An efficient synthesis of 7α,12α-dihydroxy-4-cholesten-3-one and its biological precursor 7α-hydroxy-4-cholesten-3-one: Key intermediates in bile acid biosynthesis

Article abstract of DOI:10.1016/j.steroids.2013.05.011

This paper describes a method for the chemical synthesis of 7α,12α-dihydroxy-4-cholesten-3-one (1a) and its biological precursor, 7α-hydroxy-4-cholesten-3-one (1b), both of which are key intermediates in the major pathway of bile acid biosynthesis from cholesterol. The principal reactions involved were (1) building of the cholesterol (iso-octane) side chain by 3-carbon elongation of the cholane (iso-pentane) one, (2) oxidation sequence to transform the 3a-hydroxy group of the steroidal A/B-ring to the desired 4- en-3-one system, and (3) appropriate protection strategy for hydroxy groups in the positions at C-7 and C-12 in the steroid nucleus. The absolute structure of 1a and 1b were confirmed by NMR and X-ray crystallography. The targeted compounds 1a and 1b, prepared in 11 steps from 2a and 2b respectively, should be useful for biochemical studies of bile acid biosynthesis or clinical studies of bile acid metabolism, as plasma levels of 1b (also termed C4) have been shown to correlate highly with the rate of bile acid biosynthesis in man.

Full text of DOI:10.1016/j.steroids.2013.05.011

Steroids 78 (2013) 927–937
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Steroids
journal homepage: www.elsevier.com/locate/steroids
An efficient synthesis of 7a,12a-dihydroxy-4-cholesten-3-one
and its biological precursor 7a-hydroxy-4-cholesten-3-one:
Key intermediates in bile acid biosynthesis q
Shoujiro Ogawa a, Biao Zhou b, Yusuke Kimoto b, Kaoru Omura b, Akiko Kobayashi b, Tatsuya Higashi a,
Kuniko Mitamura c, Shigeo Ikegawa c, Lee R. Hagey d, Alan F. Hofmann d, Takashi Iida b,
⇑
a Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510, Japan
b Department of Chemistry, College of Humanities & Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan
c Faculty of Pharmaceutical Sciences, Kinki University, Kowakae, Higashi-Osaka 577-8502, Japan
d Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0063, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes a method for the chemical synthesis of 7
and its biological precursor, 7a-hydroxy-4-cholesten-3-one (1b), both of which are key intermediates
in the major pathway of bile acid biosynthesis from cholesterol. The principal reactions involved were
(1) building of the cholesterol (iso-octane) side chain by 3-carbon elongation of the cholane (iso-pentane)
a
,12
a
-dihydroxy-4-cholesten-3-one (1a)
Received 20 December 2012
Received in revised form 10 April 2013
Accepted 16 May 2013
Available online 23 May 2013
one, (2) oxidation sequence to transform the 3a-hydroxy group of the steroidal A/B-ring to the desired 4-
en-3-one system, and (3) appropriate protection strategy for hydroxy groups in the positions at C-7 and
C-12 in the steroid nucleus. The absolute structure of 1a and 1b were confirmed by NMR and X-ray crys-
tallography. The targeted compounds 1a and 1b, prepared in 11 steps from 2a and 2b respectively, should
be useful for biochemical studies of bile acid biosynthesis or clinical studies of bile acid metabolism, as
plasma levels of 1b (also termed C4) have been shown to correlate highly with the rate of bile acid bio-
synthesis in man.
Keywords:
Bile acid biosynthesis
7
a
a
-Hydroxy-4-cholesten-3-one
,12a-Dihydroxy-4-cholesten-3-one
7
Hydroxy-D5 steroid dehydrogenase,
Cholesterol oxidase
X-ray analysis
Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved.
1. Introduction
sequential 7
ylase, Cyp7A1) followed by C-3 oxidation and concomitant double
bond migration (catalyzed by a C27 3b-hydroxy-
5-C27-steroid oxi-
doreductase, HSD3B7) to form 7 -hydroxy-4-cholesten-3-one (1b),
which is a precursor of CDCA (2b) [1c]. The transformation of 1a
catalyzed by the microsomal steroid 12 -hydroxylase Cyp8b1
leads to the formation of 7 ,12 -dihydroxy-4-cholesten-3-one
a-hydroxylation (catalyzed by cholesterol 7a-hydrox-
Bile acids are synthesized by the liver from cholesterol by a
complex series of reactions involving at least 14 enzymatic steps
[1a,1b]. In humans, a failure of any of these enzymatic reactions
will result in accumulation of intermediates in the bile acid biosyn-
thesis pathway, as well as a deficiency in the normal primary bile
acids, cholic acid (CA; 2a) and chenodeoxycholic acid (CDCA; 2b).
Defective bile acid biosynthesis presents clinically as liver disease
in infants and may be fatal unless treated by bile acid replacement
therapy; such therapy is life-saving [2,3].
D
a
a
a
a
(1a), which is ultimately converted to CA (2a) [1d].
The two unsaturated oxysterols (1a and 1b) are thus pivotal
biosynthetic intermediates in the so-called ‘‘neutral’’ pathway of
primary bile acid biosynthesis and are of sustained interest in
the biosynthetic, biological, physiological, and metabolic studies
of bile acids [3]. In addition, the plasma level of 1b has been shown
to correlate highly with the rate of bile acid biosynthesis [4], and is
thus a useful non-invasive biomarker for detecting increased bile
acid biosynthesis. Such a determination is useful in patients with
idiopathic diarrhea to determine whether bile acid malabsorption
is present, as the compensatory increase in bile acid synthesis
may lead to chronic diarrhea, because of bile acid induced secre-
tion in the large intestine [5].
As shown in Fig. 1, the initial biosynthetic transformation of
cholesterol to the primary bile acids (2a and 2b) involves
Abbreviations: CYP7A1, cholesterol 7
27-steroid oxidoreductase; TEMPO, 2,2,6,6-tetramethylpiperidine 1-oxyl free rad-
a -
-hydroxylase; HSD3B7, 3b-hydroxy-D5
C
ical; IBX, 2-iodoxybenzoic acid.
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
For the accurate analysis of 1a and 1b in plasma, authentic
reference compounds are needed. Although 1b is available from
⇑
Corresponding author. Tel.: +81 3 5317 9365.
E-mail address: takaiida@chs.nihon-u.ac.jp (T. Iida).
0039-128X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.05.011

928
S. Ogawa et al. / Steroids 78 (2013) 927–937
H
CYP7A1
H
H
HO
HO
OH
Cholesterol
HSD3B7
OH
CYP8B1
O
OH
O
OH
7α-Hydroxy-4-cholesten-3-one (1b)
7α,12α-Dihydroxy-4-cholesten-3-one (1a)
AKR1D1
AKR1D1
OH
O
OH
O
OH
H
H
side chain oxidation
OH
COOH
COOH
HO
OH
HO
OH
H
H
CDCA (2b)
CA (2a)
CYP7A1: Cholesterol 7α-hydroxylase/ HSD3B7: 3β-Hydroxy-Δ5-C27-steroid oxidoreductase
CYP8B1: Sterol 12α-hydroxylase/ AKR1D1: Δ4-3-Ketosteroid 5β-reductase
Fig. 1. Major biosynthetic pathway from cholesterol of the two primary bile acids in man – CA (2a) and CDCA (2b).
commercial sources, it is extremely expensive. 1a is not marketed.
To our knowledge, however, the chemical synthesis of 1a and 1b,
preparation of D a-hydroxy steroids [7]. In preliminary
4-3-oxo-7
work, we developed an improved enzymatic method for the prepa-
ration of 1b and its 7b-isomer, by the route shown in Fig. 2. However,
such an enzymatic synthesis is expensive, does not yield a pure
product, and is useful only on a micromolar scale. Furthermore, a
major shortcoming of this enzymatic synthesis is its inability to gen-
which have a D a-hydroxy structure in the steroid nucleus
4-3-oxo-7
and the iso-octane (cholestane) side chain at the C-17 position, has
not yet been reported. During the course of our studies on bile acid
metabolism in vertebrates, we required 1a and 1b as authentic ref-
erence standards. We report here the chemical synthesis of 1a and
1b using inexpensive reagents and readily available steroid sources.
Very recently, Björkhem have reviewed his five decades with oxys-
terols, particularly for 1b [Biochimie 2013;95:448-54].
erate 7a,12a-dihydroxy-4-cholesten-3-one (1a), the metabolic pre-
cursor of cholic acid, a major primary bile acid in vertebrates.
Thus, we described herein an alternative, non-enzymatic chem-
ical preparation of 1a and 1b, starting from easily available com-
mercial materials.
2. Results and discussion
2.2. Chemical synthesis
2.1. Enzymatic synthesis
Wereportheresynthesisof1a, thetargetcompound, from 2ain11
steps, according to the route shown in Fig. 3. The principal steps con-
sisted of (1) the elongation (by three carbon atoms) of the C5 cholane
(iso-pentane) side-chain in 2a to the C8 cholestane (iso-octane) side
chaininfive steps and (2)the transformation of the A/B-ring structure
Alexander and Fisher reported some years ago a convenient
enzymatic synthesis of 7
hydroxypropyl-b-cyclodextrin-facilitated cholesterol oxidase oxi-
dation of 5-cholestene-3b,7 -diol (7 -hydroxycholesterol) [6]. In
a-hydroxy-4-cholesten-3-one (1b) by the
a
a
this enzymatic synthesis using cholesterol oxidase (from Brevibacte-
rium sp.) and catalase, there is a simultaneous oxidation of the
3b-hydroxy group to the 3-oxo group and migration of the double
into the 3-oxo-D a,12a-dihydroxy steroid nucleus in six steps.
4-7
Our initial effort was directed to the construction of the choles-
tane side-chain from 2a. Reduction of the terminal carboxyl group
to the corresponding primary alcohol is usually carried out with
bond from D5 to
D
4; this procedure has been used for the

S. Ogawa et al. / Steroids 78 (2013) 927–937
929
H
H
H
AcO
AcO
O
(i)
HO
OH
O
OH
1b
Reagents and conditions: (i) hydropropyl-β-cyclodextrin/ cholesterol oxidase/ catalase/ phosphate
buffer pH 7/H2O, at 20 oC for 24 h.
