Chemical Synthesis of Uncommon Natural Bile Acids: The 9α-Hydroxy Derivatives of Chenodeoxycholic and Lithocholic Acids

Abstract

The chemical synthesis of the 9α-hydroxy derivatives of chenodeoxycholic and lithocholic acids is reported. For initiating the synthesis of the 9α-hydroxy derivative of chenodeoxycholic acid, cholic acid was used; for the synthesis of the 9α-hydroxy derivative of lithocholic acid, deoxycholic acid was used. The principal reactions involved were (1) decarbonylation of conjugated 12-oxo-Δ⁹⁽¹¹⁾-derivatives using in situ generated monochloroalane (AlH₂Cl) prepared from LiAlH₄ and AlCl₃, (2) epoxidation of the deoxygenated Δ⁹⁽¹¹⁾-enes using m-chloroperbenzoic acid catalyzed by 4,4′-thiobis-(6-tert-butyl-3-methylphenol), (3) subsequent Markovnikov 9α-hydroxylation of the Δ⁹⁽¹¹⁾-enes with AlH₂Cl, and (4) selective oxidation of the primary hydroxyl group at C-24 in the resulting 3α,9α,24-triol and 3α,7α,9α,24-tetrol to the corresponding C-24 carboxylic acids using sodium chlorite (NaClO₂) in the presence of a catalytic amount of 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and sodium hypochlorite (NaOCl). The ¹H- and ¹³C-NMR spectra are reported. The 3α,7α,9α-trihydroxy-5β-cholan-24-oic acid has been reported to be present in the bile of the Asian bear, and its 7-deoxy derivative is likely to be a bacterial metabolite. These bile acids are now available as authentic reference standards, permitting their identification in vertebrate bile acids.

The great variety of natural C₂₄ and C₂₇ bile acids, as well as C₂₇ bile alcohols, (together called bile salts) occurring in vertebrates can be explained by the evolution of differing biochemical pathways that serve to convert cholesterol into these multifunctional amphipathic compounds. Bile salt composition shows significant variation between orders but not between families, genera, or species, suggesting a biochemical trait providing clues to evolutionary relationships that complement anatomical and genetic analyses.^1–3) Naturally occurring bile salts differ markedly in their chemical structures, particularly the number, position and stereochemistry of hydroxyl groups on the steroid nucleus and on the branched C₅ or C₈ side-chain. Since some of the uncommon bile acids are present in the biliary and/or urinary bile salts of specific vertebrates or patients with hepatobiliary diseases, the bile acid profile can be used as a biomarker for clinical purposes, as well as for suggesting phylogenetic relationships.⁴⁾

Nuclear hydroxylation of bile acids in the liver of vertebrates is the result of CYP-mediated hydroxylation. If one accepts the concept of a default steroid nucleus with hydroxyl groups at C-3 and C-7, then further modifications can be considered as additions to the default structure. The dominant sites of additional hydroxylation are at C-6 (α or β), C-12 (α), or C-16 (α). Other sites of additional nuclear hydroxylation in bile acids are at C-1 (1α-/1β-), C-2 (2β-), C-4 (4β-), C-5 (5β-), and C-15 (15α-).^1–3)

Recently, a new natural bile acid having a hydroxylation site at C-9 (9α-) in the 5β-steroid nucleus (cis A/B-ring juncture) was reported by Bi et al.,⁵⁾ to be present in the bile of the Asian black bear (Ursus thibetanus). The semitrivial name, selocholic acid, was proposed for this novel bile acid. Its structure was considered to be 3α,7α,9α-trihydroxy-5β-cholan-24-oyl taurine by MS and one and two dimensional (1D)- and (2D)-NMR spectroscopy. Nonetheless, validation of the proposed structure awaits the synthesis of an authentic reference standard.

Microbial or enzymatic biotransformation are important tools for structural modification of organic compounds, especially natural products with complex structures like steroids.^6–8) This synthetic method can be used to prepare chemical structures that are difficult to obtain by ordinary chemical methods. The observed biotransformations may suggest metabolic pathways in mammals, due to similarity between mammalian and microbial systems. However, the procedure is of limited use, because of the substrate specificity.

As part of our ongoing program of chemical synthesis of novel, uncommon natural or potentially natural bile acid metabolites for use as authentic reference standards, we now describe the chemical synthesis of 9α-hydroxy-chenodeoxycholic acid [9α-OH-CDCA; 3α,7α,11α-trihydroxy-5β-cholan-24-oic acid (1b)] starting from cholic acid (2b; CA, 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid), and 9α-hydroxy-lithocholic acid [9α-OH-LCA; 3α,9α-dihydroxy-5β-cholan-24-oic acid (1a)] starting from deoxycholic acid (2a; DCA; 3α,12α-dihydroxy-5β-cholan-24-oic acid) (Fig. 1).

Fig. 1. Chemical Structures of Synthetic 9α-Hydroxylated Bile Acids (1a, b)

Results and Discussion

Figure 2 shows the synthetic route of 9α-OH-CDCA (1b) from CA (2b) and 9α-OH-LCA (1a) from DCA (2a), respectively. A one step procedure for the introduction of an α-hydroxyl group at C-9 on the 5β-steroid nucleus is known.^9,10) Thus, the most feasible approach for preparing 1a and b on a substantial scale appeared to be the 9α-hydroxylation of 9α,11α-epoxy intermediates (8a, b) derived from Δ⁹⁽¹¹⁾-enes (7a, b), obtainable from 2a and b in several steps.

