Synthesis and antitumor evaluation of methyl spongoate analogs

Article abstract of DOI:10.1016/j.steroids.2010.08.002

A series of novel methyl spongoate (1) analogs has been synthesized and evaluated for their in vitro cytotoxic properties. It was found that the nature of the C-20 side chain had significant effects on their bioactivities and some analogs showed higher cytotoxicity than 1 against A549, HCT-116, HepG2, SW-1990, MCF-7 and NCI-H460 tumor cell lines. The pharmacological results confirmed that the α,β-unsaturated carbonyl moiety, a Michael acceptor in ring A, plays a pivotal role in the cytotoxic effect of these derivatives. The compiled pharmacological data may be useful for the design of novel analogous anticancer drugs.

Full text of DOI:10.1016/j.steroids.2010.08.002

Steroids 75 (2010) 1153–1163
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antitumor evaluation of methyl spongoate analogs
Cheng-Shi Jianga, Cai-Guo Huangb, Bo Fengb, Jia Lia, Jing-Xu Gonga,∗, Tibor Kurtánc, Yue-Wei Guoa,∗
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road,
Zhang Jiang High-Tech Park, Shanghai 201203, PR China
b Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, PR China
c Department of Organic Chemistry, University of Debrecen, PO Box 20, 4010 Debrecen, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2010
Received in revised form 31 July 2010
Accepted 6 August 2010
Available online 20 August 2010
A series of novel methyl spongoate (1) analogs has been synthesized and evaluated for their in vitro
cytotoxic properties. It was found that the nature of the C-20 side chain had significant effects on
their bioactivities and some analogs showed higher cytotoxicity than 1 against A549, HCT-116, HepG2,
SW-1990, MCF-7 and NCI-H460 tumor cell lines. The pharmacological results confirmed that the ␣,␤-
unsaturated carbonyl moiety, a Michael acceptor in ring A, plays a pivotal role in the cytotoxic effect of
these derivatives. The compiled pharmacological data may be useful for the design of novel analogous
anticancer drugs.
Keywords:
Synthesis
Methyl spongoate analogs
Cytotoxicity
© 2010 Elsevier Inc. All rights reserved.
Michael acceptor
1. Introduction
revealed that many different types of compounds [4,6,8–15] with
␣,␤-unsaturated carbonyl function have antitumor activity. It was
Marine organisms have been proven to be a prolific source
of unique steroids [1]. In the last few decades, numerous novel
steroids with remarkable bioactivities were isolated and charac-
terized from various soft corals and sponges [2,3]. Steroids with
C-20 methoxycarbonyl and carboxyl group are rare and of pharma-
cological interest, although their biosynthetic origin has not been
revealed yet. Meanwhile, some of them have been found to exhibit
excellent cytotoxic and anti-inflammatory activities [4–6]. Methyl
spongoate (1) (Fig. 1) is a novel steroid with an uncommon C-20
methoxycarbonyl group isolated recently from the Sanya soft coral
Spongodes sp., which showed significant cytotoxicities against BEL-
7402 human hepatic carcinoma cell lines with an IC50 value of
0.33 ␮M, P388 murine lymphocytic leukemia tumor cell lines with
an IC50 value of 8.9 ␮M, A-549 lung carcinoma and HT-29 colon
adenocarcinoma cell lines with an IC50 value of 11.7 ␮M [6]. Its
first stereoselective synthesis was reported by Gong et al. from our
laboratory [7].
revealed that compounds with Michael acceptor moieties can form
adducts with reactive thiol groups of proteins to induce protein
modification and misfolding, which might be responsible for the
observed antitumor activities. A Michael acceptor is defined as a
molecule containing an ␣,␤-unsaturated carbonyl function, which
is able to form covalent bonds with sulfur nucleophiles (thiols) at
the ␤ position [8]. For example, 4-(benzothiazol-2-yl)-4-hydroxy-
2,5-cyclohexadien-1-one, an electrophilic dual-Michael acceptor,
displays significant cytotoxic activity against many human cancer
cell lines, since it reacts with cysteine residues 32 and 35 of the
thioredoxin active site as revealed by mass spectrometry of the
covalently bound target protein [16]. Modification of thiols in mito-
chondrial proteins [17] or intracellular depletion of glutathione
[18] leads to the generation of reactive oxygen species (ROS) and
disruption of the redox balance which are critical for apoptosis
induction via caspase-dependent or caspase-independent path-
ways and subsequent cell death. Some ␣,␤-unsaturated carbonyl
compounds such as 6-shogaol can bind to tubulin via reactions with
SH groups of cysteine residues, resulting in microtubule damage
[19]. In addition, Shiraki et al. reported that the ␣,␤-unsaturated
ketone is the core moiety of natural ligands for covalent binding
to peroxisome proliferator-activated receptor ␥ (PPAR␥) by react-
ing with a cystein residue through a Michael addition reaction
[20]. Meanwhile, it was also reported that the side chain played an
important role in the activity of steroids [21]. Based on this infor-
mation, the C-20 methoxycarbonyl group and the ␣,␤-unsaturated
carbonyl function of ring A were preserved and fourteen methyl
Although the mechanism of its antitumor activity is not fully
understood, its structural novelty and potent antitumor activity
sparked our interest in marine anticancer drug research. How-
ever, the structural modification of 1 related to its anticancer
activity has not been studied yet in detail. A literature survey
∗
Corresponding authors. Tel.: +86 21 50805813; fax: +86 21 50805813.
E-mail addresses: jxgong@mail.shcnc.ac.cn (J.-X. Gong),
ywguo@mail.shcnc.ac.cn (Y.-W. Guo).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.08.002

1154
C.-S. Jiang et al. / Steroids 75 (2010) 1153–1163
with petroleum ether/EtOAc (4:1) to give the desired products 4
as white solids (4.97 g, 95% yield, m.p. 136–138 ◦C, Ref. [23] m.p.
141–143 ◦C). 1H NMR(CDCl3) ı: 0.57 (3H, s, H3-18), 0.80 (3H, s, H3-
19), 2.10 (1H, dd, J = 4.8, 14.8 Hz, H-20), 2.35 (1H, dd, J = 9.8, 14.8 Hz,
H-20), 3.58 (1H, m, H-3), 3.64 (3H, s, H3-22). 13C NMR(CDCl3) ı:
174.4 (C-22), 71.3 (C-3), 55.3 (C-14), 54.5 (C-17), 51.4 (C-22), 46.9,
44.8, 42.1, 38.1, 37.3, 37.0, 35.5, 35.5, 35.0, 32.0, 31.4 (C-7), 28.6
(C-16), 28.1 (C-6), 24.4 (C-15), 20.9 (C-11), 12.5 (C-19), 12.3 (C-18).
EI-MS: m/z 348 [M]+.
Fig. 1. Structure of methyl spongoate (1).
2.4. Methyl 3ˇ-(tert-butyldimethylsilyloxy)-pregnan-21-oate (5)
spongoate analogs with different C-20 side chains were synthesized
to investigate their cytotoxic effects on tumor cell lines. Herein,
we present our ongoing research on the structural modification of
methyl spongoate in order to increase its antitumor activity and
study the structure–antitumor activity relationship with regard to
the nature of the C-20 substituent.
To
a solution of compound 4 (4.95 g, 14.20 mmol) in
DMF (30 mL), TBSCl (2.35 g, 15.62 mmol) and imidazole (1.92 g,
28.20 mmol) was added [26]. The reaction mixture was stirred at
room temperature for 10 h. Then the reaction was quenched with a
saturated solution of NH4Cl and extracted with CH2Cl2. The organic
layer was dried with MgSO4 and concentrated. The residue was sub-
jected to silica gel chromatography with petroleum ether/EtOAc
(40:1) to give the desired products 5 as white solids (6.44 g, 98%
yield, m.p. 116–118 ◦C). 1H NMR(CDCl3) ı: 0.04 (6H, s, –Si(CH3)2),
0.58 (3H, s, H3-18), 0.80 (3H, s, H3-19), 0.88 (9H, s, –C(CH3)3), 2.10
(1H, dd, J = 4.8, 14.5 Hz, H-20), 2.35 (1H, dd, J = 9.5, 14.5 Hz, H-20),
3.54 (1H, m, H-3), 3.65 (3H, s, H3-22). 13C NMR(CDCl3) ı: 174.4 (C-
22), 72.1 (C-3), 55.3 (C-14), 54.6 (C-17), 51.5 (C-17), 46.9, 45.0, 42.1,
38.6, 37.4, 37.2, 35.5, 35.5, 35.0, 32.1, 31.9 (C-7), 28.7 (C-16), 28.2
(C-6), 25.9, 25.9, 25.9, 24.5 (C-15), 20.9 (C-11), 18.3, 12.5 (C-19),
12.4 (C-18), −4.6, −4.6. EI-MS: m/z 462 [M]+.
2. Experimental
2.1. General methods
LiN(iPr)2 (LDA, 2.0 M solution in THF) and RCH2Br were pur-
chased from the J&K Co. Chemical were analytical grade and solvent
werepurified bygeneral methods beforebeingused. TheNMR spec-
tra were measured on Bruker Avance spectrometer (300 MHz for 1H
and 100 MHz for 13C), using the residual CHCl3 signal (ıH 7.26 ppm)
as an internal standard for 1H NMR and CDCl3 signal (ıC 77.0 ppm)
for 13C NMR. Chemical shifts are expressed in ı (ppm) and cou-
pling constants (J) in Hz. Commercial Silica gel (Qing Dao Hai Yang
Chemical Group Co., 200–300 mesh) was used for column chro-
matography and precoated silica gel plates (Yan Tai Zi Fu Chemical
Group Co., G60 F-254) were used for analytical TLC. Melting points
were determined on Büchi 510 Melting point apparatus. ESI-MS
spectra were recorded on a Q-TOF Micro LC–MS–MS mass spec-
trometer and EI-MS data was obtained on a Finnigan-MAT-95 mass
spectrometer.
2.5. General procedure for preparation of compounds 6a-n
To a solution of compound 5 (300 mg, 0.648 mmol) in dry THF
(4 mL) under N2 at −78 ◦C, LDA (0.486 mL, 0.972 mmol) was added
dropwise with stirring. After stirring the mixture for 1 h at −78 ◦C,
RCH2Br (0.972 mmol) with HMPA (0.2 mL) was added slowly [27].
Then the reaction mixture was allowed to reach room temper-
ature in approximate 2 h, and the reaction was quenched with
a saturated solution of NH4Cl. The mixture was extracted with
CH2Cl2. The organic layer was dried with MgSO4 and concen-
trated. The residue was subjected to silica gel chromatography with
petroleum ether/EtOAc (60:1 to 40:1) to give the desired products
6a-n, respectively.
