A concise synthesis of β-sitosterol and other phytosterols

Abstract

A convenient synthesis of sidechain-modified phytosterols is achieved via a temporary masking of the stigmasterol 5,6-alkene as an epoxide. Following performance of the desired modification, the alkene is regenerated through a mild deoxygenation. The approach is applied to the syntheses of β-sitosterol and campesterol acetate, and suggests a facile route to the (Z)-isomers of Δ^22–23 phytosterols.

1. Introduction

Phytosterols and their derivatives are widely applied in the food and cosmetic industries, and have recently received a great deal of attention as nutraceutical additives [1,2,3]. Phytosterols have also attracted attention as inhibitors of sarcoplasmic reticulum calcium ATPase and potassium ion channels [4,5]. As part of a collaboration investigating the structural influences on uptake and processing of sterol esters[6], we required semipreparative amounts of β-sitosterol. However, β –sitosterol is commercially available in preparative amounts only as mixtures with other phytosterols, including stigmasterol, campesterol, and/or brassicasterol (Figure 1); reported separations are relatively laborious [7,8].

Figure 1.

Structural relationship of phytosterols

Two routes have been reported for synthesis of β –sitosterol from stigmasterol, which is available in pure form. The first, selective hydrogenation of the sidechain Δ^22–23 alkene [9], was found to produce β –sitosterol contaminated with varying amounts of recovered stigmasterol as well as the fully saturated stigmastanol [10]. The second approach, which has been applied to the synthesis of sitosterol and related sterols (Figure 2), circumvents the need for selective hydrogenation by protecting the Δ^5–6 alkene as a cyclopropyl carbinyl ether [11,12]. Following hydrogenation of the Δ^22–23 double bond, solvolysis of the cyclopropane reintroduces both the C₃-alcohol and the Δ^5–6 alkene. Although we found the latter approach very useful as a means of obtaining very pure samples of β –sitosterol, semipreparative applications were challenging in terms of removal of sterol methyl ether byproducts.

Figure 2.

Selective saturation of cyclopropyl carbinyl ether

We now report a new strategy for the synthesis of side-chain modified phytosterols based upon protection of the Δ^5–6 alkene as an epoxide. The approach is illustrated with syntheses of β-sitosterol and campesterol acetate.

2. Experimental

2.1. General Experimental Procedures

AlI₃ and Cu(MnO₄)₂ were prepared by literature procedures [13,14]. All other reagents and solvents were used as supplied commercially, except CH₂Cl₂ (CaH₂) and THF (Na, Ph₂CO) which were distilled from the indicated reagent under an atmosphere of N₂. Melting points are uncorrected. Unless noted, NMR spectra were acquired at 400 MHz (¹H) or 100 MHz (¹³C) in CDCl₃; individual peaks are reported as: multiplicity, integration, coupling constant in Hz. IR spectra were recorded as neat films on a ZnSe crystal with selected absorbances reported in cm⁻¹. Mass spectroscopy was conducted at the Nebraska Center for Mass Spectrometry.

2.2. Stigmasterol acetate (2)

Stigmasterol acetate was prepared as a white solid (97%, mp 138–140 °C) by a variant of the procedure of Wang [15]. Other physical and spectral data were identical to literature values.

2.3. 5α , 6α - and 5β , 6β-Epoxides of stigmasterol acetate (6a, 6b)

A mixture of KMnO₄ (10 g, 60 mmol) and CuSO₄ · 5H₂O (5.0 g, 20 mmol) was finely ground in a mortar and pestle [14]. Water (0.5 mL) was added, and the slightly wet mixture was transferred to the reaction flask. To the stirred suspension of this mixture in 25 mL CH₂Cl₂ was added stigmasterol acetate (2, 2.12 g, 4.51 mmol), followed by t-BuOH (2.5 mL). The reaction was heated to reflux for 1 hour and cooled to room temperature. The reaction mixture was filtered through a silica pad, which was washed with ether. The residue obtained after concentration was recrystallized from CH₃OH to give a white solid (1.59 g, 75%) with mp 125–126 °C. NMR data indicated the product was a 1 : 6 mixture of the α - (6a) and β - (6b) epoxides of stigmasterol acetate [14]. Repeating this reaction with 2.23 g of stigmasterol acetate afforded 1.80 g (78%) of a 1:6 mixture of 6a and 6b.

