Chemical synthesis of uncommon natural bile acids: The 9α-hydroxy derivatives of chenodeoxycholic and lithocholic acids

Article abstract of DOI:10.1248/cpb.c16-00247

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 9a-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.

Full text of DOI:10.1248/cpb.c16-00247

Vol. 64, No. 9
Chem. Pharm. Bull. 64, 1397–1402 (2016)
1397
Note
Chemical Synthesis of Uncommon Natural Bile Acids: The 9α-Hydroxy
Derivatives of Chenodeoxycholic and Lithocholic Acids
,a
Takashi Iida,* Kazunari Namegawa,a Naoya Nakane,a Kyoko Iida,a
Alan Frederick Hofmann,b and Kaoru Omuraa
a College of Humanities & Sciences, Nihon University; Sakurajousui, Setagaya-ku, Tokyo 156–8550, Japan: and
b Department of Medicine, University of California at San Diego; La Jolla, CA 92093–0063, U.S.A.
Received March 10, 2016; accepted June 6, 2016; advance publication released online June 18, 2016
The chemical synthesis of the 9α-hydroxy derivatives of chenodeoxycholic and lithocholic acids is report-
ed. 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 4,4ꢀ-thiobis-(6-tert-butyl-3-methylphenol), (3) subsequent Mar-
kovnikov 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
1-oxyl free radical (TEMPO) and sodium hypochlorite (NaOCl). The H- 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 authen-
tic reference standards, permitting their identification in vertebrate bile acids.
Key words 9α-hydroxy-bile acid; C-9 5β-steroid hydroxylation; decarbonylation; monochloroalane; Mar-
kovnikov-hydroxylation; 2,2,6,6-tetramethylpiperidine 1-oxyl free radical
The great variety of natural C24 and C27 bile acids, as well cholan-24-oyl taurine by MS and one and two dimensional
as C27 bile alcohols, (together called bile salts) occurring in (1D)- and (2D)-NMR spectroscopy. Nonetheless, validation
vertebrates can be explained by the evolution of differing bio- of the proposed structure awaits the synthesis of an authentic
chemical pathways that serve to convert cholesterol into these reference standard.
multifunctional amphipathic compounds. Bile salt composition
Microbial or enzymatic biotransformation are important
shows significant variation between orders but not between tools for structural modification of organic compounds, es-
families, genera, or species, suggesting a biochemical trait pecially natural products with complex structures like ste-
providing clues to evolutionary relationships that complement roids.6–8) This synthetic method can be used to prepare chemi-
anatomical and genetic analyses.1–3) Naturally occurring bile cal structures that are difficult to obtain by ordinary chemical
salts differ markedly in their chemical structures, particularly methods. The observed biotransformations may suggest meta-
the number, position and stereochemistry of hydroxyl groups bolic pathways in mammals, due to similarity between mam-
on the steroid nucleus and on the branched C5 or C8 side- malian and microbial systems. However, the procedure is of
chain. Since some of the uncommon bile acids are present in limited use, because of the substrate specificity.
the biliary and/or urinary bile salts of specific vertebrates or
As part of our ongoing program of chemical synthesis of
patients with hepatobiliary diseases, the bile acid profile can novel, uncommon natural or potentially natural bile acid me-
be used as a biomarker for clinical purposes, as well as for tabolites for use as authentic reference standards, we now de-
suggesting phylogenetic relationships.4)
scribe the chemical synthesis of 9α-hydroxy-chenodeoxycholic
Nuclear hydroxylation of bile acids in the liver of verte-
brates 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 con-
sidered 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 junc-
ture) 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-
Fig. 1. Chemical Structures of Synthetic 9α-Hydroxylated Bile Acids
(1a, b)
*To whom correspondence should be addressed. e-mail: takaiida@chs.nihon-u.ac.jp
© 2016 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 64, No. 9 (2016)
acid [9α-OH-CDCA; 3α,7α,11α-trihydroxy-5β-cholan-24-oic δ 121.9 (C-11) and 137.1 (C-9) in 7b suggest that the decarbon-
acid (1b)] starting from cholic acid (2b; CA, 3α,7α,12α- ylation at C-12 occurred successfully.
trihydroxy-5β-cholan-24-oic acid), and 9α-hydroxy-lithocholic
acid [9α-OH-LCA; 3α,9α-dihydroxy-5β-cholan-24-oic acid
Subsequent epoxidation of the Δ9(11)-3α,24-diol (7a) and
Δ
9(11)-3α,7α,24-triol (7b) with m-chloroperbenzoic acid (m-
(1a)] starting from deoxycholic acid (2a; DCA; 3α,12α- CPBA) in the presence of a catalytic amount of 4,4′-thio-
dihydroxy-5β-cholan-24-oic acid) (Fig. 1).
