Syntheses of stable isotope-labeled 6β-hydroxycortisol, 6β-hydroxycortisone, and 6β-hydroxytestosterone

Article abstract of DOI:10.1016/S0039-128X(03)00102-8

A method is described for the preparation of two types of multi-labeled 6β-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four ¹³C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-²H₅]cortisone and [1,2,4,19- ¹³C₄,1,1,19,19,19-²H₅]cortisone (cortisone-²H₅-BMD and cortisone-¹³C ₄,²H₅-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-²H₅]- or [1,3- ¹³C₂,1,1,3,3,3-²H₅]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-²H₆]- or [1,3-¹³C ₂,1,1,1,3,3,3-²H₆]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6β-hydroxy-[1,1,19,19,19- ²H₅]cortisone-BMD and 6β-hydroxy-[1,2,4,19- ¹³C₄,1,1,19,19,19-²H₅]cortisone-BMD, respectively, as a mixture of 6β- and 6α-epimers in a ratio of 4:1. Separation of 6β- and 6α-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6β-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6β-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH₄ and subsequent removal of the C-3 and C-17 protecting groups gave 6β-hydroxy-[1,1,19,19,19-²H₅]cortisol (6β-hydroxycortisol-²H₅) and 6β -hydroxy-[1,2,4,19-¹³C₄,1,1,19,19,19-²H ₅]cortisol (6β-hydroxycortisol-¹³C₄, ²H₅), respectively, as a mixture of 6β- and 6α-epimers (6β:6α=4.4:1). The isotopic compositions of 6β-hydroxycortisol-²H₅ and 6β -hydroxycortisol-¹³C₄,²H₅ were 90.9 and 92.1 at.%, respectively. Furthermore, 6β-hydroxy-[1α ,16,16,17α-²H₄]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1α,16,16,17α- ²H₄]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.

Full text of DOI:10.1016/S0039-128X(03)00102-8

Steroids 68 (2003) 693–703
Syntheses of stable isotope-labeled 6␤-hydroxycortisol,
6␤-hydroxycortisone, and 6␤-hydroxytestosterone
Takashi Furuta∗, Atsushi Suzuki, Mitsuhiro Matsuzawa, Hiromi Shibasaki, Yasuji Kasuya
Department of Medicinal Chemistry and Clinical Pharmacy, School of Pharmacy, Tokyo University of Pharmacy and Life Science,
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received 15 November 2002; received in revised form 28 May 2003; accepted 4 June 2003
Abstract
A method is described for the preparation of two types of multi-labeled 6␤-hydroxycortisol containing either five deuterium atoms
at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as
analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H5]cortisone and
[1,2,4,19-13C4,1,1,19,19,19-2H5]cortisone (cortisone-2H5-BMD and cortisone-13C4,2H5-BMD) were first synthesized via indan synthon
methodstartingfromopticalactive11-oxoindanylpropionicacidandlabeledisopropenylanion([1,1,3,3,3-2H5]-or[1,3-13C2,1,1,3,3,3-2H5]
isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H6]- or [1,3-13C2,1,1,1,3,3,3-
2H6]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs
gave 6␤-hydroxy-[1,1,19,19,19-2H5]cortisone-BMD and 6␤-hydroxy-[1,2,4,19-13C4,1,1,19,19,19-2H5]cortisone-BMD, respectively, as a
mixture of 6␤- and 6␣-epimers in a ratio of 4:1. Separation of 6␤- and 6␣-epimers by thin-layer chromatography (TLC) and subsequent
hydrolysis of the BMD group at C-17 gave pure labeled 6␤-hydroxycortisone. After protecting the keto group at C-3 of the labeled
6␤-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH4 and subsequent removal of the C-3 and C-17 pro-
tecting groups gave 6␤-hydroxy-[1,1,19,19,19-2H5]cortisol (6␤-hydroxycortisol-2H5) and 6␤-hydroxy-[1,2,4,19-13C4,1,1,19,19,19-2H5]
cortisol (6␤-hydroxycortisol-13C4,2H5), respectively, as a mixture of 6␤- and 6␣-epimers (6␤:6␣ = 4.4:1). The isotopic compositions of
6␤-hydroxycortisol-2H5 and 6␤-hydroxycortisol-13C4,2H5 were 90.9 and 92.1 at.%, respectively. Furthermore, 6␤-hydroxy-[1␣,16,16,17␣-
2H4]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-
labeledtestosterone([1␣,16,16,17␣-2H4]testosterone)obtainedbyusingcatalyticdeuterationandhydrogen–deuteriumexchangereactions.
© 2003 Elsevier Inc. All rights reserved.
Keywords: 6␤-Hydroxycortisol; 6␤-Hydroxycortisone; 6␤-Hydroxytestosterone; Deuterium-label; 13C-Label; Mass spectrometry
1. Introduction
idation of testosterone to 6␤-hydroxyltestosterone [4]. Be-
cause of wide interindividual variations in the CYP3A ac-
tivity in humans, evaluation of the in vivo 6␤-hydroxylation
activity of cortisol and testosterone in human has been re-
quired for simply and accurately predicting or assessing
the CYP3A-metabolizing capacity of an individual in the
fields of drug development and clinical research and practice
[5–7]. Recently, we have proposed a new and reliable index
for phenotyping the in vivo CYP3A activity using cortisol
6␤-hydroxylation clearance [24,25]. The 6␤-hydroxylation
of synthetic and naturally occurring steroid compounds has
also drawn attention, due to the physiological significance
in the hypertension [8–10]. These findings led us to synthe-
size stable isotope-labeled 6␤-hydroxyl compounds for use
as internal standards for mass spectrometry.