Fig. 2. Synthetic Route to 9α-Hydroxylated Bile Acids (1a, b) from DCA (2a) and CA (2b)

Reagents and conditions: (i) p-Toluenesulfonic acid/MeOH, at r.t. for 12 h. (ii) Ac₂O/pyridine/benzene, at r.t. for 12 h. (iii) PCC/CH₂Cl₂, at r.t. for 12 h. (iv) SeO₂/AcOH, at reflux for 18 h. (v) AlCl₃/LiAlH₄/THF, ar reflux for 4 h. (vi) m-Chloroperbenzoic acid/4,4′-thiobis(6-tert-butyl-3-methylphenol)/chloroform, at r.t. for 1 h (at reflux for 20 min). (vii) AlCl₃/LiAlH₄/THF, at reflux for 48 h (at reflux for 12 h). (viii) NaC1O₂/TEMPO/NaClO/CH₃CN/THF, at 35°C for 3 h (at 35°C for overnight).

Regioselective acetylation of methyl deoxycholate (3a) and methyl cholate (3b) under mild conditions gave the corresponding 3α-acetate (4a) and 3α,7α-diacetate (4b), respectively, which in turn were oxidized with pyridinium chlorochromate (PCC) to yield the 3α-acetoxy-12-oxo and 3α,7α-diacetoxy-12-oxo esters (5a, b) nearly quantitatively.¹¹⁾ When 5a and b were subjected to the selective dehydration with SeO₂ in acetic acid,^12–14) the corresponding conjugated enones, Δ⁹⁽¹¹⁾-3α-acetoxy-12-oxo and Δ⁹⁽¹¹⁾-3α,7α-diacetoxy-12-oxo esters (6a, b), were obtained in high yields (78–80%). The formation of the conjugated 9(11)-ene-12-oxo structure was confirmed by the ¹H-NMR signals arising from the 19-H₃ at δ 1.19 in 6a and at 1.20 in 6b and by the ¹³C-NMR signals from the C-11 (δ 123.8) and C-9 (δ 164.1) in 6a and the C-11 (δ 125.1) and C-9 (δ 159.8) in 6b.

Decarbonylation at C-12 of 6a and b in tetrahydrofuran (THF) for 4 h at reflux temperature with monochloroalane (AlH₂Cl), generated in situ from LiAlH₄ and AlCl₃,^13,15–17) gave the Δ⁹⁽¹¹⁾-3α,24-diol (7a) and Δ⁹⁽¹¹⁾-3α,7α,24-triol (7b) in moderate isolated yields of 52–64%, respectively, accompanied by simultaneous hydrolysis of the methyl ester at C-24 and the acetoxy groups at C-3 and C-7. The 11-H olefinic ¹H signal occurring at δ 5.31 in 7a and at 5.50 in 7b and the ¹³C signals appearing at δ 119.6 (C-11) and 140.1 (C-9) in 7a and at δ 121.9 (C-11) and 137.1 (C-9) in 7b suggest that the decarbonylation at C-12 occurred successfully.

Subsequent epoxidation of the Δ⁹⁽¹¹⁾-3α,24-diol (7a) and Δ⁹⁽¹¹⁾-3α,7α,24-triol (7b) with m-chloroperbenzoic acid (m-CPBA) in the presence of a catalytic amount of 4,4′-thiobis-(6-tert-butyl-3-methylphenol)¹⁸⁾ afforded the corresponding 9α,11α-epoxy derivatives (8a, b) stereoselectively in reasonable isolated yields (88 and 64%), presumably by the attack of m-CPBA on the less sterically hindered α-face of the substrates; the β-face is more sterically crowded by the axially-oriented 18- and 19-methyl groups. The relatively low-yield (64%) of 8b, compared to that (88%) of 8a, is probably ascribed to the steric hindrance of an axially-oriented 7α-hydroxyl group. The stereochemical configuration of the 9α,11α-epoxy ring in 8a was determined by measuring the nuclear Overhauser enhanced differential spectroscopy (NOEDS), in which the irradiation of the 19-H₃ signal (δ 1.15) in 8a resulted in the formation of the correlating peaks with the 18-H₃ (δ 0.68), 5β-H (δ 1.51), 12β-H (δ 1.68), 8β-H (δ 1.93) and 11β-H (δ 3.21), thus indicating the α-configuration. Similar correlations were also observed in the NOEDS of 8b: the 19-H₃ (δ 1.18) were correlated with the 18-H₃ (δ 0.68), 5β-H (δ 1.56), 12β-H (δ 1.69), 8β-H (δ 1.99) and 11β-H (δ 3.08).

