Total synthesis of isoneoantimycin

Abstract

Antimycins are one of the well-known antifungal metabolites produced by Streptomyces bacteria. Neoantimycin and its analogues, the ring-expanded antimycins featuring a 15-membered tetraester ring, have been shown to be effective regulators of the oncogenic proteins GRP78/BiP and K-Ras. Isoneoantimycin was isolated from Streptomyces fradiae IFO12773 (ISP 5063) as a minor metabolite during the fermentation of neoantimycin and is the first reported antibiotic of the antimycin family without the macrolide core. In this study, we explored the total synthesis and stereochemical assignment of isoneoantimycin as an approach to perform structure–activity studies on neoantimycins. Taking the neoantimycin biosynthesis pathway into account, we presumed that the stereochemistry of isoneoantimycin is the same as that of neoantimycin. The synthesis of our target molecule with the (1S,2R,5S,6S,14R,15R,17S) configuration has been achieved by using chiral-pool building blocks. A comparison of the spectroscopic data between the synthetic and natural samples verified our presumption of the stereochemistry of natural isoneoantimycin.

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Introduction

Isoneoantimycin (1) was isolated from Streptomyces fradiae IFO12773 (ISP 5063) as a minor metabolite produced during the fermentation of neoantimycin (2) by Takeda and co-workers in 1998.¹ 2D NMR analyses and EIMS fragmentation revealed that the planar structure of compound 1 comprises units identical to those of neoantimycin; isoneoantimycin is the first reported antibiotic of the antimycin family without the macrolide core and consists of five components, namely γ-butyrolactone, α-hydroxy-isovaleric acid, L-threonine, α-hydroxy-β-methyl-valeric acid, and 3-formylaminosalicylic acid, as shown in Fig. 1. However, the absolute configuration of 1 could not be elucidated at that time.

Fig. 1 Structures of isoneoantimycin (1), neoantimycin (2), and antimycin A (3).

After the discovery of antimycin A in 1949,² many antimycin-class depsipeptides have been reported.³ However, very few papers on compounds without a macrolactone structure have been published.^1,4 The diverse biological activities of these antibiotics, including anti-cancer, antifungal, and immunosuppressant properties, have intrigued researchers and attracted significant attention from them.⁵ Neoantimycin and its analogues, the ring-expanded antimycins, have been shown to be effective regulators of the oncogenic proteins GRP78/BiP and K-Ras.⁶

During our exploration of antimycin-type antibiotics,⁷ we were engaged in studies directed toward the total synthesis and stereochemical assignment of 1 as an approach to perform structure–activity studies on neoantimycins. Herein, we report our success in these endeavours.

Results and discussion

The absolute configuration of 2 has been confirmed, as shown in Fig. 1, by total synthesis.^7b Recently, the biosynthetic pathway for neoantimycin production was proposed, wherein 3-formylaminosalicylic acid is employed as the starting substrate in the hybrid NRPS (nonribosomal peptide synthetase)/PKS (polyketide synthase) machinery for the growth of the depsipepitidal chain.⁸ The subsequent macrolactonization and ketoreductase reduction provide neoantimycin. Taking the neoantimycin biosynthesis pathway into account, we presumed that the stereochemistry of 1 is the same as that of neoantimycin. Compound 4 was set as the target molecule for our synthesis (Scheme 1). Our synthetic strategy involves amide formation between amine 6 (four components linearly connected) and the protected 3-formylaminosalicylic acid 5 ⁹ to afford the desired compound (1S,2R,5S,6S,14R,15R,17S)-4. Conceptually, 6 could be divided into left and right units; the left part 8 would be obtained by the condensation of N-Cbz L-threonine tert-butyl ester 9 ¹⁰ and α-hydroxy carboxylic acid derivative 10.¹¹ The right part 7 would be obtained by the condensation of α-hydroxy carboxylic acid 11 ¹² with 3-hydoxy-γ-butyrolactone 12, which is derived from D-phenylalanine.

Scheme 1 Retrosynthesis of the target molecule (4).

