WO1998017302A1 - Total synthesis of the amino hip analogue of didemnin a - Google Patents

Abstract

A synthetic method for intermediates and their use in preparing didemnin A analogs, pharmaceuticals and the use thereof is disclosed.

Description

Total Synthesis of the Amino Hip Analogue of Didemnin A

SUMMARY OF THE INVENTION

Didemnins were isolated from the Caribbean tunicate Trididemnum solidum. ' These cyclic depsipeptides possess a variety of biological activities including in vitro and in vivo antiviral, antilumor, and immunosuppressive activities.2-5 They are potent inhibitors of L1210 leukemia cells in vitro, and are also active in vivo against P388 leukemia and D 16 melanoma.3 Didemnin B, a more active compound of this class, is approximately twenty times more cytotoxic than didemnin A in vitro and has undergone phase II clinical trials for antituinor activity.- Both didemnins A and B exhibit antiviral activity against DNA and RNA viruses, with didemnin B being more active * The structures of didemnins A and B have been established as 1 and 2, respectively.^

The Structures of Didemnins A and B. Structure-activity relationship studies have been somewhat limited due to the restricted number of available modifications of the extracted natural compounds. Although the bioactivity of didemnin B has been attributed to its side chain, lb few other structural features have been examined. An X-ray crystal structure of didemnin B by Hossain, et al. ' shows that the β-turn side chain, the isostatine hydroxyl group, and the tyrosine residue extend outward from the rest of the molecule, leading to speculation about their importance for biological activity. Slmctural changes in those areas have shown these features to be essential for activity."

Although many studies have shed light on the pharmacology and chemistry of didemnins, little is known about their mechanism of action. However, recent biochemical studies of possible binding sites have provided promising results. Studies performed by Shen et al.9 have shown that didemnin B binds to a site on Nb2 node lymphoma cells and that this binding may be responsible for the immunosuppressive activity. Schreiber and co-workers ¹^ have reported that didemnin A binds elongation factor-lα (EF-lα) in a GTP-dependent manner wliich suggests EF- lα may be the target responsible for the ability of didemnins to inhibit protein synthesis.

We present here synthetic studies toward a modified macrocycle which possesses an amide bond in place of an ester bond (3). A modification such as this is likely to result in an increase in hydrogen bonding at the active site, and thus, provide a more active compound. In addition, the facile nature of the C-0 bond leads us to believe replacement of these C-0 bonds with C-N bonds may improve the stability of these compounds.

Synthetic Strategy. The retrosynthetic disconnections which formed the basis of our plan for the preparation of the protected amino-Hip analogue 3 of didemnin A are illustrated in Scheme I. We envisaged disconnection of the amide function between /V,0-Me2-L-tyrosine and L-proline to give the linear heptapeptide 4 and disconnection between L-threonine and isostatine (3S, 4R, 5S) to afford the two units: a tripeptide unit 5 comprised of /V-Me-leucine, threoninc, and /V,0 -Me2-tyrosine; and a tetrapeptide unit 6 comprised of isostatine, α -(α'- aminoisovalery propionyl (Aip), leucine, and proline. Scheme I

Synthesis of Tripeptide 5. Preparation of the diprotected tripeptide unit is shown in Scheme II. Our approach began with methylation of the uncommon amino acid, Cbz-D-lcucine, 7, with CH3l/NaH. l 1 Coupling of the derivative Cbz-D-MeLeuOH with the hydroxyl group of the threonine derivative L-TlirOEt¹^ was accomplished with dicyclohexylcarbodiimide (DCC) ' 3 to provide the dipeptide 8. Ester hydrolysis with potassium hydroxide afforded the desired carboxylic acid wlύch was then protected as a phenacyl (Pac) ester 9. Coupling with the tyrosine derivative BocMe2TyrOH ^χ4 followed by removal of the Boc protecting group^ afforded 10. Removal of the phenacyl function^ provided the key fragment 5.

Sclieme II

Reagents: a) (i) CH3I, NaH, THF; (ii) L-T rOEt, DCC, CH CI_2; b) (i) KOH, MeOH; (ii) plienacylBr, Et3N, EtOΛc; c) BocMe₂TyrOH, DCC. DMΛP, CH₂C1₂; d) Zn, HOΛc/H₂0.

Synthesis of Tetrapeptidc 6. The construction of fragment 6 involves two novel subunits (25,45)-aminoisovalerylpropionic acid (Aip) and (35, 4R, 55)-isostatine (1st). The synthesis of the required isostatine derivative involves (2R, 35)-α//o-isoleucine. The expensive

(2R, 35)-α//o-isoleucine can be obtained from commercially available (25, 35)-isoleucine, 11, by conversion to the hydroxy acid with retention and its conversion in two steps to the amino acid with inversion (Scheme III). ' 8 Conversion of (25, 35)-isoleucine to the corresponding α- hydroxy acid 12 was accomplished by using a well-known procedure^ that allows overall retention of configuration via a double inversion. Esterification was carried out with acetyl chloride in methanol, and the corresponding α-hydroxy methyl ester was transformed into the tosyloxy methyl ester 13. Treatment of the tosylate with sodium azide in D F provided the α- azido ester 14 stereoselectively. Saponification of the ester afforded the α-azido acid 15.

Hydrogenation of the azide to the free amine proceeded readily in methanol at atmospheric pressure using Pearlman's catalyst (20% palladium hydroxide on carbon),²⁰ to afford (25, 35)- α//o-isoleucine 16.