The most promising method for preparing 9α-hydroxylated bile acids is seemed to be the application of the so-called “reductive hydroxylation” of the 9α,11α-epoxides (8a, b) with lithium-ethylamine^9,10) or LiAlH₄^19–21) known to a favor α-hydroxylated product to obey the Marnovnikov rule. Attempted reductive cleavage of 8a and b with lithium-ethylamine or with LiAlH₄ alone was unsuccessful; both the reactions did not proceed at all. However, when 8a and b were subjected to AlH₂Cl, generated in situ from LiAlH₄ and AlCl₃,²²⁾ to afford the expected 3α,9α,24-triol (9a) and 3α,7α,9α,24-tetrol (9b) with good selectivity in fairly low isolated yields of 18 and 36%, respectively, after preparative high-performance liquid chromatography with a refractive index detector (HPLC-RI) chromatographic purification of the reaction products; the remaining compound recovered was the unreacted one (69% for 8a and 51% for 8b). AlH₂Cl was therefore found to be effective for the regio- and stereoselective hydroxylation of the sterically crowded epoxides. An appreciable difference in the isolated yields of 9a (18%) and 9b (36%) suggests that the substrate 8b is more unstable and reactive than 8a.

The complete ¹H- and ¹³C-NMR signal assignments for compounds 9a and b, which were confirmed by comparison of bile acid analogs reported previously,^23–25) were compiled in Table 1. The ¹H-NMR spectra of the compounds exhibited the ¹H-signals arising from the 3β-H (br m) at δ 3.57 (9a) and 3.47 (9b) and the 24-H₂ (br m) at δ 3.50 (9a) and 3.49 (9b), both of which showed essentially identical ¹H signal patterns. The stereochemical configuration of the 9α-hydroxyl group in 9b were determined by measuring the nuclear Overhauser effect spectroscopy (NOESY), in which the distinct correlation peaks were detected between the 19-H₃ (δ 0.96)/18-H₃ (δ 0.68), 19-H₃/5β-H (δ 1.58), 19-H₃/8β-H (δ 1.62), and 19-H₃/12β-H (δ 1.74). Essentially identical NOESY was also observed for 9a: 19-H₃ (δ 0.96)/18-H₃ (δ 0.69), 19-H₃/5β-H (δ 1.42), 19-H₃/8β-H (δ 1.62) and 19-H₃/12β-H (δ 1.72). Meanwhile, the ¹³C-NMR signals were appeared at δ 71.6 (9a) and 71.8 (9b) for the methine C-3, at δ 77.0 (9a) and 79.5 (9b) for the quaternary C-9, and at δ 62.2 (9a, b) for the methylene C-24 in the distortionless enhancement by polarization transfer (DEPT) spectra. These observations provide strong evidence for the presence of an axially-oriented 9α-hydroxyl group in the cis 5β-steroid nucleus (see below).

Table 1. Complete ¹H- and ¹³C-NMR Spectral Data for Synthetic 9α-Hydroxylated Compounds (9a, b, 1a, b)^a)

No	9a				9b				1a				1b
	Type	Carbon	Proton		Type	Carbon	Proton		Type	Carbon	Proton		Type	Carbon	Proton
			α	β			α	β			α	β			α	β
1	CH2	34.31	2.09	1.02	CH2	35.07	2.03	1.09	CH2	34.34	2.09	1.03	CH2	35.04	2.05	1.09
2	CH2	33.14	1.99	1.63	CH2	32.92	2.01	1.58	CH2	33.14	1.99	1.63	CH2	32.92	2.02	1.62
3	CH	71.55	—	3.57	CH	71.75	—	3.47	CH	71.55	—	3.57	CH	71.76	—	3.48
4	CH2	38.54	2.26	1.41	CH2	41.85	2.23	1.77	CH2	38.55	2.26	1.41	CH2	41.88	2.23	1.72
5	CH	43.50	—	1.42	CH	42.05	—	1.58	CH	43.49	—	1.42	CH	42.05	—	1.58
6	CH2	28.13b)	1.37c)	1.90c)	CH2	34.21	2.06	1.73	CH2	28.16b)	1.36c)	1.89c)	CH2	34.20	2.06	1.73
7	CH2	21.27	1.56	1.19	CH	69.73	—	3.91	CH2	21.28	1.56	1.19	CH	69.72	—	3.91
8	CH	39.75	—	1.62	CH	40.73	—	1.62	CH	39.76	—	1.63	CH	40.70	—	1.64
9	C	77.03	—	—	C	79.49	—	—	C	77.00	—	—	C	79.47	—	—
10	C	38.01	—	—	C	38.56	—	—	C	38.01	—	—	C	38.54	—	—
11	CH2	28.06b)	1.56c)	1.63c)	CH2	27.80b)	1.60c)	1.71c)	CH2	27.95b)	1.56c)	1.62c)	CH2	27.77b)	1.60c)	1.70c)
12	CH2	34.97	1.56	1.72	CH2	35.07	1.59	1.74	CH2	34.94	1.56	1.73	CH2	35.04	1.59	1.77
13	C	42.41	—	—	C	42.39	—	—	C	42.46	—	—	C	42.42	—	—
14	CH	47.99	1.61	—	CH	44.72	2.00	—	CH	47.99	1.62	—	CH	44.72	2.00	—
15	CH2	23.84	1.56	1.06	CH2	23.09	1.68	1.10	CH2	23.85	1.56	1.08	CH2	23.03	1.69	1.09
16	CH2	28.49b)	1.94c)	1.33c)	CH2	28.03b)	1.91c)	1.30c)	CH2	28.50b)	1.93c)	1.32c)	CH2	27.91b)	1.95c)	1.35c)
17	CH	56.10	1.20	—	CH	56.10	1.23	—	CH	55.90	1.20	—	CH	55.95	1.23	—
18	CH3	10.28	0.69		CH3	10.20	0.68		CH3	10.33	0.70		CH3	10.18	0.70
19	CH3	27.15	0.96		CH3	27.75	0.96		CH3	27.16	0.96		CH3	27.17	0.96
20	CH	35.68	1.43		CH	35.74	1.43		CH	35.41	1.45		CH	35.53	1.43
21	CH3	17.81	0.95		CH3	17.83	0.97		CH3	17.43	0.95		CH3	17.42	0.94
22	CH2	31.84	1.04/1.45		CH2	31.85	1.07/1.48		CH2	30.99	1.28/1.79		CH2	31.24	1.35/1.78
23	CH2	28.92	1.33/1.38		CH2	28.92	1.37/1.43		CH2	30.69	2.16/2.33		CH2	31.24	2.17/2.34
24	CH2	62.24	3.50		C	62.23	3.49		C	176.87	—		C	175.23	—

