Short-Step Synthesis of Chenodiol from Stigmasterol

Biosci. Biotechnol. Biochem., 68 (6), 1332–1337, 2004

Short-step Synthesis of Chenodiol from Stigmasterol

y Toru UEKAWA, Ken ISHIGAMI, and Takeshi KITAHARA

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received February 3, 2004; Accepted March 11, 2004

Chenodiol is an important bile acid widely used for the hydroxyl group (C-3 position), 2) construction of gallstone dissolution and cholestatic liver diseases. We cis-fused rings by hydrogenation (C-5 position), 3) succeeded in a short-step synthesis of chenodiol, starting allylic oxidation and stereoselective reduction (C-7 from the safer phytosterol, stigmasterol. position), and 4) ozonolysis of the side chain and subsequent transformation, including the Wittig reac- Key words: chenodiol; stigmasterol; gallstone dissolu- tion. tion; bovine spongiform encephalopathy Our first synthetic route is outlined in Scheme 2. The hydroxyl group of stigmasterol (2) was inverted by Chenodiol is an important bile acid contained in many mesylation4,5) and the subsequent treatment with cesium vertebrates. This compound is widely used in clinical acetate.6) Inversion under Mitsunobu conditions gave a applications for the dissolution of cholesterol gallstones diastereomixture at the C-3 position, and elimination of and cholestatic liver diseases.1) At present, chenodiol is the hydroxyl group was also observed. Allylic oxidation industrially synthesized from cholic acid,2,3) a major of the C-7 position was accomplished by N-hydroxy- component of bovine bile. BSE (bovine spongiform phthalimide-catalyzed air oxidation, using benzoyl per- encephalopathy) has recently become a global problem oxide as a radical initiator.7,8) The resulting hydroper- and it is now prohibited to use the specified bovine risk oxide was dehydrated to give enone 4. Regioselective materials (encephalon, marrow, intestines, lien, etc.) for ozonolysis of the side chain and the subsequent Wittig medicinal use. Although the use of bovine bile is not reaction afforded ,-unsaturated ester 5 in a good yield. actually prohibited, it would be much better for This compound was subjected to stereoselective reduc- chenodiol to be prepared from safer starting material tion at the C-7 position by using L-SelectrideÒ to give than cholic acid. We report in this paper the synthesis of alcohol 6 (: = 2:1). After separation -6, the two chenodiol starting from stigmasterol which is the main double bonds were reduced together by using a platinum component of the phytosterol mixture from such beans catalyst to selectively afford a cis-fused ring system (7). as soy. Hydrolysis of the acetate and ethyl ester gave chenodiol (1) in good yield. The synthesis of chenodiol could be Result and Discussion achieved in this way, but the total yield (7.1% in 8 steps) was not satisfactory and the introduction of hydroxyl Our synthetic plan is shown in Scheme 1. In order to groups at the C-3,7 positions was not efficient. We then convert stigmasterol to chenodiol, the transformation of decided to examine another route from stigmasterol. four parts would be required as follows: 1) inversion of The revised synthetic route is shown in Scheme 3.

Scheme 1.

y To whom correspondence should be addressed. Tel: +81-3-5841-5119; Fax: +81-3-5841-8019; E-mail: [email protected] Synthesis of Chenodiol 1333

Scheme 2. Reagents and conditions: a) MsCl, Et3N, CH2Cl2, quant. b) CsOAc, 18-c-6, toluene, reflux, 48 h, 52%. c) N-hydroxyphthalimide, benzoyl peroxide, air, iso-butyl methyl ketone, 55 C, 48 h, then Ac2O, pyridine, overnight, 56%. d) O3, pyridine, CH2Cl2, then Me2S. e) Ò Ph3P=CHCO2Et, benzene, reflux, 3 h, 69% in 2 steps. f) L-Selectride , THF, 78 C, 5 h, 75%, : = 2:1. g) H2, PtO2, 2-propanol, overnight, 90%, h) 5% aq. NaOH, MeOH, reflux, 3 h, 80%.

i Scheme 3. Reagents and conditions: a) Al(OPr )3, cyclohexanone, benzene, reflux, 5 h, 79%. b) O3, pyridine, CH2Cl2, 78 C, then Me2S. c) Ph3P=CHCO2Et, benzene, reflux, 3 h, 80% in 2 steps. d) chloranil, AcOH, toluene, reflux, 4 h, 80%. e) magnetism monoperoxyphthalate hexahydrate, Et2O, CH2Cl2, 14 d, 60%. f) mCPBA, BHT, CHCl3, reflux, 6 h, 55%. g) H2, Pd–CaCO3, MeCN, overnight, 79%. h) NaBH4, MeOH, 0 C, 78%. i) 5% aq. NaOH, MeOH, reflux, 3 h, 80%.

