Design and synthesis of herboxidiene derivatives that potently inhibit in vitro splicing

Abstract

Herboxidiene is a potent antitumor agent that targets the SF3B subunit of the spliceosome. Herboxidiene possesses a complex structural architecture with nine stereocenters and design of potent less complex structures would be of interest as a drug lead as well as a tool for studying SF3B1 function in splicing. We investigated a number of C-6 modified herboxidiene derivatives in an effort to eliminate this stereocenter and, also to understand the importance of this functionality. The syntheses of structural variants involved a Suzuki–Miyaura cross-coupling reaction as the key step. The functionalized tetrahydrofuran core has been constructed from commercially available optically active tri-O-acetyl-d-glucal. We investigated the effect of these derivatives on splicing chemistry. The C-6 alkene derivative showed very potent splicing inhibitory activity similar to herboxidiene. Furthermore, the C-6 gem-dimethyl derivative also exhibited very potent in vitro splicing inhibitory activity comparable to herboxidiene.

Information transfer from genes to proteins is a critical event for life. For nearly all human genes, precursor messenger RNA (pre-mRNA) splicing is an essential step in the process. Splicing removes intron sequences from gene transcripts to create functional messenger RNA (mRNA) for protein translation.^1,2 Recent studies have shown that defects in pre-mRNA splicing and acquired mutations of pre-mRNA splicing factors are associated with many human cancers.^3,4

Splicing is carried out by a large and highly dynamic ribo-nucleoprotein complex called the spliceosome, which is assembled from uridine-rich small nuclear RNAs (snRNA) and dozens of specific proteins.^5,6 Splicing involves complex and highly regulated multi-step reactions.^7,8 Significant progress has been made in recent years in terms of understanding bio-chemical activity, protein composition, and structures of several distinct forms of the spliceosome as they occur during the reaction pathways.^9,10

A core component of spliceosome is the SF3B1 protein, which is the largest subunit of the spliceosome factor 3b (SF3B) complex.^11,12 SF3B1 is an important pre-mRNA splicing factor with a role in the earliest stages of spliceosome assembly.¹³ It participates in a series of structural and compositional rearrangements of spliceosome snRNAs and proteins that ultimately position the intron into the spliceosome’s active site.¹⁴ Increasing evidence points to mutations in SF3B1 of the spliceosome and their involvement in various types of human cancers, including haematological malignancies and solid tumors.^15,16 The pre-mRNA splicing factor SF3B1 has emerged as an outstanding target for anticancer drug development.^17,18 Currently, a number of natural products and their derivatives have been shown to potently inhibit spliceosome function by binding to the SF3B subunit of U2 snRNP.^19,20 These include, herboxidiene (1, Fig. 1) pladienolide B (2), a semisynthetic derivative E71O7 (3), FR901464 (4), and spliceostatin A (5). These compounds display potent in vitro splicing inhibition by interfering with the SF3B subunit of spliceosome.^21–24 The semisynthetic derivative of pladienolide B and semisynthetic derivative, E71O7 have undergone advanced human clinical trials.²⁵ Herboxidiene is a very potent inhibitor of spliceosome. It exhibits potent antitumor properties and it induces both G1 and G2/M cell cycle arrest in a human normal fibroblast cell line WI-38.²⁶

Fig. 1.

Structures of herboxidiene, pladienolide B, E71O7, FR901464, and spliceostatin A.

As a result of its important structural and biological properties, herboxidiene attracted considerable synthetic attention. Several total syntheses of herboxidiene have been reported by a number of research groups.^26–30 Also, synthesis and biological evaluation of structural variants of herboxidiene have been reported by us and others. As outlined in Fig. 2, 5(R)-hydroxy herboxidiene 6 is a natural product which showed slightly lower splicing inhibition than herboxidiene. Interestingly, compound 7, the C5 epimer also showed comparable inhibitory activity.³¹ The C6 desmethyl herboxidiene 8 was synthesized, however, this compound exhibited slightly lower splicing inhibitory activity as well as anticancer activity in human cells.^29,32 The corresponding carba analog 9 showed substantial reduction in splicing inhibitory activity.³² An interesting pladienolide-herboxidiene hybrid 10 was designed. However, it was significantly less potent.²⁹ Furthermore, a carbohydrate-based herboxidiene-pladienolide hybrid molecule 11 has been designed and synthesized to modulate splicing.³³

Fig. 2.

Structures of herboxidiene derivatives 6–11.

Herboxidiene contains nine stereocenters and a number of sensitive functionalities. Therefore, it is imperative to elucidate structure–activity relationships of herboxidiene and design less complex and more potent herboxidiene derivatives. In our continuing interests in chemistry and biology of splicing inhibitors, we have designed a number of novel C6-modified, herboxidiene derivatives. Herein, we report enantioselective syntheses of C-6 modified methylene, (R)-methyl, cyclopropyl, and gem-dimethyl derivatives of herboxidiene and their in vitro evaluation of splicing inhibitory activity.

Results and discussion

We recently showed that incorporation of hydroxyl group at C-5 and removal of the C-6 methyl group resulted in only a small reduction of splicing activity. In an effort to probe the importance of C-6 methyl group, we have designed four C-6 modified derivatives including C-6 methylene derivative 12, C-6 (R)-methyl derivative 13, cyclopropyl derivative 14, and gem-dimethyl derivative 15. Our synthetic strategy for these derivatives is shown in Scheme 1. Our plan was to carry out Suzuki–Miyaura cross-coupling reaction of vinyl iodides 16 and pinacol boronate 17 to provide herboxidiene derivatives 12–15. The pinacol boronate 17 was then be synthesized in optically active form using our previously reported procedure.^30,31 Various vinyl iodides 16a–d were constructed from the known,^34,35 optically active aldehyde derivative 18, which was obtained from commercially available, optically active triacetoxy-d-glucal 19 as the key starting material.

Scheme 1.

Synthetic strategies of C-6 modified derivatives of herboxidiene.

For the synthesis of C-6 vinyl derivative 12, the synthesis of requisite vinyl iodide 16a is shown in Schemes 2 and 3. Commercially available triacetoxy-d-glucal 19 was converted to acetaldehyde derivative 18 in multigram scale as reported in the literature.^35,36 Aldehyde derivative 18 was converted to enone 20 in a four-step sequence involving, (1) reaction of aldehyde with ethylene glycol in the presence of a catalytic amount of p-TSA in toluene, heated at reflux for 3 h; (2) treatment of the resulting di-t-butylsilyl acetal with nBu₄N⁺F⁻ in THF at 0 °C to 23 °C for 12 h to give diol; (3) selective protection of primary alcohol as TBDPS ether with TBDPSCl in the presence of imidazole in CH₂Cl₂ at 0 °C to 23 °C for 3 h; and (4) oxidation of the resulting allylic alcohol with Dess–Martin periodinane to provide 20 in 60% yield over 4-steps. Catalytic hydrogenation of 20 over 10% Pd-C in EtOAc at 23 °C under a hydrogen-filled balloon afforded the corresponding ketone. Wittig olefination of the resulting ketone with methylenetriphenylphosphorane, generated from this reaction of Ph₃PCH₃Br and ^tBuOK in THF at 0 °C to 23 °C afforded the corresponding methylene derivative. Reaction of the resulting alkene derivative with nBu₄N⁺F⁻ in THF at 0 °C to 23 °C for 12 h furnished alcohol 21 in 87% yield over 3-steps. To install the vinyl iodide, we first converted alcohol 21 to its acetylene derivative by oxidation with Dess–Martin periodinane at 0 °C to 23 °C for 6 h followed by reaction of the resulting aldehyde with Bestmann–Ohira reagent³⁶ in MeOH in the presence of K₂CO₃ at 0 °C to 23 °C for 4 h to provide alkyne derivative 22 in 66% yield over 2-steps. The 1,3-dioxalane functionality of 22 was converted to the corresponding methyl ester in a three-step sequence involving, (1) treatment of 1N aqueous HCl in THF at 23 °C for 36 h; (2) oxidation of the resulting aldehyde with NaClO₂ in aqueous t-butanol in the presence of 2-methyl-2-butene and NaH₂PO₄ at 0 °C to 23 °C for 5 h and (3) esterification of the resulting carboxylic acid with MeOH using EDC in the presence of a catalytic amount of DMAP in CH₂Cl₂ at 0 °C to 23 °C for 12 h to afford methyl ester 23 in 68% yield over 3-steps.

Scheme 2.

Reagents and conditions: (a) Ethylene glycol, PTSA, toluene, reflux, 3 h, 90%; (b) TBAF, THF, 0 °C to 23 °C, 12 h; (c) TBDPSCl, imidazole, DCM, 0 °C to 23 °C, 3 h, (d) DMP, NaHCO₃, DCM, 23 °C, 4 h, 60% over four steps; (e) H₂ balloon, Pd/C, EtOAc, 23 °C, 12 h; (f) Ph₃PCH₃Br, KO^tBu, THF, 0 °C to 23 °C, 1 h; (g) TBAF, THF, 0 °C to 23 °C, 12 h, 87% over three steps; (h) DMP, DCM, 0 °C to 23 °C, 6 h; (i) Bestmann–Ohira reagent, K₂CO₃, MeOH, 0 °C to 23 °C, 4 h, 66% over two steps; (j) 1N HCl, THF, 23 °C, 36 h; (k) NaClO₂, NaH₂PO₄, tBuOH/H₂O, 0 °C to 23 °C, 5 h; (l) EDC-HCl, DMAP, MeOH, DCM, 0 °C to 23 °C, 12 h.

