Synthesis and cytotoxicity of some novel 21E-benzylidene steroidal derivatives

Article abstract of DOI:10.1016/j.steroids.2013.04.016

A series of novel derivatives of 21E-benzylidene-pregn-1,4-diene-3,20-dione 7a-g and 21E-benzylidene-4-chloro-pregn-1,4-diene-3,20-dione 8a-g was synthesized from the commercially available progesterone. These title compounds were evaluated for their cyto

Full text of DOI:10.1016/j.steroids.2013.04.016

Steroids 78 (2013) 874–879
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and cytotoxicity of some novel 21E-benzylidene steroidal
derivatives
Ning-Juan Fan a,1, Yu-Bin Bai a,1, Fei-Yu Zhang a, Bo Luo a, Jiang-Jiang Tang a, Qiang-Zhe Zhang b,
Jin-Ming Gao a,
⇑
a Shaanxi Engineering Center of Bio-Resource Chemistry & Sustainable Utilization, College of Science, Northwest A&F University, Yangling, Shaanxi 712100, China
b Life Science Platform, Centre of Chemical Genomics and Medicine Technology, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel derivatives of 21E-benzylidene-pregn-1,4-diene-3,20-dione 7a–g and 21E-benzylidene-
4-chloro-pregn-1,4-diene-3,20-dione 8a–g was synthesized from the commercially available progester-
one. These title compounds were evaluated for their cytotoxic activity against brine shrimp (Artemia sal-
ina) and murine Lewis lung carcinoma cells (LLC). It was found that compounds 7a–g exhibited stronger
activities than 8a–g against the brine shrimps, and some of the tested compounds possessed weak inhi-
bition of LLC cells.
Received 7 January 2013
Received in revised form 23 April 2013
Accepted 27 April 2013
Available online 9 May 2013
Keywords:
Progesterone
Ó 2013 Elsevier Inc. All rights reserved.
Aldol reaction
Benzylidene steroidal derivatives
Cytotoxicity
1. Introduction
two intermediates 2 and 5, respectively, with various aromatic
aldehydes by aldol condensation reactions, and their cytotoxic
Steroids have always attracted considerable interest because of
their biological signaling molecules. They can regulate a variety of
biological processes and have a potential of being drugs for the
treatment of a large number of diseases including cardiovascular
[1], autoimmune diseases [2], breast cancer, prostate cancer, osteo-
arthritis [3,4], etc. Recent studies have shown that benzylidene
derivatives are a range of versatile physiologically active com-
pounds and are used as substrates for various organic syntheses.
These compounds also possess a variety of biological activities
such as cytotoxic [5–8], antimalarial [9,10], antileishmanial
[11,12], anti-inflammatory [13,14], anti-HIV [15], antifungal [16],
and tyrosine kinase inhibition [17]. More recently, some steroidal
benzylidene derivatives have been shown to serve as dehydroge-
nase inhibitors [18], antimicrobial agents [19] and antineoplastic
agents [20–22].
activities against brine shrimp (Artemia salina) and murine Lewis
lung carcinoma cells (LLC) were also evaluated, which we wish to
report herein.
2. Experimental
2.1. General methods
Melting points were measured on an X-4 micromelting point
apparatus. NMR spectra were recorded on a Bruker Advance III
500 instrument in CDCl3 with TMS as internal standard for protons
and solvent signals as internal standard for carbon spectra. Chem-
ical shift values are mentioned in d (ppm) and coupling constants
are given in Hz. Mass spectra were obtained on either a ESI-esquire
3000 or maXis 4G Bruker Daltonics instrument.
Column chromatography (CC) was performed over silica gel
(200–300 mesh, Qingdao Marine Chemical Ltd.). All solvents were
distilled prior to use. The progress of all reactions was monitored
by TLC on 2 cm ꢀ 5 cm precoated silica gel 60 F254 plates of thick-
ness of 0.25 mm (Qingdao Marine Chemical Group, Co.). The chro-
matograms were visualized under UV 254–366 nm and iodine.
In order to obtain biologically potent compounds with diverse
structures, the present studies prepared a series of new 21E-ben-
zylidene derivatives from the commercially available progesterone
1 by structural modifications.
otal intermediate pregn-1,4-diene-3,20-dione 2, and introduction
of a chlorine atom at C-4 of 1 and then
1-dehydrogenation gave
D
1-Dehydrogenation of 1 gave a piv-
D
another intermediate, 4-chloro-pregn-1,4-diene-3,20-dione 5. The
benzylidene derivatives 7a–g and 8a–g were prepared from the
2.2. Chemical synthesis
2.2.1. 4a,5a- and 4b,5b-Epoxypregnan-3,20-dione 3 (3a and 3b)
A mixture of 10% NaOH (2.8 mL) and 30% H2O2 (5.6 mL) was
added to a solution of progesterone 1 (3.14 g, 10 mmol) in CH2Cl2
⇑
Corresponding author. Tel.: +86 29 87092515.
E-mail address: jinminggao@nwsuaf.edu.cn (J.-M. Gao).
These authors contributed equally to this work.
1
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.04.016

N.-J. Fan et al. / Steroids 78 (2013) 874–879
875
(30 mL) and methanol (40 mL). The reaction mixture was stirred at
room temperature for 24 h, at which time TLC indicated reaction
completion. The solvent was evaporated in vacuum, and then CH2-
Cl2 (150 mL) was added. The organic layer was washed with 1 mol/
L HCl solution, H2O and brine, and dried over anhydrous Na2SO4.
