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, À78 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, À78 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 À27 (c 0.37, CHCl3). IR max hydroxyl group. As the final step, ethyl ester in the side (KBr) cmÀ1: 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 À77:0 (c 0.53, CHCl3). IR À1 (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).