The Mukaiyama aldol reaction of aldehyde 14,¹³ derived from D-phenylalanine, with the commercially available silyl ketene acetal 13 in the presence of SnCl₄ proceeded exclusively to afford the corresponding syn-15 in 83% yield.¹⁴ Hydrogenolysis of 15 with Pd(OH)₂ in EtOH resulted in the removal of the benzyl ether protecting group and this was followed by a spontaneous intramolecular trans-esterification to provide 12 ^7b in 97% yield. The O-Bn protected acid 11,¹² prepared from L-valine, was treated with oxalyl chloride and a catalytic amount of DMF to produce the corresponding acid chloride, and the subsequent condensation with 12 provided the corresponding 16 in 54% yield. Reductive deprotection of the benzyl group with Pd(OH)₂ in AcOEt under a hydrogen atmosphere afforded the desired right fragment 7 in 90% yield (Scheme 2).

Scheme 2 Synthesis of the left fragment 7.

The commercially available N-Cbz L-threonine was converted into tert-Bu ester 9 ¹⁰ in 97% yield. Bis-TBS protected 10, prepared from L-isoleucine, was treated with oxalyl chloride and a catalytic amount of DMF to produce the corresponding acid chloride intermediate and the subsequent condensation with 9 provided 17 in 93% yield. The tert-Bu group of 17 was selectively removed with TESOTf-2,6-lutidine in 1,2-dichloroethane to afford the left fragment 8 in 75% yield (Scheme 3).¹⁵

Scheme 3 Synthesis of the right fragment 6.

Condensation of 7 and 8 with EDCI and DMAP provided the corresponding 18 in 67% yield (Scheme 4). In the endgame of the total synthesis of 4, the TBS group of 18 was removed with HF·Pyr in THF-Pyr to afford 19 in 95% yield. The subsequent hydrogenolysis with Pd(OH)₂ in EtOAc provided amine 6. The subsequent condensation of 6 with benzyl ether 5 ⁹ using EDCI, HOBt, and NMM in DCM provided the corresponding amide 20. Finally, the reductive removal of the benzyl ether protecting group was accomplished in the presence of a catalytic amount of Pd(OH)₂ in EtOAc under a hydrogen atmosphere to provide synthetic isoneoantimycin (4) in 31% yield from 19.¹⁶

Scheme 4 Endgame in the synthesis of 4.

The optical rotation of synthetic 4 ([α]_D +40.9, c 0.51, EtOH) was consistent with that of the natural product ([α]_D +53.8, c 0.26, EtOH).¹ A comparison of the ¹H NMR chemical shifts of synthetic 4 with those reported for the natural sample 1 showed that the chemical shifts of the two methine CH protons at C6 and C18 were misperceived in ref. 1 (Fig. 2(a) red bar). Swapping the Δδ_H of C6 and the Δδ_H of C18 resulted in a good agreement (Fig. 2(a) blue bar). The ¹³C NMR spectra of synthetic 4 was almost identical to those of the natural product (Fig. 2(b)). Two fragment ions were observed at m/z 320.1625 and 566.2268 in the HREIMS spectrum of 4, as before (Fig. S1†). However, chromatographic profiles have not been reported for the natural sample of isoneoantimycin as they not available at this moment. We could not ensure the absolute configuration of natural 1 any further.

Fig. 2 Differences between the NMR chemical shifts of natural isoneoantimycin (1) and synthetic 4. (a) Δδ_H (in ppm) = δ_H of 4 – originally reported δ_H of 1 (red bar) or corrected δ_H of 1 (blue bar). (b) Δδ_C (in ppm) = δ_C of 4 – originally reported δ_C of 1.

Conclusions

In summary, the total synthesis of isoneoantimycin with the (1S,2R,5S,6S,14R,15R,17S) configuration has been achieved. A comparison of the spectroscopic data between synthetic 4 and natural 1 verified our presumption. Further studies on the biological activities of synthetic 4 are now in progress, and the results will be reported in due course.

