Synthesis of hupehenols A, B, and E from protopanaxadiol

Abstract

Hupehenols A–E (3–7) are bioactive octanordammarane triterpenoids, among which hupehenols B (4) and E (7) are the most potent and selective human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors (IC₅₀ = 15 and 34 nM, respectively). Herein, the hupehenols were synthesized from protopanaxadiol, the main component of Panax notoginseng. The different synthetic attempts resulted in the stereoselective synthesis of hupehenols A, B, and E for further biological exploration. Notable features of the synthesis included a regioselective epoxide-opening reaction, regioselective acetylation, and a late-stage stereoselective oxa-Michael addition. Semi-synthetic derivatization of these natural products led to the determination of their absolute configurations, a better understanding of their inherent reactivity patterns, and the required C-17 configuration for murine 11β-HSD1 inhibition. These studies provide the basis for the synthesis of 11β-HSD1 inhibitors as potential targets for the treatment of type 2 diabetes.

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Introduction

Type 2 diabetes has become a serious global health burden. An estimated 171 million people worldwide suffer from diabetes, and this number is expected to increase to 366 million by 2030.^1,2 Moreover, the morbidity and mortality associated with secondary complications of diabetes, such as cardiovascular diseases, kidney failure, and retinopathy, have similarly increased. Thus, an urgent need exists for new approaches to prevent and treat diabetes and its related complications.²

11β-Hydroxysteroid dehydrogenases (11β-HSD) are pre-receptor enzymes that regulate the intracellular availability of active glucocorticoids to bind and activate the glucocorticoid receptor. Cortisol is an active glucocorticoid in humans that is predominantly expressed in the liver, adipose, and brain. Excess glucocorticoid, as observed in cases of Cushing's syndrome, leads to preferentially increased fat accumulation in the visceral depot, hepatic triacylglycerol accumulation, insulin resistance, and hyperglycemia.³ 11β-HSD1 and 11β-HSD2 are two isozymes of 11β-HSD. In tissues and intact cells, 11β-HSD1 is predominantly an oxo-reductase that converts inert cortisone into active cortisol in humans or 11-dehydrocorticosterone into corticosterone in rodents. 11β-HSD2 is expressed in only the kidneys and acts as a dehydrogenase.⁴ Various investigations have suggested that 11β-HSD1 could be a drug target for the treatment of type 2 diabetes,^5–9 which has led to the development of many well-known 11β-HSD1 inhibitors.^3,4

Glycyrrhetinic acid (1, Fig. 1) was the first triterpenoid-type 11β-HSD1 inhibitor, although carbenoxolone (2, Fig. 1) is a more potent derivative of glycyrrhetinic acid.¹⁰ Inspired by natural products, new 11β-HSD1 inhibitors have been reported by our research group.¹¹ Hupehenols A–E (3–7) are recently published natural 11β-HSD1 inhibitors with nanomolar IC₅₀ values and excellent selectivity toward 11β-HSD1.¹² However, these compounds exist in plants in trace quantities, limiting their biological exploration and medicinal development. Thus, a unified approach to the synthesis of hupehenols A–E is required. Corey et al. reported a polyene cyclization approach to dammarane triterpenoid without C-12 functionality in 17 steps from farnesol.¹³ Up to now, synthetic research of dammarane triterpenoid with both C-3 and C-12 functionalities is still limited. In this study, the first successful synthesis of nordammarane triterpenoid hupehenols A, B, and E from protopanaxadiol (PPD) through different synthetic approaches and the determination of their absolute configurations are reported.

Fig. 1 Natural 11β-HSD1 inhibitors.

Results and discussion

Chemistry

Hupehenols A–E (3–7) are rare types of 20,21,22,23,24,25,26,27-octanordammarane triterpenoids,^12,14 and their critical stereogenic centers are incorporated into the dammarane skeleton. PPD is a main component of Panax notoginseng (Burk.) F. H. Chen, which has been highly industrialized in China. Therefore, the structure and availability of PPD has allowed its use as a starting material for the synthesis of hupehenols A–E, and the synthesis plan is shown in Scheme 1. Hupehenols A–C (3–5) could be obtained from hupehenol E (7) through an oxa-Michael addition, and 7 could be synthesized via two routes. In route one, 7 could be prepared from olefin 8 through a Riley oxidation. In route two, 7 could be derived from enone 9 through an ene-one transfer. Olefin 8 could be prepared by palladium-catalyzed reduction of the trifluoromethanesulfonate obtained from ketone 11, and enone 9 could be synthesized directly from ketone 11. Additionally, 11 could be formed from 10 through reduction, elimination, and subsequent ozonolysis. Methyl ketone 10 was prepared from PPD based upon a previously reported procedure.¹⁵

Scheme 1 Synthetic plan for hupehenols A–E.

Natural PPD always exists in saponin form, and the hydrolysis of saponins is necessary to accumulate PPD. Oxidative cleavage of the glycosidic bonds would avoid cyclization of the C-20 hydroxy group with C-25,¹⁶ and PPD (12) was thus prepared using an improved oxidative alkaline hydrolysis process to perform the hydrolysis on a large scale (see Experimental section). To avoid cyclization of the side chain of 12, the double bond of the side chain was initially hydrogenated using Raney Ni to afford 13 in quantitative yield (Scheme 2). After protecting the C-3 and C-12 secondary hydroxy groups of 13 as diacetate (14a), dimethoxymethyl (MOM) ether (14b), C-3 OTBS and C-12 OPiv ester¹⁷ (14c), the elimination conditions were examined to prepare the Δ^(17,20) olefin from these compounds (Table S1, ESI†). However, these conditions (pTSA, SOCl₂, POCl₃, MsCl, Burgess' reagent,¹⁸ BF₃·Et₂O, and PPh₃/diethyl azodicarboxylate (DEAD); entries 1–8, Table S1, ESI†) failed to give the target products and instead yielded a mixture of Δ^(20,21) and Δ^(20,22) olefins 15a based upon the ozonolysis products 16a and 10a (Scheme 2). Therefore, isomerization of the Δ^(20,21) and Δ^(20,22) olefins 15a to the Δ^(17,20) olefin was attempted using catalytic RuCl₃ in boiling ethanol.¹⁹ The results were also unexpected, as most of the Δ^(20,22) olefin was converted into the thermodynamically less stable Δ^(20,21) olefin based upon the ozonolysis product. Finally, the conditions listed in entry 3 were applied to obtain 15b (entry 9, Table S1, ESI†) and 15c (entry 10, Table S1, ESI†), and subsequent ozonolysis of the Δ^(20,21) and Δ^(20,22) olefins 15 yielded ketones 16 and ketones 10.

Scheme 2 Synthesis of ketone 11^a. ^aReagents and conditions: (a) Raney Ni, H₂ (atmospheric pressure), MeOH, rt, quant.; (b) Ac₂O, triethylamine (TEA), 4-dimethylaminopyridine (DMAP), DCM, 0 °C-rt, 94%; (c) MOMCl, N,N-diisopropylethylamine (DIPEA), DCM, 0 °C-rt, 90%; (d) pivaloyl chloride (PivCl), TEA, DCM, −20 °C; (e) t-butyldimethylsilyl chloride (TBSCl), imidazole, DMAP, DCM, reflux, 82% in 2 steps; (f) O₃, DCM/MeOH, −78 °C, 80–85% over 2 steps, 16 : 10 = 1 : 3–1 : 4.7; (g) NaBH₄, THF, H₂O (10 eq.), 0 °C, 0.5 h for 10a and 16a; NaBH₄, MeOH, 0 °C, 2 h for 10b–c and 16b–c; (h) Burgess' reagent, toluene, reflux, 2 h; (i) O₃, DCM/MeOH, −78 °C, 56, 8, and 39% from 10a, 10b, and 10c, respectively, 45, 3, and 30% from 16a, 16b, and 16c, respectively, over 3 steps.

For the synthesis of ketone 11, a series of Baeyer–Villiger (BV) oxidation conditions, including 3-chloroperoxybenzoic acid (mCPBA), urea hydrogen peroxide (UHP), UHP/trifluoroacetic anhydride (TFAA),^20,21 CH₃CO₃H, acid-mediated BV reactions,^22,23 base-mediated BV reactions,^24,25 H₂O₂,²⁶ and microwaving, were utilized to introduce an oxygen functionality at the C-17 position of methyl ketone 10a.²⁷ The failure to achieve this was due to the steric bulk of C-12 and the low reactivity of the carbonyl group.²⁸ Finally, ketone 11 was synthesized from both methyl ketone 10 and ketone 16 in three scalable steps. Specifically, reduction of 10 by NaBH₄ yielded the corresponding alcohols, which were dehydrated with the Burgess reagent and subsequently ozonolyzed to afford 11. However, a low yield (<10%) was obtained when 10b and 16b were used as substrates in similar procedures (Scheme 2).

Focusing on the synthesis of olefin 8, compound 11c was deprotonated by lithium bis(trimethylsilyl)amide (LiHMDS) to form an enolate anion that was subsequently trapped by PhNTf₂ to afford trifluoromethanesulfonate 17 in 96% yield (Scheme 3). The 3D structure of the tetracyclic skeleton was determined by single-crystal X-ray diffraction analysis of 17. Compound 17 was susceptible to Pd-catalyzed reduction using HCO₂NH₄/Pd(OAc)₂/PPh₃, resulting in the formation of olefin 8 in 90% yield.²⁹

Scheme 3 Synthesis of olefin 8^a. ^aReagents and conditions: (a) LiHMDS, PhNTf₂, THF, −78 °C-rt, overnight, 96%; (b) HCO₂NH₄, Pd(OAc)₂, PPh₃, DMF, 50 °C, 1 h, 90%.

Next, the allylic oxidation of olefin 8 was studied. Several reported methods^30–47 failed to affect the allylic oxidations in good yield (Table S2, ESI†). Although allylic bromide may be converted into an enone in the presence of AgBF₄/DMSO,⁴⁸ the allylic bromide of 8 could not be formed using the classic conditions of N-bromosuccinimide (NBS)/benzoyl peroxide (BPO). Instead, conjugate diene 19 was detected as the single product.

Plausible mechanisms to explain the allylic oxidation products of olefin 8 are depicted in Scheme 4. The C-15 β-hydrogen atom of 8 was most likely preferably attacked by SeO₂ on its β-face to form the allylseleninic acid intermediate A as a favored product or alternatively the C-15 α-hydrogen atom was attacked by SeO₂ on its α-face to form allylseleninic acid intermediate B as a disfavored product due to its bulky, axial C-8 and C-14 methyl groups. Regarding A, a [2,3]-sigmatropic shift should proceed spontaneously to form a selenite ester via an envelope-like transition state,⁴³ and the selenite ester would be further converted to 18. Instead, the [2,3]-sigmatropic shift did not proceed as expected. A β-elimination occurred because of the rigid allylseleninic acid fragment of the five-membered ring, resulting in conjugate diene 19. Regarding B, neither [2,3]-sigmatropic shift nor β-elimination proceeded to form 18 or 19. Moreover, according to the literature,^34,36 when olefin 8 was treated with TBHP in the presence of transition metal, allylic radicals C and D likely formed. Then, C and D interconverted into allylic tert-butylperoxy ether intermediates E and F, ultimately resulting in enones 20 and 9c. Allylic tert-butyl oxide 21 was detected as a major product only under the condition of SeO₂/TBHP (entry 4, Table S3, ESI†), indicating that 21 might be resulted from the coupling reaction of the tert-butyl oxide radical³⁵ with intermediate C.