bis-(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-
Results and Discussion
Figure 2 shows the synthetic route of 9α-OH-CDCA (1b) CPBA on the less sterically hindered α-face of the substrates;
from CA (2b) and 9α-OH-LCA (1a) from DCA (2a), respective- the β-face is more sterically crowded by the axially-oriented
ly. A one step procedure for the introduction of an α-hydroxyl 18- and 19-methyl groups. The relatively low-yield (64%) of
group at C-9 on the 5β-steroid nucleus is known.9,10) Thus, the 8b, compared to that (88%) of 8a, is probably ascribed to the
most feasible approach for preparing 1a and b on a substantial steric hindrance of an axially-oriented 7α-hydroxyl group.
scale appeared to be the 9α-hydroxylation of 9α,11α-epoxy in- The stereochemical configuration of the 9α,11α-epoxy ring
termediates (8a, b) derived from Δ9(11)-enes (7a, b), obtainable in 8a was determined by measuring the nuclear Overhauser
from 2a and b in several steps.
enhanced differential spectroscopy (NOEDS), in which the ir-
Regioselective acetylation of methyl deoxycholate (3a) radiation of the 19-H3 signal (δ 1.15) in 8a resulted in the for-
and methyl cholate (3b) under mild conditions gave the mation of the correlating peaks with the 18-H3 (δ 0.68), 5β-H
corresponding 3α-acetate (4a) and 3α,7α-diacetate (4b), re- (δ 1.51), 12β-H (δ 1.68), 8β-H (δ 1.93) and 11β-H (δ 3.21), thus
spectively, which in turn were oxidized with pyridinium indicating the α-configuration. Similar correlations were also
chlorochromate (PCC) to yield the 3α-acetoxy-12-oxo and observed in the NOEDS of 8b: the 19-H3 (δ 1.18) were cor-
3α,7α-diacetoxy-12-oxo esters (5a, b) nearly quantitatively.11) related with the 18-H3 (δ 0.68), 5β-H (δ 1.56), 12β-H (δ 1.69),
When 5a and b were subjected to the selective dehydration 8β-H (δ 1.99) and 11β-H (δ 3.08).
with SeO2 in acetic acid,12–14) the corresponding conjugated
The most promising method for preparing 9α-hydroxylated
enones, Δ9(11)-3α-acetoxy-12-oxo and Δ9(11)-3α,7α-diacetoxy-12- bile acids is seemed to be the application of the so-called
oxo esters (6a, b), were obtained in high yields (78–80%). The “reductive hydroxylation” of the 9α,11α-epoxides (8a, b)
formation of the conjugated 9(11)-ene-12-oxo structure was with lithium-ethylamine9,10) or LiAlH419–21) known to a favor
1
confirmed by the H-NMR signals arising from the 19-H3 at α-hydroxylated product to obey the Marnovnikov rule.
δ 1.19 in 6a and at 1.20 in 6b and by the 13C-NMR signals Attempted reductive cleavage of 8a and b with lithium-
from the C-11 (δ 123.8) and C-9 (δ 164.1) in 6a and the C-11 (δ ethylamine or with LiAlH4 alone was unsuccessful; both
125.1) and C-9 (δ 159.8) in 6b.
the reactions did not proceed at all. However, when 8a and
Decarbonylation at C-12 of 6a and b in tetrahydrofuran b were subjected to AlH2Cl, generated in situ from LiAlH4
(THF) for 4h at reflux temperature with monochloroalane and AlCl3,22) to afford the expected 3α,9α,24-triol (9a) and
(AlH2Cl), generated in situ from LiAlH4 and AlCl3,13,15–17) 3α,7α,9α,24-tetrol (9b) with good selectivity in fairly low
gave the Δ9(11)-3α,24-diol (7a) and Δ9(11)-3α,7α,24-triol (7b) in isolated yields of 18 and 36%, respectively, after preparative
moderate isolated yields of 52–64%, respectively, accompa- high-performance liquid chromatography with a refractive
nied by simultaneous hydrolysis of the methyl ester at C-24 index detector (HPLC-RI) chromatographic purification of
1
and the acetoxy groups at C-3 and C-7. The 11-H olefinic H the reaction products; the remaining compound recovered was
signal occurring at δ 5.31 in 7a and at 5.50 in 7b and the 13C the unreacted one (69% for 8a and 51% for 8b). AlH2Cl was
signals appearing at δ 119.6 (C-11) and 140.1 (C-9) in 7a and at therefore found to be effective for the regio- and stereoselec-
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 12h. (ii) Ac2O/pyridine/benzene, at r.t. for 12h. (iii) PCC/CH2Cl2, at r.t. for 12h. (iv) SeO2/AcOH,
at reflux for 18h. (v) AlCl3/LiAlH4/THF, ar reflux for 4h. (vi) m-Chloroperbenzoic acid/4,4′-thiobis(6-tert-butyl-3-methylphenol)/chloroform, at r.t. for 1h (at reflux for
20min). (vii) AlCl3/LiAlH4/THF, at reflux for 48h (at reflux for 12h). (viii) NaC1O2/TEMPO/NaClO/CH3CN/THF, at 35°C for 3h (at 35°C for overnight).