Experimental

General information and materials

¹H NMR and ¹³C NMR spectra were recorded on a Bruker Biospin AVANCE III HD 400 (400 MHz) or JEOL JNM-ECZ400S (400 and 100 MHz) or Bruker Avance III 600 (600 and 150 MHz) instrument. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and br = broad), coupling constant in Hz, integration. Coupling constants were determined directly from ¹H NMR and ¹³C NMR spectra. The chemical shifts are reported in δ (ppm) values relative to CHCl₃ (δ 7.26 ppm for ¹H NMR and δ 77.0 ppm for ¹³C NMR) and to Me₄Si (δ 0.00 ppm for ¹H NMR). Mass spectra were obtained on a JEOL AccuTOF LC-plus JMS-T100LP (DART, ESI) spectrometer. HREIMS data were collected using a JEOL JMS-700 mass spectrometer. Infrared absorption spectra (IR) were measured using a JASCO FT/IR-4600 Fourier transform infrared spectrometer. UV spectra were recorded on a Shimadzu UV-1900 spectrophotometer. Optical rotations were measured on a JASCO P-2200 with a path length of 0.1 dm at ambient temperature; the concentrations are reported in g dL⁻¹.

All air and moisture-sensitive reactions were carried out in an argon-flushed 2-necked flask sealed with a rubber septum, and dried solvents and reagents were introduced using a syringe. Tetrahydrofuran (THF) was freshly distilled under an argon atmosphere from sodium benzophenone ketyl. Flash column chromatography was carried out using Kanto Chemical silica gel 60N (spherical, neutral, 40–50 μm), and pre-coated Merck silica gel plates (Art5715 Kieselgel 60F₂₅₄, 0.25 mm) were used for thin-layer chromatography (TLC). TLC visualization was performed using UV (254 nm) or a charring solution (ethanoic p-anisaldehyde, ethanoic phosphomolybdic acid).

Methyl (4R,3R)-4-benzyloxy-3-hydroxy-2,2-dimethyl-5-phenylpentanoate (15). To a solution of 14 ¹³ (1.47 g, 6.1 mmol) in CH₂Cl₂ (26 mL) was added SnCl₄ (1.0 M CH₂Cl₂ solution, 7.4 mL, 7.4 mmol) dropwise at −78 °C. After being stirred for 40 min at −78 °C, commercially available ketene silyl acetal 13 (1.61 g, 9.2 mmol) was added and then the mixture was stirred for 4 h at −78 °C. The reaction mixture was allowed to warm up to room temperature and quenched with sat. aq. NaHCO₃ (40 mL). The aqueous layer was extracted with Et₂O. The combined organic layer was dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash column chromatography (5% AcOEt in n-hexane) to give ester 15 (1.743 g, 5.1 mmol, 83%) as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.10 (m, 10H), 4.55 (d, J = 10.7 Hz, 1H), 4.36 (d, J = 10.7 Hz, 1H), 3.70 (ddd, J = 9.0, 5.2, 1.3 Hz, 1H), 3.39 (d, J = 11.0 Hz, 1H), 3.32 (s, 3H), 3.26 (dd, J = 10.9, 1.3 Hz, 1H), 3.08 (dd, J = 13.1, 5.2 Hz, 1H), 3.00 (dd, J = 13.1, 9.0 Hz, 1H), 1.21 (s, 3H), 1.05 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 177.34, 138.10, 137.70, 129.51, 128.53, 128.27, 128.16, 127.75, 126.30, 79.21, 77.36, 71.75, 51.52, 44.79, 36.95, 23.79, 22.21; HRMS (ESI⁺) m/z calcd for C₂₁H₂₆NaO₄ [M + Na]⁺ 365.17288, found 365.17324. IR (KBr): 3448, 3029, 2948, 2926, 1735, 1142, 1065, 743, 699 cm⁻¹; [α]_D = +14.0 (c 1.20, CHCl₃).