Scheme 4 Plausible mechanism of allylic oxidation.

After failing to achieve allylic oxidation of olefin 8, the focus was shifted to preparing enones 6 and 7 via the ene-one transfer of enone 9. Treating ketone 11 (11a, and 11c) with IBX⁴⁹ was unsuccessful, and the Saegusa oxidation did not proceed when the trimethylsilyl enol ether of ketone 11 (11a, and 11c) was heated with Pd(OAc)₂ in MeCN or DMSO.^50,51 Enone 9c was obtained in 78% yield by oxidative elimination of the α-phenylselenide of 11c. However, the enone 9a was synthesized from ketone 11a in low yield (24%) (Scheme 5).⁵² Introduction of a 15,16-epoxide onto 9c for the synthesis of 28 via a Wharton transposition⁵³ failed. The di-epoxide 27 was obtained instead under the base-mediated reaction with H₂O₂ or TBHP.⁵⁴ Stereoselective Luche reduction of 9c afforded the allylic alcohol, which upon treatment with mCPBA afforded the epoxide alcohol as a single product. Methylation of the 17-hydroxy group with NaH/MeI and the proton sponge/Me₃OBF₄ (ref. 55) led to decomposition of the substrate. Treatment under milder conditions using Ag₂O/MeI gave the C-17 methyl ether as a single product. Formation of the C-15 ketone involved selective opening of the 15,l6-epoxide at the C-16 position using hydride. Among the tested conditions (NaBH₄/BF₃·OEt₂, Et₃SiH/BF₃·OEt₂, Red-Al, and LiAlH₄), the use of LiAlH₄ in boiling 1,4-dioxane afforded the target diol 25 as a single product in 66% yield over 4 steps. The regioselective acetylation of diol 25 with AcCl/pyridine provided a >10 : 1 mixture of 26b and 26a in 90% yield. The absolute configuration of 26a was unambiguously established by single-crystal X-ray diffraction.

Scheme 5 Synthesis of C-15 hydroxy compound. ^aReagents and conditions: (a) LiHMDS, PhSeCl, THF, −78 °C-rt, 12 h, then NaIO₄, THF/H₂O, rt, overnight, 78% for 9c and 24% for 9a; (b) NaBH₄, CeCl₃·7H₂O, MeOH, 0 °C, 2 h; (c) 3-chloroperoxybenzoic acid (mCPBA), DCM, 0 °C-rt, 4 h; (d) Ag₂O, MeI, 60 °C, 48 h; (e) LiAlH₄, dioxane, reflux, 4 h, 66% over 4 steps; (f) AcCl, pyridine, 0 °C, overnight, 90%; (g) Triton B, 70% TBHP, MeOH/THF (4 : 1), 0 °C-rt, 12 h, 78%.

Oxidation of 26b with DMP quantitatively resulted in 29 (Scheme 6). Treating 29 with catalytic pTSA in boiling toluene resulted in the natural product hupehenol E (7) in 90% yield. Oxa-Michael addition of methanol to 7 was conducted in the presence of concentrated HCl, producing hupehenol B (4) and its C-17 isomer 40 in a ratio of 5 : 1 and in 87% yield. Treating 7 with K₂CO₃ in warm (40 °C) methanol resulted in hupehenol A (3) and its C-17 isomer 31 in a ratio of 4 : 1 and in 92% yield. Moreover, 3 was obtained in 50% yield (97% based on recovery of starting material) by heating 31 with K₂CO₃ at 60 °C in methanol through equilibrium of the retro-Michael and Michael addition of 31. This phenomenon was also observed when 3 was heated with K₂CO₃ at 60 °C in methanol. All of the analytical data for the synthetic hupehenols A, B, and E were identical to those from the natural source.

Scheme 6 Syntheses of hupehenols A, B, and E^a. ^aReagents and conditions: (a) Dess–Martin periodinane (DMP), DCM, 0 °C, 2 h, quant.; (b) pTSA, benzene, reflux, 2 h, 90%; (c) conc. HCl, MeOH, 50 °C, 3 h, 87%; (d) K₂CO₃, MeOH, 40 °C, 8 h, 92%; (e) K₂CO₃, MeOH, 60 °C, 24 h, 50%, 97% based on the recovery of starting material.

After completing the synthesis of hupehenols A, B, and E, most of the synthetic compounds were subjected to inhibition tests of both human and mouse 11β-HSD1 (Table S3, ESI†). All tested compounds with 11β-HSD1 inhibitory activities showed no cytotoxicity except compounds 6 and 7 which exhibited weak cytotoxicity (data not showed). Surprisingly, compound 30, the C-17 epimer of hupehenol B, retained its inhibitory activity toward human 11β-HSD1 (IC₅₀ = 44 nM), and, more importantly, the inhibitory activity toward mouse 11β-HSD1 was increased by almost 10-fold (IC₅₀ = 0.41 μM) compared with that of hupehenol B. Albeit the huge difference between human and murine 11β-HSD1 inhibition of hupehenols may be resulted from the specificity of hupehenols to human 11β-HSD1. Moreover, a primary structure–activity relationship of hupehenols could be summarized: C-15 carbonyl group plays an important role in inhibitory activity against human 11β-HSD1; a β-type methoxyl group at C-17 position is favourable to murine 11β-HSD1 inhibition.

Conclusions

In summary, a synthetic route was developed to obtain the bioactive octanordammarane triterpenoids hupehenols A, B, and E from PPD. A regioselective epoxide-opening reaction, regioselective acetylation, and late-stage stereoselective oxa-Michael addition were used in the synthesis. 17-epi Hupehenol B (30) was more potent than hupehenol B in its inhibitory function toward mouse 11β-HSD1. The synthetic route established here will accelerate structure–activity relationship studies for the identification of effective 11β-HSD inhibitors of tetracyclic octanordammarane triterpenoid scaffolds for potential use in bioassays in vivo.

Experimental section

General experimental procedures

Melting points were obtained on a WRX-4 apparatus and were uncorrected. Optical rotations were measured with a JASCO P-1020 polarimeter. All NMR spectra were recorded with a Bruker AVANCE III 400 MHz, 500 MHz, AVANCE III 600 MHz, and 800 MHz (¹H NMR) spectrometer, and 100 MHz, 125 MHz, 150 MHz, and 200 MHz (¹³C NMR) in CDCl₃: chemical shifts (δ) are given in ppm, coupling constants (J) in Hz, the solvent signals were used as references (CDCl₃: δ_C = 77.2 ppm; residual CHCl₃ in CDCl₃: δ_H = 7.26 ppm). High-resolution mass spectra were recorded on an Agilent 6540 Q-Tof (ESIMS) or Waters AutoSpec Premier P776 spectrometer (EIMS). All reactions were carried out under an atmosphere of argon and dry conditions, and were monitored by analytical thin-layer chromatography (TLC), which was visualized by ultraviolet light (254 nm). All solvents were obtained from commercial sources and were purified according to standard procedures.

(20/R)-Protopanaxadiol (12). 12 was obtained from the hydrolysis of Panax notoginseng saponins described in the literature¹⁵ with our improvement. In detail, we used air as oxidant, increased the reaction temperature to 120 °C, prolonged the reaction time to 72 h, adjusted the base concentration to 1.2 mol L⁻¹, and the final reaction concentration was increased to 72 g L⁻¹. With this method, Panax notoginseng saponins could be hydrolyzed in 200 g scale, and up to 10% of 12 was obtained after flash chromatography on silica gel (petroleum ether/EtOAc 1 : 1 v/v). Colorless needles; mp 245–247 °C; ¹H NMR (400 MHz, CDCl₃) δ 5.16 (1H, t, J = 7.0 Hz, H-24), 3.59 (1H, td, J = 10.4, 5.2 Hz, H-12), 3.20 (1H, dd, J = 11.6, 4.7 Hz, H-3), 1.69 (3H, s, H-26), 1.63 (3H, s, H-27), 1.19 (3H, s, H-21), 0.98 (3H, s, H-19), 0.97 (3H, s, H-28), 0.88 (3H, s, H-18), 0.87 (3H, s, H-30), 0.78 (3H, s, H-29), 0.72 (1H, brd, J = 10.8 Hz, H-5); ¹³C NMR (100 MHz, CDCl₃) δ 132.2 (C-25), 125.0 (C-24), 79.0 (C-3), 74.8 (C-20), 71.1 (C-12), 55.9 (C-5), 53.5 (C-17), 51.8 (C-14), 50.2 (C-9), 48.0 (C-13), 39.9 (C-4), 39.1 (C-1), 37.2 (C-8), 34.9 (C-7, C-10), 34.5 (C-22), 31.3 (C-15), 31.1 (C-11), 28.2 (C-28), 27.5 (C-2), 27.2 (C-21), 26.6 (C-16), 25.9 (C-26), 22.5 (C-23), 18.4 (C-6), 17.9 (C-27), 17.0 (C-30), 16.3 (C-18), 15.8 (C-19), 15.5 (C-29); positive HRESIMS [M + Na]⁺ m/z 483.3827 (calcd for C₃₀H₅₂O₃Na, 483.3809).

Diol 13. To a solution of 12 (40.0 g, 86.8 mmol) in MeOH (500 mL) was added 80 g of wet Raney Ni. The reaction was connected to a balloon of hydrogen, then degassed three times, and the resulting mixture was allowed to stir at RT overnight. The mixture was passed through a pad of Celite to remove the Raney Ni, the filter cake was washed with MeOH (3 × 200 mL), the filtrate was evaporated under vacuum to give pure 13 (40.1 g, 86.8 mmol) in 100% yield. White powder; mp 247–248 °C; [α]¹⁸_D +16 (c 3.1, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 3.57 (1H, td, J = 10.3, 5.2 Hz, H-12), 3.19 (dd, J = 11.2, 4.9 Hz, 1H), 2.02 (1H, m, H-17), 1.16 (3H, s, H-21), 0.97 (3H, s, H-19), 0.96 (3H, s, H-28), 0.88 (3H, s, H-18), 0.87 (6H, s, H-26, H-27), 0.77 (3H, s, H-29), 0.71 (1H, brd, J = 10.4 Hz, H-5); ¹³C NMR (100 MHz, CDCl₃) δ 79.0 (C-3), 74.3 (C-20), 71.0 (C-12), 56.0 (C-5), 53.6 (C-17), 51.7 (C-14), 50.2 (C-9), 47.8 (C-13), 39.9 (C-8), 39.8 (C-24), 39.1 (C-1, C-4), 37.2 (C-10), 35.3 (C-22), 34.9 (C-7), 31.4 (C-11), 31.1 (C-15), 28.3 (C-25), 28.2 (C-28), 27.5 (C-2), 27.1 (C-21), 26.7 (C-16), 22.9 (C-26), 22.8 (C-27), 21.4 (C-23), 18.4 (C-6), 17.0 (C-30), 16.3 (C-18), 15.8 (C-19), 15.5 (C-29); positive HRESIMS [M + Na]⁺ m/z 485.3969 (calcd for C₃₀H₅₄O₃Na, 485.3965).

