Synthesis of Side-Chain Oxysterols and their Enantiomers through Cross-Metathesis Reactions of Δ²² Steroids

Abstract

A synthetic route that utilizes a cross-metathesis reaction with Δ²² steroids has been developed to prepare sterols with varying C-27 side-chains. Natural sterols containing hydroxyl groups at the 25 and (25R)-26 positions were prepared. Enantiomers of cholesterol and (3β,25R)-26-hydroxycholesterol (27-hydroxycholesterol) trideuterated at C-19 were prepared for future biological studies.

1. Introduction

Side-chain oxysterols have been implicated in multiple important biological processes, including: bile acid synthesis [1], cholesterol regulation and transport [2-6], modulation of estrogen receptor function [7], and apoptosis [8]. Further, these oxygenated cholesterol derivatives are believed to be significant in diseases such as Alzheimer's [9, 10], Parkinson's [10, 11], multiple sclerosis [10, 12], Niemann-Pick type C disease [13], and cataracts [14]. With regards to cholesterol homeostasis, (3β)-cholest-5-ene-3,25-diol (25-hydroxycholesterol, 25-HC, 1) and (3β,25R)-cholest-5-ene-3,26-diol (27-hydroxycholesterol, 27-HC, 3) are known to be modulators of sterol regulatory element binding proteins (SREBP), liver X receptors (LXR) and hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase (Figure 1) [4].

Figure 1.

Structures of 25-HC (1), 25-HC-d₆ (2), 27-HC (3), ent-Chol-d₃ (4) and ent-27-HC-d₃ (5).

We are focused on the importance of the absolute configuration of 25-HC and 27-HC for their modes of action on cholesterol homeostatic-regulating proteins. We previously prepared the enantiomer of 25-HC (ent-25-HC) and have compared its biophysical and pharmacological properties with those of 25-HC [15-18]. We are also interested in performing similar comparative activity studies with 27-HC and its enantiomer, ent-27-HC. Because ent-27-HC has not been prepared previously, we evaluated synthetic strategies for its preparation.

Generally, synthetic methods used for the total synthesis of a natural steroid can be used to make the corresponding ent-steroid simply by using a chiral starting material with the opposite absolute configuration of the natural steroid. Thus, for synthesizing ent-27-HC, methods used to synthesize 27-HC are relevant. 27-HC has been prepared from the commercially available natural products diosgenin or kryptogenin [19-22]. However, these synthetic methods are not useful for preparing ent-27-HC because neither ent-diosgenin nor ent-kyptogenin occurs naturally and it is impractical to first prepare either of them for subsequent conversion into ent-27-HC.

A literature precedent for the synthesis of 27-HC from (3β)-hydroxychol-5-en-24-oic acid has been reported [23]. We have previously reported the total synthesis of ent-lithocholic acid [24] and we could have modified this synthesis to prepare the enantiomer of (3β)-3-hydroxychol-5-en-24-oic acid, then converted it to ent-27-HC. While this route could have enabled the total synthesis of ent-27-HC, we also sought a method that could prepare a variety of side-chains on the steroid. Therefore, we modified our ent-lithocholic acid synthesis to prepare an ent-Δ²² steroid which allowed us to utilize a cross-metathesis reaction with either 2^nd generation Grubbs catalyst or Stewart-Grubbs catalyst to install the remaining portions of the side-chain and complete the synthesis of ent-27-HC. We used this synthetic approach to prepare ent-27-HC-d₃ (5). We also demonstrated the generality of this approach for making side chain modified sterols by preparing 25-HC (1), 25-HC-d₆, (2), 27-HC (3) and ent-cholesterol-d₃ (ent-Chol-d₃, 4). The deuterated ent-sterols 4 and 5 will be of utility in future biological studies.

2. Experimental

General Methods

Solvents were either used as purchased or dried and purified by standard methods. All air and/or moisture sensitive reactions were carried out under nitrogen environments using oven-dried glassware, which was cooled under nitrogen gas. Flash chromatography was performed using silica gel (32-63 μm) purchased from Scientific Adsorbants (Atlanta, GA). Optical rotations were determined at room temperature on a Perkin-Elmer Model 341 polarimeter. Melting points were determined utilizing a Kofler micro hot stage and are uncorrected. IR spectra were recorded on a NaCl plate with a Perkin-Elmer 1710 FT-IR spectrophotometer. NMR spectra were recorded at ambient temperature in CDCl₃ with a 5 mm probe on a Varian Gemini 2000 operating at 300 MHz (¹H) or 75 MHz (¹³C) and were referenced to CDCl₃ (7.27 ppm or 77.00 ppm, respectively). Elemental analyses were performed by M-H-W laboratories (Phoenix, AZ). Lithocholic acid was purchased from Steraloids, Newport, RI, USA.

2.1 (3β)-Cholest-5-ene-3,25-diol (25-hydroxycholesterol, 1)

Compound 15 (23 mg, 0.057 mmol) was dissolved in Et₂O (10 mL) and cooled to 0 °C under N₂. CH₃MgBr (0.5 mL, 3 M in Et₂O) was added dropwise. The reaction was allowed to slowly reach room temperature and stirred overnight under N₂. Additional CH₃MgBr (0.2 mL) was added in the morning and the reaction stirred at room temperature for another 4 h. The solution was quenched with satd. aqueous NH₄Cl and the product extracted into Et₂O (3 × 40 mL). The combined extracts were dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 5:1) to obtain compound 1 (21 mg, 60%, 3 steps from compound 14) as a white solid: mp 172-175 °C; [α]²⁵_D -37.0 (c = 0.08, CHCl₃); ¹H NMR δ 0.66 (3H, s), 0.90 (3H, d, J = 6.6 Hz), 3.47 (1H, m), 5.32 (1H, d, J = 4.9 Hz); ¹³C NMR δ 140.9, 121.8, 71.7, 71.2, 56.9, 56.2, 50.3, 44.5, 42.5, 42.3, 39.9, 37.4, 36.6, 36.6, 35.9, 32.0, 31.6, 29.3, 29.2, 28.4, 24.4, 21.2, 20.9, 19.5, 18.8, 12.0; IR υ_max 3292, 2933, 2864, 1465, 1377 cm^-1.