2.2. Methyl 3-oxo-pregnan-21-oate (3)
To a solution of compound 2 (5.68 g, 17.72 mmol) in acetone
(30 mL), Jones reagent [22] was added slowly with stirring at
room temperature until reaction was completed. The reaction was
quenched with a saturated solution of NaHCO3 and then extracted
with CH2Cl2. The organic layer was dried with MgSO4 and con-
centrated. The residue was dissolved with Et2O (20 mL) and then
CH2N2/Et2O [23] was added until the reaction was completed. The
reaction mixture was evaporated to dryness and then subjected
to silica gel chromatography with petroleum ether/EtOAc (5:1)
to give the desired product 3 as white solids (5.22 g, 85% yield,
m.p. 143–145 ◦C, Ref. [24] m.p. 138.5–140 ◦C). 1H NMR(CDCl3) ı:
0.60 (3H, s, H3-18), 1.00 (3H, s, H3-19), 3.64 (3H, s, H3-22). 13C
NMR(CDCl3) ı: 212.1 (C-3), 174.3 (C-21), 55.5 (C-14), 53.9 (C-17),
51.4 (C-22), 46.8, 46.6, 44.7, 42.1, 38.5, 38.1, 37.2, 35.7, 35.4, 35.0,
31.7 (C-7), 28.9 (C-16), 28.1 (C-6), 24.4 (C-15), 21.1 (C-11), 12.5
(C-19), 11.4 (C-18). EI-MS: m/z 346 [M]+.
2.5.1. Methyl (20R)-20-ethyl-3ˇ-(tert-butyldimethylsilyloxy)-
pregnan-21-oate (6a)
238 mg white solid, 75% yield, m.p. 174–175 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.66 (3H, s, H3-18), 0.77 (3H, s, H3-19),
0.82 (3H, t, J = 7.3 Hz, H3-23), 0.87 (9H, s, –C(CH3)3), 1.75-1.91 (1H,
m, H-17), 2.16 (1H, dt, J = 3.8, 11.0 Hz, H-20), 3.47-3.59 (1H, m, H-3),
3.64 (3H, s, H3-24).
2.5.2. Methyl (20R)-20-propyl-3ˇ-(tert-butyldimethylsilyloxy)
-pregnan-21-oate (6b)
245 mg white solid, 75% yield, m.p. 164–167 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.66 (3H, s, H3-18), 0.77 (3H, s, H3-
19), 0.87 (12H, m, –C(CH3)3, H3-24), 1.77–1.91 (1H, m, H-17), 2.25
(1H, dt, J = 6.3, 8.7 Hz, H-20), 3.47-3.59 (1H, m, H-3), 3.63 (3H,
s, H3-25).
2.3. Methyl 3ˇ-hydroxy-pregnan-21-oate (4)
To a solution of compound 3 (5.20 g, 15.01 mmol) in MeOH
(30 mL) at 4 ◦C, NaBH4 (0.85 g, 22.52 mmol) was added with stir-
ring [25]. After stirring the reaction mixture for 30 min at 4 ◦C, the
reaction was quenched with 10% HCl solution and then extracted
with CH2Cl2. The organic layer was dried with MgSO4 and con-
centrated. The residue was subjected to silica gel chromatography
2.5.3. Methyl (20R)-20-allyl-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6c)
248 mg white solid, 76% yield, m.p. 153–155 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.68 (3H, s, H3-18), 0.77 (3H, s, H3-19), 0.87
(9H, s, –C(CH3)3), 1.84 (1H, td, J = 8.6, 4.4 Hz, H-17), 2.09–2.39 (3H,
m, H-20, H2-22), 3.49–3.57 (1H, m, H-3), 3.61 (3H, s, H3-25), 4.97

C.-S. Jiang et al. / Steroids 75 (2010) 1153–1163
1155
(1H, d, J = 10.4 Hz, Ha-24), 5.01 (1H, d, J = 19.7 Hz, Hb-24), 5.62–5.77
0.87 (9H, s, –C(CH3)3, H3-29), 1.77–1.87 (1H, m, H-17), 2.22 (1H, dt,
(1H, m, H-23).
J = 7.9, 10.3 Hz, H-20), 3.47-3.59 (1H, m, H-3), 3.36 (3H, s, H3-30).
2.5.12. Methyl (20S)-20-(2ꢀ-chlorothizol-5ꢀ-methenyl)-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6l)
2.5.4. Methyl (20R)-20-butyl-3ˇ-(tert-butyldimethylsilyloxy)
-pregnan-21-oate (6d)
235 mg white solid, 70% yield, m.p. 162–164 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.66 (3H, s, H3-18), 0.77 (3H, s, H3-19),
0.85 (3H, t, J = 6.9 Hz, H-25), 0.87 (9H, s, –C(CH3)3), 1.76–1.91 (1H,
m, H-17), 2.22 (1H, dt, J = 6.2, 14.0 Hz, H-20), 3.47-3.58 (1H, m, H-3),
3.64 (3H, s, H3-26).
331 mg white solid, 86% yield, m.p. 165–168 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.70 (3H, s, H3-18), 0.77 (3H, s, H3-19),
0.87 (9H, s, –C(CH3)3), 1.85–2.00 (1H, m, H-17), 2.51 (1H, dt, J = 7.7,
10.9 Hz, H-20), 2.92 (2H, d, J = 7.7 Hz, H2-22), 3.64–3.57 (1H, m, H-3),
3.58 (3H, s, H3-23), 7.21 (1H, s, H-4ꢀ).
2.5.5. Methyl (20R)-20-isobutyl-3ˇ-(tert-butyldimethylsilyloxy)
-pregnan-21-oate (6e)
2.5.13. Methyl (20S)-20-benzyl-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6m)
239 mg white solid, 71% yield, m.p. 200–208 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.66 (3H, s, H3-18) 0.77 (3H, s, H3-19),
0.85–0.87 (15H, m, H3-24, H3-25, –C(CH3)3), 1.76–1.87 (1H, m, H-
17), 2.34 (1H, td, J = 3.52, 11.43 Hz, H-20), 3.47–3.58 (1H, m, H-3),
3.63 (3H, s, H3-26).
322 mg white solid, 90% yield, m.p. 210–212 ◦C. 1H NMR(CDCl3)
ı: 0.04(6H, s, –Si(CH3)2), 0.70 (3H, s, H3-18), 0.77 (3H, s, H3-19), 0.87
(9H, s, –C(CH3)3), 1.91–2.08 (1H, m, H-17), 2.45 (1H, m, H-20), 2.69
(1H, dd, J = 12.1, 12.9 Hz, Ha-22), 2.86 (1H, dd, J = 3.9, 12.9 Hz, Hb-22),
3.43 (3H, s, H3-23), 3.48–3.60 (1H, m, H-3), 7.11 (2H, d, J = 6.8 Hz,
H-2ꢀ, H-6ꢀ), 7.18 (1H, d, J = 7.0 Hz, H-2ꢀ), 7.23 (2H, dd, J = 6.8, 7.0 Hz,
H-3ꢀ, H-5ꢀ).
2.5.6. Methyl (20R)-20-pentyl-3ˇ-(tert-butyldimethylsilyloxy)
-pregnan-21-oate (6f)
242 mg white solid, 70% yield, m.p. 144–145 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.66 (3H, s, H3-18), 0.77 (3H, s, H3-19),
0.86 (3H, t, J = 6.7 Hz, H3-26), 0.87 (9H, s, –C(CH3)3), 1.77–1.86 (1H,
m, H-17), 2.22 (1H, dt, J = 6.8, 11.2 Hz, H-20), 3.47-3.59 (1H, m, H-3),
3.63 (3H, s, H3-27)
2.5.14. Methyl (20R)-20-(2-phenoxyethylidenyl)-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6n)
246 mg white solid, 65% yield, m.p. 154–160 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.70 (3H, s, H3-18), 0.78 (3H, s, H3-19), 0.88
(9H, s, –C(CH3)3), 1.81–1.94 (1H, m, H-17), 2.43–2.54 (1H, m, H-20),
3.50-3.57 (1H, m, H-3), 3.64 (3H, s, H3-24), 3.89 (2H, t, J = 3.89 Hz,
H-23), 6.85 (2H, d, J = 7.62 Hz, Hꢀ-2, Hꢀ-6), 6.92 (1H, d, J = 7.33 Hz,
H-4ꢀ), 7.30 (2H, dd, J = 7.33, 7.62 Hz, H-3ꢀ, H-5ꢀ).
2.5.7. Methyl (20R)-20-(3-methyl-2-butenyl)-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6g)
292 mg white solid, 85% yield, m.p. 137–139 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.66 (3H, s, H3-18), 0.77 (3H, s, H3-19), 0.87
(9H, s, –C(CH3)3), 1.61 (3H, s, H3-25), 1.65 (3H, s, H3-26), 3.50–3.58
(1H, m, H-3), 3.60 (3H, s, H3-27), 4.97–5.09 (1H, m, H-23)
2.6. General procedure for preparation of compounds 7a-n
To a solution of compounds 6a-n in acetone (5 mL), 10% HCl
solution (3 mL) was added with stirring [28]. As the reaction was
completed, a saturated solution of NaHCO3 was added. The mix-
ture was extracted with CH2Cl2. The organic layer was dried with
MgSO4 and concentrated. The residue was subjected to silica gel
chromatography with petroleum ether/EtOAc (4:1 to 3:1) to give
the desired products 7a-n, respectively.
2.5.8. Methyl (20R)-20-(4-methyl-3-pentenyl)-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6h)
247 mg white solid, 70% yield, m.p. 154–156 ◦C. 1H NMR(CDCl3)
␦: 0.04 (6H, s, –Si(CH3)2), 0.67 (3H, s, H3-18), 0.77 (3H, s, H3-19),
0.87 (9H, s, –C(CH3)3), 1.56 (3H, s, H3-26), 1.67 (3H, s, H3-27),
1.76–1.92 (3H, m, H-17, H2-23), 2.24 (1H, dt, J = 5.6, 11.3 Hz, H-20),
3.48–3.59 (1H, m, H-3), 3.60 (3H, s, H3-28), 5.05 (1H, t, J = 6.0 Hz,
H-24).
2.6.1. Methyl (20R)-20-ethyl-3ˇ-hydroxy-pregnan-21-oate (7a)
151 mg white solid, 90% yield, m.p. 141–142 ◦C. 1H NMR(CDCl3)
ı: 0.67 (3H, s, H3-18), 0.78 (3H, s, H3-19), 0.82 (3H, t, J = 7.5 Hz, H3-
23), 2.16 (1H, dt, J = 4.0, 11.1 Hz, H-20), 3.53–3.61 (1H, m, H-3), 3.64
(3H, s, H3-24). ESI-MS: m/z 377.4 [M+H]+, 399.3 [M+Na]+, 775.7
[2M+Na]+. Anal: Calcd. for C24H40O3: C 76.55, H 10.71; found: C
76.46, H 10.82.