Upon evaporation, 4,5-epoxypregnan-3,20-dione 3 was obtained
127.61 (C-2), 155.57 (C-1), 168.83 (C-5), 186.27 (C-3), 209.09 (C-
20). MS (ESI): m/z 313 [M + H]+.
2.2.3.2. 4-Chloro-pregn-1,4-diene-3,20-dione (5). White crystals
(624 mg, 45%): mp 191–193 °C; 1H NMR (500 MHz, CDCl3) d
ppm: 0.72 (3H, s, 18-CH3), 1.31 (3H, s, 19-CH3), 2.13 (3H, s, 21-
CH3), 3.33 (1H, dq, J = 3.5, 4.5, 16.0, 6-H), 6.38 (1H, d, J = 10.0 Hz,
2-H), 7.09 (1H, d, J = 10.5 Hz, 1-H); 13C NMR (500 MHz, CDCl3) d
ppm 13.42, 19.14, 22.89, 23.16, 24.50, 28.88, 31.41, 32.40, 35.41,
38.46, 44.01, 46.18, 52.90, 55.47, 63.29, 126.34 (C-2), 128.32 (C-
4), 155.18 (C-1), 162.33 (C-5), 178.27 (C-3), 209.02 (C-20). MS
(ESI): m/z 347 [M + H]+. HR-MS (ESI): m/z 347.1775 [M + H]+ (calcd.
for C21H28ClO2, 347.1772).
(2.58 g, yield 78%) as a mixture of 4
epoxides (3a and 3b) (ratio
a,5a and 4b,5b stereomeric
a
:b = ca.1:3). mp 119–122 °C (Ref.
[23]: mp 122–124 °C); MS (ESI): m/z 331 [M + H]+. The diastereo-
meric mixture ratio was determined by proton integration of the
4-H of the product.
4a,5a
-Epoxypregnan-3,20-dione (3a): 1H NMR (500 MHz, CDCl3)
d ppm 0.67 (3H, s, 18-CH3), 1.07 (3H, s, 19-CH3), 2.13 (3H, s, 21-
CH3), 3.05 (1H, s, 4b-H); 13C NMR (500 MHz, CDCl3) d ppm 13.34,
16.48, 21.39, 24.39, 28.87, 29.12, 29.62, 29.68, 31.48, 33.07,
35.40, 36.73, 38.75, 44.02, 50.57, 55.82, 62.84, 63.61, 69.98,
206.72 (C-3), 209.27 (C-20).
2.2.4. General procedure for the synthesis of benzylidene derivatives
(7a–g and 8a–g)
To a solution of 2 or 5 (0.25 mmol) in ethanol (2.5 mL) was
added 0.06 mL solution of 50% KOH. Then benzaldehyde 6a–g
(0.3 mmol) was charged into the reaction mixture and the solution
was stirred at room temperature for 12 h. The reaction mixture
was neutralized with 1 mol/L HCl solution, and then ethyl acetate
(60 mL) was added. The organic layer was washed with brine and
dried over anhydrous Na2SO4. After evaporation of the solvent, the
corresponding compounds 7a–g and 8a–g were obtained as solid
powder by crystallization from ethyl acetate/petroleum ether.
4b,5b-Epoxypregnan-3,20-dione (3b): 1H NMR (500 MHz, CDCl3)
d ppm 0.66 (3H, s, 18-CH3), 1.16 (3H, s, 19-CH3), 2.12 (3H, s, 21-
CH3), 2.99 (1H, s, 4a
-H); 13C NMR (500 MHz, CDCl3) d ppm 13.36,
18.92, 21.53, 22.83, 24.37, 26.20, 29.77, 30.31, 31.44, 32.53,
35.02, 37.23, 38.49, 44.09, 46.43, 56.00, 62.68, 63.45, 70.20,
206.68 (C-3), 209.16 (C-20).
2.2.2. 4-Chloro-pregn-4-ene-3,20-dione (4)
According to the procedure described in Ref. [24], to a solution
of the diastereomeric mixture 3 (2.57 g, 7.8 mmol) in acetone
(100 mL) was added concentrated HCl (2 mL). The solution was al-
lowed to reflux for 1 h. The organic solvent was evaporated in vac-
cum and the desired crude product 4 was precipitated upon
addition of cold water (200 mL). Recrystallization from methanol
afforded pure compound 4 as white solid (1.68 g, 62%). mp 216–
218 °C (Ref. [23]: mp 214–217 °C). 1H NMR (500 MHz, CDCl3) d
ppm 0.68 (3H, s, 18-CH3), 1.24 (3H, s, 19-CH3), 2.13 (3H, s, 21-
CH3), 3.26 (1H, dq, J = 3.0, 4.0, 15.5, 6-H); 13C NMR (500 MHz,
CDCl3) d ppm 13.32, 17.76, 21.10, 22.91, 24.29, 28.92, 31.05,
31.44, 33.98, 34.46, 35.09, 38.60, 41.34, 43.86, 53.84, 55.93,
63.41, 127.37 (C-4), 164.38 (C-5), 190.59 (C-3), 209.14 (C-20). MS
(ESI): m/z 349 [M + H]+.