2.2 (3β)-Cholest-5-ene-26,26,26,27,27,27-d₆-3,25-diol (25-hydroxycholesterol-d₆, 2)

Compound 15 (27 mg, 0.067 mmol) was dissolved in Et₂O (10 mL) and CD₃Li (1.5 mL, 0.5M in Et₂O, stabilized with LiI) was added slowly while stirring under N₂ for 4 h. Additional CD₃Li solution (1.5 mL) was added and the reaction stirred at room temperature overnight under N₂. A third addition of CD₃Li solution (1.5 mL) was then added and stirring continued for another 4 h. The reaction was quenched with water, then 0.5 M HCl, and the product extracted into Et₂O (3 × 40 mL). The combined extracts were dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with CH₂Cl₂:EtOAc, 8:1) to obtain compound 2 (24 mg, 59%, 3 steps from compound 13) as a white solid: mp 172-174 °C; [α]²⁵_D -33.3 (c = 0.08, CHCl₃); ¹H NMR δ 0.69 (3H, s), 0.94 (3H, d, J = 6.3 Hz), 1.01 (3H, s), 2.01 (2H, m), 2.28 (2H, m), 3.53 (1H, m), 5.36 (1H, d, J = 5.1 Hz); ¹³C NMR δ 12.0, 18.8, 19.5, 20.9, 21.2, 24.4, 28.4, 31.8, 32.0, 35.9, 36.6, 36.6, 37.4, 39.9, 42.5, 42.5, 44.5, 50.3, 56.2, 56.9, 71.0, 72.0, 121.8, 140.9; IR υ_max 3307, 2934, 2902, 2865, 1465, 1376 cm^-1. HRMS (ESI) calcd. for C₂₇H₄₀D₆O₂ (M-H₂O+H⁺) 391.3847; Found 391.3852.

2.3 (3β,25R)-Cholest-5-ene-3,26-diol (27-hydroxycholesterol, 3)

Compound 22 (85 mg, 0.191 mmol) was dissolved in acetic anhydride (20 mL). NaI (530 mg) was added, N₂ was bubbled through the solution for 30 min, and then the reaction was cooled to 0 °C. SiMe₃Cl (0.4 mL) was added dropwise and the reaction was stirred at 0 °C under N₂ for 30 min, then at room temperature for 3 h. The acetic anhydride was removed in vacuo and satd. aqueous NaHCO₃ was added to the resulting residue. The product was extracted into EtOAc (3 × 40 mL) and the combined extracts were washed with 5% sodium thiosulfate solution, dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The dienol acetate intermediate was partially purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 9:1). EtOH (50 mL) was added to the partially purified intermediate, followed by NaBH₄ (197 mg, 5.20 mmol) and the reaction was stirred overnight. The EtOH was removed in vacuo and the product dissolved in EtOAc and washed with 1 M HCl, satd. aqueous NaHCO₃, dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure to obtain compound 23 which was further purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 6:1 to 3:1). Compound 23 was dissolved in MeOH (50 mL), K₂CO₃ (250 mg, 1.81 mmol) was added and the reaction was refluxed overnight under N₂. The MeOH was removed in vacuo and 1 M HCl (50 mL) was poured over the residue. The product was extracted into EtOAc (3 × 40 mL) and the combined extracts were dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with CH₂Cl₂:EtOAc, 3:1) to obtain compound 3 (44 mg, 57%, 3 steps from compound 22) as a white solid: ¹H NMR δ 0.68 (3H, s), 2.27 (2H, m), 3.50 (3H, m), 5.37 (1H, m); ¹³C NMR δ 140.9, 121.8, 72.0, 68.9, 56.9, 56.3, 50.3, 42.5, 39.9, 37.4, 36.7, 36.3, 36.0, 35.9, 33.7, 32.1, 31.8, 28.4, 24.4, 23.6, 21.2, 19.5, 18.8, 16.5, 12.0.

2.4 ent-[(3β)-Cholest-5-en-19,19,19-d₃-3-ol] (ent-cholesterol-d₃, 4)

Compound 4 (80 mg, 65%) was prepared from compound 35 (117 mg, 0.302 mmol) using the two step deconjugation procedure described as part of three step procedure for the preparation of compound 3. Compound 4 was a white solid: ¹H NMR δ 0.68 (3H, s), 0.86 (6H, d, J = 6.9 Hz), 0.99 (3H, d, J = 9.9 Hz), 2.28 (2H, m), 3.51 (1H, m), 5.35 (1H, s); ¹³C NMR δ 12.0, 18.9, 21.2, 22.7, 23.0, 24.0, 24.4, 28.2, 28.4, 31.8, 32.1, 36.0, 36.3, 36.4, 37.3, 39.7, 39.9, 42.5, 50.2, 56.3, 56.9, 72.0, 121.9, 140.9; IR υ_max 3369, 2931, 2867, 2901, 1459, 1378 cm^-1. HRMS (ESI) calcd. for C₂₇H₄₃D₃O (M+H⁺) 390.3810; Found. 390.3813.

2.5 ent-[(3β,25R)-Cholest-5-ene-19,19,19-d₃-3,26-diol] (ent-27-hydroxy cholesterol-d₃, 5)

Compound 5 (65 mg, 60%) was prepared from compound 36 (120 mg, 0.261 mmol) using the three step procedure described for the preparation of compound 3. Compound 5 was a white solid: mp 174-175 °C; [α]²⁵_D +33.5 (c = 0.25, CHCl₃); ¹H NMR δ 0.69 (3H, s), 0.92 (6H, d, J = 6.3 Hz), 2.28 (2H, m), 3.46 (3H, m), 5.35 (1H, m); ¹³C NMR δ 12.0, 16.7, 18.8, 21.2, 23.6, 24.4, 28.4, 31.8 (2 × C), 32.1, 33.7, 35.9, 36.0, 36.3, 36.5, 37.3, 39.9, 42.6, 50.2, 56.3, 56.9, 68.7, 72.0, 121.9, 140.9; HRMS (ESI) calcd. for C₂₇H₄₃D₃O₂ (2M+H⁺) 811.7445; Found. 811.7446.

