrate to dryness;

dissolving said dry filtrate in ether;

cooling said solution to -60°

C.;

adding an ethereal solution of hydrogen chloride to said cooled solution under anhydrous conditions to precipitate a hydrochloride salt therefrom;

passing argon into said mixture to remove excess hydrogen chloride therefrom; and

collecting said hydrochloride salt consisting of Dolaisoleuine tert-butyl ester hydrochloride [Dil-OBu^t HCl].

6. The method of claim 1 in which said N-Boc-S-prolinal is prepared by the steps of admixing triethylamine with a cooled solution of S-prolinol at 0°

C. in dichloromethane;

stirring said admixture;

adding di-tert-butyldicarbonate to said cool stirred admixture with stirring;

adding citric acid to said stirred mixture to form an organic layer;

washing said organic layer with water; and

removing the solvent from said washed organic layer to yield a clear oil consisting of N-Boc-S-prolinol.

7. The method of claim 1 in which Boc-(2S,2'"'"'R,3'"'"'R)-Dap is prepared by forming a mixture of the benzyl ester produced by claim 1 in ethyl acetate-methanol (3:

1) and 10% Pd/C at ambient temperature;

passing hydrogen through said mixture at ambient pressure for 48 hours;

collecting said Pd/C catalyst on celite;

washing said catalyst-containing celite with ethyl acetate to form a filtrate;

evaporating said filtrate to form an oil;

chromatographing said oil on a silica gel column;

eluting said column with hexane-acetone (7;

3) to form N-Boc-2S-3'"'"'-(2'"'"'S-methyl-3'"'"'R-methoxy-propionic acid)-pyrrolidine;

[Boc-(2S,2'"'"'S,3'"'"'R)-Dap].

8. The method of claim 1 in which Boc-(2S,2'"'"'R,3'"'"'R)-Dap is prepared by forming a first solution of the (1'"'"'R,2'"'"'R)benzyl ester produced by claim 1 in methanol;

forming a mixture of methanol and 10% Pd/C under nitrogen;

adding said first solution to said mixture with stirring in a system to form a second solution;

evacuating said system;

flushing said system with hydrogen;

removing said Pd/C catalyst from said second solution;

removing the solvent from said second solution to form a glassy solid;

dissolving said glassy solid in acetone;

filtering said acetone solution through silica gel to form a filtrate;

removing the solvent from said filtrate to form N-Boc-2S-3'"'"'-(2'"'"'R-methyl-3'"'"'R-methoxy-propionic acid) pyrrolidine;

[Boc-(2S,2'"'"'R,3'"'"'R)-Dap].

9. The method of claim 1 in which said S-Dov-S-Val-oBu^t is prepared by adding S-Val-OBu^t. hydrochloride to a solution of dolavaline in dimethylformamide to form a second solution;

warming said second solution to effect dissolution;

adding hydroxybenzotriazole and N-methylmorpholine to said warmed second solution to form a third solution;

cooling said third solution to 0°

C. over two hours;

adding dicyclohexylcarbodiimide to said cooled third solution with stirring at room temperature for about fifteen hours to form a precipitate and a filtrate;

separating said filtrate from said precipitate;

concentrating said filtrate;

adding said concentrated filtrate into ether to form an ethereal solution;

washing said ethereal solution with aqueous sodium bicarbonate in cold water;

solvent evaporating said washed ethereal solution to form a residue;

chromatographing said residue on a flash column; and

eluting said flash column with hexane-acetone (85;

15) to isolate S-Dov-S-Val-OBu^t.

10. The method according to claim 9 including the following steps:

forming a solution of S-Dov-S-Val-OBu^t in dichloromethane-trifluoroacetic acid at 0°

C.;

stirring said solution at room temperature for one hour;

evaporating the solvent from said solution;

adding ethanol to said solvent evaporated solution;

evaporating said ethanol-continuing solution to form a residue;

adding ether-hexane to said residue; and

crystallizing said residue from said ether-hexane to form S-Dov-S-Val.