Vol. 64, No. 9 (2016)
Chem. Pharm. Bull.
1399
tive hydroxylation of the sterically crowded epoxides. An oxidation of alcohols26–30) suggested that a selective oxidation
appreciable difference in the isolated yields of 9a (18%) and of a primary hydroxyl group to its carboxyl group without
9b (36%) suggests that the substrate 8b is more unstable and oxidizing a secondary hydroxyl group might be feasible. As
reactive than 8a.
expected, mild reaction of 9a and b with TEMPO in the pres-
The complete 1H- and 13C-NMR signal assignments for ence of the co-reagents resulted in successful selective oxida-
compounds 9a and b, which were confirmed by comparison tion of the primary 24-hydroxyl group to afford the desired
of bile acid analogs reported previously,23–25) were compiled in 3α,9α-dihydroxy and 3α,7α,9α-trihydroxy acids (1a; 58%, 1b;
1
Table 1. The H-NMR spectra of the compounds exhibited the 71%), respectively. The presence of a hydroxyl group at the
1H-signals arising from the 3β-H (brm) at δ 3.57 (9a) and 3.47 C-9 position and its stereochemical configuration as α in 1a
(9b) and the 24-H2 (brm) at δ 3.50 (9a) and 3.49 (9b), both and b was determined by a combined use of several 1- and
of which showed essentially identical H signal patterns. The 2D-NMR techniques (Table 1). The 13C signals appearing at
1
stereochemical configuration of the 9α-hydroxyl group in 9b δ 71.6, 77.0, and 176.9 in 1a were tentatively assigned to the
were determined by measuring the nuclear Overhauser effect C-3, C-9, and C-24 bearing hydroxyl groups, respectively: δ
spectroscopy (NOESY), in which the distinct correlation 71.8, 69.7, 79.5, and 175.2 in 1b were due to the C-3, C-7, C-9,
peaks were detected between the 19-H3 (δ 0.96)/18-H3 (δ 0.68), and C-24, respectively. The presence of a tertiary hydroxyl
1
19-H3/5β-H (δ 1.58), 19-H3/8β-H (δ 1.62), and 19-H3/12β-H (δ group at C-9 was confirmed by the appearance of the H/13C
1
1.74). Essentially identical NOESY was also observed for 9a: correlated peak arising from the 19-H3/C-9 in the H-detected
19-H3 (δ 0.96)/18-H3 (δ 0.69), 19-H3/5β-H (δ 1.42), 19-H3/8β-H heteronuclear multiple bond connectivity (HMBC) and DEPT
(δ 1.62) and 19-H3/12β-H (δ 1.72). Meanwhile, the 13C-NMR spectra of 1a and b. In addition, the correlation peaks ob-
signals were appeared at δ 71.6 (9a) and 71.8 (9b) for the me- served for the 18-H3/C-12 (δ 34.9 in 1a and 35.0 in 1b) in the
thine C-3, at δ 77.0 (9a) and 79.5 (9b) for the quaternary C-9, HMBC, the C-12/12-H2 [δ 1.56 (α) and 1.73 (β) in 1a and δ
1
and at δ 62.2 (9a, b) for the methylene C-24 in the distortion- 1.59 (α) and 1.77 (β) in 1b] in the H-detected heteronuclear
less enhancement by polarization transfer (DEPT) spectra. multiple quantum coherence (HMQC), and then the 12-H2/C-9
These observations provide strong evidence for the presence in the HMBC provided further confirmatory evidence for the
of an axially-oriented 9α-hydroxyl group in the cis 5β-steroid presence of the 9-hydroxyl group. In the NOESY of 1a, the
nucleus (see below).
