Synthesis of multiply deuterated 3- and 21-monosulfates of allo-tetrahydrocorticosteroids as internal standards for mass spectrometry

Article abstract of DOI:10.1016/j.steroids.2012.08.007

The accurate analysis of trace components in complex biological matrices requires the use of reliable internal standards. For liquid chromatography/mass spectrometry analysis, the stable isotope-labeled analogues of the analyte molecules are the most appropriate internal standards. In this paper the synthesis of the 3- and 21-monosulfates of allo-tetrahydrocorticosteroids labeled with four or five deuterium atoms is described. The principal reactions used were (1) hydrogen-deuterium exchange reaction of active methylene groups adjacent to 3- and 11-oxo group of 17,20;20,21-bismethylenedioxy derivatives of 5α-3-ketosteroids and/or 5α-11-ketosteroids with NaOD in CH ₃OD followed by reduction with NaBD₄, (2) epimerization of the 3β-hydroxy group into a 3α configuration, (3) sulfation of hydroxy groups at C-3 or C-21 in the resulting substrates with sulfur trioxide-trimethylamine complex, and (4) removal of 17,20;20,21- bismethylenedioxy groups with hydrogen fluoride in ethanol. Isotopic purity was found to be satisfactory by MS, and NMR properties of the new compounds were tabulated. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.

Full text of DOI:10.1016/j.steroids.2012.08.007

Steroids 77 (2012) 1423–1437
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of multiply deuterated 3- and 21-monosulfates
of allo-tetrahydrocorticosteroids as internal standards for mass spectrometry
Kuniko Mitamura a, Takayuki Mabuchi a, Kaori Nagae a, Masataka Nakajima a, Rina Matsumoto a,
Sachi Fujioka a, Kanta Sato a, Rika Satoh (née Okihara) b, Takashi Iida b, Shoujiro Ogawa c,
Alan F. Hofmann d, Shigeo Ikegawa a,
⇑
a Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan
b Department of Chemistry, College of Humanities & Sciences, Nihon University, Sakurajousui, Setagaya-ku, Tokyo 156-8550, Japan
c Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan
d Department of Medicine, University of California, San Diego, La Jolla, CA 92093-063, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2012
Received in revised form 20 August 2012
Accepted 24 August 2012
Available online 31 August 2012
The accurate analysis of trace components in complex biological matrices requires the use of reliable
internal standards. For liquid chromatography/mass spectrometry analysis, the stable isotope-labeled
analogues of the analyte molecules are the most appropriate internal standards. In this paper the synthe-
sis of the 3- and 21-monosulfates of allo-tetrahydrocorticosteroids labeled with four or five deuterium
atoms is described. The principal reactions used were (1) hydrogen–deuterium exchange reaction of
active methylene groups adjacent to 3- and 11-oxo group of 17,20;20,21-bismethylenedioxy derivatives
Keywords:
of 5
a
-3-ketosteroids and/or 5
a-11-ketosteroids with NaOD in CH3OD followed by reduction with NaBD4,
Multiple deuteration
Hydrogen–deuterium exchange
Sodium borodeuteride
Walden inversion
Electrospray ionization
LC/MS
(2) epimerization of the 3b-hydroxy group into a 3
a
configuration, (3) sulfation of hydroxy groups at C-3
or C-21 in the resulting substrates with sulfur trioxide–trimethylamine complex, and (4) removal of
17,20;20,21-bismethylenedioxy groups with hydrogen fluoride in ethanol. Isotopic purity was found to
be satisfactory by MS, and NMR properties of the new compounds were tabulated. The labeled com-
pounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical
and biochemical studies.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
well as the corresponding stable isotope-labeled conjugates as
internal standards.
The direct analysis of steroid conjugates in urine has long been a
goal for endocrinologists [1]. Historically, steroid analysis has in-
volved removal of the conjugating moiety by enzymatic hydrolysis
or acidic solvolysis, followed by gas chromatography/mass spec-
trometry (MS) of the steroid moiety. This analytical approach has
several potential limitations, such as incomplete hydrolysis
(mainly due to urine matrix effect), as well as interconversion of
steroids by the enzyme preparation [2]. In addition, information
as to the type and site of conjugation is not generated by this ana-
lytical approach. Electrospray ionization (ESI) has tremendously
expanded the analytical utility of MS. With the development of
interfacing liquid chromatography (LC) to MS, LC/ESI-MS has be-
come widely accepted as a powerful tool for directly measuring
the trace amount of the steroid conjugates in biological fluids. Di-
rect quantitative analysis of steroid conjugates require the avail-
ability of authentic steroid conjugates as reference standards as
Glucocorticoids (GC), cortisol and cortisone, and 11-deoxycorti-
sol (11-DOC, a biosynthetic precursor of cortisol) are largely
excreted in conjugated form as glucuronides and sulfates of
5a- and 5b-tetrahydro-reduced metabolites. Measurement of the
urinary output of the 5 - and 5b-tetrahydro-reduced metabolites
a
of GC and 11-DOC should be important in that such an analysis
would provide a non-invasive, integrated evaluation of adrenocor-
tical GC secretion, that in turn would permit the diagnosis of GC
hypertension as occurs in Cushing’s syndrome or the metabolic
syndrome [3–7]. However, the diagnostic value of measurement
of these conjugates for detection of GC hypertension is unknown,
because they have never been measured precisely due to the lack
of a reliable method for direct measurement.
We have previously reported a highly sensitive and specific LC/
ESI-linear ion trap MS method using a deuterium-labeled internal
standard for the direct measurement of 12 tetrahydrocorticoster-
oid glucuronides in human urine [8]. In addition, we have reported
the synthesis of the 18 sulfated conjugates of tetrahydrocorticos-
teroids as reference standards [9], as well as 3- and 21-monosulfat-
es of [2,2,3b,4,4-d5]-5b-tetrahydro-reduced metabolites as internal
⇑
Corresponding author.
E-mail address: ikegawa@phar.kindai.ac.jp (S. Ikegawa).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.08.007

1424
K. Mitamura et al. / Steroids 77 (2012) 1423–1437
standards [10]. The availability of such standards and internal
standards should permit the identification and measurement of
all sulfate conjugates in the urine using LC/ESI-MS that in turn
would permit assessment of the dynamics of their formation and
disposal. One limitation of such sulfate conjugate analysis, how-
ever, is the lack of stable isotope labeled 3- and 21-monosulfated
2.2. Instruments
All melting points (mp) were determined on a micro hot-stage
apparatus and are uncorrected. Mixed melting point of the all
deuterated compounds with their corresponding non-deuterated
compounds showed no depression. 1H NMR spectra were re-
corded on a JNM-AL400 or JNM-ECA700 (JEOL Ltd., Tokyo, Japan)
at 400 or 700 MHz, with CDCl3 or CD3OD containing 0.1% Me4Si
as the solvent; chemical shifts were expressed as d ppm relative
to Me4Si. The following abbreviations are used: s = singlet,
d = doublet, m = multiplet. 13C NMR spectra were obtained on a
JEOL JNM-ECA700 instrument at 176 MHz. The 13C distortionless
enhancement by polarization transfer (DEPT; 23 °C) spectra were
also measured to determine the 1H signal multiplicity and to dif-
ferentiate between CH3, CH2, CH, and C based on their proton
environments. The 1H NMR chemical shift assignments of non-
deuterated compounds and the 1H and 13C NMR chemical shift
assignments of multideuterated 3- and 21-monosulfates of allo-
tetrahydrocorticosteroids are tabulated in Tables 1 and 2, respec-
tively. The LC/ESI-MS analyses were carried out using a Finnigan
LTQ linear ion trap mass spectrometer (Thermo Fischer Scientific
Inc., Waltham, MS, USA) equipped with an ESI source and coupled
to a Paradigm MS4 pump (Michrom Bioresources Inc., Auburn,
CA, USA) and an autosampler (HTC PAL, CTC Analytics, Zwingen,
Switzerland). The ionization conditions for verifying the struc-
tures were as follows: ion source voltage, ꢀ4 kV; capillary tem-
perature, 270 °C; capillary voltage, ꢀ20 V; sheath gas (N2 gas)
flow rate, 50 arbitrary units; auxiliary gas (N2 gas) flow rate, 5
arbitrary units; tube lens offset voltage, ꢀ100 V. For collision in-
duced dissociation (CID) analysis, helium gas was used as the col-
lision gas. The normalized collision energy and the activation Q
value were set at 35% and 0.18, respectively. LC separations were
conducted on a reversed-phase (RP) semi-micro column, TSKgel
conjugates of
standards.
5a-tetrahydro-reduced metabolites as internal
The present study describes the synthesis of 3- and 21-mono-
sulfates of allo-tetrahydro-11-deoxycortisol (allo-THS), allo-tetra-
hydrocortisol (allo-THF), and allo-tetrahydrocortisone (allo-THE)
labeled with multiple deuterium atoms (Fig. 1) to be used for iso-
tope dilution MS.
2. Experimental
2.1. Materials
17,20;20,21-Bismethylenedioxypregn-4-en-3-one (1) and 17,
20;20,21-bismethylenedioxy-11b-hydroxypregn-4-en-3-one (13)
were synthesized by a previously described method [10]. Silica
gel plates (Merck; F254) were used for thin layer chromatography
and silica gel (Merck; 70–230 mesh) was used for column chroma-
tography. Aluminum oxide 90 active basic (particle size, 63–
200 lm) was purchased from Merck KGaA (Darmstadt, Germany).
Forty percent of NaOD in D2O (99.5 at.% D), CH3OD (99.5 at.% D),
and sulfur trioxide–trimethylamine (SO3–TMA) complex were sup-
plied by Sigma–Aldrich Japan K.K. (Tokyo, Japan). LiAlD4 and
NaBD4 (99% isotopic purity) were obtained from Cambridge Iso-
tope Laboratories, Inc. (Andover, MA, USA). Acetonitrile and ammo-
nium acetate of HPLC grade were purchased from Nacalai Tesque,
Inc. (Kyoto, Japan), and distilled H2O of HPLC grade was purchased
from Wako Pure Chemical Industries, Ltd. OasisÒ HLB cartridges
(adsorbent weight, 1 g) were provided by Waters Co. (Milford,
MS, USA) and conditioned by successive washings with MeOH
and H2O prior to use. All other chemicals and solvents were analyt-
ical grade and obtained from Nacalai Tesque Inc.
ODS-100S (5
l
m, 150 ꢁ 2.0 mm I.D.) from Tosoh Co. (Tokyo,
Japan) by a linear gradient elution: 15% solvent B (acetonitrile)
over 5 min and then 15% solvent B to 50% B against solvent A
(5 mM ammonium acetate buffer, pH 6.0) over 30 min at a flow
rate of 200 ll/min.
Fig. 1. Structures of 3- and 21-monosulfated conjugates of multiply deuterated allo-tetrahydrocorticosteroids.

