Potential corticoid metabolites: Chemical synthesis of 3- and 21-monosulfates and their double-conjugates of tetrahydrocorticosteroids in the 5 α- and 5 β-series

Article abstract of DOI:10.1248/cpb.58.344

Here, we describe the chemical synthesis of the complete sets of 18 novel 3- and 21-monosulfates and their double-conjugated form of tetrahydrocortisol (THF), tetrahydro-11-deoxycortisol (THS), and tetrahydrocortisone (THE) in the 5 α- and 5 β-series. The principal reactions involved are: (1) selective protection of a specific hydroxy group in substrates; (2) catalytic hydrogenation at C-5 of Δ⁴-3-ketosteroids with 10% Pd(OH) ₂/C to yield 3-oxo-5 β-steroids and reductive allomerization with 10% Pd/C to yield 3-oxo-5 α-isomers; (3) reduction of the resulting 3-oxo-5 β- and 3-oxo-5 α-steroids to the corresponding 3 α-hydroxy-compounds with Zn(BH₄)₂ and K-Selectride, respectively; and (4) sulfation of hydroxy groups at C-3 and/or C-21 in the tetrahydrocorticosteroid derivatives with sulfur trioxide-triethylamine complex.

Full text of DOI:10.1248/cpb.58.344

344
Chem. Pharm. Bull. 58(3) 344—353 (2010)
Vol. 58, No. 3
Potential Corticoid Metabolites: Chemical Synthesis of 3- and
21-Monosulfates and Their Double-Conjugates of
a
b
Tetrahydrocorticosteroids in the 5 - and 5 -Series
Rika OKIHARA,a,1) Kuniko MITAMURA,a Maki HASEGAWA,a,2) Megumi MORI,a Akina MUTO,b
Genta KAKIYAMA,b Shoujiro OGAWA,b Takashi IIDA,b Miki SHIMADA,c
Nariyasu MANO,c and Shigeo IKEGAWA*,a
b
a Faculty of Pharmaceutical Sciences, Kinki University; 3–4–1 Kowakae, Higashi-Osaka 577–8502, Japan: Department
of Chemistry, College of Humanities & Sciences, Nihon University; 3–25–40 Sakurajousui, Setagaya-ku, Tokyo 156–8550,
c
Japan: and Department of Pharmaceutical Sciences, Tohoku University Hospital; 1–1 Seiryou-machi, Aoba-ku, Sendai
980–8574, Japan. Received September 30, 2009; accepted December 14, 2009; published online December 17, 2009
Here, we describe the chemical synthesis of the complete sets of 18 novel 3- and 21-monosulfates and their
double-conjugated form of tetrahydrocortisol (THF), tetrahydro-11-deoxycortisol (THS), and tetrahydrocorti-
sone (THE) in the 5a- and 5b-series. The principal reactions involved are: (1) selective protection of a specific
hydroxy group in substrates; (2) catalytic hydrogenation at C-5 of D4-3-ketosteroids with 10% Pd(OH)2/C to
yield 3-oxo-5b-steroids and reductive allomerization with 10% Pd/C to yield 3-oxo-5a-isomers; (3) reduction of
the resulting 3-oxo-5b- and 3-oxo-5a-steroids to the corresponding 3a-hydroxy-compounds with Zn(BH4)2 and
K-Selectride®, respectively; and (4) sulfation of hydroxy groups at C-3 and/or C-21 in the tetrahydrocortico-
steroid derivatives with sulfur trioxide-triethylamine complex.
Key words sulfate; catalytic reduction; sulfur trioxide-triethylamine complex; tetrahydrocortisol; tetrahydrocortisone; tetrahy-
dro-11-deoxycortisol
As the principal glucocorticoid in humans, cortisol plays a put is relatively low. Thus the development or more straight-
crucial role in modulating the metabolic and homeostatic forward methods based on the direct analysis of steroid con-
process that protects against stress, shock, inflammation, jugates without the need for a deconjugation process is of
etc.3—7) It is synthesized primarily in the zona fasciculata great interest. The application of liquid chromatographic sep-
with a small contribution from the zona reticularis.3) While aration interfaced by soft ionization techniques, such as elec-
cortisol is essentially secreted by the adrenal glands, corti- trospray ionization (ESI) with tandem mass spectrometry
sone is mainly produced using 11b-hydroxysteroid dehydro- and/or ion trap mass spectrometry, offers an effective analyti-
genase isoenzymes, which interconvert cortisol to hormon- cal tool for the direct monitoring of the urinary conjugates of
ally inactive cortisone.3) Cortisol and cortisone are exten- tetrahydrocorticosteroids.19,20) Recently, we reported a highly
sively metabolized to tetrahydro-reduced derivatives: tetrahy- sensitive and specific liquid chromatography/ESI-linear ion
drocortisol (THF) and tetrahydrocortisone (THE), and their trap mass spectrometry method for the direct measurement
5a-stereoisomer (allo-THF and allo-THE, respectively).3,7—9) of the glucuronide conjugates of THF, THE, THS, and their
Metabolism decreases the biological activity of hormones 5a-stereoisomer in human urine.21) The authentic glucuronides
and increases their water solubility by converting them to had been chemically synthesized by Hosoda et al.22—25) One
hydrophilic compounds that can be excreted in urine. Unme- significant drawback of sulfate conjugate analysis, however,
tabolized cortisol and cortisone comprise only ca. 0.1% of is still the lack of reference materials, which are essential for
the total urinary cortisol metabolites. At least 90% of the the development and application of analytical methods. In
tetrahydro-derivatives of cortisol and cortisone metabolites addition, conjugated reference standards are also needed
are excreted into the urine as sulfate or glucuronide conju- within the field of steroid metabolism research. The objective
gates.10—13) Tetrahydro-11-deoxycortisol (THS) and its 5a- of this study was to synthesize 18 sulfated conjugates of
stereoisomer (allo-THS) which are tetrahydro-reduced tetrahydrocorticosteroids as shown in Fig. 1. The compounds
metabolites of 11-deoxycortisol (11-DOC), a biosynthetic selected in this study include possible and potential metabo-
precursor of cortisol, are also excreted in urine as conjugated lites, some of which have already been detected in human
forms.8,10,12) In order to obtain information regarding the urine.3,8,10)
related biochemistry, physiology, and pathophysiology of
endocrine disorders, it is necessary to identify all the corti- Results and Discussion
costeroid metabolites found in urine, and determine the exact
structure of their conjugates, their concentration, and the dy- curate determination of the polar and hydrophilic sulfated
namics of their formation and disposal. conjugates of tetrahydrocorticosteroids, the best analytical
For the purpose of a highly sensitive, selective, and ac-
At present, analytical methods for the detection of conju- method seemed to be the use of LC-MS/MS coupled with the
gated tetrahydrocorticosteroids are based on gas chromatog- available authentic reference standards. As part of our ongo-
raphy and mass spectrometric determination of hydrolyzed ing program to prepare new or scarce corticosteroid conju-
and derivatized compounds.14—18) Although these methods gates as potential metabolites that have clinical utility, we
are robust and sensitive, sample preparation in these indirect carried out the chemical synthesis of the 18 variants of the
methods is time consuming, and the GC-MS sample through- 3- and 21-monosulfate conjugates as well as the double-con-
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: ikegawa@phar.kindai.ac.jp

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Fig. 1. Chemical Structures of the Desired Sulfates of Tetrahydrocorticosteroids
Fig. 2. Synthetic Route to the Sulfate Conjugates of THF
jugated 3,21-disulfates of THF, THS, and THE, and their dione (2) and 21-acetoxy-17a-hydroxy-5b-pregnane-3,20-
corresponding “allo” series of 5a-stereoisomers (see Fig. 1). dione (8) from 1b and the 21-acetoxy derivative (7b) of 11-
The desired THF-3- and THF-21-sulfates (4b, 5d) and DOC (7a). Our exploratory experiments revealed that the
THF-3,21-disulfate (6b) were prepared from the 21-acetoxy catalytic hydrogenation of 1b and 7b in the presence of 5%
derivative (1b) of cortisol (1a), according to the route de- Pd/CaCO3 was unsatisfactory, and the reduction reaction did
picted in Fig. 2. A key intermediate in the synthesis of the not proceed at all. However, by changing the catalyst to 10%
sulfated THFs was 21-acetoxy-3a,11b,17a-trihydroxy-20- Pd(OH)2/C, reduction of 1b and 7b proceeded smoothly to
one (3), which was prepared from 1a in three steps. The yield the desired 2 and 8, respectively. The use of Pd(OH)2/C
transformation of conjugated D4-3-ketosteroids into the was found to be much preferred over that of Pd/CaCO3.
stereoisomeric 3-oxo-5b- (normal cis A/B-ring juncture) and
Conversion of 3,20-dioxo-5b-steroid (2) to a key inter-
3-oxo-5a-steroids (allo trans A/B-ring juncture) has been mediate, 3a-hydroxy-20-oxo-5b-steroid (3), has previously
improved by many groups of workers. It is now well recog- been achieved by the reduction of 2 using a Raney nickel cat-
nized that the formation ratio of 3-oxo-5b-/3-oxo-5a- alyst.22) However, the reduction of 2 by zinc borohydride
steroids is significantly influenced not only by the structures (Zn(BH4)2)27,28) offered an easier and more practical proce-
of the D4-3-ketosteroids but also the reducing agents that are dure for the preparation of 3. This less basic, mild zinc
employed. Combe et al.26) have recommended the use of reagent, compared to common NaBH4, leaves the oxo group
Pd/CaCO3 as a hydrogenation catalyst for the reduction of at C-20 in 2 intact at room temperature for 45 min during the
D4-3-ketosteroids into their corresponding 3-oxo-5b-steroids. reduction at C-3. In addition, Zn(BH4)2 reduction proceeds
Hosoda et al.22,23) have carried out a procedure for the prepa- more simply and efficiently than with catalytic hydrogenation
ration of 21-acetoxy-11b,17a-dihydroxy-5b-pregnane-3,20- by Raney nickel. The a-configuration of the C-3 hydroxy

346
Vol. 58, No. 3
Fig. 3. Synthetic Route to the Sulfate Conjugates of THS
Fig. 4. Synthetic Route to the Sulfate Conjugates of THE
group in 3 was characterized by a 3b-H signal appearing at Treatment of 5b with SO3-TEA complex yielded 3a-
3.64—3.73 ppm as a broad multiplet with the half-band TBDMSO-11b,17a-hydroxy-21-sulfate (5c). Subsequent
width of ca. 20 Hz in the 1H-NMR spectrum.
