Synthesis of novel 4′-acylamino modified 21E-benzylidene steroidal derivatives and their cytotoxic activities

Article abstract of DOI:10.1016/j.steroids.2017.04.006

A series of 4′-acylamino modified Δ^1,4-pregnadien-21E-benzylidene-3,20-dione derivatives (6a–v) was synthesized from the commercially available progesterone (1). These title compounds were evaluated for their toxicity against brine shrimp (Arte

Full text of DOI:10.1016/j.steroids.2017.04.006

Accepted Manuscript
Synthesis of novel 4′-acylamino modified 21E-benzylidene steroidal derivatives
and their cytotoxic activities
Ning-Juan Fan, Yang-Yang Han, Yuan-Feng Li, Jin-Ming Gao, Jiang-Jiang
Tang
PII:
S0039-128X(17)30069-7
http://dx.doi.org/10.1016/j.steroids.2017.04.006
STE 8096
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 May 2016
19 April 2017
21 April 2017
Please cite this article as: Fan, N-J., Han, Y-Y., Li, Y-F., Gao, J-M., Tang, J-J., Synthesis of novel 4′-acylamino
modified 21E-benzylidene steroidal derivatives and their cytotoxic activities, Steroids (2017), doi: http://dx.doi.org/
10.1016/j.steroids.2017.04.006
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Synthesis of novel 4′-acylamino modified 21E-benzylidene steroidal
derivatives and their cytotoxic activities
Ning-Juan Fana, Yang-Yang Hana, Yuan-Feng Lia, Jin-Ming Gaob, Jiang-Jiang Tangb,*
a Biochemistry and Molecular Biology Research Platform, College of Life Science, Northwest
A&F University, Yangling, Shaanxi 712100, China
b Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry &
Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, China
*Corresponding author. E-mail address: fannj1980@nwsuaf.edu.cn (N.J. Fan),
tangjiang11@nwsuaf.edu.cn (J.J. Tang)
1

Abstract
A series of 4′-acylamino modified ∆1,4-pregnadien-21E-benzylidene-3,20-dione
derivatives (6a-v) was synthesized from the commercially available progesterone
(1). These title compounds were evaluated for their toxicity against brine shrimp
(Artemia salina) and cytotoxic activities against two human cancer cell lines (HeLa
and MCF-7). The results revealed that compound 6f exhibited promising in vitro
cytotoxic activity to the two cancer cell lines and the nature of acylamino functional
group in the benzylidene moiety had a significant influence on cytotoxicity.
Keywords: progesterone; acylamino functional group; benzylidene steroidal
derivatives; cytotoxicity
2

1. Introduction
Extensive research has been carried out to synthesize and characterize substituted
steroids [1]. A variety of steroidal compounds with unusual and interesting structures
have been synthesized and evaluated for their biological activities [2-5]. Their
preparation remains a stimulating challenge to the organic chemistry community,
often requiring the development of new synthetic approaches and associated chemical
reactions. Moreover, the biological properties of modified steroids have proved to be
of interest. For example, progesterone as a precursor for some pharmaceutical steroids,
has been widely explored in synthesis of some new derivatives with biological
activities [6-9].
Benzylidene derivatives are well known for their diverse array of
pharmacological activities [10-17]. Their structure consists of two aromatic rings and
an α,β-unsaturated carbonyl system joining them. In some specific study it was found
that variations leading to conformational changes like epoxidation or substitution on
the double bond would result in a decreased bioactivity [18]. Steroidal benzylidene
derivatives, which were synthetized by the aldol condensation of certain steroids with
various benzaldehyde derivatives, have been reported to act as dehydrogenase
inhibitors [19], antimicrobial [20, 21] and antineoplastic agents [22-25]. Those studies
have proved an important role of the benzylidene steroidal scaffold in diverse
biological activities, and it is therefore worth an effort to further investigate this class
of compounds.
Amides are one of important functional groups prevalent in organic biomolecules.
3

Recently, a series of acylamino-substituted derivative has exhibited strong inhibitory
activities on several human cancer cell lines [26]. Though there are some reports for
other such analogs, very few efforts have been done relating to the synthesis of
acylamino modified benzylidene steroidal derivatives as well as their biological
screening. Taking inspiration from the number of reported biological activities
associated with structurally related analogs, and as a part of our commitment to search
for novel potential anticancer agents related to steroidal derivatives [27, 28], we
efficiently synthesized a series of 4′-acylamino modified benzylidene compounds
(6a-v), the steroidal derivatives from the commercially available progesterone. Brine
shrimp lethality assay has been considered as a screening system for various extracts
and synthetic compounds with antitumor activities [29]. In order to evaluate toxicity
of these synthetic compounds, their median lethal concentration (LC50) values were
determined against Artemia salina as described previously [28], as well as their
cytotoxic activities against two human cancer cell lines in vitro have been investigated
in order to find potent and selective chemotherapy agents.
2. Experimental
2.1. General methods
The melting points of the products were determined on an X-4 apparatus (Beijing
Tech Instrument Co., Beijing, P. R. China) and are uncorrected. Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker Avance spectrometer (Unity
plus 400 MHz) (Bruker Bios pin, Rheinstetten, Germany) with tetramethylsilane
4

