A novel synthetic approach to (20R)- and (20S)-21-hydroxy steroids. Synthesis of a marine sterol 21-hydroxycholesterol

Article abstract of DOI:10.1016/j.steroids.2011.01.010

A short and efficient synthesis of steroid synthons, di(tert- butyldimethylsilyl) ethers of 3,21-dihydroxy-24-nor-chol-5-en-23-al (8 and 10) and of ethyl 3,21-dihydroxy-25-homo-chola-5,23-dien-25-oate (9 and 11), having natural (20R) and unnatural (20S) c

Full text of DOI:10.1016/j.steroids.2011.01.010

Steroids 76 (2011) 517–523
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
A novel synthetic approach to (20R)- and (20S)-21-hydroxy steroids.
Synthesis of a marine sterol 21-hydroxycholesterol
Hanna Koenig, Iwona Skiera, Krzysztof Błaszczyk, Zdzisław Paryzek∗
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan´, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A short and efficient synthesis of steroid synthons, di(tert-butyldimethylsilyl) ethers of 3,21-dihydroxy-
24-nor-chol-5-en-23-al (8 and 10) and of ethyl 3,21-dihydroxy-25-homo-chola-5,23-dien-25-oate (9
and 11), having natural (20R) and unnatural (20S) configuration from 3␤-(tert-butyldimethylsilyloxy)-
14␣,20␰-card-5-enolide (2) is reported. Further elongation of the side chain of these synthons provides
a new method for the synthesis of (20R) and (20S)-21-hydroxy steroids. The utility of the method was
exemplified by the synthesis of a natural marine sterol – 21-hydroxycholesterol (18).
© 2011 Elsevier Inc. All rights reserved.
Received 23 December 2010
Received in revised form 25 January 2011
Accepted 27 January 2011
Available online 3 February 2011
Keywords:
Natural product
Synthesis
Marine sterol
Side chain
1. Introduction
also been isolated [15]. Sulfated sterols isolated from marine organ-
isms (sponges, ophiuroids and starfish) exhibit diverse biological
Marine organisms are a rich source of natural products [1,2]
with interesting biological and pharmacological properties and
there continues to be much interest in this area [3]. The investi-
gations of synthetic aspects of marine natural products chemistry
have recently been reviewed [4]. In natural products of marine
origin, a large number of marine sterols have so far been identi-
fied from diverse marine sources [1,3,5,6]. The biosynthesis of the
marine sterol side chain has also been intensely investigated and
reviewed [7].
activities [6,8]. For example, a group of unique sterols contain-
ing the 3␤,21-dihydroxy function isolated from ophiuroids (brittle
stars), which were sulfated in position C-21 of the side chain,
were protective against HIV-1 and HIV-2 [16]. Recently, anti-
inflammatory 21-hydroxy steroids named griffinisterones have
been isolated from an octocoral of the genus Dendronephthya griffini
[17] and mycalosides from marine sponge Mycale laxissima [18].
Also, the 21-hydroxylanostane-type triterpenes have been iso-
lated as minor constituents from the sclerotia of Inonotus obliquus
[19].
Synthetic methods which provide access to 21-hydroxy steroids
are scarce. The first synthesis of 21-hydroxycholesterol from 3␤-
hydroxyandrost-5-en-17-one was reported by Wicha and Bal [20].
Only a few, multi-step and low-yield syntheses of steroids with
the 21-hydroxy group have been described so far [21–25]. Natural
cardiotonic steroids are potential substrates and have been applied
in the synthesis of ring D functionalized 21-hydroxy steroids from
digitoxigenin [26,27].
Our approach to the synthesis of 21-hydroxysteroids takes the
advantage of the ready availability of 14␣-card-5-enolide 1, which
was prepared in few steps from the commercial 3␤-acetoxypregn-
5-en-20-one as a 1:1 mixture of C-20 epimers [28]. The outline
of the synthesis of 21-hydroxysteroids with natural and unnatu-
ral configurations at C-20 from lactone 1 is shown in Scheme 1.
The crucial step of the planned synthesis was the separation of
the appropriate C-20 epimeric intermediates derived from 1. The
results of this investigation are presented.
Steroid metabolites isolated from sponges, starfish and ophi-
uroids have characteristic structural features. Additional hydroxyl
groups and/or unsaturation in various positions of the steroid
nucleus are found. Some compounds have mono [8]
– or
dimethylated [9] side chains and a number of sterols with “non-
conventional” side chains have been reported [1,3]. A special group
of these steroids bear oxygenated C-21, which is rather rare in
marine sterols. Occasionally, 21-hydroxy steroids have also been
isolated as 3-oligoglycosides [10]. Interestingly, (20R,24S)-3␤-
hydroxyergost-5-ene-21-oic acid, isolated from the Indian Ocean
Sclerophytum sp. soft coral is the first example of a marine sterol to
have the side chain carboxylated in position C-21 [11]. The other
20-carboxy steroids of marine origin have recently been reported
[12–14]. 21-Hydroxy-24-nor-5␤-cholest-22-ene derivatives have
∗
Corresponding author. Tel.: +48 61 8291324; fax: +48 61 8291505.
