Chemical synthesis of 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid: An internal standard for mass spectrometric analysis of the abnormal Δ5-bile acids occurring in Niemann-Pick disease

Article abstract of DOI:10.1016/j.steroids.2009.04.007

In Niemann-Pick disease, type C1, increased amounts of 3β,7β-dihydroxy-5-cholenoic acid are reported to be present in urinary bile acids. The compound occurs as a tri-conjugate, sulfated at C-3, N-acetylglucosamidated at C-7, and N-acylamidated with taurine or glycine at C-24. For sensitive LC-MS/MS analysis of this bile acid, a suitable internal standard is needed. We report here the synthesis of a satisfactory internal standard, 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid (as the disodium salt). The key reactions involved were (1) the so-called "second order" Beckmann rearrangement (one-carbon degradation at C-24) of hyodeoxycholic acid (HDCA) 3,6-diformate with sodium nitrite in a mixture of trifluoroacetic anhydride and trifluoroacetic acid, (2) simultaneous inversion at C-3 and elimination at C-6 of the ditosylate derivatives of the resulting 3α,6α-dihydroxy-24-nor-5β-cholanoic acid with potassium acetate in aqueous N,N-dimethylformamide, and (3) regioselective sulfation at C-3 of an intermediary 3β,7β-dihydroxy-24-nor-Δ⁵ derivative using sulfur trioxide-trimethylamine complex. Overall yield of the desired compound was 1.8% in 12 steps from HDCA.

Full text of DOI:10.1016/j.steroids.2009.04.007

Steroids 74 (2009) 766–772
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Steroids
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Chemical synthesis of 3␤-sulfooxy-7␤-hydroxy-24-nor-5-cholenoic acid: An
internal standard for mass spectrometric analysis of the abnormal ꢀ5-bile
acids occurring in Niemann-Pick disease
Genta Kakiyamaa, Akina Mutoa, Miki Shimadab, Nariyasu Manob, Junichi Gotob,
Alan F. Hofmannc, Takashi Iidaa,∗
a Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya-ku, Tokyo 156-8550, Japan
b Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryou-machi, Aoba-ku, Sendai 980-8574, Japan
c 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:
Received 7 March 2009
Received in revised form 8 April 2009
Accepted 15 April 2009
Available online 24 April 2009
In Niemann-Pick disease, type C1, increased amounts of 3␤,7␤-dihydroxy-5-cholenoic acid are reported
to be present in urinary bile acids. The compound occurs as a tri-conjugate, sulfated at C-3, N-
acetylglucosamidated at C-7, and N-acylamidated with taurine or glycine at C-24. For sensitive LC–MS/MS
analysis of this bile acid, a suitable internal standard is needed. We report here the synthesis of a satis-
factory internal standard, 3␤-sulfooxy-7␤-hydroxy-24-nor-5-cholenoic acid (as the disodium salt). The
key reactions involved were (1) the so-called “second order” Beckmann rearrangement (one-carbon
degradation at C-24) of hyodeoxycholic acid (HDCA) 3,6-diformate with sodium nitrite in a mixture of tri-
fluoroacetic anhydride and trifluoroacetic acid, (2) simultaneous inversion at C-3 and elimination at C-6
of the ditosylate derivatives of the resulting 3␣,6␣-dihydroxy-24-nor-5␤-cholanoic acid with potassium
acetate in aqueous N,N-dimethylformamide, and (3) regioselective sulfation at C-3 of an intermediary
3␤,7␤-dihydroxy-24-nor-ꢀ5 derivative using sulfur trioxide–trimethylamine complex. Overall yield of
the desired compound was 1.8% in 12 steps from HDCA.
Keywords:
LC–MS/MS
Niemann-Pick disease type C1
Bile acid multi-conjugates
Internal standard
Nor-bile acid
24-Nor-hyodeoxycholic acid
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
use of LC–MS/MS, coupled with the availability of a suitable internal
reference standard, seems to be the best analytical method [3–7].
Alvelius et al. recently reported that the urine of a patient with
Niemann-Pick disease type C1 (NP-C1) had a unique bile acid pat-
tern as evidenced by the presence of ꢀ5-bile acid constituents when
analyzed by liquid chromatography-tandem mass spectrometry
(LC–MS/MS) [1]. Increased excretion of the 3␤,7␤-dihydroxychol-
5-en-24-oic acid and its 7-oxo derivative (which occur in conju-
gated form) might be pathognomonic for this disease and its detec-
tion in urine could well aid in its diagnosis.
In a previous paper, we have prepared a series of the multi-
conjugates of naturally occurring 3␤,7␤-dihydroxy-5-cholen-24-
oic acid and 3␤-hydroxy-7-oxo-5-cholen-24-oic acid as authentic
reference compounds (1a–1c and 2a–2c). These compounds were
prepared as “multi-conjugates” in that they were esterified with
sulfuric acid at C-3, with N-acetylglucosamine (GlcNAc) at C-7,
and/or with glycine (or taurine) at C-24 (Fig. 1) [2].