K. Mitamura et al. / Steroids 77 (2012) 1423–1437
1425
Table 1
1H NMR chemical shifts of non-deuterated compounds prepared.a
Compounds 2- and
3-H
3a
-H
3b-H
6-H
11
a-H
18-
CH3
19-
CH3
21-CH2
CH2O
OCOCH3
Si(CH3)3
C6H4CH3 C6H4CH3
2
3.50–3.58
(m)
5.35–
5.36
(m)
0.84
(s)
1.02
(s)
3.98 and 4.01 5.05–5.20
(d, J = 8.8 Hz) (m)
3a
3b
3.55–3.63
(m)
4.37–4.44
(m)
0.82
(s)
0.79
(s)
0.82
(s)
0.79
(s)
3.96 and 4.01 5.04–5.19
(d, J = 9.2 Hz) (m)
3.96 and
3.99
5.03–5.18
(m)
2.44 (s)
7.33 and
7.79
(d, J = 9.2 Hz)
(d,
J = 8.4 Hz)
4
0.84
(s)
1.02
(s)
3.97 and 4.01 5.04–5.20
(d, J = 9.2 Hz) (m)
5a
5b
5c
4.02–4.06
(m)
4.99–5.03
(m)
4.56–4.60
(m)
0.79
(s)
0.80
(s)
0.83
(s)
0.81
(s)
0.82
(s)
0.84
(s)
3.96 and 4.00 5.03–5.19
(d, J = 9.2 Hz) (m)
3.97 and 4.00 5.04–5.19
(d, J = 9.2 Hz) (m)
3.90 and 4.05 4.96–5.15
(d, J = 9.2 Hz) (m)
2.05 (s)
6
7
5.53–5.63
(m)
3.10–3.16
(m)
0.76
(s)
0.76
(s)
0.83
(s)
0.80
(s)
3.97 and 4.01 5.05–5.19
(d, J = 9.2 Hz) (m)
3.96 and
3.99
5.04–5.19
(m)
(d, J = 9.2 Hz)
14
3.48–3.58
(m)
5.19–
5.22
(m)
4.42–4.44
(m)
1.07
(s)
1.19
(s)
3.97 and 4.02 5.02–5.20
(d, J = 9.0 Hz) (m)
0.12 (s)
15a
15b
15c
3.54–3.59
(m)
3.48–3.54
(m)
4.38–4.45
(m)
4.33–4.34
(m)
4.28–4.34
(m)
4.28–4.32
(m)
0.96
(s)
0.98
(s)
1.02
(s)
1.04
(s)
1.00
(s)
1.05
(s)
3.96 and 4.01 5.02–5.19
(d, J = 9.0 Hz) (m)
3.90 and 3.93 4.96–5.15
(d, J = 9.0 Hz) (m)
0.11 (s)
0.11 (s)
3.96 and
3.99
5.02–5.20
(m)
2.44 (s)
7.33 and
7.79
(d, J = 9.0 Hz)
(d,
J = 8.4 Hz)
16a
16b
17a
17b
18
4.3604.37
(m)
4.39–4.40
(m)
4.40–4.41
(m)
4.40–4.41
(m)
1.06
(s)
1.09
(s)
1.03
(s)
1.04
(s)
1.19
(s)
1.27
(s)
1.07
(s)
1.08
(s)
3.96 and 4.01 5.03–5.22
(d, J = 9.0 Hz) (m)
3.98 and 4.01 5.03–5.22
(d, J = 9.0 Hz) (m)
3.97 and 4.00 5.03–5.22
(d, J = 9.0 Hz) (m)
3.97 and 4.00 5.03–5.22
(d, J = 9.0 Hz) (m)
0.13 (s)
4.02–4.07
(m)
5.00–5.01
(m)
5.53–5.63
(m)
4.38–4.40
(m)
1.03
(s)
1.09
(s)
3.96 and 4.02 5.03–5.23
(d, J = 9.0 Hz) (m)
19
3.15–3.16
(m)
4.34–4.38
(m)
1.06
(s)
1.06
(s)
3.96 and 4.00 5.02–5.22
(d, J = 9.0 Hz) (m)
27a
4.99–5.01
(m)
0.78
(s)
1.02
(s)
3.94 and
3.97
5.01–5.19
(m)
2.04 (s)
(d, J = 9.0 Hz)
3.97 and 4.0
(d, J = 9.0 Hz) (m)
27b
4.01–4.06
(m)
0.78
(s)
1.01
(s)
5.01–5.19
a
Measured in CDCl3 at 400 MHz; chemical shifts were expressed as d ppm relative to Me4Si; abbreviations used: s, singlet; d, doublet; m, multiplet; values in parentheses
refer to signal multiplicity and coupling constant (J in Hz).
2.3. Chemical synthesis
to dryness. Purification of the product by column chromatography
on silica gel with toluene–acetone (10:1, v/v) and crystallization of
a homogeneous effluent from acetone gave 2 as colorless needles:
yield, 128 mg (64%); mp 241–242 °C (lit. [11], mp 245–246 °C).
2.3.1. 17,20;20,21-Bismethylenedioxypregn-5-en-3b-ol (2)
To a solution of 17,20;20,21-bismethylenedioxypregn-4-en-3-
one (1, 200 mg) in dry pyridine (8 ml) was successively added
bromotrimethylsilane (1.2 ml) and hexamethyldisilazane (6.4 ml),
and the mixture was stirred at room temperature for 20 min. The
resulting solution was diluted with Et2O, washed with cold 2 M
HCl and H2O, and dried over anhydrous Na2SO4. Evaporation of
the solvent under reduced pressure afforded the intermediary
2.3.2. 17,20;20,21-Bismethylenedioxy-5a-pregnan-3b-ol (3a)
The compound (510 mg) was hydrogenated with 10%
2
Pd(OH)2/C catalyst (1.9 g) in EtOAc (60 ml) under a stream of
H2 gas at room temperature overnight. After removal of the cata-
lyst by filtration, the filtrate was concentrated to dryness under
reduced pressure. Purification of the product by column chroma-
tography on silica gel with toluene–acetone (10:1, v/v) as an elu-
ent and crystallization of a homogeneous effluent from CH2Cl2–
MeOH gave 3a as colorless needles: yield, 448 mg (88%); mp
255–257 °C.
D
5-3-keto derivative, which without isolation was subjected to
reduction with NaBH4 (176 mg) in tetrahydrofuran (7 ml)–MeOH
(1 ml) at room temperature for 15 min. After addition of 10% acetic
acid to decompose the excess reagent, the resulting solution was
extracted with EtOAc. The organic layer was washed with 5%
NaHCO3 and H2O, dried over anhydrous Na2SO4, and evaporated

Table 2
1H and 13C NMR chemical shifts of multiply deuterated sulfates of allo-tetrahydrocorticosteroids.a
Carbon No.
12a
12d
24a
24d
Type
13C
1H
Type
13C
1H
Type
13C
1H
Type
13C
1H
1
2
3
4
5
6
7
8
CH2
CD2
CD
CD2
CH
CH2
CH2
CH
CH
C
CH2
CH2
C
CH
CH2
CH2
C
33.8
–
–
CH2
CD2
CD
CD2
CH
CH2
CH2
CH
CH
C
CH2
CH2
C
CH
CH2
CH2
C
33.5
–
–
CH2
CD2
CD
CD2
CH
CH2
CH2
CH
CH
C
CH
CH2
C
CH
CH2
CH2
C
33.1
–
–
CH2
CD2
CD
CD2
CH
CH2
CH2
CH
CH
C
CH
CH2
C
CH
CH2
CH2
C
32.8
–
–
–
–
–
–
40.7
29.5
33.5
37.1
55.5
36.8
21.7
32.0
48.9
52.3
24.7
35.1
90.7
11.9
15.6
213.8
67.9
40.3
29.6
33.5
37.1
55.5
36.8
21.6
31.8
48.7
52.3
24.6
34.9
90.9
11.7
15.4
208.3
72.1
41.4
29.0
34.0
32.9
59.4
36.9
68.4
40.8
36.9
54.1
24.7
34.7
90.6
14.7
18.0
213.3
67.7
41.1
29.3
34.0
32.8
59.3
37.0
68.7
40.5
49.3
54.1
24.7
34.5
90.8
14.4
17.8
208.0
72.0
9
10
11
12
13
14
15
16
17
18
19
20
21
4.36–4.37 (m)
4.38–4.80 (m)
CH3
CH3
C
0.61 (s)
0.82 (s)
CH3
CH3
C
0.62 (s)
0.81 (s)
CH3
CH3
C
0.82 (s)
1.04 (s)
CH3
CH3
C
0.83 (s)
1.02 (s)
CH2
4.28 and 4.62 (d, J = 19.3 Hz)
CH2
4.82 and 5.04 (d, J = 18.3 Hz)
CH2
4.26 and 4.63 (d, J = 19.2 Hz)
CH2
4.79 and 5.02 (d, J = 18.0 Hz)
Carbon No.
26a
26d
29a
29d
Type
13C
1H
Type
13C
1H
Type
13C
1H
Type
13C
1H
1
2
3
4
5
6
7
8
CH2
CD2
CD
CD2
CH
CH2
CH2
CH
CH
C
CH2
CH2
C
CH
CH2
CH2
C
32.3
–
–
CH2
CD2
CD
CD2
CH
CH2
CH2
CH
CH
C
CH2
CH2
C
CH
CH2
CH2
C
31.9
–
–
CH2
CH2
CH
CH2
CH
CH2
CH2
CH
CD
C
CD
CD2
C
CH
CH2
CH2
C
33.3
27.7
76.7
34.2
41.6
29.1
34.0
32.8
–
CH2
CD2
CD
CD2
CH
CH2
CH2
CH
CD
C
CD
CD2
C
CH
CH2
CH2
C
32.8
29.3
67.3
36.3
41.1
29.4
34
32.8
–
37.3
–
4.59–4.64 (m)
3.94–4.00 (m)
–
–
40.4
28.9
34.0
38.0
65.3
36.5
214.0
51.6
52.6
51.8
24.2
35.4
89.5
11.7
16.2
213.0
67.9
40.0
29.1
34.1
37.9
65.4
36.8
213.5
51.4
52.6
51.8
24.1
35.3
89.7
11.5
16.1
207.7
72.0
9
10
11
12
13
14
15
16
17
18
19
20
21
36.9
–
–
–
49.6
54.1
24.7
34.7
90.6
14.6
17.9
213.4
67.9
49.3
54.1
24.7
34.6
90.9
14.4
17.8
208.2
72.1
CH3
CH3
C
0.54 (s)
1.01 (s)
CH3
CH3
C
0.56 (s)
1.00 (s)
CH3
CH3
C
0.83 (s)
1.04 (s)
CH3
CH3
C
0.83 (s)
1.03 (s)
CH2
4.21 and 4.60 (d, J = 19.6 Hz)
CH2
4.74 and 5.03 (d, J = 18.2 Hz)
CH2
4.28 and 4.65 (d, J = 18.8 Hz)
CH2
4.80 and 5.05 (d, J = 18.8 Hz)
a
Measured in CD3OD at 700 MHz in 1H NMR and at 176 MHz in 13C NMR; chemical shifts were expressed as d ppm relative to Me4Si; abbreviations used: s, singlet; d, doublet; m, multiplet; values in parentheses to signal
multiplicity and coupling constant (J in Hz).

