Total synthesis of amidepsine B and revision of its absolute configuration

Abstract

The total synthesis of (−)-amidepsine B, a potent diacylglycerol acyltransferase inhibitor, has been achieved. This synthetic study resulted in the revision of the previously assigned stereostructure of the natural amidepsine B and determined the absolute configuration.

Introduction

Amidepsines A–E were isolated from the culture broth of fungal strains FO-2942 and FO-5969 and found to be inhibitors of diacylglycerol acyltransferase (DGAT),^{1, 2, 3} which is exclusively involved in triacylglycerol formation. Excessive accumulation of triacylglycerol can cause fatty liver, obesity and hypertriglyceridemia, which leads to serious diseases, such as atherosclerosis, diabetes and metabolic disorders. Therefore, DGAT inhibitors have the potential to become drugs. Spectroscopic analyses of the amidepsines elucidated depsipeptide structures consisting of three 4,6-dihydroxy-2-methylbenzoic acid derivatives and an amino acid (except for amidepsine D), as shown in Figure 1. The DGAT inhibitory activity of the amidepsines was tested by a cellular assay using Raji cells, and the results showed that amidepsine B (1) was the most potent inhibitor. Amidepsine B (1) was previously determined to be a mixture of stereoisomers at its single chiral center, the alanine alpha carbon. A 3:2 mixture of L- and D-alanines was revealed by acid hydrolysis followed by HPLC analyses using a chiral column (Amidepsines A and C were also reported as a 3:2 mixture of L- and D-amino acids²). Herein, the total synthesis of amidepsine B and a revision of its absolute configuration will be described.

Figure 1

Structure of amidepsines A–E. ^*The original isomeric mixture reported for amidepsine B has been corrected by this work. ^**The actual configurations of amidepsines A and C are now in question.²

Results and discussion

The synthesis used a known aldehyde 2, which is a common starting material for the preparation of key substrates 5 and 10 (scheme 1).⁴ Regioselective p-O-methylation (In this reaction, reactivity of the hydroxy group adjacent to the formyl group in 3 would be precluded by the formation of an intramolecular hydrogen bond. Similar reactivity was observed.⁵) followed by benzylation gave 4 quantitatively, which was treated with NaClO₂ to afford carboxylic acid 5 quantitatively. Furthermore, similar conversion of 2 to 8, including the formation of a p-O-methoxymethyl ether instead of methylation, was also accomplished quantitatively. Esterification of 8 with allyl bromide followed by deprotection of the methoxymethyl ether afforded 10 quantitatively.

scheme 1

Synthesis of depside 15. Reagents and conditions: (a) CH₃I, K₂CO₃, acetone, rt, quant.; (b) Benzyl chloride, K₂CO₃, DMF, rt, quant. for 4 and 7; (c) NaClO₂, 2-methyl-2-butene, NaH₂PO₄·2H₂O, t-BuOH/H₂O, 0°C to rt, quant. for 5 and 8; (d) Methoxymethyl chloride, i-Pr₂NEt, CH₂Cl₂, 0°C to rt, quant.; (e) Allyl Br, NaH, DMF, 0°C to rt, quant.; (f) 6N HCl, MeOH, 60°C, quant.; (g) Trifluoroacetic anhydride, toluene, rt, quant.; (h) HCOOH, Pd(PPh₃)₄, Et₃N, THF, 0°C to rt, 98% for 12, quant. for 15; (i) (Diethylamino)sulfur trifluoride, Et₂O/CH₂Cl₂, rt, 89%; (j) 10, NaH, THF, 0°C, then 13, rt, quant.

Next, the key substrates 5 and 10 were coupled by treatment with trifluoroacetic anhydride (TFAA) to give rise to 11 quantitatively. Deprotection of the allyl ester in the presence of a palladium catalyst gave carboxylic acid 12 in 98% yield.

Subsequent esterification of 12 with 10 was troublesome. Esterification conditions, such as TFAA, 1-[(3-dimethylamino)propyl]-3-ethylcarbodiimide/dimethylaminopyridine (DMAP) and benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP)/Et₃N/DMAP led to a low yield of the desired product 14 with side products, such as the anhydride of 12. Therefore, we next focused on esterification through the acid fluoride.⁶ Reaction of 12 with (diethylamino)sulfur trifluoride (DAST) proceeded in 89% yield to furnish the stable acid fluoride 13. Treatment of 13 with sodium alkoxide, derived from 10 and sodium hydride, was effected to give the desired allyl ester 14 quantitatively. The allyl ester was then deprotected under palladium-catalyzed conditions to afford 15 quantitatively.