E-mail address: zparyzek@amu.edu.pl (Z. Paryzek).
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.01.010

518
H. Koenig et al. / Steroids 76 (2011) 517–523
Scheme 1.
2. Experimental
mixture (212 mg) of lactone 2 and acid 5 in the 7:3 ratio, estimated
from the 1H NMR spectrum, which showed signals characteristic
of 2 (four triplets at ı 3.8–4.5) and 5 (characteristic multiplet at ı
3.55–3.88).
2.1. General
M.p. values were determined on a Kofler hot-stage apparatus
and are uncorrected. IR spectra were determined with an FT-IR
Bruker FS 113V spectrophotometer for solutions in chloroform or
as KBr pellets. 1H and 13C NMR spectra were recorded with a Varian
Gemini 300 VT spectrometer (300 and 75.5 MHz, respectively) and
a Bruker Avance-DRX 600 spectrometer (600 and 151 MHz, respec-
tively) operating in the Fourier transform mode using solutions in
deuteriochloroform. Chemical shifts (ı) are expressed in ppm rela-
tive to tetramethylsilane as the internal standard. Electron impact
(ionization energy of 70 eV) and FAB mass spectra were recorded
with an AMD 402 or AMD 604 spectrometer. Solvents were dried
and distilled according to standard procedures. Reaction progress
and the purity of compounds were monitored by TLC using pre-
coated aluminum-backed silica plates (E. Merck, no. 5554). Silica
gel 60 (Merck 70–230 mesh, no. 7734) was used for flash chro-
matography and silica gel (E. Merck, no. 13895) for preparative TLC
separation.
4, oil. IR: ꢀmax 1706, 1255, 1090, 837 cm−1 1H NMR ı 5.31
.
(brd, J = 5.1 Hz, 1H, H-6), 3.69–3.58 (m, 2H, CH2O), 3.45 (m, 1H, H-
3␣), 0.996 (s, 3H, CH3-19), 0.925, 0.888, 0.882, and 0.879 [s, 27H,
SiC(CH3)3], 0.701 (s, 3H, CH3-18), 0.254, 0.057, 0.014, and 0.010 [4s,
18H, Si(CH3)2]. MS (FAB): m/z (%) 719 ([M+H]+, 25), 661 (33), 587
(28), 145 (100). HRMS calcd for C41H78O4Si3 + H: 719.5286; found
719.5301.
2.4.
(20␰)-3ˇ,21-Bis(t-butyldimethylsilyloxy)-24-norchol-5-en-23-oic
acid (5)
To a solution of silyl ester 4 (97 mg, 0.13 mmol) in THF–MeOH
(2:1, 15 ml) a solution of K2CO3 (10%, 1 ml) was added and the
mixture was stirred at room temp. for 1 h. A solution of KHSO4
(10%) was added to adjust to pH 4 and the product was extracted
with ether-benzene. The organic layer was washed with water and
brine, dried (MgSO4) and evaporated under reduced pressure to
give a crude product (87.5 mg), which was chromatographed on
silica gel column with benzene as the eluent. Acid 5 (69.3 mg, 85%)
was obtained, mp 153–155 ◦C (benzene). IR: ꢀmax 3529, 3400–2600,
2.2. (20␰)-3ˇ-(t-Butyldimethylsilyloxy)-21-hydroxy-24-norchol-
5-en-23-oic acid
(3)
1736, 1706, 1256, 1090, 836 cm−1 1H NMR: ı 5.31 (brd, J = 4.12 Hz,
.
To a solution of lactone 2 [28] (400 mg, 0.84 mmol) in ethanol
(20 ml) a solution of potassium hydroxide in ethanol (40 ml, 1 M)
was added at room temperature and the mixture was stirred for
30 min under argon. Ether (20 ml) was added and the solution was
washed with HCl (1%) to pH = 7, brine and dried (MgSO4). Evapo-
ration of the solvent under reduced pressure gave the hydroxyacid
as a white solid (410 mg); mp 188–189 ◦C (Et2O). IR (KBr): ꢀmax
1H, H-6), 3.85–3.42 (m, 3H, H-3␣, and 21-H), 0.994 (s, 3H, CH3-19),
0.904, and 0.902 [2s, 9H, C(CH3)3], 0.886 [s, 9H, C(CH3)3], 0.712 (s,
3H, CH3-18), 0.072 and 0.055 [s, 6H, Si(CH3)2]. MS (FAB): m/z (%)
605 (20) (M++H), 587 (25), 547 (22), 339 (32), 171 (39). HRMS calcd
for C35H65O4Si2: 605.4421; found 605.4431.
2.5.
3471–2655, 1704, 1253, 1092, 880, 870, 838, 776 cm−1 1H NMR ı
.
(20R)-3ˇ,21-Bis(t-butyldimethylsilyloxy)-24-norchol-5-en-23-ol
5.31 (brd, J = 4.6 Hz, 1H, H-6), 3.93–3.42 (m, 3H, H-3␣ and H-21),
2.77–2.48 (m, 2H, H-22), 1.00 (s, 3H, CH3-19), 0.88 [s, 9H, C(CH3)3],
0.73 (s, 3H, CH3-18), 0.06 [s, 6H, Si(CH3)2]. MS (FAB): m/z (%) 491
([M+1]+, 4), 473 (100), 341 (22), 154 (82), 136 (93). HRMS calcd for
(6) and
(20S)-3ˇ,21-bis(t-butyldimethylsilyloxy)-24-norchol-5-en-23-ol
(7)
C
29H51O4Si: 491.3556; found 491.3559.