Cortisol and cortisone are metabolized to 6␤-hydroxy-
cortisol, 6␣-hydroxycortisol, and 6␤-hydroxycortisone by
cytochrome P450 3A (CYP3A4 and CYP3A5) isozymes in
humans [1–3]. The CYP3A isozymes also catalyze the ox-
Abbreviations: GC-MS, gas chromatography-mass spectrometry; UV,
ultraviolet; TLC, thin-layer chromatography; BMD, bismethylenedioxy;
CYP3A, cytochrome P450 3A; MO-TMS, methoxime-trimethylsilyl;
NMR, nuclear magnetic resonance; 6␤-Hydroxycortisol-2H5, 6␤-hydroxy-
[1,1,19,19,19-2H5]cortisol; 6␤-Hydroxycortisone-2H5, 6␤-hydroxy-[1,1,
19,19,19-2H5]cortisone; 6␤-Hydroxycortisol-13C4,2H5, 6␤-hydroxy-[1,2,
4,19-13C4,11,19,19,19-2H5]cortisol; Cortisol-2H5, [1,1,19,19,19-2H5]
cortisol; Cortisol-13C4, [1,2,4,19-13C4]cortisol; Cortisol-13C4,2H5, [1,2,
4,19-13C4,1,1,19,19,19-2H5]cortisol; 6␤-Hydroxytestosterone-2H4, 6␤-
hydroxy-[1␣,16,16,17␣-2H4]testosterone
Stable isotope dilution mass spectrometry is widely
accepted as the most accurate and specific method for
estimation of the small amounts of endogenous and
∗
Corresponding author. Tel.: +81-426-76-5803; fax: +81-426-76-8969.
E-mail address: furutat@ps.toyaku.ac.jp (T. Furuta).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0039-128X(03)00102-8

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T. Furuta et al. / Steroids 68 (2003) 693–703
O
O
OH
OH
OH
OH
HO
O
D
D
D
D
CD3
CD3
O
O
OH
OH
2
2
6β or 6α-hydroxy-[1,1,19,19,19- H ]cortisol
6β-hydroxy-[1,1,19,19,19- H ]cortisone
5
5
2
2
( 6β or 6α-hydroxycortisol- H )
( 6β-hydroxycortisone- H )
5
5
O
OH
OH
D
OH
HO
D
D
D
H
D
*
CD3
D
*
*
*
13C
*
O
O
OH
OH
13
2
2
6β or 6α-hydroxy-[1,2,4,19- C ,1,1,19,19,19- H ]cortisol
6β-hydroxy-[1α,16,16,17α- H ]testosterone
4
5
4
13
2
2
( 6β or 6α-hydroxycortisol- C , H )
( 6β-hydroxytestosterone-
H )
4
4
5
Fig. 1. Structures of stable isotopically labeled 6-hydroxylated steroids.
synthetic steroids in biological fluids [11]. Palermo et al.
[12] employed 6␤-hydroxy-[1,2-2H2]cortisol as internal
standard to determine 6␤-hydroxycortisol in urine by
gas chromatography-mass spectrometry (GC-MS). The
dideutero internal standard showed significant disadvantage,
because there was a major ion contribution by the analyte
to the internal standards and vice versa. The extents of the
ion contributions were augmented by the four trimethylsi-
lyloxy groups at C-6␤, C-11␤, C-17␣, and C-21 of
the 3,20-dimethoxime-6␤,11␤,17␣,21-tetra(trimethylsilyl)
derivative (MO-TMS derivative) of 6␤-hydroxycortisol be-
cause of the high abundance of silicon isotopes. The analyte
and its labeled internal standard, therefore, should differ
by at least four or five mass units to accurately determine
6␤-hydroxycortisol. However, there have been no reports
for the synthesis of 6-hydroxylated derivatives of cortisol,
cortisone, and testosterone labeled with stable isotopes,
suitable for use as internal standards in the study of eluci-
dating metabolism of cortisol and testosterone concerning
the 6␤-hydroxylation in vivo.
We have previously developed a method for a concise
11-step total synthesis of optically pure (+)cortisol by using
the chiral hydrindan as a starting material and applied it to
the introduction of five deuterium atoms at C-19 methyl and
C-1 methylene and/or four 13C atoms at C-1, C-2, C-4, C-
19 of cortisol and cortisone, i.e. [1,1,19,19,19-2H5]cortisol,
[1,1,19,19,19-2H5]cortisone, [1,2,4,19-13C4,1,1,19,19,19-
2H5]cortisol, and [1,2,4,19-13C4,1,1,19,19,19-2H5]cortisone
[13,14]. In this study, we synthesize two types of multi-
labeled 6␤- and 6␣-hydroxycortisols, i.e. 6␤- and 6␣-
hydroxy-[1,1,19,19,19-2H5]cortisol and 6␤- and 6␣-
hydroxy-[1,2,4,19-13C4,1,1,19,19,19-2H5]cortisol, based on
the use of the corresponding labeled cortisone. 6␤-Hydroxy-
[1,1,19,19,19-2H5]cortisone was also prepared by this
procedure. Furthermore, 6␤-hydroxy-[1␣,16,16,17␣-2H4]
testosterone was synthesized from deuterium-labeled
testosterone obtained by using catalytic deuteration and
hydrogen–deuterium exchange reactions. By using the la-
beled internal standards, we recently developed a capillary
GC-MS method for the simultaneous determination of 6␤-
and 6␣-hydroxycortisols and 6␤-hydroxycortisone in urine
with good accuracy and precision [15]. The structures of 6-
hydroxylated corticosteroids and testosterone labeled with
2
13C and H are given in Fig. 1.
2. Experimental
1H NMR spectra were determined on Bruker DPX-
400 400-MHz and Bruker DPX-500 500-MHz spectrom-
eters for samples in CDCl3, CD3OD, and [2H5]pyridine
with tetramethylsilane as internal reference. Capillary gas
chromatographic-mass spectrometric (GC-MS) analysis
was done on a Shimadzu QP1000EX GC-MS equipped
with a data-processing system. GC-MS employed an SPB-1
fused-silica capillary column (15 m × 0.25 mm, i.d.) with
the stationary phase coated at a 0.25-␮m film thickness
(Supelco, Bellefonte, PA).