(4R,5R)-5-Benzyl-4-hydroxy-3,3-dimethylhydrofran-2-one (12). To a solution of 15 (731 mg, 2.13 mmol) in 99.5% EtOH (37 mL) was added 20% Pd(OH)₂ (273 mg). The inner atmosphere was purged with H₂. After being stirred overnight at room temperature, the resulting mixture was filtered through a pad of Celite® and the filtrate was concentrated. The residue was purified by flash column chromatography (30% AcOEt in n-hexane) to give 12 (455 mg, 2.07 mmol, 97%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.39–7.21 (m, 5H), 4.70 (td, J = 7.2, 3.4 Hz, 1H), 3.97–3.85 (m, 1H), 3.20 (dd, J = 13.9, 7.1 Hz, 1H), 3.10 (dd, J = 13.9, 7.4 Hz, 1H), 1.88 (br s, 1H), 1.28 (s, 3H), 1.23 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 180.76, 136.65, 129.28, 128.67, 126.84, 81.58, 76.48, 45.86, 34.66, 22.57, 17.78; HRMS (ESI⁺) m/z calcd for C₁₃H₁₇O₃ [M + H]⁺ 221.11777, found 221.11857; IR (KBr): 3369, 3027, 2977, 2942, 1752, 1092, 1007, 717, 697 cm⁻¹; [α]_D = +63.8 (c 0.91, CHCl₃). Lit^7b [α]_D = +70.5 (c 0.12, CHCl₃).

(2R,3R)-2-Benzyl-4,4-dimethyl-5-oxotetrahydrofuran-3-yl (S)-2-benzyloxy-3-methylbutanoate (16). To a mixture of 11 ¹² (565 mg, 2.7 mmol) and DMF (2 drops) in CH₂Cl₂ (5 mL) was added oxalyl chloride (390 μL, 4.5 mmol) dropwise at 0 °C. The mixture was stirred at room temperature for 1.5 h. Then, the volatiles were removed in vacuo. To the crude carboxylic acid chloride thus obtained were added CH₂Cl₂ (1.0 mL), pyridine (370 μL, 4.5 mmol) and DMAP (30 mg, 0.25 mmol) at room temperature. After being stirred for 10 min, a solution of 12 (198 mg, 0.90 mmol) in CH₂Cl₂ (1.0 mL) was added and then stirred at room temperature for 3 h. The reaction mixture was quenched by adding brine (30 mL). The aqueous layer was extracted with AcOEt (60 mL × 3). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash column chromatography (30% AcOEt in n-hexane) to give 16 (0.200 g, 0.488 mmol, 54%) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 7.38–7.17 (m, 10H), 5.40 (d, J = 3.8 Hz, 1H), 4.80 (dt, J = 9.1, 4.0 Hz, 1H), 4.71 (d, J = 11.6 Hz, 1H), 4.40 (d, J = 11.6 Hz, 1H), 3.85 (d, J = 4.4 Hz, 1H), 2.99 (dd, J = 14.5, 9.1 Hz, 1H), 2.83 (dd, J = 14.5, 4.2 Hz, 1H), 2.23–2.11 (m, 1H), 1.34 (s, 3H), 1.21 (s, 3H), 1.04 (d, J = 6.9 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 179.24, 171.63, 137.38, 136.52, 129.22, 128.89, 128.59, 128.15, 128.10, 127.25, 82.70, 80.08, 78.26, 73.12, 44.84, 35.58, 31.62, 22.98, 19.57, 18.51, 17.01; HRMS (ESI⁺) m/z calcd for C₂₅H₃₀NaO₅ [M + Na]⁺ 433.19909, found 433.19734; IR (KBr): 2965, 1750, 1497, 1457, 1387, 1347, 1190, 1133, 1109, 1073, 1003, 733, 697, 620 cm⁻¹; [α]_D = +32.9 (c 0.54, CH₂Cl₂).

(2R,3R)-2-Benzyl-4,4-dimethyl-5-oxotetrahydrofuran-3-yl (S)-2-hydroxy-3-methylbutanoate (7). To a solution of 16 (312 mg, 0.759 mmol) in AcOEt (13 mL) was added 20% Pd(OH)₂ (83 mg). The inner atmosphere was purged with H₂. After being stirred overnight at room temperature, the resulting mixture was filtered through a pad of Celite® and the filtrate was concentrated. The residue was purified by flash column chromatography (30% AcOEt in n-hexane) to give 7 (218 mg, 0.68 mmol, 90%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.34–7.04 (m, 5H), 5.31 (d, J = 3.7 Hz, 1H), 4.76 (dt, J = 8.6, 4.2 Hz, 1H), 4.12 (dd, J = 6.4, 3.0 Hz, 1H), 2.99 (dd, J = 14.4, 8.8 Hz, 1H), 2.79 (dd, J = 14.4, 4.8 Hz, 1H), 2.49 (d, J = 6.9 Hz, 1H), 2.08 (pd, J = 6.8, 3.0 Hz, 1H), 1.35 (s, 3H), 1.21 (s, 3H), 1.11 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 179.04, 174.30, 136.09, 129.16, 128.94, 127.31, 79.98, 79.04, 75.24, 44.77, 35.37, 32.01, 22.96, 19.52, 18.41, 15.54. HRMS (ESI⁺) m/z calcd for C₁₈H₂₄NaO₅ [M + Na]⁺ 343.15214, found 343.15006.

