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Synthesis of [26-2H3]brassinosteroids
Steroids 67 (2002) 587–595
Synthesis of [26-2H3]brassinosteroids Vladimir A. Khripacha,*, Vladimir N. Zhabinskiia, Olga V. Konstantinovaa, Andrey P. Antonchicka, Bernd Schneiderb a
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich str., 5/2, 220141 Minsk, Belarus b Max-Planck-Institute for Chemical Ecology, Beutenberg Campus Winzerlaer Str. 10, D-07745 Jena, Germany Received 28 June 2001; received in revised form 2 November 2001; accepted 9 November 2001
Abstract A number of [26-2H3]brassinosteroids were prepared for biochemical studies. The parent, nondeuterated compounds were considered to be biosynthetic intermediates in brassinosteroid biosynthesis. Claisen rearrangement was used to construct the steroidal side chain. Deuterium was introduced by reducing the corresponding intermediates with lithium aluminium deuteride. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Brassinosteroids; Labeled compounds; Claisen rearrangement; Brassinolide; Plant growth hormone
1. Introduction Brassinosteroids represent a new class of unique naturally occurring plant-growth-regulating substances with high biologic activity at very low concentrations. Recent research efforts have been directed toward the search for related compounds in the plant kingdom and their chemical synthesis, biologic mode of action, and practical application in agriculture and horticulture [1,2]. The elucidation of the biosynthesis of brassinosteroids is an important contribution to brassinosteroid research in general. Research on the regulation of brassinosteroid biosynthesis and their mode of action requires detailed knowledge of the pathways and enzymes responsible for brassinosteroid formation in plants. The brassinosteroid skeleton is biosynthetically formed before campestanol in early steps of the pathway. This intermediate is converted into brassinolide by a series of oxidation and reduction steps without further changes to the skeleton. These oxidation/reduction reactions may be interchanged in sequence. For example, two alternative biosynthetic routes from campestanol to castasterone, the so-called early C-6 oxidation pathway [3,4] and the late C-6 oxidation
* Corresponding author. Tel.: ⫹375-2-648-647; fax: ⫹375-2-648-647. E-mail address: [email protected] (V.A. Khripach).
pathway [5,6], have been identified in Catharanthus roseus by Japanese research groups. We hypothesized that in different plant families or even in one species, brassinosteroids are formed by a biosynthetic network of alternative pathways and subpathways. A number of feeding experiments using labelled precursors were necessary to confirm this. Deuterated compounds were the preferred substrates because detection of brassinosteroids by mass spectrometry is an established analytical method [7]. A number of labelled brassinosteroids have been synthesized by various groups and used as internal standards for quantitative analysis of endogenous brassinosteroids, and they have also been employed in biosynthetic experiments [8 –12]. [26,28-2H6]Brassinosteroids, for example, were prepared in a multistep procedure by introducing two deuterated methyl groups stepwise into the side chain of crinosterol [13–15]. [5,7,7-2H3]Epibrassinolide was synthesized by base-catalyzed enolization [16]. A similar procedure was used to prepare [5,7,7]-tritium [17,18] and -deuterium [19] labelled brassinosteroids. This enolization method yields incomplete labeling and therefore is not suitable for quantitative analysis. The main objective of the present investigation was to elaborate synthetic approaches to labelled brassinosteroid precursors with high isotopic purity and with at least three deuteriums in non-exchangable positions.
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 9 - 1 2 8 X ( 0 2 ) 0 0 0 0 4 - 1
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Scheme 1.
2. Experimental 2.1. General Melting points were taken on a Boetius micro-melting point apparatus and are uncorrected. IR spectra were recorded on an UR-20 spectrophotometer. 1H and 13C NMR spectra were taken on a Bruker AC-200 (200 MHz for 1H, 50 MHz for 13C) spectrometer using TMS as an internal standard in CDCl3 (if not stated otherwise). The exact mass measurements were carried out on a Micromass MasSpec mass spectrometer, operating in the 70 eV-EI mode. Samples were introduced by direct probe for accurate mass measurement by peak matching. Reactions were monitored by TLC using aluminium or plastic sheets precoated with silica gel 60 F254 (Merck Art. 5715). Column chromatography was carried out on Kieselgel 60 (Merck Art. 7734).
Scheme 2.
2.2. (22E,24S)-[26-2H3]-6-Methoxy-24-methyl-3␣,5cyclo-5␣-cholest-22-ene (4)
56.6, 82.4, 131.8, 136.1. HRMS Calc. for C29H45D3O: 415.38904; Found: 415.38854.
A mixture of (22E,24R)-6-methoxy-24-methyl-3␣,5cyclo-5␣-cholest-22-en-26-oic acid ethyl ester 2 (5.5 g, 11.7 mmol, prepared according to [20] and LiAlD4 (1.55 g, 37 mmol) in ether (100 ml) was stirred at room temperature for 1 h. The excess reagent was decomposed by treatment with a 15% solution of NaOH. The precipitate was filtered off, and the filtrate was evaporated and dissolved in pyridine (20 ml). TsCl (4.7 g, 24.6 mmol) was added, and the mixture was left at room temperature for 30 h. Afterwards it was diluted with water and extracted with CHCl3. The extract was dried (Na2SO4) and evaporated. The residue was dissolved in ether (150 ml) and LiAlD4 (2.05 g, 48.9 mmol) was added. The mixture was stirred for 1 h and then treated with a 15% solution of NaOH. The precipitate was filtered off, and the filtrate was evaporated. The residue was chromatographed on SiO2 with cyclohexane-EtOAc (30:1 f 12:1), resulting in compound 4 (3.1 g, 76%). 1H NMR ␦: 0.44 (dd, 2H, J ⫽ 8, 5 Hz, C4-H), 0.66 (t, 1H, J ⫽ 4.3 Hz, C3-H), 0.75 (s, 3H, 18-Me), 1.04 (s, 3H, 19-Me), 2.78 (t, 1H, J ⫽ 2.8 Hz, C6-H), 3.33 (s, 3H, OMe), 5.16 (m, 2H, C22- and C23-H). 13C NMR ␦: 12.4, 13.1, 18.1, 19.3, 19.6, 20.1, 21.0, 21.5, 22.8, 24.2, 25.0, 28.9, 30.5, 33.0, 33.4, 35.1, 35.3, 40.2, 40.3, 42.7, 43.0, 43.4, 48.1, 56.1, 56.5,
2.3. (22E,24S)-[26-2H3]-24-Methyl-3␣,5-cyclo-5␣-cholest22-en-6-one (6) A mixture of 4 (3.1 g, 7.5 mmol), TsOH (0.45 g, 2.4 mmol), water (35 ml), and dioxane (106 ml) was heated at 70°C for 2.5 h. Pyridine (1 ml) was then added, and the mixture was evaporated. The residue was dissolved in pyridine (20 ml), and TsCl (5.4 g, 28.3 mmol) was added. The mixture was left at room temperature for 30 h, then diluted with water, and extracted with CHCl3. The organic layer was dried (Na2SO4) and evaporated. The residue was dissolved in acetone (0.5 l) and then, AcOK (1.9 g, 19.4 mmol) and water (37.5 ml) were added. The mixture was refluxed for 24 h, after which Jones reagent (5 ml) was added at room temperature. The mixture was stirred for 20 min, iPrOH (10 ml) was then added, and the stirring was continued for 15 min. The mixture was diluted with water and extracted with CHCl3. The solvent was evaporated, and the residue was purified by column chromatography on SiO2 (cyclohexaneEtOAc, 40:1 f 10:1), resulting in the ketone 6 (2.6 g, 82%). Mp 87–91°C (hexane). IR (cm⫺1): 2970, 2875, 2220, 1705, 1465, 1385, 1300, 1175, 1125, 980. 1H NMR ␦: 0.74 (s, 3H, 18-Me), 1.02 (s, 3H, 19-Me), 5.16 (m, 2H, C22- and C23-H).
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C NMR ␦: 11.7, 12.3, 18.1, 19.7, 20.1, 21.0, 22.9, 24.1, 25.9, 28.8, 33.0, 33.5, 34.8, 35.3, 39.7, 40.2, 42.6, 43.1, 44.8, 46.1, 46.4, 46.8, 55.9, 57.1, 132.1, 135.8, 209.7. HRMS Calc. for C28H41D3O: 399.35774; Found: 399.35760. 13
2.4. Hydroxylation of the olefin (6) 2.4.1. Variant A A mixture of the olefine 6 (2.6 g, 6.5 mmol), AD-mix (16 g), methanesulfonamide (1.2 g, 12.6 g), K2OsO4.2H2O (0.12 g, 0.33 mmol), (DHQD)2PHAL (0.5 g, 0.64 mmol), tert-butanol (110 ml), and water (110 ml) was stirred at room temperature for 48 h. Then, Na2SO3 (6.5 g, 51.6 mmol) was added, and stirring was continued for 3 h. The mixture was partly evaporated and extracted with CHCl3. The organic layer was evaporated, and the residue was dissolved in CHCl3 and treated successively with 0.25 M H2SO4 and saturated NaHCO3. After evaporation, the residue was chromatographed on SiO2 with cyclohexane-EtOAc (9:1 f 1:1) to give a) (24S)-[262 H3]-24-methyl-3␣,5-cyclo-5␣-cholesta-6,22,23-trione 9 (0.7 g, 25%). Mp 119 –122°C (hexane). IR (cm⫺1): 2970, 2880, 2225, 1715, 1700, 1465, 1375, 1300, 925. 1H NMR ␦: 0.80 (s, 3H, 18-Me), 0.86 (d, 3H, J ⫽ 7 Hz, 28-Me), 0.91 (d, 3H, J ⫽ 6.5 Hz, 21-Me), 1.00 (d, 3H, J ⫽ 6.7 Hz, 27-Me), 1.02 (s, 3H, 19-Me), 3.24 (m, 1H, C20- or C24H), 3.45 (m, 1H, C24- or C20-H). 13C NMR ␦: 11.7, 11.9, 12.3, 16.4, 18.4, 19.7, 21.2, 22.8, 24.4, 25.9, 27.5, 29.2, 33.5, 34.7, 35.4, 39.6, 40.9, 43.1, 44.3, 44.7, 46.1, 46.3, 46.7, 51.8, 56.2, 202.4, 203.1, 209.3. HRMS Calc. for C28H39D3O3: 429.33192; Found: 429.33246; b) (22R,23R,24S)-[26-2H3]-22,23-dihydroxy-24-methyl3␣,5-cyclo-5␣-cholestan-6-one 7 (1.05 g, 37%). Mp 157– 159°C (hexane-EtOAc). IR (cm⫺1): 2945, 2875, 2375, 2235, 2210, 1690, 1465, 1385, 1315, 1175, 990. 1H NMR ␦: 0.75 (s, 3H, 18-Me), 1.02 (s, 3H, 19-Me), 3.57 (d, 1H, J ⫽ 8 Hz, C22-H), 3.74 (d, 1H, J ⫽ 8 Hz, C23-H). 13C NMR ␦: 10.1, 11.7, 12.0, 19.7, 20.7, 20.8, 22.9, 23.9, 25.9, 27.8, 30.5, 33.5, 34.9, 35.4, 36.9, 39.8, 40.1, 42.6, 44.8, 46.1, 46.4, 46.8, 52.3, 56.9, 73.5, 74.7, 209.8. HRMS Calc. for C28H43D3O: 433.3632; Found: 433.36318. 2.4.2. Variant B A mixture of the olefine 6 (2.2 g, 5.5 mmol), K3Fe(CN)6 (10.9 g, 33 mmol), K2CO3 (4.6 g, 33 mmol), methanesulfonamide (1.05 g, 11 mmol), K2OsO4.2H2O (0.04 g, 0.11 mmol), DHQD CLB (0.26 g, 0.56 mmol), tert-butanol (90 ml), and water (70 ml) was stirred for 3 days. Then, Na2SO3 (5 g, 40 mmol) was added, and stirring was continued for 24 h. The mixture was evaporated partly in vacuo and extracted with CHCl3. The organic layer was washed with 0.25 M H2SO4 and brine and then evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 5:1) to
Scheme 3.
