Novel and efficient synthesis of 22-alkynyl-13,24(23)-cyclo-18,21- dinorchol-22-en-20(23)-one analogues

Article abstract of DOI:10.1016/j.steroids.2011.01.005

The efficient synthesis of some 22-alkynyl-13,24(23)-cyclo-18,21-dinorchol- 22-en-20(23)-ones was investigated. 22-Iodocyclo-18,21-dinorcholenones were prepared from cyclo-18,21-dinorcholenones using I₂/DMAP/pyridine system firstly. The cross c

Full text of DOI:10.1016/j.steroids.2011.01.005

Steroids 76 (2011) 491–496
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Novel and efficient synthesis of
22-alkynyl-13,24(23)-cyclo-18,21-dinorchol-22-en-20(23)-one analogues
Cunde Wang∗, Lanhai Liu, Hangxian Xu, Zonglei Zhang, Xingbin Wang, Hui Liu
School of Chemistry and Chemical Engineering, Yangzhou University, 180 Siwangting Street, Yangzhou 225002, Jiangsu, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 August 2010
Received in revised form 10 January 2011
Accepted 15 January 2011
Available online 22 January 2011
The efficient synthesis of some 22-alkynyl-13,24(23)-cyclo-18,21-dinorchol-22-en-20(23)-ones was
investigated. 22-Iodocyclo-18,21-dinorcholenones were prepared from cyclo-18,21-dinorcholenones
using I2/DMAP/pyridine system firstly. The cross coupling reaction of 22-iodocyclo-18,21-
dinorcholenones and 1-alkynes was carried out efficiently catalyzed by tetrakis(triphenylphosphine)
palladium/cuprous iodide in the presence of base diisopropylethylamine. This strategy offered a very
straightforward and efficient method for access to conjugated alkynyl cyclo-18,21-dinorcholenones
from the cyclo-18,21-dinorcholenones and 1-alkynes in excellent overall yields. Evaluation of the
synthesized compounds for cytotoxicity against KB, HeLa, MKN-28 and MCF-7 cell lines showed that the
22-alkynylcyclodinorchoenones possessing hydroxylethyl and hydroxylmethyl mono-substituted side
chain at the end of alkynyl group have significantly inhibition activity.
Keywords:
Cyclo-18,21-dinorcholenones
Iodine
Palladium(0) catalyst
Cuprous iodide
© 2011 Elsevier Inc. All rights reserved.
Sonogashira reaction
1. Introduction
ing mode of action and high potency [8,9], and play important
ecological roles [10,11]. Our group has been involved in several
Pentacyclic steroids and their derivatives are a very important
class of steroids. They can be categorized as follows: the fusion of
carbocyclic ring such as benzene or cyclohexane or cyclopentane
to the steroid nucleus, the fusion of a carbocyclic ring to a heteros-
teroid skeleton, the fusion of heterocycle ring containing nitrogen,
oxygen or sulfur to the steroid nucleus, the spiro-pentacyclic
steroids and the bridged-pentacyclic steroids.
More and more studies showed pentacyclic steroids and their
derivatives display very important pharmacological and biological
properties. Many of them were tested and/or evaluated in potential
drug discovery such as ␣-substituted-␣,␤-unsaturated pentacyclic
steroid ketone [1–5]. The structure–activity relationship of the
pentacyclic steroids indicates the carbocyclic or heterocyclic ring
fused plays an important role of biological activity. Therefore,
the preparation of pentacyclic steroids has attracted considerable
attention from medicinal and synthetic organic chemists. A num-
ber of preparations for pentacyclic steroids and their derivatives
were reported recently. Especially, when pentacyclic steroids were
further modified, the unexpectedly special biologically properties
were observed [1,2,6,7]. It is known that natural products which
contain conjugated eneynes or enediynes exhibit an exceptional
biological profile due to their unique molecular structure, strik-
projects aimed at the development of highly convergent syntheses
of functionalized steroids, privileged structures in drug discovery.
Our contribution in this field has been attained mainly by coupling
reactions, as Sonogashira reaction that typically lead to conju-
gated enyne skeletons, with a secondary transformation in steroid
nucleus. This methodology enables generation of various hetero-
cyclic systems or fused rings of steroids. In our previous work,
we reported the preparation of D-ring unsaturated 17-alkynyl
steroids by Pd(PPh3)4/AgOAc-catalyzed coupling of steroidal 17-
triflates and alkynes [12]. Since D-ring unsaturated 17-alkynyl
steroids with conjugated double and triplet bond can be sub-
sequently converted into pentacyclic steroids and 17-oxosteroid
derivatives at the side chain of D-ring, this general method provides
a highly efficient route to these biologically important compounds.