K. Mitamura et al. / Steroids 77 (2012) 1423–1437
1427
2.3.3. 17,20;20,21-Bismethylenedioxy-5
toluenesulfonate (3b)
a
-pregnan-3b-yl p-
num oxide and allowed to stand at room temperature overnight.
Elution with toluene–acetone (250:1, v/v) and crystallization of a
less polar effluent from acetone–n-hexane gave 17,20;20,21-
To a solution of compound 3a (90 mg) in dry pyridine (2 ml)
was added p-toluenesulfonylchloride (195 mg), and the reaction
mixture was stirred at room temperature for 30 h. The resulting
solution was diluted with EtOAc, washed with 5% HCl, 5% NaHCO3,
and H2O, dried over anhydrous Na2SO4, and evaporated to dryness.
Purification of the product by column chromatography on silica gel
with toluene–acetone (15:1, v/v) as an eluent and crystallization of
a homogeneous effluent from acetone–n-hexane gave 3b as color-
less needles: yield, 126 mg (99%); mp 169–174 °C.
bismethylenedioxy-5a-pregn-2-ene (6) as colorless needles: yield,
16 mg (19%); mp 166–167 °C. Further elution with toluene–
acetone (50:1, v/v) and crystallization of a more polar effluent from
CH2Cl2–MeOH gave 5a as colorless needles: yield, 93 mg (35%); mp
184–186 °C.
2.3.8. 17,20;20,21-Bismethylenedioxy-2a,3a-epoxy-5a-pregnane (7)
To a solution of compound 6 (19 mg) in EtOAc (0.6 ml) was
added m-chloroperbenzoic acid (21 mg), and the mixture was stir-
red at room temperature for 5 h. The resulting solution was diluted
with EtOAc, and washed successively with 5% Na2S2O3, 5% NaHCO3,
and H2O. The organic phase was dried over anhydrous Na2SO4, and
evaporated to dryness. Purification of the product by column chro-
matography on silica gel with n-hexane–EtOAc (10:1, v/v) and
crystallization of a homogeneous effluent from acetone gave 7 as
colorless leaflets: yield, 14 mg (71%); mp 204–206 °C.
2.3.4. 17,20;20,21-Bismethylenedioxy-5a-pregnan-3-one (4)
(i) To a solution of compound 3a (118 mg) in acetone (5 ml)
was added Jones’ reagent (1 ml), and the mixture was stirred
at room temperature for 30 min. After addition of MeOH to
decompose the excess reagent, the organic solvent was
evaporated under reduced pressure. The residue was diluted
with EtOAc, washed with 5% NaHCO3 and H2O, dried over
anhydrous Na2SO4, and evaporated to dryness. Purification
of the product by column chromatography on silica gel with
toluene–acetone (20:1, v/v) as an eluent and crystallization
of a homogeneous effluent from CH2Cl2–MeOH gave 4 as col-
orless leaflets: yield, 111 mg (95%); mp 239–240 °C.
(ii) To a blue colored suspension of metallic Li (200 mg) dis-
persed in liq. NH3 (80 ml) was added a solution of the com-
pound 1 (1.0 g) in tetrahydrofuran (15 ml), and the mixture
was stirred at room temperature for 20 min with a dry-ice
condenser. The reaction was quenched by adding an excess
of NH4Cl, and then most of NH3 and solvent was evaporated.
The residue, dissolved in H2O, was acidified with 10% H2SO4
and extracted with EtOAc. The combined extract was
washed with saturated brine, dried over anhydrous MgSO4,
and evaporated to dryness under reduced pressure. The
crude product was subjected to column chromatography
on silica gel with n-hexane–EtOAc (65:35, v/v) as an eluent.
Crystallization of a homogeneous effluent from CH2Cl2–
MeOH gave 4 as colorless leaflets: yield, 594 mg (59%); mp
239–240 °C.
2.3.9. [2,2,3a,4,4-d5]-17,20;20,21-Bismethylenedioxy-5a-pregnan-3b-
ol (8a)
To a solution of compound 4 (200 mg) in CH3OD (10 ml) and dry
dioxane (9 ml) was added 40% NaOD (1.7 ml) and D2O (1.7 ml), and
the mixture was stirred at 80 °C for 22 h under a gentle stream of
N2 gas. After being cooled in ice bath, NaBD4 (85 mg) was added to
the solution and the reaction mixture was stirred at room temper-
ature for 40 min. After addition of 10% AcOH to decompose the ex-
cess reagents, the organic solvent was evaporated under reduced
pressure. The residue was diluted with EtOAc, washed with 5%
NaHCO3 and H2O, dried over anhydrous Na2SO4, and evaporated
to dryness. Purification of the product by column chromatography
on silica gel with toluene–acetone (20:1, v/v) as an eluent and crys-
tallization of a homogeneous effluent from CH2Cl2–MeOH gave 8a
as colorless needles: yield, 152 mg (76%); mp 255–257 °C.
2.3.10. [2,2,3a,4,4-d5]-17,20;20,21-Bismethylenedioxy-5a-pregnan-
3b-yl p-toluenesulfonate (8b)
The compound 8a (152 mg) was treated with p-toluenesulfonyl
chloride (292 mg) in dry pyridine (3 ml) at ice temperature for
24 h, as described for preparation of 3b. After being processed in
an analogous manner, the product was subjected to column chro-
matography on silica gel with n-hexane–EtOAc (25:1, v/v) as an
eluent. Crystallization of a homogeneous effluent from acetone–
n-hexane gave 8b as colorless needles: yield, 200 mg (95%); mp
175–179 °C.
2.3.5. 17,20;20,21-Bismethylenedioxy-5a-pregnan-3a-ol (5a)
To a solution of compound 4 (105 mg) in dry tetrahydrofuran
(3.5 ml) at ꢀ70 °C under a stream of N2 gas was added slowly a
1 M solution of L-SelectrideÒ in tetrahydrofuran (1 ml), and the
mixture was stirred at ꢀ70 °C for 2 h. After addition of 30% H2O2
(1 ml), the resulting solution was extracted with EtOAc. The com-
bined extract was washed with saturated brine, dried over anhy-
drous Na2SO4, and evaporated to dryness. Purification of the
product by column chromatography on silica gel with toluene–
acetone (20:1, v/v) and crystallization of a homogeneous effluent
from CH2Cl2–MeOH gave 5a as colorless needles: yield, 92 mg
(87%); mp 184–186 °C.
2.3.11. [2,2,3b,4,4-d5]-17,20;20,21-Bismethylenedioxy-5
-ol (10a)
A solution of compound 8b (150 mg) in toluene (1 ml) was
a-pregnan-
3a
loaded onto a column (1.4 ꢁ 26 cm) packed with basic aluminum
oxide and allowed to stand at room temperature overnight. Elution
with toluene–acetone (250:1, v/v) and crystallization of a less polar
effluent from acetone–n-hexane gave [2,3,4,4-d4]-17,20;20,21-
2.3.6. 17,20;20,21-Bismethylenedioxy-5
a
-pregnan-3a-yl acetate (5b)
bismethylenedioxy-5a-pregn-2-ene (9) as colorless needles: yield,
The compound 5a (36 mg) was acetylated with Ac2O (0.25 ml)
in dry pyridine (0.5 ml) at room temperature for 20 h. After addi-
tion of H2O, the resulting solution was extracted with EtOAc. The
organic layer was washed with 5% HCl, 5% NaHCO3, and H2O, dried
over anhydrous Na2SO4, and evaporated to dryness. Crystallization
of the product from MeOH gave 5b as colorless plates: yield, 40 mg
(100%); mp 129–131 °C.
22 mg (22%); mp 166–167 °C. Further elution with toluene–
acetone (50:1, v/v) and crystallization of a more polar effluent from
CH2Cl2–MeOH gave 10a as colorless needles: yield, 39 mg (36%);
mp 184–186 °C.
2.3.12. [2b,3b,4,4-d4]-17,20;20,21-Bismethylenedioxy-2
-pregnane (11)
The compound 9 (58 mg) was submitted to epoxidation with
a,3a-epoxy-
5a
2.3.7. Walden inversion of 3b with basic Al2O3
A solution of compound 3b (370 mg) in toluene (5 ml) was
loaded onto a glass column (1.5 ꢁ 22 cm) packed with basic alumi-
m-chloroperbenzoic acid (80 mg) in EtOAc (12 ml) at room
temperature for 5 h, as described for preparation of 7. After being
processed in an analogous manner, the product was subjected to