Oppenauer oxidation of stigmasterol (2) gave conjugat- reaction.12) Preparatory to oxidation of the C-7 position, ed enone 8 in a good yield.9,10) The side chain of resulting enone 9 was treated with chloranil to efficiently compound 8 was selectively ozonolyzed to afford an give dienone 10. Stereoselective epoxidation of 10 was aldehyde,11) this then being subjected to the Wittig examined under several conditions. Treatment of 10 1334 T. UEKAWA et al. with monoperoxyphthalate afforded desired -epoxide 8.5 Hz), 5.16 (1H, dd, J ¼ 15:2, 8.4 Hz), 5.42 (1H, d, 11 in a moderate yield, but this oxidation involved a J ¼ 5:1 Hz). long reaction time. On the other hand, oxidation with A suspension of stigmasteryl mesylate (949 mg, mCPBA took place easily in the presence of BHT as a 2.05 mmol), cesium acetate (1.97 g, 10.3 mmol) and stabilizer under refluxing conditions. This epoxide was 18-crown-6 (538 mg, 2.05 mmol) in toluene (35 ml) was hydrogenated together with both double bonds by using refluxed for 48 h. After cooling to room temperature, the a palladium catalyst to give 12; that is, stereoselective reaction mixture was poured into brine and extracted introduction of a hydroxyl group at the C-7 position and with ether. The organic layer was dried with anhydrous construction of a cis-fused ring system were simulta- magnesium sulfate and concentrated in vacuo. The neously achieved in a good yield. The reduction of residue was chromatographed over silica gel, and elution ketone 12 with sodium borohydride selectively proceed- with toluene gave 3 (485 mg, 52.0%) as a white solid. 21 ed from its convex side to afford 13, containing an - Mp 107–109 C. ½ D 27 (c 0.37, CHCl3). IR max hydroxyl group. As the final step, ethyl ester in the side (KBr) cm1: 2959 (s), 2887 (m), 2867 (m), 1732 (s), chain was hydrolyzed into carboxylic acid to effectively 1380 (m), 1358 (m), 1258 (m), 1238(s), 1150 (m), 1074 1 complete the revised synthesis of chenodiol (15% total (m), 1022 (m), 986 (m), 970 (m). H-NMR (CDCl3) yield in 8 steps). ppm: 0.70 (3H, s), 0.81 (6H, t, J ¼ 6:7 Hz), 0.85 (3H, d, In conclusion, we accomplished a short-step synthesis J ¼ 6:4 Hz), 1.0–1.8 (20H, m), 1.01 (3H, s), 1.03 (3H, d, of chenodiol starting from the phytogenic sterol, J ¼ 6:4 Hz), 1.9–2.1 (3H, m), 2.02 (3H, s), 2.48 (1H, dt, stigmasterol. It will be necessary to refine several steps J ¼ 15:4, 2.6 Hz), 4.98–5.00 (1H, m), 5.07 (1H, dd, to achieve an industrial process, but this synthesis shows J ¼ 15:1, 8.5 Hz), 5.16 (1H, dd, J ¼ 15:1, 8.5 Hz), 5.26– þ the possibility of using a phytosterol as a safer starting 5.28 (1H, m). EIMS m=z: 394(M –CH3CO2H), þ þ material for chenodiol. 351(M –CH3CO2H and C3H7), 255(M –CH3CO2H and C12H19 (C20-C29 fragment)). Anal. Calcd. for Experimental C31H50O2: C, 81.9; 11.1%. Found: C, 82.6; H, 11.1%.