Scheme 3.

Reagents and conditions: (a) HgSO₄, H₂SO₄, aq. THF, 23 °C, 3 h, 84%; (b) CrCl₂, CHl_3, THF, 23 °C, 12 h, 79% (brsm) for 16a; 82% (brsm) for 16b; (c) H₂, 10% Pd-C, EtOAc, 23 °C, 12 h (97%).

Methyl ester 23 was converted to vinyl iodide derivatives 16a and 16b as shown in Scheme 3. Reaction of alkyne derivative 23 with a catalytic amount of HgSO₄ in the presence of 3 M H₂SO₄ in aqueous THF at 23 °C for 3 h resulted in methyl ketone derivative 24 in 84% yield.³⁷ Treatment of ketone 24 with CrCl₂ and CHI₃ in THF at 23 °C for 12 h using protocol described by Takai and co-workers,³⁸ afforded vinyl iodide 16a in 79% yield as a E/Z mixture (20 : 1 by ¹H NMR analysis) which was used directly for the coupling reaction. Similarly, methyl ketone 24 was also converted to vinyl iodide derivative 16b. Catalytic hydrogenation of 24 over 10% Pd-C in EtOAc under hydrogen-filled balloon at 23 °C provided 1 : 1 mixture of diastereomeric methyl ketones 25 and 26 in 97% yield. These diastereomers were separated by silica gel column chromatography using 25% ethyl acetate in hexanes. The C6 (R)-diastereomer 26 was converted to vinyl iodide 16b by exposure to CrCl₂ and CHI₃ in THF at 23 °C as described above.

The synthesis of vinyl iodides 16c and 16d was carried out from alkene derivative 21 as shown in Scheme 4. Simmons–Smith cyclopropanation³⁹ of 21 was sluggish. We decided to carry out dichlorocyclopropanation.^40,41 Thus, exposure of alkene 21 to CHCl₃ and 50% aqueous NaOH in the presence of a catalytic amount of benzyltriethylammonium chloride at 80 °C for 48 h furnished dichlorocyclopropane derivative 27 in 80% yield. Reduction of 27 with LiAlH₄ in THF at 0 °C to 64 °C for 24 h afforded cyclopropane derivative 28 in 90% yield. Oxidation of alcohol 28 with Dess–Martin periodinane and reaction of the resulting aldehyde with Ohira–Bestmann reagent³⁶ in MeOH furnished alkyne derivative 29. The alkyne was converted to keto ester 30. Deprotection of 1,3-dioxolane with 1N HCl provided aldehyde which was oxidized with NaClO₂ to carboxylic acid. Esterification of the resulting carboxylic acid with MeOH using EDC and a catalytic amount of DMAP as described for compound 23, furnished the corresponding methyl ester. Reaction of the resulting alkyne with a catalytic amount of HgSO₄ in the presence of 3 M H₂SO₄ in aqueous THF provided methyl ketone derivative 30 in 70% yield over 4-steps starting from alkyne 29. Treatment of methyl ketone 30 with CrCl₂ and CHI₃ in THF as described above, resulted in vinyl iodide 16c in 82% yield. For installation of C-6 gem-dimethyl group, cyclopropane derivative, we planned to hydrogenate the cyclopropane derivative over PtO₂.⁴² Thus, cyclopropane derivative 30 was subjected to catalytic hydrogenation over PtO₂ in acetic acid under hydrogen-filled balloon at 40 °C for 30 min to provide cyclopropane ring opened product 31 in 85% yield. Methyl ketone functionality of 31 was transformed into vinyl iodide 16d as described above to provide 16d in 75% yield. Various vinyl iodides 16a-d so synthesized were immediately used for Suzuki–Miyaura cross-coupling reaction^43,44 with boronate 17.

Scheme 4.

Reagents and conditions: (a) Pd(PPh₃)₄ (5 mol%), Cs₂CO₃, THF, 23 °C to 55 °C, 5 h, 47% for 32; 51% for 34; (b) 0.16 M HCl, MeOH, 23 °C, 45 min; (c) Vo(acac)₂, t-BuOOH, DCM, −20 °C, 36 h, 62% over two steps for 33; 58% over two steps for 35; (d) Me₃SnOH, DCE, 80 °C, 24 h, 85% for 12; 84% for 13.

Synthesis of herboxidiene C-6 methylene derivative 12 is shown in Scheme 5. Suzuki–Miyaura cross-coupling^43,44 of pinacol boronate 17 and vinyl iodide 16a was carried out with a catalytic amount of Pd(PPh₃)₄ (5 mol%) and Cs₂CO₃ in THF at 23 °C to 55 °C for 5 h to provide coupling product 32 in 47% yield. For regio- and stereoselective installation of epoxide functionality at C14–C15, the TBS-ether of 32 was removed by exposure to 0.2 M HCl in methanol at 23 °C for 45 min. Directed epoxidation⁴⁵ of the resulting alcohol was carried out with a catalytic amount of VO(acac)₂ in the presence of t-BuOOH in CH₂Cl₂ at −20 °C for 36 h to provide the epoxide 33 in 62% yield over 2-steps. Methyl ester hydrolysis with Me₃SnOH in dichloroethane at 80 °C for 24 h using the protocol described by Nicolaou and co-workers,^46,47 provided herboxidiene C-6 methylene derivative 12 in 85% yield. For the synthesis of herboxidiene C-6 (R)-methyl derivative 13, Suzuki–Miyaura cross-coupling of pinacol boronate 17 and vinyl iodide 16b provided coupling product 34 in 51% yield. Removal of TBS group followed by directed epoxidation as described above, afforded epoxide 29 in 58% yield over 2-steps. Me₃SnOH promoted hydrolysis of the methyl ester as described above, furnished herboxidiene C-6 (R)-methyl derivative 7 in 84% yield. The syntheses of herboxidiene C-6 cyclopropyl and gem-dimethyl derivatives 14 and 15 is shown are Scheme 6. Suzuki–Miyaura cross-coupling of pinacol boronate 17 and vinyl iodides 16c and 16d provided coupling products 36 and 38 in 44% and 47% yield, respectively. Removal of TBS group followed by directed epoxidation as described above, afforded epoxide derivatives 37 and 39 in 57% and 66% yield, respectively over 2-steps. Me₃SnOH promoted hydrolysis of the methyl ester as described above, resulted in C-6 cyclopropyl and gem-dimethyl derivatives 14 and 15 in 82% and 87% yield, respectively.

Scheme 5.

Reagents and conditions: (a) Benzyltriethyl, ammonium chloride, 50% aq. NaOH, CHCl₃, 80 °C, 80% brsm; (b) LAH, THF, 64 °C, 24 h, 90%; (c) DMP, NaHCO₃, DCM, 0 °C to 23 °C; (d) Ohira–Bestmann, K₂CO₃, MeOH, over two steps, 71%; (e) 1N HCl, THF, 23 °C, 36 h; (f) 2-methyl-2-butene, NaClO₂, NaH₂PO₄, ^tBuOH/H₂O, 0 °C to 23 °C; (g) EDC-HCl, DMAP, MeOH, DCM, 0 °C to 23 °C, 66% over three steps; (h) HgSO₄, H₂SO₄, aq. THF, 23 °C, 3 h, 70%; (i) CrCl₂, CHI₃, THF, 23 °C, 12 h, 82% (brsm) for 16c and 75% (brsm) for 16d; (j) H₂, PtO₂, AcOH, 40 °C, 30 min (85%).

Scheme 6.

Reagents and conditions: (a) Pd(PPh₃)₄ (5 mol%), Cs₂CO₃, THF, 23 °C to 55 °C, 5 h, 44% for 36; 47% for 38; (b) 0.16 M HCl, MeOH, 23 °C, 1 h; (c) Vo(acac)₂, t-BuOOH, DCM, −20 °C, 36 h, 57% over two steps for 37; 66% over two steps for 39; (d) Me₃SnOH, DCE, 80 °C, 24 h, 82% for 14; 87% for 15.

We carried out dose response analysis for all synthetic derivatives in an in vitro splicing system.²³ For splicing efficiency, we measured spliced product produced after incubating an RNA splicing substrate in extracts prepared from the nuclei of human cells, which also contained herboxidiene derivative or DMSO as a control. IC₅₀ values were determined as the concentration required to reduce in vitro splicing efficiency by half compared to DMSO control (Fig. 3). Of the four C-6 modified herboxidiene derivatives evaluated, C-6 alkene derivative 12 exhibited the most potent in vitro splicing inhibitory activity. The IC₅₀ value of alkene 12 is 0.4 μM, which within the variability of the assay is comparable to the 0.3 μM IC₅₀ that we previously measured for herboxidiene.²⁷ Derivative 13 with a C-6 (R)-methyl group shows good IC₅₀ value of 2.5 μM, however, it is about 6-fold less potent than herboxidiene. Essentially, both C-6 methyl isomers shows potent splicing inhibitory activity. In an effort to remove the C-6 stereocenter, we therefore designed both C-6 cyclopropyl derivative 14 and gem-dimethyl derivative 15. The C-6 cyclopropyl derivative 14 exhibited an average IC₅₀ value of 5.2 μM. Interestingly, the gem-dimethyl derivative 15 showed 3-fold reduction in potency compared to herboxidiene.