1428
K. Mitamura et al. / Steroids 77 (2012) 1423–1437
column chromatography on silica gel with n-hexane–EtOAc (10:1,
v/v) as an eluent. Crystallization of a homogeneous effluent from
acetone gave 11 as colorless leaflets: yield, 60 mg (99%); mp
204–206 °C.
2.3.16. 17,20;20,21-Bismethylenedioxy-11b-trimethylsilyloxypregn-5-
en-3b-ol (14)
The compound 13 (500 mg) was treated with bromotrimeth-
ylsilane (0.5 ml) and hexamethyldisilazane (1 ml) in dry pyridine
(5 ml) at room temperature for 30 min, followed by reduction of
2.3.13. Reduction 11 with LiAlD4
the intermediary D
5-3-keto derivative with NaBH4 (150 mg) in
To a solution of compound 11 (59 mg) in dry tetrahydrofuran
(3 ml) was added LiAlD4 (118 mg), and the mixture was refluxed
for 5 h. After careful addition of H2O to decompose the excess re-
agent, the resulting solution was extracted with EtOAc, and the or-
ganic phase was washed successively with 5% HCl, 5% NaHCO3, and
H2O, dried over anhydrous Na2SO4, and evaporated to dryness.
Purification of the product by column chromatography on silica
gel with n-hexane–EtOAc (4:1, v/v) and crystallization of a homo-
geneous effluent from CH2Cl2–MeOH gave 10a as colorless nee-
dles: yield, 26 mg (44%); mp 184–186 °C.
EtOH (3.5 ml)–tetrahydrofuran (7 ml) at room temperature for
1 h, as described for preparation of 2. After being processed in an
analogous manner, the product was subjected to column chroma-
tography on silica gel with toluene–acetone (30:1, v/v) as an
eluent. Crystallization of a homogeneous effluent from CH2Cl2–
MeOH gave 14 as colorless needles: yield, 349 mg (60%); mp
190–194 °C.
2.3.17. 17,20;20,21-Bismethylenedioxy-11b-trimethylsilyloxy-5
pregnan-3b-ol (15a)
a-
The compound 14 (330 mg) was hydrogenated with 5% Pd/C
catalyst (100 mg) in EtOH (7 ml)–EtOAc (14 ml) under a stream
of H2 gas at room temperature overnight, as described for prepara-
tion of 3a. After being processed in an analogous manner, the prod-
uct was subjected to column chromatography on silica gel with n-
hexane–EtOAc (3:1, v/v) as an eluent. Crystallization of a homoge-
neous effluent from acetone–n-hexane gave 15a as colorless nee-
dles: yield, 287 mg (87%); mp 202–205 °C.
2.3.14. [2,2,3b,4,4-d5]-17a,21-Dihydroxy-3a-sulfooxy-5a-pregnan-
20-one sodium salt ([2,2,3b,4,4-d5]-allo-THS-3-sulfate sodium salt;
12a)
The compound 10a (21 mg) was esterified with sulfate using
SO3–TMA complex (33 mg) in dry pyridine (0.8 ml) at room tem-
perature for 2 h, as described for preparation of 5c. After being pro-
cessed in an analogous manner, the product was subjected to
column chromatography on silica gel with CHCl3–MeOH (4:1, v/v)
as an eluent. Crystallization of a homogeneous effluent Et2O–MeOH
gave the intermediary 3-sulfate (10b), which was then treated with
46% HF (1 ml) in EtOH (0.4 ml)–tetrahydrofuran (0.4 ml) at ice tem-
perature overnight. Saturated Na2CO3 was added to adjust the solu-
tion to pH 8. The solution was then diluted with H2O and
centrifuged at 3000 rpm for 10 min. The supernatant was loaded
onto an OasisÒ HLB cartridge. After being washed with H2O, the de-
sired sulfate was eluted with MeOH. Purification of the product by
column chromatography on silica gel with CHCl3–MeOH (5:1, v/v)
as an eluent and attempted crystallization of a homogeneous efflu-
ent from Et2O–MeOH gave 12a as a colorless amorphous product:
yield, 16 mg (79%); mp 149–150 °C. ESI-MS analysis indicated d0
0.01%, d1 0.00%, d2 0.00%, d3 0.16%, d4 9.80% and d5 89.99%.
2.3.18. 17,20;20,21-Bismethylenedioxy-5a-pregnan-3b,11b-diol (15b)
To a solution of compound 15a (20 mg) in acetone (3 ml) was
added 5 M H2SO4 (0.1 ml), and the mixture was stirred at room
temperature for 1 h. After evaporation of the solvent under re-
duced pressure, the residue was dissolved in EtOAc, which in turn
was washed with 5% NaHCO3 and H2O, dried over anhydrous Na2-
SO4, and evaporated to dryness. Crystallization of the product from
acetone gave 15b as colorless needles: yield, 17 mg (99%); mp
238–240 °C (lit. [12], mp 238–241 °C).
2.3.19. 17,20;20,21-Bismethylenedioxy-3b-p-toluenesulfooxy-5
pregnan-11b-ol (15c)
a-
The compound 15b (100 mg) was treated with p-toluenesulfo-
nyl chloride (310 mg) in dry pyridine (4 ml) at room temperature
overnight, as described for preparation of 3b. After being processed
in an analogous manner, the product was subjected to column
chromatography on silica gel with n-hexane–EtOAc (3:1, v/v) as
an eluent. Crystallization of a homogeneous effluent from ace-
tone–n-hexane gave 15c as colorless needles: yield, 137 mg
(100%); mp 183–185 °C (lit. [12], mp 170–172 °C (cyclohexane–
acetone)).
2.3.15. [2,2,3b,4,4-d5]-3a,17a-Dihydroxy-21-sulfooxy-5a-pregnan-
20-one sodium salt ([2,2,3b,4,4-d5]-allo-THS-21-sulfate sodium salt;
12d)
The compound 10a (22 mg) was acetylated with Ac2O (0.5 ml)
in dry pyridine (1 ml) at room temperature, as described for prep-
aration of 5b. After being processed in an analogous manner, the
resulting intermediary 3-acetate (10c), which without isolation
was treated with 46% HF (0.5 ml) in EtOH–tetrahydrofuran
(0.14 ml; 1:1, v/v) at ice temperature overnight, as described for
preparation of 12a. After being processed in an analogous manner,
the crude product was purified by column chromatography on sil-
ica gel with toluene–acetone (3:1, v/v) as an eluent to give the
2.3.20. 17,20;20,21-Bismethylenedioxy-11b-trimethylsilyloxy-5
pregnan-3-one (16a)
a-
To a solution of 15a (50 mg) in CH2Cl2 (2 ml) were added pyrid-
inium chlorochromate (28 mg), sodium acetate trihydrate (1 mg),
and Celite (20 mg), and the mixture was stirred at room tempera-
ture overnight. EtOAc was added to the resulting suspension. After
removal of insoluble materials by filtration on a short pad of Celite,
the filtrate was evaporated under reduced pressure. Crystallization
of the product from CH2Cl2–MeOH gave 16a as colorless plates:
yield, 48 mg (96%); mp 209–211 °C.
intermediary 3a-acetoxy-21-hydroxy derivative (12b), which in
turn was treated with SO3–TMA complex (8 mg) in dry pyridine
(0.4 ml) at room temperature overnight, as described for prepara-
tion of 10b. After being processed in an analogous manner, the
21-sulfate (12c) obtained was hydrolyzed with 30% NaOH
(0.05 ml) in MeOH (0.45 ml) at room temperature overnight. After
removal of MeOH under a gentle stream of N2 gas, the residue was
dissolved in H2O and loaded onto an OasisÒ HLB cartridge. The car-
tridge was washed with H2O, and the crude sulfate was eluted with
MeOH. Purification of the product by column chromatography on
silica gel with CHCl3–MeOH (5:1, v/v) and attempted crystalliza-
tion of a homogeneous effluent from MeOH gave 12d as a colorless
amorphous product: yield, 4 mg (16%), mp 203–204 °C. ESI-MS
analysis indicated d0 0.05%, d1 0.01%, d2 0.04%, d3 0.36%, d4
11.00% and d5 88.54%.
2.3.21. 17,20;20,21-Bismethylenedioxy-11b-hydroxy-5
one (16b)
a-pregnan-3-
(i) The compound 16a (500 mg) was treated with 5 M H2SO4
(0.3 ml) in acetone (8 ml), as described for preparation of
15b. After being processed in an analogous manner, the prod-
uct was subjected to column chromatography on silica gel