Melting point (mp) data were measured with (3,22E)-3-Acetoxystigmasta-5,22-dien-7-one (4). To Yanagimoto micro-melting point hot-stage apparatus a solution of 3 (909 mg, 2.00 mmol) and N-hydroxy- and are not corrected. Infrared spectra were obtained phthalimide (163 mg, 1.00 mmol) in iso-butyl methyl with a Hitachi 270-30 spectrophotometer, and optical ketone (45 ml) was added dibenzoyl peroxide (10 mg) at rotation values were measured with a Perkin-Elmer 241 55 C. The reaction mixture was stirred by a rapid polarimeter. 1H-NMR spectra were recorded with the stream of air at 55–57 C for 48 h. After cooling to room solvent peak as an internal standard by a Jeol JNM- temperature, the reaction mixture was concentrated in 13 AL300 spectrometer at 300 MHz. C-NMR spectra vacuo. The residue was suspended in CCl4 (30 ml), the were recorded with the solvent peak as an internal precipitate being removed by filtration and concentrated standard by a Jeol JNM-AL400 instrument at 100 MHz. in vacuo. Pyridine (5.0 ml) and acetic anhydride (0.5 ml) EI-HRMS and ESI-HRMS data were respectively were added to the residue at 0 C, and the mixture was recorded with Jeol JMS-AX505W and Mariner (Applied stirred overnight at room temperature. The reaction Biosystems) spectrometer. Column chromatography was mixture was concentrated in vacuo, and the residue was carried out in columns packed with silica gel 60 N 63– chromatographed over silica gel (hexane:EtOAc = 4:1) 210 mm (Kanto Chemical Co.). Preparative TLC was to give 4 (525 mg, 56.0%) as a white solid and 3 carried out with Merck Kieselgel 60F254 1.05744. (recovered, 30 mg). 21 Mp 109–111 C. ½ D 77:0 (c 0.53, CHCl3). IR 1 (3,22E)-3-Acetoxystigmasta-5,22-diene (3). To the max (KBr) cm : 2959 (s), 2871 (m), 1740 (s), 1672 (s), solution of stigmasterol (2.06 g, 5.00 mmol) and triethyl- 1632 (m), 1458 (m), 1388 (m), 1366 (m), 1256 (s), 1228 amine (2.53 g, 25.0 mmol) in toluene (30 ml) was added (m), 1188 (m), 1152 (m), 1022 (m), 1022 (m), 994 (m). 1 MsCl (1.15 g, 10.0 mmol) at 0 C under argon. The H-NMR (CDCl3) ppm: 0.71 (3H, s), 0.78–0.85 (6H, mixture was stirred at 0 C for 15 min and then at room m), 0.8–2.5 (22H, m), 0.85 (3H, d, J ¼ 6:5 Hz), 1.03 temperature for 2 h. The reaction mixture was poured (3H, d, J ¼ 6:6 Hz), 1.21 (3H, s), 2.01 (3H, s), 2.56–2.66 into brine and extracted with ether. The organic layer (1H, m), 5.02 (1H, dd, J ¼ 15:1, 8.7 Hz), 5.11–5.14 (1H, was successively washed with 1 N HCl and saturated m), 5.17 (1H, dd, J ¼ 15:1, 8.4 Hz), 5.66 (1H, d, NaHCO3, dried with anhydrous magnesium sulfate J ¼ 1:5 Hz). ESI-HRMS m=z: calcd. for C31H48NaO3 and concentrated in vacuo. The resulting stigmasteryl ½M þ Naþ, 491.3501; found, 491.3538. mesylate (2.31 g, 99%) was used in the next step without purification. (3,22E)-3-Acetoxy-7-oxo-5,22-choladien-24-oic acid 1 H-NMR (CDCl3) ppm: 0.70 (3H, s), 0.79–0.83 ethyl ester (5). Ozone was bubbled into a stirred solution (6H, m), 0.85 (3H, d, J ¼ 6:4 Hz), 1.02 (3H, d, of 4 (399 mg, 0.852 mmol) and pyridine (0.25 ml) in J ¼ 6:4 Hz), 0.9–2.1 (23H, m), 2.44–2.59 (2H, m), CH2Cl2 (25 ml) at 78 C. After the solution had turned 3.01 (3H, s), 4.47–4.58 (1H, m), 5.02 (1H, dd, J ¼ 15:2, slightly blue, nitrogen was bubbled into the solution to Synthesis of