2.5.9. Methyl (20R)-20-heptyl-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6i)
273 mg white solid, 75% yield, m.p. 128–129 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.67 (3H, s, H3-18), 0.77 (3H, s, H3-19),
0.86 (3H, t, J = 7.2 Hz, H3-28), 0.87 (9H, s, –C(CH3)3), 1.76–1.90 (1H,
m, H-17), 2.22 (1H, dt, J = 7.6, 10.9 Hz, H-20), 3.48–3.58 (1H, m, H-3),
3.63 (3H, s, H3-29).
2.6.2. Methyl (20R)-3ˇ-hydroxy-20-propyl-pregnan-21-oate
(7b)
164 mg white solid, 94% yield, m.p. 157–158 ◦C. 1H NMR(CDCl3)
ı: 0.66 (3H, s, H3-18), 0.78 (3H, s, H3-19), 0.87 (3H, t, J = 7.0 Hz,
H3-24), 2.20–2.29 (1H, m, H-20), 3.52–3.60 (1H, m, H-3), 3.63 (3H,
s, H3-25). ESI-MS: m/z 391.3 [M+H]+. Anal: Calcd. for C25H42O3: C
76.86, H 10.84; found: C 76.80, H 10.92.
2.5.10. Methyl (20R)-20-(4-methyl-3-hexenyl)-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6j)
290 mg white solid, 80% yield, m.p. 150–160 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.67 (3H, s, H3-18), 0.77 (3H, s, H3-19),
0.87 (9H, s, –C(CH3)3), 0.97 (3H, t, J = 7.5 Hz, H3-28), 1.56 (3H, s, H3-
26), 1.84–1.89 (3H, m, H-17, H2-23), 1.96 (2H, q, J = 7.5 Hz, H2-27),
2.25 (1H, dt, J = 7.2, 10.5 Hz, H-20), 3.47–3.59 (1H, m, H-3), 3.64 (3H,
s, H3-29), 5.05 (1H, t, J = 6.8 Hz, H-24).
2.6.3. Methyl (20R)-20-allyl-3ˇ-hydroxy-pregnan-21-oate (7c)
171 mg white solid, 97% yield, m.p. 162–165 ◦C. 1H NMR(CDCl3)
ı: 0.68 (3H, s, H3-18), 0.78 (3H, s, H3-19), 2.09–2.40 (3H, m, H-20,
H2-22), 3.51–3.61 (1H, m, H-3), 3.61 (3H, s, H3-25), 4.96 (1H, d,
J = 10.8 Hz, Ha-24), 5.00 (1H, d, J = 17.2 Hz, Hb-24), 5.61–5.77 (1H,
m, H-23). ESI-MS: m/z 389.3 [M+H]+, 411.2 [M+Na]+. Anal: Calcd.
for C25H40O3: C 77.27, H 10.38; found: C 76.47, H 10.29.
2.5.11. Methyl (20R)-20-octyl-3ˇ-
(tert-butyldimethylsilyloxy)-pregnan-21-oate (6k)
261 mg white solid, 70% yield, m.p. 121–122 ◦C. 1H NMR(CDCl3)
ı: 0.04 (6H, s, –Si(CH3)2), 0.67 (3H, s, H3-18), 0.77 (3H, s, H3-19),

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2.6.4. Methyl (20R)-20-butyl-3ˇ-hydroxy-pregnan-21-oate (7d)
154 mg white solid, 92% yield, m.p. 119–120 ◦C. 1H NMR(CDCl3)
ı: 0.66 (3H, s, H3-18), 0.77 (3H, s, H3-19), 0.85 (3H, t, J = 7.0 Hz, H-
25), 2.18–2.27 (1H, m, H-20), 3.51–3.62 (1H, m, H-3), 3.64 (3H, s,
H3-26). ESI-MS: m/z 405.3 [M+H]+, 427.3 [M+Na]+, 831.7 [2M+Na]+.
2.6.12. Methyl (20S)-20-(2ꢀ-chlorothizol-5ꢀ-methenyl)-3ˇ
-hydroxy-pregnan-21-oate (7l)
216 mg white solid, 86% yield, m.p. 129–132 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 0.78 (3H, s, H3-19), 1.86–2.01 (1H, m, H-17),
2.51 (1H, dt, J = 7.7, 10.9 Hz, H-20), 2.93 (2H, d, J = 7.6 Hz, H2-22), 3.58
(4H, m, H-3, H3-23), 7.20 (1H, s, H-4ꢀ). ESI-MS: m/z 480.5 [M+H]+,
502.2 [M+Na]+. Anal: Calcd. for C26H38ClNO3S: C 65.04, H 7.98, N
2.92; found: C 65.19, H 8.08, N 2.97.
Anal: Calcd. for C26H44O3: C 77.18, H 10.96; found: C 77.09, H 10.98.
2.6.5. Methyl (20R)-3ˇ-hydroxy-20-isobutyl-pregnan-21-oate
(7e)
169 mg white solid, 99% yield, m.p. 175–176 ◦C. 1H NMR(CDCl3)
ı: 0.68 (3H, s, H3-18) 0.78 (3H, s, H3-19), 0.84–0.88 (6H, m, H3-24,
H3-25), 2.34 (1H, dt, J = 3.37, 11.2 Hz, H-20), 3.51–3.61 (1H, m, H-
3), 3.63 (3H, s, H3-26). ESI-MS: m/z 405.3 [M+H]+, 427.3 [M+Na]+,
831.6 [2M+Na]+. Anal: Calcd. for C26H44O3: C 77.18, H 10.96; found:
C 77.01, H 11.16.
2.6.13. Methyl (20S)-20-benzyl-3ˇ-hydroxy-pregnan-21-oate
(7m)
228 mg white solid, 95% yield, m.p. 205–208 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.77 (3H, s, H3-19), 1.91–2.06 (1H, m, H-17),
2.48 (1H, m, H-20), 2.69 (1H, dd, J = 12.0, 12.6 Hz, Ha-22), 2.86 (1H,
dd, J = 3.7, 12.6 Hz, Hb-22), 3.43 (3H, s, H3-23), 3.52–3.64 (1H, m,
H-3), 7.10 (2H, d, J = 6.7 Hz, H-2ꢀ, H-6ꢀ), 7.18 (1H, d, J = 7.3 Hz, H-4ꢀ),
7.23 (2H, dd, J = 6.7, 7.3 Hz, H-3ꢀ, H-5ꢀ). ESI-MS: m/z 439.4 [M+H]+,
461.4 [M+Na]+. Anal: Calcd. for C29H42O3: C 79.41, H 9.65; found:
C 79.62, H 9.73.
2.6.6. Methyl (20R)-3ˇ-hydroxy-20-pentyl-pregnan-21-oate (7f)
157 mg white solid, 90% yield, m.p. 122–123 ◦C. 1H NMR(CDCl3)
ı: 0.66 (3H, s, H3-18), 0.78 (3H, s, H3-19), 0.85 (3H, t, J = 6.7 Hz, H3-
26), 2.17–2.28 (1H, m, H-20), 3.52–3.62 (1H, m, H-3), 3.63 (3H, s,
H3-27). EI-MS: m/z 418[M]+, 419[M+H]+. Anal: Calcd. forC27H46O3:
C 77.46, H 11.07; found: C 77.35, H 10.89.
2.6.14. Methyl
(20R)-3ˇ-hydroxy-20-(2-phenoxyethylidenyl)-pregnan-21-oate
(7n)
164 mg white solid, 90% yield, m.p. 169–171 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 0.79 (3H, s, H3-19), 2.43–2.54 (1H, m, H-20),
3.54–3.61 (1H, m, H-3), 3.64 (3H, s, H3-24), 3.88 (2H, t, J = 6.6 Hz,
H-23), 6.85 (2H, d, J = 7.62 Hz, Hꢀ-2, Hꢀ-6), 6.92 (1H, d, J = 7.33 Hz,
H-4ꢀ), 7.30 (2H, dd, J = 7.33, 7.62 Hz, H-3ꢀ, H-5ꢀ). ESI-MS: m/z 491.5
[M+Na]+. Anal: Calcd. for C30H44O4: C 76.88, H 9.46; found: C 76.60,
H 9.45.
2.6.7. Methyl (20R)-3ˇ-hydroxy-20-
(3-methyl-2-butenyl)-pregnan-21-oate (7g)
203 mg white solid, 95% yield, m.p. 132–134 ◦C. 1H NMR(CDCl3)
ı: 0.67 (3H, s, H3-18), 0.78 (3H, s, H3-19), 1.57 (3H, s, H3-25), 1.65
(3H, s, H3-26), 3.59 (1H, m, H-3), 3.60 (3H, s, H3-27), 5.01 (1H, t,
J = 7.0 Hz, H-23). ESI-MS: m/z 417.4 [M+H]+, 439.3 [M+Na]+, 855.7
[2M+Na]+. Anal: Calcd. for C27H44O3: C 77.83, H 10.64; found: C
77.66, H 10.86.
2.7. General procedure for preparation of compounds 8a-n
2.6.8. Methyl (20R)-3ˇ-hydroxy-20-
To a solution of compounds 7a-n in acetone (5 mL) was added
Jones reagent slowly with stirring [29]. The reaction progress was
followed by TLC and as the reaction was completed, a saturated
solution of NaHCO3 was added. The mixture was extracted with
CH2Cl2. The organic layer was dried with MgSO4 and concen-
trated. The residue was subjected to silica gel chromatography with
petroleum ether/EtOAc (10:1 to 6:1) to give the desired products
8a-n, respectively.
(4-methyl-3-pentenyl)-pregnan-21-oate (7h)
167 mg white solid, 93% yield, m.p. 116–118 ◦C. 1H NMR(CDCl3)
ı: 0.67 (3H, s, H3-18), 0.78 (3H, s, H3-19), 1.56 (3H, s, H3-26), 1.66
(3H, s, H3-27), 2.18–2.32 (1H, m, H-20), 3.52–3.61 (1H, m, H-3),
3.63 (3H, s, H3-28), 5.05 (1H, t, J = 7.18 Hz, H-24). ESI-MS: m/z 431.7
[M+H]+, 453.3 [M+Na]+. Anal: Calcd. for C28H46O3: C 78.09, H 10.77;
found: C 78.00, H 10.62.
2.6.9. Methyl (20R)-20-heptyl-3ˇ-hydroxy-pregnan-21-oate (7i)
181 mg white solid, 90% yield, m.p. 126–127 ◦C. 1H NMR(CDCl3)
ı: 0.66 (3H, s, H3-18), 0.78 (3H, s, H3-19), 0.86 (3H, t, J = 6.6 Hz,
H3-28), 2.22 (1H, dt, J = 7.48, 10.9 Hz, H-20), 3.52–3.61 (1H, m, H-
3), 3.63 (3H, s, H3-29). ESI-MS: m/z 447.4 [M+H]+. Anal: Calcd. for
C29H50O3: C 77.97, H 11.28; found: C 77.90, H 11.39.