desilylation at C-3 in 5c with HCl resulted in the formation
The 3a-hydroxy group in 3 was selectively sulfated with of THF-21-sulfate (5d). Furthermore, alkaline hydrolysis
sulfur trioxide-triethylamine (SO3-TEA) complex in pyri- of 3 with sodium methoxide, followed by sulfation of the
dine. The sulfation reaction proceeded cleanly and rapidly resulting THF (6a) with SO3-TEA complex, afforded THF-
under mild experimental conditions. The resulting 3-sulfated 3,21-disulfate (6b).
compound (4a) was then hydrolyzed with sodium methoxide
Essentially identical procedures were used to prepare the
to remove the C-21 acetoxy group. To purify the crude THF- 3- and 21-monosulfates and 3,21-disulfates of THS (10b,
3-sulfate (4b), it was loaded onto an Oasis® HLB cartridge 11d, 12b) and THE (14b, 15d, 16b), starting from 11-DOC
for reversed-phase solid phase extraction. After the cartridge (7a) and THE-21-acetate (13), respectively, as outlined in
was washed with water to remove excess reagents and inor- Figs. 3 and 4.
ganic salts, elution with methanol yielded a homogeneous
The focus of our next synthetic targets was the “allo”
effluent, which was characterized as THF-3-sulfate (4b).
series of the sulfate conjugates of tetrahydrocorticosteroids,
Meanwhile, compound 3 was selectively silylated at C-3 which are the 5a-stereoisomers of sulfate-conjugated THF,
by tert-butyldimethylsilyl chloride (TBDMSCl) to obtain THS, and THE, as shown in Fig. 5. Of various methods for
21-acetoxy-3a-TBDMSO-11b,17a-dihydroxy-5b-20-one the reductive allomerization at C-5 of D4-3-ketosteroids
(5a), which in turn was hydrolyzed with sodium methoxide reported previously,24,25,29,30) it seemed to us that the most
to afford 3a-TBDMSO-11b,17a,21-trihydroxy-20-one (5b). feasible route, starting from the readily available 1b through

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347
Fig. 5. Synthetic Route to the Sulfate Conjugates of Allo-THF and Allo-THE
the inversion of the configuration at C-5, offered a better way protected with TBDMSCl to give 19c. Treatment of 20a with
to obtain the 3-ethoxy-3,5-diene intermediate (17).24,25) The SO3-TEA complex, followed by desilylation of the resulting
procedures employed were (1) the etherification of 1b with 3a-sulfooxy derivative (20b) with HCl, afforded the desired
triethyl orthoformate in the presence of a catalytic amount of allo-THF-3-sulfate (20c).
p-toluenesulfonic acid and (2) selective reduction of the D3,5-
Allo-THF-21-sulfate (21d) was prepared by the following
bond in the resulting 17 over 10% Pd/C catalyst, yielding sequence steps: (1) acetylation at C-3 in 20a, (2) desilylation
enol ether (18), which after treatment with HCl gave the de- at C-21 in 21a, (3) sulfation at C-21 in 21b, and then (4)
sired 19a. After chromatographic purification, the allomer- alkaline hydrolysis at C-3 in 21c. Allo-THF-3,21-disulfate
ized 3-oxo-5a compound (19a) was isolated in a fairly good (20e) was similarly obtained by desilylation at C-21 in 20a
yield.
and sulfation at both C-3 and C-21 in 20d. The preparation
A sterically bulky hydride reducing agent, potassium of allo-THE-21-sulfate (22d) and allo-THF-3,21-disulfate
tri-sec-butylborohydride (K-Selectride®), is known to have (22e) was also attained starting from 3a-acetoxy-21-TB-
high stereoselectivity in the reduction of oxo to the hydroxy DMSO-17a-hydroxy-5a-11,20-dione (22a) by the essen-
group.24,25,30,31) For instance, 3-oxo-5b- and 3-oxo-5a-steroids tially identical procedures described for the preparation
are reduced to 3b-hydroxy-5b- and 3a-hydroxy-5a-isomers, of THF-21-sulfate (5d) and THF-3,21-disulfate (6b). Selec-
respectively.32) In fact, when 21-TBDMSO-11b,17a-dihy- tive oxidation of the 3a-acetoxy-21-TBDMSO-11b,17a-di-
droxy-5a-3,20-dione (19c) was subjected to reduction with hydroxy-5a-20-one (21a) with pyridinium chlorochromate
K-Selectride®, the major isolated product was the axially 3a- (PCC) under mild experimental conditions provided 22a in
hydroxylated product (20a). The a-configuration at C-3 in good isolated yield.
20a was confirmed by the equatorial 3b-H signal appearing
Based on the above findings, allo-THE-3-sulfate (23d)
at 4.03—4.08 ppm as a multiplet with the half-band width of could be expected to be prepared from 22a in several steps.
1
ca. 8 Hz in the H-NMR spectrum. Prior to the reduction re- Unexpectedly, a preliminary experiment revealed that the at-
action of 19c, the C-21 acetoxy group in 19a was hydrolyzed tempted alkaline hydrolysis of the C-3 acetoxy group in 22a
with sodium methoxide, then the C-21 hydroxy group of the yields a complex mixture, probably arising from the decom-
resulting 11b,17a,21-trihydroxy-5a-3,20-dione (19b) was position products, from which the expected 21-TBDMSO-

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Vol. 58, No. 3
Fig. 6. Synthetic Route to the Sulfate Conjugates of Allo-THS
Table 1. 13C-NMR Chemical Shifts for the 3- and 21-Mono- and 3,21-Disulfate Conjugatesa)
Carbon 4b
5d
6b
10b
11d
12b
14b
15d
16b
20c
21d
20e
23d
22d
22e
27c
28d
27e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
34.7
34.6
31.7
72.5
37.2
44.9
27.6
28.6
33.1
45.9
36.0
68.3
40.8
49.3
54.1
24.7
36.3
90.9
17.7
27.2
34.6
29.4
80.5
34.6
44.9
27.5
28.5
33.1
46.0
35.9
68.3
40.8
49.3
54.0
24.7
36.2
90.9
17.8
27.1
35.1
28.9
80.3
34.6
43.6
27.7
28.2
37.3
41.5
35.6
21.7
32.1
48.4
52.2
24.7
36.4
90.7
15.5
23.8
35.0
31.2
72.4
37.2
43.6
27.8
28.3
37.4
41.6
35.7
21.7
31.9
48.4
52.3
24.7
36.5
91.0
15.3
23.9
35.0
28.9
80.4
34.6
43.7
27.8
28.2
37.4
41.5
35.6
35.7 35.8
29.4 31.8
80.0 72.0
34.7 37.3
44.6 44.6
27.7 27.8
28.3 28.4
38.2 38.2
52.6 52.7
35.2 35.3
35.8
29.4
80.2
34.8
44.7
27.7
28.3
38.2
52.6
35.3
33.2
27.7
76.5
34.2
41.6
29.0
33.9
32.8
59.3
36.9
68.4
40.7
49.3
54.0
24.7
34.6
90.5
14.6
17.9
32.8 33.3
29.3 27.7
67.2 76.5
37.4 34.2
41.1 41.6
29.3 29.1
34.0 34.0
32.8 32.8
59.3 59.3
37.0 37.0
32.5
27.6
76.1
34.1
40.6
28.9
34.1
37.9
65.3
36.4
32.0
29.3
67.0
36.9
40.1
29.1
34.1
37.8
65.4
36.2
32.5
27.6
76.2
34.1
40.6
28.9
34.1
37.8
65.2
36.5
33.9
27.9
76.3
34.6
40.8
29.5
33.4
37.0
55.4
36.8
21.6
32.0
48.7
52.3
24.7
35.0
90.7
11.8
15.6
33.5
29.7
67.2
37.2
40.3
29.6
33.5
37.0
55.5
36.7
21.6
31.8
48.7
52.3
24.6
34.9
90.9
11.7
15.4
33.9
27.9
76.4
34.6
40.8
29.5
33.4
37.0
55.4
36.8
21.6
31.8
48.7
52.3
24.6
34.9
90.9
11.8
15.4
29.3
80.4
34.6
44.9
27.5
28.6
33.1
46.0
36.2
68.3
41.0
49.3
54.0
24.8
36.2
90.6
17.9
17.1
21.7 213.8 213.4 213.8
68.4 68.4 213.9 213.4 213.4
32.0
48.4
52.3
24.7
36.5
91.0
15.3
23.8
51.6 51.4
52.5 52.6
51.6 51.7
24.2 24.1
35.3 35.3
89.5 89.7
16.2 16.0
23.6 23.7
51.5
52.6
51.6
24.2
35.3
89.8
16.0
23.6
40.5 40.6
49.3 49.3
54.1 54.1
24.7 24.7
34.5 34.6
90.8 90.9
14.4 14.6
17.8 17.8
51.5
52.5
51.7
24.1
35.3
89.4
11.6
16.1
51.4
52.5
51.8
24.1
35.3
89.7
11.4
16.0
51.4
52.5
51.8
24.1
35.3
89.7
11.6
16.1
213.1 207.9 207.7 213.5 208.6 208.4 213.0 207.7 207.8 213.2 208.0 207.8 212.9 208.0 207.4 213.6 208.3 208.5
67.8 72.0 72.2 67.9 72.2 72.1 67.9 72.2 72.1 67.7 72.0 72.0 67.9 71.9 71.9 67.9 72.1 72.1
a) In ppm downfield from TMS.
