New cinnamic acid-pregenolone hybrids as potential antiproliferative agents: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.steroids.2019.108499

A series of new cinnamic acid-pregenolone hybrids (5a-5o) was designed, synthesized and evaluated for their in vitro antiproliferative activity. Some of them showed potential antiproliferative activity and selectivity towards a panel of cancer cell lines,

Full text of DOI:10.1016/j.steroids.2019.108499

Steroids152(2019)108499
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
New cinnamic acid-pregenolone hybrids as potential antiproliferative
agents: Design, synthesis and biological evaluation
Yong-Xi Ge, Yan-Hong Wang, Juan Zhang⁎, Zhi-Pu Yu, Xin Mu, Jia-Li Song, Yin-Yin Wang,
Feifei Yang, Ning Meng, Cheng-Shi Jiang⁎, Hua Zhang⁎
School of Biological Science and Technology, University of Jinan, Jinan 250022, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of new cinnamic acid-pregenolone hybrids (5a-5o) was designed, synthesized and evaluated for their in vitro
antiproliferative activity. Some of them showed potential antiproliferative activity and selectivity towards a panel of
cancer cell lines, including A549, H157, HepG2, MCF-7, and HL-60. Among these analogs, compound 5f showed the
most promising activity with IC50 values ranging from 3.2 to 6.8 μM, and it was taken as a model compound in the
following antiproliferative mechanism study. In Hoechst 33258 staining assay, 5f-treated A549 cells displayed sig-
nificant apoptosis characteristics. Flow cytometry analysis revealed that 5f showed the antiproliferative activity
against A549 via G1 cell cycle arrest and inducing apoptosis. Western blotting analysis demonstrated that 5f en-
hanced apoptosis of A549 cells by down-regulating Bcl-2 and up-regulating Bax protein expression. The present study
highlighted this series of cinnamic acid-pregenolone hybrids as a new antiproliferative lead prototype.
Cinnamic acid
Pregenolone
Molecular hybridization
Antiproliferative
Apoptosis
1. Introduction
Steroids have been also an especially attractive class of NP to medicinal
chemists, owing not only to their fascinating structural framework but also
Cancer is a class of diseases involving uncontrolled cell growth with
the potential to invade or spread to other parts of the body [1]. Despite
important advances in anticancer therapy, cancer is still the second
killer to human following cardiovascular diseases, and it accounts for
an estimated 9.6 million deaths worldwide in 2018 [2]. Therefore,
there is an agent need to discover more effective anticancer drugs.
Natural products (NP) have long been a rich source of many significant
drugs, especially in the area of anti-cancer drugs. From the 1940s to the
end of 2014, approximate 49% of 175 small-molecular anticancer drugs
approved by U.S. FDA actually are either NP or derived from therefrom [3].
Natural cinnamic acids from different sources (coffee beans, olives, fruits,
vegetables, etc.) have be reported as one of attractive types of anticancer
candidates [4]. Cinnamic acid and its natural analogs, including caffeic
acid, ferulic acid, isoferulic acid, and sinapic acid, were frequently used as
versatile pharmacophores in anticancer drug design because of their anti-
proliferative and antitumorigenic potency [5,6]. As shown in Fig. 1, cyano-
hydroxycinnamate AG490 (1a) is the first small Jak2 inhibitor that is used
as an anticancer agent in clinic [7]; compound 1b showed comparable
inhibitory activity with erlotinib against EGFR and HER-2, and it could be
developed into a valuable anticancer candidate [8]; and caffeic acid deri-
vative 1c displayed significant antiproliferative activity, being 10-fold more
potent than cisplatin against K562 cancer cells [9].
to their diverse pharmacological properties [10]. As an important member
of steroids, pregnenolone (Fig. 1) is particularly known as the endogenous
precursor to most steroid hormones, such as estrogen, progesterone, tes-
tosterone, and glucocorticoids [11]. The unique structural features and di-
verse bioactivities of pregnenolone quietly make it to be a starting com-
pound of anticancer drugs discovery [12,13]. For example, chlorambucil
ester of pregnenolone 2a was cytotoxic to varying degrees against MDA468,
MDANQ01, MCF-7, H460, OVCA-3 and A375 cancer cells [14], and 21-
arylidenepregnenolone derivatives 2b showed remarkable antiproliferative
effect against EC109 cells [15]. In the last few years, we have successfully
identified several pregnenolone-derived antiproliferative agents [16–19]. In
our continuous project to discover new anticancer leads, a set of novel
antiproliferative cinnamic acid-pregnenolone hybrids I (Fig. 1) were re-
cently achieved based on molecular hybridization approach. Herein, we
describe the synthesis, in vitro antiproliferative evaluation and mechanism
study of these hybrids as antiproliferative and apoptosis-inducing agents.