Approach to 5α , 6α - and 5β , 6β-Epoxides of stigmasterol acetate (6a, 6b) via peracid epoxidation: To a 0° C solution of 2 (0.308 g, 0.66 mmol) in CH₂Cl₂ (20 mL) was added mCPBA (0.170 g, 0.76 mmol). After 4 h at 0 °C, the reaction was diluted with sat. aq K₂CO₃ (80mL) and the aqueous layer was extracted with CH₂Cl₂ (50 mL x 3). The combined organic layers were dried over anhydrous Na₂SO₄ and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography (hexane/EtOAc, 90:10) to afford 0.257 g (83%) of a white solid which was a 2.6:1 mixture of α - (6a) and β-isomers (6b) according to ¹H NMR.

2.4. 5α , 6α - and 5β , 6β-Epoxy sitosterol acetate (7a, 7b)

To a solution of the 1 : 6 mixture of 6a and 6b (1.35 g, 3.0 mmol) in EtOAc (150 mL) was added 10% Pd/C (0.32 g), and the stirred reaction mixture was placed under an atmosphere of H₂ (balloon) for 12 h. The reaction mixture was filtered through a Celite pad, and the filtrate evaporated to furnish white solid (1.28 g, 96%, mp 113–114 °C) as a 1:6 mixture of epoxides 7a and 7b [8]. Repeating this reaction on 1.76 g of 6a/6b afforded 1.75 g (99%) of a 1:6 mixture of 7a and 7b.

2.5. β-Sitosterol acetate (4)

The 1 : 6 mixture of epoxides 7a and 7b (470 mg, 1.0 mmol) was dissolved in 2:1 CH₃CN/CH₂Cl₂ (30 mL). Aluminum triiodide was added (610 mg, 1.5 mmol) and the resulting mixture was stirred at room temperature for 10 minutes. The reaction was quenched with aq. 10% Na₂S₂O₃ (100 mL) and the resulting mixture was extracted with CH₂Cl₂ (3 x 100 mL). The combined organic layers were dried over Na₂SO₄, and the residue from the concentrated filtrate was purified by flash chromatography (hexane/EtOAc, 95:5) to give 360 mg (80%) of 4 as a white solid: mp 111–112 °C, [α ]_D = −34.5 (CHCl₃, c = 1.0). Other physical data were identical to values reported in the literature [11]. Repeating this reaction on 1.70 g of 7a/7b afforded 1.40 g (85%) of 4.

2.6. β-Sitosterol (3)

To a solution of β-sitosterol acetate (4, 240 mg, 0.47 mmol) in 1:1 CH₃OH:CH₂Cl₂ (30 mL) was added K₂CO₃ (140 mg, 1.01 mmol). The reaction mixture was stirred at room temperature for 12 hours and then concentrated under vacuum. The residue was extracted with 30 mL CH₂Cl₂. The organic layer was washed with 30 mL water and dried over Na₂SO₄. The filtered organic layer was concentrated and the residue was purified through flash chromatography (hexane/EtOAc, 80:20) to give 220 mg (93%) of β -sitosterol 3 as a white solid. Mp 134–135 °C, [α ]_D = −37.0 (CHCl₃, c = 1.0). Elemental analysis calculated for C₂₉H₅₀O: C 83.60, H 11.96; found: 83.99, 12.15. Other spectral properties were identical to values reported in the literature [11]. Repeating this reaction on 1.35 g of 8 afforded 1.20 g (98%) of beta sitosterol (3).

2.7. (S)-2,3-Dimethylbutan-1-ol (8)

(S)-2,3-Dimethylbutan-1-ol 8 was prepared as a colorless liquid (overall yield 60%, [α ]_D = 4.4 (CHCl₃, c = 1.0) by the procedure of Tietze, affording a product with spectral data identical to literature values [16].

2.8. (S)-2-(2,3-Dimethylbutylthio)benzothiazole (9)

To a mixture of dimethylbutanol 8 (102 mg, 1.00 mmol), 2-mercaptobenzothiazole (183 mg, 1.10 mmol) and PPh₃ (288 mg, 1.10 mmol) in freshly distilled THF (4 mL) was added diisopropy azodicarboxylate (DIAD, 0.21 mL, 1.10 mmol) dropwise at 0°C under argon. The reaction was stirred for 3 h at 0 °C and then quenched with water. The aqueous layer was extracted with EtOAc (10mL x 3) and the combined organic layers were dried over anhydrous Na₂SO₄. The filtered organic layer was concentrated in vacuo and the residue purified by flash chromatography (hexane/EtOAc, 99:1) to afford thioether 9 (228 mg, 91%) as a light yellow oil. [α ]_D = 42.3 (CHCl₃, c = 1.6); IR 2957, 1455, 1426, 1057, 991, 752 cm⁻¹; ¹H NMR: Δ 7.89 (d, J= 8.1 , 1H), 7.75 (d, J= 8.1 , 1H), 7.42 (t, J= 7.2 , 1H), 7.29 (t, J= 7.2 , 1H), 3.50 (dd, J= 12.7 , 4.8 , 1H), 3.18 (dd, J= 12.7, 8.2 , 1H), 1.88–1.77 (m, 2H), 1.04 (d, J= 6.7 , 3H), 0.99 (d, J= 6.6 , 3H), 0.94 (d, J= 6.6 , 3H); ¹³C NMR: Δ 167.75, 153.38, 135.15, 125.99, 124.08, 121.44, 120.91, 38.87, 38.78, 31.62, 20.37, 17.96, 15.29; HRFAB-MS (m/z) [M-H]⁺ calcd for [C₁₃H₁₈NS₂]⁺: 252.0881, found: 252.0875.