3a,17a-dihydroxy-5a-11,20-dione was isolated in only poor and subsequent alkaline hydrolysis of the resulting 3a-
yield after tedious chromatographic separation. However, by acetoxy-17a-hydroxy-21-trityl-5a-11,20-dione (23a) with
changing the starting material to 3a-acetoxy-17a,21-dihy- NaOH to afford 3a,17a-dihydroxy-21-trityl-5a-11,20-dione
droxy-5a-11,20-dione (22b), the reactions proceeded cleanly, (23b). Sulfation at C-3 in 23b with SO3-TEA complex and
and the desired 23d was obtained in three steps. The reac- desilylation of the resulting 17a-hydroxy-3a-sulfooxy-21-
tions involved are tritylation of the primary hydroxy group at trityl ether (23c) with HCl yielded allo-THE-3-sulfate (23d).
C-21 in 22b with triphenylmethyl chloride (trityl chloride)
Figure 6 shows the synthetic route to allo-THS-3-sulfate

March 2010
349
methanol (1 ml) at room temperature for 1.5 h. After evaporation of the sol-
vent, the residue dissolved in water was loaded onto an Oasis® HLB car-
tridge and washed with water. Elution with methanol and recrystallization of
a homogeneous effluent from methanol–Et2O gave 4b as colorless amor-
phous substances: yield, 30 mg (52.3 %); mp 196 °C (dec.). 1H-NMR
(CD3OD) d: 0.82 (3H, s, 18-CH3), 1.17 (3H, s, 19-CH3), 4.25 (1H, m, 11a-
H), 4.24—4.38 (1H, br m, 3b-H), 4.29 and 4.66 (each 1H, d, Jꢀ19.0 Hz, 21-
CH2). HR-MS (ESIꢁ), Calcd for C21H33O8S [MꢁNaꢂHꢁH]ꢁ: 445.1896;
Found, m/z: 445.1926.
21-Acetoxy-3a-tert-butyldimethylsilyloxy-11b,17a-dihydroxy-5b-
pregnan-20-one (5a) To a solution of 3 (90 mg) in pyridine (0.2 ml)–N,Nꢃ-
dimethylformamide (DMF, 0.3 ml) was successively added imidazole
(45 mg) and TBDMSCl (100 mg), and the mixture was stirred at room tem-
perature for 4 h. The resulting slotuion was diluted with Et2O, washed with
water, dried with anhydrous Na2SO4, and evaporated to dryness. Recrystal-
lization of the product from acetone gave 5a as colorless leaflets: yield,
115 mg (ca. 100%); mp 200—204 °C (lit.,22) mp 197—199 °C). 1H-NMR
(CDCl3) d: 0.07 (6H, s, 3-OSi(CH3)2), 0.90 (12H, s, 18-CH3 and 3-OSi-tert-
Bu), 1.15 (3H, s, 19-CH3), 2.17 (3H, s, 21-OCOCH3), 3.57—3.67 (1H, br m,
3b-H), 4.31 (1H, m, 11a-H), 4.80 and 5.06 (each 1H, d, Jꢀ16.0 Hz, 21-
CH2).
3a-tert-Butyldimethylsilyloxy-11b,17a,21-trihydroxy-5b-pregnan-20-
one (5b) A mixture of 5a (200 mg) and sodium methoxide (50 mg) in
methanol (5 ml) was stirred at room temperature for 1 h. After evaporation
of the solvent, the residue was diluted with EtOAc, washed with water, dried
over anhydrous Na2SO4, and evaporated to dryness. Purification of the crude
product by column chromatography on silica gel with toluene–acetone (5 : 1,
v/v) as an eluent and recrystallization of a homogeneous effluent from
ether–hexane gave 5b as colorless plates: yield, 142 mg (77%); mp 179—
181 °C (lit.,22) mp 187—189 °C). 1H-NMR (CDCl3) d: 0.07 (6H, s, 3-
OSi(CH3)2), 0.88 (3H, s, 18-CH3), 0.90 (9H, s, 3-OSi-tert-Bu), 1.15 (3H, s,
19-CH3), 3.57—3.67 (1H, br m, 3b-H), 4.32 (1H, m, 11a-H), 4.29 and 4.66
(each 1H, d, Jꢀ19.4 Hz, 21-CH2).
(27c), allo-THS-21-sulfate (28d), and allo-THS-3,21-disul-
fate (27e), starting from 11-DOC-21-acetate (7b). Synthetic
procedures for these compounds are essentially identical to
those for the analogous THF sulfates (20c, 21d, 20e) men-
tioned above.
In conclusion, a series of the 18 variants of the 3- and 21-
monosulfates and 3,21-disulfates of THF, THS, and THE, as
well as their 5a-stereoisomers, are now available. These au-
thentic reference standards would be useful for the highly
sensitive, selective, and accurate LC-MS/MS determination
of the sulfate conjugates of tetrahydrocorticosteroids present
in human urine. A further 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.
Experimental
Materials Tetrahydrocortisone 21-acetate (THE-21-acetate: 13) was
obtained from Steraloids Inc. (Newport, RI, U.S.A.). Cortisol and 11-deoxy-
cortisol (11-DOC) were purchased from Wako Pure Chemical Industries,
Ltd. (Osaka, Japan). Silica gel plates (Merck; F254) and silica gel 60 (Merck;
70—230 mesh) were used for analytical and column chromatography,
respectively. A reversed-phase adsorbent, Cosmosil 140C18-OPN, was avail-
able from Nacalai Tesque Inc. (Kyoto, Japan). Oasis® HLB cartridges (ad-
sorbent weight, 1 g) and Sep-Pak® tC18 cartridges (adsorbent weight, 5 g)
were provided by Waters Co. (Milford, MS, U.S.A.) and successively condi-
tioned by washings with methanol and water prior to use. All other chemi-
cals and solvents were analytical grade and obtained from Nacalai Tesque
Inc.
Instruments All melting points (mp) were determined on a micro hot-
1
stage apparatus and are uncorrected. H-NMR spectra were recorded on a
3a,11b,17a-Trihydroxy-21-sulfooxy-5b-pregnan-20-one Sodium Salt
(THF-21-sulfate; 5d) The compound 5b (50 mg) was treated with SO3-
TEA complex (40 mg) at room temperature for 70 min, as described for the
preparation of 4b. Evaporation of the solvent under reduced pressure af-
forded the intermediary 21-sulfate (5c). Without isolation of 5c, it was sub-
jected to the desilylation at C-3 with 5% HCl (0.2 ml) in acetone (2 ml) at
room temperature for 3 h. After evaporation of acetone under reduced pres-
sure, the resulting solution was adjusted to pH 8 with 1 M NaOH and loaded
onto an Oasis® HLB cartridge. After being washed with water, the crude sul-
fated product was eluted with methanol. Recrystallization of the product
from methanol–Et2O gave 5d as colorless amorphous substances: yield,
10 mg (21%); mp 164 °C (dec.). 1H-NMR (CDCl3–CD3OD; 5 : 1, v/v) d:
0.82 (3H, s, 18-CH3), 1.15 (3H, s, 19-CH3), 3.57—3.67 (1H, br m, 3b-H),
4.28 (1H, m, 11a-H), 4.83 and 5.16 (each 1H, d, Jꢀ20.0 Hz, 21-CH2). HR-
MS (ESIꢁ), Calcd for C21H33O8S [MꢁNaꢂHꢁH]ꢁ: 445.1896; Found, m/z:
445.1876.
3a,11b,17a,21-Tetrahydroxy-5b-pregnan-20-one (THF; 6a) The
compound 3 (150 mg) was hydrolyzed with sodium methoxide (43 mg) as
described for the preparation of 5b. After being processed analogously, the
crude hydrolysis product was subjected to column chromatography on silica
gel with toluene–acetone (3 : 1, v/v) as an eluent. Recrystallization of a ho-
mogeneous effluent from acetone gave 6a as colorless needles: yield, 110 mg
(82%); mp 215—217 °C (lit.,29) mp 202—204 °C). 1H-NMR (CDCl3–
CD3OD; 10 : 1, v/v) d: 0.83 (3H, s, 18-CH3), 1.16 (3H, s, 19-CH3), 3.55—
3.66 (1H, br m, 3b-H), 4.28 (1H, m, 11a-H), 4.27 and 4.64 (each 1H, d, Jꢀ
20.0 Hz, 21-CH2).
JEOL JNM-EX270 (Tokyo, Japan) at 270 MHz or JNM-AL400 instrument
at 400 MHz, with CDCl3 or CD3OD containing 0.1% Me4Si as the solvent;
chemical shifts are expressed as d ppm relative to Me4Si. The following ab-
breviations are used: sꢀsinglet, dꢀdoublet, mꢀmultiplet, br mꢀbroad mul-
tiplet. 13C-NMR spectra were obtained on a JEOL JNM-EX 270 instrument
at 68.8 MHz. The 13C distortionless enhancement by polarization transfer
(DEPT; 135°, 90°, 45°) spectra were also measured to determine the 1H sig-
nal multiplicity and to differentiate between CH3, CH2, CH, and C based on
their proton environments. High-resolution mass spectra (HR-MS) were
recorded on Shimadzu LC-MS-IT-TOF with electrospray ionization source
under the negative ion mode (ESIꢁ).
Chemical Synthesis. 21-Acetoxy-11b,17a-dihydroxy-5b-pregnane-
3,20-dione (2) A solution of cortisol 21-acetate (1b, 1.0 g) (prepared from
cortisol by the usual acetic anhydride-pyridine method) in pyridine (14.5 ml)
was hydrogenated overnight at room temperature in the presence of 10%
Pd(OH)2/C catalyst (102 mg). After filtration of the catalyst on Celite, the fil-
trate was concentrated to dryness under reduced pressure. Recrystallization
of the product from ethanol afforded 2 as colorless needles: yield, 445 mg
(44 %); mp 220—223 °C (lit.,22) mp 210—212 °C). 1H-NMR (CDCl3) d:
0.88 (3H, s, 18-CH3), 1.27 (3H, s, 19-CH3), 2.19 (3H, s, 21-OCOCH3), 4.37
(1H, m, 11a-H), 4.88 and 5.06 (each 1H, d, Jꢀ17.6 Hz, 21-CH2).