11. The method of claim 1 in which said N-Z-S-Val-(3R,4S,5S)-Dil-OBu^t is prepared by forming a solution of N-Z-S-valine in anhydrous chloroform at -23°

C.;

adding to said solution under argon with stirring N-methylmorpholine followed by pivaloyl chloride to form a mixture;

stirring said mixture at -23°

C. for three hours;

adding (3R,4S,5S)-Dil-OBu^t.HCl and N-methyl morpholine to said stirred mixture with continuous stirring;

concentrating said stirred mixture by sweeping with argon at room temperature;

adding chloroform to said stirred solution;

washing said chloroform containing solution sequentially with saturated aqueous citric acid, water, aqueous sodium bicarbonate and water;

evaporating said washed solution to form a residue;

chromatographing said residue on a silica gel column; and

eluting said column with hexane-acetone to provide N-Z-S-Val-(3R,4S,5S)-Dil-OBu^t.

12. The method according to claim 4 including the steps of:

forming a solution of dolavaline in pyridine;

cooling said solution to 0°

C.;

adding pentafluorophenol trifluoroacetate and trifluoroacetic anhydride under argon to said stirred solution with stirring;

adding ethyl acetate to said stirred solution to form an organic phase;

washing said organic phase sequentially with aqueous sodium bicarbonate and water;

solvent evaporating said washed solution to form an oil;

purifying said oil by chromatography on a silica gel column; and

eluting said column with hexane-acetone (99;

1) to provide dolavaline pentafluorophenol ester.

13. The method of claim 12 including the steps of forming a solution of N-Z-(S)-Val-(3R,4S,5S)-Dil-OBu^t and dolavaline pentafluorophenol ester in anhydrous dioxane;

adding 10% Pd/C catalyst to said solution to form a mixture;

saturating said mixture with hydrogen under anhydrous conditions and ambient temperature and pressure;

filtering said hydrogenated mixture to remove said catalyst therefrom and provide a filtrate;

evaporating the solvent from said filtrate to provide a residue;

dissolving said residue in a benzene solvent to form a second solution;

adding trifluoroacetic acid at 0°

C. under argon to said second solution with stirring;

removing said solvent from said second solution to leave a second residue;

chromatographing said second residue on a SEPHADEX®

LH-20 column in 3;

2 dichloromethane-methanol; and

isolating, by means of flash chromatography on a silica gel column with a hexane-acetone (4;

1) eluant, a powder consisting of S-Dov-S-Val-(3R,4S,5S)-Dil-OBu^t.

16. The method according to claim 6 including the steps of:

dissolving N-Boc-S-prolinol in anhydrous dimethylsulfoxide at room temperature to form a solution;

adding triethylamine to said solution to form a mixture;

stirring said mixture at room temperature;

cooling said stirred mixture to 0°

C.;

adding sulfur trioxide-pyridine complex to said cooled mixture with stirring at 0°

C. for two hours;

adding ice water to said mixture;

pouring said iced mixture into water;

extracted said water mixture with dichloromethane to form an extract;

washing said extract sequentially with water, saturated aqueous citric acid, water, saturated aqueous sodium bicarbonate and water;

drying said washed extract and removing the solvent therefrom to leave a residue;

drying said residue under high vacuum to provide N-Boc-S-Prolinal as a yellow oil.

17. The method of claim 1 in which said 2-S-propionyloxy-1,1,2-triphenylethanol is prepared by forming a solution of 2S-hydroxy-1,1,2-triphenylethanol in pyridine;

adding propionic anhydride to said solution to form a mixture;

stirring said mixture to form a precipitate;

filtering said mixture to collect said precipitate and form a filtrate;

concentrating said filtrate by azeotropic distillation with pyridine to form a concentrate; and

triturating said concentrate with methanol to form 2-S-propionyloxy-1,1,2-triphenylethanol.

18. The method of claim 1 in which said N-Boc-S-dolaphenine is prepared by forming a slurry of activated manganese dioxide with dry dioxane;

introducing said slurry into a dry glass column;

eluting said slurry containing glass column with dioxane under positive nitrogen pressure;

flowing N-Boc-2(3),4(5)-tetrahydro-dolaphenine in dry dioxane through said column for four hours;

discarding the initial fraction from said column;

collecting all subsequent fractions and recovering N-Boc-S-dolaphenine therefrom.