To a solution of ester 4 (505 mg, 0.70 mmol) in toluene (50 ml) a
1 M solution of diisobutylaluminum hydride (DIBALH) in hexanes
(2 ml, 2 mmol) was added at −78 ◦C and the solution was stirred for
1 h. An additional portion of DIBALH (2 ml) was added and the stir-
ring continued for another 0.5 h. Acetic acid (1 ml) was added and
the mixture was extracted with ether, washed with water, Na2CO3
(5%) and brine. The organic layer was dried (MgSO4) and the solvent
was evaporated under reduced pressure to give a crude product
(362.6 mg), which was chromatographed on a silica gel column
(18 g) with benzene–ethyl acetate (20:1) as the eluent. The com-
bined pure fractions gave compound 6 (145 mg, 40%) and 7 (120 mg,
33%).
2.3. t-Butyldimethylsilyl (20␰)-3ˇ,21-bis(t-
butyldimethylsilyloxy)-24-norchol-5-en-23-oate
(4)
To
a solution of hydroxyacid 3 (412.8 mg, 0.84 mmol) in
dimethylformamide (30 ml) imidazole (458 mg, 6.74 mmol) and t-
butyldimethylsilyl chloride (TBDMSCl; 506 mg, 3.34 mmol) were
added and the solution was stirred at room temperature for 1 h.
Ether and water was added and the organic layer was dried (MgSO4)
and evaporated to give the residue (680 mg), which was chro-
matographed on a silica gel (16 g) column with hexane and benzene
as eluents. Ester 4 (278 mg, 46% yield) was obtained along with the
6. White solid, mp 71–73 ◦C (ethanol–water). IR (KBr): ꢀmax
3360, 1255, 1093, 888, 871, 853, 773, 666 cm−1 1H NMR (CDCl3):
.

H. Koenig et al. / Steroids 76 (2011) 517–523
519
ı 5.32 (brd, J = 4.95 Hz, 1H, H-6), 3.86–3.39 (m, 5H, H-3␣, H-21 and
H-23), 0.998 (s, 3H, CH3-19), 0.912 [s, 9H, C(CH3)3], 0.888 [s, 9H,
C(CH3)3], 0.699 (s, 3H, CH3-18), 0.083 [s, 6H, Si(CH3)2], 0.056 [s,
6H, Si(CH3)2]. 13C NMR (C6D6): ı 141.9, 122.0, 73.4, 65.3, 61.6, 57.3,
51.8, 51.1, 44.0, 43.0, 41.5, 40.4, 38.1, 37.4, 35.2, 33.2, 32.8, 32.7,
28.5, 26.7, 25.0, 22.0, 20.1, 19.0, 18.9, 12.9, −3.74, −4.64, −4.70. MS
(FAB): m/z (%) 591 ([M+H]+, 70), 327 (100), 159 (58), 133 (64). Anal-
ysis calcd for: C35H67O3Si2: C, 71.12; H, 11.25. Found: C, 71.20; H,
11.37.
(4), 75 (14), 17 (21). HRMS calcd for C35H61O4Si2: 601.4108; found
601.4094.
2.8.
(20S)-3ˇ,21-Bis(t-butyldimethylsilyloxy)-24-norchol-5-en-23-al
(10)
The oxidation of compound 7 (65 mg, 0.11 mmol) was carried
out as for alcohol 6 (vide supra) to give a crude product (52 mg),
which was chromatographed on a silica gel column (1.5 g) with
benzene as the eluent. Compound 10 (35.5 mg, 54%) was obtained
as a white solid; mp 154–157 ◦C (benzene). IR: ꢀmax 3031–282,
7. White solid, mp 148–149 ◦C (acetone). IR (KBr): ꢀmax 3495,
1254, 1097, 1029, 864, 839, 776 cm−1 1H NMR (CDCl3): ı 5.31 (brd,
.
J = 5.21 Hz, 1H, H-6), 3.77–3.60 (m, 3H) and 3.51–3.42 (m, 2H), (3␣-
H, H-21, and H-23), 0.996 (s, 3H, H-19), 0.907 [s, 9H, C(CH3)3], 0.888
[s, 9H, C(CH3)3], 0.703 (s, 3H, H-18), 0.077 [s, 6H, Si(CH3)2], 0.057
[s, 6H, Si(CH3)2]. 13C NMR: ı 142.0, 121.9, 73.4, 65.8, 61.1, 57.2,
51.3, 50.9, 44.0, 43.0, 41.5, 40.5, 38.1, 37.3, 35.1, 33.1, 32.7, 28.4,
26.6, 26.5, 24.9, 21.8, 20.0, 18.9, 18.8, 12.6, −3.85, −4.88, −4.92.
MS (FAB): m/z (%) 591 ([M+H]+, 46), 327 (88), 145 (82), 133 (100).