2.2.4.1. 21E-Benzylidene-pregn-1,4-diene-3,20-dione (7a). White
powder (82 mg, 82%), mp 80–81 °C (ethyl acetate-petroleum
ether); 1H NMR (500 MHz, CDCl3) d ppm 0.71 (3H, s, 18-CH3),
1.23 (3H, s, 19-CH3), 6.09 (1H, s, 4-H), 6.24 (1H, dd, J = 1.5,
10.0 Hz, 2-H), 6.76 (1H, d, J = 15.5 Hz, 21-H), 7.04 (1H, d,
J = 10.0 Hz, 1-H), 7.39–7.40 (3H, m, 3H of Ar–H), 7.54–7.57 (3H,
m, 2H of Ar–H and 22-H); 13C NMR (500 MHz, CDCl3): d ppm
13.67 (C-18), 18.69 (C-19), 22.72, 22.87, 24.77, 32.82, 33.62,
35.63, 38.81, 43.57, 45.04, 52.33, 55.87, 61.72, 123.89 (C-4),
126.52 (C-21), 127.54 (C-2), 128.31, 128.96, 130.44, 134.67,
141.84 (C-22), 155.82 (C-1), 169.16 (C-5), 186.37 (C-3), 199.94
(C-20). MS (ESI): m/z 401 [M + H]+. HR-MS (ESI): m/z 401.2476
[M + H]+ (calcd. for C28H33O2, 401.2475).
2.2.4.2. 21E-(2-Chlorobenzylidene) pregn-1,4-diene-3,20-dione (7b).
White powder (93 mg, 86%), mp 98–99 °C(ethyl acetate-petroleum
ether); 1H NMR (500 MHz, CDCl3) d ppm 0.71 (3H, s, 18-CH3), 1.23
(3H, s, 19-CH3), 6.09 (1H, s, 4-H), 6.24 (1H, dd, J = 1.5, 10.0 Hz, 2-H),
6.73 (1H, d, J = 16.0 Hz, 21-H), 7.05 (1H, d, J = 10.0 Hz, 1-H), 7.29–
7.33 (2H, m, Ar–H), 7.42 (1H, d, J = 8.0 Hz, Ar–H), 7.63 (1H d,
J = 7.5 Hz, Ar–H), 7.93 (1H d, J = 16.0 Hz, 22-H); 13C NMR
(500 MHz, CDCl3): d ppm 13.75 (C-18), 18.69 (C-19), 22.88, 22.88,
24.78, 32.81, 33.60, 35.62, 38.92, 43.56, 45.03, 52.24, 55.86,
61.02, 123.89 (C-4), 127.14 (C-21), 127.55 (C-2), 127.55, 129.31,
130.27, 131.16, 132.91, 135.40, 137.83 (C-22), 155.82 (C-1),
169.14 (C-5), 186.41 (C-3), 200.02 (C-20). MS (ESI): m/z 435
[M + H]+. HR-MS (ESI): m/z 435.2087 [M + H]+ (calcd. for C28H32-
ClO2, 435.2085).
2.2.3. Preparation of pregn-1,4-diene-3,20-dione (2) and 4-chloro-
pregn-1,4-diene-3,20-dione (5)
According to a procedure described by Chen et al. [25]. To a
solution of steroid 1 or 4 (4 mmol) and tert-butyldimethylchloros-
ilane (TBDMSCl, 30.4 mg, 0.2 mmol) in dioxane (20 mL) was added
2,3-dichloro-5,6-dicyano-benzoquinone (DDQ, 1.18 g, 5.2 mmol) in
two portions at 0 °C. The reaction mixture was warmed to room
temperature and then refluxed for 2 h with stirring. After the solu-
tion cooled down to room temperature, the reaction mixture was
diluted with chloroform (100 mL) and was subjected to vacuum fil-
tration over Al2O3 layer, which was washed with chloroform
(100 mL). The filtrate was washed with saturated Na2CO3 solution
(2 ꢀ 50 mL) and brine (50 mL). The combined organic extracts
were dried over anhydrous Na2SO4 and the solvent was removed
under reduced pressure to yield a crude product as yellow oil.
Recrystallization from petroleum ether/ethyl acetate afforded
compounds 2 and 5, respectively.
2.2.4.3. 21E-(4-Chlorobenzylidene) pregn-1,4-diene-3,20-dione (7c).