14. The method of synthesizing dolastatin 10 comprising the steps of:

forming a cooled first solution (0°

C.) of 2-(N-Boc-1'"'"'S-amino-2'"'"'-phenyl-ethyl)-2-thiazole (N-Boc-S- dolaphenine), in dichloromethane;

adding trifluoroacetic acid to said first solution to form a second solution;

stirring said second solution at 0°

C. for one hour and at room temperature for one hour;

evaporating the solvent from said second solution in a stream of nitrogen to leave a residue;

triturating said residue with hexane;

drying said triturated residue in a high vacuum over phosphorous pentoxide for 24 hours to form the trifluoroacetate salt of dolaphenine as a gum;

mixing said trifluoroacetate salt of dolaphenine in dimethoxyethane and Boc-(2S,2'"'"'R,3'"'"'R)-Dap to form a first mixture;

cooling said mixture to 0°

C.;

treating said cooled first mixture with triethylamine followed by diethylphosphorocyanidate to form a second mixture;

stirring said second mixture under argon for three hours at 0°

C.;

evaporating the solvent from said second mixture;

chromatographing said second mixture successfully on SEPHADEX®

LH-20 in hexane-methanol-dichloromethane (2;

2.5;

7.5) and on silica gel with hexane-acetone (9;

1) to yield Boc-(2S,2'"'"'R,3'"'"'R)-Dap-Doe;

concurrently therewith, forming a third solution of S-Dov-S-Val-(3R,4S,5S)-Dil-OBu^t in dichloromethane;

cooling said third solution to 0°

C.;

adding trifluoracetic acid to said cooled third solution;

holding said cooled third solution under argon for two hours;

evaporating said third solution with a stream of nitrogen;

adding carbon tetrachloride to said evaporated third solution;

evaporating said carbon tetrachloride from said third solution to provide a tripeptide [S-Dov-S-Val-(3R,4S,5S)-Dil-OH] trifluoroacetic acid salt;

drying said tripeptide trifluoroacetic acid salt;

adding trifluoracetic acid to said Boc-(2S,2'"'"'R,3'"'"'R)-Dap-S-Doe in dichloromethane at 0°

C. to form a fourth solution;

holding said fourth solution under argon for one hour;

evaporating said fourth solution with a stream of nitrogen;

adding carbon tetrachloride to said evaporated fourth solution;

evaporating said carbon tetrachloride from said fourth solution to form a dipeptide trifluoroacetic acid salt;

forming a fifth solution of said dipeptide [(2S,2'"'"'R,3'"'"'R)-Dap-S-Doe] trifluoroacetic acid salt and a trifluoroacetic acid salt of S-Dov-S-Val-(3R,4S,5S)-Dil in dimethoxyethane;

cooling said fifth solution under argon to 0°

C.;

adding triethylamine and diethylphosphorocyanidate to said cooled fifth solution;

stirring said fifth solution at 0°

C. for one hour and then at room temperature for two hours to complete the peptide bond formation;

evaporating the solvent from said fifth solution with a stream of nitrogen to form a crude mixture;

chromatographing said crude mixture successfully on a column of SEPHADEX®

LH-20 in hexane-dichloromethane- methanol (2;

7.5;

2.5) and on a column of silica gel;

eluting said silica gel column with dichloromethane to dichloromethane-methanol gradient of from one to five percent to yield dolastatin 10 as an amorphous powder.

15. The method according to claim 14 including the further steps of dissolving said amorphous powder in dimethoxyethane to form a sixth solution;

adding ammonium hydroxide to said sixth solution;

concentrating said sixth solution to dryness;

rechromatographing said dry solution successfully on columns of SEPHADEX®

LH-20, silica gel, and SEPHADEX®

LH-20 in methanol to produce pure Dolastatin 10.

19. The method of claim 14 in which said N-Boc-S-dolaphine is prepared by forming a solution of N-Boc-S- Phenylalaninol in anhydrous dimethylsulfoxide-triethylamine;

oxidizing said solution with sulfur trioxide pyridine complex;

adding water to said oxidized solution to precipitate N-Boc-S-phenylalaninol;

forming a solution of said N-Boc-S-phenylalaninal and 2-aminoethanethiol in anhydrous benzene;

stirring said solution under argon for four hours;

removing the solvents and excess reagents from said stirred solution by filtration through a silica gel pad to form a residue;

crystallizing said residue to form 2-(N-Boc-1'"'"'S-amino-2'"'"'-phenyl-ethyl)-2-(R,S)-thiazolidine [N-Boc-2(3),4(5)-tetrahydro-dolaphenine];

forming a suspension of battery grade manganese dioxide in anhydrous benzene;

adding 1'"'"'-ethyl (N-Boc-1'"'"'S-amino-2'"'"'-phenyl)-2 (R,S)-thiazolidine [N-Boc-2(3),4(5)-tetrahydro-dolaphenine] in benzene to said suspension to form a mixture;

stirring said mixture under argon at 55°

C. for 24 hours;

adding additional manganese dioxide with continued stirring;

filtering said stirred solution through silica gel;

washing said silica gel sequentially with benzene, ethyl acetate, chloroform and chloroform acetone (1;