21-Acetoxy-3a,11b,17a-trihydroxy-5b-pregnan-20-one (3) To a freshly
prepared solution of Zn(BH4)2 in Et2O (7 ml)27) was added slowly a solution
of 2 (700 mg) in dry tetrahydrofuran (8 ml) under N2 gas, and the mixture
was stirred at room temperature for 45 min. The resulting solution was di-
luted with EtOAc, washed with 5% HCl and water, dried over anhydrous
Na2SO4, and evaporated to dryness. Purification of the product by column
chromatography on silica gel with hexane–EtOAc (1 : 1, v/v) as an eluent
and recrystallization of a homogeneous effluent from hexane–EtOAc gave 3
as colorless prisms: yield, 553 mg (79%); mp 197—200 °C (lit.,22) mp 196—
11b,17a-Dihydroxy-3a,21-disulfooxy-5b-pregnan-20-one Disodium Salt
(THF-3,21-disulfate; 6b) The compound 6a (50 mg) was treated with
SO3-TEA complex (50 mg) at room temperature for 1 h, followed by 1 M
NaOH, as described for the preparation of 4b. After passing through an
Oasis® HLB cartridge and being washed with water, the desired sulfate was
eluted with methanol. Evaporation of the solvent and recrystallization of the
product from methanol afforded 6b as colorless amorphous substances:
1
198 °C). H-NMR (CDCl3) d: 0.89 (3H, s, 18-CH3), 1.17 (3H, s, 19-CH3),
2.18 (3H, s, 21-OCOCH3), 3.62—3.73 (1H, br m, 3b-H), 4.33 (1H, m, 11a-
H), 4.81 and 5.10 (each 1H, d, Jꢀ20.0 Hz, 21-CH2).
1
yield, 61 mg (78%); mp 139—141 °C. H-NMR (CD3OD) d: 0.74 (3H, s,
11b,17a,21-Trihydroxy-3a-sulfooxy-5b-pregnan-20-one Sodium Salt
(THF-3-sulfate; 4b) To a solution of 3 (50 mg) in dry pyridine (0.5 ml)
was added SO3-TEA complex (30 mg), and the mixture was stirred at room
temperature for 30 min. After evaporation of the solvent under reduced pres-
sure, the residue dissolved in a small amount of water was adjusted to pH 8
with 1 M NaOH and loaded onto an Oasis® HLB cartridge. After being
washed with water, elution with methanol gave the intermediary 21-acetoxy-
3a-sulfate (4a). This was hydrolyzed with sodium methoxide (36 mg) in
18-CH3), 1.07 (3H, s, 19-CH3), 4.16 (1H, m, 11a-H), 4.14—4.27 (1H, br m,
3b-H), 4.74 and 4.95 (each 1H, d, Jꢀ18.0 Hz, 21-CH2). HR-MS (ESIꢁ),
Calcd for C21H32O11S2 [Mꢁ2Naꢂ2Hꢁ2H]2ꢁ
: 262.0693; Found, m/z:
262.0676.
21-Acetoxy-3a,17a-dihydroxy-5b-pregnan-20-one (9) 11-Deoxycor-
tisol 21-acetate (7b, 500 mg) (prepared from 11-DOC (7a) by the usual
acetic anhydride-pyridine method) in pyridine (22 ml) was catalytically hy-

350
Vol. 58, No. 3
drogenated in the presence of 10% Pd(OH)2/C (50 mg) at room temperature eluted with methanol. This compound was recrystallized from methanol as
overnight. After filtration of the catalyst on Celite, the filtrate was concen- colorless amorphous substances: yield, 7.3 mg (22%); mp 184 °C (dec.). 1H-
trated to dryness under reduced pressure, yielding the intermediary 3-oxo-5b NMR (CDCl3–CD3OD; 1 : 1, v/v) d: 0.62 (3H, s, 18-CH3), 0.93 (3H, s, 19-
derivative (8). Without purification of 8, it was reduced with Zn(BH4)2/Et2O CH3), 4.40—4.50 (1H, br m, 3b-H), 4.80 and 5.15 (each 1H, d, Jꢀ18.2 Hz,
solution (1.25 ml) at room temperature for 3 h and processed as described
for the preparation of 3. Purification of the crude product by column chro-
matography on silica gel with toluene–acetone (3 : 1, v/v) as an eluent and
21-CH2). HR-MS (ESIꢁ), Calcd for C21H32O10S2 [Mꢁ2Naꢂ2Hꢁ2H]2ꢁ
254.0719; Found, m/z: 254.0708.
17a,21-Dihydroxy-3a-sulfooxy-5b-pregnane-11,20-dione Sodium Salt
:
recrystallization of a homogeneous effluent from EtOAc gave 9 as colorless (THE-3-sulfate; 14b) A mixture of THE-21-acetate (13, 50 mg) and SO3-
needles: yield, 196 mg (39%); mp 222—225 °C (lit., mp 229—232 °C23)
;
TEA complex (50 mg) in pyridine (1 ml) was stirred at room temperature
221—224 °C33)). 1H-NMR (CDCl3) d: 0.64 (3H, s, 18-CH3), 0.90 (3H, s, 19- overnight. The solvent was evaporated under reduced pressure, and the
CH3), 2.15 (3H, s, 21-OCOCH3), 3.56—3.68 (1H, br m, 3b-H), 4.79 and
5.09 (each 1H, d, Jꢀ17.7 Hz, 21-CH2).
residue was extracted with EtOAc. The combined extract was washed with
water, dried over anhydrous Na2SO4, and evaporated to give a first crop of
the intermediary 3-sulfate (14a). The aqueous layer was passed through Am-
berlite XAD-2 resin (10 g) and washed with water. Elution with methanol
gave a second crop of 14a: total yield, 35 mg (58.5%). Without further pu-
17a,21-Dihydroxy-3a-sulfooxy-5b-pregnan-20-one Sodium Salt (THS-
3-sulfate; 10b) The compound 9 (50 mg) was subjected to the sulfation
with SO3-TEA complex (30 mg) at 45 °C for 4 h, as described for the prepa-
ration of 4a. After evaporation of the solvent under reduced pressure, the rification, it was hydrolyzed with sodium methoxide (6 mg) in methanol
residue dissolved in water was loaded onto an Oasis® HLB cartridge. After
(6 ml) at room temperature for 2 h, as described for the preparation of 5b.
being washed with water, the desired sulfate (10a) was eluted with After evaporation of the solvent, the reaction product dissolved in water was
methanol. Subsequent hydrolysis of the condensed methanolic solution (ca. passed through a reversed-phase column on Cosmosil 140C18-OPN (10 g)
1 ml) was attained with sodium methoxide (10 mg) at room temperature for and washed with water. Elution with methanol afforded the crude product,
1 h. The solvent was evaporated and the residue dissolved in water was which in turn was chromatographed on silica gel with CHCl3–methanol
loaded onto an Oasis® HLB cartridge. After being washed with water, (3 : 1, v/v) as an eluent. Recrystallization of a homogeneous effluent from
elution with methanol afforded 10b which was recrystallized from methanol–Et2O gave 14b as colorless amorphous substances: yield, 26 mg
methanol–Et2O as colorless amorphous substances: yield, 56 mg (97%); mp
(45%); mp 177 °C (dec.). 1H-NMR (CDCl3–CD3OD; 5 : 1, v/v) d: 0.55 (3H,
196 °C (dec.). 1H-NMR (CDCl3–CD3ODꢀ10 : 1, v/v) d: 0.61 (3H, s, 18- s, 18-CH3) 1.14 (3H, s, 19-CH3), 4.21 and 4.65 (each 1H, d, Jꢀ20.0 Hz, 21-
CH3), 0.93 (3H, s, 19-CH3), 4.18—4.31 (1H, br m, 3b-H), 4.28 and 4.68 CH2), 4.34—4.44 (1H, br m, 3b-H). HR-MS (ESIꢁ), Calcd for C21H31O8S
(each 1H, d, Jꢀ19.4 Hz, 21-CH2). HR-MS (ESIꢁ), Calcd for C21H33O7S [MꢁNaꢂHꢁH]ꢁ: 443.1740; Found, m/z: 443.1730.
[MꢁNaꢂHꢁH]ꢁ: 429.1947; Found, m/z: 429.1926.
21-Acetoxy-3a-tert-butyldimethylsilyloxy-17a-hydroxy-5b-pregnane-
21-Acetoxy-3a-tert-butyldimethylsilyloxy-17a-hydroxy-5b-pregnan- 11,20-dione (15a) THE-21-acetate (13, 100 mg) was treated with TBDM-
20-one (11a) The compound 9 (100 mg) was treated with TBDMSCl SCl (66 mg) at room temperature for 4 h, as described for the preparation of
(90 mg) overnight at room temperature, as described for the preparation of 5a. After being processed analogously, the product was recrystallized from
5a. After being processed analogously, the product was recrystallized from methanol to give 15a as colorless leaflets: yield, 128 mg (ca. 100%); mp
1
ethanol to give 11a as colorless leaflets: yield, 129 mg (ca. 100%); mp 218—221 °C (lit.,22) mp 214—216 °C). H-NMR (CDCl3) d: 0.06 (6H, s, 3-
1
191—193 °C (lit.,23) mp 188—189 °C). H-NMR (CDCl3) d: 0.06 (6H, s, 3-
OSi(CH3)2), 0.60 (3H, s, 18-CH3), 0.89 (9H, s, 3-OSi-tert-Bu), 1.13 (3H, s,
OSi(CH3)2), 0.65 (3H, s, 18-CH3), 0.90 (12H, s, 19-CH3 and 3-OSi-tert-Bu), 19-CH3), 2.16 (3H, s, 21-OCOCH3), 3.54—3.63 (1H, br m, 3b-H), 4.62 and
2.17 (3H, s, 21-OCOCH3), 3.54—3.63 (1H, br m, 3b-H), 4.82 and 5.09 5.13 (each 1H, d, Jꢀ17.6 Hz, 21-CH2).
(each 1H, d, Jꢀ17.6 Hz, 21-CH2).