distinct correlation peaks were observed between the 19-H3
Recently successful use of sodium chlorite (NaClO2) (δ 0.96)/18-H3 (δ 0.70), 19-H3/5β-H (δ 1.42), 19-H3/8β-H (δ
catalyzed by 2,2,6,6-tetramethylpiperidine 1-oxyl free radi- 1.63) and 19-H3/12β-H (δ 1.73), strongly indicating that the
cal (TEMPO) and sodium hypochlorite (NaClO; bleach) for configuration of the hydroxyl group at C-9 is α. As shown
1
Table 1. Complete H- and 13C-NMR Spectral Data for Synthetic 9α-Hydroxylated Compounds (9a, b, 1a, b)a)
9a
9b
1a
1b
No
Proton
Proton
Proton
Proton
Type Carbon
Type Carbon
Type Carbon
Type Carbon
α
β
α
β
α
β
α
β
1
2
CH2
CH2
CH
CH2
CH
CH2
CH2
CH
C
34.31
33.14
71.55
38.54
43.50
2.09
1.99
—
1.02 CH2
1.63 CH2
35.07
32.92
71.75
41.85
42.05
34.21
69.73
40.73
79.49
38.56
2.03
2.01
—
1.09
1.58
3.47
1.77
1.58
1.73
3.91
1.62
—
CH2
CH2
CH
CH2
CH
CH2
CH2
CH
C
34.34
33.14
71.55
38.55
43.49
2.09
1.99
—
1.03
1.63
3.57
1.41
1.42
CH2
CH2
CH
35.04
32.92
71.76
41.88
42.05
34.20
69.72
40.70
79.47
38.54
2.05
2.02
—
1.09
1.62
3.48
1.72
1.58
1.73
3.91
1.64
—
3
3.57
1.41 CH2
1.42 CH
1.90c) CH2
CH
4
2.26
—
1.37c)
1.56
—
2.23
—
2.26
—
28.16b) 1.36c)
CH2
CH
1.89c) CH2
2.23
—
5
6
28.13b)
21.27
39.75
77.03
38.01
28.06b)
34.97
42.41
47.99
23.84
28.49b)
56.10
10.28
27.15
35.68
17.81
31.84
28.92
62.24
2.06
—
2.06
—
7
1.19
1.62
—
CH
CH
C
21.28
39.76
77.00
38.01
1.56
—
1.19
1.63
—
CH
CH
C
8
—
—
9
—
—
—
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
C
—
—
C
—
—
C
—
—
C
—
—
CH2
CH2
C
1.56c)
1.56
—
1.63c) CH2
27.80b) 1.60c)
1.71c) CH2
27.95b) 1.56c)
1.62c) CH2
27.77b) 1.60c)
1.70c)
1.77
—
1.72 CH2
35.07
42.39
44.72
23.09
1.59
—
1.74
—
CH2
C
34.94
42.46
47.99
23.85
1.56
—
1.73
—
CH2
C
35.04
42.42
44.72
23.03
1.59
—
—
—
C
CH
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH2
CH2
1.61
1.56
1.94c)
1.20
CH
2.00
—
CH
1.62
—
CH
2.00
—
1.06 CH2
1.33c) CH2
1.68
1.10
1.30c) CH2
CH2
1.56
1.08
1.32c) CH2
CH2
1.69
1.09
1.35c)
—
28.03b) 1.91c)
28.50b) 1.93c)
27.91b) 1.95c)
—
CH
CH3
CH3
CH
CH3
CH2
CH2
C
56.10
10.20
27.75
35.74
17.83
31.85
28.92
62.23
1.23
—
CH
CH3
CH3
CH
CH3
CH2
CH2
C
55.90
10.33
27.16
35.41
17.43
30.99
30.69
176.87
1.20
—
CH
CH3
CH3
CH
CH3
CH2
CH2
C
55.95
10.18
27.17
35.53
17.42
31.24
31.24
175.23
1.23
0.69
0.68
0.96
1.43
0.97
0.70
0.96
1.45
0.95
0.70
0.96
1.43
0.94
0.96
1.43
0.95
1.04/1.45
1.33/1.38
3.50
1.07/1.48
1.37/1.43
3.49
1.28/1.79
2.16/2.33
—
1.35/1.78
2.17/2.34
—
1
a) Measured in CD3OD at 500.2MHz in H-NMR and at 125.8MHz in 13C-NMR. Chemical shifts were expressed as δ ppm relative to TMS. b, c) Assignments along a verti-
cal column bearing the same superscript may be interchanged.

1400
Chem. Pharm. Bull.
Vol. 64, No. 9 (2016)
matography-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 (250mm×10mm i.d.; particle
size 5µm; Shiseido).