Fig. 2. Enzymatic synthesis of 1b from cholesterol.
R
R3
H
R
CH2OH
COOH
(b)
CHO
(c)
H
H
R2
AcO
OAc
R1
AcO
H
OAc
H
H
R1 R2 R3
H (2a)
=
=
=
=
R
R
OAc (4a)
H (4b)
OAc (5a)
H (5b)
H
H
O
O
O
H
H
H
(2b)
O
O
R1 R2 R3
(a)
c
c
c
c (3a)
(3b)
OA OA OA
c
H
OA OA
R
R
(d)
(e)
AcO
OAc
AcO
OAc
H
H
=
=
=
R
R
OAc (6a)
H (6b)
OAc (7a)
H (7b)
=
R
R
R
(f)
(g)
(h)
c
A
O
O
OAc
H
O
O
OH
H
H
H
=
=
=
=
R
Ac (9a)
R
Ac (8a)
O
O
=
=
R
H (10a)
O
H (9b)
H (8b)
H (10b)
R
R
R
(i)
(j)
(k)
H
O
O
O
OCHO
O
OCHO
H
=
=
=
=
R
=
=
R
R
OH (1a)
H (1b)
OCHO (11a)
H (11b)
OCHO (12a)
H (12b)
pyridine, at 40 oC for 1 h.
Reagents and conditions: (a) acetic anhydride/ 4-dimethylaminopyridine/
(b) NaBH Et N/ ethyl
/
4
3
chloroformate/ THF, at 0 o for 2 h. (c) 2,2,6,6-tetramethylpiperidine 1-oxyl free radical/ KBr/ NaOCl aq./ Na CO aq./
C
2
3
NaHCO3 aq. buffer (pH 8.6), at 0 oC for 20 min. (d) isopropyltriphenylphosphonium iodide/ 1.6M n-BuLi in hexane/ THF,
at 0 oC for 30 min, then tris[2-(2-methoxyethoxy)ethyl]amine/ THF, at 0 oC for 15 min. (e) H2/ 10% Pd/C/ MeOH:AcOH:
o
EtOAc (10:10:1), at r.t. for 3 h. (f) HCl/ MeOH, at
for 6 h. (g) CrO
acetone/ CH Cl , at 0 o C for 30 min.
H SO /
4 2 2
30 C
/
3
2
(h)
at r.t. for 1 h. (j) IBX/ trifluoroacetic acid/ DMSO at 40 oC for 40 h.
10% KOH/ MeOH, at r.t. 16 h. (i) HCOOH/ HClO
,
4
,
(k) 0.2N NaOH aq., at r.t. 30 min.
Fig. 3. Synthetic route to the target compounds (1a and 1b) from bile acids (2a and 2b).

930
S. Ogawa et al. / Steroids 78 (2013) 927–937
LiAlH4 under strong alkaline conditions. However, this reduction
technique has a disadvantage, as it also causes partial or complete
hydrolysis of the acetyl protecting groups at C-3, C-7 and C-12 in
3a. Therefore, we explored the use of less basic NaBH4, combined
with ethyl chloroformate and triethylamine (Et3N) [8]. Thus, CA
peracetate, 3a, prepared from 2a by the usual method, was treated
with NaBH4/ethyl chloroformate/Et3N in dry THF to give the corre-
Recently the report of a new, powerful dehydrogenation oxi-
dant, o-iodoxybenzoic acid (IBX), prompted us to apply this re-
agent for the preparation of the targeted 12a from 11a. Nicolaou
et al. [12] have reported that IBX, a readily available hypervalent
iodine (V) reagent, oxidizes ketones efficiently to the conjugated
enones or dienones, and benzylamines to the amines. In addition,
IBX was found to be highly effective in mediating dehydrogena-
sponding 24-hydroxy-3
a,7a
,12a-triacetate 4a. As expected, the
tions adjacent to carbonyl functionalities (to form a,b-unsaturated
reduction reaction proceeded smoothly via the anhydride deriva-
tive of 3a as an intermediate without hydrolysis of the acetyl
groups.
carbonyl compounds) as well as at benzylic and related carbon
centers (to form conjugated aromatic carbonyl systems). In the
IBX-mediated dehydration, some 3-oxo-5a-steroids (trans A/B-ring
Preliminary experiments revealed that the conversion of 4a to
the corresponding 3a,7a,12a-triacetoxy-24-aldehyde (5a) by the
juncture) have been shown to be converted to the corresponding 3-
oxo-D1 enones or 3-oxo-D1,4 dienones, depending on the experi-
mental conditions. However, Li and Tochtrop [13] have recently re-
ported that IBX smoothly and regioselectively performs the
dehydrogenation of 3-oxo-5b-steroids (cis A/B-ring juncture) to
give the 3-oxo-D4 enones, probably owing to the stereochemical
difference of the A/B-rings in the steroid nucleus.
When 11a was treated with freshly prepared IBX (see Section 3)
[14] in DMSO containing trifluoroacetic acid at 40 °C for 40 h, the
dehydrogenation reaction took place as expected at the hydrogen
use of pyridinium chlorochromate (PCC) and pyridinium dichro-
mate (PDC) as oxidants was unsatisfactory; the yield was less than
60% and required tedious purification of the desired product by
chromatography. However, when 4a was subjected to oxidation
with sodium hypochlorite (NaClOÁnH2O) in the presence of cata-
lytic amounts of 2,2,6,6-tetramethylpiperidine1-oxyl (as free radi-
cal; TEMPO) in a NaHCO3 buffer solution [9], mild, selective
oxidation of the C-24 primary alcohol 4a took place, to afford the
corresponding C-24 aldehyde 5a in good isolated yield of 79%.
Subsequent Wittig reaction of 5a with isopropylphenylphos-
phonium iodide in the presence of n-BuLi at 0 °C for 15 min,
atoms attached to C-4 and C-5 to give the desired
ketone, 7 ,12 -diformyloxy-4-cholesten-3-one (12a), in moderate
yield (50%), without being accompanied by the elimination of the
-formyl group. Also, the undesirable products – 3-oxo-D1
7,12-diformate and 3-oxo-
1,4-7,12-diformate – as well as the
a,b-unsaturated
a
a
yielded the unsaturated
D
24-3,7,12-triacetate 6a having the C8
7a
-
branched side chain. Although the basic conditions resulted con-
currently in partial or complete deprotection of the acetyl groups
at C-7 and C-12 in 6a, these mixtures were re-acetylated without
difficulty. Catalytic hydrogenation of 6a using 10% Palladium on
carbon (Pd/C) catalyst in a solvent mixture of EtOAc–methanol–
acetic acid under slightly positive pressure proceeded smoothly
D
other by-products, were not formed at all. Based on a suggestion
of Nicolaou et al. [12], a possible ionic-mechanism for the dehydro-
genation of 11a by IBX suggests that the reaction is initiated by sin-
gle electron transfer from the substrate 11a to IBX to form a radical
cation which reacts further to give the desired 12a.
to afford 5b-cholestan-3
a
,7
a
,12
a
-triacetate (7a) exclusively. Thus,
We found that the use of commercially available stabilized IBX
(SIBX), which contains 55 wt.% of 2-iodobenzoic acid as a stabilizer,
was unsatisfactory, because it lacked reproducibility, and often re-
sulted in a complicated mixture of products. Isolation of 12a was
tedious and time-consuming, and as a result, was obtained in
low yield.
the elongation of the C5 side chain by three carbon atoms to form
the C8 cholestane side chain was accomplished in 5 steps from 2a;
the total yield of 7a from 2a was ca. 40%.
The C27 triacetate 7a, when treated with methanolic HCl at
30 °C, was deprotected regioselectively at the equatorially-ori-
ented 3
-ol (8a), Subsequent oxidation of 8a with Jones reagent, fol-
lowed by complete alkaline hydrolysis (using 10% methanolic
KOH) of the resulting 3-oxo-7 ,12 -diacetoxy derivative (9a) pro-
,12 -dihydroxy-5b-cholestan-3-one
a
-acetyl group to yield 7
a
,12
a
-diacetoxy-5b-cholestan-
In the final step, the usual alkaline hydrolysis of 12a with aque-
ous NaOH at room temperature for 30 min gave the targeted
7a,12a-dihydroxy-4-cholesten-3-one (1a). In this hydrolysis reac-
3a
a
a
tion of 12a, the elimination product – 3-oxo-4,6-diene – was pro-
duced (2%). On the other hands, when 12b was treated with
aqueous NaOH, 3-oxo-4,6-diene was detected 2% as by-product
(yields were established by HPLC). Thus, our successful strategy
for obtaining the desired changes in the steroid nucleus was based
on solving two problems – first was selection of the appropriate
protecting groups for the hydroxy groups of 2a and 2b and the sec-
ond was the use of a modified IBX as the dehydrogenation reagent.
ceeded cleanly to give
7
a
a
(10a) nearly quantitatively.
The choice of a suitable protecting group for the 7a- and 12a-
hydroxy groups in 10a was an essential factor for subsequent
reactions. In our exploratory works, attempted dehydrogenation
of 9a with IBX (see below) was successful, but subsequent hydro-
lysis of the resulting 7
Under the various alkaline hydrolysis conditions that were exam-
ined, allylic elimination of the 7 -acetyl group took place to give
a mixture of 12 -hydroxy-4,6-cholestadien-3-one and 12 -acet-
oxy-4,6-cholestadien-3-one as the major products. To prevent
the elimination, we protected the 7 - and 12 -hydroxy groups
a,12a-diacetoxy-4-cholesten-3-one failed.
Analogous 7a-hydroxy-4-cholesten-3-one (1b) (lacking a 12a-hy-
a
droxy group) was also prepared starting from CDCA (2b) by essen-
tially the same procedures as described in detail for the
preparation of 1a from 2a. Overall yields of 1a and 1b in 11 steps
were ca. 6.5% and 7.4%, respectively.
a
a
a
a
as formate derivatives. The diformyloxy-5b-cholestan-3-one
(11a) was found to be preferable to 9a as a substrate for the
IBX reaction, because the formate is more labile and the deprotec-
tion is much easy under mild conditions. The compound 11a was
prepared nearly quantitatively from 10a by the usual formic acid/
HClO4 method [10].