a) Measured in CD₃OD at 500.2 MHz in ¹H-NMR and at 125.8 MHz in ¹³C-NMR. Chemical shifts were expressed as δ ppm relative to TMS. b, c) Assignments along a vertical column bearing the same superscript may be interchanged.

Recently successful use of sodium chlorite (NaClO₂) catalyzed by 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and sodium hypochlorite (NaClO; bleach) for oxidation of alcohols^26–30) suggested that a selective oxidation of a primary hydroxyl group to its carboxyl group without oxidizing a secondary hydroxyl group might be feasible. As expected, mild reaction of 9a and b with TEMPO in the presence of the co-reagents resulted in successful selective oxidation of the primary 24-hydroxyl group to afford the desired 3α,9α-dihydroxy and 3α,7α,9α-trihydroxy acids (1a; 58%, 1b; 71%), respectively. The presence of a hydroxyl group at the C-9 position and its stereochemical configuration as α in 1a and b was determined by a combined use of several 1- and 2D-NMR techniques (Table 1). The ¹³C signals appearing at δ 71.6, 77.0, and 176.9 in 1a were tentatively assigned to the C-3, C-9, and C-24 bearing hydroxyl groups, respectively: δ 71.8, 69.7, 79.5, and 175.2 in 1b were due to the C-3, C-7, C-9, and C-24, respectively. The presence of a tertiary hydroxyl group at C-9 was confirmed by the appearance of the ¹H/¹³C correlated peak arising from the 19-H₃/C-9 in the ¹H-detected heteronuclear multiple bond connectivity (HMBC) and DEPT spectra of 1a and b. In addition, the correlation peaks observed for the 18-H₃/C-12 (δ 34.9 in 1a and 35.0 in 1b) in the HMBC, the C-12/12-H₂ [δ 1.56 (α) and 1.73 (β) in 1a and δ 1.59 (α) and 1.77 (β) in 1b] in the ¹H-detected heteronuclear multiple quantum coherence (HMQC), and then the 12-H₂/C-9 in the HMBC provided further confirmatory evidence for the presence of the 9-hydroxyl group. In the NOESY of 1a, the distinct correlation peaks were observed between the 19-H₃ (δ 0.96)/18-H₃ (δ 0.70), 19-H₃/5β-H (δ 1.42), 19-H₃/8β-H (δ 1.63) and 19-H₃/12β-H (δ 1.73), strongly indicating that the configuration of the hydroxyl group at C-9 is α. As shown in Fig. 3, essentially identical NOESY correlations were also observed for 1b: 19-H₃ (δ 0.96)/18-H₃ (δ 0.70), 19-H₃/5β-H (δ 1.58), 19-H₃/8β-H (δ 1.64) and 19-H₃/12β-H (δ 1.77). To conclude, the 3α,9α,24-triol (9a) and 3α,7α,9α,24-tetrol (9b) were successfully converted to the desired 9α-OH-LCA (1a) and 9α-OH-CDCA (1b), respectively.

Fig. 3. NOESY Correlations Observed for 9α-OH-CDCA (1b)

As note 1b has been reported to be present in the biliary bile acids of the Asian black bear. 1a has not been reported to occur naturally, but 7-dehydroxylation is a dominant bacterial biotransformation in the anaerobic colon,³¹⁾ leading to the prediction that 1a should be present in the fecal bile acids of Ursus thibetanus. Hydroxylation of steroids at C-9 by microbial enzymes³²⁾ or by simulated microsomal oxidation³³⁾ has also been reported.

Thus, the availability of 9α-OH-LCA (1a) and 9α-OH-CDCA (1b) should facilitate the identification of these uncommon bile acids in body fluids of vertebrates and also highlights an unusual site of steroid hydroxylation.

Experimental

Materials

DCA (2a) and CA (2b) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals and solvents were of analytical reagent grade and available from commercial sources. All compounds were dried by azeotropic distillation before use in reactions.