A satisfactory internal reference standard for the LC–MS/MS anal-
ysis is, therefore, desirable. As part of a program in our laboratory
to prepare novel bile acids that have clinical utility, we undertook
the chemical synthesis of the four variants of unnatural bile acid
conjugates (Fig. 2). Of the four candidate compounds prepared,
3␤-sulfooxy-7␤-hydroxy-24-nor-5-cholenoic acid (IV) was found
to be the most suitable for the LC–MS/MS analysis of a complicated
mixture of naturally occurring 1a–1c and 2a–2c. We also report
here the mobility of IV on C18 reversed-phase high-performance
liquid chromatography (RP-HPLC) and its suitability as an internal
standard.
2. Results and discussion
The four variants of candidate compounds (I–IV) were designed
on the basis of the hydrophilic–hydrophobic balance in steroid
molecules. The literature indicates that multiple factors influence
the mobility of bile acids when separated by RP-HPLC. These
include the position, number, and stereochemical configuration
of functional groups [3,8], as well as their mode of conjugation,
i.e., with amino acid (glycine or taurine) [8–10], sulfuric acid [5,6]
For the purpose of a highly selective, sensitive, accurate analy-
sis of such polar and hydrophilic ꢀ5-bile acid multi-conjugates, the
∗
Corresponding author. Tel.: +81 3 3329 1151; fax: +81 3 3303 9800.
E-mail address: takaiida@chs.nihon-u.ac.jp (T. Iida).
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.04.007

G. Kakiyama et al. / Steroids 74 (2009) 766–772
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Fig. 1. Structures of the 3␤,7␤-dihydroxy-5-cholen-24-oic acid and 3␤-hydroxy-7-oxo-5-cholen-24-oic acid multi-conjugates in a patient with NP-C1.
and/or monosaccharide (d-glucose, N-acetyl-d-glucosamine or d-
glucuronic acid) [11–13]. The ꢀ5 bile acid has an A/B-ring juncture
that is pseudo-trans; as a result, the 3␤-hydroxy group is equato-
rial, diminishing the hydrophilicity of the ␣-side of the bile acid
molecule; on the other hand, the 7␤-hydroxy group of ursodeoxy-
cholic acid (3␣,7␤-dihydroxy-5␤-cholan-24-oic acid, UDCA) also
impinges on the hydrophobic ␤-side of the molecule, greatly
diminishing the retention time, as compared to chenodeoxycholic
acid (3␣,7␣-dihydroxy-5␤-cholan-24-oic acid, CDCA) in which the
hydroxy group at C-7 is ␣. Such polar substituents have a marked
effect on bile acid hydrophilicity which in turn influences partition-
ing between the mobile and stationery phases during RP-HPLC. In
addition, shape factors such as the A/B-ring juncture can influence
binding to the C18 stationary phase. The number of carbon atoms at
the C-17 side chain also influences binding to the stationery phase
[3].
Based on above the findings and the structures of naturally
occurring C24 ꢀ5-bile acid multi-conjugates (1a–1c and 2a–2c),
we designed four variants of the unnatural types of analogous
bile acid conjugates (I–IV), which differed from one another in
the position and number of the conjugation modes, the presence
or absence of a ꢀ5-bond, and/or the length of the cholane side
chain (C3–C5) (Conventional C24 bile acids have the isopentanoic
(C5H9O2) side chain). The chemical syntheses of I–IV have been
attained and their synthetic routes were shown in Figs. 3–6; in the
case of the compounds I–III, the physical and spectral data are given
in Section 4, but those of the intermediary compounds have been
omitted.
Our results showed that 3␣-N-acetylglucosaminyl-7␣-sulfooxy-
5␤-cholan-24-oic acid (I as sodium salt) and 3␣-N-
acetylglucosaminyl-7␤-sulfooxy-5␤-cholan-24-oic acid (II as
sodium salt) were unsuitable as an internal reference standard,
because both I and II had too long retention times using RP-HPLC.
Variant
3␤-sulfooxy-7␤-hydroxy-23,24-bisnor-5-cholen-22-oic
acid (III as sodium salt) was also unsuitable because it generated an
unknown interfering ion when added to urine samples that were
analyzed by LC–MS/MS (unpublished observations). On the other
hand, 3␤-sulfooxy-7␤-hydroxy-24-nor-5-cholen-23-oic acid (IV
as sodium salt) when added as an internal standard permitted an
LC–MS/MS analysis of the targeted molecules (1a–1c and 2a–2c)
that was both sensitive and specific and thus useful for clinical
application.
As shown in Fig. 3, the desired IV was prepared start-
ing from hyodeoxycholic acid (3␣,6␣-dihydroxy-5␤-cholan-24-oic
acid; HDCA, 3). A key reaction involved is the so-called “second
order” Beckmann rearrangement of the performate derivative (3a)
of 3 with sodium nitrite in a mixture of trifluoroacetic anhydride
and trifluoroacetic acid [14]. Subsequent alkaline hydrolysis of
Fig. 2. Structures of the candidate compounds for an internal standard for the LC–MS/MS analysis of the multi-conjugates (1a–1c and 2a–2c) reported to be present in urinary
bile acids in a patient with NP-C1.

