Studies on Taxol Biosynthesis: Preparation of Taxadiene-diol- and triol-Derivatives by Deoxygenation of Taxusin

Abstract

The putative taxol biosynthesis metabolites, taxa-4(20),11(12)-diene-5α, 13α -diol (7), taxa-4(20),11(12)-diene-5α, 9α, 13α-triol (9), and taxa-4(20),11(12)-diene-5α, 10β, 13α-triol (10), have been prepared by Barton deoxygenation of the C-9 and C10-hydroxyl groups of protected derivatives of taxusin, a major taxoid metabolite isolated from Yew heart wood. The synthetic protocol devised, is amenable for the preparation of isotopically labeled congeners that will be useful to probe further intermediate steps in the biosynthesis of taxol.

Introduction

The approval of taxol (Paclitaxel, 1) as a clinically useful therapeutic agent against ovarian and other types of cancer continues to stimulate interest in both the chemical synthesis and biogenesis of this agent.^1,2 Numerous approaches for the synthesis of the taxane framework³ and for taxol itself⁴ have been reported but the architectural complexity of the taxol core mandates lengthy syntheses, with low overall yields rendering chemical synthesis approaches to this drug impractical for large-scale manufacturing of clinical-grade material. The bark of Taxus brevifolia Nutt., the Pacific yew tree, served as the initial source for commercial-scale production of taxol.¹ However, this species grows in environmentally sensitive areas of the Pacific Northwest and has become an untenable source for large-scale taxol production. Alternative approaches for taxol production such as semi-synthesis from 10-deacetylbaccatin III, which can be isolated from the needles of the European yew Taxus baccata, a renewable resource, were subsequently adopted as the current commercial method for taxol production.⁵ Additionally, it is reasonable to anticipate that taxol will become utilized for other types of cancer and as a consequence, pressure on the population of yew species worldwide will increase significantly. Alternative sources for taxol production based on biological systems have become an important goal and our laboratories have sought to employ emerging technologies based on the genetic manipulation of Taxus sp. cell cultures to address this problem. In order to meet these objectives, we have sought to elucidate a detailed understanding of the steps of taxol biosynthesis and the identification of the associated genes.

Previously, we have reported a combination of in vivo feeding studies and investigations with cell-free enzyme systems, using Taxus sp. stem tissue or suspension cultured Taxus sp. cells as bioconversion vectors. The experimental results of these investigations have led us to surmise that the early steps of taxol biosynthesis proceed in sequence from the initial conversion of geranylgeranyl pyrophosphate (2) to the parent diene 3 catalyzed by taxadiene synthase (Scheme 1).^6,7 Following this first committed step in taxol biosynthesis, taxadiene hydroxylase, a cytochrome P-450-dependent enzyme, regioselectively hydroxylates 3 with allylic transposition of the 4,5-double bond, to the first oxygenated metabolite, taxa-4(20),11(12)-diene-5α-ol (4).⁸ The resulting intermediate taxadienol 4 can be subsequently acetylated through the action of taxadienol-O-acetyltransferase to provide the corresponding acetate 5.^9a Studies have shown that taxa-4(20),11(12)-diene-5α-acetate (5) was converted to taxa-4(20),11(12)-diene-5α,10β-diol-5-acetate (6) by taxoid 10β-hydroxylase.^10,12a Experiments also demonstrated that taxa-4(20),11(12)-diene-5α-ol (4) was converted by a 13α-hydroxylase to taxa-4(20),11(12)-diene-5α,13α-diol (7, Scheme 1),^12b thereby introducing uncertainties as to the precise order of the early hydroxylation and C5-acetylation diverging from the confirmed intermediate taxadienol (4). The complementary but opposite substrate selectivities of the 13α-hydroxylase and the 10β-hydroxylase, which favors taxadien-5α-ol and taxadien-5α-acetate as substrates respectively, as well as feeding studies of Taxus sp. cells with the relevant intermediates,¹³ suggest a bifurcation in the early stages of Taxol biosynthesis.¹⁴ Based on the relative abundance of the several hundreds defined taxoids bearing oxy-functional groups at various positions, the subsequent order of hydroxylation reactions downstream appears likely to occur at C10, followed by C13 and C9, and later C7 and C2, the precise timing of acetylation at C5 is, however, uncertain.¹⁴ Considering numerous possible parallel sequences of oxygenation, we have decided to investigate a series of plausible intermediates downstream from 4 by way of the C13-oxygenated species⁷ that can be converted into taxa-4(20), 11(12)-diene-5α,9α,13α-triol (9) or taxa-4(20), 11(12)-diene-5α,10β,13α-triol (10). In order to evaluate the possible intermediacy of such species, we required access to authentic specimens of 7, 9 and 10 since we have found that sufficiently good substrates for subsequent hydroxylation reactions are often produced in trace amounts and whose structures are very difficult to confirm. We report herein, the semi-synthesis of 7, 9 and 10 by radical deoxygenation of taxusin.