Instruments

All melting points (mp) were determined on a micro hot stage apparatus and are uncorrected. Specific rotations were measured on a ATA GO AP-300 and ATA GO DP-63 auto-recording polarimeter. IR spectra, on a JASCO FT-IR-4100 spectrophotometer (samples were absorbed on a powdered KBr surface and measured with the diffusion reflection method).

¹H- and ¹³C-NMR spectra were obtained on a JEOL ECA 500 FT instrument operated at 500 and 125 MHz, respectively, with CDCl₃ or CD₃OD containing 0.1% Me₄Si as the solvent; chemical shifts were expressed in δ (ppm) relative to Me₄Si (0 ppm). The ¹³C DEPT spectra were measured to determine the exact ¹³C signal multiplicity and to differentiate among CH₃, CH₂, CH, and C based on their proton environments. In order to further confirm the ¹H and ¹³C signal assignments for some of compounds, NOEDS, and 2D, ¹H–¹H correlation spectroscopy (COSY), ¹H–¹H NOESY, HMQC (¹H/¹³C coupling) and HMBC (long-range ¹H–¹³C coupling) spectra were also performed.

High-resolution liquid chromatography-mass spectrometry by electrospray ionization (HR-LC/ESI-MS) source was carried out using a JEOL AccuTOF JMS-T100LC liquid chromatography-mass spectrometer (JEOL, Tokyo, Japan) coupled to an Agilent 1200 series binary pump (Agilent Technologies Inc., Santa Clara, CA, U.S.A.) operated in the negative ion mode or positive ion mode.

The preparative HPLC-RI apparatus consisted of a Hitachi L-7100 pump (Tokyo, Japan), a Shodex RI-102 detector, and a Capcell Pak AQ RP-C₁₈ column (250 mm×10 mm i.d.; particle size 5 µm; Shiseido).

Normal-phase TLC was performed on pre-coated Kieselgel 60F₂₅₄ plates (E. Merck, Darmstad, Germany) using a mixture of EtOAc–hexane (8 : 2, v/v), EtOAc–hexane–acetic acid (9 : 1 : 0.1, v/v/v), EtOAc–methanol (95 : 5, v/v) or EtOAc–methanol–acetic acid (95 : 5 : 0.1, v/v/v) as the developing solvent.

Methyl 3α-Acetoxy-12-oxo-5β-chol-9(11)-en-24-oate (6a)

A mixture of the 3α-acetoxy-12-oxo 5a (1.0 g; 2.2 mmol) and selenium dioxide (SeO₂) (500 mg, 4.6 mmol) dissolved in acetic acid (40 mL) was refluxed for 18 h. After cooling at room temperature, the insoluble matter was filtered off, and the mother liquor was extracted with EtOAc. The combined extract was washed with 5% NaHCO₃ solution and water, dried with Drierite, and evaporated to dryness. Chromatography of the crude residue over a column of silica gel (50 g) eluting with EtOAc–hexane (3 : 7, v/v) resulted in a single component, which was identified as the conjugated Δ⁹⁽¹¹⁾-12-ketone 6a which was recrystallized from methanol as colorless needles: mp 149–152°C; yield, 835 mg (78%). [α]_D²³+140.0 (c 0.10, MeOH). FT-IR 1740, 1726 (C=O), 1645 (C=C) cm⁻¹. ¹H-NMR (500 MHz, CDCl₃) δ: 0.90 (s, 3H, 18-H₃), 1.00 (d, 3H, J=6.2 Hz, 21-H₃), 1.19 (s, 3H, 19-H₃), 1.99 (s, 3H, 3-OCOCH₃), 3.66 (s, 3H, 24-COOCH₃), 4.71 (brm, 1H, 3β-H), 5.70 (s, 1H, 11-H). ¹³C-NMR (125.8 MHz, CDCl₃) δ: 10.8 (C-18), 19.6 (C-21), 21.4 (C-19), 24.3, 26.3, 26.6, 27.5, 27.8, 29.8, 30.7, 31.6, 34.1, 35.1, 35.4, 37.9, 40.1, 41.9, 47.4, 51.5 (C-25), 53.2, 53.6, 73.9 (C-3), 123.8 (C-11), 164.1 (C-9), 170.7 (3-OCOCH₃), 174.8 (C-24), 205.2 (C-12). HR-ESI-MS, Calcd for C₂₇H₄₀O₅Na [M+Na]⁺, 467.2773. Found, m/z 467.2746.

