Patent Application: US-28490599-A

Abstract:
disclosed is a synthetic method for the preparation of analogs of didemnin a , particularly the amino - hip analog of didemnin a , also known as “ aipdidemnin a ” . these compounds have the following structures :

Description:
we have described the total syntheses of the amino hip analogue of didemnin a . previous studies have shown that didemnins are subject to hydrolysis and undergo decomposition due to the instability of the ester bonds . replacement of the ester bond with an amide linkage should increase the maintenance of the active cyclic conformation and , thus , provide a compound of greater activity . 1 h nmr spectra were recorded on varian xl - 200 , general electric qe - 300 , varian xl - 400 , and general electric qn - 500 spectrometers . 1 h chemical shifts are references in cdcl 3 and methanol - d 4 to residual chcl 3 ( 7 . 26 ppm ) and cd 2 hod ( 3 . 34 ppm ). electron impact ( ei ) mass spectra were recorded on a finnigan mat ch - 5 df spectrometer . high resolution ( hrfab ) and fast atom bombardment ( fab ) mass spectra were recorded on a vg zab - se mass spectrometer operating in the fab mode sing magic bullet matrix . 27 microanalytical results were obtained from the school of chemical sciences microanalytical laboratory . infrared ( ir ) spectra were obtained on an ir / 32 ftir spectrophotometer . solid samples were analyzed as chloroform solutions in sodium chloride cells . liquids or oils were analyzed as neat films between sodium chloride plates . optical rotations ( in degrees ) were measures with a dip 360 or a dip 370 digital polarimeter with an na lamp ( 589 nm ) using a 5 × 0 . 33 - cm ( 1 . 0 ml ) cell . melting points were determined on a capillary melting point apparatus and are not corrected . normal phase column chromatography was performed using merck - kieselgel silica gel ( 70 - 230 mesh ). fuji - davison c18 gel ( 100 - 200 mesh was used for reversed phase column chromatography . all solvents were spectral grade . analytical thin layer chromatography was performed on precoated plates ( merck , f - 254 indicator ). these plates were developed by various methods including exposure to ninhydrin , iodine , and uv light ( 254 nm ). hplc was performed with a waters 900 instrument and an econosil c 18 column ( alltech / applied science ) and a phenomenex c 18 column . thf was distilled from sodium benzophenone ketyl and ch 2 cl 2 from p 2 o 5 . dimethylformamide ( dmf ), triethylamine ( et 3 n ), and n - methylmorpholine ( nmm ) were distilled from calcium hydride and stored over koh pellets . pyridine was distilled from koh and stored over molecular sieves . other solvents used in reactions were reagent grade without purification . di - tert - butyl dicarbonate [( boco ) 2 o ], dicyclohexycarboniimide ( dcc ), i -( 3 - dimethylaminopropyl )- 3 - ethylcarboniimide hydrochloride ( edci ), dimethylaminophtidine ( dmap ). i - hydrozybenzotriazole ( hobt ), d - and l - isoleucine , l - tyrosine , l - isoleucine , l - threonin , d - valine , and l - proline were obtained from the aldrich chemical company . all reactions requiring anhydrous conditions were performed under an atmosphere of nitrogen . sodium hydride ( 60 % dispersion , 6 . 47 g , 162 . 9 mmol ) was added portionwise , with cooling , to a solution of cbz - d - leuoh ( 14 . 4 g , 54 . 3 mmol ) in thf ( 21 . 4 ml ) was added portionwise , with cooling . methyl iodide ( 27 . 0 ml , 435 mmol ) was then added via a dropping funnel . the reaction was allowed to stand at room temperature for 24 hours . ethyl acetate ( 70 ml ) was slowly added to the reaction mixture , followed by water , to destroy the excess sodium hydride . the solution was then evaporated to dryness and theoily residue partitioned between ether ( 30 ml ) and water ( 60 ml ). the ether layer was washed the aqueous sodium bicarbonate ( 5 ml ) and the combined aqueous layers were acidified with 4n hcl to ph 3 . the solution was extracted with ethyl acetate ( 3 × 15 ml ) and the extract was washed with 5 % aqueous sodium thiosulfate ( 2 × 10 ml ) and water ( 10 ml ). the solution was dried over sodium sulfate and the solvent evaporated to give an oily residue which crystallized overnight . recrystallization from petroleum ether produced a white solid ( 12 . 7 g , 84 %); [ α ] 29 na + 24 . 7 ° ( c 0 . 02 , chcl 3 ), lit . 11b [ α ] 29 d + 26 . 9 ° ( c 0 . 02 , chcl 3 ); m . p . 71 - 72 ° c . ( lit . 11b 72 - 73 ° c . ); 1 h nmr ( 300 mhz , cdcl 3 δ 7 . 40 - 7 . 27 ( 5h , m ), 5 . 17 ( 2h , s ), 4 . 74 ( 1h , m ), 2 . 87 ( 3h , s ), 1 . 78 - 1 . 76 ( 2h , m ), 1 . 62 - 1 . 57 ( 1h , m ), 0 . 92 - 0 . 80 ( 6h , m ); fabms 280 . 2 ( m + h ), 236 . 2 ( m − co 2 ); hrfabms cacd for c 15 h 22 no 4 ( m + h ) 280 . 1549 , found 280 . 1556 ; anal . calcd for c 15 h 21 no 4 ; c , 64 . 48 ; h , 7 . 58 ; n , 5 . 02 . found : c , 64 . 30 ; h , 7 . 65 ; n , 4 . 93 . a current of dry hcl was passed through a suspension of l - threonine ( 35 . 0 g , 0 . 