give: a) (24S)-[26-2H3]-24-methyl-3␣,5-cyclo-5␣-cholesta-6,22,23-trione 9 (150 mg, 6%); b) (22S,23S,24S)[26- 2 H 3 ]-22,23-dihydroxy-24-methyl-3 ␣ ,5-cyclo-5 ␣ cholestan-6-one 8 (0.55 g, 23%). Mp. 179 –182°C (EtOAc-hexane). IR (cm⫺1): 2970, 2880, 2220, 1690, 1470, 1385, 1300, 1175, 1135, 1030, 970. 1H NMR ␦: 0.78 (s, 3H, 18-Me), 1.03 (s, 3H, 19-Me), 3.45 (d, 1H, J ⫽ 9.3 Hz, C22-H), 3.69 (d, 1H, J ⫽ 4.5 Hz, C23-H). 13C NMR ␦: 10.1, 11.7, 12.0, 14.1, 15.7, 19.7, 21.6, 22.9, 24.3, 25.9, 26.2, 27.8, 33.5, 34.8, 35.5, 39.7, 41.7, 42.3, 43.3, 44.7, 46.1, 46.4, 46.7, 52.7, 56.6, 71.1, 71.6, 209.8. HRMS Calc. for C 28 H 43 D 3 O 3 : 433.36353; Found: 433.36340; c) (22R,23R,24S)-[26-2H3]-22,23-dihydroxy24-methyl-3 ␣ ,5-cyclo-5 ␣ -cholestan-6-one 7 (0.85 g, 36%).
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2.5. (22R,23R,24S)-[26-2H3]-22,23-Diacetoxy-24-methyl3␣,5-cyclo-5␣-cholestan-6-one (10) A mixture of the diol 7 (0.85 g, 1.96 mmol), acetic anhydride (0.74 ml, 7.8 mmol), DMAP (0.02 g, 0.2 mmol), and pyridine (7 ml) was heated at 45°C for 4.5 h, after which it was diluted with water and extracted with CHCl3. The organic layer was dried (Na2SO4), and the solvent was evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (15:1 f 6:1) to give the diacetate 10 (0.8 g, 75%). Mp 172–174°C (hexane-EtOAc). IR (cm⫺1): 2965, 2880, 1750, 1700, 1460, 1380, 1300, 1250, 1030, 980. 1H NMR ␦: 0.77 (s, 3H, 18-Me), 1.03 (s, 3H, 19-Me), 2.02, 2.05 (s, 3H, OAc), 5.18 (d, 1H, J ⫽ 8.5 Hz, C22-H), 5.35 (d, 1H, J ⫽ 8.5 Hz, C23-H). HRMS Calc. for C32H47D3O5: 517.38466; Found: 517.38441. 2.6. (22R,23R,24S)-[26-2H3]-3-Bromo-22,23-diacetoxy24-methyl-5␣-cholestan-6-one (11) A solution of HBr (48%, 3 ml) was added to a stirred solution of 10 (0.9 g, 1.7 mmol) in acetic acid (10 ml). The mixture was stirred for 10 min and then diluted with water. The precipitate was filtered, washed, and air-dried to give the bromide 11 (1.0 g, 97%). Mp 221–222°C (MeOHEtOAc). IR (cm⫺1): 2975, 2955, 2880, 2380, 2215, 1750, 1720, 1470, 1380, 1255, 1030, 980. 1H NMR ␦: 0.72 (s, 3H, 18-Me), 0.82 (s, 3H, 19-Me), 2.02, 2.05 (s, 3H, OAc), 3.85– 4.05 (m, 1H, C3-H), 5.16 (d, 1H, J ⫽ 9 Hz, C22-H), 5.33 (d, 1H, J ⫽ 9 Hz, C23-H). 13C NMR ␦: 11.0, 11.8, 12.8, 13.1, 20.2, 20.9, 21.3, 23.8, 28.0, 30.1, 32.3, 33.3, 36.9, 37.7, 39.1, 39.3, 39.7, 40.6, 42.8, 46.4, 50.5, 52.3, 53.7, 56.5, 58.9, 74.1, 75.7, 170.5, 209.4. HRMS Calc. for C32H49D3O5Br: 598.31864; Found: 598.31850. 2.7. Reaction of the bromide (11) with Hg(OAc)2 A mixture of the bromide 11 (257 mg, 0.43 mmol), Hg(OAc)2 (260 mg, 0.82 mmol), and acetic acid (4 ml) was heated at 70°C for 35 h. The solvent was evaporated in vacuo, and the residue was dissolved in CHCl3 and filtered through SiO2. The filtrate was evaporated and chromatographed on SiO2 with petroleum ether-EtOAc (20:1 f 2:1) to give: a) (22R,23R,24S)-[26-2H3]-22,23-diacetoxy-24methyl-5␣-cholest-2-en-6-one 15 (33 mg, 15%). Mp 214 – 216°C (hexane-EtOAc). IR (cm⫺1): 2975, 2945, 2875, 2215, 1755, 1720, 1445, 1385, 1250, 1060, 1030. 1H NMR ␦: 0.72 (s, 3H, 18-Me), 2.01, 2.03 (s, 3H, OAc), 5.17 (d, 1H, J ⫽ 9 Hz, C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H), 5.50 –5.76 (m, 2H, C2- and C3-H). 13C NMR ␦: 11.1, 11.8, 12.8, 13.5, 20.3, 20.89, 20.94, 21.1, 21.7, 23.9, 28.1, 30.2, 37.0, 37.7, 39.38, 39.46, 39.79, 39.99, 42.7, 46.9, 52.4, 53.4, 53.8, 56.7, 74.2, 75.8, 124.5, 125.0, 170.5, 211.7. HRMS Calc. for C32H49D3O5: 517.384655; Found: 517.384598; b) (22R,23R,24S)-[26-2H3]-3,22,23-triacetoxy-24-methyl-5␣cholestan-6-one 12a (130 mg, 52%). Mp 94 –97°C (EtOAc-
MeOH). 1H NMR ␦: 0.72 (s, 3H, 18-Me), 0.78 (s, 3H, 19-Me), 0.85–1.09 (m, 9H, 21-, 27-, and 28-Me), 2.03, 2.06, 2.07 (s, 3H, OAc), 5.07–5.22 (m, 2H, C3- and C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H). IR (cm⫺1): 2955, 2880, 1750, 1720, 1375, 1255, 1030. HRMS Calc. for C34H51D3O7: 577.405785; Found: 577.404312. 2.8. Saponification of the triacetate (12a) The triacetate 12a (58 mg, 0.1 mmol) was added to a solution of KOH in MeOH (15%, 15 ml). After the mixture was refluxed for 1 h, then it was treated with brine and extracted with CHCl3. The extract was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 1:4) to give: a) (22R,23R,24S)-[26-2H3]-3␣,22,23-trihydroxy-24-methyl5␣-cholestan-6-one 12b (9 mg, 20%). Mp 230 –233°C (EtOAc-MeOH). IR (cm⫺1): 2945, 2880, 2215, 1715, 1390, 1255, 985. 1H NMR ␦: 0.70 (s, 3H, 18-Me), 0.75 (s, 3H, 19-Me), 2.32 (dd, 1H, J ⫽ 13, 4.5 Hz, C7-H), 3.56 (d, 1H, J ⫽ 8 Hz, C22-H), 3.73 (d, 1H, J ⫽ 8 Hz, C23-H), 4.18 (br.s, 1H, C3-H). HRMS Calc. for C28H45D3O4: 451.37409; Found: 451.37289; b) a mixture of 12b and 12c (13 mg, 29%); c) (22R,23R,24S)-[26-2H3]-3,22,23-trihydroxy-24methyl-5␣-cholestan-6-one 12c. Mp 251–254°C (EtOAcMeOH). IR (cm⫺1): 2955, 2880, 2235, 2210, 1715, 1470, 1430, 1390, 1250, 1130, 1120, 1060, 980. 1H NMR ␦: 0.69 (s, 3H, 18-Me), 3.56 (d, 1H, J ⫽ 8.5 Hz, C22-H), 3.64 (m, 1H, C3-H), 3.73 (d, 1H, J ⫽ 8.5 Hz, C23-H). HRMS Calc. for C28H45D3O4 (M⫹-H2O): 433.363526; Found: 433.362503. 2.9. Dehydrobromination of the bromide (11) A mixture of the bromide 11 (555 mg, 0.93 mmol), LiCO3 (1.1 g, 14.7 mmol), and DMF (10 ml) was refluxed for 3.5 h, after which it was cooled down, diluted with water, and extracted with CHCl3. The organic layer was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (25:1 f 10:1) to give: a) (22R,23R,24S)-[26-2H3]-22,23-diacetoxy24-methyl-5␣-cholest-2-en-6-one 15 (254 mg, 52%); b) (22R,23R,24S)-[26-2H3]-3-formyloxy-22,23-diacetoxy-24methyl-5␣-cholestan-6-one 14 (72 mg, 16%). IR (cm⫺1): 2950, 2880, 2215, 1750, 1730, 1375, 1255, 1030. 1H NMR ␦: 0.72 (s, 3H, 18-Me), 2.01, 2.03 (s, 3H, OAc), 5.17 (d, 1H, J ⫽ 9 Hz, C22-H), 5.28 (br.s, 1H, C3-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H), 8.04 (s, 1H, CHO). 2.10. (22R,23R,24S)-[26-2H3]-3,22,23-Triacetoxy-24methyl-B-homo-7-oxa-5␣-cholestan-6-one (13a) A solution of H2O2 (30%, 2 ml) was added to a stirred solution of (CF3CO)2O (9 ml, 63.7 mol) in CH2Cl2 (6 ml), at ⫺5°C. The mixture was stirred for 15 min, and then a solution of the triacetoxyketone 12a (135 mg, 0.22 mmol) in
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CH2Cl2 (4 ml) was added. The mixture was kept for 3 h, then diluted with water, and extracted with CHCl3. The extract was washed with saturated NaHCO3, dried (Na2SO4), and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (15:1 f 2:1) to give the lactone 13a (40 mg, 29%). IR (cm⫺1): 2960, 1745, 1380, 1255, 1190, 1030. 1H NMR ␦: 0.75 (s, 3H, 18-Me), 0.92 (s, 3H, 19-Me), 2.01, 2.03, 2.08 (s, 3H, OAc), 4.09 (m, 2H, C7-H), 5.11 (m, 1H, C3-H), 5.16 (d, 1H, J ⫽ 9 Hz, C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H). HRMS Calc. for C34H51D3O8 (M⫹-AcOH): 533.379570; Found: 533.380447. 2.11. (22R,23R,24S)-[26-2H3]-3␣,22,23-Trihydroxy-24methyl-B-homo-7-oxa-5␣-cholestan-6-one (13b) The triacetate 13a (40 mg, 0.069 mmol) was added to a solution of KOH in MeOH (15%, 2 ml). The mixture was refluxed for 1 h, then cooled down, and a solution of HCl (25%, 1 ml) was added. After being stirred at room temperature for 2 h, the mixture was diluted with water and extracted with CHCl3. The organic layer was dried (Na2SO4), and evaporated, and the residue was purified on SiO2 (CHCl3-MeOH, 100:1 f 50:1) to give the trihydroxylactone 13b (25 mg, 79%). Mp 225–227°C (hexaneEtOAc). HRMS Calc. for C28H43D3O4 (M⫹-H2O): 449.358441; Found: 449.358147. 2.12. (22R,23R,24S)-[26-2H3]-22,23-Diacetoxy-24-methyl5␣-cholesta-3,6-dione (17) A solution of HCl (36%, 0.2 ml) in MeOH (1.8 ml) was added to a stirred solution of the ester 14 (72 mg, 0.13 mmol) and stirring was continued for 0.5 h. The solvent was removed in vacuo, after which the residue was dissolved in CHCl3 and washed with saturated NaHCO3. The organic layer was dried (Na2SO4) and evaporated. The residue was dissolved in acetone (6 ml), and Jones reagent (0.1 ml) was added. The mixture was stirred for 10 min, treated with i PrOH (0.5 ml), and then diluted with brine. Extraction with CHCl3 and purification on SiO2 (petroleum ether-EtOAc, 15:1 f 2:1) resulted in the diketone 17 (54 mg, 79%). IR (cm⫺1): 2965, 2880, 1755, 1725, 1380, 1255, 1030. 1H NMR ␦: 0.74 (s, 3H, 18-Me), 0.98 (s, 3H, 19-Me), 2.01, 2.04 (s, 3H, OAc), 5.17 (d, 1H, J ⫽ 9 Hz, C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H). 13C NMR ␦: 11.1, 11.8, 12.6, 12.8, 20.2, 20.89, 20.94, 21.7, 23.9, 28.1, 30.2, 37.0, 37.4, 38.0, 38.1, 39.3, 39.8, 41.2, 42.9, 46.5, 52.4, 53.4, 56.5, 57.5, 74.1, 75.7, 170.6, 208.9, 211.1. HRMS Calc. for C32H47D3O6: 533.379570; Found: 533.378830. 2.13. (22R,23R,24S)-[26-2H3]-2␣,3␣-Epoxy-22,23diacetoxy-24-methyl-5␣-cholestan-6-one (18) A mixture of the olefin 15 (175 mg, 0.34 mmol), MCPBA (175 mg, 1.01 mmol), and CHCl3 (4 ml) was
Scheme 4.
