Chemical synthesis of (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid and its taurine and glycine conjugates
2004; Elsevier BV; Volume: 45; Issue: 3 Linguagem: Inglês
10.1194/jlr.d300027-jlr200
ISSN1539-7262
AutoresGenta Kakiyama, Takashi Iida, Atsushi Yoshimoto, Takaaki Goto, Nariyasu Mano, Junichi Goto, Toshio Nambara, Lee R. Hagey, Alan F. Hofmann,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoA method for the synthesis of Δ22-β-muricholic acid (Δ22-β-MCA), (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid, and its taurine and glycine conjugates (Δ22-β-muricholyltaurine and Δ22-β-muricholylglycine) is described. The key intermediate, 3α,6β,7β-triformyloxy-23,24-dinor-5β-cholan-22-al, was prepared from β-muricholic acid (β-MCA) via the 24-nor-22-ene and 24-nor-22,23-diol derivatives. Wittig reaction of the aldehyde with (carbomethoxymethylene) triphenylphosphorane and subsequent hydrolysis gave (unconjugated) Δ22-β-MCA. Condensation reaction of the unconjugated acid with taurine or glycine methyl ester using diethylphosphorocyanide yielded the naturally occurring taurine or glycine conjugate (N-acylamidate) of Δ22-β-MCA.These synthetic reference compounds are now available for investigation of the metabolism of β-MCA by bacterial and hepatic enzymes in the rat and should also be useful as substrates for reductive deuteration or tritiation to give the 22,23-2H or 3H-β-MCA. A method for the synthesis of Δ22-β-muricholic acid (Δ22-β-MCA), (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid, and its taurine and glycine conjugates (Δ22-β-muricholyltaurine and Δ22-β-muricholylglycine) is described. The key intermediate, 3α,6β,7β-triformyloxy-23,24-dinor-5β-cholan-22-al, was prepared from β-muricholic acid (β-MCA) via the 24-nor-22-ene and 24-nor-22,23-diol derivatives. Wittig reaction of the aldehyde with (carbomethoxymethylene) triphenylphosphorane and subsequent hydrolysis gave (unconjugated) Δ22-β-MCA. Condensation reaction of the unconjugated acid with taurine or glycine methyl ester using diethylphosphorocyanide yielded the naturally occurring taurine or glycine conjugate (N-acylamidate) of Δ22-β-MCA. These synthetic reference compounds are now available for investigation of the metabolism of β-MCA by bacterial and hepatic enzymes in the rat and should also be useful as substrates for reductive deuteration or tritiation to give the 22,23-2H or 3H-β-MCA. When the chemical structure of the common natural C24 bile acids was deduced, it was assumed that the C5 side chain of all natural C24 bile acids was saturated. However, in 1973, while analyzing urinary bile acids from bile duct ligated rats, Danielsson (1Danielsson H. Effect of biliary obstruction on formation and metabolism of bile acids in rat.Steroids. 1973; 22: 567-577Crossref PubMed Scopus (47) Google Scholar) noted a bile acid with chromatographic behavior slightly different from that of β-muricholic acid (β-MCA; 3α,6β,7β-trihydroxy-5β-cholan-24-oic acid) and with two fewer mass units by mass spectroscopy. He proposed that this bile acid was β-MCA with a double bond in the side chain. Similar observations were made by Kern et al. (2Kern F. Eriksson H. Curstedt T. Sjövall J. Effect of ethynylestradiol on biliary excretion of bile acids, phosphatidylcholines, and cholesterol in bile fistula rat.J. Lipid Res. 1977; 18: 623-634Abstract Full Text PDF PubMed Google Scholar), who characterized the biliary bile acids of rats in whom cholestasis was induced by ethynylestradiol. In 1980, Kuriyama et al. (3Kuriyama K. Ban Y. Nakashima T. Murata T. Simultaneous determination of biliary bile acids in rat: electron impact and ammonia chemical ionization mass spectrometric analysis of bile acids.Steroids. 