(TMS) as internal standard. Chemical shift values (δ) were given in parts per million
(ppm). Thin-layer chromatography (TLC) was performed on silica gel GF254
(Qingdao Marine Chemical Ltd., P. R. China). Column chromatography (CC) was
performed over silica gel (200-300 mesh, Qingdao Marine Chemical Ltd.), High
resolution electrospray ionization mass spectrometry (HRESIMS) data were recorded
on LCMS-IT-TOF (Shimadzu, Kyoto, Japan). Commercial solvents and reagents were
of reagent grade.
2.2. Chemical synthesis
2.2.1. 21E-(4-nitrobenzylidene)pregna-1,4-diene-3,20-dione (3)
To a solution of 2 (1.56 g, 5 mmol) in ethanol (20 mL) was added 1.2 mL
solution of 50% KOH. Then 4-nitrobenzaldehyde (6 mmol) was charged into the
reaction mixture with stirring 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 (150 mL) was added. The organic layer was washed with brine and dried over
anhydrous Na2SO4. After evaporation of the solvent, the corresponding compound 3
was obtained as solid powder by crystallization from ethyl acetate/petroleum ether.
1
Yellow solid (1.82 g, 82%), mp 151-153°C; H NMR (400 MHz, CDCl3) δ ppm 0.71
(3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.08 (1H, s, 4-H), 6.24 (1H, dd, J =4.0, 12.0
Hz, 2-H), 6.84 (1H, d, J = 16.0 Hz, 21-H), 7.03 (1H, d, J = 8.0 Hz, 1-H), 7.56 (1H, d,
J = 16.0 Hz, 22-H), 7.68 (2H, d, J = 12.0 Hz, Ar-H), 8.24 (2H, d, J = 8.0 Hz, Ar-H);
13C NMR (100MHz, CDCl3): δ ppm 13.74, 18.69, 22.70, 22.83, 24.71, 32.76, 33.56,
5

35.62, 38.86, 43.47, 45.24, 52.25, 55.88, 62.27, 124.00, 124.21, 127.66, 128.83,
129.79, 138.71, 140.91, 148.51, 155.52, 168.79, 186.30, 199.44. HR-MS (ESI): m/z
446.2321 [M+H]+ (calcd. for C28H32NO4, 446.2326).
2.2.2. 21E-(4-aminobenzylidene)pregna-1,4-diene-3,20-dione (4)
A mixture of compound 3 (1.78 g, 4 mmol), Tin(II) chloride (4.512 g, 20 mmol),
concentrated hydrochloric acid (2 mL), and ethyl acetate (40 mL) was stirred under
reflux for 1 h. After cooled to room temperature, the mixture was treated with 40 mL
water, and neutralized with a solution of 1 mol/L NaOH to pH was 8-9. The aqueous
phase was extracted with of ethyl acetate (3×80 mL). The combined organic phase
was dried with Na2SO4 and filtered. The filtrate was concentrated to give 4 as yellow
1
powder (1.23 g, 74%). mp 223-225°C; H NMR (400 MHz, CDCl3) δ ppm 0.69 (3H,
s, 18-CH3), 1.23 (3H, s, 19-CH3), 4.01 (2H, s, 4′-NH2), 6.09 (1H, s, 4-H), 6.24 (1H, dd,
J =4.0, 12.0 Hz, 2-H), 6.58 (1H, d, J = 16.0 Hz, 21-H), 6.66 (2H, d, J = 8.0 Hz, Ar-H),
7.05 (1H, d, J = 8.0 Hz, 1-H), 7.38 (2H, d, J = 8.0 Hz, Ar-H), 7.49 (1H, d, J = 16.0 Hz,
22-H); 13C NMR (100MHz, CDCl3): δ ppm 13.62, 18.71, 22.78, 22.89, 24.81, 32.85,
33.66, 35.66, 38.82, 43.59, 44.98, 52.40, 55.89, 61.49, 114.87, 122.66, 123.92, 124.78,
127.56, 130.26, 142.35, 148.97, 155.83, 169.17, 186.42, 199.89. HR-MS (ESI): m/z
416.2589 [M+H]+ (calcd for C28H34NO2, 416.2584).
2.2.3. Synthesis of 4-acylaminobenzylidene derivatives (6a-v)
A mixture of compound 4 (42 mg, 0.1 mmol) and a series of acyl chloride 5a-v
( 0.2 mmol) in dry pyridine (2.0 mL) was stirred at room temperature. When the
reaction was completed (monitored by TLC), the solvent was removed under reduced
6

pressure and the residue obtained was purified by thin-layer chromatography over
silica gel GF254 (chloroform/acetone = 8:1) to afford 4′-acylamino derivative 6a-v in
70-88% yield. The spectral data of various compounds is given as below.
2.2.3.1. 21E-(4-acetamidobenzylidene)pregna-1,4-diene-3,20-dione (6a):
White powder (39mg, 86%), mp 135-137°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.69 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 2.19 (3H, s, CH3 of acetyl), 6.08 (1H, s,
4-H), 6.24 (1H, dd, J =4.0, 12.0 Hz, 2-H), 6.68 (1H, d, J = 16.0 Hz, 21-H), 7.03 (1H, d,
J = 12.0 Hz, 1-H), 7.48-7.51 (3H, m), 7.58 (2H, d, J = 8.0 Hz, Ar-H), 7.97 (1H, s,
4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.46, 18.48, 22.53, 22.68, 24.50, 24.57,
32.64, 33.43, 35.42, 38.60, 43.43, 44.87, 52.15, 55.66, 61.50, 119.53, 123.67, 125.16,
127.30, 129.06, 130.05, 140.03, 141.10, 155.84, 168.47, 169.22, 186.31, 199.76.
HR-MS (ESI): m/z 458.2698 [M+H]+ (calcd for C30H36NO3, 458.2690).
2.2.3.2. 21E-(4-n-propionamidobenzylidene)pregna-1,4-diene-3,20-dione (6b)
White powder (38mg, 81%), mp 142-144°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 1.25 (3H, t, J = 4.0 Hz, CH3 of propionyl),
6.08 (1H, s, 4-H), 6.24 (1H, dd, J =4.0, 12.0 Hz, 2-H), 6.68 (1H, d, J = 16.0 Hz, 21-H),
7.05 (1H, d, J = 8.0 Hz, 1-H), 7.48-7.59 (6H, m); 13C NMR (100MHz, CDCl3): δ ppm
9.45, 13.56, 18.60, 22.67, 22.78, 24.68, 30.71, 32.73, 33.54, 35.56, 38.73, 43.48,
44.96, 52.27, 55.80, 61.63, 119.55, 123.83, 125.29, 127.46, 129.18, 130.18, 140.05,
141.15, 155.72, 169.05, 172.06, 186.31, 199.78. HR-MS (ESI): m/z 472.2852 [M+H]+
(calcd for C31H38NO3, 472.2847).
2.2.3.3. 21E-(4-n-butyrylamidobenzylidene)pregna-1,4-diene-3,20-dione (6c)
7

