Chemical Synthesis of Rare Natural Bile Acids: 11α-Hydroxy Derivatives of Lithocholic and Chenodeoxycholic Acids

Article abstract of DOI:10.1002/lipd.12013

A method for the preparation of 11α-hydroxy derivatives of lithocholic and chenodeoxycholic acids, recently discovered to be natural bile acids, is described. The principal reactions involved were (1) elimination of the 12α-mesyloxy group of the methyl esters of 3α-acetate-12α-mesylate and 3α,7α-diacetate-12α-mesylate derivatives of deoxycholic acid and cholic acid with potassium acetate/hexamethylphosphoramide; (2) simultaneous reduction/hydrolysis of the resulting △¹¹-3α-acetoxy and △¹¹-3α,7α-diacetoxy methyl esters with lithium aluminum hydride; (3) stereoselective 11α-hydroxylation of the △¹¹-3α,24-diol and △¹¹-3α,7α,24-triol intermediates with B₂H₆/tetrahydrofuran (THF); and (4) selective oxidation at C-24 of the resulting 3α,11α,24-triol and 3α,7α,11α,24-tetrol to the corresponding C-24 carboxylic acids with NaClO₂ catalyzed by 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and NaClO. In summary, 3α,11α-dihydroxy-5β-cholan-24-oic acid and 3α,7α,11α-trihydroxy-5β-cholan-24-oic acid have been synthesized and their nuclear magnetic resonance (NMR) spectra characterized. These compounds are now available as reference standards to be used in biliary bile acid analysis.

Full text of DOI:10.1002/lipd.12013

Lipids (2018)
DOI 10.1002/lipd.12013
ORIGINAL ARTICLE
Chemical Synthesis of Rare Natural Bile Acids: 11α-Hydroxy
Derivatives of Lithocholic and Chenodeoxycholic Acids
Kazunari Namegawa1 · Kyoko Iida1 · Kaoru Omura1 · Shoujiro Ogawa2 · Alan F. Hofmann3 ·
Takashi Iida1
Received: 20 September 2017 / Revised: 19 November 2017 / Accepted: 22 November 2017
© 2018 AOCS
Abstract A method for the preparation of 11α-hydroxy
derivatives of lithocholic and chenodeoxycholic acids,
recently discovered to be natural bile acids, is described.
The principal reactions involved were (1) elimination of the
12α-mesyloxy group of the methyl esters of 3α-acetate-
12α-mesylate and 3α,7α-diacetate-12α-mesylate derivatives
of deoxycholic acid and cholic acid with potassium acetate/
hexamethylphosphoramide; (2) simultaneous reduction/
2,2,6,6-Tetramethylpiperidine 1-oxyl free radical ꢀ
3α,7α,11α-Trihydroxy-5β-cholan-24-oic acid
Lipids (2018).
Abbreviations
CA
cholic acid
11
11
CDCA
DCA
chenodeoxycholic acid
deoxycholic acid
hydrolysis of the resulting 4 -3α-acetoxy and 4 -3α,7α-
diacetoxy methyl esters with lithium aluminum hydride;
11
HMPA
HR-LC/
ESI-MS
LAH
hexamethylphosphoramide
high-resolution liquid chromatograph-mass
spectrometry with an electrospray ionization
lithium aluminum hydride
lithocholic acid
(3) stereoselective 11α-hydroxylation of the 4 -3α,24-diol
11
and 4 -3α,7α,24-triol intermediates with B2H6/tetrahy-
drofuran (THF); and (4) selective oxidation at C-24 of the
resulting 3α,11α,24-triol and 3α,7α,11α,24-tetrol to the cor-
responding C-24 carboxylic acids with NaClO2 catalyzed
by 2,2,6,6-tetramethylpiperidine 1-oxyl free radical
(TEMPO) and NaClO. In summary, 3α,11α-dihydroxy-5β-
cholan-24-oic acid and 3α,7α,11α-trihydroxy-5β-cholan-24-
oic acid have been synthesized and their nuclear magnetic
resonance (NMR) spectra characterized. These compounds
are now available as reference standards to be used in biliary
bile acid analysis.
LCA
NMR
RP-
HPLC/RI
nuclear magnetic resonance
reversed-phase high-performance liquid
chromatograph with a refractive index
detector
RP-TLC
TEMPO
reversed-phase thin-layer chromatograph
2,2,6,6-tetramethylpiperidine 1-oxyl free
radical
TLC
thin-layer chromatograph
Keywords 3α,11α-Dihydroxy-5β-cholan-24-oic acid ꢀ
11α-Hydroxy-bile acid ꢀ 11α-Hydroxylation ꢀ NMR ꢀ
Introduction
The great variety of bile acids and bile alcohols occurring
in vertebrates can be explained by the evolution of differing
biochemical pathways that serve to convert cholesterol into
amphipathic conjugated bile acids or bile alcohols. Bile
acid or bile alcohol composition shows significant variation
between orders but not between families, genera, or spe-
cies. Biliary bile acid composition is a biochemical trait that
may provide clues to evolutionary relationships,
* Takashi Iida
takaiida1101@gmail.com
1
College of Humanities & Sciences, Nihon University,
Sakurajousui, Setagaya, Tokyo 156-8550, Japan
2
Faculty of Pharmaceutical Sciences, Tokyo University of Science,
Noda, Chiba 278-8510, Japan
3
Department of Medicine, University of California San Diego, La
Jolla, CA 92093-8200, USA
Published online:
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Fig. 1 Chemical structures of 11α-hydroxy-bile acids
complementing anatomical and genetic analyses
(Hofmann & Hagey, 2008, 2014; Hofmann, Hagey, & Kra-
sowski, 2010). Naturally occurring bile acids and bile alco-
hols differ markedly in their chemical structures,
particularly in the number, position, and stereochemistry of
hydroxyl groups affixed to the 5β- or 5α-steroid nucleus
and in the branched side chain. In inborn errors of bile acid
biosynthesis, uncommon bile acids may be formed and
excreted into bile or urine. These abnormal bile acids may
provide diagnostic information (Sjövall, 2004).