29 mol ) in absolute ethanol ( 350 ml ), with shaking , until a clear solution formed . the solution then refluxed for 30 minutes , and was evaporated to dryness under reduced pressure , and the oily residue was taken up in absolute ethanol ( 175 ml ) and , again , taken to dryness under reduced pressure . the oily residue was then treated with a saturated solution of ammonia in chloroform . the ammonium chloride was filtered off and the filtrate was taken to dryness at 0 ° c . under reduced pressure . a yellow solid was isolated ( 36 . 2 g . 85 %); [ α ] 29 na + 0 . 82 ° ( c 5 . 0 , etoh ). lit . 12 [ α ] 29 d + 0 . 95 ° ( c 5 . 0 etoh ); m . p . 51 - 53 ° c . ( lit 12 52 - 54 ° c . ); 1 h nmr ( 200 mhz , cdcl 3 w / tms ) δ 4 . 82 ( 1h . m ), 4 . 40 ( 1h , d ), 4 . 05 ( 2h , q ), 1 . 62 ( 3h , d ), 1 . 21 ( 3h ; t ); fabms 148 . 2 ( m + h ); hrfabms calcd for c 6 h 14 no 3 ( m + h ) 148 - 0974 , found 148 . 0972 . z - d - meleuoh ( 2 . 12 g . 7 . 59 mmol ) was dissolved in 100 ml of ch 2 cl 2 and cooled to 0 ° c . dcc ( 1 . 72 g , 8 . 35 mmol ) was added and the solution was stirred at 0 ° c . for 10 minutes . l - throet ( 1 . 12 g . 7 . 59 mmol ) in a ml of ch 2 cl 2 was added and the solution was allowed to warm to rt . after approximately 15 hours , dicyclohexylurea was removed by filtration and washed with ch 2 cl 2 . the filtrate was washed with 10 % citric acid , 5 % sodium bicarbonate , and water and dried over sodium sulfate . the solution was evaporated to dryness and the product purified by silica gel column chromatography ( hexane / etoac = 65 / 35 ) to afford the product as a yellow oil ( 2 . 35 g . 76 %); 1 h nmr ( 400 mhz , cdcl 3 ) δ 7 . 36 - 7 . 42 ( 5h , m ), 6 . 73 ( 1h . d ), 6 . 50 ( 1h , br s ), 5 . 18 ( 2h , s ), 4 . 83 ( 1h , m ), 4 . 51 ( 1h , m ), 4 . 30 ( 1h , m ), 4 . 17 ( 2h , q ), 2 . 81 ( 3h , s ), 1 . 74 ( 2h , m ), 1 . 70 ( 3h , d ), 1 . 68 ( 2h , m ), 1 . 20 ( 3h , t ), 0 . 82 - 0 . 90 ( 6h , m ); fabms 431 . 4 ( m + na ), 409 . 2 ( m + h ); hbfabms calcd for c 21 h 33 n 2 o 6 ( m + h ) 409 . 2344 . found 409 . 2339 ; anal . calcd for c 21 h 32 n 2 o 6 : c , 61 . 73 ; h , 7 . 90 ; n , 6 . 86 . found =: c , 62 . 00 ; h , 8 . 08 ; n , 7 . 07 . z - d - meleuthroet ( 1 . 80 g , 4 . 42 mmol ) was dissolved in methanol and 2n koh was slowly added to the mixture at 0 ° c . the solution was stirred for 2 hours . tlc analysis ( chcl 3 / meoh 95 : 5 ) showed the reaction to be complete . the mixture was neutralized using 2n chi . the solvent was then evaporated . the solution was partitioned between ethyl acetate and water and the organic layer separated . aqueous hcl was added to the aqueous layer to ph 3 . this was extracted with ethyl acetate and all o the ethyl acetate extracts were combined . the solution was dried over mgso 4 and the solvent evaporated to give a dark orange oil ( 1 . 77 g . 98 %) which was used for the next reaction without purification ; 1 h nmr ( 200 mhz , cdcl 3 ) α 7 . 36 - 7 . 41 ( 5h , m ), 6 . 72 ( 1h , s ), 6 . 52 ( 1h , br s ), 5 . 18 ( 2h , s ), 4 . 83 ( 1h , m ), 4 . 51 ( 1h ., m ), 4 . 30 ( 1h , m ) 2 . 81 ( 3h , s ), 1 . 74 ( 2h , m ), 1 . 70 ( 3h , d ), 1 . 68 ( 1h , m ), 0 . 82 - 0 . 90 ( 6h , m ); fabms 381 . 2 ( m + h ); hrfabms calcd for c 19 h 29 n 2 o 6 ( m + h ) 381 . 2026 , found 381 . 2021 ; anal . calcd for c 19 h 28 n 2 o 6 : c , 59 . 97 ; h , 7 . 42 ; n , 7 . 37 , found . c , 60 . 53 ; h , 7 . 06 ; n , 7 . 11 . z - d - meleuthroh ( 1 . 50 g . 3 . 95 mmol ) was dissolved in ethyl acetate ( 25 ml ). triethylamine ( 0 . 39 g . 3 . 95 mmol ) and phenacyl bromide ( 0 . 079 g ., 3 . 98 mmol ) were added and , within a few minutes , a precipitate formed . the mixture was stirred overnight . at this time , water and ether were added and the two layers separated . the organic layer was washed with 0 . 1n hcl , saturated sodium bicarbonate , and brine , and then dried over mgso 4 . the residue was chromatographed on silica gel ( hexane / etoac = 4 / 1 ) to give a clear oil ( 1 . 71 g , 87 %); 1 h nmr ( 200 mhz , cdcl 3 ) δ 7 . 34 - 7 . 41 ( 10h , m ), 6 . 71 ( 1h , s ), 6 . 52 ( 1h , br s ), 5 . 27 ( 2h , s ), 5 . 18 ( 2h , s ), 4 . 83 ( 1h , m ), 4 . 61 ( 1h , m ), 4 . 50 ( 1h , m )., 2 . 81 ( 3h , s ), 1 . 74 ( 2h . m ), 1 . 70 ( 3h , d ), 1 . 68 ( 1h , m ), 0 . 82 - 0 . 90 ( 6h , m ); fabms 537 . 1 ( m + k ), 499 . 1 ( m + h ); hrfabms calcd for c 27 h 35 n 2 o 7 ( m + h ) 499 . 2444 . found 499 . 2450 . tyrosine ethyl ester ( 5 . 06 g , 25 mmol ) was dissolved in 25 ml of water and solid sodium hydroxide was added until litmus paper indicated a neutral ph . diozane ( 50 ml ) and ( boc ) 2 o ( 6 . 12 g , 27 . 5 mmol ) were added with cooling . the reaction was allowed to stir for 2 hours . water and ether were added and the two layers separated . the organic layer was extracted three times with aqueous sodium hydroxide ( in ). the aqueous layers were allowed to sit overnight then neutralized with aqueous hcl and extracted with ether , which was washed with brine and dried over mgso 4 . a yellow oil was obtained ( 6 . 02 g , 86 %); 1 h nmr ( 200 mhz , cdcl 3 ) δ 7 . 10 ( 2h , d ), 6 . 84 ( 2h , d ), 4 . 92 - 5 . 00 ( 1h , m ), 4 . 47 - 4 . 52 ( 1h , m ), 3 . 00 - 3 . 