stirred at room temperature for 1.5 h, then washed successively with 5% Na2SO3, 25% NH3, and brine. The organic layer was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (15:1 f 3:1) to give the epoxide 18 (130 mg, 72%). Mp 207–209°C (hexane-EtOAc). IR (cm⫺1): 2975, 2955, 2880, 1745, 1715, 1380, 1260, 1240, 1030. 1H NMR ␦: 0.69 (s, 3H, 18-Me), 0.72 (s, 3H, 19-Me), 2.00, 2.03 (s, 3H, OAc), 3.13 (t, 1H, J ⫽ 4.4 Hz, C2-H), 3.27 (br.s, 1H, C3-H), 5.15 (d, 1H, J ⫽ 8.7 Hz, C22-H), 5.33 (d, 1H, J ⫽ 8.7 Hz, C23-H). 13 C NMR ␦: 11.0, 11.7, 12.8, 15.0, 20.2, 20.86, 20.91, 21.0, 23.8, 28.0, 30.2, 36.9, 37.5, 37.9, 38.4, 39.3, 39.8, 42.6, 46.8, 49.9, 50.1, 52.3, 53.0, 56.4, 74.1, 75.7, 77.3, 170.5, 211.1. HRMS Calc. for C32H47D3O6: 533.379570; Found: 533.379532. 2.14. (22R,23R,24S)-[26-2H3]-2␣,3␣-Epoxy-22,23dihydroxy-24-methyl-5␣-cholestan-6-one (19) A mixture of 18 (130 mg, 0.24 mmol), MeONa (2.3 mmol, prepared from 54 mg of Na), and MeOH (9 ml) was left at room temperature for 7 days and subsequently diluted with brine and extracted with CHCl3. The resulting extract was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (6:1 f 1:1) to yield the epoxydiol 19 (92 mg, 83%). Mp 223– 224°C (hexane-EtOAc). IR (cm⫺1): 2975, 2950, 2875, 1715, 1395, 1265, 1100, 985. 1H NMR ␦: 0.69 (s, 3H, 18-Me), 0.71 (s, 3H, 19-Me), 3.14 (t, 1H, J ⫽ 4.5 Hz, C2-H), 3.28 (br.s, 1H, C3-H), 3.55 (d, 1H, J ⫽ 8.5 Hz, C22-H), 3.72 (d, 1H, J ⫽ 8.5 Hz, C23-H). 13C NMR ␦: 10.1, 11.8, 11.9, 15.0, 20.7, 20.8, 21.1, 23.8, 27.6, 30.5, 36.8, 37.5, 37.8, 38.4, 39.4, 40.1, 42.6, 46.8, 49.9, 50.2, 52.3, 53.0, 56.5, 73.5, 74.6, 211.5. HRMS Calc. for C28H43D3O4: 449.358441; Found: 449.358874.
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2.16. (22R,23R,24S)-[26-2H3]-2,3-Epoxy-22,23dihydroxy-24-methyl-5␣-cholestan-6-one (21) A mixture of the bromohydrine 20 (123 mg, 0.19 mmol), MeONa (3.9 mmol, prepared from 90 mg of Na), and MeOH (6 ml) was kept at room temperature for 2 days, then diluted with water, and extracted with CHCl3. The organic layer was dried (Na2SO4) and evaporated, and the residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 1:2) to give the epoxide 21 (50 mg, 54%). Mp 194 –197°C (hexane-EtOAc). IR (cm⫺1): 2875, 2850, 2215, 1720, 1390, 1260, 1240, 1100, 1030, 990. 1H NMR ␦: 0.68 (s, 3H, 18-Me), 0.81 (s, 3H, 19-Me), 3.12–3.29 (m, 2H, C2and C3-H), 3.56 (d, 1H, J ⫽ 8.5 Hz, C22-H), 3.73 (d, 1H, J ⫽ 8.5 Hz, C23-H). 13C NMR ␦: 10.1, 11.87, 11.91, 15.3, 20.4, 20.7, 20.8, 21.1, 23.8, 27.6, 30.5, 36.8, 37.2, 37.5, 39.5, 40.0, 40.3, 42.6, 46.8, 50.6, 52.3, 52.4, 53.2, 54.8, 56.5, 73.5, 74.7, 210.9. HRMS Calc. for C28H43D3O4: 449.358441; Found: 449.358068. 2.17. (22R,23R,24S)-[26-2H3]-2-Bromo-3␣-hydroxy22,23-diacetoxy-24-methyl-5␣-cholestan-6-one (22)
Scheme 5.
2.15. (22R,23R,24S)-[26-2H3]-3␣-Bromo-2-hydroxy22,23-diacetoxy-24-methyl-5␣-cholestan-6-one (20) NBS (43 mg, 0.25 mmol) and water (1 ml) were added to a stirred solution of the olefine 15 (62 mg, 0.12 mmol) in DME (6 ml). The mixture was stirred at room temperature for 1 h, then diluted with 5% Na2S2O3, and extracted with EtOAc. The extract was dried (Na2SO4) and evaporated. The residue was purified on SiO2 (petroleum ether-EtOAc, 10:1 f 4:1) to give the bromohydrine 20 (63 mg, 86%). IR (cm⫺1): 2940, 2375, 1755, 1725, 1380, 1255, 1030. 1H NMR ␦: 0.72 (s, 3H, 18-Me), 0.98 (s, 3H, 19-Me), 2.01, 2.04 (s, 3H, OAc), 4.22 (br.s, 1H, C2-H), 4.40 (br.s, 1H, C3-H), 5.16 (d, 1H, J ⫽ 9 Hz, C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H). 13C NMR ␦: 11.1, 11.9, 12.8, 15.5, 20.2, 20.88, 20.94, 21.1, 23.8, 23.9, 28.1, 29.7, 30.2, 36.9, 37.2, 38.0, 39.4, 39.8, 41.0, 42.8, 46.4, 52.4, 53.0, 53.8, 54.3, 56.5, 70.9, 74.2, 75.8, 170.7, 211.5. HRMS Calc. for C32H48D3BrO6: 613.305731; Found: 613.307098.
A solution of the epoxide 18 (370 mg, 0.65 mmol) and HBr (48%, 4.8 ml) in CHCl3 (4.8 ml) and acetic acid (12 ml) was stirred at room temperature for 30 min and subsequently, diluted with water and extracted with CHCl3. The organic layer was washed with water, dried (Na2SO4), and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 1:1), resulting in the bromohydrine 22 (400 mg, 95%). IR (cm⫺1): 2960, 2880, 2220, 1755, 1725, 1380, 1255, 1025, 985. 1H NMR ␦: 0.61 (s, 3H, 18-Me), 0.98 (s, 3H, 19-Me), 1.88, 1.92 (s, 3H, OAc), 4.19 (br.s, 1H, C3-H), 4.25 (br.s, 1H, C2-H), 5.09 (d, 1H, J ⫽ 9 Hz, C22-H), 5.26 (d, 1H, J ⫽ 9 Hz, C23-H). 13C NMR ␦: 11.0, 11.9, 12.8, 15.4, 20.2, 20.7, 20.9, 21.1, 22.5, 22.7, 23.8, 28.0, 30.1, 36.9, 37.2, 39.4, 39.7, 40.5, 41.4, 42.8, 46.3, 49.6, 50.8, 52.3, 54.8, 56.4, 70.3, 74.2, 75.8, 77.3, 170.7, 212.1. HRMS Calc. for C32H48D3BrO6: 613.305731; Found: 613.306763. 2.18. (22R,23R,24S)-[26-2H3]-2␣-Bromo-22,23-diacetoxy24-methyl-5␣-cholesta-3,6-dione (23) Jones reagent (1.1 ml) was added to a solution of the bromohydrine 22 (400 mg, 0.62 mmol) in acetone (20 ml). After the mixture was stirred for 10 min, iPrOH (1.5 ml) was added, and stirring was continued for 5 min. The mixture was then diluted with water and extracted with CHCl3. The extract was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 1:1) to yield the diketone 23 (270 mg, 67%). IR (cm⫺1): 2965, 2880, 1750, 1380, 1255, 1030. 1H NMR ␦: 0.73 (s, 3H, 18-Me), 1.04 (s, 3H, 19-Me), 2.01, 2.03 (s, 3H, OAc), 4.76 (dd, 1H, J ⫽ 13, 6 Hz, C2-H), 5.16 (d, 1H, J ⫽ 8.7 Hz, C22-H), 5.34 (d, 1H, J ⫽ 8.7 Hz, C23-H).