Methyl 3α,7α-Diacetoxy-12-oxo-5β-chol-9(11)-en-24-oate (6b)

The 3,7-diacetoxy-12-oxo 5b (1.0 g; 2.0 mmol), subjected to the dehydrogenation with SeO₂ in acetic acid and processed as described for the preparation of 6a, gave a crude product. Chromatography of the product on a column of silica gel (50 g) and elution with EtOAc–hexane (3 : 7, v/v) afforded the title compound 6b which was recrystallized from methanol as colorless needles: mp 159–160°C (159–161°C)¹¹⁾; yield, 797 mg (80%). [α]_D³¹+80.0 (c 0.10, MeOH). FT-IR 1735 (C=O), 1671 (C=C) cm⁻¹. ¹H-NMR (500 MHz, CDCl₃) δ: 0.92 (s, 3H, 18-H₃), 1.00 (d, 3H, J=6.3 Hz, 21-H₃), 1.20 (s, 3H, 19-H₃), 1.99 (s, 6H, 3- and 7-OCOCH₃), 3.65 (s, 3H, 24-COOCH₃), 4.59 (br m, 1H, 3β-H), 5.15 (m, 1H, 7β-H), 5.82 (s, 1H, 11-H). ¹³C-NMR (125.8 MHz, CDCl₃) δ: 10.8 (C-18), 19.4 (C-21), 21.4 (C-19), 21.5, 23.7, 27.5, 27.8, 30.1, 30.7, 30.9, 31.5, 35.0, 35.4, 36.2, 40.1, 40.8, 41.1, 46.8, 47.1, 51.6 (C-25), 53.3, 70.2 (C-7), 73.8 (C-3), 125.1 (C-11), 159.8 (C-9), 170.1 (7-OCOCH₃), 170.7 (3-OCOCH₃), 174.7 (C-24), 204.6 (C-12). HR-ESI-MS, Calcd for C₂₉H₄₃O₇ [M+H]⁺, 503.3009. Found, m/z 503.3086.

5β-Chol-9(11)-en-3α,24-diol (7a)

To an ice-cooled solution of AlCl₃ (4.2 g; 31.5 mmol) in dry THF (25 mL) was added LiAlH₄ (0.5 g; 13.2 mmol) gradually, and the resulting solution was stirred 10 min at room temperature and was subsequently refluxed during 30 min. A solution of the Δ⁹⁽¹¹⁾-3α-acetoxy-12-ketone 6a (1.0 g, 2.25 mmol) in THF (25 mL) was added dropwise to the AlH₂Cl solution, and after the addition was complete, reflux was maintained during 4 h: the reaction was monitored by TLC. The reaction mixture was cooled with an ice-bath, and water was carefully added. The reaction product was extracted with CH₂Cl₂, and the combined extract was washed with 5% H₂SO₄ and water, dried with Drierite, and evaporated to dryness. The crude residue, which consisted essentially of a single spot on TLC was chromatographed on a column of silica gel (50 g). Elution with EtOAc–hexane (1 : 1, v/v) gave the desired Δ⁹⁽¹¹⁾-3α,24-diol 7a which was recrystallized from EtOAc–hexane as colorless needles: mp 179–180°C; yield, 510 mg (64%). [α]_D²³+40.0 (c 0.10, MeOH). FT-IR 3324 (OH), 1647 (C=C) cm⁻¹. ¹H-NMR (500 MHz, CDCl₃) δ: 0.58 (s, 3H, 18-H₃), 0.92 (d, 3H, J=6.2 Hz, 21-H₃), 1.05 (s, 3H, 19-H₃), 3.61 (br m, 3H, 3β-H and 24-H₂), 5.31 (d, 1H, J=5.8 Hz, 11-H). ¹³C-NMR (125.8 MHz, CDCl₃) δ: 11.7 (C-18), 18.4 (C-21), 25.4 (C-19), 27.0 (×2), 28.5, 29.5, 29.7, 31.9, 32.0, 35.5, 35.8, 36.7, 38.1, 38.6, 41.0, 42.1, 42.2, 53.4, 56.4, 63.7 (C-24), 72.4 (C-3), 119.6 (C-11), 140.1 (C-9). HR-ESI-MS, Calcd for C₂₄H₄₀O₂Na [M+Na]⁺, 383.2926. Found, m/z 383.2943.

5β-Chol-9(11)-en-3α,7α,24-triol (7b)

The Δ⁹⁽¹¹⁾-3α,7α-diacetoxy-12-ketone 6b (1.0 g, 2.0 mmol) was subjected to the deoxygenation reaction with LiAlH₄ and AlCl₃ in THF and processed as described for the preparation of 7a to yield an oily residue. The oil was chromatographed on a column of silica gel (50 g) and eluted with EtOAc–hexane (1 : 1, v/v). Recrystallization of the product from EtOAc–hexane gave the Δ⁹⁽¹¹⁾-3α,12α,24-diol 7b as colorless needles: mp 154–156°C; yield, 390 mg (52%). [α]_D³¹+20.0 (c 0.10, MeOH). FT-IR 3270 (OH), 1639 (C=C) cm⁻¹. ¹H-NMR (500 MHz, CDCl₃) δ: 0.61 (s, 3H, 18-H₃), 0.99 (d, 3H, J=6.2 Hz, 21-H₃), 1.06 (s, 3H, 19-H₃), 3.48 (br m, 1H, 3β-H), 3.62 (br m, 2H, 24-H₂), 3.97 (m, 1H, 7β-H), 5.51 (d, 1H, J=5.8 Hz, 11-H). ¹³C-NMR (125.8 MHz, CDCl₃) δ: 11.4 (C-18), 18.3 (C-21), 24.7 (C-19), 28.4, 29.4, 29.9, 31.8, 31.9, 34.3, 35.5, 35.6, 38.7, 41.1 (×2), 41.4, 41.6, 41.7, 46.5, 56.1, 63.6 (C-24), 68.8 (C-7), 72.4 (C-3), 121.9 (C-11), 137.1 (C-9). HR-ESI-MS, Calcd for C₂₄H₃₉O₃ [M−H]⁻, 375.2899. Found, m/z 375.3173.