2.6 (3α,5β)-24-norchol-22-en-3-ol (10)

(3α,5β)-24-Norchol-22-en-3-ol, 3-acetate (6, 6.17 g, 16.56 mmol, 1 eq) prepared according to the literature [25], was dissolved in hot MeOH (300 mL) and benzene (50 mL). K₂CO₃ (10.3 g, 74.5 mmol, 4.5 eq) was added and the mixture was heated to reflux and stirred overnight. The solvent was removed in vacuo and 1 M HCl was added. The product was extracted into CH₂Cl₂ (3 × 200 mL), dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 8:1 to 6:1) to give compound 10 (4.75 g, 87%) as a white solid: mp 137-140 °C; [α]²⁵_D +123.7 (c = 0.40, CHCl₃); ¹H NMR δ 0.67 (3H, s), 0.93 (3H, s), 1.02 (3H, d, J = 6.6 Hz), 3.61 (1H, m), 4.92 (2H, m), 5.69 (1H, s); ¹³C NMR δ 145.5, 111.6, 72.0, 56.7, 55.8, 42.3, 41.4, 40.7, 40.3, 36.6, 36.0, 35.5, 34.8, 30.7, 28.6, 27.3, 26.6, 24.4, 23.5, 21.0, 20.2, 12.4; IR υ_max 3307, 2931, 2865, 1637, 1449, 1261 cm^-1. Anal. Calcd for C₂₃H₃₈O: C, 83.57; H, 11.59; Found: C, 83.74; H, 11.43.

2.7 (3α,5β)-3-Hydroxychol-22-en-24-oic acid, methyl ester (11)

Compound 10 (2.52 g, 7.62 mmol) was dissolved in CH₂Cl₂ (200 mL), trans-3-hexenedioic acid dimethyl ester (1.2 mL, 7.67 mmol) and Grubbs catalyst, 2^nd generation (660 mg, 0.78 mmol) were added. The reaction was heated to reflux and allowed to stir for 40 h. CH₂Cl₂ was removed in vacuo and the product was partially purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 6:1 to 4:1) to recover a mixture of cis/trans isomers to give compound 11 (2.17 g) as a light brown solid which was converted to compound 12 without further purification or characterization.

2.8 (3α,5β)-3-Hydroxycholane-24-carboxylic acid, methyl ester (12)

Compound 11 (2.01 g, 4.99 mmol) was dissolved in EtOAc (150 mL) and Pd/C (1.0 g, 10% on charcoal) was added to a round-bottomed flask equipped with a balloon and evacuated and purged with H₂ (3 ×). The reaction was stirred while bubbling H₂ through the solvent for 3 h. The solution was filtered through celite and the solvent removed in vacuo to yield compound 11 (2.01 g, 70%, 2 steps from compound 6): mp 105-108 °C; [α]²⁵_D +265.8 (c = 0.32, CHCl₃); ¹H NMR 5 0.63 (3H, s), 0.91 (6H, 2s), 2.28 (2H, m), 3.64 (1H, m), 3.66 (3H, s); ¹³C NMR δ 12.2, 18.7, 21.0, 21.7, 23.5, 24.4, 26.6, 27.4, 28.4, 30.7, 34.7, 34.7, 35.5, 35.6, 35.7, 36.0, 36.6, 40.3, 40.6, 42.3, 42.8, 51.6, 56.2, 56.7, 72.0, 174.5; IR υ_max 3368, 2934, 2864, 1742, 1448 cm^-1. Anal. Calcd for C₂₆H₄₄O₃: C, 77.18; H, 10.96; Found: C, 77.20; H, 11.05.

2.9 (5β)-3-Oxocholane-24-carboxylic acid, methyl ester (13)

Compound 12 (2.01 g) was dissolved in acetone (250 mL) and N₂ was bubbled through the solution for 30 min. Jones reagent (30% H₂SO₄, 30% chromic acid) was added dropwise until a persistent yellow color remained. The reaction was allowed to stir at room temperature under N₂ for 2 h. Water was added and the acetone removed in vacuo. Brine (100 mL) was added and the product was extracted into EtOAc (3 × 250 mL). The combined extracts were washed with brine (200 mL), dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 9:1) to obtain compound 13 (1.93 g, 96%) as a white solid: mp 92-93 °C; [α]²⁵_D +28.4 (c = 0.50, CHCl₃); ¹H NMR δ 0.67 (3H, s), 0.91 (3H, d, J = 6.3 Hz), 1.01 (3H, s), 2.69 (1H, t, J = 14.3 Hz), 3.66 (3 H, s); ¹³C NMR δ 213.5, 174.5, 56.6, 56.2, 51.6, 44.5, 42.9, 42.5, 40.9, 40.2, 37.4, 37.2, 35.7, 35.6, 35.5, 35.0, 34.6, 28.3, 26.8, 25.9, 24.3, 22.8, 21.7, 21.4, 18.7, 12.2; IR υ_max 2932, 2865, 1736, 1716, 1445, 1377 cm^-1. Anal. Calcd for C₂₆H₄₂O₃: C, 77.56; H, 10.51; Found: C, 77.69; H, 10.35.

2.10 3-Oxochol-4-ene-24-carboxylic acid, methyl ester (14)

Compound 13 (110 mg, 0.273 mmol) was dissolved in AcOH (50 mL) and pyridinium tribromide (102 mg, 0.319 mmol) was added. The mixture was stirred, under N₂ at room temperature for 1 h. The AcOH was removed in vacuo and satd. aqueous NaHCO₃ was added to the residue. The product was extracted into EtOAc (3 × 40 mL), dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The brominated intermediate was partially purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 6:1). The partially purified intermediate was dissolved in DMF (40 mL) and LiCl (450 mg, 10.62 mmol) was added. The solution was heated to 100 °C and stirred for 2 h under N₂. DMF was removed in vacuo and 1 M HCl (50 mL) was added. The product was extracted into EtOAc (3 × 40 mL) and the combined extracts dried over anhydrous Na₂SO₄, and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 8:1) to obtain compound 14 (50 mg, 46%) as a white solid: mp 118-122 °C; [α]²⁵_D +47.6 (c = 0.24, CHCl₃); ¹H NMR δ 0.71 (3H, s), 0.90 (3H, d, J = 1.9 Hz), 3.67 (3H, s), 5.73 (1H, s); ¹³C NMR δ 199.7, 174.4, 171.7, 123.9, 56.0, 55.9, 53.9, 51.6, 42.5, 39.7, 38.7, 35.8, 35.7, 35.6, 35.5, 34.6, 34.1, 33.1, 32.2, 28.2, 24.3, 21.7, 21.2, 18.7, 17.5, 12.1; IR υ_max 2937, 1736, 1671, 1616 cm^-1. Anal. Calcd for C₂₆H₄₀O₃: C, 77.95; H, 10.06; Found: C, 78.23; H, 10.21.