12 ( 2h , m ), 1 . 43 ( 9h , s ); eims 282 . 0 . a solution of boctyroh ( 5 . 30 g , 18 . 8 mmol ) and methyl iodide ( 2 . 57 ml , 41 . 4 mmol ) in 80 ml of dry thf was cooled at 0 ° c . and sodium hydride ( 60 % dispersion , 2 . 47 g , 62 . 0 mmol ) was added . the reaction was allowed to stir at 0 ° c . for 1 hour , then at rt overnight . excess sodium hydride was quenched by the dropwise addition of 10 ml of thf / h 2 o ( 1 : 1 ) and the solvents were removed in vacuo . after removal of the solvents , the deep orange gel was diluted with 30 ml of water and washed with pentane ( 2 × 30 ml ). the aqueous phase was acidified with solid citric acrd ( ph 2 ). ethyl acetate was used for extraction . the combined extracts were washed with brine , dried ( mgso 4 ) and concentrated in vacuo . the crude residue was purified by silica gel column chromatography eluting with ethyl ether to afford the desired compound as a yellow oil ( 5 . 22 g , 90 %); [ α ] 29 d - 15 . 7 ° ( c 1 . 0 , meoh ), lit . 14b [ α ] 22 d - 16 . 9 ° ( c 1 . 0 , meoh ) 1 h nmr 300 mhz , cdcl 3 ) δ 7 . 18 & amp ; 7 . 12 ( 2h , two d ), 6 . 85 ( 2h , d ), 4 , 58 ( 1h , two t ), 3 . 80 ( 3h , s ), 3 . 24 & amp ; 3 . 13 ( 1h , 2dd ), 2 . 76 & amp ; 2 . 68 ( 3h , 2s ), 1 . 43 & amp ; 1 . 38 ( 9h , 2s ); fabms 619 . 3 ( 2m + h ), 310 . 2 ( m + h ), 210 . 2 ( m − boc ); hrfabms calcd for c 16 h 24 mo 5 ( m + h ) 310 . 1654 , found 310 . 1648 . bocme 2 tyroh ( 0 . 27 g , 0 . 91 mmol ) in ch 2 cl 2 ( 20 ml ), dcc ( 19 . 5 mg , 0 . 95 mmol ) and dmap ( 41 . 3 mg ) were added at 0 ° to a solution of cbz - d - meleuthropac ( 0 . 45 g , 0 . 91 mmol ). the solution was allowed to warm to room temperature and stirred for 12 h . dicycloheylurea was filtered and washed with ethyl acetate . the filtrate and washings were combined and washed with 10 % citric acid , 5 % sodium bicarbonate and water , dried over mgso 4 and concentrated . the crude residue was purified by flash column chromatography eluting with hexane and ethyl acetate ( 4 : 1 ) to obtain the product ( 0 . 53 g , 74 %) as an orange oil ; 1 h nmr ( 500 mhz , cdcl 3 δ 7 . 30 - 8 . 00 ( 10h , m ), 6 . 82 & amp ; 7 . 10 ( 2h , d ), 5 . 31 ( 1h , s ), 5 . 20 - 4 . 8 . 5 ( 1h , m ), 3 . 74 ( 3h , s ), 3 . 10 & amp ; 3 . 07 ( 1h , 2 dd ) 2 . 92 ( 3h , s ), 2 . 73 ( 3h , s ), 1 . 71 ( 3h , d ), 1 . 44 & amp ; 1 . 37 ( 9h , 2 s ), 0 . 92 ( 6h , m ); fabms 828 . 4 ( m + k ), 812 . 4 ( m + na ), 790 . 3 ( m + h ), 690 . 4 m − boc ); hrfabms calcd for d 43 h 57 n 3 o 11 ( m + h ) 790 . 3915 , found 790 . 3916 . the tripeptide 10 ( 30 . 0 mg , 38 . 0 μmol ) was treated with zn ( 60 mg ) in acoh / h 2 o ( 70 : 30 ) and the mixture was stirred at rt overnight , zn was filtered off using celite and the solution was partitioned between ether and water . the organic layer was separated and dried over na 2 so 4 . purification by reversed phase column chromatography ( ch 3 ch / h 2 o gradient system ) afforded the product as a clear oil ( 21 . 3 mg , 92 %); fabms 710 . 4 ( m + k ), 694 . 3 ( m + na ), 672 . 3 ( m + h ), 572 . 3 ( m − boc ), see fig1 ; hrfabms calcd for c 35 h 52 n 4 o 9 ( m + h ) 672 . 3734 , found 672 , 3674 . acetyl chloride ( 6 . 13 ml ) was added dropwise to meoh ( 90 ml ) cooled in an ice bath . after addition was complete , a solution of the α - hydroxy acid ( 23 . 0 g , 0 . 17 mol ) in meoh ( 60 ml ) was added . the solution was stirred at 0 ° c . for 1 h , then at rt oversight , concentrated and diluted with ether . the either solution was washed with saturated nahco 3 , brine , bried over mgso 4 and concentrated to give a yellow oil ( 19 . 8 g , 80 %); [ α ] 29 d + 27 . 3 ( c 0 . 95 , chcl 3 ), lit . 18 [ λ ] 20 d + 28 . 5 ( c 0 . 95 , chcl 3 ); 1 h nmr ( 300 mhz , cdcl 3 ) δ 0 . 91 ( t , 3h ), 0 . 97 ( d , 3h ), 1 . 21 & amp ; 1 . 37 ( m , 2h ), 1 . 78 ( m , 1h ), 2 . 92 ( br s , 1h ), 3 . 82 ( s , 3h ), 4 . 08 ( d , 1h ); cims 147 . 1 ( m + h ). the hydroxypentanoate ( 4 . 44 g , 30 . 6 mmol ) was dissolved in dry ch 2 cl 2 and cooled in an ice bath to 0 ° c . pyridine ( 45 . 0 ml ) was added followed by p - toluenesulfonyl chloride ( 11 . 5 g , 60 . 8 mmol ) in small portions with constant stirring . the mixture was stirred at rt overnight , then heated at 40 ° c . for 1 h . the solvent was evaporated and the residue dissolved in etoac and washed with 1n h 2 so 4 and 1n khco 3 . the extracts were dried over mgso 4 and evaporated in vacuo to give a dark orange oil ( 8 . 32 g , 88 %); 1 h nmr ( 300 mhz , cdcl 3 ) δ 0 . 93 ( t , 3h ), 0 . 97 ( d , 3h ), 1 . 21 & amp ; 1 . 41 ( m , 1h ), 1 . 91 ( m , 1h ), 2 . 41 ( s , 2h ), 3 . 60 ( s , 3h ), 4 . 63 ( d , 2h ), 7 . 24 & amp ; 7 . 80 ( d , 2h ); fabms 339 . 2 ( m + k ), 323 . 2 ( m + na ), 301 . 1 ( m + h ), 241 . 2 ( m − co 2 ch 3 ); hrfabms calcd for c 14 h 21 o 5 s ( m + h ) 301 . 11 10 , found 301 . 1109 . sodium azide ( 1 . 20 g , 18 . 6 mmol ) was added to a stirred solution of methyl 2 - tosyloxy - 3 - methylpentanoate ( 3 . 29 g , 10 . 