K. Mitamura et al. / Steroids 77 (2012) 1423–1437
1429
with n-hexane–EtOAc (3:2, v/v) as an eluent. Crystallization
of a homogeneous effluent from CH2Cl2–MeOH gave 16b as
colorless needles: yield, 422 mg (98%); mp 220–221 °C.
(ii) The compound 13 (1.0 g) was subjected to Birch reduction
with metallic Li in liq. NH3 and tetrahydrofuran as described
for preparation of compound 4. Crystallization of the puri-
fied product from CH2Cl2–MeOH gave 16b as colorless nee-
dles: yield, 511 mg (51%); mp 220–221 °C.
and the reaction mixture was stirred at room temperature for
3 h. After being processed in an analogous manner, the product
was subjected to column chromatography on silica gel with tolu-
ene–acetone (1:1, v/v) as an eluent. Crystallization of a less polar
effluent from acetone gave 20a as colorless plates: yield, 138 mg
(68%); mp 238–240 °C. Crystallization of a more polar effluent from
acetone–n-hexane gave [2,2,3b,4,4-d5]-17,20;20,21-bismethylen-
edioxy-5a-pregnan-3a,11b-diol (22a) as colorless needles: yield,
16 mg (8%); mp 239–241 °C.
2.3.22. 17,20;20,21-Bismethylenedioxy-5a-pregnane-3a,11b-diol
(17a)
2.3.27. [2,2,3a,4,4-d5]-17,20;20,21-Bismethylenedioxy-3b-p-
The compound 16b (500 mg) was reduced with a 1 M solution
of L-SelectrideÒ in tetrahydrofuran (7.7 ml) at ꢀ78 °C for 3 h, as de-
scribed for preparation of 5a. After being processed in an analogous
manner, the crude product was subjected to column chromatogra-
phy on silica gel with n-hexane–EtOAc (1:1, v/v) as an eluent. Crys-
tallization of a homogeneous effluent from EtOAc–MeOH gave 17a
as colorless pillars: yield, 443 mg (88%); mp 239–241 °C (lit. [12],
mp 232–236 °C).
toluenesulfooxy-5
a
-pregnan-11b-ol (20b)
The compound 20a (113 mg) was treated with p-toluenesulfo-
nyl chloride (275 mg) in dry pyridine (4 ml), as described for prep-
aration of 3b. After being processed in an analogous manner, the
product was subjected to column chromatography on silica gel
with n-hexane–EtOAc (3:1, v/v) as an eluent. Crystallization of a
homogeneous effluent from acetone–n-hexane gave 20b as color-
less needles: yield, 155 mg (100%); mp 193–195 °C.
2.3.23. 3
ol (17b)
a
-Acetoxy-17,20;20,21-bismethylenedioxy-5
a
-pregnan-11b-
2.3.28. [2,2,3b,4,4-d5]-17,20;20,21-Bismethylenedioxy-5
,11b-diol (22a)
The compound 20b (57 mg) was epimerized by heating it with
a-pregnan-
3a
The compound 17a (140 mg) was acetylated with Ac2O (1.1 ml)
in pyridine (1.4 ml) at room temperature for 1 h, as described for
preparation of 5b. After being processed in an analogous manner,
crystallization of the product from acetone–n-hexane gave 17b as
colorless pillars: yield, 154 mg (100%); mp 176–177 °C.
CH3COOK (160 mg) in DMF (1.5 ml)–H2O (0.5 ml) at 150 °C for
6 h. The resulting solution was diluted with EtOAc, washed with
H2O, dried over anhydrous Na2SO4, and evaporated to dryness.
The residue obtained was hydrolyzed with 20% NaOH in MeOH
(4.5 ml)–tetrahydrofuran (1.5 ml) at room temperature for 1 h.
After evaporation of the solvent, the residue dissolved in EtOAc
was washed with H2O, dried over anhydrous Na2SO4, and
evaporated to dryness. Purification of the product by column
chromatography on silica gel with n-hexane–EtOAc (10:1, v/v) as
an eluent and crystallization of a less polar effluent from acetone-
n-hexane gave [2,2,4,4-d4]-17,20;20,21-bismethylenedioxy-5a-
pregn-2-ene-11b-ol (21) as colorless needles: yield, 9 mg (24%),
mp 172 °C. This compound was also obtained by elimination reac-
tion in which 20b (157 mg) in c-collidine was refluxed for 4 h with
2.3.24. Walden inversion of 15c with CH3COOK
To a solution of 15c (30 mg) in N,N0-dimethylformamide (DMF)
(4 ml) and H2O (1.5 ml) was added CH3COOK (95 mg), and the mix-
ture was heated at 150 °C for 6 h. The resulting mixture was di-
luted with EtOAc, washed with H2O, dried over anhydrous
Na2SO4, and evaporated to dryness. The residue was hydrolyzed
with 20% NaOH (0.2 ml) in MeOH (0.5 ml)–tetrahydrofuran
(0.5 ml) at room temperature for 1 h. The resulting solution was di-
luted with EtOAc, washed with H2O, dried over anhydrous Na2SO4,
and evaporated to dryness. Purification of the product by column
chromatography on silica gel with n-hexane–EtOAc (101:1, v/v)
and crystallization of a homogeneous effluent from acetone–n-
a yield of 109 mg (100%). Further elution with n-hexane–EtOAc
(3:2, v/v) as an eluent and crystallization of a more polar effluent
from EtOAc–MeOH gave 22a as colorless pillars: yield, 28 mg
(68%); mp 239–241 °C,
hexane gave 17,20;20,21-bismethylenedioxy-5a-pregn-2-en-11b-
ol (18) as colorless needles: yield, 6 mg (20%); mp 172 °C. (lit.
[12], mp 170–173 °C). This compound was also obtained by an
2.3.29. [2b,3b,4,4-d4]-17,20;20,21-Bismethylenedioxy-2
-pregnan-11b-ol (23)
The compound 21 (128 mg) was treated with m-chloroperben-
a,3a-epoxy-
elimination reaction in which 15c (76 mg) in
c-collidine (4 ml)
5a
was refluxed for 4 h with a yield of 42 mg (80%).
Further elution with n-hexane–EtOAc (1:1, v/v) and crystalliza-
tion of a homogeneous effluent from EtOAc–MeOH gave 17a as col-
orless pillars: yield, 15 mg (50%). This compound was also obtained
by stereoselective reduction of compound 16b.
zoic acid (143 mg) in EtOAc (3.5 ml), as described for preparation
of 19. After being processed in an analogous manner, the product
was subjected to column chromatography on silica gel with tolu-
ene–acetone (6:1, v/v). Crystallization of a homogeneous effluent
from acetone–n-hexane gave 23 as colorless needles: yield,
53 mg (40%); mp 210–211 °C.
2.3.25. 17,20;20,21-Bismethylenedioxy-2a,3a-epoxy-5a-pregnan-
11b-ol (19)
The compound 18 (17 mg) was treated with m-chloroperben-
zoic acid (25 mg) in EtOAc (0.5 ml) at room temperature for 2 h,
as described for preparation of 7. After being processed in an anal-
ogous manner, the product was subjected to column chromatogra-
phy on silica gel with n-hexane–EtOAc (2:1, v/v) as an eluent.
Crystallization of a homogeneous effluent from acetone–n-hexane
gave 19 as colorless needles: yield, 14 mg (81%), mp 210–211 °C.
2.3.30. Reduction of 23 with LiAlD4
The compound 23 (53 mg) was reduced by refluxing it with
LiAlD4 (106 mg) in dry tetrahydrofuran (3 ml) for 1.5 h, as de-
scribed for preparation of 10a. After being processed in an analo-
gous manner, the product was subjected to column
chromatography on silica gel with toluene–acetone (4:1, v/v) as
an eluent. Crystallization of a homogeneous effluent from ace-
tone–n-hexane gave 22a: yield, 46 mg (85%); mp 245–247 °C.
2.3.26. [2,2,3a,4,4-d5]-17,20;20,21-Bismethylenedioxy-5a-pregnan-
3b,11b-diol (20a)
2.3.31. [2,2,3b,4,4-d5]-3
-pregnan-11b-ol (22b)
The compound 22a (46 mg) was acetylated with Ac2O (0.5 ml)
in dry pyridine (1 ml) at room temperature overnight, as described
for preparation of 17b. After being processed in an analogous
a-Acetoxy-17,20;20,21-bismethylenedioxy-
To a solution of compound 16b (200 mg) in CH3OD (20 ml) and
dry dioxane (1 ml) were added 40% NaOD (1 ml) and D2O (1.4 ml),
and the mixture was stirred at 60 °C for 22 h under a gentle stream
of N2 gas. After cooling, NaBD4 (20 mg) was added to the solution
5a

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K. Mitamura et al. / Steroids 77 (2012) 1423–1437
Fig. 2. Synthetic route to 3- and 21-monosulfated conjugates of [2,2,3b,4,4-d5]-allo-THS. Ts = –SO2C6H4CH3, Ac = –COCH3. Reagents and conditions: (i) TMSBr, HMDS, dry
pyridine, r.t. (ii) 2 M HCl. (iii) NaBH4, MeOH–tetrahydrofuran, r.t. (iv) H2, 10% Pd(OH)2/C, EtOAc, r.t. (v) Jones’ reagent, acetone, r.t. (vi) p-TsCl, dry pyridine, r.t. (vii) L-
Selectride, dry tetrahydrofuran, ꢀ70 °C, 2 h. (viii) Al2O3. (ix) MCPBA, EtOAc, r.t. (x) NaOD/MeOD/dioxane/D2O, 80 °C, 22 h. (xi) NaBD4 r.t., 40 min. (xii) LiAlD4, dry
tetrahydrofuran. (xiii) SOꢀ3 NþðCH3Þ , dry pyridine. (xiv) Ac2O, dry pyridine, r.t. (xv) 46% HF, EtOH–tetrahydrofuran, 0 °C, 7 h. (xvi) 30% NaOH, MeOH, r.t.
3
manner, crystallization of the product from acetone–n-hexane
gave 22b as colorless needles: yield, 50 mg (100%); mp 176–187 °C.
pyridine (0.2 ml) at room temperature for 2 h. After evaporation
of the solvent in vacuo, the residue was hydrolyzed with 20% NaOH
(0.2 ml) in MeOH (1 ml), as described for preparation of 12d. After
being processed in an analogous manner, the product was sub-
jected to column chromatography on silica gel with CHCl3–MeOH
(3:1, v/v) as an eluent. Attempted crystallization of a homogeneous
effluent from Et2O–MeOH gave 24d as colorless amorphous prod-
uct: yield, 4.7 mg (16%); mp 175 °C. ESI-MS analysis indicated d0
0.01%, d1 0.02%, d2 0.10%, d3 0.23%, d4 0.55% and d5 99.09%.
2.3.32. [2,2,3b,4,4-d5]-11b,17a,21-Trihydroxy-3a-sulfooxy-5a-
pregnan-20-one sodium salt ([2,2,3b,4,4-d5]-allo-THF-3-sulfate
sodium salt; 24a)
The compound 22a (35 mg) was esterified with sulfate using
SO3–TMA complex (60 mg) in dry pyridine at room temperature
for 3 h, as described for preparation of 10b. After being processed
in an analogous manner, the intermediary 3-sulfate (22c), which
without purification was treated with 46% HF (4 ml) in EtOH
(1.5 ml)–tetrahydrofuran (1.5 ml) at ice temperature overnight,
as described for preparation of 12a. After being processed in an
analogous manner, the product was subjected to column chroma-
tography on silica gel with CHCl3–MeOH (3:1, v/v) as an eluent. At-
tempted crystallization of a homogeneous effluent from CH2Cl2–
MeOH gave 24a as a colorless amorphous product: yield, 23 mg
(58%); mp 142 °C. ESI-MS analysis indicated d0 0.01%, d1 0.04%, d2
0.05%, d3 0.26%, d4 2.90% and d5 96.74%.
2.3.34. [2,2,3b,4,4-d5]-17,20;20,21-Bismethylenedioxy-3
-pregnan-11-one (25b)
The compound 22b (50 mg) was treated with pyridinium chlo-
a-hydroxy-
5a
rochromate (30 mg), sodium acetate trihydrate (2 mg), and Celite
(100 mg) in CH2Cl2 (0.5 ml) at room temperature overnight, as de-
scribed for preparation of 16a. After being processed in an analo-
gous manner, the intermediary 11-ketone (25a), which without
purification was hydrolyzed with 20% NaOH (0.4 ml) in MeOH
(1.8 ml)–tetrahydrofuran (1.8 ml) at room temperature for 1 h.
After evaporation of the solvent, the residue was diluted with
EtOAc, washed with H2O, dried over anhydrous Na2SO4, and evapo-
rated to dryness. Crystallization of the product from Et2O–n-hexane
gave 25b as colorless needles: yield, 40 mg (93%); mp 199–200 °C.
2.3.33. [2,2,3b,4,4-d5]-3a,11b,17a-Trihydroxy-21-sulfooxy-5a-
pregnan-20-one sodium salt ([2,2,3b,4,4-d5]-allo-THF-21-sulfate
sodium salt; 24d)
The compound 22b (28 mg) was treated with 46% HF (4 ml) in
EtOH (1 ml)–tetrahydrofuran (1 ml) at ice temperature overnight,
as described for preparation of 12b. After being processed in an
analogous manner, the product was subjected to column chroma-
tography on silica gel with toluene–acetone (6:1, v/v) as an eluent
2.3.35. [2,2,3b,4,4-d5]-17a,21-Dihydroxy-3a-sulfooxy-5a-pregnan-
11,20-dione sodium salt ([2,2,3b,4,4-d5]-allo-THE-3-sulfate sodium
salt; 26a)
The compound 25b (35 mg) was esterified with sulfate using
SO3–TMA (30 mg) in dry pyridine (1 ml) at room temperature for
4 h, as described for preparation of 22c. After being processed in
to give the intermediary 3
a-acetoxy-21-hydroxyl derivative (24c),
which was esterified with sulfate using SO3–TMA (15 mg) in dry

