Total Syntheses of Cathepsin D Inhibitory Izenamides A, B, and C and Structural Confirmation of Izenamide B

The first total syntheses of izenamides A, B, and C, which are depsipeptides inhibitor of cathepsin D, were accomplished. In addition, the stereochemistry of izenamide B was confirmed by our syntheses. The key features of our synthetic route involve the avoidance of critical 2,5-diketopiperazine (DKP) formation and the minimization of epimerization during the coupling of amino acids for the target peptides.

Recently, three new depsipeptides, namely, izenamides A (1), B (2), and C (3), were isolated from the marine cyanobacterium 1605-5 by Suenaga and coworkers ( Figure 1) [25]. Interestingly, izenamides A (1) and B (2) were shown to inhibit cathepsin D in vitro. These two depsipeptides consist of seven monomers, including four amino acids, two hydroxy acids, and a γ-amino-β-hydroxy acid called statine. Izenamide C (3), which lacks a statine moiety and possesses a glycine unit instead of the alanine seen in izenamides A (1) and B (2), has no inhibitory activity against cathepsin D. The absolute configuration of the statine moiety in izenamide B (2) was indirectly determined by numerous efforts by Suenaga and coworkers [25].
In our pursuit of developing novel cathepsin D inhibitors, we recently became interested in establishing an efficient synthetic route towards izenamides A, B, and C. We were also interested in confirming the structure of izenamide B to ensure the stereochemistry of the statine moiety before we started medicinal chemistry studies. We herein report the first total syntheses of izenamides A, B, and C as well as the structural confirmation of izenamide B.

Synthetic Strategy for Izenamides A (1), B (2), and C (3)
Our retrosynthetic analysis is outlined in Scheme 1. We envisioned that the target izenamides could be accessed via a versatile convergent approach. Izenamides A (1) and B (2) would be synthesized by assembling three fragments, tetrapeptide 4, statine 6, and esters 7 or 8. Considering that izenamide C (3) has a very similar structure except for the statine moiety, it could also be convergently synthesized by the amide coupling of tetrapeptide 5 and ester 7. Tetrapeptides 4 and 5 were anticipated to be derived from commercially available amino acids and NMe-d-Phe, which could be obtained from d-Phe (9). Protected statine 6 could be prepared from Boc-l-leucinal (10) through the known procedure [26,27]. Esters 7 and 8 can be conveniently obtained by the esterification of protected hydroxy acids 11 and 12 (or 13). Scheme 1. Retrosynthetic analysis for the synthesis of izenamides 1, 2 and 3.

Syntheses of Fragments
The synthesis of izenamides commenced with synthesizing tetrapeptides 4 and 5. Initially, NMe-d-Phe 16 was prepared from commercially available d-Phe (9) as shown in Scheme 2. Amine protection of d-Phe 9 with Boc anhydride and N-methylation of resulting carbamate 14 afforded acid 15, which was esterified to produce desired monomer 16 in 84% yield over three steps. With monomer 16 in hand, we turned our attention to the synthesis of tetrapeptides 4 and 5 (Scheme 3). To avoid facile DKP formation upon sequential elongation from the C-terminal NMe-Phe and Pro residue, we synthesized tetrapeptides 4 and 5 according to the optimized sequence of fragment couplings. Amidation of l-alanine methyl ester hydrochloride (17) and glycine methyl ester hydrochloride (18) with Boc-Ile-OH followed by ester hydrolysis with LiOH afforded dipeptides 21 and 22 in high yields. Deprotection of 16 and coupling of resulting free amine 23 with acids 21 and 22 produced tripeptides 24 and 25 in 77 and 87% yields over two steps, respectively. Unfortunately, epimerization was observed during the amidation to form 24. After thorough optimization of the coupling conditions, amidation in the presence of DEPBT in CH 2 Cl 2 at 0 • C provided the best results in terms of high chemical yield and minimal epimerization (2.8:1). Tripeptides 24 and 25 were subjected to hydrolysis and then amidation with l-proline methyl ester hydrochloride to afford tetrapeptides 4 and 5 in 84 and 81% yield over two steps, respectively. In general, the N-Me peptide backbone near the C-terminal is prone to α-epimerization upon carboxy activation due to steric hindrance and electronic effects [28]. However, we could obtain the desired tetrapeptides without epimerization under the optimized conditions (DEPBT, CH 2 Cl 2 , 0 • C). Optically pure statine 6 was prepared by the known procedure [26,27] (Scheme 4). An elegant highly diastereoselective allyl addition to N-Boc-l-leucinal in the presence of SnCl 4 [26] provided desired threo isomer 27. The reaction of homoallylic alcohol 27 with 2,2-dimethoxypropane in the presence of catalytic PPTS followed by RuO 4 -mediated oxidative degradation [27] afforded monomer acid 6. Scheme 4. Synthesis of statine 6.
The synthesis of esters 7 and 8, which are the C-terminal fragments of izenamides, began with the preparation of silyl-protected valic acid 11 (Scheme 5). Global TBS-protection of commercially available l-valic acid (29) and hydrolysis of resulting silyl ester 30 produced C-terminal acid 11 in 94% yield over 2 steps. d-Allo-isoleucic acid 32 [29], which was prepared through the diazotization of commercially available d-allo-Ile (31), and d-valic acid (33) were converted to corresponding allyl esters 12 (64% for 2 steps) and 13 (91%), respectively. The EDC-mediated esterification of acid 11 with alcohol 12 afforded ester 7. Unfortunately, a satisfactory yield for this esterification was not achieved due the production of undesired esters by inevitable silyl transfer between hydroxyl groups. Therefore, we decided to change the TBS protecting group to a bulkier TBDPS group, which was anticipated to minimize silyl transfer (Scheme 6). To our delight, the silyl transfer was minimized in the coupling reactions, and fragments 36 and 37 were obtained in 92% and 94% yields, respectively. Finally, allyl deprotection of esters 36 and 37 with N-Me-aniline in the presence of Pd(0) produced acids 38 in 88% and 39 in 92% yield, respectively. Scheme 6. Synthesis of fragments 38 and 39 with minimized silyl transfer.