C3,12-Diacetate 14a and Di-MOM ether 14b. To a stirred solution of 13 (10.0 g, 21.6 mmol) in TEA (18.0 mL, 129.6 mmol) or DIPEA (15.0 mL, 86.4 mmol) in DCM (200 mL), Ac₂O (8.2 mL, 86.4 mmol), and DMAP (263.8 mg, 2.16 mmol) or MOMCl (4.9 mL, 64.8 mmol) in DCM (100 mL) was added dropwise by a constant pressure funnel at 0 °C. After completion of the addition, the resulting mixture was allowed to stir at RT overnight. The reaction was quenched (saturated aqueous NaCl) at 0 °C, then the mixture was extracted with DCM (3 × 200 mL), and dried over Na₂SO₄ (s). The solvent was evaporated and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 10 : 1 v/v) to afford 14a (11.0 g, 20.3 mmol) in 94% yield; 14b (10.6 g, 19.4 mmol) in 90% yield.

14a: white foam; [α]¹⁸_D −1 (c 1.1, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.73 (1H, td, J = 10.3, 4.8 Hz, H-12), 4.48 (1H, dd, J = 11.3, 4.5 Hz, H-3), 2.04 (3H, s, C-12 OAc), 2.03 (3H, s, C-3 OAc), 1.11 (3H, s, H-21), 1.01 (3H, s, H-19), 0.95 (3H, s, H-30), 0.90 (3H, s, H-26), 0.89 (3H, s, H-28), 0.85 (6H, s, H-27, H-29); ¹³C NMR (100 MHz, CDCl₃) δ 171.0 (C-3 OAc), 169.8 (C-12 OAc), 80.7 (C-3), 76.7 (C-12), 73.9 (C-20), 56.0 (C-5), 52.9 (C-14), 52.8 (C-17), 50.1 (C-9), 45.0 (C-13), 40.2 (C-4), 39.9 (C-24), 38.7 (C-1), 38.0 (C-8), 37.2 (C-10), 36.6 (C-22), 34.6 (C-7), 31.7 (C-15), 28.4 (C-25), 28.3 (C-11), 28.1 (C-28), 27.4 (C-16), 26.5 (C-21), 23.7 (C-2), 22.9 (C-26), 22.8 (C-27), 21.7 (C-23), 21.5 (C-12 OAc), 21.4(C-3 OAc), 18.3 (C-6), 17.5 (C-30), 16.6 (C-29), 16.4 (C-18), 15.7 (C-19); positive HRESIMS [M + Na]⁺ m/z 569.4176 (calcd for C₃₄H₅₈O₅Na, 569.4176).

14b: white foam; [α]¹⁸_D −1 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.05 (1H, s, C-20-OH), 4.83 (1H, d, J = 6.9 Hz, C-12 OMOM), 4.74 (1H, d, J = 6.8 Hz, C-3 OMOM), 4.61 (2H, t, J = 7.2 Hz, C-3, C-12 OMOM), 3.56 (1H, td, J = 10.5, 4.6 Hz, H-12), 3.40 (3H, s, C-12 OMOM), 3.38 (3H, s, C-3 OMOM), 3.07 (1H, dd, J = 11.7, 4.1 Hz, H-3), 1.10 (3H, s, H-21), 0.99 (3H, s, H-19), 0.96 (3H, s, H-28), 0.89 (9H, s, H-26, H-27, H-30), 0.87 (3H, s, H-18), 0.81 (3H, s, H-29), 0.74 (1H, brd, J = 9.1 Hz, H-5); ¹³C NMR (100 MHz, CDCl₃) δ 96.2 (C-3 OMOM), 94.3 (C-12 OMOM), 85.1 (C-3), 77.8 (C-12), 73.0 (C-20), 56.5 (C-5), 55.7 (C-12 OMOM), 55.6 (C-3 OMOM), 53.5 (C-17), 52.3 (C-14), 50.3 (C-9), 46.3 (C-13), 40.3 (C-8), 39.9 (C-24), 39.1 (C-4), 38.9 (C-1), 37.3 (C-10), 36.4 (C-22), 34.9 (C-7), 31.7 (C-15), 28.4 (C-25), 28.2 (C-28), 27.9 (C-11), 27.3 (C-16), 26.6 (C-21), 24.3 (C-2), 22.9 (C-26, C-27), 21.6 (C-23), 18.4 (C-6), 17.4 (C-30), 16.4 (C-18, C-29), 15.7 (C-19); positive HRESIMS [M + Na]⁺ m/z 573.4491 (calcd for C₃₄H₆₂O₅Na, 573.4489).

C3-TBS ether, C12-Piv ester 14c. To a stirred solution of 13 (10.0 g, 21.6 mmol) and TEA (18.0 mL, 129.6 mmol) in DCM (200 mL), PivCl (8.0 mL, 64.8 mmol) was added dropwise at −20 °C. After completion of the addition, the resulting mixture was stirred at −20 °C overnight. The reaction was quenched (saturated aqueous NaCl) at 0 °C, then mixture was extracted with DCM (3 × 200 mL), and dried over Na₂SO₄ (s). The solvent was evaporated to afford crude C12-Piv ester which was used without purification. The crude C12-Piv ester was dissolved in DCM (200 mL), to this solution was added imidazole (8.8 g, 129.6 mmol), DMAP (2.6 g, 21.6 mmol), and TBSCl (13.0 g, 86.4 mmol), then the resulting mixture was refluxed for 8 h. The reaction was quenched (saturated aqueous NaCl), extracted with DCM (3 × 200 mL), and dried over Na₂SO₄ (s). The solvent was evaporated and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 15 : 1 v/v) to afford 14c (11.7 g, 17.7 mmol) in 82% yield over 2 steps: slightly yellow foam; [α]¹⁸_D −0.4 (c 6.5, CHCl₃) ¹H NMR (400 MHz, CDCl₃) δ 4.80 (1H, td, J = 10.1, 4.7 Hz, H-12), 3.16 (1H, dd, J = 10.8, 4.6 Hz, H-3), 1.19 (9H, s, OPiv), 1.09 (3H, s, H-21), 1.00 (3H, s, H-19), 0.95 (3H, s, H-30), 0.89 (12H, s, H-26, H-27, H-28), 0.87 (12H, s, OTBS), 0.84 (3H, s, H-18), 0.73 (3H, s, H-29), 0.02 (6H, s, OTBS); ¹³C NMR (100 MHz, CDCl₃) δ 177.5 (OPiv), 79.3 (C-3), 77.0 (C-12), 73.4 (C-20), 55.9 (C-5), 53.4 (C-17), 52.9 (C-14), 49.9 (C-9), 44.5 (C-13), 40.1 (C-8), 39.7 (C-24), 39.5 (OPiv), 39.2 (C-4), 38.8 (C-1), 37.0 (C-10), 36.2 (C-22), 34.7 (C-7), 31.1 (C-15), 28.4 (C-25), 28.2 (C-28), 27.8 (C-2), 27.6 (C-11), 27.2 (OPiv), 26.9 (C-16), 26.2 (OTBS), 25.9 (C-21), 22.8 (C-26), 22.6 (C-27), 21.3 (C-23), 18.4 (OTBS), 18.1 (C-6), 17.3 (C-30), 16.2 (C-18), 15.8 (C-29), 15.6 (C-19), −3.8 (OTBS), −4.9 (OTBS); positive HRESIMS [M + NH₄]⁺ m/z 678.5863 (calcd for C₄₁H₈₀O₄NSi, 678.5851).

General method for the preparation of methyl ketone (10a, 10b, 10c). To a solution of C3,C12-diprotected substrate 14a, 14b, 14c (80 mmol) in pyridine (300 mL), POCl₃ (320 mmol) in pyridine (100 mL) was added dropwise at 0 °C by a constant pressure funnel. After completion of the addition, the mixture was allowed to warm to 40 °C and stirred for another 12 h. The reaction was quenched (saturated aqueous NaCl) at 0 °C, then the mixture was extracted with EtOAc (3 × 200 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum to give a dark oily product which was passed through a short pad of silica gel with petroleum ether/EtOAc (20 : 1, v/v) as eluent to afford the crude olefin as a colorless oil. The crude olefin was dissolved in DCM/MeOH (250 mL, 5 : 1, v/v) and stirred at −78 °C for 30 min, then ozone was bubbled through the solution until a pale blue color persisted. Nitrogen was bubbled for 5 min, methyl sulfide (260 mmol) was added, and the mixture was allowed to reach RT and stirred overnight. The solvent was evaporated and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 20 : 1–10 : 1 v/v) to afford methyl ketone 10a, 10b, and 10c along with ketone 16a, 16b, and 16c.

10a (70% yield): colorless flakes; mp 266–268 °C; [α]¹⁸_D +21 (c 0.23, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.77 (1H, td, J = 11.0, 5.2 Hz, H-12), 4.47 (1H, dd, J = 11.3, 4.7 Hz, H-3), 2.87 (1H, td, J = 10.9, 6.1 Hz, H-17), 2.32 (1H, t, J = 11.0 Hz, H-13), 2.12 (3H, s, H-21), 2.04 (3H, s, C-12 OAc), 1.92 (3H, s, C-3 OAc), 1.06 (3H, s, H-30), 0.93 (3H, s, H-19), 0.89 (3H, s, H-18), 0.84 (6H, s, H-28, H-29); ¹³C NMR (100 MHz, CDCl₃) δ 211.8 (C-20), 170.9 (C-3 OAc), 170.8 (C-12 OAc), 80.5 (C-5), 74.6 (C-12), 55.8 (C-5), 51.9 (C-17), 51.5 (C-14), 50.2 (C-9), 48.9 (C-13), 39.9 (C-8), 38.6 (C-1), 37.8 (C-4), 37.1 (C-10), 34.9 (C-15), 32.2 (C-7), 29.5 (C-21), 27.9 (C-28), 27.8 (C-11), 27.3 (C-16), 23.5 (C-2), 21.3 (C-3 OAc), 21.1 (C-12 OAc), 18.0 (C-6), 16.7 (C-19), 16.5 (C-18), 16.3 (C-29), 15.6 (C-30); positive HRESIMS [M + Na]⁺ m/z 483.3078 (calcd for C₂₈H₄₄O₅Na, 483.3081).

16a (15% yield): white foam; [α]¹⁸_D +37 (c 0.45, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.76 (1H, td, J = 10.9, 5.1 Hz, H-12), 4.47 (1H, dd, J = 11.3, 4.6 Hz, H-3), 2.84 (1H, td, J = 10.8, 5.9 Hz, H-17), 2.04 (3H, s, C-12 OAc), 1.90 (3H, s, C-3 OAc), 1.06 (3H, s, H-30), 0.93 (3H, s, H-19), 0.88 (3H, s, H-18), 0.87 (3H, s, H-26), 0.86 (3H, s, H-27), 0.85 (3H, s, H-28), 0.84 (3H, s, H-29); ¹³C NMR (100 MHz, CDCl₃) δ 214.0 (C-20), 171.1 (C-3 OAc), 170.9 (C-12 OAc), 80.7 (C-3), 74.9 (C-12), 56.1 (C-5), 51.7 (C-14), 51.1 (C-17), 50.4 (C-9), 48.9 (C-13), 43.4 (C-22), 40.1 (C-8), 38.8 (C-1, C-24), 38.0 (C-4), 37.3 (C-10), 35.1 (C-15), 32.5 (C-7), 28.1 (C-11, C-28), 27.9 (C-16), 23.7 (C-2), 22.7 (C-26), 22.6 (C-27), 21.6 (C-23), 21.4 (C-3 OAc), 21.3 (C-12 OAc), 18.2 (C-6), 16.9 (C-19), 16.6 (C-18), 16.5 (C-29), 15.8 (C-30); positive HRESIMS [M + Na]⁺ m/z 553.3863 (calcd for C₃₃H₅₄O₅Na, 553.3863).