Scheme 1.

Possible divergence of taxadiene to lightly oxygenated taxoids and convergence to a putative pentaol.

Results and Discussion

Taxusin was selected as a starting substrate due to its structural relatedness to a variety of putative pathway metabolites and its high natural abundance in European and Japanese yew heart woods. Previous studies from our laboratory have revealed that taxusin can be deacetylated efficiently to the corresponding tetraol and re-protected with modest efficiency to form a series of differentially protected polyols that can be selectively deoxygenated by classical radical deoxygenation methods with the exception of the C13 alcohol.¹⁵ All attempts to remove the C13 alcohol results in rearrangement of the C11(12)-bridgehead alkene to the C12(13) alkene. Despite our failure to deoxygenate taxusin at C-13, this substrate proved to be useful for making lightly oxygenated taxoids with oxygenation at C13.

Taxusin was partially deacylated by treatment with K₂CO₃ in THF/MeOH at 0 °C for 3 days, which gave the 5,13-diol 12 predominantly with some 9,10-diol. A small amount of the more polar taxusin tetraol was produced but easily separated (Scheme 2). The diol 12 was treated with TESCl in the presence of imidazole in DMF, followed by deacetylation of the 9,10-diacetyl groups using lithium aluminum hydride (LAH) in THF, which provided diol 13 in high yield. Exposure of 13 to LHMDS followed by addition of carbon disulfide and methyl iodide, gave an inseparable mixture of the xanthates 14a and 14b in approximately 1:1 ratio. The C-9 and C-10-hydroxyl residues were successfully removed by Barton deoxygenation respectively to give an inseparable mixture of 15a and 15b. Finally, removal of the O-TES ethers by treatment with TBAF in the presence of acetic acid in THF, gave the desired triols taxa-4(20), 11(12)-diene-5α, 10β, 13α-triol (10) and taxa-4(20), 11(12)-diene-5α, 9α, 13α-triol (9), which were separable by silica gel chromatography (Scheme 2).

Scheme 2.

Synthesis of taxa-4(20), 11(12)-diene-5α, 10β, 13α-triol (10) and taxa-4(20), 11(12)-diene-5α, 9α, 13α-triol (9).

The mixture of 15a/15b, also proved useful for the synthesis of the corresponding taxa-4(20), 11(12)-diene-5α, 13α-diol. As shown in Scheme 3, 15a/15b was subjected to treatment with LHMDS in THF followed by the addition of carbon disulfide and methyl iodide to give a mixture of the xanthates 16a/16b, which could be separated on column but was used as a mixture in the following step. Barton deoxygenation of 16a/16b yielded the protected diol 17 in 78% yield. Final treatment of 17 with HF-pyridine complex afforded taxa-4(20), 11(12)-diene-5α, 13α-diol (7) (Scheme 3).

Scheme 3.

Synthesis of taxa-4(20), 11(12)-diene-5α, 13α-diol.

While this route provided the first authentic specimens of 9 and 10, it proved to be capricious due to the lability of the O-TES ethers. As such, a more robust, second-generation synthesis was devised as shown in Scheme 4 where O-diethyl-iso-propylsilyl ethers (ODEIPS) were employed.

Scheme 4.

Cyclic carbonate route to 9 and 10.

In addition, we found that conversion of taxusin into the corresponding tetraol, followed by protection of the 9,10-diol unit as its cyclic carbonate proved advantageous and reproducible.

