Further study on synthesis and evaluation of 3,16,20-polyoxygenated steroids of marine origin and their analogs as potent cytotoxic agents

Article abstract of DOI:10.1016/j.steroids.2010.02.011

A series of new polyoxygenated steroid derivatives with various steroid skeleton moieties were synthesized. Antitumor activity of the compounds against three tumor cell lines (Breast cancer MCF7, lung cancer NCI and oral cancer KB) were evaluated. Compoun

Full text of DOI:10.1016/j.steroids.2010.02.011

Steroids 75 (2010) 432–444
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Further study on synthesis and evaluation of 3,16,20-polyoxygenated steroids of
marine origin and their analogs as potent cytotoxic agents
Potjamarn Bunyathaworn, Suthinee Boonananwong, Boonsong Kongkathip∗, Ngampong Kongkathip
Natural Products and Organic Synthesis Research Unit (NPOS), Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science,
Kasetsart University, 50 Phaholyotin Road, Chatuchak, Bangkok 10900, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new polyoxygenated steroid derivatives with various steroid skeleton moieties were syn-
thesized. Antitumor activity of the compounds against three tumor cell lines (Breast cancer MCF7, lung
cancer NCI and oral cancer KB) were evaluated. Compounds with aromatic A ring of this series exhibited
the most potent cytotoxicities in all tested cells. The absence of OH at C-16 or lack of cholesterol like
side chain at C-20 in the steroid skeleton apparently result in decreased cytotoxicity. The compound
became inactive when the side chain contains double bond at C-24–C-25. When hydroxyl group at C-3
was protected no cytotoxicities against MCF7 and NCI and considerable low cytotoxicity against KB cell
lines were observed.
Received 5 November 2009
Received in revised form 11 February 2010
Accepted 11 February 2010
Available online 24 February 2010
Keywords:
Biological activity
Anticancer
Polyoxygenated cholestanes
Cancer cells (MCF7, NCI, KB)
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
cer cell lines (MCF7), human lung cancer cell lines (NCI) and human
epidermoid carcinoma cell lines (KB)]. In our study it was found
Steroids isolated from various marine organisms (marine
steroids) manifest diverse biological activities [1–3]. Some of
them are extremely toxic against tumor cells [4–7] and show
anti-inflammatory [8] and other effects [9,10]. It is therefore not
surprising that marine steroids arouse considerable interest in not
only chemists, but also among pharmacologists and physicians.
A large number of polyoxygenated steroids have been found
in algae, and virtually in all marine invertebrate phyla, such as
Porifera, Coelenterata, Bryozoa, Molusca, Echinodermata, Arthro-
poda and Tunicata as well as in fish [1]. A series of polyoxygenated
steroids with uncommonly present oxidation both at C-16 and
C-20 were isolated from the anthozoan Antipathes subpinnata
[11,12] and the gorgonian Leptogorgia sarmentosa [13–16] and
two of these polyoxygenated substances were reported to exhibit
cytotoxicity against P388 or mouse lymphoid neoplasma human
lung cancinoma (A549), human colon carcinoma (HTG) and
human metanoma (MEL 28) with ED50 value of 1 mg/mL in all
tested cells [16]. Recently [17] we described the synthesis of
four naturally occurring 3,16,20-trioxygenated cholestanes, (20S)-
20-hydroxycholestane-3,16-dione (1), (16S,20S)-16,20-dihydroxy
cholestan-3-one (2), (20S)-20-hydroxycholest-1-ene-3,16-dione
(3) and (20S)-20-hydroxy cholest-4-ene-3,16-dione (4) (Fig. 1) and
their antitumor activity against three cell lines [human breast can-
that compound 1 containing keto functional group at both C-3
and C-16 lack of activity to all tested cell lines and compound 2
which contains keto functional group at C-3 and a hydroxyl group
at C-16 showed activity against only NCI, whereas compounds 3
and 4 containing ␣,␤-unsaturated ketone at ring A showed sig-
nificant cytotoxicity for NCI and moderated activity for MCF7
and KB cell lines. These results provided useful information for
future design. Therefore, in order to determine the role of the type
and degree of unsaturation in A ring and also alkyl side chain
of polyoxygenated steroids in the anticancer activity we decided
to proceed in our investigation by synthesizing various types of
3,16,20-trioxygenated steroids in which, ring A was modified as
quinone or aromatic ring, different functionality at C-16 and dif-
ferent degrees of oxidation at the cholesterol side chain were also
prepared and evaluated the anticancer activity of these compounds
and gain insight into structure–activity relationships (SARs).
2. Experimental
2.1. General
Proton nuclear magnetic resonance (1H NMR) spectra and car-
bon nuclear magnetic resonance (13C NMR) spectra were recorded
on a Varian Gemini 300 spectrophotometer and on 400 and
100 MHz Brucker Advance DPX-400. Chemical shifts were recorded
as ı values in ppm. Spectra were acquired in CDCl3 unless oth-
erwise stated. The peak due to residual CHCl3 (7.26 ppm for 1H
∗
Corresponding author. Tel.: +662 5625444x2235; fax: +662 5793955.
E-mail address: fscibsk@ku.ac.th (B. Kongkathip).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.02.011

P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
433
(C-16), 59.8 (C-17), 53.3 (C-14), 43.6 (C-13), 43.5 (C-9), 43.1 (C-22),
40.4 (C-12), 37.6 (C-8), 36.5 (C-15), 29.4 (C-6), 27.4 (C-7), 26.3 (C-
11), 26.0 (C-27), 25.4 (C-21), 23.2 (C-23), 17.4 (C-26), 14.6 (C-18).
MS (APCI), m/z (relative intensity): 399 (4), 381 (98), 363 (100).
HRMS m/z: C26H38O3Na [M+Na]+, cald 421.2719, found 421.2721.
2.2.2. 3-Benzyloxy, 16R,20S-dihydroxy-24-cholestene-ꢀ1,3,5(10)
-estratriene (29)
The crude product was purified by flash column chromatogra-
phy eluting with 3:7 ethyl acetate:hexane to provide 29 (204 mg,
38.9%) as a colorless wax. FTIR (neat) ꢁmax 3411 (OH) cm−1 1H
.
NMR (400 MHz, CDCl3 and CD3OD) ı 7.31 (m, 5H, CH-Ar), 7.09
(d, J = 8.6 Hz, 1H, CH-1), 6.70 (dd, J = 8.6, 2.8 Hz, 1H, CH-2), 6.64 (d,
J = 2.8 Hz, 1H, CH-4), 5.08 (m, 1H, H-24), 4.96 (s, 2H, CH2OBn), 4.49
(m, 1H, CH-16), 2.79 (m, 2H, CH2-6), 2.25 (m, 1H, H-9), 1.90 (m, 1H,
H-11), 1.86 (m, 1H, H-12), 1.75 (m, 1H, H-15), 1.69 (m, 2H, H-7),
1.64 (s, 3H, H-26), 1.60 (m, 1H, H-11), 1.59 (m, 2H, H-14), 1.58 (s,
3H, H-27), 1.49 (m, 1H, H-15, H-8), 1.27 (s, 3H, H-21), 1.24 (m, 1H,
H-12), 0.72 (s, 3H, H-18). 13C NMR (100 MHz, CDCl3 and CD3OD)
ı 156.7 (C-3), 138.0 (C-Ar), 137.3 (C-5), 133.0 (C-25), 131.8 (C-10),
128.5 (Ar), 127.8 (Ar), 127.4 (Ar), 126.1 (C-1), 124.4 (C-24), 114.8
(C-4), 112.3 (C-2), 76.3 (C-20), 73.4 (C-16), 69.9 (C-OBn), 69.5 (C-
17), 52.8 (C-14), 45.2 (C-13), 43.9 (C-22), 43.6 (C-9), 40.0 (C-12),
37.7 (C-8), 34.6 (C-15), 29.7 (C-6), 27.2 (C-7), 26.2 (C-11), 25.7 (C-
21), 25.6 (C-27), 22.1 (C-23), 17.7 (C-26), 15.2 (C-18). MS (APCI),
m/z (relative intensity): 488 (28), 471 (100), 453 (73).
Fig. 1. Synthetic natural marine steroids.
and 77.23 ppm for 13C) was used as the internal reference. Cou-
pling constants (J) are given in Hz, and multiplicity is defined as
follows: br = broad, s = singlet, d = doublet, dd = double of doublets,
dt = double of triplet, t = triplet, q = quartet, m = multiplet. Infrared
(IR) spectra were recorded in cm−1 on a Perkin-Elmer 2000 Fourier
transform infrared spectrophotometer at the Chemistry Depart-
ment, Faculty of Science, Kasetsart University. Samples were ana-
lyzed as KBr disks. Mass spectra were obtained on a Agilent Tech-
nology 1100 series LL/MSD Trap. Melting points (mp) were deter-
mined on a Fisher John apparatus and MEI-TEMP capillary melting
point apparatus at the Chemistry Department, Kasetsart Univer-
sity and are reported uncorrected in ◦C. All chemicals and solvents
were purchased from the Fluka Co. Ltd. as analytical grade and sol-
vents were purified by general method before being used. m-IBX
was prepared as described by Santagostino and co-workers [18].
2.2.3. 3ˇ,20S-Dihydroxycholestane (35)
The crude product was filtered through a short column (eluting
with 3:7 ethyl acetate:hexane) to provide the product 35 as a white
solid (67.2 mg, 60%) mp 141–143 ◦C [lit-141–143 ◦C] [19]. FTIR (KBr)
ꢂ
max 3366 (OH) cm−1 1H NMR (CDCl3, 400 MHz). ı 3.15 (m, 1H, H-
.
3), 1.25–1.72 (m, 20H, CH2) 1.40–1.50 (m, 6H, CH) 1.23 (s, 3H, H-21),
1.18 (m, 2H, H-23), 1.11 (m, 2H, H-15), 1.07 (s, 3H, H-18), 0.98 (m,
1H, H-14), 0.81 (s, 3H, H-19), 0.82 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.60
(m, 1H, H-9), 13C NMR (CDCl3) ı 75.8 (C-20), 72.4 (C-3), 59.1 (C-17),
54.9 (C-14), 54.1 (C-9), 45.9 (C-5), 43.3 (C-13), 43.2 (C-22), 42.1 (C-
4), 40.4 (C-12), 39.7 (C-24), 36.6 (C-1), 36.9 (C-2), 35.7 (C-10), 35.0
(C-8), 31.8 (C-7), 28.8 (C-6), 28.1 (C-25), 26.4 (C-21), 25.0 (C-16),
24.6 (C-15), 23.1 (C-27); 22.9 (C-26), 21.2 (C-23), 20.9 (C-11), 18.0
(C-18), 11.6 (C-19). MS (APCI), m/z (relative intensity): 404 (M+,3),
389 (7), 386 (27), 319 (100), 301 (75), 276 (48), 258 (38). HRMS m/z:
C27H48O2Na [M+Na]+, cald 427.3552, found 427.3561.
2.2. General procedure for preparation of compounds 10, 29 and
35
The mixture containing of Mg turning (338 mg), I2 (cat-
alytic amount) and dry THF (30 mL) was added 5-bromo-2-
methylpentane or 5-bromo-2-methyl-2-pentene (9.39 mmol) at
room temperature under N2. The mixture was stirred at room tem-
perature for 3 h and then the solution of 9, 28 or 34 (1.87 mmol) in
dry THF (20 mL) was added drowised at 0 ◦C. The reaction mixture
was stirred at 0 ◦C for 20 min and then quenched with ice-water
followed by neutralized with diluted HCl. The reaction mixture
was then extracted with methylene chloride. The combined organic
layers were washed with water, dried over anhydrous sodium sul-
phate, filtered and concentrated in vacuo. The crude product was
purified by flash column chromatography.
2.3. General procedure for preparation of compound 18, 31
To a stirred solution of 17 or 29 (0.15 mmol)) in CH2Cl2 (50 mL)
was added NaOAc (7.27 mmol) and PCC (8.0 mmol) at room tem-
perature. After stirring for 1 or 3 h, the reaction mixture was filtered
through celite pad. The filtrate was evaporated and purified by flash
column chromatography.
2.2.1. Synthesis of 3,16S,20S-trihydroxy-24-cholestene-ꢀ1,3,5(10)
-estratriene (10)
2.3.1. Synthesis of 3-tert-butyl-dimethylsiloxy-(16S,20S)-
The crude product was purified by flash column chromatogra-
phy eluting with 3:7 ethyl acetate:hexane to provide 10 as a white
solid (164.5 mg, 60%), mp 220–222 ◦C. FTIR (KBr), ꢁmax 3427 (OH)
16,20-acetonide-24-one-chlorestane-ꢀ1,3,5(10)-estratriene (18)
The crude product was purified by flash column chromatogra-
phy eluting with 1:19 ethyl acetate:hexane to provide 18 as a white
cm−1
.
1H NMR (CDCl3, 400 MHz) ı 7.00 (d, J = 8.32 Hz, 1H, H-1), 6.53
solid (100 mg, 60%) mp 54–55 ◦C. FTIR (KBr) ꢁmax 1708 (C O) cm−1
.
(dd, J = 8.32, 2.56 Hz, 1H, H-2), 6.47 (d, J = 2.56 Hz, 1H, H-4), 5.05 (m,
1H, H-24), 4.54 (m, 1H, H-16), 2.73 (m, 2H, H-6), 2.66 (m, 1H, H-
15), 2.16 (m, 2H, H-9, H-12), 1.93 (m, 1H, H-11), 1.91 (m, 2H, H-23),
1.77 (m, 1H, H-22), 1.74 (m, 1H, H-7), 1.66 (s, 3H, H-26), 1.57 (m,
1H, H-22), 1.55 (s, 3H, H-27), 1.43 (m, 1H, H-8), 1.32 (m, 1H, H-17),
1.31 (m, 3H, H-7, H-11, H-15), 1.27 (m, 1H, H-12), 1.25 (s, 3H, H-21),
1.07 (s, 3H, H-18), 0.97 (m, 1H, H-14). 13C NMR (CDCl3, 100 MHz)
ı 154.1 (C-3), 137.7 (C-5), 131.7 (Me-25), 131.3 (C-10), 126.0 (C-1),
124.4 (C-24), 112.5 (C-2), 115.0 (C-4), C-20 overlap with CDCl3, 73.4
1H NMR (CDCl3, 400 MHz) ı 6.91 (d, J = 8.44 Hz, 1H, H-1), 6.41 (dd,
J = 8.44, 2.64 Hz, 1H, H-2), 6.36 (d, J = 2.64 Hz, 1H, H-4), 4.36 (m, 1H,
H-16), 2.26 (m, 2H, H-6), 2.43 (d, J = 6.90 Hz, 1H, H-25), 2.42 (m, 2H,
H-23), 2.10 (m, 1H, H-22), 2.09 (m, 1H, H-15), 2.03 (m, 1H, H-11),
2.00 (m, 1H, H-9), 1.92 (m, 1H, H-12), 1.68 (m, 1H, H-7), 1.46 (m, 1H,
H-22), 1.42 (m, 1H, H-11), 1.32 (m, 1H, H-8), 1.25 (m, 1H, H-15), 1.22
(s, 3H, Me), 1.19 (m, 1H, H-12), 1.15 (s, 3H, Me), 1.10 (s, 3H, H-21),
0.93 (s, 3H, H-18), 0.92 (d, J = 6.9 Hz, 6H, H-26, H-27), 0.90 (m, 1H,
H-14), 0.80 (m, 1H, H-17), 0.79 (s, 6H, Me3CSi), 0.0 (s, 3H, Me2Si).