10b (60% yield): colorless needles; mp 119–121 °C; [α]¹⁸_D +12 (c 1.67, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.73 (1H, d, J = 6.8 Hz, C-12 OMOM), 4.60 (2H, dd, J = 6.7, 2.5 Hz, C-3, 12 OMOM), 4.50 (1H, d, J = 6.8 Hz, C-3 OMOM), 3.46 (1H, td, J = 10.6, 5.0 Hz, H-12), 3.38 (3H, s, C-12 OMOM), 3.30 (3H, s, C-3 OMOM), 3.07 (1H, dd, J = 11.6, 4.2 Hz, H-3), 2.74 (1H, td, J = 10.8, 5.9 Hz, H-17), 2.21 (1H, t, J = 10.9 Hz, H-13), 2.16 (3H, s, H-21), 1.05 (3H, s, H-30), 0.95 (3H, s, H-28), 0.89 (3H, s, H-18), 0.87 (3H, s, H-19), 0.80 (3H, s, H-29), 0.73 (1H, brd, J = 10.8 Hz, H-5); ¹³C NMR (100 MHz, CDCl₃) δ 212.7 (C-20), 96.2 (C-3 OMOM), 95.0 (C-12 OMOM), 85.0 (C-3), 77.2 (C-12), 56.4 (C-5), 55.7 (C-3 OMOM), 55.6 (C-12 OMOM), 53.2 (C-17), 51.4 (C-14), 51.3 (C-13), 50.7 (C-9), 40.1 (C-8), 39.2 (C-4), 38.9 (C-1), 37.3 (C-10), 35.3 (C-15), 32.7 (C-7), 28.6 (C-11), 28.3 (C-21), 28.2 (C-28), 27.3 (C-16), 24.3 (C-2), 18.4 (C-6), 17.0 (C-19), 16.5 (C-18), 16.4 (C-29), 15.8 (C-30); positive HRESIMS [M + Na]⁺ m/z 487.3396 (calcd for C₂₈H₄₈O₅Na, 487.3394).

16b (20% yield): white foam; [α]¹⁸_D +17 (c 1.50, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.74 (1H, d, J = 6.7 Hz, C-12 OMOM), 4.60 (2H, d, J = 6.7 Hz, C-3, 12 OMOM), 4.50 (1H, d, J = 6.7 Hz, C-3 OMOM), 3.44 (1H, td, J = 10.6, 4.8 Hz, H-12), 3.38 (3H, s, C-12 OMOM), 3.30 (3H, s, C-3 OMOM), 3.07 (1H, dd, J = 11.4, 3.7 Hz, H-3), 2.75 (1H, td, J = 10.7, 5.7 Hz, H-17), 2.28 (1H, dd, J = 13.5, 8.3 Hz, H-13), 1.05 (3H, s, H-30), 0.96 (3H, s, H-28), 0.88 (6H, s, H-18, H-19), 0.86 (6H, s, H-26, H-27), 0.80 (3H, s, H-29), 0.73 (1H, brd, J = 10.4 Hz, H-5); ¹³C NMR (100 MHz, CDCl₃) δ 214.8 (C-20), 96.2 (C-3 OMOM), 95.1 (C-12 OMOM), 85.0 (C-3), 77.8 (C-12), 56.5 (C-5), 55.7 (C-3 OMOM), 55.6 (C-12 OMOM), 52.1 (C-17), 51.3 (C-13, C-14), 50.7 (C-9), 42.2 (C-22), 40.1 (C-8), 39.2 (C-4), 38.9 (C-1), 38.8 (C-24), 37.3 (C-10), 35.4 (C-15), 32.8 (C-7), 28.6 (C-25), 28.2 (C-28), 28.1 (C-11), 27.6 (C-16), 24.3 (C-2), 22.7 (C-26, C-27), 21.6 (C-23), 18.4 (C-6), 17.0 (C-19), 16.5 (C-18), 16.4 (C-29), 15.8 (C-30); positive HRESIMS [M + Na]⁺ m/z 557.4179 (calcd for C₃₃H₅₈O₅Na, 557.4176).

10c (66% yield): white foam; [α]¹⁸_D +13 (c 0.79, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 4.70 (1H, td, J = 10.9, 5.0 Hz, H-12), 3.16 (1H, dd, J = 10.9, 4.6 Hz, H-3), 2.83 (1H, td, J = 11.0, 5.9 Hz, H-17), 2.43 (1H, t, J = 11.0 Hz, H-13), 2.11 (3H, s, H-21), 1.11 (9H, s, OPiv), 1.08 (3H, s, H-30), 0.92 (3H, s, H-19), 0.87 (3H, s, H-28), 0.86 (9H, s, OTBS), 0.85 (3H, s, H-18), 0.72 (3H, s, H-29), 0.02 (6H, s, OTBS); ¹³C NMR (100 MHz, CDCl₃) δ 211.0 (C-20), 178.6 (OPiv), 79.4 (C-3), 75.0 (C-12), 56.1 (C-5), 52.0 (C-14), 51.9 (C-17), 50.6 (C-9), 48.1 (C-13), 40.1 (C-8), 39.7 (C-4), 39.2 (C-1), 38.9 (OPiv), 37.3 (C-10), 35.3 (C-15), 32.5 (C-7), 29.5 (C-21), 28.5(C-28), 28.0 (C-11), 27.8 (C-2), 27.7 (C-16), 27.2 (OPiv), 26.1 (OTBS), 18.6 (C-6), 18.3 (OTBS), 17.0 (C-19), 16.5 (C-18), 16.0 (C-29), 15.8 (C-30), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + Na]⁺ m/z 597.4307 (calcd for C₃₅H₆₂O₄SiNa, 597.4310).

16c (14% yield): white foam; [α]¹⁸_D +18 (c 0.21, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 4.67 (1H, td, J = 11.0, 5.0 Hz, H-12), 3.15 (1H, dd, J = 11.4, 4.5 Hz, H-3), 2.80 (1H, td, J = 11.0, 5.7 Hz, H-17), 2.48 (1H, t, J = 10.9 Hz, H-13), 1.09 (9H, s, OPiv), 1.07 (3H, s, H-30), 0.93 (3H, s, H-19), 0.88 (3H, s, H-28), 0.87 (9H, s, OTBS), 0.86 (6H, d, J = 1.2 Hz, H-18, H-26), 0.85 (3H, d, J = 1.7 Hz, H-27), 0.73 (3H, s, H-29), 0.70 (1H, dd, J = 11.5, 1.5 Hz, H-5), 0.02 (6H, d, J = 1.6 Hz, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 212.9 (C-20), 178.6 (OPiv), 79.3 (C-3), 75.1 (C-12), 56.1 (C-5), 51.9 (C-14), 50.9 (C-17), 50.6 (C-9), 47.6 (C-13), 43.1 (C-22), 40.0 (C-8), 39.6 (C-4), 39.2 (C-1), 38.8 (OPiv), 38.7 (C-24), 37.3 (C-10), 35.3 (C-15), 32.5 (C-7), 28.5 (C-28), 28.1 (C-16), 28.0 (C-25), 27.9 (C-11), 27.8 (C-2), 27.2 (OPiv), 26.1 (OTBS), 22.7 (C-26, C-27), 21.3 (C-23), 18.5 (C-6), 18.3 (OTBS), 17.0 (C-19), 16.6 (C-18), 16.0 (C-29), 15.8 (C-30), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + NH₄]⁺ m/z 662.5534 (calcd for C₄₀H₇₆O₄NSi, 662.5538).

General method for the preparation of ketone (11a, 11c) from methyl ketone 10a and 10c. To a solution of methyl ketone 10a (5 g, 10.87 mmol) or 10c (5 g, 8.71 mmol) in THF (100 mL, contained 10 equiv. of H₂O) or MeOH (100 mL) was added portionwise of NaBH₄ (0.82 g, 21.74 mmol) or (0.66 g, 17.42 mmol) at 0 °C, the resulting mixture was stirred at 0 °C for 0.5 or 2 h. The reaction was quenched (saturated aqueous NH₄Cl, 10 mL), the solvent was evaporated and the residue was dissolved in water and EtOAc (200 mL, 1 : 1, v/v), the organic layer was washed with brine (50 mL × 3), and dried over Na₂SO₄ (s). The solvent was evaporated to give the crude C-20 hydroxy product.

The crude C-20 hydroxy product was dissolved in toluene (10 mL), and to this solution was added Burgess Reagent (7.76 g, 32.61 mmol) or (6.22 g, 26.13 mmol), the resulting mixture was heated to reflux for 2 h. The reaction was quenched (water, 10 mL), extracted by ethyl acetate (100 mL), the organic layer was washed with brine (3 × 50 mL), dried over Na₂SO₄ (s). The solvent was evaporated, and the brown oily residue was passed a short pad of silica column (petroleum ether/ethyl acetate 20 : 1 v/v) to give crude olefin product as a colorless oil.

The crude olefin product was dissolved in DCM/MeOH (100 mL, 5 : 1, v/v) and stirred at −78 °C for 30 min, then ozone was bubbled through the solution until a pale blue color persisted. Nitrogen was bubbled for 5 min, and methyl sulfide (40 mmol) was added, the mixture was allowed to reach RT and stir overnight. The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 20 : 1–10 : 1 v/v) to afford ketone 11a (2.63 g, 6.08 mmol, 56% yield over 3 steps) and 11c (1.85 g, 3.40 mmol, 39% yield over 3 steps).

General method for the preparation of ketone (11a, 11c) from ketone 16a and 16c. Follow the method for the preparation of ketone (11a, 11c) from ketone 10a and 10c. Using 16a (5 g, 9.43 mmol) or 16c (5 g, 7.76 mmol) as starting material afford ketone 11a (1.83 g, 4.24 mmol, 45% yield over 3 steps) and 11c (1.27 g, 2.33 mmol, 30% yield over 3 steps).