In vertebrates, C24 bile acids that are synthesized directly
from cholesterol in the hepatocyte via C27 bile alcohols and
C27 higher bile acids are termed primary bile acids. The
dominant primary bile acid is chenodeoxycholic acid
(CDCA, 3α,7α-dihydroxy-5β-cholan-24-oic acid); an addi-
tional major site of hydroxylation was at C-12 to yield cho-
lic acid (CA, 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid).
In the large intestine, anaerobic modifications of the
hydroxyl groups in the primary bile acids occur to
(3α,7α,11α-trihydroxy-5β-cholan-24-oic acid, 1b) (Fig. 1).
We have also re-examined methods of synthesis of C-11
hydroxy-bile acids that were reported in the distant past
when DCA was used as starting material for the synthesis
of cortisone.
form secondary bile acids. The most striking change is
4,6
7-dehydroxylation that occurs via a 3-oxo-4
(Ishida
Experimental
Materials
et al., 1998; Sjövall, 2004) nucleotide intermediate. The
7-dehydroxylation product of CA is deoxycholic acid (DCA,
3α,12α-dihydroxy-5β-cholan-24-oic acid); that of CDCA is
lithocholic acid (LCA, 3α-hydroxy-5β-cholan-24-oic acid).
If one accepts the concept of a default steroid nucleus with
hydroxyl groups at C-3, C-7, and/or C-12, then further modi-
fications can be considered as additions to the default struc-
ture. The other sites of nuclear hydroxylation in uncommon
naturally occurring bile acids are at C-1 (1α-/1β-), C-2 (2α-/
2β-), C-4 (4β-), C-5 (5β-), C-6 (6α-/6β-), C-10 (19β-), C-15
(15α-), and C-16 (16α) (Hofmann et al., 2010; Hofmann &
Hagey, 2008, 2014).
DCA (2a) and CA (2b) were obtained from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). All other chemi-
cals and solvents were of analytical reagent grade and
available from commercial sources. All compounds were
dried by azeotropic distillation before use in reactions.
Instruments
Recently, bile acids hydroxylated at the rare hydroxyl-
ation sites of C-9 (9α-) (Bi, Chai, Song, Lei, & Tu, 2009)
and C-11 (11α-) (Ishida et al., 1998) have been identified.
A 9α-hydroxy-bile acid (in the form of its taurine conju-
gate) was isolated from the bile of the Asian black bear
(Selenarctos thibetanus) (Bi et al., 2009). A bile acid hav-
ing the structure of 3α,7α,11α-trihydroxy-5β-cholan-24-oic
acid (as the taurine conjugate) was isolated from the biliary
bile of the sunfish (Mola mola) (Ishida et al., 1998).
For some years, we have had an ongoing program of
chemical synthesis of rare or potential bile acid metabolites
and thus to make them available as reference standards.
The conclusive evidence for the assigned structure is based
on NMR spectra and some cases on X-ray diffraction. As
an example of our work, we have recently reported the
preparation of 3α,9α-dihydroxy- and 3α,7α,9α-trihydroxy-
5β-cholan-24-oic acids (Iida et al., 2016).
All melting points (mp) were determined on a micro hot
stage apparatus and are uncorrected. 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 (tetramethylsilane, TMS)
as the solvent; chemical shifts were expressed in σ (ppm)
relative to TMS (0 ppm). The 13C distortionless enhance-
ment by polarization transfer (DEPT; 135 ꢁ, 90 ꢁ, and 45 ꢁ)
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
1
confirm the H and 13C signal assignments for some of the
1
1
compounds, H–1H correlation spectroscopy (COSY), H
nuclear Overhauser and exchange spectroscopy (NOESY),
1H detected heteronuclear multiple quantum coherence
1
1
(HMQC; H–13C coupling), and H detected heteronuclear
multiple bond connectivity (HMBC; long-range 1H–13C
coupling) experiments were also done. These 2D–NMR
spectra were recorded using standard pulse sequences and
parameters recommended by the manufacture.
Here, we report the successful synthesis of the two
hitherto unreported 11α-hydroxy-LCA (3α,11α-dihydroxy-
5β-cholan-24-oic acid, 1a) and 11-hydroxy-CDCA
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High-resolution liquid chromatograph-mass spectrome-
12α-OSO2CH3), 3.65 (s, 3H, 24-COOCH3), 4.69 (brm, 1H,
3β-H), 5.10 (m, 1H, 12β-H). 13C NMR (125.8 MHz,
CDCl3) σ: 12.5 (C-18), 17.8 (C-21), 21.6 (3-OCOCH3),
23.3 (C-19), 23.6, 25.9, 26.6, 27.1, 27.2, 27.6, 30.9, 31.1,
32.5, 34.1, 34.3, 34.9, 35.3, 35.9, 39.8 (12-OSO2CH3),
41.9, 46.1, 47.3, 48.8, 51.6 (C-25), 74.2 (C-3), 85.3 (C-12),
170.8 (3-OCOCH3), 174.6 (C-24). HR-LC/ESI-MS, Calcd
for C28H46O7SNa [M + Na]+, 549.2862; found, m/z
549.2862.