Fig. 3.

Impact of herboxidiene derivatives on in vitro splicing. Quantification of normalized splicing efficiency vs. inhibitor concentration for in vitro splicing reactions with compounds 12–15. IC₅₀ values are estimated from nonlinear regression fit of triplicate measurements with 95% confidence interval values indicated in parentheses.

These results were nicely supported in terms of spliceosome assembly. Spliceosome assembly intermediates were visualized as the stereotypical change in mobility of radiolabeled splicing substrate in native gels. The herboxidiene derivatives disrupted the transition from A to B complex at the same concentration at which they inhibited splicing efficiency (Fig. 4). This result is consistent with the requirement for the inhibitor target SF3B in the spliceosome assembly pathway.

Fig. 4.

Native gel analysis of spliceosome assembly. The first five lanes show a time course of splicing reactions in 1% DMSO using a radiolabeled pre-mRNA substrate which were separated under native conditions. The following lanes show 30 minutes time points of splicing reactions incubated with the indicated compound concentration. The identity of splicing complexes is denoted with assembly occurring in the following order: H/E → A → B → C.

Conclusion

In summary, we have designed, synthesized, and evaluated the splicing properties of C-6 modified derivatives of herboxidine. These compounds were designed to investigate the importance of the C-6 substituent, possible replacement of the C-6 stereocenter with a new substituent, and improve splicing inhibitory activity. The syntheses of derivatives involved the Suzuki–Miyaura cross-coupling reaction of a pinacol boronate derivative with an appropriate vinyl iodide partner. All vinyl iodide derivatives were synthesized in optically active form using triacetoxy-d-glucal as the key starting material. The C-6 cyclopropane derivatives were synthesized by cyclopropanation of the C-6 methylene derivative with dichlorocarbene followed by LAH reduction. Reductive opening of the cyclopropane ring provided the corresponding C-6-gem-dimethyl derivatives. We evaluated all synthetic derivatives in in vitro spliceosome inhibitory assay. The C-6 alkene derivative 12 showed very potent splicing inhibitory activity comparable to herboxidiene, indicating that C-6(S)-methyl group may not be critical to herboxidiene splicing inhibitory activity. The C-6 (R)-methyl derivative 13 resulted in about 6-fold loss in potency. Interestingly, the C-6 gem-dimethyl derivative 15, which eliminated C-6 stereochemistry, exhibited very potent splicing inhibition with slight loss in activity compared to herboxidiene. The corresponding cyclopropyl derivative 14 showed 5-fold loss of activity compared to its gem-dimethyl derivative. The present work indicated that both C-6 alkene and the corresponding gem-dimethyl derivative are able to maintain comparable splicing inhibitory activity as herboxidiene. Further design and synthesis of novel derivatives are in progress.

Experimental section

All reactions were carried out under an inert atmosphere, either nitrogen or argon, using magnetic stirring and oven-dried glassware. All solvents were anhydrous and distilled prior to use. Dichloromethane and triethylamine were distilled from calcium hydride. Tetrahydrofuran, diethyl ether, and benzene were distilled from sodium/benzophenone. All other solvents were HPLC grade. Flash column chromatography was performed using EM Science 60–200 mesh silica gel. Thin-layer chromatography was performed using 60 F-254 E. Merck silica gel plates. ¹H- and ¹³C-NMR were recorded using Bruker AV-400 MHz, Avance DRX-500, Varian Mercury-Vx-300, and Gemini-2300 spectrometers and use Me₄Si as an internal standard. Optical rotations were recorded on a PerkinElmer 341 polarimeter. A Thermo Finnigan LCQ classic mass was used for HRMS analysis. The purity of test compounds was determined by HRMS and HPLC analysis.

(2R,6S)-6-((1,3-Dioxolan-2-yl)methyl)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-2H-pyran-3(6H)-one (20)

To a solution of aldehyde 18 (4.0 g, 12.81 mmol) in toluene (40 mL) at 23 °C were added ethylene glycol (2.0 mL) and p-TSA (441 mg, 2.56 mmol). The reaction was then connected to Dean–Stark apparatus and refluxed for 3 h. After this period, the reaction mixture was cooled to 23 °C, and quenched with saturated NaHCO₃ solution. The resulting solution was extracted with EtOAc (2×), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (10% EtOAc in hexanes) to give desired product as an amorphous solid (4.1 g, 90%).

To a solution of di-t-butylsilyl acetate (2.7 g, 7.58 mmol) in THF (30 mL) was added TBAF (1 M in THF, 11.4 mL, 11.37 mmol) at 0 °C. The resulting reaction mixture was allowed to warm to 23 °C and stirred for 12 h. The reaction mixture was quenched with H₂O and extracted with EtOAc (3×). The combined organic extracts were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude was used for the next step without any further purification.

To a solution of above diol compound (1.5 g, 6.94 mmol) in CH₂Cl₂ (25 mL) at 0 °C were added imidazole (566 mg, 8.33 mmol) and TBDPSCl (1.9 g, 6.94 mmol). The reaction mixture was allowed to warm to 23 °C and stirred for 3 h. The reaction was diluted with H₂O and extracted with CH₂Cl₂ (2×), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (20% EtOAc in hexanes) to give allylic the alcohol as a colorless oil (2.5 g, 80%).

To a solution of the above allylic alcohol (1.2 g, 2.64 mmol) in CH₂Cl₂ (20 mL) were added DMP (2.2 g, 5.28 mmol) and NaHCO₃ (666 mg, 7.93 mmol) at 0 °C. The resulting reaction mixture was allowed to warm to 23 °C and stirred for 4 h. After this period, the reaction mixture was quenched with saturated Na₂S₂O₃ followed by saturated NaHCO₃ solution. The mixture was stirred vigorously for 20 min and extracted with CH₂Cl₂ (2×). The combined organic extracts were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (10% EtOAc in hexanes) to give enone 20 as an amorphous solid (1.0 g, 60% over 4 steps). ¹H NMR (400 MHz, CDCl₃) δ 7.71 (ddd, J = 6.2, 3.1, 10.8 Hz, 4H), 7.45–7.33 (m, 6H), 7.04 (dd, J = 10.3, 1.5 Hz, 1H), 6.10 (dd, J = 10.3, 2.4 Hz, 1H), 5.11 (dd, J = 6.2, 3.6 Hz, 1H), 4.60 (ddd, J = 7.5, 5.6, 2.0 Hz, 1H), 4.16–4.05 (m, 3H), 4.04–3.96 (m, 2H), 3.92–3.82 (m, 2H), 2.14 (ddd, J = 14.0, 7.8, 3.6 Hz, 1H), 1.98 (ddd, J = 14.1, 6.1, 5.4 Hz, 1H), 1.02 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 194.5, 151.3, 135.6, 133.6, 133.5, 129.5, 127.5, 126.9, 101.3, 81.5, 70.3, 64.9, 64.7, 63.0, 38.9, 26.6, 19.2; ${[\alpha ]}_{\text{D}}^{20}=-18.6\left(c0.22,{\text{CHCl}}_3\right)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₂₆H₃₂O₅SiNa 475.1919, found 475.1923.

((2S,6R)-6-((1,3-Dioxolan-2-yl)methyl)-3-methylenetetrahydro-2H-pyran-2-yl)methanol (21)

To a solution of enone 20 (960 mg, 2.12 mmol) in EtOAc (10 mL) was added Pd-C (100 mg, 10 wt%). The resulting reaction mixture was stirred under a hydrogen-filled balloon for 12 h. After this period, the reaction mixture was filtered through Celite, washed with EtOAc, and concentrated under reduced pressure. The crude was used for the next step without any further purification.

To a suspension of Ph₃P⁺CH₃Br⁻ (2.2 g, 6.14 mmol) in dry THF (10 mL) was added potassium tert-butoxide (5.1 mL, 1 M, 5.12 mmol) at 0 °C and the reaction mixture was continued to stir for another 30 min. A solution of ketone (930 mg, 2.05 mmol) in THF (10 mL) was added at 0 °C and the resulting mixture was continued to stir for another 1 h. The reaction was quenched with NH₄Cl solution and extracted with EtOAc (2×). The combined organic extracts were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude was used for the next step without any further purification.

To a solution of alkene compound (800 mg, 1.77 mmol) in dry THF (10 mL) at 0 °C was added a solution of TBAF (1.0 M in THF, 2.65 mL, 2.65 mmol). The resulting reaction mixture was allowed to warm to 23 °C and stirred for 12 h. The reaction mixture was diluted with H₂O and extracted with EtOAc (2×). The combined organic extracts were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (30% EtOAc in hexanes) to give alcohol 21 as a colorless oil (348 mg, 87% over 3 steps). ¹H NMR (400 MHz, CDCl₃) δ 5.04 (dd, J = 5.9, 4.1 Hz, 1H), 4.84 (d, J = 0.8 Hz, 1H), 4.69 (s, 1H), 4.04–3.95 (m, 2H), 3.95–3.84 (m, 4H), 3.83–3.73 (m, 2H), 2.42 (ddd, J = 13.9, 4.8, 2.7 Hz, 1H), 2.37–2.23 (m, 2H), 1.98 (ddd, J = 14.1, 8.3, 4.1 Hz, 1H), 1.80 (dddd, J = 14.1, 10.1, 5.4, 3.3 Hz, 2H), 1.50 (tdd, J = 12.9, 11.4, 4.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 143.4, 107.5, 102.3, 77.9, 74.0, 64.9, 64.7, 63.0, 40.4, 33.7, 32.8; ${[\alpha ]}_{\text{D}}^{20}=+5.3\left(c0.28,{\text{ CHCl}}_3\right)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₁H₁₈O₄Na 237.1106, found 237.1103.