2.11 (3β)-3-Hydroxychol-5-ene-24-carboxylic acid, ethyl and methyl ester (15)

Compound 15 (27 mg, a 3:1 mixture of ethyl and methyl esters as determined by ¹H NMR) was prepared from compound 14 (41 mg, 0.102 mmol) using the procedure described for the preparation of compound 3. Compound 15 was characterized only by its ¹H NMR spectrum and was subsequently converted to either compound 1 or compound 2.

2.12 (2R,7R)-2,7-bis(methyl)-1,8-bis[(4S)-2-oxo-4-(phenylmethyl)-3-oxazolidinyl]-4-octene-1,8-dione (17)

Compound 16 [26] (5.6 g, 20.5 mmol) was dissolved in CH₂Cl₂ (250 mL) and Grubbs catalyst, 1^st generation (1.5 g, 1.8 mmol) was added. The reaction was heated to reflux and stirred under N₂ for 40 h. The CH₂Cl₂ was removed in vacuo and the product purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 4:1 to 2:1) to obtain partially purified compound 17 (4.6 g, 86%) as a grey solid: ¹H NMR δ 1.18 (6H, m), 2.23 (2H, m), 2.33 (2H, m), 2.48 (2H, m), 2.71 (2H, m), 3.31 (2H, d, J = 13.2 Hz), 4.21 (4H, m), 4.69 (2H, m), 5.55 (2H, m), 7.31 (10H, m).

2.13 (2R,7R)-2,7-dimethyl-oct-4-ene-1,8-diol (18)

Partially purified compound 17 (4.45 g, 8.58 mmol) was dissolved in dry THF and cooled to 0 °C while stirring under N₂. LiAlH₄ (55 mL, 2 M in THF) was added and the reaction was allowed to reach room temperature and stirred overnight. The LiAlH₄ was quenched by sequentially adding water (4.2 mL), 8.4 mL 10% NaOH (8.4 mL) and 12.6 mL water (12.6 mL). Precipitates were removed by filtration and the product extracted into EtOAc (3 × 200 mL). The combined extracts were washed with brine, dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was partially purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 2:1) to obtain compound 18 (1.4 g, 65%) as a mixture of E and Z isomers which was used directly in the Grubbs cross-metathesis reaction with compound 19.

2.14 (5β)-24-Norchol-22-en-3-one (19)

Compound 19 (1.37 g, 93%) was prepared from compound 6 (1.49 g, 4.51 mmol) using the procedure described for the preparation of compound 13. Compound 19 was a white solid: mp 126-127 °C; [α]²⁵_D +154.6 (c = 0.49, CHCl₃); ¹H NMR δ 0.70 (3H, s), 1.01 (3H, s), 1.03 (3H, s), 2.69 (1H, t, J = 14.3 Hz), 4.86 (2H, m), 5.65 (1H, m); ¹³C NMR δ 213.3, 145.2, 111.8, 56.6, 55.8, 44.5, 42.9, 42.5, 41.3, 41.0, 40.1, 37.3, 37.2, 35.7, 35.1, 28.6, 26.8, 25.9, 24.3, 22.8, 21.3, 20.2, 12.4; IR υ_max 2939, 2857, 1716, 1445 cm^-1. Anal. Calcd for C₂₃H₃₆O: C, 84.09; H, 11.04; Found: C, 84.01; H, 10.91.

2.15 (5β,25R)-26-Hydroxycholestan-3-one (20)

Compound 19 (1.16 g, 3.53 mmol), compound 17 (610 mg, 3.56 mmol) and Grubbs catalyst, 2^nd generation (300 mg, 0.25 mmol) were dissolved CH₂Cl₂ (300 mL), heated to reflux, and stirred for 72 h. Additional Grubbs catalyst, 2^nd generation (300 mg, 0.25 mmol) was added, heated to reflux, and stirred for 24 h. The solvent was removed in vacuo, and partially purified by flash column chromatography (silica gel eluted with a 6:1 hexanes:EtOAc, 6:1). A portion of this product (740 mg, 1.84 mmol) was dissolved in 150 mL EtOAc (150 mL) and hydrogenated (70 psi) using 10% Pd/C as catalyst. The hydrogenation was run overnight. After filtration through celite and solvent removal under reduced pressure, the product was purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 4:1) to yield compound 20 (660 mg, 54%, 2 steps from compound 19) as a white solid: mp 87-90 °C; [α]²⁵_D +36.6 (c = 0.47, CHCl₃); ¹H NMR δ 0.68 (3H, s), 2.30 (1H, m), 2.70 (1H, t, J = 14.0 Hz), 3.49 (2H, m); ¹³C NMR δ 213.6, 68.6, 56.6, 56.5, 44.5, 42.9, 42.5, 40.9, 40.2, 37.3, 37.2, 36.3, 35.9, 35.8, 35.7, 35.0, 33.7, 28.4, 26.8, 25.9, 24.3, 23.6, 22.8, 21.4, 18.8, 16.7, 12.2; IR υ_max 3418, 2932, 2865, 1715, 1455, 1378 cm^-1. Anal. Calcd for C₂₇H₄₆O₂: C, 80.54; H, 11.52; Found: C, 80.59; H, 11.28.