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C NMR ␦: 11.7, 12.5, 13.5, 13.8, 20.9, 21.6, 22.3, 24.5, 28.7, 30.8, 37.2, 37.6, 38.4, 39.8, 40.5, 43.5, 44.7, 46.9, 51.5, 53.0, 53.6, 53.8, 57.0, 58.4, 74.8, 77.7, 171.2, 201.5, 208.2. 13
2.19. (22R,23R,24S)-[26-2H3]-22,23-Diacetoxy-2␣hydroxy-24-methyl-5␣-cholesta-3,6-dione (24) A mixture of the bromohydrine 23 (0.25 g, 0.41 mmol), K2CO3 (1.0 g, 7.2 mmol), acetone (15 ml) and water was heated at 45°C for 15 h, diluted with water, and subsequently partly evaporated in vacuo. The residue was extracted with CHCl3. The extract was dried (Na2SO4) and evaporated to give 0.25 g of crude hydroxy ketone 24. 1H NMR ␦: 0.74 (s, 3H, 18-Me), 1.06 (s, 3H, 19-Me), 2.01, 2.04 (s, 3H, OAc), 4.20– 4.34 (dd, 1H, J ⫽ 12, 7 Hz, C2-H), 5.15 (d, 1H, J ⫽ 8.5 Hz, C22-H), 5.33 (d, 1H, J ⫽ 8.5 Hz, C23-H). 2.20. Reduction of the hydroxy ketone (24) with NaBH4 A solution of NaBH4 (21 mg, 0.55 mmol) was added dropwise to a stirred mixture of the hydroxy ketone 24 (0.24 g, 0.44 mmol) in EtOH (10.5 ml) at ⫺10°C. The mixture was stirred for another hour at ⫺10°C (TLC control) and then, neutralized with 5% H2SO4. The organic solvent was partially removed in vacuo, after which the mixture was extracted with CHCl3. The organic layer was dried (Na2SO4) and evaporated, and the residue was dissolved in 15% KOH in MeOH (12 ml). The mixture was refluxed for 1.5 h, then diluted with water, and extracted with CHCl3. The extract was dried (Na2SO4), evaporated, and chromatographed on SiO2 (CHCl3-MeOH ⫽ 100:1 f 10:1) to give: a) (22R,23R,24S)-[26-2H3]-2␣,3␣,22,23-tetrahydroxy-24methyl-5␣-cholestan-6-one 29 (30 mg, 16% based on 23). 1 H NMR ␦: 0.70 (s, 3H, 18-Me), 0.94 (s, 3H, 19-Me), 3.56 (d, 1H, J ⫽ 8.5 Hz, C22-H), 3.64 –3.84 (m, 2H, C23- and C2-H), 4.05 (br.s, 1H, C3-H). HRMS Calc. for C22H36O4 (split at bond C22-C23): 364.261360; Found: 364.261215; b) (22R,23R,24S)-[26- 2 H 3 ]-2 ␣ ,3  ,22,23-tetrahydroxy-24methyl-5␣-cholestan-6-one 26 (80 mg, 42% based on 23). Mp 243–246°C (EtOAc-MeOH). 1H NMR (C5D5N) ␦: 0.71 (s, 3H, 18-Me), 0.80 (s, 3H, 19-Me), 3.68 – 4.14 (m, 4H, C2-, C3-, C22-, and C23-H). 13C NMR (C5D5N) ␦: 10.8, 12.0, 12.7, 14.4, 20.9, 21.2, 21.9, 24.0, 28.1, 29.3, 31.0, 37.7, 37.9, 39.9, 41.0, 42.7, 42.9, 45.8, 46.6, 52.9, 56.75, 56.80, 72.2, 73.0, 74.2, 75.9, 210.0. HRMS Calc. for C28H43D3O4 (M⫹-H2O): 449.358441; Found: 449.359949. 2.21. (22R,23R,24S)-[26-2H3]-2␣,3,22,23-Tetrahydroxy24-methyl-B-homo-7-oxa-5␣-cholestan-6-one (27) A solution of H2O2 (30%, 0.2 ml) was added to a solution of trifluoroacetic anhydride (0.5 ml, 3.54 mmol) in CH2Cl2 (3 ml) at 0°C. The mixture was stirred for 15 min, then cooled to ⫺20°C, and a solution of the ketone 26 (73 mg, 0.16 mmol) in CH2Cl2 (30 ml) was added. The mixture
593
was kept at ⫺10°C for 10 h and treated first with an excess of NaHSO3 to destroy CF3CO3H and then with Na2CO3. The target compound was extracted with CHCl3, after which the organic layer was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 (CHCl3MeOH ⫽ 30:1 f 8:1) to give the lactone 27 (68 mg, 91%). Mp. 255–256°C (EtOAc-EtOH). 1H NMR (C5D5N) ␦: 0.73 (s, 3H, 18-Me), 1.05 (s, 3H, 19-Me), 1.14 (d, 3H, J ⫽ 7 Hz, 21-Me), 1.22 (d, 3H, J ⫽ 6.5 Hz, 27-Me), 3.21 (dd, 1H, J ⫽ 12, 4.5 Hz, C5-H), 3.80 – 4.21 (m, 4H, C2-, C3-, C22-, and C23-H). 13C NMR (C5D5N) ␦: 10.9, 11.8, 12.7, 16.2, 20.9, 21.2, 23.1, 24.9, 28.1, 31.1, 34.7, 38.0, 38.8, 39.7, 40.1, 41.1, 42.6, 46.8, 48.2, 51.6, 52.9, 58.4, 70.4, 71.9, 73.1, 74.2, 74.5, 175.6. HRMS Calc. for C28H43D3O5 (M⫹-H2O): 465.353355; Found: 465.355033.
3. Results and discussion Initially, the steroidal side chain had to be constructed with a carbon skeleton characteristic of brassinosteroids. This was achieved using the Claisen rearrangement. Compound 2 contained a methyl group at C-24 with the necessary stereochemistry and a ⌬22-double bond ensuring further introduction of the 22R,23R-diol function. In addition, the ester function in 2 allowed easy introduction of three deuteriums at C-26 by successive reduction with LiAlD4, tosylation, and again, reduction with LiAlD4. Starting with 4, all the deuterated compounds were present as mixtures of diastereomers because of formation of a new chiral center at C-25. This result clearly followed from the 13C NMR spectra, which showed signals of 26and 27-methyl groups approx. 50% as intensive as other signals. However, this fact was not important for the biosynthetic studies involving steroidal A and B cycles. Treatment of the i-steroidal ether 4 with acid led to regeneration of the 3-hydroxy-⌬5-system, resulting in compound 5, which was then transformed into the ketone 6. The further synthetic route implied introduction of a 22R,23R-diol function. The best solution starting from ⌬22olefins is known to be Sharpless hydroxylation [21] in the presence of chiral alkaloids of the quinidine series. Natural 22R,23R-diols are major products of this oxidation, although minor 22S,23S-diols are also formed in various ratios depending on the reaction conditions. We studied two variants of this reaction. The first one made use of p-chlorobenzoate dihydroquinidine. A mixture (1.6:1) of diols 7 and 8 was isolated. Oxidation by a commercial ADmix containing hydroquinidine 1,4-phthalazinediyl diether ((DHQD)2-PHAL) proceeded very slowly, which was why an additional amount of K2OsO4.2H2O and (DHQD)2-PHAL was needed. The resulting ratio of 22R,23R- and 22S,23S-isomers was much better (only a small amount of the latter was isolated, together with unindentified products); the problem in this case was overoxidation with formation of the diketone 9. A
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similar reaction has been described for hydroxylation of stigmastane derivatives [22]. Compound 7 was acetylated and then treated with HBr to give 11. Transformation of 3-bromides like 11 into typhasterol derivatives has been reported in a moderate yield by treatment with AgOAc [23]. We carried out the nucleophilic substitution of bromide with an acetoxy group by using Hg(OAc)2 and had similar results. Nevertheless, the reaction gave enough acetate 12a to allow saponification and formation of [26-2H3]typhasterol 12b. The Baeyer-Villiger oxidation of the latter furnished another deuterated brassinosteroid, [26-2H3]-2-deoxybrassinolide 13. Heating of lithium carbonate and the bromide 11 in DMF led to the dehydrobromination of 11 to give the ⌬2-olefine 15. Another product 14 resulted from nucleophilic substitution of bromine under these reaction conditions. Hydrolysis of the formyl ester 14, followed by Jones oxidation, gave the 22,23diacetate of [26-2H3]-3-dehydroteasterone 17. There have been no data until now about the role of secasterone [24], containing a 2,3-epoxy group, in the brassinosteroid biosynthetic network. For the majority of brassinosteroids, the presence of a 2␣,3␣-diol function is characteristic. However, there are also examples having 2␣,3- or 2,3␣ configurations, which is why we decided to prepare both 2␣,3␣- and 2,3-epoxides. Epoxidation of the olefine 15, followed by removal of the acetoxy groups, led to [26-2H3]-2,3-episecasterone 19. Treatment of the olefine with bromosuccinimide gave the transdiaxial bromohydrine 20, which was further transformed into [26-2H3]secasterone 21 by treatment with base. Synthesis of brassinosteroids 26 and 27, bearing two equatorial hydroxy groups, proved to be the most difficult part of the investigation [25]. The epoxide 18 proved to be a convenient intermediate for this purpose. Its opening with HBr, followed by Jones oxidation, led to the bromoketone 23. The nucleophilic substitution of the bromine at the next step gave the hydroxy ketone 24. Treatment of compound 24 with NaBH4 proceeded with preferential reduction of the C-3 keto group. Saponification of the resulting reaction mixture followed by column chromatography gave [262 H3]-3-epicastasterone 26 and [26-2H3]castasterone 29. The Baeyer-Villiger oxidation of 26 afforded the [26-2H3]-3epibrassinolide 27.
Acknowledgments We thank Dr. N.B. Khripach (Minsk) for recording the NMR spectra and Dr. Neil J. Oldham (Jena) for the exact mass measurements. Emily Wheeler (Jena) is thanked for linguistic support in the preparation of this manuscript. The authors are grateful to the Belorussian Foundation for Fundamental Research for financial support.
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V.A. Khripach et al. / Steroids 67 (2002) 587–595 [21] Amberg W, Bennani YL, Chadha RK, Crispino GA, Davis WD, Hartung J, Jeong KS, Ogino Y, Shibata T, Sharpless KB. Syntheses and crystal structures of the cinchona alkaloid derivatives used as ligands in the osmium-catalyzed asymmetric dihydroxylation of olefins. J Org Chem 1993;58:844 –9. [22] Hellrung B, Voigt B, Schmidt J, Adam G. Synthesis of new ⌬D5,7oxygenated and ⌬D5,7-unsaturated brassinosteroid analogs. Steroids 1997;62:415–21. [23] Aburatani M, Takeuchi T, Mori K. Facile syntheses of brassino-
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steroids: brassinolide, castasterone, teasterone and typhasterol. Agric Biol Chem 1987;51:1909 –13. [24] Schmidt J, Spengler B, Yokota T, Nakayama M, Takatsuto S, Voigt B, Adam G. Secasterone, the first naturally occurring 2,3epoxybrassinosteroid from Secale cereale. Phytochemistry 1995; 38:1095–7. [25] Levinson EE, Traven VF. Synthesis of 3,24-diepicastasterone: a natural brassinosteroid with 2,3-trans-diol function. J Chem Res Synop 1996:196 –7.