Having constructed the depside 15, we next turned to condensation with L-alanine before preparing the 3:2 mixture of L- and D-alanines toward the total synthesis (scheme 2). Treatment of 15 with L-alanine benzyl ester in the presence of BOP and Et₃N produced benzyl ester (+)-16 in 79% yield. Finally, global deprotection by hydrogenolysis completed the total synthesis of (−)-amidepsine B [(−)-1] in 83% yield.

scheme 2

Completion of the total synthesis of (–)-amidepsine B [(–)-1]. Reagents and conditions: (k) L-alanine benzyl ester, BOP, Et₃N, DMF, rt, 79%. For (–)-16: D-alanine benzyl ester, BOP, Et₃N, DMF, rt, 89%; (l) H₂, Pd(OH)₂, THF/EtOH, rt, 83%. For (+)-1: 94%.

The physical properties (¹H and ¹³C NMR, m.p., IR and MS) of synthetic (−)-amidepsine B (1) were completely identical to those of a natural sample, and the optical rotation of synthetic (−)-1 ([α]²³_D –17.3°; c 0.10, MeOH) also corresponded with that of the authentic sample of (1) ([α]²⁵_D –16°; c 0.1, MeOH). This result triggered the need for the re-assignment of the absolute configuration of natural amidepsine B (1). According to the procedure shown above, (+)-amidepsine B [(+)-1] by condensation of 15 with D-alanine was also synthesized.

We next analyzed the synthetic amidepsines (+)-1 and (−)-1 and the natural amidepsine B (1) with a chiral HPLC column. When the 1:1 mixture of the synthetic amidepsines (+)-1 and (−)-1 was injected onto the chiral HPLC system, each enantiomer was separated completely as shown in Figure 2a. Figures 2b and c show the HPLC analytical data of (+)-1 and (−)-1, respectively. Next, the natural amidepsine B (1) was also subjected to HPLC analysis and it was determined that the retention time was identical to that of (−)-amidepsine B [(−)-1] as shown in Figure 2d.

Figure 2

HPLC analysis of natural and synthetic amidepsines B. The abscissa axis indicates retention time. (a) a 1:1 mixture of synthetic amidepsines B (+)-1 and (−)-1; (b) synthetic amidepsine B [(+)-1] (derived from D-alanine); (c) synthetic amidepsine B [(−)-1] (derived from L-alanine); (d) natural amidepsine B.

This result shows that the absolute configuration of amidepsine B is S, derived from L-alanine, and that the previous determination of the absolute configuration of amidepsine B was inaccurate. It is likely that the acid hydrolysis of amidepsine B must have led to the epimerization of the resulting alanine. It is highly likely that amidepsines A and C are also L-amino acids, which will be reported in due course.

In conclusion, we have achieved the total synthesis of amidepsine B. Moreover, we have revised the absolute configuration of natural amidepsine B. Extension of this chemistry to the synthesis of structural analogs of amidepsine B so that the structure–activity relationships can be explored is currently under way and will be reported in due course.

Experimental Procedure

Commercially available solvents were dried and distilled before use. Reactions were monitored by TLC using Merck F60₂₅₄ silica gel plates (0.25 mm). Spots were visualized with ultraviolet (UV) light (254 nm) and stained with phosphomolybdic acid. Flash column chromatography was performed on silica gel 60N (spherical, neutral, particle size 40–50 μm). Preparative TLC was performed on Merck F60₂₅₄ silica gel plates (0.50 mm). The chiral HPLC system was comprised of a Daicel-CHIRALPAK IA column (0.46 cm φ × 25 cm), and a mobile phase of hexane/THF (tetrahydrofuran)/TFAA (80:20:0.1) (Senshu HPLC system; 10 μl injection; UV, 254 nm; flow rate, 1.0 ml min⁻¹).

Melting points were determined on a Yanagimoto micro melting apparatus and uncorrected. Optical rotations were obtained with a JASCO DIP-1000 polarimeter. Mass spectra were recorded on a JEOL JMS-700 mass spectrometer. FT-IR spectra were recorded on a Horiba FT-710 spectrometer. ¹H-NMR spectra were recorded on a JEOL JNM-EX270 (270 MHz), MERCURY-300 (300 MHz) and UNITY-400 (400 MHz) spectrometers in CDCl₃ or DMSO-d₆. ¹H-NMR spectral data are reported according to the following conventions: chemical shifts relative to CDCl₃ (7.26 p.p.m.) or DMSO-d₆ (2.48 p.p.m.), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br s=broad singlet, br d=broad doublet and dd=double doublet), coupling constant and integration. ¹³C-NMR spectral data are reported in p.p.m. relative to CDCl₃ (77.0 p.p.m.) or DMSO-d₆ (39.5 p.p.m.).