1980; 34: 717-728Google Scholar) used gas chromatography-mass spectrometry to show that an unsaturated derivative of β-MCA was present as a minor constituent in the biliary bile acids of the healthy Wistar rat. In 1993, Davis and Thompson (4Davis D.G. Thompson M.B. Nuclear magnetic resonance identification of the taurine conjugate of 3α,6β,7β-trihydroxy-5β,22-cholen-24-oic acid (tauro-Δ22-β-muricholate) in the serum of female rats treated with α-naphthylisothiocyanate.J. Lipid Res. 1993; 34: 651-661Abstract Full Text PDF PubMed Google Scholar) used 1H- and 13C-NMR to establish the chemical structure of the unsaturated bile acids as Δ22-β-muricholic acid [Δ22-β-MCA; (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid], and Thompson, Davis, and Morris (5Thompson M.B. Davis D.G. Morris R.W. Taurine conjugate of 3α,6β,7β-trihydroxy-5β,22-cholen-24-oic acid (tauro-Δ22-β-muricholate): the major bile acid in the serum of female rats treated with α-naphthylisothiocyanate and its secretion by liver slices.J. Lipid Res. 1993; 34: 553-561Abstract Full Text PDF PubMed Google Scholar) reported that its plasma concentration in female Fischer rats exceeded that of β-MCA. This observation was confirmed for Sprague-Dawley rats in 1996 by Rodrigues et al. (6Rodrigues C.M.P. Kren B.T. Steer C.J. Setchell K.D.R. Formation of Δ22-bile acids in rats is not gender specific and occurs in the peroxisome.J. Lipid Res. 1996; 37: 540-550Abstract Full Text PDF PubMed Google Scholar). Subsequently, this group reported that the concentration of Δ22-β-MCA exceeded that of β-MCA in liver homogenates (7Setchell K.D.R. Rodrigues C.M.P. Clerici C. Solinas A. Morelli A. Gartung C. Boyer J. Bile acid concentrations in human and rat liver tissue and in hepatocyte nuclei.Gastroenterology. 1997; 112: 226-235Abstract Full Text PDF PubMed Scopus (156) Google Scholar), although no correction was made for entrapped blood, in which the bile acid concentration was likely to be much higher than in hepatocytes. In the early studies in which Δ22-β-MCA was identified in plasma or bile, it was inferred that the Δ22-β-MCA had been formed by the enteric flora. This assumption was based on the finding that the compound disappeared from bile after prolonged biliary drainage (1Danielsson H. Effect of biliary obstruction on formation and metabolism of bile acids in rat.Steroids. 1973; 22: 567-577Crossref PubMed Scopus (47) Google Scholar, 8Eriksson H. Taylor W. Sjövall J. Occurrence of sulfated 5α-cholanoates in rat bile.J. Lipid Res. 1978; 19: 177-186Abstract Full Text PDF PubMed Google Scholar) as well as on reports from the Eyssen group that this acid was formed in vitro by bacteria (9Eyssen H. De Pauw G. Stragier J. Verhulst A. Cooperative formation of ω-muricholic acid by intestinal microorganisms.Appl. Environ. Microbiol. 1983; 45: 141-147Crossref PubMed Google Scholar) and in vivo in gnotobiotic rats (10Robben J. Caenepeel P. Eldere J.V. Eyssen E. Effects of intestinal microbial bile salt sulfatase activity on bile salt kinetics in gnotobiotic rats.Gastroenterology. 1988; 94: 494-502Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Kayahara et al. (11Kayahara T. Tamura T. Amuro Y. Higashino K. Igimi H. Uchida K. Δ22-β-Muricholic acid in monoassociated rats and conventional rats.Lipids. 1994; 29: 289-296Crossref PubMed Scopus (12) Google Scholar) confirmed and extended this finding by using gnotobiotic rats to show that several bacterial species form Δ22-β-MCA. This group, working independently of the Eyssen group, also established its chemical structure by 1H- and 13C-NMR spectroscopy. The first evidence that Δ22-β-MCA could also be formed in the liver was presented by Thompson, Davis, and Morris (5Thompson M.B. Davis D.G. Morris R.W. Taurine conjugate of 3α,6β,7β-trihydroxy-5β,22-cholen-24-oic acid (tauro-Δ22-β-muricholate): the major bile acid in the serum of female rats treated with α-naphthylisothiocyanate and its secretion by liver slices.J. Lipid Res. 1993; 34: 553-561Abstract Full Text PDF PubMed Google Scholar), who in 1993 showed that Δ22-β-MCA could be formed by rat liver slices. Two years later, Setchell et al. (12Setchell K.D.R. Yamashita H. Rodrigues C.M.P. O'Connell N.C. Kren B.T. Steer C.J. Δ22-Ursodeoxycholic acid, a unique metabolite of administered ursodeoxycholic acid in rats, indicating partial β-oxidation as a major pathway for bile acid metabolism.Biochemistry. 