Synthesis of [26-2H3]brassinosteroids Vladimir A. Khripacha,*, Vladimir N. Zhabinskiia, Olga V. Konstantinovaa, Andrey P. Antonchicka, Bernd Schneiderb a
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich str., 5/2, 220141 Minsk, Belarus b Max-Planck-Institute for Chemical Ecology, Beutenberg Campus Winzerlaer Str. 10, D-07745 Jena, Germany Received 28 June 2001; received in revised form 2 November 2001; accepted 9 November 2001
Abstract A number of [26-2H3]brassinosteroids were prepared for biochemical studies. The parent, nondeuterated compounds were considered to be biosynthetic intermediates in brassinosteroid biosynthesis. Claisen rearrangement was used to construct the steroidal side chain. Deuterium was introduced by reducing the corresponding intermediates with lithium aluminium deuteride. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Brassinosteroids; Labeled compounds; Claisen rearrangement; Brassinolide; Plant growth hormone
1. Introduction Brassinosteroids represent a new class of unique naturally occurring plant-growth-regulating substances with high biologic activity at very low concentrations. Recent research efforts have been directed toward the search for related compounds in the plant kingdom and their chemical synthesis, biologic mode of action, and practical application in agriculture and horticulture [1,2]. The elucidation of the biosynthesis of brassinosteroids is an important contribution to brassinosteroid research in general. Research on the regulation of brassinosteroid biosynthesis and their mode of action requires detailed knowledge of the pathways and enzymes responsible for brassinosteroid formation in plants. The brassinosteroid skeleton is biosynthetically formed before campestanol in early steps of the pathway. This intermediate is converted into brassinolide by a series of oxidation and reduction steps without further changes to the skeleton. These oxidation/reduction reactions may be interchanged in sequence. For example, two alternative biosynthetic routes from campestanol to castasterone, the so-called early C-6 oxidation pathway [3,4] and the late C-6 oxidation
* Corresponding author. Tel.: ⫹375-2-648-647; fax: ⫹375-2-648-647. E-mail address: [email protected] (V.A. Khripach).
pathway [5,6], have been identified in Catharanthus roseus by Japanese research groups. We hypothesized that in different plant families or even in one species, brassinosteroids are formed by a biosynthetic network of alternative pathways and subpathways. A number of feeding experiments using labelled precursors were necessary to confirm this. Deuterated compounds were the preferred substrates because detection of brassinosteroids by mass spectrometry is an established analytical method [7]. A number of labelled brassinosteroids have been synthesized by various groups and used as internal standards for quantitative analysis of endogenous brassinosteroids, and they have also been employed in biosynthetic experiments [8 –12]. [26,28-2H6]Brassinosteroids, for example, were prepared in a multistep procedure by introducing two deuterated methyl groups stepwise into the side chain of crinosterol [13–15]. [5,7,7-2H3]Epibrassinolide was synthesized by base-catalyzed enolization [16]. A similar procedure was used to prepare [5,7,7]-tritium [17,18] and -deuterium [19] labelled brassinosteroids. This enolization method yields incomplete labeling and therefore is not suitable for quantitative analysis. The main objective of the present investigation was to elaborate synthetic approaches to labelled brassinosteroid precursors with high isotopic purity and with at least three deuteriums in non-exchangable positions.
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 9 - 1 2 8 X ( 0 2 ) 0 0 0 0 4 - 1
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Scheme 1.
2. Experimental 2.1. General Melting points were taken on a Boetius micro-melting point apparatus and are uncorrected. IR spectra were recorded on an UR-20 spectrophotometer. 1H and 13C NMR spectra were taken on a Bruker AC-200 (200 MHz for 1H, 50 MHz for 13C) spectrometer using TMS as an internal standard in CDCl3 (if not stated otherwise). The exact mass measurements were carried out on a Micromass MasSpec mass spectrometer, operating in the 70 eV-EI mode. Samples were introduced by direct probe for accurate mass measurement by peak matching. Reactions were monitored by TLC using aluminium or plastic sheets precoated with silica gel 60 F254 (Merck Art. 5715). Column chromatography was carried out on Kieselgel 60 (Merck Art. 7734).
Scheme 2.
2.2. (22E,24S)-[26-2H3]-6-Methoxy-24-methyl-3␣,5cyclo-5␣-cholest-22-ene (4)
56.6, 82.4, 131.8, 136.1. HRMS Calc. for C29H45D3O: 415.38904; Found: 415.38854.
A mixture of (22E,24R)-6-methoxy-24-methyl-3␣,5cyclo-5␣-cholest-22-en-26-oic acid ethyl ester 2 (5.5 g, 11.7 mmol, prepared according to [20] and LiAlD4 (1.55 g, 37 mmol) in ether (100 ml) was stirred at room temperature for 1 h. The excess reagent was decomposed by treatment with a 15% solution of NaOH. The precipitate was filtered off, and the filtrate was evaporated and dissolved in pyridine (20 ml). TsCl (4.7 g, 24.6 mmol) was added, and the mixture was left at room temperature for 30 h. Afterwards it was diluted with water and extracted with CHCl3. The extract was dried (Na2SO4) and evaporated. The residue was dissolved in ether (150 ml) and LiAlD4 (2.05 g, 48.9 mmol) was added. The mixture was stirred for 1 h and then treated with a 15% solution of NaOH. The precipitate was filtered off, and the filtrate was evaporated. The residue was chromatographed on SiO2 with cyclohexane-EtOAc (30:1 f 12:1), resulting in compound 4 (3.1 g, 76%). 1H NMR ␦: 0.44 (dd, 2H, J ⫽ 8, 5 Hz, C4-H), 0.66 (t, 1H, J ⫽ 4.3 Hz, C3-H), 0.75 (s, 3H, 18-Me), 1.04 (s, 3H, 19-Me), 2.78 (t, 1H, J ⫽ 2.8 Hz, C6-H), 3.33 (s, 3H, OMe), 5.16 (m, 2H, C22- and C23-H). 13C NMR ␦: 12.4, 13.1, 18.1, 19.3, 19.6, 20.1, 21.0, 21.5, 22.8, 24.2, 25.0, 28.9, 30.5, 33.0, 33.4, 35.1, 35.3, 40.2, 40.3, 42.7, 43.0, 43.4, 48.1, 56.1, 56.5,
2.3. (22E,24S)-[26-2H3]-24-Methyl-3␣,5-cyclo-5␣-cholest22-en-6-one (6) A mixture of 4 (3.1 g, 7.5 mmol), TsOH (0.45 g, 2.4 mmol), water (35 ml), and dioxane (106 ml) was heated at 70°C for 2.5 h. Pyridine (1 ml) was then added, and the mixture was evaporated. The residue was dissolved in pyridine (20 ml), and TsCl (5.4 g, 28.3 mmol) was added. The mixture was left at room temperature for 30 h, then diluted with water, and extracted with CHCl3. The organic layer was dried (Na2SO4) and evaporated. The residue was dissolved in acetone (0.5 l) and then, AcOK (1.9 g, 19.4 mmol) and water (37.5 ml) were added. The mixture was refluxed for 24 h, after which Jones reagent (5 ml) was added at room temperature. The mixture was stirred for 20 min, iPrOH (10 ml) was then added, and the stirring was continued for 15 min. The mixture was diluted with water and extracted with CHCl3. The solvent was evaporated, and the residue was purified by column chromatography on SiO2 (cyclohexaneEtOAc, 40:1 f 10:1), resulting in the ketone 6 (2.6 g, 82%). Mp 87–91°C (hexane). IR (cm⫺1): 2970, 2875, 2220, 1705, 1465, 1385, 1300, 1175, 1125, 980. 1H NMR ␦: 0.74 (s, 3H, 18-Me), 1.02 (s, 3H, 19-Me), 5.16 (m, 2H, C22- and C23-H).
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C NMR ␦: 11.7, 12.3, 18.1, 19.7, 20.1, 21.0, 22.9, 24.1, 25.9, 28.8, 33.0, 33.5, 34.8, 35.3, 39.7, 40.2, 42.6, 43.1, 44.8, 46.1, 46.4, 46.8, 55.9, 57.1, 132.1, 135.8, 209.7. HRMS Calc. for C28H41D3O: 399.35774; Found: 399.35760. 13
2.4. Hydroxylation of the olefin (6) 2.4.1. Variant A A mixture of the olefine 6 (2.6 g, 6.5 mmol), AD-mix (16 g), methanesulfonamide (1.2 g, 12.6 g), K2OsO4.2H2O (0.12 g, 0.33 mmol), (DHQD)2PHAL (0.5 g, 0.64 mmol), tert-butanol (110 ml), and water (110 ml) was stirred at room temperature for 48 h. Then, Na2SO3 (6.5 g, 51.6 mmol) was added, and stirring was continued for 3 h. The mixture was partly evaporated and extracted with CHCl3. The organic layer was evaporated, and the residue was dissolved in CHCl3 and treated successively with 0.25 M H2SO4 and saturated NaHCO3. After evaporation, the residue was chromatographed on SiO2 with cyclohexane-EtOAc (9:1 f 1:1) to give a) (24S)-[262 H3]-24-methyl-3␣,5-cyclo-5␣-cholesta-6,22,23-trione 9 (0.7 g, 25%). Mp 119 –122°C (hexane). IR (cm⫺1): 2970, 2880, 2225, 1715, 1700, 1465, 1375, 1300, 925. 1H NMR ␦: 0.80 (s, 3H, 18-Me), 0.86 (d, 3H, J ⫽ 7 Hz, 28-Me), 0.91 (d, 3H, J ⫽ 6.5 Hz, 21-Me), 1.00 (d, 3H, J ⫽ 6.7 Hz, 27-Me), 1.02 (s, 3H, 19-Me), 3.24 (m, 1H, C20- or C24H), 3.45 (m, 1H, C24- or C20-H). 13C NMR ␦: 11.7, 11.9, 12.3, 16.4, 18.4, 19.7, 21.2, 22.8, 24.4, 25.9, 27.5, 29.2, 33.5, 34.7, 35.4, 39.6, 40.9, 43.1, 44.3, 44.7, 46.1, 46.3, 46.7, 51.8, 56.2, 202.4, 203.1, 209.3. HRMS Calc. for C28H39D3O3: 429.33192; Found: 429.33246; b) (22R,23R,24S)-[26-2H3]-22,23-dihydroxy-24-methyl3␣,5-cyclo-5␣-cholestan-6-one 7 (1.05 g, 37%). Mp 157– 159°C (hexane-EtOAc). IR (cm⫺1): 2945, 2875, 2375, 2235, 2210, 1690, 1465, 1385, 1315, 1175, 990. 1H NMR ␦: 0.75 (s, 3H, 18-Me), 1.02 (s, 3H, 19-Me), 3.57 (d, 1H, J ⫽ 8 Hz, C22-H), 3.74 (d, 1H, J ⫽ 8 Hz, C23-H). 13C NMR ␦: 10.1, 11.7, 12.0, 19.7, 20.7, 20.8, 22.9, 23.9, 25.9, 27.8, 30.5, 33.5, 34.9, 35.4, 36.9, 39.8, 40.1, 42.6, 44.8, 46.1, 46.4, 46.8, 52.3, 56.9, 73.5, 74.7, 209.8. HRMS Calc. for C28H43D3O: 433.3632; Found: 433.36318. 2.4.2. Variant B A mixture of the olefine 6 (2.2 g, 5.5 mmol), K3Fe(CN)6 (10.9 g, 33 mmol), K2CO3 (4.6 g, 33 mmol), methanesulfonamide (1.05 g, 11 mmol), K2OsO4.2H2O (0.04 g, 0.11 mmol), DHQD CLB (0.26 g, 0.56 mmol), tert-butanol (90 ml), and water (70 ml) was stirred for 3 days. Then, Na2SO3 (5 g, 40 mmol) was added, and stirring was continued for 24 h. The mixture was evaporated partly in vacuo and extracted with CHCl3. The organic layer was washed with 0.25 M H2SO4 and brine and then evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 5:1) to
Scheme 3.