White powder (41mg, 85%), mp 129-131°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.69 (3H, s, 18-CH3), 0.99 (3H, t, J = 8.0 Hz, CH3 of butyryl), 1.22 (3H, s, 19-CH3),
6.08 (1H, s, 4-H), 6.24 (1H, dd, J =4.0, 12.0 Hz, 2-H), 6.67 (1H, d, J = 16.0 Hz, 21-H),
7.05 (1H, d, J = 8.0 Hz, 1-H), 7.47-7.51 (3H, m), 7.59 (2H, d, J = 8.0 Hz, Ar-H), 7.87
(1H, br, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.45, 13.56, 18.47, 18.78,
22.53, 22.66, 24.55, 32.63, 33.41, 35.41, 38.58, 39.44, 43.42, 44.85, 52.13, 55.65,
61.48, 119.48, 123.64, 125.09, 127.27, 129.05, 129.95, 140.05, 141.14, 155.87,
169.27, 171.47, 186.33, 199.78. MS (ESI): m/z: 486[M+H]+; HR-MS (ESI): m/z
486.3009 [M+H]+ (calcd for C32H40NO3, 486.3003).
2.2.3.4. 21E-(4-n-Pentanamidobenzylidene)pregna-1,4-diene-3,20-dione (6d)
White powder (43mg, 86%), mp 117-119°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.69 (3H, s, 18-CH3), 0.93 (3H, t, J = 8.0 Hz, CH3 of valeryl), 1.22 (3H, s, 19-CH3),
6.08 (1H, s, 4-H), 6.24 (1H, dd, J =4.0, 12.0 Hz, 2-H), 6.67 (1H, d, J = 16.0 Hz, 21-H),
7.05 (1H, d, J = 8.0 Hz, 1-H), 7.47-7.51 (3H, m), 7.59 (2H, d, J = 8.0 Hz, Ar-H), 7.85
(1H, br, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.46, 13.64, 18.48, 22.19,
22.54, 22.68, 24.57, 27.40, 32.64, 33.43, 35.42, 37.34, 38.60, 43.43, 44.87, 52.15,
55.66, 61.50, 119.48, 123.66, 125.09, 127.30, 129.07, 129.94, 140.11, 141.15, 155.85,
169.23, 171.62, 186.32, 199.78. HR-MS (ESI): m/z 500.3166 [M+H]+ (calcd for
C33H42NO3, 500.3160).
2.2.3.5. 21E-(4-benzamidobenzylidene)pregna-1,4-diene-3,20-dione (6e)
White powder (41mg, 79%), mp 234-236°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.07 (1H, s, 4-H), 6.23 (1H, dd, J =4.0,
8

12.0 Hz, 2-H), 6.71 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 8.0 Hz, 1-H),
7.47-7.51 (3H, m), 7.54-7.56 (3H, m), 7.72 (2H, d, J = 8.0 Hz, Ar-H), 7.88 (2H, d, J =
8.0 Hz, Ar-H), 8.18 (1H, s, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.57, 18.60,
22.68, 22.78, 24.68, 32.73, 33.53, 35.56, 38.74, 43.47, 44.97, 52.26, 55.80, 61.67,
120.08, 123.83, 125.48, 127.04, 127.47, 128.77, 129.23, 130.60, 132.04, 134.53,
140.01, 141.09, 155.68, 165.68, 169.00, 186.30, 199.80. HR-MS (ESI): m/z 520.2857
[M+H]+ (calcd for C35H38NO3, 520.2846).
2.2.3.6. 21E-(4-(2-fluorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6f)
White powder (44mg, 82%), mp 223-225°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.08 (1H, s, 4-H), 6.23 (1H, dd, J =4.0,
12.0 Hz, 2-H), 6.71 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.17-7.22 (1H, m), 7.31-7.35 (1H, m), 7.51-7.57 (4H, m), 7.72 (2H, d, J = 8.0 Hz,
Ar-H), 8.14-8.19 (1H, m), 8.60 (1H, d, J = 16.0 Hz, 4′-NH); 13C NMR (100MHz,
CDCl3): δ ppm 13.68, 18.72, 22.76, 22.88, 24.79, 32.83, 33.63, 35.65, 38.83, 43.56,
45.08, 52.34, 55.89, 61.76, 116.16, 120.46, 123.89, 125.23, 125.64, 127.54, 129.30,
130.93, 132.26, 134.09, 139.66, 141.07, 152.80, 155.73, 160.30 (d, J = 246 Hz, C-F),
161.30, 169.08, 186.36, 199.90. HR-MS (ESI): m/z 538.2763 [M+H]+ (calcd for
C35H37NO3F, 538.2752).
2.2.3.7. 21E-(4-(3-fluorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6g)
1
White powder (46 mg, 85%), mp 218-220°C; H NMR (400 MHz, CDCl3) δ
ppm 0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.06 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.71 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
9