2. Materials and methods
2.1. Chemistry
Commercially available reagents were used without further purification.
⁎ Corresponding authors.
E-mail addresses: bio_zhangj@ujn.edu.cn (J. Zhang), bio_jiangcs@ujn.edu.cn (C.-S. Jiang), bio_zhangh@ujn.edu.cn (H. Zhang).
https://doi.org/10.1016/j.steroids.2019.108499
Received 16 July 2019; Received in revised form 28 August 2019; Accepted 10 September 2019
Availableonline16September2019
0039-128X/©2019ElsevierInc.Allrightsreserved.

Y.-X. Ge, et al.
Steroids152(2019)108499
Fig. 1. Design of novel cinnamic acid-pregenolone hybrids (I).
Organic solvents were evaporated with reduced pressure using a Büchi R-
100 evaporator (Büchi, Flawil, Switzerland). Reactions were monitored by
thin-layer chromatography (TLC) using Yantai JingYou (Yantai, China)
GF254 silica gel plates. Silica gel column chromatography was performed
on Biotage Isolera One Flash Chromatography System (Biotage, Uppsala,
Sweden) using silica gel (200–300 mesh) from Qingdao Hailang Inc.
(Qingdao, China). NMR spectra were measured on Bruker Avance III
600 MHz spectrometer (Bruker, Fällanden, Switzerland). Chemical shifts
were expressed in δ (ppm) and coupling constants (J) in Hz with residual
solvent signals as references (CDCl3, δ H 7.26 ppm and δC 77.0 ppm; CD3OD,
δH 3.31 ppm and δC 49.0 ppm). ESIMS (electrospray ionization mass spec-
trometry) analyses were performed on an Agilent 1260-6460 Triple Quard
LC-MS instrument (Agilent, Waldbronn, Germany). HR-ESIMS data were
acquired on an Agilent Q-TOF 6520 spectrometer (Agilent, Waldbronn,
Germany).
J = 5.9 Hz, 2H), 7.41–7.32 (m, 3H), 6.45 (s, –NH), 6.41 (d, J = 15.6 Hz,
1H), 5.32 (s, 1H), 5.17 (s, –NH), 4.48 (m, 1H), 3.55–3.49 (m, 2H),
3.42–3.35 (m, 2H), 2.52 (t, J = 9.0 Hz, 1H), 2.33 (m, 1H), 2.26 (m, 1H),
2.19–2.16 (m, 1H), 2.12 (s, 3H), 2.06–1.92 (m, 2H), 1.87 (m, 1H),
1.85–1.80 (m, 1H), 1.71–1.65 (m, 2H), 1.60–1.50 (m, 3H), 1.48–1.40
(m, 3H), 1.27–1.23 (m, 1H), 1.17–1.10 (m, 2H), 0.98 (s, 3H), 0.62 (s,
3H). 13C NMR (150 MHz, CDCl3) δ 209.8, 166.8, 157.4, 141.3, 139.8,
134.9, 129.8, 128.9, 128.0, 122.4, 120.6, 74.8, 63.8, 57.0, 50.0, 44.1,
41.0, 40.9, 38.9, 38.6, 37.1, 36.7, 31.9, 31.9, 31.8, 28.2, 24.6, 22.9,
21.2, 19.4, 13.4. ESI-MS m/z: 555.2 [M+Na]+. HR-ESIMS: [M+H]+
+
calcd for C33H45N2O4 533.3379, found 533.3391.