Chenodiol 1335 eject the excess ozone. After adding dimethyl sulfide (3,5,7)-3-Acetoxy-7-hydroxycholan-24-oic acid i (0.15 ml), the reaction mixture was allowed to warm to ethyl ester (7). A suspension of PtO2 (5 mg) in Pr OH room temperature and then stirred for 3 h. After (3.0 ml) was stirred vigorously under hydrogen. Com- concentrating in vacuo, the residue was dissolved pound 6 was added to the suspension, and the mixture in benzene (25 ml), and Ph3P=CHCO2Et (593 mg, was stirred overnight under hydrogen. After filtration 1.70 mmol) was added. The mixture was refluxed for and concentration, the residue was chromatographed 3 h and then concentrated in vacuo. The residue was over silica gel (hexane:EtOAc = 1:1) to give 7 (20.8 mg, chromatographed over silica gel (hexane:EtOAc = 4:1– 90.0%) as a white solid. 21 3:1) to give 5 (268 mg, 68.8%) as a white solid. Mp 115–116 C. ½ D +28.8 (c 0.88, CHCl3). IR 22 1 Mp 165–167 C. ½ D 72:7 (c 0.42, CHCl3). IR max (KBr) cm : 3539 (m), 2939 (s), 2867 (m), 1743 1 max (KBr) cm : 2947 (m), 2871 (m), 1736 (s), 1718 (s), 1728 (s), 1468 (m), 1378 (m), 1362 (m), 1248 (s), (s), 1672 (s), 1650 (m), 1372 (m), 1332 (m), 1290 (m), 1130 (m), 1096 (m), 1074 (m), 1024 (m), 976 (m), 892 1 1262 (s), 1234 (s), 1186 (m), 1178 (m), 1146 (m), 1072 (m). H-NMR (CDCl3): 0.66 (3H, s), 0.9–2.0 (27H, 1 (m), 1030 (m), 992 (m). H-NMR (CDCl3) ppm: 0.72 m), 0.93 (3H, d, J ¼ 7:7 Hz), 1.25 (3H, t, J ¼ 7:2 Hz), (3H, s), 1.10 (3H, d, J ¼ 6:6 Hz), 1.2–2.1 (16H, m), 1.21 2.01 (3H, s), 2.15–2.39 (3H, m), 3.85(1H, q, J ¼ (3H, s), 1.29 (3H, t, J ¼ 7:14 Hz), 2.01 (3H, s), 2.20– 2:6 Hz), 4.12 (2H, q, J ¼ 7:1 Hz), 4.58 (1H, tt, J ¼ 11:5, 2.47 (2H, m), 2.62 (1H, ddd, J ¼ 15:8, 3.3, 2.0 Hz), 4.18 4.5 Hz). ESI-HRMS m=z: calcd. for C28H46NaO5 (2H, q, J ¼ 7:2 Hz), 5.13 (1H, t, J ¼ 2:8 Hz), 5.67 (1H, ½M þ Naþ, 485.3243; found, 485.3285. d, J ¼ 1:8 Hz), 5.81 (1H, dd, J ¼ 15:6, 0.7 Hz), 6.85 (1H, dd, J ¼ 15:6, 9.1 Hz), ESI-HRMS m=z: calcd. for (3,5,7)-3,7-Dihydroxycholan-24-oic acid (cheno- þ C28H40NaO5 ½M þ Na , 479.2773; found, 479.2732. diol 1). A solution of 7 (18.2 mg, 39.3 mol) in 5% NaOH (0.5 ml) and MeOH (2.0 ml) was refluxed for 3 h. (3,7,22E)-3-Acetoxy-7-hydroxy-5,22-choladien-24- After removing MeOH by evaporation, 0.5 N HCl was oic acid ethyl ester (6). To a solution of 5 (114 mg, added to the mixture, before it was extracted with 0.250 mmol) in THF (10 ml) was added L-SelectrideÒ EtOAc. The organic layer was dried with anhydrous (1.0 M in THF, 750 l, 0.750 mmol) at 78C under magnesium sulfate and concentrated in vacuo. The argon, and the mixture was stirred for 3 h. After adding residue was purified by preparative TLC (CHCl3:MeOH water (1.0 ml), the reaction mixture was warmed to 0 C = 9:1) to give chenodiol (12.4 mg, 80.0%) as a white and adjusted to pH 3 with 1 N HCl. The mixture was solid. poured into brine and extracted with EtOAc. The Mp 119–120 C (recrystd. from EtOAc–hexane), lit. 3) 21 organic layer was dried with anhydrous magnesium mp 119.5–121 C. ½ D +10 (c 0.14, 1,4-dioxane), 25 2) 21 sulfate and concentrated in vacuo. The residue was lit. ½ D +11 (c 1.00, 1,4-dioxane), ½ D +10 (c chromatographed over silica gel (hexane:EtOAc = 2:1) 0.14, 1,4-dioxane, commercially available chenodiol 1 to give a mixture of 6 and 7-epi-6 (86.0 mg, 75.0%, from Acros Organics). IR max (KBr) cm : 3443 (m), : = 2:1 by 1H-NMR). This mixture was purified by 2935 (s), 2867 (s), 1712 (s), 1374 (m), 1242 (m), 1198 1 preparative TLC (CH2Cl2:acetone = 20:1 3) to give 6 (m), 1075 (m) 1002 (m), 978 (m). H-NMR (CDCl3): (45.0 mg, 39.1%) as a white solid. 