3a-tert-Butyldimethylsilyloxy-17a,21-dihydroxy-5b-pregnane-11,20-
3a-tert-Butyldimethylsilyloxy-17a,21-dihydroxy-5b-pregnan-20-one dione (15b) The compound 15a (30 mg) was hydrolyzed with sodium
(11b) The compound 11a (50 mg) in methanol (3 ml)–tetrahydrofuran methoxide (5 mg) at room temperature for 50 min, as described for the
(0.5 ml) was treated with sodium methoxide (8 mg) at room temperature for preparation of 5b. After being processed analogously, the crude product was
30 min, as described for the preparation of 5b. After being processed analo-
chromatographed on silica gel with hexane–EtOAc (2 : 1, v/v) as an eluent.
gously, the product was recrystallized from Et2O–hexane to give 11b as col- Recrystallization of a homogeneous effluent from acetone gave 15b as color-
orless needles: yield, 45 mg (ca. 100%); mp 184—186 °C (lit.,23) mp 187— less leaflets: yield, 22 mg (80%); mp 209—211 °C (lit.,22) mp 208—210 °C).
1
189 °C). H-NMR (CDCl3) d: 0.06 (6H, s, 3-OSi(CH3)2), 0.63 (3H, s, 18- 1H-NMR (CDCl3) d: 0.06 (6H, s, 3-OSi(CH3)2), 0.58 (3H, s, l8-CH3), 0.89
CH3), 0.89 (9H, s, 3-OSi-tert-Bu), 0.90 (3H, s, 19-CH3), 3.54—3.63 (1H, (9H, s, 3-OSi-tert-Bu), 1.13 (3H, s, 19-CH3), 3.52—3.63 (1H, br m, 3b-H),
br m, 3b-H), 4.29 and 4.66 (each 1H, d, Jꢀ20.0 Hz, 21-CH2).
4.24 and 4.64 (each 1H, d, Jꢀ20.0 Hz, 21-CH2).
3a,17a-Dihydroxy-21-sulfooxy-5b-pregnan-20-one Sodium Salt (THS-
21-sulfate; 11d) The compound 11b (30 mg) was treated with SO3-TEA
complex (50 mg) at 50 °C for 26 h, as described for the preparation of 5d.
3a,17a-Dihydroxy-21-sulfooxy-5b-pregnane-11,20-dione Sodium Salt
(THE-21-sulfate; 15d) The compound 15b (25 mg) was treated with SO3-
TEA complex (25 mg) at room temperature overnight, as described for the
After being processed analogously, the resulting 21-sulfate (11c) was sub- preparation of 4b. After evaporation of the solvent under reduced pressure,
jected to the desilylation at C-3 with 5% HCl (0.1 ml) in acetone (1 ml) at
room temperature for 15 min. The resulting solution was adjusted to pH 8
with 2.5 M NaOH and poured onto an Oasis® HLB cartridge. After being
the resulting 21-sulfate (15c) dissolved in acetone (5 ml)–methanol (0.5 ml)
was treated with 5% HCl (0.5 ml) at room temperature for 10 min. The mix-
ture was adjusted to pH 8 with 5% NaHCO3, poured onto a column of Cos-
washed with water, the product eluted with methanol was subjected to col- mosil (5 g), and washed with water. Elution with methanol gave the crude
umn chromatography on silica gel. Elution with CHCl3–methanol (6 : 1, v/v)
desilylated product, which in turn was chromatographed on silica gel with
CHCl3–methanol (2 : 1, v/v) as an eluent. Recrystallization of a homoge-
yielded 11d as colorless amorphous substances, 21 mg (72%); mp 180 °C
1
(dec.). H-NMR (CDCl3–CD3OD; 5 : 1, v/v) d: 0.53 (3H, s, 18-CH3), 0.84 neous effluent from methanol–Et2O gave 15d as colorless amorphous sub-
1
(3H, s, 19-CH3), 3.43—3.55 (1H, br m, 3b-H), 4.73 and 5.09 (each 1H, d,
stances: yield, 20 mg (82%); mp 187 °C (dec.). H-NMR (CDCl3–CD3OD;
Jꢀ18.2 Hz, 21-CH2). HR-MS (ESIꢁ), Calcd for C21H33O7S [MꢁNaꢂHꢁH]ꢁ: 5 : 1, v/v) d: 0.56 (3H, s, 18-CH3), 1.13 (3H, s, 19-CH3), 3.55—3.67 (1H,
429.1947; Found, m/z 429.1928.
br m, 3b-H), 4.70 and 5.15 (each 1H, d, Jꢀ18.5 Hz, 21-CH2). HR-MS
3a,17a,21-Trihydroxy-5b-pregnan-20-one (THS; 12a) The com- (ESIꢁ), Calcd for C21H31O8S [MꢁNaꢂHꢁH]ꢁ: 443.1740; Found, m/z:
pound 9 (60 mg) was hydrolyzed with sodium methoxide (21 mg) at room 443.1729.
temperature for 1.5 h, as described for the preparation of 5b. After being
processed analogously, the crude product was subjected to column chro- 21-acetate (13, 200 mg) was hydrolyzed with sodium methoxide (52 mg) and
matography on silica gel with toluene–acetone (3 : 1, v/v) as an eluent. Re- processed as described for the preparation of 5b. Recrystallization of the
3a,17a,21-Trihydroxy-5b-pregnane-11,20-dione (THE; 16a) THE-
crystallization of a homogeneous effluent from methanol gave 12a as color- product from acetone–hexane gave 16a as colorless prisms: yield, 165 mg
1
less needles: yield, 40 mg (75%); mp 197—199 °C (lit.,34) mp 222—230 °C). (92%); mp 190—191 °C (lit.,35) mp 195—196.5 °C). H-NMR d: 0.55 (3H,
1H-NMR (CDCl3) d: 0.62 (3H, s, 18-CH3), 0.92 (3H, s, 19-CH3), 3.54—3.66 s, 18-CH3), 1.14 (3H, s, 19-CH3), 3.40—3.52 (1H, br m, 3b-H), 4.19 and
(1H, br m, 3b-H), 4.26 and 4.66 (each 1H, d, Jꢀ19.7 Hz, 21-CH2).
4.62 (each 1H, d, Jꢀ19.7 Hz, 21-CH2).
17a-Hydroxy-3a,21-disulfooxy-5b-pregnane-11,20-dione
17a-Hydroxy-3a,21-disulfooxy-5b-pregnan-20-one Disodium Salt
Disodium
(THS-3,21-disulfate; 12b) The compound 12a (21 mg) was treated with Salt (THE-3,21-disulfate; 16b) THE (16a, 38 mg) was treated with SO3-
SO3-TEA complex (20 mg) overnight at 40 °C, followed by 1 M NaOH, as
TEA complex (30 mg) at 50 °C overnight, followed by 1 M NaOH, as de-
described for the preparation of 4b. After being loaded onto an Oasis® HLB scribed for the preparation of 4b. The reaction mixture was passed through a
cartridge and washed with water, the desired THS-3,21-disulfate (12b) was column of Cosmosil (10 g) and washed with water. Elution with methanol

March 2010
351
A
3a,11b,17a,21-Tetrahydroxy-5a-pregnan-20-one (Allo-THF; 20d)
gave the crude product, which was then chromatographed on silica gel with
CHCl3–methanol (2 : 1, v/v) as an eluent to give 16b as colorless amorphous
substances: yield, 43 mg (73%); mp 167 °C (dec.). H-NMR d: 0.58 (3H, s,
18-CH3), 1.15 (3H, s, 19-CH3), 3.97—4.09 (1H, br m, 3b-H), 4.93 and 5.32
(each 1H, d, Jꢀ18.4 Hz, 21-CH2). HR-MS (ESIꢁ), Calcd for C21H30O11S2
[Mꢁ2Naꢂ2Hꢁ2H]2ꢁ: 261.0615; Found, m/z: 261.0604.
mixture of the compound 20a (45 mg) and 5% HCl (2 ml) in acetone
(0.5 ml) was stirred at room temperature for 10 min. The desilylation product
was extracted with EtOAc, and the combined extract was washed with water,
dried over anhydrous Na2SO4. After evaporation of the solvent, the product
was recrystallized from methanol to give 20d as colorless needles: yield,
33 mg (96%); mp 252—258 °C (lit.,29) mp 248—256 °C). 1H-NMR
(CDCl3–CD3OD; 1 : 10, v/v) d: 0.83 (3H, s, 18-CH3), 1.03 (3H, s, 19-CH3),
3.95—4.00 (1H, m, 3b-H), 4.28 and 4.64 (each 1H, d, Jꢀ19.6 Hz, 21-CH2),
4.39 (1H, m, 11a-H).
11b,17a-Dihydroxy-3a,21-disulfooxy-5a-pregnan-20-one Disodium
Salt (Allo-THF-3,21-disulfate; 20e) The compound 20d (24 mg) was
treated with SO3-TEA complex (58 mg) at room temperature for 2 h, fol-
lowed by 1 M NaOH, as described for the preparation of 4b. After loading
onto an Oasis® HLB cartridge and being washed with water, the crude prod-
uct was eluted with methanol. Chromatography of the product on a column
of silica gel and elution with CHCl3–methanol (2 : 1, v/v) afforded 20e
as colorless amorphous substances which was recrystallized from
methanol–Et2O: yield, 38 mg (ca. 100%); mp 142—143 °C. 1H-NMR
(CDCl3–CD3OD; 2 : 3, v/v) d: 0.84 (3H, s, 18-CH3), 1.04 (3H, s, 19-CH3),
4.39 (1H, m, 11a-H), 4.62—4.67 (1H, m, 3b-H), 4.87 and 5.06 (each 1H,
d, Jꢀ18.0 Hz, 21-CH2). HR-MS (ESIꢁ), Calcd for C21H32O11S2
[Mꢁ2Naꢂ2Hꢁ2H]2ꢁ: 262.0693; Found, m/z: 262.0683.