11a: white powder; mp 202–204 °C; [α]¹⁸_D +47 (c 0.14, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.01 (1H, td, J = 10.8, 5.4 Hz, H-12), 4.48 (1H, dd, J = 11.6, 4.6 Hz, H-3), 2.37 (1H, d, J = 10.8 Hz, H-13), 2.05 (3H, s, C-12 OAc), 2.04 (3H, s, C-3 OAc), 1.10 (3H, s, H-30), 0.95 (3H, s, H-19), 0.90 (3H, s, H-18), 0.86 (3H, s, H-28), 0.85 (3H, s, H-29); ¹³C NMR (125 MHz, CDCl₃) δ 214.2 (C-17), 171.0 (C-3 OAc), 170.5 (C-12 OAc), 80.5 (C-3), 69.0 (C-12), 56.1 (C-5), 55.8 (C-13), 50.2 (C-9), 47.6 (C-14), 39.8 (C-8), 38.9 (C-1), 38.0 (C-4), 37.4 (C-10), 35.0 (C-15), 33.9 (C-7), 28.1 (C-28), 28.0 (C-16), 27.8 (C-11), 23.7 (C-2), 21.4 (C-3,12 OAc), 18.2 (C-6), 17.5 (C-19), 16.7 (C-29), 16.5 (C-18), 15.9 (C-30); positive HRESIMS [M + H]⁺ m/z 433.2946 (calcd for C₂₆H₄₁O₅, 433.2949).

11c: white foam; [α]¹⁸_D +2 (c 0.49, CHCl₃); ¹H NMR (800 MHz, CDCl₃) δ 4.96 (1H, td, J = 10.8, 5.3 Hz, H-12), 3.17 (1H, dd, J = 11.4, 4.5 Hz, H-3), 2.38 (1H, d, J = 10.8 Hz, H-13), 1.20 (9H, s, OPiv), 1.11 (3H, s, H-30), 0.95 (3H, s, H-19), 0.90 (3H, s, H-18), 0.88 (9H, s, OTBS), 0.75 (3H, s, H-29), 0.03 (6H, s, OTBS); ¹³C NMR (200 MHz, CDCl₃) δ 214.0 (C-17), 178.1 (OPiv), 79.2 (C-3), 69.0 (C-12), 56.1 (C-13), 55.9 (C-5), 50.4 (C-9), 47.7 (C-14), 39.8 (C-8), 39.7 (C-4), 39.2 (C-1), 38.8 (OPiv), 37.4 (C-10), 35.1 (C-15), 34.1 (C-7), 28.5 (C-28), 28.0 (C-2), 27.8 (C-11), 27.7 (C-16), 27.3 (OPiv), 26.1 (OTBS), 18.5 (C-6), 18.3 (OTBS), 17.6 (C-19), 16.6 (C-18), 16.0 (C-29, C-30), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + H]⁺ m/z 547.4176 (calcd for C₃₃H₅₉O₄Si, 547.4177).

General method for the preparation of ketone 11b from ketone 16b and 10b. Follow the method for the preparation of ketone (11a, 11c) from ketone 10a and 10c. Using 16b (2 g, 3.74 mmol) or 10b (2 g, 4.31 mmol) as starting material afford ketone 11b (131 mg, 0.30 mmol, 8% yield over 3 steps) or 11b (148 mg, 0.34 mmol, 3% yield over 3 steps).

11b: white foam; [α]¹⁸_D +18 (c 0.21, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 4.94 (1H, d, J = 6.9 Hz, C-12 OMOM), 4.73 (1H, d, J = 6.7 Hz, C-3 OMOM), 4.71 (1H, d, J = 7.0 Hz, C-12 OMOM), 4.60 (1H, d, J = 6.7 Hz, C-3 OMOM), 3.46 (1H, td, J = 10.7, 4.9 Hz, H-12), 3.43 (3H, s, C-12 OMOM), 3.38 (3H, s, C-3 OMOM), 3.07 (1H, dd, J = 11.7, 4.0 Hz, H-3), 2.32 (1H, d, J = 10.2 Hz, H-13), 1.08 (3H, s, H-30), 0.97 (3H, s, H-28), 0.90 (3H, s, H-19), 0.88 (3H, s, H-18), 0.80 (3H, s, H-29), 0.74 (1H, brd, J = 10.1 Hz, H-5); ¹³C NMR (150 MHz, CDCl₃) δ 215.4 (C-17), 96.2 (C-3, 12 OMOM), 84.9 (C-3), 72.4 (C-12), 57.9 (C-13), 56.4 (C-5), 55.7 (C-3,12 OMOM), 50.2 (C-9), 47.4 (C-14), 39.6 (C-8), 39.1 (C-1), 38.9 (C-4), 37.3 (C-10), 35.0 (C-15), 34.0 (C-7), 28.9 (C-11), 28.1 (C-28), 27.8 (C-16), 24.2 (C-2), 18.3 (C-6), 17.5 (C-19), 16.4 (C-18, C-29), 15.9 (C-30); positive HRESIMS [M + Na]⁺ m/z 459.3089 (calcd for C₂₆H₄₄O₅Na, 459.3086).

Trifluoromethanesulfonyl enolate 17. To a solution of ketone 11c (1.00 g, 1.83 mmol) in THF (20 mL) was added portionwise of LiHMDS (1 M in THF, 2.00 mL, 2.00 mmol) at −78 °C, the resulting mixture was stirred at −78 °C for 30 min. PhNTf₂ (0.71 g, 2.00 mmol) in THF (10 mL) was added to the mixture, and the reaction was allowed warm to 0 °C and stirred for 12 h. The reaction was quenched (saturated aqueous NH₄Cl, 5 mL) at 0 °C, then the mixture was extracted with EtOAc (3 × 30 mL). The organic layer was washed with saturated aqueous CuSO₄ (2 × 50 mL), brine (3 × 50 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum, and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 50 : 1 v/v) to afford 17 (1.19 g, 1.76 mmol) in 96% yield. Colorless prismatic crystal; mp 194–195 °C; [α]¹⁸_D −37 (c 0.15, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.63 (1H, d, J = 1.2 Hz, H-16), 5.10 (1H, td, J = 10.8, 5.1 Hz, H-12), 3.24 (1H, d, J = 11.6 Hz, H-13), 3.16 (1H, dd, J = 11.0, 4.8 Hz, H-3), 1.21 (3H, s, H-30), 1.19 (9H, s, OPiv), 1.08 (3H, s, H-19), 0.89 (3H, s, H-28), 0.88 (9H, s, OTBS), 0.86 (3H, s, H-18), 0.74 (3H, s, H-29), 0.03 (6H, s, OTBS-Me); ¹³C NMR (100 MHz, CDCl₃) δ 178.0 (OPiv), 151.2 (C-17), 115.9 (C-16), 79.2 (C-3), 70.1 (C-12), 56.2 (C-5), 53.2 (C-14), 50.3 (C-9), 49.1 (C-13), 39.7 (C-8), 39.3 (C-1), 39.0 (C-4), 38.9 (OPiv), 37.4 (C-10), 36.0 (C-15), 34.1 (C-7), 28.5 (C-28), 28.1 (C-11), 27.7 (C-2), 27.2 (OPiv), 26.1 (OTBS), 18.6 (C-19), 18.5 (C-6), 18.3 (C-30, OTBS), 16.2 (C-18), 16.0 (C-29), −3.6 (OTBS), −4.8 (OTBS); positive HREIMS [M]⁺ m/z 678.3588 (calcd for C₃₄H₅₇F₃O₆SSi, 678.3597).

Olefin 8. A solution of 17 (0.22 g, 0.32 mmol), Pd(OAc)₂ (14 mg, 0.06 mmol), and PPh₃ (34 mg, 0.12 mmol) in DMSO (5 mL) was bubbled through argon for 15 min. To this solution was added HCO₂NH₄ (40 mg, 0.64 mmol), and the resulting mixture was allowed warm to 50 °C and stirred for 12 h. Water (10 mL) and ether (20 mL) were added, and the organic layer was washed with brine (3 × 20 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 100 : 0 v/v) to afford 8 (0.15 g, 0.29 mmol) in 90% yield. White foam; [α]¹⁸_D −12 (c 0.40, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 5.71 (1H, dd, J = 5.9, 2.5 Hz, H-16), 5.61 (1H, d, J = 5.9 Hz, H-17), 4.91 (1H, td, J = 10.9, 5.1 Hz, H-17), 3.16 (1H, dd, J = 11.2, 4.5 Hz, H-3), 2.84 (1H, d, J = 11.7 Hz, H-13), 1.19 (9H, s, OPiv), 1.06 (3H, s, H-30), 1.05 (3H, s, H-19), 0.88 (12H, s, H-28, OTBS), 0.87 (3H, s, H-18), 0.74 (3H, s, H-29), 0.03 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 178.5 (OPiv), 131.3 (C-16), 130.2 (C-17), 79.3 (C-3), 72.3 (C-12), 56.2 (C-5), 54.1 (C-14), 52.3 (C-13), 50.9 (C-9), 40.5 (C-15), 39.7 (C-8), 39.3 (C-4), 39.1 (C-1), 38.9 (OPiv), 37.4 (C-10), 35.1 (C-7), 28.5 (C-28), 28.3 (C-11), 27.8 (C-2), 27.3 (OPiv), 26.1 (OTBS), 18.6 (C-6), 18.5 (C-30), 18.3 (OTBS), 17.9 (C-19), 16.3 (C-18), 16.0 (C-29), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + NH₄]⁺ m/z 548.4490 (calcd for C₃₃H₆₂O₃NSi, 548.4493).

Diene 19. A solution of 8 (20 mg, 0.038 mmol) and SeO₂ (17 mg, 0.151 mmol) in dioxane/benzene (2 mL, 1 : 1, v/v) was heated at 80 °C for 2 h. The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 100 : 0 v/v) to afford 19 (18 mg, 0.034 mmol) in 90% yield. White foam; [α]¹⁸_D −2 (c 0.43, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 6.22 (1H, dd, J = 5.3, 1.8 Hz, H-16), 6.18 (1H, d, J = 5.3 Hz, H-15), 5.90 (1H, d, J = 1.8 Hz, H-17), 5.48 (1H, dd, J = 9.4, 6.4 Hz, H-12), 3.20 (1H, dd, J = 11.5, 4.6 Hz, H-3), 1.27 (9H, s, OPiv), 1.07 (3H, s, H-30), 0.92 (3H, s, H-28), 0.88 (9H, s, OTBS), 0.84 (3H, s, H-18), 0.73 (3H, s, H-29), 0.66 (3H, s, H-19), 0.04 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 178.1 (OPiv), 153.5 (C-13), 143.1 (C-15), 129.2 (C-16), 119.1 (C-17), 79.1 (C-3), 71.4 (C-12), 62.6 (C-14), 56.1 (C-5), 50.2 (C-9), 40.9 (C-8), 39.7 (C-4), 39.5 (C-1), 39.1 (OPiv), 37.7 (C-10), 37.4 (C-7), 29.6 (C-11), 28.6 (C-28), 27.8 (C-2), 27.4 (OPiv), 26.1 (OTBS), 18.6 (C-6), 18.3 (OTBS), 17.3 (C-30), 16.7 (C-18), 16.1 (C-29), 15.6 (C-19), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + Na]⁺ m/z 551.3882 (calcd for C₃₃H₅₆O₃SiNa, 551.3891).

Enone 20 and t-butyl ether 21. A solution of 8 (20 mg, 0.038 mmol), SeO₂ (21 mg, 0.19 mmol), and tBuOOH (5 M in DCM, 38 μL, 0.19 mmol) in benzene (2 mL) was heated under argon at 60 °C for 2 h. The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 50 : 1–30 : 1 v/v) to afford 20 (6 mg, 0.011 mmol) in 30% yield and 21 (8 mg, 0.013 mmol) in 35% yield.