Scheme III H₂ OH L AcCI, QTs

H₂5Q₄ MeOH _^

C0₂H C0₂H C0₂CH₃ NaN0₂ 2. TsCI, pyridinβ

11 90% 12 70% 13

The major portion of the isostatine subunit, D-α//σ-isoleucine, 16, was transformed into the /err-butoxycarbonyl (Boc) acid under standard conditions.¹6 After activation of its carboxyl group as the imidazolide by use of carbonyldiimidazole, treatment with the magnesium cnolatc of ethyl hydrogen malonate afforded the required β-keto ester 18. The reduction by NaBI j of the carbonyl group of the β-keto ester was effectively stereospecific, generating the desired (35, <\R, 55)-19a as the major product (>10: 1) after chromatographic separation. Saponification afforded the required Boc-(35, 4R, 5S)-Ist-OII, 20 (Scheme IV). Scheme IV

The next step toward the synthesis of the tripeptide fragment (6) involved formation of the amino Hip subunit. This unit was synthesized from Cbz-L-valine, 21, utilizing a procedure based in part on the work of Nagarajan.21 After activation of its carboxyl group as the imidazolide by use of carbonyldiimidazole, treatment with the magnesium enolate of ethyl hydrogen methyl malonate (EHMM) afforded the required β-keto ester 22. Sodium borohydride reduction of the β-keto ester produced a diastereomeric mixture of alcohols which were separable by column chromatography. Following saponification and coupling with L-leucine methyl ester (L-LeuOMe), flash chromatography afforded the desired compound 23. Saponification produced the acid and treatment with phenacyl (Pac) bromide provided the protected derivative 24. Oxidation of the secondary alcohol with pyridinium chlorochromate on alumina²² provided the β-keto amide. Removal of the phenacyl protecting group provided the free acid which was coupled with L-proline trimethylsilylester. Catalytic hydrogenation removed the Cbz protecting group and coupling of the isostatine subunit 20 with the amine produced the diprotected tetrapeptide. The Boc protecting group was then removed under standard conditions^ to afford the key tetrapeptide unit 6 (Scheme V). Scheme V

Reagents: a) (i) CO(imiti)₂, THF; (ii) EHMM, iPrMgBr; l>) (i) NaBH , ElOII; (ii) KOH, MeOH; (iii) LeuOMe, DCC, CH₂CI₂; c) (i) KOH, MeOH; (ii) phenacytBr. Et₃N. EtOΛc; (iii) PCC, ΛI θ3, CH₂Cl₂; cl) (i) Zn/HOΛc; (ii) ProOTMSe, DCC. CH₂Cl₂; (iii) H , Pd/C, MeOH; (iv) BocIslOH lfi, DCC, CH₂CI₂; (v) HCI. dioxane.

Synthesis of Linear Heptapeptide 4. The synthesis of the linear heptapeptide involved coupling of the two subunits, Cbz-D-MeLeuThr(OMe2TyrBoc)OH, 5 , and. II-

IstAipLeuProOTMSe, 6. A variety of coupling methods (BopCl,²⁴ DCC, EEDQ²⁵) were attempted, however, the EDCI method²⁶ was shown to be the most efficient. Deprotections of the Iriniethylsilyl ester and the Boc functions were performed under standard conditions to give the linear heptapeptide 7. Cyclization was achieved by treatment with EDCI and catalytic hydrogenation provided the amino Hip (Aip) analog of didemnin A, 8 (Scheme VI). Scheme VI

Reagents: a) (i)EDC, NMM. DMF; (ii) TBΛF. THF; (iii) TFA, THF; b) EDC, DMF.

DETAILED DESCRIPTION OP THE PREFERRED EMBODIMENTS

We have described the total synthesis of the amino Hip analogue of didemnin Λ. Previous studies have shown that didemnins arc 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. General Experimental Procedures. H NMR spectra were recorded on Varian XL-200, General Electric QE-300, Varian XL-400, and General Electric QN-500 spectrometers, i chemical shifts are referenced in CDCI3 and methanol-^ to residual CHCI3 (7.26 ppm) and CD2HOD (3.34 ppm). Electron impact (El) 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 using magic bullet matrix.²^ 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 measured with a DIP 360 or a DIP 370 digital polarimeter with an Na lamp (589 nm) using a 5- X 0.35-cm (1.0 mL) cell. Melting points were determined on a capillary melting point apparatus and are not corrected. Nonnal phase column _t chromatography was performed using Merck-kieselgel silica gel (70 - 230 mesh). Fuji-Davison C 18 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 990 instrument and an Econosil Cj 8 column (AUtech/ Applied Science) and a Phenomenex Ci 8 column.

THF was distilled from sodium benzophenone ketyl and CH2CI2 from P2O5. Dimethylformamide (DMF), triethylamine (E13N), and TV-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-rerr-butyl dicarbonate [(BocO)2θ], dicyclohexylcarbodiimide (DCC), l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), dimethylaminopyridinc (DMAP), 1-hydroxybenzotriazole (HOBT), D- and L-isoleucine, L-tyrosine, L-isoleucine, L- threonine, D-valine, and L-proline were obtained from the Aldrich Chemical Company. All reactions requiring anhydrous conditions were performed under an atmosphere of nitrogen.

N-Benzylαxycarbonyl-N-melhyl-D-Ieucine (Cbz-D-MeLeuOH). 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 h. 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 the oily residue partitioned between ether (30 mL) and water (60 mL). The ether layer was washed with aqueous sodium bicarbonate (5 mL) and the combined aqueous layers were acidified with 4N IIC1 to pH 3. The solution was extracted with ethyl acetate (3 X 15 mL) and the extract washed with 5% aqueous sodium thiosulfate (2 X 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%); [u Na + 24.7' (c 0.02, CHCI3), Lit. l ^ [α]²⁹D+ 26.9' (c 0.02, CHCI3); m.p. 71-72'C (Lit. ^{l l b} 72-73"C); ^[H NMR (300 MHz, CDCI3) δ 7.40-7.27 (5H, m), 5.17 (2H, s), 4.74 (I II, m), 2.87 (3H, s), 1.78- 1.76 (2H, m), 1.62-1.57 (III, ), 0.92-0.80 (6H, m); FABMS 280.2 (M + H), 236.2 (M - CO2); HRFABMS Calcd for C15H22NO4 (M + H) 280.1549. Found 280.1556; Anal. Calcd for C15H21NO4 : C, 64.48; H, 7.58; N, 5.02. Found : C, 64.30; II, 7.65; N. 4.93.