2.1.1.2. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(2-methoxybenzamido)ethylcarbamate (5b). White
solid (28 mg, 33%). 1H NMR (600 MHz, CDCl3)
δ 7.87 (d,
2.1.1. General procedure for preparing targets compounds 5a-5o
J = 15.8 Hz, 1H), 7.46 (d, J = 7.4 Hz, 1H), 7.31 (dd, J = 8.3, 7.4 Hz,
1H), 6.93 (dd, J = 7.4, 7.4 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.52 (d,
J = 15.8 Hz, 1H), 6.34 (s, –NH), 5.32 (s, 1H), 5.22 (s, –NH), 4.48 (m,
1H), 3.86 (s, 3H), 3.56–3.48 (m, 2H), 3.41–3.45 (m, 2H), 2.52 (t,
J = 8.9 Hz, 1H), 2.38–2.30 (m, 1H), 2.26 (t, J = 11.8 Hz, 1H),
2.20–2.15 (m, 1H), 2.12 (s, 3H), 2.07–2.02 (m, 1H), 1.96 (m, 1H),
1.87 (m, 1H), 1.82 (m, 1H), 1.69–1.61 (m, 3H), 1.59–1.52 (m, 2H), 1.45
(dd, J = 16.1, 7.0 Hz, 3H), 1.27–1.18 (m, 2H), 1.18–1.09 (m, 2H), 0.98
(s, 3H), 0.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 209.8, 167.5, 158.4,
157.4, 139.9, 136.8, 131.0, 129.1, 123.8, 122.3, 121.4, 120.8, 111.2,
74.7, 63.8, 57.0, 55.6, 50.0, 44.1, 41.1, 40.7, 38.9, 38.6, 37.1, 36.7,
31.9, 31.9, 31.7, 28.2, 24.6, 22.9, 21.2, 19.4, 13.4. ESI-MS m/z: 585.2
Compounds 3 and 4 were prepared according to our previously re-
ported protocol [17]. To a stirred solution of prognenolone derivative 4
(2.67 g, 5.3 mmol) in CH2Cl2 (30 mL) was added excessive amount of
trifluoroacetic acid under N2 at 0 °C. When the thin-Layer chromato-
graphy (TLC) indicated the completion of reaction, the solvent was re-
moved under reduced pressure to yield de-protected intermediate as light
yellow oil, which was used in next step without further purification.
To a solution of corresponding cinnamic acid (1.1 equiv) in dry
CH2Cl2 (3 mL) were added 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate (HATU) (29 mg, 0.15 mmol) and
N,N-diisopropylethylamine (DIPEA) (29 mg, 0.225 mmol) at room
temperature. After 0.5 h, the above yellow oil (77 mg) was added, and
the reaction mixture was stirred at room temperature for 16 h. The
volatiles were removed under reduced pressure, and the residue was
partitioned between EtOAc and H2O. The EtOAc layer was dried over
MgSO4 and condensed to afford a crude residue. The residue was pur-
ified by silica gel column chromatography with petroleum ether/
acetone (2:1) to produce the desired products 5a-5o, respectively.
[M+Na]+. HR-ESIMS: [M+H]+ calcd for C34H47N2O5 563.3485,
+
found 563.3464.
2.1.1.3. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(3-methoxybenzamido)ethylcarbamate (5c). White
solid (36 mg, 43%). 1H NMR (600 MHz, CDCl3)
δ 7.58 (d,
J = 15.6 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H),
7.01 (s, 1H), 6.90 (dd, J = 7.6, 7.9 Hz, 1H), 6.43 (s, –NH), 6.39 (d,
J = 15.6 Hz, 1H), 5.32 (s, 1H), 5.17–5.09 (s, –NH), 4.48 (m, 1H), 3.82
(s, 3H), 3.56–3.46 (m, 2H), 3.45–3.32 (m, 2H), 2.53 (t, J = 8.9 Hz, 1H),
2.33 (m, 1H), 2.27 (m, 1H), 2.17 (m, 1H), 2.12 (s, 3H), 2.04 (m, 1H),
2.1.1.1. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-benzamidoethylcarbamate (5a). White solid (26 mg,
33%). 1H NMR (600 MHz, CDCl3) δ 7.61 (d, J = 15.6 Hz, 1H), 7.49 (d,
2

Y.-X. Ge, et al.