1995; 34: 4169-4178Crossref PubMed Scopus (24) Google Scholar) provided additional indirect evidence for the formation of Δ22-β-MCA in hepatocytes by showing that ursodeoxycholic acid (UDCA; 3α,7β-dihydroxy-5β-cholan-24-oic acid) was converted to Δ22-UDCA when incubated with rat peroxisomes. Thus, the consensus of the work to date is that Δ22-β-MCA can be formed not only by the enteric flora of the distal intestine but also by hepatocyte peroxisomal enzymes, at least in the rat. In the rat, the compound is present in plasma, bile, and intestinal content in both conjugated and unconjugated forms (5Thompson M.B. Davis D.G. Morris R.W. Taurine conjugate of 3α,6β,7β-trihydroxy-5β,22-cholen-24-oic acid (tauro-Δ22-β-muricholate): the major bile acid in the serum of female rats treated with α-naphthylisothiocyanate and its secretion by liver slices.J. Lipid Res. 1993; 34: 553-561Abstract Full Text PDF PubMed Google Scholar), suggesting that it is absorbed from the distal intestine after formation by bacteria and then conjugated with taurine or glycine during transport through the hepatocyte; the conjugates then undergo enterohepatic circulation. However, Δ22-β-MCA is sometimes absent in fecal bile acids of the rat despite being present in intestinal content (10Robben J. Caenepeel P. Eldere J.V. Eyssen E. Effects of intestinal microbial bile salt sulfatase activity on bile salt kinetics in gnotobiotic rats.Gastroenterology. 1988; 94: 494-502Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 11Kayahara T. Tamura T. Amuro Y. Higashino K. Igimi H. Uchida K. Δ22-β-Muricholic acid in monoassociated rats and conventional rats.Lipids. 1994; 29: 289-296Crossref PubMed Scopus (12) Google Scholar), indicating that intestinal bacteria can both desaturate and saturate the side chain of β-MCA. No studies have been performed on the metabolism of Δ22-β-MCA because of the absence of synthetic material. Kihira and Hoshita (13Kihira K. Hoshita T. Synthesis of α,β-unsaturated C24 bile acids.Steroids. 1985; 46: 767-774Crossref PubMed Scopus (12) Google Scholar) reported the synthesis of the Δ22 derivatives of the common natural bile acids (cholic, deoxycholic, and chenodeoxycholic acids and UDCA) but did not extend their work to β-MCA, presumably because of the difficulty of the synthesis. Our laboratory has previously developed an improved synthesis of β-MCA and its congeners (α- and ω-muricholic acid) (14Iida T. Momose T. Tamura T. Matsumoto T. Chang F.C. Goto J. Nambara T. Potential bile acid metabolites. 14. Hyocholic and muricholic acid stereoisomers.J. Lipid Res. 1989; 30: 1267-1279Abstract Full Text PDF PubMed Google Scholar). In this paper, we report the chemical synthesis of Δ22-β-MCA [1a] and its taurine [1b] and glycine [1c] conjugates (Δ22-β-muricholyltaurine and Δ22-β-muricholylglycine). Chemical structures are shown in Fig. 1. Melting points (mp) were determined on a micro hot-stage apparatus and are uncorrected. Infrared (IR) spectra were obtained on a Bio-Rad FTS-7 FT-IR spectrometer (Philadelphia, PA) as KBr discs. 