L-Threonine Ethyl Ester (L-ThrOEt). A current of dry HC1 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 min, 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 O'C under reduced pressure. A yellow solid was isolated (36.2 g, 85%); [<x]²⁹Na + 0.82° (c 5.0, EtOH). Lit. I² [α]²^ + 0.95' (c 5.0, EtOH); m.p. 5 l-53'C (Lit.12 52-54'C); 1 H NMR (200 MHz, CDCI3 w/ TMS) δ 4.82 (IH, m), 4.40 (IH, d), 4.05 (2H, q), 1.62 (3H, d), 1.21 (3H.- t); FABMS 148.2 (M + H); HRFABMS Calcd for C6H14NO3 (M + H) 148.0974, Found 148O972.

Z-D-Methylleucylthreonine Ethyl Ester (8). Z-D-MeLeuOH (2.12 g, 7.59 mmol) was dissolved in 100 mL of CH2CI2 and cooled to O'C. DCC (1.72 g, 8.35 mmol) was added and the solution was stirred at O'C for 10 min. L-ThrOEt (1.12 g, 7.59 m ol) in 5 mL of CH2CI2 was added and the solution was allowed to warm to rt. After approximately 15 h, dicyclohcxylurea was removed by filtration and washed with CH2CI2. 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/ElOΛc = 65/35) to afford the product as a yellow oil (2.35 g, 76%); ^lH NMR (400 MHz, CDCI3) δ 7.36-7.42 (511, m), 6.73 ( IH, d), 6.50 (I H, br s), 5.18 (2H, s), 4.83 ( I H, m), 4.51 , ( IH. m), 4.30 (IH, m), 4.17 (211, q), 2.81 (3H, s). 1.74 (211, m), 1.70 (3H, d), 1.68 (IH, m), 1.20 (311, t), 0.82-0.90 (6H, m); FABMS 431.4 (M + Na). 409.2 (M + H); HRFABMS Calcd for C21II33N206 (M + H) 409.2344. Found 409.2339; Anal. Calcd for C21II32N2O6 : C, 61.73; II, 7.90; N, 6.86. Found : C, 62.00; H, 8.08; N, 7.07.

Z-D-Methylleucylthreonine (Z-D-MeLcuThrOH). Z-D-MeLeuThrOEt ( 1.80 g, 4.42 mmol) was dissolved in methanol and 2N KOH was slowly added to the mixture at O'C. The solution was stirred for 2 h. TLC analysis (CHCl3 MeOH 95:5) showed the reaction to be complete. The mixture was neutralized using 2N HC1. The solvent was then evaporated. The solution was partitioned between ethyl acetate and water and the organic layer separated. Aqueous HC1 was added to the aqueous layer to pH 3. This was extracted with ethyl acetate and all of the ethyl acetate extracts were combined. The solution was dried over MgS04 and the solvent evaporated to give a dark orange oil (1.77 g, 98%) which was used for the next reaction without purification; ^lH NMR (200 MHz, CDCI3) δ 7.36-7.41 (5H, m), 6.72 (IH, s), 6.52 (IH, br s), 5.18 (2H, s), 4.83 ( IH, m), 4.51 (IH, m), 4.30 (IH, m), 2.81 (3H, s), 1.74 (2H, m). 1.70 (3H. d), 1.68 (IH, m), 0.82-0.90 (6H, m); FABMS 381.2 (M + H); HRFABMS Calcd for C19H29N2O6 (M + H) 381.2026, Found 381.2021; Anal. Calcd for C19H28N2O6 : C, 59.97; H, 7.42; N, 7.37. _, Found : C, 60.53; H, 7.06; N, 7.1 1.

Z-D-Methylleucylthreonine Phenacyl Ester (9). Z-D-MeLeuThrOH (1.50 g, 3.95 mmol) was dissolved in ethyl acetate (25 L). Triethylamine (0.39 g, 3.95 mmol) and phenacyl bromide (0.79 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 HO, saturated sodium bicarbonate, and brine, and then dried over MgS04. The residue was chromatographed on silica gel (hexane/EtOAc = 4/1) to give a clear oil (1.71 g, 87%); III NMR (200 MHz, CDCI3) δ 7.34- 7.41 ( 10H, m), 6.71 (III, s), 6.52 ( IH, br s), 5.27 (2H, s), 5.18 (211, s), 4.83 (IH, m), 4.61 ( I H, m), 4.50 ( IH, m), 2.81 (3H, s), 1.74 (2H, m), 1.70 (3H, d), 1.68 (III, m), 0.82-0.90 (6H, m); FABMS 537.1 (M + K), 499.1 (M + II); HRFABMS Calcd for C27H35N2O7 (M + H) , 499.2444. Found 499.2450.

N- rr-Butoxycarbonyl-tyrosine (BocTyrOH).16 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 pll. Dioxane (50 mL) and (Boc)2θ (6.12 g, 27.5 mmol) were added with cooling. The reaction was allowed to stir for 2 h. 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 HC1 and extracted with ether, which was washed with brine and dried over MgSθ4. A yellow oil was obtained (6.02 g, 86%); *H NMR (200 MHz, CDCI3) δ 7.10 (211, d), 6.84 (2H, d), 4.92-5.00 ( IH, m), 4.47-4.52 (IH, m), 3.00-3.12 (2H, m), 1.43 (9H, s); EIMS 282.0.

N-/err-ButoxycarbonyI-N,0-dimethyltyrosine (BocMe2TyrOII). 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 to 0 "C and sodium hydride (60% dispersion, 2.47 g, 62.0 mmol) was added. The reaction mixture was allowed to stir at 0 ^*C for 1 h, then at rt overnight. Excess sodium hydride was quenched by the dropwise addition of 10 mL of THF/H2O (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 X 30 mL). The aqueous phase was acidified with solid citric acid (pH 2). Ethyl acetate was used for extraction. The combined extracts were washed with brine, dried (MgSθ4) 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]²⁹D - 15.7° (c 1.0, MeOH), Lit.14b [α]²²D -16.9^* (c 1.0, MeOH); 1 H NMR (300 MHz, CDCI3) δ 7.18 & 7.12 (2H, two d), 6.85 (2H, d), 4.58 (III, two t), 3.80 (3H, s), 3.24 & 3.13 (111, 2dd), 2.76 & 2.68 (3H, 2s), 1.43 & 1.38 (9H, 2s); FABMS 619.3 (2M + II), 310.2 (M + H), 210.2 (M - Boc); HRFABMS Calcd for C16H24NO5 (M + H) 310.1654, Found 310.1648.