In continuation of our studies on developing new steroids with
conjugated enynes, our attention was focused on functionalized
pentacyclic steroid scaffolds, which produce a variety of pentacyclic
steroid derivatives with cyclo-18,21-dinorcholenone core struc-
ture (Fig. 1). For this reason, simple methods that can install this
unsaturated hydrocarbon moiety are highly desirable. Thus, some
conjugated alkynyl cyclo-18,21-dinorcholenones were obtained
from the cyclo-18,21-dinorcholenones and 1-alkynes as a useful
precursor for the preparation of many interesting steroids here
(Scheme 1 and Tables 1 and 2). Evaluation of the synthesized com-
pounds for cytotoxicity against KB, HeLa, MKN-28 and MCF-7 cell
lines showed that the 22-alkynylcyclodinorchoenones possessing
∗
Corresponding author. Tel.: +86 514 8797 5568; fax: +86 514 8797 5244.
E-mail address: wangcd@yzu.edu.cn (C. Wang).
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.01.005

492
C. Wang et al. / Steroids 76 (2011) 491–496
22
20
19
23
24
22
17
23
13
20
16
19
17
13
16
14
14
8
15
8
15
3
3
13,24-cyclo-18,21-dinorcholane
13,23-cyclo-18,21-dinorcholane
Fig. 1. Structures of 13,24-cyclo- and 13,23-cyclo-18,21-dinorcholane.
X2
R2
X1
Y
R1
Y
Z
Z
Y
Z
i
ii
HO
HO
HO
H
H
H
2a-c
1a-c
3-10
Reaction conditions: i. I2, DMAP, pyridine, CCl4, rt, 15 h; ii. Pd[(C6H5)3P]4, CuI,
1-Alkyne, DIPEA, THF, N2, reflux, 4 h.
Scheme 1. Synthesis of 22-alkynyl-13,24(23)-cyclo-18,21-dinorcholenones 3–10. Reaction conditions: (i) I2, DMAP, pyridine, CCl4, rt, 15 h; (ii) Pd[(C6H5)3P]4, CuI, 1-alkyne,
DIPEA, THF, N2, reflux, 4 h.
Table 1
Preparation of 22-iodocyclo-18,21-dinorcholenones 2a–c.
Entry
3-OH
5-H
Y
Z
X1
X2
2 (yield, %)
1
2
3
␣
␤
␣
␤
␣
␣
CH2
CH2
C
C
N/A
O
O
H
H
I
I
I
H
2a
2b
2c
98
93
98
C
O
hydroxylethyl and hydroxylmethyl mono-substituted side chain at
the end of alkynyl group have significantly inhibition activity.
dinorchol-22-en-20-one were prepared using Dr. Covey’s method
(Washington University School of Medicine in St. Louis) [2].
2. Experimental
2.2.2. Preparation of
3-hydroxy-22-iodo-13,24-cyclo-18,21-dinorchol-22-en-20-one
and
2.1. General
3-hydroxy-22-iodo-13,23-cyclo-18,21-dinorchol-20-en-23-one
2.2.2.1. (3˛,5ˇ)-3-Hydroxy-22-iodo-13,24-cyclo-
All reactions under nitrogen were run. All melting points
were determined in a Yanaco melting point apparatus and are
uncorrected. IR spectra were recorded in a Nicolet FT-IR 5DX spec-
trometer. The 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra
were recorded in a Bruker ACF-300 spectrometer with TMS as inter-
nal reference in CDCl3 solutions. The J values are given in hertz.
Only discrete or characteristic signals for the 1H NMR are reported.
The elemental analyses were performed in a Perkin-Elmer 240C
instrument. Optical rotations were determined in a Perkin-Elmer
343 polarimeter. Flash chromatography was performed on silica
gel (230–400 mesh) eluting with ethyl acetate–hexanes mixture.