2.3. NMR properties
In the 1H-NMR spectrum of 1a, a pair of doublets appearing at
0.86 and 0.87 ppm were assigned to the three protons at C-26
and C-27 in the terminal isopropyl methyl groups, providing evi-
dence for the structure of the C8 cholestane side-chain. The 19-
H3 signal (singlet) in 1a was appreciably shifted downfield by
0.19 ppm and resonated at 1.19 ppm, compared to that in 10a
(1.00 ppm), whereas a signal appearing at 5.82 ppm as a singlet
was assigned to the 4-H. These observations indicated that 1a
Our preliminary experiments revealed that dehydrogenation of
11a with iodoxybenzene catalyzed by benzeneselenic anhydride
was successful [10], but that selective hydrogenation of the D1
-
bond of the resulting 7
a,12a-diformyloxy-1,4-choladien-3-one
was unsuccessful. An attempt at direct oxidation of 11a with sele-
nium dioxide [11] to give 7
a,12a
-diformyloxy-4-cholesten-3-one
has the 3-oxo-
D a-hydroxy structure in the A/B-ring juncture
4-7
(12a) also failed.
[15]. Furthermore, the occurrence of the two quaternary 13C signals

S. Ogawa et al. / Steroids 78 (2013) 927–937
931
at 198.7 and 167.4 ppm and the tertiary 13C signal at 126.9 ppm in
Table 2
Atomic coordinates for 1b.
the 13C-NMR spectrum of 1a also indicated the presence of the con-
jugated enone moiety. The 2D HMBC spectrum of 1a showed the
correlation peaks between 19-H3 vs C-5 and 4-H vs C-5, strongly
Atom
x
y
z
O1
O2
C1
C2
C3
C4
C5
C6
0.3802(4)
0.4221(3)
0.1216(5)
0.1304(6)
0.2649(5)
0.2480(5)
0.1334(4)
0.0990(6)
0.1829(5)
0.0593(4)
0.0939(4)
0.0296(4)
0.59455(13)
0.24336(10)
0.47800(13)
0.55708(15)
0.53955(17)
0.45526(15)
0.39204(15)
0.31000(15)
0.23469(15)
0.23467(13)
0.31865(12)
0.39988(14)
0.31593(15)
0.23932(13)
0.15567(13)
0.16165(12)
0.07132(13)
0.01642(15)
0.07567(12)
0.13839(17)
0.40816(17)
0.03032(13)
0.08980(17)
0.24964(10)
0.28138(7)
0.34909(10)
0.31535(12)
0.26951(13)
0.24879(11)
0.27063(10)
0.24375(10)
0.27502(9)
0.32505(8)
0.35279(8)
0.32228(10)
0.40415(10)
0.43621(9)
0.40896(9)
0.35876(8)
0.33866(10)
0.38624(9)
0.43173(8)
0.40204(11)
0.31629(12)
0.47663(9)
0.52038(10)
0.49369(9)
0.53488(10)
0.54577(11)
0.58762(14)
0.6351(3)
indicating the presence of the
D
4-bond.
Essentially identical 1H- and 13C-NMR characteristics were ob-
served in the spectra of 1b.
2.4. X-ray analysis of 1b
C7
C8
C9
The molecular structure of 7a-hydroxy-4-cholesten-3-one (1b)
was determined by X-ray analysis. Data for crystallographic de-
tails, atomic coordinates, and selected bond lengths (Å) and angles
(°) for 1b are presented in Tables 1–3, respectively. Thus, 1b crys-
tallizes in a P212121 space group having four molecules to the unit
cell. The molecular structure and the crystal structure of 1b are
presented in Figs. 4 and 5, respectively. The bond distances and
bond angles have the expected values for this kind of steroid. In
particular, the enone O@C–C@C fragment in ring A is almost
anti-periplanar with a small dihedral angle of 3.55° (see Table 3).
The bond distances are 1.222(4) for O1-C3, 1.440(4) for C3-C4,
and 1.336(4) Å for C4–C5, respectively.
The X-ray crystal structure of 1b reveals that it is aligned as a
zigzag array connected by intermolecular hydrogen bonds be-
tween the oxygen atom at C-3 (O1) and the hydroxy-group (O2)
of the neighboring molecule along the b axis with an O1ÁÁÁO2 dis-
tance of 2.743(3) Å. These arrays are arranged in a head-to-tail
manner, and form a molecular layer parallel to the bc-plane. Also
shown in Fig. 5, the rigid steroid rings create a large free space into
which the side chain is located. Furthermore, because of the ab-
sence of strong intermolecular interactions, the C8 alkyl (choles-
tane) side chain is quite flexible and, therefore, the thermal
motion in that part of the molecule is large. As a consequence, C-
26, C-27 and the methine hydrogen atom at the terminal isopropyl
group are disordered mutually over three positions due to the free
rotation around the C-24–C-25 bond. As a result of the refine-
ments, the site occupancy factor of C-27 was almost 1.0, and C-
26 was divided in C-26-1 and C-26-2 with site occupancy factors
of 0.60 and 0.40, respectively. The methine hydrogen atom of the
isopropyl group was not included in the refinement process. Thus,
X-ray analysis provided conclusive evidence for the stereochemical
structure of 1b, resolving the A/B-ring juncture in the steroid nu-
cleus and the terminal isopropyl group at the C8 cholestane
side-chain.
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C261
C262
C27
À0.0210(5)
0.0523(5)
0.0132(4)
0.1386(4)
0.1387(6)
0.1649(5)
0.1299(4)
À0.2409(4)
À0.2306(4)
0.0231(4)
À0.0243(6)
0.1677(5)
0.0704(5)
0.1985(6)
0.1081(9)
0.1332(15)
À0.097(2)
0.2296(11)
À0.04384(15)
À0.09800(16)
À0.17933(17)
À0.2340(3)
À0.1910(4)
À0.2454(6)
À0.3163(3)
0.6(occ.)
0.5982(4)
0.4(occ.)
0.59058(15)
Table 3
Selected bond lengths (Å) and angles (°) for 1b.
O1-C3
C3-C4
C4-C5
C5-C6
C6-C7
C7-C8
C7-O2
O2-H1
1.222(4)
1.440(4)
1.336(4)
1.491(4)
1.531(4)
1.521(4)
1.426(4)
0.840
O1-C3-C2
121.6(3)
121.6(3)
116.8(3)
123.6(3)
118.3
O1-C3-C4
C2-C3-C4
C3-C4-C5
C3-C4-H6
C5-C4-H6
C4-C5-C6
C5-C6-C7
C6-C7-C8
C6-C7-O2
C7-O2-H1
118.1
120.2(3)
111.3(3)
108.9(2)
108.0(2)
109.5
Table 1
Crystallographic details for 1b.
1b
Formula
M
Crystal system
Space group
a (Å)
C27H44O2
400.64
Orthorhombic
P212121
5.893(1)
15.734(3)
26.699(6)
2475.6(9)
4
b (Å)
Fig. 4. The molecular structure of 1b with numbered atoms. Thermal ellipsoids are
drawn at 30% of probability.
c (Å)
V (Å3)
Z value
Dc/g cmÀ3
F(000)
In conclusion, we report herein the chemical synthesis of 1a and
1b from 2a and 2b, respectively. The synthesis proceeded through
easily prepared, well defined intermediates and provided analyti-
cally pure products. The availability of 1a and 1b will permit fur-
ther studies on their plasma levels and facilitate studies on the
mechanism by which 1b (and possibly 1a) enters plasma, presum-
ably as a component of secreted lipoproteins. It will be of interest
to determine whether plasma levels of 1a also correlate with the
rate of bile acid synthesis, and if so, which intermediate (1a or
1.075
888
0.650
l
(Mo K
a
)/cmÀ1
No. reflns measured
No. unique reflns.
29,147
5678
3249
0.049
0.059
No. observed (I > 3
r
)
R1 (I > 3
r
)
wR2 (I > 3
r
)
Goodness of fit
0.943

932
S. Ogawa et al. / Steroids 78 (2013) 927–937
Fig. 5. Crystal structure of 1b viewed along the a axis.
1b) shows a better correlation. In addition, the compounds are now
dimensional (2D) 1H detected heteronuclear multiple quantum
(HMQC; 1H-13C coupling) and 1H detected heteronuclear multiple
bond correlation (HMBC; long-range 1H-13C coupling) experiments
were also performed.
available as substrates for ensuing biosynthetic transformations
catalyzed by 12a-hydroxylase (acting on 1b) as well as the D
4-3-
oxosteroid 5b-reductase (acting on both 1a and 1b) [16].
High-resolution mass spectra by electrospray ionization
(HR-ESI-MS) or by atmospheric pressure chemical ionization
3. Experimental
(HR-APCI-MS) were carried out using
a JEOL AccuTOF JMS-
T100LC liquid chromatography-mass spectrometer equipped with
an ESI source or an APCI source and coupled to a Agilent 1200 ser-
ies binary pump (Agilent Technologies Inc., Santa Clara, CA, USA)
operated in the negative ion mode or positive ion mode.
High-performance liquid chromatography (HPLC) was ob-
tained a Jasco LC-2000 plus HPLC system, consisting of two
PU-2085 high-pressure pumps, a MX-2080–32 solvent mixing
module, and a CO-2060 column heater equipped with a Chrom-
NAV data processing system (Tokyo, Japan). The column was a
3.1. Materials
CA (2a) was obtained from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). CDCA (2b) was supplied from Mitsubishi Tanabe
Pharma Co. (Tokyo, Japan). All other chemicals and solvents were
of analytical reagent grade and available from commercial sources.
All compounds were dried by azeotropic distillation prior to use.