Analysis calcd for C35H67O3Si2: C, 71.12; H, 11.25. Found: C, 70.93;
H, 11.14.
2733, 1722 (C O), 1360, 1259, 1196, 950, 959, 938, 891, 779 cm−1
.
1H NMR (CDCl3): ı 9.77 (dd, J1 = 1 Hz, J2 = 3.4 Hz, 1H, H-23), 5.32
(brd, J = 5.2 Hz, 1H, H-6), 3.68 (dd, J1 = 3.6 Hz, J2 = 9.9 Hz, 1H, HA-
21), 3.48 (m, 1H, H-3␣) 3.42 (dd, J1 = 7.6 Hz, J2 = 9.9 Hz, 1H, HB-21),
0.996 (s, 3H, CH3-19), 0.889 [s, 9H, C(CH3)3], 0.870 [s, 9H, C(CH3)3],
0.727 (s, 3H, CH3-18), 0.058, 0.015 [s, 12H, Si(CH3)2]. MS (EI):
m/z (%) 531 (100) (M+−t-Bu), 399 (85), 307 (44), 253 (30). HRMS
calcd for C31H55O3Si2: 531.3690; found 531.3671. Analysis calcd
for C35H64O3Si2: C, 71.12; H, 11.25. Found: C, 71.20; H, 11.37.
2.6.
2.9. Ethyl (20S)-3ˇ,21-bis(t-butyldimethylsilyloxy)-25-
homochola-5,23-dien-25-oate
(11)
(20R)-3ˇ,21-Bis(t-butyldimethylsilyloxy)-24-norchol-5-en-23-al
(8)
To a solution of DMSO (235 ␮l, 3.30 mmol) in methylene chlo-
ride (5 ml) stirred under argon at −78 ◦C oxalyl chloride (170 ␮l,
0.2 mmol) was added and the mixture was stirred for 5 min. A
solution of compound 6 (147 mg, 0.25 mmol) in CH2Cl2 (5 ml) was
added dropwise over 10 min and the resulting solution was stirred
at −78 ◦C for 0.5 h. Triethylamine (1.35 ml, 9.7 mmol) was added
and the stirring was continued for another 5 min. The mixture was
warmed to room temperature, NaHCO3 (10%, 2.5 ml) was added
followed by ether. The organic layer was washed with water, dried
(MgSO4) and evaporated under reduced pressure to give a crude
product (178.5 mg), which was chromatographed on silica gel col-
umn (5 g) with benzene as the eluent. Compound 8 (107.5 mg, 73%)
was obtained as an oil. 1H NMR (CDCl3): ı 9.75 (t, J = 2.4 Hz, 1H, H-
23), 5.31 (brd, J = 5.4 Hz, 1H, H-6), 3.84 (dd, J1 = 4.2 Hz, J2 = 9.9 Hz,
1H, H-21), 3.48 (m, 1H, H-3␣), 3.41 (dd, J1 = 8.4 Hz, J2 = 9.9 Hz,
1H, H-21), 1.00 (s, 3H, CH3-19), 0.889 [s, 9H, C(CH3)3], 0.878
[s, 9H, C(CH3)3], 0.732 [s, 3H, CH3-18], 0.058 [s, 6H, SiC(CH3)2].
MS (EI): m/z (%) 531 ([M−t-Bu]+, 100), 399 (54), 307 (26), 253
(14), 145 (20). HRMS calcd for C31H55O3Si2: 531.3690; found
531.3698.
To
a solution of aldehyde 10 (29.2 mg, 0.0496 mmol) in
anhydrous benzene (1 ml) Ph3P = CHCOOEt (21.3 mg, 0.061 mmol,
1.2 eq) was added and the mixture was refluxed under argon for
8 h. An additional portion of the ylide (42.5 mg, 2.4 eq) was added
and refluxing continued for 5 h. The solvent was evaporated under
reduced pressure and the residue was chromatographed on a silica
gel column (3 g) with hexane and a mixture of hexane–benzene
(1:1) as the eluent to give ester 11 (27.8 mg, 85%) as an oil. IR:
ꢀmax 3394, 2953, 2932, 2857, 1721, 1673, 1655, 1471, 1463, 1386,
1257, 1095, 1005, 939, 836, 775, 758, 666 cm−1 1H NMR: ı 6.91
.
(dt, J23–24 = 15.5, J23–22 = 7.4 Hz, 1H, H-23), 5.82 (d, J24–23 = 15.7 Hz,
1H, H-24), 5.31 (brd, J = 4.9 Hz, 1H, H-6), 4.17 (q, J = 7.1 Hz, 2H,
OCH2CH3), 3.55 (dd, J = 3.0 Hz, 1H, H-21), 3.38–3.50 (m, 2H, H-3␣,
H-21), 1.28 (t, J = 7.1 Hz, 3H, OCH2CH3), 0.99 (s, 3H, CH3-19), 0.88
(s, 9H, t-BuSi), 0.89 (s, 9H, t-BuSi), 0.70 (s, 3H, CH3-18), 0.06 [s, 6H,
(CH3)2Si], 0.01[s, 6H, (CH3)2Si]. MS (EI): m/z (%) 601 (100) (M+−t-
Bu), 469 (10), 75 (12). HRMS calcd for C35H61O4Si2: 601.4108; found
601.4107.