2.16 (5β,25R)- 26-(Acetyloxy)-cholestan-3-one (21)

Compound 20 (170 mg, 0.420 mmol) was dissolved in pyridine (30 mL). Acetic anhydride (0.6 mL, 6.35 mmol) and 4-(dimethylamino)pyridine (20 mg, 0.16 mmol) were added and the mixture stirred under N₂ at room temperature for 3.5 h. The pyridine was removed in vacuo. Water was poured over the residue and the product extracted into EtOAc (3 × 40 mL). The combined extracts were washed with brine, dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 12:1) to obtain compound 21 (164 mg, 87%) as a white solid: mp 62-65 °C; [α]²⁵_D +21.1 (c = 0.19, CHCl₃); ¹H NMR δ 0.65 (3H, s), 2.31 (1H, m), 2.71 (1H, t, J = 14.1 Hz), 3.90 (2H, m); ¹³C NMR δ 213.3, 171.3, 69.7, 56.6, 56.4, 44.5, 42.9, 42.5, 40.9, 40.2, 37.3, 37.2, 36.1, 35.8, 35.7, 35.0, 33.9, 32.6, 28.4, 26.8, 25.9, 24.3, 23.4, 22.8, 21.3, 21.1, 18.8, 16.9, 12.2; IR υ_max 2936, 2865, 1740, 1716, 1467, 1377, 1238 cm^-1. Anal. Calcd for C₂₉H₄₈O₃: C, 78.33; H, 10.88; Found: C, 78.57; H, 10.69.

2.17 (25R)-26-(Acetyloxy)-cholest-4-en-3-one (22)

Compound 22 (74 mg, 51%) was prepared from compound 21 (146 mg, 0.323 mmol) using the procedure described for the preparation of compound 14. Compound 22 was an oil: [α]²⁵_D +53.5 (c = 0.12, CHCl₃); ¹H NMR δ 0.68 (3H, s), 0.89 (3H, d, J = 4.9 Hz), 3.91 (2H, m), 5.69 (1H, s); IR υ_max 2935, 2868, 1740, 1676, 1465, 1375, 1236 cm^-1. Anal. Calcd for C₂₉H₄₆O₃: C, 78.68; H, 10.47; Found: C, 78.58; H, 10.69.

2.18 2,7-dimethyloct-4-ene (24)

4-Methyl-1-pentene (20 mL, 157.8 mmol) was dissolved in CH₂Cl₂. Grubbs catalyst, 2nd generation (189 mg, 0.23 mmol) was added and the reaction was stirred under N₂ at room temperature for 12 h. The product was purified by flash column chromatography (silica gel eluted with hexanes) to obtain compound 24 as an oil (5.7 g, 51%): ¹H NMR δ 0.88 (12H, m), 1.56 (2H, septet), 1.90 (2H, m), 5.38 (2H, m); IR υ_max. 2899, 2927, 2870, 1466, 1384, 1367 cm^-1.

2.19 (2S,7S)-2,7-bis(methyl)-1,8-bis[(4R)-2-oxo-4-(phenylmethyl)-3-oxazolidinyl]-4-octene-1,8-dione (26)

Compound 25 (5.02 g, 21.5 mmol) was dissolved in CH₂Cl₂ (250 mL) and Grubbs catalyst, 1^st generation (1.5 g, 1.8 mmol) was added. The reaction was heated to reflux and stirred under N₂ for 40 h. The CH₂Cl₂ was removed in vacuo and the product partially purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 4:1 to 2:1) to obtain compound 26 (3.94 g, 83%) as a gray solid, which was not characterized before being converted to compound 27.

2.20 (2S,7S)-2,7-dimethyl-oct-4-ene-1,8-diol (27)

Compound 27 (1.13 g, 82%) was prepared from compound 26 (3.94 g, 8.03 mmol) using the procedure described for the preparation of compound 18. Compound 27 was not characterized before being converted to compound 28.

2.21 (2S,7S)-2,7-dimethyl-4-octene-1,8-diol, diacetate (28)

Compound 28 (1.11 g, 66%) was prepared from compound 27 (1.13 g, 6.56 mmol) using the procedure described for the preparation of compound 21. Compound 28 had: ¹H NMR δ 0.89 (6H, m), 2.03 (6H, s), 3.88 (4H, m), 5.37 (2H, m); ¹³C NMR δ 16.5, 16.6, 20.8, 30.8, 32.7, 32.9, 36.3, 68.7, 128.6, 129.6, 171.1; IR υ_max 2962, 1739, 1463, 1367, 1239 cm^-1; HRMS (ESI) calcd. for C₁₄H₂₄O₄ (M+H⁺) 257.1747; Found: 257.1748.

2.22 ent-[(3α,5β)-chola-16,22-diene-3,24-diol] (30)

Compound 29 (1.09 g, 2.32 mmol), was prepared by the same procedure reported previously for the natural abundance form [24], then dissolved in dry THF and cooled to -78 °C. DIBAL-H (1.5 M in THF, 25 mL) was added and the reaction was stirred under N₂ at -78 °C for 1 h. Sodium potassium tartrate was added slowly until bubbling ceased. The product was extracted into EtOAc (2 × 250 mL), then washed with brine (100 mL), dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 2:1) to yield compound 30 (840 mg, 97%) as a white solid: [α]²⁵_D -28.0 (c = 0.49, CHCl₃); ¹H NMR δ 0.75 (3H, s), 1.12 (3H, d, J = 6.9 Hz), 2.85 (1H, m), 3.61 (1H, m), 4.10 (2H, d, J = 4.5 Hz), 5.34 (1H, t, J = 1.65 Hz), 5.64 (2H, m); ¹³C NMR δ 16.6, 20.7, 26.6, 27.3, 30.6, 31.1, 34.6, 34.7, 35.3 (2 × C), 35.6, 36.6, 41.2, 42.3, 47.4, 57.6, 63.9, 72.0, 122.5, 126.8, 138.5, 159.1; HRMS (ESI) calcd. for C₂₉H₄₅D₃O₃ (M-H₂O+H⁺) 344.3033; Found: 344.3025.