Normal-phase TLC was performed on pre-coated Kieselgel
60F254 plates (E. Merck, Darmstad, Germany) using a mix-
ture 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–meth-
anol–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)
Fig. 3. NOESY Correlations Observed for 9α-OH-CDCA (1b)
in Fig. 3, essentially identical NOESY correlations were also A mixture of the 3α-acetoxy-12-oxo 5a (1.0g; 2.2mmol)
observed for 1b: 19-H3 (δ 0.96)/18-H3 (δ 0.70), 19-H3/5β-H (δ and selenium dioxide (SeO2) (500mg, 4.6mmol) dissolved
1.58), 19-H3/8β-H (δ 1.64) and 19-H3/12β-H (δ 1.77). To con- in acetic acid (40mL) was refluxed for 18h. After cooling at
clude, the 3α,9α,24-triol (9a) and 3α,7α,9α,24-tetrol (9b) were room temperature, the insoluble matter was filtered off, and
successfully converted to the desired 9α-OH-LCA (1a) and the mother liquor was extracted with EtOAc. The combined
9α-OH-CDCA (1b), respectively.
extract was washed with 5% NaHCO3 solution and water,
As note 1b has been reported to be present in the biliary dried with Drierite, and evaporated to dryness. Chroma-
bile acids of the Asian black bear. 1a has not been reported tography of the crude residue over a column of silica gel
to occur naturally, but 7-dehydroxylation is a dominant bacte- (50g) eluting with EtOAc–hexane (3:7, v/v) resulted in a
rial biotransformation in the anaerobic colon,31) leading to the single component, which was identified as the conjugated
prediction that 1a should be present in the fecal bile acids of
Δ
9(11)-12-ketone 6a which was recrystallized from methanol
Ursus thibetanus. Hydroxylation of steroids at C-9 by micro- as colorless needles: mp 149–152°C; yield, 835mg (78%).
bial enzymes32) or by simulated microsomal oxidation33) has [α]D23+140.0 (c 0.10, MeOH). FT-IR 1740, 1726 (C=O), 1645
also been reported.
(C=C) cm−1. 1H-NMR (500MHz, CDCl3) δ: 0.90 (s, 3H,
Thus, the availability of 9α-OH-LCA (1a) and 9α-OH-CDCA 18-H3), 1.00 (d, 3H, J=6.2Hz, 21-H3), 1.19 (s, 3H, 19-H3), 1.99
(1b) should facilitate the identification of these uncommon bile (s, 3H, 3-OCOCH3), 3.66 (s, 3H, 24-COOCH3), 4.71 (brm,
acids in body fluids of vertebrates and also highlights an un- 1H, 3β-H), 5.70 (s, 1H, 11-H). 13C-NMR (125.8MHz, CDCl3)
usual site of steroid hydroxylation.
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
Experimental
Materials DCA (2a) and CA (2b) were obtained from (3-OCOCH3), 174.8 (C-24), 205.2 (C-12). HR-ESI-MS, Calcd
Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other for C27H40O5Na [M+Na]+, 467.2773. Found, m/z 467.2746.
chemicals and solvents were of analytical reagent grade and
available from commercial sources. All compounds were dried (6b) The 3,7-diacetoxy-12-oxo 5b (1.0g; 2.0mmol), subjected
by azeotropic distillation before use in reactions. to the dehydrogenation with SeO2 in acetic acid and processed
Methyl 3α,7α-Diacetoxy-12-oxo-5β-chol-9(11)-en-24-oate
Instruments All melting points (mp) were determined as described for the preparation of 6a, gave a crude product.
on a micro hot stage apparatus and are uncorrected. Specific Chromatography of the product on a column of silica gel
rotations were measured on a ATAGO AP-300 and ATAGO (50g) and elution with EtOAc–hexane (3:7, v/v) afforded the
DP-63 auto-recording polarimeter. IR spectra, on a JASCO title compound 6b which was recrystallized from methanol
FT-IR-4100 spectrophotometer (samples were absorbed on a as colorless needles: mp 159–160°C (159–161°C)11); yield,
powdered KBr surface and measured with the diffusion reflec- 797mg (80%). [α]D311+80.0 (c 0.10, MeOH). FT-IR 1735 (C=O),
tion method).
1671 (C=C) cm−1. H-NMR (500MHz, CDCl3) δ: 0.92 (s, 3H,
1H- and 13C-NMR spectra were obtained on a JEOL ECA 18-H3), 1.00 (d, 3H, J=6.3Hz, 21-H3), 1.20 (s, 3H, 19-H3),
500 FT instrument operated at 500 and 125MHz, respectively, 1.99 (s, 6H, 3- and 7-OCOCH3), 3.65 (s, 3H, 24-COOCH3),
with CDCl3 or CD3OD containing 0.1% Me4Si as the solvent; 4.59 (brm, 1H, 3β-H), 5.15 (m, 1H, 7β-H), 5.82 (s, 1H, 11-H).