try with electrospray ionization (HR-LC/ESI-MS) source
was carried out using a AccuTOF JMS-T100LC liquid
chromatograph-mass spectrometer (Jeol, Tokyo, Japan)
coupled to a Agilent 1200 series binary pump (Agilent
Technologies Inc., Santa Clara, CA, USA) operated in the
positive or negative ion mode. The ionization conditions
were as follows: needle voltage, −2 kV in the positive and
negative ion modes; ionizatꢁion guide peak voltage, 2 kV;
ion source temperature, 80 C; desolvating plate tempera-
ture, 250 ꢁC; absolute ring-lens voltage, −15 V; orifice
voltage, 75 V, mass range m/z 50–1000; and nebulizing
gas, N2.
Methyl 3α,7α-Diacetoxy-12α-Mesyloxy-5β-Cholan-24-
Oate (5b)
The preparative reversed-phase high-performance liquid
chromatograph with refractive index detector (RP-HPLC/
RI) consisted of a Hitachi L-7100 pump (Tokyo, Japan),
Shodex RI-102 detector (Tokyo, Japan), and Sugai U620
column heater (Wakayama, Japan). Preparative RP-HPLC/
RI was carried out by isocratic elution on a Capcell Pak
MGII RP-C18 column (250 mm × 10 mm internal diameter
(I.D.); particle size, 5 μm; Shiseido, Tokyo, Japan) using a
mixture of methanol–water–acetic acid (80:20:0.05, v/v/v)
as the mobile phase at the flow rate 7.4 mL/min.
Normal-phase thin-layer chromatograph (TLC) was per-
formed on precoated Kieselgel 60F254 plates (E. Merck,
Darmstad, Germany) using EtOAc-hexane mixtures as the
developing solvent. Reversed-phase TLC was performed
on precoated RP-18F254s plates (E. Merck, Darmstad, Ger-
many) using methanol–water mixtures as the developing
solvent.
The 3α,7α-diacetoxy-12α-hydroxy ester 4b (2.0 g,
3.9 mmol), prepared in two steps from CA (2b), was sub-
jected to mesylation with methanesulfonylchloride
(3.2 mL, 41 mmol) and processed as described for the
preparation of 5a. The product was an oily residue. Chro-
matography of the oily residue on a silica gel column
(200 g) and elution with EtOAc-hexane (4:6, v/v) afforded
the 12α-mesyloxy derivative 5b, which was recrystallized
from EtOAc-hexane as colorless thin plates (a single spot
on TLC): mp, 137–139 C, yield, 2.14 g (93%). H NMR
(500 MHz, CDCl3) σ: 0.76 (s, 3H, 18-H3), 0.92 (s, 3H,
19-H3), 0.99 (d, 3H, J = 6.3 Hz, 21-H3), 2.02 (s, 3H, 3α-
OCOCH3), 2.08 (s, 3H, 7α-OCOCH3), 3.08 (s, 3H,
12α-OSO2CH3), 3.65 (s, 3H, 24-COOCH3), 4.56 (brm, 1H,
3β-H), 4.90 (m, 1H, 7β-H), 5.10 (m, 1H, 12β-H). 13C NMR
(125.8 MHz, CDCl3) σ: 12.4 (C-18), 17.8 (C-21), 21.6 (3-
OCOCH3), 22.7 (7-OCOCH3), 22.7 (C-19), 22.9, 26.8,
27.1, 27.4, 28.6, 30.9, 31.0, 31.3, 34.5, 34.8, 34.8, 35.2,
37.9, 39.8 (12-OSO2CH3), 41.0, 43.1, 46.1, 47.1, 51.6
(C-25), 70.6 (C-7), 74.1 (C-3), 84.6 (C-12), 170.5
(7-OCOCH3), 170.8 (3-OCOCH3), 174.5 (C-24). HR-LC/
ESI-MS, Calcd for C30H48O9SNa [M + Na]+, 607.2917;
found, m/z 607.2910.