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Fig. 3. Synthetic route to 3␤-sulfooxy-7␤-hydroxy-24-nor-5-cholen-23-oic acid disodium salts (IV) from HDCA (3).
the resulting 24-nor-23-nitrile intermediate afforded 24-nor-HDCA
(4a) in isolated yield of 44% [14]. The procedure was found to be an
easy, efficient method for one-carbon degradation of the isopen-
tanoic (C5) side chain in natural C24 bile acids.
Esterification of 4a with methanol/p-toluenesulfonic acid, fol-
lowed by tosylation of the resulting methyl ester 4b with tosyl
chloride in pyridine yielded readily 3␣,6␣-ditosyloxy-24-nor-
5␤-cholan-23-oate (5). When 5 was refluxed in aqueous N,Nꢀ-
dimethylformamide (DMF) in the presence of potassium acetate,
simultaneous SN2 inversion at C-3 and E2 elimination at C-6 [2,15]
took place smoothly to give methyl 3␤-hydroxy-24-nor-5-cholen-
23-oate (6a) in 38% isolated yield after chromatographic purifica-
tion on silica gel.
tert-Butyldimethylsilylation (TBDMSi) of 6a with tert-
butyldimethylsilyl chloride in dry DMF and pyridine and
subsequent allylic oxidation of the resulting 3␤-TBDMSi-24-nor-ꢀ5
ester 6b with pyridinium dichromate and tert-butylhydroperoxide
(TBHP) [16] yielded the corresponding 3␤-TBDMSi-7-oxo-24-nor-
ꢀ5 ester 7 in 84% isolated yield after chromatographic purification
on silica gel.
Reduction of the 7-ketone 7 with Zn(BH4)2 [2,17] afforded
exclusively the equatorially-oriented 3␤-TBDMSi-7␤-hydroxy-24-
Fig. 4. Synthetic route to 3␣-(2-acetamide-2-deoxy-␤-d-glucopyranosyl)-7␣-sulfooxy-5␤-cholan-24-oic acid disodium salts (I) from CDCA.

G. Kakiyama et al. / Steroids 74 (2009) 766–772
769
Fig. 5. Synthetic route to 3␣-(2-acetamide-2-deoxy-␤-d-glucopyranosyl)-7␤-sulfooxy-5␤-cholan-24-oic acid disodium salts (II) from UDCA.
nor-ꢀ5 ester 8a (50%), which in turn was acetylated by the usual
method to yield the corresponding 3␤-TBDMSi-7␤-acetoxy-24-
nor-ꢀ5 ester 8b. Selective deprotection of the TBDMSi group at C-3
in 8b with HCl gave the corresponding 3␤-hydroxy-7␤-acetoxy-24-
nor-ꢀ5 ester 8c.
Regioselective sulfation at C-3 in 8c was successfully attained
with sulfur trioxide–trimethylamine complex in pyridine [18]. After
the reaction, the product was purified by passing through a Sep-
Pak C18 cartridge for solid-phase extraction, and then eluting with
a mixture of methanol and water. Alkaline hydrolysis of the effluent
with NaOH afforded the desired 3␤-sulfooxy-7␤-hydroxy-ꢀ5-24-
nor-5␤-cholan-23-oic acid (IV) in 76% isolated yield. Thus, IV was
obtained from HDCA (3) in 12 steps with total yield of 1.8%.
3. Characterization of the four synthetic compounds by
HPLC
Fig. 7 shows a typical chromatogram of a mixture of the four
synthetic compounds I–IV, together with some naturally occurring
1a and 2a on RP-HPLC with an ELSD. They showed much different
retention times from one another, emerging from a C18 column in
the order of III, IV, II, and then I. Of these compounds, IV was found
to be the best choice as an internal reference standard. In addition,
the sulfooxy compound IV was also found to be stable after addition
to urine and during the LC–MS analysis.
The desired compound IV is now available for the highly sen-
sitive, selective, and accurate LC–MS/MS determination of the
Fig. 6. Synthetic route to 3␤-sulfooxy-7␤-hydroxy-23,24-bisnor-5-cholen-22-oic acid (III) from 3␤-hydroxy-23,24-bisnor-5-cholen-22-oic acid.

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Pak-type MGII column [250 mm × 3.0 mm inner diameter (ID); par-
ticle size, 5 ␮m; Shiseido, Tokyo, Japan] was employed and kept at
37 ◦C. An Alltech 2000ES ELSD (Deerfield, IL) was used under the
following conditions; the flow rate of purified compressed air as
nebulizing gas was 1.6 L/min, the temperature of the heated drift
tube was 82 ◦C, and the gain was 8. A mixture of two solvent sys-
tems, which is composed of methanol for the organic solvent A
and 20 mM ammonium acetate (pH 5.4) for the aqueous buffer
solution B, was used as the mobile phases [9]. The composition of
the solvent A was gradually increased as follows: initial—12.0 min,
60% A (constant); 12.1–30.0 min, 60% A→75% A (linear gradient);
30.1 min-end, 75% A (constant). The flow rate was kept constant at
0.3 mL/min during analysis.