1
21-Acetoxy-11b,17a-dihydroxy-5a-pregnane-3,20-dione (19a) To a
stirred solution of cortisol 21-acetate (1b, 2.0 g) in tetrahydrofuran (70 ml)–
ethanol (5 ml) was added triethyl orthoformate (7 ml) and p-toluenesulfonic
acid (75 mg), and the mixture was stirred at room temperature for 2 h. After
addition of pyridine (2 ml), the mixture was diluted with EtOAc. The organic
layer was washed with 5% NaHCO3 and saturated brine, dried over anhy-
drous Na2SO4, and evaporated to dryness. Purification of the crude product
by column chromatography on silica gel with hexane–EtOAc (7 : 3, v/v) as
an eluent gave the intermediary 3-ethoxy-3,5-diene (17). This was dissolved
in EtOAc–ethanol (12 ml; 1 : 1, v/v) and the solution was catalytically hydro-
genated on 10% Pd/C (30 mg) overnight at room temperature. After addition
of EtOAc (50 ml) followed by removal of the catalyst by filtration on Celite,
the filtrate was acidified with 7.3% HCl. The organic layer was washed with
5% NaHCO3 and saturated brine, dried over anhydrous Na2SO4, and evapo-
rated to dryness. Purification of the product by column chromatography on
silica gel with hexane–EtOAc (3 : 2, v/v) as an eluent and recrystallization of
a homogeneous effluent from acetone gave 19a as colorless prisms: yield,
497 mg (25%); mp 195—197 °C (lit.,29) mp 220—224 °C). 1H-NMR
(CDCl3) d: 0.94 (3H, s, 18-CH3), 1.26 (3H, s, 19-CH3), 2.18 (3H, s, 21-
OCOCH3), 4.45 (1H, m, 11a-H), 4.83 and 5.05 (each 1H, d, Jꢀ17.6 Hz, 21-
CH2).
11b,17a,21-Trihydroxy-5a-pregnane-3,20-dione (19b) The com-
pound 19a (205 mg) was hydrolyzed with sodium methoxide (35 mg) as de-
scribed for the preparation of 5b. Recrystallization of the crude product
from acetone gave 19b as colorless amorphous substances: yield, 164 mg
(89%); mp 229—231 °C (lit.,25) mp 231—235 °C). 1H-NMR (CDCl3) d:
0.88 (3H, s, 18-CH3), 1.26 (3H, s, 19-CH3), 4.27 and 4.63 (each 1H, d,
Jꢀ19.6 Hz, 2I-CH2), 4.41 (1H, m, 11a-H).
21-tert-Butyldimethylsilyloxy-11b,17a-dihydroxy-5a-pregnane-3,20-
dione (19c) The compound 19b (450 mg) was treated with TBDMSCl
(324 mg) at room temperature for 5 h and processed as described for the
preparation of 5a. Recrystallization of the product from acetone gave 19c as
colorless needles: yield, 597 mg (ca. 100%); mp 198—200 °C. 1H-NMR
(CDCl3) d: 0.12 (6H, m, 21-OSi(CH3)2), 0.93 (12H, s, 18-CH3 and 21-OSi-
tert-Bu), 1.26 (3H, s, 19-CH3), 4.42 and 4.55 (each 1H, d, Jꢀ18.0 Hz, 21-
CH2), 4.44 (1H, m, 11a-H).
3a-Acetoxy-21-tert-butyldimethylsilyloxy-11b,17a-dihydroxy-5a-
pregnan-20-one (21a) A solution of 20a (404 mg) in pyridine–acetic an-
hydride (3 ml; 2 : 1, v/v) was stirred overnight at room temperature. After ex-
traction of the acetylation product with EtOAc, the combined extract was
washed with 5% NaHCO3 and water, dried over anhydrous Na2SO4, and
evaporated to give 21a which was recrystallized from Et2O–hexane as color-
less amorphous substances: yield, 418 mg (91%); mp, 163—164 °C. 1H-
NMR (CDCl3) d: 0.12 (6H, m, 21-OSi(CH3)2), 0.91 (3H, s, 18-CH3), 0.94
(9H, s, 21-OSi-tert-Bu), 1.04 (3H, s, 19-CH3), 2.06 (3H, s, 3-OCOCH3),
4.42 and 4.54 (each 1H, d, Jꢀ17.6 Hz, 21-CH2), 4.44 (1H, m, 11a-H),
4.98—5.04 (1H, m, 3b-H).
3a-Acetoxy-11b,17a,21-trihydroxy-5a-pregnan-20-one
(21b) The
compond 21a (90 mg), treated with 5% HCl (0.5 ml) at room temperature for
10 min and processed as described for the preparation of 20d, yielded the
desilylation product. Recrystallization from EtOAc gave 21b as colorless
needles: yield, 64 mg (91%); mp 227—229 °C (lit.,25) mp 228—230 °C). 1H-
NMR (CDCl3–CD3OD; 20 : 1, v/v) d: 0.86 (3H, s, 18-CH3), 1.03 (3H, s, 19-
CH3), 2.06 (3H, s, 3-OCOCH3), 4.27 and 4.63 (each 1H, d, Jꢀ19.6 Hz, 21-
CH2), 4.42 (1H, m, 11a-H), 4.98—5.04 (1H, m, 3b-H).
3a,11b,17a-Trihydroxy-21-sulfooxy-5a-pregnan-20-one Sodium Salt
(Allo-THF-21-sulfate; 21d) The compound 21b (50 mg) was treated with
SO3-TEA complex (60 mg) at room temperature for 3 h, followed by 1 M
NaOH, as described for the preparation of 4b. After being processed analo-
gously, the resulting 21-sulfate (21c) dissolved in methanol (1 ml) was hy-
drolyzed with 5 M NaOH (0.3 ml) at room temperature for 20 min. The sol-
vent was evaporated under reduced pressure and the residue dissolved in
water was applied onto an Oasis® HLB cartridge. After being washed with
water, the crude product was eluted with methanol. Chromatography of the
product on a column of silica gel and elution with CHCl3–methanol (4 : 1,
v/v) afforded 21d as colorless amorphous substances which was recrystal-
lized from methanol–Et2O: yield, 19 mg (33%); mp 176 °C. 1H-NMR
(CDCl3–CD3OD; 1 : 10, v/v) d: 0.84 (3H, s, 18-CH3), 1.03 (3H, s, 19-CH3),
3.94—4.00 (1H, m, 3b-H), 4.39 (1H, m, 11a-H), 4.80 and 5.05 (each 1H, d,
Jꢀ18.0 Hz, 21-CH2). HR-MS (ESIꢁ), Calcd for C21H33O8S [MꢁNaꢂHꢁ
H]ꢁ: 445.1896; Found, m/z: 445.1881.
21-tert-Butyldimethylsilyloxy-3a,11b,17a-trihydroxy-5a-pregnan-20-
one (20a) To a stirred solution of 19c (497 mg) in tetrahydrofuran (3.5 ml)
at ꢁ78 °C was added slowly a 1 M solution of K-Selectride® in tetrahydrofu-
ran (1.5 ml), and the mixture was further stirred at ꢁ78 °C for 1.2 h. After
gradual addition of 30% H2O2 (1.5 ml), the resulting solution was extracted
with EtOAc. The combined extract was washed with saturated brine, dried
over anhydrous Na2SO4, and evaporated. Purification of the product by col-
umn chromatography on silica gel with hexane–EtOAc (2 : 1, v/v) as an elu-
ent and recrystallization of a homogeneous effluent from acetone gave 20a
as colorless prisms: yield, 450 mg (90%). mp 173—176 °C. 1H-NMR
(CDCl3) d: 0.12 (6H, m, 21-OSi(CH3)2), 0.90 (3H, s, 18-CH3), 0.93 (9H, s,
21-OSi-tert-Bu), 1.03 (3H, s, 19-CH3), 4.03—4.08 (1H, m, 3b-H), 4.41 and
4.54 (each 1H, d, Jꢀ17.8 Hz, 21-CH2), 4.46 (1H, m, 11a-H).
11b,17a,21-Trihydroxy-3a-sulfooxy-5a-pregnan-20-one Sodium Salt
(Allo-THF-3-sulfate; 20c) The compound 20a (60 mg), treated with SO3-
TEA complex (30 mg) at room temperature for 2 h and processed as de-
scribed for the preparation of 5d, yielded the crude 3a-sulfate (20b). With-
out isolation of 20b, it was subjected to the desilylation at C-21 with 5%
HCl (0.2 ml) at room temperature for 1.5 h. The resulting solution was ad-
justed to pH 8 with 2.5 M NaOH and extracted with EtOAc. The organic
layer was dried over anhydrous Na2SO4 and evaporated to give a first crop of
the reaction product. Meanwhile, the aqueous layer was passed through an
Oasis® HLB cartridge, washed with water, and elution with methanol af-
forded a second crop. After evaporation of the solvent of the combined ex-
tract, the residue was subjected to column chromatography on silica gel.
Elution with CHCl3–methanol (3 : 1, v/v) afforded a homogeneous fraction.
After evaporation of the solvent, the residue was recrystallized from
methanol–Et2O to give 20c as colorless amorphous substances: yield, 55 mg
(94%); mp 143 °C (dec.). 1H-NMR (CDCl3–CD3OD; 1 : 5, v/v) d: 0.83 (3H,
s, 18-CH3), 1.04 (3H, s, 19-CH3), 4.28 and 4.65 (each 1H, d, Jꢀ18.8 Hz, 21-
CH2), 4.38 (1H, m, 11a-H), 4.59—4.64 (1H, m, 3b-H). HR-MS (ESIꢁ),
Calcd for C21H33O8S [MꢁNaꢂHꢁH]ꢁ: 445.1896; Found, m/z: 445.1882.
3a-Acetoxy-21-tert-butyldimethylsilyloxy-17a-hydroxy-5a-pregnane-
11,20-dione (22a) To a solution of 21a (372 mg) in CH2Cl2 (4 ml) was
added Celite (500 mg), sodium acetate (19 mg) and PCC (230 mg), and the
mixture was stirred at room temperature for 1.5 h. The mixture was diluted
with Et2O and filtered on silica gel. After evaporation of the solvent in the
filtrate, the residue was subjected to column chromatography on silica gel
with hexane–EtOAc (5 : 1, v/v) as an eluent. Recrystallization of a homoge-
neous effluent from acetone–hexane gave 22a as colorless amorphous sub-
1
stances: yield, 283 mg (76%); mp 185—188 °C. H-NMR (CDCl3) d: 0.12
and 0.13 (each 3H, s, 21-OSi(CH3)2), 0.61 (3H, s, 18-CH3), 0.93 (9H, s, 21-
OSi-tert-Bu), 1.01 (3H, s, 19-CH3), 2.05 (3H, s, 3-OCOCH3), 4.38 and 4.45
(each 1H, d, Jꢀ17.6 Hz, 21-CH2), 4.98—5.03 (1H, m, 3b-H).