Taxusin was converted into tetraol 18 by global reductive removal of the acetates with LAH. Cyclic carbonate formation followed by protection of the C5 and C13 hydroxyl groups as O-DEIPS ethers proceeded in 62% overall yield. Reductive removal of the cyclic carbonate followed by xanthate formation, gave the inseparable xanthates 22a/22b, which were directly subjected to Barton deoxygenation conditions. Final removal of the silyl ethers with tetra-n-butylammonium fluoride (TBAF) produced 9 and 10 (73% and 10% yields, respectively). Similarly, 23a and 23b were converted to 7 in good overall yield as shown in Scheme 5.

Scheme 5.

Conversion of 23b to 7.

The synthesis of these substrates with deuterium labeling is enabled through the use of tri-n-butyltin deuteride in the radical deoxygenation step.

Experimental

5,13-Dideacetyltaxusin (12)

To a solution of taxusin (120 mg, 0.24 mmol) in THF/MeOH (4 mL, 1:1) was added K₂CO₃ (49 mg, 0.36 mmol) at 0 °C. The mixture was stirred for 3 days at 0 °C, after which time, EtOAc (20 mL) and sat. NH₄Cl (aq) (5 ml) were added to the mixture. The organic layer was separated and washed with brine. The organic layer was concentrated and purified by chromatography (silica gel, hexanes/EtOAc 1:1), yielding 12 (80 mg, 80%). ¹H NMR (300 MHz, CDCl₃) δ 6.05 (d, J = 10.2 Hz, 1 H), 5.69 (d, J = 10.2 Hz, 1 H), 5.04 (s, 1 H), 4.69 (s, 1 H) 4.40-4.24 (br s, 1 H), 4.28 (s, 1 H), 3.32 (d, J = 8.4 Hz, 1 H), 3.24 (d, J = 4.5 Hz, 1 H), 2.90-2.70 (comp, 2 H), 2.20 (d, J = 1.2 Hz, 3 H), 2.02 (s, 3 H), 1.98 (s, 3 H), 1.94-1.54 (comp, 7 H), 1.49 (s, 3 H), 1.24-1.16 (m, 1 H), 0.88 (s, 3 H), 0.68 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 170.5, 170.1, 152.3, 141.7, 134.8, 110.7, 77.7, 74.3, 73.6, 68.5, 43.4, 39.5, 38.6, 36.3, 35.8, 32.8, 30.5, 27.2, 26.6, 25.9, 21.2, 20.9, 17.1, 16.7; IR (NaCl) 3450 (br), 2944, 1724, 1242 cm⁻¹; HR-MS calcd for C₂₄H₃₅O₆ (M⁺-H) 419.2434, found 419.2422.

5α,13α-Bis(triethylsilyloxy)-taxa-4(20),11(12)-diene-9α,10β-diol (13)

To the solution of 12 (1.32 g, 3.13 mmol) and imidazole (534 mg, 7.85 mmol) in DMF (5 ml), TESCl (991 mg, 6.57 mmol) was added at 0 °C. The mixture was stirred for 24 h at rt. The mixture was diluted by diethyl ether (40 ml) and washed with water, NH₄Cl (aq), and brine and dried over Na₂SO₄, yielding a crude mixture, which was used in the next reaction without further purification. Thus, a solution of the crude diacetate prepared above and LAH (238 mg, 6.26 mmol) in THF (10 ml) was stirred at rt for 15 h. The mixture was diluted by THF-H₂O (3:1, 10 ml) at 0 °C. The mixture was filtered and the filtrate was diluted with EtOAc. The mixture was washed by NH₄Cl (aq) and brine, dried over Na₂SO₄, and condensed in vacuo, yielding diol 13 (1.60 g, 90 %). ¹H NMR (400 MHz, CDCl₃) δ 4.91 (s, 1 H), 4.80 (d, J = 9.8 Hz, 1 H), 4.82-4.75 (m, 1 H), 4.58 (s, 1 H), 4.15 (t, J = 2.6 Hz, 1 H), 4.12 (d, J = 9.8 Hz, 1 H), 2.98 (d, J = 5.6 Hz, 1 H), 2.36 (td. J = 9.2, 14.0 Hz, 1 H), 2.48-2.22 (br s, 2 H), 1.99 (s, 3 H), 1.76-1.50 (comp, 7 H), 1.50 (s, 3 H), 1.17 (s, 3 H), 1.18-1.10 (m, 1 H), 1.2-0.88 (comp, 21 H), 0.70-0.55 (comp, 12 H); ¹³C NMR (100 MHz, CDCl₃) δ 155.4, 140.0, 135.5, 109.4, 79.6, 74.7, 73.0, 69.4, 43.1, 42.0, 39.7, 36.7, 35.9, 32.0, 31.6, 29.0, 28.5, 25.6, 18.4, 14.9, 7.2, 5.1, 4.7.