T. Furuta et al. / Steroids 68 (2003) 693–703
695
6␤-Hydroxycortisol (6␤,11␤,17␣,21-tetrahydroxypregn-
4-ene-3,20-dione) and 6␣-hydroxycortisol (6␣,11␤,17␣,21-
tetrahydroxypregn-4-ene-3,20-dione) were purchased from
Steraloids (Wilton, NH). 6␤-Hydroxycortisone (6␤,17␣,21-
trihydroxypregn-4-ene-3,11,20-trione) was purchased from
Sigma (St. Louis, MO). 6␤-Hydroxytestosterone (Ultrafine,
Manchester, UK), testosterone (Tokyo Kasei Kogyo, Tokyo,
Japan), and androsta-1,4-diene-3,17-dione (Tokyo Kasei
Kogyo) were obtained commercially. Sodium borodeu-
teride (NaB2H4) (99 at.% 2H) and 30% sodium deuteroxide
(NaOD) (99 at.% 2H) were purchased from Acros (Fair
13C4,2H5-BMD-3-ethyl-3,5-dienol ether (2b) was synthe-
sized from ca. 6.75 mg of cortisone-13C4,2H5-BMD (1b).
Cortisone-13C4,2H5-BMD-3-ethyl-3,5-dienol ether (2b);
m/z 439 (M+). Unlabeled compound; m/z 430 (M+).
2.1.3. 17α,20;20,21-Bismethylenedioxy-
[1,1,19,19,19-2H5]pregn-4-ene-6β-ol-11-one
(6β-hydroxycortisone-2H5-BMD) (3a)
A solution of crude cortisone-2H5-BMD-3-ethyl-3,5-
dienol ether (2a) in absolute ethanol (10 ml) placed in a
50 ml round-bottomed flask was irradiated with a 100 W
high pressure mercury arc lamp through a Pyrex filter for
4 h at room temperature [16]. The light source was placed
at a distance of 10 cm from the reaction vessel. The reaction
was monitored by TLC. Evaporation of the solvent under
reduced pressure at room temperature gave a crude labeled
compound as a mixture of 6␤- and 6␣-hydroxycortisones-
2H5-BMD (6␤:6␣ = 4:1, determined by GC-MS). Separa-
tion of the epimers by preparative TLC (CHCl3/CH3OH =
100:6, v/v) gave pure 6␤-hydroxycortisone-2H5-BMD (3a)
in a 25% yield (ca. 2.1 mg); m/z 425 (M+). Unlabeled
6␤-hydroxycortisone-BMD; m/z 420 (M+). TLC: RF 0.31
for 6␤-hydroxycortisone-2H5-BMD and RF 0.26 for 6␣-
hydroxycortisone-2H5-BMD.
2
Lawn, NJ). Deuterium gas (99.5 at.% H, Showa Denko,
Tokyo, Japan), benzene-2H6 (99.5 at.% H, Merck, Darm-
2
2
stadt, Germany), CH3OD (99.5 at.% H, Aldrich, Milwau-
2
kee, WI), and deuterium chloride (99.5 at.% H, Aldrich)
were obtained commercially. Preparative thin-layer chro-
matography (TLC) was performed on glass plates coated
with a 0.25-mm layer of silica gel 60 F254 (Merck).
17␣,20;20,21-Bismethylenedioxy-[1,1,19,19,19-2H5 ]
pregn-4-ene-3,11-dione (cortisone-2H5-BMD), 17␣,21-di-
hydroxy-[1,1,19,19,19-2H5]pregn-4-ene-3,11,20-trione (cor-
tisone-2H5), and 11␤,17␣,21-trihydroxy-[1,1,19,19,19-2H5]
pregn-4-ene-3,20-dione (cortisol-2H5) were synthesized in
our laboratory [13]. 17␣,20;20,21-Bismethylenedioxy-[1,2,
4,19-13C4,1,1,19,19,19-2H5]pregn-4-ene-3,11-dione (corti-
sone-13C4,2H5-BMD), 17␣,21-dihydroxy-[1,2,4,19-13C4,1,
1,19,19,19-2H5]pregn-4-ene-3,11,20-trione (cortisone-13C4,
2H5), and 11␤,17␣,21-trihydroxy-[1,2,4,19-13C4,1,1,19,19,
19-2H5]pregn-4-ene-3,20-dione (cortisol-13C4,2H5) were
also synthesized in our laboratory [14].
2.1.4. 17α,20;20,21-Bismethylenedioxy-
[1,2,4,19-13C4,1,1,19,19,19-2H5]pregn-4-ene-6β
-ol-11-one (6β-hydroxycortisone-13C4,2H5-BMD) (3b)
According to the procedure for the preparation of 6␤-
hydroxycortisone-2H5-BMD (3a), 6␤-hydroxycortisone-
13C4,2H5-BMD (3b) was synthesized from ca. 6.75 mg
of cortisone-13C4,2H5-BMD (1b). Separation of 6␤- and
6␣-epimers (4:1) by preparative TLC (CHCl3/CH3OH =
100:6, v/v) gave pure 6␤-hydroxycortisone-13C4,2H5-BMD
(3b) in a 24% yield (ca. 1.6 mg); m/z 429 (M+). Unlabeled
6␤-hydroxycortisone-BMD; m/z 420 (M+). TLC: RF 0.31
for 6␤-hydroxycortisone-13C4,2H5-BMD and RF 0.26 for
6␣-hydroxycortisone-13C4,2H5-BMD.