1H- and 13C-NMR spectra were obtained on a JEOL JNM-EX 270 FT NMR instrument (Tokyo, Japan) at 270 and 68.80 MHz, respectively, with CDCl3 containing 10% CD3OD or CD3OD as the solvent; chemical shifts were expressed as δ ppm relative to tetramethylsilane. Low-resolution mass spectra (LR-MS) were recorded on a JEOL JMS-303 mass spectrometer with electron ionization (EI) at 70 eV under the positive ion mode (PIM). LR-MS was also obtained on a JEOL JMS-LCmate equipped with electrospray ionization (ESI) under the negative ion mode (NIM). High-resolution mass spectra (HR-MS) were recorded on a JEOL JMS-LCmate double-focusing magnetic mass spectrometer equipped with an ESI probe under the PIM or the NIM. HR-MS were also obtained on a JEOL JMS-700 mass spectrometer with an EI probe under the PIM. Sep-Pak Vac tC18 cartridges (adsorbent weight, 5 g) were purchased from Waters Associates (Milford, MA). Thin-layer chromatography was performed on precoated silica gel (0.25 mm layer thickness; E. Merck) using hexane-ethyl acetate (EtOAc)-acetic acid mixtures (80:20:1∼20:80:1, v/v/v) or EtOAc-methanol-acetic acid mixtures (7:2:1, v/v/v) as the developing solvents. A solution of β-MCA [3] (1.0 g, 2.4 mmol), prepared from chenodeoxycholic acid [2] (see Results and Discussion), in 99% formic acid (7 ml) containing 60% perchloric acid (100 mg) was stirred at 50°C for 1.5 h. Acetic anhydride (3 ml) was added slowly with ice-bath cooling, and the mixture was poured into water. The reaction product was extracted with CH2Cl2, and the combined extract was washed with water, dried with anhydrous calcium sulfate (Drierite), and evaporated to dryness. The residue was recrystallized from benzene-hexane as colorless crystals: yield, 1.21 g (100%); mp, 90–92°C. IR (KBr), νmaxcm−1: 1,713 (C=O). 1H-NMR (CDCl3), δ: 0.72 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.2 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 4.85 (1H, brm, 3β-H), 5.06 (1H, dd, J = 11.1 and 3.8 Hz, 7α-H), 5.14 (1H, m, 6α-H), 7.93, 8.02, and 8.10 (each 1H, s, 3α-, 6β-, and 7β-OCHO). LR-MS (EI-PIM), m/z: 492 (M+, 4%), 446 (M-HCOOH, 5%), 418 (9%), 400 (M-2HCOOH, 80%), 372 (39%), 354 (M-3HCOOH, 90%), 339 (27%), 299 (100%), 272 (22%), 253 [M-3HCOOH-side chain (S.C.), 66%], 213 (22%), 211 (M-3HCOOH-S.C.-ring D, 18%), 159 (31%). HR-MS (EI-PIM), calculated for C27H40O8 [M]+: 492.2723; found, m/z: 492.2692. To a solution of the triformate [4] (1.0 g, 2.0 mmol) in dry benzene (18 ml), lead tetraacetate (1.44 g, 3.24 mmol), cuprous acetate (0.1 g, 0.55 mmol), and dry pyridine (0.1 ml) were added successively, and the mixture was refluxed for 12 h. Most of benzene was evaporated under reduced pressure, and the residual solution was poured onto a column of silica gel (30 g). Elution with benzene-EtOAc (9:1, v/v) afforded the title compound [5], which recrystallized from ethanol-water as colorless crystals: yield, 450 mg (49%); mp, 71–73°C. IR (KBr), νmaxcm−1: 1,728 (C=O). 1H-NMR (CDCl3), δ: 0.74 (3H, s, 18-CH3), 1.04 (3H, d, J = 6.5 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 4.77–4.87 (3H, brm, 3β- and 23-H), 5.06 (1H, dd, J = 11.1 and 4.0 Hz, 7α-H), 5.13 (1H, m, 6α-H), 5.65 (1H, m, 22-H), 7.92, 8.02, and 8.10 (each 1H, s, 3α-, 6β-, and 7β-OCHO). LR-MS (EI-PIM), m/z: 446 (M+, 2%), 431 (M-CH3, 9%), 418 (18%), 400 (M-HCOOH, 4%), 389 (16%), 372 (16%), 354 (M-2HCOOH, 37%), 343 (33%), 326 (31%), 308 (M-3HCOOH, 14%), 299 (69%), 271 (21%), 253 (M-3HCOOH-S.C., 100%), 211 (M-3HCOOH-S.C-ring D, 11%), 197 (8%), 159 (21%). HR-MS (EI-PIM), calculated for C26H38O6 [M]+: 446.2669; found, m/z: 446.2665. To the 24-nor-22-ene [5] (1.38 g, 3.0 mmol) dissolved in tert-butyl alcohol-tetrahydrofuran-water (34.5 ml; 7:2:1, v/v/v) was added N-methylmorpholine N-oxide (2 ml) and osmium tetroxide (30 mg, 0.12 mmol), and the mixture was allowed to stand at room temperature for 12 h. The reaction product was extracted with CHCl3, and the combined extract was washed with water, dried with Drierite, and evaporated to a dark brown oily residue. Chromatography of the oil on a column of silica gel (50 g) and elution with benzene-EtOAc (6:4–1:9, v/v) afforded the desired 24-nor-22ξ,23-diol [6] as viscous oil, which resisted crystallization attempts: yield, 950 mg (66%). IR (KBr) νmaxcm−1: 1,713 (C=O), 3,427 (OH). 