7.22-7.27 (1H, m), 7.43-7.50 (2H, m), 7.55 (2H, d, J = 8.0 Hz, Ar-H), 7.61 (1H, d, J =
12.0 Hz, Ar-H), 7.66 (1H, d, J = 8.0 Hz, Ar-H), 7.72 (2H, d, J = 8.0 Hz, Ar-H), 8.34
(1H, s, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.56, 18.58, 22.64, 22.77, 24.66,
32.72, 33.51, 35.52, 38.72, 43.49, 44.99, 52.24, 55.77, 61.66, 114.63, 119.10, 120.29,
122.63, 123.84, 125.56, 127.42, 129.22, 130.49, 130.85, 139.81, 141.11, 152.92,
155.91, 162.78 (d, J = 245 Hz, C-F), 164.51, 169.26, 186.44, 199.93. HR-MS (ESI):
m/z 538.2759 [M+H]+ (calcd for C35H37NO3F, 538.2752).
2.2.3.8. 21E-(4-(4-fluorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6h)
1
White powder (47 mg, 88%), mp 187-189°C; H NMR (400 MHz, CDCl3) δ
ppm 0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.06 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.12-7.16 (2H, m), 7.49-7.55 (3H, m), 7.72 (2H, d, J = 8.0 Hz, Ar-H), 7.89-7.93 (2H,
m), 8.37 (1H, s, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.47, 18.47, 22.54,
22.68, 24.56, 32.63, 33.42, 35.42, 38.61, 43.42, 44.89, 52.15, 55.67, 61.55, 115.85,
120.25, 123.81, 125.54, 127.45, 129.28, 129.63, 130.67, 140.04, 141.17, 155.97,
162.64, 164.80, 164.98 (d, J = 252 Hz, C-F), 169.35, 186.47, 199.94. HR-MS (ESI):
m/z 538.2760 [M+H]+ (calcd for C35H37NO3F, 538.2752).
2.2.3.9. 21E-(4-(3,4-difluorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6i)
1
White powder (44 mg, 79%), mp 211-213°C; H NMR (400 MHz, CDCl3) δ
ppm 0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.06 (1H, s, 4-H), 6.23 (1H, dd, J =
4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.22-7.29 (1H, m), 7.49-7.55 (3H, m), 7.64-7.69 (1H, m), 7.72 (2H, d, J = 8.0 Hz,
10

Ar-H), 7.76-7.81 (1H, m), 8.52 (1H, s, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm
13.47, 18.46, 22.54, 22.70, 24.56, 32.64, 33.44, 35.42, 38.62, 43.46, 44.90, 52.19,
55.68, 61.56, 117.15, 117.65, 119.62, 120.38, 123.79, 125.66, 127.43, 129.27, 130.88,
131.63, 139.82, 141.12, 148.92, 151.48, 156.11, 163.67, 169.53, 186.53, 199.97.
HR-MS (ESI): m/z 556.2683 [M+H]+ (calcd for C35H36NO3F2, 556.2658).
2.2.3.10. 21E-(4-(2-chlorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6j)
1
White powder (44 mg, 80%), mp 216-218°C; H NMR (400 MHz, CDCl3) δ
ppm 0.69 (3H, s, 18-CH3), 1.21 (3H, s, 19-CH3), 6.06 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.36-7.45 (3H, m), 7.50-7.56 (3H, m), 7.70-7.74 (3H, m), 8.31 (1H, s, 4′-NH); 13C
NMR (100MHz, CDCl3): δ ppm 13.46, 18.47, 22.53, 22.66, 24.56, 32.62, 33.40,
35.41, 38.59, 43.39, 44.87, 52.10, 55.65, 61.53, 119.92, 123.64, 125.47, 127.14,
127.28, 129.14, 130.14, 130.23, 130.45, 130.77, 131.71, 134.70, 139.44, 140.93,
155.77, 164.43, 169.13, 186.30, 199.76. HR-MS (ESI): m/z 554.2464 [M+H]+ (calcd
for C35H37NO3Cl, 554.2456).
2.2.3.11. 21E-(4-(3-chlorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6k)
1
White powder (44 mg, 80%), mp 140-141°C; H NMR (400 MHz, CDCl3) δ
ppm 0.69 (3H, s, 18-CH3), 1.21 (3H, s, 19-CH3), 6.05 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.38-7.42 (1H, m), 7.48-7.54 (4H, m), 7.72-7.78 (3H, m), 7.87-7.88 (1H, m), 8.58 (1H,
s, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.45, 18.45, 22.52, 22.67, 24.55,
32.61, 33.40, 35.40, 38.60, 43.41, 44.88, 52.13, 55.65, 61.53, 120.17, 123.63, 125.22,
11