Capcell Pack type AQ C18 (particle size, 3
l
m, 250 mm  3.0 mm
3.2. Instruments
I.D.; Shiseido, Tokyo, Japan) kept at 37 °C. The detector was an
Alltech 2000ES evaporative light-scattering detector (Deerfield,
IL, USA) operated under the following conditions: the flow rate
of purified compressed air used as a nebulizing gas was 1.8 L/
min, and the temperature of the heated drift was 68.3 °C (1a)
or 65.5 °C (1b). The mobile phase used was
15 mM-ammonium acetate/acetic acid buffer solution (pH 5.4)
and methanol [15:85 (1a) or 1:9 (1b), v/v]; the flow rate was
kept at 0.4 lL/min during the analysis. Analytical thin-layer
All melting points (mp) were determined on a micro hot stage
apparatus and are uncorrected. 1H- and 13C-NMR spectra were ob-
tained on a JEOL ECA 500 FT instrument operated at 500 and
125.8 MHz, respectively, with CDCl3 or CD3OD containing 0.1%
Me4Si as the solvent, except where otherwise indicated; chemical
shifts were expressed in d (ppm) relative to Me4Si. The 13C distor-
tionless enhancement by polarization transfer (DEPT; 135°, 90°,
and 45°) spectra were measured to determine the exact 13C signal
multiplicity and to differentiate between 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, two-
a mixture of
chromatography (TLC) was performed on pre-coated silica gel
plates (E. Merck, Darmstad, Germany) using EtOAc–hexane mix-
tures as the developing solvent.

S. Ogawa et al. / Steroids 78 (2013) 927–937
933
3.3. Chemical synthesis
developing solvent. After adding methanol (1 mL), the reaction
product was extracted with CH2Cl2. The combined extract was
washed with water, dried over Drierite, and evaporated to dryness
to give the crude 24-aldehyde-3,7,12-triacetate 5a. Recrystalliza-
tion from methanol afforded the analytically pure 5a as colorless
thin plates; yield, 790 mg (80%); mp, 86–87 °C (literature value:
mp, 55 °C) [18]. 1H-NMR (500 MHz, CDCl3): d = 0.73 (s, 3H, 18-
H3), 0.82 (d, 3H, J = 5.4 Hz, 21-H3), 0.92 (s, 3H, 19-H3), 2.05, 2.09,
2.14 (each s, 3H, OCOCH3), 4.58 (m, 1H, 3b-H), 4.91 (brs, 1H, 7b-
H), 5.09 (brs, 1H, 12b-H), 9.76 (s, 1H, 24-CHO). 13C-NMR
(125.8 MHz, CDCl3): d = 12.2 (C-18), 17.6 (C-21), 21.4, 21.5, 21.6
(each OCOCH3), 22.5 (C-19), 22.9 (C-15), 25.7 (C-11), 27.0 (C-2),
27.3 (C-16), 27.7 (C-22), 29.0 (C-9), 31.3 (C-6), 34.2 (C-10), 34.5
(C-4), 34.7 (C-1), 34.4 (C-20), 37.9 (C-8), 40.9 (C-23), 41.0 (C-5),
43.5 (C-14), 45.2 (C-13), 47.5 (C-17), 70.7 (C-7), 74.1 (C-3), 75.3
(C-12), 170.3, 170.5, 170.5 (each OCOCH3), 202.8 (C-24). HR-
ESI-MS, calcd. for C30H47O7 [M + H]+, 519.3322; found 519.3315.
3.3.1. 2-Iodoxybenzoic acid (IBX)
IBX was prepared, according to the following procedure [14]. In
short, to a solution of 2-iodobenzene (1.0 g, 4 mmol) in water
(13 mL) was added OXONEÒ (monopersulfate compound; 2KHSO5/
KHSO4/K2SO4; 3.62 g, 5.8 mmol). The resulting suspension was
stirred at 70 °C for 3.5 h and then at 5 °C for 1.5 h. The precipitated
semi-crystalline solid was filtered, washed with water and acetone,
and dried at room temperature for 16 h to afford IBX, which was
directly used without further purification. ‘‘Caution! IBX is explo-
sive at high temperature.’’
3.3.2. 3a,7a,12a-Triacetoxy-5b-cholan-24-ol (4a)
A mixture of CA peracetate 3a (11.8 g, 22.1 mmol), Et3N (4 mL,
28.7 mmol) in dry THF (100 mL), and ethyl chloroformate
(2.8 mL, 29.4 mmol) was stirred at room temperature for 2 h. To
the resulting milky turbid suspension, NaBH4 (4.0 g, 106 mmol)
and then methanol (10 mL) were added gradually with ice-bath
cooling, until a clear solution was obtained. After further stirring
at 0 °C for 2 h, the reaction product was extracted with EtOAc.
The combined extract was washed with saturated brine, dried over
Drierite, and evaporated to dryness. The oily residue was
chromatographed on a silica gel column (250 g). Elution with hex-
ane–EtOAc (7:3, v/v) afforded the corresponding 24-hydroxy-
3,7,12-triacetate 4a which was recrystallized from Et2O–hexane
as colorless thin plates; yield, 6.9 g (60%); mp, 134–135 °C. 1H-
NMR (500 MHz, CDCl3): d = 0.74 (s, 3H, 18-H3), 0.84 (d, 3H,
J = 6.3 Hz, 21-H3), 0.93 (s, 3H, 19-H3), 2.05, 2.09, 2.14 (each s, 3H,
OCOCH3), 3.60 (m, 2H, 24-H2), 4.56 (m, 1H, 3b-H), 4.91 (brs, 1H,
7b-H), 5.10 (brs, 1H, 12b-H). 13C-NMR (125.8 MHz, CDCl3):
d = 12.1 (C-18), 17.7 (C-21), 21.3, 21.4, 21.5 (each OCOCH3), 22.4
(C-19), 22.7 (C-15), 25.4 (C-11), 26.8 (C-2), 27.1 (C-16), 28.8 (C-
9), 29.0 (C-22), 31.1 (C-6), 31.5 (C-23), 34.2 (C-10), 34.5 (C-4),
34.6 (C-1), 34.7 (C-20), 37.6 (C-8), 40.8 (C-5), 43.3 (C-14), 44.9
(C-13), 47.4 (C-17), 63.2 (C-24), 70.6 (C-7), 74.0 (C-3), 75.4 (C-
12), 170.3, 170.5, 170.5 (each OCOCH3). HR-ESI-MS, calcd. for
3.3.5. 3a,7a-Diacetoxy-5b-cholan-24-al (5b)
The 24-hydroxy-3,7-diacetate 4b (1.0 g, 2.16 mmol) was con-
verted to the corresponding 24-aldehyde-3,7-diacetate 5b by the
TEMPO oxidation as described for the preparation of 5a. The crude
5b was recrystallized from Et2O as colorless thin plates; yield,
882 mg (89%); mp, 153–155 °C (literature value: mp, 153–154 °C)
[17]. 1H-NMR (500 MHz, CDCl3): d = 0.63 (s, 3H, 18-H3), 0.92 (d,
3H, J = 6.3 Hz, 21-H3), 0.93 (s, 3H, 19-H3), 2.03, 2.06 (each s, 3H,
OCOCH3), 4.59 (m, 1H, 3b-H), 4.88 (brs, 1H, 7b-H), 9.76 (s, 1H,
24-CHO). 13C-NMR (125.8 MHz, CDCl3): d = 11.7 (C-18), 18.4 (C-
21), 20.6 (C-11), 21.5, 21.6 (each OCOCH3), 22.7 (C-19), 23.5 (C-
15), 26.8 (C-2), 28.0 (C-16), 28.1 (C-22), 31.3 (C-6), 34.1 (C-20),
34.6 (C-10), 34.8 (C-4), 34.9 (C-1), 35.3 (C-9), 37.9 (C-8), 39.5 (C-
12), 40.9 (C-23), 41.0 (C-5), 42.7 (C-13), 50.4 (C-14), 55.8 (C-17),
71.2 (C-7), 74.1 (C-3), 170.4, 170.6 (each OCOCH3), 203.1 (C-24).
HR-APCI-MS, calcd. for
461.3221.
C
28H45O5 [M + H]+, 461.3267; found
3.3.6. 3a,7a,12a-Triacetoxy-5b-cholest-24-ene (6a)
C
30H49O7 [M + H]+, 521.3478; found 521.3431.
To a magnetically stirred solution of isopropyltriphenylphos-
phonium iodide (230 mg, 0.53 mmol) in dry THF (5 mL), a solution
3.3.3. 3 ,7 -Diacetoxy-5b-cholan-24-ol (4b)
a
a
of 1.6 M n-BuLi in hexane (330
cooling. The mixture was stirred at 0 °C for 30 min. To this mixture,
solution of the 24-aldehyde-3,7,12-triacetate 5a (100 mg,
0.19 mmol) and tri[2-(2-methoxyethoxy)ethyl]amine (10 L) dis-
lL) was added slowly with ice-bath
The C-24 carboxyl group of CDCA diacetate 3b (12.0 g,
25 mmol) [obtained from CDCA (2b)], was reduced by the proce-
dure described for the preparation of 4a to give 24-hydroxy-3,
7-diacetate 4b, which was recrystallized from Et2O-hexane as col-
orless thin plates; yield, 6.9 g (59%); mp, 160–162 °C (literature va-
lue: mp, 159 °C) [17]. 1H-NMR (500 MHz, CDCl3): = d 0.65 (s, 3H,
18-H3), 0.93 (s, 3H, 19-H3), 0.94 (d, 3H, J = 5.2 Hz, 21-H3), 2.05,
2.09 (each s, 3H, OCOCH3), 3.61 (m, 2H, 24-H2), 4.59 (m, 1H, 3b-
H), 4.88 (brs, 1H, 7b-H). 13C-NMR (125.8 MHz, CDCl3): d = 11.7
(C-18), 18.5 (C-21), 20.6 (C-11), 21.4, 21.6 (each OCOCH3), 22.6
(C-19), 23.5 (C-15), 26.7 (C-2), 28.1 (C-16), 29.2 (C-23), 31.4 (C-
6), 31.7 (C-22), 34.0 (C-10), 34.7 (C-8), 34.8 (C-4), 34.9 (C-1), 35.4
(C-20), 37.9 (C-9), 39.5 (C-12), 40.9 (C-5), 42.6 (C-13), 50.4 (C-
14), 55.9 (C-17), 63.5 (C-24), 71.2 (C-7), 74.2 (C-3), 170.5, 170.6
(OCOCH3). HR-APCI-MS, calcd. for C28H47O5 [M + H]+, 463.3424;
found 463.3439.