20: white foam; [α]¹⁸_D −22 (c 0.10, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 5.88 (1H, s, H-17), 5.63 (1H, dd, J = 9.8, 7.3 Hz, H-12), 3.20 (1H, dd, J = 11.4, 4.6 Hz, H-3), 2.57 (1H, d, J = 19.2 Hz, H-15a), 1.89 (1H, d, J = 19.2 Hz, H-15b), 1.25 (9H, s, OPiv), 0.92 (3H, s, H-28), 0.89 (9H, s, OTBS), 0.88 (3H, s, H-18), 0.83 (3H, s, H-19), 0.77 (1H, brd, J = 11.6 Hz, H-5), 0.74 (3H, s, H-29), 0.04 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 207.8 (C-16), 183.6 (C-13), 177.7 (OPiv), 124.4 (C-17), 79.2 (C-3), 70.0 (C-12), 56.0 (C-5), 51.9 (C-14), 49.5 (C-9), 46.4 (C-15), 41.3 (C-8), 39.7 (C-4), 39.1 (C-1, OPiv), 37.6 (C-10), 35.6 (C-7), 28.8 (C-11), 28.6 (C-28), 27.7 (C-2), 27.3 (OPiv), 26.1 (OTBS), 22.6 (C-30), 18.5 (C-6), 18.3 (OTBS), 16.6 (C-18), 16.1 (C-19, C-29), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + H]⁺ m/z 545.4023 (calcd for C₃₃H₅₇O₄Si, 545.4021).

21: white foam; [α]¹⁸_D +1 (c 0.50, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 5.38 (1H, dd, J = 11.0, 6.3 Hz, H-12), 5.30 (1H, s, H-17), 4.51 (1H, m, H-16), 3.18 (1H, dd, J = 11.4, 4.6 Hz, H-3), 1.22 (9H, s, OPiv), 1.19 (9H, s, OtButyl), 0.90 (3H, s, H-28), 0.88 (9H, s, OTBS), 0.84 (3H, s, H-18), 0.81 (3H, s, H-19), 0.73 (3H, s, H-29), 0.03 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 177.7 (OPiv), 150.0 (C-13), 121.3 (C-17), 79.3 (C-3), 76.2 (C-16), 73.2 (OtButyl), 70.3 (C-12), 56.0 (C-5), 55.6 (C-14), 50.1 (C-9), 44.6 (C-15), 40.9 (C-8), 39.7 (C-4), 39.1 (C-1), 39.0 (OPiv), 37.5 (C-10), 35.1 (C-7), 28.9 (C-11), 28.7 (OtButyl), 28.6 (C-28), 27.8 (C-2), 27.4 (OPiv), 26.1 (OTBS), 24.7 (C-30), 18.6 (C-6), 18.3 (OTBS), 16.7 (C-18), 16.6 (C-19), 16.1 (C-29), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + Na]⁺ m/z 625.4622 (calcd for C₃₇H₆₆O₄SiNa, 625.4623).

Epoxide 22. CrO₃ (76 mg, 0.76 mmol) was suspended in DCM (2 mL) at −20 °C, and the 3,5-dimethylpyrazole (75 mg, 0.76 mmol) was added in one portion. After stirring at −20 °C for 15 min, 8 (20 mg, 0.038 mmol) in DCM (1 mL) was added and the mixture was allowed warm to room temperature and stirred for 12 h. The reaction was quenched (water, 5 mL), the mixture was extracted with DCM (3 × 10 mL). The organic layer was washed with brine (3 × 20 mL), dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 40 : 1 v/v) to afford unreacted 8 (8 mg) and 22 (9 mg, 0.016 mmol) in 42% yield. White foam; [α]¹⁸_D −0.3 (c 0.50, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 4.93 (1H, td, J = 10.9, 5.1 Hz, H-12), 3.53 (1H, d, J = 3.4 Hz, H-16), 3.32 (1H, d, J = 2.9 Hz, H-17), 3.15 (1H, dd, J = 11.1, 4.5 Hz, H-3), 1.21 (9H, s, OPiv), 1.11 (3H, s, H-30), 0.94 (3H, s, H-19), 0.87 (12H, s, H-28, OTBS), 0.83 (3H, s, H-18), 0.72 (3H, s, H-29), 0.69 (1H, brd, J = 9.4 Hz, H-5), 0.02 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 178.9 (OPiv), 79.2 (C-3), 71.1 (C-12), 60.7 (C-14), 60.4 (C-16), 59.4 (C-17), 56.1 (C-5), 50.9 (C-13), 50.5 (C-9), 39.6 (C-4), 39.0 (C-8), 38.9 (OPiv), 37.3 (C-10), 36.8 (C-15), 35.0 (C-7), 28.5 (C-28), 28.0 (C-11), 27.7 (C-2), 27.3 (OPiv), 26.0 (OTBS), 20.4 (C-30), 18.4 (C-6), 18.2 (OTBS), 16.4 (C-19), 16.3 (C-18), 15.9 (C-29), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + K]⁺ m/z 585.3746 (calcd for C₃₃H₅₈O₄SiK, 585.3736).

Ketone 23. A solution of 8 (20 mg, 0.038 mmol), CrO₃ (7.6 mg, 0.076 mmol), and tBuOOH (70% aqueous solution, 98 mg, 0.19 mmol) in DCM (2 mL) was stirred at room temperature for 8 h. The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 30 : 1 v/v) to afford 23 (11 mg, 0.027 mmol) in 70% yield. White foam; [α]¹⁸_D −8 (c 0.32, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 5.73 (1H, dd, J = 4.7, 2.5 Hz, H-17), 5.63 (1H, d, J = 5.7 Hz, H-16), 4.93 (1H, td, J = 11.1, 4.8 Hz, H-12), 2.88 (1H, d, J = 11.8 Hz, H-13), 1.20 (9H, s, OPiv), 1.10 (3H, s, H-19), 1.09 (3H, s, H-28), 1.08 (3H, s, H-30), 1.05 (3H, s, H-29), 0.96 (3H, s, H-18); ¹³C NMR (150 MHz, CDCl₃) δ 217.9 (C-3), 178.5 (OPiv), 131.3 (C-17), 130.2 (C-16), 72.0 (C-12), 55.4 (C-5), 54.1 (C-14), 52.4 (C-13), 50.2 (C-9), 47.5 (C-4), 40.5 (C-15), 39.8 (C-1), 39.3 (C-8), 38.9 (OPiv), 37.2 (C-10), 34.3 (C-7), 34.0 (C-2), 28.7 (C-11), 27.3 (OPiv), 27.0 (C-28), 21.1 (C-29), 19.8 (C-6), 18.1 (C-19), 17.9 (C-30), 16.1 (C-18); positive HRESIMS [M + H]⁺ m/z 415.3207 (calcd for C₂₇H₄₃O₃, 415.3207).

Enone 9c (prepared from olefin 8). A mixture of 8 (20 mg, 0.038 mmol), Al₂O₃ (basic, 50 mg), PDC (56 mg, 0.15 mmol), and tBuOOH (70% aqueous solution, 20 mg, 0.15 mmol) in benzene (2 mL) was stirred at room temperature for 48 h. The mixture was passed through a short pad of Celite, the filter cake was washed with ethyl acetate (3 × 10 mL) and the filtrate was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 40 : 1 v/v) to afford 9c (8 mg, 0.014 mmol) in 38% yield along with 22 (<1 mg, <5% yield) and 20 (9 mg, 0.016 mmol, 41% yield). White foam; [α]¹⁸_D −10 (c 0.74, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 7.54 (1H, d, J = 5.9 Hz, H-15), 5.87 (1H, d, J = 5.9 Hz, H-16), 5.15 (1H, td, J = 10.7, 4.9 Hz, H-12), 3.17 (1H, dd, J = 11.0, 4.6 Hz, H-3), 2.66 (1H, d, J = 11.2 Hz, H-13), 1.25 (9H, s, OPiv), 1.07 (6H, s, H-19, H-30), 0.91 (3H, s, H-28), 0.89 (9H, s, OTBS), 0.86 (3H, s, H-18), 0.76 (3H, s, H-29), 0.03 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 205.1 (C-17), 178.2 (OPiv), 164.4 (C-15), 131.3 (C-16), 79.2 (C-3), 68.9 (C-12), 56.8 (C-5), 56.2 (C-13), 54.3 (C-14), 50.7 (C-9), 39.7 (C-8), 38.9 (OPiv), 38.8 (C-1), 38.3 (C-4), 37.6 (C-10), 33.6 (C-7), 28.9 (C-11), 28.6 (C-28), 27.7 (C-2), 27.3 (OPiv), 26.1 (OTBS), 23.5 (C-30), 19.0 (C-19), 18.4 (C-6), 18.3 (OTBS), 16.1 (C-18), 16.0 (C-29), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + H]⁺ m/z 545.4028 (calcd for C₃₃H₅₇O₄Si, 545.4021).

Olefin 24. To a solution of 8 (10 mg, 0.019 mmol) in DMF (1 mL) at 120 °C was added DDQ (9 mg, 0.038 mmol) in DMF (1 mL, contained 10 equiv. of H₂O), the resulting mixture was stirred at 120 °C for 3 h. The mixture was passed through a short pad of basic alumina with EtOAc (30 mL) as eluent, the filtrate was evaporated under vacuum to give 24 (8 mg, 0.019 mmol) in 100% yield. Colorless oil; [α]¹⁸_D −15 (c 1.00, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.72 (1H, d, J = 2.7 Hz, H-17), 5.63 (1H, brs, H-16), 4.92 (1H, td, J = 11.4, 4.9 Hz, H-12), 3.21 (1H, dd, J = 11.3, 4.7 Hz, H-3), 2.85 (1H, d, J = 11.7 Hz, H-13), 1.20 (9H, s, OPiv), 1.06 (3H, s, H-30), 0.98 (3H, s, H-28), 0.88 (3H, s, H-18), 0.79 (3H, s, H-29); ¹³C NMR (100 MHz, CDCl₃) δ 178.4 (OPiv), 131.3 (C-17), 130.3 (C-16), 78.9 (C-3), 72.2 (C-12), 56.2 (C-5), 54.1 (C-14), 52.3 (C-13), 50.9 (C-9), 40.5 (C-15), 39.3 (C-8), 39.1 (C-4, OPiv), 38.9 (C-1), 37.6 (C-10), 35.1 (C-7), 28.4 (C-11), 28.2 (C-28), 27.4 (C-2), 27.3 (OPiv), 18.5 (C-6), 18.4 (C-19), 18.0 (C-30), 16.3 (C-18), 15.5 (C-29); positive HRESIMS [M + Na]⁺ m/z 439.3185 (calcd for C₂₇H₄₄O₃Na, 439.3183).