The synthetic internal standards I–IV and naturally occurring
ꢀ5 compounds 1a and 2a were dissolved in methanol at the con-
centration of 200 ␮g/mL as stock solutions, respectively. An aliquot
(100 ␮L) of each stock solution was mixed, and then 7.5 ␮L of the
resulting mixture was injected into the above HPLC-ELSD system.
Fig. 7. A typical RP-HPLC chromatogram of a mixture of the four candidates for inter-
nal standards (compounds I–IV), together with two of the ꢀ5-bile acid conjugates
(1a and 2a) identified from the urine of a patient with NP-C1.
4.4. Preparation of I–IV
multi-conjugates of 3␤,7␤-dihydroxy-5-cholen-24-oic acid and
3␤-hydroxy-7-oxo-5-cholen-24-oic acid, compounds reported to
be present in the urine of a patient with NP-C1 [1,2]. A further
detailed study on a method for the clinical application is now pro-
gressing in our laboratory and the result will be reported elsewhere.
4.4.1. 3˛,6˛-Dihydroxy-24-nor-5ˇ-cholan-23-oic acid
(24-Nor-hyodeoxycholicacid)(4a) and its methyl ester (4b)
Compound 4a was prepared from HDCA 3 (25 g), accord-
ing to the procedure of Schteingart and Hofmann [14]: yield,
10 g (44%); mp, 197–198 ◦C (EtOAc–hexane) [lit. [14] 218–219.5 ◦C
(EtOAc)]. IR ꢁmax cm−1: 1728 (C O), 3297 (OH). 1H NMR (CD3OD)
ı: 0.72 (3H, s, 18-CH3), 0.93 (3H, s, 19-CH3), 1.01 (3H, d, J
5.4, 21-CH3), 3.51 (1H, brm, 3␤-H), 4.01 (1H, brm, 6␤-H). LR-
MS (EI), m/z: 360 (M–H2O, 100%), 345 (M–H2O–CH3, 41%), 342
(M–2H2O, 97%), 327 (M–2H2O–CH3, 40%), 255 (M–2H2O–SC,
16%), 246 (M–H2O–side chain (SC)–part of ring D, 27%), 231
(M–H2O–CH3–SC–part of ring D, 46%), 228 (M–2H2O–SC–part of
ring D, 24%), 213 (M–2H2O–CH3–SC–part of ring D, 60%). HR-
MS (ESI−), calculated as C23H37O4 [M−H]−: 377.2692; found, m/z:
377.2707.
4. Experimental
4.1. Materials and reagents
HDCA (3) and 3␤-hydroxy-23,24-bisnor-5-cholen-24-oic acid
were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo,
Japan) and Steraloids Inc. (Newport, RI, USA), respectively. UDCA
and CDCA were kindly donated from Mitsubishi Tanabe Pharma Co.
Ltd. (Osaka, Japan). All other reagents and solvents used were of
analytical reagent grade.
A mixture of the 24-nor-hyodeoxycholic acid (4a) (10 g) and p-
toluenesulfonic acid (1.2 g) in methanol (120 mL) was left to stand
at room temperature for over night. Most of the solvent was evap-
orated, and the residue was extracted with EtOAc. The combined
extract was washed with brine, dried with Drierite, and evaporated.
Recrystallization of the oily residue from EtOAc–hexane gave the
desired methyl ester 4b as colorless amorphous solids: yield 6 g
(60%); mp, 82.5–84 ◦C. IR ꢁmax cm−1: 1735 (C O), 3309 (O H). 1H
NMR (CDCl3) ı: 0.64 (3H, s, 18-CH3), 0.86 (3H, s, 19-CH3), 0.93 (3H,
d, J 5.4, 21-CH3), 3.57 (1H, brm, 3␤-H), 3.63 (3H, s, COOCH3), 4.00
(1H, brm, 6␤-H). 13C NMR (CDCl3) ı: 12.0 (C-18), 19.5 (C-21), 20.7 (C-
11), 23.5 (C-19), 24.1 (C-15), 28.2 (C-16), 29.2 (C-4), 30.1 (C-2), 33.7
(C-20), 34.8 (C-8), 35.5 (C-1), 35.9 (C-10), 39.8 (C-9), 41.4 (C-22), 42.9
(C-13), 48.4 (C-5), 51.3 ( COOOCH3), 56.0 (C-17), 56.1 (C-14), 68.0
(C-6), 71.5 (C-3), 174.0 (C-24). LR-MS (EI), m/z: 374 (M–H2O, 100%),
359 (M–H2O–CH3, 39%), 356 (M–2H2O, 81%), 341 (M–2H2O–CH3,
39%), 301 (26%), 255 (M–2H2O–SC, 28%), 246 (M–H2O–SC–part
of ring D, 32%), 231 (M–H2O–CH3–SC–part of ring D, 45%), 228
(M–2H2O–SC–part of ring D, 28%), 213 (M–2H2O–CH3–SC–part
of ring D, 71%). HR-MS (EI), calculated as C24H38O3 [M−H2O]:
374.2821; found, m/z: 374.2804.