Steroids152(2019)108499
1.96 (m, 1H), 1.87 (m, 1H), 1.87 (m, 1H), 1.71–1.62 (m, 3H), 1.60–1.51
(m, 2H), 1.48–1.38 (m, 3H), 1.31–1.19 (m, 2H), 1.17–1.07 (m, 2H),
0.98 (s, 3H), 0.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 209.8, 166.7,
160.0, 157.4, 141.2, 139.8, 136.3, 129.9, 122.4, 121.0, 120.6, 115.6,
111.3, 74.8, 63.8, 57.0, 55.4, 50.0, 44.1, 41.1, 40.9, 38.9, 38.6, 37.1,
2.37–2.24 (m, 2H), 2.12 (s, 3H), 2.05 (m, 1H), 1.97 (m, 1H), 1.85
(m, 2H), 1.70 (m, 1H), 1.68–1.62 (m, 2H), 1.61–1.54 (m, 3H), 1.49 (m,
3H), 1.23–1.20 (m, 2H), 1.13 (m, 1H), 1.01 (s, 3H), 0.90 (m, 2H), 0.62
(s, 3H). 13C NMR (150 MHz, MeOD) δ 212.4, 169.6, 160.6, 158.9,
142.0, 141.2, 130.6, 127.7, 123.2, 118.4, 116.7, 75.7, 64.7, 58.0, 51.4,
45.1, 41.4, 40.6, 39.8, 39.6, 38.2, 37.7, 33.2, 32.8, 31.7, 29.2, 25.5,
23.8, 22.2, 19.7, 13.6. ESI-MS m/z: 571.3 [M+Na]+. HR-ESIMS: [M
36.7, 31.9, 31.9, 31.7, 28.2, 24.6, 22.9, 21.2, 19.4, 13.4. ESI-MS m/z:
+
585.2 [M+Na]+
.
HR-ESIMS: [M+H]+ calcd for C34H47N2O5
+H]+ calcd for C33H45N2O5 549.3328, found 549.3293.
+
563.3485, found 563.3466.
2.1.1.4. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(2-methoxybenzamido)ethylcarbamate (5d). White
solid (17 mg, 20%). 1H NMR (600 MHz, CDCl3) δ 7.57 (d, J =
15.6 Hz, 1H), 7.44 (d, J = 7.7 Hz, 2H), 6.88 (d, J = 7.7 Hz, 2H), 6.38
(s, –NH), 6.27 (d, J = 15.6 Hz, 1H), 5.31 (s, 1H), 5.20 (s, –NH), 4.48 (m,
1H), 3.82 (s, 3H), 3.51 (m, 2H), 3.37 (m, 2H), 2.52 (t, J = 8.9 Hz, 1H),
2.33 (m, 1H), 2.26 (m, 1H), 2.17 (m, 1H), 2.12 (s, 3H), 2.03 (m, 1H),
1.96 (m, 1H), 1.87 (m, 1H), 1.82 (m, 1H), 1.70–1.65 (m, 2H), 1.61–1.51
(m, 3H), 1.49–1.41 (m, 3H), 1.27–1.18 (m, 2H), 1.17–1.09 (m, 2H),
0.97 (s, 3H), 0.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 209.8, 167.1,
161.0, 157.3, 140.9, 139.8, 129.5, 127.6, 122.4, 118.2, 114.4, 74.8,
63.8, 57.0, 55.5, 50.0, 44.1, 41.0, 40.9, 38.9, 38.6, 37.1, 36.7, 31.9,
31.9, 31.7, 28.2, 24.6, 22.9, 21.1, 19.4, 13.4. ESI-MS m/z: 585.2 [M
+Na]+. HR-ESIMS: [M+H]+ calcd for C34H47N2O5+ 563.3485, found
563.3478.