2.10. (20␰)-3ˇ-(t-Butyldimethylsilyloxy)-21,23-epoxy-24-
norchol-5-en-23␰-ol
(12)
2.7. Ethyl (20R)-3ˇ,21-bis(t-butyldimethylsilyloxy)-25-
homochola-5,23-dien-25-oate
(9)
To a solution of lactone 2 (167 mg, 0.35 mmol) in anhydrous
toluene (5 ml) stirred at −78 ◦C under argon a solution of DIBALH
(1 M, 2.2 ml) in hexanes was added and stirring was continued for
1 h. Acetic acid (2 ml) was added and the mixture was stirred at
room temp. for 0.5 h. Extraction with ether was followed by usual
workup to give a crude product (160.9 mg) as a white solid, which
was chromatographed on a silica gel column with benzene–AcOEt
(20:1) as the eluent. The combined pure fractions gave lactol 12
(141.2 mg, 84%), mp 178–183 ◦C (benzene). IR (KBr): ꢀmax 3600,
To a solution of aldehyde 8 (55.5 mg, 0.093 mmol) in anhydrous
benzene (3 ml) (carbethoxymethylene)triphenylphosphorane
(Ph3P = CHCOOEt) (120.0 mg, 0.33 mmol, 10.8 eq) was added and
the mixture was refluxed under argon for 4.5 h. The solvent
was evaporated under reduced pressure and the residue was
chromatographed on a silica gel column (6 g) with hexane as
the eluent to give ester 9 (47.1 mg, 76%) as an oil. IR: ꢀmax 2952,
2931, 2902, 2856, 1722, 1650, 1471, 1462, 1382, 1368, 1309, 1256,
3410, 1255, 1094, 1069, 888, 869, 837 cm−1 1H NMR (CDCl3): ı
.
1204, 1165, 1093, 1047, 1005, 959, 939, 836, 774,666 cm−1 1H
;
5.56–5.42 (m, 1H, H-23), 5.32 (brd, J = 5.5 Hz, 1H, H-6), 4.24–3.86
(m, 1H, H-21), 3.62–3.38 (m, 1H, H-21), 3.48 (m, 1H, H-3␣), 1.005
and 0.998, (2s, 3H, CH3-19), 0.89 [s, 9H, SiC(CH3)3], 0.71, 0.69, 0.67,
and 0.66 (2s, 3H, CH3-18), 0.06 [s, 6H, Si(CH3)2]. MS (EI): m/z (%)
474 (1) (M+), 417 (100) (M+−t-Bu), 399 (69), 331 (12), 255 (15),
171 (11), 159 (23), 145 (23), 75 (94). HRMS calcd for C28H47O3Si
(M+−CH3): 459.3294; found 459.3287.
NMR: ı 6.98 (dt, J23–24 = 15.4, J23–22 = 6.7 Hz, 1H, H-23), 5.85 (d,
J24–23 = 15.7 Hz, 1H, H-24), 5.31 (brd, J = 4.9 Hz, 1H, H-6), 4.18 (q,
J = 7.1 Hz, 2H, OCH2CH3), 3.67 (dd, J = 4.1, J = 3.6 Hz, 1H, H-21),
3.44–3.53 (m, 2H, H-3␣, H-21), 1.28 (t, J = 7.1 Hz, 3H, OCH2CH3),
0.99 (s, 3H, CH3-19), 0.88 (s, 9H, t-BuSi), 0.89 (s, 9H, t-BuSi), 0.68
(s, 3H, CH3-18), 0.06 [s, 6H, (CH3)2Si], 0.03 [s, 6H, (CH3)2Si]. MS
(EI): m/z (%) 601 ([M−t-Bu]+, 100), 469 (25), 272 (5), 249 (2), 159

520
H. Koenig et al. / Steroids 76 (2011) 517–523
0.06 [s, 6H, Si(CH3)2]. 13C NMR: ı 173.1 and 172.9 (C O), 141.5 and
141.3, 138.0, 128.6, 127.8, 127.7, 127.4, 120.9, 120.8, 72.6, 65.2, 64.3,
56.7, 56.4, 50.2, 50.1 and 50.08, 43.8. MS (EI): m/z (%) 579 (M+) (1),
522 (M+−t-Bu) (11), 504 (20), 415 (100), 397 (2), 339 (5), 281 (3),
159 (16), 107 (27), 75 (96). HRMS calcd for C32H48O3NSi (M+−t-Bu):
522.3403; found 522.3371.
2.11. Reaction of lactol 12 with (carboethoxymethylene)triphenyl
phosphorane
To a solution of lactol 12 (256 mg, 0.54 mmol) in anhydrous ace-
tonitrile (28 ml) Ph3P = CHCO2Et (585 mg, 1.68 mmol) was added
and the solution was refluxed under argon for 23 h. The solvent
was evaporated under reduced pressure to give an oily residue,
which was dissolved in DMF (7 ml). To this solution imidazole
(300 mg, 4.41 mmol) and TBDMSCl (300 mg, 1.99 mmol) was added
and the mixture was stirred at room temperature for 40 min.