434
P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
13C NMR (CDCl3, 100 MHz) ı 214.8 (C-24), 153.3 (C-3), 137.8 (C-5),
133.1 (C-10), 126.0 (C-1), 117.1 (C-4), 120.0 (C-2), 97.15 (C), 74.3
(C-20), 68.7 (C-16), 56.0 (C-17), 53.8 (C-14), 44.0 (C-9), 42.7 (C-13),
41.9 (C-25), 40.3 (C-12), 37.9 (C-8), 36.8 (C-22), 34.7 (C-23), 33.5
(C-15), 31.7 (Me), 29.6 (C-6), 27.6 (C-7), 26.1 (C-11), 25.8 (C-21),
25.7 (MeCSi), 25.7 (MeCSi), 25.7 (MeCSi), 23.9 (Me), 18.5 (C-27),
18.4 (C-26), 18.2 (CSi), 14.9 (C-18), −0.40 (MeSi), −0.40 (MeSi). MS
(APCI), m/z (relative intensity): 569 (100), 493 (87).
(m, 2H, H-7), 1.46 (m, 2H, H-8, H-11), 1.43 (m, 1H, H-25), 1.42 (m,
1H, H-15) 1.40 (m, 2H, H-22), 1.29 (m, 2H, H-24), 1.27 (s, 9H, Me,
Me, H-21), 1.18 (m, 2H, H-23), 1.12 (s, 3H, Me-18), 1.02 (m, 1H, H-
14), 0.85 (m, 1H, H-17). 0.80 (d, J = 6.6 Hz, 6H, H-26, H-27), 13C NMR
(CDCl3) ı 153.4 (C-3), 138.1 (C-5), 132.6 (C-10), 127.6 (C-1), 115.2
(C-4), 112.3 (C-2), 78.3 (C-20), 74.1 (C-16), 60.3 (C-17), 53.5 (C-14),
44.4 (C-13), 43.7 (C-9), 43.3 (C-22), 40.5 (C-12), 39.6 (C-15), 37.6
(C-8), 37.1 (C-24), 29.5 (C-6), 27.9 (C-25), 27.5 (C-7), 26.9 (C-11),
26.4 (C-21), 22.7 (Me), 22.6 (Me), 22.4 (C-23), 15.1 (C-18). HRMS
m/z: C29H44O3Na [M+Na]+, cald 463.3188, found 463.3191.
2.3.2. 3-Benzyloxy, 20S-hydroxyl-24-cholestene-
16-one-ꢀ1,3,5(10)-estratriene (31)
2.4.3. 3-tert-Butyl-dimethylsiloxy-
The crude product was purified by flash column chromatog-
raphy (3:17 ethyl acetate:hexane) to yield 31 (39.8 mg, 80%) as
(16S,20S)-16,20-acetonide-
cholestane-ꢀ1,3,5(10)-estratriene (21)
a white solid. 85–86 ◦C FTIR (neat) ꢁmax 3439, 1727 cm−1 1H
,
The crude product was purified by flash column chromatog-
raphy (3:7 ethyl acetate:hexane) to afford 21 (70 mg. 99%) as a
NMR (400 MHz, CDCl3 and CD3OD) ı 7.30 (m, 5H, CH-Ar), 7.08
(d, J = 8.4 Hz, 1H, H-1), 6.70 (dd, J = 8.4, 2.7 Hz, 1H, H-2), 6.65 (d,
J = 2.7 Hz, 1H, H-4), 5.10 (m, 1H, H-24), 5.01 (s, 2H, CH2OBn), 2.80
(m, 2H, H-6), 1.65 (s, 3H, H-26), 1.59 (s, 3H, H-27), 1.31 (s, 3H, H-21),
1.00 (s, 3H, H-18). 13C NMR (100 MHz, CDCl3 and CD3OD) ı 220.7
(C-16), 156.7 (C-3), 138.2 (C-Ar), 137.2 (C-5), 133.3 (C-25), 132.0
(C-10), 128.6 (CH-Ar), 127.7 (CH-Ar), 127.3 (CH-Ar), 126.2 (C-1),
124.8 (C-24), 114.7 (C-4), 112.4 (C-2), 73.6 (C-20), 220.7 (C-16),
70.1 (CH2OBn), 71.9 (C-17), 52.6 (C-14), 45.2 (C-13), 43.4 (C-22),
43.5 (C-9), 40.1 (C-12), 37.8 (C-8), 39.1 (C-15), 30.0 (C-6), 27.3 (C-
7), 26.5 (C-11), 25.8 (C-21), 24.6 (C-27), 22.2 (C-23), 17.6 (C-26),
15.0 (C-18).
colorless syrup. FTIR (KBr), ꢂmax 1497, 1246 cm−1 1H NMR (CDCl3,
.
400 MHz) ı 7.02 (d, J = 8.45 Hz, 1H, H-1), 6.52 (dd, J = 8.45, 2.66 Hz,
1H, H-2), 6.47 (d, J = 2.66 Hz, 1H, H-4), 4.47 (m, 1H, H-16), 2.78 (m,
2H, H-6), 2.20 (m, 1H, H-9), 1.95 (m, 1H, H-11),1.90 (m, 1H, H-15),
1.85 (m, 1H, H-12), 1.82 (m, 2H, H-7), 1.81 (m, 1H, H-22), 1.61 (m,
1H, H-11), 1.48 (m, 1H, H-15), 1.45 (m, 1H, H-8), 1.38 (m, 1H, H-
22), 1.31 (m, 2H, H-21), 1.28 (m, 2H, H-24), 1.24 (m, 1H, H-12),
1.19 (m, 2H, H-23), 1.13 (s, 1H, H-18), 1.10 (m, 1H, H-14), 1.00 (s,
9H, Me3CSi), 0.86 (m, 1H, H-17), 0.85 (d, J = 6.6 Hz, 6H, H-26, H-27),
0.82 (s, 6H, Me2Si). 13C NMR (CDCl3, 100 MHz) ı 153.4 (C-3), 137.3
(C-5), 131.9 (C-10), 126.2 (C-1), 119.9 (C-4), 117.1 (C-2), 97.2 (C),
79.0 (C-20), 74.9 (C-16), 60.6 (C-17), 53.8 (C-14), 44.2 (C-9), 43.8
(C-13), 43.0 (C-22), 40.0 (C-12), 37.8 (C-8), 37.2 (C-24), 33.7 (C-15),
31.8 (Me), 29.9 (C-6), 28.1 (C-25), 27.4 (C-7), 27.1 (C-11), 26.5 (C-
21), 25.8 (MeCSi), 24.2 (Me), 22.7 (C-27), 22.6 (C-26), 22.5 (C-23),
17.7 (CSi), 15.3 (C-18), −0.40 (MeSi), −0.4 (MeSi). MS (APCI), m/z
(relative intensity): 554 (100), 494 (30).
2.4. General procedure for preparation of compounds 11, 13, 21,
27, 30, 32 and 34
To a stirred suspension of 5% Pd/C (0.0064 mmol) in ethanol
(3 mL) was added a solution of 10, 12, 16, 26, 29, 31 or 33
(0.126 mmol) in ethyl acetate (3 mL). The reaction mixture was
treated with hydrogen ballon and stirred for 16 h. The reaction mix-
ture was filtered through celite pad and rinsed with ethyl acetate.
The filtrate was concentrated by using rotary evaporator. The crude
product was purified by flash column chromatography.
2.4.4. 3,16˛-Dihydroxy-17-acetyl-ꢀ1,3,5(10)-estratriene (27)
The crude product was purified by flash column chromatog-
raphy (3:7 ethyl acetate:hexane) to afford 27 (19.24 mg, 99%) as
a white solid, mp 94–96 ◦C. FTIR (KBr), ꢁmax 3436 (OH), 1699
2.4.1. Synthesis of 3,16S,20S-trihydroxycholestene-ꢀ1,3,5(10)
estratriene (11)
-
(C O) cm−1 1H NMR (CDCl3, 400 MHz) ı 7.16 (d, J = 8.53 Hz, 1H,
.
H-1), 6.66 (dd, J = 8.53, 2.74 Hz, 1H, H-2), 6.48 (d, J = 2.74 Hz, 1H, H-
4), 4.87 (m, 1H, H-16), 2.85 (m, 2H, H-6), 2.83 (m, 1H, H-9), 2.63 (m,
1H, H-17), 2.20 (m, 1H, H-15), 2.16 (s, 3H, H-21), 2.10 (m, 1H, H-
11), 2.08 (m, 1H, H-12), 1.77 (m, 2H, H-7), 1.62 (s, 3H, Me), 1.55 (s,
3H, Me), 1.48 (m, 1H, H-11), 1.47 (m, 1H, H-8), 1.43 (m, 1H, H-15),
1.29 (m, 1H, H-12), 1.23 (s, 3H, Me), 1.15 (s, 3H, Me), 1.08 (s, 3H,
H-18), 1.02 (m, 1H, H-14), 13C NMR (CDCl3, 100 MHz) ı 179.6 (C-
20), 156.9 (C-3), 137.8 (C-10), 132,2 (C-5), 126.2 (C-1), 115.3 (C-4),
112.8 (C-2), 71.8 (C-16), 55.4 (C-14), 46.3 (C-13), 44.5 (C-9), 39.2 (C-
8), 37.1 (C-17), 34.6 (C-12), 32.1 (C-15), 28.2 (C-6), 27.5 (C-11), 26.2
(C-21), 26.1 (C-7), 15.8 (C-18). HRMS m/z: C20H26O3Na [M+Na]+,
cald 337.18780, found 337.18786.
The crude product was purified by flash column chromatogra-
phy (3:7 ethyl acetate:hexane) to afford 11 (11 mg, 100%) as a white
solid, mp 245–246 ◦C. FTIR (KBr) ꢂmax 3389 (OH) cm−1 1H NMR
.
(CDCl3) ı 7.02 (d, J = 8.4 Hz, 1H, H-1), 6.54 (dd, J = 8.4, 2.7 Hz, 1H, H-
2), 6.47 (d, J = 2.6 Hz, 1H, H-4), 4.52 (m, 1H, H-16), 2.71 (m, 2H, H-6),
2.23 (m, 1H, H-12), 2.15 (m, 1H, H-11), 2.07 (m, 1H, H-9), 1,74 (m,
2H, H-7), 1.49 (m, 1H, H-11), 1.48 (m, 1H, H-8), 1.43 (m, 1H, H-25),
1.40 (m, 2H, H-22), 1.29 (m, 2H, H-24), 1.27 (m, 1H, H-12), 1.23 (m,
1H, H-17), 1.22 (s, 3H, H-21), 1.18 (m, 2H, H-23), 1.11 (m, 2H, H-15),
1.07 (s, 3H, H-18), 0.98 (m, 1H, H-14), 0.80 (d, J = 6.6 Hz, 6H, H-26, H-
27). 13C NMR (CDCl3) ı 154.0 (C-3), 137.5 (C-5), 131.4 (C-10), 125.8
(C-1), 114.8 (C-4), 112.3 (C-2), 76.7 (C-20), 73.1 (C-16), 59.4 (C-17),
53.1 (C-14), 44.0 (C-13), 43.5 (C-9), 42.9 (C-22), 40.2 (C-12), 39.4
(C-15), 37.5 (C-8), 36.3 (C-24), 29.3 (C-6), 27.6 (C-25), 27.3 (C-7),
26.1 (C-11), 25.7 (C-21), 22.2 (Me × 2), 22.1 (C-23), 14.3 (C-18). MS
(APCI), m/z (relative intensity): 401 (7), 383 (90), 365 (100). HRMS
m/z: C26H40O3Na [M+Na]+, cald 423.2875, found 423.2862.
2.4.5. 3-Hydroxy, 16R,20S-trihydroxy-cholestane-ꢀ1,3,5(10)
estratriene (30)
-
The crude product was purified by flash column chromatogra-
phy (3:7 ethyl acetate:hexane) to afford 30 (50 mg, 99%) as a white
solid, mp 94–96 ◦C. FTIR (KBr), ꢂmax 3347 (OH) cm−1 1H NMR
.
(CDCl3 and CD3OD) ı 7.00 (d, J = 8.48, Hz, 1H, H-1), 6.53 (dd, J = 8.36,
2.64 Hz, 1H, H-2), 6.47 (d, J = 2.52 Hz, 1H, H-4), 4.42 (m, 1H, H-16),
2.72 (m, 2H, H-6), 2.16 (m, 1H, H-9), 2.14 (m, 1H, H-11), 1.94 (m,
1H, H-12), 1.74 (m, 1H, H-7), 1.62 (m, 2H, H-23), 1.61 (m, 1H, H-17),
1.60 (m, 2H, H-14), 1.58 (m, 3H, H-22), 1.47 (m, 1H, H-25), 1.43 (m,
2H, H-24), 1.38 (m, 1H, H-12), 1.34 (m, 1H, H-8), 1.29 (m, 1H, H-7),
1.22 (s, 3H, H-21), 1.20 (m, 2H, H-15), 0.81 (d, J = 6.64 Hz, 6H, H-26,
H-27), 0.71 (s, 3H, H-18). 13C NMR (CDCl3 and CD3OD) ı 154.9 (C-
3), 137.6 (C-5), 131.3 (C-10), 125.8 (C-1), 114.9 (C-4), 112.4 (C-2),
2.4.2. 3-Hydroxy-(16S,20S)-16,20-acetonide-cholestan-
ꢀ
1,3,5(10)-estratriene (13)
The crude product was purified by flash column chromatogra-
phy (3:7 ethyl acetate:hexane) to afford 13 (8 mg. 99%) as a colorless
syrup. FTIR (KBr) ꢂmax 3380 (OH) cm−1 1H NMR (CDCl3) ı 7.06 (m,
1.6H, H-1), 6.55 (dd, J = 8.4, 2.76 Hz, 1.6H, H-2), 6.49 (d, J = 2.7 Hz,
1.6H, H-4), 4.45 (m, 1H, H-16), 2.73 (m, 2H, H-6), 2.19 (m, 1H, H-
15) 2.14 (m, 1H, H-9), 2.12 (m, 1H, H-11), 2.09 (m, 1H, H-12), 1.82