2-Hydroxy-4-methoxy-6-methylbenzaldehyde (3)

To a solution of 2,4-dihydroxy-6-methylbenzaldehyde (2) (0.56 g, 3.68 mmol) in acetone (36.8 ml), K₂CO₃ (0.51 g, 3.68 mmol) and CH₃I (3.44 ml, 55.2 mmol) were added. The reaction mixture was stirred for 2.5 h at room temperature, quenched with H₂O and extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (20:1 hexanes/EtOAc) to give 3 (0.61 g, quant.) as a white powder; m.p. 65–67 °C; IR (KBr): 3084, 2966, 2906, 2846, 1635, 1576, 1473 and 1429 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 12.45 (s, 1H), 10.05 (s, 1H), 6.23 (s, 2H), 3.80 (s, 3H), 2.49 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 192.8, 166.6, 166.4, 143.7, 113.2, 110.4, 98.5, 55.4 and 18.2; HRMS (FAB, m–NBA): calcd. for C₉H₁₁O₃: 167.0708 [M+H]⁺, found: m/z 167.0703.

2-Benzyloxy-4-methoxy-6-methylbenzaldehyde (4)

To a solution of 3 (0.60 g, 3.64 mmol) in N,N-dimethylformamide (DMF) (18.2 ml), K₂CO₃ (5.03 g, 36.4 mmol) and BnCl (6.29 ml, 56.4 mmol) were added at 0 °C. After stirring for 2.5 h at room temperature, the reaction was quenched with H₂O and extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (20:1 to 10:1 hexanes/EtOAc) to give 4 (0.93 g, quant.) as a white powder; m.p. 78–80 °C; IR (KBr): 3321, 3091, 3049, 2966, 2925, 2871, 2789, 1666, 1603 and 1448 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 10.59 (s, 1H), 7.38 (m, 5H), 6.40 (d, J=1.8 Hz, 1H), 6.34 (d, J=1.8 Hz, 1H), 5.14 (s, 2H), 3.84 (s, 3H), 2.60 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 190.4, 164.2, 164.2, 144.6, 136.0, 128.6, 128.1, 127.2, 117.5, 109.1, 97.0, 70.5, 55.3 and 22.3; HRMS (FAB, m–NBA): calcd. for C₁₆H₁₇O₃: 257.1178 [M+H]⁺, found: m/z 257.1176.

2-Benzyloxy-4-methoxy-6-methylbenzoic acid (5)

To a solution of 4 (0.16 g, 0.61 mmol) in t-BuOH/H₂O (1:1) (6.2 ml) 2-methyl-2-butene (0.26 ml, 2.45 mmol), NaH₂PO₄·2H₂O (0.29 g, 1.84 mmol) and NaClO₂ (0.17 ml, 1.84 mmol) were added at 0 °C. The reaction mixture was stirred for 24 h at room temperature, diluted with H₂O and extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (5:1 to 3:1 hexanes/EtOAc) to give 5 (0.18 g, quant.) as a white powder; m.p. 98–100 °C; IR (KBr): 3298, 3022, 2935, 2877, 2837, 1705, 1604 and 1448 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 10.22 (br s, 1 H), 7.40 (m, 5H), 6.48 (s, 2H), 5.20 (s, 2H), 3.83 (s, 3H), 2.61 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 169.9, 162.0, 158.4, 142.7, 135.6, 128.7, 128.3, 127.3, 113.5, 108.9, 97.9, 71.3, 55.3 and 22.0; HRMS (FAB, m–NBA): calcd. for C₁₆H₁₆O₄: 272.1049 [M]⁺, found: m/z 272.1049.

2-Hydroxy-4-methyoxymethoxy-6-methylbenzaldehyde (6)

To a solution of 5 (0.56 g, 3.70 mmol) in CH₂Cl₂ (37.0 ml) i-Pr₂NEt (0.71 ml, 4.07 mmol) and chloromethyl methyl ether (MOMCl) (0.56 ml, 7.39 mmol) were added at 0 °C. After stirring for 45 min at room temperature, the reaction was quenched with H₂O and extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (30:1 hexanes/EtOAc) to give 6 (0.72 g, quant.) as a white powder; m.p. 46–48 °C; IR (KBr): 3089, 2964, 1631, 1493 and 1431 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 12.31 (s, 1H), 10.11 (s, 1H), 6.42 (d, J=2.2 Hz, 1H), 6.36 (d, J=2.2 Hz, 1H), 5.19 (s, 2H), 3.46 (s, 3H), 2.53 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 193.1, 165.9, 164.1, 144.0, 113.9, 110.9, 101.3, 93.8, 56.3 and 183.3; HRMS (FAB, m–NBA): calcd. for C₁₀H₁₃O₄: 197.0814 [M+H]⁺, found: m/z 197.0808.