IR (KBr): 3490, 2973, 2936, 2876, 1765, 1458, 1393, 1207, 1148, 2048, 1025, 1005, 979, 711, 624 cm⁻¹; [α]_D = +108.8 (c 1.78, CH₂Cl₂).

(2R,3S)-3-Benzyloxycarbonylamino-4-tert-butoxy-4-oxobutan-2-yl (2S,3S)-2-tert-butyldimethylsilyloxy-3-methylpentanoate (17). To a mixture of freshly prepared 10 ¹¹ (1.10 g) and DMF (35 μL, 0.45 mmol) in CH₂Cl₂ (9.0 mL) was added oxalyl chloride (0.55 mL, 6.41 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 2 h and then warmed to room temperature and stirred for an additional 1 h. Then, the volatiles were removed in vacuo. To the crude carboxylic acid chloride thus obtained were added a solution of alcohol 9 ¹⁰ (269 mg, 0.869 mmol) in CH₂Cl₂ (0.5 mL) and pyridine (3.7 mL) dropwise at room temperature. After being stirred for 14 h, the reaction mixture was diluted with THF (120 mL) and filtered through a pad of Celite® and the filtrate was concentrated in vacuo. The crude residue was dissolved in AcOEt (50 mL) and then washed with H₂O, sat. aq. NaHCO₃ and brine, dried over Na₂SO₄, and concentrated in vacuo. Purification by flash column chromatography (10% AcOEt in n-hexane) afforded ester 17 as a colorless oil (436 mg, 0.81 mmol, 93%). ¹H NMR (400 MHz, CDCl₃) δ 7.42–7.28 (m, 5H), 5.43 (td, J = 6.4, 2.3 Hz, 1H), 5.39 (d, J = 9.8 Hz, 1H), 5.21–5.07 (m, 2H), 4.37 (dd, J = 9.7, 2.3 Hz, 1H), 4.18 (d, J = 3.7 Hz, 1H), 3.99 (d, J = 4.2 Hz, 1H), 1.83–1.69 (m, 1H), 1.57–1.46 (m, 1H), 1.44 (s, 9H), 1.29 (d, J = 6.4 Hz, 3H), 1.23–1.11 (m, 1H), 0.94 (s, 9H), 0.91 (s, 3H), 0.89 (s, 9H), 0.85 (t, J = 7.4 Hz, 3H), 0.13 (d, J = 7.3 Hz, 3H), 0.10 (s, 3H), 0.02 (s, 3H), 0.00 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.29, 168.82, 156.75, 136.36, 128.68, 128.35, 128.21, 82.91, 77.36, 76.41, 71.08, 67.36, 58.28, 39.65, 28.11, 25.85, 24.00, 18.33, 17.41, 15.78, 11.76, −4.67, −5.20; HRMS (ESI⁺) m/z calcd for C₂₈H₄₇NaO₇Si [M + Na]⁺ 560.30197, found 560.29795. IR (KBr): 2960, 2933, 2858, 1734, 1517, 1461, 1255, 1143, 1065, 838, 778 cm⁻¹; [α]_D = −2.6 (c 1.06, CH₂Cl₂).