Taxa 4(20), 11(12)-diene-5α, 10β, 13α-triol (10)

Procedure A

To the solution of 13 (137 mg, 0.24 mmol) in THF (3 mL), LHMDS (0.25 mmol, 1 M THF solution) was added at 0 °C. After stirred for 5 min, CS₂ (0.1 mL) was added to the mixture. The mixture was stirred for 30 min at rt, then MeI (16 µL, 36 mg, 0.25 mmol) was added. The mixture was stirred for another 14 h at rt. The mixture was diluted with CH₂Cl₂ (20 mL) and washed with water. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by preparative TLC (hexanes/EtOAc 30:1), yielding a inseparable mixture of 14a and 14b (75 mg, 47 %).

To a solution of 14a and 14b (75 mg, 0.11 mmol) obtained above in toluene (2 mL), AIBN (cat.) and nBu₃SnH (63 µL, 0.23 mmol) were added. The mixture was stirred at reflux for 3 h. The mixture was concentrated and purified by flash chromatography (silica gel, hexanes/EtOAc 11:1), yielding an inseparable mixture of 15a and 15b (54 mg, 86 %).

To a solution of 15a and 15b (67 mg, 0.12 mmol) in THF were added TBAF (0.26 mmol, 1 M THF solution) and AcOH (1 drop). After 9 h additional TBAF (0.52 mmol) was added and the mixture was stirred for 72 h. The mixture was diluted with EtOAc and washed with NH₄Cl (aq) and brine. The organic layer was concentrated and purified by preparative TLC (silica gel, hexanes/EtOAc 1:1, then CHCl₃/MeOH 20:1), yielding triol 10 (20 mg, 51 %) and triol 9 (15 mg, 38 %).

Procedure B

A solution of 23a and 23b (280 mg, 0.485 mmol) and TBAF (651 mg, 2.50 mmol) in DMF (30 mL) was stirred at 55°C for 12 h. The mixture was diluted with EtOAc (75 mL) and washed with H₂O (3 × 50 mL) and brine (50 mL). The organic layer was concentrated and purified by flash chromatography (silica gel, eluted with hexanes/EtOAc 3:2 then 1:1), yielding triol 9 (120 mg, 77%) and triol 10 (27 mg, 17%). ¹H NMR (300 MHz, CDCl₃) δ 5.15 (dd, J = 5.7, 11.7 Hz, 1 H), 4.97 (s, 1 H), 4.64 (s, 1 H), 4.38-4.22 (comp, 2 H), 3.22 (s, 1 H), 2.88-2.60 (comp, 3 H), 2.18 (dd, J = 11.7, 14.4 Hz, 1 H), 2.12-2.00 (m,1 H), 2.01 (d, J = 1.2 Hz, 3 H), 1.72-1.52 (comp, 7 H), 1.48 (s, 3 H), 1.38-1.10 (comp, 2 H), 0.93 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (100 MHz, CD₃CN) 156.2, 142.5, 136.9, 109.6, 75.2, 69.1, 68.7, 48.4, 41.5, 39.8, 39.7, 37.6, 35.9, 34.1, 34.0, 32.3, 25.9, 22.5, 21.5, 17.2; IR (KBr) 3349 (br), 2927, 1652, 1456, 1004, 894 cm⁻¹; HR-MS (M⁺+Na) calcd for C₂₀H₃₂O₃Na 343.2244, found 343.2240.