9 mmol ) in dmf ( 30 ml ). the solution was kept at 50 ° c . for 24 h , then partitioned between etoac and water . the aqueous layer was separated and extracted with etoac ( 3 × 50 ml ). the combined organic layers were dried over mgso 4 and concentrated in vacuo to give a deep yellow oil ( 1 . 51 g , 81 %), ir ( neat ) v 3500 - 3000 ( very br m ), 2970 ( s ), 2939 ( br m ), 2111 ( s ), 1736 ( s ), 1472 ( w ), 1387 ( w ), 1225 ( br m ), 1175 ( w ), 1086 ( w ), 732 ( s ) cm − 1 ; 1 h nmr ( 300 mhz , cdcl 3 ) δ 0 . 93 ( t , 3h ), 0 . 97 ( d , 3h ), 1 . 22 & amp ; 1 . 43 ( m , 1h ), 1 . 96 ( m , 1h ), 3 . 78 ( s , 3h ), 3 . 85 ( d , 2h ); cims 172 . 1 ( m + h ). to a solution of a α - azido ester ( 6 . 56 g , 38 . 3 mmol ) in thf ( 58 ml ) at 0 ° c . was added 1n naoh ( 52 ml ). the reaction mixture was stirred at 0 ° c . for 1 h and then at rt overnight . the mixture was diluted with ether ( 30 ml ), the organic layer separated , and the aqueous phase extracted with ether ( 30 ml ). the aqueous layer was then cooled to 0 ° c ., acidified to ph 2 by dropwise addition of conc . hcl , and extracted with ethyl acetate ( 3 × 25 ml ). the combined ethyl acetate extracts were dried ( mgso 4 ) and concentrated in vacuo to furnish 10 . 9 g ( 95 %); ir ( neat ) v max 3500 - 3000 ( very br m ), 2974 ( s ), 2942 ( br m ), 2090 ( s ), 1464 ( w ), 1382 ( w ), 1222 ( br m ), 1168 ( w ), 1088 ( w ), 721 ( s ) cm − 1 ; 1 h nmr ( 300 mhz , cdcl 3 ) δ 0 . 93 ( 3h , t ), 0 . 97 ( 3h , d ), 1 . 21 & amp ; 1 . 41 ( 1h , m ), 1 . 97 ( 1h , m ), 3 . 90 ( 2h , m ); fabms 158 . 2 ( m + h ); hrfabms calcd for c 14 h 21 o 5 s ( m + h ), 301 . 1110 , found 301 . 1109 . to a solution of the α - azido acid ( 6 . 01 g , 38 . 2 mmol ) in meoh ( 25 ml ) was added 20 % pd ( oh ) 2 on carbon ( 1 . 89 g ). the reaction flask was purged with h 2 gas and the contents vigorously stirred at rt and atmospheric pressure for 15 h , filtered and the filter pad washed with distilled water and ethanol . the filtrate was concentrated in vacuo to afford the product as a white solid . recrystallization from etoac provided the compound as colorless needles ( 4 . 75 g , 95 %); 1 h nmr ( 300 mhz , meoh - d 4 ) δ 0 . 93 ( t , 3h ), 0 . 97 ( d , 3h ), 1 . 32 & amp ; 1 . 46 ( m , 1h ), 2 . 47 ( m , 1h ), 3 . 58 ( d , 2h ); fabms 132 . 1 ( m + h ); anal . calcd for c 6 h 13 no 2 : c , 54 . 92 ; h , 9 . 99 ; n , 10 . 64 . found : c , 54 . 79 ; h , 10 . 17 ; n , 10 . 26 a solution of d - allo - isoleucine ( 120 mg , 0 . 916 mmol ) was dissolved in water ( 2 . 5 ml ) and 1n naoh ( 1 . 83 ml ) and stirred at rt for 48 h . di - tert - butyl dicarbonate ( 200 mg , 0 . 916 mmol ) in dioxane ( 5 , 00 ml ) was added to the stirred mixture at 0 ° c . after 12 h the dioxane was evaporated , the aqueous residue washed with et 2 o , mixed with etoac , and the rapidly stirred mixture acidified with 2 n h 2 so 4 at 0 ° c . this solution was extracted with etoac . and the combined organic extracts were dried ( na 2 so 4 ) and coned in vacuo to a crystalline material ( 179 mg , 85 %); mp 35 - 37 ° c . ( lit . 28 34 - 36 ° c . ); [ α ] 29 d − 42 . 7 ° ( c 2 . 04 , chcl 3 ), [ lit . 28 [ α ] 27 d − 40 . 7 ° ( c 2 . 06 , chcl 3 ]); 1 h nmr ( 300 mhz . cdcl 3 ) δ 5 . 52 ( br s , 1h ), 3 . 72 - 3 . 54 ( m , 1h ), 1 . 92 - 2 . 01 ( m , 1h ), 1 . 43 ( s , 9h ). 1 . 37 - 1 . 12 ( m , 3h ), 0 . 97 ( t , 3h ), 0 . 93 ( d , 3h ); fabms 463 . 2 ( 2m + h ), 232 . 1 ( m + h ), 132 . 1 m − boc ); hrfabms calcd for c : 1 h 21 no 4 ( m + h ) 232 . 1551 , found 232 . 1548 . a previously reported procedure was used . 29 potassium hydroxide ( 10 . 02 g , 85 % koh , 156 mmol ) in ethanol ( 99 ml ) was added dropwise to a stirred solution of diethyl malonate ( 23 . 69 ml , 156 mmol ) in ethanol ( 108 ml ), and the solution was stirred at rt overnight . the mixture refluxed for 1 h and the solid was filtered off . the ethanolic solution on cooling gave the monopotassium salt . water ( 5 ml ) was added to the dried potassium salt , and the solution was cooled to 0 ° c . concentrated hydrochloric acid ( 3 . 45 ml ) was added , keeping the temperature below 5 ° c . the solid was filtered and washed with ether . the filtrate was extracted with ch 2 cl 2 , dried ( mgso 4 ), and concentrated to give a yellow oil ( 9 . 96 g , 48 %; lit . 29b 51 %); 1 h nmr ( 300 mhz , cdcl 3 ) δ 4 . 37 ( s , 2h ), 4 . 24 ( q , 2h ), 1 . 34 ( t , 3h ); fabms 133 . 0 ( m + h ). a tetrahydrofuran solution of isoproprylmagnesium chloride ( 1 . 42 ml , 13 . 5 mmol ) was added dropwise to a solution of ethyl hydrogen malonate ( 891 mg , 6 . 75 mmol ) in dry ch 2 cl 2 ( 5 . 62 ml ). the reaction was then cooled in an ice - salt bath while a solution of boc - d - allo - isoleucine ( 520 mg , 2 . 25 mmol ) and n , n ′- carbonyldiimidazole ( 360 mg , 2 . 25 mmol ) in dry thf ( 1 . 20 ml ) was added . the mixture was stirred overnight at rt , then poured into cold hydrochloric acid ( 10 %, 100 ml ). the ethyl ester was extracted with ether , washed with aqueous nahco 3 , dried ( mgso 4 ) and concentrated to give 475 mg ( 70 %) of a pale yellow oil ; ir ( neat ) v max 3355 , 1750 , 1700cm − 1 ; 1 h nmr ( 300 mhz , cdcl 3 ) δ 4 . 98 ( d , 1h ), 3 . 90 - 4 . 57 ( m , 1h ), 4 . 18 ( q , 2h ), 3 . 