18,21-dinorchol-22-en-20-one
(2a). The
mixture
of
(3␣,5␤)-3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-20-
one (328 mg, 1.0 mmol), 4-dimethylaminopyridine (9.8 mg,
0.08 mmol) and iodine (508 mg, 2.0 mmol) in pyridine (3.0 mL)
and CCl4 (10 mL) was stirred for 12 h at room temperature. After
the mixture was diluted with EtOAc (15 ml), the resulting mixture
was washed with sat. Na2S2O3, water, 10% HCl and brine, dried
over Na2SO4. Removal of solvent, the residue was purified by flash
chromatography (silica gel, EtOAc/hexanes, 1/6) to give product
(3␣,5␤)-3-hydroxy-22-iodo-13,24-cyclo-18,21-dinorchol-22-
en-20-one (2a) (445 mg, 98%) as a white solid, mp 210–212 ◦C
(EtOAc–hexanes); [␣]D20 = +47.47◦ (c = 0.990, CHCl3); 1H NMR
(300 MHz, CDCl3) 7.59 (m, 1H), 3.63 (m, 1H), 2.50 (t, J = 10.2 Hz,
1H), 0.92 (s, 3H); 13C NMR (75 MHz, CDCl3): 194.38, 157.02, 101.62,
71.29, 56.84, 55.72, 47.96, 41.74, 39.81, 36.06, 35.64, 35.15, 34.41,
33.89, 30.28, 29.95, 27.23, 26.68, 26.26, 26.01, 23.18, 20.08; IR
(NaCl, film, cm−1): 3392, 2246, 1674, 1597, 1447, 1330, 910, 731;
2.2. Organic synthesis
2.2.1. Preparation of the starting material
3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-20-one and
3-hydroxy-13,23-cyclo-18,21-dinorchol-20-en-23-one
(3␣,5␣)-3-Hydroxy-13,23-cyclo-18,21-dinorchol-20-en-
23-one, (3␣,5␤) and (3␤,5␣)-3-hydroxy-13,24-cyclo-18,21-

C. Wang et al. / Steroids 76 (2011) 491–496
493
Table 2
Synthesis of 22-alkynyl-13,24(23)-cyclo-18,21-dinorcholenones 3–10.
Entry
3-OH
5-H
Y
Z
R1
H
R2
Product (yield, %)
1
␣
␤
CH2
C
O
O
O
3
4
5
6
92
OH
OH
2
3
4
␣
␤
␣
␤
␣
␣
CH2
CH2
C
C
H
H
92
93
86
OH
OH
C
O
N/A
H
5
6
7
␣
␣
␣
␣
␣
␣
C
C
C
O
O
O
N/A
N/A
N/A
H
H
H
7
8
9
90
80
92
OH
OH
OH
8
␣
␣
C
O
N/A
H
10
94
Anal. Calcd. for C22H31IO2: C, 58.15; H, 6.88; Found C, 58.10; H,
7.02.
mixture was diluted with DCM (15 ml) and the solid was removed
by filtered, the residue was purified by flash chromatography (silica
gel, EtOAc/hexanes, 1/10–1/4) to give product 3–10.
2.2.2.2. (3ˇ,5˛)-3-Hydroxy-22-iodo-13,24-cyclo-18,21-dinorchol-
22-en-20-one (2b). Following the above procedure using
(3␤,5␣)-3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-20-one as
a starting material, the title compound 2b (93%) was obtained as
a white solid, mp 188–190 ◦C (EtOAc–hexanes); [␣]2D0 = +13.95◦
(c = 0.43, CHCl3); 1H NMR (300 MHz, CDCl3) 7.59 (q, J = 3.0 Hz, 1H),
3.60 (m, 1H), 2.49 (t, J = 10.2 Hz, 1H), 0.80 (s, 3H); 13C NMR (75 MHz,
CDCl3): 194.39, 156.90, 101.80, 71.05, 56.98, 55.84, 53.69, 48.05,
44.75, 38.05, 36.93, 35.50(2C), 33.89, 31.98, 31.39, 30.11, 28.37,
27.29, 26.12, 20.66, 12.34; IR (NaCl, film, cm−1): 3400, 2245, 1672,
1596, 1444, 1331, 907, 731; Anal. Calcd. for C22H31IO2: C, 58.15; H,
6.88; Found C, 58.03; H, 7.10.
2.2.3.1. (3˛,5ˇ)-3-Hydroxy-22-(3-(3-hydroxybut-1-ynyl))-13,24-
cyclo-18,21-dinorchol-22-en-20-one (3). Following the general
procedure, the title compound 3 was obtained as needles solid,
92% in yield, mp 220–222 ◦C (EtOAc–hexanes); [␣]2D0 = +13.79◦
(c = 0.435, CHCl3); 1H NMR (300 MHz, CDCl3) 7.11 (m, 1H), 4.67 (m,
1H), 3.63 (bs, 1H), 3.28 (dd, J = 4.8, 15.3 Hz, 1H), 1.48 (d, J = 6.3 Hz,
6H), 0.92 (s, 3H); 13C NMR (75 MHz, CDCl3): 198.35, 152.39, 122.34,
94.59, 78.10, 71.60, 58.41, 58.35, 57.60, 56.40, 47.74, 41.95, 40.04,
36.02, 35.59, 35.40, 34.52, 34.27, 30.39, 27.20, 26.77, 26.40, 26.15,
24.12, 24.01, 23.22, 20.25; IR (NaCl, film, cm−1): 3400, 2244, 1670,
1448, 1365, 1165, 1081, 911, 731; Anal. Calcd. for C27H38O3: C,
78.98; H, 9.33; Found C, 78.88; H, 9.06.