give: a) (24S)-[26-2H3]-24-methyl-3␣,5-cyclo-5␣-cholesta-6,22,23-trione 9 (150 mg, 6%); b) (22S,23S,24S)[26- 2 H 3 ]-22,23-dihydroxy-24-methyl-3 ␣ ,5-cyclo-5 ␣ cholestan-6-one 8 (0.55 g, 23%). Mp. 179 –182°C (EtOAc-hexane). IR (cm⫺1): 2970, 2880, 2220, 1690, 1470, 1385, 1300, 1175, 1135, 1030, 970. 1H NMR ␦: 0.78 (s, 3H, 18-Me), 1.03 (s, 3H, 19-Me), 3.45 (d, 1H, J ⫽ 9.3 Hz, C22-H), 3.69 (d, 1H, J ⫽ 4.5 Hz, C23-H). 13C NMR ␦: 10.1, 11.7, 12.0, 14.1, 15.7, 19.7, 21.6, 22.9, 24.3, 25.9, 26.2, 27.8, 33.5, 34.8, 35.5, 39.7, 41.7, 42.3, 43.3, 44.7, 46.1, 46.4, 46.7, 52.7, 56.6, 71.1, 71.6, 209.8. HRMS Calc. for C 28 H 43 D 3 O 3 : 433.36353; Found: 433.36340; c) (22R,23R,24S)-[26-2H3]-22,23-dihydroxy24-methyl-3 ␣ ,5-cyclo-5 ␣ -cholestan-6-one 7 (0.85 g, 36%).
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2.5. (22R,23R,24S)-[26-2H3]-22,23-Diacetoxy-24-methyl3␣,5-cyclo-5␣-cholestan-6-one (10) A mixture of the diol 7 (0.85 g, 1.96 mmol), acetic anhydride (0.74 ml, 7.8 mmol), DMAP (0.02 g, 0.2 mmol), and pyridine (7 ml) was heated at 45°C for 4.5 h, after which it was diluted with water and extracted with CHCl3. The organic layer was dried (Na2SO4), and the solvent was evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (15:1 f 6:1) to give the diacetate 10 (0.8 g, 75%). Mp 172–174°C (hexane-EtOAc). IR (cm⫺1): 2965, 2880, 1750, 1700, 1460, 1380, 1300, 1250, 1030, 980. 1H NMR ␦: 0.77 (s, 3H, 18-Me), 1.03 (s, 3H, 19-Me), 2.02, 2.05 (s, 3H, OAc), 5.18 (d, 1H, J ⫽ 8.5 Hz, C22-H), 5.35 (d, 1H, J ⫽ 8.5 Hz, C23-H). HRMS Calc. for C32H47D3O5: 517.38466; Found: 517.38441. 2.6. (22R,23R,24S)-[26-2H3]-3-Bromo-22,23-diacetoxy24-methyl-5␣-cholestan-6-one (11) A solution of HBr (48%, 3 ml) was added to a stirred solution of 10 (0.9 g, 1.7 mmol) in acetic acid (10 ml). The mixture was stirred for 10 min and then diluted with water. The precipitate was filtered, washed, and air-dried to give the bromide 11 (1.0 g, 97%). Mp 221–222°C (MeOHEtOAc). IR (cm⫺1): 2975, 2955, 2880, 2380, 2215, 1750, 1720, 1470, 1380, 1255, 1030, 980. 1H NMR ␦: 0.72 (s, 3H, 18-Me), 0.82 (s, 3H, 19-Me), 2.02, 2.05 (s, 3H, OAc), 3.85– 4.05 (m, 1H, C3-H), 5.16 (d, 1H, J ⫽ 9 Hz, C22-H), 5.33 (d, 1H, J ⫽ 9 Hz, C23-H). 13C NMR ␦: 11.0, 11.8, 12.8, 13.1, 20.2, 20.9, 21.3, 23.8, 28.0, 30.1, 32.3, 33.3, 36.9, 37.7, 39.1, 39.3, 39.7, 40.6, 42.8, 46.4, 50.5, 52.3, 53.7, 56.5, 58.9, 74.1, 75.7, 170.5, 209.4. HRMS Calc. for C32H49D3O5Br: 598.31864; Found: 598.31850. 2.7. Reaction of the bromide (11) with Hg(OAc)2 A mixture of the bromide 11 (257 mg, 0.43 mmol), Hg(OAc)2 (260 mg, 0.82 mmol), and acetic acid (4 ml) was heated at 70°C for 35 h. The solvent was evaporated in vacuo, and the residue was dissolved in CHCl3 and filtered through SiO2. The filtrate was evaporated and chromatographed on SiO2 with petroleum ether-EtOAc (20:1 f 2:1) to give: a) (22R,23R,24S)-[26-2H3]-22,23-diacetoxy-24methyl-5␣-cholest-2-en-6-one 15 (33 mg, 15%). Mp 214 – 216°C (hexane-EtOAc). IR (cm⫺1): 2975, 2945, 2875, 2215, 1755, 1720, 1445, 1385, 1250, 1060, 1030. 1H NMR ␦: 0.72 (s, 3H, 18-Me), 2.01, 2.03 (s, 3H, OAc), 5.17 (d, 1H, J ⫽ 9 Hz, C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H), 5.50 –5.76 (m, 2H, C2- and C3-H). 13C NMR ␦: 11.1, 11.8, 12.8, 13.5, 20.3, 20.89, 20.94, 21.1, 21.7, 23.9, 28.1, 30.2, 37.0, 37.7, 39.38, 39.46, 39.79, 39.99, 42.7, 46.9, 52.4, 53.4, 53.8, 56.7, 74.2, 75.8, 124.5, 125.0, 170.5, 211.7. HRMS Calc. for C32H49D3O5: 517.384655; Found: 517.384598; b) (22R,23R,24S)-[26-2H3]-3,22,23-triacetoxy-24-methyl-5␣cholestan-6-one 12a (130 mg, 52%). Mp 94 –97°C (EtOAc-
MeOH). 1H NMR ␦: 0.72 (s, 3H, 18-Me), 0.78 (s, 3H, 19-Me), 0.85–1.09 (m, 9H, 21-, 27-, and 28-Me), 2.03, 2.06, 2.07 (s, 3H, OAc), 5.07–5.22 (m, 2H, C3- and C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H). IR (cm⫺1): 2955, 2880, 1750, 1720, 1375, 1255, 1030. HRMS Calc. for C34H51D3O7: 577.405785; Found: 577.404312. 2.8. Saponification of the triacetate (12a) The triacetate 12a (58 mg, 0.1 mmol) was added to a solution of KOH in MeOH (15%, 15 ml). After the mixture was refluxed for 1 h, then it was treated with brine and extracted with CHCl3. The extract was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 1:4) to give: a) (22R,23R,24S)-[26-2H3]-3␣,22,23-trihydroxy-24-methyl5␣-cholestan-6-one 12b (9 mg, 20%). Mp 230 –233°C (EtOAc-MeOH). IR (cm⫺1): 2945, 2880, 2215, 1715, 1390, 1255, 985. 1H NMR ␦: 0.70 (s, 3H, 18-Me), 0.75 (s, 3H, 19-Me), 2.32 (dd, 1H, J ⫽ 13, 4.5 Hz, C7-H), 3.56 (d, 1H, J ⫽ 8 Hz, C22-H), 3.73 (d, 1H, J ⫽ 8 Hz, C23-H), 4.18 (br.s, 1H, C3-H). HRMS Calc. for C28H45D3O4: 451.37409; Found: 451.37289; b) a mixture of 12b and 12c (13 mg, 29%); c) (22R,23R,24S)-[26-2H3]-3,22,23-trihydroxy-24methyl-5␣-cholestan-6-one 12c. Mp 251–254°C (EtOAcMeOH). IR (cm⫺1): 2955, 2880, 2235, 2210, 1715, 1470, 1430, 1390, 1250, 1130, 1120, 1060, 980. 1H NMR ␦: 0.69 (s, 3H, 18-Me), 3.56 (d, 1H, J ⫽ 8.5 Hz, C22-H), 3.64 (m, 1H, C3-H), 3.73 (d, 1H, J ⫽ 8.5 Hz, C23-H). HRMS Calc. for C28H45D3O4 (M⫹-H2O): 433.363526; Found: 433.362503. 2.9. Dehydrobromination of the bromide (11) A mixture of the bromide 11 (555 mg, 0.93 mmol), LiCO3 (1.1 g, 14.7 mmol), and DMF (10 ml) was refluxed for 3.5 h, after which it was cooled down, diluted with water, and extracted with CHCl3. The organic layer was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (25:1 f 10:1) to give: a) (22R,23R,24S)-[26-2H3]-22,23-diacetoxy24-methyl-5␣-cholest-2-en-6-one 15 (254 mg, 52%); b) (22R,23R,24S)-[26-2H3]-3-formyloxy-22,23-diacetoxy-24methyl-5␣-cholestan-6-one 14 (72 mg, 16%). IR (cm⫺1): 2950, 2880, 2215, 1750, 1730, 1375, 1255, 1030. 1H NMR ␦: 0.72 (s, 3H, 18-Me), 2.01, 2.03 (s, 3H, OAc), 5.17 (d, 1H, J ⫽ 9 Hz, C22-H), 5.28 (br.s, 1H, C3-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H), 8.04 (s, 1H, CHO). 2.10. (22R,23R,24S)-[26-2H3]-3,22,23-Triacetoxy-24methyl-B-homo-7-oxa-5␣-cholestan-6-one (13a) A solution of H2O2 (30%, 2 ml) was added to a stirred solution of (CF3CO)2O (9 ml, 63.7 mol) in CH2Cl2 (6 ml), at ⫺5°C. The mixture was stirred for 15 min, and then a solution of the triacetoxyketone 12a (135 mg, 0.22 mmol) in