Cbz-D-MeLeu-Thr(OMe2TyrBoc)-OPac (10). BocMe2TyrOH (0.27 g, 0.91 mmol) in CH2CI2 (20 mL), DCC (19.5 mg, 0.95 mmol) and DMAP (41.3 g) were added at O'C 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. Dicyclohexylurea 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 MgS04 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; 111 NMR (500 MHz, CDCI3) δ 7.30-8.00 (10H, ), 6.82 & 7.10 (2H, d), 5.31 (IH. s), 5.20-4.85 (IH, m), 3.74 (3H, s), 3.10 & 3.07 (IH, 2 dd) 2.92 (3H, s), 2.73 (3H, s), 1.71 (3H, d), 1.44 & 1.37 (9H, 2 s), 0.92 (6H, ); FABMS 828.4 (M + K), 812.4 (M + Na), 790.3 (M + H), 690.4 (M - Boc); HRFABMS Calcd for C43H57N3O11 (M + H) 790.3915. Found 790.3916.

Cbz-D-MeLeu-Thr(OMc2TyrBoc)-OII (5). The tripeptide 10 (30.0 mg, 38.0 μmol) was treated with Zn (60 mg) in AcOH/H2θ (70:30) and the mixture was stirred at rt overnight, Zn was filtered off using celite and die solution was partitioned between ether and water. The organic layer was separated and dried over Na2S04. Purification by reversed phase column chromatography (CH3CN/H2O 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 supplementary material, S-l ; HRFABMS Calcd for C35H52N4O9 (M + II) 672.3734, Found 672.3674.

(2S, 3S)-2-IIydroxy-3-methylρentanoic acid (12). A previously reported procedure was used.19 A suspension of L-isoleucine (30.0 g, 0.23 mol) in distilled water (180 mL) was stirred vigorously while solutions of 2N sodium nitrite (123.6 mL) and 2N H2SO4 (123.6 mL) were added simultaneously from dropping funnels. After the addition was complete, the mixture became colorless after 30 min. The solution was stirred at rt for 15 h then extracted with EtOAc. The EtOAc extracts were dried (Na2Sθ4), filtered, and concentrated. The resulting crude solid was recrystallized from ether/petroleum ether to provide the desired compound as a white crystalline solid (28.2 g, 93%); l NMR (300 MHz, CDCI3) δ 0.90 (t. 3H), 0.97 (d, 3H), 1.23 & 1.38 (m, 2H), 1.70 (m, III), 2.80 (br s, IH), 4.10 (d, IH); FABMS 269.2 (2M + H), 135.1 (M , + H).

Methyl (25, 3S)-2-IIydroxy-3-methylpentanoate. 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 O'C for 1 h, then at rt overnight, concentrated and diluted with ether. The ether solution was washed with saturated N.1HCO3, brine, dried over MgSθ4 and concentrated to give a yellow oil (19.8 g, 80%); [α]29_D +27.3 (c 0.95, CIICI3), Lit.18 [α] 0_D +28.5 (c 0.95, CHCI3); III NMR (300 MHz, CDCI3) δ 0.91 (t, 3H), 0.97 (d, 3H), 1.21 & 1.37 (m, 2H). 1.78 ( , IH), 2.92 (br s, IH), 3.82 (s, 3H), 4.08 (d, IH); CIMS 147.1 (M + H).

Methyl (2S, 3S)-2-Tosyloxy-3-methylμentanoate (13). The hydroxypentanoate (4.44 g, 30.6 mmol) was dissolved in dry CH2CI2 and cooled in an ice bath to O'C. Pyridine (45.0 mL) was added followed by /7-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 IN H2SO4 and IN KHCO3. The extracts were dried over MgSθ4 and evaporated in vacuo to give a dark orange oil (8.32g, 88%); i NMR (300 MHz, CDCI3) δ 0.93 (t, 3H), 0.97 (d, 3H), 1.21 & 1.41 (m, IH), 1.91 (m, IH), 2.41 (s, 2H), 3.60 (s, 3H), 4.63 (d. 2H). 7.24 & 7.80 (d, 2H); FABMS 339.2 (M + K). 323.2 (M + Na), 301.1 (M + H), 241.2 (M - CO2CH3); HRFABMS Calcd for C14H21O5S (M + H) 301.1110, Found 301.1 109.

Methyl (2R , 3S)-2-Azido-3-mclhyIpentanoale (14). 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 X 50 mL). The combined organic layers were dried over MgSθ4 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). 21 1 1 (s), 1736 (s), 1472 (w), 1387 (w), 1225 (br m), 1 175 (w), 1086 (w), 732 (s) cm"¹ ; IH NMR (300 MHz, CDCI3) δ 0.93 (t, 3H), 0.97 (d, 3H), 1.22 & 1.43 (m, I H), 1.96 (m, IH). 3.78 (s, 3H), 3.85 (d. 2H); CIMS 172.1 (M + H).

(2R, 3S)-2-Azido-3-ιnethyIpentanoic acid (15). To a solution of α-azido ester (6.56 g, 38.3 mmol) in TIIF (58 mL) at O'C was added IN NaOH (52 L). The reaction mixture was stirred at O'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 O'C, acidified to pH 2 by dropwise addition of cone. HC1, and extracted with ethyl acetate (3 X 25 mL). The combined ethyl acetate extracts were dried (MgSθ4) and concentrated in vacuo to furnish 10.9 g (95%); IR (neat) v_maχ 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" ! ; i NMR (300 MHz. CDCI3) δ 0.93 (3H, t). 0.97 (311, d). 1.21 & 1.41 (IH. m), 1.97 (IH, m). 3.90 (2H, m); FABMS 158.2 (M + H); HRFABMS Calcd for C14H21O5S (M + H),301.1 110, Found 301.1 109.

D-α//ø-isoleucine (16). 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 H2 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 ι>ι vacuo to afford the product as a white solid. Recrystallization from EtOAc provided the compound as colorless needles (4.75 g, 95%); hi NMR (300 MHz, MeOH-< ) δ 0.93 (t, 3H), 0.97 (d, 3H), 1.32 & 1.46 (m, IH), 2.47 (m, IH). 3.58 (d, 2H); FABMS 132.1 (M + H); Anal. Calcd for C6H 13NO2 : C, 54.92; H, 9.99; N, 10.64. Found : C. 54.79; H, 10.17; N. 10.26.