a
l
solved in dry THF (5 mL) was then added, and the mixture was fur-
ther stirred at 0 °C for 15 min. After quenching the reaction by the
addition of acetone (2 mL), the reaction product was extracted
with CH2Cl2. The combined extract was washed with water, dried
over Drierite, and evaporated to give a light yellow residue. Acety-
lation of the residue by the usual method and subsequent work-up
by solvent extraction gave the crude reaction product. Chromatog-
raphy of the product on a column of silica gel (5 g) and elution with
hexane–EtOAc (4:1, v/v) afforded the desired
D
24-3,7,12-triacetate
6a which recrystallized from methanol as colorless needles; yield,
63 mg (60%); mp, 84–86 °C. 1H-NMR (500 MHz, CDCl3): d = 0.73 (s,
3H, 18-H3), 0.82 (d, 3H, J = 8.1 Hz, 21-H3), 0.92 (s, 3H, 19-H3), 1.59
(s, 3H, 26-H3), 1.68 (s, 3H, 27-H3), 2.05, 2.09, 2.14 (each s, 3H,
OCOCH3), 4.58 (m, 1H, 3b-H), 4.92 (brs, 1H, 7b-H), 5.07 (t, 1H,
J = 3.4 Hz, 24-H), 5.10 (brs, 1H, 12b-H). 13C-NMR (125.8 MHz,
CDCl3): d = 12.2 (C-18), 17.6 (C-26), 17.8 (C-21), 21.4, 21.4, 21.6
(each OCOCH3), 22.5 (C-19), 22.8 (C-15), 24.6 (C-23), 25.5 (C-11),
25.7 (C-27), 26.8 (C-2), 27.2 (C-16), 28.8 (C-9), 31.2 (C-6), 34.3
(C-10), 34.6 (C-4), 34.7 (C-1), 34.8 (C-20), 35.8 (C-22), 37.7 (C-8),
40.9 (C-5), 43.3 (C-14), 45.0 (C-13), 47.7 (C-17), 70.7 (C-7), 74.1
(C-3), 75.4 (C-12), 124.8 (C-24), 131.1 (C-25), 170.3, 170.5, 170.5
(each OCOCH3). HR-APCI-MS, calcd. for C33H53O6 [M + H]+,
545.3842; found 545.3824.
3.3.4. 3a,7a,12a-Triacetoxy-5b-cholan-24-al (5a)
To a solution of the 24-hydroxy-3,7,12-triacetate 4a (1.0 g,
1.9 mmol) in CH2Cl2 (50 mL), 2,2,6,6-tetramethylpiperidine1-oxyl
free radical (TEMPO) (7 mg, 45 lmol), and KBr (24 mg) were added
gradually with ice-bath cooling. To the mixture, a solution of NaO-
Cl (2 mL) dissolved in a buffer solution (pH 8.6, 14 mL) prepared
from 0.5 M sodium bicarbonate, and 0.05 M sodium carbonate
was added. The mixture was stirred at 0 °C for 20 min; the reaction
was monitored by TLC using hexane–EtOAc (1:1, v/v) as the

934
S. Ogawa et al. / Steroids 78 (2013) 927–937
3.3.7. 3
a
,7
a
-Diacetoxy-5b-cholest-24-ene (6b)
EtOAc. The combined extract was washed with saturated brine,
dried over Drierite, and evaporated to dryness. The oily residue
was chromatographed on a column of silica gel (15 g). Elution with
EtOAc-hexane (6:4, v/v) gave the 3-hydroxy-7,12-diacetate 8a,
which was crystallized from methanol as colorless needles; yield,
334 mg (72%); mp, 138–141 °C. 1H-NMR (500 MHz, CDCl3):
d = 0.72 (s, 3H, 18-H3), 0.79 (d, 3H, J = 6.3 Hz, 21-H3), 0.86 (d, 6H,
J = 6.8 Hz, 26-H3 and 27-H3), 0.91 (s, 3H, 19-H3), 2.09, 2.13 (each
s, 3H, OCOCH3), 3.50 (m, 1H, 3b-H), 4.90 (brs, 1H, 7b-H), 5.10
(brs, 1H, 12b-H). 13C-NMR (125.8 MHz, CDCl3): d = 12.2 (C-18),
17.8 (C-21), 21.4, 21.6 (each OCOCH3), 22.5 (C-19 and C-26), 22.8
(C-23 and C-27), 23.8 (C-15), 25.5 (C-11), 27.3 (C-16), 28.0 (C-
25), 28.9 (C-9), 30.4 (C-2), 31.3 (C-6), 34.3 (C-10), 34.8 (C-1), 35.1
(C-20), 35.9 (C-22), 37.7 (C-8), 38.6 (C-4), 39.4 (C-24), 41.0 (C-5),
43.3 (C-14), 45.0 (C-13), 47.7 (C-17), 70.9 (C-7), 71.7 (C-3), 75.6
(C-12), 170.6, 170.7 (each OCOCH3). HR-APCI-MS calcd. for
C31H51O5 [MÀH]À, 503.3737; found, 503.3741.
The 24-aldehyde-3,7-diacete 5b (900 mg 1.95 mmol), was sub-
jected to the Wittig reaction with isopropyltriphenylphosphonium
iodide and processed as described for the preparation of 6a. The
procedure afforded the
D
24-3,7-diacetate 6b which crystallized
from methanol as colorless needles; yield, 552 mg (58%); mp,
74–76 °C (literature value: mp, 67–69 °C) [19]. 1H-NMR
(500 MHz, CDCl3): d = 0.64 (s, 3H, 18-H3), 0.92 (d, 3H, J = 5.0 Hz,
21-H3), 0.93 (s, 3H, 19-H3), 1.60 (s, 3H, 27-H3), 1.68 (s, 3H, 26-
H3), 2.03, 2.05 (each s, 3H, OCOCH3), 4.59 (m, 1H, 3b-H), 4.88
(brs, 1H, 7b-H), 5.09 (m, 1H, 24-H). 13C-NMR (125.8 MHz, CDCl3):
d = 11.6 (C-18), 17.6 (C-26), 18.5 (C-21), 20.5 (C-11), 21.4, 21.5
(each OCOCH3), 22.6 (C-19), 23.5 (C-15), 24.7 (C-23), 25.7 (C-27),
26.7 (C-2), 28.2 (C-16), 31.3 (C-6), 34.0 (C-9), 34.6 (C-4), 34.8 (C-
10), 34.9 (C-1), 35.6 (C-20), 36.1 (C-22), 37.9 (C-8), 39.5 (C-12),
40.9 (C-5), 42.7 (C-13), 50.4 (C-14), 56.1 (C-17), 71.3 (C-7), 74.2
(C-3), 125.1 (C-24), 131.0 (C-25), 170.5, 170.6 (each OCOCH3).
HR-ESI-MS, calcd. for
487.3753.
C
31H51O4 [M + H]+, 487.3787; found
3.3.11. 7a-Acetoxy-5b-cholestan-3a-ol (8b)
3-Hydroxy-7-acetate 8b was prepared from the 5b-cholestane
3,7-diacetate 7b (600 mg, 1.23 mmol) by selective hydrolysis at
C-3 using concentrated HCl as described for the preparation of
8a. Crystallization of the crude product from EtOAc gave colorless
needles; yield, 437 mg (80%); mp, 145–146 °C. 1H-NMR (500 MHz,
CDCl3): d = 0.64 (s, 3H, 18-H3), 0.87 (d, 6H, J = 6.8 Hz, 26-H3 and 27-
H3), 0.92 (d, 3H, J = 5.0 Hz, 21-H3), 0.93 (s, 3H, 19-H3), 2.05 (s, 3H,
OCOCH3), 3.50 (m, 1H, 3b-H), 4.88 (brs, 1H, 7b-H). 13C-NMR
(125.8 MHz, CDCl3): d = 11.6 (C-18), 18.6 (C-21), 20.6 (C-11), 21.6
(OCOCH3), 22.5 (C-26), 22.7 (C-19), 22.8 (C-27), 23.6 (C-23), 23.8
(C-15), 28.0 (C-16), 28.1 (C-25), 30.6 (C-2), 31.4 (C-6), 34.1 (C-9),
34.7 (C-10), 35.2 (C-1), 35.7 (C-20), 36.1 (C-22), 37.9 (C-8), 38.9
(C-4), 39.4 (C-12), 39.5 (C-24), 41.1 (C-5), 42.6 (C-13), 50.4 (C-
14), 56.1 (C-17), 71.4 (C-7), 71.8 (C-3), 170.7 (OCOCH3). HR-ESI-
MS, calcd. for C29H49O3 [MÀH]À, 445.3682; found, 445.3647.
3.3.8. 3
a a,12a-Triacetoxy-5b-cholestane (7a)
D
,7
The
24-3,7,12-triacetate 6a (60 mg, 0.11 mmol) in a mixed sol-
vent (20 mL) of EtOAc-methanol-acetic acid (10:10:1, v/v/v) was
hydrogenated in the presence of 10% Pd/C catalyst (6 mg) under
a slight positive pressure. After stirring at room temperature for
3 h, the catalyst was removed by filtration through Celite, and
the solvent of the mother liquor was evaporated under reduced
pressure to give a pale yellow oil of 5b-cholestane 3,7,12-triacetate
(7a). Although 7a was found to be homogeneous according to TLC,
HPLC, and NMR analyses, it resisted crystallization attempts; yield,
58.8 mg (98%). 1H-NMR (500 MHz, CDCl3): d = 0.72 (s, 3H, 18-H3),
0.79 (d, 3H, J = 6.8 Hz, 21-H3), 0.84 (d, 6H, J = 6.8 Hz, 26-H3 and
27-H3), 0.91 (s, 3H, 19-H3), 2.03, 2.07, 2.12 (each s, 3H, OCOCH3),
4.56 (m, 1H, 3b-H), 4.89 (brs, 1H, 7b-H), 5.09 (brs, 1H, 12b-H).