1H-NMR (CDCl3), δ: 0.72 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.2 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 3.65–3.75 (3H, brm, 22ξ- and 23-H), 4.81 (1H, brm, 3β-H), 5.09–5.13 (2H, m, 6α- and 7α-H), 7.93, 8.02, and 8.10 (each 1H, s, 3α-, 6β-, and 7β-OCHO). LR-MS (EI-PIM), m/z: 462 (M-H2O, 2%), 444 (M-2H2O, 5%), 434 (M-HCOOH, 8%), 416 (M-H2O-HCOOH, 15%), 403 (48%), 388 (M-2HCOOH, 22%), 357 (69%), 342 (M-3HCOOH, 10%), 329 (39%), 311 (100%), 299 (32%), 281 (34%), 253 (M-3HCOOH-S.C., 29%), 211 (M-3HCOOH-S.C.-ring D, 18%), 159 (26%), 107 (25%). HR-MS (ESI-PIM), calculated for C26H40O8Na [M+Na]+: 503.2621; found, m/z: 503.2632. To a magnetically stirred solution of sodium periodate (NaIO4) (1.5 g, 7.0 mmol) dissolved in methanol (14 ml) and water (7 ml), a solution of the 24-nor-22ξ,23-diol [6] (900 mg, 1.9 mmol) in methanol (15 ml) was added, and the mixture was stirred at room temperature for 12 h. The reaction product was extracted with CHCl3, and the combined extract was washed with water, dried with Drierite, and evaporated to dryness. Although the residual oil [7] was found to be homogeneous according to TLC, it resisted crystallization attempts: yield, 800 mg (95%). IR (KBr), νmaxcm−1: 1,726 (C=O). 1H-NMR (CDCl3), δ: 0.77 (3H, s, 18-CH3), 0.96 (3H, d, J = 6.8 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 4.81 (1H, brm, 3β-H), 5.07 (1H, dd, J = 11.3 and 3.5 Hz, 7α-H), 5.14 (1H, m, 6α-H), 7.92, 8.02, and 8.10 (each 1H, s, 3α-, 6β-, and 7β-OCHO), 9.57 (1H, d, J = 3.0 Hz, 22-CHO). LR-MS (EI-PIM), m/z: 448 (M+, 3%), 416 (6%), 402 (M-HCOOH, 7%), 374 (M-HCOOH-CHO, 17%), 356 (M-2HCOOH, 93%), 328 (M-2HCOOH-CHO, 70%), 310 (M-3HCOOH, 42%), 282 (M-3HCOOH-CHO, 20%), 272 (37%), 253 (M-3HCOOH-S.C., 45%), 211 (M-3HCOOH-S.C.-ring D, 4%), 159 (41%), 111 (100%). HR-MS (EI-PIM), calculated for C25H36O7 [M]+: 448.2461; found, m/z: 448.2459. To a stirred solution of the 23,24-dinor-22-aldehyde [7] (370 mg, 0.6 mmol) in dry benzene (28 ml), methyl (triphenylphosphoranylidene)acetate (500 mg, 1.5 mmol) was added, and the mixture was refluxed for 12 h under a stream of N2. After cooling at room temperature, most of the solvent was evaporated under reduced pressure, and the reaction product was poured onto a column of silica gel (20 g). Elution with benzene-EtOAc (95:5, v/v) gave the title compound [8] as viscous oil, which resisted crystallization attempts: yield, 350 mg (84%). IR (neat), νmaxcm−1: 1,186 (C-O), 1,651 (C=C), 1,715, 1,732 (C=O). 1H-NMR (CDCl3), δ: 0.75 (3H, s, 18-CH3), 1.09 (3H, d, J = 7.0 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 3.72 (3H, s, COOCH3), 4.80 (1H, brm, 3β-H), 5.06 (1H, dd, J = 10.8 and 3.8 Hz, 7α-H), 5.14 (1H, m, 6α-H), 5.74 (1H, d, J = 15.7 Hz, 23-H), 6.82 (1H, dd, J = 15.7 and 8.9 Hz, 22-H), 7.91, 8.02, and 8.10 (each 1H, s, 3α-, 6β-, and 7β-OCHO). LR-MS (EI-PIM), m/z: 504 (M+, 2%), 473 (M-CH3O, 2%), 458 (M-HCOOH, 1%), 412 (M-2HCOOH, 11%), 366 (M-3HCOOH, 7%), 343 (34%), 299 (M-2HCOOH-S.C., 30%), 271 (11%), 253 (M-3HCOOH-S.C., 50%), 211 (M-3HCOOH-S.C.-ring D, 6%), 159 (12%), 114 (100%). HR-MS (EI-PIM), calculated for C28H40O8 [M]+: 504.2723; found, m/z: 504.2722. A solution of the triformyloxy-22-ene ester [8] (500 mg, 0.99 mmol) in 5% methanolic KOH (5 ml) was refluxed for 6 h. After evaporation of most of the solvent, the residue was dissolved in water and then acidified with 10% H2SO4 with ice-bath cooling. The precipitated solid was filtered off, washed with water, and recrystallized from aqueous methanol to give the desired Δ22-β-MCA as colorless prisms: yield, 370 mg (92%); mp, 205–209°C. IR (KBr), νmaxcm−1: 1,707 (C=O), 3,342 (OH). 