Benzene–ether (1:1) was added and the organic layer was washed
with water, NaHCO3 (5%) and brine, dried (MgSO4) and evaporated
under reduced pressure to give a crude product which was chro-
matographed on a silica gel column (20 g) with hexane–benzene
(2:1) as eluent. A mixture of ester (9) and (11) (313 mg, 85%) was
obtained as an oil. IR (CHCl3): ꢀmax 3016, 2954, 2931, 2904, 2856,
2.14. (20␰)-3ˇ,21-Bis(t-butyldimethylsilyloxy)-23-[(R)-(1-
phenylethyl)amino]-23-oxo-24-norchol-5-en
(16)
To a solution of lactone 2 (50.8 mg, 0.11 mmol) in anhydrous
toluene (0.5 ml) 2-hydroxypyridine (23.4 mg, 0.25 mmol), and (R)-
␣-methylbenzylamine (130 ␮l) was added and the mixture was
refluxed under argon for four days. Dilution with methylene chlo-
ride and usual workup gave a crude product as an oil, which was
purified on a silica gel column (2.5 g) with chloroform–hexane (5:1)
as the eluent. The combined pure fractions (31.5 mg, 0.048 mmol)
were dissolved in DMF (2.5 ml), imidazole (31.7 mg, 0.21 mmol) and
TBDMSCl (36.3 mg, 0.53 mmol) were added and the mixture was
stirred at room temperature for 2 h. Benzene (5 ml) was added and
the organic layer was washed with water, brine, dried (MgSO4)
and evaporated under reduced pressure. The crude product was
chromatographed on a silica gel column (1.5 g) with CH2Cl2 as the
eluent to give amide 16 (17.7 mg) as an oil. 1H NMR (CDCl3): ı
7.31 (m, 5H, C6H5), 6.16 (d, J = 7.97 Hz, 1H, NH), 6.06 (d, J = 8.24 Hz,
1H, NH), 5.31 (brd, J = 4.67 Hz, 1H, H-6), 5.18–5.08 (m, 1H, NHCH),
3.75–3.69 (m, 2H, H-21), 3.46 (m, 1H, H-3␣), 1.47 [d, J = 6.87 Hz, 3H,
C(CH3)C6H5], 0.98, 0.92, 0.89, 0.69, 0.67, 0.06 (methyl groups). MS
(FAB): m/z (%) 708 (M+), 650 (M+−t-Bu), 587 [M+−NHCH(CH3)Ph],
576 (M+−TBDMSO). HRMS calcd for C43H74NO3Si2: 708.52075;
found 708.52142.
1708, 1650, 1471, 1463, 1382, 1369, 1310, 1257, 1093 cm−1 1H
.
NMR (CDCl3): ı 6.99–6.92 (m, 1H, H-23), 5.81 (d, J = 15.6 Hz, 1H,
H-24), 5.31 (brd, J = 5.2 Hz, 1H, H-6), 4.16 (q, J = 7.1 Hz, 2H, OCH2
CH3), 3.68–3.63 (m, 3H, H2-21, H-3␣), 1.27 (t, J = 7.1 Hz, 3H, OCH2
CH3), 0.98 (s, 3H, CH3-19), 0.883 and 0.875 [s, 9H, C(CH3)3], 0.688
and 0.672 (2s, 3H, CH3-18), 0.047 and 0.045 [s, 6H, Si(CH3)2]. MS
(EI): m/z (%) 601 (33) (M+−t-Bu), 469 (10), 75 (100). HRMS calcd for
C
35H61O4Si2 (M+−t-Bu): 601.4108; found 601.4099.
2.12. Ethyl (20␰,23␰)-3ˇ-(t-butyldimethylsilyloxy)-21,23-epoxy-
25-homochol-5-en-25-oate
(14)
To a solution of lactol 12 (141.2 mg, 0.30 mmol) in anhydrous
benzene (20 ml) Ph3P = CHCO2Et (363 mg, 1.04 mmol) was added
and the solution was refluxed for 23 h under argon and stirred at
room temperature for another 24 h. The solvent was evaporated
under reduced pressure and the residue was chromatographed on
silica gel column (5 g) with benzene–CH2Cl2 and CH2Cl2 as the
eluent. Compound 14 was obtained as an oil (120.1 mg, 74%). IR
2.15. Cholest-5-ene-3ˇ,21-diol (18)
To
a
solution of i-butyltriphenylphosphonium bromide
(KBr): ꢀmax 3150, 2925, 2250, 1750, 1450, 1350, 1100 cm−1 1H
.
(71.1 mg, 0.18 mmol) in anhydrous THF (3 ml) stirred under
argon at 0 ◦C 1.6 M n-butyl lithium in hexane (0.27 ml, 0.43 mmol)
was added and the solution was stirred at room temperature for
1 h to become orange colored. A solution of aldehyde 8 (56.4 mg,
0.09 mmol) in THF (3.6 ml) was added and the reaction mixture
was stirred for 1 h. The usual workup gave a crude product 17,
which was dissolved in an AcOH:THF:H2O mixture (3:1:1, 6 ml)
and the solution was kept at 65 ◦C for 3 h (disappearance of the
substrate on the TLC plate). The product was extracted with
benzene, the organic layer was washed as usual and the solvent
evaporated under reduced pressure to give a residue as an oil.