2.7.1. Methyl (20R)-20-ethyl-3-oxo-pregnan-21-oate (8a)
112 mg white solid, 86% yield, m.p. 145–146 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.83 (3H, t, J = 7.5 Hz, H3-23), 0.99 (3H, s, H3-
19), 3.64 (3H, s, H3-24). ESI-MS: m/z 375.4 [M+H]+. Anal: Calcd. for
C24H38O3: C 76.96, H 10.23; found: C 76.68, H 10.21.
2.7.2. Methyl (20R)-20-propyl-3-oxo-pregnan-21-oate (8b)
138 mg white solid, 96% yield, m.p. 128–130 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.87 (3H, t, J = 7.2 Hz, H3-24), 0.98 (3H, s, H3-
19), 3.63 (3H, s, H3-25). ESI-MS: m/z 389.3 [M+H]+, 411.3 [M+Na]+.
Anal: Calcd. for C25H40O3: C 77.27, H 10.38; found: C 77.16,
H 10.21.
2.6.10. Methyl
(20R)-3ˇ-hydroxy-20-(4-methyl-3-hexenyl)-pregnan-21-oate
(7j)
213 mg white solid, 99% yield, m.p. 106–108 ◦C. 1H NMR(CDCl3)
ı: 0.67 (3H, s, H3-18), 0.78 (3H, s, H3-19), 0.96 (3H, t, J = 7.8 Hz,
H3-28), 1.96 (2H, q, J = 7.62 Hz, H2-27), 2.20–2.29 (1H, m, H-20),
3.51–3.62 (1H, m, H-3), 3.64 (3H, s, H3-29), 5.05 (1H, t, J = 7.0 Hz,
H-24). ESI-MS: m/z 445.3 [M+H]+, 467.3 [M+Na]+. Anal: Calcd. for
C29H48O3: C 78.33, H 10.88; found: C 78.03, H 10.99.
2.7.3. Methyl (20R)-20-allyl-3-oxo-pregnan-21-oate (8c)
147 mg white solid, 98% yield, m.p. 145–147 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 0.99 (3H, s, H3-19), 3.62 (3H, s, H3-25), 4.97
(1H, d, J = 9.6 Hz, Ha-24), 5.01 (1H, d, J = 17.0 Hz, Hb-24), 5.63–5.76
(1H, m, H-23). ESI-MS: m/z 387.3 [M+H]+, 409.3 [M+Na]+. Anal:
Calcd. for C25H38O3: C 77.68, H 9.91; found: C 77.41, H 9.92.
2.6.11. Methyl (20R)-3ˇ-hydroxy-20-octyl-pregnan-21-oate
(7k)
191 mg white solid, 99% yield, m.p. 124–125 ◦C. 1H NMR(CDCl3)
ı: 0.67 (3H, s, H3-18), 0.78 (3H, s, H3-19), 0.87 (3H, t, J = 6.4 Hz,
H3-29), 2.22 (1H, dt, J = 8.1, 10.5 Hz, H-20), 3.52–3.62 (1H, m, H-3),
3.36 (3H, s, H3-30). ESI-MS: m/z 461.4 [M+H]+, 483.4 [M+Na]+. Anal:
Calcd. for C30H52O3: C 78.21, H 11.38; found: C 78.11, H 11.31.
2.7.4. Methyl (20R)-20-butyl-3-oxo-pregnan-21-oate (8d)
119 mg white solid, 89% yield, m.p. 166–167 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.86 (3H, t, J = 7.2 Hz, H-25), 0.98 (3H, s, H3-

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19), 3.64 (3H, s, H3-26). ESI-MS: m/z 403.4 [M+H]+. Anal: Calcd. for
26H42O3: C 77.56, H 10.51; found: C 77.44, H 10.47.
Hb-22), 3.44 (3H, s, H3-23), 7.11 (2H, d, J = 7.3 Hz, H-2ꢀ, H-6ꢀ), 7.18
(1H, d, J = 6.7 Hz, H-4ꢀ), 7.23 (2H, dd, J = 6.7, 7.3 Hz, H-3ꢀ, H-5ꢀ). ESI-
MS: m/z 437.4 [M+H]+, 459.3 [M+Na]+. Anal: Calcd. for C29H40O3:
C 79.77, H 9.23; found: C 79.39, H 9.25.
C
2.7.5. Methyl (20R)-20-isobutyl-3-oxo-pregnan-21-oate (8e)
126 mg white solid, 85% yield, m.p. 209–211 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 0.86–0.88 (6H, m, H3-24, H3-25), 0.99 (3H,
s, H3-19), 3.63 (3H, s, H3-26). ESI-MS: m/z 403.4 [M+H]+, 425.3
[M+Na]+. Anal: Calcd. for C26H42O3: C 77.56, H 10.51; found: C
77.32, H 10.61.
2.7.14. Methyl
(20R)-3-oxo-20-(2-phenoxyethylidenyl)-pregnan-21-oate (8n)
133 mg white solid, 93% yield, m.p. 176–178 ◦C. 1H NMR(CDCl3)
ı: 0.74 (3H, s, H3-18), 0.99 (3H, s, H3-19), 3.64 (3H, s, H3-24), 3.89
(2H, t, J = 6.8 Hz, H-23), 6.85 (2H, d, J = 7.9 Hz, Hꢀ-2, Hꢀ-6), 6.92 (1H, d,
J = 7.0 Hz, H-4ꢀ), 7.30 (2H, dd, J = 7.0, 7.9 Hz, H-3ꢀ, H-5ꢀ). ESI-MS: m/z
489.4 [M+Na]+. Anal: Calcd. for C30H42O4: C 77.21, H 9.07; found:
C 77.10, H 8.89.
2.7.6. Methyl (20R)-3-oxo-20-pentyl-pregnan-21-oate (8f)
115 mg white solid, 84% yield, m.p. 122–124 ◦C. 1H NMR(CDCl3)
ı: 0.69 (3H, s, H3-18), 0.85 (3H, t, J = 6.6 Hz, H3-26), 0.98 (3H, s,
H3-19), 3.63 (3H, s, H3-27). ESI-MS: m/z 439.3 [M+Na]+, 855.7
[2M+Na]+. Anal: Calcd. for C27H44O3: C 77.83, H 10.64; found: C
77.60, H 10.58.
2.8. General procedure for preparation of compounds 9a-l
To a solution of compounds 8a-l (1 equiv.) in THF (3 mL), pyri-
dinium hydrobromide perbromide (1 equiv.) was added at room
temperature with stirring [30], as the reaction was completed, a
saturated solution of NaHCO3 was added and the mixture was
extracted with CH2Cl2. The organic layer was dried with MgSO4 and
concentrated. The residue was subjected to silica gel chromatogra-
phy with petroleum ether/EtOAc (12:1 to 8:1) to give the desired
products 9a-l, respectively.
2.7.7. Methyl
(20R)-20-(3-methyl-2-butenyl)-3-oxo-pregnan-21-oate (8g)
155 mg white solid, 85% yield, m.p. 139–142 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.98 (3H, s, H3-19), 1.57 (3H, s, H3-25), 1.65
(3H, s, H3-26), 3.60 (3H, s, H3-27), 5.01 (1H, t, J = 6.88 Hz, H-23). ESI-
MS: m/z 415.4 [M+H]+, 437.3 [M+H]+, 855.7 [2M+Na]+. Anal: Calcd.
for C27H42O3: C 78.21, H 10.21; found: C 78.39, H 10.38.
2.7.8. Methyl
2.8.1. Methyl (20R)-2˛-bromo-20-ethyl-3-oxo-pregnan-21-oate
(9a)
(20R)-20-(4-methyl-3-pentenyl)-3-oxo-pregnan-21-oate (8h)
117 mg white solid, 80% yield, m.p. 122–125 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.98 (3H, s, H3-19), 1.56 (3H, s, H3-26), 1.67
(3H, s, H3-27), 3.63 (3H, s, H3-28), 5.05 (1H, t, J = 6.89 Hz, H-24). ESI-
MS: m/z 429.3 [M+H]+, 451.3 [M+Na]+. Anal: Calcd. for C28H44O3:
C 78.46, H 10.35; found: C 78.57, H 10.49.
80 mg white solid, 72% yield, m.p. 128–131 ◦C. 1H NMR(CDCl3) ı:
0.69 (3H, s, H3-18), 0.83 (3H, t, J = 7.3 Hz, H3-23), 1.06 (3H, s, H3-19),
2.17 (1H, td, J = 3.3, 11.0 Hz, H-20), 2.61 (1H, dd, J = 6.3, 12.8 Hz, H␣-
4), 3.64 (3H, s, –OCH3), 4.73 (1H, dd, J = 6.3, 13.3 Hz, H␤-2). ESI-MS:
m/z 475.2 [M+Na]+, 929.5 [2M+Na]+. Anal: Calcd. for C24H37BrO3:
C 63.57, H 8.22; found: C 63.50, H 8.03.
2.7.9. Methyl (20R)-20-heptyl-3-oxo-pregnan-21-oate (8i)
136 mg white solid, 85% yield, m.p. 120–121 ◦C. 1H NMR(CDCl3)
ı: 0.69 (3H, s, H3-18), 0.86 (3H, t, J = 6.5 Hz, H3-28), 0.98 (3H, s, H3-
19), 3.63 (3H, s, H3-29). ESI-MS: m/z 444.5 [M+H]+. Anal: Calcd. for
2.8.2. Methyl (20R)-2˛-bromo-3-oxo-20-propyl-21-oate (9b)
116 mg white solid, 82% yield, m.p. 108–109 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.87 (3H, t, J = 7.2 Hz, H3-21), 1.06 (3H, s,
H3-19), 2.20–2.30 (1H, m, H-20), 2.61 (1H, dd, J = 6.4, 12.9 Hz, H␣-
4), 3.64 (3H, s, H3-25), 4.73 (1H, dd, J = 6.3, 13.3 Hz, H␤-2). ESI-MS:
m/z 489.3 [M+Na]+, 957.5 [2M+Na]+. Anal: Calcd. for C25H39BrO3:
C 64.23, H 8.41; found: C 64.12, H 8.50.
C29H48O3: C 78.33, H 10.88; found: C 78.20, H 10.80.
2.7.10. Methyl
(20R)-20-(4-methyl-3-hexenyl)-3-oxo-pregnan-21-oate (8j)
173 mg white solid, 90% yield, m.p. 104–106 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.96 (3H, t, J = 6.4 Hz, H3-28), 0.98 (3H, s, H3-
19), 3.64 (3H, s, H3-29), 5.05 (1H, t, J = 6.4 Hz, H-24). ESI-MS: m/z
443.3 [M+H]+, 465.3 [M+Na]+. Anal: Calcd. for C29H46O3: C 78.68,
H 10.47; found: C 78.45, H 10.55.