2-Benzyloxy-4-methoxymethoxy-6-methylbenzaldehyde (7)

To a solution of 6 (0.76 g, 3.86 mmol) in DMF (19.3 ml) K₂CO₃ (5.33 g, 38.6 mmol) and BnCl (4.44 ml, 38.6 mmol) were added at 0 °C. After stirring for 45 min at room temperature, the mixture was warmed to 50 °C and stirred for 30 min. The mixture was quenched with H₂O and extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (20:1 hexanes/EtOAc) to give 7 (1.09 g, 99%) as a white powder; m.p. 43–46°C; IR (KBr): 2927, 1674, 1601, 1450 and 1415 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 10.59 (s, 1H), 7.39 (m, 5H), 6.57 (d, J=2.0 Hz, 1H), 6.48 (d, J=2.0 Hz, 1H), 5.19 (s, 2H), 5.13 (s, 2H), 3.48 (s, 3H), 2.58 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 190.6, 164.1, 161.9, 144.5, 135.9, 128.6, 128.1, 127.3, 118.2, 111.3, 98.5, 93.9, 70.5, 56.2 and 22.1; HRMS (FAB, m–NBA): calcd. for C₁₇H₁₇O₄: 285.1132 [M-H]^–, found: m/z 285.1119.

2-Benzyloxy-4-methoxymethoxy-6-methylbenzoic acid (8)

To a solution of 7 (0.70 g, 2.45 mmol) in t-BuOH/H₂O (1:1) (24.4 ml), 2-methyl-2-butene (1.04 ml, 9.79 mmol), NaH₂PO₄·2H₂O (1.10 g, 7.34 mmol) and NaClO₂ (0.66 ml, 7.34 mmol) were added at 0 °C. The reaction mixture was stirred for 24 h at room temperature, diluted with H₂O and extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (5:1 hexanes/EtOAc) to give 8 (0.74 g, quant.) as a yellow oil. IR (KBr): 3168, 2927, 1697, 1601 and 1448 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 9.75 (br s, 1H), 7.41 (m, 5H), 6.59 (d, J=2.2 Hz, 1H), 6.58 (d, J=2.2 Hz, 1H), 5.17 (s, 2H), 5.16 (s, 2H), 3.47 (s, 3H), 2.51 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 169.3, 159.6, 158.3, 142.7, 135.5, 128.7, 128.3, 127.4, 114.4, 111.5, 99.4, 94.1,71.4, 56.1 and 21.9; HRMS (FAB, m–NBA): calcd. for C₁₇H₁₉O₅: 303.1232 [M+H]⁺, found: m/z 303.1244.

2-Benzyloxy-4-methoxymethoxy-6-methylbenzoic acid allyl ester (9)

To a solution of 8 (1.06 g, 3.51 mmol) in DMF (35.1 ml), NaH (0.21 g, 5.27 mmol) and allyl bromide (0.91 ml, 10.5 mmol) were added at 0 °C under N₂. After stirring for 30 min, the reaction mixture was warmed to room temperature and stirred for 1.5 h. The mixture was quenched with H₂O and extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (15:1 hexanes/EtOAc) to give 9 (1.14 g, quant.) as a colorless oil. IR (KBr): 2945, 1726, 1599 and 1446 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 7.33 (m, 5H), 6.50 (d, J=2.3 Hz, 2H), 5.95 (m, 1 H), 5.35 (dd, J=17.2, 1.5 Hz, 1H), 5.19 (dd, J=10.2, 1.5 Hz, 1H), 5.13 (s, 2H), 5.06 (s, 2H), 4.78 (br d, J=5.6 Hz, 2H), 3.44 (s, 3H), 2.30 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 167.7, 158.8, 157.0, 138.2, 136.5, 132.1, 128.3, 127.7, 127.1, 118.4, 117.8, 109.6, 99.1, 94.2, 70.3, 65.6, 55.9 and 19.7; HRMS (FAB, m–NBA): calcd. for C₂₀H₂₃O₅: 343.1545 [M+H]⁺, found: m/z 343.1556.