Taxa 4(20), 11(12)-diene-5α, 9α, 13α-triol (9)

¹H NMR (300 MHz, CDCl₃) δ 5.03 (s, 1 H), 4.69 (s, 1 H), 4.40-4.30 (br s, 1 H), 4.32 (s, 1 H), 4.14-4.02 (m, 1 H), 3.55 (s, 1 H), 2.96-2.70 (comp, 3 H), 2.54 (dt, J = 3.8, 13.2 Hz, 1 H), 2.30-2.15 (br s, 1 H), 2.00 (s, 3 H), 1.93 (dt, J = 5.1, 13.2 Hz, 1 H), 1.80-1.60 (comp, 5 H), 1.55-1.45 (m, 1 H), 1.38-1.32 (m, 1 H), 1.33 (s, 3 H), 1.20 (dd, J = 3.9, 15.3 Hz, 1 H), 0.92 (s, 3 H), 0.83 (s, 3 H); ¹³C NMR (75 MHz, acetone-d₆) δ 156.4, 138.2, 136.3, 109.6, 76.8, 75.9, 69.6, 46.7, 40.9, 40.6, 38.1, 37.6, 37.1, 32.8, 32.5, 28.4, 26.6, 26.3, 19.0, 17.6; IR (NaCl) 3350 (br), 2941, 1448, 1101 cm⁻¹; HR-MS calcd for C₂₀H₃₂O₃Na (M⁺+Na) 343.2244, found 343.2240.

5α, 13α-Bis(triethylsilyloxy)-10β-S-methyl dithiocarboxytaxa-4(20), 11(12)-diene (16a) and 5α, 13α-di-(triethylsilyloxy)-9α-S-methyl dithiocarboxytaxa-4 (20), 11(12)-diene-10β-ol (16b)

To the solution of the mixture of 15a and 15b (62 mg, 0.11 mmol) in THF (2 mL), LHMDS (0.12 mL, 1 M THF solution) was added at 0 °C. After stirring for 10 min, CS₂ (0.1 mL) was added to the mixture. The mixture was stirred for 2 h at rt, then MeI (7 µL, 0.12 mmol) was added. The mixture was stirred for 3 h at rt. The mixture was diluted with CH₂Cl₂ (20 mL) and washed with water. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by PTLC (hexanes/EtOAc 100:1), yielding 16a (41 mg, 57%) and 16b (20 mg, 28 %). These were used for the next step without further purification.

5α, 13α-Bis(triethylsilyloxy)-taxa-4(20), 11(12)-diene (17)

To a solution of the mixture of 16a and 16b (61 mg, 0.095 mmol) obtained above in toluene (2 mL), AIBN (cat.) and nBu₃SnH (52 µL, 0.19 mmol) were added. The mixture was stirred at reflux for 3 h. The mixture was concentrated and purified by chromatography (silica gel, heptane), yielding 17 (40 mg, 79 %). ¹H NMR (400 MHz, benzne-d₆) δ 4.96-4.78 (m, 1 H), 4.64 (s, 1 H), 4.184 (t, J = 3.0 Hz, 1 H), 3.33 (m, 1 H), 2.90 (dt, J = 3.6, 12.8 Hz, 1 H), 2.44 (td, J = 9.2, 13.6 Hz, 1 H), 2.41 (dt, J = 4.8, 13.2 Hz, 1 H), 2.14 (s, 3 H), 2.15-2.00 (comp, 2 H), 1.80-1.60 (comp, 5 H), 1.38 (s, 3 H), 1.44-1.34 (m, 1 H), 1.30-1.20 (m, 1 H), 1.16 (s, 3 H), 1.14-1.00 (comp, 18 H), 1.00-0.90 (comp, 2 H), 0.80-0.62 (comp, 15 H); ¹³C NMR (100 MHz, benzne-d₆) 156.8, 137.0, 133.8, 108.7, 75.0, 70.1, 42.6, 41.7, 40.8, 40.5, 37.0, 36.5, 35.0, 32.9, 31.1, 30.1, 28.2, 26.3, 23.3, 15.3, 7.8, 7.7, 5.8, 5.4; IR (NaCl): 2951, 2361, 1457, 1239, 1118, 1062, 1007, 886, 838, 739 cm⁻¹;

Taxa 4(20), 11(12)-diene-5α, 13α-diol 7

Procedure A

To a solution of 17 (25 mg, 0.047 mmol) in pyridine (2 mL) was added HF-pyridine (0.2 mL, 70 % solution in pyridine) at 0 °C. The mixture was stirred for 3 h at room temperature and was diluted with EtOAc (30 mL) and washed with saturated CuSO₄, water, NH₄Cl (aq) and brine. The organic layer was dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by chromatography (silica gel, CHCl₃/MeOH 9:1), yielding 7 (12 mg, 84 %).