13C-NMR (125.8 MHz, CDCl3): d = 12.0 (C-18), 17.7 (C-21), 21.2,
21.3, 21.4 (each OCOCH3), 22.4 (C-19 and C-26), 22.6 (C-27), 22.7
(C-23), 23.7 (C-15), 25.4 (C-11), 26.9 (C-2), 27.1 (C-16), 27.8 (C-
25), 28.7 (C-9), 31.1 (C-6), 34.2 (C-10), 34.5 (C-1 and C-4), 34.9
(C-20), 35.8 (C-22), 37.6 (C-8), 39.2 (C-24), 40.8 (C-5), 43.2 (C-
14), 44.8 (C-13), 47.6 (C-17), 70.6 (C-7), 73.9 (C-3), 75.3 (C-12),
170.2, 170.3, 170.3 (each OCOCH3). HR-APCI-MS, cald. for
3.3.12. 7a,12a-Diacetoxy-5b-cholestan-3-one (9a)
Jones reagent (0.6 mL) was added gradually to a stirred solution
of the 3-hydroxy-7,12-diacetate 8a (300 mg, 0.59 mmol) in ace-
tone (8 mL) and CH2Cl2 (1.5 mL) until the solution became dark
brown. After stirring at 0 °C for 30 min, 2-propanol (0.8 mL) was
added to the mixture. The reaction product was extracted with
CH2Cl2. The combined extract was washed with water, dried over
Drierite, and evaporated to afford the 3-oxo-7,12-diacetate 9a
which crystallized from EtOAc as colorless needles; yield, 248 mg
(83%); mp, 171–172 °C. 1H-NMR (500 MHz, CDCl3): d = 0.77 (s,
3H, 18-H3), 0.82 (d, 3H, J = 5.4 Hz, 21-H3), 0.86 (d, 6H, J = 5.4 Hz,
26-H3 and 27-H3), 1.02 (s, 3H, 19-H3), 2.07, 2.12 (each s, 3H,
OCOCH3), 5.00 (brs, 1H, 7b-H), 5.15 (brs, 1H, 12b-H). 13C-NMR
(125.8 MHz, CDCl3): d = 12.2 (C-18), 17.8 (C-21), 21.3, 21.5 (each
OCOCH3), 21.6 (C-19), 22.5 (C-26), 22.8 (C-27), 23.8 (C-15 and C-
23), 25.8 (C-11), 27.2 (C-16), 27.9 (C-25), 29.8 (C-9), 30.9 (C-6),
34.4 (C-10), 35.0 (C-20), 35.9 (C-22), 36.1 (C-1), 36.6 (C-2), 37.7
(C-8), 39.3 (C-24), 42.4 (C-5), 43.2 (C-14), 44.6 (C-4), 45.0 (C-13),
47.8 (C-17), 70.6 (C-7), 75.3 (C-12), 170.4, 170.5 (each OCOCH3),
212.4 (C-3). HR-APCI-MS, calcd. for C31H51O5 [M + H]+, 503.3737;
found, 503.3701.
C
33H55O6 [M + H]+, 547.3999; found 547.3988.
3.3.9. 3
a
,7a
-Diacetoxy-5b-cholestane (7b)
24-3,7-diacetate 6b (100 mg,
Catalytic hydrogenation of
D
0.21 mmol) by the procedure as described for the preparation of
7a gave the 5b-cholestane 3,7-diacetate (7b); yield, 98 mg (98%);
mp, 85–87 °C (recrystallized from methanol as colorless thin
plates) (literature value: mp, 83–87 °C) [20]. 1H-NMR (500 MHz,
CDCl3): d = 0.64 (s, 3H, 18-H3), 0.87 (d, 6H, J = 6.8 Hz, 26-H3 and
27-H3), 0.92 (d, 3H, J = 5.0 Hz, 21-H3), 0.93 (s, 3H, 19-H3), 2.04,
2.06 (each s, 3H, OCOCH3), 4.59 (m, 1H, 3b-H), 4.88 (brs, 1H, 7b-
H). 13C-NMR (125.8 MHz, CDCl3): d = 11.7 (C-18), 18.6 (C-21),
20.6 (C-11), 21.5, 21.6 (each OCOCH3), 22.5 (C-26), 22.7 (C-19),
22.8 (C-27), 23.6 (C-23), 23.8 (C-15), 26.8 (C-2), 28.0 (C-25), 28.1
(C-16), 31.3 (C-6), 34.0 (C-9), 34.6 (C-4), 34.8 (C-10), 34.9 (C-1),
35.7 (C-20), 36.1 (C-22), 37.9 (C-8), 39.4 (C-12), 39.5 (C-24), 40.9
(C-5), 42.6 (C-13), 50.4 (C-14), 56.2 (C-17), 71.3 (C-7), 74.2 (C-3),
170.4, 170.6 (each OCOCH3). HR-APCI-MS calcd. for C31H53O4
[M + H]+, 489.3944; found 489.3938.
3.3.13. 7a-Acetoxy-5b-cholestan-3-one (9b)
The 3-hydroxy-7-acetate 8b (400 mg, 0.90 mmol), subjected to
the oxidation reaction with Jones reagent and processed as de-
scribed for the preparation of 9a, afforded a homogeneous oily
product of the 3-oxo-7-acetate 9b, which resisted crystallization
attempts; yield, 372 mg (93%). 1H-NMR (500 MHz, CDCl3):
d = 0.68 (s, 3H, 18-H3), 0.87 (d, 6H, J = 6.8 Hz, 26-H3 and 27-H3),
0.92 (d, 3H, J = 5.0 Hz, 21-H3), 1.04 (s, 3H, 19-H3), 2.04 (s, 3H,
OCOCH3), 4.96 (brs, 1H, 7b-H). 13C-NMR (125.8 MHz, CDCl3):
3.3.10. 7a,12a-Diacetoxy-5b-cholestan-3a-ol (8a)
A mixture of the 5b-cholestane 3,7,12-triacetate 7a (500 mg,
0.91 mmol) and conc. HCl (6 drops) in methanol (15 mL) was slow
stirred at 30 °C for 6 h. Most of the solvent was evaporated under
reduced pressure, and the reaction product was extracted with

S. Ogawa et al. / Steroids 78 (2013) 927–937
935
d = 11.7 (C-18), 18.7 (C-21), 21.0 (C-11), 21.5 (OCOCH3), 21.8 (C-
19), 22.5 (C-26), 22.8 (C-27), 23.6 (C-23), 23.8 (C-15), 28.0 (C-16),
28.1 (C-25), 31.0 (C-6), 34.9 (C-9) 35.0 (C-10), 35.7 (C-20), 36.1
(C-22), 36.6 (C-2), 36.9 (C-1), 37.9 (C-8), 39.4 (C-12 and C-24),
42.6 (C-5), 42.7 (C-13), 44.7 (C-4), 50.3 (C-14), 56.1 (C-17), 71.2
(C-7), 170.3 (OCOCH3), 212.6 (C-3). HR-APCI-MS, calcd. for
3.3.17. 7a-Formyloxy-5b-cholestan-3-one (11b)
The 7-hydroxy-3-one 10b (250 mg, 0.62 mmol) was converted
to the 3-oxo-7-formate 11b by the method described for the prep-
aration of 11a. Although this compound was analytically pure by
TLC, and NMR, it resisted crystallization attempts; yield, 182 mg
(68%). 1H-NMR (500 MHz, CDCl3): d = 0.69 (s, 3H, 18-H3), 0.86 (d,
3H, J = 6.0 Hz, 27-H3), 0.87 (d, 3H, J = 6.0 Hz, 26-H3), 0.92 (d, 3H,
J = 6.6 Hz, 21-H3), 1.04 (s, 3H, 19-H3), 5.12 (brs, 1H, 7b-H) 8.06 (s,
1H, OCHO). 13C-NMR (125.8 MHz, CDCl3): d = 11.7 (C-18), 18.7
(C-21), 21.0 (C-11), 21.8 (C-19), 22.5 (C-26), 22.8 (C-27), 23.5 (C-
23), 23.7 (C-15), 28.0 (C-25), 28.1 (C-16), 31.2 (C-6), 34.8 (C-9),
35.0 (C-10), 35.7 (C-20), 36.1 (C-22), 36.5 (C-1), 36.8 (C-2), 37.9
(C-8), 39.4 (C-12 and C-24), 42.6 (C-5), 42.7 (C-13), 44.7 (C-4),
50.0 (C-14), 56.0 (C-17), 71.3 (C-7), 160.5 (OCHO), 212.1 (C-3).
C
29H49O3 [M + H]+, 445.3682; found, 445.3680.
3.3.14. 7a,12a-Dihydroxy-5b-cholestan-3-one (10a)
The 3-oxo-7,12-diacetate 9a (200 mg, 0.40 mmol) was com-
pletely hydrolyzed in 10% methanolic KOH (3.5 g) at 30 °C for
16 h. Most of the solvent was evaporated off, and the residue
was dissolved in water and acidified with 10% HCl with stirring
and ice-bath cooling. The precipitate was collected by filtration
and washed with water. Recrystallization from methanol gave
the 7,12-dihydroxy-3-one 10a as colorless thin plates; yield,
149 mg (90%); mp, 195–197 °C (literature value: mp, 209–210 °C)
[21]. 1H-NMR (500 MHz, CDCl3): d = 0.74 (s, 3H, 18-H3), 0.86 (d,
3H, J = 6.0 Hz, 27-H3), 0.87 (d, 3H, J = 6.0 Hz, 26-H3), 0.98 (d, 3H,
J = 6.5 Hz, 21-H3), 1.00 (s, 3H, 19-H3), 3.93 (brs, 1H, 7b-H), 4.06
(brs, 1H, 12b-H). 13C-NMR (125.8 MHz, CDCl3): d = 12.6 (C-18),
17.8 (C-21), 21.7 (C-19), 22.5 (C-26), 22.8 (C-27), 23.1 (C-23),
23.8 (C-15), 27.3 (C-9), 27.5 (C-16), 28.0 (C-25), 28.5 (C-11), 33.7
(C-6), 34.9 (C-10), 35.4 (C-20), 36.0 (C-22), 36.6 (C-1), 36.8 (C-2),
39.4 (C-24), 39.6 (C-8), 41.9 (C-14), 43.1 (C-5), 45.6 (C-4), 46.6
(C-13), 47.7 (C-17), 68.3 (C-7), 72.9 (C-12), 213.1 (C-3). HR-APCI-
MS, calcd. for C27H45O3 [MÀH]À, 417.3369; found, 417.3361.