/V-rerf-ButoxycarbonyI-D-α//ø-isolcucine (17). A solution of D-α//σ-isoleucinc (120 mg, 0.916 mmol) was dissolved in water (2.5 mL) and IN NaOH (1.83 mL) and stirred at rt for 48 h. Dι^'-tert-bulyl dicarbonate (200 mg, 0.916 mmol) in dioxane (5.00 mL) was added to the stirred mixture at O'C. After 12 h the dioxane wass evaporated, the aqueous residue washed with Et₂0, mixed with EtOAc, and the rapidly stirred mixture acidified with 2 N H2SO4 at O'C. This solution was extracted with EtOAc, and the combined organic extracts were dried (Na2S04) and coned in vacuo to a crystalline material (179 mg, 85%); mp 35-37'C (Lit.²⁸ 34-36'C); [α]²⁹D , -42.7° (c 2.04, CHCI3), [Lit.²⁸ [α]27_D -40.7^* (c 2.06, CHCI3)]; III NMR (300 MHz, CDCI3) δ 5.52 (br s, I II), 3.72-3.54 (m, IH), 1.92-2.01 (in, IH), 1.43 (s, 9H), 1.37- 1.12 (m, 311), 0.97 (t. 3H), 0.93 (d. 3H); FABMS 463.2 (2M + H), 232.1 (M + II), 132.1 (M - Boc); HRFABMS Calcd for Ci 1H21NO4 (M + H) 232.1551, Found 232.1548.

Ethyl Hydrogen Malonate. A previously reported procedure was used.²⁹ Potassium hydroxide (10.02 g, 85% KOH, 156 mmol) in ethanol (99 mL) was added drop wise 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 O'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 CH2CI2, dried (MgS04), and concentrated to give a yellow oil (9.96 g. 48%; Lit.29b _{51 %);} 1_{H NMR (30}o _MHz, CDCI3) δ 4.37 (s, 2H). 4.24 (q, 2H), 1.34 (t, 3H); FABMS 133.0 (M + H).

Ethyl (4Λ,5S)-3-ferr-Butoxycarbonylamino-5-methyl-3-oxoheptanoate (18). A tetrahydrofuran solution of isopropylmagnesium chloride (1.42 mL, 13.5 mmol) was added dropvvise to a solution of ethyl hydrogen malonate (891 mg, 6.75 mmol) in dry CH2CI2 (5.62 mL). The reaction was then cooled in an ice-salt bath while a solution of Boc-D-α//σ-isoleucine (520 mg, 2.25 mmol) and N, /V'-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 NaHCθ3, dried (MgSθ4) and concentrated to give 475 mg (70%) of a pale yellow oil; IR (neat) vmax 3355, 1750. 1700 cm- 1 ; 111 NMR (300 MHz, CDCI3) δ 4.98 (d, IH), 3.90-4.57 (m, IH), 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).

(3S, 4Λ, 5S)- V-/er/-Butoxycarbonyl-isostatine Ethyl Ester (19a). To a stirred solution of 18 (500 mg. 1.66 mmol) in Et2θ (2.90 mL) and EtOH (6.80 mL) cooled in an ice-salt bath was added NaBH4 (60 mg, 1.58 mmol). The solution wass allowed to stir at -20 ^*C for 2 h then poured into ice water, extracted with EtOAc and dried over MgS04. 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: Rf 0.20 (hexane/EtOAc = 3/1); [α]² o -6.7' (c 0.5, MeOH), Lit.30 [α]²3_D -6.4' (c 0.5, MeOH); IR (neat) v_maχ 3350, 1740, 1700 curl ; In NMR (300 MHz, CDCI3) δ 4.43 (d, III). 4.20 (q, 211), 3.90 - 3.61 (m, 2H), 3.30 (br s, HI), 2.50 (d, 2H), 1.90 - 1.98 (m, IH). 1.41 - 1.30 (m, 2H), 1.40 (s, 911). 1.24 (t, 3H), 0.97 (d, 311), 0.90 (t, 311); FABMS 304.2 (M + H), 204.2 (M - Boc); HRFABMS Calcd for C15II29NO5 (M + H) 304.21 17, Found 304.2123; Anal. Calcd for C15H28NO5 : C, 59.37; H. 9.64; N, 4.62. Found : C, 59.03; H, 9.38; N. 4.88. 19b: Rf 0.22 (hexane/EtOAc = 3/1); [α]²⁹p +26.9 (c 0.5, MeOH), Lit.30 [α]²3_D +26.4 (c 0.5, MeOH); IR (neat) v_maχ 3410, 1740, 1710.cnr l ; 'H NMR (300 MHz, CDCI3) δ 4.42 (d, IH), 4.20 (q, 2H), 3.87 - 3.61 (m. 2H), 3.32 (br s, IH). 2.49 (d. 2H), 1.92 - 1.98 (m, IH), 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 C15H29NO5 (M + H) 304.21 17. Found 304.2123; Anal. Calcd for C15H28NO5 : C. 59.37; H, 9.64; N. 4.62. Found : C, 59.03; H, 9.38; N. 4.88.

(3S, 4R, 5S)-V-rer/-Butoxycarbonyl-isostatine (20). 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 slowly added to the mixture at O'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 MgS04 and the solvent evaporated to give a yellow oil (215 mg, 78%) which was used for the next reaction without purification; [α]²⁹β -4.6' (c 0.0014, CHCI3), Lit.^{1 l b} [α]²⁰D -5.7" (c 0.0014. CHCI3); IH NMR (300 MHz, CDCI3) δ 4.43 (d, IH), 3.85 - 3.63 (m, 2H), 2.76 (br, IH), 2.41 ( , 211). 2.00 - 1.93 (m, III), 1.43 - 1.35 (m, 2H), 1.43 (s, 911). 0.91 (t, 3H), 0.87 (d, , 3H); FABMS 276.1 (M + H), 176.1 (M - Boc); HRFABMS Calcd for C13H25NO5 (M + H) 276.1806, Found 276.1810.