125.41, 127.27, 127.35, 129.08, 129.87, 130.60, 131.81, 134.69, 136.22, 139.77,
140.99, 155.83, 164.40, 169.21, 186.30, 199.79. HR-MS (ESI): m/z 554.2466 [M+H]+
(calcd for C35H37NO3Cl, 554.2456).
2.2.3.12. 21E-(4-(4-chlorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6l)
1
White powder (47 mg, 86%), mp 197-199°C; H NMR (400 MHz, CDCl3) δ
ppm 0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.06 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.44 (2H d, J = 8.0 Hz, Ar-H), 7.51 (1H, d, J = 16.0 Hz, 22-H), 7.54 (2H, d, J = 8.0 Hz,
Ar-H), 7.72 (2H, d, J = 8.0 Hz, Ar-H), 7.84 (2H, d, J = 8.0 Hz, Ar-H), 8.41 (1H, s,
4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.45, 18.46, 22.53, 22.67, 24.55, 32.62,
33.41, 35.40, 38.60, 43.41, 44.87, 52.14, 55.65, 61.54, 120.10, 123.63, 125.40, 127.27,
128.48, 128.84, 129.10, 130.57, 132.77, 138.16, 139.77, 140.97, 155.84, 164.65,
169.23, 186.31, 199.76. HR-MS (ESI): m/z 554.2462 [M+H]+ (calcd for C35H37NO3Cl,
554.2456).
2.2.3.13. 21E-(4-(2,4-dichlorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione
(6m)
1
White powder (45 mg, 76%), mp 153-155°C; H NMR (400 MHz, CDCl3) δ
ppm 0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.06 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.32-7.34 (1H, m), 7.44 (1H, s, Ar-H), 7.51 (1H, d, J = 16.0 Hz, 22-H), 7.55 (2H, d, J
= 8.0 Hz, Ar-H), 7.65-7.72 (3H, m), 8.50 (1H, s, 4′-NH); 13C NMR (100MHz, CDCl3):
δ ppm 13.55, 18.57, 22.63, 22.76, 24.66, 32.71, 33.50, 35.51, 38.69, 43.48, 44.96,
12

52.20, 55.75, 61.64, 120.08, 123.71, 125.65, 127.36, 127.59, 129.22, 130.09, 131.00,
131.19, 131.50, 133.30, 137.21, 139.41, 140.95, 155.81, 163.61, 169.18, 186.32,
199.82. HR-MS (ESI): m/z 588.2058 [M+H]+ (calcd for C35H36NO3Cl2, 588.2067).
2.2.3.14. 21E-(4-(3,5-dichlorobenzamido)benzylidene)pregna-1,4-diene-3,20-dione
(6n)
1
White powder (41 mg, 70%), mp 144-146°C; H NMR (400 MHz, CDCl3) δ
ppm 0.69 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.06 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.05 (1H, d, J = 12.0 Hz, 1-H),
7.48-7.54 (4H, m), 7.74 (2H, d, J = 8.0 Hz, Ar-H), 7.78 (2H, s, Ar-H), 8.83 (1H, s,
4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.46, 18.43, 22.52, 22.70, 24.55, 32.63,
33.42, 35.39, 38.61, 43.48, 44.90, 52.18, 55.66, 61.54, 120.32, 123.59, 125.51, 125.81,
127.22, 129.06, 130.78, 131.51, 135.35, 137.35, 139.60, 140.97, 156.07, 163.23,
169.52, 186.42, 199.84. HR-MS (ESI): m/z 588.2061 [M+H]+ (calcd for
C35H36NO3Cl2, 588.2067).
2.2.3.15. 21E-(4-(2-bromobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6o)
1
White powder (48 mg, 80%), mp 211-213°C; H NMR (400 MHz, CDCl3) δ
ppm 0.69 (3H, s, 18-CH3), 1.21 (3H, s, 19-CH3), 6.06 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.30-7.34 (1H, m), 7.38-7.42 (1H, m), 7.49-7.56 (3H, m), 7.62 (2H, d, J = 8.0 Hz,
Ar-H), 7.71 (2H, d, J = 8.0 Hz, Ar-H), 8.16 (1H, s, 4′-NH); 13C NMR (100MHz,
CDCl3): δ ppm 13.47, 18.49, 22.54, 22.67, 24.58, 32.62, 33.41, 35.43, 38.61, 43.37,
44.88, 52.11, 55.67, 61.55, 119.11, 119.88, 123.67, 125.50, 127.32, 127.60, 129.15,
13

129.64, 130.80, 131.65, 133.38, 137.32, 139.43, 140.92, 155.65, 165.45, 168.98,
186.22, 199.74. HR-MS (ESI): m/z 598.1938 [M+H]+ (calcd for C35H37NO3Br,
598.1951).
2.2.3.16. 21E-(4-(3-bromobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6p)
1
White powder (46 mg, 77%), mp 132-134°C; H NMR (400 MHz, CDCl3) δ
ppm 0.69 (3H, s, 18-CH3), 1.21 (3H, s, 19-CH3), 6.05 (1H, s, 4-H), 6.23 (1H, dd, J
=4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.31-7.35 (1H, m), 7.48-7.54 (3H, m), 7.65 (1H, d, J = 8.0 Hz, Ar-H), 7.73 (2H, d, J =
8.0 Hz, Ar-H), 7.82 (1H, d, J = 8.0 Hz, Ar-H), 8.03 (1H, s, Ar-H), 8.58 (1H, s, 4′-NH);
13C NMR (100MHz, CDCl3): δ ppm 13.46, 18.46, 22.54, 22.68, 24.56, 32.62, 33.41,
35.41, 38.60, 43.43, 44.89, 52.14, 55.66, 61.54, 120.19, 122.69, 123.63, 125.42,
125.72, 127.27, 129.09, 130.12, 130.22, 130.62, 134.74, 136.43, 139.77, 141.00,
155.88, 164.31, 169.27, 186.34, 199.81. HR-MS (ESI): m/z 598.1928 [M+H]+ (calcd
for C35H37NO3Br, 598.1951).
2.2.3.17. 21E-(4-(4-bromobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6q)
White powder (49mg, 82%), mp 205-207°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.69 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 6.05 (1H, s, 4-H), 6.22 (1H, dd, J =4.0,
12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.49-7.54 (3H, m), 7.59 (2H d, J = 8.0 Hz, Ar-H), 7.72 (2H, d, J = 8.0 Hz, Ar-H), 7.76
(2H, d, J = 8.0 Hz, Ar-H), 8.47 (1H, s, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm
13.45, 18.45, 22.53, 22.66, 24.54, 32.61, 33.40, 35.39, 38.59, 43.40, 44.86, 52.13,
55.64, 61.52, 120.11, 123.61, 125.38, 126.61, 127.26, 128.64, 129.09, 130.55, 131.78,
14