Methanol (3 ml), ammonium formate (15 mg) and Pd on carbon
(10%, 9 mg) were added and the mixture was refluxed for 10 h. The
solvent was evaporated under reduced pressure, CHCl3 (6 ml) was
added, the solution was filtered and evaporated under reduced
pressure to give a crystalline residue, which was crystallized from
an ether-hexane mixture to give compound 18 mp 156–160 ◦C
(lit. [20] mp 154–157 ◦C). The 1H NMR spectrum of this sample
was identical with that of the original sample [20] (characteristic
signals at ı: 5.35 (d, J = 5.49 Hz, 1H, H-6), 3.69 (brs, 2H, H-21), 3.53
(brs, 1H, H-3␣), 1.01 (s, 3H, CH3-19), 0.70 (s, 3H, CH3-18)).
NMR (CDCl3): ı 5.32 (brd, J = 4.9 Hz, 1H, H-6), 4.21–4.38 (m, 1H, H-
23), 4.12–4.19 (q, J1 = 7.1 Hz, J2 = 14.3 Hz, 2H, OCH2CH3), 3.88–4.03
(m, 1H, H-21), 3.36–3.52 (m, 2H, H-3␣, H-21), 2.42–2.64 (m, 2H,
CH2CO2Et), 1.24–1.29 (t, J = 7.09 Hz, 3H, OCH2CH3), 0.99, 1.01 (2s,
3H, CH3-19), 0.89 [s, 9H, SiC(CH3)3], 0.66, 0.69 (2s, 3H, CH3-18),
0.06 [s, 6H, Si(CH3)2]. MS (EI): m/z (%) 529 [(M+−15) (3)], 487 (100),
469 (15), 159 (14), 75 (86). HRMS calcd for C29H47O4Si: 487.3244;
found 487.3260.
2.13. (20␰)-3ˇ-(t-Butyldimethylsilyloxy)-23-(benzylamino)-23-
oxo-24-norchol-5-en-21-ol
(15)
To a solution of aluminum trichloride (56.2 mg, 0.42 mmol) in
methylene chloride–benzylamine (1:1, 7.5 ml) a solution of lactone
2 (120 mg, 0.25 mmol) in benzylamine (2.2 ml) was added drop-
wise and the mixture was stirred at room temperature for 4 h, then
poured on ice-water. The product was extracted with benzene, the
organic layer was washed with brine, HCl (0.1 N) and brine to neu-
tral, dried (MgSO4) and evaporated under reduced pressure to give
a crude product (143.5 mg) which was chromatographed on a sil-
ica gel column (3.8 g) with benzene and benzene–AcOEt (2:1) as
eluent. Amide 15 (127 mg, 86%) was obtained as a white solid,
mp 185–188 ◦C (EtOH). IR (CHCl3): ꢀmax 3026, 3023, 3020, 3017,
3013, 3011, 2935, 2904, 2856, 2400, 1722, 1654, 1518, 1471, 1435,
1254, 1232, 1091 cm−1. 1H NMR (CDCl3): ı 7.37–7.28 (m, 5H, C6H5),
6.08 (brs, 1H, NH), 5.03 (brd, J = 4.9 Hz, 1H, H-6), 4.46–4.42 (m, 2H,
NHCH2), 3.83–3.53 (m, 2H, H-21), 3.49 (m, 1H, H-3␣), 0.987 and
0.994 (s, 3H, CH3-19), 0.88 [s, 9H, C(CH3)3], 0.71 (s, 3H, CH3-18),
3. Results and discussion
The basic hydrolysis of lactone 2 [28] afforded hydroxy acid 3 as
a mixture of (20R) and (20S) isomers in the ratio close to 1:1. The
reaction required very careful work-up to avoid recyclization. Thus,
the immediate treatment of the crude 3 with t-butyldimethylsilyl
chloride (TBDMSCl) and imidazole in DMF gave the stable protected

H. Koenig et al. / Steroids 76 (2011) 517–523
521
Scheme 2.
labile and the attempted oxidation of these alcohols with PCC in
methylene chloride gave negative results. Under these conditions
cleavage of the protecting silyl groups occurred. However, mild
oxidation of alcohols 6 (Scheme 4) and 7 under Swern conditions
[30,31] enabled isolation of pure 3,21-diprotected aldehydes 8 and
10 in 73 and 84% yield, respectively.
The two carbon side chain elongation was achieved when
aldehydes 8 and 10 reacted cleanly with (carboethoxymethy-
lene)triphenyl phosphorane in refluxing benzene to give unsatu-
rated esters 9 (Scheme 4) and 11 in 76 and 85% yield, respectively.
The configuration of the ꢁ23,24 double bond in esters 9 and 11 was
trans as assigned from the 1H NMR spectra [25,32]. The spectra also
indicated the formation of traces of the cis isomer (less than 5%).
Compounds 8–11 are important and versatile synthons which can
be used in further transformations leading to 21-hydroxy steroids
with diversely functionalized side chains (Fig. 1).