Ethyl Hydrogen Methylmalonate. 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 h 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 O'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 CII2C12, then dried and concentrated to give a yellow oil (4.86 g, 58%, Lit.²⁹ 60%); 111 NMR (200 MHz, CDCI3) δ 4.23 (q, 2H), 3.47 (q, IH), 1.42_,(d. 3H), 1.27 (t, 3H); FABMS 147.1 (M + H).

Cbz-AipOEt (22). A tetrahydrofuran solution of isopropylmagnesium chloride (9.57 L, 90.8 mmol) was added dropwise to a solution of ethyl hydrogen methylmalonate (6.63 g, 45.4 mmol) in dry CH2CI2 (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 Λ/^V'-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 NaHCθ3, brine, dried (MgS04) and concentrated. Purification by flash column chromatography (hexane/EtOAc = 10/1) gave the desired product as a yellow oil (4.50 g, 89%); lH NMR (200 MHz. CDCI3) δ 7.32-7.38 (s, 5H), 5.24-5.37 (m. IH), 5.17 (s, 2H), 4.20 (q, 211), 3.41 (q, HI), 2.18-2.21 (m, IH), 1.45 (d, 3H), 1.32-1.37 (m, IH), 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 I 8H26NO5 (M + H) 336.181 1 , Found 336.1817; Anal. Calcd for C18H25NO5 : C, 64.44; H, 7.52; N, 4.18. Found : C, 64.70; H, 7.62; N. 4.37.

Cbz-DihydroAipOEt. To a stirred solution of Cbz-AipOEt (6.54 g, 19.5 mmol) in Et2θ (15 mL) and EtOH (35 mL) at -20'C, NaBH4 (0.74 g, 19.5 mmol) was added over a period of 15 , min. The reaction mixture was stirred 15 nun at -20 ^*C and poured into ice water (50 mL). After extraction with ethyl acetate (30 mL), the combined organic extracts were dried (MgS04) and concentrated to give a yellow oil (6.12 g, 93%); lH NMR (300 MHz, CDCI3) δ 7.39 (s, 5H), 7.01 (br s, IH), 5.13 (s, 211), 4.72-4.53 (m, IH), 4.18 (q, 2H), 3.80-3.71 (m. IH). 3.38 (br s, IH), 2.32-2.20 (m, IH), 1.98 ( , IH), 1.40 (d, 3H), 1.25 (t, 3H), 0.92-0.80 (m, 6H); FABMS 338.1 (M + H); Anal. Calcd for C18H27NO5 : C, 64.06; H, 8.07; N, 4.15. Found : C, 64.21 ; H. 8.36; N, 4.29.

Cbz-DihydroAipOH. 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 IIC1 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 MgSθ4 and the solvent was evaporated to give a pale yellow oil (4.59 g, 84 %); i NMR (300 MHz. CDCI3) δ 7.39 (s. 5H), 7.04 (br s, IH), 5.13 (s, 2H). 4.40-4.20 (m. IH), 3.92-3.71 (m. IH), 3.33 (br s, IH), 2.32-2.20 (m, IH), 1.98 ( , IH). 1.40 (d, 3H), 0.92-0.80 (m. 6H); FABMS 310.2 (M + H); HRFABMS Calcd for C16H24NO5 (M + H) 310.1654, Found 310.1651; Anal. Calcd for C16H23NO5 : C, 62.10; II. 7.51 ; N, 4.53. Found : C, 62.50; H, 7.71; N, 4.37.

Cbz-DihydroΛip-LeuOMe (23). Cbz-DihydroAipOH ( 1.17 g, 3.82 mmol) was dissolved in dry CH2CI2 (25 mL) and cooled to O'C. DCC (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 dicyclohexylurea, 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 MgSθ4, and concentrated. The residue was purified by column cliromatography eluting with hexane/ethyl acetate (50:50) to give a clear oil (1.23 g, 74%); lH NMR (300 MHz, CDCI3) δ 7.43 (s, 5H), 6.81 (br s, IH), 6.42 (br s, IH), 4.78-4.72 (m, IH), 4.48-4.43 (in, IH), 3.78-3.72 (m, IH), 3.67 (s, 311), 3.51 (br s, IH), 2.50-2.40 (m, IH), 2.37-2.20 (m, IH), 1.40-1.30 (dd, 6H), 1.10-0.90 ( . 9H); FABMS 437.2 (M + H); HRFABMS Calcd for C23H37N2O6 (M + H) 437.2652, Found 437.2653; Anal. Calcd for C23H36N2O6 : C, 63.27; H, 8.32; N, 6.42. Found : C, 63.65; II, 8.35; N, 6.49.

Cbz-DihydroAip-LeuOH. 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 the reaction to be complete. The solution was neutralized with 2N HCl and extracted with ethyl acetate. The aqueous layer was adjusted to pH 3 and extracted with ethyl acetate. The combined ethyl acetate extracts were then dried over MgSθ4. Evaporation of the solvent left a yellow oil (270 mg, 92%); III NMR (200 MHz, CDCI3) δ 7.42 (s, 5H). 6.81 (br s. IH), 6.42 (br s. IH), 4.78-4.72 (m, IH), 4.48-4.43 (m, IH), 3.78-3.72 (m. IH). 3.51 (br s. IH), 2.50-2.40 (m, IH). 2.37-2.20 (m, IH), 1.40- 1.30 (dd. 6H), 1.10-0.90 (m, 9H); FABMS 423.2 (M + H); HRFABMS Calcd for C22H35N2O6 (M + H) 423.2495, Found 423.2493.