9α,11α-Epoxy-5β-cholan-3α,24-diol (8a)

A mixture of the 9(11)-en-3α,24-diol 7a (300 mg, 0.84 mmol), 4,4′-thiobis-(6-tert-butyl-3-methylphenol) (10 mg, 0.03 mmol) and 75% m-chloroperbenzoic acid (m-CPBA, 400 mg, 1.74 mmol) in CHCl₃ (30 mL) was stirred at 35°C for 1 h; the reaction was monitored by TLC. The organic layer was washed with 5% Na₂S₂O₃ solution, 5% NaHCO₃ solution, and water, dried with Drierite, and evaporated to dryness. Chromatography of the residue using a column of silica gel (60 g) and elution with EtOAc–hexane (1 : 1, v/v) gave the 9α,11α-epoxide 9a which was crystallized from methanol as colorless needles: mp 187–189°C; yield, 280 mg (88%). [α]_D²³+40.0 (c 0.10, MeOH). FT-IR 3357 (OH) cm⁻¹. ¹H-NMR (500 MHz, CD₃OD) δ: 0.68 (s, 3H,18-H₃), 0.93 (d, 3H, J=6.3 Hz, 21-H₃), 1.15 (s, 3H, 19-H₃), 3.21 (d, J=5.2, 11β-H), 3.49 (br m, 3H, 3β-H and 24-H₂). ¹³C-NMR (125.8 MHz, CD₃OD) δ: 13.1 (C-18), 17.4 (C-21), 23.9 (×2), 26.0 (C-19), 28.1, 28.3, 28.9, 31.7, 32.2, 34.8, 35.3, 35.4, 36.6, 37.1, 40.0, 40.4, 42.1, 45.1, 51.3, 56.5 (C-11), 62.2 (C-24), 67.4 (C-9), 70.8 (C-3). HR-ESI-MS, Calcd for C₂₄H₄₀O₃Na [M+Na]⁺, 399.2875. Found, m/z 399.2884.

9α,11α-Epoxy-5β-cholan-3α,7α,24-triol (8b)

The 9(11)-en-3α,7α,24-triol (100 mg, 0.27 mmol) was converted to its 9α,11α-epoxy-3α,7α,24-triol (8b) (at reflux for 20 min) by the method described for the preparation of 8a. After chromatographic separation on silica gel (30 g) eluting with EtOAc–hexane (75 : 25, v/v), the major reaction product was recrystallized from methanol–water as colorless needles: mp 170–173°C; yield, 69 mg (64%). [α]_D³⁰+10.0 (c 0.10, MeOH). FT-IR 3244 (OH) cm⁻¹. ¹H-NMR (500 MHz, CD₃OD) δ: 0.68 (s, 3H, 18-H₃), 0.93 (d, 3H, J=6.9 Hz, 21-H₃), 1.18 (s, 3H, 19-H₃), 3.08 (d, 1H, J=5.7 Hz, 11β-H), 3.36 (br m, 1H, 3β-H), 3.48 (br m, 2H, 24-H₂), 3.97 (d, 1H, J=2.8 Hz, 7β-H). ¹³C-NMR (125.8 MHz, CD₃OD) δ: 12.9 (C-18), 17.9 (C-21), 23.1, 26.3 (C-19), 28.1, 28.8, 31.7, 32.3, 34.3, 35.2, 35.4, 35.5, 39.4, 39.9 (×2), 40.6, 41.1, 41.2, 48.6 (C-11), 56.4, 62.2 (C-24), 65.4 (C-9), 68.5 (C-7), 71.3 (C-3). HR-ESI-MS, Calcd for C₂₄ H₄₀O₄Na [M+Na]⁺, 415.2824. Found, m/z 415.2805.

5β-Cholan-3α,9α,24-triol (9a)

To a magnetically stirred dry THF solution, at −5°C, was added slowly AlCl₃ (4.41 g, 33 mmol). Then, LiAlH₄ (500 mg, 13 mmol) was added slowly, and the mixture was stirred at room temperature for 30 min. A solution of the 3α,24-dihydroxy-9α,11α-epoxide 8a (850 mg, 2.26 mmol) in dry THF (20 mL) was added dropwise to the AlH₂Cl solution, and the mixture was refluxed for 48 h. After cooling the solution at room temperature, the reaction product was extracted with CH₂Cl₂. The combined extract was washed with 5% H₂SO₄ and water, dried with Drierite, and evaporated. The residue was subjected to preparative HPLC-RI on a Capcell Pak AQ RP-C₁₈ column. Elution with methanol–H₂O (85 : 15, v/v) afforded the desired compound 9a which crystallized from methanol as colorless needles; mp 174–177°C: yield, 154 mg (18%); the remaining compound recovered was the unreacted one 8a (584 mg, 69%). [α]_D²³+50.0 (c 0.10, MeOH). FT-IR 3321 (OH) cm⁻¹, 1709. ¹H-NMR (500 MHz, CD₃OD) δ: 0.69 (s, 3H, 18-H₃), 0.95 (d, 3H, J=5.2 Hz, 21-H₃), 0.96 (s, 3H, 19-H₃), 3.50 (br m, 2H, 24-H₂), 3.57 (br m, 1H, 3β-H). ¹³C-NMR: see Table 1. HR-ESI-MS, Calcd for C₂₄H₄₂O₃Na [M+Na]⁺, 401.3032. Found, m/z 401.3000.