3a-Acetoxy-17a,21-dihydroxy-5a-pregnane-11,20-dione (22b) The
compound 22a (60 mg), treated with 5% HCl (0.5 ml) and processed as
described for the preparation of 20d, yielded the crude desilylation prod-
uct. Recrystallization from acetone–hexane gave 22b as colorless needles:

352
Vol. 58, No. 3
yield, 45 mg (96%); mp 187—190 °C (lit.,25) mp 187—190 °C). 1H-NMR
(CDCl3) d: 0.61 (3H, s, 18-CH3), 1.01 (3H, s, 19-CH3), 2.04 (3H, s, 3-
OCOCH3), 4.25 and 4.64 (each 1H, d, Jꢀ20.0 Hz, 21-CH2), 4.98—5.03 (1H,
m, 3b-H).
dried over anhydrous Na2SO4, and evaporated to dryness. The residue was
chromatographed on a column of silica gel. Elution with EtOAc–hexane
(1 : 2, v/v) afforded the intermediary 3-ethoxy-3,5-diene (24). This com-
pound 24 dissolved in EtOAc (100 ml) and ethanol (100 ml) was catalyti-
cally hydrogenated over 10% Pd/C (98 mg) at room temperature for 1 h.
After filtration of the catalyst on Celite, the filtrate was acidified with 12%
HCl. The precipitate was filtered off to give a first fraction. The mother
liquor was extracted with EtOAc, and the combined extract was washed with
5% NaHCO3 and saturated brine, dried over anhydrous Na2SO4, and evapo-
rated to give a second fraction. Recrystallization of the combined fraction
from EtOAc gave the title compound 26a as colorless amorphous sub-
stances: yield, 3.11 g (57%); mp 246—248 °C (lit.,24) mp 251—252 °C). 1H-
NMR (CDCl3) d: 0.70 (3H, s, 18-CH3), 1.02 (3H, s, 19-CH3), 2.17 (2H, s,
21-OCOCH3), 4.83 and 5.09 (each 1H, d, Jꢀ17.6 Hz, 21-CH2).
3a,17a-Dihydroxy-21-sulfooxy-5a-pregnane-11,20-dione Sodium Salt
(Allo-THE-21-sulfate; 22d) The compound 22b (35 mg), subjected to the
sulfation (at room temperature for 1.5 h) with SO3-TEA complex (60 mg),
followed by 1 M NaOH, and processed as described for the preparation of 4b,
gave the intermediary 3a-acetoxy-21-sulfate (22c). Without isolation of 22c,
it was hydrolyzed with 2.5 M NaOH (0.8 ml) in methanol (2 ml) at room tem-
perature for 1 h and then the solution was adjusted to pH 8 with 5% HCl.
After being loaded onto an Oasis® HLB cartridge and washed with water,
the crude hydrolysis product was eluted with methanol. Chromatography of
the product on a column of silica gel and elution with CHCl3–methanol
(3 : 1, v/v) afforded the desired allo-THE-21-sulfate (22d) which recrystal-
lized from methanol–Et2O as colorless powders: yield, 47 mg (92%); mp
169 °C (dec.). 1H-NMR (CDCl3–CD3OD; 2 : 1, v/v) d: 0.57 (3H, s, 18-CH3),
0.99 (3H, s, 19-CH3), 3.95—4.01 (1H, m, 3b-H), 4.70 and 5.12 (each 1H, d,
Jꢀ18.6 Hz, 21-CH2). HR-MS (ESIꢁ), Calcd for C21H31O7S [MꢁNaꢂHꢁ
H]ꢁ: 443.1740; Found, m/z: 443.1725.
17a-Hydroxy-3a,21-disulfooxy-5a-pregnane-11,20-dione Disodium
Salt (Allo-THE-3,21-disulfate; 22e) Allo-THE-21-sulfate (22d, 25 mg),
subjected to the sulfation (at room temperature for 20 h) with SO3-TEA
complex (80 mg), followed by 1 M NaOH, and processed as described for the
preparation of 4b, gave the crude product. Chromatography of the product
on a column of silica gel and elution with CHCl3–methanol (2 : 1,
v/v) afforded allo-THE-3,21-disulfate (22e) which recrystallized from ace-
tone–methanol–Et2O as colorless amorphous substances: yield, 30 mg
(99%); mp 145 °C (dec.). 1H-NMR (CDCl3–CD3OD; 1 : 1, v/v) d: 0.57 (3H,
s, 18-CH3), 1.01 (3H, s, 19-CH3), 4.58—4.63 (1H, m, 3b-H), 4.73 and 5.09
(each 1H, d, Jꢀ18.4 Hz, 21-CH2). HR-MS (ESIꢁ), Calcd for C21H30O11S2
[Mꢁ2Naꢂ2Hꢁ2H]2ꢁ: 261.0615; Found, m/z: 261.0600.
3a-Acetoxy-17a-hydroxy-21-trityloxy-5a-pregnane-11,20-dione (23a)
A mixture of 22b (113 mg) and trityl chloride (254 mg) in pyridine (0.5 ml)
was stirred at 60 °C for 3 h. After extraction of the reaction product with
Et2O, the combined extract was washed with water, dried over anhydrous
Na2SO4, and evaporated. The oily residue was subjected to column chro-
matography on silica gel. Elution with hexane–EtOAc (2 : 1, v/v) gave a ho-
mogenous yellow oil which was characterized as the 21-trityl ether (23a):
yield, 149 mg (83%). 1H-NMR (CDCl3) d: 0.46 (3H, s, 18-CH3), 0.98 (3H, s,
19-CH3), 2.03 (3H, m, 3-OCOCH3), 3.92 and 4.13 (each 1H, d, Jꢀ17.6 Hz, 21-
CH2), 4.96—5.01 (1H, m, 3b-H), 7.26—7.46 (15H, m, Ar-H).
3a,17a-Dihydroxy-21-trityloxy-5a-pregnane-11,20-dione (23b) To a
solution of 23a (30 mg) in methanol–tetrahydrofuran (0.6 ml; 1 : 1, v/v) was
added 0.75 M NaOH (0.3 ml), and the mixture was stirred at room tempera-
ture for 6 h. The resulting solution was neutralized with 5% HCl and ex-
tracted with EtOAc. The combined extract was washed with water, dried
over anhydrous Na2SO4, and evaporated. Chromatography of the residue on
a column of silica gel and elution with hexane–EtOAc (1 : 1, v/v) afforded
23b as colorless amorphous substances: yield, 2.4 mg (43%); mp 145 °C.
1H-NMR (CDCl3) d: 0.47 (3H, s, 18-CH3), 0.97 (3H, s, 19-CH3), 3.92 and
4.13 (each 1H, d, Jꢀ17.6 Hz, 21-CH2), 4.00—4.05 (1H, m, 3b-H), 7.25—
7.46 (15H, m, Ar-H).
21-tert-Butyldimethylsilyloxy-17a-hydroxy-5a-pregnane-3,20-dione
(26c) A solution of the compound 26a (501 mg) in 1,4-dioxane (60 ml)
was hydrolyzed with 1% methanolic NaOH (3 ml) at room temperature for
1 h. The hydrolysis product was extracted with EtOAc, and the combined ex-
tract was washed with saturated brine, dried over anhydrous Na2SO4, and
evaporated. The residue was recrystallized from methanol to give the
17a,21-dihydroxy-3,20-dioxo intermediate (26b), which was treated with
TBDMSCl (586 mg) at room temperature for 1.5 h and processed as de-
scribed for the preparation of 5a, yielded the silylation product. Recrystal-
lization from methanol afforded the title compound 26c as colorless leaflets:
1
yield, 501 mg (91%); mp 190—192 °C (lit.,24) mp 191—192 °C). H-NMR
(CDCl3) d: 0.12 (6H, s, 21-OSi(CH3)2), 0.69 (3H, s, 18-CH3), 0.93 (9H, s,
21-OSi-tert-Bu). 1.03 (3H, s, 19-CH3), 4.42 and 4.55 (each 1H, d, Jꢀ
18.0 Hz, 21-CH2).
21-tert-Butyldimethylsilyloxy-3a,17a-dihydroxy-5a-pregnan-20-one
(27a) The compound 26c (320 mg) was reduced with 1 M K-Selectride® in
tetrahydrofuran (1.3 ml) at ꢁ80 °C for 30 min and processed as described for
the preparation of 20a. The crude product was recrystallized from methanol
to give 27a as colorless leaflets: yield, 285 mg (89%); mp 171—172 °C
1
(lit.,24) mp 175—177 °C). H-NMR (CDCl3) d: 0.11 (6H, s, 21-OSi(CH3)2),
0.66 (3H, s, 18-CH3), 0.78 (3H, s, 19-CH3), 0.93 (9H, s, 21-OSi-tert-Bu),
4.02—4.07 (1H, m, 3b-H).