1) to form a combined eluant;

evaporating said combined eluant to form a residue;

chromatographing said residue on a silica gel column;

eluting said column with ethyl acetate-hexane; and

crystallizing from said eluant granules consisting of 2-(1'"'"'S-Boc-amino-2'"'"'-phenyl-ethyl)-2-thiazole [N-Boc-S-dolaphenine].

20. The method of claim 14 in which said Boc-(2S,2'"'"'R,3R)-Dap is prepared by admixing trifluoroacetic anhydride in dichloromethane into a cooled -78°

C. solution of dimethylsulfoxide in dichloromethane with stirring;

adding N-Boc-S-prolinol in dichloromethane to said cool stirred mixture and continue stirring for about an hour;

adding triethylamine to said solution;

warming said solution to -20°

C.;

extracting said solution with ether to form an ethereal solution;

washing and concentrating said ethereal solution to form an oil;

dissolving said oil in acetone;

filtering said oil-acetone solution through a silica-gel column;

eluting said column with hexane-acetone (93;

7) to yield N-Boc-S-prolinal;

adding 2S-Propionyloxy-1,1,2-triphenylethanol to a solution of lithium diisopropylamide in tetrahydrofuran at -78°

C. under argon to form a reaction mixture;

permitting said reaction mixture to warm to room temperature over two hours to form a yellow solution;

cooling the yellow solution to -95°

C.;

adding freshly synthesized magnesium bromide to said cooled yellow solution;

thereafter adding a solution of Boc-S-prolinal in tetrahydrofuran to said yellow solution and allowing the reaction therebetween to proceed for two hours at -95°

C. under methanol-liquid nitrogen;

terminating said reaction with saturated aqueous ammonium chloride;

warming said mixture to 0°

C.;

adding ether to said mixture to form an ethereal solution;

washing, drying and solvent evaporating said ethereal solution to provide a glassy solid;

dissolving said glassy solid to form a solution;

adsorbing said solution on silica gel;

chromatographing said adsorbed silica gel;

eluting said chromatographed silica gel to provide N-Boc-2S-3'"'"'-[2"-ethoxy-(1"-hydroxy-1",1",2"-triphenyl) 2'"'"'S-methyl-3'"'"'R-hydroxy-propionate]-pyrrolidine;

methylating said N-Boc-2S-3'"'"' propionate-pyrrolidine in dichloromethane with boron trifluoride etherate and diazomethane in anhydrous dichloromethane;

eluting said methylated pyrrolidine with hexane-acetone (9;

1) to provide N-Boc-2S-3'"'"'-[2"-ethoxy(1"-hydroxy-1",1",2"-triphenyl) 2'"'"'S-methyl-3'"'"'R-methoxy-propionate]-pyrrolidine;

adding potassium tert-butoxide to a solution of said (2'"'"'S,3'"'"'R) ester in tetrahydrofuran with stirring until the solution turns bright yellow;

warming the bright yellow solution to -15°

C. and stopping the epimerization thereof by the addition of sufficient saturated aqueous citric acid thereto to dilute said solution;

extracting said solution with dichloromethane to form a combined extract;

washing said extract and removing the solvent therefrom to provide a clear yellow oil;

chromatographing said yellow oil on silica gel;

eluting said silica gel to provide N-Boc-2S-3'"'"'-[2"ethoxy(1"-hydroxy-1",1",2"-triphenyl) 2'"'"'S- methyl-3'"'"'R-methoxy-propionate] -pyrrolidine.