White powder (93 mg, 86%), mp 76–77 °C (ethyl acetate-petro-
leum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.71 (3H, s, 18-
CH3), 1.23 (3H, s, 19-CH3), 6.09 (1H, s, 4-H), 6.24 (1H, dd, J = 1.5,
10.0 Hz, 2-H), 6.72 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d,
J = 10.5 Hz, 1-H), 7.36 (2H, d, J = 8.5 Hz, Ar–H), 7.48 (2H, d,
J = 8.5 Hz, Ar–H), 7.50 (1H d, J = 16.0 Hz, 22-H); 13C NMR
(500 MHz, CDCl3): d ppm 13.67 (C-18), 18.70 (C-19), 22.73, 22.86,
24.75, 32.80, 33.61, 35.65, 38.84, 43.52, 45.09, 52.33, 55.89,
61.91, 123.97 (C-4), 126.85 (C-21), 127.61 (C-2), 129.24, 129.45,
133.18, 136.31, 140.36 (C-22), 155.62 (C-1), 168.92 (C-5), 186.32
2.2.3.1. Pregn-1,4-diene-3,20-dione (2). White solid (810 mg, 65%):
mp 149–150 °C (Ref. [25]: 149–151 °C); 1H NMR (500 MHz, CDCl3)
d ppm 0.70 (3H, s, 18-CH3), 1.24 (3H, s, 19-CH3), 2.12 (3H, s, 21-
CH3), 6.08 (1H, s, 4-H), 6.24 (1H, dd, J = 2.0, 10.0 Hz, 2-H), 7.05
(1H d, J = 10.0 Hz, 1-H); 13C NMR (500 MHz, CDCl3): d ppm 13.42
(C-18), 18.69 (C-19), 22.81, 22.84, 24.55, 31.43, 32.77, 33.52
35.48, 38.52, 43.48, 44.07; 52.21, 55.61, 63.38, 123.95 (C-4),

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N.-J. Fan et al. / Steroids 78 (2013) 874–879
(C-3), 199.71 (C-20). MS (ESI): m/z 435 [M + H]+. HR-MS (ESI): m/z
d, J = 16.0 Hz, 21-H), 7.07 (1H, d, J = 10.0 Hz, 1-H), 7.39–7.40 (3H,
m, 3H of Ar–H), 7.54–7.57 (3H, m, 2H of Ar–H and 22-H); 13C
NMR (500 MHz, CDCl3): d ppm 13.67 (C-18), 19.14 (C-19), 22.75,
23.21, 24.72, 28.93, 32.51, 35.55, 38.75, 44.99, 46.24, 53.01,
55.74, 61.65, 126.30 (C-2), 126.46 (C-21), 128.29, 128.32 (C-4),
128.98, 130.48, 134.64, 141.93 (C-22), 155.33 (C-1), 162.52 (C-5),
178.35 (C-3), 199.89 (C-20). MS (ESI): m/z 435 [M + H]+. HR-MS
(ESI): m/z 435.2089 [M + H]+ (calcd. for C28H32ClO2, 435.2085).
435.2088 [M + H]+ (calcd. for C28H32ClO2, 435.2085).
2.2.4.4. 21E-(2-Methoxybenzylidene) pregn-1,4-diene-3,20-dione
(7d). White powder (90 mg, 84%), mp 66–67 °C (ethyl acetate-
petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.70 (3H, s,
18-CH3), 1.23 (3H, s, 19-CH3), 3.89 (3H, s, AOCH3), 6.09 (1H, s, 4-
H), 6.25 (1H, dd, J = 1.5, 10.0 Hz, 2-H), 6.83(1H, d, J = 16.0 Hz, 21-
H), 6.91–6.95 (2H, m, Ar–H), 7.05 (1H, d, J = 10.5 Hz, 1-H),
7.36(1H, d, J = 7.5 Hz, Ar–H), 7.54 (1H, d, J = 7.5 Hz, Ar–H), 7.87
(1H, d, J = 16.0 Hz, 22-H); 13C NMR (500 MHz, CDCl3): d ppm
13.67 (C-18), 18.69 (C-19), 22.79, 22.93, 24.80, 32.85, 33.66,
35.65, 38.75, 43.63, 44.91, 52.37, 55.55, 55.86, 61.22, 111.21,
120.75, 123.64, 123.85 (C-4), 127.41 (C-21), 127.50 (C-2), 128.74,
131.63, 137.27 (C-22), 155.94 (C-1), 158.63, 169.32 (C-5), 186.40
(C-3), 200.47 (C-20). MS (ESI): m/z 431 [M + H]+. HR-MS (ESI): m/
z 431.2586 [M + H]+ (calcd. for C29H35O3, 431.2581).
2.2.4.9. 21E-(2-Chlorobenzylidene) -4-chloro-pregn-1,4-diene-3,20-
dione (8b). White powder (103 mg, 88%), mp 200–202 °C (ethyl
acetate-petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.72
(3H, s, 18-CH3), 1.29 (3H, s, 19-CH3), 6.37 (1H, d, J = 9.5 Hz, 2-H),
6.73 (1H, d, J = 16.0 Hz, 21-H), 7.07 (1H, d, J = 10.0 Hz, 1-H), 7.29–
7.33 (2H, m, Ar–H), 7.43 (1H, d, J = 8.0 Hz, Ar–H), 7.63 (1H d,
J = 8.0 Hz, Ar–H), 7.94 (1H d, J = 16.0 Hz, 22-H); 13C NMR
(500 MHz, CDCl3): d ppm 13.77 (C-18), 19.16 (C-19), 22.93, 23.24,
24.75, 28.93, 32.50, 35.56, 38.88, 44.98, 46.24, 52.92, 55.74,
60.88, 126.34 (C-2), 127.14 (C-21), 127.56, 128.31 (C-4), 129.30,
130.31, 131.19, 132.91, 135.44, 137.95 (C-22), 155.33 (C-1),
162.50 (C-5), 178.38 (C-3), 199.96 (C-20). MS (ESI): m/z 469
[M + H]+. HR-MS (ESI): m/z 469.1697 [M + H]+ (calcd. for C28H31Cl2-
O2, 469.1696).
2.2.4.5. 21E-(4-Methoxybenzylidene) pregn-1,4-diene-3,20-dione
(7e). White powder (88 mg, 82%), mp 87–88 °C (ethyl acetate-
petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.70 (3H, s,
18-CH3), 1.23 (3H, s, 19-CH3), 3.84 (3H, s, AOCH3), 6.09 (1H, s, 4-
H), 6.24 (1H, dd, J = 1.5, 10.0 Hz, 2-H), 6.65 (1H, d, J = 15.5 Hz, 21-
H), 6.91 (2H, d, J = 8.0 Hz, Ar–H), 7.05 (1H, d, J = 10.5 Hz, 1-H),
7.49 (2H, d, J = 7.5 Hz, Ar–H), 7.52 (1H, d, J = 15.5 Hz, 22-H); 13C
NMR (500 MHz, CDCl3): d ppm 13.63 (C-18), 18.68 (C-19), 22.75,
22.87, 24.77, 32.88, 33.63, 35.62, 38.79, 43.60, 44.99, 52.36,
55.42, 55.85, 61.60, 114.41, 123.88 (C-4), 124.37, 127.31 (C-21),
127.54 (C-2), 130.02, 141.62 (C-22), 155.90 (C-1), 161.55, 169.28
(C-5), 186.38 (C-3), 199.84 (C-20). MS (ESI): m/z 431 [M + H]+.