133.24, 139.78, 140.97, 155.82, 164.77, 169.21, 186.29, 199.75. HR-MS (ESI): m/z
598.1941 [M+H]+ (calcd for C35H37NO3Br, 598.1951).
2.2.3.18. 21E-(4-(2′-iodobenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6r)
White powder (51mg, 79%), mp 144-146°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.69 (3H, s, 18-CH3), 1.21 (3H, s, 19-CH3), 6.01 (1H, s, 4-H), 6.20 (1H, dd, J =4.0,
12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J = 12.0 Hz, 1-H),
7.11-7.15 (1H, m), 7.38-7.42 (1H, m), 7.49-7.56 (4H, m), 7.73 (2H, d, J = 8.0 Hz,
Ar-H), 7.88 (1H, d, J = 8.0 Hz, Ar-H), 8.24 (1H, s, 4′-NH); 13C NMR (100MHz,
CDCl3): δ ppm 13.54, 18.54, 22.61, 22.73, 24.64, 32.68, 33.47, 35.48, 38.66, 43.45,
44.94, 52.16, 55.72, 61.58, 92.35, 120.00, 123.69, 125.52, 127.34, 128.19, 128.43,
129.20, 130.79, 131.47, 139.68, 139.89, 141.05, 141.68, 155.81, 167.30, 169.16,
186.31, 199.85. HR-MS (ESI): m/z 646.1815 [M+H]+ (calcd for C35H37NO3I,
646.1813).
2.2.3.19. 21E-(4-(3-methylbenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6s)
1
White powder (41 mg, 77%), mp 117-118°C; H NMR (400 MHz, CDCl3) δ
ppm 0.70 (3H, s, 18-CH3), 1.22 (3H, s, 19-CH3), 2.41 (3H, s, Ar-CH3), 6.06 (1H, s,
4-H), 6.23 (1H, dd, J =4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d,
J = 8.0 Hz, 1-H), 7.35 (2H, d, J = 4.0 Hz, Ar-H), 7.50-7.55 (3H, m), 7.65 (1H, d, J =
4.0 Hz, Ar-H), 7.67-7.74 (3H, m), 8.23 (1H, s, 4′-NH); 13C NMR (100MHz, CDCl3):
δ ppm 13.47, 18.50, 21.23, 22.55, 22.68, 24.58, 32.63, 33.42, 35.44, 38.62, 43.38,
44.88, 52.14, 55.68, 61.55, 119.96, 123.70, 123.92, 125.29, 127.34, 127.70, 128.51,
129.13, 130.37, 132.68, 134.40, 138.57, 140.02, 141.06, 155.67, 165.83, 169.00,
15

186.25, 199.75. HR-MS (ESI): m/z 534.3015 [M+H]+ (calcd for C36H40NO3,
534.3003).
2.2.3.20. 21E-(4-(4-methylbenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6t)
White powder (40mg, 75%), mp 126-128°C; 1H NMR (400 MHz, CDCl3) δ ppm
0.69 (3H, s, 18-CH3), 1.21 (3H, s, 19-CH3), 2.41 (3H, s, Ar-CH3), 6.06 (1H, s, 4-H),
6.23 (1H, dd, J =4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d, J =
8.0 Hz, 1-H), 7.27 (2H d, J = 8.0 Hz, Ar-H), 7.49-7.54 (3H, m), 7.72 (2H, d, J = 8.0
13
Hz, Ar-H), 7.78 (2H, d, J = 8.0 Hz, Ar-H), 8.21 (1H, s, 4′-NH); C NMR (100MHz,
CDCl3): δ ppm 13.45, 18.48, 21.35, 22.53, 22.66, 24.56, 32.61, 33.40, 35.42, 38.60,
43.37, 44.86, 52.12, 55.66, 61.53, 119.93, 123.68, 125.23, 126.96, 127.32, 129.11,
129.28, 130.27, 131.49, 140.06, 141.07, 142.52, 155.67, 165.55, 169.00, 186.24,
199.74. HR-MS (ESI): m/z 534.3011 [M+H]+ (calcd for C36H40NO3, 534.3003).
2.2.3.21. 21E-(4-(3-methoxybenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6u)
1
White powder (39 mg, 71%), mp 119-120°C; H NMR (400 MHz, CDCl3) δ
ppm 0.69 (3H, s, 18-CH3), 1.21 (3H, s, 19-CH3), 3.85 (3H, s, Ar-OCH3), 6.06 (1H, s,
4-H), 6.23 (1H, dd, J =4.0, 12.0 Hz, 2-H), 6.70 (1H, d, J = 16.0 Hz, 21-H), 7.04 (1H, d,
J = 8.0 Hz, 1-H), 7.06-7.08 (1H, m), 7.34-7.44 (3H, m), 7.49-7.55 (3H, m), 7.73 (2H,
d, J = 8.0 Hz, Ar-H), 8.33 (1H, s, 4′-NH); 13C NMR (100MHz, CDCl3): δ ppm 13.44,
18.46, 22.52, 22.65, 24.55, 32.60, 33.39, 35.40, 38.59, 43.37, 44.85, 52.11, 55.29,
55.64, 61.52, 112.38, 118.02, 118.67, 119.98, 123.65, 125.29, 127.29, 129.10, 129.59,
130.40, 135.85, 139.94, 141.02, 155.71, 159.75, 165.49, 169.06, 186.25, 199.73.
HR-MS (ESI): m/z 550.2957 [M+H]+ (calcd for C36H40NO4, 550.2952).
16