2.1. Syntheses of 6β- and 6α-hydroxycortisols and
6β-hydroxycortisone labeled with 13C and H
2
2.1.1. 17α,20;20,21-Bismethylenedioxy-3-ethoxy-
[1,1,19,19,19-2H5]pregna-3,5-diene-11-one
(cortisone-2H5-BMD-3-ethyl-3,5-dienol ether) (2a)
To a suspension of 17␣,20;20,21-bismethylenedioxy-
[1,1,19,19,19-2H5]pregn-4-ene-3,11-dione (cortisone-2H5-
BMD) (1a; ca. 8 mg, 20 ␮mol) in dioxane (300 ␮l) were
added triethyl orthoformate (100 ␮l, 0.60 mmol) and ab-
solute ethanol (5 ␮l), followed by addition of 20 ␮l of
H2SO4/dioxane (1:20, v/v). The mixture was then stirred
vigorously for 10 min at room temperature. To the dark
reaction mixture were added pyridine (10 ␮l) and water
(0.5 ml). The reaction mixture was extracted with CHCl3
(5 × 1 ml), the CHCl3 extract washed with water, and the
solvent evaporated to dryness to give the crude cortisone-
2H5-BMD-3-ethyl-3,5-dienol ether (2a); m/z 435 (M+).
Unlabeled compound; m/z 430 (M+).
2.1.5. 6β,17α,21-Trihydroxy-[1,1,19,19,19-2H5]pregn-
4-ene-3,11,20-trione (6β-hydroxycortisone-2H5) (4a)
A solution of pure 6␤-hydroxycortisone-2H5-BMD (3a)
(ca. 85 ␮g) in ethanol/tetrahydrofuran (0.2 ml, 1:1, v/v) was
added dropwise to 46% HF (0.6 ml) in a polyethylene test-
tube at 0 ◦C with stirring. The mixture was stirred vigorously
at 0 ◦C for 8 h and then kept at 2–5 ◦C for 15 h. The mixture
was neutralized to pH ca. 7 by careful addition of chilled
saturated Na2CO3 solution and then extracted with ethyl ac-
etate (4 × 10 ml). The extracts were washed with water and
the solvent was evaporated under reduced pressure. Purifi-
cation of the residue by TLC (RF 0.31, CHCl3/CH3OH =
100:6, v/v) gave pure 6␤-hydroxycortisone-2H5 (4a) in
2.1.2. 17α,20;20,21-Bismethylenedioxy-3-ethoxy-
1
[1,2,4,19-13C4,1,1,19,19,19-2H5]pregna-3,5-diene-11-one
(cortisone-13C4,2H5-BMD-3-ethyl-3,5-dienol ether) (2b)
According to the procedure for the preparation of
cortisone-2H5-BMD-3-ethyl-3,5-dienol ether (2a), cortisone-
a 34.8% yield (26.6 ␮g). H NMR spectrum of (4a) was
not measured. The labeled compound (4a) was converted
to and characterized as 3,20-dimethoxime-6␤,17␣,21-
tri(trimethylsilyl) derivative (MO-triTMS derivative); m/z

696
T. Furuta et al. / Steroids 68 (2003) 693–703
655 (M+) [15]. Unlabeled 6␤-hydroxycortisone; m/z 650
(M+) as MO-triTMS derivative [15]; δH (400 MHz, CDCl3):
0.67 (3H, s, 13-CH3), 1.62 (3H, s, 10-CH3), 4.33–4.34 (1H,
t, 6␣-CH), and 5.83 (1H, s, 4-CH).
androsta-1,4-diene-3,17-dione (9) dissolved in benzene-2H6
(19.5 ml) for 10 min. The mixture was stirred for 1 h under
the deuterium atmosphere. After the reaction (absorption
of 78.4 ml (3.5 mmol) of deuterium gas), the solution was
evaporated to dryness, and the residue was taken up in
a mixture of petroleum ether and CHCl3 (1:1, v/v) and
filtered through alumina B (50 mg). Evaporation of the sol-
vent gave 910.2 mg of androstenedione-2H2 (10) in a 90.2%
yield. Androstenedione-2H2; m/z 288 (M+). Unlabeled
compound; m/z 286 (M+).
2.1.6. 6β (or 6α),11β,17α,21-Tetrahydroxy-
[1,1,19,19,19-2H5]pregn-4-ene-3,20-dione (6β- and
6α-hydroxycortisols-2H5) (6a)
The conversion of cortisone-BMD into cortisol was
achieved by a known route [13,14]. The 3-keto group of
6␤-hydroxycortisone-2H5-BMD (3a) (ca. 2.1 mg) was pro-
tected with semicarbazide and the 11-keto group was then
reduced with NaBH4. Removal of the protecting group
at C-3 with pyruvic acid, followed by hydrolysis of the
BMD protecting group with 46% HF afforded crude 6␤-
and 6␣-hydroxycortisols-2H5 (6a). The epimers of the
labeled compounds (6a) (RF 0.20) could not be sepa-
rated by TLC. Purification of the crude materials by TLC
(RF 0.20, CHC13/CH3OH = 9:1, v/v) gave 6␤- and 6␣-
hydroxycortisols-2H5 (6a) (ca. 25 ␮g) as a mixture of two
epimers (6␤:6␣ = 4.4:1, determined by GC-MS). 1H NMR
spectra of 6␤- and 6␣-hydroxycortisols-2H5 (6a) were not
measured. The labeled compounds (6a) were converted
to and characterized as 3,20-dimethoxime-6␤,11␤,17␣,21-
tetra(trimethylsilyl) derivatives (MO-tetraTMS derivatives);
m/z 730 (M+) [15]. Unlabeled 6␤-hydroxycortisol; m/z
725 (M+) as MO-tetraTMS derivative [15]. δH (400 MHz,
CDCl3): 0.90 (3H, s, 13-CH3), 1.62 (3H, s, 10-CH3), 4.39
(1H, s, 11␣-CH), 4.25 (1H, t, 6␣-CH), and 5.74 (1H, s,
4-CH).