K. Mitamura et al. / Steroids 77 (2012) 1423–1437
1431
Fig. 3. Synthetic route to 3- and 21-monosulfated conjugates of [2,2,3b,4,4-d5]-allo-THF. Ts = –SO2C6H4CH3, Ac = –COCH3. Reagents and conditions: (i) TMSBr, HMDS, dry
pyridine. (ii) 2 M HCl. (iii) NaBH4, EtOH–tetrahydrofuran, r.t. (iv) H2, 5% Pd/C, EtOH–EtOAc. (vi) p-TsCl, dry pyridine. (vii) PCC, CH2Cl2. (viii) L-Selectride, dry tetrahydrofuran.
(ix) Ac2O, dry pyridine. (x) CH3COOK, DMF, 150 °C, 6 h. (xi) MCPBA, EtOAc. (xii) NaOD/MeOD/dioxane/D2O, 60 °C, 22 h. (xiii) NaBD4, r.t., 3 h. (xiv) c-collidine, reflux. (xv)
LiAlD4, dry tetrahydrofuran. (xvi) SOꢀ3 NþðCH3Þ , dry py, r.t. (xvii) 46% HF, EtOH–tetrahydrofuran, 0 °C, 7 h. (xviii) 20% NaOH, MeOH.
3
Fig. 4. Synthetic route to 3- and 21-monosulfated conjugates of [2,2,3b,4,4-d5]-allo-THE. Ac = –COCH3. Reagents and conditions: (i) PCC, CH2Cl2. (ii) 20% NaOH, MeOH. (iii)
SO3ꢀN+(CH3)3, dry pyridine, r.t. (iv) 46% HF, EtOH–tetrahydrofurane, 0 °C, 7 h.
an analogous manner, the intermediary 3-sulfate (25c), which
without purification was treated with 46% HF (3.5 ml) in EtOH
(0.5 ml)–tetrahydrofuran (0.5 ml) at ice temperature overnight,
as described for preparation of 24a. After being processed in an
analogous manner, the product was subjected to column chroma-
tography on silica gel with CHCl3–MeOH (2:1, v/v). Attempted
crystallization of a homogeneous effluent from Et2O–MeOH gave
26a as colorless amorphous product: yield, 16 mg (51%); mp
154 °C. ESI-MS analysis indicated d0 0.00%, d1 0.00%, d2 0.10%,
d31.64%, d4 21.89% and d5 76.37%.
2.3.36. [2,2,3b,4,4-d5]-3
one (26b)
a-Acetoxy-17a,21-dihydroxy-5a-pregnan-11-
The compound 25a (27 mg) was treated with 46% HF (4 ml) in
EtOH (0.4 ml)–tetrahydrofuran (0.4 ml) at ice temperature over-
night, as described for preparation of 24b. After being processed
in an analogous manner, the crude product was subjected to col-
umn chromatography on silica gel with toluene–acetone (4:1, v/
v) as an eluent. Crystallization of a homogeneous effluent from ace-
tone–n-hexane gave 26b as colorless pillars: yield, 15 mg (61%);
mp 187–188 °C.

1432
K. Mitamura et al. / Steroids 77 (2012) 1423–1437
Fig. 5. Synthetic route to 3- and 21-monosulfated conjugates of [9a,11a,12,12-d4]-allo-THF. Ac = –COCH3. Reagents and conditions: (i) Jones’ reagent, acetone, r.t. (ii) 30%
NaOH, MeOH, r.t. (iii) NaOD/MeOD/dioxane/D2O, 60 °C, 96 h. (iv) NaBD4, r.t., 24 h. (v) SO3ꢀN+(CH3)3, dry pyridine, r.t. (vi) Ac2O, dry pyridine, r.t. (vii) 46% HF, EtOH–
tetrahydrofurane, 0 °C, 7 h.
2.3.37. [2,2,3b,4,4-d5]-3
11,20-dione sodium salt ([2,2,3b,4,4-d5]-allo-THE-21-sulfate sodium
salt; 26d)
a
,17
a
-Dihydroxy-21-sulfooxy-5
a
-pregnane-
(5 ml) and dry dioxane (0.4 m) at 60 °C for 96 h under a gentle
stream of N2 gas, followed by reduction with NaBD4 (80 mg) at
room temperature for 24 h, as described for preparation of 8a.
After being processed in an analogous manner, the product was
subjected to column chromatography on silica gel with toluene–
acetone (8:1, v/v) as an eluent. Crystallization of a homogeneous
effluent from acetone–n-hexane gave 28a as colorless plates: yield,
102 mg (67%). mp 241 °C.
The compound 26b (15 mg) was esterified with sulfate using
SO3–TMA complex (15 mg) in dry pyridine (0.2 ml) at room tem-
perature, as described for preparation of 10b. After being processed
in an analogous manner, the intermediary 21-sulfate (26c), which
without purification was hydrolyzed with 20% NaOH (0.1 ml) in
MeOH (0.5 ml) at room temperature for 1 h, as described for prep-
aration of 24d. After being processed in an analogous manner, at-
tempted crystallization of the product from Et2O–MeOH gave
26d as colorless amorphous product: yield, 7 mg (40%); mp
169 °C. ESI-MS analysis indicated d0 0.00%, d1 0.00%, d2 0.03%, d3
1.67%, d4 22.39% and d5 77.73%.
2.3.40. [9
a
,11
a
,12,12-d4]–3
a
- Sulfooxy-11b,17
a
,21-trihydroxy-5
a-
pregnane-20-one sodium salt ([9a,11a,12,12-d4]-allo-THF-3-sulfate
sodium salt; 29a)
The compound 28a (50 mg) was esterified with sulfate using
SO3–TMA complex (50 mg) in dry pyridine (4 ml) at room temper-
ature, as described for preparation of 22c. After being processed in
an analogous manner, the intermediary 3-sulfate (28b), which
without purification was treated with 46% HF (3 ml) in EtOH
(1.5 ml)–tetrahydrofuran (1.5 ml) at ice temperature for 24 h, as
described for preparation of 24a. After being processed in the anal-
ogous manner, the product was subjected to column chromatogra-
phy on silica gel with CHCl3–MeOH (5:1, v/v) as an eluent.
Attempted crystallization of a homogeneous effluent from Et2O–
MeOH gave 29a as a colorless amorphous product: yield, 32 mg
(56%); mp 142 °C. ESI-MS analysis indicated d0 0.04%, d1 0.08%, d2
1.37%, d3 21.71%, d4 78.08%.
2.3.38. 17,20;20,21-Bismethylenedioxy-3a-hydroxy-5a-pregnan-11-
one (27b)
The compound 17b (172 mg) was oxidized with Jones’ reagent
(1 ml) in acetone (3 ml) at room temperature for 30 min, as de-
scribed for preparation of 4. After being processed in an analogous
manner, the intermediary 3a-acetoxy-11-ketone (27a) was hydro-
lyzed with 30% NaOH (0.2 ml) in MeOH–tetrahydrofuran (1:1, v/v)
(2.8 ml) at room temperature for 1 h. After the usual work-up,
purification of the product by column chromatography on silica
gel with toluene–acetone (3:1, v/v) and crystallization of a homo-
geneous effluent from Et2O–n-hexane gave 27b as colorless nee-
dles: yield, 139 mg (90%). mp 199–200 °C.
2.3.41. [9a,11a,12,12-d4]-3a-Acetoxy-17,20;20,21-
bismethylenedioxy-5a-pregnan-11b-ol (28c)
2.3.39. [9
,11b-diol (28a)
The compound 27b (150 mg) was submitted to H-D exchange
reaction with 40% NaOD (0.6 ml) and D2O (0.8 ml) in CH3OD
a
,11
a
,12,12-d4]-17,20;20,21-Bismethylenediox-5
a
-pregnane-
The compound 28a (50 mg) was acetylated with Ac2O in pyri-
dine, as described for 6c. After being processed analogously, crys-
tallization of the product from acetone–n-hexane gave 28c as
colorless needles: yield, 55 mg (100%); mp 176–177 °C.
3a