1
ꢁ
Methyl 3α-Acetoxy-12α-Mesyloxy-5β-Cholan-24-
Oate (5a)
To methyl 3α-acetoxy-12α-hydroxy-5β-cholan-24-oate 4a
(2.0 g, 4.5 mmol), prepared from DCA (2a) in two steps
(methyl esterification and then partial acetylation), dis-
solved in dry pyridine (10 mL) and magnetically stirred,
methanesulfonylchloride (3.5 mL, 45 mmol) was added
dropwise. After stirring at room temperature for 16 h, the
dark pyridine solution was stirred, diluted with ice chips,
and extracted with EtOAc; the reaction was monitored by
TLC. The combined EtOAc extract was washed succes-
sively with cold, dilute HCl, NaHCO3, and water, decolor-
ized with Norite, dried with anhydrous MgSO4, and
evaporated under reduced pressure. The residue was chro-
matographed on a column of silica gel (150 g) eluting with
EtOAc-hexane (35:65, v/v). Recrystallization of the prod-
uct from EtOAc-hexane gave the title compound 5a as col-
Methyl 3α-Acetoxy-5β-Chol-11-Ene-24-Oate (6a)
To a solution of the 3α-acetoxy-12α-mesyloxy ester 5a
(1.5 g, 2.8 mmol) dissolved in hexamethylphosphoramide
(HMPA, 15 mL) was added potassium acetate (6 g). The
resulting solution was stirred for 48 h at 100 ꢁC. After
cooling at room temperature, the reaction was quenched by
adding water gradually and the product was extracted with
EtOAc. The combined EtOAc extract was washed with
10% HCl and saturated brine, dried with Drierite, and evap-
orated to dryness. Chromatography of the crude residue
over a column of silica gel (150 g) eluting with EtOAc-
hexane (2:8, v/v) resulted in a single component, which
was identified as the desired 3α-acetoxy-11-ene 6a, which
was recrystallized from EtOAc-hexane as colorless thin
plates (a single spot on TLC): mp, 118–120 ꢁC; yield,
ꢁ
orless thin plates (a single spot on TLC): mp, 104–105 C;
1
2.15 g (91%). H NMR (500 MHz, CDCl3) σ: 0.75 (s, 3H,
18-H3), 0.91 (s, 3H, 19-H3), 0.98 (d, 3H, J = 6.3 Hz,
21-H3), 2.03 (s, 3H, 3α-OCOCH3), 3.05 (s, 3H,
Lipids (2018)

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1
0.96 g (78%). H NMR (500 MHz, CDCl3) σ: 0.72 (s, 3H,
184–186 ꢁC; yield, 0.72 g, 90%. 1H NMR (500 MHz,
CDCl3) σ: 0.73 (s, 3H, 18-H3), 0.88 (s, 3H, 19-H3), 1.01
(d, 3H, J = 6.9 Hz, 21-H3), 3.61–3.65 (brm, 3H, 3β-H and
24-H2), 5.41 (dd, 1H, J = 1.2, 10.3 Hz, 11-H), 6.11 (dd,
1H, J = 2.9, 10.3 Hz, 12-H). 13C NMR (125.8 MHz,
CDCl3) σ: 16.8 (C-18), 18.8 (C-21), 23.1, 23.8 (C-19),
25.6, 28.2, 28.6, 29.5, 30.6, 31.8, 34.5, 35.1, 35.3, 36.3,
37.2, 41.2, 43.2, 45.1, 52.2, 53.7, 63.7 (C-24), 71.8 (C-3),
125.5 (C-11), 139.0 (C-12). HR-LC/ESI-MS, Calcd for
C24H40O2Na [M + Na]+, 383.2926; found, m/z 383.2897.
18-H3), 0.88 (s, 3H, 19-H3), 0.99 (d, 3H, J = 6.3 Hz, 21-
H3), 2.01 (s, 3H, 3α-OCOCH3), 3.66 (s, 3H, 24-COOCH3),
4.72 (brm, 1H, 3β-H), 5.39 (dd, 1H, J = 1.7, 10.3 Hz, 11-
H), 6.09 (dd, 1H, J = 3.4, 10.3 Hz, 12-H). 13C NMR
(125.8 MHz, CDCl3) σ: 16.7 (C-18), 18.4 (C-21), 21.5 (3-
OCOCH3), 23.1, 23.7 (C-19), 25.5, 26.7, 28.0, 28.5, 31.0,
31.1, 33.0, 34.4, 34.9, 35.1, 36.0, 41.0, 43.2, 45.1, 51.6 (C-
25), 52.0, 53.7, 74.3 (C-3), 125.5 (C-11), 138.9 (C-12),
170.8 (3-OCOCH3), 174.6 (C-24). HR-LC/ESI-MS, Calcd
for C27H42O4Na [M + Na]+, 453.2981; found, m/z
453.2969.
5β-Chol-11-Ene-3α,7α,24-Triol (7b)
Methyl 3α,7α-Diacetoxy-5β-Chol-11-Ene-24-Oate (6b)
The 3α,7α-diacetoxy-11-ene 6b (1.96 g, 4.0 mmol), sub-
jected to reductive cleavage with LAH in dry THF at room
temperature for 30 min and processed as described for the
preparation of 7a, afforded a crude reaction product. Chro-
matography of the product on a column of silica gel (50 g)
and elution with EtOAc-hexane (9:1, v/v)~EtOAc-
methanol (9:1, v/v) afforded the 3α,7α,24-trihydroxy-11-
ene 7b, which was recrystallized from methanol–water as
colorless thin plates (a single spot on TLC): mp,
175–177 ꢁC; yield, 1.43 g (95%). 1H NMR (500 MHz,
CD3OD) σ: 0.77 (s, 3H, 18-H3), 0.88 (s, 3H, 19-H3), 1.05
(d, 3H, J = 6.3 Hz, 21-H3), 3.40 (brm, 1H, 3β-H), 3.52
(brm, 2H, 24-H2), 3.91 (m, 1H, 7β-H), 5.50 (dd, 1H,
J = 1.7, 10.3 Hz, 11-H), 6.17 (dd, 1H, J = 2.9, 10.3 Hz,
12-H). 13C NMR (125.8 MHz, CD3OD) σ: 17.0 (C-18),
19.3 (C-21), 23.4, 23.9 (C-19), 29.7, 30.3, 31.4, 33.1, 36.4,
36.5, 36.7, 36.7, 37.6, 39.2, 41.1, 42.4, 46.1, 50.6, 53.3,
63.5 (C-24), 69.4 (C-7), 72.7 (C-3), 126.6 (C-11), 140.1
(C-12). HR-LC/ESI-MS, Calcd for C24H40O3Na
[M + Na]+, 399.2875; found, m/z 399.2853.