chemical shifts were expressed in δ (ppm) relative to Me4Si 13C-NMR (125.8MHz, CDCl3) δ: 10.8 (C-18), 19.4 (C-21), 21.4
(0ppm). The 13C DEPT spectra were measured to determine (C-19), 21.5, 23.7, 27.5, 27.8, 30.1, 30.7, 30.9, 31.5, 35.0, 35.4,
the exact 13C signal multiplicity and to differentiate among 36.2, 40.1, 40.8, 41.1, 46.8, 47.1, 51.6 (C-25), 53.3, 70.2 (C-7),
CH3, CH2, CH, and C based on their proton environments. In 73.8 (C-3), 125.1 (C-11), 159.8 (C-9), 170.1 (7-OCOCH3), 170.7
order to further confirm the H and 13C signal assignments for (3-OCOCH3), 174.7 (C-24), 204.6 (C-12). HR-ESI-MS, Calcd
1
some of compounds, NOEDS, and 2D, H–1H correlation spec- for C29H43O7 [M+H]+, 503.3009. Found, m/z 503.3086.
1
troscopy (COSY), H–1H NOESY, HMQC (1H/13C coupling)
5β-Chol-9(11)-en-3α,24-diol (7a) To an ice-cooled solu-
1
and HMBC (long-range H–13C coupling) spectra were also tion of AlCl3 (4.2g; 31.5mmol) in dry THF (25mL) was added
1
performed.
LiAlH4 (0.5g; 13.2mmol) gradually, and the resulting solution
High-resolution liquid chromatography-mass spectrometry was stirred 10min at room temperature and was subsequently
by electrospray ionization (HR-LC/ESI-MS) source was car- refluxed during 30min. A solution of the Δ9(11)-3α-acetoxy-12-
ried out using a JEOL AccuTOF JMS-T100LC liquid chro- ketone 6a (1.0g, 2.25mmol) in THF (25mL) was added drop-

Vol. 64, No. 9 (2016)
Chem. Pharm. Bull.
1401
wise to the AlH2Cl solution, and after the addition was com- the method described for the preparation of 8a. After chro-
plete, reflux was maintained during 4h: the reaction was mon- matographic separation on silica gel (30g) eluting with
itored by TLC. The reaction mixture was cooled with an ice- EtOAc–hexane (75:25, v/v), the major reaction product was
bath, and water was carefully added. The reaction product was recrystallized from methanol–water as colorless needles: mp
extracted with CH2Cl2, and the combined extract was washed 170–173°C; yield, 69mg (64%). [α]D30+10.0 (c 0.10, MeOH).
with 5% H2SO4 and water, dried with Drierite, and evaporated FT-IR 3244 (OH) cm−1. 1H-NMR (500MHz, CD3OD) δ:
to dryness. The crude residue, which consisted essentially of 0.68 (s, 3H, 18-H3), 0.93 (d, 3H, J=6.9Hz, 21-H3), 1.18 (s,
a single spot on TLC was chromatographed on a column of 3H, 19-H3), 3.08 (d, 1H, J=5.7Hz, 11β-H), 3.36 (brm, 1H,
silica gel (50g). Elution with EtOAc–hexane (1:1, v/v) gave 3β-H), 3.48 (brm, 2H, 24-H2), 3.97 (d, 1H, J=2.8Hz, 7β-H).
the desired Δ9(11)-3α,24-diol 7a which was recrystallized from 13C-NMR (125.8MHz, CD3OD) δ: 12.9 (C-18), 17.9 (C-21),
EtOAc–hexane as colorless needles: mp 179–180°C; yield, 23.1, 26.3 (C-19), 28.1, 28.8, 31.7, 32.3, 34.3, 35.2, 35.4, 35.5,
510mg (64%). [α]D231+40.0 (c 0.10, MeOH). FT-IR 3324 (OH), 39.4, 39.9 (×2), 40.6, 41.1, 41.2, 48.6 (C-11), 56.4, 62.2 (C-24),
1647 (C=C) cm−1. H-NMR (500MHz, CDCl3) δ: 0.58 (s, 3H, 65.4 (C-9), 68.5 (C-7), 71.3 (C-3). HR-ESI-MS, Calcd for C24
18-H3), 0.92 (d, 3H, J=6.2Hz, 21-H3), 1.05 (s, 3H, 19-H3), 3.61 H40O4Na [M+Na]+, 415.2824. Found, m/z 415.2805.
(brm, 3H, 3β-H and 24-H2), 5.31 (d, 1H, J=5.8Hz, 11-H).