46 ( s , 2h ), 0 . 70 - 2 . 00 ( m , 6h ), 1 . 43 ( s , 9h ), 1 . 26 ( t , 3h ), 0 . 78 ( d , 3h ); fabms 302 . 2 ( m + h ), 202 . 2 ( m − boc ). to a stirred solution of 18 ( 500 mg , 1 . 66 mmol ) in et 2 o ( 2 . 90 ml ) and etoh ( 6 . 80 ml ) cooled in an ice - salt bath was added nabh 4 ( 60 mg , 1 . 58 mmol ). the solution was allowed to stir at − 20 ° c . for 2 h then poured into ice water . extracted with etoac and dried over mgso 4 . the residue was chromatographed on silica gel ( hexane / etoac = 4 / 1 ) to give 325 mg ( 65 %) of the desired isomer 19a and 25 mg ( 5 %) of the minor isomer 19b . 19a : r f 0 . 20 ( hexane / etoac = 3 / 1 ); [ α ] 29 d − 6 . 7 ° ( c . 0 . 5 , meoh ), lit . 30 [ α ] 23 d − 6 . 4 ° ( c 0 . 5 , meoh ); ir ( neat ) v max 3350 , 1740 , 1700 cm − 1 ; 1 h nmr ( 300 mhz , cdcl 3 ) δ 4 . 43 ( d , 1h ), 4 . 20 ( q , 2h ), 3 . 90 - 3 . 61 ( m , 2h ), 3 . 30 ( br s , 1h ), 2 . 50 ( d , 2h ), 1 . 90 - 1 . 98 ( m , 1h ), 1 . 41 - 1 . 30 ( m , 2h ), 1 . 40 ( s , 9h ), 1 . 24 ( t , 3h ), 0 . 97 ( d , 3h ), 0 . 90 ( t , 3h ); fabms 304 . 2 ( m + h ) 204 . 2 ( m − boc ); hrfabms calcd for c 15 h 29 no 5 ( m + h ) 304 . 2117 , found 304 . 2123 ; anal . calcd for c 15 h 28 no 5 : c , 59 . 37 ; h , 9 . 64 ; n , 4 . 62 . found : c , 59 . 03 ; h , 9 . 38 ; n , 4 . 88 . 19b : r f 0 . 22 ( hexane / etoac = 3 / 1 ); [ α ] 29 d + 26 . 9 ( c 0 . 5 , meoh ), lit . 30 [ α ] 23 d + 26 . 4 ( c 0 . 5 , meoh ); ir ( neat ) v max 3410 , 1740 , 1710 . cm − 1 ; 1 h nmr ( 300 mhz , cdcl 3 ) δ 4 . 42 ( d , 1h ), 4 . 20 ( q , 2h ), 3 . 87 - 3 . 61 ( m , 2h ), 3 . 32 ( br s , 1h ), 2 . 49 ( d , 2h ), 1 . 92 - 1 . 98 ( m , 1h ), 1 . 41 - 1 . 30 ( m , 2h ), 1 . 40 ( s , 9h ), 1 . 24 ( t , 3h ), 0 . 98 ( d , 3h ), 0 . 88 ( t , 3h ); fabms 304 . 2 ( m + h ), 204 . 2 ( m − boc ); hrfabms calcd for c 15 h 29 no 5 ( m + h ) 304 . 2117 , found 304 . 2123 ; anal . calcd for c 15 h 28 no 5 : c , 59 . 37 ; h , 9 . 64 ; n , 4 . 62 . found : c , 59 . 03 ; h , 9 . 38 ; n , 488 . boc -( 3s , 4r , 5s )- ist - oet ( 300 mg , 1 . 00 mmol ) was dissolved in methanol ( 5 . 00 ml ) and 2n naoh ( 2 . 00 ml ) was alowly added to the mixture at 0 ° c . the solution was stirred at rt overnight at which time tlc analysis ( hexane / etoac = 4 / 1 ) showed the presence of a carboxylic acid . the mixture was neutralized using 2n hcl . the solvent was evaporated and the solution was partitioned between etoac and water and the organic layer separated . aqueous hcl was added to the aqueous layer to ph 3 . this was extracted with etoac and the etoac extracts were combined . the solution was dried over mgso 4 and the solvent evaporated to give a yellow oil ( 215 mg , 78 %) which was used for the next reaction without purification ; [ α ] 29 d − 4 . 6 ° ( c 0 . 0014 , chcl 3 ), lit . 11b [ α ] 20 d − 57 ° ( c 0 . 0014 , chcl 3 ); 1 h nmr ( 300 mhz , cdcl 3 ) 4 . 43 ( d , 1h ), 3 . 85 - 3 . 63 ( m , 2h ), 2 . 76 ( br , 1h ), 2 . 41 ( m , 2h ), 2 . 00 - 1 . 93 ( m , 1h ), 1 . 43 - 1 . 35 ( m , 2h ), 1 . 43 ( s , 9h ), 0 . 91 ( t , 3h ), 0 . 87 ( d , 3h ); fabms 276 . 1 ( m + h ), 176 . 1 ( m − boc ); hrfabms calcd for c 13 h 25 no 5 ( m + h ) 276 . 1806 , found 276 . 1810 . potassium hydroxide ( 3 . 53 g , 90 % koh , 57 . 4 mmol ) in ethanol ( 35 ml ) was added dropwise to a stirred solution of diethyl methylmalonate ( 9 . 87 ml , 57 . 4 mmol ) in ethanol ( 40 ml ), and the solution was stirred at rt overnight . the mixture was heated at reflux for 1 hr and the solid filtered off . the ethanolic solution on cooling gave the monopotassium salt . water ( 5 ml ) was added to the dried potassium salt , and the solution was cooled to 0 ° c . concentrated hydrochloric acid ( 3 . 45 ml ) was added , keeping the temperature below 5 ° c . the solid was filtered and washed with ether and the filtrate was extracted with ch 2 cl 2 , then dried and concentrated to give a yellow oil ( 4 . 86 g , 58 %, lit . 29 60 %); 1 h nmr ( 200 mhz , cdcl 3 ) δ 4 . 23 ( q , 2h ), 3 . 47 ( q , 1h ), 1 . 42 ( d , 3h ), 1 . 27 ( t , 3h ); fabms 147 . 1 ( m + h ). a tetrahydrofuran solution of isopropylmagensium chloride ( 9 . 57 ml , 90 . 8 mmol ) was added dropwise to a solution of ethyl hydrogen methylmalonate ( 6 . 63 g , 45 . 4 mmol ) in dry ch 2 cl 2 ( 35 ml ). the reaction was then cooled in an ice - salt bath while a solution of cbz - l - valine ( 3 . 87 g , 15 . 1 mmol ) and n , n ′- carbonyldiimidazole ( 2 . 44 g , 15 . 1 mmol ) in dry thf ( 15 ml ) was added . the mixture was stirred overnight at rt , then poured into cold hydrochloric acid ( 10 %, 200 ml ). the ethyl ester was extracted with ether , washed with aqueous nahco 3 , brine , dried ( mgso 4 ) and concentrated . purification by flash column chromatography ( hexane / etoac = 10 / 1 ) gave the desired product as a yellow oil ( 4 . 50 g , 89 %); 1 h nmr ( 200 mhz , cdcl 3 ) δ 7 . 32 - 7 . 38 ( s , 5h ), 5 . 24 - 5 . 37 ( m , 1h ), 5 . 17 ( s , 2h ), 4 . 20 ( q , 2h ), 3 . 41 ( q , 1h ), 218 - 2 . 21 ( m , 1h ), 1 . 45 ( d , 3h ), 1 . 32 - 1 . 37 ( m , 1h ), 1 . 24 ( t , 3h ), 0 . 94 ( d , 3h ), 0 . 81 ( d , 3h ); fabms 374 . 0 ( m + k ), 336 . 