17a,21-Dihydroxy-3a-sulfooxy-5a-pregnan-20-one Sodium Salt (Allo-
THS-3-sulfate; 27c) The compound 27a (182 mg) was treated with SO3-
TEA (253 mg) at room temperature for 3.5 h, as described for the prepara-
tion of 4b. After removal of pyridine by decantation of the mixture diluted
with petroleum ether, the precipitated solids (21-TBDMSO-17a-hydroxy-
3a-sulfooxy-5a-20-one intermediate, 27b) were filtered off. To the crude
27b dissolved in a small amount of methanol was added 10% HCl (0.5 ml),
and the mixture was left standing at room temperature for 30 min. The re-
sulting solution was loaded onto a Sep-Pack® tC18 cartridge. Elution with
methanol–water (3 : 7, v/v) gave a homogenous fraction which was then ad-
justed to pH 8 with 10% methanolic NaOH. The resulting solution was
passed through a Sep-Pak® tC18 cartridge and washed with 5% aqueous
methanol. Elution with 25% methanol gave 27c which recrystallized from
methanol–EtOAc as colorless amorphous substances: yield, 33 mg (28%);
mp 141—144 °C. 1H-NMR (CD3OD) d: 0.61 (3H, s, 18-CH3), 0.82 (3H,
s, 19-CH3), 4.28 and 4.62 (each 1H, d, Jꢀ18.9 Hz, 21-CH2), 4.58—4.62
(1H, m, 3b-H). HR-MS (ESIꢁ), Calcd for C21H33O7S [MꢁNaꢂHꢁH]ꢁ:
429.1947; Found, m/z: 429.1932.
17a,21-Dihydroxy-3a-sulfooxy-5a-pregnane-11,20-dione Sodium Salt
(Allo-THE-3-sulfate; 23d) The compound 23b (8 mg) was subjected to
the sulfation (at room temperature for 4 h) with SO3-TEA complex (18 mg)
as described for the preparation of 4b. After evaporation of the solvent under
reduced pressure, the crude product, 3a-sulfooxy-21-trityl ether 23c, was
treated with ethanol (0.9 ml) containing conc. HCl (0.05 ml) at room temper-
ature for 2 h, and then the solution was adjusted to pH 8 with 0.75 M NaOH.
After evaporation of the solvent, the residue was diluted with EtOAc. The
aqueous layer was loaded onto an Oasis® HLB cartridge and washed with
water. The desired allo-THE-3-sulfate (23d) was eluted with methanol and
recrystallized from methanol–Et2O as colorless amorphous substances:
yield, 2.4 mg (39%); mp 154 °C. 1H-NMR (CDCl3–CD3OD; 1 : 1, v/v) d:
0.56 (3H, s, 18-CH3), 1.01 (3H, s, 19-CH3), 4.22 and 4.60 (each 1H, d, Jꢀ
19.6 Hz, 21-CH2), 4.61—4.66 (1H, m, 3b-H). HR-MS (ESIꢁ), Calcd for
C21H31O8S [MꢁNaꢂHꢁH]ꢁ: 443.1740; Found, m/z: 443.1723.
3a,17a,21-Trihydroxy-5a-pregnan-20-one (Allo-THS; 27d) The
compound 27a (285 mg), treated with 10% HCl (50 ml) at room temperature
for 30 min and processed as described for the preparation of 20d, yielded the
desilylation product. The crude residue was dissolved in water was loaded
onto a Sep-Pak® tC18 cartridge and elution with 80% methanol afforded 27d
which recrystallized from methanol as colorless amorphous substances:
yield, 215 mg (ca. 100%); mp 188—189 °C (lit., mp 225—226 °C24); mp
203—213 °C36)). 1H-NMR (CDCl3) d: 0.66 (3H, s, 18-CH3), 0.84 (3H, s, 19-
CH3), 4.03—4.07 (1H, m, 3b-H), 4.28 and 4.66 (each 1H, d, Jꢀ18.9 Hz, 21-
CH2).
17a-Hydroxy-3a,21-disulfooxy-5a-pregnan-20-one Disodium Salt
(Allo-THS-3,21-disulfate; 27e) The compound 27d (35 mg) was treated
with SO3-TEA (94 mg) at room temperature for 4 h, followed by 1%
methanolic NaOH, as described for the preparation of 27c. After being
loaded onto a Sep-pak® tC18 cartridge and washed with 5% aqueous
methanol, the desired allo-THS-3,21-disulfate (27e) was eluted with 20%
methanol. This compound was recrystallized from methanol as colorless
21-Acetoxy-17a-hydroxy-5a-pregnane-3,20-dione (26a) To a solution
of the 11-DOC-21-acetate (7b, 5.47 g) in 1,4-dioxane (110 ml) was added
triethyl orthoformate (6 ml) and conc. H2SO4 (23 ml) in ethanol (4 ml), and
the mixture was stirred at room temperature for 1 h. Pyridine (5 ml) was
added to the mixture and the reaction product was extracted with EtOAc.
The combined extract was washed with 5% NaHCO3 and saturated brine,
1
leaflets: yield, 25 mg (45%); mp 148—151 °C. H-NMR (CD3OD) d: 0.62
(3H, s, 18-CH3), 0.83 (3H, s, 19-CH3), 4.57—4.61 (1H, m, 3b-H), 4.73 and
4.94 (each 1H, d, Jꢀ18.9 Hz, 21-CH2). HR-MS (ESIꢁ), Calcd for

March 2010
353
C21H32O10S2 [Mꢁ2Naꢂ2Hꢁ2H]2ꢁ: 254.0719; Found, m/z: 254.0710.
3a-Acetoxy-21-tert-butyldimethylsilyloxy-17a-hydroxy-5a-pregnan-
20-one (28a) The compound 27a (235 mg), treated with acetic anhy-
dride–pyridine at room temperature overnight and processed as described for
the preparation of 21a, yielded the crude acetylation product. Recrystalliza-
tion from methanol–water gave the title compound 28a as colorless needles:
Saunders Elsevia, Philadelphia, PA, 2008, pp. 445—503.
8) Bongiovanni A. M., Cohn R. M., “Chemical and Biological Aspects
on Steroid Conjugation,” ed. by Bernstein S., Solomon S., Springer-
Verlag, New York, 1970, pp. 409—453.
9) White P. C., “Principles and Practice of Endocrinology and Metabo-
lism,” 3rd ed., ed. by Becker, K. L., Lippincott Williams & Wilkins,
Philadelphia, 2001, pp. 704—714.
1
yield, 239 mg (93%); mp 172—175 °C (lit.,24) mp 179—180 °C). H-NMR
10) Kornel L., Miyabo S., Takeda R., Steroidologia, 2, 197—236 (1971).
11) Kornel L., Saito Z., J. Steroid Biochem., 6, 1267—1284 (1975).
12) Kornel L., Miyabo S., Steroids, 25, 697—706 (1975).
13) Aranoff G., Rösler A., Acta Endocrinol., 94, 371—375 (1980).
14) Ulick S., Tedde R., Wang J. Z., J. Clin. Endocrinol. Metab., 74, 593—
599 (1992).
15) Shackleton C. H., J. Steroid Biochem. Mol. Biol., 45, 127—140
(1993).
16) Kasuya Y., Shibasaki H., Furuta T., Steroids, 65, 89—97 (2000).
17) Shibasaki H., Tanabe C., Furuta T., Kasuya Y., Steroids, 66, 795—801
(2001).
18) Rivero-Marabé J. J., Maynar-Mariño J. I., García-de-Tiedra, M. P.,
Galán-Martín A. M., Caballero-Loscos M. J., Maynar-Mariño M., J.
Chromatogr. B, 761, 77—84 (2001).
19) Raffaelli A., Saba A., Vignali E., Marcocci C., Salvadori P., J. Chro-
matogr. B, 830, 278—285 (2006).
20) Antignac J. P., Le Bizec B., Monteau F., André F., Steroids, 67, 873—
882 (2002).
21) Ikegawa S., Hasegawa M., Okihara R., Shimidzu C., Chiba H., Iida T.,
Mitamura K., Anal. Chem., 81, 10124—10134 (2009).
22) Hosoda H., Saito K., Ito Y., Yokohama H., Ishii K., Nambara T.,
Chem. Pharm. Bull., 30, 2110—2118 (1982).
(CDCl3) d: 0.11 (6H, s, 21-OSi(CH3)2), 0.66 (3H, s, 18-CH3), 0.79 (3H, s,
19-CH3), 0.93 (9H, s, 21-OSi-tert-Bu), 2.05 (3H, s, 3-OCOCH3), 4.38 and
4.43 (each 1H, d, Jꢀ18.0 Hz, 21-CH2), 4.92—4.97 (1H, m, 3b-H).
3a,17a-Dihydroxy-21-sulfooxy-5a-pregnan-20-one Sodium Salt (Allo-
THS-21-sulfate, 28d) The compound 28a (53 mg), treated with 10% HCl
(8 ml) at room temperature for 1 h and processed as described for the prepa-
ration of 20d, yielded the desilylation product (28b). Without purification, it
was treated with SO3-TEA (30 mg) at room temperature overnight, as de-
scribed for the preparation of 4b, afforded the intermediary 21-sulfate (28c),
which was then hydrolyzed with 10% methanolic NaOH (1 ml) overnight at
50 °C. The resulting solution was diluted with water and adjusted to pH 8
with 10% HCl. After being loaded onto a Sep-pak® tC18 cartridge and
washed with 5% aqueous methanol, the desired allo-THS-21-sulfate (28d)
was eluted with 40% methanol. This compound was recrystallized from
methanol as colorless amorphous substances: yield, 17 mg (51%); mp 201—
1
203 °C. H-NMR (CD3OD) d: 0.62 (3H, s, 18-CH3), 0.81 (3H, s, 19-CH3),
3.93—3.98 (1H, m, 3b-H), 4.82 and 5.04 (each 1H, d, Jꢀ18.9 Hz, 21-CH2).
HR-MS (ESIꢁ), Calcd for C21H33O7S [MꢁNaꢂHꢁH]ꢁ: 429.1947; Found,
m/z: 429.1933.
The 13C-NMR chemical shifts of the eighteen 3- and 21-monosulfates and
their double-conjugates of 3,21-disulfates in the 5a- and 5b-series, assigned
on the basis of the parent non-sulfated corticoid analogs,37) are compiled in
Table 1.
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Acknowledgments This work was supported by a Grant-in Aid for Sci-
entific Research (C) (to K. M., Grant 20590164) for 2008—2010, (to S. I.,
Grant 21590184) for 2009—2011, and (to T. I., Grant 21550091) for 2009—
2011 from the Japan Society for the Promotion of Science, and Culture of
Japan, and High-Tech Research Center Project for Private Universities:
matching fund subsidy from the Ministry of Education, Culture, Sports, Sci-
ence and Technology (to S. I.) for 2007—2011.
References and Notes
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