HR-MS (ESI): m/z 431.2575 [M + H]+ (calcd. for C29H35O3,
431.2581).
2.2.4.10. 21E-(4-Chlorobenzylidene)-4-chloro-pregn-1,4-diene-3,20-
dione (8c). White powder (103 mg, 88%), mp 198–200 °C (ethyl
acetate-petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.70
(3H, s, 18-CH3), 1.29 (3H, s, 19-CH3), 6.36 (1H, d, J = 10.0 Hz, 2-
H), 6.71 (1H, d, J = 16.5 Hz, 21-H), 7.07 (1H, d, J = 10.0 Hz, 1-H),
7.36 (2H, d, J = 8.5 Hz, Ar–H), 7.47 (2H, d, J = 8.0 Hz, Ar–H), 7.52
(1H, d, J = 16.5 Hz, 22-H); 13C NMR (500 MHz, CDCl3): d ppm
13.68 (C-18), 19.14 (C-19), 22.75, 23.20, 24.69, 28.91, 32.49,
35.53, 38.75, 45.03, 46.22, 52.98, 55.72, 61.80, 126.30 (C-2),
126.78 (C-21) 128.27 (C-4), 129.24, 129.47, 133.14, 136.33,
140.43 (C-22), 155.30 (C-1), 162.49 (C-5), 178.33 (C-3), 199.68
(C-20). MS (ESI): m/z 469 [M + H]+. HR-MS (ESI): m/z 469.1690
[M + H]+ (calcd. for C28H31Cl2O2, 469.1696).
2.2.4.6. 21E-(2-Nitrobenzylidene) pregn-1,4-diene-3,20-dione (7f).
Pale yellow powder (94 mg, 84%), mp 150–152 °C (ethyl acetate-
petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.73 (3H, s,
18-CH3), 1.24 (3H, s, 19-CH3), 6.08 (1H, s, 4-H), 6.23 (1H, dd,
J = 1.5, 10.0 Hz, 2-H), 6.64 (1H, d, J = 16.0 Hz, 21-H), 7.05 (1H, d,
J = 10.0 Hz, 1-H), 7.57 (1H, d, J = 7.0 Hz, Ar–H), 7.64–7.69 (2H, m,
Ar–H), 7.94 (1H, d, J = 16.0 Hz, 22-H), 8.05 (1H, d, J = 8.0 Hz, Ar–
H); 13C NMR (500 MHz, CDCl3): d ppm 13.78 (C-18), 18.71 (C-19),
22.85, 22.96, 24.77, 32.80, 33.57, 35.61, 38.83, 43.52, 45.17,
52.19, 55.82, 60.92, 123.91 (C-4), 125.00, 127.55 (C-2), 129.11 (C-
21), 130.35, 131.02, 131.61, 133.58, 137.22 (C-22), 148.57, 155.74
(C-1), 169.01 (C-5), 186.33 (C-3), 199.76 (C-20). MS (ESI): m/z
446 [M + H]+. HR-MS (ESI): m/z 446.2321 [M + H]+ (calcd. for
2.2.4.11.
21E-(2-Methoxybenzylidene)-4-chloro-pregn-1,4-diene-
3,20-dione (8d). White powder (97 mg, 84%), mp 152–154 °C (ethyl
acetate-petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.70
(3H, s, 18-CH3), 1.28 (3H, s, 19-CH3), 3.88 (3H, s, -OCH3), 6.36
(1H, d, J = 8.5 Hz, 2-H), 6.83(1H, d, J = 16.0 Hz, 21-H)), 6.92–6.96
(2H, m, Ar–H), 7.07 (1H, d, J = 10.0 Hz, 1-H), 7.36(1H, d, J = 8.0 Hz,
Ar–H), 7.53 (1H, d, J = 7.5 Hz, Ar–H), 7.88 (1H, d, J = 16.0 Hz, 22-
H); 13C NMR (500 MHz, CDCl3): d ppm 13.68 (C-18), 19.14 (C-19),
22.80, 23.26, 24.75, 28.96, 32.54, 35.54, 38.67, 44.87, 46.30,
53.01, 55.56, 55.69, 61.12, 111.21, 120.76, 123.55, 126.25 (C-2),
127.32 (C-21), 128.74 (C-4), 128.97, 131.71, 137.35 (C-22),
155.51 (C-1), 158.63, 162.72 (C-5), 178.41 (C-3), 200.45 (C-20).
MS (ESI): m/z 465 [M + H]+. HR-MS (ESI): m/z 465.2193 [M + H]+
(calcd. for C29H34ClO3, 465.2191).
C28H32NO4, 446.2326).
2.2.4.7. 21E-(4-Methylbenzylidene) pregn-1,4-diene-3,20-dione (7g).