Completion of the Syntheses
We finally assembled the prepared fragments of izenamide C as shown in Scheme 7. The EDC-mediated amide coupling of amine 40, which was prepared from tetrapeptide 5, with acid 38 followed by silyl-deprotection produced izenamide C (3) in 81% over 3 steps. The completed synthesis of izenamides A (1) and B (2) is illustrated in Scheme 8. Boc-deprotection of tetrapeptide 4 with TFA and amide coupling with acid 6 afforded pentapeptide 42 in 77% yield over two steps. Global deprotection under acidic conditions followed by amidation of the resulting amine with acids 39 or 38 produced the corresponding heptapeptides. Finally, desilylation of heptapeptides afforded izenamides A (1) and B (2) in 66% and 61% over 3 steps, respectively. Spectral data of the synthesized depsipeptides 1, 2, and 3 were all identical with the reported data of natural 1, 2 and 3 [25]. Moreover, the absolute configuration of the statine moiety in 2 was identical to that of 1. The 1 H and 13 C NMR spectra of some compounds are in the Supplementary Materials.

General Information
Unless noted otherwise, all starting materials and reagents were obtained from commercial suppliers (Sigma-Aldrich, St. Louis, MO, USA; TCI, Tokyo, Japan; Combi-Blocks, San Diego, CA, USA) and were used without further purification. Tetrahydrofuran and Et 2 O were distilled from sodium benzophenone ketyl. Dichloromethane, chloroform and acetonitrile were freshly distilled from calcium hydride. All solvents used for routine isolation of products and chromatography were reagent grade and glass distilled. Reaction flasks were dried at 100 • C. Air and moisture sensitive reactions were performed under argon atmosphere. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck, Kenilworth, NJ, USA) with the indicated solvents. Thin-layer chromatography was performed using 0.25 mm silica gel plates (Merck, Kenilworth, NJ, USA). Optical rotations were measured with JASCO P-2000 digital polarimeter (Tokyo, Japan) at ambient temperature using cylindrical cell of 10 mm or 100 mm pathlength. Infrared spectra were recorded on a JASCO FT-IR-4200 spectrometer (Tokyo, Japan). High resolution mass spectra were obtained with JEOL JMS-700 (Tokyo, Japan) and Agilent Q TOF 6530 (Santa Clara, CA, USA) instruments. 1 H and 13 C NMR spectra were recorded using BRUKER AVANCE-800 (Billerica, MA, USA). Chemical shifts are expressed in parts per million (ppm, δ) downfield from tetramethylsilane and are referenced to the deuterated solvent (CHCl 3 , 1 H δ 7.24, 13 C δ 77.0; MeOH-d 4 , 1 H δ 3.30, 13 C δ 49.00). 1 H-NMR data were reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet and/or multiple resonances; br, broad signal), coupling constant in hertz (Hz) and number of protons.