2.8.3. Methyl (20R)-20-allyl-2˛-bromo-3-oxo-pregnan-21-oate
(9c)
133 mg white solid, 87% yield, m.p. 108–110 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 1.05 (3H, s, H3-19), 2.61 (1H, dd, J = 6.3,
12.8 Hz, H␣-4), 3.61 (3H, s, H3-25), 4.73 (1H, dd, J = 6.2, 13.5 Hz,
H␤-2), 5.62–5.76 (1H, m, H-23). ESI-MS: m/z 487.2 [M+Na]+, 953.5
[2M+Na]+. Anal: Calcd. for C25H37BrO3: C 64.51, H 8.01; found: C
64.40, H 8.30.
2.7.11. Methyl (20R)-20-octyl-3-oxo-pregnan-21-oate (8k)
153 mg white solid, 90% yield, m.p. 115–116 ◦C. 1H NMR(CDCl3)
ı: 0.70 (3H, s, H3-18), 0.87 (3H, t, J = 6.6 Hz, H3-29), 0.99 (3H, s, H3-
19), 3.36 (3H, s, H3-30). ESI-MS: m/z 459.4 [M+H]+. Anal: Calcd. for
C30H50O3: C 78.55, H 10.99; found: C 78.60, H 11.12.
2.8.4. Methyl (20R)-2˛-bromo-20-butyl-3-oxo-pregnan-21-oate
(9d)
2.7.12. Methyl
95 mg white solid, 80% yield, m.p. 95–96 ◦C. 1H NMR(CDCl3) ı:
0.69 (3H, s, H3-18), 0.85 (3H, t, J = 7.2 Hz, H3-25), 1.06 (3H, s, H3-19),
2.17–2.28 (1H, m, H-20), 2.60 (1H, dd, J = 6.3, 12.8 Hz, H␣-4), 3.63
(3H, s, H3-26), 4.73 (1H, dd, J = 6.3, 13.3 Hz, H␤-2). ESI-MS: m/z 503.5
[M+Na]+, 985.6 [2M+Na]+. Anal: Calcd. for C26H41BrO3: C 64.85, H
8.58; found: C 64.94, H 8.50.
(20S)-20-(2ꢀ-chlorothizolyl-5ꢀ-methenyl)-3-oxo-pregnan-21-oate
(8l)
191 mg white solid, 98% yield, m.p. 166–170 ◦C. 1H NMR(CDCl3)
ı: 0.74 (3H, s, H3-18), 0.99 (3H, s, H3-19), 2.52 (1H, dt, J = 7.7, 11.0 Hz,
H-20), 2.94 (2H, d, J = 7.7 Hz, H2-22), 3.58 (3H, s, H3-23), 7.20 (1H,
s, H-4ꢀ). ESI-MS: m/z 478.3 [M+H]+. Anal: Calcd. for C26H36ClNO3S:
C 65.32, H 7.59, N 2.93; found: C 65.21, H 7.67, N 2.89.
2.8.5. Methyl
(20R)-2˛-bromo-20-isobutyl-3-oxo-pregnan-21-oate (9e)
117 mg white solid, 92% yield, m.p. 150–153 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 0.85–0.88 (6H, m, H3-24, H3-25), 1.06 (3H, s,
H3-19), 2.61 (1H, dd, J = 6.4, 13.0 Hz, H␣-4), 3.64 (3H, s, H3-26), 4.73
(1H, dd, J = 6.3, 13.3 Hz, H␤-2). ESI-MS: m/z 505.3 [M+Na]+, 985.5
2.7.13. Methyl (20S)-20-benzyl-3-oxo-pregnan-21-oate (8m)
186 mg white solid, 90% yield, m.p. 281–284 ◦C. 1H NMR(CDCl3)
ı: 0.73 (3H, s, H3-18), 0.99 (3H, s, H3-19), 2.49–2.60 (1H, m, H-20),
2.69 (1H, dd, J = 12.7, 12.7 Hz, Ha-22), 2.87 (1H, dd, J = 3.9, 12.7 Hz,

1158
C.-S. Jiang et al. / Steroids 75 (2010) 1153–1163
[2M+Na]+. Anal: Calcd. for C26H41BrO3: C 64.85, H 8.58; found: C
64.72, H 8.60.
2.9. General procedure for preparation of compounds 10a-n
Method A: To a solution of compounds 9a-l (30 mg, 1 equiv.) in
dry DMF (2 mL), Li2CO3 (1.5 equiv.) and LiBr (1 equiv.) was added
with stirring [31]. After refluxing the reaction mixture for 2 h, 10 mL
of H2O was added. The mixture was extracted with CH2Cl2 and
the organic layer was dried with MgSO4 and concentrated. The
residue was subjected to silica gel chromatography with petroleum
ether/EtOAc (10:1 to 5:1) to give the desired products 10a-l, respec-
tively.
Method B: To a solution of compounds 8m,n (30 mg, 1 equiv.)
in 5 mL toluene/DMSO (v:v = 2:1), IBX (1.2 equiv.) was added
with stirring [32,33]. After refluxing the reaction mixture for 12
h, 10 mL of H2O was added. The mixture was extracted with
CH2Cl2. The organic layer was dried with MgSO4 and concen-
trated. The residue was subjected to silica gel chromatography with
petroleum ether/EtOAc (6:1) to give the desired products 10m,n,
respectively.
2.8.6. Methyl
(20R)-2˛-bromo-3-oxo-20-pentyl-pregnan-21-oate (9f)
87 mg white solid, 77% yield, m.p. 88–89 ◦C. 1H NMR(CDCl3) ı:
0.69 (3H, s, H3-18), 0.86 (3H, t, J = 6.7 Hz, H3-26), 1.06 (3H, s, H3-19),
2.19–2.27 (1H, m, H-20), 2.61 (1H, dd, J = 6.5, 13.0 Hz, H␣-4), 3.63
(3H, s, H3-27), 4.73 (1H, dd, J = 6.5, 13.3 Hz, H␤-2). ESI-MS: m/z 519.3
[M+Na]+, 1013.5 [2M+Na]+. Anal: Calcd. for C27H43BrO3: C 65.44, H
8.75; found: C 65.40, H 8.84.
2.8.7. Methyl (20R)-2˛-bromo-20-(3-methyl-2-butenyl)-
3-oxo-pregnan-21-oate (9g)
129 mg amorphous form, 80% yield. 1H NMR(CDCl3) ı: 0.70 (3H,
s, H3-18), 1.06 (3H, s, H3-19), 1.57 (3H, s, H3-25), 1.66 (3H, s, H3-
26), 2.61 (1H, dd, J = 5.4, 11.9 Hz, H␣-4), 3.60 (3H, s, H3-27), 4.73
(1H, dd, J = 6.2, 13.2 Hz, H␤-2). 5.01 (1H, t, J = 8.0 Hz, H-23). ESI-MS:
m/z 495.3 [M+H]+, 1009.6 [2M+Na]+. Anal: Calcd. for C27
H41BrO3:
C 65.71, H 8.37; found: C 65.90, H 8.49.
2.9.1. Methyl (20R)-20-ethyl-3-oxo-pregnan-1-en-21-oate (10a)
14 mg white solid, 57% yield, m.p. 155–158 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 0.83 (3H, t, J = 7.3 Hz, H3-23), 0.98 (3H, s,
H3-19), 3.64 (3H, s, H3-24), 5.83 (1H, d, J = 10.3 Hz, H-2), 7.10 (1H,
d, J = 10.3 Hz, H-1). 13C NMR(CDCl3) ı: 200.3 (C-3), 176.6 (C-21),
158.5 (C-1), 127.3 (C-2), 55.5 (C-14), 52.2 (C-17), 51.0 (C-24), 49.8
(C-9), 49.0 (C-20), 44.2 (C-5), 42.2 (C-13), 40.9 (C-4), 38.9 (C-10),
37.3 (C-12), 35.6 (C-8), 31.2 (C-7), 27.5 (C-16), 26.9 (C-6), 25.0
(C-22), 23.6 (C-15), 21.0 (C-11), 12.9 (C-19), 12.3 (C-18), 11.8 (C-
23). ESI-MS: m/z 373.4 [M+H]+, 395.3 [M+Na]+, 767.6 [2M+Na]+.
Anal: Calcd. for C24H36O3: C 77.38, H 9.74; found: C 77.21,
H 9.77.
2.8.8. Methyl (20R)-2˛-bromo-20-(4-methyl-3-pentenyl)
-3-oxo-pregnan-21-oate (9h)
103 mg white solid, 90% yield, m.p. 98–100 ◦C. 1H NMR(CDCl3) ı:
0.70 (3H, s, H3-18), 1.06 (3H, s, H3-19), 1.56 (3H, s, H3-26), 1.67 (3H,
s, H3-27), 2.61 (1H, dd, J = 6.4, 12.9 Hz, H␣-4), 3.64 (3H, s, H3-28),
4.73 (1H, dd, J = 6.3, 13.3 Hz, H␤-2). 5.05 (1H, t, J = 7.0 Hz, H-23). ESI-
MS: m/z 507.2 [M+H]+, 529.3 [M+Na]+. Anal: Calcd. for C28H43BrO3:
C 66.26, H 8.54; found: C 66.05, H 8.62.
2.8.9. Methyl
(20R)-2˛-bromo-20-heptyl-3-oxo-pregnan-21-oate (9i)
116 mg white solid, 85% yield, m.p. 137–138 ◦C. 1H NMR(CDCl3)
ı: 0.69 (3H, s, H3-18), 0.86 (3H, t, J = 6.7 Hz, H3-28), 1.06 (3H, s, H3-
19), 2.23 (1H, dt, J = 7.5, 11.1 Hz, H-20), 2.61 (1H, dd, J = 6.3, 12.8 Hz,
H␣-4), 3.63 (3H, s, H3-29), 4.73 (1H, dd, J = 6.3, 13.3 Hz, H␤-2). ESI-
MS: m/z 523.3 [M+H]+. Anal: Calcd. for C29H47BrO3: C 66.52, H 9.05;
found: C 66.48, H 8.96.
2.9.2. Methyl (20R)-3-oxo-20-propyl-pregnan-1-en-21-oate
(10b)
15 mg white solid, 62% yield, m.p. 146–148 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 0.87 (3H, t, J = 7.0 Hz, H3-24), 0.98 (3H, s,
H3-19), 3.64 (3H, s, H3-25), 5.83 (1H, d, J = 10.3 Hz, H-2), 7.10 (1H, d,
J = 10.3 Hz, H-1). 13C NMR(CDCl3) ı: 200.3 (C-3), 176.7 (C-21), 158.5
(C-1), 127.3 (C-2), 55.6 (C-14), 52.7 (C-17), 51.0 (C-25), 49.9 (C-9),
47.1 (C-20), 44.2 (C-5), 42.2 (C-13), 40.9 (C-4), 38.9 (C-10), 37.3 (C-
12), 35.6 (C-8), 34.2 (C-22), 31.2 (C-7), 27.5 (C-16), 27.0 (C-6), 23.6
(C-15), 21.1 (C-11), 20.6 (C-23) 14.0 (C-24), 12.9 (C-19), 12.3 (C-18).