2.1.1.8. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl
2-(3-hydroxy-4-methoxybenzamido)ethylcarbamate
(5h). White solid (41 mg, 47%). 1H NMR (600 MHz, MeOD) δ 7.41
(d, J = 15.7 Hz, 1H), 7.04 (d, J = 1.8 Hz, 1H), 7.00 (dd, J = 8.4,
1.8 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.40 (d, J = 15.7 Hz, 1H), 5.32
(s, 1H), 4.37 (m, 1H), 3.88 (s, 3H), 3.39 (t, J = 5.8 Hz, 2H), 3.26 (t,
J = 5.8 Hz, 2H), 2.64 (t, J = 9.0 Hz, 1H), 2.36–2.23 (m, 2H), 2.12 (s,
3H), 2.05 (m, 1H), 1.96 (m, 1H), 1.91–1.82 (m, 2H), 1.70 (m, 1H),
1.68–1.62 (m, 2H), 1.61–1.53 (m, 2H), 1.49 (d, J = 9.4 Hz, 2H), 1.29
(s, 1H), 1.23 – 1.18 (m, 2H), 1.13 (m, 1H), 1.01 (s, 3H), 0.62 (s, 3H).
13C NMR (150 MHz, MeOD) δ 212.4, 169.4, 158.9, 150.9, 148.0, 142.0,
141.2, 129.4, 123.2, 122.2, 119.3, 114.5, 112.5, 75.7, 64.7, 58.0, 56.4,
51.4, 45.1, 41.4, 40.6, 39.8, 39.6, 38.2, 37.7, 33.2, 32.8, 31.7, 29.2,
25.5, 23.8, 22.2, 19.7, 13.6. ESI-MS m/z: 601.3 [M + Na]+. HR-ESIMS:
[M + H]+ calcd for C34H47N2O6 579.3434, found 579.3437.
+
2.1.1.5. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2.1.1.9. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(2-hydroxybenzamido)ethylcarbamate (5e). White
solid (14 mg, 17%). 1H NMR (600 MHz, MeOD) δ 7.82 (d, J =
15.9 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.16 (dd, J = 7.8, 7.7 Hz, 1H),
6.85–6.81 (m, 2H), 6.71 (d, J = 15.9 Hz, 1H), 5.33 (s, 1H), 4.38 (m,
1H), 3.39 (t, J = 6.0 Hz, 2H), 3.27 (t, J = 6.0 Hz, 2H), 2.61 (m, 1H),
2.35–2.24 (m, 2H), 2.11 (s, 3H), 2.12 (m, 1H), 2.05 (m, 1H), 1.96 (m,
1H), 1.90–1.82 (m, 2H), 1.70–1.65 (m, 3H), 1.60–1.53 (m, 2H),
1.52–1.42 (m, 3H), 1.25–1.15 (m, 2H), 1.11 (m, 1H), 1.01 (s, 3H),
0.98–0.93 (m, 1H), 0.61 (s, 3H). 13C NMR (150 MHz, MeOD) δ 212.3,
169.9, 158.8, 158.0, 141.2, 138.0, 131.8, 129.8, 123.2, 123.1, 121.5,
120.6, 116.9, 75.7, 64.6, 58.0, 51.4, 45.1, 41.4, 40.6, 39.8, 39.6, 38.2,
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(3,4-dimethoxybenzamido)ethylcarbamate (5i). White
solid (50 mg, 56%). 1H NMR (600 MHz, CDCl3) δ 7.55 (d, J = 15.6 Hz,
1H), 7.07 (d, J = 8.2 Hz, 1H), 7.01 (s, 1H), 6.83 (d, J = 8.2 Hz, 1H),
6.40 (s, –NH), 6.28 (d, J = 15.6 Hz, 1H), 5.31 (s, 1H), 5.18 (s, –NH),
4.47 (m, 1H), 3.89 (s, 6H), 3.51 (t, J = 5.3 Hz, 2H), 3.38 (t, J = 5.3 Hz,
2H), 2.52 (t, J = 8.9 Hz, 1H), 2.33 (m, 1H), 2.26 (m, 1H), 2.16 (s, 2H),
2.11 (s, 3H), 2.03 (m, 1H), 1.95 (m, 1H), 1.87 (m, 1H), 1.82 (m, 1H),
1.70–1.62 (m, 2H), 1.60–1.50 (m, 3H), 1.47–1.40 (m, 3H), 1.28–1.17
(m, 2H), 1.17–1.07 (m, 2H), 0.97 (s, 3H), 0.61 (s, 3H). 13C NMR
(150 MHz, CDCl3) δ 209.8, 167.0, 157.4, 150.7, 149.2, 141.2, 139.8,
127.9, 122.4, 122.2, 118.4, 111.1, 109.7, 74.8, 63.8, 56.9, 56.1, 56.0,
50.0, 44.1, 40.9, 38.9, 38.6, 37.1, 36.7, 31.9, 31.8, 31.7, 31.1, 28.2,
24.6, 22.9, 21.2, 19.4, 13.3. ESI-MS m/z: 615.3 [M + Na]+. HR-ESIMS:
37.7, 33.1, 32.8, 31.7, 29.2, 25.5, 23.8, 22.2, 19.7, 13.6. ESI-MS m/z:
571.3 [M+Na]+
.