4.2. General experimental procedure
Melting points (mp) were determined on a micro hot-stage
apparatus and are uncorrected. IR spectra were obtained in KBr
discs on a JASCO FT-IR 460 plus spectrometer (Tokyo, Japan). 1H and
13C NMR spectra were obtained on a JEOL JNM-EX 270 FT instru-
ment at 270 and 68.8 MHz, respectively. Low-resolution mass spec-
tra (LR-MS) were recorded on a JEOL JMS-DX 303 mass spectrometer
with an electron ionization (EI) source at 70 eV. High-resolution
mass spectra (HR-MS) were measured using a JEOL JMS-700 with
an EI source under the positive ion mode. For some polar com-
pounds, HR-MS were also recorded on Shimadzu LC–MS-IT-TOF
with ESI source under the negative ion mode. Normal phase TLC
for nonsulfated compounds was performed on pre-coated silica
gel plates (0.25 mm layer thickness; E. Merck, Darmstadt, Ger-
many) using hexane–ethyl acetate mixtures (95:5–60:40, v/v) as
the developing solvent. Reversed-phase (RP) TLC for the sulfated
compounds was carried out on pre-coated RP-18F254S plates using
methanol–water–acetic acid mixtures (90:10:1, v/v/v) as the devel-
oping solvent. Sep-Pak Vac tC18 cartridges (5 g; Waters, Milford, MA,
USA) were used for solid-phase extraction.
4.4.2. Methyl 3˛,6˛-ditosyloxy-24-nor-5ˇ-cholan-23-oate (5)
To a solution of methyl 24-nor-hyodeoxycholate 4b (5.2 g,
13.2 mmol) in dry pyridine (15 mL), a solution of tosyl chloride
(7.5 g, 39.6 mmol) in dry pyridine (8 mL) was added, and the mix-
ture was stirred at room temperature for 4 h. Ice chips were added
gradually to the mixture, and the precipitated solid was filtered.
The precipitate was redissolved in CH2Cl2, washed with water,
dried with Drierite, and then evaporated to dryness. The yellow
4.3. RP-HPLC analysis of the synthetic internal standards
The apparatus used was a LC-2000plus HPLC system (two
PU-2085 high-pressure pumps, an MX-2080-32 solvent mixing
module, and a CO-2060 column heater) equipped with a Chrom-
NAV data-processing system (JASCO Co., Tokyo, Japan). A Capcell

G. Kakiyama et al. / Steroids 74 (2009) 766–772
771
oily residue was passed through a short column of silica gel (120 g)
and eluted with EtOAc–hexane (6:4, v/v). Although the ditosylate 5
was pure according to the TLC and NMR analyses, it resisted crystal-
lization attempts: yield, 6.5 g (70%). IR ꢁmax cm−1: 1733 (C O). 1H
NMR (CDCl3) ı: 0.63 (3H, s, 18-CH3), 0.80 (3H, s, 19-CH3), 0.95 (3H,
d, J 5.4, 21-CH3), 2.47 (6H, s, 3␤- and 6␤-O3SC6H4CH3), 4.30 (1H,
brm, 3␤-H), 4.78 (1H, brm, 6␤-H), 7.32–7.37, and 7.71–7.80 (8H, m,
3␤- and 6␤-O3SC6H4CH3). LR-MS (EI), m/z: 356 (M-2TsOH, 100%),
341 (M–2TsOH–CH3, 23%), 255 (M–2TsOH–SC, 23%), 248 (17%), 235
(30%), 213(M–2TsOH–CH3–SC–part of ring D, 15%). HR-MS (EI), cal-
culated for C24H36O2 [M−2TsOH]: 356.2715; found m/z: 356.2693.
MS (EI), m/z: 487 (M–CH3, 3%), 459 (11%), 445 (M–C(CH3)3, 100%),
413 (M–C(CH3)3–CH4O, 36%) 371 (M–TBDMSiOH, 27%). HR-MS (EI),
calculated for C30H50O4Si [M]: 502.3478; found m/z: 502.3468.