2.23 ent-[(3α,5β)-Cholesta-16,22-diene-19,19,19-d₃-3-ol](31)

Compound 30 (780 mg, 2.08 mmol) was dissolved in CH₂Cl₂ (300 mL) and compound 24 (945 mg, 6.74 mmol) was added, followed by the addition of 1,3-bis(2-methylphenyl)-2-imidazolidinylidene]dichloro(2-isopropoxyphenylmethylene)ruthenium (II) (478 mg, 0.84 mmol). The reaction was heated to reflux and stirred under N₂ for 72 h. The solvent was removed in vacuo and the product partially purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 9:1 to 2:1) to obtain semi-purified compound 31 (630 mg, 78%) which was not characterized before being converted to compound 33.

2.24 ent-[(3α,5β,25R)-Cholesta-16,22-diene-19,19,19-d₃-3,26-diol, 26-acetate] (32)

Partially purified compound 32 (630 mg, 63%) was prepared from compound 30 (840 mg, 2.24 mmol) and compound 28 (650 mg, 2.53 mmol) using the procedure described for the preparation of compound 31. The product was not characterized before being converted to compound 34.

2.25 ent-[(5β)-cholestan-3-one-19,19,19-d3] (33)

Compound 31 (630 mg, 1.63 mmol) was dissolved in EtOAc (150 mL) and hydrogenated (70 psi) using 10% Pd/C (414 mg) as catalyst. The hydrogenation was run overnight. The product was filtered through celite, and the solvent removed in vacuo. This product was dissolved in acetone, and Jones reagent added until a persistent yellow color was achieved. This reaction was stirred under N₂ for 30 min, brine was added and the product extracted into EtOAc (3 × 100 mL). The combined extracts were washed with brine (100 mL), dried over anhydrous Na₂SO₄ and the solvents removed under reduced pressure. The product was purified by flash column chromatography (silica gel eluted with hexanes:EtOAc, 18:1) to obtain compound 33 (440 mg, 54%, 3 steps from compound 30) which had: [α]²⁵_D -32.5 (c = 0.82, CHCl₃); ¹H NMR δ 0.69 (3H, s), 0.91 (3H, d, J = 6.3 Hz), 0.91 (3H, d, J = 6.9 Hz), 2.29 (1H, t of d, J = 5.4 Hz, J = 9.0 Hz), 2.70 (1H, t, J = 14.1 Hz); ¹³C NMR δ 12.2, 18.8, 21.4, 22.7, 23.0, 24.0, 24.3, 25.9, 26.8, 28.2, 28.4, 34.8, 35.7, 35.9, 36.3, 37.1, 37.4, 39.7, 40.2, 40.9, 42.5, 42.9, 44.5, 56.5, 56.6, 213.7; IR υ_max 2950, 2867, 1717, 1455, cm^-1; HRMS (ESI) calcd. for C₂₇H₄₃D₃O (M+H⁺) 390.3810; Found: 390.3814.

2.26 ent-[(5β,25R)-26-(Acetyloxy)-cholestan-3-one-19,19,19-d3] (34)

Compound 34 (350 mg, 35%) was prepared from compound 32 (630 mg, 1.41 mmol) using the procedure described for the preparation of compound 33. Compound 34 had: [α]²⁵_D -64.9 (c = 0.68, CHCl₃); ¹H NMR δ 0.68 (3H, s), 0.92 (3H, d, J = 6.6 Hz), 0.92 (3H, d, J = 6.9 Hz), 2.06 (3H, s), 2.33 (1H, m), 2.65 (1H, t, J = 14.4 Hz), 3.96 (2H, m); ¹³C NMR δ 12.2, 16.9, 18.8, 21.1, 31.3, 23.4, 24.3, 25.9, 26.8, 28.4, 32.6, 33.9, 34.8, 35.7, 35.8, 36.1, 37.1, 37.3, 40.2, 40.8, 42.5, 42.9, 44.4, 56.4, 56.6, 69.7, 171.4, 213.6; IR υ_max 3369, 2933, 2865, 1740, 1716, 1455, 1377, 1238 cm^-1; HRMS (ESI) calcd. for C₂₉H₄₅D₃O₃ (M+H⁺) 448.3865; Found: 448.3866.

2.27 ent-[Cholest-4-ene-3-one-19,19,19-d3] (35)

Compound 35 (177 mg, 45%) was prepared from compound 33 (400 mg, 1.03 mmol) using the procedure described for the preparation of compound 14. Compound 35 was a white solid: mp 58-60 °C; [α]²⁵_D -89.6 (c = 0.34, CHCl₃); ¹H NMR δ 0.71 (3H, s), 0.86 (6H, d, J = 6.6 Hz), 0.91 (3H, d, J = 6.6 Hz), 2.03 (2H, m), 2.40 (4H, m), 5.73 (1H, s); ¹³C NMR δ 12.1, 18.8, 21.2, 22.7, 23.0, 24.0, 24.3, 28.2, 28.3, 32.2, 33.1, 34.1, 35.8, 35.9, 36.3, 38.6, 39.6, 39.8, 42.5, 53.9, 56.0, 56.2, 123.9, 171.9, 199.9; IR υ_max 2950, 2867, 1675, 1467, 1448, 1379, 1264, 1226 cm^-1; HRMS (ESI) calcd. for C₂₇H₄₁D₃O (M+H⁺) 388.3653; Found: 388.3654.

2.28 ent-[(25R)-26-(Acetyloxy)-cholest-4-ene-3-one-19,19,19-d3] (36)

Compound 36 (125 mg, 42%,) was prepared from compound 34 (324 mg, 0.73 mmol) using the procedure described for the preparation of compound 14. Compound 36 was a white solid: [α]²⁵_D -64.9 (c = 0.43, CHCl₃); ¹H NMR δ 0.70 (3H, s), 0.91 (3H, d, J = 6.9 Hz), 0.91 (3H, d, J = 6.3), 2.05 (3H, s), 3.86 (2H, m), 5.72 (1H, s); ¹³C NMR δ 12.1, 16.9, 18.7, 21.2, 23.4, 24.3, 28.3, 32.2, 32.6, 33.1, 33.9, 34.1, 35.8, 36.1, 38.5, 39.8, 42.5, 53.9, 56.0, 56.2, 69.7, 123.9, 171.5, 171.9, 199.8; IR υ_max 3459, 2935, 2870, 1739, 1674, 1617, 1449, 1376, 1240 cm^-1; HRMS (ESI) calcd. for C₂₉H₄₃D₃O₃ (M+H⁺) 446.3708; Found: 446.3699.