2.2.2.3. (3˛,5˛)-3-Hydroxy-22-iodo-13,23-cyclo-18,21-dinorchol-
20-en-23-one (2c). Following the above procedure using
(3␣,5␣)-3-hydroxy-13,23-cyclo-18,21-dinorchol-20-en-23-one as
a starting material, the title compound 2c (98%) was obtained as
a white solid, mp 215–217 ◦C (EtOAc–hexanes); [␣]2D0 = +65.73◦
(c = 0.25, CHCl3); 1H NMR (300 MHz, CDCl3) 7.93 (d, J = 3.6 Hz, 1H),
4.05 (s, 1H), 2.86 (dd, J = 3.3, 9.9 Hz, 1H), 2.30 (m, 1H), 0.92 (s, 3H);
13C NMR (75 MHz, CDCl3): 206.75, 170.23, 101.86, 66.10, 56.80,
54.88, 53.82, 53.37, 38.93, 36.15, 35.64, 32.91, 32.77, 32.09, 32.01,
29.72, 28.75, 28.14, 25.29, 19.41, 10.94; IR (NaCl, film, cm−1): 3392,
2248, 1702, 1578, 1447, 1271, 909, 731; Anal. Calcd. for C21H29IO2:
C, 57.28; H, 6.64; Found C, 57.10; H, 6.52.
2.2.3.2. (3˛,5ˇ)-3-Hydroxy-22-(3-(2-(1-
hydroxycyclohexyl)ethynyl))-13,24-cyclo-18,21-dinorchol-22-en-
20-one (4). Following the general procedure, the title compound
4 was obtained as needles solid, 90% in yield, mp 222–224 ◦C
(MeOH); [␣]2D0 = +12.01◦ (c = 0.267, CHCl3); 1H NMR (300 MHz,
CD3OD) 7.24 (m, 1H), 3.59 (m, 1H), 1.01 (s, 3H); 13C NMR (75 MHz,
DMSO-d6): 196.96, 152.91, 121.38, 96.63, 77.82, 69.72, 66.85,
56.94, 55.37, 46.89, 41.42, 39.63, 36.14, 35.11, 34.97, 34.18(2C),
33.58, 30.27, 26.56(2C), 26.09, 25.79, 25.47, 24.83, 23.13(2C),
22.57(2C), 19.69; IR (NaCl, film, cm−1): 3402, 2244, 1680, 1448,
1365, 1165, 1081, 911, 731; Anal. Calcd. for C30H42O3: C, 79.96; H,
9.39; Found C, 79.78; H, 9.20.
2.2.3. General procedure for the coupling reaction catalyzed by
Pd[(C6H5)3P]4/CuI from 22-iodocyclo-18,21-dinorcholenones and
1-alkynes
2.2.3.3. (3ˇ,5˛)-3-Hydroxy-22-(3-(3-hydroxy-3-methylbut-1-
ynyl))-13,24-cyclo-18,21-dinorchol-22-en-20-one
(5). Following
To the mixture of compound 2a–c (1.00 mmol), CuI (19 mg,
0.10 mmol), Pd[(C6H5)3P]4 (60 mg, 0.05 mmol) and DIPEA (390 mg,
3.00 mmol) in dried THF (10 mL) was added 1-alkyne (1.50 mmol)
by syringe at room temperature under nitrogen. The resultant mix-
ture was stirred under refluxing and nitrogen for 4 h. After the
the general procedure, the title compound 5 was obtained as
needles solid, 89% in yield, mp 216–218 ◦C (EtOAc–hexanes);
[␣]2D0 = −4.75◦ (c = 0.36, CHCl3); 1H NMR (300 MHz, CDCl3) 7.09
(m, 1H), 3.62 (m, 1H), 3.34 (bs, 1H), 1.55 (s, 6H), 0.81 (s, 3H); 13C
NMR (75 MHz, CDCl3): 198.11, 151.95, 122.51, 97.16, 76.41, 71.04,

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C. Wang et al. / Steroids 76 (2011) 491–496
65.12, 57.47, 56.10, 53.75, 47.55, 44.79, 37.98, 36.97, 35.53, 35.33,
34.00, 32.01, 31.30(3C), 28.41, 27.08, 26.36, 26.11, 20.63, 12.35; IR
(NaCl, film, cm−1): 3414, 2243, 1670, 1445, 731; Anal. Calcd. for
C27H38O3: C, 78.98; H, 9.33; Found C, 79.22; H, 9.18.