Scheme 3.
diol ester 4. The mild hydrolysis of 4 with methanolic potassium
carbonate gave acid 5 in 85% yield (Scheme 2).
An alternative approach to 21-hydroxy steroids from lactone
2 was also investigated. Thus, 2 was reduced with diisobutyla-
luminum hydride to lactol 12, isolated in 84% yield. This gave a
mixture of unsaturated esters 9 and 11 in 85% yield upon reaction
with (carboethoxymethylene)triphenyl phosphorane in acetoni-
trile followed by immediate silylation. These two diastereoisomers
gave poorly resolved spots on a TLC plate and could not be effec-
tively separated. When the crude product of the previous reaction,
hydroxy ester 13, was chromatographed on a silica gel column
eluted with methylene chloride, a cyclization product, the substi-
tuted tetrahydrofuran derivative 14 was isolated in 74% yield as the
only product. Another approach to 21-hydroxysteroids was based
on the aminolysis of lactone 2. The reaction of 2 with benzylamine
in the presence of aluminum trichloride as a catalyst afforded ben-
zylamide 15 in 78% yield as a mixture of C-20 epimers which could
not be resolved chromatographically. Also, the reaction of lactone
Unfortunately, the polarity of (20R) and (20S) diastereoisomers
of acid 5 and its methyl ester was identical in various solvent sys-
tems; therefore they could not be separated at this stage of the
synthesis. The reduction of the protected ester 4 with diisobutylalu-
minum hydride (DIBALH) in toluene at −78 ◦C (Scheme 3) afforded
a mixture of C-20 epimeric alcohols which gave two clearly differ-
ent spots on a TLC plate. Indeed, this mixture was separated on a
silica gel column and pure diastereoisomers 6 and 7 were obtained
in 35 and 29% yield, respectively. The tentative assignment of the
configuration of alcohols 6 and 7 was based on the observed differ-
ences in the region of ı 3.86–3.39 in the 1H NMR spectra of both
isomers, compared with the published data for compounds of sim-
ilar structure [23,29]. The natural (20R) configuration was assumed
for the more polar isomer 6.
All further transformations could be performed on pure C-20-
epimers. The protecting groups in alcohols 6 and 7 were very
Scheme 4.

522
H. Koenig et al. / Steroids 76 (2011) 517–523
Fig. 1. Chemical structures of compounds 10–16.
Scheme 5.
2 with chiral amines, (R) and (S)-␣-phenylethylamine, gave unsep-
arable mixtures of respective C-20 epimeric benzylamides. The
product of the reaction of 2 with (R)-␣-phenylethylamine was fully
characterized as 21-tert-butyldimethylsilyl derivative 16.
According to our approach to 21-hydroxy steroids, 21-
hydroxycholesterol 18 was synthesized (Scheme 5). This sterol
was isolated from the Pacific starfish Euretaster insignis as
out using Prediction of Activity Spectra for Substances (PASS) pro-
gram [34]. The predicted activity spectrum for compounds 4–11,
in which TBDMS protecting groups were replaced by hydrogen,
includes antihypercholesterolemic, embryotoxic, interleukin ago-
nist and cholesterol synthesis inhibition activity, and reproductive
dysfunction treatment with the estimated probability for com-
pounds to be active higher than 70%.
a
21-sulfate ester [29]. Thus, the Wittig reaction of alde-
hyde 8 with isobutyltriphenylphosphonium bromide and n-butyl
lithium gave 3␤,21-dihydroxycholesta-5,23-diene as 3,21-bis[tert-
butyl(dimethyl)silyl] ether 17. This was a mixture of cis and trans
4. Conclusion
In summary, we have developed a short and efficient synthesis
of steroid synthons 8–11 from 14␣-cardenolide 2. The synthons
can be further transformed to other 21-hydroxy-steroids with a
functionalized side chain of various lengths. The method allows the
synthesis of both natural (20R)- and unnatural (20S)-21-hydroxy
steroids for the evaluation of their biological activity. The utility of
this method was exemplified by the synthesis of a natural marine
sterol, 21-hydroxycholesterol.
ꢁ
23-olefins, as evidenced by the multiplicity of the ı 5.45–5.18 sig-
nal of protons H-23, H-24 and H-6 in the 1H NMR spectrum. The
deprotection of 17 (AcOH/THF/H2O) followed by catalytic trans-
fer hydrogenation [33] afforded 21-hydroxycholesterol 18. The 1
H
NMR, IR and mass spectra of 18 were in full agreement with those of
the original sample of (20R)-cholest-5-ene-3␤,21-diol [20]. More-
over, the synthesis of diol 18 confirmed the assignment of the
configuration of carbon atom C-20 in (20R) and (20S) steroids 8,
9 and 10, 11, respectively.
Polyhydroxylated sterols of marine origin have shown a broad
spectrum of biological activities including cytotoxic [1,3] and
antiviral properties [16]. The computer-assisted prediction of the
biological activity for synthetic 21-hydroxysteroids was carried
Acknowledgements
Partial financial support by the Ministry of Science and Higher
Education (project No. 7 T09A 110 21) is gratefully acknowledged.
We warmly thank Professor Jerzy Wicha for providing a sample of

H. Koenig et al. / Steroids 76 (2011) 517–523
523
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