ESI-MS: m/z 387.3 [M+H]+, 409.3 [M+Na]+, 795.6 [2M+Na]+. Anal:
Calcd. for C25H38O3: C 77.68, H 9.91; found: C 77.45, H 9.80.
2.8.10. Methyl (20R)-2˛-bromo-20-
(4-methyl-3-hexenyl)-3-oxo-pregnan-21-oate (9j)
162 mg amorphous form, 90% yield. 1H NMR(CDCl3) ı: 0.69 (3H,
s, H3-18), 0.96 (3H, t, J = 7.5 Hz, H3-28), 1.06 (3H, s, H3-19), 2.21–2.29
(1H, m, H-20), 2.61 (1H, dd, J = 6.4, 12.6 Hz, H␣-4), 3.62 (3H, s, H3-
29), 4.73 (1H, dd, J = 6.2, 13.2 Hz, H␤-2), 5.05 (1H, t, J = 7.18 Hz, H-24).
ESI-MS: m/z 543.3 [M+Na]+. Anal: Calcd. for C29
H45BrO3: C 66.78,
2.9.3. Methyl (20R)-20-allyl-3-oxo-pregnan-1-en-21-oate (10c)
15 mg white solid, 60% yield, m.p. 115–118 ◦C. 1H NMR(CDCl3)
ı: 0.73 (3H, s, H3-18), 0.98 (3H, s, H3-19), 3.63 (3H, s, H3-25), 4.97
(1H, d, J = 10.1 Hz, Ha-24), 5.02 (1H, d, J = 17.2 Hz, Hb-25), 5.61-5.78
(1H, m, H-23), 5.83 (1H, d, J = 10.3 Hz, H-2), 7.10 (1H, d, J = 10.3 Hz,
H-1). 13C NMR(CDCl3) ı: 200.2 (C-3), 175.7 (C-21), 158.4 (C-1),
135.3 (C-2), 127.4 (C-23), 116.6 (C-24), 55.6 (C-14), 52.3 (C-17),
51.0 (C-25), 49.9 (C-9), 47.4 (C-20), 44.2 (C-5), 42.2 (C-13), 40.9 (C-
4), 38.9 (C-10), 37.2 (C-12), 36.3 (C-22), 35.6 (C-8), 31.2 (C-7), 27.5
(C-16), 26.9 (C-6), 23.6 (C-15), 21.0 (C-11), 12.9 (C-19), 12.3 (C-
18). ESI-MS: m/z 385.3 [M+H]+, 407.2 [M+Na]+, 791.6 [2M+Na]+.
Anal: Calcd. for C25H36O3: C 78.08, H 9.44; found: C 78.12,
H 9.53.
H 8.70; found: C 66.89, H 8.90.
2.8.11. Methyl
(20R)-2˛-bromo-20-octyl-3-oxo-pregnan-21-oate (9k)
140 mg white solid, 90% yield, m.p. 97–98 ◦C. 1H NMR(CDCl3) ı:
0.69 (3H, s, H3-18), 0.86 (3H, t, J = 6.3 Hz, H3-29), 1.06 (3H, s, H3-19),
2.23 (1H, dt, J = 7.5, 11.1 Hz, H-20), 2.61 (1H, dd, J = 6.2, 12.9 Hz, H␣-
4), 3.63 (3H, s, H3-30), 4.73 (1H, dd, J = 6.3, 13.3 Hz, H␤-2). ESI-MS:
m/z 539.3 [M+H]+, 559.2 [M+Na]+. Anal: Calcd. for C30H49BrO3: C
67.02, H 9.19; found: C 67.13, H 9.14.
2.8.12. Methyl (20S)-2˛-bromo-20-(2ꢀ-chlorothizol-
5ꢀ-methenyl)-3-oxo-pregnan-21-oate (9l)
189 mg white solid, 95% yield, m.p. 190–194 ◦C. 1H NMR(CDCl3)
ı: 0.74 (3H, s, H3-18), 1.06 (3H, s, H3-19), 2.49–2.54 (1H, m, H-20),
2.55 (1H, dt, J = 6.1, 12.9 Hz, H␣-4), 2.94 (2H, d, J = 7.6 Hz, H2-22),
3.58 (3H, s, H3-23), 4.73 (1H, dd, J = 6.3, 13.3 Hz, H␤-2), 7.20 (1H,
s, H-4ꢀ). ESI-MS: m/z 558.2 [M+Na]+, 1137.2 [2M+Na]+. Anal: Calcd.
for C26H35BrClNO3S: C 56.07, H 6.33, N 2.51; found: C 56.21, H 6.40,
N 2.59.
2.9.4. Methyl (20R)-20-butyl-3-oxo-pregnan-1-en-21-oate (10d)
15 mg white solid, 60% yield, m.p. 125–126 ◦C. 1H NMR(CDCl3)
ı: 0.72 (3H, s, H3-18), 0.86 (3H, t, J = 7.0 Hz, H3-25), 0.99 (3H, s,
H3-19), 3.65 (3H, s, H3-26), 5.84 (1H, d, J = 10.3 Hz, H-2), 7.10 (1H, d,
J = 10.3 Hz, H-1). 13C NMR(CDCl3) ı: 200.2 (C-3), 176.7 (C-21), 158.5
(C-1), 127.3 (C-2), 55.5 (C-14), 52.7 (C-17), 51.0 (C-26), 49.8 (C-9),

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47.3 (C-20), 44.2 (C-5), 42.1 (C-13), 40.9 (C-4), 38.9 (C-10), 37.3 (C-
12), 35.5 (C-8), 31.7, 31.1 (C-7), 29.5, 27.5 (C-16), 27.0 (C-6), 23.6
(C-15), 22.6, 21.0 (C-11), 13.9 (C-25), 12.9 (C-19), 12.2 (C-18). ESI-
MS: m/z 401.4 [M+H]+, 423.4 [M+Na]+, 823.7 [2M+Na]+. Anal: Calcd.
for C26H40O3: C 77.95, H 10.06; found: C 77.70, H 9.92.
19), 3.65 (3H, s, H3-29), 5.83 (1H, d, J = 10.3 Hz, H-2), 7.10 (1H, d,
J = 10.3 Hz, H-1). 13C NMR(CDCl3) ı: 200.3 (C-3), 176.7 (C-21), 158.5
(C-1), 127.3 (C-2), 55.6 (C-14), 52.7 (C-17), 51.0 (C-29), 49.8 (C-9),
47.3 (C-20), 44.2 (C-5), 42.2 (C-13), 40.9 (C-4), 38.9 (C-10), 37.3 (C-
12), 35.6 (C-8), 32.0, 31.7, 31.2 (C-7), 29.5, 29.1, 27.5 (C-16), 27.4,
27.0 (C-6), 23.6 (C-15), 22.6, 21.0 (C-11), 14.1 (C-28), 12.9 (C-19),
12.2 (C-18). ESI-MS: m/z 443.3 [M+H]+. Anal: Calcd. for C29H46O3:
C 78.68, H 10.47; found: C 78.35, H 10.50.
2.9.5. Methyl (20R)-20-isobutyl-3-oxo-pregnan-1-en-21-oate
(10e)
16 mg white solid, 65% yield, m.p. 178–180 ◦C. 1H NMR(CDCl3)
ı: 0.72 (3H, s, H3-18), 0.86–0.88 (6H, m, H3-24, H3-25), 0.98 (3H, s,
H3-19), 3.63 (3H, s, H3-26), 5.83 (1H, d, J = 10.3 Hz, H-2), 7.10 (1H, d,
J = 10.3 Hz, H-1). 13C NMR(CDCl3) ı: 200.3 (C-3), 176.7 (C-21), 158.5
(C-1), 127.3 (C-2), 55.6 (C-14), 52.9 (C-17), 51.0 (C-26), 49.8 (C-9),
45.3 (C-20), 44.2 (C-5), 42.2 (C-13), 41.3 (C-22), 40.9 (C-4), 38.9 (C-
10), 37.2 (C-12), 35.5 (C-8), 31.1 (C-7), 27.5 (C-16), 27.2 (C-6), 26.2
(C-23), 24.0 (C-25), 23.6 (C-15), 21.3 (C-24), 21.0 (C-11), 12.9 (C-
19), 12.2 (C-18). ESI-MS: m/z 401.3 [M+H]+, 423.3 [M+Na]+, 823.7
2.9.10. Methyl
(20R)-20-(4-methyl-3-hexenyl)-3-oxo-pregnan-1-en-21-oate
(10j)
15 mg white solid, 60% yield, m.p. 78–80 ◦C. 1H NMR(CDCl3) ı:
0.69 (3H, s, H3-18), 0.96 (3H, t, J = 7.8 Hz, H3-28), 1.06 (3H, s, H3-
19), 3.65 (3H, s, H3-29), 5.05 (1H, t, J = 10.3 Hz, H-24), 5.83 (1H,
d, J = 10.3 Hz, H-2), 7.10 (1H, d, J = 10.3 Hz, H-1). 13C NMR(CDCl3)
ı: 200.2 (C-3), 176.6 (C-21), 158.5 (C-1), 137.6 (C-25), 127.4 (C-
2), 122.0 (C-24), 55.6 (C-14), 52.7 (C-17), 51.0 (C-29), 49.9 (C-9),
47.1 (C-20), 44.2 (C-5), 42.2 (C-13), 40.9 (C-4), 38.9 (C-10), 37.3 (C-
12), 35.6 (C-8), 32.2, 32.3, 31.2 (C-7), 27.5 (C-16), 27.1 (C-6), 25.8,
23.6 (C-15), 21.1 (C-11), 15.8 (C-26), 12.9 (C-19), 12.7 (C-28), 12.2
(C-18). ESI-MS: m/z 441.4 [M+H]+, 463.3 [M+Na]+. Anal: Calcd. for
[2M+Na]+. Anal: Calcd. for C26
77.82, H 10.01.