1 ( m + h ), 292 . 1 ( m − oet ); hrfabms calcd for c 18 h 26 no 5 ( m + h ) 336 . 1811 , found 336 . 1817 ; anal . calcd for c 18 h 25 no 5 : c , 64 . 44 ; h , 7 . 52 ; n , 4 . 18 . found : c , 64 . 70 ; h , 7 . 62 ; n , 4 . 37 . to a stirred solution of cbz - aipoet ( 6 . 54 g , 19 . 5 mmol ) in et 2 o ( 15 ml ) and etoh ( 35 ml ) at − 20 ° c ., nabh 4 ( 0 . 74 g , 19 . 5 mmol ) was added over a period of 15 min . the reactioin mixture was stirred 15 min at − 20 ° c . and poured into ice water ( 50 ml ). after extraction with ethyl acetate ( 30 ml ), the combined organic extracts were dried ( mgso 4 ) and concentrated to give a yellow oil ( 6 . 12 g , 93 %); 1 h nmr ( 300 mhz , cdcl 3 ) δ 7 . 39 ( s , 5h ), 7 . 01 ( br s , 1h ), 5 . 13 ( s , 2h ), 4 . 72 - 4 . 53 ( m , 1h ), 4 . 18 ( q , 2h ), 3 . 80 - 3 . 71 ( m , 1h ), 3 . 38 ( br s , 1h ), 2 . 32 - 2 . 20 ( m , 1h ), 1 . 98 ( m , 1h ), 1 . 40 ( d , 3h ), 1 . 25 ( t , 3h ), 9 . 92 - 0 . 80 ( m , 6h ); fabms 338 . 1 ( m + h ); anal . calcd for c 18 h 27 no 5 : c , 64 . 06 ; h , 8 . 07 ; n , 4 . 15 . found : c , 64 . 21 ; h , 8 . 36 ; n , 4 . 29 . cbz - dihydroaipoet ( 5 . 99 g , 17 . 7 mmol ) was dissolved in methanol and 2n koh was slowly added to the mixture at 0 ° c . the solution was allowed to stir for 2 h . tlc analysis ( hexane / ethyl acetate 10 : 1 ) showed the reaction to be complete . at this time , 2n hcl was added to neutralization . the solvent was evaporated and the solution was partitioned between ethyl acetate and water . the organic layer was separated . aqueous hcl was added to bring the aqueous layer to ph 3 which was then extracted with ethyl acetate . the ethyl acetate extracts were combined , the solution was dried over mgso 4 and the solvent was evaporated to give a pale yellow oil ( 4 . 59 g , 84 %); 1 h nmr ( 300 mhz , cdcl 3 ) δ 7 . 39 ( s , 5h ), 7 . 04 ( br s , 1h ), 5 . 13 ( s , 2h ), 4 . 40 - 4 . 20 ( m . 1h ), 3 . 92 - 3 . 71 ( m , 1h ), 3 . 33 ( br s , 1h ), 2 . 32 - 2 . 20 ( m , 1h ), 1 . 98 ( m , 1h ), 1 . 40 ( d , 3h ), 0 . 92 - 0 . 80 ( m , 6h ); fabms 310 . 2 ( m + h ); hrfabms calcd for c 16 h 24 no 5 ( m + h ) 310 . 1654 , found 310 . 1651 ; anal . calcd for c 16 h 23 no 5 : c , 62 . 10 ; h , 7 . 51 ; n , 4 . 53 . found : c , 62 . 50 ; h , 7 . 71 ; n , 4 . 37 . cbz - dihydroaip - leuome ( 23 ). cbz - dihydroaipoh ( 1 . 17 g , 3 . 82 mmol ) was dissolved in dry ch 2 cl 2 ( 25 ml ) and cooled to 0 ° c . ddc ( 0 . 87 g , 4 . 20 mmol ) and dmap ( 0 . 32 g , 2 . 90 mmol ) were added with stirring and the mixture was stirred for 1 h . after filtration of dicycloheylurea , leucine methyl ester ( 0 . 56 g , 3 . 82 mmol ) was added and the mixture was stirred overnight . the residue was concentrated and taken up in ethyl acetate . the solution was washed with aqueous citric acid , aqueous sodium bicarbonate , dried over mgso 4 , and concentrated . the residue was purified by column chromatography eluting with hexane / ethyl acetate ( 50 : 50 ) to give a clear oil ( 1 . 23 g , 74 %); 1 h nmr ( 300 mhz , cdcl 3 ) δ 7 . 43 ( s , 5h ), 6 . 81 ( br s , 1h ), 6 , 42 ( br s , 1h ), 4 . 78 - 4 . 72 ( m , 1h ), 4 . 48 - 4 . 43 ( m , 1h ), 3 . 78 - 3 . 72 ( m , 1h ), 3 . 67 ( s , 3h ), 3 . 51 ( br s , 1h ), 2 . 50 - 2 . 40 ( m , 1h ), 2 . 37 - 2 . 20 ( m , 1h ), 1 . 40 - 1 . 30 ( dd , 6h ), 1 . 10 - 0 . 90 ( m . 9h ), fabms 437 . 2 ( m + h ); hrfabms calcd for c 23 h 37 n 2 o 6 ( m + h ) 437 . 2652 , found 437 . 2653 ; anal . calcd for c 23 h 36 n 2 o 6 : c , 63 . 27 ; h , 8 . 32 ; n . 6 . 42 . found : c , 63 . 65 ; h , 8 . 35 ; n , 6 . 49 . cbz - dihydroaip - leuome ( 303 mg , 0 . 70 mmol ) was dissolved in meoh and 2n koh was slowly added with cooling . after approximately 3 h stirring , tlc analysis ( hexane / ethyl acetate 6 : 1 ) showed reaction to be complete . the solution was neutralized with 2n hcl and extracted with ethyl actate . the aqueous layer was adjusted to ph 3 and extracted with ethyl acetate . the combined ethyl acetate extracts were then dried over mgso 4 . evaporation of the solvent left a yellow oil ( 270 mg , 92 %); 1 h nmr ( 200 mhz , cdcl 3 ) δ 7 . 42 ( s , 5h ), 6 . 81 ( br s , 1h ), 6 . 42 ( br s 1h ), 4 . 78 - 4 . 72 ( m , 1h ), 4 . 48 - 4 . 42 ( m , 1h ), 3 . 78 - 3 . 72 ( m , 1h ), 3 . 51 ( br s , 1h ), 2 . 50 - 2 . 40 ( m , 1h ), 2 . 37 - 2 . 30 ( m , 1h ), 1 . 40 - 1 . 30 ( dd , 6h ), 1 . 10 - 0 . 90 ( m , 9h ); fabms 423 . 2 ( m + h ); hrfabms calcd for c 22 h 35 n 2 o 6 ( m + h ) 423 . 2495 , found 423 . 2493 . cbz - dihydroaip - leuoh ( 2 . 03 g , 4 . 81 mmol ) was dissolved in ethyl acetate ( 33 ml ), triethylamine ( 0 . 66 ml ) and phenacyl ( pac ) bromide ( 0 . 97 mg , 6 . 85 mmol ) were added to the mixture was stirred at rt overnight . water and ether were added and the two layers separated . the organic layer was washed with 0 . 1n hcl saturated sodium bicarbonate , and brine , then dried over mgso 4 . concentration by evaporation of the solvent gave a tan oil . the residue was chromatographed on silica gel ( hexane / etoac = 4 / 1 ) to give 1 . 27 g ( 53 %) of one isomer and 0 . 96 g ( 40 %) of the other isomer ; a : r f 0 . 