39.17, 36.35, 35.81, 33.10, 32.83, 32.25, 30.22, 28.93, 28.63, 28.34,
27.97, 25.47, 22.01(3C), 19.61, 11.09; IR (NaCl, film, cm−1): 3393,
2247, 1701, 1612, 1448, 909, 732; Anal. Calcd. for C27H38O2: C,
82.18; H, 9.71; Found C, 82.12; H, 9.58.
2.2.3.4. (3˛,5˛)-3-Hydroxy-22-(3-(3-hydroxy-3-methylbut-1-
2.3. Cytotoxic activity against human epidermoid carcinoma cell
line KB, human cervical carcinoma cell line HeLa, human gastric
carcinoma cell line MKN-28 and human breast carcinoma cell line
MCF-7
ynyl))-13,23-cyclo-18,21-dinorchol-22-en-23-one
(6). Following
the general procedure, the title compound 6 was obtained as
needles solid, 86% in yield, mp 120–122 ◦C (EtOAc–hexanes);
[␣]2D0 = +60.29◦ (c = 0.59, CHCl3); 1H NMR (300 MHz, CDCl3) 7.60
(d, J = 3.3 Hz, 1H), 4.09 (s, 1H), 3.82 (s, 1H), 2.74 (m, 1H), 1.56 (s, 6H),
0.912 (s, 3H); 13C NMR (75 MHz, CDCl3): 209.81, 165.48, 128.24,
100.50, 73.04, 66.42, 64.98, 57.28, 57.04, 54.10, 49.86, 39.16, 36.35,
35.81, 33.09, 32.95, 32.31, 32.25, 31.13, 30.98, 30.16, 28.84, 28.34,
25.50, 19.66, 11.22; IR (NaCl, film, cm−1): 3401, 2245, 1687, 1448,
1360, 1165, 908, 732; Anal. Calcd. for C26H36O3: C, 78.75; H, 9.15;
Found C, 78.48; H, 8.96.
All tumor cell lines tested were purchased from Shanghai Insti-
tute of Cell Biology, Chinese Academy of Science. The cell lines were
cultured in RPMI 1640 medium with 10% newborn calf serum. It
was maintained in a humidified incubator with an atmosphere of
95% air and 5% CO2 at 37 ◦C. The cells were continuously passaged
once every 3–4 days. Growing cells were collected on experiments.
DMSO was used as latent solvent with the highest concentration
less than 0.1% in solution of the drug. The control groups of dox-
orubicin, blank (1640) and DMSO solvent were set up at the same
time. Proliferative activity was evaluated by colorimetric sulforho-
damine B (SRB) assay. Briefly, cells were plated in 96-well plates.
After cell adhering, they were treated with different compounds in
a dose-dependent way for 44 h. Then the cells were fixed by 10%
TDA for 1 h and stained by SRB for 10 min. After washed with acetic
acid to remove the excess dye, protein bounding dye was dissolved
in 10 mM Tris and detected by a Model Elx 800 Autoplate reader
(Bio-Tek Instruments, USA).
2.2.3.5. (3˛,5˛)-3-Hydroxy-22-(3-(3-hydroxybut-1-ynyl))-13,23-
cyclo-18,21-dinorchol-22-en-23-one (7). Following the general
procedure, the title compound 7 was obtained as needles solid,
90% in yield, mp 115–117 ◦C (EtOAc–hexanes); [␣]2D0 = +82.21◦
(c = 0.62, CHCl3); 1H NMR (300 MHz, CDCl3) 7.61 (d, J = 3.9 Hz, 1H),
4.69 (m, 1H), 4.08 (s, 1H), 3.63 (d, J = 4.2 Hz, 1H), 2.75 (m, 1H), 2.30
(m, 1H), 2.20 (s, 1H), 1.49 (d, J = 6.6 Hz, 3H), 0.91 (s, 3H); 13C NMR
(75 MHz, CDCl3): 209.98, 165.74, 128.08, 97.80, 74.78, 66.44, 58.24,
57.30, 56.90, 53.94, 49.86, 39.13, 36.34, 35.74, 33.01, 32.92, 32.27,
32.24, 30.10, 28.79, 28.32, 25.53, 23.91, 19.58, 11.11; IR (NaCl, film,
cm−1): 3418, 2247, 1694, 1613, 1447, 1115, 908, 732; Anal. Calcd.
for C25H34O3: C, 78.49; H, 8.96; Found C, 78.40; H, 8.76.