Procedure B

To a solution of the mixture of 26 (60 mg, 0.107 mmol) in THF (10 mL) were added TBAF (140 mg, 0.535 mmol). The mixture was stirred at 55°C overnight and concentrated and purified by flash chromatography (silica gel, hexanes/EtOAc 3:2), yielding diol 7 (27 mg, 83%). ¹H NMR (300 MHz, CDCl₃) δ 4.96 (s, 1 H), 4.63 (s, 1 H), 4.38-4.24 (br s, 1 H), 4.27 (t, J = 2.6 Hz, 1 H), 3.51 (br s, 1 H), 2.85 (dd, J = 5.4, 13.2 Hz, 1 H), 2.83-2.70 (comp, 2 H), 2.23 (dt, J = 5.7, 12.9 Hz, 1 H), 2.18-2.09 (br s, 2 H), 1.95 (s, 3 H), 2.00-1.90 (m, 1 H), 1.82-1.60 (comp, 5 H), 1.32 (s, 3 H), 1.28-1.16 (comp, 2 H), 1.00 (ddd, J = 2.4, 4.5, 13.2 Hz, 1 H), 0.88 (s, 3 H), 0.60 (s, 3 H); ¹³C NMR (75 MHz, CDCl₃) δ 153.7, 139.5, 132.5, 109.5, 74.7, 68.7, 40.4, 39.6, 39.24, 39.18, 36.2, 35.8, 32.4, 32.1, 31.4, 27.4, 25.4, 24.9, 22.2, 16.3; IR (NaCl): 3359 (br), 2921, 1439, 1008 cm⁻¹. HR-MS calcd for C₂₀H₃₂O₂Na (M⁺+Na) 327.2294, found 327.2292.

Cyclic carbonate (19)

A solution of tetraol 18 (6.41 g, 19.1 mmol) and CDI (5.0 g, 30.8 mmol) in toluene (300 ml) was heated at 70 °C overnight. After cooled to room temperature, the mixture was concentrated and the residue was purified by flash chromatography (silica, 1:1 EtOAc/hexanes) to give 19 as a white powder (4.80 g, 70 %). ¹H NMR (300 MHz, acetone-d₆) δ 5.79 (d, J = 12.0 Hz, 1 H), 5.09 (s, 1 H), 4.95 (d, J = 12.0 Hz, 1 H), 4.72 (s, 1 H), 4.45 (app dt, J = 4.5, 9.8 Hz, 1 H), 4.31 (d, J = 2.4 Hz, 1 H), 4.17 (d, J = 2.1 Hz, 1 H), 3.76 (d, J = 9.6 Hz, 1 H), 3.16-3.10 (br s 1 H), 2.84 (ddd, J = 9.0. 10.2, 15.3 Hz, 1 H), 2.18 (d, J = 1.2 H, 3 H), 1.99 (dd, J = 4.5, 13.5 Hz, 1 H), 1.86-1.80 (comp, 5 H), 1.46 (s, 3 H), 1.48-1.40 (m, 1 H), 1.18 (d, J = 4.8, 15.3 Hz, 1 H), 1.03 (s, 3 H), 0.92 (s, 3 H); ¹³C NMR (75 MHz, acetone-d₆) δ 155.7, 154.0, 150.5, 133.7, 110.8, 88.2, 81.1, 75.1, 69.3, 41.5, 41.4, 40.2, 37.6, 36.8, 33.9, 31.7, 28.8, 27.1, 26.3, 18.3, 17.8; IR (NaCl): 3407 (br), 2944, 2877, 1801, 1190, 1016 cm⁻¹. HR-MS calcd for C₂₁H₃₀O₅Na (M⁺+Na) 385.1991, found 385.1982.

Cyclic carbonate (20)