4.4.6. Methyl 3ˇ-tert-butyldimethylsilyloxy-7ˇ-hydroxy-24-nor-
5-cholen-23-oate
(8a)
To a freshly prepared solution of Zn(BH4)2 in Et2O (ca. 1 M,
6 mL) [16], a solution of the 3␤-TBDMSi-7-oxo ester 7 (490 mg,
0.97 mmol) in benzene (6 mL) was added slowly. After standing
at room temperature for 2 h, the mixture was diluted with ben-
zene, and the combined organic layer was washed with water, dried
with Drierite, and evaporated to dryness. The residue was sub-
jected to a column of silica gel (30 g) and eluted with EtOAc–hexane
(1:9, v/v). Recrystallization of the residue from aqueous methanol
gave the 3␤-TBDMSi-7␤-hydroxy ester 8a in the form of color-
less amorphous solids: yield, 240 mg (50%); mp, 131–132 ◦C. IR
ꢁmax cm−1: 1734 (C O), 3310 (O H). 1H NMR (CDCl3) ı: 0.06
(6H, s, Si(CH3)2C(CH3)3), 0.73 (3H, s, 18-CH3), 0.89 (9H, s,
Si(CH3)2C(CH3)3), 1.00 (3H, d, J 8.1, 21-CH3), 1.04 (3H, s, 19-CH3),
3.50 (1H, brm, 3␣-H), 3.83 (1H, m, 7␣-H), 5.25 (1H, brs, 6-H). LR-
MS (EI), m/z: 486 (M–H2O, 100%), 429 (M–H2O–C(CH3)3, 12%), 354
(M–H2O–TBDMSiOH, 69%), 339 (M–H2O–TBDMSiOH–CH3, 75%),
313 (69%), 281 (11%), 253 (M–H2O–TBDMSiOH–SC, 12%), 235 (14%),
211 (M–H2O–TBDMSiOH–CH3–part of ring D, 10%). HR-MS (EI), cal-
culated for C30H52O4Si [M]: 504.3635; found m/z: 504.3620.
4.4.3. Methyl 3ˇ-hydroxy-24-nor-5-cholen-23-oate (6a)
A solution of the ditosylate 5 (6 g, 8.6 mmol) and CH3COOK
(840 mg) dissolved in DMF (65 mL) and water (7 mL) was refluxed
for 12 h. The solution was cooled at room temperature, and the
reaction product was extracted with EtOAc. The organic layer was
washed with brine, dried over Mg2SO4, and evaporated under
reduced pressure. The brown oily residue was passed through a
column of silica gel (140 g) and eluted with EtOAc–hexane (2:8,
v/v). Recrystallization of the oil from methanol gave the desired 3␤-
hydroxy-ꢀ5-ester 6a in the form of colorless needles: yield, 1.2 g
(38%); mp, 123–124 ◦C. IR ꢁmax cm−1: 1739 (C O), 3391 (O H). 1
H
NMR (CDCl3) ı: 0.72 (3H, s, 18-CH3), 0.99 (3H, d, J 5.4, 21-CH3),
1.01 (3H, s, 19-CH3), 3.53 (1H, brm, 3␣-H), 3.66 (3H, s, COOCH3),
5.36 (1H, brs, 6-H). LR-MS (EI), m/z: 374 (M, 100%), 359 (M–CH3,
40%), 356 (M–H2O, 74%), 341 (M–H2O–CH3, 47%), 289 (46%), 263
(M–CH3–ring A, 70%), 255 (M–H2O–SC, 28%), 231 (M–CH3–SC–part
of ring D, 30%), 213 (M–H2O–CH3–SC–part of ring D, 47%). HR-MS
(EI), calculated for C24H38O3 [M]: 374.2821; found m/z: 374.2808.
4.4.7. Methyl 3ˇ-tert-butyldimethylsilyloxy-7ˇ-acetoxy-24-nor-
5-cholen-23-oate (8b)
The 7␤-hydroxy ester 8a was converted into the correspond-
ing 7␤-acetate 8b by the acetic anhydride-pyridine method.
After the usual work-up, 8b was recrystallized from methanol
in the form of colorless needles: yield, 180 mg (88%); mp,
146–147 ◦C. IR ꢁmax cm−1: 1742 (C O). 1H NMR (CDCl3) ı: 0.04
(6H, s, Si(CH3)2C(CH3)3), 0.73 (3H, s, 18-CH3), 0.88 (9H, s,
4.4.4. Methyl
3ˇ-tert-butyldimethylsilyloxy-24-nor-5-cholen-23-oate (6b)
To a solution of the 3␤-hydroxy-ꢀ5 ester 6a (750 mg, 2.0 mmol)
in anhydrous DMF (11 mL) and pyridine (0.5 mL), imidazole (1.8 g)
and tert-butyldimethylsilyl chloride (TBDMSiCl; 1.0 g, 6.6 mmol)
were added. The mixture was stirred at room temperature for 1.5 h.
The reaction product was extracted with EtOAc, and the combined
extract was washed with brine, dried with Drierite, and evaporated.
The residue was passed through a short column of silica gel (30 g)
by elution with EtOAc–hexane (2:98, v/v) and recrystallized from
methanol to give the 3␤-TBDMSi derivative 6b in the form of col-
Si(CH3)2C(CH3)3), 0.99 (3H, d,
J 8.1, 21-CH3), 1.06 (3H, s,
19-CH3), 2.02 (3H, s, OCOCH3), 3.49 (1H, brm, 3␣-H), 5.02
(1H, d, J 8.1, 7␣-H), 5.18 (1H, brs, 6-H). LR-MS (EI), m/z: 486
(M–CH3COOH, 100%), 429 (M–CH3COOH–C(CH3)3, 7%), 355 (49%),
354 (M–CH3COOH–TBDMSiOH, 32%), 339 (29%), 313 (25%). HR-MS
(EI), calculated for C30H50O3Si [M−CH3COOH]: 486.3529; found
m/z: 486.3520.