3. Results and discussion
2.2.3.6. (3˛,5˛)-3-Hydroxy-22-(3-(3-hydroxyprop-1-ynyl))-13,23-
cyclo-18,21-dinorchol-22-en-23-one (8). Following the general
procedure, the title compound 8 was obtained as needles solid,
80% in yield, mp 110–113 ◦C (EtOAc–hexanes); [␣]2D0 = +55.71◦
(c = 0.70, CHCl3); 1H NMR (300 MHz, CDCl3) 7.64 (d, J = 3.9 Hz, 1H),
4.44 (s, 2H), 4.07 (s, 1H), 2.76 (m, 1H), 2.49 (bs, 1H), 2.30 (m, 1H),
0.91 (s, 3H); 13C NMR (75 MHz, CDCl3): 209.75, 166.13, 128.11,
93.79, 76.73, 66.60, 57.40, 56.93, 54.03, 51.38, 49.97, 39.28, 36.44,
35.85, 33.09, 33.00, 32.33(2C), 30.16, 28.97, 28.40, 25.64, 19.62,
11.90; IR (NaCl, film, cm−1): 3400, 2248, 1694, 1446, 908, 731;
Anal. Calcd. for C24H32O3: C, 78.22; H, 8.75; Found C, 78.20; H,
8.66.
3.1. Chemistry
To initiate our studies, we first prepared three 22-iodo-
18,21-dinorcholenones in 93–98% yields through iodination of
the corresponding 18,21-dinorcholenones using iodine in the
presence of DMAP and pyridine in CCl4 at room temperature
for 15 h (Scheme 1 and Table 1). Recently, the construction
of conjugated enynes via palladium-catalyzed coupling reac-
tion of 1-alkynes with 1-haloalkenes or alkenyl triflates has
attracted considerable interest [13,14]. Among of them, it is
convenient of palladium-catalyzed cross-coupling reactions of
terminal alkynes with ␣-halo-␣,␤-unsaturated ketones. Typi-
cally these coupling reactions are carried out using a palladium
catalyst such as Pd(PPh3)2Cl2, Pd(PPh3)4, Pd(OAc)2 and a cop-
per salt as co-catalyst in the presence of an amine base.
To make a detailed investigation for the cross-coupling reac-
tion of 22-iodo cyclo-18,21-dinorcholenone 2a and 3-butyn-2-ol,
firstly, (3␣,5␤)-3-hydroxy-22-iodo-13,24-cyclo-18,21-dinorchol-
22-en-20-one 2a was treated with 1.5 equiv. of 3-butyn-2-ol
in THF in the presence of 10% Pd(PPh3)2Cl2 (0.05 equiv.), CuI
(0.10 equiv.), and DIPEA (3.00 mmol) under a nitrogen atmosphere
to give (3␣,5␤)-3-hydroxy-22-(3-(3-hydroxybut-1-ynyl))-13,24-
cyclo-18,21-dinorchol-22-en-20-one 3 in 85% yield (Table 3, entry
1). The use of Pd(PPh3)4 (0.05 equiv.) or Pd/C (0.05 equiv.)-PPh3
(0.12 equiv.) in the place of Pd(PPh3)2Cl2 gave the yield of 3 to 92%,
72% respectively (Table 3, entries 2 and 3). Therefore, it was evi-
dent that Pd(PPh3)4 (5 mol%) was the most effective catalyst for
this transformation. But Pd/C–CuI–PPh3 is a less expensive catalytic
system than Pd(PPh3)4–CuI or Pd(PPh3)2Cl2–CuI catalytic system.
When copper iodide was replaced with silver halides for the cou-
pling reaction of sensitive vinyl triflates and 1-alkyne [15], the
desired conjugated enyne product was efficiently obtained in good
yield. Thus, we tested this reaction using the above said 22-iodo-
18,21-dinorcholenone 2a and 3-butyn-2-ol in THF under nitrogen
in the presence of Pd(PPh3)4 using silver iodide as a co-catalyst. It
2.2.3.7. (3˛,5˛)-3-Hydroxy-22-(3-(2-(1-
hydroxycyclohexyl)ethynyl))-13,23-cyclo-18,21-dinorchol-22-en-
23-one (9). Following the general procedure, the title compound
9 was obtained as needles solid, 92% in yield, mp 150–152 ◦C
(EtOAc–hexanes); [␣]2D0 = +74.43◦ (c = 0.46, CHCl3); 1H NMR
(300 MHz, CDCl3) 7.60 (d, J = 3.6 Hz, 1H), 4.08 (s, 1H), 3.75 (m, 1H),
2.73 (m, 1H), 2.33–2.29 (m, 2H), 0.91 (s, 3H); 13C NMR (75 MHz,
CDCl3): 209.85, 165.42, 128.32, 99.69, 74.78, 68.42, 66.38, 57.24,
57.03, 54.05, 49.82, 39.58(2C), 39.10, 36.31, 35.76, 33.06, 32.89,
32.30, 32.24, 30.16, 28.81, 28.32, 25.50, 25.16, 23.15(2C), 19.66,
11.23; IR (NaCl, film, cm−1): 3419, 2243, 1689, 1614, 1447, 907,
732; Anal. Calcd. for C29H40O3: C, 79.77; H, 9.23; Found C, 79.42;
H, 9.18.