HR-APCI-MS, calcd. for
431.3545.
C
28H47O3 [M + H]+, 431.3525; found
3.3.18. 7a,12a-Diformyloxy-4-cholesten-3-one (12a)
To a magnetically stirred solution of the 3-oxo-7,12-diformate
11a (50 mg, 0.11 mmol) in DMSO (5 mL) was added freshly pre-
pared IBX (40 mg, 0.28 mmol) and two drops of trifluoroacetic
acid; the mixture was stirred at 40 °C for 40 h. After cooling to
room temperature, the reaction product was extracted with EtOAc,
and the combined extract was washed with saturated brine, dried
over Drierite, and evaporated to dryness. The crude product was
purified on a silica gel column (3 g). Elution with EtOAc-hexane
(1:4, v/v) yielded the conjugated 3-oxo-D
4-7,12-diformate 12a
which was recrystallized from methanol as colorless prisms; yield,
28 mg (56%); mp, 134–136 °C. 1H-NMR (500 MHz, CDCl3): d = 0.81
(s, 3H, 18-H3), 0.86 (d, 9H, J = 6.5 Hz, 21-H3 and 26-H3 and 27-H3),
1.21 (s, 3H, 19-H3), 5.19 (brs, 1H, 7b-H), 5.30 (brs, 1H, 12b-H), 5.71
(s, 1H, 4-H), 8.08, 8.11 (each s, 1H, OCHO). 13C-NMR (125.8 MHz,
CDCl3): d = 12.1 (C-18), 16.8 (C-19), 17.8 (C-21), 22.5 (C-26), 22.8
(C-15 and C-27), 23.7 (C-23), 25.6 (C-11), 27.1 (C-16), 27.9 (C-
25), 33.7 (C-2), 35.1 (C-1), 35.2 (C-20), 35.8 (C-22), 37.3 (C-6),
37.6 (C-10), 38.2 (C-9), 39.4 (C-24), 40.6 (C-8), 42.8 (C-14), 44.8
(C-13), 47.4 (C-17), 70.6 (C-7), 74.8 (C-12), 126.8 (C-4), 160.3,
160.4 (each OCHO), 165.0 (C-5), 198.4 (C-3). HR-APCI-MS, calcd.
for C29H45O5 [M + H]+, 473.3267; found 473.3307.
3.3.15. 7a-Hydroxy-5b-cholestan-3-one (10b)
The 3-oxo-7-acetate 9b (300 mg, 0.67 mmol), subjected to com-
plete hydrolysis with 10% methanolic KOH (6 g) at 30 °C for 16 h
and processed as described above, afforded the 7-hydroxy-3-one
10b; yield, 266 mg (98%); mp, 62–65 °C (colorless needles from
petroleum ether) (literature value: mp, 122–123 °C) [22]. 1H-
NMR (500 MHz, CDCl3): d = 0.70 (s, 3H, 18-H3), 0.87 (d, 6H,
J = 5.4 Hz, 26-H3 and 27-H3), 0.93 (d, 3H, J = 5.4 Hz, 21-H3), 1.01
(s, 3H, 19-H3), 3.93 (brs, 1H, 7b-H). 13C-NMR (125.8 MHz, CDCl3):
d = 11.8 (C-18), 18.7 (C-21), 21.0 (C-11), 21.9 (C-19), 22.6 (C-26),
22.8 (C-27), 23.7 (C-23), 23.8 (C-15), 28.0 (C-25), 28.2 (C-16),
33.4 (C-9), 33.8 (C-6), 35.3 (C-10), 35.8 (C-20), 36.2 (C-22), 36.8
(C-1), 37.0 (C-2), 39.4 (C-8), 39.5 (C-12), 39.6 (C-24), 42.7 (C-13),
43.2 (C-5), 45.6 (C-4), 50.4 (C-14), 56.2 (C-17), 68.6 (C-7), 213.2
(C-3). HR-ESI-MS, calcd. for C27H45O2 [MÀH]À, 401.3420; found
401.3434.
3.3.19. 7a-Formyloxy-4-cholesten-3-one (12b)
The 3-oxo-7-formate 11b (200 mg, 0.46 mmol), subjected to the
dehydration with IBX and processed as described for the prepara-
tion of 12a, afforded an oily residue. Chromatography of the oily
product on a silica gel column (10 g) and elution with EtOAc-hex-
ane (1:9, v/v) gave the conjugated 3-oxo-D
4-7-formate 12b which
recrystallized from acetone as colorless thin plates; yield, 127 mg
(64%); mp, 152–155 °C. 1H-NMR (500 MHz, CDCl3): d = 0.71 (s,
3H, 18-H3), 0.87 (d, 6H, J = 6.8 Hz, 26-H3 and 27-H3), 0.92 (d, 3H,
J = 5.0 Hz, 21-H3), 1.22 (s, 3H, 19-H3), 5.17 (brs, 1H, 7b-H) 5.70 (s,
1H, 4-H), 8.05 (s, 1H, OCHO). 13C-NMR (125.8 MHz, CDCl3):
d = 11.7 (C-18), 17.1 (C-19), 18.6 (C-21), 20.8 (C-11), 22.5 (C-26),
22.8 (C-27), 23.5 (C-23), 23.7 (C-15), 28.0 (C-16 and C-25), 33.9
(C-2), 35.4 (C-1), 35.6 (C-20), 36.0 (C-22), 37.4 (C-6), 38.2 (C-10),
38.3 (C-8), 39.0 (C-12), 39.4 (C-24), 42.4 (C-13), 46.2 (C-9), 50.0
(C-14), 55.9 (C-17), 71.2 (C-7), 126.5 (C-4), 160.4 (OCHO), 166.1
(C-5), 198.8 (C-3). HR-APCI-MS, calcd. for C28H45O3 [M + H]+,
429.3369; found 429.3411.
3.3.16. 7
a,12a-Diformyloxy-5b-cholestan-3-one (11a)
A
solution of the 7,12-dihydroxy-3-one 10a (200 mg,
0.48 mmol) in 98% formic acid (2 mL) containing one drop of 60%
perchloric acid was stirred at room temperature for 1 h. Acetic
anhydride (2 mL) was added slowly with ice-bath cooling, and
then the mixture was poured into ice. The precipitated solid was
filtered, washed with water, and the product (11a) was recrystal-
lized from methanol 11a as colorless thin plates; yield, 218 mg
(96%); mp, 176–178 °C. 1H-NMR (500 MHz, CDCl3): d = 0.79 (s,
3H, 18-H3), 0.86 (d, 9H, J = 6.5 Hz, 21-H3 and 26-H3 and 27-H3),
1.04 (s, 3H, 19-H3), 5.16 (brs, 1H, 7b-H), 5.32 (brs, 1H, 12b-H),
8.10, 8.16 (each s, 1H, OCHO). 13C-NMR (125.8 MHz, CDCl3):
d = 12.2 (C-18), 17.8 (C-21), 21.6 (C-19), 22.5 (C-26), 22.8 (C-15
and C-27), 23.7 (C-23), 25.9 (C-11), 27.2 (C-16), 27.9 (C-25), 29.5
(C-9), 31.0 (C-6), 34.4 (C-10), 35.2 (C-20), 35.8 (C-22), 36.1 (C-1),
36.5 (C-2), 37.7 (C-8), 39.4 (C-24), 42.2 (C-5), 42.8 (C-14), 44.6
(C-4), 45.0 (C-13), 47.5 (C-17), 70.6 (C-7), 75.3 (C-12), 160.3,
160.5 (each OCHO), 211.7 (C-3). HR-APCI-MS, calcd for C29H47O5
[M + H]+, 475.3424; found 475.3458.
3.3.20. 7
a
,12
solution of the 3-oxo-
mol) in THF (1 mL) and 0.8% aqueous NaOH (1 mL) was left
a
-Dihydroxy-4-cholesten-3-one (1a)
A
D
4-7,12-diformate 12a (15 mg,
32
l
to stand at room temperature for 30 min; the reaction was moni-
tored by TLC. The solution was diluted with water, acidified by
10% HCl with ice-bath cooling, and the precipitated solid was col-
lected by filtration, washed with water. Recrystallization from

936
S. Ogawa et al. / Steroids 78 (2013) 927–937
methanol gave the desired 3-oxo-D
4-7,12-dihydroxy compound 1a
References
as colorless needles; yield, 11 mg (80%); mp, 147–148 °C. 1H-NMR
(500 MHz, CDCl3): d = 0.75 (s, 3H, 18-H3), 0.86 (d, 3H, J = 6.8 Hz, 27-
H3), 0.87 (d, 3H, J = 6.8 Hz, 26-H3), 0.98 (d, 3H, J = 6.0 Hz, 21-H3),
1.19 (s, 3H, 19-H3), 3.98 (brs, 1H, 7b-H), 4.05 (brs, 1H, 12b-H),
5.82 (s, 1H, 4-H). 13C-NMR (125.8 MHz, CDCl3): d = 12.6 (C-18),
16.9 (C-19), 17.8 (C-21), 22.5 (C-26), 22.8 (C-27), 23.0 (C-15),
23.8 (C-23), 27.4 (C-16), 28.0 (C-25), 28.3 (C-11), 33.9 (C-2), 35.2
(C-1), 35.4 (C-20), 36.0 (C-22), 38.0 (C-10), 38.9 (C-9), 39.5 (C-
24), 40.0 (C-8), 40.8 (C-6), 42.0 (C-14), 46.4 (C-13), 47.6 (C-17),
68.3 (C-7), 72.5 (C-12), 126.9 (C-4), 167.4 (C-5), 198.7 (C-3). HR-
ESI-MS, calcd. for C27H43O3 [MÀH]À, 415.3212; found 415.3211.
[1] a Princen GM. Regulation of bile acid biosynthesis. Curr Pharm Des
1997;3:59–84;
b Russell WD. The enzymes, regulation, and genetics of bile acid synthesis.
Annu Rev Biochem 2003;72:137–74;
c Norlin M, Wikvall K. Enzymes in the conversion of cholesterol into bile acids.
Curr Mol Med 2007;7:199–218;
d Bernhardsson C, Björkhem I, Danielsson H, Wikvall K. 12
a-Hydroxylation of
7a
-hydroxy-4-cholesten-3-one by reconstituted system from rat liver
a
microsomes. Biochem Biophys Res Commun 1973;54:1030–8.