0.66 (3H, s), 0.9–2.0 (26H, m), 0.90 (3H, s), 0.94 (3H, d, 21 Mp 123–124 C. ½ D 64:6 (c 0.52, CHCl3). IR J ¼ 6:2 Hz), 2.1–2.5 (3H, m), 3.47 (1 H, tt, J ¼ 10:8, 1 13 max (KBr) cm : 3435 (m), 2939 (s), 2891 (m), 1738 4.6 Hz), 3.86 (1H, br.d, J ¼ 2:4 Hz). C-NMR (s), 1720 (s), 1648 (m), 1462 (m), 1364 (m), 1330 (m), (CDCl3) ppm: 11.9, 18.4, 20.7, 22.9, 23.8, 28.3, 30.6, 1252 (s), 1240 (s), 1196 (m), 1178 (m), 1144 (m), 1114 31.0, 31.3, 32.9, 34.6, 35.1, 35.4, 35.6, 39.5, 39.7, 41.6, (m), 1024 (m), 868 (m), 816 (m), 742 (m). 1H-NMR 42.8, 50.4, 55.9, 68.6, 72.0, 178.6. ESI-HRMS m=z: þ (CDCl3) ppm: 0.72 (3H, s), 1.0–1.8 (16H, m), 1.05 (3H, calcd. for C24H40NaO4 ½M þ Na , 415.2824; found, s), 1.10 (3H, d, J ¼ 6:6 Hz), 1.28 (3H, t, J ¼ 7:1 Hz), 415.2827. 1.9–2.0 (1H, m), 2.01 (3H, s), 2.1–2.4 (2H, m), 2.51 (1H, dd, J ¼ 15:2, 1.5 Hz), 3.80 (1H, br.s), 4.17 (2H, q, (22E)-3-Oxostigmasta-4,22-diene (8). A solution of J ¼ 7:2 Hz), 5.04 (1H, s), 5.55 (1H, d, J ¼ 5:4 Hz), 5.73 stigmasterol (825 mg, 2.00 mmol) and cyclohexanone (1H, d, J ¼ 15:6 Hz), 6.83 (1H, dd, J ¼ 15:6, 9.0 Hz). (3.11 ml, 30.0 mmol) in toluene (20 ml) was refluxed þ ESI-HRMS m=z: calcd. for C28H42NaO5 ½M þ Na , under argon, and toluene (5 ml) was distilled off. After i 481.2930; found, 481.2943. cooling to 50 C, Al(OPr )3 (817 mg, 4.00 mmol) was 1 7-epi-6: H-NMR (CDCl3) ppm: 0.73 (3H, s), 1.05 added to the mixture. After refluxing for 5 h, the reaction (3H, s), 1.1–1.9 (16H, m), 1.10 (3H, d, J ¼ 6:6 Hz), 1.28 mixture was concentrated in vacuo. Ether was added to (3H, t, J ¼ 7:2 Hz), 1.9–2.1 (1H, m), 2.01 (3H, s), 2.2– the residue, and the precipitate was removed by 2.4 (2H, m), 2.49 (1H, ddd, J ¼ 15:4, 5.3, 2.4 Hz), 3.88 filtration. The filtrate was concentrated in vacuo, and (1H, dt, J ¼ 7:7, 2.1 Hz), 4.17 (2H, q, J ¼ 7:2 Hz), 5.02 the residue was chromatographed over silica gel (1H, t, J ¼ 2:8 Hz), 5.22 (1H, t, J ¼ 1:9 Hz), 5.74 (1H, (hexane:EtOAc = 2:1) to give 8 (649 mg, 79.0%) as a dd, J ¼ 15:6, 0.6 Hz), 6.83 (1H, dd, J ¼ 15:6, 9.0 Hz). white solid. 21 Mp 127–128 C. ½ D +52.1 (c 0.78, CHCl3). IR 1336 T. UEKAWA et al. 1 max (KBr) cm : 2951 (s), 2867 (s), 1674 (s), 1620 (m), (3H, d, J ¼ 7:0 Hz), 1.11 (3H, s), 1.29 (3H, t, J ¼ 1462 (m), 1444 (m), 1382 (m), 1368 (m), 1354 (m), 7:1 Hz), 1.95–2.05 (2H, m), 1.15–1.85 (11H, m), 2.18– 1330 (m), 1270 (m), 1246 (m), 1228 (m), 972 (m), 962 2.65 (4H, m), 4.18 (2H, q, J ¼ 7:1 Hz), 5.67 (1H, s), (m), 950 (m), 934 (m), 864 (m), 684 (m). 1H-NMR 5.75 (1H, dd, J ¼ 15:5, 0.7 Hz), 6.08 (1H, d, J ¼ (CDCl3) ppm: 0.73 (3H, s), 0.80 (3H, d, J ¼ 6:5 Hz), 10:3 Hz), 6.13 (1H, d, J ¼ 10:3 Hz), 6.82 (1H, dd, J ¼ þ 0.81 (3H, t, J ¼ 7:5 Hz), 0.85 (3H, d, J ¼ 6:4 Hz), 0.9– 15:5, 8.9 Hz). EI-HRMS m=z (M ): calcd. for C26H36O3, 1.9 (20H, m), 1.02 (3H, d, J ¼ 6:6 Hz), 1.18 (3H, s), 396.2664; found, 396.2671. 