White powder (86 mg, 83%), mp 92–93 °C (ethyl acetate-petro-
leum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.70 (3H, s, 18-
CH3), 1.22 (3H, s, 19-CH3), 2.38 (3H, s, CH3-Ph), 6.09 (1H, s, 4-H),
6.23 (1H, dd, J = 1.5, 10.0 Hz, 2-H), 6.72 (1H, d, J = 16.0 Hz, 21-H),
7.04 (1H, d, J = 10.0 Hz, 1-H), 7.20 (2H, d, J = 7.5 Hz, Ar–H), 7.45
(2H, d, J = 7.5 Hz, Ar–H), 7.53 (1H d, J = 16.0 Hz, 22-H); 13C NMR
(500 MHz, CDCl3): d ppm 13.66 (C-18), 18.70 (C-19), 21.53, 22.73,
22.87, 24.78, 32.83, 33.63, 35.64, 38.81, 43.55, 45.03, 52.35,
55.88, 61.65, 123.93 (C-4), 125.62 (C-21), 127.58 (C-2), 128.33,
129.70, 131.91, 140.96, 141.91 (C-22), 155.75 (C-1), 169.07 (C-5),
186.38 (C-3), 200.00 (C-20). MS (ESI): m/z 415 [M + H]+. HR-MS
(ESI): m/z 415.2629 [M + H]+ (calcd. for C29H35O2, 415.2632).
2.2.4.12. 21E-(4-Methoxybenzylidene)-4-chloro- pregn-1,4-diene-
3,20-dione (8e). White powder (95 mg, 82%), mp 124–125 °C (ethyl
acetate-petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.70
(3H, s, 18-CH3), 1.29 (3H, s, 19-CH3), 3.84 (3H, s, -OCH3), 6.36
(1H, d, J = 9.5 Hz, 2-H), 6.64 (1H, d, J = 15.0 Hz, 21-H), 6.91 (2H, d,
J = 7.5 Hz, Ar–H), 7.07 (1H, d, J = 9.5 Hz, 1-H), 7.49 (2H, d,
J = 8.0 Hz, Ar–H), 7.52 (1H, d, J = 15.0 Hz, 22-H); 13C NMR
(500 MHz, CDCl3): d ppm 13.65 (C-18), 19.14 (C-19), 22.76, 23.21,
24.73, 28.94, 32.52, 35.53, 38.73, 44.96, 46.27, 53.01, 55.45,
55.71, 61.53, 114.42, 124.29, 126.28 (C-2), 127.27 (C-21), 128.67
(C-4), 130.06, 141.73 (C-22), 155.45 (C-1), 161.57, 162.66 (C-5),
178.41 (C-3), 199.85 (C-20). MS (ESI): m/z 465[M + H]+. HR-MS
(ESI): m/z 465.2189 [M + H]+ (calcd. for C29H34ClO3, 465.2191).
2.2.4.8. 21E-Benzylidene-4-chloro-pregn-1,4-diene-3,20-dione (8a).
White powder (90 mg, 83%), mp 91–92 °C (ethyl acetate-petro-
leum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.71 (3H, s, 18-
CH3), 1.29 (3H, s, 19-CH3), 6.36 (1H, d, J = 10.0 Hz, 2-H), 6.76 (1H,

N.-J. Fan et al. / Steroids 78 (2013) 874–879
877
Table 1
2.2.4.13.
21E-(2-Nitrobenzylidene)-4-chloro-pregn-1,4-diene-3,20-
Cytotoxic activity of the tested compounds against brine shrimps and murine Lewis
lung carcinoma cells (LLC) at 30 g/mL.
dione (8f). White powder (99 mg, 83%), mp 205–207 °C (ethyl ace-
tate-petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.74 (3H,
s, 18-CH3), 1.30 (3H, s, 19-CH3), 6.36 (1H, d, J = 10.0 Hz, 2-H), 6.63
(1H, d, J = 16.0 Hz, 21-H), 7.06 (1H, d, J = 9.5 Hz, 1-H), 7.57 (1H, d,
J = 7.0 Hz, Ar–H), 7.63–7.68 (2H, m, Ar–H), 7.94 (1H, d,
J = 16.0 Hz, 22-H), 8.05 (1H, d, J = 8.0 Hz, Ar–H); 13C NMR
(500 MHz, CDCl3): d ppm 13.81 (C-18), 19.17 (C-19), 23.03, 23.21,
24.75, 28.92, 32.48, 35.54, 38.77, 45.14, 46.23, 52.86, 55.70,
60.74, 125.04, 126.31 (C-2), 128.28 (C-4), 129.14 (C-21), 130.38,
131.05, 131.64, 133.61, 137.36 (C-22), 148.54, 155.35 (C-1),
162.49 (C-5), 178.35 (C-3), 199.74 (C-20). MS (ESI): m/z 480
[M + H]+. HR-MS (ESI): m/z 480.1936 [M + H]+ (calcd. for C28H31-
ClNO4, 480.1936).