K. Mitamura et al. / Steroids 77 (2012) 1423–1437
1433
[M-H-CH2O]-
404.2
[M-H]-
434.3
100
50
100
m/z 404
O
CH2OH
OH
m/z 374
D
D
D
+D
-H2O
m/z 98
m/z 416
-O3SO
H
50
D
D
[M-H-CH2CO-H2O]-
374.3
[M-H-H2O]-
416.3
435.2
433.4
432.4
[M-H]-
434.3
[DSO4]-
98.1
436.2
437.3
429.3
420
0
0
410
430
440
450
100
150
200
250
300
350
400
450
500
m/z
m/z
[M-H]-
434.3
100
433.3
100
[M-H]-
434.3
50
50
[M-H-CH2OSO3]-
324.3
[HSO4]-
97.1
435.2
[M-H-H2O]-
416.4
433.4
430
436.2
437.2
0
0
100
150
200
250
300
350
400
450
500
410
420
440
450
m/z
m/z
Fig. 6. ESI-MS (left) and product ion spectra (right) obtained by CID of [MꢀH]ꢀ of 3- (upper) and 21-monosulfated (lower) conjugates of [2,2,3b,4,4-d5]-allo-THS. Proposed
structures of major fragments are depicted.
2.3.42. [9a,11a,12,12-d4]-3a-Acetoxy-11b,17a,21-trihydroxy-5a-
3. Results and discussion
pregnan-20-one (29b)
The use of stable isotopically labeled (13C, 2H, 15N and 18O)
internal standards for LC/MS analysis offers major advantages in
that they behave in an almost identical manner to the analytes
through all steps in the isolation and chromatographic procedure,
thereby allowing procedural losses to be compensated for. The dif-
ference between the molecular masses of the analyte and isotopi-
cally labeled internal standard should be at least three mass units
to avoid overlapping of the monitored mass peaks. For obtaining
labeled 3- and 21-monosulfates of allo-THS, allo-THF, and allo-
THE with at least three non-exchangeable stable isotopes, direct
sulfation with a sulfating agent such as SO3–TMA complex and/or
enzyme assisted sulfation using sulfotransferase can be consid-
ered. However, these methods are inadequate for preparation of
the targeted sulfates due to the unavailability of the allo-tetrahy-
drocorticosteroids labeled at predesigned positions. The other
method that can be considered is the known synthetic method
[9] starting from commercial sources of stable isotopically labeled
cortisol and cortisone. Here, the difficulties encountered are the
low yields due to the multiple reaction steps and the great ex-
penses of stable isotopically labeled steroids. Moreover, these syn-
thetic methods are inadequate for preparation of the multiply
deuterated sulfates of allo-THS due to the unavailability of the sta-
ble isotope labeled 11-DOC containing at least three or four non-
exchangeable stable isotopes.
The compound 28c (60 mg) was treated with 46% HF (1.5 ml) in
EtOH (0.3 ml)–tetrahydrofuran (0.3 ml) at ice temperature for 24 h,
as described for 24b. After being processed in an analogous man-
ner, the crude product was purified by column chromatography
on silica gel with n-hexane–EtOAc (1:1, v/v) as an eluent. Crystal-
lization of a homogeneous effluent from acetone–n-hexane gave
29b as colorless needles: yield, 31 mg (57%); mp 227–229 °C.
2.3.43. [9a,11
a,12,12-d4]-3
a
,11b,17
a
-Trihydroxy-21-sulfooxy-5
a-
pregnan-20-one sodium salt ([9a,11a,12,12-d4]-allo-THF-21-sulfate
sodium salt; 29d)
The compound 29b (34 mg) was esterified with sulfate using
SO3–TMA complex (40 mg) in dry pyridine (2 ml) at room temper-
ature for 24 h, as described for preparation of 12c. After being pro-
cessed analogously, the resulting 21-sulfate (29c) dissolved in
MeOH (0.5 ml) was hydrolyzed with 30% NaOH (0.5 ml) at room
temperature for 1 h, as described for preparation of 24d. After
being processed in an analogous manner, purification of the prod-
uct by column chromatography on silica gel with CHCl3–MeOH
(4:1, v/v) as an eluent and attempted crystallization of a homoge-
neous effluent from Et2O–MeOH gave 29d as a colorless amor-
phous product: yield, 23 mg (59%), mp 175 °C. ESI-MS analysis
indicated d0 0.04%, d1 0.04%, d2 1.67%, d3 20.17%, d4 78.08%.

1434
K. Mitamura et al. / Steroids 77 (2012) 1423–1437
[M-H]-
450.4
[M-H-CH2O]-
420.2
100
100
50
m/z 420
O
CH2OH
OH
m/z 390
HO
D
D
D
+D
-H2O
m/z 98
m/z 432
-O3SO
H
D
D
50
[M-H-CH2CO-H2O]-
390.2
451.3
[M-H]-
450.2
[DSO4]-
98.0
[M-H-H2O]-
432.3
445.5
430
448.7
440
452.2
453.3
449.8
450
0
0
100
150
200
250
300
m/z
350
400
450
500
460
470
m/z
m/z 306
-H2O
m/z 324
[M-H]-
450.4
[M-H-CH2OSO3]-
340.3
100
100
-16
+H
m/z 97
m/z 340
O
-
CH2 SO3
[HSO4]-
97.0
m/z 139
HO
OH
D
D
50
50
D
[M-H-CH2OSO3-16-H2O]-
306.3
-H2O
m/z 432
HO
H
D
D
[M-H]-
450.3
[M-H-CH2OSO3-16]-
449.3
450
[M-H2O]-
432.5
[COCH2OSO3H]-
139.1
324.3
451.2
452.2
447.5
449.7
453.2
0
0
100
150
200
250
300
350
400
500
430
440
450
460
470
m/z
m/z
Fig. 7. ESI-MS (left) and product ion spectra (right) obtained by CID of [MꢀH]ꢀ of 3- (upper) and 21-monosulfated (lower) conjugates of [2,2,3b,4,4-d5]-allo-THF. Proposed
structures of major fragments are depicted.
In our previous paper, we reported an efficient synthetic meth-
od of the sulfates of [2,2,3b,4,4-d5]-tetrahydrocorticosteroids in the
5b-series starting from bismethylenedioxy (BMD) derivatives of
cortisol and 11-DOC [10]. The method involved the introduction
of multiple deuterium atoms into the active methylene adjacent
to the carbonyl group of the 5b-3-keto steroid derivative by a
keto-enol exchange reaction in active solvent (D2O and CH3OD) un-
der alkaline conditions followed by stereoselective reduction with
14 with Pd(OH)2/C and Pd/C, respectively, under a stream of H2
gas engendered an attack from less hindered -side to afford 3b-
hydroxy-5 -reduced derivatives (3a and 15a) in a high yield. Oxi-
a
a
dation of 3b-hydroxy group in compound 3a with Jones’ reagent in
acetone and in compound 15a with pyridinium chlorochromate in
CH2Cl2 afforded the 3-keto derivatives (4 and 16a). Removal of the
11b-trimethylsilyloxy group of compound 16a was attained by
treatment with 5 M H2SO4 in acetone to give the desired 11b-hy-
droxy-3-keto derivative (16b). The key compounds 4 and 16b were
also obtained from compounds 1 and 13 by Birch reduction using
Li in liq. ammonia and tetrahydrofuran with the yields of 59%
and 51%, respectively.
NaBD4 leading to the 3b-deuterio-3
a
-hydroxy-5b-steroids. It is
well known that the reduction of 5
a-3-keto-steroids with steri-
cally less demanding hydride like NaBH4 yields predominantly
3b-hydroxy steroids. Thus, the applied protocol for the preparation
of 3b-deuterio-3
sion of the alcohol functionality of 3
a
-hydroxy-5
a
-steroids requires subsequent inver-
Accordingly, H–D exchange reaction of the compound 4 with
40% sodium deuterium oxide in CH3OD-dioxane followed by
reduction with lithium tri-sec-butylborodeuteride was attempted
a-deuterio-3b-hydroxy ste-
roids by one of the standard procedures, preferably under
Mitsunobu conditions [13], necessitating at least one additional
linear synthetic steps. This problem prompted us to employ a ste-
rically congested reagent like lithium tri-sec-butylborodeuteride
[14] providing direct access to 3
configuration.
to give the desired [2,2,3b,4,4-d5]-3a-alcohol with an axial hydroxy
group. However, difficulties were encountered in the preparation
of the deuteride reagent which would fulfill the criteria of a steri-
cally hindered deuterated reagent. Therefore, an alternative meth-
a-hydroxy-5a-steroids with axial
od which involves the reduction of 5
a-3-keto steroid with NaBD4
We therefore prepared 5
a
-3-keto BMD derivatives (4 and 16b)
generating the 3 -d-3b-alcohol and subsequent inversion of the
a
as the key compounds starting from the BMD derivative of 11-DOC
(1) and cortisol (13) (Figs. 2 and 3). Deconjugation reaction of com-
pounds 1 and 13 with trimethylsilylbromide and hexamethyldisi-
alcohol functionality was undertaken. Thus, the compound 4 after
H–D exchange reaction was submitted to reduction with NaBD4
yielding the [2,2,3a,4,4-d5]-3b-hydroxy (8a) and the desired
lazane in pyridine followed by reduction of the intermediary D5
-
[2,2,3b,4,4-d5]-3 -hydroxy (10a) derivative in a ratio of 8:1. The
a
3-ketosteroids with NaBH4 afforded the corresponding
D
5-3b-hy-
major product of 3b-hydroxy derivative (8a) was transformed into
droxy steroids (2 and 14). Catalytic reduction of compound 2 and
p-toluenesulfonate (8b), which in turn was submitted to Walden