2-Benzyloxy-4-hydroxy-6-methylbenzoic acid allyl ester (10)

To a solution of 9 (2.10 g, 6.13 mmol) in CH₃OH (30.5 ml), 6 N HCl (30.5 ml) was added. After stirring for 30 min at 60 °C, the reaction mixture was neutralized with a saturated aqueous NaHCO₃ solution and extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (10:1 hexanes/EtOAc) to give 10 (1.83 g, quant.) as a yellow oil. IR (KBr): 3381, 3080, 3033, 2937, 2875, 1697, 1603 and 1458 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 7.31 (m, 5H), 6.26 (d, J=2.0 Hz, 1H), 6.20 (d, J=2.0 Hz, 1H), 5.93 (m, 1H), 5.35 (dd, J=17.2, 1.3 Hz, 1H), 5.19 (dd, J=10.6, 1.3 Hz, 1H), 4.92 (s, 2H), 4.77 (br d, J=5.6 Hz, 2H), 2.21 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 169.0, 158.1, 157.5, 138.6, 136.4, 131.8, 128.3, 127.8, 127.1, 118.7, 115.7, 109.6, 98.2, 70.2, 66.0 and 19.6; HRMS (FAB, m–NBA): calcd. for C₁₈H₁₉O₄: 299.1283 [M+H]⁺, found: m/z 299.1296.

2-Benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methylbenzoic acid allyl ester (11)

To a solution of 5 (0.19 g, 0.69 mmol) and 10 (0.17 g, 0.57 mmol) in toluene (2.3 ml), TFAA (0.57 ml) was added. The reaction mixture was stirred for 1 h at room temperature, quenched with H₂O and extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (40:1 to 20:1 hexanes/EtOAc) to give 11 (0.32 g, quant.) as a white powder; m.p. 122–125 °C; IR (KBr): 2927, 2856, 1738, 1597 and 1437 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 7.33 (m, 10H), 6.54 (d, J=1.7 Hz, 1H), 6.52 (d, J=1.7 Hz, 1H), 6.45 (d, J=1.7 Hz, 1H), 6.40 (d, J=2.0 Hz, 1H), 5.91 (m, 1H), 5.34 (dd, J=17.2, 1.2 Hz, 1H), 5.19 (dd, J=10.2, 1.2 Hz, 1H), 5.09 (s, 2H), 4.86 (s, 2H), 4.77 (br d, J=5.6 Hz, 2H), 3.82 (s, 3H), 2.43 (s, 3H), 2.26 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 167.3, 166.4, 161.8, 157.8, 156.6, 152.2, 139.0, 137.9, 136.3, 136.2, 131.9, 128.5, 128.3, 128.1, 127.8, 127.7, 127.3, 121.6, 118.5, 115.8, 115.5, 107.2, 104.2, 97.4, 70.7, 70.5, 65.8, 55.4, 20.0 and 19.4; HRMS (FAB, m–NBA+NaI): calcd. for C₃₄H₃₂O₇Na: 575.2046 [M+Na]⁺, found: m/z 575.2072.

2-Benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methylbenzoic acid (12)

To a solution of 11 (0.32 g, 0.58 mmol) in THF (5.8 ml), HCOOH (0.10 ml, 2.90 mmol), Et₃N (0.40 ml, 2.90 mmol) and Pd(PPh₃)₄ (34.0 mg, 29.0 μmol) were added at 0 °C. The reaction mixture was stirred for 24 h at room temperature, quenched with H₂O and extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (1:1 hexanes/EtOAc) to give 12 (0.29 g, 98%) as a white powder; m.p. 155–158 °C; IR (KBr): 3475, 2991, 2958, 2931, 2873, 2659, 2561, 1741, 1691, 1597 and 1442 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.36 (m, 10H), 6.62 (d, J=1.8 Hz, 1H), 6.61 (d, 1H, J=1.8 Hz), 6.47 (d, 1H, J=1.9 Hz), 6.43 (d, J=2.1 Hz, 1H), 5.10 (s, 2H), 4.93 (s, 2H), 3.83 (s, 3H), 2.45 (s, 3H), 2.43 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 170.1, 166.3, 161.9, 157.9, 157.3, 152.9, 140.7, 139.2, 136.3, 135.5, 128.6, 128.5, 128.2, 128.2, 127.7, 127.4, 119.2, 117.0, 115.3, 107.3, 104.7, 97.4, 71.2, 70.7, 55.4, 20.8 and 20.1; HRMS (FAB, m–NBA+NaI): calcd. for C₃₁H₂₈O₇Na: 535.1733 [M+Na]⁺, found: m/z 535.1758.