The 3α,7α-diacetoxy-12α-mesyloxy ester 5b (1.4 g,
2.4 mmol), subjected to the elimination reaction (reaction
time, 24 h) with potassium acetate in HMPA and processed
as described for the preparation of 6a, gave a crude prod-
uct. Chromatography of the product on a column of silica
gel (100 g) and elution with EtOAc-hexane (2:8, v/v)
afforded the 3α,7α-diactoxy-11-ene 6b, which was recrys-
tallized from EtOAc-hexane as colorless thin plates
(a single spot on TLC): mp, 142–143 ꢁC; yield, 0.95 g
(81%). 1H NMR (500 MHz, CDCl3) σ: 0.73 (s, 3H, 18-H3),
0.89 (s, 3H, 19-H3), 1.00 (d, 3H, J = 6.3 Hz, 21-H3), 2.02
(s, 3H, 3α-OCOCH3), 2.05 (s, 3H, 7α-OCOCH3), 3.66 (s,
3H, 24-COOCH3), 4.60 (brm, 1H, 3β-H), 4.96 (m, 1H, 7β-
H), 5.45 (dd, 1H, J = 1.7, 10.3 Hz, 11-H), 6.15 (dd, 1H,
J = 2.9, 10.3 Hz, 12-H). 13C NMR (125.8 MHz, CDCl3) σ:
16.8 (C-18), 18.4 (C-21), 21.5 (3-OCOCH3), 22.7
(7-OCOCH3), 22.5, 23.1 (C-19), 26.8, 28.5, 30.9, 31.0,
31.9, 34.9, 35.3, 35.3, 36.0, 36.4, 36.7, 40.3, 45.2, 49.3,
51.6 (C-25), 51.6, 70.4 (C-7), 74.0 (C-3), 124.9 (C-11),
139.2 (C-12), 170.5 (7-OCOCH3), 170.8 (3-OCOCH3),
174.7 (C-24). HR-LC/ESI-MS, Calcd for C29H44O6Na
[M + Na]+, 511.3036; found, m/z 511.2989.
5β-Cholane-3α,11α,24-Triol (8a) and 5β-Cholane-
3α,12α,24-Triol
A solution of the 3α,24-dihydroxy-11-ene 7a ꢁ(500 mg,
1.4 mmol) in dry THF (20 mL) was cooled to 0 C. Under
a N2 atmosphere, a solution of diborane complex (B2H6) in
THF (10 mL of 1.0 mol/L solution) was injected slowly to
the stirred solution of the olefin. The reaction mixture was
stirred for 16 h at room temperature; the reaction was mon-
itored by TLC. During ice-bath cooling and stirring, water
(1 mL), 3 N NaOH (10 mL), and 30% H2O2 (10 mL) were
added cautiously. After further stirring for 3 h at room tem-
perature, the mixture was neutralized with 10% HCl and
extracted with EtOAc. The combined extract was washed
with saturated brine, dried with Drierite, and evaporated to
dryness. The oily residue, which consisted of two compo-
nents, was separated by preparative RP-HPLC eluting with
a mixture of EtOAc methanol–water (8:2, v/v) as the devel-
oping solvent.
5β-Chol-11-Ene-3α,24-Diol (7a)
To a magnetically stirred solution of 3α-acetoxy-11-ene 6a
(0.96 g, 2.2 mmol) in dry tetrahydrofuran (THF) (20 mL)
was added gradually lithium aluminum hydride (LAH,
200 mg, 5.3 mmol) with ice-bath cooling. The mixture was
further stirred at room temperature for 30 min; the reaction
was monitored by TLC. The reaction was quenched by
adding gradually 30% H2O2 (0.2 mL), 3 N NaOH (0.6
mL), and water (0.6 mL). After evaporation of the solvents,
the residue was chromatographed on a column of silica gel
(25 g) eluting with EtOAc-hexane (7:3, v/v)ꢂEtOAc-meth-
anol (9:1, v/v). Recrystallization of the major product from
methanol–water gave the 3α,24-dihydroxy-11-ene 7a as
colorless thin plates (a single spot on TLC): mp,
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The more polar fraction was identified as the desired
(0.5 mL) was added 2,2,6,6-tetramethylpiperidine 1-oxyl
free radical (TEMPO; 5 mg, 30 μmol) and 0.2 M sodium
phosphate buffer (pH, 6.7; 0.5 mL). To this solution was
very slowly added 70% sodium chlorite (NaClO2, 80 mg,
0.62 mmol) dissolved in water (0.6 mL) and then 5% sodium
hypochlorite (bleach, NaClO, 15 μL) diluted in water
(0.3 mL). The mixture was further stirred at 35 ꢁC for 16 h,
with the course of the reaction being monitored by TLC. The
reaction was quenched by adding an ice-cold solution of sat-
urated Na2S2O3, and the product was extracted with EtOAc.
The combined EtOAc extract was washed with saturated
brine, dried with Drierite, and evaporated to dryness. The
desired product, 3α,11α-dihydroxy acid 1a, was purified by
passing through preparative RP-HPLC/RI eluting with
methanol–water-acetic acid (80:20:0.05, v/v/v) and recrystal-
lized from methanol–water as colorless needles (a single spot
on RP-TLC): mp, 103–106 ꢁC [lit. (Long & Gallagher,
1946), mp, 125–145 ꢁC]; yield, 96 mg (84%). 1H NMR and
13C NMR: see Table 1. HR-LC/ESI-MS, Calcd for
C24H39O4 [M-H]−, 391.2848; found, m/z 391.2890.