2.2.2. [1α,2,2,4,6,6,16,16-2H8]Androst-4-ene-3,17-dione
(androstenedione-2H8) (11)
Androstenedione-2H2 (10) (200 mg, 0.7 mmol) was dis-
solved in CH3OD (6 ml) in order to replace the hydroxyl pro-
tons of trace H2O contained in crystals with deuterium. The
solvent was immediately evaporated under reduced pressure
at room temperature. To a solution of androstenedione-2H2
(10) in 3.0 ml of CH3OD/D2O (5:1, v/v) was added 12.5%
NaOD in D2O (650 ␮l), and the mixture was heated at 65 ◦C
for 24 h under the argon atmosphere [17]. After the reaction,
the mixture was neutralized with 7% DCl (pD 6–7) at room
temperature, and the solvent was evaporated under reduced
pressure. The residue was dissolved in D2O (5 ml) and then
extracted with CHCl3 (4 × 10 ml). The organic solvent was
dried over anhydrous sodium sulfate, and then removed un-
der reduced pressure to give 127 mg of androstenedione-2H8
(11) as colorless crystals in a 63.5% yield. Androstenedione-
2H8; m/z 294 (M+). Unlabeled compound; m/z 286 (M+).
2.2.3. [1α,2,2,4,6,6,16,16,17α-2H9]Androst-4-en-
2.1.7. 6β (or 6α),11β,17α,21-Tetrahydroxy-
17β-ol-3-one (testosterone-2H9) (12)
[1,2,4,19-13C4,1,1,19,19,19-2H5]-pregn-4-ene-3,20-dione
Androstenedione-2H8 (11) (102 mg) was dissolved in
CH3OD (3 ml) in order to replace the hydroxyl protons
of trace H2O in crystals with deuterium. The solvent was
immediately evaporated under reduced pressure at room
temperature. To a solution of androstenedione-2H8 (11) in
15 ml of n-C3H7OD/D2O (4:1, v/v) was added approxi-
mately 10 mg of NaB2H4, and the mixture was stirred at
0 ◦C for 2 h. After the reaction, the mixture was neutralized
with 2% CH3COOD in D2O (pD 6–7), and the solvent
was evaporated under reduced pressure. The residue was
dissolved in D2O (5 ml) and then extracted with CHCl3
(3 × 8 ml). The organic solvent was dried over anhydrous
sodium sulfate, and then removed under reduced pressure to
give 102 mg of testosterone-2H9 (12) as colorless crystals
in a 99% yield. Testosterone-2H9; m/z 297 (M+). Unlabeled
compound; m/z 288 (M+).
(6β- and 6α-hydroxycortisol-13C4,2H5) (6b)
According to the procedure for the preparation of 6␤-
hydroxycortisol-2H5, 6␤-hydroxycortisol-13C4,2H5 was
synthesized from ca. 1.62 mg of 6␤-hydroxycortisone-
13C4,2H5-BMD (3b). The resulting material was subjected
to preparative TLC (RF 0.20, CHC13/CH3OH = 9:1,
v/v), giving 6␤- and 6␣-hydroxycortisols-13C4,2H5 (6b)
(ca. 27 ␮g) as a mixture of two epimers (6␤:6␣ = 4.4:1,
1
determined by GC-MS). H NMR spectra of 6␤- and 6␣-
hydroxycortisols-13C4,2H5 (6b) were not measured. The la-
beled compounds (6b) were converted to and characterized
as MO-tetraTMS derivatives; m/z 734 (M+) [15]. Unla-
beled 6␤-hydroxycortisol; m/z 725 (M+) as MO-tetraTMS
derivative.
2
2.2. Synthesis of 6β-hydroxytestosterone labeled with H
2.2.4. [1α,16,16,17α-2H4]Androst-4-en-17β-ol-3-one
(testosterone-2H4) (13)
2.2.1. [1α,2α-2H2]Androst-4-ene-3,17-dione
(androstenedione-2H2) (10)
Testosterone-2H9 (12) (100 mg) dissolved in a 5%
methanolic solution of H2SO4 (2 ml) was heated at 55 ◦C
for 48 h with constant stirring. After the reaction, the mix-
ture was neutralized to pH 7–8 with 4.6 ml of 2.5% KOH in
H2O at room temperature, and then extracted with CHCl3
A solution of chlorotris(triphenylphosphine)rhodium (I)
(250 mg) in benzene-2H6 (19.5 ml) was stirred for 1 h
at room temperature under the deuterium atmosphere.
To the solution was added dropwise 1 g (3.5 mmol) of

T. Furuta et al. / Steroids 68 (2003) 693–703
697
(4 × 6 ml). The organic solvent was dried over anhydrous
sodium sulfate, and then removed under reduced pressure.
The residue was subjected to column chromatography on
silica gel. The column (60 cm × 2.5 cm, i.d.) was eluted
with toluene, followed by n-hexane/CHCl3/acetone (5:4:1,
v/v/v) to give 57.6 mg of [1␣,16,16,17␣-2H4]androst-4-
en-17␤-ol-3-one (testosterone-2H4) (13) as colorless crys-
tals in a 58.5% yield. Testosterone-2H4; m/z 292 (M+).
1H NMR (400 MHz, CDCl3) proton signals at 1.48 ppm
(16␤-CH), 1.70 ppm (1␣-CH), 2.05 ppm (16␣-CH), and
3.65 ppm (17␣-CH) disappeared, and 13C NMR δ (400 MHz,
CDCl3): 30.42 ppm (s, 16-CH2), 35.71 ppm (s, 1-CH2),
and 81.58 ppm (s, 17␣-CH) disappeared. Unlabeled testos-
terone; m/z 288 (M+). δH (500 MHz, CDCl3): 1.48 (1H, m,
16␤-CH), 1.70 (1H, m, 1␣-CH), 2.05 (1H, m, 16␣-CH),
and 3.65 (1H, t, 17␣-CH). 13C NMR δ (500 MHz, CDCl3):
30.42 (s, 16-CH2), 35.71 (s, 1-CH2), and 81.58 (s, 17␣-CH).