HR-ESIMS: [M+H]+ calcd for C33H45N2O5
+
+
549.3328, found 549.3328.
[M + H]+ calcd for C35H49N2O6 593.3591, found 593.3591.
2.1.1.6. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(3-hydroxybenzamido)ethylcarbamate (5f). White
2.1.1.10. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(benzo[d][1,3]dioxole-5-carboxamido)ethylcarbamate
(5j). White solid (30 mg, 35%). 1H NMR (600 MHz, CDCl3) δ 7.51 (d,
J = 15.5 Hz, 1H), 6.98 (s, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.77 (d,
J = 8.0 Hz, 1H), 6.48 (s, –NH), 6.23 (d, J = 15.5 Hz, 1H), 5.98 (s, 2H),
5.31 (s, 1H), 5.23 (s, –NH), 4.47 (m, 1H), 3.49 (t, J = 5.2 Hz, 2H), 3.37
(t, J = 5.2 Hz, 2H), 2.52 (t, J = 8.9 Hz, 1H), 2.32 (d, J = 8.4 Hz, 1H),
2.25 (t, J = 11.6 Hz, 1H), 2.17 (m, 1H), 2.11 (s, 3H), 2.08 (m, 1H),
1.99–1.92 (m, 1H), 1.80 (s, 3H), 1.65 (m, 1H), 1.60–1.50 (m, 3H),
1.46–1.41 (m, 3H), 1.27–1.17 (m, 2H), 1.15–1.10 (m, 2H), 0.97 (s, 3H),
0.61 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 209.8, 167.0, 157.4, 149.2,
148.3, 141.0, 139.8, 129.3, 124.1, 122.4, 118.7, 108.6, 106.5, 101.6,
74.8, 63.8, 56.9, 50.0, 44.1, 40.9, 40.9, 38.9, 38.6, 37.1, 36.7, 31.9,
31.8, 31.7, 28.2, 24.6, 22.9, 21.1, 19.4, 13.3. ESI-MS m/z: 599.3 [M
+Na]+. HR-ESIMS: [M+H]+ calcd for C34H45N2O6+ 577.3278, found
577.3284.
solid (19 mg, 23%). 1H NMR (600 MHz, MeOD)
δ 7.45 (d,
J = 15.7 Hz, 1H), 7.19 (dd, J = 8.0, 7.7 Hz, 1H), 7.01 (d, J = 7.7 Hz,
1H), 6.96 (s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.53 (d, J = 15.7 Hz, 1H),
5.32 (s, 1H), 4.38 (m, 1H), 3.43–3.36 (m, 2H), 3.29–3.24 (m, 2H), 2.61
(t, J = 8.9 Hz, 1H), 2.37–2.24 (m, 2H), 2.13 (m, 1H), 2.11 (s, 3H), 2.04
(m, 1H), 1.95 (m, 1H), 1.86 (m, 1H), 1.74–1.51 (m, 5H), 1.47 (m, 3H),
1.25–1.05 (m, 3H), 1.00 (s, 3H), 0.95 (m, 1H), 0.60 (s, 3H). 13C NMR
(150 MHz, MeOD) δ 212.2, 169.0, 159.0, 158.8, 142.0, 141.2, 137.5,
130.9, 123.3, 121.7, 120.4, 118.0, 115.2, 75.7, 64.6, 58.0, 51.4, 45.1,
41.3, 40.6, 39.8, 39.6, 38.2, 37.7, 33.1, 32.8, 31.7, 29.2, 25.5, 23.8,
22.2, 19.7, 13.6. ESI-MS m/z: 571.3 [M+Na]+. HR-ESIMS: [M+H]+
+
calcd for C33H45N2O5 549.3328, found 549.3345.