2.2.3.22. 21E-(4-(4-methoxybenzamido)benzylidene)pregna-1,4-diene-3,20-dione (6v)
1
White powder (39 mg, 71%), mp 242-244°C; H NMR (100 MHz, CDCl3) δ
ppm 0.69 (3H, s, 18-CH3), 1.21 (3H, s, 19-CH3), 3.86 (3H, s, Ar-OCH3), 6.06 (1H, s,
4-H), 6.23 (1H, dd, J =4.0, 12.0 Hz, 2-H), 6.69 (1H, d, J = 16.0 Hz, 21-H), 6.95 (2H d,
J = 8.0 Hz, Ar-H), 7.04 (1H, d, J = 8.0 Hz, 1-H), 7.49-7.54 (3H, m), 7.71 (2H, d, J =
8.0 Hz, Ar-H), 7.86 (2H, d, J = 8.0 Hz, Ar-H), 8.21 (1H, s, 4′-NH); 13C NMR
(400MHz, CDCl3): δ ppm 13.47, 18.49, 22.55, 22.67, 24.57, 32.63, 33.42, 35.43,
38.61, 43.39, 44.87, 52.14, 55.31, 55.67, 61.53, 113.81, 119.93, 123.69, 125.18,
126.52, 127.33, 128.91, 129.12, 130.17, 140.21, 141.11, 155.70, 162.48, 165.13,
169.04, 186.26, 199.76. HR-MS (ESI): m/z 550.2959 [M+H]+ (calcd for C36H40NO4,
550.2952).
2.3. Biological activity assay
2.3.1 Brine shrimp toxicity assay
The brine shrimp toxicity assay was assayed by microtiter-plate method described
by our group [28]. Brine shrimp larvae (Artemia salina L.) were hatched in a small
partitioned tank in artificial seawater. Illumination was provided on one side to attract
newly hatched larvae. Brine shrimp larvae with second instar stage were used in this
assay. Two milligrams of compounds 2, 3, 4 and 6a-v were made up to 2 mg/mL in
DMSO. Serial dilutions were made in the wells of 96-well microplates (Nunc,
Denmark), to each microplates twenty to thirty brine shrimp larvae were added and
some more artificial seawater were put to make exact 200 µL. Negative control was
17

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 minutes 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 program to estimate
LC50 values of the tested compounds.
The lethality rate was calculated using the formula: L = [(A-B-N) / (G-N)]×100,
where L = percent of the dead larvae after 24 h; A = number of the dead larvae after
24 h; B = average 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.
2.3.2 Cell cytotoxicity assay
Human cervical cancer cell line (HeLa) and breast cancer cell line (MCF-7）were
o
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 inactivated fetal bovine
serum, 10 µg/mL penicillin, and 10 µg/mL 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 [30]. Cells were plated in 96-well flat-microtiter plates at a density of
18

3000-10000 cells/well for adherence before 6-24 h, and then were treated with various
concentrations of 21E-benzylidene steroidal derivatives for 48 h. After the indicated
time, the media was aspirated, and 100 µL media cantaining 10 µL MTT solution (5
mg/mL in PBS) stored at -20 °C in the dark was added to each well and plates were
incubated for 4 h at 37 °C. Then, the media was moved and 110 µL of DMSO was
added to each well to solubilize the formant crystals. The absorbance was measured at
490 nm using an ELISA microplate reader. The viability of treated cells was
normalized to that of the control cells set as 100%.Each sample was performed in
four replicate wells, and the entire experiment was repeated four times. Cisplatin, an
agent administered clinically in the treatment of numerous malignancies, was used as
a positive control.
3. Results and discussion
3.1. Chemistry
The target 4′-acylamino modified 21E-benzylidene steroidal compounds (6a-v)
were prepared as shown in Scheme 1. The pivotal intermediate
pregn-1,4-diene-3,20-dione (2) was prepared from progesterone (1) according to our
published procedure [27]. In the presence of basic catalyst (50% KOH) in ethanol,
aldol condensation of the intermediate 2 with 4-nitrobenzaldehyde afforded the crude
4′-nitrobenzylidene derivative 3. After crystallized from ethyl acetate/petroleum ether,
the purity of the condensed products was tested with TLC. The starting material was
completely consumed, the reaction led to a single product and no further purification
was needed. The condensation formed a new double bond at position 21. Based on the
19

1H NMR spectroscopic data of crystallized product, there are two characteristic
signals of 6.84 and 7.56 ppm which can be assigned to the coupling protons of the
double bond at C-21 and C-22. Their coupling constant was 16.0 Hz, thereby
indicating the geometry of the double bond is E, which was in agreement with that of
21E-benzylidene steroidal derivatives [25, 31]. Thereafter, compound 3 was readily
reduced with tin (II) chloride in ethyl acetate and concentrated hydrochloric acid to
afford 4′-aminobenzylidene derivative 4 in 74% yield. The signals of the 21-H and
22-H are at 6.58 and 7.49 ppm respectively, while the two protons at lower fields (δ =
4.01 ppm) are the -NH2 protons. Finally, amino derivative 4 underwent acylation with
various acyl chloride (5a-v) in pyridine to provide the target products 6a-v with good
yields (70-88%). All the new compounds synthesized were characterized by 1H NMR,
13C NMR and HR-ESIMS analyses.
Scheme 1 here
3.2. Biology
Firstly, all the synthesized compounds were evaluated their median lethal
concentration (LC50) against brine shrimp larvae (Artemia salina). As showed in
Table 1, when 4′-nitro compound 3 was reduced to 4′-amino derivative 4, the LC50
value changed from 27.6 µg/mL to 15.9 µg/mL, which were stronger than 2 (LC50
>
50 µg/mL). In contrast, all of the 4′-acylaminobenzylidene derivatives 6a-v displayed
weaker toxicity against the brine shrimp with LC50 value about 50 µg/mL than their
parent compound 4. The results indicate that introducing benzylidene groups into 2
20