[1,2,4,19-13C4,1,1,19,19,19-2H5]cortisol (6␤-hydroxycorti-
sol-13C4,2H5) shown in Fig. 1. Deuterium-labeled 6␤-
hydroxycortisone, 6␤-hydroxy-[1,1,19,19,19-2H5]cortisone
(6␤-hydroxycortisone-2H5), was also synthesized. A very
short synthesis of multiply labeled 6␤-hydroxycortisone
would be possible if an oxidative step of introducing a hy-
droxyl group at C-6 could be set after the preparation of
labeled cortisone [13,14], i.e. [1,1,19,19,19-2H5]cortisone
(cortisone-2H5) and [1,2,4,19-13C4,1,1,19,19,19-2H5]corti-
sone (cortisone-13C4,2H5) (Scheme 1). In this study, oxi-
dation of C-6 was performed by using a photochemically
induced autoxidation reported by Gardi and Lusignani [16].
Dihydroxyacetone moiety of cortisone-2H5 was first pro-
tected as bismethylenedioxy (BMD) with 37% HCHO to
give cortisone-2H5-BMD (1a) in 70–75% yields. The intro-
duction of a hydroxyl group at C-6 of cortisone-BMD was
conducted by ultraviolet (UV)-irradiated autoxidation of the
3-ethyl-3,5-dienol ether (2a) in ethanol to give a mixture
of 6␣- and 6␤-hydroxycortisones-2H5-BMD (3a) (route A,
Scheme 1). The ratio of 6␤- and 6␣-epimers was approxi-
mately 4:1, determined by GC-MS. Purification and sepa-
ration of the epimers by TLC (RF 0.31, CHCl3/CH3OH =
100:6, v/v) and subsequent hydrolysis of the BMD acetals
with 46% HF afforded pure 6␤-hydroxycortisone-2H5 (4a).
6␤-Hydroxycortisol-2H5 (6a) was then prepared via the re-
duction of the 11-keto group of 6␤-hydroxycortisone-2H5-
BMD (3a). The 3-keto group of pure 6␤-hydroxycortisone-
2H5-BMD (3a) was protected with semicarbazide and the
11-keto group was then reduced with NaBH4. Removal of
the protecting group at C-3 with pyruvic acid followed by
hydrolysis of the BMD acetals with 46% HF afforded a
mixture of 6␤- and 6␣-hydroxycortisols-2H5 (6a) (6␤:6␣ =
4.4:1), accompanied by the epimerization of hydroxyl
group at C-6. The ratio of 6␤- and 6␣-epimers (4.4:1) was
determined by GC-MS. It is likely that 6␣-hydroxylated
compound is thermodynamically more stable than the cor-
responding 6␤-hydroxylated steroid, where the axially po-
sitioned 6␤ group is sterically hindered by the 10␤-methyl
group [18]. The ␤- and ␣-epimers of 6-hydroxycortisol
could not be separated by TLC (RF 0.20, CHCl3/CH3OH =
9:1, v/v), and the mixture was then used as analytical
internal standards for simultaneously measuring 6␤- and
6␣-hydroxycortisols in human urine by GC-MS [15].
2.2.5. [1α,16,16,17α-2H4]Androst-4-en-6β,17β-diol-3-one
(6β-hydroxytestosterone-2H4) (15)
To a solution of 39 mg (0.13 mmol) of testosterone-2H4
(13) in dioxane (2.1 ml) was added triethyl orthoformate
(390 ␮l) and absolute ethanol (40 ␮l). After adding a solu-
tion of 4.8% H2SO4 in dioxane (200 ␮l; 10:200, v/v), the
reaction mixture was stirred vigorously for 20 min. After
adding pyridine (40 ␮l) dropwise, followed by H2O (1 ml)
at room temperature, the mixture was extracted with di-
ethyl ether (4 × 4 ml). The extracts were washed with water
and the solvent was evaporated under reduced pressure to
give a crude testosterone-2H4-3-ethyl-3,5-dienol ether (14).
A solution of the crude compound (14) in ethanol (30 ml)
was irradiated with a 100 W high-pressure mercury arc
lamp through a Pyrex filter for 1 h [16]. After the reac-
tion, the solvent was evaporated under reduced pressure,
and the residue was subjected to column chromatogra-
phy on silica gel. The column (20 cm × 2.5 cm, i.d.) was
eluted with CHCl3/CH3OH (9:1, v/v) to give 0.46 mg of
6␤-hydroxytestosterone-2H4 (15) as colorless crystals in a
1.1% yield. 6␤-Hydroxytestosterone-2H4; m/z 308 (M+).
Neither 1H nor 13C NMR spectra of labeled compound
(15) were measured. Unlabeled 6␤-hydroxytestosterone;
m/z 304 (M+). δH (500 MHz, pyridine-2H5): 1.59–1.65
(2H, m, 1␣-CH and 15␣-CH), 1.77 (1H, m, 16␤-CH), 2.07
(1H, m, 16␣-CH), and 3.91 (1H, m, 17␣-CH). 13C NMR
δ (500 MHz, CDCl3): 31.09 (s, 16-CH2), 35.68 (s, 1-CH2),
and 81.35 (s, 17␣-CH).
6␤-Hydroxy-[1,2,4,19-13C4,1,1,19,19,19-2H5]cortisol (6␤-
hydroxycortisol-13C4,2H5) (6b) was synthesized via route
A (Scheme 1) starting from [1,2,4,19-13C4,1,1,19,19,19-
2H5]cortisone-BMD (1b), since autoxidation of cortisol-
BMD-3-ethyl-3,5-dienol ether (8a) (route B, Scheme 1)
resulted in less formation of 6␤-hydroxycortisol (5a)
(6␤:6␣ = 1:3.5, determined by GC-MS). The labeled 6␤-
hydroxycortisol (6b) has been used as an analytical internal
standard [26] for simultaneously determining endogenous
and exogenous (labeled) 6␤-hydroxycortisols in urine after
the administration of stable isotopically labeled cortisol
(cortisol-2H5 or cortisol-13C4) as biological internal stan-
dard (tracer) in human subjects [24,25].