21. The method of claim 14 in which said S-Dov-S-Val-(3R,4S,5S)-Dil-OBu^t is prepared by admixing said N-Z-N-methyl-(S,S)- isoleucinol with triethylamine in dimethylsulfoxide at room temperature;

cooling the mixed solution to 0°

C.;

adding sulfur trioxide-pyridine complex to said solution under argon;

extracting the aldehyde from said solution with ether;

washing said ethereal solution successively with citric acid, water, and sodium bicarbonate to yield chromatographing pure N-Z-N-methyl-(S,S)-isoleucinal;

preparing a solution of lithium diisopropylamide from n-butyllithium and diisopropylamine in tetrahydrofuran at -78°

C.;

warming said solution to -20°

C.;

recooling said solution to -78°

C.;

adding tert-butyl acetate to said recooled solution;

stirring said solution while rewarming said solution to -20°

C.;

recooling said solution to -78°

C. and adding said chromatographically pure N-Z-N-methyl-(S,S)-isoleucinal thereto;

treating the resulting mixture with ice water;

extracting the mixture with ether;

drying said extract;

solvent evaporating said dry extract to yield a viscous oil;

chromatographing and eluting said viscous oil with hexane-acetone (47;

3) to yield tert-butyl(3S,4S), 5S)-3-hydroxy-4-(N,Z-N-methyl)-amino-5-methyl-heptanoate and tert-butyl (3R,4S,5S)-3- hydroxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate;

adding boron trifluoride etherate to a cooled and stirred solution of tert-butyl (3R,4S,5S)-3- hydroxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate in dichloromethane under argon;

thereafter adding an anhydrous solution of diazomethane in dichloromethane to said solution;

filtering the polymethylene side products from said solution to form a filtrate;

concentrating said filtrate;

chromatographing said concentrated filtrate on a silica gel column;

eluting said column with hexane-acetone (97;

3) to yield tert-butyl (3R,4S,5S)-3- methoxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate.

22. The method of claim 19 in which said Boc-(2S,2'"'"'R,3'"'"'R)-Dap is prepared by admixing trifluoroacetic anhydride in dichloromethane into a cooled -78°

C. solution of dimethylsulfoxide in dichloromethane with stirring;

adding N-Boc-S-prolinol in dichloromethane to said cool stirred mixture and continue stirring for about an hour;

adding triethylamine to said solution;

warming said solution to -20°

C.;

extracting said solution with ether to form an ethereal solution;

washing and concentrating said ethereal solution to form an oil;

dissolving said oil in acetone;

filtering said oil-acetone solution through a silica-gel column;

eluting said column with hexane-acetone (93;

7) to yield N-Boc-S-prolinal;

adding 2S-Propionyloxy-1,1,2-triphenylethanol to a solution of lithium diisopropylamide in tetrahydrofuran at -78°

C. under argon to form a reaction mixture;

permitting said reaction mixture to warm to room temperature over two hours to form a yellow solution;

cooling the yellow solution to -95°

C.;

adding freshly synthesized magnesium bromide to said cooled yellow solution;

thereafter adding a solution of Boc-S-prolinal in tetrahydrofuran to said yellow solution and allowing the reaction therebetween to proceed for two hours at -95°

C. under methanol-liquid nitrogen;

terminating said reaction with saturated aqueous ammonium chloride;

warming said mixture to 0°

C.;

adding ether to said mixture to form an ethereal solution;

washing, drying and solvent evaporating said ethereal solution to provide a glassy solid;

dissolving said glassy solid to form a solution;

adsorbing said solution on silica gel;

chromatographing said adsorbed silica gel;

eluting said chromatographed silica gel to provide N-Boc-2S-3'"'"'-[2"-ethoxy-(1"-hydroxy-1",1",2"-triphenyl) 2'"'"'S-methyl-3'"'"'R-hydroxy-propionate]-pyrrolidine;

methylating said N-Boc-2S-3'"'"' propionate-pyrrolidine in dichloromethane with boron trifluoride etherate and diazomethane in anhydrous dichloromethane;

eluting said methylated pyrrolidine with hexane-acetone (9;

1) to provide N-Boc-2S-3'"'"'-[2"-ethoxy(1"-hydroxy-1",1",2"-triphenyl) 2'"'"'S-methyl-3'"'"'R-methoxy-propionate]-pyrrolidine;

adding potassium tert-butoxide to a solution of said (2'"'"'S,3'"'"'R) ester in tetrahydrofuran with stirring until the solution turns bright yellow;

warming the bright yellow solution to -15°

C. and stopping the epimerization thereof by the addition of sufficient saturated aqueous citric acid thereto to dilute said solution;

extracting said solution with dichloromethane to form a combined extract;

washing said extract and removing the solvent therefrom to provide a clear yellow oil;

chromatographing said yellow oil on silica gel;

eluting said silica gel to provide N-Boc-2S-3'"'"'[2"ethoxy(1"-hydroxy-1",1",2"-triphenyl) 2'"'"'S-methyl-3'"'"'R-methoxy-propionate]-pyrrolidine.