2.2.3.8. (3˛,5˛)-3-Hydroxy-22-(3-(4-methylpent-1-ynyl))-13,23-
cyclo-18,21-dinorchol-22-en-23-one (10). Following the general
procedure, the title compound 10 was obtained as needles solid,
94% in yield, mp 110–112 ◦C (EtOAc–hexanes); [␣]2D0 = +87.70◦
(c = 0.37, CHCl3); 1H NMR (300 MHz, CDCl3) 7.55 (d, J = 3.6 Hz, 1H),
4.05 (s, 1H), 2.73 (m, 1H), 2.35 (m, 1H), 2.27 (d, J = 6.6 Hz, 2H), 1.00
(d, J = 6.6 Hz, 6H), 0.92 (s, 3H); 13C NMR (75 MHz, CDCl3): 209.72,
164.49, 129.09, 95.55, 72.36, 66.45, 57.00, 56.95, 54.08, 49.57,

C. Wang et al. / Steroids 76 (2011) 491–496
495
Table 3
Pd/Ag or Cu salt-catalyzed cross-coupling reaction of 22-iodo cyclo-18,21-dinorcholenone 2a with 3-butyn-2-ol to give compound 3a.
Entry
[Pd] catalyst
Co-catalyst
Base
Solvent
Yield (%)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
PdCl2(PPh3)2
Pd(PPh3)4
Pd/C-PPh3
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
CuI (10 mol%)
CuI (10 mol%)
CuI (10 mol%)
AgI (20 mol%)
AgBF4 (20 mol%)
AgOAc (20 mol%)
CuI (10 mol%)
CuI (10 mol%)
CuI (10 mol%)
CuI (10 mol%)
CuI (10 mol%)
CuI (10 mol%)
CuI (20 mol%)
CuI (5 mol%)
iPr2NEt
iPr2NEt
iPr2NEt
iPr2NEt
iPr2NEt
iPr2NEt
iPr2NEt
iPr2NEt
Et3N
Pyridine
K2CO3
Cs2CO3
iPr2NEt
iPr2NEt
THF
THF
THF
THF
THF
THF
DMF
MeCN
THF
THF
THF
THF
THF
THF
85
92
72
76
82
86
86
80
88
62
70
87
92
80
a
Reaction conditions: 22-iodo cyclo-18,21-dinorcholenone 2a/3-butyn-2-ol/base = 1/1.5/3.0. Refluxing, nitrogen; 4 h
Isolated yield based on 22-iodo cyclo-18,21-dinorcholenone 2a.
b
was found that Pd(PPh3)4/AgI catalysts in THF gave product 3 in
studies showed those natural and synthetic steroids with ␣␤
unsaturated ketone or enyne structural core give the potency
against human cancer cell lines [17]. Thus, all compounds syn-
thesized as described above were subjected to in vitro cytotoxic
evaluation against KB (human epidermoid carcinoma), HeLa
(human cervical carcinoma), MKN-28 (human gastric cancer) and
MCF-7 (human breast carcinoma) cell lines. Doxorubicin which
exhibits cytotoxicity against KB, HeLa, MKN-28 and MCF-7 cell
lines, was used as reference. The results are summarized in Table 4.
From the data shown in Table 4, most of the new com-
pounds showed a measurable anti-cancer activity against KB,
HeLa, MKN-28 and MCF-7 cell lines tested. Although this is a
preliminary screening, the results showed compound 10 con-
taining only isobutyl side chain without hydroxyl was found
inactive to all the tumor cells tested (IC50 value > 80 ␮g/ml).
Similar results were found for the compounds 4–6 and 9 contain-
ing hydroxyl multi-substituted side chain at the end of alkynyl
group for all cell lines. However, the cytotoxicity data of 22-
alkynylcyclodinorchoenone possessing hydroxylethyl side chain at
the end of alkynyl group showed significant cytotoxicity activity
(IC50 value 23.2 0.8, 32.0 2.6, 21.6 2.4 and 46.8 3.5 ␮g/ml
for KB, HeLa, MKN-28 and MCF-7, respectively). Similar results
were also found for the compounds 3 and 7 containing hydroxyl
mono-substituted side chain at the end of alkynyl group for all
cell lines. The above results indicated that the end of 22-alkynyl
group with hydroxyl mono-substituted side chain must be present
to retain cytotoxic activity. This may indicate that the anticancer
properties depend not only on hydroxyl group of the end of 22-
alkynyl side chain but also on the moieties attached to the side
chain.