[2] Heubi EJ, Setchell DRK, Bove EK. Inborn errors of bile acid metabolism. Liver
Dis 2007;27(3):282–94.
[3] a Setchell DRK, Suchy JF, Welsh BM, Zimmer-Nechemias L, Heubi J, Balistreri
FW.
D
4-3-Oxosteroid 5b-reductase deficiency described in identical twins with
neonatal hepatitis. J Clin Invest 1988;82:2148–57;
b Shneider LB, Setchell DRK, Whitington FP, Neilson AK, Suchy JF.
D
4-3-
Oxosteroid 5b-reductase deficiency causing neonatal liver failure and
hemochromatosis. J Pediatr 1994;124:234–8.
[4] a Sauter G, Berr F, Beuers U, Fischer S, Paumgartner G. Serum concentrations of
3.3.21. 7
a
-Hydroxy-4-cholesten-3-one (1b)
The 3-oxo-
D
4-diformate 12b (100 mg, 0.23 mmol) in THF
7a-hydroxy-4-cholesten-3-one reflect bile acid synthesis in humans.
Hepatology 1996;24:123–6;
b Gälman C, Arvidsson I, Angelin B, Rudling M. Monitoring hepatic cholesterol
7a-hydroxylase activity by assay of the stable bile acid intermediate 7a-
(5 mL) was hydrolyzed with 0.8% aqueous NaOH, followed by acid-
ification with 10% HCl as described for the preparation of 1a. After
being processed analogously, the crude product dissolved in ben-
zene was purified using a column of silica gel (5 g), and eluting
with EtOAc-hexane (3:7, v/v). Recrystallization from methanol
hydroxy-4-cholesten-3-one in peripheral blood.
859–66.
J Lipid Res 2003;44:
[5] Wedlake L, A’Hern R, Russell D, Thomas K, Walters RFJ, Andreyev JNH.
Systematic review: the prevalence of idiopathic bile acid malabsorption as
diagnosed by SeHCAT scanning in patients with diarrhoea-predominant
irritable bowel syndrome. Aliment Pharmacol Ther 2009;30:707–17.
gave the 3-oxo-D
4-7-hydroxy compound 1b as colorless needles;
yield, 73 mg (78%); mp, 180–182 °C (literature value: mp, 183–
184 °C) [23]. 1H-NMR (500 MHz, CDCl3): d = 0.71 (s, 3H, 18-H3),
0.89 (d, 3H, J = 6.8 Hz, 27-H3), 0.90 (d, 3H, J = 6.8 Hz, 26-H3), 0.91
(d, 3H, J = 5.4 Hz, 21-H3), 1.20 (s, 3H, 19-CH3), 3.97 (brs, 1H, 7b-
H), 5.80 (s, 1H, 4-H). 13C-NMR (125.8 MHz, CDCl3): d = 11.8 (C-
18), 17.0 (C-19), 18.6 (C-21), 20.8 (C-11), 22.5 (C-26), 22.8 (C-27),
23.5 (C-23), 23.7 (C-15), 28.0 (C-25), 28.1 (C-16), 33.9 (C-2), 35.3
(C-1), 35.7 (C-20), 36.0 (C-22), 38.4 (C-10), 39.1 (C-6), 39.4 (C-
24), 39.7 (C-8), 40.9 (C-12), 42.4 (C-13), 45.1 (C-9), 50.4 (C-14),
55.9 (C-17), 68.4 (C-7), 126.7 (C-4), 168.0 (C-5), 198.9 (C-3). HR-
ESI-MS, calcd. for C27H43O2 [MÀH]À, 399.3263; found 399.3280.
[6] Alexander LD, Fisher FJ. A convenient synthesis of 7
one by the hydroxypropyl-b-cyclodextrin-facilitated cholesterol oxidase
oxidation of 3b,7 -cholest-5-ene-3,7-diol. Steroids 1995;60:290–4.
a-hydroxy-cholest-4-en-3-
a
[7] a Labaree D, Hoyte MR, Hochberg BR. A direct steroselective synthesis of 7b-
hydroxytestosterone. Steroids 1997;62:482–6;
Li S, Pang J, Wilson KW, Schroepfer Jr JG. Sterol synthesis. Preparation and
characterization of fluorinated and deuterated analogs of oxygenated
derivatives of cholesterol. Chem Phys Lipids 1999;99:33–71.
[8] a Kuramoto T, Kihira K, Matsumoto N, Hoshita T. Determination of sulfated
position in 5b-bufol sulfate by a carbon-13 nuclear magnetic resonance study.
Chem Pharm Bull 1981;29:1136–9;
b Babu P, Maitra U. Synthesis and in vitro cholesterol dissolution by 23- and
24-phosphonoblie acids. Steroids 2005;70:681–9.
[9] a Anelli LP, Biffi C, Montanari F, Quici S. Fast and selective oxidation of primary
alcohols to aldehydes or to carboxylic acids and secondary alcohols to ketones
mediated by oxoammonium salts under two-phase conditions. J Org Chem
1987;52:2559–62;
3.4. X-ray crystal structure determination of 1b
b de Nooy AJE, Besemer CA, van Bekkum H. On the use of stable organic
nitroxyl radicals for the oxidation of primary and secondary alcohols.
Synthesis 1996:1153–74;
Colorless crystals of 1b suitable for X-ray analysis were grown
by recrystallization from methanol. A crystal having dimensions
of 0.25 Â 0.25 Â 0.30 mm3 was selected. X-ray intensity data for
1b were collected on a Rigaku AFC-8 diffractometer equipped with
c Bode WJ, Carreira ME. Stereoselective syntheses of epothilones A and B via
nitrile oxide cycloadditions and related studies.
6410–24.
J Org Chem 2001;66:
a Mercury CCD detector using monochromated Mo K
a radiation
[10] Iida T, Nishida S, Chang CF, Niwa T, Goto J, Nambara T. Potential bile acid
metabolites. XXI. A new synthesis of allochenodeoxycholic and allocholic
acids. Chem Pharm Bull 1993;41(4):763–5.
(k = 0.71070 Å) at 193 K. The structure was solved using direct
methods and refined by a full-matrix least-squares procedure
based on F2 using the Crystal Structure crystallographic software
package. The crystal structures were refined with anisotropic tem-
perature factors for all non-hydrogen atoms. The positions of
hydrogen atoms were generated theoretically, and were refined
using the riding model. Crystallographic details and atomic coordi-
nates are summarized in Tables 1 and 2, respectively.
[11] Hoshita T, Shefer S, Mosbach HE. Conversion of 7
en-3-one to 5 -cholestane-3 ,7 ,12 -triol by iguana liver microsomes. J Lipid
Res 1968;9:237–43.
a,12a-dihydroxycholest-4-
a
a
a
a
[12] Nicolaou CK, Montagnon T, Baran SP, Zhong LY. Iodine(V) reagents in organic
synthesis. Part 4. o-Iodoxybenzoic acid as a chemospecific tool for single
electron transfer-based oxidation processes.
2002;124:2245–58.
J
Am Chem Soc
[13] Li Q, Tochtrop PG. A stereoselective synthesis of the allo-bile acids from the 5b-
isomers. Tetrahedron Lett. 2011;52:4137–9.
[14] Frigerio M, Santagostino M, Sputore S.
A user-friendly entry to 2-
iodoxybenzoic acid (IBX). J Org Chem 1999;64:4537–8.
[15] Marat K, Templeton FJ, Kumar PSV. An NMR study of A-ring conformation in
some 4-en-3-one steroids. Magn Reson Chem 1987;25:25–30.
Acknowledgements
[16] Faucher F, Cantin L, Luu-The V, Labrie F, Breton R. The crystal structure of
This work was supported in part by a Grant-in-Aid for Scientific
Research (C) from the Japan Society for Promotion of Science (to T.I.
21550091) for 2012–2014 and the Supported Program for the Stra-
tegic Research Foundation at Private Universities subsidized MEXT
2009 (S0901022) for 2009–2013.
human
D4-3-ketosteroid 5b-reductase defines the functional role of the
residues of the catalytic tetrad in the steroid double bond reduction
mechanism. Biochemistry 2008;47:8261–70.
[17] Une M, Shinonaga Y, Matoba N, Kuroki S, Kihira K, Hoshita T. Identification of
new bile alocohols, 5b-cholestane-3
,7 ,26,27-tetrol, and 5b-cholestane-3
gallbladder bile. J Lipid Res 1986;27:1318–23.
[18] Kannan A, Clercq DE, Pannecouque C, Witvrouw M, Hartman LT, Turpin AJ,
a
,7
a
,24,26-tetrol, 5b-cholestane-
3a
a
a,7a,26,27-tetrol in human
Appendix A. Supplementary data
et al. Synthesis and anti-HIV activity of
Tetrahedron 2001;57:9385–91.
a bile acid analog of cosalane.
[19] Dayal B, Batta KA, Shefer S, Tint SG, Salen G, Mosbach HE. Preparation of
24(R)- and 24(S)-5b-cholestane-3 ,7 ,24-triols and 25(R)- and 25(S)-5b-
cholestane-3 ,7 ,26-triols by hydroboration procedure. Lipid Res
1978;19:191–5.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2013.05.
011.
a
a
a
a
a
J

S. Ogawa et al. / Steroids 78 (2013) 927–937
937
¯
[20] Yamazaki K, Kawahara T, Shimizu K. Metabolic studies of bile acids XXVII
partial synthesis of some 3-keto-coprostane derivatives. Yonago Acta Medica
1959;4:37–9.
[22] Takada K, Osaka H, Horiki A. Bile acids and steroids. XIX. Sythesis of some of 7-
oxygenated 5b-cholestane. Yakugaku Zasshi 1961;81:325–30.
[23] Danielsson H. Synthesis and metabolism of
Chem Scand 1961;15:242–8.
D a-ol-3-one. Acta
4-cholestene-7
[21] Berséus O, Danielsson H, Kallner A. Synthesis and metabolism of cholest-4-
ene-7a,12a-diol-3-one and 5b-cholestane-7a,12a-diol-3-one. J Biol Chem
1965;240:2396–401.