5.02 (1H, dd, J ¼ 15:2, 8.4 Hz), 5.15 (1H, dd, J ¼ 15:2, 8.4 Hz), 5.72 (1H, s). EI-HRMS m=z (Mþ): calcd. for (6,7,22E)-6,7-Epoxy-3-oxo-4,22-choladien-24-oic C29H46O, 410.3549; found, 410.3554. acid ethyl ester (11). Method A: To a refluxed solution of 10 (200 mg, 0.504 mmol) and BHT (11.1 mg) in (22E)-3-Oxo-4,22-choladien-24-oic acid ethyl ester CHCl3 (15 ml) was added three portions of mCPBA (9). Ozone was bubbled into a stirred solution of 8 (80%, 82 mg 3, 0.38 mmol 3) at 2-h intervals. After (2.19 g, 5.33 mmol) and pyridine (1.4 ml) in CH2Cl2 refluxing for 6 h, the reaction mixture was cooled to (140 ml) at 78 C. After the solution had turned room temperature and poured into saturated NaHCO3. slightly blue, nitrogen was bubbled into it to eject the The mixture was extracted with ether. After washing excess ozone. After adding dimethyl sulfide (0.9 ml), the with brine, the organic layer was dried with anhydrous reaction mixture was allowed to warm to room temper- magnesium sulfate and concentrated in vacuo. The ature, and then stirred for 3 h. After washing with 1 N residue was chromatographed over silica gel HCl, the organic layer was dried with anhydrous (hexane:EtOAc = 3:1) to give 11 (114 mg, 55.0%) as magnesium sulfate and concentrated in vacuo. The a white solid. residue was dissolved in benzene (25 ml), and Method B: A solution of magnesium monoperoxy- Ph3P=CHCO2Et (2.79 g, 8.00 mmol) was added. The phthalate hexahydrate (80%, 77.0 mg, 0.250 mmol) in mixture was refluxed for 4 h and then concentrated in distilled water (1 ml) was adjusted to pH 1 with 3 N HCl vacuo. The residue was chromatographed over silica gel and extracted with ether (1 ml). The organic layer was (hexane:EtOAc = 3:1) to give 9 (1.71 g, 80.4%) as a dried with anhydrous magnesium sulfate, and the white solid. magnesium sulfate was removed by filtration. The 21 Mp 162–164 C. ½ D +60.9 (c 1.04, CHCl3). IR filtrate was added to a solution of 10 (19.8 mg, 1 max (KBr) cm : 2943 (s), 2871 (m), 2851 (m), 1722 50.0 mol) in CHCl3 (2.0 ml), and the mixture was (s), 1676 (s), 1652 (m), 1614 (m), 1458 (m), 1446 (m), stirred in darkness at room temperature for 14 days. The 1432 (m), 1386 (m), 1368 (m), 1328 (m), 1294 (m), reaction mixture was poured into saturated NaHCO3 and 1268 (m), 1236 (s), 1206 (m), 1180 (m), 1166 (m), 1142 extracted with ether. The organic layer was dried with (m), 1032 (m), 984 (m), 950 (m), 864 (m). 1H-NMR anhydrous magnesium sulfate and concentrated in (CDCl3) ppm: 0.75 (3H, s), 0.9–1.9 (16H, m), 1.09 (3H, vacuo. The residue was purified by preparative TLC d, J ¼ 6:6 Hz), 1.19 (3H, s), 1.29 (3H, t, J ¼ 7:1 Hz), (hexane:EtOAc = 1:1) to give 11 (12.3 mg, 59.6%) as a 1.98–2.05 (2H, m), 2.21–2.49 (5H, m), 4.18 (2H, q, white solid. 21 J ¼ 7:1 Hz), 5.73 (1H, d, J ¼ 1:3 Hz), 5.74 (1H, dd, Mp 171–172 C. ½ D +16 (c 0.53, CHCl3). IR max J ¼ 15:6, 0.7 Hz), 6.82 (1H, dd, J ¼ 15:6, 9.0 Hz). EI- (KBr) cm1: 2991 (m), 2947 (m), 2871 (m), 1722 (s), þ HRMS m=z (M ): calcd. for C29H38O3, 398.2821; 1678 (s), 1650 (m), 1624 (m), 1460 (m), 1446 (m), 1386 found, 398.2821. (m), 1368 (m), 1332 (m), 1294 (m), 1268 (m), 1234 (s), 1206 (m), 1186 (m), 1142 (m), 1114 (m), 1030 (m), 984 1 (22E)-3-Oxo-4,6,22-cholatrien-24-oic acid ethyl ester (m), 946 (m), 868 (m). H-NMR (CDCl3): 0.77 (3H, (10). A mixture of 9 (1.65 g, 4.14 mmol), chloranil s), 1.09 (3H, d, J ¼ 6:6 Hz), 1.2–2.05 (13H, m), 1.10 (1.12 g, 4.55 mmol), acetic acid (17.0 ml) and toluene (3H, s), 2.23–2.46 (1H, m), 2.40–2.63 (3H, m), 1.29 (3H, (4.25 ml) was refluxed for 2 h. After cooling to room t, J ¼ 7:2 Hz), 3.33 (1H, br.d, J ¼ 3:7 Hz), 3.45 (1H, d, temperature, the resulting precipitate was removed by J ¼ 3:7 Hz), 4.18 (2H, q, J ¼ 7:2 Hz), 5.75 (1H, dd, filtration and concentrated in vacuo. The residue was J ¼ 15:6, 0.74 Hz), 6.11 (1H, s), 6.82 (1H, dd, J ¼ 15:6, þ dissolved in EtOAc and washed with saturated NaHCO3. 