A solution of diol 19 (475 mg, 1.31 mmol), imidazole (700 mg, 10.2 mmol) and DEIPSCl (0.63 ml, 0.34 mmol) in DMF (7 ml) was stirred at rt for 40 h. The mixture was diluted with EtOAc (25 mL), washed with H₂O (3 × 25 mL) and brine (25 mL). The organic layer was dried over Na₂SO₄. After filtration, the filtrate was concentrated and the residue was purified by flash chromatography (silica, 1:20 then 1:10 EtOAc/hexanes) to give 20 as a clear liquid (726 mg, 89 %). ¹H NMR (300 MHz, CDCl₃) δ 5.58 (d, J = 12.0 Hz, 1 H), 5.06 (d, J = 12.0 Hz, 1 H), 4.96 (s, 1 H), 4.84 (app t, J = 9.0 Hz, 1 H), 4.60 (s, 1 H), 4.18 (t, J = 2.3 Hz, 1 H), 2.70-2.63 (s, 1 H), 2.41 (td, J = 9.3. 14.1 Hz, 1 H), 2.03 (d, J = 0.6 Hz, 3 H), 1.88-1.73 (comp, 2 H), 1.67-1.55 (comp, 4 H), 1.50 (s, 3 H), 1.53-1.44 (m, 1 H), 1.18 (s, 3 H), 1.10-0.83 (comp, 30 H), 0.70-0.54 (comp, 8 H); ¹³C NMR (75 MHz, CDCl₃) δ 155.1, 152.2, 149.2, 129.2, 110.1, 87.1, 79.6, 77.0, 74.1, 69.4, 42.0, 39.6, 39.4, 35.7, 35.4, 32.4, 30.9, 28.2, 28.0, 25.3, 17.6, 17.5, 17.4, 15.4, 13.0, 12.8, 7.20, 7.18, 7.15, 7.10, 3.7, 3.5, 3.4, 3.1; IR (NaCl): 2944, 2877, 1809, 1463, 1184 cm⁻¹. HR-MS calcd for C₃₅H₆₂O₅Si₂Na (M⁺+Na) 641.4034, found 641.4050.

5α, 13α-Bis(diethylisopropylsilyloxy)-taxa-4(20),11(12)-diene-9α, 10α-diol (21)

A mixture of the cyclic carbonate 20 (1.5 g, 2.42 mmol) and LiAlH₄ (184 mg, 4.85 mmol) in THF (150 ml) was stirred at rt for 6 h whereupon EtOAc (10 mL) was added slowly to quench excess LiAlH₄. The mixture was partitioned between EtOAc (80 mL), H₂O (40 mL) and brine (40 mL). The aqueous layer was extracted with EtOAc (2 × 80 mL) and the combined organic layers were washed with brine (150 mL). The organic layer was dried over Na₂SO₄ and filtered. The filtrate was concentrated and the residue was purified by flash chromatography (silica gel, 1:7 then 1:3 EtOAc/hexanes) to give 21 as a white solid (1.44 g, 100%). ¹H NMR (300 MHz, CDCl₃) δ 4.89 (s, 1 H), 4.80 (t, J = 1.2 Hz, 1 H), 4.76 (d, J = 9.9 Hz, 1 H), 4.57 (s, 1 H), 4.15 (s, 1 H), 4.10 (d, J = 9.9 Hz, 1 H), 2.90 (d, J = 5.4 Hz, 1 H), 2.50-2.20 (br s, 2 H), 2.33 (td, J = 9.3, 13.2, Hz, 1 H), 1.97 (d, J = 1.2 Hz, 3 H), 1.72-1.52 (comp, 7 H), 1.47 (s, 3 H), 1.15 (s, 3 H), 1.04-0.84 (comp, 27 H), 0.84 (s, 3 H), 0.70-0.54 (comp, 8 H); ¹³C NMR (75 MHz, CDCl₃) δ 155.1, 139.7, 135.3, 109.6, 79.4, 74.7, 72.7, 69.3, 42.9, 42.0, 39.4, 36.6, 35.5, 31.9, 31.4, 29.0, 28.5, 25.5, 18.3, 17.6, 17.54, 17.52, 17.49, 14.5, 13.1, 12.9, 7.24 7.21, 7.17, 3.8, 3.6, 3.5, 3.2; IR (NaCl): 3402 (br), 2944, 1463, 1072, 1014 cm⁻¹. HR-MS calcd for C₃₄H₆₄O₄Si₂Na (M⁺+Na) 615.4241, found 615.4238.