2-Benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methylbenzoyl fluoride (13)

To a solution of 12 (0.03 g, 0.06 mmol) in Et₂O/CH₂Cl₂ (2:1) (0.9 ml) DAST (18.6 μl, 0.14 mmol) was added. The reaction mixture was stirred for 1.5 h at room temperature, quenched with H₂O and extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (25:1 hexanes/EtOAc) to give 13 (27.0 mg, 89%) as a white powder; m.p. 111–114 °C; IR (KBr): 3624, 3458, 3035, 2924, 2852, 1817, 1732, 1593 and 1442 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.38 (m, 10H), 6.62 (s, 1H), 6.48 (d, J=2.0 Hz, 1H), 6.44 (d, J=1.9 Hz, 1H), 5.10 (s, 2H), 4.92 (s, 2H), 3.84 (s, 3H), 2.46 (s, 3H), 2.41 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 166.0, 162.0, 158.9 (³J_CF=2.2 Hz), 158.0, 156.6 (¹J_CF=351.7 Hz), 154.5, 141.8 (³J_CF=2.4 Hz), 139.2, 136.2, 135.6, 128.5, 128.2, 128.0, 127.7, 127.1, 116.6 (⁴J_CF=2.4 Hz), 115.0, 114.5 (²J_CF=56.2 Hz), 107.3, 104.7, 97.4, 70.7, 70.6, 55.3, 20.5 (⁴J_CF=1.2 Hz), 20.0; HR-MS (FAB, m–NBA+NaI): calcd. for C₃₁H₂₇FO₆Na: 537.1684 [M+Na]⁺, found: m/z 537.1687.

2-Benzyloxy-4-[2-benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methyl]benzoyloxy-6-methylbenzoic acid allyl ester (14)

To a solution of 10 (22.0 mg, 0.07 mmol) in THF (0.5 ml), NaH (6.0 mg, 0.15 mmol) was added at 0 °C under N₂. After stirring for 5 min at room temperature, a solution of 13 (26.0 mg, 0.05 mmol) in THF (0.5 ml) was added dropwise. After 30 min, the mixture was treated with MOMCl (3.8 μl, 0.05 mmol) for the conversion of an excess amount of 10 into 9 and then stirred for 30 min. The resulting mixture was quenched with H₂O and extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (10:1 to 1:1 hexanes/EtOAc) to give 14 (40.0 mg, quant.) as a white powder; m.p. 183–187 °C; IR (KBr): 2925, 2858, 1741, 1597 and 1439 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 7.33 (m, 15H), 6.59 (d, J=2.0 Hz, 1H), 6.58 (d, J=2.0 Hz, 1H), 6.49 (d, J=2.1 Hz, 1H), 6.47 (d, J=2.0 Hz, 1H), 6.46 (d, J=2.1 Hz, 1H), 6.41 (d, J=2.0 Hz, 1H), 5.91 (m, 1H), 5.33 (dd, J=17.2, 1.5 Hz, 1H), 5.18 (dd, J=10.5, 1.5 Hz, 1H), 5.10 (s, 2H), 4.85 (s, 2H), 4.82 (s, 2H), 4.76 (br d, J=5.9 Hz, 2H), 3.83 (s, 3H), 2.45 (s, 3H), 2.37 (s, 3H), 2.24 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 167.3, 166.4, 166.0, 161.9, 157.9, 157.0, 156.6, 152.8, 152.0, 139.1, 138.2, 137.9, 136.3, 136.2, 136.0, 132.0, 128.5, 128.5, 128.4, 128.2, 128.2, 127.9, 127.9, 127.8, 127.3, 121.8, 120.5, 118.6, 116.0, 115.7, 115.4, 107.2, 104.1, 97.4, 70.8, 70.7, 70.5, 65.8, 55.4, 20.1, 19.4 and 19.3; HRMS (FAB, m–NBA+NaI): calcd. for C₄₉H₄₄O₁₀Na: 815.2832 [M+Na]⁺, found: m/z 815.2806.

2-Benzyloxy-4-[2-benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methyl]benzoyloxy-6-methylbenzoic acid (15)