46 ( hexane / etoac = 1 / 1 ); 1 h nmr ( 300 mhz , cdcl 3 ) δ 7 . 90 ( d , 2h ), 7 . 61 ( m , 1h ), 7 . 50 ( m , 2h ), 7 . 40 ( s , 5h ), 6 . 03 ( br s , 1h ), 6 . 00 ( br s , 1h ), 5 . 40 ( ab q , 2h ), 5 . 10 ( s , 2h ), 4 . 78 - 4 . 72 ( m , 1h ), 4 . 45 - 4 . 53 ( m , 1h ), 4 . 05 - 4 . 10 ( m , 1h ), 3 . 70 ( br s , 1h ), 2 . 50 ( q , 1h ), 2 . 40 - 2 . 32 ( n , 1h ), 2 . 00 - 1 . 85 ( m , 3h ), 1 . 25 ( dd , 6h ), 1 . 06 ( d , 3h ), 1 . 02 - 0 . 80 ( dd , 6h ); fabms 541 . 2 ( m + h ); hrfabms calcd for c 30 h 41 n 2 o 7 ( m + h ) 541 . 2916 , found 541 . 2916 ; anal . calcd for c 30 h 40 n 2 o 7 : c , 66 . 63 ; h , 7 . 46 ; n , 5 . 18 . found : c , 66 . 61 ; h , 7 . 44 ; n , 5 . 26 . b : r f 0 . 30 ( hexane / etoac = 1 / 1 ); 1 h nmr ( 300 mhz , cdcl 3 ) δ 7 . 90 ( d , 2h ), 7 . 62 ( m , 1h ), 7 . 50 ( m , 2h ), 7 . 40 ( s , 5h ), 6 . 03 ( br s , 1h ), 6 . 00 ( br s , 1h ), 5 . 40 ( ab q , 2h ), 5 . 10 ( s , 2h ), 4 . 78 - 4 . 73 ( m , 1h ), 4 . 44 - 4 . 55 ( m , 1h ), 4 . 06 - 4 . 12 ( m , 1h ), 3 . 72 ( br s , 1h ), 2 . 51 ( q 1h ), 2 . 39 - 2 . 31 ( m , 1h ), 2 . 00 - 1 . 85 ( m , 3h ), 1 . 24 ( dd , 6h ), 1 . 06 ( d . 3h ), 1 . 05 - 0 . 83 ( dd , 6h ); fabms m / z 541 . 2 ( m + h ); hrfabms calcd for c 30 h 41 n 2 o 7 ( m + h ) 541 . 2916 , found 541 . 2914 ; anal . calcd for c 30 h 40 n 2 o 7 : c , 66 . 63 ; h , 7 . 46 ; n , 5 . 18 . found : c , 66 . 61 ; h , 7 . 44 ; n , 5 . 26 . a solution of cbz - dihydro - aip - leuo - pac ( 0 . 44 g , 0 . 81 mmol ) in ch 2 cl 2 ( 2 . 10 ml ) was stirred while pyridinium chlorochromate on alumina reagent 16 ( 1 . 57 g ) was added . after 2 h stirring at rt , the solution was filtered and washed with ether . the combined filtrates were combined and the solvent evaporated . the residue was chromatographed on silica gel hexane / etoac = 4 / 1 ) to give 0 . 37 g ( 87 %) of the desired product as a white solid ; r f 0 . 42 hexane / etoac = 1 / 1 ); 1 h nmr ( 300 mhz , cdcl 3 ) δ 7 . 90 ( d , 2h ), 7 . 61 ( m , 1h ), 7 . 50 ( m , 2h ), 7 . 40 ( s , 5h ), 6 . 90 ( br s , 1h ), 6 . 88 ( br s , 1h ), 5 . 40 ( ab q , 2h ), 5 . 18 ( s , 2h ), 4 . 78 - 4 . 72 ( m , 1h ), 4 . 45 - 4 . 53 ( m , 1h ), 3 . 68 ( q , 1h ), 2 . 25 - 2 . 38 ( m , 1h ), 1 . 92 - 1 . 71 ( m , 3h ), 1 . 45 ( d , 3h ), 1 . 10 - 1 . 00 ( dd , 6h ), 0 . 80 - 0 . 75 ( dd , 6h ); fabms 1077 . 3 ( 2m + h ), 577 . 3 ( m + k ), 561 . 2 ( m + na ), 539 . 3 ( m + h ); hrfabms calcd for c 30 h 39 n 2 o 7 ( m + h ) 539 . 2757 , found 539 . 2762 ; anal . calcd for c 30 h 38 n 2 o 7 : c , 66 . 88 ; h , 7 . 11 ; n , 5 . 18 . found : c , 66 . 92 ; h , 7 . 33 ; n , 478 . the protected dipeptide ( 167 mg , 0 . 31 mmol ) was treated with zn ( 500 mg ) in acoh / h 2 o ( 70 : 30 ). the mixture was allowed to stir at rt overnight , zn was filtered off using celite and the solution was partitioned between ether and water . the organic layer was separated and dried over na 2 so 4 . purification by column chromatography ( chcl 3 / meoh ) afforded the product as a white powder . the reaction flask was protected with a cacl 2 tube and the mixture allowed to stir at rt for 1½ h . solvent was evaporated and the remaining oil was placed under vacuum to give a yellow solid ( 87 . 7 mg , 70 %); 1 h nmr ( 300 mhz , cdcl 3 ) δ 7 . 42 ( s , 5h ), 6 . 88 ( br s , 1h ), 6 . 85 ( br s , 1h ), 5 . 42 ( ab q , 2h ), 5 . 15 ( s , 2h ), 4 . 79 - 4 . 72 ( m , 1h ), 4 . 55 - 4 . 47 ( m , 1h ), 3 . 67 ( q , 1h ), 2 . 37 - 2 . 26 ( m , 1h ), 1 . 94 - 1 . 75 ( m , 3h ), 1 . 46 ( d , 3h ), 1 . 12 - 1 . 01 ( dd , 6h ), 0 . 80 - 0 . 74 ( dd , 6h ); fabms 460 . 3 ( m + k ), 443 . 2 ( m + na ), 421 . 3 ( m + h ). cbz - aip - leu - oh ( 36 . 7 mg , 85 . 0 μmol ) was dissolved in dry ch 2 cl 2 ( 1 . 0 ml ) and the solution was cooled to 0 ° c . dcc ( 26 . 1 mg , 0 . 13 mmol ) was added and the mixture was stirred for 30 min at 0 ° c . pro - otmse ( 18 . 5 mg , 85 . 0 μmol ) in ch 2 cl 2 ( 1 . 0 ml ) was added and the solution was stirred for 30 min at 0 ° c . and at rt overnight . the residue was concentrated and taken up in ethyl acetate . the solution was washed with aqueous citric acid , aqueous sodium bicarbonate , dried over mgso 4 , and concentrated . the residue was purified by column chromatography , eluting with ch 2 cl 2 / meoh ( 95 : 5 ) to give a yellow oil ( 34 . 0 mg , 65 %); 1 h nmr ( 300 mhz , cdcl 3 ) δ 7 . 47 ( s , 5h ), 6 . 85 ( br s , 1h ), 6 . 84 ( br s , 1h ), 5 . 45 ( ab q , 2h ), 5 . 13 ( s 2h ), 4 . 80 - 4 . 73 ( m , 1h ), 4 . 53 - 4 . 47 ( m , 1h ), 4 . 24 - 4 . 01 ( dt , 4h ), 3 . 63 ( q , 1h ), 2 . 35 - 2 . 23 ( m , 1h ), 1 . 94 - 1 . 75 ( m , 3h ), 1 . 46 ( d , 3h ), 1 . 12 - 1 . 01 ( dd , 6h ), 0 . 80 - 0 . 74 ( dd , 6h ), 0 . 00 ( s , 9h ); fabms 656 . 3 ( m + k ), 640 . 2 ( m + na ), 618 . 3 ( m + h ). the protected tripeptide ( 24 . 9 mg , 40 . 3 μmol ) was dissolved in isopropyl alcohol ( 1 . 00 ml ) and 10 % pd / c catalyst ( 0 . 99 mg ) was added . the solution was hydrogenated for 3 h , the catalyst was removed by filtration over celite , and the solvent removed to afford the desired product ( 15 . 6 mg . 82 %), 1 h nmr ( 300 mhz , cdcl 3 ) δ 6 . 