1H-NMR (CD3OD), δ: 0.73 (3H, s, 18-CH3), 1.09 (3H, s, 19-CH3), 1.11 (3H, d, J = 6.5 Hz, 21-CH3), 3.49–3.61 (3H, brm, 3β-, 6α, and 7α-H), 5.72 (1H, d, J = 15.4 Hz, 23-H), 6.86 (2H, dd, J = 15.4 and 6.8 Hz, 22-H). 13C-NMR (CDCl3+10% CD3OD), δ: 12.1 (C-18), 19.1 (C-21), 20.6 (C-11), 25.2 (C-19), 26.9 (C-15), 28.2 (C-16), 29.4 (C-2), 33.6 (C-10), 35.0 (C-1), 35.3 (C-4), 38.2 (C-8), 39.4 (C-2), 39.5 (C-9), 39.7 (C-12), 43.7 (C-13), 47.2 (C-5), 54.1 (C-17), 55.3 (C-14), 70.4 (C-3), 73.0 (C-7), 75.4 (C-6), 118.7 (C-23), 155.6 (C-22), 169.2 (C-24). LR-MS (ESI-NIM), m/z: 405 ([M-H]−, 100%), 111 (42.3%). HR-MS (ESI-NIM), calculated for C24H37O5 [M-H]−: 405.2641; found, m/z: 405.2682. A solution of the free acid [1a] (100 mg, 0.25 mmol) and p-toluenesulfonic acid (30 mg) in methanol (5 ml) was left overnight at room temperature. After evaporation of most of the solvent, the reaction product was extracted with EtOAc. The combined extract was washed with saturated brine, dried with Drierite, and evaporated to dryness. The resulting C-24 methyl ester, although homogeneous according to TLC, resisted crystallization attempts: yield, 93 mg (86%). IR (neat), νmaxcm−1: 1,651 (C=C), 1,715 (C=O), 3,364 (OH). 1H-NMR (CDCl3), δ: 0.73 (3H, s, 18-CH3), 1.10 (3H, s, 19-CH3), 1.10 (3H, d, J = 6.5 Hz, 21-CH3), 3.44 (1H, dd, J = 10.3 and 3.8 Hz, 7α-H), 3.51 (1H, brm, 3β-H), 3.57 (1H, m, 6α-H), 3.72 (3H, s, COOCH3), 5.75 (1H, d, J = 16.2 Hz, 23-H), 6.90 (1H, dd, J = 16.2 and 8.1 Hz, 22-H). 13C-NMR (CDCl3), δ: 12.4 (C-18), 19.4 (C-21), 20.7 (C-11), 25.4 (C-19), 27.1 (C-15), 28.4 (C-16), 29.9 (C-2), 33.8 (C-10), 35.4 (C-1), 35.4 (C-4), 38.5 (C-8), 39.6 (C-2), 39.6 (C-9), 39.8 (C-12), 44.0 (C-13), 47.2 (C-5), 51.4 (COOCH3), 54.1 (C-17), 55.3 (C-14), 71.0 (C-3), 73.4 (C-7), 75.5 (C-6), 118.6 (C-23), 154.9 (C-22), 167.5 (C-24). LR-MS (EI-PIM), m/z: 420 (M+, 2%), 402 (M-H2O, 100%), 384 (M-2H2O, 52%), 369 (M-2H2O-CH3, 14%), 366 (M-3H2O, 4%), 347 (10%), 305 (22%), 289 (M-H2O-S.C., 22%), 271 (M-2H2O-S.C., 80%), 253 (M-3H2O-S.C., 53%), 229 (11%), 211 (M-3H2O-S.C.-ring D, 9%), 175 (12%), 147 (20%), 114 (52%). HR-MS (EI-PIM), calculated for C25H40O5 [M]+: 420.2876; found, m/z: 420.2893. To a magnetically stirred solution of the nonamidated Δ22-β-MCA [1a] (100 mg, 0.25 mmol) in dry N,N-dimethylformamide (DMF) (9 ml) were successively added powdered taurine (80 mg, 0.64 mmol), diethylphosphorocyanide (DEPC) (75 μl), and anhydrous Et3N (150 μl), and the resulting suspension was stirred at room temperature for 60 min. The reaction mixture was adjusted to pH 12–14 with 1 M NaOH and then to pH 8–9 with 10% HCl. The resulting solution was diluted with water (90 ml), passed through a preconditioned Sep-Pak Vac tC18 cartridge, and eluted successively with water (20 ml), 20% methanol (20 ml), and 50% methanol (25 ml). The last fraction, which contains the desired component, was evaporated under reduced pressure, and the residue was recrystallized from methanol-ether to give the taurine-conjugated Δ22-β-MCA sodium salt [1b] as colorless crystals: yield, 82 mg (62%); mp, 283–285°C. IR (KBr), νmaxcm−1: 1,624 (C=C), 1,664 (C=O), 3,385 (OH). 1H-NMR (CD3OD), δ: 0.75 (3H, s, 18-CH3), 1.10 (3H, s, 19-CH3), 1.11 (3H, d, J = 7.0 Hz, 21-CH3), 3.03 (2H, t, J = 7.0 Hz, CH2S), 3.48 (1H, dd, J = 10.0 and 3.8 Hz, 7α-H), 3.55 (1H, brm, 3β-H), 3.62 (1H, m, 6α-H), 5.83 (1H, d, J = 15.1 Hz, 23-H), 6.70 (2H, dd, J = 15.1 and 8.9 Hz, 22-H). 