Enone 9c (prepared from ketone 11c). To a solution of ketone 11c (1.0 g, 1.83 mmol) in THF (10 mL) was added dropwise of LiHMDS (1 M in THF, 2.20 mL, 2.20 mmol) at −78 °C, the resulting mixture was stirred at −78 °C for 30 min. PhSeCl (0.42 g, 2.20 mmol) in THF (5 mL) was added dropwise to the mixture, and the reaction was allowed warm to room temperature and stirred for 12 h. The reaction was quenched (saturated aqueous NH₄Cl, 5 mL) at 0 °C, then mixture was extracted with EtOAc (3 × 30 mL). The organic layer was washed with brine (3 × 50 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was passed through a short pad of Celite with petroleum ether/EtOAc (20 : 1, v/v, 100 mL) as eluent, the filtrate was evaporated under vacuum to give crude selenide. This crude product was dissolved in THF/H₂O (20 mL, 1 : 1, v/v), and NaIO₄ (3.9 g, 18.30 mmol) was added to this solution, the resulting mixture was stirred vigorously at room temperature overnight. The reaction mixture was passed through a short pad of Celite with EtOAc (100 mL) as eluent, the filtrate was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 30 : 1–20 : 1 v/v) to afford 9c (0.78 g, 1.43 mmol) in 78% yield over 2 steps (the spectra data were listed above).

Enone 9a. Follow the method for the preparation of enone 9c from ketone 11c. Using 11a (1.0 g, 2.31 mmol) as starting material afford ketone 9a (0.24 g, 0.55 mmol, 24% yield over 2 steps). White foam; [α]²³_D −16 (c 0.20, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.54 (1H, d, J = 5.9 Hz, H-15), 5.89 (1H, d, J = 5.9 Hz, H-16), 5.19 (1H, td, J = 10.7, 4.9 Hz, H-12), 4.48 (1H, dd, J = 11.2, 4.4 Hz, H-3), 2.11 (3H, s, C-12 OAc), 2.04 (3H, s, C-3 OAc), 1.07 (3H, s, H-30), 1.06 (3H, s, H-19), 0.88 (3H, s, H-28), 0.87 (3H, s, H-29); ¹³C NMR (100 MHz, CDCl₃) δ 205.1 (C-17), 171.0 (C-3 OAc), 170.7 (C-12 OAc), 164.3 (C-15), 131.4 (C-16), 80.4 (C-3), 68.9 (C-12), 56.6 (C-5), 56.2 (C-13), 54.2 (C-14), 50.6 (C-9), 38.4 (C-8), 38.3 (C-1), 38.1 (C-4), 37.6 (C-10), 33.4 (C-7), 28.9 (C-11), 28.1 (C-28), 28.0 (C-2), 23.6 (C-30), 21.4 (C-3,12 OAc), 18.9 (C-6), 18.1 (C-19), 16.6 (C-29), 16.0 (C-18); positive HRESIMS [M + H]⁺ m/z 431.2793 (calcd for C₂₆H₃₉O₅, 431.2792).

Epoxide 27. To a solution of 9c (10 mg, 0.018 mmol) and TBHP (70% aqueous solution, 3 mg, 0.036 mmol) in MeOH/THF (3 mL, 4 : 1, v/v) at 0 °C was added Triton B (40% MeOH solution, 2 μL), the resulting mixture was allowed warm to room temperature and stirred for 12 h. The reaction was quenched (saturated aqueous NH₄Cl, 1 mL) at 0 °C, then mixture was extracted with EtOAc (3 × 5 mL). The organic layer was washed with brine (3 × 5 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 50 : 1 v/v) to afford 27 (7 mg, 0.014 mmol) in 78% yield. White foam; [α]¹⁸_D −10 (c 0.23, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 3.75 (1H, d, J = 2.3 Hz, H-15), 3.58 (1H, d, J = 2.3 Hz, H-16), 3.43 (1H, d, J = 3.7 Hz, H-12), 3.16 (1H, dd, J = 11.3, 4.6 Hz, H-3), 1.17 (1H, dd, J = 12.1, 5.8 Hz, H-9), 1.13 (3H, s, H-30), 0.92 (3H, s, H-28), 0.91 (6H, s, H-18, H-19), 0.88 (9H, s, OTBS), 0.76 (3H, s, H-29), 0.71 (1H, brd, J = 11.1 Hz, H-5), 0.03 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 203.3 (C-17), 79.0 (C-3), 63.2 (C-13), 61.8 (C-12), 60.2 (C-15), 55.6 (C-5), 55.5 (C-16), 44.6 (C-9), 39.5 (C-4), 38.4 (C-1), 37.1 (C-8), 36.9 (C-10), 33.8 (C-7), 28.7 (C-28), 27.5 (C-2), 26.0 (OTBS), 21.7 (C-11), 18.2 (C-6), 18.0 (OTBS), 17.2 (C-19), 16.4 (C-29), 16.0 (C-18), 14.7 (C-30), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + NH₄]⁺ m/z 492.3506 (calcd for C₂₈H₅₀O₄NSi, 492.3504).

Diol 25. To a solution of ketone 9c (0.50 g, 0.92 mmol) and CeCl₃·7H₂O (0.69 g, 1.84 mmol) in MeOH (10 mL) was added NaBH₄ (70 mg, 1.84 mmol) in portions at 0 °C, the resulting mixture was stirred at 0 °C for 2 h. The reaction was quenched (saturated aqueous NH₄Cl solution, 5 mL) at 0 °C, and the mixture was extracted with EtOAc (3 × 30 mL). The organic layer was washed with water (3 × 50 mL), brine (3 × 50 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum to give crude allylic alcohol. This crude product was dissolved in DCM (10 mL) at 0 °C, and MCPBA (77%, 0.62 g, 2.76 mmol) was added to this solution in portions, the resulting mixture was allowed to warm to room temperature and stirred for 4 h. The reaction was quenched (saturated aqueous Na₂S₂O₃, 5 mL) at 0 °C, and the mixture was extracted with DCM (30 mL × 3). The organic layer was washed with 5% aqueous NaOH (50 mL × 3), brine (50 mL × 3), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum to give crude epoxide alcohol.

The crude epoxide alcohol was dissolved in MeI (10 mL), to this solution was added freshly prepared Ag₂O (2.1 g, 9.2 mmol), the resulting suspension was sealed and heated at 40 °C for 48 h. The mixture was passed through a short pad of Celite with EtOAc (100 mL) as eluent, the filtrate was evaporated under vacuum to give crude epoxide methylether. This crude epoxide methylether was dissolved in dioxane (20 mL), to this solution was added LiAlH₄ (70 mg, 1.84 mmol) in portions at 0 °C, the resulting mixture was refluxed for 4 h. The reaction was cooled to 0 °C, wet Na₂SO₄ (500 mg) was added in portions and the mixture was stirred vigorously for 1 h. The mixture was passed through a short pad of Celite with EtOAc (200 mL) as eluent, the filtrate was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 1 : 1 v/v) to afford 25 (0.30 g, 0.61 mmol) in 66% yield over 4 steps. White foam; [α]¹⁸_D −9 (c 0.20, CHCl₃); ¹H NMR (800 MHz, CDCl₃) δ 3.97 (1H, brs, H-15), 3.79 (1H, m, H-12), 3.77 (1H, m, H-17), 3.34 (3H, s, OMe), 3.15 (1H, dd, J = 11.4, 4.4 Hz, H-3), 2.28 (1H, t, J = 9.5 Hz, H-13), 1.35 (3H, s, H-30), 0.91 (3H, s, H-19), 0.89 (3H, s, H-28), 0.88 (9H, s, OTBS), 0.74 (3H, s, H-18), 0.72 (3H, s, H-29), 0.67 (1H, brd, J = 11.1 Hz, H-5), 0.03 (6H, s, OTBS); ¹³C NMR (200 MHz, CDCl₃) δ 84.4 (C-17), 79.9 (C-15), 79.4 (C-3), 72.2 (C-12), 57.4 (OMe), 56.1 (C-5), 51.6 (C-14), 51.4 (C-9), 50.6 (C-13), 41.8 (C-16), 41.4 (C-8), 39.7 (C-4), 39.5 (C-1), 37.4 (C-10), 35.0 (C-7), 30.9 (C-11), 28.6 (C-28), 27.8 (C-2), 26.1 (OTBS), 19.1 (C-30), 18.3 (C-19, OTBS), 16.9 (C-18), 16.0 (C-29), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + Na]⁺ m/z 517.3687 (calcd for C₂₉H₅₄O₄SiNa, 517.3684).

Acetyl ester 26a and 26b. To a solution of 32 (0.20 g, 0.40 mmol) in pyridine (5 mL) at 0 °C was added dropwise of AcCl (0.23 mL, 3.2 mmol), the resulting mixture was stirred at 0 °C overnight. The reaction was quenched (water, 1 mL) at 0 °C, and the mixture was extracted with EtOAc (3 × 10 mL). The organic layer was washed with saturated aqueous CuSO₄ (3 × 10 mL), brine (3 × 10 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 8 : 1 v/v) to afford 26a (14 mg, 0.024 mmol) and 26b (0.18 g, 0.34 mmol) in 90% yield.

26a: colorless needles; mp 194–196 °C; [α]¹⁸_D +11 (c 0.20, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 5.11 (1H, td, J = 11.0, 5.4 Hz, H-12), 4.88 (1H, d, J = 5.9 Hz, H-15), 3.78 (1H, td, J = 8.4, 3.9 Hz, H-17), 3.20 (3H, s, OMe), 3.15 (1H, dd, J = 11.3, 4.5 Hz, H-3), 2.42 (1H, dd, J = 10.8, 8.5 Hz, H-13), 2.05 (3H, s, C-12 OAc), 2.00 (3H, s, C-15 OAc), 1.25 (3H, s, H-30), 0.90 (3H, s, H-19), 0.88 (9H, s, OTBS), 0.74 (3H, s, H-29), 0.66 (1H, d, J = 11.4 Hz, H-5), 0.03 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 171.3 (C-12 OAc), 170.3 (C-15 OAc), 82.7 (C-17), 79.4 (C-15), 79.3 (C-3), 73.3 (C-12), 57.4 (OMe), 55.9 (C-5), 52.0 (C-14), 51.2 (C-9), 49.9 (C-13), 41.1 (C-8), 40.0 (C-16), 39.6 (C-4), 39.5 (C-1), 37.3 (C-10), 34.7 (C-7), 28.5 (C-28), 27.8 (C-2), 27.7 (C-11), 26.0 (OTBS), 22.0 (C-15 OAc), 21.5 (C-12 OAc), 18.7 (C-30), 18.3 (C-6), 18.2 (C-19, OTBS), 18.0 (C-19), 16.9 (C-18), 16.0 (C-29), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + Na]⁺ m/z 601.3898 (calcd for C₃₃H₅₈O₆SiNa, 601.3895).