3. Results and discussion
3.1. Syntheses of 6β-hydroxycortisol and
6β-hydroxycortisone labeled with 13C and H
2
The present study was designed to synthesize two types
of labeled 6␤-hydroxycortisols, 6␤-hydroxy-[1,1,19,19,19-
2H5]cortisol (6␤-hydroxycortisol-2H5) and 6␤-hydroxy-

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T. Furuta et al. / Steroids 68 (2003) 693–703
Scheme 1. Synthesis of stable isotopically labeled 6-hydroxylated corticosteroids (6␤- and 6␣-hydroxycortisols-2H5, 6␤- and 6␣-hydroxycortisols-13C4,2H5,
and 6␤-hydroxycortisone-2H5).

T. Furuta et al. / Steroids 68 (2003) 693–703
699
Fig. 2. EI mass spectra of MO-TMS derivatives of unlabeled 6␤-hydroxycortisol (A), 6␤-hydroxycortisol-2H5 (B), and 6␤-hydroxycortisol-13C4,2H5 (C).
Fig. 2 shows the electron-impact (EI) mass spectra of
the MO-TMS derivatives of unlabeled 6␤-hydroxycortisol
and labeled 6␤-hydroxycortisols (6␤-hydroxycortisol-2H5
and 6␤-hydroxycortisol-13C4,2H5) (6a and 6b). The molec-
ular ions of MO-TMS derivatives of 6␤-hydroxycortisol-
2H5 (m/z 730) (6a) and 6␤-hydroxycortisol-13C4,2H5
(m/z 734) (6b) were five and nine mass unit higher,
respectively, than the molecular ion (m/z 725) of un-
labeled 6␤-hydroxycortisol. In the mass spectra of the
MO-TMS derivatives of labeled 6␤-hydroxycortisone (6␤-
hydroxycortisone-2H5) (4a), the molecular ion (m/z 655)
was five mass units higher than that (m/z 650) of unlabeled
6␤-hydroxycortisone (the mass spectra are not shown).
The isotopic purity was calculated to be >97% for 6␤-
hydroxycortisol-2H5 (6a), 6␤-hydroxycortisol-13C4,2H5
(6b), and 6␤-hydroxycortisone-2H5 (4a), based on the ion
intensities in the region of the molecular ion of the respective
compound.
2
3.2. Synthesis of 6β-hydroxytestosterone labeled with H
Synthesis of multiply labeled 6␤-hydroxytestosterone
was carried out by the oxidation of C-6␤ position of
deuterium-labeled testosterone by using a photochemically

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T. Furuta et al. / Steroids 68 (2003) 693–703
Scheme 2. Synthesis of deuterium-labeled 6␤-hydroxytestosterone (6␤-hydroxytestosterone-2H4).
induced autoxidation. We synthesized [1␣,16,16,17␣-2H4]
testosterone (15) containing four non-exchangeable deu-
terium atoms at C-1, C-16, and C-17, starting from
androsta-1,4-diene-3,17-dione (9). As shown in Scheme 2,
selective reduction of the 1,2-double bond of steroidal
1,4-diene-3-one (9) in benzene-2H6 with chlorotris(triphe-
nylphosphine)rhodium (I) as catalyst under the deuterium
atmosphere gave [1␣,2␣-2H2]androst-4-ene-3,17-dione
(androstenedione-2H2) (10). The stereochemistry of the
deuterium atoms was established by Djerassi and Gutzwiller
[19], who reported that reduction of the C-1,2 double bond
of the steroidal 1,4-diene-3-one with homogenous rhodium
catalyst occurred from the ␣ side, while heterogeneous
palladium-catalyzed tritiation [20] or deuteration [21] pro-
ceeds from the ␤-side.
exchange reaction. Treatment of [1␣,2␣-2H2]androst-4-ene-
3,17-dione (androstenedione-2H2) (10) with 12.5% NaOD
at 65 ◦C for 24 h gave [1␣,2,2,4,6,6,16,16]androstenedione
(androstenedione-2H8) (11) by displacing active hydrogens
at C-2, C-4, C-6, and C-16 with deuteriums. Reduction of the
C-17 keto group of androstenedione-2H8 (11) with NaBD4
at 0 ◦C for 2 h gave [1␣,2,2,4,6,6,16,16,17␣]testosterone
(testosterone-2H9) (12). Subsequent back-exchange of five
deuterium atoms at C-2, C-4, and C-6 of testosterone-2H9
(12) with a 5% methanolic solution of H2SO4 at 55 ◦C
for 48 h gave [1␣,16,16,17␣-2H4]androst-4-en-17␤-ol-3-
one (testosterone-2H4) (13). Proton signals at 1.48 ppm
(16␤-CH), 1.70 ppm (1␣-CH), 2.05 ppm (16␣-CH), and
1
3.65 ppm (17␣-CH) disappeared in the H NMR spectrum
of testosterone-2H4 (Fig. 3). EI mass spectra of labeled
testosterone (testosterone-2H4) (13) (the mass spectra are
not shown) showed that the molecular ion (m/z 292) was
four mass units higher than that (m/z 288) of unlabeled
testosterone.
The introduction of deuterium at C-16 and C-17 of
testosterone was then achieved according to the procedure
reported by Sanaullah and Bowers [17] for synthesizing
[16,16,17-2H3]testosterone using a hydrogen–deuterium

T. Furuta et al. / Steroids 68 (2003) 693–703
701
Fig. 3. 1H NMR spectra of unlabeled testosterone (A) and testosterone-2H4 (B).