23. The method of claim 19 in which said S-Dov-S-Val-(3R,4S,5S)-Dil-OBu^t is prepared by admixing said N-Z-N-methyl-(S,S)- isoleucinol with triethylamine in dimethylsulfoxide at room temperature;

cooling the mixed solution to 0°

C.;

adding sulfur trioxide-pyridine complex to said solution under argon;

extracting the aldehyde from said solution with ether;

washing said ethereal solution successively with citric acid, water, and sodium bicarbonate to yield chormatographically pure N-Z-N-methyl-(S,S)-isoleucinal;

preparing a solution of lithium diisopropylamide from n-butyllithium and diisopropylamine in tetrahydrofuran at -78°

C.;

warming said solution to -20°

C.;

recooling said solution to -78°

C.;

adding tert-butyl acetate to said recooled solution;

stirring said solution while rewarming said solution to -20°

C.;

recooling said solution to -78°

C. and adding said chromatographically pure N-Z-N-methyl-(S,S)-isoleucinal thereto;

treating the resulting mixture with ice water;

extracting the mixture with ether;

drying said extract;

solvent evaporating said dry extract to yield a viscous oil;

chromatographing and eluting said viscous oil with hexane-acetone (47;

3) to yield tert-butyl(3S,4S,5S)-3-hydroxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate and tert-butyl (3R,4S,5S)-3-hydroxy-4-(N-Z-N-methyl)amino-5-methyl-heptanoate;

adding boron trifluoride etherate to a cooled and stirred solution of tert-butyl (3R,4S,5S)-3-hydroxy-4-(N-Z-N-methyl)amino-5-methyl-heptanoate in dichloromethane under argon;

thereafter adding an anhydrous solution of diazomethane in dichloromethane to said solution;

filtering the polymethylene side products from said solution to form a filtrate;

concentrating said filtrate;

chromatographing said concentrated filtrate on a silica gel column;

eluting said column with hexane-acetone (97;

3) to yield tert-butyl (3R,4S,5S)-3-methoxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate [N-Z-Dolaisoleuine tert-butyl ester].

24. The method of claim 20 in which said S-Dov-S-Val-(3R,4S,5S)-Dil-OBu^t is prepared by admixing said N-Z-N-methyl-(S,S)-isoleucinol with triethylamine in dimethylsulfoxide at room temperature;

cooling the mixed solution to 0°

C.;

adding sulfur trioxide-pyridine complex to said solution under argon;

extracting the aldehyde from said solution with ether;

washing said ethereal solution successively with citric acid, water, and sodium bicarbonate to yield chromatographically pure N-Z-N-methyl-(S,S)-isoleucinal;

preparing a solution of lithium diisopropylamide from n-butyllithium and diisopropylamine in tetrahydrofuran at -78°

C.;

warming said solution to -20°

C.;

recooling said solution to -78°

C.;

adding tert-butyl acetate to said recooled solution;

stirring said solution while rewarming said solution to -20°

C.;

recooling said solution to -78°

C. and adding said chormatographically pure N-Z-N-methyl-(S,S)- isoleucinal thereto;

treating the resulting mixture ether;

drying said extract;

solvent evaporating said dry extract to yield a viscous oil;

chromatographing and eluting said viscous oil with hexane-acetone (47;

3) to yield tert-butyl(3S,4S, 5S)-3-hydroxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate and tert-butyl (3R,4S,5S)-3-hydroxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate;

adding boron trifluoride etherate to a cooled and stirred solution of tert-butyl (3R,4S,5S)-3-hydroxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate in dichloromethane under argon;

thereafter adding an anhydrous solution of diazomethane in dichloromethane to said solution;

filtering the polymethylene side products from said solution to form a filtrate;

concentrating said filtrate;

chromatographing said concentrated filtrate on a silica gel column;

eluting said column with hexane-acetone (97;

3) to yield tert-butyl (3R,4S,5S)-3-methoxy-4-(N-Z-N-methyl)-amino-5-methyl-heptanoate.