3α,11α,24-triol 8a and recrystallized from methanol–water
as colorless needles (a single spot on RP-TLC): mp,
109–112 ꢁC; yield, 226 mg (43%). 1H NMR and 13C
NMR: see Table 1. HR-LC/ESI-MS, Calcd for
C24H42O3Na [M + Na]+, 401.3032; found, m/z 401.3034.
The less polar fraction, which was eluted with the same
solvent system was identified as the 3α,12α,24-triol
ꢁ
(a single spot on RP-TLC): mp, 96–99 C (recrystallized
from methanol–water); yield, 236 mg (45%). 1H NMR
(500 MHz, CDCl3) σ: 0.67 (s, 3H, 18-H3), 0.89 (s, 3H, 19-
H3), 0.97 (d, 3H, J = 6.9 Hz, 21-H3), 3.58–3.62 (brm, 3H,
3β-H and 24-H2), 3.98 (m, 1H, 12β-H). 13C NMR
(125.8 MHz, CDCl3) σ: 12.8 (C-18), 17.8 (C-21), 23.2
(C-19), 23.8, 26.2, 27.2, 27.7, 28.6, 29.6, 30.5, 31.9, 33.7,
34.2, 35.3, 35.5, 36.1, 36.5, 42.2, 46.6, 47.6, 48.3, 63.6
(C-24), 71.9 (C-3), 73.3 (C-12). HR-LC/ESI-MS, Calcd for
C24H42O3Na [M + Na]+, 401.3032; found, m/z 401.2984.
5β-Cholane-3α,7α,11α,24-Tetrol (8b) and 5β-Cholane-
3α,7α,12α,24-Tetrol
3α,7α,11α-Trihydroxy-5β-Cholan-24-Oic Acid (1b)
The 3α,7α,24-trihydroxy-11-ene 7b (500 mg, 1.3 mmol)
was subjected to the hydroboration/oxidation reaction with
B2H6-NaOH/H2O2 and processed as described for the prep-
aration of 8a. The procedure gave a mixture of two compo-
nents, which were separated by RP-HPLC/RI.
The first, eluted with a mixture of methanol–water (8:2,
v/v), consisted of a homogeneous fraction that was recrys-
tallized from methanol–water as colorless needles and char-
acterized as the desired 3α,7α,1ꢁ1α,24-tetrol 8b (a single
spot on RP-TLC): mp, 212–213 C; yield, 210 mg (40%).
1H NMR and 13C NMR: see Table 1. HR-LC/ESI-MS,
Calcd for C24H42O4Na [M + Na]+, 417.2981; found, m/z
417.3006.
The 3α,7α,11α,24-triol 8b (220 mg, 0.58 mmol) was sub-
jected to selective oxidation with TEMPO/NaClO2/NaClO
at 35 ꢁC for 16 h and processed as described for the prepa-
ration of 1a to yield a crude 3α,7α,11α-trihydroxy acid 1b.
Preparative RP-HPLC/RI of the crude 1b and elution with
methanol–water–acetic acid (80:20:0.05) afforded the ana-
lytically pure compound, which was recrystallized from
methanol–water as colorless needles (a single spot on RP-
1
ꢁ
TLC): mp, 203–206 C; yield, 188 mg (82%). H NMR
and 13C NMR: see Table 1. HR-LC/ESI-MS, Calcd for
C24H39O5 [M-H]−, 407.2798; found, m/z 407.2799.
The second fraction, which was eluted with the same sol-
vent system, was characterized as the 3α,7α,12α,24-tetrol
(a single spot on RP-TLC): mp, 232–233 ꢁC (recrystallized
from methanol–water); yield, 225 mg (43%). 1H NMR
(500 MHz, CD3OD) σ: 0.72 (s, 3H, 18-H3), 0.92 (s, 3H,
19-H3), 1.02 (d, 3H, J = 6.3 Hz, 21-H3), 3.37 (brm, 1H,
3β-H), 3.51 (brm, 2H, 24-H2), 3.80 (m, 1H, 7β-H), 3.96
(m, 1H, 12β-H). 13C NMR (125.8 MHz, CD3OD) σ: 13.0
(C-18), 18.0 (C-21), 23.2 (C-19), 24.2, 27.9, 28.8, 29.6,
30.4, 31.2, 33.2, 35.8, 35.9, 37.1, 40.5, 41.0, 43.0, 43.2,
47.4, 48.3, 49.8, 63.6 (C-24), 69.1 (C-7), 72.9 (C-3), 74.1
(C-12). HR-LC/ESI-MS, Calcd for C24H42O4Na
[M + Na]+, 417.2981; found, m/z 417.2987.
Results and Discussion
Nuclear 11α-hydroxylation of bile acids in the liver of the
sunfish (Mola mola) is the result of specific Cytochrome
P450 (CYP)-mediated hydroxylation. According to Ishida
et al. (1998), the sunfish has gained an enzyme that can
introduce an α-hydroxyl group directly at C-11 of the 5β-
cholestane skeleton to produce the 11α-hydroxy-bile salts.
Another possibility is that the sunfish can catalyze 11β-
hydroxylation of the 5β-cholestane skeleton and then
convert the configuration of the product from β to α with
the help of intestinal flora, as in the case of formation of
ursodeoxycholic acid (UDCA) from CDCA.