13C-NMR (CD3OD) δ: 12.9 (C-18), 20.2 (C-21), 21.9 (C-11), 26.1 (C-19), 28.1 (C-15), 29.5 (C-16), 30.6 (C-2), 34.9 (C-10), 36.4 (CH2N), 36.5 (C-1), 39.4 (C-8), 40.7 (C-20), 41.1 (C-9 and C-12), 44.9 (C-13), 49.1 (C-5), 51.5 (CH2S), 55.8 (C-17), 56.9 (C-14), 71.7 (C-3), 74.2 (C-7), 77.1 (C-6), 122.2 (C-23), 151.8 (C-22), 168.9 (C-24). LR-MS (ESI-NIM), m/z: 512 ([M-H]−). HR-MS (ESI-NIM), calculated for C26H41NO7S [M-H]−: 512.2682; found, m/z: 512.2665. To a magnetically stirred solution of Δ22-β-MCA [1a] (120 mg, 0.29 mmol) in dry DMF (4 ml), glycine methyl ester hydrochloride (100 mg, 0.65 mmol), DEPC (120 μl), and Et3N (0.5 ml) were added successively, and the resulting suspension was stirred at room temperature for 1 h. The reaction product was extracted with EtOAc, and the combined extract was washed with water, dried with Drierite, and evaporated to dryness. The residue was then refluxed in 5% methanolic KOH (10 ml) for 30 min. Most of the solvent was evaporated under reduced pressure, and the hydrolysis product dissolved in water (5 ml) was acidified by 5% H2SO4 with ice-bath cooling. After stirring for 10 min at room temperature, the precipitated solid was filtered, washed with water, and dried to give the glycine-conjugated Δ22-β-MCA [1c], which was recrystallized from 1,4-dioxane-ether as colorless crystals: yield 120 mg (89%); mp, 234–235°C. IR (KBr), νmaxcm−1: 1,624 (C=C), 1,666, 1,732 (C=O), 3,375 (OH). 1H-NMR (as the methyl ester in CDCl3), δ: 0.73 (3H, s, 18-CH3), 1.10 (3H, d, J = 7.0 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 3.76 (2H, d, J = 4.1 Hz, CH2N), 3.78 (1H, s, COOCH3), 5.77 (1H, d, J = 15.4 Hz, 23-H), 6.06 (1H, t, J = 8.9 Hz, -NH-), 6.76 (2H, dd, J = 15.4 and 6.22 Hz, 22-H). 13C-NMR (as the methyl ester in CDCl3), δ: 12.3 (C-18), 19.5 (C-21), 20.7 (C-11), 25.4 (C-19), 27.1 (C-15), 28.5 (C-16), 29.3 (C-2), 33.8 (C-10), 35.4 (C-1), 35.6 (C-4), 39.4 (C-2), 39.6 (C-9), 39.8 (C-12), 41.3 (-CH2COOCH3), 43.9 (C-13), 47.2 (C-5), 52.4 (-CH2COOCH3), 54.2 (C-17), 55.3 (C-14), 71.0 (C-3), 73.5 (C-7), 75.5 (C-6), 120.5 (C-23), 151.2(C-22), 166.2 (C-24), 170.6 (-CH2COOCH3). LR-MS (ESI-NIM), m/z: 462 ([M-H]−). HR-MS (ESI-NIM), calculated for C26H40O6N [M-H]−: 462.2855; found: m/z, 462.2827. The synthetic route to Δ22-β-MCA [1a] is shown in Fig. 2. The key starting compound, β-MCA [3], was prepared according to the established procedures (14Iida T. Momose T. Tamura T. Matsumoto T. Chang F.C. Goto J. Nambara T. Potential bile acid metabolites. 14. Hyocholic and muricholic acid stereoisomers.J. Lipid Res. 1989; 30: 1267-1279Abstract Full Text PDF PubMed Google Scholar) reported previously by us from chenodeoxycholic acid [2] as follows: chenodeoxycholic acid [2] was converted to its 3α-cathyloxy-7-oxo ester; bromination of the resulting ketone with bromine in the presence of HBr afforded the 6α-bromo-3α-cathyloxy-7-oxo ester; reduction of the bromo-ketone with Zn(BH4)2 gave 6α-bromo-3α-cathyloxy-7α-hydroxy ester; treatment of the bromohydrin in acetic acid with zinc powder yielded 3α-cathyloxy-Δ6 ester; β-face cis-dihydroxylation of the Δ6 ester with osmium tetroxide-N-methylmorpholine N-oxide afforded the 3α-cathyloxy-6β,7β-dihydroxy ester, which in turn was hydrolyzed to give the corresponding free acid [3] (total yield of 30% from chenodeoxycholic acid [2]). Two methods have been reported for the introduction of a (22E)-double bond in the bile acid side chain (C4H8COOH) of β-MCA [3]. The first method involves an ene reaction with (Z)-ethylidene-(Δ17(20))-steroids and methyl propiolate (HC≡CCOOCH3) under the presence of ethyl aluminum dichloride (EtAlCl2) or diethyl aluminum chloride as Lewis acid-catalyzed conditions (15Batcho A.D. Berger D.E. Uskoković M.R. C-20 stereospecific introduction of a steroid side chain.J. Am. Chem. Soc. 1981; 103: 1293-1295Crossref Scopus (50) Google Scholar, 16Dauben W.G. Brookhart T. Stereocontrolled synthesis of steroid side chains.J. Am. Chem. Soc. 1981; 103: 237-238Crossref Scopus (54) Google Scholar). The second method consists of condensation of (20S)-20-methyaldehyde pregnane derivatives with a Wittig reagent, (carbomethoxymethylene)triphenylphosphorane [(C6H5)3P=CHCOOCH3] (17Vanderah D.J. Djerassi C. Marine natural products. Synthesis of four naturally occurring 20β-H cholanoic acid derivatives.J. Org. Chem. 1978; 43: 1442-1448Crossref Scopus (77) Google Scholar). Of the two methods, the latter appeared to be much more practical than the former, because of easy preparation of the key intermediate, (20S)-23,24-dinoraldehyde [7]. Based on the above assumption, our next effort was directed to the preparation of the 24-nor-triformyloxy-22-ene intermediate [5] from β-MCA [3]. Oxidative decarboxylation of the β-MCA triformate derivative [4] with lead tetraacetate and cuprous acetate in refluxing pyridine (18Carlson C.L. Belobaba D.T.E. Hofmann A.F. Wedmid Y. 24-Nor-5β-chol-22-enes derived from the major bile acids by oxidative decarboxylation.Steroids. 1977; 30: 787-793Crossref PubMed Scopus (17) Google Scholar) yielded 24-nor-triformyloxy-22-ene [5] in an isolated yield of 46% after chromatographic purification on a column of silica gel. The triformate [4] was obtained in nearly quantitative yield by treatment of [3] with 99% formic acid in the presence of 60% perchloric acid (19Tserng K-Y. Klein P.D. Formylated bile acids: improved synthesis, properties, and partial deformylation.Steroids. 1977; 29: 635-648Crossref PubMed Scopus (56) Google Scholar). Vicinal dihydroxylation of the unsaturated 22-ene [5] in tert-butyl alcohol-tetrahydrofuran-water mixtures with osmium tetroxide and N-methylmorphorine N-oxide produced exclusively an epimeric mixture (22R and 22S) of vicinal 22ξ,23-diols [6], without being accompanied by a simultaneous hydrolysis of the formyloxy protecting groups at the C-3, C-6, and C-7 positions, as indicated by the 13C-NMR spectrum. The one-step dihydroxylation is much more simple and straightforward than the two-step procedure reported by Kihira and Hoshita (13Kihira K. Hoshita T. Synthesis of α,β-unsaturated C24 bile acids.Steroids. 1985; 46: 767-774Crossref PubMed Scopus (12) Google Scholar) and Kihira and others (20Kihira K. Kuramoto T. Hoshita T. New bile alcohols: synthesis of (22R)- and (22S)-5β-cholestane-3α,7α,12α,22,25-pentols.Steroids. 1976; 27: 383-393Crossref PubMed Scopus (16) Google Scholar, 21Kihira K. Morioka Y. Hoshita T. Synthesis of (22R and 22S)-3α,7α,22-trihydroxy-5β-cholan-24-oic acids and structure of haemulcholic acid, a unique bile acid isolated from fish bile.J. Lipid Res. 1981; 22: 1181-1187Abstract Full Text PDF PubMed Google Scholar), in which epoxidation of the 22-ene [5] with hydrogen peroxide and subsequent methanolic KOH cleavage of the resulting epoxide were used. When the triformyloxy-22ξ,23-diol [6] (both epimers) was subjected to oxidation with NaIO4 in methanol-water mixtures (13Kihira K. Hoshita T. Synthesis of α,β-unsaturated C24 bile acids.Steroids. 1985; 46: 767-774Crossref PubMed Scopus (12) Google Scholar), both the 22R- and 22S-epimers were quantitatively converted into the 23,24-dinor-triformyloxy-22-aldehyde [7] in an excellent yield of 92%. Subsequent Wittig reaction of the triformyloxy-22-aldehyde [7] in benzene with a reagent, methyl (triphenylphosphoranylidene)acetate, led to the formation of the (E)-isomer of the 3α,6β,7β-triformyloxy-22-ene methyl ester [8]. The resulting reaction product, which consisted essentially of a single component, was purified by passing through a column of silica gel and eluting with a mixture of benzene-EtOAc (95:5, v/v). Alkaline hydrolysis of 3α,6β,7β-triformyloxy-22-ene methyl ester [8] with 5% methanolic KOH followed by acidification with 10% H2SO4 resulted in the simultaneous hydrolysis of the methyl ester at C-24 and the formyloxy protecting group at the C-3, C-6, and C-7 positions to give the desired (unconjugated) Δ22-β-MCA [1a]. Es
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