2.9 (S)-2-(2,3-Dimethylbutylsulfonyl)benzothiazole (10)

A 0°C solution of 9 (183 mg, 0.73 mmol) in EtOH (10 mL) was oxidized with ammonium heptamolybdate tetrahydrate (1.8 g, 1.46 mmol) and 30% H₂O₂ (2.5 mL, 21.9 mmol) for 2 hours. The mixture was extracted with EtOAc (10 mL x 3) and the combined organic extracts were washed with brine (10 mL x 3). The dried organic layers was filtered and the residue obtained upon concentration was purified by flash chromatography (hexane/ EtOAc, 90:10) to afford sulfone 10 (177mg, 86%) as a pale yellow oil. [α ]_D = 15.5 (CHCl₃, c = 3.4); IR 2961, 1470, 1324, 1140, 1085, 758 cm⁻¹; ¹H NMR: Δ 8.23 (d, J= 7.9, 1H), 8.03 (d, J= 7.9, 1H), 7.68–7.59 (m, 2H), 3.59(dd, J= 14.4, 3.5, 1H), 3.31(dd, J= 14.1, 8.9, 1H), 2.29–2.19(m, 1H), 1.82–1.73(m, 1H), 1.10(d, J= 6.9, 3H), 0.89(d, J= 6.8, 3H), 0.85(d, J= 6.9, 3H); ¹³C NMR: Δ 166.66, 152.70, 136.74, 128.00, 127.67, 125.43, 122.38, 58.83, 38.68, 32.47, 19.23, 17.89, 15.93; HRFAB-MS (m/z) [M-H]⁺ calcd for [C₁₃H₁₈NO₂S₂]⁺: 284.0779, found: 284.0778.

2.10. (3β ,5α ,6α )- and (3β ,5β ,6β )- Pregnane-20α-carboxaldehyde-5,6-epoxy-3-yl acetate (11a, b)

A −78 °C solution of 6a, 6b (~ 1:6 mixture, 100 mg, 0.21 mmol) in 10 mL of 50/50 CH₂Cl₂/MeOH was treated with a gaseous stream of ozone (2% O₃/O₂) for 5 minutes. The solution was purged with pure oxygen and then solvent was removed under vacuum. The residue was redissolved in 10 mL of 10/90 H₂O/AcOH and treated with zinc powder (55 mg, 0.84 mmol). The reaction mixture was stirred for 2 hours at room temperature and then extracted with 50 mL CH₂Cl₂. The organic layer was washed with water (25 mL x 3), then dried over anhydrous Na₂SO₄. The filtered organic layer was concentrated and the residue purified by flash chromatography (hexane/ EtOAc, 90:10) to afford a 1:6 mixture of epoxides 11a and 11b as a white solid (81 mg, 99%), mp 87–8 °C. IR: 2950, 1727, 1367, 1262, 1238, 1042, 783 cm⁻¹; ¹H NMR: Δ 9.57 (d, J= 3.3 , 0.76H, β ), 9.55 (d, J= 3.3 , 0.16H, α ), 4.99–4.91 (m, 0.14H, α ), 4.81–4.73(m, 0.87H, β ), 3.09(d, J= 2.2, 0.88H, β ), 2.90 (d, J= 4.2, 0.13H, α ), 2.38–2.31(m, 1H), 2.13–1.82(m, 9H), 1.54–0.89(m, 20H), 0.7(s, 3H); ¹³C NMR: Δ 204.95, 170.52, 71.25, 63.41, 62.48, 55.39, 51.05, 50.93, 49.42, 42.89, 39.43, 37.95, 36.67, 35.06, 32.41, 29.74, 27.17, 26.97, 24.54, 21.84, 21.30, 17.03, 13.40, 12.11; HRFAB-MS (m/z) [M-Li]⁺ calcd for [C₂₄H₃₆LiO₄]⁺: 395.2774, found: 395.2778.