5β-Cholan-3α,7α,9α,24-tetrol (9b)

The 3α,7α,24-trihydroxy-9α,11α-epoxide 8b (100 mg, 0.25 mmol), subjected to reductive cleavage with AlH₂Cl at reflux condition for 12 h and processed as described for the preparation of 9a, afforded an oily residue. Preparative HPLC-RI of the oily residue on a Capcell Pak AQ RP-C₁₈ column and elution with methanol–water (80 : 20, v/v) afforded the desired compound 9b which crystallized from methanol–water as colorless needles; mp 175–176°C; yield, 43 mg (36%); the remaining compound recovered was the unreacted one 8b (51 mg, 51%). [α]_D³⁰+20.0 (c 0.10, MeOH). FT-IR 3293 (OH) cm⁻¹. ¹H-NMR (500 MHz, CD₃OD) δ: 0.68 (s, 3H, 18-H₃), 0.95 (d, 3H, J=6.9 Hz, 21-H₃), 0.97 (s, 3H, 19-H₃), 3.47 (br m, 1H, 3β-H), 3.49 (br m, 2H, 24-H₂), 3.91 (m, 1H, 7β-H). ¹³C-NMR: see Table 1. HR-ESI-MS, Calcd for C₂₄H₄₂O₄Na [M+Na]⁺, 417.2981. Found, m/z 417.2998.

3α,9α-Dihydroxy-5β-cholan-24-oic Acid (1a)

To a magnetically stirred solution of the 3α,11α,24-triol 9a (20 mg, 53 µmol) in dry THF (1 mL) and CH₃CN (0.25 mL) was added TEMPO (1 mg, 6 µmol) and a 0.2 M sodium phosphate buffer (pH, 6.7; 40 µL). Solutions of 2% NaClO (25 µL) and NaClO₂ (7 mg dissolved in 50 µL of H₂O) were added simultaneously at 35°C to the solution over 0.5 h, and the reaction mixture was further stirred at 35°C for 3 h; the reaction was monitored by TLC. The reaction was quenched by adding a cold saturated solution of Na₂S₂O₃. After stirring for 0.5 h at room temperature, the reaction product was extracted with EtOAc. The combined extract was washed with a saturated brine, dried with Drierite, and evaporated to dryness. The residue was chromatographed on a column of silica gel (10 g) eluting with EtOAc–hexane–acetic acid (8 : 2 : 0.1, v/v/v). Recrystallization of the product from methanol–water gave the titled compound 1a as colorless needles: mp 88–91°C; yield, 12 mg (58%). [α]_D²³+40.0 (c 0.10, MeOH). FT-IR 3447 (OH), 1709 (C=O) cm⁻¹. ¹H-NMR (500 MHz, CD₃OD) δ: 0.70 (s, 3H, 18-H₃), 0.95 (d, 3H, J=6.3 Hz, 21-H₃), 0.96 (s, 3H, 19-H₃), 3.57 (br m, 1H, 3β-H). ¹³C-NMR: see Table 1. HR-ESI-MS, Calcd for C₂₄H₃₉O₄ [M−H]⁻, 391.2848. Found, m/z 391.2847.

3α,7α,9α-Trihydroxy-5β-cholan-24-oic Acid (1b)

The 3α,7α,9α,24-terol 9b (40 mg, 0.10 mmol) was subjected to the selective oxidation (at 35°C overnight) with NaClO₂ in the presence of TEMPO and NaClO in sodium phosphate buffer and processed as described for the preparation of 1a; the reaction product was an oily residue. Chromatography of the oil on a preparative HPLC-RI on a Capcell Pak AQ RP-C₁₈ column and elution with a mixture of methanol–water–acetic acid (8 : 2 : 0.2, v/v/v) afforded the desired compound 1b which was crystallized from methanol–EtOAc as colorless needles; mp 139–142°C; yield, 29 mg (71%). [α]_D²⁹+40.0 (c 0.10, MeOH). FT-IR 3320 (OH), 1713 (C=O) cm⁻¹. ¹H-NMR (500 MHz, CD₃OD) δ: 0.70 (s, 3H, 18-H₃), 0.94 (d, 3H, J=6.9 Hz, 21-H₃), 0.96 (s, 3H, 19-H₃), 3.48 (br m, 1H, 3β-H), 3.91 (m, 1H, 7β-H). ¹³C-NMR: see Table 1. HR-ESI-MS, Calcd for C₂₄H₃₉O₅ [M−H]⁻, 407.2796. Found, m/z 407.2822.

Acknowledgment

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.I., 15K01809) for 2015–2017.

Conflict of Interest

The authors declare no conflict of interest.

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