H40O3: C 77.95, H 10.06; found: C
2.9.6. Methyl (20R)-3-oxo-20-pentyl-pregnan-1-en-21-oate
(10f)
15 mg white solid, 60% yield, m.p. 109–110 ◦C. 1H NMR(CDCl3)
ı: 0.71 (3H, s, H3-18), 0.85 (3H, t, J = 6.6 Hz, H3-26), 0.98 (3H, s,
H3-19), 3.64 (3H, s, H3-27), 5.83 (1H, d, J = 10.3 Hz, H-2), 7.10 (1H, d,
J = 10.3 Hz, H-1). 13C NMR(CDCl3) ı: 200.2 (C-3), 176.7 (C-21), 158.5
(C-1), 127.3 (C-2), 55.6 (C-14), 52.7 (C-17), 51.0 (C-27), 49.8 (C-9),
47.3 (C-20), 44.2 (C-5), 42.1 (C-13), 40.9 (C-4), 38.9 (C-10), 37.3 (C-
12), 35.6 (C-8), 31.9, 31.7, 31.2 (C-7), 27.5 (C-16), 27.0 (C-6), 27.0,
23.6 (C-15), 22.5, 21.0 (C-11), 14.0 (C-26), 12.9 (C-19), 12.2 (C-18).
ESI-MS: m/z 415.4 [M+H]+, 437.4 [M+Na]+, 851.7 [2M+Na]+. Anal:
Calcd. for C27H42O3: C 78.21, H 10.21; found: C 78.10, H 10.12.
C
29H44O3: C 79.04, H 10.06; found: C 79.15, H 10.20.
2.9.11. Methyl (20R)-20-octyl-3-oxo-pregnan-1-en-21-oate
(10k)
15 mg white solid, 60% yield, m.p. 84–85 ◦C. 1H NMR(CDCl3) ı:
0.71 (3H, s, H3-18), 0.86 (3H, t, J = 7.0 Hz, H3-29), 0.98 (3H, s, H3-
19), 3.65 (3H, s, H3-30), 5.83 (1H, d, J = 10.3 Hz, H-2), 7.10 (1H,
d, J = 10.3 Hz, H-1). 13C NMR(CDCl3) ı: 200.3 (C-3), 176.7 (C-21),
158.5 (C-1), 127.3 (C-2), 55.6 (C-14), 52.7 (C-17), 51.0 (C-30), 49.9
(C-9), 47.3 (C-20), 44.2 (C-5), 42.2 (C-13), 40.9 (C-4), 38.9 (C-10),
37.3 (C-12), 35.6 (C-8), 32.0, 31.8, 31.2 (C-7), 29.5, 29.4, 29.2, 27.5
(C-16), 27.3, 27.0 (C-6), 23.6 (C-15), 22.6, 21.1 (C-11), 14.1 (C-29),
12.9 (C-19), 12.2 (C-18). ESI-MS: m/z 457.3 [M+H]+. Anal: Calcd. for
2.9.7. Methyl (20R)-20-(3-methyl-2-butenyl)
-3-oxo-pregnan-1-en-21-oate (10g)
18 mg white solid, 70% yield, m.p. 128–130 ◦C. 1H NMR(CDCl3) ı:
0.72 (3H, s, H3-18), 0.98 (3H, s, H3-19), 1.57 (3H, s, H3-25), 1.66 (3H,
s, H3-26), 3.61 (3H, s, H3-27), 5.01 (1H, t, J = 7.0 Hz, H-23), 5.83 (1H,
d, J = 10.3 Hz, H-2), 7.10 (1H, d, J = 10.3 Hz, H-1). 13C NMR(CDCl3)
ı: 200.3 (C-3), 176.1 (C-21), 158.5 (C-1), 133.6 (C-24), 127.3 (C-2),
120.8 (C-23), 55.6 (C-14), 52.4 (C-17), 51.0 (C-27), 49.9 (C-9), 47.8
(C-20), 44.2 (C-5), 42.2 (C-13), 40.9 (C-4), 38.9 (C-10), 37.2 (C-12),
35.6 (C-8), 31.2 (C-7), 30.7 (C-22), 27.5 (C-16), 27.0 (C-6), 25.8 (C-
26), 23.6 (C-15), 21.0 (C-11), 17.6 (C-25), 12.9 (C-19), 12.2 (C-18).
ESI-MS: m/z 413.3 [M+H]+, 435.3 [M+Na]+, 847.7 [2M+Na]+. Anal:
Calcd. for C27H40O3: C 78.60, H 9.77; found: C 78.38, H 9.63.
C30H48O3: C 78.90, H 10.59; found: C 78.78, H 10.50.
2.9.12. Methyl (20S)-20-(2ꢀ-chlorothizol-5ꢀ-methenyl)
-3-oxo-pregnan-1-en-21-oate (10l)
15 mg white solid, 60% yield, m.p. 213–217 ◦C. 1H NMR(CDCl3)
ı: 0.75 (3H, s, H3-18), 0.99 (3H, s, H3-19), 2.52 (1H, dt, J = 7.7,
10.9 Hz, H-20), 2.94 (2H, d, J = 7.6 Hz, H2-22), 3.59 (3H, s, H3-23),
5.84 (1H, d, J = 10.3 Hz, H-2), 7.09 (1H, d, J = 10.3 Hz, H-1), 7.21
(1H, s, H-4ꢀ). 13C NMR(CDCl3) ı: 200.1 (C-3), 174.7 (C-21), 158.2
(C-1), 150.2 (C-2ꢀ), 139.1 (C-4ꢀ), 138.1 (C-5ꢀ), 127.4 (C-2), 55.6 (C-
14), 52.6 (C-17), 51.5, 49.8, 49.7, 44.2 (C-5), 42.3 (C-13), 40.9 (C-4),
38.9 (C-10), 37.0 (C-12), 35.5 (C-8), 31.1 (C-7), 29.7 (C-22), 27.5
(C-16), 27.2 (C-6), 23.6 (C-15), 21.0 (C-11), 12.9 (C-19), 12.2 (C-
18). ESI-MS: m/z 476.4 [M+H]+, 487.2 [M+Na]+. Anal: Calcd. for
C26H34ClNO3S: C 65.59, H 7.20, N 2.94; found: C 65.34, H 7.13,
N 3.01.
2.9.8. Methyl (20R)-20-
(4-methyl-3-pentenyl)-3-oxo-pregnan-1-en-21-oate (10h)
16 mg white solid, 65% yield, m.p. 100–102 ◦C. 1H NMR(CDCl3) ı:
0.72 (3H, s, H3-18), 0.98 (3H, s, H3-19), 1.56 (3H, s, H3-26), 1.67 (3H,
s, H3-27), 3.65 (3H, s, H3-28), 5.05 (1H, t, J = 7.0 Hz, H-23), 5.83 (1H,
d, J = 10.3 Hz, H-2), 7.10 (1H, d, J = 10.3 Hz, H-1). 13C NMR(CDCl3)
ı: 200.2 (C-3), 176.5 (C-21), 158.5 (C-1), 132.1 (C-25), 127.3 (C-
2), 123.6 (C-24), 55.6 (C-14), 52.7 (C-17), 51.0 (C-28), 49.9 (C-9),
47.1 (C-20), 44.2 (C-5), 42.2 (C-13), 40.9 (C-4), 38.9 (C-10), 37.3 (C-
12), 35.6 (C-8), 32.2 (C-22), 31.2 (C-7), 27.5 (C-16), 27.1 (C-6), 25.9
(C-23), 25.7 (C-27), 23.6 (C-15), 21.1 (C-11), 17.6 (C-26), 12.9 (C-
19), 12.2 (C-18). ESI-MS: m/z 427.4 [M+H]+, 429.3 [M+Na]+, 875.7
[2M+Na]+. Anal: Calcd. for C28H42O3: C 78.83, H 9.92; found: C
78.62, H 9.98.
2.9.13. Methyl (20S)-20-benzyl-3-oxo-pregnan-1-en-21-oate
(10m)
12 mg white solid, 40% yield, m.p. 155–158 ◦C. 1H NMR(CDCl3)
ı: 0.75 (3H, s, H3-18), 0.99 (3H, s, H3-19), 2.49-2.61 (1H, m, H-20),
2.70 (1H, dd, J = 11.8, 12.7 Hz, Ha-22), 2.88 (1H, dd, J = 3.9, 12.7 Hz,
Hb-22), 3.45 (3H, s, H3-23), 5.84 (1H, d, J = 10.3 Hz, H-2), 7.08-7.13
(3H, m, H-2, H-2ꢀ, H-6ꢀ), 7.18 (1H, d, J = 10.3 Hz, H-4ꢀ), 7.23 (2H, m,
H-3ꢀ, H-5ꢀ). 13C NMR(CDCl3) ı: 200.2 (C-3), 175.5 (C-21), 158.4 (C-
1), 139.2 (C-1ꢀ), 128.6 (C-3ꢀ, C-5ꢀ), 128.6 (C-2ꢀ, C-6ꢀ), 127.4 (C-2),
126.3 (C-4ꢀ), 55.7 (C-14), 52.8 (C-17), 50.9 (C-23), 50.1 (C-9), 49.9
(C-20), 44.2 (C-5), 42.3 (C-13), 40.9 (C-4), 38.9 (C-10), 38.4 (C-22),
37.2 (C-12), 35.6 (C-8), 31.2 (C-7), 27.5 (C-16), 27.4 (C-6), 23.7 (C-
15), 21.1 (C-11), 12.9 (C-19), 12.2 (C-18). ESI-MS: m/z 435.4 [M+H]+,
2.9.9. Methyl (20R)-20-heptyl-3-oxo-pregnan-1-en-21-oate
(10i)
17 mg white solid, 67% yield, m.p. 92–93 ◦C. 1H NMR(CDCl3) ı:
0.71 (3H, s, H3-18), 0.86 (3H, t, J = 6.6 Hz, H3-28), 0.98 (3H, s, H3-

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C.-S. Jiang et al. / Steroids 75 (2010) 1153–1163
Scheme 1. Synthesis of compounds 10a-n. Reagents and conditions: (a) reaction conditions are specified in Ref. [6]; (b) NaBH4, MeOH, 4 ◦C, 30 min, 95%; (c) TBSCl, imidazole,
DMF, r.t., 10 h, 98%; (d) LDA, RCH2Br, dry THF, −78 ◦C, 1 h, −78 ◦C to r.t., 2 h; (e) acetone, 10% HCl, 5 min, r.t.; (f) Jones reagent, acetone, 0.5 h, r.t.; (g) C5H5N+HBr3−, THF, 0.5 h,
r.t.; (h) LiBr, Li2CO3, dry DMF, reflux, 2 h; (i) IBX, DMSO, toluene, reflux, 12 h.
457.3 [M+Na]+, 891.7 [2M+Na]+. Anal: Calcd. for C29H38O3: C 80.14,
H 8.81; found: C 80.35, H 8.90.
2.10. Biological assays
The cancer cell lines A549 (human lung adenocarcinoma cell
line), HCT-116 (human colon carcinoma cell line), HepG2 (human
hepatocellular carcinoma cell line), SW-1990 (human pancreatic
adenocarcinoma cell line), MCF-7 (human breast adenocarcinoma
cell line) and NCI-H460 (human large lung carcinoma cell line)
were cultured in a proper medium supplemented with 10% fetal
bovine serum in a humidified atmosphere of 5% CO2 at 37 ◦C.