P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
435
75.5 (C-20), 72.8 (C-16), 67.6 (C-17), 52.6 (C-14), 43.5 (C-13), 43.4
(C-9), 43.3 (C-22), 39.7 (C-12), 39.3 (C-15), 37.6 (C-8), 34.3 (C-24),
29.3 (C-6), 27.8 (C-25), 27.3 (C-7), 25.8 (C-11), 24.7 (C-21), 22.3 (C-
26), 22.2 (C-27), 20.6 (C-23), 14.8 (C-18). HRMS m/z: C26H40O3Na
[M+Na]+, cald 423.2875, found 423.2874.
(C-20), 73.2 (C-16), 61.1 (C-17), 53.2 (C-14), 43.6 (C-9), 43.2 (C-13),
40.4 (C-12), 39.87 (C-22), 37.6 (C-8), 36.5 (C-15), 33.6 (C-25), 29.5
(C-6), 27.5 (C-7), 26.3 (C-11), 25.5 (C-21), 23.3 (C-23), 18.7 (C-27),
17.7 (C-26), 14.6 (C-18). MS (APCI), m/z (relative intensity): 417 (4),
381 (100). HRMS m/z: C26H40O4Na [M+Na]+, cald 439.2824, found
439.2804.
2.4.6. 3-Hydroxy, 20S-hydroxyl-cholestane-16-one-ꢀ1,3,5(10)
estratriene (32)
-
2.5.2. 3-tert-Butyl-dimethylsiloxy-16S,20S-trihydroxycholestene-
The crude product was purified by flash column chromatogra-
phy (3:7 ethyl acetate:hexane) to afford 32 (47.0 mg, 99%) as a white
solid, mp 94–96 ◦C. FTIR (KBr), ꢂmax 3347 (OH), 1727 cm−1. 1H NMR
(CDCl3 and CD3OD) ı 7.06 (d, J = 8.34, Hz, 1H, H-1), 6.52 (dd, J = 8.34,
2.57 Hz, 1H, H-2), 6.48 (d, J = 2.57 Hz, 1H, H-4), 2.81 (m, 2H, H-6),
2.21 (dd, J = 18.4, 13.4, 1H, H-15), 2.18 (m, 1H, H-9), 2.12 (m, 1H, H-
11), 2.12 (d, J = 6.8 Hz, 1H, H-17), 1.81 (dd, J = 13.4, 8.8 Hz 1H, H-15),
1.63 (m, 2H, H-14), 1.46 (m, 1H, H-25), 1.46 (m, 1H, H-11), 1.43 (m,
2H, H-22), 1.39 (m, 2H, H-12), 1.36 (m, 1H, H-8), 1.31 (m, 1H, H-7),
1.31 (s, 3H, H-21), 1.29 (m, 2H, H-23), 1.09 (m, 1H, H-24), 0.82 (d,
J = 6.60 Hz, 6H, H-26, H-27), 0.90 (s, 3H, H-18). 13C NMR (CDCl3 and
CD3OD) ı 220.5 (C-16), 154.9 (C-3), 137.6 (C-5), 131.3 (C-10), 125.8
(C-1), 114.9 (C-4), 112.4 (C-2), 75.5 (C-20), 72.8 (C-16), 67.6 (C-17),
52.6 (C-14), 43.5 (C-13), 43.4 (C-9), 43.3 (C-22), 39.7 (C-12), 39.3
(C-15), 37.6 (C-8), 34.3 (C-24), 29.3 (C-6), 27.8 (C-25), 27.3 (C-7),
25.8 (C-11), 24.7 (C-21), 22.3 (C-26), 22.2 (C-27), 20.6 (C-23), 14.8
(C-18) MS (APCI), m/z (relative intensity): HRMS m/z: C26H38O3Na
[M+Na]+, cald 421.2719, found 421.2721.
ꢀ
1,3,5(10)-estratriene (22)
The crude residue was purified by flash column chromatography
eluting with 1:1 ethyl acetate:hexane to provide 22 (19.4 mg, 60%)
as a colorless syrup. FTIR (neat) ꢁmax 3411 (OH) cm−1 1H NMR
.
(CDCl3, 400 MHz) ı 7.02 (d, J = 8.42 Hz, 1H, H-1), 6.52 (dd, J = 8.42,
2.59 Hz, 1H, H-2), 6.49 (d, J = 2.59 Hz, 1H, H-4), 3.73 (m, 1H, H-16),
2.80 (m, 2H, H-6), 2.15 (m, 1H, H-9), 1.96 (m, 1H, H-12), 1.92 (m,
1H, H-11), 1.74 (m, 1H, H-15), 1.68 (m, 2H, H-7), 1.62 (m, 1H, H-17),
1.61 (m, 2H, H-14, H-23), 1.60 (m, 1H, H-11, H-22), 1.47 (m, 1H, H-
8), 1.45 (m, 2H, H-15,H-24), 1.40 (m, 2H, H-11, H-21), 1.24 (m, 1H,
H-12), 1.00 (s, 9H, Me3CSi), 0.80 (d, J = 6.65 Hz, 6H, H-26, H-27), 0.79
(s, 6H, Me2Si), 0.73 (s, 3H, H-18), 13C NMR (CDCl3, 100 MHz) ı 153.1
(C-3), 137.7 (C-5), 131.6 (C-10), 125.9 (C-1), 119.7 (C-4), 117.3 (C-2),
97.2 (C), 75.3 (C-20), 72.5 (C-16), 67.5 (C-17), 52.5 (C-14), 44.1 (C-
9), 43.5 (C-22), 43.1 (C-13), 39.8 (C-12), 39.2 (C-15), 37.7 (C-8), 34.2
(C-24), 31.9 (Me), 29.5 (C-6), 27.6 (C-25), 27.4 (C-7), 27.2 (MeCSi),
25.8 (C-11), 25.5 (MeCSi), 25.5 (MeCSi), 24.9 (C-21), 22.2 (C-26),
22.1 (C-27),20.5 (C-23), 17.8 (CSi), 14.9 (C-18), −0.40 (MeSi), −0.4
(MeSi). HRMS m/z: C32H54O3SiNa [M+Na]+, cald 537.3740, found
537.3715.
2.4.7. 3ˇ-Acetoxyallopregnan-20-one (34)
The crude product was purified by flash column chromatogra-
phy (3:7 ethyl acetate:hexane) to afford 34 (150 mg, 99%) as a white
solid, mp 146–147 ◦C. FTIR (KBr), ꢁmax 1738, 1691 cm−1
.
1H NMR
2.6. (20S)-20-Hydrooxycholest-1-ene-3,16-dione (3),
(20S)-20-hydroxy cholest-4-ene-3,16-dione (4) and
(20S)-20-hydroxy cholest-1,4-diene-3,16-dione (5)
(CDCl3, 400 MHz) ı 4.59–4.73 (m, 1H, C3), 2.29–2.40 (m, 17H, CH),
2.26 (s, 3H, CH3), 2.11 (s, 3H, CH3), 1.15–1.86 (m, 17H, CH and CH2)
0.91–1.06 (m, 2H, CH), 0.7–0.77 (m, 1H, CH2), 0.81 (s, 3H, CH3).
13C NMR (CDCl3, 100 MHz) ı 209.7 (C O), 170.7 (C O), 155.5 (C-
3), 148.4 (C-10), 73.6 (CH), 70.8 (CH), 56.3 (C-13), 54.7 (CH), 46.3
(CH), 44.9 (CH), 36.6 (CH), 35.7 (CH2), 34.7 (CH2), 29.3 (CH2), 27.5
(CH2), 27.1 (CH3), 26.2 (CH2), 21.0 (CH3), 15.8 (CH3). MS (APCI), m/z
(relative intensity): 360 (M+,39), 315 (49), 43 (100).
A mixture of meta-iodoxybenzoic acid (76 mg, 0.027 mmol)
and diphenyl diselenide (8.0 mg, 0.02 mmol) in toluene (5 mL)
was refluxed until the yellow color of diphenyl diselenide dis-
appeared. The solution of (20S)-20-hydroxycholestane-3,16-dione
(1) (95 mg, 0.23 mmol) in toluene (3 mL) was introduced to this
mixture. The mixture was further heated under reflux for 3 h.
The reaction mixture was cooled to room temperature and par-
titioned with methylene chloride and water. The organic layer
was separated and dried over anhydrous sodium sulphate, fil-
tered and concentrated in vacuo. The crude residue was purified by
flash column chromatography (2:8 ethyl acetate:hexane) to yield
(20S)-20-hydrooxycholest-1-ene-3,16-dione (3) (17.8 mg, 18.9%),
(20S)-20-hydroxy cholest-4-ene-3,16-dione (4) (12.1 mg, 12.9%),
and (20S)-20-hydroxy cholest-1,4-diene-3,16-dione (5) (29.2 mg,
31.2%) as a pale yellow wax.
2.5. General procedure for preparation of compounds 15 and 22
To a stirred solution of compounds 14 or 21 (0.083 mmol) in
dioxane (4 mL) was added 70% aqueous acetic acid (4 mL) and the
mixture was heated at 50 ◦C for 24 h. The reaction mixture was
neutralized with saturated aqueous sodium hydrogen carbonate
and extracted with ethyl acetate. The combined organic layer was
washed with water and the solvent was removed under reduced
pressure. The crude residue was purified by flash column chro-
matography.
Compound 3. FTIR (KBr), ꢂmax 3510, 1729, 1679 cm−1 1H NMR
.
(CDCl3) ı 7.05 (1H, d, J = 10.2 Hz, H-1), 5.80 (1H, d, J = 10.2 Hz, H-2),
2.18 (m, 2H, H-4), 2.20 (m, 1H, H-15), 2.14 (s, 1H, H-17), 2.08 (m,
1H, H-12), 1.87 (m, 1H, H-5), 1.85 (m, 1H, H-11), 1.83 (dd, J = 18.5,
14.1 Hz, 1H, H-15), 1.82 (m, 1H, H-8), 1.59 (m, 2H, H-7), 1.55 (m, 2H,
H-22), 1.48 (m, 1H, H-25), 1.47 (m, 2H, H-11, H-12), 1.45 (m, 2H, H-
6), 1.40 (m, 1H, H-14), 1.30 (m, 2H, H-23), 1.19 (s, 3H, H-21), 1.10 (m,
1H, H-9), 1.08 (m, 2H, H-24), 1.03 (m, 1H, H-8), 0.98 (s, 3H, H-19),
0.89 (s, 3H, H-18), 0.81 (d, J = 6.6 Hz, 3H, H-27), 0.80 (d, J = 6.6 Hz,
3H, H-26). 13C NMR (CDCl3) ı 220.8 (C-16), 199.9 (C-3); 157.3 (C-
1), 127.7 (C-2), 73.9 (C-20), 71.4 (C-17), 50.8 (C-14), 49.7 (C-9), 44.2
(C-5), 43.1 (C-13), 42.4 (C-22), 40.8 (C-4), 39.5 (C-24), 39.3 (C-12),
39.2 (C-15), 39.0 (C-10), 34.81 (C-8), 31.3 (C-7), 28.0 (C-25), 27.3
(C-6), 25.4 (C-21), 22.7 (C-27), 22.6 (C-26), 20.6 (C-23), 21.0 (C-11),
14.8 (C-18), 13.40 (C-19). MS (APCI), m/z (relative intensity): 415
[(M+H)+, 65], 397 [(M+H)+−H2O, 100], 329 (6), 313 (M+−C6H13, 5),
287 (33).
2.5.1. 3,16S,20S,24-Tetrahydroxy-24-cholestan-ꢀ1,3,5(10)
-estratriene (15)
The crude residue was purified by flash column chromatogra-
phy eluting with 1:1 ethyl acetate:hexane to provide 15 (19.4 mg,
56%) as a white solid, mp 224–226 ◦C. FTIR (KBr) ꢂmax 3366 (OH)
cm−1. 1H NMR (CDCl3) ı 7.02 (d, J = 8.44 Hz, 1H, H-2), 6.54 (dd, J = 8.4,
2.6 Hz, 1H, H-2), 6.48 (d, J = 2.64, Hz, 1H, H-4), 4.55 (m, 1H, H-16),
3.24 (m, 1H, H-24), 2.76 (m, 2H, H-6), 2.22 (m, 1H, H-15), 2.16 (m,
1H, H-12), 2.11 (m, 1H, H-11), 2.08 (m, 1H, H-9), 1.52 (m, 1H, H-
22), 1.75 (m, 1H, H-7), 1.60 (m, 1H, H-25), 1.58 (m, 1H, H-22), 1.52
(m, 1H, H-11), 1.45 (m, 1H, H-8), 1.40 (m, 1H, H-15), 1.30 (m, 1H,
H-12), 1.29 (m, 2H, H-7, H-23), 1.27 (m, 1H, H-17), 1.25 (s, 3H, H-
21), 1.20 (m, 1H, H-23), 1.07 (s, 1H, H-18), 0.95 (m, 1H, H-14), 0.85
(m, 6H, H-26, H-27). 13C NMR (CDCl3) ı: 154.0 (C-3), 137.8 (C-5),
131.7 (C-10), 126.1 (C-1), 115.1 (C-4), 112.6 (C-2), 77.2 (C-24), 76.5