(2S,6R)-6-((1,3-Dioxolan-2-yl)methyl)-2-ethynyl-3-methylenetetrahydro-2H-pyran (22)

To a solution of alcohol 21 (1.0 g, 4.67 mmol) in CH₂Cl₂ (20 mL) at 0 °C was added DMP (2.97 g, 7.0 mmol). The reaction temperature was allowed to warm to 23 °C and stirred for 6 h. After this period, the reaction mixture was quenched with saturated Na₂S₂O₃ followed by saturated NaHCO₃ solution. The mixture was stirred vigorously for 30 min and extracted with CH₂Cl₂ (2×). The combined organic extracts were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude was used for the next step without any further purification.

To a solution of aldehyde in MeOH (10 mL) at 0 °C were added Bestmann–Ohira reagent (1.08 g, 5.60 mmol) and K₂CO₃ (973.8 mg, 7.0 mmol). The flask was wrapped with aluminium foil. The suspension was warmed to 23 °C and stirred for 4 h. The reaction was then diluted with EtOAc, quenched with saturated NaHCO₃ solution. The aqueous layer was extracted with EtOAc (2×). The combined organic extracts were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (10% EtOAc in hexanes) to give alkyne 22 as a colorless oil (646 mg, 66% over 2 steps). ¹H NMR (400 MHz, CDCl₃) δ 5.39 (d, J = 1.4 Hz, 1H), 5.08 (dd, J = 6.8, 3.6 Hz, 1H), 4.96 (s, 1H), 4.69 (s, 1H), 4.03–3.94 (m, 2H), 3.94–3.84 (m, 2H), 3.81–3.73 (m, 1H), 2.66 (d, J = 2.2 Hz, 1H), 2.54 (ddd, J = 13.9, 4.4, 2.5 Hz, 1H), 2.36–2.25 (m, 1H), 2.10–2.03 (m, 1H), 1.87–1.81 (m, 1H), 1.73 (ddd, J = 14.1, 6.9, 4.6 Hz, 1H), 1.55–1.46 (m, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 141.9, 110.6, 101.8, 79.9, 75.6, 75.0, 70.7, 64.8, 64.7, 40.3, 33.6, 31.7; ${[\alpha ]}_{\text{D}}^{20}=-36.0\left(c0.13,{\text{ CHCl}}_3\right)$.

Methyl 2-((2R,6S)-6-ethynyl-5-methylenetetrahydro-2H-pyran-2-yl)acetate (23)

To a solution of alkyne 22 (140 mg, 0.67 mmol) in THF (1.5 mL) at 23 °C was added 1N HCl (1.5 mL), and the resulting mixture was stirred for 36 h. After this period, the reaction mixture was quenched with saturated NaHCO₃ solution and the aqueous layer was extracted with CH₂Cl₂ (2×). The combined organic extracts were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude aldehyde was used for the next step without any further purification.

To a solution of above crude aldehyde (106 mg, 0.64 mmol) in tert-butanol and water (2 : 1, 6.0 mL) at 0 °C were added 2-methyl-2-butene (450 mg, 6.43 mmol), NaClO₂ (80% purity, 175 mg, 1.93 mmol) and NaH₂PO₄·H₂O (264 mg, 1.93 mmol). The reaction was allowed to warm to 23 °C. The solution was continued to stir until the aldehyde disappeared (5 h). The biphasic mixture was separated and the aqueous layer was extracted with EtOAc (2×), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude acid was used for the next step without any further purification.

The above crude acid (110 mg, 0.61 mmol) was dissolved in CH₂Cl₂ (5 mL) at 0 °C. To this solution was added EDC·HCl (141 mg, 0.73 mmol), DMAP (90 mg, 0.73 mmol) and MeOH (59 mg, 1.84 mmol). The reaction temperature was allowed to warm to 23 °C and stirred for 12 h. After this period, the reaction was diluted with H₂O, extracted with CH₂Cl₂ (2×), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (20% EtOAc in hexanes) to afford methyl ester 23 as a colorless oil (89 mg, 68% over 3 steps). ¹H NMR (400 MHz, CDCl₃) δ 5.40 (d, J = 1.4 Hz, 1H), 4.97 (s, 1H), 4.72 (d, J = 1.0 Hz, 1H), 4.02 (dtd, J = 11.2, 6.5, 2.0 Hz, 1H), 3.71 (s, 3H), 2.74–2.68 (m, 1H), 2.66 (d, J = 2.2 Hz, 1H), 2.58–2.52 (m, 1H), 2.45 (dd, J = 15.8, 6.3 Hz, 1H), 2.38–2.27 (m, 1H), 1.87 (ddt, J = 12.9, 4.6, 2.2 Hz, 1H), 1.49 (tdd, J = 13.1, 11.3, 4.5 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 171.3, 141.4, 111.0, 79.6, 75.8, 74.6, 70.9, 51.7, 40.7, 32.8, 31.5; ${[\alpha ]}_{\text{D}}^{20}=-6.1(c0.13,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₁H₁₄O₃Na 217.0844, found 217.0840.

Methyl 2-((2R,6S)-6-acetyl-5-methylenetetrahydro-2H-pyran-2-yl)acetate (24)

To a solution of methyl ester 23 (65 mg, 0.34 mmol) in THF (3 mL) was added HgSO₄ (20 mg, 0.07 mmol in 1 mL of 3 M H₂SO₄) at 23 °C. This solution was stirred at 23 °C until the starting material disappeared (3 h). The reaction mixture was diluted with EtOAc and neutralized with saturated NaHCO₃ solution. The aqueous layer was extracted with EtOAc (2×), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (20% EtOAc in hexanes) to afford methyl ketone 24 as a colorless oil (60 mg, 84%). ¹H NMR (400 MHz, CDCl₃) δ 4.89 (s, 1H), 4.62 (s, 1H), 4.35 (s, 1H), 4.07–3.95 (m, 1H), 3.69 (s, 3H), 2.63 (dd, J = 15.4, 7.6 Hz, 1H), 2.53–2.43 (m, 2H), 2.35 (ddd, J = 13.0, 4.2, 2.1 Hz, 1H), 2.20 (s, 3H), 1.86 (ddt, J = 12.9, 5.0, 2.5 Hz, 1H), 1.52 (ddd, J = 17.7, 12.1, 6.4 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 207.5, 171.4, 140.8, 109.8, 84.7, 73.4, 51.8, 40.9, 32.4, 32.4, 26.7; ${[\alpha ]}_{\text{D}}^{20}=-42.3(c0.13,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₁H₁₆O₄Na 235.0950, found 235.0948.

Methyl 2-((2R,6S)-6-((E)-1-iodoprop-1-en-2-yl)-5-methylenetetrahydro-2H-pyran-2-yl)acetate (16a)

A suspension of CrCl₂ (203 mg, 1.70 mmol) in THF (3 mL) was stirred at 23 °C. To this suspension was added a mixture of ketone 24 (35 mg, 0.17 mmol) and CHI₃ (195 mg, 0.51 mmol) in THF (3 mL) with the use of a cannula. The resulting dark brown solution was stirred for 12 h. After this period, EtOAc and H₂O were added to this mixture. The organic layer was separated and the aqueous layer was extracted with EtOAc (2×). The combined organic extracts were dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (10% EtOAc in hexanes) to afford vinyl iodide 16a as a yellow oil (25 mg, 79% brsm, E/Z: 20 : 1). ¹H NMR (400 MHz, CDCl₃) δ 6.27 (s, 1H), 4.83 (s, 1H), 4.57 (d, J = 1.1 Hz, 1H), 4.41 (s, 1H), 4.07–3.93 (m, 1H), 3.68 (s, 3H), 2.59 (dd, J = 15.4, 7.0 Hz, 1H), 2.50–2.39 (m, 2H), 2.39–2.26 (m, 1H), 1.87–1.82 (m, 1H), 1.81 (d, J = 1.0 Hz, 3H), 1.52–1.40 (m, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 171.5, 145.7, 143.8, 110.0, 83.5, 80.2, 73.9, 51.7, 40.9, 32.9, 32.3, 21.9; ${[\alpha ]}_{\text{D}}^{20}=-14.0(c0.1,{\text{ CHCl}}_3)$.