To a solution of 14 (40.0 mg, 0.05 mmol) in THF (0.5 ml), HCOOH (9.4 μl, 0.25 mmol), Et₃N (34.8 μl, 0.25 mmol) and Pd(PPh₃)₄ (29.0 mg, 3.00 μmol) were added at 0 °C. The reaction mixture was stirred for 24 h at room temperature, quenched with H₂O and extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (2:1 hexanes/EtOAc) to give 15 (38.0 mg, quant.) as a white powder; m.p. 215–216 °C; IR (KBr): 3446, 2924, 2858, 1745, 1691, 1597 and 1441 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 7.40 (m, 15H), 6.60 (br s, 2H), 6.58 (d, J=2.0 Hz, 1H), 6.56 (d, J=2.1 Hz, 1H), 6.47 (d, J=2.2 Hz, 1H), 6.42 (d, J=2.3 Hz, 1H), 5.10 (s, 2H), 4.88 (s, 2H), 4.85 (s, 2H), 3.83 (s, 3H), 2.47 (s, 3H), 2.44 (s, 3H), 2.38 (s, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 167.2, 166.4, 165.9, 161.9, 158.0, 157.5, 157.2, 153.0, 152.9, 142.4, 139.2, 138.4, 136.3, 136.0, 135.1, 128.8, 128.6, 128.6, 128.5, 128.3, 128.2, 128.0, 127.9, 127.7, 120.2, 118.4, 117.6, 116.1, 115.4, 107.3, 104.6, 104.2, 97.5, 71.6, 70.9, 70.8, 55.5, 21.5, 20.1 and 19.5; HRMS (FAB, m–NBA+NaI): calcd. for C₄₆H₄₀O₁₀Na: 775.2519 [M+Na]⁺, found: m/z 775.2510.

(2S)-2{2-Benzyloxy-4-[2-benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methyl]benzoyloxy-6-methylbenzoy}aminopropionic acid benzyl ester [(+)-(16)]

To a solution of 15 (92.0 mg, 0.12 mmol) in DMF (1.2 ml), L-Ala-OBn·HCl (58.0 mg, 0.27 mmol), Et₃N (68.0 μl, 0.49 mmol) and BOP (135.0 mg, 0.30 mmol) were added. The reaction mixture was stirred for 3.5 h at room temperature, quenched with H₂O and extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with an eluent (3:1 to 2:1 hexanes/EtOAc) to give (+)-(16) (88.0 mg, 79%) as a white powder; m.p. 179–181 °C; [α]²⁷_D +21.5 (c 0.1, CHCl₃); IR (KBr): 2925, 1741, 1595 and 1446 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 7.38 (m, 20H), 6.61 (br s, 2H), 6.51 (d, J=1.3 Hz, 1H), 6.48 (br s, 2H), 6.43 (d, J=1.6 Hz, 1H), 6.41 (d, J=7.6 Hz, 1H), 5.18 (d, J=12.3 Hz, 1H), 5.13 (d, J=12.3 Hz, 1H), 5.11 (s, 2H), 4.87 (s, 2H), 4.79 (m, 1H), 4.78 (s, 2H), 3.84 (s, 3H), 2.47 (s, 3H), 2.39 (s, 3H), 2.28 (s, 3H), 1.35 (d, J=7.0 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 172.5, 166.4, 166.4, 166.2, 161.9, 157.9, 157.1, 156.2, 152.8, 151.8, 139.1, 139.1, 138.3, 136.3, 136.0, 136.0, 135.3, 128.6, 128.6, 128.6, 128.4, 128.4, 128.3, 128.2, 128.2, 128.0, 128.0, 127.8, 127.6, 123.8, 120.5, 116.2, 116.0, 115.4, 107.3, 104.2, 104.0, 97.4, 70.8, 70.8, 70.5, 67.0, 55.4, 48.1, 20.1, 19.5 and 18.3; HRMS (FAB, m–NBA): calcd. for C₅₆H₅₂NO₁₁: 914.3540 [M+H]⁺, found: m/z 914.3497.

(2S)-2{2-Benzyloxy-4-[2-benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methyl]benzoyloxy-6-methylbenzoy}aminopropionic acid{(−)-Amidepsine B [(−)-1]}

A mixture of (+)-(16) (25.1 mg, 0.03 mmol) and Pd(OH)₂ (12.2 mg) in THF/EtOH (1:1) (2.8 ml) was stirred under H₂ atmosphere for 16 h. The mixture was filtered through a pad of Celite and the filtrate was concentrated. The residue was purified by preparative TLC on silica gel (5:1 CHCl₃/CH₃OH) with an eluent (3:1 CHCl₃/CH₃OH) to give (−)-(1) as a white powder (13.0 mg, 87%). Moreover, this sample was dissolved in CHCl₃/CH₃OH (25:1) (5 ml) and washed with 1% aqueous H₃PO₄ (1 ml). The organic phase was dried over anhydrous Na₂SO₄ and concentrated in vacuo to give (−)-(1) (12.4 mg, 83%) as a white powder; m.p. 153–156 °C; [α]²⁹_D –17.3 (c 0.1, CH₃OH); IR (KBr): 3087, 2937, 2858, 1668, 1610 and 1454 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 10.4 (br s, 1H), 8.44 (d, J=6.8 Hz, 1 H), 6.68 (br s, 1H), 6.66 (br s, 1H), 6.56 (br s, 1H), 6.53 (br s, 1H), 6.39 (br s, 1H), 6.36 (br s, 1H), 4.35 (m, 1H), 3.74 (s, 3H), 2.45 (s, 3H), 2.38 (s, 3H), 2.35 (s, 3H), 1.31 (d, J=7.1 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 174.1, 166.7, 166.4, 165.8, 162.1, 159.1, 156.1, 155.0, 152.0, 150.5, 139.6, 137.8, 137.6, 123.5, 118.4, 114.1, 113.3, 110.7, 108.1, 107.1, 106.4, 99.0, 55.2, 47.6, 20.8, 19.2, 18.8 and 16.9; HRMS (FAB, m–NBA): calcd. for C₂₈H₂₆NO₁₁: 552.1506 [M-H]^–, found: m/z 552.1510.