436
P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
Compound 4. FTIR (KBr), ꢂmax 3447, 1725, 1676 cm−1 1H NMR
.
added to destroy the excess lithium. The solvent was removed and
the residue was dissolved in 10% aq. HCl and extracted with methy-
lene chloride. The organic layer was washed with water, dried over
anhydrous sodium sulphate and the solvent was removed in vacuo.
Purification of the crude product by flash column chromatography
eluting with ethyl acetate:hexane (1:9) provided the desired prod-
uct 8 as a white solid (2.47 g, 44%), mp 244–246 ◦C [lit.145–147 ◦C]
(CDCl3) ı 5.68 (s, 1H, H-4), 2.37 (m, 1H, H-2), 2.27 (m, 1H, H-2), 2.24
(m, 1H, H-15), 2.22 (m, 2H, H-6), 2.13 (s, 1H, H-17), 2.08 (m, 1H, H-
12), 1.96 (m, 1H, H-1), 1.85 (dd, J = 18.4, 13.4 Hz, 1H, H-15), 1.68
(m, 1H, H-11), 1.58 (m, 1H, H-7), 1.48 (m, 2H, H-22), 1.47 (m, 3H,
H-8, H-12, H-25), 1.44 (m, 1H, H-11), 1.40 (m, 1H, H-14), 1.30 (m,
2H, H-23), 1.29 (m,1H, H-1), 1.19 (s, 3H, H-21), 1.15 (s, 3H, H-19),
1.09 (m, 1H, H-9), 1.08 (m, 2H, H-24, 1.02 (m, 1H, H-7), 0.90 (s, 3H,
H-18), 0.80 (d, 6H, J = 6.6 Hz, H-26, H-27). 13C NMR (CDCl3) ı 220.8
(C-16), 199.7 (C-3), 169.9 (C-5), 124.1 (C-4), 73.9 (C-20), 71.2 (C-
17), 50.3 (C-14), 49.7 (C-9), 43.1 (C-13), 42.4 (C-22), 40.9 (C-2), 39.4
(C-24), 39.2 (C-12), 39.1 (C-15), 38.8 (C-1), 38.5 (C-10), 34.0 (C-8),
33.8 (C-6), 32.5 (C-7), 28.0 (C-25), 25.4 (C-21), 22.7 (C-27), 22.6 (C-
26), 20.9 (C-23), 21.0 (C-11), 14.8 (C-18), 13.40 (C-19). MS (APCI),
m/z (relative intensity): 415 [(M+H)+, 100)], 397 [(M+H)+−H2O, 93],
345 (45), 313 (M+−C6H13, 21), 287 (23).
[20]. FTIR (KBr), ꢁmax 3402 (OH), 3062 (CH-Ar), 1609 cm−1 1H NMR
.
(CDCl3, 400 MHz) ı 7.06 (d, J = 8.36 Hz, 1H, H-1), 6.54 (dd, J = 2.76 Hz,
8.36 Hz, 1H, CH-2), 6.48 (d, J = 2.76 Hz, 1H, CH-4), 4.67 (s, OH), 4.37
(m, 1H, H-16), 3.44 (m, 1H, H-26), 3.32 (t, J = 10.83 Hz, 1H, H-26),
2.76 (m, 2H, H-6), 2.01 (m, 1H, H-15), 1.83 (m, 1H, H-17), 1.30 (m,
1H, H-15), 0.92 (d, J = 6.84 Hz, 3H, H-27,), 0.75 (s, 3H, H-18), 0.72
(d, J = 6.32 Hz, 3H, H-21). 13C NMR (CDCl3, 100 MHz) ı 153.7 (C-3),
138.3 (C-5), 132.8 (C-10), 126.3 (C-1), 115.2 (C-4), 112.6 (C-2), 109.3
(C-22), 80.9 (C-16), 66.9 (C-26), 62.3 (CH), 55.3 (C-14), 43.7 (C-9),
41.6 (CH), 40.8 (C-13), 39.9 (C-12), 38.3 (C-8), 31.53 (C-15), 31.40
(CH2), 30.3 (CH), 29.5 (C-6), 28.8 (CH2), 27.8 (C-7), 26.4 (C-11), 17.1
(C-21), 16.4 (C-18), 14.5 (C-21). MS (EI), m/z (relative intensity):
396 (13), 282 (48), 139 (100).
Compound 5. FTIR (KBr), ꢂmax 3455 (OH), 1726 (C O), 1662
(C O) cm−1 1H NMR (CDCl3) ı 6.97 (d, J = 10.2 Hz, 1H, H-1), 6.19
.
(dd, J = 10.12, 1.88 Hz, 1H, H-2), 6.02 (d, m, 1H, H-4), 0.93 (s,
3H, H-18), 0.80 (d, 6H, J = 6.6 Hz, H-26, H-27). 13C NMR (CDCl3):
ı: 220.8 (C-16), 186.1 (C-3); 170.7 (C-5), 154.9 (CH-1), 127.8
(CH-4), 124.3 (CH-2), 73.8 (C-20), 71.4 (CH-17), 51.9 (CH-14),
49.9 (CH-9), 43.4 (C-10), 43.1 (C-13), 42.4 (CH2-22), 39.4 (CH2-
24), 39.3 (C H2-12), 38.9 (CH2-15), 33.9 (C-8), 33.4 (CH2-6),
32.0 (C-7), 28.0 (C-25); 25.3 (C-21), 22.7 (C-27), 22.6 (C-26),
22.2 (C-11), 21.1 (C-23), 18.7 (C-19), 14.6 (C-18). MS (APCI),
m/z (relative intensity): 413 (62), 412 (13), 395.2 (100), 285.2
(89). HRMS m/z: C27H40O3Na [M+Na]+, cald 435.2875, found
435.2883.
2.9. 3-Acetoxy-16ˇ-acetoxymethylvaleroyloxy-17-acetyl-
ꢀ
1,3,5(10)-estratriene (9)
A mixture of 19-nor-ꢀ1,3,5(10)-spirostatriene-3-ol (8) (300 mg,
0.78 mmol), acetic anhydride (5.4 mL), ammonium chloride
(83.14 mg, 1.56 mmol) and pyridine (0.06 mL) was heated to
125–135 ◦C and kept at that temperature for 12 h. After cooling
down, the reaction mixture was neutralized with saturated sodium
hydrogen carbonate then extracted with methylene chloride. The
organic layer was washed with water, dried over anhydrous sodium
sulphate, filtered and concentrated in vacuo. The crude residue was
used in the next step without further purification.
2.7. Spirosta-ꢀ1,4-diene-3-one (7)
A mixture of meta-iodoxybenzoic acid (43 g, 153.85 mmol) and
diphenyl diselenide (972 mg, 3.08 mmol) in toluene (50 mL) was
refluxed until the yellow color of diphenyl diselenide disappeared.
The solution of tigogenin (6) (6.4 g, 15.38 mmol) in toluene (50 mL)
was introduced to this mixture. The mixture was further heated
under reflux for 7 h. The reaction mixture was cooled to room
temperature and partitioned with methylene chloride and water.
The organic layer was separated and dried over anhydrous sodium
sulphate and concentrated under vacuo. The residue was purified
by flash column chromatography to provide 7 as a white solid
The crude residue was dissolved in 1,2-dichloroethane (0.7 mL),
water (0.1 mL) and acetic acid (0.25 mL). The mixture was cooled
to 0 ◦C. A solution of chromium trioxide (105 mg, 1.05 mmol) in
water (2.19 mL) and acetic acid (0.25 mL) was added (the temper-
ature was kept below 7 ◦C). The mixture was allowed to warm to
room temperature and stirred for another 2 h. A solution of sodium
chloride (124 mg) in water (1.66 mL) and methanol (1.66 mL) was
introduced and the mixture was stirred for 1 h. The reaction mix-
ture was neutralized using sodium bicarbonate and extracted with
methylene chloride and the organic phase was washed with water,
dried over anhydrous sodium sulphate and filtered. The filtrate was
evaporated off solvent and the crude residue was purified by flash
column chromatography eluting with ethyl acetate:hexane (1:9)
to provide 9 (148 mg, 38%) as a pale yellow syrup. FTIR (neat),
(4.37 g, 70%), mp 182–184 ◦C. FTIR (KBr), ꢁmax 1664 (C O) cm−1
.
1H NMR (CDCl3, 400 MHz) ı 6.97 (d, J = 10.14 Hz, 1H, H-1), 6.2 (dd,
J = 10.2 Hz, 1.99 Hz, 1H, H-2), 5.99 (m, 1H, H-4), 4.31 (m, 1H, H-
16), 3.39 (m, 1H, H-26), 3.28 (m, 1H, H-26), 1.94 (m,1H, H-15),
1.27 (m, 1H, H-15), 1.18 (s, 3H, H-19), 0.89 (d, J = 7 Hz, 3H, H-27,),
0.78 (s, 3H, H-18), 0.71 (d, J = 6.36 Hz, 3H, H-21). 13C NMR (CDCl3,
100 MHz) ı 186.1 (C-3), 168.8 (C-10), 155.6 (C-1), 127.3 (C-4), 123.7
(C-2), 109.6 (C-5), 80.3 (C-16), 66.7 (C-26), 61.9 (CH), 55.1 (CH),
52.2 (CH), 43.4 (C), 42.5 (CH), 40.3 (C-15), 39.3 (C), 34.9 (CH), 33.6
(CH2), 32.6 (CH2), 31.8 (CH2), 31.2 (CH2), 30.1 (CH), 28.7 (CH2), 22.6
(CH2), 18.6 (C-19), 16.9 (C-21), 16.7 (C-18), 14.3 (C-27). MS (EI),
m/z (relative intensity): 410 (18), 351 (14), 289 (10), 181 (28), 139
(100).
ꢂmax 1756 (C O), 1734 (C O), 1712 (C O) cm−1 1H NMR (CDCl3,
.
400 MHz) ı 7.19 (d, J = 8.48, 1H, CH-1), 6.76 (dd, J = 8.48, 2.56, 1H,
CH-2), 6.70 (d, J = 2.48, 1H, CH-4), 5.49 (m, 1H, CH-16), 3.81 (d, 6.12,
2H, CH2-26), 2.78 (m, 2H, CH2-6), 2.46 (m, 1H, CHAHB-15), 2.40 (d,
7.60, 1H, CH-17), 2.21 (m, 1H, CH-9), 2.20 (s, 3H, CH3C O), 2.19 (m,
1H, CHAHB-11), 2.15 (m, 1H, CHAHB-7), 2.09 (m, 1H, CHAHB-12),
2.01 (s, 3H, Me-21), 1.96 (s, 3H, CH3C O), 1.77 (m, 3H, CH2-22, CH-
27), 1.63 (m, 1H, CHAHB-23), 1.50 (m, 2H, CHAHB-7, CH-8), 1.37 (m,
1H, CHAHB-15, CHAHB-23), 1.35 (m, 1H, CHAHB-11), 1.19 (m, 1H,
CHAHB-12), 1.05 (m, 1H, CH-14), 0.99 (s, 3H, Me-18), 0.85 (s, 3H,
Me-25). 13C NMR (CDCl3, 100 MHz) ı 260.0 (CO), 172.9 (CO), 171.1
(CO), 169.8 (CO), 148.45 (C-3), 137.9 (C-5), 137.6 (C-10), 126.24 (C-
1), 121.5 (C-4), 118.61 (C-2), 74.3 (C-16), 68.7 (C-26), 66.7 (C-17),
53.0 (C-14), 43.9 (C-9), 42.6 (C-13), 37.97 (C-12), 37.1 (C-8), 34.8
(C-15), 32.0 (CH3C O), 31.9 (C-22), 30.5 (C-24), 29.3 (C-6), 28.2 (C-
23), 27.3 (C-7), 25.7 (C-11), 21.0 (C-21), 20.8 (CH3C O), 16.3 (C-25),
13.4 (C-18). MS (APCI), m/z (relative intensity): 512 (52), 339 (100),
297 (34). HRMS m/z C30H40O7Na [M+Na]+, calcd 535.2672, found
535.2673.
2.8. 19-Nor-ꢀ1,3,5(10)-22˛-spirostatriene-3-ol (8)
A lithium was cut into small pieces and introduced to the
mixture of biphenyl (17.3 g, 112.1 mmol) in dry tetrahydrofuran
(100 mL) under N2 atmosphere at room temperature. The mixture
was stirred under reflux for 0.5 h and the solution had turned blue.
The mixture was treated dropwise, under N2, with the solution of
7 (6.5 g, 16.0 mmol) and diphenyl methane (7.8 mL) in dry tetrahy-
drofuran. After refluxing for 2 h and cooled down, methanol was