l
Entry
Ar
LC50a
(lg/mL)
Inhibitory rateb
2
4
5
7a
–
–
–
>100
>100
>100
22.6
0
0
0
31.29
7b
7c
26.5
24.0
27.44
24.36
7d
7e
7f
17.7
19.8
28.3
22.23
14.04
66.18
2.2.4.14. 21E-(4-Methylbenzylidene)-4-chloro-pregn-1,4-diene-3,20-
dione (8g). White powder (93 mg, 83%), mp 184–186 °C (ethyl ace-
tate-petroleum ether); 1H NMR (500 MHz, CDCl3) d ppm 0.70 (3H,
s, 18-CH3), 1.28 (3H, s, 19-CH3), 2.38 (3H, s, CH3-Ph), 6.35 (1H, d,
J = 10.0 Hz, 2-H), 6.72 (1H, d, J = 15.5 Hz, 21-H), 7.07 (1H, d,
J = 10.0 Hz, 1-H), 7.20 (2H, d, J = 8.0 Hz, Ar–H), 7.44 (2H, d,
J = 7.5 Hz, Ar–H), 7.53 (1H, d, J = 15.5 Hz, 22-H); 13C NMR
(500 MHz, CDCl3): d ppm 13.66 (C-18), 19.15 (C-19), 21.54, 22.76,
23.21, 24.73, 28.93, 32.51, 35.55, 38.74, 44.96, 46.25, 53.02,
55.73, 61.56, 125.55, 126.28 (C-2), 128.34 (C-4), 128.34, 129.22,
129.71, 131.88, 140.99, 141.97 (C-22), 155.36 (C-1), 162.56 (C-5),
178.34 (C-3), 199.91 (C-20). MS (ESI): m/z 449 [M + H]+. HR-MS
(ESI): m/z 449.2241 [M + H]+ (calcd. for C29H34ClO2, 449.2242).
7g
21.1
17.08
CH3
8a
8b
>100
>100
51.12
12.02
8c
>100
12.96
2.3. Biology
2.3.1. Brine shrimp cytotoxicity assay
8d
8e
8f
>100
>100
>100
8.34
5.96
25.63
The procedure for brine shrimp lethality assay was modified
from the assay previously described by our group [26]. Brine
shrimp larvae (A. salina) were hatched in a small partitioned tank
in artificial seawater. Illumination was provided on one side to at-
tract newly hatched larvae. Brine shrimp larvae with second instar
stage were used in this assay. Two milligrams of compounds and 5,
7a–g and 8a–g were made up to 2 mg/mL in DMSO. Through anal-
ysis and filtration, five different concentrations (50, 40, 30, 20 and
8g
>100
<1
14.22
CH3
10 lg/mL) of the compounds were used in this assay. Serial dilu-
Toosendanin
tions were made in the wells of 96-well microplates (Nunc, Den-
mark), to each microplates twenty to thirty brine shrimp larvae
were added and some more artificial seawater were put to make
a
The LC50 represents the concentration for the compound to cause 50% lethality
of brine shrimps.
All data are the average of four determinations, which were reproducible with
deviation less than 10%.
b
exact 200 lL. Negative control was prepared with corresponding
concentrations of DMSO in artificial seawater. Following 24 h of
incubation at room temperature, casualties were counted under
microscope. One hundred microliters of methanol were then added
to each well to immobilize the brine shrimp larvae and after
15 min the total numbers of brine shrimp in each well were
counted. The experiment was repeated three times, lethality data
were transformed by probit analysis in SPSS 16.0 computer pro-
gram to estimate LC50 values. Table 1 gives LC50 values of different
compounds.
The lethality rate was calculated using the formula:
L = [(A ꢁ B ꢁ N)/(G ꢁ N)] ꢀ 100, where L = percent of the dead lar-
vae after 24 h; A = number of the dead larvae after 24 h; B = aver-
age number of the dead larvae in the blind samples after 24 h;
N = number of the dead larvae before starting the test; G = number
of selected larvae for test.
streptomycin. Exponentially growing cells were used throughout.
The cell cytotoxicity was assessed by Solarbio MTT kit (Beijing
Solarbio Science & Technology Co., LTD) based on the reduction
of MTT by the mitochondrial succinate dehydrogenase of intact
cells to a purple formazan product [27]. LLC cells were plated in
96-well flat-microtiter plates at a density of 3000–10,000 cells/
well for adherence before 6–24 h, then the media was replaced
by fresh media and cells were incubated with various concentra-
tions of 21E-benzylidene steroidal derivatives for 48 h. After the
indicated time, the media was aspirated, and 100 lL media can-
taining 10
l
L MTT solution (5 mg mLꢁ1 in PBS) stored at ꢁ20 °C
in the dark was added to each well and plates were incubated for
2.3.2. Cell cytotoxicity assay
4 h at 37 °C. Then, the media was moved and 110 lL of DMSO
Murine Lewis lung carcinoma cells (LLC) were grown in 100 mm
culture flask at 37 °C under 5% CO2 in high glucose Dulbecco’s
modified Eagle’s medium supplemented with 10% (v/v) heat inac-
was added to each well to solubilize the formant crystals. The
absorbance was measured at 490 nm using an ELISA microplate
reader. Each sample was performed in four replicate wells, and
the entire experiment was repeated two or three times.
tivated fetal bovine serum, 10 lg/mL penicillin, and 10 lg/mL

878
N.-J. Fan et al. / Steroids 78 (2013) 874–879
O
O
O
i
ii
O
O
O
O
l
C
1
(
3
3b
)
b
a
n
a
4
4
3
3
3
d
,
,
4
a:
a
5
b
iii
iii
:
a
5
b
21
r
A
O
O
O
22
v
i
v
i
O
O
O
R
l
C
a-
7
a-
8
=
H
=
Cl
7
8
R
R
,
,
g
g
5
2
Scheme 1. Synthesis of 21E-benzylidene derivatives. Reagents: (i) H2O2, NaOH, MeOH–CH2Cl2; (ii) conc. HCl, acetone, reflux; (iii) DDQ, TBDMSCl, Dioxane, reflux; (iv)
benzaldehydes 6a–g, EtOH–KOH.
coupling constant was about 16 Hz, thereby indicating the geome-
try of the double bond is E, which was in agreement with that of
21E-benzylidene steroidal derivatives reported [22,31].