(2R)-2{2-Benzyloxy-4-[2-benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methyl]benzoyloxy-6-methylbenzoy}aminopropionic acid benzyl ester [(−)-(16)]

According to the conversion of 15 into (+)-16, 15 (30.0 mg, 0.04 mmol) was subjected to a condensation reaction with D-Ala-OBn·p-TsOH (21.8 mg, 0.060 mmol) to afford (−)-(16) (32.6 mg, 89 %) as a white powder; m.p. 181–183 °C; [α]²³_D –8.7 (c 0.1, CHCl₃); IR (KBr): 2925, 1741, 1595 and 1446 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 7.38 (m, 20H), 6.61 (br s, 2H), 6.51 (d, J=1.3 Hz, 1H), 6.48 (br s, 2H), 6.43 (d, J=1.6 Hz, 1H), 6.41 (d, J=7.6 Hz, 1H), 5.18 (d, J=12.3 Hz, 1H), 5.13 (d, J=12.3 Hz, 1H), 5.11 (s, 2H), 4.87 (s, 2H), 4.79 (m, 1H), 4.78 (s, 2H), 3.84 (s, 3H), 2.47 (s, 3H), 2.39 (s, 3H), 2.28 (s, 3H), 1.35 (d, J=7.0 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 172.5, 166.4, 166.4, 166.2, 161.9, 157.9, 157.1, 156.2, 152.8, 151.8, 139.1, 139.1, 138.3, 136.3, 136.0, 136.0, 135.3, 128.6, 128.6, 128.6, 128.4, 128.4, 128.3, 128.2, 128.2, 128.0, 128.0, 127.8, 127.6, 123.8, 120.5, 116.2, 116.0, 115.4, 107.3, 104.2, 104.0, 97.4, 70.8, 70.8, 70.5, 67.0, 55.4, 48.1, 20.1, 19.5 and 18.3; HRMS (FAB, m–NBA+NaI): calcd. for C₅₆H₅₁NO₁₁Na: 936.3360 [M+Na]⁺, found: m/z 936.3383.

(2R)-2{2-Benzyloxy-4-[2-benzyloxy-4-(2-benzyloxy-4-methoxy-6-methyl)benzoyloxy-6-methyl]benzoyloxy-6-methylbenzoy}aminopropionic acid{(+)-Amidepsine B [(+)-1]}

According to the conversion of (+)-16 into (−)-1, (−)-(16) (30.8 mg, 0.03 mmol) gave (+)-(1) (17.7 mg, 94%) as a white powder; m.p. 156–159°C; [α]²⁵_D +10.4 (c 0.1, CH₃OH); IR (KBr): 3087, 2937, 2858, 1668, 1610 and 1454 cm⁻¹; ¹H-NMR (270 MHz, CDCl₃) δ 10.4 (br s, 1H), 8.44 (d, J=6.8 Hz, 1H), 6.68 (br s, 1H), 6.66 (br s, 1H), 6.56 (br s, 1H), 6.53 (br s, 1H), 6.39 (br s, 1H), 6.36 (br s, 1H), 4.35 (m, 1H), 3.74 (s, 3H), 2.45 (s, 3H), 2.38 (s, 3H), 2.35 (s, 3H), 1.31 (d, J=7.1 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 174.1, 166.7, 166.4, 165.8, 162.1, 159.1, 156.1, 155.0, 152.0, 150.5, 139.6, 137.8, 137.6, 123.5, 118.4, 114.1, 113.3, 110.7, 108.1, 107.1, 106.4, 99.0, 55.2, 47.6, 20.8, 19.2, 18.8 and 16.9; HRMS (FAB, m–NBA): calcd. for C₂₈H₂₈NO₁₁: 554.1662 [M+H]⁺, found: m/z 554.1663.