N-Benzyloxycarbonyl-O-(2S,3S)-2-tert-butyldimethylsilyloxy-3-methylpentanoyl-L-threonine (8). To a stirred solution of tert-butyl ester 17 (491 mg, 0.913 mmol) in CH₂ClCH₂Cl (9.8 mL) were added 2,6-lutidine (0.60 mL, 5.18 mmol) and TESOTf (0.65 mL, 3.02 mmol) successively at 0 °C. The reaction mixture was stirred at 45 °C for 20 h at room temperature, diluted with phosphate buffer (pH 7), acidified with 1 M aq. KHSO₄ and then extracted with AcOEt (23 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. Purification by flash column chromatography (30% to 100% AcOEt in n-hexane) afforded carboxylic acid 8 as a colorless oil (328 mg, 0.68 mmol, 75%). ¹H NMR (400 MHz, CDCl₃) δ 9.74 (br s, 1H), 7.41–7.29 (m, 5H), 5.50 (dq, J = 6.3, 2.5 Hz, 1H), 5.42 (d, J = 9.6 Hz, 1H), 5.23–5.08 (m, 2H), 4.56 (dd, J = 9.7, 2.6 Hz, 1H), 3.97 (d, J = 4.9 Hz, 1H), 1.80–1.63 (m, 1H), 1.47–1.36 (m, 1H), 1.33 (d, J = 6.4 Hz, 3H), 1.15 (m, 1H), 0.89 (s, 9H), 1.29 (d, J = 6.3 Hz, 3H), 1.25–1.10 (m, 1H), 0.94 (s, 6H), 0.89 (s, 9H), 0.87–0.82 (m, 3H), 0.00 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 179.04, 174.30, 136.09, 129.16, 128.94, 127.31, 79.98, 79.04, 77.36, 75.24, 44.77, 35.37, 32.01, 22.96, 19.52, 18.41, 15.54; HRMS (ESI⁺) m/z calcd for C₂₄H₃₉NNaO₇Si [M + Na]⁺ 504.23935, found 504.23448; IR (KBr): 2961, 2933, 1751, 1735, 1710, 1509, 1253, 1155, 1144, 1089, 1065, 839, 777 cm⁻¹; [α]_D = +4.14 (c 1.85, CH₂Cl₂).

(2R,3S)-4-(((S)-1-(((2R,3R)-2-Benzyl-4,4-dimethyl-5-oxotetrahydrofuran-3-yl)oxy)-3-methyl-1-oxobutan-2-yl)oxy)-3-benzyloxycarbonylamino-4-oxobutan-2-yl-(2S,3S)-2-tert-butyldimethylsilyloxy-3-methylpentanoate (18). To a stirred solution of 8 (228 mg, 0.47 mmol), 7 (167 mg, 0.52 mmol) and DMAP (25 mg, 0.21 mmol) in CH₂Cl₂ (2.8 mL) was added a solution of EDCI (125 mg, 0.81 mmol) in CH₂Cl₂ (2.9 mL) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 20 h. The crude residue was diluted with water (40 mL) and then extracted with CH₂Cl₂ (12 mL × 3); the combined organic layer was dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (30% AcOEt in n-hexane) to give compound 18 as a colorless oil (249 mg, 0.32 mmol, 67%); ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.21 (m, 10H), 5.51–5.43 (m, 1H), 5.38 (d, J = 9.6 Hz, 1H), 5.32–5.27 (m, 1H), 5.14 (s, 2H), 4.96 (d, J = 2.9 Hz, 1H), 4.75 (dt, J = 9.1, 3.8 Hz, 1H), 4.61 (dd, J = 9.6, 3.2 Hz, 1H), 4.06 (d, J = 3.9 Hz, 1H), 3.07 (dd, J = 14.7, 9.1 Hz, 1H), 2.88 (dd, J = 14.7, 3.9 Hz, 1H), 2.35–2.29 (m, 1H), 1.82–1.71 (m, 1H), 1.38 (d, J = 6.4 Hz, 3H), 1.30 (s, 3H), 1.18 (s, 3H), 1.10 (d, J = 6.9 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.90 (s, 9H), 0.84 (t, J = 7.4 Hz, 3H), 0.02 (s, 3H), −0.01 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.29, 168.82, 156.75, 136.36, 128.68, 128.35, 128.21, 82.91, 77.36, 76.41, 71.08, 67.36, 58.28, 39.65, 28.11, 25.85, 24.00, 18.33, 17.41, 15.78, 11.76, −4.67, −5.20; HRMS (ESI⁺) m/z calcd for C₄₂H₆₁NO₁₁Si [M + Na]⁺ 806.3912, found 806.3883; IR (KBr): 3435, 1655, 1560, 1459, 1088, 820 cm⁻¹; [α]_D = +26.9 (c 0.88, CH₂Cl₂).