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CH2Cl2 (4 ml) was added. The mixture was kept for 3 h, then diluted with water, and extracted with CHCl3. The extract was washed with saturated NaHCO3, dried (Na2SO4), and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (15:1 f 2:1) to give the lactone 13a (40 mg, 29%). IR (cm⫺1): 2960, 1745, 1380, 1255, 1190, 1030. 1H NMR ␦: 0.75 (s, 3H, 18-Me), 0.92 (s, 3H, 19-Me), 2.01, 2.03, 2.08 (s, 3H, OAc), 4.09 (m, 2H, C7-H), 5.11 (m, 1H, C3-H), 5.16 (d, 1H, J ⫽ 9 Hz, C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H). HRMS Calc. for C34H51D3O8 (M⫹-AcOH): 533.379570; Found: 533.380447. 2.11. (22R,23R,24S)-[26-2H3]-3␣,22,23-Trihydroxy-24methyl-B-homo-7-oxa-5␣-cholestan-6-one (13b) The triacetate 13a (40 mg, 0.069 mmol) was added to a solution of KOH in MeOH (15%, 2 ml). The mixture was refluxed for 1 h, then cooled down, and a solution of HCl (25%, 1 ml) was added. After being stirred at room temperature for 2 h, the mixture was diluted with water and extracted with CHCl3. The organic layer was dried (Na2SO4), and evaporated, and the residue was purified on SiO2 (CHCl3-MeOH, 100:1 f 50:1) to give the trihydroxylactone 13b (25 mg, 79%). Mp 225–227°C (hexaneEtOAc). HRMS Calc. for C28H43D3O4 (M⫹-H2O): 449.358441; Found: 449.358147. 2.12. (22R,23R,24S)-[26-2H3]-22,23-Diacetoxy-24-methyl5␣-cholesta-3,6-dione (17) A solution of HCl (36%, 0.2 ml) in MeOH (1.8 ml) was added to a stirred solution of the ester 14 (72 mg, 0.13 mmol) and stirring was continued for 0.5 h. The solvent was removed in vacuo, after which the residue was dissolved in CHCl3 and washed with saturated NaHCO3. The organic layer was dried (Na2SO4) and evaporated. The residue was dissolved in acetone (6 ml), and Jones reagent (0.1 ml) was added. The mixture was stirred for 10 min, treated with i PrOH (0.5 ml), and then diluted with brine. Extraction with CHCl3 and purification on SiO2 (petroleum ether-EtOAc, 15:1 f 2:1) resulted in the diketone 17 (54 mg, 79%). IR (cm⫺1): 2965, 2880, 1755, 1725, 1380, 1255, 1030. 1H NMR ␦: 0.74 (s, 3H, 18-Me), 0.98 (s, 3H, 19-Me), 2.01, 2.04 (s, 3H, OAc), 5.17 (d, 1H, J ⫽ 9 Hz, C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H). 13C NMR ␦: 11.1, 11.8, 12.6, 12.8, 20.2, 20.89, 20.94, 21.7, 23.9, 28.1, 30.2, 37.0, 37.4, 38.0, 38.1, 39.3, 39.8, 41.2, 42.9, 46.5, 52.4, 53.4, 56.5, 57.5, 74.1, 75.7, 170.6, 208.9, 211.1. HRMS Calc. for C32H47D3O6: 533.379570; Found: 533.378830. 2.13. (22R,23R,24S)-[26-2H3]-2␣,3␣-Epoxy-22,23diacetoxy-24-methyl-5␣-cholestan-6-one (18) A mixture of the olefin 15 (175 mg, 0.34 mmol), MCPBA (175 mg, 1.01 mmol), and CHCl3 (4 ml) was
Scheme 4.
stirred at room temperature for 1.5 h, then washed successively with 5% Na2SO3, 25% NH3, and brine. The organic layer was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (15:1 f 3:1) to give the epoxide 18 (130 mg, 72%). Mp 207–209°C (hexane-EtOAc). IR (cm⫺1): 2975, 2955, 2880, 1745, 1715, 1380, 1260, 1240, 1030. 1H NMR ␦: 0.69 (s, 3H, 18-Me), 0.72 (s, 3H, 19-Me), 2.00, 2.03 (s, 3H, OAc), 3.13 (t, 1H, J ⫽ 4.4 Hz, C2-H), 3.27 (br.s, 1H, C3-H), 5.15 (d, 1H, J ⫽ 8.7 Hz, C22-H), 5.33 (d, 1H, J ⫽ 8.7 Hz, C23-H). 13 C NMR ␦: 11.0, 11.7, 12.8, 15.0, 20.2, 20.86, 20.91, 21.0, 23.8, 28.0, 30.2, 36.9, 37.5, 37.9, 38.4, 39.3, 39.8, 42.6, 46.8, 49.9, 50.1, 52.3, 53.0, 56.4, 74.1, 75.7, 77.3, 170.5, 211.1. HRMS Calc. for C32H47D3O6: 533.379570; Found: 533.379532. 2.14. (22R,23R,24S)-[26-2H3]-2␣,3␣-Epoxy-22,23dihydroxy-24-methyl-5␣-cholestan-6-one (19) A mixture of 18 (130 mg, 0.24 mmol), MeONa (2.3 mmol, prepared from 54 mg of Na), and MeOH (9 ml) was left at room temperature for 7 days and subsequently diluted with brine and extracted with CHCl3. The resulting extract was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (6:1 f 1:1) to yield the epoxydiol 19 (92 mg, 83%). Mp 223– 224°C (hexane-EtOAc). IR (cm⫺1): 2975, 2950, 2875, 1715, 1395, 1265, 1100, 985. 1H NMR ␦: 0.69 (s, 3H, 18-Me), 0.71 (s, 3H, 19-Me), 3.14 (t, 1H, J ⫽ 4.5 Hz, C2-H), 3.28 (br.s, 1H, C3-H), 3.55 (d, 1H, J ⫽ 8.5 Hz, C22-H), 3.72 (d, 1H, J ⫽ 8.5 Hz, C23-H). 13C NMR ␦: 10.1, 11.8, 11.9, 15.0, 20.7, 20.8, 21.1, 23.8, 27.6, 30.5, 36.8, 37.5, 37.8, 38.4, 39.4, 40.1, 42.6, 46.8, 49.9, 50.2, 52.3, 53.0, 56.5, 73.5, 74.6, 211.5. HRMS Calc. for C28H43D3O4: 449.358441; Found: 449.358874.
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2.16. (22R,23R,24S)-[26-2H3]-2,3-Epoxy-22,23dihydroxy-24-methyl-5␣-cholestan-6-one (21) A mixture of the bromohydrine 20 (123 mg, 0.19 mmol), MeONa (3.9 mmol, prepared from 90 mg of Na), and MeOH (6 ml) was kept at room temperature for 2 days, then diluted with water, and extracted with CHCl3. The organic layer was dried (Na2SO4) and evaporated, and the residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 1:2) to give the epoxide 21 (50 mg, 54%). Mp 194 –197°C (hexane-EtOAc). IR (cm⫺1): 2875, 2850, 2215, 1720, 1390, 1260, 1240, 1100, 1030, 990. 1H NMR ␦: 0.68 (s, 3H, 18-Me), 0.81 (s, 3H, 19-Me), 3.12–3.29 (m, 2H, C2and C3-H), 3.56 (d, 1H, J ⫽ 8.5 Hz, C22-H), 3.73 (d, 1H, J ⫽ 8.5 Hz, C23-H). 13C NMR ␦: 10.1, 11.87, 11.91, 15.3, 20.4, 20.7, 20.8, 21.1, 23.8, 27.6, 30.5, 36.8, 37.2, 37.5, 39.5, 40.0, 40.3, 42.6, 46.8, 50.6, 52.3, 52.4, 53.2, 54.8, 56.5, 73.5, 74.7, 210.9. HRMS Calc. for C28H43D3O4: 449.358441; Found: 449.358068. 2.17. (22R,23R,24S)-[26-2H3]-2-Bromo-3␣-hydroxy22,23-diacetoxy-24-methyl-5␣-cholestan-6-one (22)
Scheme 5.
2.15. (22R,23R,24S)-[26-2H3]-3␣-Bromo-2-hydroxy22,23-diacetoxy-24-methyl-5␣-cholestan-6-one (20) NBS (43 mg, 0.25 mmol) and water (1 ml) were added to a stirred solution of the olefine 15 (62 mg, 0.12 mmol) in DME (6 ml). The mixture was stirred at room temperature for 1 h, then diluted with 5% Na2S2O3, and extracted with EtOAc. The extract was dried (Na2SO4) and evaporated. The residue was purified on SiO2 (petroleum ether-EtOAc, 10:1 f 4:1) to give the bromohydrine 20 (63 mg, 86%). IR (cm⫺1): 2940, 2375, 1755, 1725, 1380, 1255, 1030. 1H NMR ␦: 0.72 (s, 3H, 18-Me), 0.98 (s, 3H, 19-Me), 2.01, 2.04 (s, 3H, OAc), 4.22 (br.s, 1H, C2-H), 4.40 (br.s, 1H, C3-H), 5.16 (d, 1H, J ⫽ 9 Hz, C22-H), 5.34 (d, 1H, J ⫽ 9 Hz, C23-H). 13C NMR ␦: 11.1, 11.9, 12.8, 15.5, 20.2, 20.88, 20.94, 21.1, 23.8, 23.9, 28.1, 29.7, 30.2, 36.9, 37.2, 38.0, 39.4, 39.8, 41.0, 42.8, 46.4, 52.4, 53.0, 53.8, 54.3, 56.5, 70.9, 74.2, 75.8, 170.7, 211.5. HRMS Calc. for C32H48D3BrO6: 613.305731; Found: 613.307098.
A solution of the epoxide 18 (370 mg, 0.65 mmol) and HBr (48%, 4.8 ml) in CHCl3 (4.8 ml) and acetic acid (12 ml) was stirred at room temperature for 30 min and subsequently, diluted with water and extracted with CHCl3. The organic layer was washed with water, dried (Na2SO4), and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 1:1), resulting in the bromohydrine 22 (400 mg, 95%). IR (cm⫺1): 2960, 2880, 2220, 1755, 1725, 1380, 1255, 1025, 985. 1H NMR ␦: 0.61 (s, 3H, 18-Me), 0.98 (s, 3H, 19-Me), 1.88, 1.92 (s, 3H, OAc), 4.19 (br.s, 1H, C3-H), 4.25 (br.s, 1H, C2-H), 5.09 (d, 1H, J ⫽ 9 Hz, C22-H), 5.26 (d, 1H, J ⫽ 9 Hz, C23-H). 13C NMR ␦: 11.0, 11.9, 12.8, 15.4, 20.2, 20.7, 20.9, 21.1, 22.5, 22.7, 23.8, 28.0, 30.1, 36.9, 37.2, 39.4, 39.7, 40.5, 41.4, 42.8, 46.3, 49.6, 50.8, 52.3, 54.8, 56.4, 70.3, 74.2, 75.8, 77.3, 170.7, 212.1. HRMS Calc. for C32H48D3BrO6: 613.305731; Found: 613.306763. 2.18. (22R,23R,24S)-[26-2H3]-2␣-Bromo-22,23-diacetoxy24-methyl-5␣-cholesta-3,6-dione (23) Jones reagent (1.1 ml) was added to a solution of the bromohydrine 22 (400 mg, 0.62 mmol) in acetone (20 ml). After the mixture was stirred for 10 min, iPrOH (1.5 ml) was added, and stirring was continued for 5 min. The mixture was then diluted with water and extracted with CHCl3. The extract was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 with petroleum ether-EtOAc (10:1 f 1:1) to yield the diketone 23 (270 mg, 67%). IR (cm⫺1): 2965, 2880, 1750, 1380, 1255, 1030. 1H NMR ␦: 0.73 (s, 3H, 18-Me), 1.04 (s, 3H, 19-Me), 2.01, 2.03 (s, 3H, OAc), 4.76 (dd, 1H, J ⫽ 13, 6 Hz, C2-H), 5.16 (d, 1H, J ⫽ 8.7 Hz, C22-H), 5.34 (d, 1H, J ⫽ 8.7 Hz, C23-H).