orless plates: yield, 890 mg (91%); mp, 163–165 ◦C. IR ꢁmax cm−1
:
1747 (C O). 1H NMR (CDCl3) ı: 0.05 (6H, s, Si(CH3)2C(CH3)3), 0.71
(3H, s, 18-CH3), 0.89 (9H, s, Si(CH3)2C(CH3)3), 0.99 (3H, d, J 5.4,
21-CH3), 1.00 (3H, s, 19-CH3), 3.48 (1H, brm, 3␤-H), 3.66 (3H, s,
COOCH3), 5.31 (1H, brs, 6-H). LR-MS (EI), m/z: 431 (M–C(CH3)3,
100%). HR-MS (EI), calculated for C30H51O3Si [M]: 487.3607; found
m/z: 487.3600.
4.4.8. Methyl 3ˇ-hydroxy-7ˇ-acetoxy-24-nor-5-cholen-23-oate
(8c)
To a solution of the 3␤-TBDMSi-7␤-acetoxy ester 8b (120 mg,
0.22 mmol) in methanol (6 mL) and acetone (2 mL), 10% HCl (40 ␮L)
was added. After being left to stand at room temperature for
1 h, the mixture was extracted with EtOAc. The EtOAc layer was
washed with brine, dried with Drierite, and evaporated to dry-
ness. Recrystallization of the residue from methanol gave the title
compound 8c in nearly quantitative yield in the form of colorless
needles: yield, 95 mg; mp, 137–138 ◦C. IR ꢁmax cm−1: 1742 (C O).
1H NMR (CDCl3) ı: 0.73 (3H, s, 18-CH3), 0.99 (3H, d, J 8.1, 21-CH3),
1.07 (3H, s, 19-CH3), 2.03 (3H, s, OCOCH3), 3.55 (1H, brm, 3␣-
H), 3.66 (3H, s, COOCH3), 5.02 (1H, d, J 10.8, 7␣-H), 5.21 (1H,
brs, 6-H). LR-MS (EI), m/z: 354 (M–H2O–CH3COOH, 100%), 339
(M–H2O–CH3COOH–CH3, 14%), 253 (M–H2O-CH3COOH–SC, 13%),
235 (24%). HR-MS (EI), calculated for C24H36O3 [M−CH3COOH]:
372.2664; found m/z: 372.2647.
4.4.5. Methyl
3ˇ-tert-butyldimethylsilyloxy-7-oxo-24-nor-5-cholen-23-oate (7)
To a magnetically stirred suspension of the 3␤-TBDMSi-ꢀ5 ester
6b (650 mg, 1.3 mmol), pyridinium dichromate (1.5 g, 4.0 mmol)
and Celite (1.3 g) in benzene (12 mL) and then 70%TBHP (1.1 mL)
was added gradually with ice-bath cooling. The whole mixture was
stirred at room temperature for 24 h. After filtration of the reaction
mixture on Celite, the mother liquor was evaporated under reduced
pressure. A dark brown residue was passed through a column of sil-
ica gel (25 g) and elution with EtOAc–hexane (5:95, v/v) gave the
title compound 7, which was recrystallized from methanol in the
form of colorless needles: yield, 550 mg (84%); mp, 216–217 ◦C. IR
ꢁmax cm−1: 1670 (C O, enone), 1741 (C O, ester). 1H NMR (CDCl3)
ı: 0.06 (6H, s, Si(CH3)2C(CH3)3), 0.72 (3H, s, 18-CH3), 0.89 (9H, s,
Si(CH3)2C(CH3)3), 0.99 (3H, d, J 5.4, 21-CH3), 1.19 (3H, s, 19-CH3),
3.60 (1H, brm, 3␤-H), 3.66 (3H, s, COOCH3), 5.66 (1H, s, 6-H). LR-
4.4.9. 3ˇ-Sulfooxy-7ˇ-hydroxy-24-nor-5-cholen-23-oic acid
disodium salts (IV)
To a solution of the 3␤-hydroxy-7␤-acetoxy ester 8c (33 mg,
0.08 mmol) in dry pyridine (1.6 mL), sulfur trioxide–trimethylamine
complex (50 mg, 0.4 mmol) was added, and the mixture was stirred

772
G. Kakiyama et al. / Steroids 74 (2009) 766–772
at room temperature for 1 h. The reaction mixture was poured into
ice-cooled petroleum ether (10 mL), and the precipitated solid was
collected by filtration. After being washed with petroleum ether,
the solid product was dissolved in 5% methanolic NaOH (10 mL) and
the solution was refluxed 7 h. The resulting solution was adjusted
to pH 8 with 10% HCl, diluted with water (90 mL), and loaded
onto a Sep-Pak® tC18 cartridge. The cartridge was washed with
water (20 mL) and 20% methanol (20 mL). The desired 3␤-sulfate
(1a) was eluted with methanol (20 mL) and recrystallized from
methanol–EtOAc in the form of colorless amorphous solids: yield,
28 mg (76%); mp, ca. 250 ◦C (decomposition). 1H NMR (CD3OD) ı:
0.76 (3H, s, 18-CH3), 1.02 (3H, d, J 5.4, 21-CH3), 1.08 (3H, s, 19-
CH3), 3.74 (1H, d, J 8.1, 7␣-H), 4.15 (1H, brm, 3␣-H). LR-MS (EI),
m/z: 340 (M–H2SO4–H2O, 100%), 325 (M–H2SO4–H2O–CH3, 9%),
253 (M–H2SO4–H2O–SC, 9%), 221 (30%). HR-MS (ESI−), calculated
for C23H34O7S [M−2Na+2H−H]: 455.2103; found m/z: 455.2122.