Results and Discussion

Our goal was to develop a synthetic route that could be utilized to synthesize a variety of side-chain oxysterols in both the natural and unnatural (ent) sterol series. We chose to start with either lithocholic acid or ent-lithocholic acid as starting materials; the former is commercially available and the latter was previously synthesized [24]. The synthetic strategy was to convert these steroid enantiomers to their corresponding Δ²² steroids and subsequently utilize a cross-metathesis reaction to convert them to our target steroids (Figure 1). Methods were first developed in the natural steroid series because of its ready availability.

In order to generate the Δ²²-steroid for the Grubbs cross-metathesis, lithocholic acid was acetylated at the 3-position and then subjected to oxidative decarboxylation using Pb(OAc)₄, Cu(OAc)₂ and catalytic pyridine to obtain steroid 6 as described previously [25]. A series of reactions were attempted to cross-metathesize steroid 6 with 2-methylpent-4-en-2-ol (7, synthesized as previously described [27]), using Grubbs catalyst, 2^nd Generation (Scheme 1). Unfortunately, the yields for steroid 8 (characterized only by ¹H NMR) were poor (31-39%), despite attempts to optimize this reaction with varying solvent conditions, temperature, reaction time and equivalents of either olefin 7 or the Grubbs catalyst. In all attempts, the main product was homo-metathesized olefin 9 (previously prepared by other methods [28]) and unreacted steroid 6. This is consistent with a recent report attempting similar cross-metathesis reactions on Δ²²-steroids [29]. However, we found that if olefin 9 was used in place of olefin 7, the cross-metathesis with the Δ²²-steroid proceeded smoothly. The high reactivity of terminal olefin 7 with the catalyst apparently resulted in the exhaustion of the catalytic cycle prior to involvement of 6. We conclude that the less reactive internal olefins perform better than terminal olefins in cross-metathesis reactions with Δ²²-steroids.

Scheme 1.

Attempts to cross-metathesize the Δ²² steroid 6 to generate compound 8. Reagents and Conditions: (a) acetic anhydride, DMAP, pyridine, 12 h, ca. 100%; (b) Pb(OAc)₄, Cu(OAc)₂, benzene, cat. pyridine, reflux, 6 h, 69%. Steroid 6 was then treated with Grubbs catalyst, 2^nd generation, CH₂Cl₂, reflux, 40 h with either compound 7 or compound 9.

In order to generate 25-HC (1) and 25-HC-d₆ (2), steroid 6 was deacetylated (higher yields were obtained with the deacetylated steroid) with K₂CO₃ to obtain steroid 10 in 87% yield (Scheme 2). Steroid 10 was then treated with trans-3-hexenedioic acid dimethyl ester and Grubbs catalyst, 2^nd generation to form the cross-metathesis product 11 as a mixture of E and Z isomers (E:Z ratio > 95:5), which upon hydrogenation generated steroid 12 in good yield (70%, 2 steps). Steroid 12 was then oxidized to the 3-ketosteroid 13 using Jones reagent (96%). The 4-position of steroid 13 was selectively brominated using pyridinium tribromide in HOAc and subsequently converted with LiCl in DMF to the known Δ⁴-3-ketosteroid 14 [30] in 46% yield. The Δ⁴-3-ketosteroid 14 was converted to the Δ⁵-3β-hydroxysteroid 15 through a dienol acetate intermediate (formed by treatment with acetic anhydride, NaI and trimethyl silyl chloride) which was then stereoselectively reduced with NaBH₄ [31]. Steroid 15 was purified as a combination of methyl and ethyl esters (the latter formed during the NaBH₄ reduction in EtOH). Lastly, steroid 15 was either treated with methyl magnesium bromide to generate 25-HC (1, 60% from 14) or trideuterated methyllithium to generate 25-HC-d₆ (2, 59% from 14).

Scheme 2.

Reagents and Conditions: (a) K₂CO₃, Methanol/benzene (6/1), reflux, 12 h, 87%; (b) trans-3-hexenedioic acid dimethyl ester, Grubbs catalyst, 2^nd generation, CH₂Cl₂, reflux, 40 h; (c) H₂, Pd/C (10%), EtOAc, 3 h, 70%, (2 steps); (d) Jones reagent, acetone, 25 °C, 2 h, 96%; (e) pyridinium tribromide, AcOH, 1 h; (f) LiCl, DMF, 100 °C, 2 h, 46% (2 steps); (g) acetic anhydride, TMSCl, NaI, 0 °C to 25 °C, 3 h; (h) NaBH₄, EtOH, 25 °C, 12 h; (i) MeMgBr, diethyl ether, 0 °C to 25 °C, 20 h, 60% (3 steps); (j) CD₃Li, ether, 20 h, 59% (3 steps).

Olefinic diol 18 (Scheme 3), used to generate the side-chain for 27-HC, was synthesized by treating commercially available (4S)-3-(1-oxopropyl)-4-(phenylmethyl)-2-oxazolidinone with allyl iodide and NaHMDS to form the diastereoselectively pure product 16 (82%) [26]. Compound 16 was then homo-metathesized with Grubbs catalyst, 1^st generation to generate compound 17 in 77% yield as a mixture of E and Z isomers. The oxazolidinone chiral auxiliary was removed from compound 17 using LiAlH₄ to form the olefinic diol 18 as a mixture of E and Z isomers (65%).

Scheme 3.

Reagents and Conditions: (a) allyl magnesium iodide, NaHMDS, THF, -78 °C, 4 h, 82%; (b) Grubbs catalyst, 1^st generation, CH₂Cl₂, reflux, 24 h, 77%; (c) LiAlH₄, THF, 0 °C, 12 h, 65%.