K. Mitamura et al. / Steroids 77 (2012) 1423–1437
1435
[M-H]-
448.3
[M-H-CH2O]-
418.3
100
100
m/z 418
m/z 388
O
CH2OH
OH
O
D
D
D
+D
m/z 98
-O3SO
50
H
50
D
D
[M-H-CH2CO-H2O]-
388.3
449.2
[M-H]-
448.3
447.4
446.3
[DSO4]-
98.1
450.2
0
0
100
200
300
m/z
400
500
430
440
450
460
470
m/z
[M-H]-
448.3
[M-H]-
448.2
100
100
50
50
447.2
[HSO4]-
97.1
[M-H-COCH2OSO3]-
310.3
[M-H-H2O]-
430.3
449.2
450.2
[M-H-CH2OSO3]-
338.3
[COCH2OSO3H]-
139.5
447.5
0
0
100
200
300
m/z
400
500
430
440
m/z
450
460
470
Fig. 8. ESI-MS (left) and product ion spectra (right) obtained by CID of [MꢀH]ꢀ of 3- (upper) and 21-monosulfated (lower) conjugates of [2,2,3b,4,4-d5]-allo-THE. Proposed
structures of major fragments are depicted.
inversion in a column packed with a basic alumina affording the
cess reagents and inorganic salts, elution with MeOH yielded a
homogenous effluent, which was characterized as [2,2,3b,4,4-d5]-
allo-THS-3-sulfate (12a) after chromatographic purification on sil-
ica gel.
desired 3a-hydroxy derivative (10a) accompanied by the genera-
tion of small amount of D2 derivative (9). Epoxidation of com-
pound 9 with m-chloroperbenzoic acid in EtOAc took place from
the less hindered
,3 -epoxide (11), which was then submitted to reductive cleav-
age of the -epoxide with lithium aluminum deuteride. As was ex-
pected the trans-diaxial opening of the oxido ring occurred to give
the [2,2,3b,4,4-d5]-3 -hydroxy derivative. To obtain non-deuter-
a
-side to afford predominant formation of the
Compound 10a was acetylated at C-3 to obtain 3-aectate (10c),
which in turn was hydrolyzed with hydrogen fluoride to afford
2a a
a
[2,2,3b,4,4-d5]-3
ment of 12b with SO3-TMA complex in pyridine yielded
[2,2,3b,4,4-d5]-3 -acetoxy-17 -hydroxy-20-one-21-sulfate (12c).
a-acetoxy-17a,21-dihydroxy-20-one (12b). Treat-
a
a
a
ated authentic samples, compounds 5a, 5b, 6, and 7 were prepared
by successive stereoselective reduction of compound 4 with com-
mercially available Li(sec-butyl)3BH, acetylation at the 3a-hydroxy
Subsequent alkaline hydrolysis at C-3 in 12c with 30% NaOH in
MeOH resulted in the formation of [2,2,3b,4,4-d5]-allo-THS-21-sul-
fate (12d) (Fig. 2).
group of compound 5a with Ac2O in pyridine, Walden inversion of
compound 3b in a basic alumina column, and epoxidation of com-
pound 6 with m-chloroperbenzoic acid in EtOAc.
Our next efforts were focused on the 3- and 21-monosulfates of
[2,2,3b,4,4-d5]-allo-THF (24a and 24d) and [2,2,3b,4,4-d5]-allo-THE
(26a and 26d). Except for Walden inversion of compound 20b with
CH3COOK in a mixed solvent of DMF–H2O at 150 °C, an essentially
identical procedures were used to prepare the [2,2,3b,4,4-d5]-allo-
THF (24a and 24d) and [2,2,3b,4,4-d5]-allo-THE (26a and 26d)
The 3a-hydroxy group in the compound 10a was esterified with
sulfate using SO3–TMA complex in pyridine. The sulfation reaction
proceeded cleanly and rapidly under mild experimental conditions.
The resulting sulfated compound (10b) was hydrolyzed with
hydrogen fluoride to remove the BMD protecting group. To purify
the crude 3-sulfate of [2,2,3b,4,4-d5]-allo-THS (12a), it was loaded
onto an OasisÒ HLB cartridge for reversed-phase solid phase
extraction. After the cartridge was washed with H2O to remove ex-
(Fig. 3), starting from [2,2,3b,4,4-d5]-3
bismethylenedioxy-5 -pregnan-11b-ol (22b) which was an inter-
mediate of preparation of 24a and 24d (Fig. 4).
Finally, preparation of the [9 ,11 ,12,12-d4]-allo-THF (29a and
29d) was also undertaken starting from compound 17b, as outlined
a-acetoxy-17,20;20,21-
a
a
a

1436
K. Mitamura et al. / Steroids 77 (2012) 1423–1437
[M-H]-
449.3
[M-H-CH2O]-
419.24
100
100
m/z 419
O
CH2OH
OH
m/z 389
D
D
HO
D
+H
-O3SO
D
m/z 97
-H2O
m/z 431
H
50
50
[M-H-CH2CO-H2O]-
389.3
[M-H]-
449.3
448.3
448.5
447.5
450.3
[HSO4]-
[M-H-H2O]-
431.3
451.2
452.2
97.1
0
0
430
440
450
460
470
100
150
200
250
300
350
400
450
500
m/z
m/z
[M-H]-
449.3
100
448.3
100
[M-H]-
449.3
50
50
[M-H-H2O]-
431.4
448.4
447.5
450.2
[M-H-CH2OSO3]-
339.3
[HSO4]-
97.1
451.2
452.2
0
0
430
440
450
m/z
460
470
100
150
200
250
300
350
400
450
500
m/z
Fig. 9. ESI-MS (left) and product ion spectra (right) obtained by CID of [MꢀH]ꢀ of 3- (upper) and 21-monosulfated (lower) conjugates of [9
a,11a,12,12-d4]-allo-THF. Proposed
structures of major fragments are depicted.
in Fig. 5. Oxidation of 17b with Jones’ reagent in acetone followed
by alkaline hydrolysis with 30% NaOH in MeOH afforded the 11-ke-
OꢀH2O]ꢀ and small [DSO4]ꢀ ions were observed. In contrast, the
CID spectra of the [MꢀH]ꢀ ion of the 21-sulfates derived from
the steroidal moiety by the cleavage of dihydroxy side chain were
shifted 4 and 5 Da, whereby [MꢀH]ꢀ ion or [MꢀHꢀCH2OSO3]ꢀ ion
as the base peak with modest or small peak corresponding to
[MꢀHꢀCH2OSO3]ꢀ, [MꢀHꢀCOCH2OSO3]ꢀ, [HSO4]ꢀ ions were ob-
served. Thus, the results of the CID analysis confirmed the struc-
tures of 3- and 21-sulfates of [2,2,3b,4,4-d5]-allo-THS, [2,2,3b,4,4-
tone (27b). Deuterium exchange at the 9
pound 27b followed by reduction with NaBD4 furnished the
desired [9 ,11 ,12,12-d4]-11b-hydroxy derivative (28a). Identical
procedures were used to prepare 3- and 21-monosaulfate of
[9 ,11 ,12,12-d4]-allo-THF using compound 28a.
a and 12 positions of com-
a
a
a
a
The structures of the obtained sulfates were confirmed by 1H
and 13C NMR signals (Table 2) along with negative-ion ESI-MS
and CID spectra (Fig. 6–9). 1H and 13C NMR chemical shifts were
identical with those of the non-deuterated compounds [9] except
d5]-allo-THF, [2,2,3b,4,4-d5]-allo-THE, and [9a,11a,12,12-d4]-allo-
THF, which gave rise to characteristic transition [MꢀH]ꢀ ? [-
MꢀHꢀCH2O]ꢀ and [MꢀH]ꢀ ? [MꢀHꢀCH2OSO3]ꢀ could be used
for monitoring ion. The isotopic purities of the labeled compounds
as [d5]- and [d4]-form are estimated to be more than 76 at.% D,
based on the ion intensities in the region of deprotonated mole-
cules of the sulfates.
for the disappearance of 3b- and 11a
-proton signals, and 13C sig-
nals at C-2, C-3, and C-4 of the sulfates of [2,2,3b,4,4-d5]-allo-
THS, [2,2,3b,4,4-d5]-allo-THF, and [2,2,3b,4,4-d5]-allo-THE, and C-
9, C-11, and C-12 signal of the sulfates of [9a,11a,12,12-d4]-allo-
THF. The negative-ion ESI-MS spectra showed [MꢀH]ꢀ ions as
the base peak at m/z 434.3 for 3- and 21-sulfate of d5-allo-THS,
at m/z 450.4 for 3- and 21-sulfates of d5-allo-THE, at m/z 448.3
for 3- and 21-sulfates, and at 449.3 for 3- and 21-sulfates of d4-
allo-THF. The CID spectra of the [MꢀH]ꢀ ion of the 3-sulfates de-
rived from the steroidal moiety by the loss of H2O and the cleavage
In conclusion, 3- and 21-monosulfates of multiply deuterated
allo-THS, allo-THF, and allo-THE are now available. These com-
pounds should be useful for the highly sensitive, selective, and
accurate LC–MS/MS determination of the sulfate conjugates of
allo-tetrahydrocorticosteroids present in biological fluids. A fur-
ther detailed study on a method for the analytical and clinical
application is now progressing in our laboratory, and the result will
be reported at a later date.
of dihydroxy side chain were shifted
4 and 5 Da, whereby
[MꢀHꢀCH2O]ꢀ ion as the base peak with modest [MꢀHꢀCH2-

K. Mitamura et al. / Steroids 77 (2012) 1423–1437
1437
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Acknowledgements
The authors acknowledge support for this work by Grant-in-
Aids for Scientific Research (C) for 2009–2011 (To S.I., Grant
21590184) and 2011–2013 (To. K.M., Grant 23590209) from the Ja-
pan Society for the Promotion of Science, and Culture of Japan, and
High-Tech Research Center Project for Private Universities: match-
ing fund subsidy from the Ministry of Education, Culture, Sports,
Science and Technology (2007–2011).
[9] Okihara R, Mitamura K, Hasegawa M, Mori M, Muto A, Kakiyama G, Ogawa S,
Iida T, Shimada M, Mano N, Ikegawa S. Potential corticosteroids metabolites:
chemical synthesis of 3- and 21-monosulfates and their double conjugates of
tetrahydrocorticosteroids in the
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