Cell suspensions were plated in 96-well plates at a density of
2 × 104 cells mL−1. 10 ␮L of the test compound solutions (in DMSO)
at various concentrations were added to each well and the cul-
ture was incubated for 72 h at 37 ◦C in a 5% CO2 incubator. After
this treatment, 20 ␮L of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) solution (5 mg/mL) was added to
each well and cells were incubated at 37 ◦C in 5% CO2. After incu-
bation for 4 h, the medium was removed and 100 ␮L of DMSO was
added. The absorbance was measured at 570 nm. Dose–response
curves were generated and the concentration of each compound
2.9.14. Methyl (20R)-3-oxo-20-
(2-phenoxyethylidenyl)-pregnan-1-en-21-oate (10n)
13 mg white solid, 45% yield, m.p. 175–178 ◦C. 1H NMR(CDCl3)
ı: 0.76 (3H, s, H3-18), 1.00 (3H, s, H3-19), 2.49–2.61 (1H, m, H-20),
3.66 (3H, s, H3-24), 3.89 (2H, t, J = 7.6 Hz, H-23), 5.84 (1H, d, J = 9.8 Hz,
H-2), 6.86 (2H, d, J = 7.9 Hz, H-2ꢀ, H-6ꢀ), 6.93 (1H, t, J = 7.9 Hz, H-4ꢀ),
7.11 (1H, d, J = 9.8 Hz, H-1), 7.27 (2H, dd, J = 7.9, 7.9 Hz, H-3ꢀ, H-5ꢀ).
13C NMR(CDCl3) ı: 200.4 (C-3), 176.1 (C-21), 158.7, 158.6, 129.4
(C-3ꢀ, C-5ꢀ), 127.4 (C-2), 120.7 (C-4ꢀ), 114.5 (C-2ꢀ, C-6ꢀ), 66.0 (C-23),
55.7 (C-14), 52.5 (C-17), 51.3 (C-24), 49.8 (C-9), 44.6 (C-20), 44.2 (C-
5), 42.3 (C-13), 40.9 (C-4), 38.9 (C-10), 37.2 (C-12), 35.6 (C-8), 31.6
(C-22), 31.2 (C-7), 27.5 (C-16), 27.2 (C-6), 23.6 (C-15), 21.1 (C-11),
13.0 (C-19), 12.2 (C-18). ESI-MS: m/z 465.3 [M+H]+, 487.4 [M+Na]+,
951.7 [2M+Na]+. Anal: Calcd. for C30H40O4: C 77.55, H 8.68; found:
C 77.32, H 8.77.

C.-S. Jiang et al. / Steroids 75 (2010) 1153–1163
1161
Table 1
Cytotoxicity of methyl spongoate analogs presented as IC50 (␮M).
Compound
A549
HCT-116
HepG2
28.7
–
–
–
28.4
–
–
–
>35
–
–
–
>35
–
–
–
16.2
–
–
–
>35
–
–
–
35.0
–
–
–
33.2
–
–
–
>35
–
–
–
6.1
–
–
SW-1990
MCF-7
NCI-H460
1
7a
8a
9a
10a
7b
8b
9b
10b
7c
8c
9c
10c
7d
8d
9d
10d
7e
8e
9e
10e
7f
8f
9f
10f
7g
8g
9g
10g
7h
8h
9h
10h
7i
8i
9i
10i
7j
8j
4.9
>35
>35
>35
13.8
>35
>35
>35
4.9
>35
>35
>35
7.8
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
17.7
16.4
>35
>35
>35
4.6
>35
>35
>35
>35
>35
>35
>35
15.8
>35
>35
–
>35a
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
24.1
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
11.2
>35
>35
>35
>35
>35
>35
>35
>35
>35
>35
–
30.3
–
–
–
8.6
–
18.2
–
–
–
16.4
–
–
–
22.5
–
–
–
27.6
–
–
–
13.7
–
–
–
15.0
–
–
–
31.4
–
–
–
13.6
–
–
–
20.6
–
–
–
9.9
–
17.3
–
–
–
25.8
–
–
–
32.9
–
–
–
32.2
–
–
–
17.2
–
–
–
23.0
–
–
–
24.1
–
–
–
15.3
–
–
–
26.7
–
–
–
8.8
–
b
–
>35
–
–
–
>35
–
–
–
25.5
–
–
–
20.7
–
–
–
44.1
–
–
–
4.1
–
–
–
20.6
–
–
–
6.6
–
–
–
–
–
–
9j
–
–
10j
7k
8k
>35
>35
>35
–
>35
>35
>35
–
14.3
–
–
28.1
–
–
25.4
–
–
27.2
–
–
9k
–
–
–
–
10k
7l
8l
>35
>35
>35
>35
16.1
>35
>35
12.1
>35
–
>35
–
8.8
–
–
–
>35
–
–
13.3
–
9.2
–
–
–
>35
–
–
8.3
–
11.4
–
–
–
>35
–
–
8.5
–
12.7
–
–
–
>35
–
–
29.2
–
>35
>35
>35
>35
>35
>35
>35
–
9l
10l
7m
8m
10m
7n
8n
–
–
–
–
10n
Fluorouracil
Doxorubicin
>35
–
0.53
>35
–
0.33
>35
109.6
–
>35
120.3
–
>35
31.2
–
>35
8.5
–
a
IC50 values greater than 35 ␮M were considered inactive.
Not determined.
b
required to inhibit cell proliferation by 50% (IC50) was calculated
from the linear portion of the log dose–response curves [34–36].
Compound 2 was first oxidized by the Jones reagent [22] and
then methylated with diazomethane [23] to give the keto steroid
derivatives 3 in 85% yield. The keto steroids 3 was reduced with
NaBH4 [25] in methanol at 4 ◦C to afford the corresponding 3␤-
hydroxyl derivative 4 in 95% yield. Then 4 was reacted at room
temperature with tert-butyldimethylsilyl (TBS) chloride and imi-
dazole in DMF to result in the protected derivative 5 in 98% [26].
In the 1H NMR spectrum of 4 and 5, the splitting pattern and
3. Results and discussion
3.1. Chemistry
3
the JH,H coupling constants of H-3 were similar with those of
For the synthesis of target compounds 10a-n, a synthetic route
was worked out starting from compound 2, whose synthesis was
realized by a literature method [7] (see Scheme 1).
pregnenolone acetate, indicating the ␣ orientation of the H-3. In
order to obtain esters 6a-n with natural R configuration at C-20,

1162
C.-S. Jiang et al. / Steroids 75 (2010) 1153–1163
we treated compound 5 with LiN(iPr)2 and RCH2Br in dry THF at
−78 ◦C to give 6a-n in 60–90% yields by following a previously
reported literature method [27], in which the stereoselectivities
of the alkylation was explained by steric hindrance and the relative
stabilities of the possible alkylation products. The same result was
found from the proton NMR data that no formation of the epimeric
product at C-20 was observed in any of the alkylation reaction.
Sterols 7a-n were obtained from esters 6a-n by TBS-deprotection
of 3␤-OH using 10% HCl in acetone [28], reducing the reaction
time compared with n-Bu4NF/THF, for about 5 min at room tem-
perature. The oxidation of the 3␤-OH by Jones reagent in acetone
[29], reducing the reaction time compared with PDC/CH2Cl2, at
room temperature afforded keto steroids 8a-n in 80–98% yields.
Keto steroids 8a-l were converted to 9a-l by ␣-bromination with
pyridinium tribromide (C5H5N+HBr3−) in THF at room tempera-
ture [30]. In 9a-l, the coupling constants of the H-2 with H2-1
potent inhibitory activity against MCF-7 and compound 10i had the
strongest inhibition against NCI-H460 with an IC50 value of 8.8 ␮M.
Clearly, the nature of the side chain has a significant influence on
the cytotoxicity and different chains selectively inhibited certain
cell lines and they were inactive against others. For instance, the
prenyl side chain in 10g showed the strongest inhibition against
A549 and SW-1990 cell lines but it was inferior to the heptyl side
chain of 10i against the other tested cell lines. By the introduction
of different C-20 side chains, analogs exhibiting larger cytotoxi-
city against selected cell lines than the lead compound 1 were
identified.
4. Conclusion
A series of methyl spongoate (1) analogs with different C-20
side chains was designed and synthesized. The cytotoxicity of 1,
target compounds 10a-n and synthetic precursors 7, 8, 9 were eval-
uated against a panel of cancer cell lines including A549, HCT-116,
HepG2, SW-1990, MCF-7, and NCI-H460. The results not only have
demonstrated that the side chain has an important role in deter-
mining antitumor activity but also confirmed the significance of the
␣,␤-unsaturated carbonyl moiety of the steroid as an electrophilic
Michael acceptor in the antitumor activity. By structural modifica-
tion, analogs showing more potent cytotoxic activity than methyl
spongoate (1) have been found. The results of the pharmacological
study may be useful for the design of novel chemotherapeutic drug
candidates.
(3J2-H␤,1-H␣ ≈ 13 Hz, J2-H␤,1-H␤ ≈ 6 Hz, see Section 2) showed that
3
the 2-H has ␤-axial orientation and hence the 2-bromo substituent
is ␣-equatorial, and no 2␤-brominations was found from proton
NMR data; i.e. the bromination took place diastereoselectively
because the ␣-equatorial substituent is more stable than 2␤-
equatorial substituent. The target compounds 10a-l were obtained
from keto steroids 9a-l by dehydrobromination using lithium car-
bonate as a base and lithium bromide as an additive in dry DMF
upon 2 h reflux [31]. The target compounds 10m,n were prepared
directly in the oxidation of keto steroids 8m,n by 1.5 equiv. of 2-
iodoxybenzoic acid (IBX) [32] in toluene-DMSO (approx. 2:1) after
a 12 h reflux [33].
Acknowledgements
3.2. Biological evaluation
This research work was financially supported by National S&T
Major Project (2009ZX09301-001, 2009ZX09103-060), National
Science Foundation of China (30730108, 20721003), STCSM Project
(09ZR1438000, 10540702900), CAS Key Project (SIMM0907KF-
09), TRF-NSFC Joint Research Project (20911140471) and partially
funded by Hungarian-Chinese Intergovernment S&T Cooperation
Programme (2009–2011).
All the target compounds 10a-n, most of the synthetic precur-
sors and methyl spongoate (1) were tested for antitumor activity
in an MTT assay [34–36] against a panel of cancer cell lines includ-
ing A549 (human lung adenocarcinoma cell line), HCT-116 (human
colon carcinoma cell line), HepG2 (human hepatocellular carci-
noma cell line), SW-1990 (human pancreatic adenocarcinoma cell
line), MCF-7 (human breast adenocarcinoma cell line), NCI-H460
(human large lung carcinoma cell line). The results are shown in
Table 1.
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