P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
437
2.10. 3-Hydroxy-(16S,20S)-16,20-acetonide-24-cholestene-
1,3,5(10)-estratriene (12)
was extracted with methylene chloride. The combined organic
layers were dried over anhydrous sodium sulphate, filtered and
concentrated in vacuo. The crude product was purified by flash col-
umn chromatography eluting with 2:98 ethyl acetate:hexane to
provide 16 (106 mg, 79%) as a colorless syrup. FTIR (KBr), ꢁmax 1607,
ꢀ
To
a stirred solution of 10 (250 mg, 0.63 mmol) in 2,2-
dimethoxypropane (7.7 mL) was added p-toluenesulphonic acid
(6 mg, 0.032 mmol) at room temperature. The mixture was stirred
for 2 h and triethylamine was added (3 drops). The mixture was
added to the ice-water and extracted with methylene chloride. The
combined organic layers were dried over anhydrous sodium sul-
phate, filtered and concentrated in vacuo. The crude product was
purified by flash column chromatography eluting with 1:9 ethyl
acetate:hexane to provide 12 as a colorless solid (192.7 mg, 70%),
1496, 1247 cm−1 1H NMR (CDCl3, 400 MHz) ı 7.03 (d, J = 8.44 Hz,
.
1H, H-1), 6.53 (dd, J = 8.44, 2.68 Hz, 1H, H-2), 6.47 (d, J = 2.56 Hz, 1H,
H-4), 5.01 (m, 1H, H-24), 4.45 (m, 1H, H-16), 2.73 (m, 2H, H-6), 2.20
(m, 1H, H-15), 2.15 (m, 1H, H-9), 2.14 (m, 2H, H-23), 1.97 (m, 1H, H-
12), 1.93 (m, 1H, H-11), 1.83 (m, 2H, H-7), 1.81 (m, 1H, H-22), 1.62 (s,
3H, H-27), 1.56 (s, 3H, H-26), 1.44 (m, 1H, H-15), 1.43 (m, 1H, H-22),
1.42 (m, 1H, H-8), 1.40 (m, 4H, H-11, H-21), 1.29 (m, 1H, H-12), 1.06
(s, 9H, H-18, MeX2), 1.02 (m, 1H, H-14), 0.90 (s, 9H, Me3CSi), 0.89
(m, 1H, H-17), 0.80 (s, 6H, Me2Si). 13C NMR (CDCl3, 100 MHz) ı 153.3
(C-3), 137.8 (C-5), 131.4 (C-10), 131.1 (C-25), 126.0 (C-1), 124.5 (C-
24), 119.9 (C-4), 117.1 (C-2), 97.1 (C), 75.1 (C-20), 68.7 (C-16), 55.6
(C-17), 53.9 (C-14), 44.2 (C-22), 44.0 (C-9), 42.6 (C-13), 40.0 (C-12),
37.9 (C-8), 33.5 (C-15), 31.8 (Me), 29.6 (C-6), 26.2 (C-11), 27.5 (C-7),
25.7 (MeCSi), 25.7 (MeCSi), 25.7 (MeCSi), 25.7 (C-27), 25.6 (C-21),
24.2 (Me), 22.6 (C-23), 18.2 (C-26), 17.7 (CSi), 14.8 (C-18), −0.40
(MeSi), −0.4 (MeSi). MS (APCI), m/z (relative intensity): 553 (100),
495 (30).
mp 68–70 ◦C. FTIR (KBr), ꢁmax 3373 (OH) cm−1 1H NMR (CDCl3,
.
400 MHz) ı 7.06 (d, J = 8.40 Hz, 1H, H-1), 6.55 (dd, J = 8.40, 2.68 Hz,
1H, H-2), 6.48 (d, J = 2.68 Hz, 1H, H-4), 5.01 (m, 1H, H-24), 4.44 (m,
1H, H-16), 2.75 (m, 2H, H-6), 2.19 (m, 1H, H-15), 2.14 (m, 1H, H-9),
2.12 (m, 1H, H-11), 2.09 (m, 1H, H-12), 1.97 (m, 2H, H-23), 1.83 (m,
1H, H-22), 1.80 (m, 2H, H-7), 1.62 (s, 3H, Me), 1.55 (s, 3H, Me), 1.50
(m, 1H, H-22), 1.46 (m, 2H, H-8, H-11), 1.42 (m, 1H, H-15), 1.41
(s, 3H, H-21), 1.29 (m, 1H, H-12), 1.28 (s, 3H, H-27), 1.27 (s, 3H,
H-26), 1.06 (s, 3H, H-18), 1.02 (m, 1H, H-14), 0.98 (m, 1H, H-17).
13C NMR (CDCl3, 100 MHz) ı 153.3 (C-3), 138.2 (C-5), 132.7 (C-10),
131.4 (C25), 126.4 (C-1), 124.4 (C-24), 115.2 (C-4), 112.6 (C-2), 97.2
(C), 75.2 (C-20), 68.7 (C-16), 55.6 (C-17), 53.7 (C-14), 44.2 (C-22),
43.9 (C-9), 42.6 (C-13), 40.0 (C-12), 37.9 (C-8), 33.5 (C-15), 31.8 (C-
26), 29.6 (C-6), 27.5 (C-7) 26.2 (C-11),), 25.7 (C-27), 25.6 (Me), 25.2
(C-21), 22.6 (C-23), 17.7 (Me), 14.8 (C-18). MS (APCI), m/z (relative
intensity): 439 (30), 381 (100), 363 (76).
2.13. Synthesis of 3-tert-butyl-dimethylsiloxy-(16S,20S)
-16,20-acetonide-24-ol-chlorestane-ꢀ1,3,5(10)-estratriene (17)
To a solution of 16 (106 mg, 0.20 mmol) in dry THF (5.6 mL) was
added portionwise, with stirring, BH3.THF complex (ca 1 M in THF,
0.2 mL) during a period of 2 h. Hydrogen peroxide solution (35%,
0.6 mL) and 1N sodium hydroxide (0.8 mL) was then added and
stirring was continued for 16 h. Water was added and the reac-
tion mixture was extracted with methylene chloride. The combined
organic layers were dried over anhydrous sodium sulphate, filtered
and concentrated in vacuo. The crude product was purified by flash
column chromatography eluting with 1:19 ethyl acetate:hexane
to provide 17 as a white solid (53 mg, 48%) mp 69–71 ◦C. FTIR
2.11. 3,24-Dihydroxy-(16S,20S)-16,20-acetonide-cholestan-
ꢀ
1,3,5(10)-estratriene (14)
To a solution of 12 (100 mg, 0.228 mmol) in dry THF (5.8 mL)
was added portionwise, with stirring, BH3.THF complex (ca. 1 M
in THF, 0.22 mL) during a period of 2 h at 0 ◦C. Hydrogen perox-
ide solution (35%, 1.2 mL) and 1N sodium hydroxide (1.6 mL) was
then added and continued stirring for 16 h. Water was added and
the mixture was extracted with methylene chloride. The combined
organic layers were dried over anhydrous sodium sulphate, filtered
and concentrated in vacuo. The crude product was purified by flash
column chromatography eluting with 1:4 ethyl acetate:hexane to
provide 14 as a colorless wax (67.1 mg, 64%). FTIR (KBr), ꢂmax 3411
(KBr), ꢁmax 3429 (OH) cm−1 1H NMR (CDCl3, 400 MHz) ı 6.90 (d,
.
J = 8.44 Hz, 1H, H-1), 6.42 (dd, J = 8.44, 2.44 Hz, 1H, H-2), 6.36 (d,
J = 2.48, 1H, H-4), 4.33 (m, 1H, H-16), 3.13 (m, 1H, H-24), 2.26 (m,
2H, H-6), 2.06 (m, 2H, H-11, H-15), 2.05 (m, 1H, H-22), 2.00 (m, 1H,
H-9), 1.93 (m, 1H, H-12), 1.67 (m, 2H, H-7), 1.49 (m, 1H, H-25), 1.42
(m, 2H, H-23), 1.37 (m, 2H, H-22), 1.30 (s, 3H, Me), 1.33 (m, 1H,
H-8), 1.27 (s, 3H, Me), 1.16 (s, 3H, H-21), 1.13 (m, 1H, H-15), 1.11
(m, 1H, H-11), 1.10 (m, 1H, H-12), 1.08 (m, 1H, H-14), 0.95 (s, 3H,
H-18), 0.86 (m, 1H, H-17), 0.79 (s, 9H, Me3CSi), 0.75 (s, 6H, H-26,
H-27), 0.66 (s, 6H, Me2Si). 13C NMR (CDCl3, 100 MHz) ı 153.3 (C-3),
137.8 (C-5), 133.1 (C-10), 125.6 (C-1), 119.9 (C-4), 117.1 (C-2), 97.5
(C), 77.6 (C-24), 74.3 (C-20), 68.8 (C-16), 56.0 (C-17), 53.7 (C-14),
44.0 (C-9), 42.7 (C-13), 44.1 (C-22), 40.1 (C-12), 37.8 (C-8), 33.9 (C-
25), 33.6 (C-15), 31.6 (Me), 29.6 (C-6), 28.4 (C-23), 27.6 (C-7), 26.1
(C-11), 25.9 (C-21), 25.7 (MeCSi), 25.7 (MeCSi), 25.7 (MeCSi), 24.4
(Me), 19.0 (C-27), 15.0 (C-18), 18.2 (CSi), 17.4 (C-26), −0.40 (MeSi),
−0.40 (MeSi). MS (APCI), m/z (relative intensity): 571 (100), 495
(40). HRMS: 593.4003 C35H58O4NaSi require 593.4002.
(OH) cm−1 1H NMR (CDCl3) ı 7.05 (d, J = 8.4 Hz, 1H, H-1), 6.55 (dd,
.
J = 8.32, 2.40 Hz, 1H, H-2), 6.49 (d, J = 2.16 Hz, 1H, H-4), 4.43 (m, 1H,
H-16), 3.26 (m, 1H, H-24), 2.73 (m, 2H, H-6), 2.18 (m, 1H, H-15),
2.15 (m, 1H, H-11), 2.08 (m, 1H, H-9), 2.04 (m, 2H, H-12), 1.64 (m,
2H, H-7), 1.60 (m, 1H, H-25), 1.52 (m, 1H, H-11), 1.43 (m, 1H, H-8),
1.41 (s, 3H, H-30), 1.34 (m, 6H, H-15, H-22, H-29), 1.17 (s, 3H, H-21),
1.05 (s, 3H, H-18), 1.02 (m, 1H, H-14), 1.0 (m, 1H, H-17), 0.87 (m,
6H, H-26, H-27). 13C NMR (CDCl3) ı 153.6 (C-3), 138.1 (C-5), 132.5
(C-10), 126.3 (C-1), 115.3 (C-4), 112.7 (C-2), 97.6 (C), 77.4 (C-24),
76.5 (C-20), 68.8 (C-16), 56.2 (C-17), 53.6 (C-14), 43.8 (C-9), 42.7
(C-13), 40.9 (C-12), 40.0 (C-22), 37.8 (C-8), 33.5 (C-25), 33.3 (C-15),
31.5 (Me), 29.5 (C-6), 28.3 (C-23), 27.5 (C-7), 26.2 (C-11), 25.8 (C-
21), 24.4 (Me), 18.9 (Me-26), 17.0 (C-27), 15.1 (C-18). MS (APCI),
m/z (relative intensity):457 (31), 456 (11), 381 (100). HRMS m/z:
2.14. Synthesis of 3-hydroxy (16S,20S)-16,20-acetonide-24-
C
29H44O4Na [M+Na]+, cald. 479.3137, found 479.3141.
ol-chlorestane-ꢀ1,3,5(10)-estratriene (19)
2.12. 3-tert-Butyl-dimethylsiloxy-(16S,20S)-16,20-acetonide-24-
To a stirred solution of 3-tert-butyl-dimethylsiloxy-(16S,20S)-
cholestene-ꢀ1,3,5(10)-estratriene (16)
16,20-acetonide-24-one-chlorestane-ꢀ1,3,5(10)-estratriene
(18)
(60 mg, 0.088 mmol) in THF was added TBAF (0.44 mmol) at room
temperature. After stirring for 10 h, water was added and the
reaction mixture was extracted with ether. The combined organic
phases were washed with water, dried over anhydrous sodium
sulphate, filtered and concentrated in vacuo. The crude product
was purified by flash column chromatography eluting with 3:97
To
a stirred solution of 12 (107 mg, 0.24 mmol) in DMF
(0.4 mL) and methylene chloride (5 mL) was added imidazole
(50 mg, 0.73 mmol) and tert-butyldimethylsilyl chloride (73 mg,
0.49 mmol). The reaction mixture was stirred at ambient tempera-
ture for 3 h, then water was added (3 drops). The reaction mixture