26b: white powder; mp 237–239 °C; [α]¹⁸_D +5 (c 0.40, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.10 (1H, td, J = 10.9, 5.4 Hz, H-12), 3.93 (1H, d, J = 5.3 Hz, H-15), 3.75 (1H, td, J = 8.3, 3.5 Hz, H-17), 3.22 (3H, s, OMe), 3.16 (1H, dd, J = 11.1, 4.8 Hz, H-3), 2.37 (1H, dd, J = 10.9, 8.1 Hz, H-13), 2.05 (3H, s, OAc), 1.36 (3H, s, H-30), 0.91 (3H, s, H-19), 0.89 (3H, s, H-28), 0.88 (9H, s, OTBS), 0.78 (3H, s, H-18), 0.74 (3H, s, H-29), 0.68 (1H, d, J = 10.6 Hz, H-5), 0.03 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 171.3 (OAc), 82.9 (C-17), 79.5 (C-15), 79.3 (C-3), 73.8 (C-12), 57.3 (OMe), 56.0 (C-5), 52.5 (C-14), 51.4 (C-9), 49.2 (C-13), 42.5 (C-16), 41.3 (C-8), 39.7 (C-4), 39.5 (C-1), 37.3 (C-10), 34.7 (C-7), 28.6 (C-28), 27.8 (C-2, C-11), 26.1 (OTBS), 21.5 (OAc), 18.8 (C-30), 18.6 (C-19), 18.3 (C-6, OTBS), 16.9 (C-18), 16.0 (C-29), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + Na]⁺ m/z 559.3787 (calcd for C₃₁H₅₆O₅SiNa, 559.3789).

Ketone 29. To a solution of 26b (20 mg, 0.037 mmol) in DCM (2 mL) at 0 °C was added DMP (31 mg, 0.074 mmol), the resulting suspension was stirred at 0 °C for 2 h. The reaction was quenched (saturated aqueous Na₂S₂O₃, 2 mL) at 0 °C, and the mixture was extracted with DCM (5 mL × 3). The organic layer was washed with brine (10 mL × 3), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 15 : 1 v/v) to afford 29 (20 mg, 0.037 mmol) in 100% yield. White powder; mp 245–246 °C; [α]¹⁸_D +62 (c 0.16, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.14 (1H, td, J = 11.2, 5.0 Hz, H-12), 3.99 (1H, dd, J = 16.7, 7.1 Hz, H-17), 3.24 (3H, s, OMe), 3.15 (1H, dd, J = 11.3, 4.9 Hz, H-3), 2.83 (1H, dd, J = 18.9, 7.1 Hz, H-16a), 2.05 (3H, s, OAc), 1.97 (1H, dd, J = 18.9, 7.0 Hz, H-16b), 1.04 (3H, s, H-30), 1.03 (3H, s, H-19), 0.89 (3H, s, H-28), 0.85 (3H, s, H-18), 0.74 (3H, s, H-29), 0.03 (6H, s, OTBS); ¹³C NMR (150 MHz, CDCl₃) δ 215.1 (C-15), 171.5 (OAc), 79.2 (C-3), 77.5 (C-17), 71.5 (C-12), 59.5 (C-14), 57.5 (OMe), 55.9 (C-5), 50.8 (C-13), 50.1 (C-9), 44.2 (C-16), 39.8 (C-8), 39.6 (C-4), 39.1 (C-1), 37.5 (C-10), 33.8 (C-7), 28.5 (C-28), 27.9 (C-2), 27.8 (C-11), 26.0 (OTBS), 21.4 (OAc), 18.7 (C-19), 18.3 (OTBS), 18.2 (C-6), 16.2 (C-18), 16.0 (C-29), 13.5 (C-30), −3.6 (OTBS), −4.8 (OTBS); positive HRESIMS [M + Na]⁺ m/z 557.3636 (calcd for C₃₁H₅₄O₅SiNa, 557.3633).

Hupehenol E (7). A solution of 29 (10 mg, 0.019 mmol) and pTSA (2 mg, 0.011 mmol) in benzene (2 mL) was refluxed for 2 h. The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 6 : 1 v/v) to afford 7 (7 mg, 0.017 mmol) in 90% yield. White amorphous powder; [α]²⁴_D −91 (c 0.10, MeOH); positive HRESIMS [M + H]⁺ m/z 389.2694 (calcd for C₂₄H₃₇O₄, 389.2686). ¹H and ¹³C NMR data were identical with the literature.¹²

Hupehenol B (4) and 17-epi-hupehenol B (30). To a solution of 7 (10 mg, 0.026 mmol) in MeOH (2 mL) at 0 °C was added concentrated HCl (1 μL), the resulting mixture was allowed warm to 50 °C stirred for 3 h. The reaction was cooled, followed by addition of EtOAc (5 mL) and water (5 mL), the organic layer was washed with brine (3 × 10 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 10 : 1–6 : 1 v/v) to afford 30 (2 mg, 0.004 mmol) and 4 (8 mg, 0.019 mmol) in 87% yield.

Hupehenol B (4): colorless needles; mp 223–224 °C; [α]²⁴_D +13 (c 0.20, MeOH); positive HRESIMS [M + H]⁺ m/z 421.2950 (calcd for C₂₅H₄₁O₅, 421.2949). ¹H and ¹³C NMR data were identical with the literature.¹²

17-epi-Hupehenol B (30): white powder; mp 197–198 °C; [α]¹⁸_D +73 (c 0.20, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 5.14 (1H, td, J = 11.1, 4.9 Hz, H-12), 3.99 (1H, dd, J = 16.4, 7.2 Hz, H-17), 3.24 (3H, s, OMe), 3.19 (1H, dd, J = 11.6, 3.8 Hz, H-3), 2.84 (1H, dd, J = 18.9, 7.1 Hz, H-16a), 2.23 (1H, t, J = 10.4 Hz, H-13), 2.05 (3H, s, OAc), 1.97 (1H, dd, J = 18.9, 6.9 Hz, H-16b), 1.04 (6H, s, H-19, H-30), 0.98 (3H, s, H-28), 0.84 (3H, s, H-18), 0.77 (3H, s, H-29), 0.73 (1H, brd, J = 10.3 Hz, H-5); ¹³C NMR (150 MHz, CDCl₃) δ 215.1 (C-15), 171.5 (OAc), 78.7 (C-3), 77.5 (C-17), 71.4 (C-12), 59.4 (C-14), 57.5 (OMe), 55.7 (C-5), 50.7 (C-9), 50.1 (C-13), 44.2 (C-16), 39.7 (C-8), 39.0 (C-1, C-4), 37.6 (C-10), 33.6 (C-7), 28.1 (C-28), 27.8 (C-11), 27.3 (C-2), 21.4 (OAc), 18.7 (C-19), 17.9 (C-6), 16.1 (C-18), 15.5 (C-29), 13.5 (C-30); positive HRESIMS [M + Na]⁺ m/z 443.2774 (calcd for C₂₅H₄₀O₅Na, 443.2768).

Hupehenol A (3) and 17-epi-hupehenol A (31). To a solution of 7 (10 mg, 0.026 mmol) in MeOH (2 mL) was added K₂CO₃ (7 mg, 0.052 mmol), the resulting mixture was allowed warm to 40 °C stirred for 8 h. The reaction was cooled, followed by addition of EtOAc (5 mL) and water (5 mL), the organic layer was washed with water (3 × 10 mL), brine (3 × 10 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 2 : 1–1 : 1 v/v, contained 5% DCM) to afford 3 (7 mg, 0.019 mmol) and 31 (2 mg, 0.005 mmol) in 92% yield.

Hupehenol A (3): white amorphous powder; [α]²⁷_D +10 (c 0.20, MeOH); positive HRESIMS [M + Na]⁺ m/z 401.2640 (calcd for C₂₃H₃₈O₄Na, 401.2662). ¹H and ¹³C NMR data were identical with the literature.¹²

17-epi-Hupehenol A (31): white amorphous powder; [α]²⁷_D +56 (c 0.40, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 4.08 (1H, dt J = 9.6, 7.1 Hz, H-17), 3.91 (1H, dd, J = 12.5, 7.6 Hz, H-12), 3.38 (3H, s, OMe), 3.18 (1H, dd, J = 11.6, 4.1 Hz, H-3), 2.83 (1H, dd, J = 18.6, 6.9 Hz, H-16a), 2.18 (1H, t, J = 10.4 Hz, H-13), 2.05 (1H, dd, J = 18.6, 7.1 Hz, H-16b), 1.05 (3H, s, H-19), 0.96 (3H, s, H-28), 0.94 (3H, s, H-30), 0.84 (3H, s, H-18), 0.77 (3H, s, H-29), 0.71 (1H, brd, J = 9.7 Hz, H-5); ¹³C NMR (150 MHz, CDCl₃) δ 214.6 (C-15), 79.1 (C-17), 78.8 (C-3), 70.4 (C-12), 59.0 (C-14), 57.7 (OMe), 55.9 (C-5), 51.3 (C-13), 50.5 (C-5), 44.1 (C-16), 39.9 (C-8), 39.0 (C-1, C-4), 37.6 (C-10), 33.8 (C-7), 31.1 (C-11), 28.2 (C-28), 27.4 (C-2), 18.7 (C-19), 18.0 (C-6), 16.1 (C-18), 15.5 (C-29), 13.3 (C-30); positive HRESIMS [M + Na]⁺ m/z 401.2661 (calcd for C₂₃H₃₈O₄Na, 401.2662).

Isomerization of hupehenol A (3) and 17-epi-hupehenol A (31) in methanol. To a solution of 31 (5 mg, 0.013 mmol) or 3 (5 mg, 0.013 mmol) in MeOH (1 mL), K₂CO₃ (3 mg) was added, and the resulting mixture was allowed to warm to 60 °C and stirred for 24 h. The reaction was cooled, followed by addition of EtOAc (5 mL) and water (5 mL), the organic layer was washed with water (3 × 10 mL), brine (3 × 10 mL), and dried over Na₂SO₄ (s). The solvent was evaporated under vacuum and the residue was purified by flash chromatography on silica gel (petroleum ether/EtOAc 2 : 1–1 : 1 v/v, contained 5% DCM) to afford 3 (2.5 mg, 0.0065 mmol) and 31 (2.4 mg, 0.0062 mmol) in 50% yield, 97% yield (brsm.).

11β-HSD1 inhibition assay. Inhibition of synthetic compounds on human or mouse 11β-HSD1 enzymatic activities was determined by the scintillation proximity assay (SPA) using microsomes containing 11β-HSD1, according to previous work.^56,57 Briefly, the full-length cDNAs of human or murine 11β-HSD1, obtained from the cDNA libraries (NIH Mammalian Gene Collection), were cloned into the pcDNA3 expression vector. HEK-293 cells were transfected with the pcDNA3-derived expression plasmid and selected by cultivation in the presence of G418 (700 μg mL⁻¹). The microsomal fraction overexpressing 11β-HSD1, which was prepared from the HEK-293 cells stably transfected with 11β-HSD1, was used as the enzyme source for SPA. Microsomes containing human or mouse 11β-HSD1 were incubated with NADPH, [³H] cortisone, and compounds at 37 °C in phosphate buffer (40 mM Na₂HPO₄, 1 mM EDTA, 5% glycerol) for 2 h (human) or 1 h (murine). 18β-Glycyrrhetinic acid (GA) was used as positive control. IC₅₀ values were calculated by using Prism Version 4 GraphPad Software.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Nos U1502223, 81172941), National Basic Research Program of China (973 Program No. 2011CB915503), and Yunnan High-End Technology Professionals Introduction Program (No. 2010CI117).

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Footnote

† Electronic supplementary information (ESI) available: C-20 hydroxy elimination of compounds 14, allylic oxidation attempts of compound 8, bioassay data, NMR spectra for all isolated compounds, X-ray crystallographic data for compounds 17, 26a, and 4. CCDC 1405681–1405683. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra04236h

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