Methyl 2-((2R,5R,6S)-6-acetyl-5-methyltetrahydro-2H-pyran-2-yl)acetate (26)

To a solution of methyl ketone 24 (200 mg, 0.94 mmol) in EtOAc (10 mL) was added Pd-C (20 mg, 10 wt%). The reaction mixture was stirred under a hydrogen-filled balloon for 12 h. After this period, the reaction mixture was filtered through Celite, washed with EtOAc (20 mL), and concentrated under reduced pressure. The residue was purified by silica gel chromatography (25% EtOAc in hexanes) and separated diastereomeric mixture of (1 : 1) methyl ketones 25 and 26 as a colorless oil (195 mg, combined yield 97%). Data for 26: ¹H NMR (400 MHz, CDCl₃) δ 3.89 (d, J = 2.4 Hz, 1H), 3.87–3.77 (m, 1H), 3.69 (s, 3H), 2.65 (dd, J = 15.1, 7.8 Hz, 1H), 2.48 (dd, J = 15.1, 5.2 Hz, 1H), 2.18 (dd, J = 9.1, 4.9 Hz, 1H), 2.12 (s, 3H), 1.81 (ddd, J = 13.0, 11.4, 4.7 Hz, 1H), 1.70 (s, 1H), 1.59–1.41 (m, 2H), 0.87 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 210.1, 171.6, 85.4, 75.1, 51.6, 41.2, 30.0, 29.2, 27.1, 25.4, 12.1; ${[\alpha ]}_{\text{D}}^{20}=-36.2(c1.0,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₁H₁₈O₄Na 237.1106, found 237.1104.

Methyl 2-((2R,5R,6S)-6-((E)-1-iodoprop-1-en-2-yl)-5-methyltetrahydro-2H-pyran-2-yl) acetate (16b)

Compound 16b was prepared from 26 (70 mg, 0.33 mmol) by following the same procedure outlined for 16a to give a colorless oil. Yield (45 mg, 82% brsm). ¹H NMR (400 MHz, CDCl₃) δ 6.14–6.12 (m, 1H), 3.92 (s, 1H), 3.85–3.77 (m, 1H), 3.67 (s, 3H), 2.58 (dd, J = 14.9, 7.4 Hz, 1H), 2.43 (dd, J = 15.0, 5.5 Hz, 1H), 1.92 (ddt, J = 9.2, 4.7, 2.4 Hz, 1H), 1.86–1.75 (m, 1H), 1.71 (s, 3H), 1.69–1.61 (m, 1H), 1.51–1.41 (m, 2H), 0.75 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 171.7, 145.7, 83.1, 77.0, 75.0, 51.7, 41.4, 30.1, 29.0, 25.7, 21.9, 11.6; ${[\alpha ]}_{\text{D}}^{20}=-49.1(c1.0,{\text{ CHCl}}_3)$.

((4S,6R)-6-((1,3-Dioxolan-2-yl)methyl)-1,1-dichloro-5-oxaspiro [2.5]octan-4-yl)methanol (27)

A solution of alcohol 21 (1.0 g, 4.67 mmol) in CHCl₃ (100 mL) was added a solution of 50% aqueous NaOH (30 mL) and benzyltriethylammonium chloride (128 mg, 0.56 mmol) at 23 °C. The resulting reaction mixture was heated to 80 °C for 48 h. The organic layer was separated and washed with H₂O, then brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (10% EtOAc/DCM) to give dichloro cyclopropane 27 as a colorless oil (801 mg, 80% brsm). ¹H NMR (400 MHz, CDCl₃) δ 5.02 (t, J = 4.9 Hz, 1H), 3.98 (ddd, J = 13.3, 8.7, 3.7 Hz, 3H), 3.91–3.71 (m, 4H), 3.55 (dd, J = 11.4, 7.6 Hz, 1H), 2.50 (s, 1H), 2.05–1.90 (m, 3H), 1.88–1.74 (m, 2H), 1.60–1.53 (m, 1H), 1.51 (d, J = 7.8 Hz, 1H), 1.31 (d, J = 7.8 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 102.4, 71.4, 65.6, 65.0, 64.8, 63.4, 40.5, 31.8, 29.4, 28.7, 28.2; ${[\alpha ]}_{\text{D}}^{20}=+32.17(c0.23,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₂H₁₈Cl₂O₄Na 319.0474, found 319.0479.

((4S,6R)-6-((1,3-Dioxolan-2-yl)methyl)-5-oxaspiro[2.5]octan-4-yl) methanol (28)

To a solution of dichloro cyclopropane 27 (200 mg, 0.68 mmol) in dry THF (5 mL) at 0 °C was added a solution of LAH (1.0 M in THF, 6.8 mL, 6.76 mmol). The reaction was allowed to warm to 23 °C and then heated at reflux for 24 h. The reaction was quenched with NH₄Cl and extracted with EtOAc (3×). The combined organic extracts were washed with H₂O, then brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (20% EtOAc/DCM) to give cyclopropane 28 as a colorless oil (141 mg, 90%). ¹H NMR (400 MHz, CDCl₃) δ 5.02 (dd, J = 5.8, 4.3 Hz, 1H), 4.02–3.89 (m, 2H), 3.89–3.78 (m, 3H), 3.75–3.62 (m, 1H), 3.32–3.18 (m, 2H), 2.21 (s, 1H), 2.11–1.91 (m, 2H), 1.78 (ddd, J = 14.1, 5.8, 4.1 Hz, 1H), 1.71–1.59 (m, 2H), 1.52 (tdd, J = 13.0, 11.2, 4.1 Hz, 1H), 0.95 (ddd, J = 13.4, 4.0, 2.8 Hz, 1H), 0.69–0.61 (m, 1H), 0.45 (dd, J = 9.1, 4.7 Hz, 1H), 0.26–0.18 (m, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 102.5, 79.7, 74.7, 64.9, 64.7, 61.7, 40.6, 35.2, 31.8, 19.2, 9.0, 8.6; ${[\alpha ]}_{\text{D}}^{20}=+11.1(c0.1,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₂H₂₀O₄Na 251.1265, found 251.1261.

(4S,6R)-6-((1,3-Dioxolan-2-yl)methyl)-4-ethynyl-5-oxaspiro[2.5] octane (29)

Compound 29 was prepared from 28 (130 mg, 0.57 mmol) by following the same procedure outlined for 22 to give a colorless oil. Yield (90 mg, 71% over 2 steps). ¹H NMR (400 MHz, CDCl₃) δ 5.05 (dd, J = 6.9, 3.6 Hz, 1H), 4.56 (s, 1H), 4.00–3.93 (m, 2H), 3.91–3.80 (m, 2H), 3.69–3.59 (m, 1H), 2.32 (d, J = 2.2 Hz, 1H), 2.10–1.94 (m, 2H), 1.78–1.59 (m, 3H), 1.59–1.46 (m, 1H), 1.09 (ddd, J = 13.5, 3.9, 2.9 Hz, 1H), 1.01–0.95 (m, 1H), 0.65 (dd, J = 8.5, 4.5 Hz, 1H), 0.27–0.24 (m, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 101.9, 79.4, 75.3, 73.5, 72.9, 64.8, 64.7, 40.6, 33.1, 31.4, 19.8, 8.9; ${[\alpha ]}_{\text{D}}^{20}=-38.3(c0.18,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₃H₁₈O₃Na 245.1160, found 245.1157.

Methyl 2-((4S,6R)-4-acetyl-5-oxaspiro[2.5]octan-6-yl)acetate (30)

Compound 30 was prepared from 29 (50 mg, 0.24 mmol) by following the same procedure outlined for 23 and 24 to give a colorless oil. Yield (36 mg, over 4 steps 70%). ¹H NMR (400 MHz, CDCl₃) δ 4.06 (d, J = 0.8 Hz, 1H), 3.90 (dddd, J = 11.1, 7.7, 5.2, 2.2 Hz, 1H), 3.68 (s, 3H), 2.64 (dd, J = 15.1, 7.9 Hz, 1H), 2.49 (dd, J = 15.1, 5.2 Hz, 1H), 2.09 (s, 3H), 2.08–2.01 (m, 1H), 1.73–1.64 (m, 1H), 1.53 (tdd, J = 13.0, 11.3, 4.1 Hz, 1H), 0.99 (ddd, J = 13.6, 4.0, 2.8 Hz, 1H), 0.72 (dd, J = 7.3, 2.6 Hz, 1H), 0.33–0.18 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 208.1, 171.6, 84.9, 74.1, 51.7, 41.2, 34.7, 30.6, 27.5, 18.2, 9.1, 8.5; ${[\alpha ]}_{\text{D}}^{20}=-74.2(c0.07,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₂H₁₈O₄Na 249.1108, found 249.1105.

Methyl 2-((4S,6R)-4-((E)-1-iodoprop-1-en-2-yl)-5-oxaspiro[2.5] octan-6-yl)acetate (16c)

Compound 16c was prepared from 30 (30 mg, 0.13 mmol) by following the same procedure outlined for 16a to give a colorless oil. Yield (18 mg, 82% brsm). ¹H NMR (400 MHz, CDCl₃) δ 6.11 (s, 1H), 4.32 (s, 1H), 3.99–3.87 (m, 1H), 3.70 (s, 3H), 2.64 (dd, J = 15.1, 7.0 Hz, 1H), 2.48 (dd, J = 15.1, 6.0 Hz, 1H), 2.08 (dd, J = 26.6, 13.5 Hz, 1H), 1.75 (s, 3H), 1.74–1.63 (m, 1H), 1.52 (ddd, J = 24.3, 12.9, 4.1 Hz, 1H), 1.09–1.01 (m, 1H), 0.48 (dd, J = 8.5, 4.7 Hz, 1H), 0.38–0.29 (m, 1H), 0.23 (dt, J = 9.8, 5.1 Hz, 1H), 0.15 (dt, J = 9.2, 4.8 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 171.8, 144.8, 84.6, 80.2, 74.7, 51.6, 41.2, 34.8, 31.0, 23.4, 20.4, 9.3, 8.5; ${[\alpha ]}_{\text{D}}^{20}=+7.4(c1.3,{\text{ CHCl}}_3)$.