5β-Cholan-3α,9α,24-triol (9a) To a magnetically stirred
13C-NMR (125.8MHz, CDCl3) δ: 11.7 (C-18), 18.4 (C-21), 25.4 dry THF solution, at −5°C, was added slowly AlCl3 (4.41g,
(C-19), 27.0 (×2), 28.5, 29.5, 29.7, 31.9, 32.0, 35.5, 35.8, 36.7, 33mmol). Then, LiAlH4 (500mg, 13mmol) was added slowly,
38.1, 38.6, 41.0, 42.1, 42.2, 53.4, 56.4, 63.7 (C-24), 72.4 (C-3), and the mixture was stirred at room temperature for 30min.
119.6 (C-11), 140.1 (C-9). HR-ESI-MS, Calcd for C24H40O2Na A solution of the 3α,24-dihydroxy-9α,11α-epoxide 8a (850mg,
[M+Na]+, 383.2926. Found, m/z 383.2943.
5β-Chol-9(11)-en-3α,7α,24-triol (7b) T h e
2.26mmol) in dry THF (20mL) was added dropwise to the
Δ
9(11)-3α,7α- AlH2Cl solution, and the mixture was refluxed for 48h. After
diacetoxy-12-ketone 6b (1.0g, 2.0mmol) was subjected to cooling the solution at room temperature, the reaction product
the deoxygenation reaction with LiAlH4 and AlCl3 in THF was extracted with CH2Cl2. The combined extract was washed
and processed as described for the preparation of 7a to yield with 5% H2SO4 and water, dried with Drierite, and evapo-
an oily residue. The oil was chromatographed on a column rated. The residue was subjected to preparative HPLC-RI on a
of silica gel (50g) and eluted with EtOAc–hexane (1:1, v/v). Capcell Pak AQ RP-C18 column. Elution with methanol–H2O
Recrystallization of the product from EtOAc–hexane gave the (85:15, v/v) afforded the desired compound 9a which crys-
Δ
9(11)-3α,12α,24-diol 7b as colorless needles: mp 154–156°C; tallized from methanol as colorless needles; mp 174–177°C:
yield, 390mg (52%). [α]D31+20.0 (c 0.10, MeOH). FT-IR 3270 yield, 154mg (18%); the remaining compound recovered
(OH), 1639 (C=C) cm−1. 1H-NMR (500MHz, CDCl3) δ: was the unreacted one 8a (584mg, 69%). [α]D23+50.0 (c 0.10,
0.61 (s, 3H, 18-H3), 0.99 (d, 3H, J=6.2Hz, 21-H3), 1.06 (s, MeOH). FT-IR 3321 (OH) cm−1, 1709. H-NMR (500MHz,
1
3H, 19-H3), 3.48 (brm, 1H, 3β-H), 3.62 (brm, 2H, 24-H2), CD3OD) δ: 0.69 (s, 3H, 18-H3), 0.95 (d, 3H, J=5.2Hz, 21-H3),
3.97 (m, 1H, 7β-H), 5.51 (d, 1H, J=5.8Hz, 11-H). 13C-NMR 0.96 (s, 3H, 19-H3), 3.50 (brm, 2H, 24-H2), 3.57 (brm,
(125.8MHz, CDCl3) δ: 11.4 (C-18), 18.3 (C-21), 24.7 (C-19), 1H, 3β-H). 13C-NMR: see Table 1. HR-ESI-MS, Calcd for
28.4, 29.4, 29.9, 31.8, 31.9, 34.3, 35.5, 35.6, 38.7, 41.1 (×2), C24H42O3Na [M+Na]+, 401.3032. Found, m/z 401.3000.
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 9α,11α-epoxide 8b (100mg, 0.25mmol), subjected to reductive
[M−H]−, 375.2899. Found, m/z 375.3173.