2.11. (3β ,5α ,6α ,22Z)- and (3β ,5β ,6β ,22Z)- Ergost-5,6-epoxy-22-en-3-yl acetate (12a, 12b)

To a 78 °C solution of sulfone 10 (62 mg, 0.22 mmol) in THF (5 mL) was dropwise added LiHMDS (0.22 mL, nominally 1M in THF, 0.22 mmol). The reaction was stirred for 1 h, whereupon the mixture of aldehydes 11a and 11b (~1:6, 85 mg, 0.22 mmol) was added in 5 mL of THF. Stirring was continued for 1 hour, and reaction was gradually warmed to room temperature. The reaction was quenched by 15 mL water and extracted with EtOAc (10 mL x 3). The combined organic layers were dried over anhydrous Na₂SO₄ and filtered. The residue obtained upon concentration was purified by flash chromatography (hexane/ EtOAc, 95:5) to afford a 1:16 mixture of epoxides 12a and 12b as a white solid (90 mg, 90%), mp 145–147 °C. IR: 2950, 2867, 1743, 1368, 1037, 764 cm⁻¹; ¹H NMR: Δ5.02(dd, J= 10.9, 9.9 , 2H), 4.83–4.73(m, 1H), 3.09(d, J= 2.0 , 0.95H, β ), 2.91(d, J= 4.4, 0.06, a), 2.43–2.33(m, 1H), 2.21–1.80(m, 9H), 1.68–0.83(m, 31H), 0.69(s, 3H); ¹³C NMR: Δ170.56, 135.13, 131.22, 71.34, 63.58, 62.52, 56.27, 56.02, 51.02, 42.18, 39.72, 38.32, 38.01, 36.68, 35.04, 34.48, 33.35, 32.43, 29.74, 28.32, 27.20, 24.15, 21.92, 21.34, 20.55, 20.36, 19.94, 18.63, 17.06, 12.06; HRFAB-MS (m/z) [M-H]⁺ calcd for [C₃₀H₄₉O₃]⁺: 457.3682, found: 457.3668.

2.12. 5α , 6α - and 5β , 6β-Epoxides of campesterol acetate (13a, 13b)

The mixture of epoxides 12a and 12b (30mg, 0.065 mmol) was dissolved in 5mL EtOAc. 10% Pd/C (7 mg) was added, and the reaction mixture was stirred at room temperature under an atmosphere of H₂ (balloon) for 12 h. The reaction mixture was filtered through a Celite pad, and the filtrate evaporated to a white solid (28 mg, 94%, mp 110–111 °C) as 1:9 mixture of epoxides 13a and 13b. IR: 2953, 2867, 1729, 1367, 1263, 1043, 784 cm⁻¹; ¹H NMR: Δ5.01–4.93(m, 0.15H, ■), 4.83–4.73(m, 0.96H, β ), 3.09(d, J=2.1 , 0.9H, β ), 2.90(d, J= 4.4 , 0.1H, ■), 2.12–1.8(m, 8H), 1.58–0.77(m, 37H), 0.65(s, 3H); ¹³C NMR: Δ 170.54, 71.34, 63.58, 62.51, 56.19, 56.14, 50.97, 42.28, 39.78, 38.81, 38.01, 36.66, 35.82, 35.03, 33.65, 32.47, 32.41, 30.26, 29.73, 28.14, 27.21, 24.18, 21.92, 21.31, 20.19, 18.66, 18.24, 17.03, 15.37, 11.76; HRFAB-MS (m/z) [M-H]⁺ calcd for [C₃₀H₅₁O₃]⁺: 459.3838, found: 459.3820.