9.0 Hz). EI-HRMS m=z (M ): calcd. for C26H36O4, The organic layer was dried with anhydrous magnesium 412.2614; found, 412.2611. sulfate and concentrated in vacuo. The residue was chromatographed over silica gel (hexane:EtOAc = 3:1) (5,7)-7-Hydroxy-3-oxocholan-24-oic acid ethyl es- to give 10 (1.32 g, 80.4%) as a white solid. ter (12). A suspension of 11 (20.0 mg, 48.5 mol) and 21 Mp 154–156 C. ½ D 23:2 (c 0.73, CHCl3). IR 5% Pd–CaCO3 (5.0 mg) in MeCN (1.0 ml) was stirred 1 max (KBr) cm : 2998 (m), 2947 (s), 1722 (s), 1666 (s), overnight under hydrogen. The reaction mixture was 1582 (m), 1460 (m), 1446 (m), 1416 (m), 1386 (m), filtered and concentrated in vacuo. The residue was 1370 (m), 1350 (m), 1330 (m), 1292 (m), 1262(m), 1234 purified by preparative TLC (hexane:EtOAc = 1:1) to (s), 1206 (m), 1184 (m), 1166 (m), 1142 (m), 1030 (m), give 12 (16.0 mg, 78.8%) as a white solid. 1 21 984 (m). H-NMR (CDCl3) ppm: 0.79 (3H, s), 1.10 Mp 146–147 C. ½ D +17 (c 0.51 CHCl3). IR max Synthesis of Chenodiol 1337 (KBr) cm1: 3503 (m), 2943 (s), 2875 (m), 1710 (s), Acknowledgments 1442 (m), 1380 (m), 1302 (m), 1256 (m), 1236 (m), 1196 (m), 1176 (m), 1158 (m), 1098 (m), 1070 (m), We sincerely thank Mr. Masafumi Moriwaki (Direc- 1 1038 (m), 996 (m). H-NMR (CDCl3): 0.70 (3H, s), tor, Research & Development Center, Nagase & Co., 0.94 (3H, d, J ¼ 6:4 Hz), 1.00 (3H, s), 1.05–2.45 (26H, Ltd.) and Dr. Masaya Ikunaka (Chief Scientist, 2nd m), 1.25 (3H, t, J ¼ 7:2 Hz), 3.39 (1H, dd, J ¼ 15:2, Laboratories, Research & Development Center, Nagase 13.6 Hz), 3.92 (1H, q, J ¼ 2:8 Hz), 4.12 (2H, q, & Co., Ltd.) for kindly providing information about J ¼ 7:2 Hz). ESI-HRMS m=z: calcd. for C26H42O4 chenodiol. ½M þ Naþ, 441.2981; found, 441.2994. References (3,5,7)-3,7-Dihydroxycholan-24-oic acid ethyl ester (13). To a solution of 12 (33.5 mg, 80.0 mol) in 1) Grundy, S. M., Lan, S. P., Lachin, J., Baum, R. A., Habig, R. L., Hanson, R. F., Hersh, T., Hightower, N. C., MeOH (1.0 ml) was added NaBH4 (3.0 mg, 79 mol) at 0 C. After stirring at 0 C for 30 min, the reaction Hofmann, A. F., Lasser, E. C., Marks, J. W., Mekhjian, H., Okun, R., Schaefer, R. A., Schoenfield, L. J., Shaw, mixture was poured into 1 N HCl and extracted with L., Soloway, R. D., Thistle, J. L., Thomas, F. P., and EtOAc. The organic layer was washed with saturated Tyor, M. P., The effects of chenodiol on biliary lipids NaHCO3 and then dried with anhydrous magnesium and their association with gallstone dissolution in the sulfate. After concentration, the residue was purified by national cooperative gallstone study (NCGS). J. Clin. preparative TLC (hexane:EtOAc = 3:8) to give 13 Invest., 73, 1156–1166 (1984). (26.3 mg, 78.2%) as an amorphous solid. 2) Fieser, L. F., and Rajagopalan, S., Oxidation of steroids. 