438
P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
ethyl acetate:hexane to provide compound 19 as a colorless syrup.
(18.6 mg, 60%), FTIR (KBr), ꢁmax 3373 (OH), 1708 (C O) cm−1 1H
bicarbonate and extracted with methylene chloride and the organic
phase was washed with water, dried over anhydrous sodium sul-
phate, filtered and the filtrate was concentrated. The residue was
dissolved in benzene (30 mL) and basic alumina (1 g) was added
and stirred for 16 h. Filtered and plenty rinsed with methylene
chloride. The filtrate was evaporated off solvent. The crude prod-
uct was purified by flash column chromatography eluting with 1:9
ethyl acetate:hexane to give 23 as a white solid (146.6 mg, 58%),
mp 154–156 ◦C [lit 161–162 ◦C] [21] FTIR (KBr), ꢁmax 3062, 1609
.
NMR (CDCl3, 400 MHz) ı 7.05 (d, J = 8.42 Hz, 1H, H-1), 6.54 (dd,
J = 8.42, 2.65 Hz, 1H, H-2), 6.48 (d, J = 2.65 Hz, 1H, H-4), 4.42 (m,
1H, H-16), 2.80 (m, 2H, H-6), 2.45 (m, J = 6.80 Hz, 1H, H-25), 2.43
(m, 2H, H-23), 2.20 (m, 1H, H-15), 2.16 (m, 1H, H-9), 2.12 (m, 1H,
H-22), 2.10 (m, 1H, H-11), 2.08 (m, 1H, H-12), 1.77 (m, 2H, H-7),
1.50 (m, 1H, H-22), 1.48 (m, 1H, H-11), 1.47 (m, 1H, H-8), 1.43 (m,
1H, H-15), 1.29 (m, 1H, H-12), 1.23 (s, 3H, Me), 1.15 (s, 3H, Me),
1.18 (s, 3H, H-21), 1.08 (s, 3H, H-18), 1.02 (m, 1H, H-14), 0.95 (m,
1H, H-17), 0.94 (d, J = 6.8 Hz, 6H, H-26, H-27). 13C NMR (CDCl3,
100 MHz) ı 214.2 (CH-24), 154.0 (C-3), 138.1 (C-5), 132.9 (C-10),
126.2 (C-1), 115.0 (C4), 112.7 (C-2), 97.0 (C-28), 74.5 (C-20), 68.8
(C-16), 56.1 (C-17), 53.8 (C-14), 43.9 (C-9), 42.8 (C-13), 41.5 (C-25),
40.1 (C-12), 37.9 (C-8), 36.5 (C-22), 34.6 (C-23), 33.5 (C-15), 29.6
(C-6), 28.7 (Me), 27.6 (C-7), 26.1 (C-11), 25.7 (C-21), 24.1 (Me), 18.3
(C-27), 18.2 (C-26), 18.3 (C-18). HRMS m/z: C29H42O4Na [M+Na]+,
cald 477.2981, found 477.2991.
(C O) cm−1 1H NMR (CDCl3, 400 MHz) ı 7.32 (d, J = 8.43 Hz, 1H,
.
CH-1), 6.88 (dd, J = 8.4, 2.6 Hz, 1H, CH-2), 6.83 (d, J = 2.4 Hz, H, CH-4),
6.78 (dd, J = 3.3, 1.8 Hz, 1H, CH-16), 2.93 (m, 2H, CH2-6), 2.32 (s, 3H,
Me), 2.32 (s, 3H, Me), 0.95 (s, 3H, Me). 13C NMR (CDCl3, 100 MHz)
ı 196.7 (C O), 169.8 (C O), 155.4 (C-3), 148.4 (C-10), 148.4 (C-17),
144.1 7 (C-16), 137.9 (C-5), 126.1 (C-1), 121.4 (C-4), 118.5 (C-2), 55.6
(C-13), 46.4 (CH), 44.3 (CH), 36.6 (CH), 34.7 (CH2), 31.9 (CH2), 29.3
(CH2), 27.5 (CH2), 27.1 (CH3), 26.2 (CH2), 21.0 (CH3), 15.8 (CH3). MS
(EI), m/z (relative intensity): 296 (100), 159 (94).
2.15. Attempted hydrolysis of 3-tert-butyl-dimethylsiloxy-
(16S,20S16S,20S)-16,20-acetonide-24-one-chlorestane-ꢀ1,3,5(10)
-estratriene (20)
2.17. 16˛,17˛-Epoxy-17-acetyl-ꢀ1,3,5(10)-estratriene (24)
To a stirred solution of 23 (45 mg) in methanol (5 mL) was added
2.5N potassium hydroxide (1 mL) at room temperature. The reac-
tion mixture was cooled to 0 ◦C and then was treated with 30%
hydrogen peroxide (1 mL). The mixture was stirred for 16 h. The sol-
vent was removed, extracted with methylene chloride, dried over
anhydrous sodium sulphate and concentrated in vacuo. The crude
product was purified by flash column chromatography eluting with
3:7 ethyl acetate:hexane to give 24 as a white solid (37 mg, 90%),
mp 224–226 ◦C [lit 234.6 ◦C] [22] FTIR (KBr), ꢁmax 3468 (OH), 3018
To a stirred solution of 3-tert-butyl-dimethylsiloxy-(16S,20S)-
16,20-acetonide-24-one-chlorestane-ꢀ1,3,5(10)-estratriene
(18)
(56 mg, 0.1045 mmol) in THF (3 mL) was added 10% aqueous
hydrochloric acid (3 drops) at room temperature. After being
stirred for 5 min, the solvent was removed. The reaction mixture
was neutralized by sodium hydrogen carbonate and extracted with
methylene chloride. The combined organic layers were washed
with water, dried over anhydrous sodium sulphate, filtered and
concentrated in vacuo. The crude product was purified by flash
column chromatography eluting with 3:97 ethyl acetate:hexane
to provide cyclic compound 20 as a pale yellow solid (25.8 mg,
(CH-Ar), 1686 (C O), 1620 cm−1 1H NMR (CDCl3, 400 MHz) ı 7.15
.
(d, J = 8.46 Hz, 1H, H-1), 6.65 (dd, J = 8.36, 2.74 Hz, 1H, H-2), 6.58 (d,
J = 2.66 Hz, 1H, H-4), 4.80 (s, 1H), 3.76 (s, 1H, H-16), 2.84 (m, 2H, H-
6), 2.08 (s, 3H, H-21), 1.08 (s, 3H, H-18). 13C NMR (CDCl3, 100 MHz)
ı 204.9 (C-20), 153.4 (C-3), 137.9 (C-10), 132,4 (C-5), 126.3 (C-1),
115.2 (C-4), 112.7 (C-2), 71.1 (CH), 60.5 (CH), 44.5 (CH), 44.0 (CH),
42.1 (C-13), 36.3 (CH), 31.5 (CH2), 29.4 (CH2), 27.4 (CH2), 27.2 (CH2),
26.0 (C-21), 25.9 (CH2), 15.3 (C-18). MS (EI), m/z (relative intensity):
312 (15), 251 (3).
48.4%), mp 65–67 ◦C. FTIR (KBr) ꢁmax1653, 1607, 1498 cm−1 1H
.
NMR (CDCl3, 400 MHz) ı 7.06 (d, J = 8.4 Hz, 1H, H-11), 6.53 (dd,
J = 8.4 Hz, 1H, H-2), 6.47 (d, J = 2.68 Hz, 1H, H-4), 4.31 (m, 1H, H-16),
2.75 (m, 2H, H-6), 2.14 (m, 1H, H-11), 2.13 (m, 2H, H-15), 2.10 (m,
1H, H-9), 2.07 (m, 1H, H-12), 1.88 (m, 2H, H-22), 1.79 (m, 2H, H-7),
1.75 (m, 1H, H-25), 1.53 (m, 1H, H-8), 1.14 (m, 1H, H-11), 1.37 (s,
3H, H-21), 1.32 (m, 1H, H-15), 1.28 (m, 2H, H-23), 1.05 (m, 1H,
H-14), 1.03 (s, 3H, H-18). 0.92 (m, 1H, H-17), 0.91 (d, J = 6.92 Hz, 3H,
H-26), 0.90 (s, 9H, Me3CSi), 0.68 (d, J = 6.9 Hz, 3H, H-27), 0.60 (s, 6H,
(CH3)2Si). 13C NMR (CDCl3, 100 MHz) ı 153.3 (C-3), 137.8 (C-5),
133.2 (C-10), 126.9 (C-1), 120.0 (C-4), 117.1 (C-2), 108.9 (C-24),
82.0 (C-20), 72.1 (C-16), 58.5 (C-17), 54.0 (C-14), 44.0 (C-2), 42.4
(C-13), 40.0 (C-12), 39.8 (C-22), 37.8 (C-8), 35.8 (C-25), 33.2 (C-15),
32.0 (C-23), 29.6 (C-6), 27.7 (C-7), 26.2 (C-11), 25.7 (Me3CSi), 25.1
(C-21), 18.2 (CSi), 17.3 (C-26), 17.0 (C-27), 14.7 (C-18), −0.4 ((C
H3)2Si).
2.18. 3-Benzyloxy-16˛,17˛-epoxy-17-acetyl-ꢀ1,3,5(10)
-estratriene (25)
To a mixture of 16␣,17␣-epoxy-17-acetyl-ꢀ1,3,5(10)-estratriene
(24) (20 mg, 0.064 mmol), K2CO3 (13 mg, 0.096 mmol) in acetone
(2 mL) was added benzyl bromide (0.01 mL, 0.076 mmol) and
refluxed for 6 h. The reaction mixture was quenched with satu-
rated aqueous ammonium chloride and the solvent was removed
under reduced pressure. The mixture was extracted with methy-
lene chloride and the combined organic layers were washed with
water, dried over anhydrous sodium sulphate, filtered and concen-
trated under reduced pressure. The crude product was purified by
flash column chromatography (2:8 ethyl acetate:hexane) to pro-
vide 25 as a colorless wax (25 mg, 97%), mp 100–101 ◦C. FTIR (KBr),
2.16. 3,17-Acethyl-ꢀ1,3,5(10),16-estratetraene (23)
A
mixture of 19-nor-ꢀ1,3,5(10)-22␣-spirostatriene-3-ol (8)
(300 mg, 0.78 mmol), acetic anhydride (5.4 mL), ammonium chlo-
ride (83.14 mg, 1.56 mmol) and pyridine (0.06 mL) was heated to
125–135 ◦C and kept at this temperature for 12 h. After cooling
down, the reaction mixture was dissolved in 1,2-dichloroethane
(0.7 mL), water (0.083 mL), acetic acid (0.25 mL) and acetone (2 mL)
and cooled to 0 ◦C. A solution of chromium trioxide (105 mg,
1.05 mmol, 2.9 eq) in water (2.19 mL) and acetic acid (0.25 mL)
was added (kept the temperature less than 7 ◦C) then the reaction
mixture was stirred in cooled water until TLC indicated the com-
pletion of the reaction. Then a solution of sodium chloride (124 mg)
in water (1.66 mL) and methanol (1.66 mL) was introduced and
stirred for 1 h. The reaction mixture was neutralized using sodium
ꢁmax 1702 (C O) cm−1 1H NMR (CDCl3, 400 MHz) ı 7.36–7.23 (m,
.
5H, ArH), 7.10 (d, J = 8.42 Hz, 1H, CH-1), 6.70 (dd, J = 8.60, 2.68 Hz,
CH-2), 1H), 6.63 (d, J = 2.72 Hz, 1H, CH-4), 4.95 (s, 2H, CH2-Ar), 3.64
(s, 1H, CH-16), 2.78 (m, 2H, CH2-6), 1.98 (s, 3H, CH3-21), 0.99 (s,
3H, CH3-18). 13C NMR (CDCl3, 100 MHz) ı 172.0 (C O), 158.2 (C-
3), 137.9 (C-10), 137.5 (C-Ar), 133.3 (C-5), 128.7 (CH-Ar), 128.1
(CH-Ar), 127.6 (CH-Ar), 126.3 (C-1), 115.1 (C-4), 112.4 (C-2), 70.1
(CH2OBn), 60.7 (C-16), 56.2 (C-16), 44.7 (CH), 44.3 (CH), 42.2 (C-
13), 36.5 (CH), 31.7 (CH2), 29.8 (C-6), 27.7 (CH2), 27.4 (CH2), 26.2
(C-21), 26.1 (CH2), 15.5 (C-18). HRMS m/z: C27H30O3Na [M+Na]+,
cald 425.2093, found 425.2098.