Methyl 2-((2R,6S)-6-acetyl-5,5-dimethyltetrahydro-2H-pyran-2-yl) acetate (31)

To a solution of cyclopropane methyl ketone 30 (30 mg, 0.13 mmol) in AcOH (2 mL) was added PtO₂ (45 mg, 0.20 mmol). The reaction mixture was stirred at 40 °C under a hydrogen-filled balloon for 30 min. After this period, the reaction mixture was filtered through Celite, washed with EtOAc (20 mL), and concentrated under reduced pressure. The residue was purified by silica gel chromatography (10% EtOAc in hexanes) to give gem-dimethyl ketone 31 as an oil (26 mg, 85%). ¹H NMR (400 MHz, CDCl₃) δ 3.77 (td, J = 8.1, 4.0 Hz, 1H), 3.68 (s, 3H), 3.51 (s, 1H), 2.61 (dd, J = 15.0, 7.8 Hz, 1H), 2.48 (dd, J = 15.0, 5.1 Hz, 1H), 2.10 (s, 3H), 1.60–1.42 (m, 4H), 0.97 (s, 3H), 0.91 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 209.6, 171.5, 89.8, 74.4, 51.6, 41.1, 39.2, 32.6, 27.9, 27.5, 26.8, 19.9; ${[\alpha ]}_{\text{D}}^{20}=-73.8(c0.93,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₁₂H₂₀O₄Na 251.1265, found 251.1263.

Methyl 2-((2R,6S)-6-((E)-1-iodoprop-1-en-2-yl)-5,5-dimethyltetrahydro-2H-pyran-2-yl)acetate (16d)

Compound 16d was prepared from 31 (40 mg, 0.18 mmol) by following the same procedure outlined for 16a to give a light yellow oil. Yield (19 mg, 75% brsm). ¹H NMR (400 MHz, CDCl₃) δ 6.09 (s, 1H), 3.76 (dd, J = 13.1, 6.6 Hz, 1H), 3.70 (s, 1H), 3.67 (s, 3H), 2.60 (dd, J = 15.0, 6.8 Hz, 1H), 2.45 (dd, J = 15.0, 6.0 Hz, 1H), 1.80 (s, 3H), 1.54–1.46 (m, 4H), 0.87 (s, 3H), 0.83 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 188.0, 171.6, 146.0, 89.4, 79.8, 74.8, 51.5, 41.1, 39.2, 33.3, 27.9, 27.7, 23.1, 20.1; ${[\alpha ]}_{\text{D}}^{20}=+13.3(c0.06,{\text{ CHCl}}_3)$.

Methyl 2-((2R,6S)-6-((2E,4E,6S,8E,10S,11R,12R)-12-((tert-butyldimethylsilyl)oxy)-11-methoxy-6,8,10-trimethyltrideca-2,4,8-trien-2-yl)-5-methylenetetrahydro-2H-pyran-2-yl)acetate (32)

A mixture of vinyl iodide 16a (25 mg, 0.07 mmol) and pinacol boronate 17 (38 mg, 0.08 mmol) in THF (3.0 mL) was stirred under argon. To this solution was added Pd(PPh₃)₄ (4.3 mg, 0.003 mmol) and Cs₂CO₃ (487 mg, 1.49 mmol). The resulting mixture was heated at 55 °C for 5 h. The reaction mixture was cooled to 23 °C. The mixture was diluted with H₂O and the aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (5% EtOAc in hexanes) to give triene compound 32 as an oil (24 mg, 47%). ¹H NMR (400 MHz, CDCl₃) δ 6.22 (dd, J = 15.1, 10.9 Hz, 1H), 5.98 (d, J = 10.9 Hz, 1H), 5.56 (dd, J = 15.1, 7.5 Hz, 1H), 5.12 (d, J = 9.0 Hz, 1H), 4.78 (s, 1H), 4.60 (d, J = 1.4 Hz, 1H), 4.28 (s, 1H), 3.96 (dtd, J = 11.1, 6.5, 2.0 Hz, 1H), 3.82 (p, J = 6.2 Hz, 1H), 3.67 (s, 3H), 3.43 (s, 3H), 2.77 (t, J = 5.4 Hz, 1H), 2.66–2.52 (m, 2H), 2.50–2.29 (m, 4H), 2.02 (dd, J = 13.2, 6.9 Hz, 1H), 1.90 (dd, J = 13.4, 7.6 Hz, 1H), 1.82 (ddd, J = 5.9, 5.0, 2.5 Hz, 1H), 1.73 (d, J = 0.8 Hz, 3H), 1.58 (d, J = 1.1 Hz, 3H), 1.51–1.39 (m, 1H), 1.12 (d, J = 6.3 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 4.7 Hz, 12H), 0.06 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 171.6, 144.4, 140.6, 133.2, 131.4, 130.8, 127.9, 123.6, 109.3, 89.7, 84.7, 73.4, 70.1, 60.7, 51.5, 47.4, 41.0, 35.0, 33.7, 32.8, 32.2, 25.9, 20.1, 19.9, 18.0, 16.1, 15.4, 13.6, −4.7; ${[\alpha ]}_{\text{D}}^{20}=-25.0(c0.06,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₃₂H₅₆O₅SiNa 571.3789, found 571.3793.

Methyl 2-((2R,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methylenetetrahydro-2H-pyran-2-yl) acetate (33)

To a stirred solution of compound 32 (50 mg, 0.09 mmol) in MeOH (2 mL) at 0 °C was added 0.2 M HCl in MeOH (1 mL). The ice-bath was removed and the stirring was continued for 1 h. The reaction was quenched with saturated NaHCO₃ solution. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were dried over Na₂SO₄, and concentrated under reduced pressure. The crude was used for the next step without any further purification.

To a mixture of alcohol (35 mg, 0.08 mmol) and VO(acac)₂ (5.4 mg, 0.02 mmol) in CH₂Cl₂ (2 mL) at −78 °C was added ^tBuOOH (63 μl, 5–6 M in PhH, 0.34 mmol). The resulting mixture was stirred at −20 °C for 36 h. The reaction was quenched by adding Me₂S (0.5 mL). The resulting mixture was stirred at 23 °C until the color turned green. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (20–30% EtOAc/hexanes) to yield the epoxy ester 33 as a colorless oil (23 mg, 62% over 2 steps). ¹H NMR (400 MHz, CDCl₃) δ 6.27 (dd, J = 14.9, 11.0 Hz, 1H), 5.95 (d, J = 10.8 Hz, 1H), 5.45 (dd, J = 15.0, 8.7 Hz, 1H), 4.77 (s, 1H), 4.56 (s, 1H), 4.28 (s, 1H), 4.02–3.91 (m, 1H), 3.85 (s, 1H), 3.67 (s, 3H), 3.53 (s, 3H), 2.96 (t, J = 5.3 Hz, 1H), 2.65–2.52 (m, 3H), 2.50–2.38 (m, 3H), 2.32 (t, J = 11.3 Hz, 1H), 1.92–1.79 (m, 2H), 1.73 (s, 3H), 1.58–1.51 (m, 1H), 1.44 (dd, J = 11.9, 4.1 Hz, 1H), 1.29 (s, 3H), 1.18 (d, J = 6.4 Hz, 3H), 1.04 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 171.6, 144.3, 139.2, 134.2, 127.6, 124.9, 109.2, 87.5, 84.7, 73.4, 68.2, 65.9, 61.2, 61.2, 51.5, 46.7, 40.9, 35.2, 35.0, 32.7, 32.2, 21.9, 18.9, 16.5, 13.5, 11.8; ${[\alpha ]}_{\text{D}}^{20}=-19.3(c0.16,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₂₆H₄₂O₆Na 473.2874, found 473.2880.

2-((2R,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methylenetetrahydro-2H-pyran-2-yl)acetic acid (12)

To a stirred solution of epoxy ester 33 (8.0 mg, 0.02 mmol) in (CH₂Cl)₂ (1 mL) was added Me₃SnOH (32 mg, 0.18 mmol). The reaction mixture was then heated at 80 °C for 24 h. The reaction mixture was diluted with CH₂Cl₂ (10 mL), washed with NaHSO₄ (0.01 M), then brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (5% MeOH/CH₂Cl₂) to give the acid 12 as an oil (6.6 mg, 85% yield). ¹H NMR (400 MHz, CD₃OD) δ 6.37 (dd, J = 15.0, 10.9 Hz, 1H), 5.99 (d, J = 10.4 Hz, 1H), 5.51 (dd, J = 15.0, 9.0 Hz, 1H), 4.80 (s, 1H), 4.59 (d, J = 1.4 Hz, 1H), 4.33 (s, 1H), 3.98 (dt, J = 11.1, 5.6 Hz, 1H), 3.82 (p, J = 6.4 Hz, 1H), 3.55 (s, 3H), 3.01 (dd, J = 6.3, 4.2 Hz, 1H), 2.68 (d, J = 9.4 Hz, 1H), 2.56–2.33 (m, 5H), 1.94 (dd, J = 13.4, 4.4 Hz, 1H), 1.89–1.82 (m, 1H), 1.75 (d, J = 0.9 Hz, 3H), 1.53 (ddd, J = 9.5, 6.9, 4.2 Hz, 1H), 1.43 (dd, J = 11.3, 4.7 Hz, 1H), 1.38 (t, J = 7.1 Hz, 1H), 1.32 (s, 3H), 1.14 (d, J = 6.5 Hz, 3H), 1.08 (d, J = 6.7 Hz, 3H), 0.86 (d, J = 7.0 Hz, 3H); ¹³C NMR (201 MHz, CD₃OD) δ 144.8, 139.1, 134.0, 130.9, 128.4, 127.6, 125.0, 108.1, 87.1, 84.8, 73.8, 68.4, 66.3, 61.2, 60.4, 46.6, 35.0, 35.0, 32.5, 31.9, 21.1, 18.4, 15.3, 12.4, 10.0; ${[\alpha ]}_{\text{D}}^{20}=-16.6(c0.12,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₂₅H₄₀O₆Na 459.2734, found 459.2729.