Acknowledgements
This work was supported by a Grant-in-Aid for Young Scien-
tists (B) (to G.K., Grant 20,750,141) for 2008–2009, a Grant-in-Aid
for Scientific Research (C) (to T.I., Grant 19,510,223) for 2007–2008,
JST Research for Promoting Technological Seeds (to T.I.) for 2007,
and a Nihon University Multidisciplinary Research Grant (to T.I.)
for 2007–2008.
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4.4.10. 3˛-(2-Acetamido-2-deoxy-ˇ-d-glucopyranosyl)-7˛-
sulfooxy-5ˇ-cholan-24-oic acid disodium salts (I)
(Chenodeoxycholic acid-3˛-N-acetylglucosaminide-7ˇ-sulfate)
This compound was prepared in 8 steps (see Fig. 4) from CDCA:
mp, 203–205 ◦C (recrystallized from methanol–EtOAc). 1H NMR
(CD3OD) ı: 0.69 (3H, s, 18-CH3), 0.93 (3H, s, 19-CH3), 0.95 (3H,
d, J 5.4, 21-CH3), 1.99 (3H, s, NHCOCH3), 3.50 (1H, brm, 3␤-H),
3.23–3.89 (7H, m, 2ꢀ-, 3ꢀ-, 4ꢀ-, 5ꢀ-, and 6ꢀ-H), 4.44 (1H, brs, 7␤-H), 4.56
(1H, d, J 8.1, 1ꢀ␣-H), 7.95 (3H, J 8.1, NHCOCH3). LR-MS (EI), m/z: 356
(M–GlcNAc–H2SO4, 100%), 341 (M–GlcNAc–H2SO4–CH3, 69%), 255
(M–GlcNAc–H2SO4–SC, 31%), 228 (M–GlcNAc–H2SO4–part of ring
D, 14%), 213 (M–GlcNAc–H2SO4–CH3–part of ring D, 28%). HR-MS
(ESI−), calculated for C32H51NO12S [M−2Na+2H−H]−: 674.3209;
found m/z: 674.3241.
4.4.11. 3˛-(2-Acetamido-2-deoxy-ˇ-d-glucopyranosyl)-7ˇ-
sulfooxy-5ˇ-cholan-24-oic acid disodium salts (II)
(Ursodeoxycholic acid-3˛-N-acetylglucosaminide-7ˇ-sulfate)
This compound was prepared in 7 steps (see Fig. 5) from
UDCA: mp, 212–214 ◦C (recrystallized from methanol–EtOAc).
1H NMR (CD3OD) ı: 0.69 (3H, s, 18-CH3), 0.96 (3H, d,
J
5.4, 21-CH3), 0.97 (3H, s, 19-CH3), 1.98 (3H, s, NHCOCH3),
3.23–3.88 (7H, m, 2ꢀ-, 3ꢀ-, 4ꢀ-, 5ꢀ-, 6ꢀ-H, and 3␤-H), 4.29
(1H, brm, 7␣-H), 4.55 (1H, d,
J J
8.1, 1ꢀ␣-H), 8.0 (1H, d,
8.1 Hz NHCOCH3). LR-MS (EI), m/z: 356 (M–GlcNAc–H2SO4,
100%), 341 (M–GlcNAc–H2SO4–CH3, 57%), 302 (26%), 255
(M–GlcNAc–H2SO4–SC, 34%), 228 (M–GlcNAc–H2SO4–part of
ring D, 14%), 213 (M–GlcNAc–H2SO4–CH3–part of ring D, 27%).
HR-MS (ESI−), calculated for C32H51NO12
674.3209; found m/z: 674.3247.
S
[M−2Na+2H−H]−:
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acid disodium salts (III)
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3␤-hydroxy-23,24-bisnor-5-cholen-22-oic acid: mp, ca. 140 ◦C
(decomposition) (recrystallized from methanol–EtOAc). 1H NMR
(CD3OD) ı: 0.73 (3H, s, 18-CH3), 1.08 (3H, s, 19-CH3), 1.15 (3H,
d, J 5.4, 21-CH3), 3.74 (1H, d, J 8.1 Hz, 7␣-H), 4.14 (1H, brm,
3␣-H), 5.29 (1H, s, 6-H). LR-MS (EI), m/z: 326 (M–H2O–H2SO4,
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