P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
439
2.19. 3-Benzyloxy-16˛-hydroxy-17-acetyl-ꢀ1,3,5(10)-estratriene
(26)
1 h. The reaction mixture was extracted with 1,2-dichloroethane
and washed with water. Solid sodium acetate (1.38 g, 16.9 mmol)
was added to the organic phase and the solvent was distilled off
azeotropically to remove 1,2-dichloroethane then washed with
water, dried over anhydrous sodium sulphate, filtered and the fil-
trate was concentrated. The crude product was purified by flash
column chromatography eluting with 1:2 ethyl acetate:hexane to
give 33 as a white solid (1.67, 55%), mp 166–168 ◦C. FTIR (KBr),
To a solution of 25 (26 mg, 0.0647 mmol) in ethanol (1 mL)
and methylene chloride (0.2 mL) was added hydrazine hydrate
(0.24 mL, 4.975 mmol) at room temperature. After stirring for 5 h,
the solvent was evaporated and stirred vigorously for 30 min with
the mixture of ethyl acetate (3 mL) 10% aqueous hydrochloric acid
(3 mL). The organic layer was separated and the aqueous layer
was washed with ethyl acetate. The combined organic layers were
washed with brine and water, dried over anhydrous sodium sul-
phate. Concentration of the organic extract under reduced pressure,
followed by purification by flash column chromatography using
ethyl acetate:hexane (3:7) afforded the product 26 (24 mg, 92%)
as a white solid, mp 224–226 ◦C. FTIR (KBr), ꢁmax 3436 (OH), 1699
ꢁmax 1738, 1665, 1592 cm−1 1H NMR (CDCl3, 400 MHz) ı 6.70 (s,
.
3H, C16), 4.59–4.73 (m, 1H, C3), 2.29–2.40 (m, 10H, CH), 2.26 (s, 3H,
CH3), 2.03 (s, 3H, CH3), 1.15–1.86 (m, 17H, CH and CH2) 0.91–1.06
(m, 2H, CH2), 0.7–0.77 (m, 1H, CH), 0.85 (s, 3H, CH3). 13C NMR
(CDCl3, 100 MHz) ı 196.8 (C O), 170.7 (C O), 155.5 (C-3), 148.4
(C-10), 145.4 (C-17), 144.1 (C-16), 73.6 (CH), 56.3 (C-13), 54.7 (CH),
46.3 (CH), 44.9 (CH), 36.6 (CH), 35.7 (CH2), 34.7 (CH2), 29.3 (CH2),
27.5 (CH2), 27.1 (CH3), 26.2 (CH2), 21.0 (CH3), 15.8 (CH3). MS (APCI),
m/z (relative intensity): 358 (M+,40), 315 (36), 43 (100).
(C O) cm−1 1H NMR (CDCl3, 400 MHz) ı 7.46–7.30 (m, 5H, Ar (H)),
.
7.19 (d, J = 8.40 Hz, 1H, H-1), 6.65 (dd, J = 8.8, 2.8 Hz, 1H, H-2), 6.55
(d, J = 2.80 Hz, 1H, H-4), 5.05 (s, 2H, CH2Ar), 4.90 (m, 1H, H-16), 2.87
(m, 2H, H-6), 2.63 (d, J = 6.8 Hz, H-17), 2.15 (s, 3H, H-21), 0.68 (s,
3H, H-18). 13C NMR (CDCl3, 100 MHz) ı 177.0 (C O), 156.8 (C-3),
137.9 (C-10), 137.3 (C-Ar), 133.1 (C-5), 128.7 (CH-Ar), 128.6 (CH-
Ar), 127.4 (CH-Ar), 126.1 (C-1), 114.9 (C-4), 112.2 (C-2), 72.1 (C-16),
70.0 (CH2OBn), 55.6 (CH), 46.5 (C-13), 44.2 (CH), 38.9 (CH), 37.0 (C-
17), 34.9 (CH2), 32.0 (CH2), 29.7 (C-6), 27.7 (CH2), 26.4 (CH3), 26.2
(CH2), 15.9 (CH3). MS (APCI), m/z (relative intensity): 387 (100).
HRMS m/z: C27H32O3Na [M+Na]+, cald 427.2249, found 427.2252.
2.22. Biological assays
KB (human epidermoid carcinoma of cavity, ATCC CCL-17),
MCF7 (human breast adenocarcinoma, ATCC HTB-22) and NCI-
H187 (human small cell lung carcinoma, ATCC CRL-5804) were
determined by resazurin microplate assay (REMA) which was a
modified method of the use of a fluorescent dye for mammalian
cell cytotoxicity according to Brien et al. [23]. Ellipticine and dox-
orubicin were used as positive controls. DMSO and sterile distilled
water were used as negative controls. Briefly, cells at a logarithmic
growth phase were harvested and diluted to105 cells/mL in fresh
medium and gently mixed. Test compounds were diluted in culture
medium in a ratio of 1:2 giving 8 concentrations. Five microliters
of test sample and 45 ␮L of cells were put into 384-well microtiter
plates in total volume of 50 ␮L/well. Plates were incubated at 37 ◦C,
5% CO2, for 72 h for KB and MCF7, and 5 days for NCI-H187. After
the incubation periods, 12.5 ␮L of resazurin solution was added to
each well and the plates were incubated at 37 ◦C for 4 h. The plates
were then processed for optical density absorbance analysis using a
Victor 3 Microplate reader at dual wavelengths of 530 and 590 nm.
2.20. Benzyloxy-16˛-acetoxy-17-acetyl-ꢀ1,3,5(10)-estratriene
(28)
To a stirred solution of 3-benzyloxy-16␣-hydroxy-17-acetyl-
ꢀ
1,3,5(10)-estratriene (26) (404 mg, 1 mmol) in pyridine (2 mL) was
added acetic anhydride (2 mL) at 0 ◦C. The resulting mixture was
continued stirring for 3 h. Ice-water was added and the reac-
tion mixture was diluted with methylene chloride and extracted
with water. The combined extracts were dried over anhydrous
sodium sulphate, filtered and concentrated under reduce pressure.
The crude product was purified by flash column chromatogra-
phy (2:8 ethyl acetate:hexane) to afford 28 (430.4 mg, 96.5%) as
a white solid, mp 144–146 ◦C. FTIR (neat), ꢁmax 1736 (C O), 1707
3. Results and discussion
(C O) cm−1 1H NMR (400 MHz) ı 7.35–7.23 (m, 5H, ArH), 7.10 (d,
.
3.1. Chemistry
J = 8.6 Hz, CH-1), 6.70 (dd, J = 8.6, 2.4 Hz, 1H, CH-2), 6.64 (d, J = 2.8 Hz,
1H, CH-4), 5.45 (m, 1H, CH-16), 4.95 (s, 2H, CH2OBn), 2.78 (m, 2H,
CH2-6), 2.67 (d, J = 6.4 Hz, 1H, CH-17), 2.11 (s, 3H, Me-21), 1.94 (s,
3H, Me), 0.61 (s, 3H, Me). 13C NMR (100 MHz) ı 206.4 (C O), 170.6
(C O), 156.8 (C-3), 137.7 (C-10), 137.2 (C-Ar), 132.2 (C-5), 128.5
(CH-Ar), 8 (CH-Ar), 3 (CH-Ar), 126.1 (C-1), 114.8 (C-4), 112.3 (C-2),
75.7 (C-16), 70.1 (C-17), 69.9 (CH2OBn), 53.3 (CH), 44.9 (CH), 43.5
(C-13), 38.8 (CH2), 38.0 (CH), 33.1 (CH2), 31.4 (Me), 25.5 (CH2), 27.5
(CH2), 26.2 (CH2), 21.1 (CH3), 14.5 (CH3). MS (APCI), m/z (relative
intensity): HRMS: 469.2363 C29H34O4Na require 469.2355.
The initial structure–activity relationship studies of 3,16,20-
polyoxygenated steroids system were undertaken. We first
investigated the different structural features of ring A and observed
the effect of these altered systems. In this respect, we prepared
two new steroids, 5 and 32, with differences in the oxidation and
unsaturation on ring A as depicted in Schemes 1 and 4. Oxidation
of 1 using meta-iodoxybenzoic acid and diphenyl diselenide [24]
in refluxed toluene until the reaction was completed (monitoring
by TLC), smoothly afforded a mixture of (20S)-20-hydroxycholest-
1-ene-3,16-dione (3), (20S)-20-hydrooxycholest-4-ene-3,16-dione
(4) and (20S)-20-hydroxycholest-1,4-diene-3,16-dione (5) in 3:2:5
ratio. The 1H NMR of 5 showed three olefinic quinone protons at
6.97, 6.19 and 6.02 ppm.
2.21. 3ˇ-Acetoxy-5˛-pregn-16-ene-20-one (33)
A mixture of tigogenin (6) (3.52 g, 8.45 mmol), acetic anhydride
(17 mL), ammonium chloride (0.45 g, 8.45 mmol) and pyridine
(1.47 mL) was heated to 125–135 ◦C and kept at this tempera-
ture for 9 h. After cooling down, the mixture was dissolved in
1,2-dichloroethane (7 mL), water (1 mL), acetic acid (1 mL) and
cooled to 0 ◦C. A solution of chromium trioxide (1.62 g, 16.2 mmol,
2.9 equiv.) in water (2 mL) and acetic acid (0.7 mL) was added (kept
the temperature less than 7 ◦C) then the reaction mixture was
stirred in cooled water until TLC indicated the completion of the
reaction. Then a solution of sodium chloride (124 mg) in water
(1.66 mL) and methanol (1.66 mL) was introduced and stirred for
Scheme 1.

440
P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
Scheme 2. Reagents and conditions: (a) m-IBX, (PhSe)2, toluene, reflux, 7 h, 70%; (b) Li, Ph2, Ph2CH2, THF, reflux, 2 h, 44%; (c) (i) NH4Cl.py, 135◦C, 12 h.; (ii) CrO3, AcOH, H2O,
(CH2Cl)2, 0 ◦C, 2 h. (d) 5-bromo-2-methyl-2-pentene, Mg, THF, 0 ◦C, 20 min, 60%; (e) H2, 5%Pd/C, EtOAc, EtOH, rt. 16 h, 100%; (f) 2,2-dimethoxypropane, TsOH, rt, 70%; (g) H2,
5% Pd/C, EtOH, rt, 16 h, 99%; (h) BH3, THF (1 M in THF), THF, 0 ◦C, 2 h, then 35% H2O2, 1N NoOH, 64%; (i) 70% AeOH, 50 ◦C, 24 h, 56%.
The aromatic steroid analogues 11 were synthesized from
tigogenin (6) (Scheme 2) by first oxidation using m-iodoxybenzoic
acid (m-IBX) and diphenyl diselenide [24] to give quinone 7. Arom-
atization of 7 by treating with lithium, biphenyl in the presence of
diphenylmethane provided compound 8 in moderate yield. Trans-
formation of the spiro ketal moiety in 8 to the keto ester moiety
in 9 was performed in a one pot preparation by the method devel-
oped by Micovic et al. [25] and modified by Fuchs. [26]. Grignard
reaction of 9 with 2-bromo-5-methyl-2-pentene and Mg in THF
gave 3,16,20-trihydroxy-1,3,5(10)-estratriene (10). Hydrogenation
of unsaturated side chain was achieved by using Pd/C catalyst in
ethanol and ethyl acetate at atmospheric pressure and room tem-
perature. The reaction provided cholesterol-like side chain of 11 in
quantitative yield.
In order to study the effect on the cytotoxicity of a ketone
or hydroxyl at C-24 of cholestane side chain, 1,3-diol of 10 was
protected as acetonide by treatment with 2,2-dimethoxy propane
and TsOH to furnish the corresponding acetonide 12 in 70% yield
(Scheme 2). Hydrogenation of 12 using 5% Pd/C in EtOAc-EtOH
at room temperature provided the corresponding 13 in almost
quantitative yield. Hydroboration of 12 followed by oxidation
using H2O2 provided alcohol 14. Removal of cyclic ketal in 14
with 70% acetic acid gave the corresponding 16,20,24-triol 15. An
attempt to oxidize 2◦ alcohol at the side chain of 14 to the cor-
responding ketone with CrO3 was unsuccessful probably due to
the phenolic hydroxyl group. Therefore the phenolic OH must be
protected.
oxidation of 16 as described above provided alcohol 17. Oxidation
of 17 to ketone 18 was accomplished by using PCC in methylene
chloride. Silyl protecting group of 18 was removed by treatment
with TBAF to give the corresponding phenol 19. Unfortunately
deprotection of cyclic ketal in 18 by 10% HCl did not give the
expected dihydroxy ketone but a new cyclic ketal 20 was formed
instead. In order to study the effect on the cytotoxicity of phenolic
hydroxyl group, the protected phenolic compound was prepared.
The unsaturated side chain of 16 was hydrogenated to give 21
and then 1,3-acetal was deprotected to provide 17,20-dihydroxy
steroid (22).
Apart from the effect of the side chain, we have also examined
the effect of functionality at C16. Therefore the synthesis of the 16␣-
ol and 16-keto analogues were undertaken as shown in Scheme 4.
Transformation of the spiro ketal moiety in 8 to acetyl side chain in
23 was performed in one pot preparation by the method developed
by Micovic et al. [25], and modified by Fuchs et al. [26]. Epoxida-
tion of ␣,␤-unsaturated ketone 23 gave epoxide 24 then protection
of hydroxyl as benzyl ether gave ␣-epoxide 25. Rigeoselective ring
opening of the epoxide by hydrazine in ethanol afforded ␣-hydroxy
ketone 26 in good yield. Debenzylation of 26 by hydrogenation
using Pd/C catalyst provided 27 in nearly quantitative yield. Acety-
lation of 26 with Ac2O gave acetate 28 in 97% yield. Steroid ketone
28 was converted to 29 by the methodology already discussed in
Scheme 2. Hydrogenation of 29 by using Pd/C catalyst gave 30.
Whereas oxidation of 29 with PCC in sodium acetate provided
16-ketosteroid 31 in good yield. Debenzylation of 31 by hydro-
genation using Pd/C catalyst provided 32 in nearly quantitative
yield.
Silyllation of 12 with tert-butyldimethylsilyl chloride, potas-
sium carbonate gave 16 in good yield (Scheme 3). Hydroboration