21 1 ½ D +12 (c 0.57, CHCl3). IR max (KBr) cm : III. Selective oxidations and acylations in the bile acid 3415 (m), 2935 (s), 2871 (s), 1740 (s), 1466 (m), 1448 series. J. Am. Chem. Soc., 72, 5530–5536 (1950). (m), 1418 (m), 1376 (m), 1326 (m), 1302 (m), 1164 (s), 3) Iida, T., and Chan, F., Potential bile acid metabolites. 3. 1 A new route to chenodeoxycholic acid. J. Org. Chem., 1078 (s), 1002 (m), 980 (m). H-NMR (CDCl3): 0.65 (3H, s), 0.89 (3H, s), 0.9–2.0 (25H, m), 0.92 (3H, d, 46, 2786–2788 (1981). 4) Takatsuto, S., and Ikekawa, N., Synthesis of (22R,23R)- J ¼ 6:2 Hz), 1.24 (3H, t, J ¼ 7:2 Hz), 2.13–2.38 (3H, 28-homobrassinolide. Chem. Pharm. Bull., 30, 4181– m), 3.45 (1H, tt, J ¼ 11:0, 4.4 Hz), 3.84 (1H, q, 4185 (1982). J ¼ 2:8 Hz), 4.11 (2H, q, J ¼ 7:2 Hz). ESI-HRMS 5) Aburatani, M., Takeuchi, T., and Mori, K., Synthesis of þ m=z: calcd. for C26H44O4 ½M þ Na , 443.3137; found, brassinolide. Part II. A simple synthesis of steroidal 443.3141. 3,5-cyclo-6-ones and their efficient transformation to steroidal 2-en-6-ones. Synthesis, 181–183 (1987). (3,5,7)-3,7-Dihydroxycholan-24-oic acid (cheno- 6) Li, L., and Yang, J., Chinese Patent 1217336 (May 5, diol 1). A solution of 13 (25.3 mg, 60.1 mol) in 5% 1999). NaOH (0.5 ml) and MeOH (2.0 ml) was refluxed for 2 h. 7) Foricher, J., Fu¨rbringer, C., and Pfoertner, K., U. S. After being cooled to room temperature, MeOH was Patent 5030739 (July 9, 1991). 8) Jones, S. R., and Selinsky, B. S., Efficient route to 7- removed by evaporation, and 1 N HCl (5 ml) was added (benzoyl)-3-dioxolane cholestan-24(R)-ol, a key inter- to the mixture. The mixture was saturated with NaCl and mediate in the synthesis of squalamine. J. Org. Chem., extracted with EtOAc. The organic layer was dried with 63, 3786–3789 (1998). anhydrous magnesium sulfate and concentrated in 9) Gavagnin, M., Ungur, N., Mollo, E., Templado, J., and vacuo. The residue was purified by preparative TLC Cimino, G., Structure and synthesis of a progesterone (CHCl3:MeOH = 9:1) to give chenodiol (19.9 mg, homologue from the skin of the dorid nudibranch Aldisa 80.4%) as a white solid. smaragdina. Eur. J. Org. Chem., 1500–1504 (2002). Mp 119–120 C (recrystd. from EtOAc-hexane). 10) Barrero, A. F., Sanchez, J. F., Alvarez-Manzaneda, E. J., 21 1 ½ D +10 (c 0.13, 1,4-dioxane). IR max (KBr) cm : Munoz, D. M., and Haidour, A., Terpenoids and sterols 3447 (m), 2935 (s), 2867 (s), 1708 (s), 1368 (m), 1074 from the wood of Abies pinsapo. Phytochemistry, 32, 1 1261–1265 (1993). (m), 1000 (m), 978 (m). H-NMR (CDCl3): 0.66 (3H, s), 0.9–2.0 (26H, m), 0.90 (3H, s), 0.94 (3H, d, 11) Shepherd, D. A., Donia, R. A., Campbell, J. A., Johnson, B. A., Holysz, R. P., Slomp Jr. G., Stafford, J. E., J ¼ 6:2 Hz), 2.1–2.5 (3H, m), 3.47 (1H, tt, J ¼ 10:8, 13 Pederson, R. L., and Ott, A. C., A synthesis of 4.6 Hz), 3.86 (1H, br.d, J ¼ 2:4 Hz). C-NMR progesterone from ergosterol. J. Am. Chem. Soc., 77, (CDCl3): 11.9, 18.4, 20.7, 22.9, 23.8, 28.3, 30.6, 31.0, 1212–1215 (1955). 31.3, 32.9, 34.6, 35.1, 35.4, 35.6, 39.5, 39.7, 41.6, 42.8, 12) Linker, M., and Kreiser, W., Synthesis of methyl 50.4, 55.9, 68.6, 72.0, 178.6. ESI-HRMS m=z: calcd. for (20R,22E)- and (20S,22E)-3-oxochola-1,4,22-trien-24- þ C24H40O4 ½M þ Na , 415.2824; found, 415.2803. oate. Helv. Chim. Acta, 85, 1096–1101 (2002).