5α, 13α-Bis(diethylisopropylsilyloxy)-9α-S-methyl dithiocarboxytaxa-4(20), 11(12)-diene (24b)

A solution of KHMDS (1.18 mL, 0.5 M in toluene, 0.59 mmol) was added to 23b (200 mg, 0.347 mmol) in THF (20 mL) at −10°C. The mixture was stirred at 0°C for 10 min and then at room temperature for 20 min, whereupon carbon disulfide (36 µL, 0.58 mmol) was added, and the solution was stirred for another 30 min. MeI (36 µL, 0.54 mmol) was then added and the mixture was allowed to stir overnight. The mixture was then partitioned between EtOAc (25 mL) and 1% AcOH in water (25 mL) and the organic layer separated. The organic layer was washed with brine (25 mL) and dried over Na₂SO₄. After filtration, the filtrate was concentrated and the residue was purified by flash chromatography (silica gel, hexanes then 1:100 EtOAc/hexanes) to give 24b as a clear oil (210 mg, 91 %). ¹H NMR (300 MHz, CDCl₃) δ 6.20 (dd, J = 5.7, 11.7 Hz, 1 H), 4.92 (s, 1 H), 4.80 (app t, J = 9.0 Hz, 1 H), 4.61 (s, 1 H), 4.18 (s, 1 H), 3.19 (d, J = 5.1 Hz, 1 H), 2.89 (t, J = 12.9 Hz, 1 H), 2.56 (s, 3 H), 2.55-2.48 (br s, 1 H), 2.32 (td, J = 9.0, 14.1 Hz, 1 H), 2.20-2.05 (m, 1 H), 1.98 (s, 3 H), 1.80-1.55 (comp, 6 H), 1.63 (s, 3 H), 1.17-1.08 (m, 1 H), 1.11 (s, 3 H), 1.05-0.90 (comp, 26 H), 0.71 (s, 3 H), 0.70-0.60 (comp, 8 H) ¹³C NMR (75 MHz, CDCl₃) δ 215.6, 154.2, 136.3, 132.1, 109.9, 90.7, 74.4, 60.4, 45.2, 41.3, 39.6, 36.8, 35.2, 21.2, 30.7, 30.5, 28.9, 28.5, 26.7, 19.0, 18.3, 17.7, 17.6, 17.55, 17.53, 14.7, 13.1, 12.9, 7.29. 7.25. 7.2. 3.8. 3.6. 3.5. 3.2; IR (NaCl): 2945, 2872, 1645, 1466, 1240, 1078 cm⁻¹; HR-MS calcd for C₃₆H₆₅O₃S₂Si₂ (M⁺-H) 665.3914, found 665.3905.

5α, 13α-Bis(diethylisopropylsilyloxy)-4(20), 11(12)-diene (25)

A solution of 24b (160 mg, 0.24 mmol), AIBN (20 mg, 0.12 mmol), Bu₃SnH (140 mg, 127 µL, 0.48 mmol) in toluene (15 mL) was heated at 115°C for 3 h. The mixture was concentrated and the residue purified by flash chromatography (silica gel, hexanes then 1:100 EtOAc /hexanes) to give 25 as clear oil (100 mg, 74%). ¹H NMR (300 MHz, CDCl₃) δ 4.81 (s, 1 H), 4.75 (t, J = 8.9 Hz, 1 H), 4.55 (s, 1 H), 4.14 (t, J = 3.5 Hz, 1 H), 3.06 (d, J = 6.3 Hz, 1 H), 2.76 (ddd, J = 5.4, 12.6, 14.1 Hz, 1 H), 2.33 (td, J = 9.3, 13.8 Hz, 1 H), 2.16 (dt, J = 4.1, 13.2 Hz, 1 H), 2.10-1.90 (comp, 2 H), 1.89 (s, 3 H), 1.80-1.40 (comp, 6 H), 1.37 (s, 3 H), 1.33-1.12 (comp, 3 H), 1.05 (s, 3 H), 1.02-0.84 (comp, 25 H), 0.65 (s, 3 h), 0.74-0.62 (comp, 8 H); ¹³C NMR (75 MHz, CDCl₃) δ 156.4, 136.4, 133.3, 107.8, 73.9, 69.3, 42.0, 41.2, 39.9, 39.7, 36.8, 35.7, 34.8, 32.1, 30.7, 30.1, 27.8, 25.4, 22.7, 17.7, 17.6, 17.5, 14.6, 13.1, 12.9, 7.33, 7.30, 7.27, 7.2, 3.9, 3.7, 3.5, 3.3; IR (NaCl): 2941, 1458, 1016 cm⁻¹; IR (NaCl): 2941, 2877, 1463, 1059, 1013 cm⁻¹; HR-MS calcd for C₃₄H₆₃O₂Si₂ (M⁺-H) 559.4365, found 559.4367.