P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
441
Scheme 3. Reagents and conditions: (a). TBDMSCI, imidazole, DMF, CH2CL2, rt, 36 h, 79%; (b) BH3. THF (1M in THF), THF, rt, 2 h, then 35% H2O2, 1N NaOH, 48%; (c) NaOAc,
PCC, CH2Cl2, rt, 1 h, 60%; (d) TBAF, THF, AcOH, CH3CN, 70%; (e) 10% HCl, CH2Cl2, rt, 5 min, 49%; (f) H2, 5% Pd/C, EtOAc, EtOH, rt, 16 h, 99%; (g) 70% AcOH, dioxane, 50◦C, 24 h,
70%.
Scheme 4. Reagents and conditions: (a) (i) NH4Cl, py, Ac2O, 135 ◦C, 12 h, (ii) CrO3, AcOH, H2O, (CH2Cl2)2, 0 ◦C, 2 h and (iii) Al2O3, benzene, rt, 16 h. 58% 3 steps; (b) H2O2,
NaOH, MeOH, rt, 16 h, 90%; (c) BnBr, K2CO3, acetone, reflux, 6 h, 97%; (d) NH2NH2·H2O, EtOH, CH2Cl2, rt, 30 min, 92%; (e) H2, 5% Pd/C, EtOAc, EtOH, rt, 16 h, 99%; (f) Ac2O, py,
0
◦C, 3 h, 97%; (g) 5-bromo-2-,ethyl-2-pentene, Mg, THF, 0 ◦C, 20 min, 39%; (h) PCC, NaOAc, CH2Cl2, rt, 3 h, 70%.
Scheme 5. Reagents and conditions: (a) (i) NH4Cl, py, Ac2O, 135 ◦C, 9 h; (ii) CrO3, AcOH H2O, (CH2Cl2)2, rt, 1 h. (iii) Al2O3, benzene, rt, 16 h. 55% 3 steps; (b) H2, 5%Pd/C, EtOAc,
EtOH, rt, 16 h, 99%; (c) 5-bromo-2-methylpentane, Mg, THF, 0 ◦C, 5 min, 87%.

442
P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
Table 1
Cytotoxicity of the synthetic steroids 5, 10, 11, 13, 15, 19, 22, 27, 30, 32 and 35 against human carcinoma cell lines (MCF7, NCI and KB).
Compound
IC50 (␮M)a
MCF7
NCI
KB
55.53
55.75
48.40
Inactiveb
Inactiveb
Inactiveb
Inactiveb
Inactiveb
11.73
12.66
51.18
16.64
42.43
Inactiveb
20.41
21.41
16.69
48.04

P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
443
Table 1 (Continued )
Compound
IC50 (␮M)a
MCF7
NCI
KB
Inactiveb
Inactiveb
56.13
Inactiveb
Inactiveb
Inactiveb
30.2
11.95
39.45
12.66
17.43
26.43
Inactiveb
53.59
53.99
Ellipticinec
ND
1.79
0.16
1.72
0.33
Doxorubicinec
1.51
MCF7, human breast adenocarcinoma; NCI, human small cell lung carcinoma; KB, human epidermoid carcinoma of cavity.
a
Data are typical values from six replicate experiments.
Inactive = inhibition < 50%.
Used as reference.
b
c
Moreover the analogue without functional group at C-16
was also synthesized as shown in Scheme 5. Transformation of
tigogenin (6) to unsaturated ketone 33 was performed by the previ-
ously described method in Scheme 4. Hydrogenation of 33 provided
the corresponding ketone 34 in nearly quantitative yield. Gridnard
reaction of 34 with 5-bromo-2-methylpentane and Mg in THF gave
3␤,20-dihydroxylcholestane (35) in good yield.
3.2. Biological activity
All synthetic compounds as described above were subjected to
an in vitro cytotoxic evaluation against KB (human epidermoid
carcinoma), NCI (human lung cancer) and MCF-7 (human breast
carcinoma) cell lines by using resazurin microplate assay (REMA)
which was a modified method of fluorescent dye for the mam-

444
P. Bunyathaworn et al. / Steroids 75 (2010) 432–444
malian cell cytotoxicity according to Brien et al. [23]. Ellipticine and
doxorubicin were used as positive control. The results are summa-
rized in Table 1.
support from the TRF, through the Royal Golden Jubilee Program.
Financial support from the Center for Innovation in Chemistry
(PERCH-CIC), Commission on Higher Education, Ministry of Educa-
tion and Kasetsart University Research and Development Institute
(KURDI) are also gratefully acknowledged.
In our previous work compound 1 bearing a ketone at C-3
showed no cytotoxicity (IC50 > 100 ␮M) against all tested cells,
whereas the analogues 3 and 4 bearing ␣,␤-unsaturated ketone
in ring A showed an increase in the cytotoxic activity against all
tested cells [MCF7 (30.65 and 31.43 ␮M), NCI (6.16 and 10.51 ␮M)
and KB (47.22 and 41.74 ␮M)] [17]. The quinone steroid 5 synthe-
sized from this work which has a higher level of oxidation on ring
A, showed a considerably lower cytotoxicity than 3 and 4 against
MCF7 and NCI but similar activity for KB. In contrast when ring A is
aromatic as steroid 32, it exhibited the most potent cytotoxicities
in all tested cells (see Table 1).
Comparison on cytotoxicity of A-ring aromatic compounds
which have different functional group or stereochemistry at C-16
(11, 30 and 32) showed that compound 32 with ketone group at C-
16 is very active to all tested cells whereas 11 bearing ␤-OH group at
C-16 showed no activity against NCI and MCF7 but two times more
active against KB cell lines than 32. Interestingly, 16␣-OH steroid
(30) exhibited higher cytotoxicity against MCF7 and NCI than the
corresponding 16␤-OH isomer (11) but lower for KB cell lines (see
Table 1).
The absence of OH at C-16 (35) and cholesterol like side chain at
C-20 (27) in the steroid skeleton apparently resulted in decreased
cytotoxicity, indicating that both OH and side chain must be present
to retain cytotoxicity against the tested cancer cell lines. However
it was found that steroids which have both hydroxyl groups at C-
16 and C-20 protected as cyclic ketal such as compounds 13 and 19
still had activities. Compound 13 showed very strong cytotoxicity
against MCF7 and KB but moderately active against NCI whereas
19 showed strong activity against MCF7, NCI and moderate activity
against KB.
Steroid analogues, compounds 10 and 15 which have functional
group on side chain and 11 which has no functional group. Com-
pound 10 containing double bond at C-24 on side chain showed no
cytotoxicity against all tested cell lines whereas compound 15 with
hydroxyl group at C-24 showed very potent cytotoxicity against
KB, moderate against MCF7 and was inactive to NCI. Compound
11 showed no activity against both MCF7 and NCI but was very
active against KB cell lines. These results indicated that functional
group on steroids side chain have some kind of cytotoxic selectiv-
ity against the tested cell lines. The importance of the presence of a
cholesterol like side chain and hydroxyl group at C-3 is illustrated
by the lack of activity in compound 27 and no cytotoxicity against
both MCF7 and NCI and considerable low cytotoxicity against KB
cell lines of compound 22 when hydroxyl group at C-3 was pro-
tected.
References
[1] D’Auria MV, Minale L, Riccio M. Polyoxygenated steroids of marine origin. Chem
Rev 1993;93:1839–95.
[2] Stonik VA. Marine polar steroids. Russian Chem Rev 2001;70:673–715.
[3] Aiello A, Fattorusso E, Menna M. Steroids from sponges: recent reports. Steroids
1999;64:687–714.
[4] Rueda A, Zubia E, Ortega MJ, Carballo JL, Salva J. New metabolites from the
sponge Spongia agaricina. J Nat Prod 1998;61:258–61.
[5] Morris LA, Christie ME, Jaspars M, van Ofwegen LP. A bioactive secosterol with
an unusual A- and B-ring oxygenation pattern isolated from an Indonesian soft
coral Lobophytum sp. J Nat Prod 1998;61:538–41.
[6] Kerr RG, Naz S, Narayanan R. New antiproliferative epoxysecosterols from
Pseudoptero-gorgia americana. Tetrahedron Lett 2000;41:6035–40.
[7] Anta C, Gonzalez N, Rodriguez J, Jimenez CA. New secosterol from
the Indonesian octocoral Pachyclavularia violacea.
J Nat Prod 2002;65:
1357–9.
[8] He H, Kulanthaivel P, Baker BJ, Kalter K, Darges J, Cofield D, et al. New
antiproliferative and antiinflammatory 9,11-secosterols from the gorgonian
Pseudopterogorgia sp. Tetrahedron 1995;51:51–8.
[9] Dopeso J, Quinoa E, Riguera R, Debitus C, Bergquist PR. Eryspongiol: ten
new highly hydroxylated 9,11-secosteroids with antihistaminic activity from
the sponge Euryspongia sp. stereochemistry and reduction. Tetrahedron
1994;50:3813–28.
[10] Capon RJ, Faulkner DJ. Herbasterol, and ichthyotoxic 9,11-secosterol from the
sponge Dysidea herbacea. J Org Chem 1985;50:4771–3.
[11] Aiello A, Fattorusso E, Menna M. Five new polar sterols from the black coral
Antipathes subpinnata. Steroids 1991;96:513–7.
[12] Aiello A, Fattorusso E, Menna M. Four new bioactive polyhydroxylated
sterols from the black coral Antipathes subpinnata.
J Nat Prod 1992;55:
321–5.
[13] Cimino G, De Rosa S, De Stefano S, ScognamiglioG, SodanoG. Cholest-4,14-dien-
15,20␰ diol-3-16-dione, a novel polyoxygenated marine steroid that easily loses
the side chain. Tetrahedron Lett 1981;22:3013–6.
[14] Benvegnu R, Cimino G, De Rosa S, De Stefano S. Guggulsterol-like steroids
from the Mediterranean gorgonian Leptogorgia sarmentosa. Experientia
1982;38:1443–4.
[15] Cimino G, De Rosa S, De Stefano S, Sodano G. C-18 hydrooxy steroids from
the Mediterranean gorgonian Leptogorgia sarmentosa. Experientia 1984;40:
246–8.
[16] Garrido L, Zubia E, Ortega MJ, Salva J. Isolation and structure elucidation of
new cytotoxic steroids from the gorgonian Leptogorgia sarmentosa. Steroids
2000;65:85–8.
[17] Kongkathip B, Boonananwong S, Kongkathip N. First synthesis of 3,16,20-
polyoxygenated cholestanes, new cytotoxic steroids from the gorgonian
Leptogorgia sarmentosa. Steroid 2008;73:1123–7.
[18] Frigerio M, Santagostino M, Sputore S. A user-friendly entry to 2-iodoxybenzoic
acid (IBX). J Org Chem 1999;64:4537–8.
[19] Chaudhuri NK, Williams JG, Nickolson R, Gut M. Stereochemistry of the addition
reactions of Grignard reagents to 20-keto steroids. Synthesis of 17a,20␣-
dihydroxy-cholesterol. J Org Chem 1969;34:3759–66.
[20] Sondheimer FF, Neumann H, Ringold J, Rosenkranz G. Steroidal sapogenins.
XXXIII. Aromatization experiments in the diosgenin series. J Am Chem Soc
1954;76:2230–3.
[21] Djerassi C, Rosenkranz G, Iriarte J, Berlin J, Romo J. Steroids. XII. Aroma-
tization experiments in the progesterone series. J Am Chem Soc 1951;73:
1523–7.
[22] Djerassi C, Zaffaroni A, Ringold HJ, Rosenkranz G, Sondheimer F, Thomas
GH, et al. Synthesis of 19-nor-ꢀ4-pregnene-11␤,17␣,21-triol-3,20-dione (19-
norhydrocortisone) and related 19-nor-adrenal hormones. J Am Chem Soc
1958;80:6110–4.
[23] Brien JO, Wilson I, Orton T, Pognan F. Investigation of the alamar blue(resazurin)
fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J
Biochem 2000;267:5421–6.
[24] Barton DHR, and Motherwell WB. Dehydrogenation agents based on deriva-
tives of selenium and their use in the dehydrogenation in the 1,4-positiion of
steroids. US Patent 4,434,080; 1984.
In conclusion we have described the chemical synthesis of a
series of polyoxygenated steroid derivatives with various steroid
skeleton moieties. Based on this SAR study, compounds with A-ring
aromatic of this series exhibited the most potent cytotoxicities in all
tested cells. The hydroxyl group at C-16 and cholesterol like side
chain at C-20 of the steroids is at least partly responsible for the
observed activity. The existence of hydroxyl group at C-3 was also
crucial for cytotoxic activity.
Acknowledgements
[25] Micovic V, Ivanovic MD, Piata DM. Simplified preparation of 16-
dehydropregnenolone acetate. Synthesis 1990:591–2.
[26] Kim S, Sutton SC, Guo C, LaCour TG, Fuchs PL. Synthesis of the north 1 cephalo-
statin family from hecogenin acetate. J Am Chem Soc 1999;121:2056–70.
P.B. is a Ph.D. student under the Royal Golden Jubilee Program,
the Thailand Research Fund (TRF). We are grateful for financial