(2R,3S)-4-(((S)-1-(((2R,3R)-2-Benzyl-4,4-dimethyl-5-oxotetrahydrofuran-3-yl)oxy)-3-methyl-1-oxobutan-2-yl)oxy)-3-benzyloxycarbonylamino-4-oxobutan-2-yl-(2S,3S)-2-hydroxy-3-methylpentanoate (19). Silyl ether 18 (246 mg, 0.314 mmol) was weighed into a Teflon test tube. THF (3.6 mL) and pyridine (0.62 mL) were added, and then HF-Py (0.60 mL) was added to the solution. The mixture was stirred for 17 h. The reaction was quenched with sat. aq. NaHCO₃ and the mixture was extracted with AcOEt. The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated in vacuo. Purification by flash column chromatography (30% AcOEt in n-hexane) afforded 19 as a colorless oil (201 mg, 0.30 mmol, 95%); ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.21 (m, 10H), 5.62 (qd, J = 6.3, 2.8 Hz, 1H), 5.39 (d, J = 9.6 Hz, 1H), 5.28 (d, J = 3.6 Hz, 1H), 5.14 (s, 2H), 4.97 (d, J = 2.9 Hz, 1H), 4.75 (td, J = 9.4, 3.7 Hz, 1H), 4.68 (dd, J = 9.7, 2.8 Hz, 1H), 4.03–3.97 (m, 1H), 3.04 (dd, J = 14.6, 9.3 Hz, 1H), 2.87–2.77 (m, 1H), 2.30 (pd, J = 6.7, 2.8 Hz, 1H), 1.77 (m, 1H), 1.68 (q, J = 1.5 Hz, 1H), 1.38 (d, J = 6.4 Hz, 3H), 1.30 (s, 3H), 1.17 (s, 3H), 1.10 (d, J = 6.9 Hz, 3H), 0.96 (t, J = 6.5 Hz, 6H), 0.89 (t, J = 7.5 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 179.00, 173.88, 169.43, 168.34, 156.66, 136.65, 135.98, 129.37, 128.81, 128.72, 128.51, 128.30, 127.11, 80.39, 79.20, 77.54, 74.61, 71.64, 67.69, 57.50, 44.67, 38.83, 35.35, 29.96, 23.98, 22.88, 19.53, 18.41, 16.81, 16.34, 15.43, 11.87; HRMS (ESI⁺) m/z calcd for C₃₆H₄₇NNaO₁₁ [M + Na]⁺ 692.30468, found 692.30167; IR (KBr): 3449, 3351, 2968, 2936, 1751, 1514, 1392, 1203, 1131, 1063, 735, 698 cm⁻¹; [α]_D = +43.2 (c 1.27, CH₂Cl₂).

(2R,3S)-3-Amino-4-(((S)-1-(((2R,3R)-2-benzyl-4,4-dimethyl-5-oxotetrahydrofuran-3-yl)oxy)-3-methyl-1-oxobutan-2-yl)oxy)-4-oxobutan-2-yl-(2S,3S)-2-hydroxy-3-methylpentanoate (6). To a solution of 19 (178 mg, 0.27 mmol) in AcOEt (17 mL) was added 20% Pd(OH)₂ (112 mg). The inner atmosphere was purged with H₂. After being stirred overnight at room temperature, the resulting mixture was filtered through a pad of Celite® and the filtrate was concentrated to give amine 6. This was used for the next reaction without further purification.

Isoneoantimycin (4, synthetic). To a solution of 6 (132 mg, 0.25 mmol) in CH₂Cl₂ (10 mL) were added 2-benzyloxy-3-formylaminobenzoic acid 5 ⁹(216 mg, 0.80 mmol), HOBt (109 mg, 0.81 mmol), EDCI·HCl (155 mg, 1.00 mmol), and NMM (87 μL, 0.79 mmol) successively. The reaction mixture was stirred at room temperature overnight, diluted with water, and extracted 3 times with AcOEt. The combined organic layers were washed with water and brine, dried over Na₂SO₄, and concentrated in vacuo. The resulting amide 20 was used for the next step without further purification.