3. Results and discussion
3.1. Chemistry
The synthetic pathways of a series of new progesterone-derived
21E-benzylidene steroids 7a–g and 8a–g are shown in Scheme 1.
Synthesis of intermediate 4-chloro-pregn-1,4-diene-3,20-dione 5
started with the commercially available progesterone 1. According
to the procedure previously described by Kirk and Hartshorn [28],
treatment of 1 with alkaline hydrogen peroxide afforded a 1:3 mix-
3.2. Biology
In order to evaluate cytotoxic effects of the compounds 2, 4, 5,
7a–g and 8a–g, their median lethal concentration (LC50) values
were determined against brine shrimp (Artemia salina L.) larvae
as reported previously [26]. As summarized in Table 1, LC50 values
of all the tested compounds were found to be quite higher than
those of the positive control toosendanin, a commercial botanical
insecticide derived from Melia azedarach. Compounds 7a–g, which
lacked a chlorine atom at C-4 position, significantly inhibited the
growth of the brine shrimp larvae in a dose-depentent manner. It
is clear that 7a–g showed similar cytotoxicity with LC50 values of
ture of epoxide 3, i.e., 4
4b,5b-epoxypregnan-3,20-dione 3b, favoring the desired isomer
3b, as the ratio of to b isomers was determined based on the pro-
ton integration of the H-4 (H
and Hb) in the 1H NMR spectra of 3
a,5a-epoxypregnan-3,20-dione 3a and
a
a
(see Supporting Information). This result is significantly different
from that of the reaction of 1 with dimethyldioxirane, which gave
a mixture of the epoxide favoring a-epoxide 3a [29]. The resulting
17.7–28.3
l
g/mL, which were stronger than the positive control
epoxides (3a and 3b) were directly utilized without further purifi-
cation for the next step. As a result, treatment of the resulting 4,5-
epoxy-steroid 3 with concentrated HCl at reflux afforded 4-chloro-
2 (LC50 > 100
l
g/mL). In contrast, compounds 8a–g, with the same
counterpart of aromatic aldehydes as 7a–g, caused a loss of activ-
ity. Among the compounds tested, compound 7d, having an ortho-
OCH3 in the aromatic ring, was found to be the most active
pregn-4-ene-3,20-dione 4 in good yield. Subsequently,
drogenation of with 2,3-dichloro-5,6-dicyano-benzoquinone
D
1-dehy-
4
(LC50 = 17.7 lg/mL). It should be noteworthy that the electron-
(DDQ) in dioxane in the presence of tert-butyldimethylchlorosilane
(TBDMSCl) [25] afforded the desired compound 5 in 45% yield.
Similarly, the other intermediate, pregn-1,4-diene-3,20-dione 2,
donating group on the benzene ring was favorable for the cytotoxic
activity (7a vs. 7d, 7e, 7g), while the electron-withdrawing group
could weaken the cytotoxicity (7a vs. 7b, 7c, 7f), as compared to
7a (Table 1). The results indicate that introducing benzylidene
groups into 2 was capable of enhancing the cytotoxicity against
the brine shrimp and that introducing a chlorine atom into C-4 po-
sition resulted in a loss of activity.
Furthermore, these synthetic compounds were tested by the
MTT assay for their in vitro cytotoxicity against the murine Lewis
lung carcinoma cells (LLC) [27]. From the results of Table 1, we
found that compounds 7a–g and 8a–g showed some inhibitive ef-
fects (inhibition rate of 5% to 66%) to LLC cells at concentrations of
can be directly obtained through
D
1-dehydrogenation of 1 using
DDQ in 65% yield. Finally, aldol condensation reactions of the inter-
mediates 2 and 5 with various benzaldehydes 6a–g, followed by
crystallization from ethyl acetate-petroleum ether without the
need of laborious chromatography, afforded the corresponding tar-
get compounds 7a–g and 8a–g (Scheme 1), respectively. The struc-
tures of these newly synthesized steroidal derivatives were
characterized by NMR and HR-ESIMS analyses. Specifically, from
the 13C NMR spectra of 3 (see Supporting Information), we can ob-
serve that each carbon signal appears in pairs, and the 13C NMR
30 lg/mL, of which 7f and 8a displayed a weak cytotoxic activity
(66.2% and 51.1%, respectively) at this concentration.
spectrum of the minor 4
sented in this study is well matched to that of
a
,5
a
-epoxypregnan-3,20-dione (3a) pre-
-epoxide recorded
a
by Carvalho et al. [30], accordingly, we assigned the epoxy config-
uration of the major isomer 3b to the b- isomer. On the other hand,
a new double bond formed was placed at the position 21 based on
the 1H NMR spectroscopic data. The 1H NMR spectroscopic data of
compounds 7a–g and 8a–g showed two characteristic signals in
the range of 6.6–6.8 and 7.5–7.9 ppm, which can be assigned to
the coupling protons of the double bond at C-21 and C-22. Their
4. Conclusions
In summary, we have described the synthesis of a series of no-
vel 21E-benzylidene derivatives of progesterone and their cytotox-
icity to the brine shrimps and LLC cells. Compounds 7a–g were
found to have significant cytotoxic effects against brine shrimps,

N.-J. Fan et al. / Steroids 78 (2013) 874–879
879
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