were capable of enhancing the toxicity against the brine shrimp and that introducing
any acylamino substituent group into C-4′ position of benzene ring resulted in a
reduction of such activity.
Furthermore, all the synthesized 4′-acylamino modified 21E-benzylidene
steroidal derivatives were tested in vitro against two human cancer cell lines HeLa
(cervical carcinoma) and MCF-7 (breast adenocarcinoma) by the MTT assay.
Cytotoxic potency of the compounds was indicated by inhibition rate on cell
proliferation at concentrations of 30 µg/mL, the results are summarized in Table 1.
Compared to 2, compounds 3, 4 and 6a-v showed some inhibitive activity (highest
inhibition rate is 64%), which were inferior to the positive control cisplatin. Among
all the tested compounds, 6b-f, 6j and 6o displayed a moderate cytotoxic activity (all
inhibition rate over 50%) to HeLa cells at the concentrations of 30 µg/mL, however,
only 6f and 6i were found to have the similar inhibition activity (64% and 50%,
respectively) to MCF-7 cells. Besides, compound 6f exhibited the strongest inhibitory
effects on both HeLa (58%) and MCF-7 (64%) at 30 µg/mL which revealed that 6f
had potent cytotoxicity for these two cancer cells.
Table 1 here
The results presented in Table 1 also implied that the nature of the substituent
groups at the acylamino play a critical role in cytotoxic potency. Compounds 6a, 6b,
6c and 6d with the aliphatic chain of methyl, ethyl, n-propyl and n-butyl, respectively,
exhibited increasing cytotoxicity against HeLa cancer cells (6a < 6b < 6c < 6d),
nevertheless, for MCF-7 cells, ethyl substitution was superior to the others (6b vs. 6a,
21

6c and 6d), suggesting that the length of the aliphatic chain of the acylamino groups is
critical for the compounds cytotoxic activity. In addition, compounds 6a-d displayed
stronger inhibitory effect against HeLa than MCF-7 cells, implying that 6a-d with
aliphatic chain substituents were more sensitive to HeLa cells.
Moreover, replacement of the aliphatic groups of the acylamino moiety with the
aromatic groups resulted in derivatives 6e-v, which had similar activity against the
two cells. The effect of different halogen monosubstitutents at ortho-, meta- or para-
position of phenyl ring is in approximate order of -F > -Cl > -Br > -I against HeLa
cells (6f > 6j > 6o > 6r, 6g > 6k > 6p and 6h > 6l > 6q). Notably, the cell growth
inhibition rates of 6e-v (except 6f and 6i) against HeLa cells were again higher than
those against MCF-7 cells, reflecting the certain selectivity for the two cancer cells.
Overall, introduction of an additional group at the phenyl ring of the acylamino
moiety had a significant influence on cytotoxic activity.
4. Conclusions
In conclusion, a series of new 4′-acylamino modified benzylidene steroidal
derivatives of progesterone have been prepared and their toxicity against brine shrimp
and cytotoxicity against two human cancer cell lines (HeLa and MCF-7) were
evaluated. The synthesis procedure was simple and efficient. From the cytotoxicity
results, we may conclude that the introduction of benzylidene groups at 21-position of
pregn-1,4-diene-3,20-dione (2) was favorable for the cytotoxicity, and a variety of
acylamino functional groups at 4′-position on the benzene ring were sensitive to the
cytotoxic activity response. This study may provide information for further designing
22

and developing more potent anticancer agents.
Acknowledgements
This project was supported by the National Natural Science Foundation of China
(21402156).
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26

Table 1. Toxicity against brine shrimp (LC50) for the synthesized compounds and their growth
inhibition rate against HeLa and MCF-7 cells at the concentration of 30 µg/mL.
Growth inhibition (%) b
LC50 a (µg/mL)
Entry
R
HeLa
N.A. c
41
MCF-7
N.A.
40
-
-
-
>50
27.6
15.9
33.5
42.7
>50
>50
2
3
39
38
4
22
N.A.
32
6a
6b
6c
6d
6e
50
53
N.A.
N.A.
57
>50
>50
>50
>50
>50
>50
>50
>50
54
58
47
43
41
53
45
28
29
64
6f
6g
6h
6i
40
38
50
N.A.
N.A.
N.A.
6j
6k
6l
>50
>50
22
33
N.A.
N.A.
6m
6n
>50
>50
>50
>50
>50
>50
>50
>50
-
51
N.A.
24
N.A.
N.A.
21
6o
6p
6q
44
N.A.
N.A.
N.A.
N.A.
N.A.
88
6r
26
6s
6t
47
36
6u
29
6v
99
Cisplatin
a The LC50 represents the concentration for the compound to cause 50% lethality of brine shrimps. b All data are
c
the average of four determinations, which were reproducible with deviation less than 10%. N.A.
means inhibition values less than 20%.
27

4' NO2
21
O
O
O
22
ii
i
O
O
O
2
progesterone (1)
3
H
N
4' NH2
4'
R
21
21
O
O
O
22
22
iii
iv
O
O
6a-v
4
Scheme 1. Synthesis of the 4′-acylamino-modified 21E-benzylidene steroidal derivatives 6a-v.
Reagents and conditions: (i) DDQ/TBDMSCl, Dioxane, reflux 2h; (ii) 4-Nitrobenzaldehyde/KOH,
EtOH, rt 12h, 82%; (iii) SnCl2.2H2O, conc. HCl/EtOAc, reflux 1h, 74%; (iv) RCOCl(5a-v),
Pyridine, rt, 70-88%.
28

Graphical abstract
H
N
R
O
O
O
steps
O
O
progesterone
6a-v
29