Cbz-DihydroAip-Leu-OPac. 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 and 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 MgSθ4. 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 : Rf 0.46 (hexane/EtOAc = 1/1); 111 NMR (300 MHz, CDCI3) δ 7.90 (d, 2H), 7.61 (m. ^'lH), 7.50 (in, 2H), 7.40 (s, 5H), 6.03 (br s, III), 6.00 (br s, HI), 5.40 (AB q, 2H), 5.10 (s, 211), 4.78-4.72 (m, III), 4.45-4.53 (m, IH), 4.05-4.10 (m, IH), 3.70 (br s, IH), 2.50 (q, IH), 2.40-2.32 (m, IH). 2.00-1.85 (m, 311), 1.25 (dd, 6H). 1.06 (d, 3H), 1.02-0.80 (dd, 6H); FABMS 541.2 (M + H); HRFABMS Calcd for C30H41N2O7 (M + H) 541.2916, Found 541.2914; Anal. Calcd for C30H40N2O7 : C, , 66.63; H, 7.46; N, 5.18. Found : C, 66.61 ; H, 7.44; N, 5.26. b: Rf 0.30 (hexane/EtOAc = 1/1); lH NMR (300 MHz, CDCI3) δ 7.90 (d, 2H), 7.62 (m, IH), 7.50 ( , 2H), 7.40 (s, 5H). 6.03 (br s, IH), 6.00 (br s, IH), 5.40 (AB q. 2H), 5.10 (s, 2H), 4.78-4.73 (m, IH), 4.44-4.55 (m, IH). 4.06- 4.12 (m, IH), 3.72 (br s, IH), 2.51 (q, IH), 2.39-2.31 (m, III), 2.00-1.85 (m. 3H). 1.24 (dd, 611), 1.06 (d, 3H), 1.05-0.83 (dd, 6H) ; FABMS m/z 541.2 (M + H); HRFABMS Calcd for C30H41N2O7 (M + H) 541.2916, Found 541.2914; Anal. Calcd for C30H40N2O7 : C, 66.63; II, 7.46; N, 5.18. Found : C, 66.61 ; H, 7.44; N. 5.26.

Cbz-Aip-Leu-OPac (24). A solution of Cbz-DihydroAip-LeuO-Pac (0.44 g, 0.81 mmol) in CH2CI2 (2.10 mL) was stirred while pyridinium chlorochromatc on alumina reagent '⁶ (1.57 g) wass added. After 2h stirring at rt, the solution wass 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; Rf 0.42 (hexane/EtOAc = 1/1); lH NMR (300 MHz, CDCI3) δ 7.90 (d, 2H), 7.61 (m, HI), 7.50 (m. 2H), 7.40 (s, 5H), 6.90 (br s, IH), 6.88 (br s, IH), 5.40 (AB q, 2H), 5.18 (s, 2H), 4.78-4.72 (m, IH). 4.45-4.53 (m, IH), 3.68 (q, IH), 2.25-2.38 (m, IH), 1.92- 1.71 (m, 3H), 1.45 (d, 3H), 1.10-1.00 (dd, 611), 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 C30H39N2O7 (M + H) 539.2757, Found 539.2762; Anal. Calcd for C30H38N2O7 : C, 66.88; H, 7.11; N, 5.18. Found : C, 66.92; H, 7.33; N, 4.78.

Cbz-Aip-Leu-OII. The protected dipeptide (167 mg, 0.31 mmol) was treated with Zn (500 mg) in AcOH/H2θ (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 Na2S04. Purification by column chromatography (CHCl3/MeOH) afforded the product as a white powder. The reaction flask was protected with a CaCl2 tube and the mixture allowed to stir at rt for 1 1/2 h. Solvent was evaporated and the remaining oil was placed under vacuum to give a yellow solid (87.7 mg, 70%); lH NMR (300 MHz, CDCI3) δ 7.42 (s, 511), 6.88 (br s, IH), 6.85 (br s, IH), 5.42 (AB q, 2H), 5.15 (s, 2H), 4.79-4.72 (m, IH), 4.55- , 4.47 (m, IH), 3.67 (q, IH), 2.37-2.26 ( , IH), 1.94-1.75 (m, 311), 1.46 (d, 3H), 1.12- 1.01 (dd, 6H), 0.80-0.74 (dd, 611); FABMS 460.3 (M + K), 443.2 (M + Na), 421.3 (M + H).

Cbz-Aip-Leu-Pro-OTMSe. Cbz-Aip-Leu-OH (35.7 mg, 85.0 μmol) was dissolved in dry CH2CI2 (1.0 mL) and the solution was cooled to O'C. DCC (26.1 mg, 0.13 mmol) was added and the mixture was stirred for 30 min at O'C. Pro-OTMSe (18.5 mg, 85.0 μmol) in CH2CI2 ( 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 MgSθ4, and concentrated. The residue was purified by column chromatography, eluting with Cl Ctø/MeOH (95:5) to give a yellow oil (34.0 mg, 65%); lH NMR (300 MHz, CDCI3) δ 7.47 (s, 5H), 6.85 (br s, IH), 6.84 (br s, III), 5.45 (AB q, 2H), 5.13 (s, 2H), 4.80-4.73 (m. IH), 4.53-4.47 (m, IH), 4.24-4.01 (dt, 4H), 3.63 (q, IH), 2.35-2.23 (m, IH), 1.94-1.75 (m, 311), 1.46 (d, 311), 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). H-Λip-Leu-Pro-OTMSe. The protected tripeptide (24.9 mg, 40.5 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%) ; lH NMR (300 MHz, CDCI3) δ 6.84 (br s, IH), 6.82 (br s, I H), 5.41 (AB q, 2H). 5.09 (s, 2H), 4.82-4.71 (m. IH), 4.56-4.48 (m, IH), 4.25-4.00 (dt, 4H), 3.62 (q, III), 2.37-2.23 (m, IH), 1.95-1.75 (m, 3H), 1.47 (d, 3H). 1.14-1.01 (dd, 611), 0.82-0.74 (dd, 6H), 0.00 (s, 9H); FABMS m/z 506.3 (M + Na); 484.3 (M + H).

Boc-Ist-Λip-Lcu-Pro-OTMSe. Boc-Ist-OH (7.51 mg, 27.3 μmol) was dissolved in dry CH2CI2 ( 1.0 mL) and the solution was cooled to O'C. DCC (10.52 mg, 0.089 mmol) was added and the mixture was stirred for 30 min at O'C. H-Aip-Leu-Pro-OTMSe (3.28 mg, 27.3 μmol) in CII2CI2 (1.0 mL) was added and the solution was stirred for 30 min at O'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 MgS04, and concentrated. The residue was purified by reversed phase HPLC using a gradient system of CH3CN/H2O (45.0 mg, , 83%); FABMS 741.5 (M + H), 641.5 (M - Boc).