Steroid 10 was oxidized with Jones reagent to form the 3-ketosteroid 19 in 93% yield (Scheme 4). Steroid 19 was cross-metathesized with Grubbs catalyst, 2^nd generation and the olefinic diol to form the Δ²²-steroid intermediate which after hydrogenation yielded steroid 20 (54%, 2 steps). Steroid 20 was acetylated to obtain steroid 21 (87%) and then converted into steroid 22 (51%) by the earlier described bromination/elimination sequence. The earlier described sequence for the conversion of Δ⁴-3-ketosteroids to Δ⁵-3β-hydroxysteroids yielded steroid 23 which was not characterized, but was immediately deacetylated to yield 27-HC (3, 57% 3 steps).

Scheme 4.

Reagents and Conditions: (a) Jones reagent, acetone, 25 °C, 2 h, 93%; (b) 18, Grubbs catalyst, 2^nd generation, CH₂Cl₂, reflux, 72 h; (c) H₂, Pd/C (10%), EtOAc, 12 h, 54%, 2 steps; (d) acetic anhydride, DMAP, pyridine, 3.5 h, 87%; (e) pyridinium tribromide, AcOH, 1.5 h; (f) LiCl, DMF, 100 °C, 2 h, 51% (2 steps); (g) acetic anhydride, TMSCl, NaI, 0 °C to 25 °C, 3 h; (h) NaBH₄, EtOH, 12 h; (i) K₂CO₃, MeOH, reflux, 12 h, 57% (3 steps).

The synthetic route utilized for the ent-steroids was very similar to that described for the natural steroids. The reagent used to generate the side-chain for ent-Chol-d₃ (4) in the cross-metathesis reaction was synthesized by treating 4-methylpent-1-ene with Grubbs catalyst, 1^st generation to obtain 2,7-dimethyloct-4-ene 24 as a mixture of E and Z isomers in 51% yield (Scheme 5). The side-chain used to construct ent-27-HC-d₃ (5) was synthesized similarly to that for 27-HC. (4R)-3-(1-oxopropyl)-4-(phenylmethyl)-2-oxazolidinone was allylated, treated with 1^st generation Grubbs catalyst to generate the disubstituted olefin, and hydrolyzed to synthesize the diol 27. This compound was then acetylated with acetic anhydride, DMAP, and pyridine to generate the bis-acetylated product 28 in 50% overall yield for the four steps.

Scheme 5.

Reagents and Conditions: (a) Grubbs catalyst, 1^st generation, CH₂Cl₂, 12 h, 51%; (b) allyl magnesium iodide, NaHMDS, THF, -78 °C, 4 h, 93%; (c) Grubbs catalyst, 1^st generation, CH₂Cl₂, reflux, 24 h, 83%; (d) LiAlH₄, THF, 0 °C, 12 h, 82%; (e) acetic anhydride, DMAP, pyridine, 2 h, 66%.

ent-Testosterone-19,19,19-d₃ was synthesized as previously reported [32]. The deuterium labeled ent-testosterone was then converted to ent-steroid 29 (Scheme 6) in the same manner that unlabeled ent-testosterone was converted to the analogous unlabeled steroid [24]. In order to simplify the synthesis, we attempted to perform the cross-metathesis reaction directly on the natural enantiomer of compound 29 using both 2^nd generation Grubbs catalyst and the more active ruthenium catalyst, 38. This simplification was unsuccessful; however, when the natural enantiomer of compound 29 was reduced to the natural enantiomer of compound 30 using DIBAL-H, we found that the ruthenium-based catalyst 38 successfully catalyzed its cross-metathesis reaction with olefin 24. Therefore, ent-steroid 29 was reduced to ent-steroid 30 with DIBAL-H (97%) and was then treated with the ruthenium-based catalyst 38 and either olefin 24 or 28 to form ent-steroids 31 or 32, respectively. Both ent-steroids were then hydrogenated and oxidized with Jones reagent to generate ent-steroids 33 and 34 in 54% and 35% yield, respectively over 3 steps. Treatment of ent-steroids 33 and 34 with pyridinium tribromide in HOAc followed by LiCl in DMF yielded ent-steroids 35 and 36 in 45% and 42% yield, respectively. These Δ⁴-3-ketones were then converted either to ent-Chol-d₃ (65%) or to the acetylated intermediate 37 as described for the preparation of steroid 15. Treatment of ent-steroid 37 with K₂CO₃ yielded ent-27-HC-d₃ in 60% yield from ent-steroid 35. ent-Chol-d₃ has previously been prepared by a different total synthesis method [33].

Scheme 6.

Reagents and Conditions: (a) DIBAL-H (1M in THF), THF, -78 °C, 1 h, 97%; (b) 38 with 24 for synthesis of 31 or 38 with 28 for synthesis of 32, CH₂Cl₂, reflux, 72 h; (c) H₂/Pd, EtOAc, 4h; (d) Jones reagent, acetone, 30 min, 35-53% for 3 steps; (e) pyridinium tribromide, AcOH, 1.5 h; (f) LiCl, DMF, 100 °C, 2 h, 42-45% (2 steps); (g) acetic anhydride, TMSCl, NaI, 0 °C to 25 °C, 3 h; (h) NaBH₄, EtOH, 12 h, 65% for 4 (2 steps); (i) K₂CO₃, MeOH, reflux, 12 h, 60% for 5 (3 steps).

In summary, a cross-metathesis reaction has been successfully utilized to synthesize 25-HC (1), 25-HC-d₆ (2), 27-HC (3), ent-Chol-d₃ (4) and ent-27-HC-d₃ (5). We believe the methodology developed will be of general use for synthesizing other analogues with side-chains that are different from those described here. Further studies utilizing the newly synthesized ent-27-HC-d₆ to probe the mechanism for its regulation of cholesterol homeostasis are anticipated.

Abbreviations

25-HC

(3β)-cholest-5-ene-3,25-diol

25-HC-d₆

(3β)-cholest-5-ene-26,26,26,27,27,27-d₆-3,25-diol

27-HC

(3β,25R)-cholest-5-ene-3,26-diol

ent-Chol-d₃

ent-[(3β)-Cholest-5-en-19,19,19-d₃-3-ol]

ent-27-HC-d₃

ent-[(3β,25R)-Cholest-5-ene-19,19,19-d₃-3,26-diol]