84 ( br s , 1h ). 6 . 82 ( br s , 1h ), 5 . 41 ( ab q , 2h ), 5 . 09 ( s , 2h ), 4 . 82 - 4 . 71 ( m , 1h ), 4 . 56 - 4 . 48 ( m , 1h ), 4 . 25 - 4 . 00 ( dt , 4h ), 3 . 62 ( q , 1h ), 2 . 37 - 2 . 23 ( m , 1h ), 1 . 95 - 1 . 75 ( m , 3h ), 1 . 47 ( d , 3h ), 1 . 14 - 1 . 01 ( dd , 6h ), 0 . 82 - 0 . 74 ( dd , 6h ), 0 . 00 ( s , 9h ); fabms m / z 506 . 3 ( m + na ); 484 . 3 ( m + h ). boc - ist - oh ( 7 . 51 mg , 27 . 3 μmol ) was dissolved in dry ch 2 cl 2 ( 1 . 0 ml ) and the solution was cooled to 0 ° c . dcc ( 10 . 52 mg , 0 . 089 mmol ) was added and then mixture was stirred for 30 min at 0 ° c . h - aip - leu - pro - otmse ( 3 . 28 mg , 27 . 3 μmol ) in ch 2 cl 2 ( 1 . 0 ml ) was added and the solution was stirred for 30 min at 0 ° c . and at rt overnight . the residue was concentrated and taken up in ethyl acetate . the solution was washed with aqueous citric acid , aqueous sodium bicarbonate , dried over mgso 4 , and concentrated . the residue was purified by reversed phase hplc using a gradient system of ch 3 ch / h 2 o ( 45 . 0 mg , 83 %); fabms 741 . 5 ( m + h ), 641 . 5 ( m − boc ). the protected tetrapeptide ( 30 . 0 mg , 0 . 045 mmol ) was dissolved in meoh ( 2 ml ) and a steady current of hcl was passed through the solution for approximately 20 min . evaporation of the solvent produced a yellow oil which was purified by reversed phase column chromatography eluting with ch 3 cn / h 2 o ( gradient system ) to give 22 . 0 mg ( 87 %) of the compound as a yellow powder ; fabms 641 . 5 ( m + h ), see fig2 . acid 5 ( 21 . 9 mg , 28 . 4 μmol ) and n - methylmorpholine ( 6 . 4 μl ) were dissolved in dry thf ( 0 . 4 ml ), and the solution was cooled to 0 ° c . a solution of amine 6 ( 16 . 2 mg , 28 . 4 μmol ) and hobt ( 0 . 81 mg ) in 1 . 5 ml of the dry thf were added . this suspension was mixed with a cold solution of edci ( 9 . 76 mg , 51 . 1 μmol ) in 0 . 5 ml of thf . the reaction mixture was stirred at 0 ° c . for ½ h . the solution was then concentrated to 0 . 50 ml , kept at 0 ° c . for 24 h , the diluted with ether . the organic layer was washed with 10 % hcl , 5 % nahco 3 , and saturated nacl solutions . the organic layer was dried ( na 2 so 4 ), filtered , and concentrated . the crude oil was purified by reversed phase hplc using a gradient system of ch 3 cn / h 2 o to give 11 . 6 mg ( 32 %) of the linear heptapeptide , see fig3 ; fabms 1294 . 2 ( m + h ); 1194 / 2 ( m − boc ), see fig4 ; hrfabms calcd for c 67 h 108 n 7 o 16 si ( m + h ) 1294 . 7649 , found 1294 . 7644 . 1m tbaf ( 2 . 1 μl ) was added to a solution of the fully protected linear heptapeptide ( 5 . 80 mg , 4 . 50 μmol ) in dry thf . after 2 h stirring at 0 ° c ., the mixture was diluted with distilled water and concentrated to a small volume . the remaining solution was diluted with etoac and 2n hcl was added to render the aqueous layer acidic . the etoac layer was washed three times with water and dried with na 2 so 4 . evaporation of the solvent gave the deprotected heptapeptide in quantitative yield ( 4 . 3 mg ); fabms 1194 . 2 ( m + h ); 1094 . 2 ( m − boc ), see fig5 ; hrfabms calcd for c 62 h 100 n 7 o 14 si ( m + h ) 1194 . 7098 , found 1194 . 7089 . the deprotected heptapeptide was subjected to a solution of 1m tfa ( 9 . 57 ) μl ). after 1 h stirring at room temperature , the solvent was evaporated . water was added to the residue and the aqueous solution was extracted with etoac . the extract was washed with 5 % nahco 3 and water , and dried over na 2 so 4 . the compound was purified by reversed phase hplc using a gradient system of ch 3 cn / h 2 o see fig6 ) to give 3 . 58 mg ( 91 %); fabms 1094 . 2 ( m + h ), see fig7 . the linear heptapeptide 7 ( 3 . 58 mg , 3 . 28 μmol ) was dissolved in dry thf ( 0 . 08 ml ), and the solution was cooled to 0 ° c . edci ( 0 . 63 mg , 3 . 28 μmol ) in 1 . 0 ml of thf was added , and the reaction mixture was stirred at 0 ° c . for 2 h . after storage in the freezer overnight , the solution was diluted with ether . the organic layer was washed with 10 % hcl , 5 % nahco 3 , and saturated nacl solutions . the organic layer was dried ( na 2 so 4 ), filtered , and concentrated . the crude oil was purified by reversed phase hplc using a gradient system of ch 3 cn / h 2 o to give 1 . 41 mg ( 40 %) of the protected analogue 3 ; fabms 1076 . 7 ( m + h ), see fig8 ; hrfabms calcd for c 57 h 86 n 7 o 13 ( m + h ) 1076 . 6284 , found 1076 . 6283 . the protected compound 3 ( 1 . 41 mg ) was dissolved in isopropyl alcohol ( 0 . 50 ml ) and 10 % pd / c catalyst ( 0 . 50 mg ) was added . the solution was hydrogenated for 3 h . at this time , the catalyst was removed by filtration over celite and the solvent removed to afford the desired compound 8 , aipdidemnin a . footnotes : a test performed by dr . g . r . wilson in this laboratory ; b 0 - least toxic to 16 ( toxic ); c +++ complete inhibition , ++ strong inhibition , + moderate inhibition , − no inhibition . footnotes : a test performed by dr . g . r . wilson in this laboratory ; b new compounds . 1 . 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