II-Ist-Aip-Leu-Pro-OTMSe (6). 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 CH3CN/H2O (gradient system) to give 22.0 mg (87%) of the compound as a yellow powder; FABMS 641.5 (M + H), see Supplementary Material, S-2.

Cbz-D-MeLeu-Thr[0-(N,0-Me2TyrBoc)]-Ist-Aip-Leu-Pro-OTMSe (4). 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 (18.2 mg, 28.4 μmol) and HOBT (0.81 mg) in 1.5 mL of 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 O'C for 1/2 h. The solution was then concentrated to 0.50 mL, kept at 0 'C for 24 h, then diluted with ether. The organic layer was washed with 10% HCl, 5% NaHCθ3, and saturated NaCl solutions. The organic layer was dried (Na2Sθ4), filtered, and concentrated. The crude oil was purified by reversed phase HPLC using a gradient system of CH3CN/H2O to give 1 1.6 mg (32%) of the linear heptapeptide, see Supplementary Material, S-3; FABMS 1294.2 (M + H); 1194.2 (M - Boc), see supplementary material, S-4; HRFABMS Calcd for C67H i08N7θiόSi (M + H) 1294.7649, Found 1294.7644.

Cbz-D-MeLeu-Thr-/V,0-Me2Tyr-Ist-Aip-Lcu-ProOII (7). IM 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 Na₂ θ4. Evaporation of the solvent gave the deprotected heptapeptide in quantitative yield (4.3 mg); FABMS 1194.2 (M + H); 1094.2 (M - Boc). see Supplementary Material, S-5; HRFABMS Calcd for C62H iθθ 7θ i4Si (M + H) 1194.7098, Found 1 194.7089. The deprotected , heptapeptide was subjected to a solution of IM 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% NaIICθ3 and water, and dried over Na2Sθ4. The compound was purified by reversed phase HPLC using a gradient system of CH3CN/H2O (see Supplementary Material, S-6) to give 3.58 mg (91%); FABMS 1094.2 (M + H), see supplementary material, S-7.

AipDidemnin A (8). The linear heptapeptide 4 (3.58 mg, 3.28 μmol) was dissolved in dry THF (0.80 mL), and the solution was cooled to O'C. EDCI (0.63 mg, 3.28 μmol) in 1.0 mL of THF was added, and the reaction mixture was stirred at O'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% NaHC03, and saturated NaCl solutions. The organic layer.was dried (Na Sθ4), filtered, and concentrated. The crude oil was purified by reversed phase HPLC using a gradient system of CH3CN/H2O to give 1.41 mg (40%) of the protected analogue; FABMS 1076.7 (M + H). see Supplementary Material, S-8; HRFABMS Calcd for C57H86N7O13 (M + H) 1076.6284, Found 1076.6283. The compound (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 rerrioved by filtration over celite and the solvent removed to afford the desired compound.

References

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Table I. Antiviral Activities of Amino Hip Didemnin Analogues' φ - ^Wew: Compounds

HSV/CV-1

Compound rtg/mT . Cytotoxocity" Activity¹"

Cbz-[Aip^J] )irferrmτn A 100 10 +

=r- 50 8 4- 20 0 + 10 0 Di emrjiii A (1) 100 0 4- 50 0 20 0 + 10 0

^»Peιfpπn?d by Dr. G. R. Wϋsoa in this laboratory. *o Qgas_t , coxic) to 16 (toxic). ^c÷++ = = complete inhibition, ++ «* strong iπ-nbitϊoi-, + = moderate inhibition, - = no inhibition. t

Table II, L1210 Cytotoxlcity of Amino Hip Didemnin Annlogucs" ^ C MΘW Compounds

Compounds Dose (ng/mL)

250 25 2.5 0.25

Inhibition (%) IC_jo (ng/mL)

Dicleinniti A (1) W 70 0 ^"75^" Cbz-tAip^-li emniu A 100 87 0 0 85 [Aip³Jdidemnin A (3) 98 20 0 0 J00

"PorFormcd by Dr. G. R. Wilson in this laboratory.

Claims

WHAT IS CLAIMED IS:

1. The compound Cbz- [Aip³] -Didemnin A.

2. The compound [Aip³]-Didemnin A.

3. The compound (2S,4S)-aminoisovalerylpropionic acid (Aip).

4. The compound (3S,4R,5S)-isostatine (1st).

5. A synthetic process for the formation of the Amino Hip analogue of Didemnin A, said process comprising the steps of:

(a) forming a linear heptapeptide by coupling two subunits, Cbz-D-MeLeuThr(OMe₂TyrBoc)OH (5) and H-IstAipLeuProOTMSe (6), followed by deprotection to yield the linear heptapeptide (7); and

(b) cyclization of heptapeptide (7) afforded the amino Hip (Aip) analog of Didemnin A.

6. The intermediate compound having the formula 5:

7. The intermediate compound having the formula 6:

8. The intermediate compound having the formula 7:

3. A pharmaceutical composition comprising Cbz- [Aip3] didemnin A and an optional pharmaceutically acceptable excipient, diluent or carrier.

4. A pharmaceutical composition comprising [Aip3]didemnin A and an optional pharmaceutically acceptable excipient, diluent or carrier.

5. A method of treating mammalian neoplastic tumors comprising administering to a mammal in need of such treatment, an effective amount of a pharmaceutical composition defined in claim 3 or 4.

6. A method of treating mammalian RNA or DNA viral infections comprising administering to a mammal in need of such treatment, an effective amount of a pharmaceutical composition defined in claim 3 or 4.

7. A method of treating mammalian bacterial infections comprising administering to a mammal in need of such treatment, an effective amount of a pharmaceutical composition defined in claim 3 or 4.

8. A method of treating mammalian fungal infections comprising administering to a mammal in need of such treatment, an effective amount of a pharmaceutical composition defined in claim 3 or 4.

9. A method of promoting immunosuppression in mammals comprising administering to a mammal in need of such treatment, an effective amount of a pharmaceutical composition defined in claim 3 or 4.