Methyl 2-((2R,5R,6S)-6-((2E,4E,6S,8E,10S,11R,12R)-12-((tert-butyldimethylsilyl)oxy)-11-methoxy-6,8,10-trimethyltrideca-2,4,8-trien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl) acetate (34)

Compound 34 was prepared from 16b (20 mg, 0.06 mmol) by following the same procedure outlined for 32 to give a yellow oil. Yield (17 mg, 51%). ¹H NMR (400 MHz, CDCl₃) δ 6.20 (dd, J = 15.0, 11.0 Hz, 1H), 5.99 (d, J = 11.0 Hz, 1H), 5.54 (dt, J = 12.4, 6.2 Hz, 1H), 5.13 (d, J = 9.2 Hz, 1H), 3.88–3.76 (m, 3H), 3.68 (s, 3H), 3.44 (d, J = 2.0 Hz, 3H), 2.78 (dd, J = 5.8, 5.0 Hz, 1H), 2.63 (dd, J = 14.8, 7.0 Hz, 1H), 2.59–2.50 (m, 1H), 2.49–2.32 (m, 2H), 2.04 (dd, J = 13.3, 6.3 Hz, 1H), 1.89 (dt, J = 13.7, 7.6 Hz, 2H), 1.83–1.75 (m, 1H), 1.70 (dt, J = 6.2, 4.3 Hz, 1H), 1.63 (s, 3H), 1.58 (d, J = 1.2 Hz, 3H), 1.52–1.42 (m, 2H), 1.12 (d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.8 Hz, 12H), 0.77 (d, J = 3.4 Hz, 3H), 0.06 (d, J = 2.7 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 171.9, 139.5, 134.0, 131.6, 130.7, 123.8, 123.2, 89.8, 82.4, 74.8, 70.1, 60.8, 51.5, 47.5, 41.4, 35.0, 33.6, 30.4, 29.2, 25.9, 20.1, 19.90, 18.0, 16.1, 15.3, 13.8, 11.6, −4.7; ${[\alpha ]}_{\text{D}}^{20}=-12.5(c0.16,{\text{ CHCl}}_3)$.

Methyl 2-((2R,5R,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-ethyltetrahydro-2H-pyran-2-yl) acetate (35)

Compound 35 was prepared from 34 (15 mg, 0.03 mmol) by following the same procedure outlined for 33 to give a light yellow oil. Yield (6.1 mg, 58% yield). ¹H NMR (400 MHz, CDCl₃) δ 6.23 (dd, J = 15.0, 11.0 Hz, 1H), 5.97 (d, J = 11.0 Hz, 1H), 5.44 (dd, J = 14.9, 8.5 Hz, 1H), 3.91–3.75 (m, 3H), 3.68 (s, 3H), 3.55 (s, 3H), 2.98 (t, J = 5.4 Hz, 1H), 2.66–2.53 (m, 3H), 2.48–2.37 (m, 2H), 1.92–1.76 (m, 3H), 1.75–1.65 (m, 2H), 1.58 (s, 3H), 1.54 (dd, J = 6.3, 3.3 Hz, 1H), 1.49–1.44 (m, 1H), 1.28 (s, 3H), 1.18 (d, J = 6.4 Hz, 3H), 1.05 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.77 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 171.8, 137.9, 135.0, 125.0, 122.9, 87.5, 82.4, 74.8, 68.2, 65.9, 61.3, 61.2, 51.5, 46.8, 41.4, 35.1, 34.7, 30.4, 29.6, 29.4, 25.8, 21.8, 18.9, 16.5, 13.8, 11.6; ${[\alpha ]}_{\text{D}}^{20}=-42.8(c0.07,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₂₆H₄₄O₆Na 475.3048, found 475.3041.

2-((2R,5R,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetic acid (13)

Compound 13 was prepared from 35 (5.2 mg, 0.01 mmol) by following the same procedure outlined for 12 to give a colorless oil. Yield (4.2 mg, 84% yield). ¹H NMR (400 MHz, CD₃OD) δ 6.28 (dd, J = 15.0, 11.0 Hz, 1H), 6.00 (d, J = 11.1 Hz, 1H), 5.41 (dd, J = 15.0, 8.8 Hz, 1H), 3.87–3.74 (m, 2H), 3.54 (d, J = 5.3 Hz, 3H), 3.35 (dd, J = 3.2, 1.6 Hz, 1H), 2.99 (dd, J = 6.4, 4.1 Hz, 1H), 2.65 (d, J = 9.4 Hz, 1H), 2.53–2.38 (m, 3H), 1.93–1.82 (m, 3H), 1.71 (d, J = 17.8 Hz, 2H), 1.64 (s, 3H), 1.54–1.42 (m, 3H), 1.27 (s, 3H), 1.23–1.20 (m, 1H), 1.12–1.10 (m, 3H), 1.04 (d, J = 6.3 Hz, 3H), 0.85–0.82 (m, 3H), 0.78 (d, J = 7.1 Hz, 3H); ¹³C NMR (201 MHz, CDCl₃) δ 139.0, 133.6, 125.0, 124.0, 87.6, 82.8, 75.6, 68.6, 65.9, 61.3, 61.0, 47.5, 41.4, 36.0, 35.5, 30.3, 29.7, 29.1, 26.0, 22.3, 19.6, 16.3, 14.04, 11.6, 11.2; ${[\alpha ]}_{\text{D}}^{20}=-35.5(c0.09,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₂₅H₄₂O₆Na 461.2890, found 461.2884.

Methyl 2-((4S,6R)-4-((2E,4E,6S,8E,10S,11R,12R)-12-((tert-butyldimethylsilyl)oxy)-11-methoxy-6,8,10-trimethyltrideca-2,4,8-trien-2-yl)-5-oxaspiro[2.5]octan-6-yl)acetate (36)

Compound 36 was prepared from 16c (17 mg, 0.05 mmol) by following the same procedure outlined for 32 to give a colorless oil. Yield (12 mg, 44%). ¹H NMR (400 MHz, CDCl₃) δ 6.14–6.04 (m, 1H), 5.78 (d, J = 10.9 Hz, 1H), 5.51 (dd, J = 15.1, 7.5 Hz, 1H), 5.11 (d, J = 9.2 Hz, 1H), 4.14 (s, 1H), 3.94–3.78 (m, 2H), 3.66 (s, 3H), 3.43 (s, 3H), 2.78 (dd, J = 12.5, 7.4 Hz, 1H), 2.64 (dt, J = 9.4, 7.7 Hz, 1H), 2.53 (ddd, J = 9.2, 6.9, 5.0 Hz, 1H), 2.46 (ddd, J = 15.0, 6.2, 3.9 Hz, 1H), 2.35 (dq, J = 14.0, 7.0 Hz, 1H), 2.12–1.96 (m, 2H), 1.94–1.81 (m, 1H), 1.65 (d, J = 0.8 Hz, 3H), 1.56 (t, J = 5.7 Hz, 3H), 1.52–1.45 (m, 1H), 1.12 (d, J = 6.3 Hz, 3H), 1.07–0.99 (m, 1H), 0.97–0.91 (m, 3H), 0.90–0.86 (m, 12H), 0.47 (d, J = 8.2 Hz, 1H), 0.34–0.24 (m, 1H), 0.22 – −0.07 (m, 8H); ¹³C NMR (101 MHz, CDCl₃) δ 171.9, 140.2, 132.0, 131.4, 130.8, 128.0, 123.5, 89.7, 85.0, 74.2, 70.1, 60.8, 51.5, 47.5, 41.3, 35.0, 33.6, 31.0, 25.9, 20.4, 20.1, 20.0, 18.0, 16.1, 15.4, 14.9, 9.1, 8.6, −4.7; ${[\alpha ]}_{\text{D}}^{20}=-10.8(c0.12,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₃₅H₅₈O₅SiNa 585.3946, found 585.3938.