3α,11α-Dihydroxy-5β-Cholan-24-Oic Acid (1a)
A need for a moderate supply of 1a and 1b as reference
specimens prompted us to examine prior literature prepara-
tions of the compounds. Microbial biotransformation
To a magnetically stirred solution of the 3α,11α,24-triol 8a
(110 mg, 0.28 mmol) in dry THF (2 mL) and CH3CN
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Fig. 2 Synthetic route to 11α-hydroxy-bile acids from DCA and CA
(Bhatti & Khera, 2012; Bortolini, Medici, & Poli, 1997)
has been an important tool for structural modification of
such steroids as 11-deoxycortisol and progesterone to the
corresponding 11α-hydroxy steroids (Kolet, Niloferjahan,
Haldar, Gonnade, & Thulasiram, 2013; Petric, Hakki,
Bernhardt, Zigon, & Cresnar, 2010; Swizdor, Panek, &
Milecka-Tronina, 2014). Although the simultaneous
11α,15β- and 11β,15β-dihydroxylations of LCA have been
reported by a fungus Cunninghamella blakesleeana ST-22
(Jodoi, Nihira, Naoki, Yamada, & Taguchi, 1987), such
biological transformation is severely restricted for the selec-
tive 11α-hydroxylation in bile acids.
Meanwhile, 3α,11α-dihydroxy-5β-cholan-24-oic acid
(1a), one of the earliest 11α-hydroxy-bile acids known, has
been synthesized chemically by several lengthy and low-
yield procedures (Gallagher & Long, 1946; Long & Galla-
gher, 1946; Turner, Mattox, Engel, Mckenzie, & Kendall,
1946). The first synthetic route involved the Wolff-Kish-
ner/isomerization reaction of the 12-hydrazone derivative
of methyl 3α,11β-diacetoxy-12-keto-5β-cholan-24-oate
(Long & Gallagher, 1946) or its 3α,11β-dihydroxy acid
(Gallagher & Long, 1946). The second approach consisted
of the catalytic hydrogenation of 3α-hydroxy-11-keto-5β-
cholanoic acid, which was obtained from methyl 3α-
hydroxy-11-keto-12ξ-bromo-5β-cholanate (Turner et al.,
1946). However, the two routes were less straightforward
and inelegant than indicated in the literatures; very low
yields of 1a were obtained only after a laborious multistep
process. Our simplified method for preparing 11α-hydroxy-
bile acids (1a and 1b), which proceeds through easily
prepared, well-defined intermediates, affords pure products
in good isolated yields.
As outlined in Fig. 2, the key intermediates in our work
11
11
were the 4 -3α,24-diol and 4 -3α,7α,24-triols (7a and
7b), which were prepared in four steps starting from DCA
(2a) and CA (2b). Thus, selective acetylation of the methyl
esters (3a and 3b) of 2a and 2b under mild conditions
resulted in the formation of the partially acetylated com-
pounds at C-3 and C-7 (4a and 4b) (Fieser & Rajagopalan,
1950; Iida, Tamura, Matsumoto, & Chang, 1985), respec-
tively, which in turn were treated with methanesulfo-
nylchloride to yield the corresponding 12α-mesyloxy
derivatives (5a and 5b) nearly quantitatively. When 5a or
5b was subjected to deoxygenation with potassium acetate
11
in HMPA at 100 ꢁC, a single olefinic product, 4 -3α-
11
acetate or 4 -3α,7α-diacetate ester (6a or 6b), was
1
obtained in a good isolated yield of 78 (or 81%). In the H
NMR spectra, the olefinic proton signals occurred at σ 5.39
(dd, 11-H) and 6.09 (dd, 12-H) in 6a and at σ 5.45 (dd, 11-
H) and 6.15 (dd, 12-H) in 6b, suggesting that an unsatu-
rated bond was introduced between the C-11 and C-12.
11
Additional evidence in support of the presence of the 4 -
bond was also provided by the 13C NMR signals arising
from the C-11 at σ 125.5 and the C-12 at σ 138.9 in 6a and
the C-11 at σ 124.9 and the C-12 at σ 139.2 in 6b.
Subsequent treatment of 6a and 6b with LAH at room
temperature resulted in simultaneous reduction/hydrolysis
11
at C-3/C-7 and C-24 to afford the key intermediates, 4 -
11
3α,24-diol (7a) and 4 -3α,7α,24-triol (7b), in high yields
1
(90 and 95%), respectively. In the H NMR spectrum of
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1
1
7b, the H signals appearing at σ 5.50 (dd, 11-H) and 6.17
to the C-12 (σ 52.6). The H/13C correlation peaks in the
(dd, 12-H), together with at σ 3.40 (brm, 3β-H), 3.91 (m,
7β-H), and 3.52 (brm, 24-H2), supported the evidence of
the structure of 7b. In addition, the 13C signals in 7b occur-
ring at σ 126.6 (C-11) and 140.1 (C-12), along with at σ
72.7 (C-3), 69.4 (C-7), and 63.5 (C-24), provided a confir-
matory evidence. Essentially, similar 1H and 13C NMR
characteristics were observed in 7a.
HMQC were observed between the 12α-H (σ 1.28)/C-12
1
and 12β-H (σ 2.23)/C-12. The H/1H correlation peaks in
the COSY were detected between the 12α-H/11β-H
(σ 3.79, brm), 12β-H/11β-H, and 9α-H (σ 1.56)/11β-H,
indicating the presence of a hydroxy group at the C-11
position. The stereochemical configuration of an equato-
rially oriented 11α-hydroxy group in 1a was confirmed by
measuring the NOESY, in which the distinct correlation
peaks were detected between the 1,3-diaxially oriented 18-
H3 (σ 0.70, s)/11β-H and 19-H3 (σ 1.06, s)/11β-H as well
as the spatially closed 12β-H/11β-H. Essentially, similar 1H
and 13C NMR correlations were also observed in 1b, and
their characteristics in the 5β-steroid nucleus were very
similar to those reported for naturally occurring 5β-choles-
tane-3α,7α,11α,26,27-pentol and 3α,7α,11α-trihydroxy-5β-
cholanoic acid taurine conjugate (Ishida et al., 1998).