To a solution of this material (401 mg) in AcOEt (15 mL) was added 20% Pd(OH)₂ (90 mg). The inner atmosphere was purged with H₂. After being stirred overnight at room temperature, the resulting mixture was filtered through a pad of Celite® and the filtrate was concentrated. The residue was purified by flash column chromatography (30% AcOEt in n-hexane) to give the white solid 4 (57.8 mg, 83 μmol, 31% for 3 steps) as a 6 : 1 mixture of rotamers; ¹H NMR (600 MHz, CDCl₃) Major isomer δ 12.54 (s, 1H), 8.54 (dd, J = 8.1, 1.3 Hz, 1H), 8.49 (d, J = 1.7 Hz, 1H), 7.91 (s, 1H), 7.34–7.24 (m, 3H), 7.22 (dd, J = 8.1, 1.3 Hz, 1H), 7.22 (dd, J = 8.1, 1.3 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.92 (t, J = 8.1 Hz, 1H), 5.68 (qd, J = 6.5, 3.0 Hz, 1H), 5.32 (d, J = 3.7 Hz, 1H), 5.14 (dd, J = 8.8, 3.1 Hz, 1H), 5.02 (d, J = 2.9 Hz, 1H), 4.77 (dt, J = 9.5, 3.5 Hz, 1H), 4.10 (d, J = 4.0 Hz, 1H), 3.02 (dd, J = 14.7, 9.5 Hz, 1H), 2.83 (dd, J = 14.6, 3.5 Hz, 1H), 2.37–2.31 (m, 1H), 1.84–1.77 (m, 1H), 1.42 (d, J = 6.4 Hz, 3H), 1.43–1.37 (m, 1H), 1.32 (s, 3H), 1.31–1.24 (m, 1H), 1.19 (s, 3H), 1.13 (d, J = 6.9 Hz, 3H), 1.00 (d, J = 6.9 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.91 (d, J = 7.4 Hz, 1H); minor isomer (diagnostic peaks only) δ 12.40 (s, 1H), 8.77 (d, J = 11.5 Hz, 1H), 7.78 (d, J = 11.4 Hz, 1H), 7.37 (d, J = 7.9 Hz, 1H), 1.51 (d, J = 6.4 Hz, 1H); ¹³C NMR (151 MHz, CDCl₃) Major isomer δ 178.95, 173.96, 170.29, 169.03, 168.31, 159.10, 150.71, 136.67, 129.36, 128.86, 127.58, 127.20, 125.02, 120.20, 119.26, 112.90, 80.34, 79.45, 77.87, 74.59, 71.86, 55.44, 44.71, 39.13, 35.45, 30.01, 24.40, 22.92, 19.56, 18.43, 17.10, 16.36, 15.29, 11.97; minor isomer (diagnostic peaks only) δ 161.07, 151.43, 129.32, 127.30, 120.97, 120.54; HRMS (ESI⁺) m/z calcd for C₃₆H₄₆N₂NaO₁₂ [M + Na]⁺ 721.29484, found 721.29106; UV λ^EtOH_max nm (ε) 346 (3842), 182 (16 384); IR (KBr): 3369, 2968, 2935, 2877, 1751, 1686, 1534, 1364, 1251, 1200, 1132, 1067 cm⁻¹; [α]²⁵_D = +42.0 (c 0.19, EtOH).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by a Grant-in-Aid for Scientific Research (C) (No. 22K06666). We are grateful to Dr Matsumi Doe, Analytical Division, Osaka Metropolitan University, for the 2D NMR measurements.

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Footnote

† Electronic supplementary information (ESI) available: Comparisons of NMR data of natural and synthetic compounds (Table S1), HREIMS fragmentation (Fig. S1), experimental details for known compounds and NMR spectra for new compounds. See DOI: https://doi.org/10.1039/d3ob00099k

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