cleavage with AlH2Cl at reflux condition for 12h and pro-
5β-Cholan-3α,7α,9α,24-tetrol (9b) The 3α,7α,24-trihydroxy-
9α,11α-Epoxy-5β-cholan-3α,24-diol (8a) A mixture of cessed as described for the preparation of 9a, afforded an
the 9(11)-en-3α,24-diol 7a (300mg, 0.84mmol), 4,4′-thio- oily residue. Preparative HPLC-RI of the oily residue on a
bis-(6-tert-butyl-3-methylphenol) (10mg, 0.03mmol) and 75% Capcell Pak AQ RP-C18 column and elution with methanol–
m-chloroperbenzoic acid (m-CPBA, 400mg, 1.74mmol) in water (80:20, v/v) afforded the desired compound 9b which
CHCl3 (30mL) was stirred at 35°C for 1h; the reaction was crystallized from methanol–water as colorless needles; mp
monitored by TLC. The organic layer was washed with 5% 175–176°C; yield, 43mg (36%); the remaining compound re-
Na2S2O3 solution, 5% NaHCO3 solution, and water, dried covered was the unreacted one 8b (51mg, 51%). [α]D30+20.0
1
with Drierite, and evaporated to dryness. Chromatography (c 0.10, MeOH). FT-IR 3293 (OH) cm−1. H-NMR (500MHz,
of the residue using a column of silica gel (60g) and elution CD3OD) δ: 0.68 (s, 3H, 18-H3), 0.95 (d, 3H, J=6.9Hz, 21-H3),
with EtOAc–hexane (1:1, v/v) gave the 9α,11α-epoxide 9a 0.97 (s, 3H, 19-H3), 3.47 (brm, 1H, 3β-H), 3.49 (brm, 2H,
which was crystallized from methanol as colorless needles: 24-H2), 3.91 (m, 1H, 7β-H). 13C-NMR: see Table 1. HR-ESI-
mp 187–189°C; yield, 280mg (88%). [α]D23+40.0 (c 0.10, MS, Calcd for C24H42O4Na [M+Na]+, 417.2981. Found, m/z
1
MeOH). FT-IR 3357 (OH) cm−1. H-NMR (500MHz, CD3OD) 417.2998.
δ: 0.68 (s, 3H,18-H3), 0.93 (d, 3H, J=6.3Hz, 21-H3), 1.15 (s,
3α,9α-Dihydroxy-5β-cholan-24-oic Acid (1a) To a mag-
3H, 19-H3), 3.21 (d, J=5.2, 11β-H), 3.49 (brm, 3H, 3β-H and netically stirred solution of the 3α,11α,24-triol 9a (20mg,
24-H2). 13C-NMR (125.8MHz, CD3OD) δ: 13.1 (C-18), 17.4 53 µmol) in dry THF (1mL) and CH3CN (0.25mL) was added
(C-21), 23.9 (×2), 26.0 (C-19), 28.1, 28.3, 28.9, 31.7, 32.2, 34.8, TEMPO (1mg, 6µmol) and a 0.2M sodium phosphate buffer
35.3, 35.4, 36.6, 37.1, 40.0, 40.4, 42.1, 45.1, 51.3, 56.5 (C-11), (pH, 6.7; 40µL). Solutions of 2% NaClO (25µL) and NaClO2
62.2 (C-24), 67.4 (C-9), 70.8 (C-3). HR-ESI-MS, Calcd for (7mg dissolved in 50µL of H2O) were added simultaneously
C24H40O3Na [M+Na]+, 399.2875. Found, m/z 399.2884.
at 35°C to the solution over 0.5h, and the reaction mixture
9α,11α-Epoxy-5β-cholan-3α,7α,24-triol(8b) The9(11)-en- was further stirred at 35°C for 3h; the reaction was moni-
3α,7α,24-triol (100mg, 0.27mmol) was converted to its tored by TLC. The reaction was quenched by adding a cold
9α,11α-epoxy-3α,7α,24-triol (8b) (at reflux for 20min) by saturated solution of Na2S2O3. After stirring for 0.5h at room

1402
Chem. Pharm. Bull.
57, 528–531 (2009).
Vol. 64, No. 9 (2016)
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 (10g) eluting
with EtOAc–hexane–acetic acid (8:2:0.1, v/v/v). Recrystal-
lization of the product from methanol–water gave the titled
compound 1a as colorless needles: mp 88–91°C; yield, 12mg
(58%). [α]D23+40.0 (c 0.10, MeOH). FT-IR 3447 (OH), 1709
(C=O) cm−1. 1H-NMR (500MHz, CD3OD) δ: 0.70 (s, 3H,
18-H3), 0.95 (d, 3H, J=6.3Hz, 21-H3), 0.96 (s, 3H, 19-H3), 3.57
(brm, 1H, 3β-H). 13C-NMR: see Table 1. HR-ESI-MS, Calcd
for C24H39O4 [M−H]−, 391.2848. Found, m/z 391.2847.
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3α,7α,9α-Trihydroxy-5β-cholan-24-oic Acid (1b) The
3α,7α,9α,24-terol 9b (40mg, 0.10mmol) 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 reac-
tion 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, 29mg (71%). [α]D29+40.0 (c 0.10, MeOH).
FT-IR 3320 (OH), 1713 (C=O) cm−1. 1H-NMR (500MHz,
CD3OD) δ: 0.70 (s, 3H, 18-H3), 0.94 (d, 3H, J=6.9Hz, 21-H3),
0.96 (s, 3H, 19-H3), 3.48 (brm, 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.
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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.
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Conflict of Interest The authors declare no conflict of
interest.
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