When 7a and 7b were subjected to the hydroboration/
oxidation with B2H6 and H2O2/NaOH, the isolated
products were the 3α,11α,24-triol (8a, 43%) and
3α,7α11α,24-tetrol (8b, 40%), accompanied by the simulta-
neous formation of the 3α,12α,24-triol (45%) and
3α,7α12α,24-tetrol (43%), respectively. The result implies
that anti-Markownikoff’s attack of B2H6 on the disubsti-
11
tuted 4 -bond occurred from the less sterically hindered
α-side and gave roughly equivalent amounts of the two
possible 11α- and 12α-hydroxy isomers, which were sepa-
rated efficiently by RP-HPLC/RI; no more sterically
crowded 11β- and 12β-hydroxy compounds were produced.
The 1H NMR spectra of 8a exhibited the 1H signals arising
from the 3β-H at σ 3.68 (brm), the 11β-H at σ 3.85 (brm)
and the 24-H2 at σ 3.61 (brm). On the other hand, the 13C
signals appeared for the methine C-3 at σ 72.2, the methine
C-11 at σ 69.6 and the methylene C-24 at σ 62.2 in the
The most promising method for preparing 11α-hydroxy-
bile acids would seem to be the application of the so-called
anti-Markownikoff hydroxylation with B2H6 and H2O2/
11
NaOH of appropriate 49(11)- or 4 -5β-cholene deriva-
tives. Nussim, Mazur, and Sondheimer (1964) reported that
the hydroxylation of 49(11)-5α-steroids with A/B-trans
junction yielded almost exclusively the corresponding 11α-
alcohols; however, the reaction did not proceed at all for
1
DEPT experiment. Essentially, identical H and 13C NMR
4
9(11)-5β-steroids with A/B-cis junction; on the other hand,
the reaction of 4 -5α-steroids yielded the corresponding
11
spectra were observed in 8b.
Recently, successful use of NaClO2 catalyzed by
TEMPO and NaClO for oxidation of alcohols suggested
that a selective oxidation of primary hydroxy group to its
carboxyl group without oxidizing a secondary hydroxy
group might be feasible (Anelli, Biffi, Montanari, & Quici,
1987; de Nooy, Besemer, & van Bekkum, 1996; Khripach
et al., 2005; Zhao et al., 1999). Expectedly, mild reaction
of 8a and 8b with NaClO2 in the presence of the coreagents
resulted in selective oxidation of the primary 24-hydroxy
group to afford the desired 3α,11α-dihydroxy and
3α,7α,11α-trihydroxy acids (1a and 1b) in excellent iso-
lated yields of 84 and 82%, respectively.
11α- and 12α-alcohols in a ratio of 1:1. In preliminary
experiments, we found that 11α-hydroxylation of 4
9(11)
-
5β-cholen-3α,24-diol (Iida et al., 2016) was unsuccessful
under various conditions, in analogy with the previous find-
ing of other 49(11)-5β-steroids (Nussim et al., 1964). Fur-
thermore, an attempted reductive cleavage with AlH2Cl
(Iida et al., 2016) of methyl 3α-acetoxy-11α,12α-epoxy-5β-
cholan-24-oate, which was easily prepared from 6a inter-
mediate, failed to afford 5β-cholan-3α,12α,24-triol as the
major product. Hydroxylation of 6a, instead of 7a, was also
unsatisfactory, yielding a complex mixture of products,
probably because of the simultaneous formation of partially
hydrolyzed products at C-3/C-7 and C-24 by the alkaline
reagent. Therefore, the use of 7a and 7b as substrates is
essential for the 11α-hydroxylation with B2H6 and
NaOH/H2O2.
1
Table 1 shows the complete H and 13C NMR signal
assignments for 11α-hydroxylated compounds (8a, 8b, 1a,
and 1b). The presence and stereochemical configuration of
the 11α-hydroxy group in the compounds were determined
1
by a combined use of the DEPT and several H/1H and
In conclusion, the availability of 3α,11α-dihydroxy-5β-
cholan-24-oic acid (1a) and 3α,7α,11α-trihydroxy-5β-cho-
lan-24-oic acid (1b) as standards should facilitate their
identification when present in vertebrates.
1H/13C shift correlated 2D–NMR techniques, which
include COSY, NOESY, HMQC, and HMBC. For exam-
ple, the structure of 1a was confirmed as follows. Thus, the
13C signals appearing at σ 72.8 and 69.9 in the DEPT
experiment, together with the 1H signals occurring at σ
3.57 (3β-H, brm) and 3.79 (11β-H, brm), were assigned to
the C-3 and C-11 methine carbons bearing a hydroxy
Acknowledgements This work was supported in part by a Grant-in-
Aid for Scientific Research (C) from the Japan Society of Promotion
of Science (to T.I., 15 K01809) for 2015-2017. Work at University
California, San Diego was also supported in part by a grant from the
American Physiological Society.
1
group. In the HMBC, the appearance of the H/13C corre-
lated peak arising from the 18-H3 (σ 0.70, s) was assigned
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Conflict of interest We declare no conflict of interest.
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