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C NMR ␦: 11.7, 12.5, 13.5, 13.8, 20.9, 21.6, 22.3, 24.5, 28.7, 30.8, 37.2, 37.6, 38.4, 39.8, 40.5, 43.5, 44.7, 46.9, 51.5, 53.0, 53.6, 53.8, 57.0, 58.4, 74.8, 77.7, 171.2, 201.5, 208.2. 13
2.19. (22R,23R,24S)-[26-2H3]-22,23-Diacetoxy-2␣hydroxy-24-methyl-5␣-cholesta-3,6-dione (24) A mixture of the bromohydrine 23 (0.25 g, 0.41 mmol), K2CO3 (1.0 g, 7.2 mmol), acetone (15 ml) and water was heated at 45°C for 15 h, diluted with water, and subsequently partly evaporated in vacuo. The residue was extracted with CHCl3. The extract was dried (Na2SO4) and evaporated to give 0.25 g of crude hydroxy ketone 24. 1H NMR ␦: 0.74 (s, 3H, 18-Me), 1.06 (s, 3H, 19-Me), 2.01, 2.04 (s, 3H, OAc), 4.20– 4.34 (dd, 1H, J ⫽ 12, 7 Hz, C2-H), 5.15 (d, 1H, J ⫽ 8.5 Hz, C22-H), 5.33 (d, 1H, J ⫽ 8.5 Hz, C23-H). 2.20. Reduction of the hydroxy ketone (24) with NaBH4 A solution of NaBH4 (21 mg, 0.55 mmol) was added dropwise to a stirred mixture of the hydroxy ketone 24 (0.24 g, 0.44 mmol) in EtOH (10.5 ml) at ⫺10°C. The mixture was stirred for another hour at ⫺10°C (TLC control) and then, neutralized with 5% H2SO4. The organic solvent was partially removed in vacuo, after which the mixture was extracted with CHCl3. The organic layer was dried (Na2SO4) and evaporated, and the residue was dissolved in 15% KOH in MeOH (12 ml). The mixture was refluxed for 1.5 h, then diluted with water, and extracted with CHCl3. The extract was dried (Na2SO4), evaporated, and chromatographed on SiO2 (CHCl3-MeOH ⫽ 100:1 f 10:1) to give: a) (22R,23R,24S)-[26-2H3]-2␣,3␣,22,23-tetrahydroxy-24methyl-5␣-cholestan-6-one 29 (30 mg, 16% based on 23). 1 H NMR ␦: 0.70 (s, 3H, 18-Me), 0.94 (s, 3H, 19-Me), 3.56 (d, 1H, J ⫽ 8.5 Hz, C22-H), 3.64 –3.84 (m, 2H, C23- and C2-H), 4.05 (br.s, 1H, C3-H). HRMS Calc. for C22H36O4 (split at bond C22-C23): 364.261360; Found: 364.261215; b) (22R,23R,24S)-[26- 2 H 3 ]-2 ␣ ,3  ,22,23-tetrahydroxy-24methyl-5␣-cholestan-6-one 26 (80 mg, 42% based on 23). Mp 243–246°C (EtOAc-MeOH). 1H NMR (C5D5N) ␦: 0.71 (s, 3H, 18-Me), 0.80 (s, 3H, 19-Me), 3.68 – 4.14 (m, 4H, C2-, C3-, C22-, and C23-H). 13C NMR (C5D5N) ␦: 10.8, 12.0, 12.7, 14.4, 20.9, 21.2, 21.9, 24.0, 28.1, 29.3, 31.0, 37.7, 37.9, 39.9, 41.0, 42.7, 42.9, 45.8, 46.6, 52.9, 56.75, 56.80, 72.2, 73.0, 74.2, 75.9, 210.0. HRMS Calc. for C28H43D3O4 (M⫹-H2O): 449.358441; Found: 449.359949. 2.21. (22R,23R,24S)-[26-2H3]-2␣,3,22,23-Tetrahydroxy24-methyl-B-homo-7-oxa-5␣-cholestan-6-one (27) A solution of H2O2 (30%, 0.2 ml) was added to a solution of trifluoroacetic anhydride (0.5 ml, 3.54 mmol) in CH2Cl2 (3 ml) at 0°C. The mixture was stirred for 15 min, then cooled to ⫺20°C, and a solution of the ketone 26 (73 mg, 0.16 mmol) in CH2Cl2 (30 ml) was added. The mixture
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was kept at ⫺10°C for 10 h and treated first with an excess of NaHSO3 to destroy CF3CO3H and then with Na2CO3. The target compound was extracted with CHCl3, after which the organic layer was dried (Na2SO4) and evaporated. The residue was chromatographed on SiO2 (CHCl3MeOH ⫽ 30:1 f 8:1) to give the lactone 27 (68 mg, 91%). Mp. 255–256°C (EtOAc-EtOH). 1H NMR (C5D5N) ␦: 0.73 (s, 3H, 18-Me), 1.05 (s, 3H, 19-Me), 1.14 (d, 3H, J ⫽ 7 Hz, 21-Me), 1.22 (d, 3H, J ⫽ 6.5 Hz, 27-Me), 3.21 (dd, 1H, J ⫽ 12, 4.5 Hz, C5-H), 3.80 – 4.21 (m, 4H, C2-, C3-, C22-, and C23-H). 13C NMR (C5D5N) ␦: 10.9, 11.8, 12.7, 16.2, 20.9, 21.2, 23.1, 24.9, 28.1, 31.1, 34.7, 38.0, 38.8, 39.7, 40.1, 41.1, 42.6, 46.8, 48.2, 51.6, 52.9, 58.4, 70.4, 71.9, 73.1, 74.2, 74.5, 175.6. HRMS Calc. for C28H43D3O5 (M⫹-H2O): 465.353355; Found: 465.355033.
3. Results and discussion Initially, the steroidal side chain had to be constructed with a carbon skeleton characteristic of brassinosteroids. This was achieved using the Claisen rearrangement. Compound 2 contained a methyl group at C-24 with the necessary stereochemistry and a ⌬22-double bond ensuring further introduction of the 22R,23R-diol function. In addition, the ester function in 2 allowed easy introduction of three deuteriums at C-26 by successive reduction with LiAlD4, tosylation, and again, reduction with LiAlD4. Starting with 4, all the deuterated compounds were present as mixtures of diastereomers because of formation of a new chiral center at C-25. This result clearly followed from the 13C NMR spectra, which showed signals of 26and 27-methyl groups approx. 50% as intensive as other signals. However, this fact was not important for the biosynthetic studies involving steroidal A and B cycles. Treatment of the i-steroidal ether 4 with acid led to regeneration of the 3-hydroxy-⌬5-system, resulting in compound 5, which was then transformed into the ketone 6. The further synthetic route implied introduction of a 22R,23R-diol function. The best solution starting from ⌬22olefins is known to be Sharpless hydroxylation [21] in the presence of chiral alkaloids of the quinidine series. Natural 22R,23R-diols are major products of this oxidation, although minor 22S,23S-diols are also formed in various ratios depending on the reaction conditions. We studied two variants of this reaction. The first one made use of p-chlorobenzoate dihydroquinidine. A mixture (1.6:1) of diols 7 and 8 was isolated. Oxidation by a commercial ADmix containing hydroquinidine 1,4-phthalazinediyl diether ((DHQD)2-PHAL) proceeded very slowly, which was why an additional amount of K2OsO4.2H2O and (DHQD)2-PHAL was needed. The resulting ratio of 22R,23R- and 22S,23S-isomers was much better (only a small amount of the latter was isolated, together with unindentified products); the problem in this case was overoxidation with formation of the diketone 9. A
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similar reaction has been described for hydroxylation of stigmastane derivatives [22]. Compound 7 was acetylated and then treated with HBr to give 11. Transformation of 3-bromides like 11 into typhasterol derivatives has been reported in a moderate yield by treatment with AgOAc [23]. We carried out the nucleophilic substitution of bromide with an acetoxy group by using Hg(OAc)2 and had similar results. Nevertheless, the reaction gave enough acetate 12a to allow saponification and formation of [26-2H3]typhasterol 12b. The Baeyer-Villiger oxidation of the latter furnished another deuterated brassinosteroid, [26-2H3]-2-deoxybrassinolide 13. Heating of lithium carbonate and the bromide 11 in DMF led to the dehydrobromination of 11 to give the ⌬2-olefine 15. Another product 14 resulted from nucleophilic substitution of bromine under these reaction conditions. Hydrolysis of the formyl ester 14, followed by Jones oxidation, gave the 22,23diacetate of [26-2H3]-3-dehydroteasterone 17. There have been no data until now about the role of secasterone [24], containing a 2,3-epoxy group, in the brassinosteroid biosynthetic network. For the majority of brassinosteroids, the presence of a 2␣,3␣-diol function is characteristic. However, there are also examples having 2␣,3- or 2,3␣ configurations, which is why we decided to prepare both 2␣,3␣- and 2,3-epoxides. Epoxidation of the olefine 15, followed by removal of the acetoxy groups, led to [26-2H3]-2,3-episecasterone 19. Treatment of the olefine with bromosuccinimide gave the transdiaxial bromohydrine 20, which was further transformed into [26-2H3]secasterone 21 by treatment with base. Synthesis of brassinosteroids 26 and 27, bearing two equatorial hydroxy groups, proved to be the most difficult part of the investigation [25]. The epoxide 18 proved to be a convenient intermediate for this purpose. Its opening with HBr, followed by Jones oxidation, led to the bromoketone 23. The nucleophilic substitution of the bromine at the next step gave the hydroxy ketone 24. Treatment of compound 24 with NaBH4 proceeded with preferential reduction of the C-3 keto group. Saponification of the resulting reaction mixture followed by column chromatography gave [262 H3]-3-epicastasterone 26 and [26-2H3]castasterone 29. The Baeyer-Villiger oxidation of 26 afforded the [26-2H3]-3epibrassinolide 27.
Acknowledgments We thank Dr. N.B. Khripach (Minsk) for recording the NMR spectra and Dr. Neil J. Oldham (Jena) for the exact mass measurements. Emily Wheeler (Jena) is thanked for linguistic support in the preparation of this manuscript. The authors are grateful to the Belorussian Foundation for Fundamental Research for financial support.
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