The introduction of a hydroxyl group at C-6␤ of
testosterone-2H4 (13) was conducted according to the pro-
cedure for 6␤-hydroxylation of cortisone and cortisol. The
3-ethyl-3,5-dienol ether (14) was first prepared by treat-
ment of testosterone-2H4 (13) in dioxane (2.1 ml) contain-
ing 40 ␮l of absolute ethanol with triethyl orthoformate
(390 ␮l), followed by adding a solution of H2SO4/dioxane
(200 ␮l; 10:200, v/v). The reaction led to the formation of
testosterone-3-ethyl-3,5-dienol ether (m/z 316; M+) (14)
and its 17␤-ethoxy derivative (m/z 344; M+) in a ratio of
2:1. Attempts to purify testosterone-3-ethyl-3,5-dienol ether
by recrystallization or by column chromatography were
unsuccessful because of instability of the 3-ethyl-3,5-dienol
ether. A solution of crude testosterone-2H4-3-ethyldienol
ether (14) thus obtained in ethanol (30 ml) was irradiated
with a 100 W high-pressure mercury arc lamp through a
Pyrex filter for 1 h. Purification by column chromatography
gave [1␣,16,16,17␣-2H4]androst-4-en-6␤,17␤-diol-3-one
(6␤-hydroxytestosterone-2H4) (15) as colorless crystals.
The 6␣-epimer was not detected by GC-MS.
Fig. 4 shows the EI mass spectra of unlabeled 6␤-
hydroxytestosterone and labeled 6␤-hydroxytestosterone

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T. Furuta et al. / Steroids 68 (2003) 693–703
Fig. 4. EI mass spectra of unlabeled 6␤-hydroxytestosterone (A) and 6␤-hydroxytestosterone-2H4 (B).
(6␤-hydroxytestosterone-2H4) (15). The molecular ions of
(15) (m/z 308) was four mass units higher than that (m/z
304) of authentic unlabeled 6␤-hydroxytestosterone. The
isotopic purity was calculated to be 78%, based on the ion
intensities in the region of the molecular ion of the com-
pound. The NMR spectral data of labeled 6-hydroxylated
compounds were not obtainable in this study because of
a small scale of the reactions employed. Assignments for
the labeled positions of 6-hydroxylated compounds (6␤-
hydroxycortisone, 6␤- and 6␣-hydroxycortisols, and 6␤-
hydroxytestosterone) were based on the NMR data of the
corresponding labeled precursors (labeled cortisone and
cortisol [13,14], and labeled testosterone shown in Fig. 3).
The chemical shift data for the full assignment of 13C and
1H NMR spectra of unlabeled 6␤-hydroxytestosterone and
6␤-hydroxycortisol are summarized in Table 1.
In general, the yield of the 6␤-hydroxylated compound
is much higher than that of 6␣-hydroxylated isomer on au-
toxidation of 3,5-dien-3-ol ether of steroidal 4-ene-3-keto
compounds [16], and the ratio between 6␤- and 6␣-epimers
(6␤/6␣) was 8–10 [22,23]. Preferential oxygenation at C-6
from the ␤ side is probably due to the planarity of the C-5,6
double bond of ring B resulting from the steroidal 3,5-dienol
moiety. The sp2 carbon at C-6 of ring B could be tilted down-
ward away from the ␤ angular methyl group at C-10 and to-
ward the ␣ axial hydrogen at C-9. Oxygenation at C-6 may
be hindered more by the C-9 hydrogen on the ␣-side than
by the methyl group on the ␤-side. In the present study, 6␣-
hydroxycortisol was predominantly formed (6␤:6␣ = 1:3.5)
on autoxidation of cortisol-BMD-3-ethyl-3,5-dienol ether,
whereas 6␤-hydroxycortisone possessing an 11-keto group
was predominantly formed (6␤:6␣ = 4:1) In the case of
cortisol possessing a hydroxyl group at C-11␤, non-bonding
interaction between the 11␤-hydroxyl and the 10␤-methyl
group may force the latter to bend toward the C-6 of the
B-ring, preventing oxygenation of C-6 from the ␤-side.
The stable isotope methodology coupled with mass
spectrometry would provide a useful tool for the phar-
macokinetic and metabolic studies of cortisol, cortisone,
and testosterone with a particular interest in evaluating
6␤- or 6␣-hydroxylation in the metabolism in human. The
present study demonstrated a synthesis of multi-labeled
6-hydroxylated cortisol, cortisone, and testosterone with
13C and 2H for use as internal standards for GC-MS
for study of phenotyping the in vivo CYP3A activity in
humans.

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Position
6␤-Hydroxycortisol
6␤-Hydroxytestosterone
13C NMR
37.83
1H NMR
13C NMR
37.68
1H NMR
1
2
␣
␤
␣
␤
1.83
2.24
2.33
2.62
␣
␤
␣
␤
1.60
1.91
2.42
2.56
34.92
34.92
3
4
5
6
7
203.21
125.32
173.00
73.18
199.75
126.00
169.00
72.64
5.74
6.05
A
second enzyme protecting mineralocorticoid receptors from
␣
␣
␤
4.25
1.33
2.12
2.35
0.92
␣
␣
␤
4.55
1.26
2.13
2.29
0.89
glucocorticoid occupancy. Am J Physiol 1998;274:C1245–52.
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40.77
39.46
8
9
10
11
28.28
57.74
40.13
68.46
30.57
54.54
38.67
21.19
␣
4.39
␣
␤
␣
␤
1.50
1.48
1.15
2.09
12
40.86
␣
␤
2.02
1.62
37.42
13
14
15
48.30
53.40
24.71
43.63
51.05
23.93
1.76
1.82
1.45
1.47
2.72
1.01
1.60
1.34
2.07
1.77
3.91
1.00
1.55
␣
␤
␣
␤
␣
␤
␣
␤
␣
16
34.59
31.09
17
18
19
20
21
90.37
17.83
22.96
212.93
67.67
81.35
11.90
19.76
ꢀ
3,5-dien-3-ol
0.90
1.62
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Acknowledgements
This study was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, and in part by
a Grant for Private Universities provided by the Promotion
and Mutual Aid Corporation for Private Schools of Japan.
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