76% yield (Table 3, entry 4). Besides silver iodide, fluoroborate or
acetate [12,16] proved to be efficient in this reaction too, although
lower yields were obtained (Table 3, entries 5 and 6). Afterwards,
we examined various bases (iPr2NEt, Et3N, pyridine, K2CO3 and
Cs2CO3) and solvents (DMF, MeCN and THF) (Table 3, entries 2
and 7–12). Among these tested conditions, iPr2NEt, Et3N and cae-
sium carbonate could promote efficiently this reaction in THF as
solvent in high yield. However, the weak base pyridine and potas-
sium carbonate promoted limitedly this coupling reaction in lower
yields (Table 3, entries 10 and 11). Furthermore, the co-catalyst
copper iodide concentration was between 10 and 20 mol% to yield
product 3 in 92% (Table 3, entry 13). But the result showed that
yields of product 3 were obviously different when cuprous iodide
concentration was reduced from 10% to 5 mol%. Under general
and optimized conditions: 22-iodo cyclo-18,21-dinorcholenones
i
2a–c, 1-alkynes, Pd(PPh3)4, CuI and Pr2NEt in the mole ratios of
1:1.5:0.05:0.10:3.0 in THF under reflux), a series of 22-iodo cyclo-
18,21-dinorcholenones 2a–c and 1-alkynes were tested toward
this coupling reaction. As indicated in Table 3, various 22-iodo
cyclo-18,21-dinorcholenones and 1-alkynes were effective for this
coupling reaction, and the desired products 3–10 could be obtained
in excellent yields (Table 3, entries 1–8).
3.2. Biological activity
Covey and coworkers [2] reported the starting material
3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-20-one
(1a
and 1b) shows modest cytotoxicity on the [35S]TBPS as IC50
(373 20–1570 140 nM) at the GABA receptors. However, some
Table 4
The in vitro cytotoxic activity (IC50, in ␮M) of compounds against human carcinoma cell lines (KB, HeLa, MKN-28, and MCF-7)a.
Compound
Cancer cell lines
KB
IC50 (␮g/ml)
HeLa
34.0 0.5
57.8 3.2
72.4 3.2
MKN-28
MCF-7
3
4
5
6
32.0 4.6
76.0 1.3
>80
22.2 3.2
52.2 0.6
70.3 2.5
>80
26.5 0.5
48.2 1.8
59.5 3.0
73.2 2.6
52.4 0.7
46.8 3.5
>80
>80
>80
7
8
9
45.2 4.5
23.2 0.8
>80
56.2 0.8
32.0 2.6
76.2 0.8
60.4 1.3
21.6 2.4
>80
10
>80
0.024
>80
0.22
>80
0.20
>80
0.23
Doxorubicinb
KB: human epidermoid carcinoma; HeLa: human cervical carcinoma; MKN-28: human gastric cancer; MCF-7: human breast cancer.
a
The results are the average mean of eight replicate determinations SD.
Used as reference.
b

496
C. Wang et al. / Steroids 76 (2011) 491–496
4. Conclusion
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In summary, we have successfully developed a novel and
operationally simple coupling reaction for highly efficient synthe-
sis of 22-alkynyl-13,24(23)-cyclo-18,21-dinorchol-22-en-20(23)-
ones. Firstly, 22-Iodocyclo-18,21-dinorcholenones were pre-
pared from cyclo-18,21-dinorcholenones using I2/DMAP/pyridine
system. The cross coupling reaction of 22-iodocyclo-18,21-
dinorcholenones and 1-alkynes was carried out efficiently cat-
alyzed by tetrakis(triphenylphosphine) palladium/cuprous iodide
in the presence of base diisopropylethylamine. This strategy
offered a very straightforward and efficient method for access to
conjugated alkynyl cyclo-18,21-dinorcholenones from the cyclo-
18,21-dinorcholenones and 1-alkynes in excellent overall yields,
which are key intermediates for the preparation of some biolog-
ically important modified 22-side chain pentacyclic steroids and
structurally related fused pentacyclic steroids.
The preliminary results showed that those 22-alkynylcyclodi-
norchoenones possessing hydroxylethyl and hydroxylmethyl
mono-substituted side chain at the end of alkynyl group have sig-
nificant impact on inhibiting human epidermoid carcinoma cell
line KB, human cervical carcinoma cell line HeLa, human gastric
cancer cell line MKN-28 and human breast carcinoma cell line
MCF-7. Due to the structural features of our novel compounds,
this mechanism of action cannot be discarded. Further research
on the structure–activity relationship, their possible mechanism
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Acknowledgements
We are grateful to the Natural Science Foundation of Jiangsu
Education Ministry of China (Grant 07KJB150135) and the NSF
of Jiangsu Province (Grant BK2008216) for financial support. We
thank Dr. Xin Jiang for helpful discussions.
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