Methyl 2-((4S,6R)-4-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-oxaspiro[2.5]octan-6-yl)acetate (37)

Compound 37 was prepared from 36 (13 mg, 0.02 mmol) by following the same procedure outlined for 33 to give a colorless oil. Yield (6.2 mg, over 2 steps 57%). ¹H NMR (400 MHz, CDCl₃) δ 6.17 (ddd, J = 16.4, 10.9, 5.9 Hz, 1H), 5.79 (d, J = 10.8 Hz, 1H), 5.45 (dd, J = 15.1, 8.7 Hz, 1H), 4.17 (s, 1H), 4.00–3.83 (m, 2H), 3.70 (s, 3H), 3.57 (s, 3H), 3.00 (t, J = 5.4 Hz, 1H), 2.69–2.36 (m, 5H), 2.07 (ddd, J = 13.4, 12.5, 10.2 Hz, 1H), 1.89 (dt, J = 13.6, 5.0 Hz, 1H), 1.76–1.65 (m, 3H), 1.65 (d, J = 0.9 Hz, 2H), 1.59–1.45 (m, 3H), 1.33–1.25 (m, 3H), 1.24–1.19 (m, 3H), 1.11–1.02 (m, 3H), 0.97–0.85 (m, 3H), 0.56–0.42 (m, 1H), 0.39–0.23 (m, 1H), 0.21–0.06 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 171.9, 138.9, 133.2, 127.7, 125.0, 87.6, 85.0, 74.3, 68.3, 66.0, 61.3, 61.3, 51.6, 46.8, 41.4, 35.3, 35.0, 31.1, 29.7, 22.0, 20.3, 19.0, 16.6, 14.8, 11.8, 9.1, 8.8.

2-((4S,6R)-4-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-oxaspiro[2.5]octan-6-yl)acetic acid (14)

Compound 14 was prepared from 37 (5 mg, 0.13 mmol) by following the same procedure outlined for 12 to give a colorless oil. Yield (3.9 mg, 82%). ¹H NMR (400 MHz, CDCl₃) δ 6.17–6.08 (m, 1H), 5.88 (d, J = 11.2 Hz, 1H), 5.41 (dd, J = 15.0, 9.2 Hz, 1H), 4.10 (s, 1H), 3.91 (dd, J = 12.2, 6.4 Hz, 2H), 3.58 (d, J = 3.4 Hz, 3H), 3.12–3.08 (m, 1H), 2.64–2.46 (m, 3H), 2.37 (dd, J = 12.4, 7.8 Hz, 1H), 2.16–2.05 (m, 1H), 1.95 (dd, J = 13.5, 4.1 Hz, 1H), 1.64 (s, 3H), 1.62 (s, 2H), 1.59 (d, J = 4.1 Hz, 2H), 1.57 (s, 2H), 1.26 (d, J = 1.9 Hz, 3H), 1.17 (d, J = 6.5 Hz, 3H), 1.04 (d, J = 6.7 Hz, 3H), 0.80 (d, J = 7.0 Hz, 3H), 0.60–0.50 (m, 1H), 0.37–0.28 (m, 1H), 0.25–0.16 (m, 1H), 0.11 (dd, J = 9.2, 4.6 Hz, 1H); ${[\alpha ]}_{\text{D}}^{20}=+16.3(c0.11,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₂₆H₄₃O₆ 451.3072, found 451.2302.

Methyl 2-((2R,6S)-6-((2E,4E,6S,8E,10S,11R,12R)-12-((tert-butyldimethylsilyl)oxy)-11-methoxy-6,8,10-trimethyltrideca-2,4,8-trien-2-yl)-5,5-dimethyltetrahydro-2H-pyran-2-yl)acetate (38)

Compound 38 was prepared from 16d (18 mg, 0.05 mmol) by following the same procedure outlined for 32 to give a colorless oil. Yield (16 mg, 47%). ¹H NMR (400 MHz, CDCl₃) δ 6.21–6.14 (m, 1H), 5.81 (d, J = 10.8 Hz, 1H), 5.51 (dd, J = 15.1, 7.5 Hz, 1H), 5.12 (d, J = 8.5 Hz, 1H), 3.83 (dd, J = 12.3, 6.2 Hz, 1H), 3.75 (dd, J = 12.6, 6.1 Hz, 1H), 3.66 (s, 3H), 3.53 (s, 1H), 3.43 (s, 3H), 2.79–2.76 (m, 1H), 2.65–2.51 (m, 2H), 2.49–2.31 (m, 2H), 2.01 (dd, J = 13.0, 7.1 Hz, 1H), 1.90 (dd, J = 13.3, 7.6 Hz, 1H), 1.72 (d, J = 0.9 Hz, 3H), 1.58 (d, J = 1.3 Hz, 3H), 1.55–1.41 (m, 4H), 1.12 (d, J = 6.1 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H), 0.92–0.86 (m, 15H), 0.79 (s, 3H), 0.06 (d, J = 3.0 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 171.8, 139.9, 133.6, 131.5, 130.8, 127.8, 123.8, 90.1, 89.8, 74.5, 70.1, 60.8, 51.4, 47.5, 41.3, 39.5, 35.0, 33.7, 33.7, 28.0, 28.0, 25.9, 20.4, 20.1, 20.0, 18.0, 16.1, 15.4, 14.5, −4.7; ${[\alpha ]}_{\text{D}}^{20}=+9.2(c0.13,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₃₃H₆₀O₅SiNa 587.4115, found 587.4110.

Methyl 2-((2R,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5,5-dimethyltetrahydro-2H-pyran-2-yl) acetate (39)

Compound 39 was prepared from 38 (18 mg, 0.02 mmol) by following the same procedure outlined for 33 to give a colorless oil. Yield (9.8 mg, 66% yield, over 2 steps). ¹H NMR (400 MHz, CDCl₃) δ 6.22 (dd, J = 15.0, 10.8 Hz, 1H), 5.79 (d, J = 10.9 Hz, 1H), 5.41 (dd, J = 15.1, 8.7 Hz, 1H), 3.85 (d, J = 4.3 Hz, 1H), 3.76 (dt, J = 13.0, 6.4 Hz, 1H), 3.66 (s, 3H), 3.57–3.51 (m, 4H), 2.97 (t, J = 5.4 Hz, 1H), 2.64–2.52 (m, 3H), 2.48–2.36 (m, 2H), 1.88 (dd, J = 13.5, 4.8 Hz, 1H), 1.72 (d, J = 1.0 Hz, 3H), 1.58–1.43 (m, 5H), 1.28 (s, 3H), 1.18 (d, J = 6.4 Hz, 3H), 1.04 (d, J = 6.7 Hz, 3H), 0.90–0.87 (m, 6H), 0.78 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 171.8, 138.4, 134.7, 127.4, 125.1, 90.1, 87.5, 74.5, 68.2, 66.0, 61.2, 51.5, 46.8, 41.3, 39.5, 35.2, 35.0, 33.6, 27.9, 22.0, 20.5, 18.9, 16.5, 14.4, 11.8; ${[\alpha ]}_{\text{D}}^{20}=-4.5(c0.20,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₂₇H₄₆O₆Na 489.3191, found 489.3188.

2-((2R,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5,5-dimethyltetrahydro-2H-pyran-2-yl)acetic acid (15)

Compound 15 was prepared from 39 (9.8 mg, 0.02 mmol) by following the same procedure outlined for 12 to give a colorless oil. Yield (8.5 mg, 87% yield). ¹H NMR (800 MHz, CD₃OD) δ 6.30 (dd, J = 15.0, 10.9 Hz, 1H), 5.86 (d, J = 10.9 Hz, 1H), 5.44 (dt, J = 15.2, 7.6 Hz, 1H), 3.84–3.80 (m, 1H), 3.79–3.76 (m, 1H), 3.61–3.52 (m, 4H), 2.99 (dd, J = 6.3, 4.2 Hz, 1H), 2.67 (d, J = 9.4 Hz, 1H), 2.54–2.41 (m, 3H), 1.93 (dt, J = 20.6, 10.3 Hz, 1H), 1.74 (s, 3H), 1.59–1.48 (m, 5H), 1.30 (s, 4H), 1.22–1.18 (m, 1H), 1.13 (d, J = 6.5 Hz, 3H), 1.06 (d, J = 6.7 Hz, 3H), 0.91 (s, 3H), 0.87 (d, J = 7.0 Hz, 3H), 0.83 (s, 3H); ¹³C NMR (201 MHz, CD₃OD) δ 138.4, 134.3, 127.4, 125.1, 90.0, 87.1, 74.8, 68.4, 66.4, 61.2, 60.4, 48.0, 46.7, 41.2, 39.3, 35.0, 35.0, 33.3, 27.7, 27.2, 21.3, 19.6, 18.4, 15.3, 13.4, 10.1; ${[\alpha ]}_{\text{D}}^{20}=-20.0(c0.27,{\text{ CHCl}}_3)$; HRMS (m/z) (ESI M + Na⁺) calc. for C₂₆H₄₄O₆Na 475.3030, found 475.3034.

In vitro splicing assays

10 nM radiolabeled pre-mRNA substrate was incubated at 30 °C for 30 minutes in 50% HeLa cell nuclear extract supplemented with 60 mM potassium glutamate, 2 mM magnesium acetate, 2 mM ATP, 5 mM creatine phosphate, 0.05 mg mL⁻¹ tRNA, and increasing concentrations of compounds dissolved in DMSO or DMSO alone. RNA was then and separated by denaturing polyacrylamide gel electrophoresis. Radiolabeled RNA was visualized by phosphorimaging, and individual bands were quantified with ImageQuant software (Molecular Dynamics). Splicing efficiency is calculated as the amount of mRNA relative to total substrate RNA and normalized to a DMSO control reaction. Values from three independent measurements were graphed and IC₅₀ values were determined by nonlinear regression using the [Inhibitor] vs. normalized response model in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California USA, http://www.graphpad.com.

Native gel analysis

Aliquots of in vitro splicing reactions were taken at different time intervals, and splicing complexes were separated native agarose gel electrophoresis as detailed in.³⁵ Radiolabeled RNA was visualized by phosphorimaging.