Experimental Part
Boc-NMe-d-Phe-OMe (16). To a solution of d-Phe 9 (1.7 g, 10.3 mmol) and Boc 2 O (3.5 mL, 15.4 mmol) in a mixture THF and H 2 O (1:1, 50 mL) was added NaOH (0.6 g, 15.4 mmol) at room temperature. After stirring overnight, the reaction mixture was quenched with 1N HCl and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. To a solution of above crude Boc-d-Phe-OH 14 (10.3 mmol) in dry THF (20 mL) was added NaH (60% dispersion in mineral oil, 2.1 g, 51.5 mmol) at room temperature. After stirring for 1 h, iodomethane (3.2 mL, 51.5 mmol) was added to the reaction mixture. The reaction mixture was stirred for 12 h, quenched with 1N HCl, and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. The residue was used in the next step without further purification. To a solution of crude acid 15 (10.3 mmol) in dry DMF (20 mL) were added iodomethane (1.3 mL, 20.6 mmol) and K 2 CO 3 (2.8 g, 20.6 mmol) at room temperature. After stirring overnight, the reaction mixture was quenched with 1N HCl and extracted with Et 2 O. The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/Hexane = 1:20) to give 2.5 g (  3 mmol) at room temperature. After stirring for 2 h, the reaction mixture was quenched with 1N HCl and extracted with EtOAc. The combined organic layer was dried over MgSO 4 and concentrated in vacuo to afford 1.9 g (99%) of Boc-l-Ile-l-Ala-OH 21 as a white solid. The free acid 21 was used in the next step without further purification. Boc-L-Ile-Gly-OH (22). To a solution of glycine methyl ester hydrochloride 18 (1.5 g, 11.9 mmol), Boc-l-Ile-OH (2.3 g, 9.9 mmol), DIPEA (5.2 mL, 29.8 mmol), and HOAt (1.4 g, 10.4 mmol) in CH 2 Cl 2 (40 mL) was added EDC·HCl (3.8 g, 19.9 mmol) at room temperature. After stirring for 5 h, the reaction mixture was quenched with 1N HCl and extracted with CH 2 Cl 2 . The combined organic layer was washed with aqueous NaHCO 3 , dried over MgSO 4 , and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/Hexane = 1:2 to 1:1) to give 2.7 g (91%) of dipeptide 20 as a white solid. [ 3 mmol) at room temperature. After stirring for 2 h, the reaction mixture was quenched with 1N HCl and extracted with EtOAc. The combined organic layer was dried over MgSO 4 and concentrated in vacuo to afford 1.9 g (99%) of Boc-l-Ile-Gly-OH 22 as white solid. The free acid 22 was used in the next step without further purification.
. To a solution of Boc-l-Ile-Gly-NMe-d-Phe-l-Pro-OMe 5 (34 mg, 0.1 mmol) in CH 2 Cl 2 (0.8 mL) was added TFA (0.2 mL) dropwise at room temperature. After stirring for 1 h, the reaction mixture was concentrated in vacuo. To a solution of above amine salt 40 (0.1 mmol), acid 38 (37 mg, 0.1 mmol), DIPEA (0.1 mL, 0.2 mmol), and HOAt (11 mg, 0.1 mmol) in CH 2 Cl 2 (1.0 mL) was added EDC·HCl (24 mg, 0.1 mmol) at room temperature. After stirring overnight, the reaction mixture was quenched with 1N HCl and extracted with CH 2 Cl 2 . The combined organic layer was washed with aqueous NaHCO 3 , dried over MgSO 4 , and concentrated in vacuo. The residue was used in the next step without further purification. To a solution of crude hexapeptide (0.1 mmol) in THF (1.0 mL) was added TBAF (1M in THF, 0.2 mL, 0.2 mmol) at room temperature. After stirring for 4 h, the reaction mixture was quenched with H 2 O and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 , and concentrated in vacuo. The residue was purified by flash column chromatography (Acetone/Hexane = 1:3) to give 33 mg (83% for 3 steps) of izenamide C (3)  . To a solution of Boc-l-Ile-l-Ala-NMe-d-Phe-l-Pro-OMe 4 (140 mg, 0.2 mmol) in CH 2 Cl 2 (1.6 mL) was added TFA (0.4 mL) dropwise at room temperature. After stirring for 1 h, the reaction mixture was concentrated in vacuo. The residue was used in the next step without further purification. To a solution of above amine salt 41 (0.2 mmol), acid 6 (100 mg, 0.3 mmol), DIPEA (0.1 mL, 0.7 mmol), and HOAt (43 mg, 0.3 mmol) in CH 2 Cl 2 (2.4 mL) was added EDC·HCl (93 mg, 0.5 mmol) at room temperature. After stirring overnight, the reaction mixture was quenched with 1N HCl and extracted with CH 2 Cl 2 . The combined organic layer was washed with aqueous NaHCO 3 , dried over MgSO 4 , and concentrated in vacuo.  Izenamide A (1). To a solution of pentapeptide 42 (41 mg, 0.1 mmol) in CH 2 Cl 2 (0.8 mL) was added TFA (0.2 mL) dropwise at room temperature. After stirring for 1 h, the reaction mixture was concentrated in vacuo. To a solution of above amine salt 43 (0.1 mmol), acid 39 (32 mg, 0.1 mmol), DIPEA (0.1 mL, 0.2 mmol), and HOAt (10 mg, 0.1 mmol) in CH 2 Cl 2 (1.0 mL) was added EDC·HCl (21 mg, 0.1 mmol) at room temperature. After stirring for 6 h, the reaction mixture was quenched with 1N HCl and extracted with CH 2 Cl 2 . The combined organic layer was washed with aqueous NaHCO 3 , dried over MgSO 4 and concentrated in vacuo. The residue was used in the next step without further purification. To a solution of crude heptapeptide (0.1 mmol) in THF (1.0 mL) was added TBAF (1M in THF, 0.2 mL, 0.2 mmol) at room temperature. After stirring for 6 h, the reaction mixture was quenched with H 2 O and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO 4 and concentrated in vacuo. The residue was purified by flash column chromatography (Acetone/Hexane = 1:3 to 1:1) to give 29 mg (66% for 3 steps) of izenamide A (1) as white solid.