2.13. Campesterol acetate (5)

The mixture of epoxides 13a and 13b (28 mg, 0.061 mmol) was dissolved in 2:1 CH₃CN/CH₂Cl₂ (3 mL). Aluminum triiodide (37 mg, 0.091 mmol) was added and the resulting mixture was stirred at room temperature for 40 minutes. The reaction was quenched with aq. 10% Na₂S₂O₃ (10 mL) and the resulting mixture was extracted with CH₂Cl₂ (3 x 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄, and the residue from the concentrated filtrate was purified by flash chromatography (hexane/EtOAc, 95:5) to give 24mg (91%) campesterol acetate (5) as a white solid. Mp 130–131 °C, [α ]_D = −32 (CHCl₃, c = 0.7); IR 2954, 1730, 1367, 1247, 1037, 735 cm⁻¹; ¹H NMR: Δ 5.39(d, J= 4.8, 1H), 4.66–4.58(m, 1H), 2.33(d, J= 7.9, 2H), 2.05(s, 3H), 1.90–1.84(m, 2H), 1.59–0.78(m, 38H), 0.68(s, 3H); ¹³C NMR: Δ170.56, 139.66, 122.66, 73.99, 56.69, 56.08, 50.02, 42.31, 39.73, 38.84, 38.12, 36.99, 36.59, 35.90, 33.70, 32.43, 31.90, 31.86, 30.27, 28.24, 27.78, 24.29, 21.46, 21.03, 20.22, 19.32, 18.70, 18.26, 15.38, 11.87 ; HRFAB–MS (m/z) [M-Na]⁺ calcd for [C₃₀H₅₀O₂Na]⁺: 465.3709, found: 465.3703. Elemental analysis calculated for C₃₀H₅₀O₂: C 81.20, H 11.38; found: 81.39, 11.39. The ¹H NMR data matched that of a literature report [17].

3. Results and Discussion

Our synthesis of β-sitosterol (3) is illustrated in Scheme 1. Selective epoxidation of the Δ^5–6 alkene of stigmasterol acetate (2) with copper permanangate formed a 6:1 mixture of the 5β ,6β - and 5α ,6α epoxides 6b and 6a [14,18]. Hydrogenation over Pd/C cleanly furnished a 1:6 mixture of sitosterol epoxides 7a and 7b. Deoxygenation of the saturated epoxides with AlI₃ [13] proceeded rapidly to furnish a good yield of β-sitosteryl acetate 4. Saponification afforded pure β-sitosterol (3), with mp 134–135 °C and [α] _D = − 37.0 [8,19].

Scheme 1.

Synthesis of sitosterol

Epoxidation with the commercially available peracid mCPBA also gave good selectivity for the Δ^5–6 alkene, but now produced a 2.6:1 mixture of stigmasterol oxides favoring the α-isomer (6a). Hydrogenation proceeded uneventfully to furnish the corresponding mixture of sitosterol oxides 7a and 7b. However, attempted deoxygenation under the same conditions as employed earlier (AlI₃, 10 min, CH₃CN/CH₂Cl₂) now furnished only 33% of β-sitosterol acetate (4), accompanied by a significant amount (estimated > 60% by mass) of a more polar product which yellowed immediately upon exposure to room light. The formation of the byproduct could be avoided almost completely by allowing the deoxygenation to proceed for 40 min. Alternatively, the byproduct could be converted to 4 by treatment with additional AlI₃. The results suggest that the deoxygenation of the α - and β - epoxides proceeds at very different rates, with the 5α , 6α diastereomer (6a) reacting via the intermediacy of a semistable iodohydrin.

As illustrated in Scheme 2, our strategy also provides a facile means of preparing other sidechain-modified phytosterols. For example, ozonolysis of the mixture of 6a/6b furnished an approximately 1:6 mixture of aldehydes 11a and 11b. Julia-Kocienski olefination, using an enantiomerically pure sulfone (10) derived from (S)-2,3-dimethylbutanol (8) [16] furnished exclusively alkene [20], corresponding to the monoepoxide of the Z-isomer of crinosterol [21]. Hydrogenation, followed by deoxygenation of the epoxide as before, furnished campesterol acetate (5).

Scheme 2.

Synthesis of campesterol acetate

The selective formation of Z-alkenes is unusual in Julia couplings [22] and we investigated the olefination of 11a,b with a known sulfone derived from isobutyl alcohol (Figure 3) [23]. This reaction also selectively furnished the Z-alkene; the lower selectivity (2:1) compared with that observed for the synthesis of 12a,b may reflect the reduced degree of steric encumbrance in this model system. The ability to readily prepare Z-isomers of phytosterols opens the door to a number of steroid analogs not available from synthetic routes based upon Claisen rearrangements of C₂₂ allylic alcohols [12].

Figure 3.

Generality of Z-selective olefination

Conclusion

The formation of β-sitosterol has been achieved in 52% overall yield from commercially available stigmasterol using relatively simple chemistry and via easily purified intermediates. The core strategy, protection of the Δ^5–6 alkene as an epoxide, holds potential for synthesis of other phytosterols as well as unnatural analogs.

Appendix A. Supplementary data

Supplementary data for this article, consisting of ¹H and ¹³C NMR spectra for compounds 3-13, can be found in the online version at doi 10.1016/j.steroids.2010.xxxx.