2.1.1.7. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(4-hydroxybenzamido)ethylcarbamate (5 g). White
2.1.1.11. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
solid (19 mg, 23%). 1H NMR (600 MHz, MeOD)
δ
7.45 (d,
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(2,3-dimethoxybenzamido)ethylcarbamate (5k). White
solid (54 mg, 61%). 1H NMR (600 MHz, CDCl3) δ 7.87 (d, J = 15.9 Hz,
1H), 7.10 (d, J = 7.8 Hz, 1H), 7.03 (dd, J = 8.1, 7.8 Hz, 1H), 6.91 (d,
J = 15.7 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 8.5 Hz, 2H),
6.40 (d, J = 15.7 Hz, 1H), 5.38 (m, 1H), 4.59 (s, 1H), 4.38 (m, 1H),
3.44–3.36 (m, 2H), 3.27–3.24 (m, 2H), 2.63 (t, J = 4.4 Hz, 1H),
3

Y.-X. Ge, et al.
Steroids152(2019)108499
Scheme 1. Synthesis of 5a-5o. Reagents and conditions: (a) p-nitrophenyl chloroformate, pyridine, CH2Cl2, rt, overnight, 79%; (b) N-Boc-ethylenediamine, Et3N,
CH2Cl2, rt, overnight, 84%; (c) i. trifluoroacetic acid, CH2Cl2, rt, overnight; ii. cinnamic acids, HATU, DIPEA, CH2Cl2, 36 h, 17–56%.
J = 8.1 Hz, 1H), 6.48 (d, J = 15.9 Hz, 1H), 6.40 (s, –NH), 5.32 (s, 1H),
5.17 (s, –NH), 4.48 (m, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.57–3.47 (m,
2H), 3.42–3.33 (m, 2H), 2.53 (t, J = 9.0 Hz, 1H), 2.33 (d, J = 12.3 Hz,
1H), 2.26 (t, J = 11.9 Hz, 1H), 2.17 (m, 1H), 2.12 (s, 3H), 2.03 (m, 1H),
1.96 (m, 1H), 1.87 (m, 1H), 1.83 (m, 1H), 1.69–1.65 (m, 2H), 1.61–1.51
(m, 3H), 1.48–1.40 (m, 3H), 1.28–1.20 (m, 2H), 1.17–1.11 (m, 2H), 0.98
(s, 3H), 0.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 209.8, 167.1, 157.3,
153.3, 148.4, 139.9, 136.0, 129.1, 124.2, 122.3, 122.3, 119.5, 113.5,
74.8, 63.8, 61.3, 57.0, 56.0, 50.0, 44.1, 41.0, 40.9, 38.9, 38.6, 37.1, 36.7,
31.9, 31.9, 31.7, 28.2, 24.6, 22.9, 21.2, 19.4, 13.4. ESI-MS m/z: 615.3 [M
2.1.1.14. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(3,4,5-trimethoxybenzamido)ethylcarbamate (5n). White
solid (22 mg, 24%). 1H NMR (600 MHz, CDCl3) δ 7.51 (d, J = 15.5 Hz,
1H), 6.70 (s, 2H), 6.58 (s, –NH), 6.33 (d, J = 15.5 Hz, 1H), 5.30 (s, 1H),
5.27 (s, –NH), 4.47 (t, J = 10.8 Hz, 1H), 3.85 (s, 9H), 3.54–3.48 (m, 2H),
3.40–3.34 (m, 2H), 2.51 (t, J = 8.8 Hz, 1H), 2.32 (m, 1H), 2.25 (m, 1H),
2.15 (m, 1H), 2.11 (s, 3H), 2.02 (m, 1H), 1.95 (m, 1H), 1.89–1.79 (m, 2H),
1.65–1.61 (m, 2H), 1.60–1.49 (m, 3H), 1.47–1.40 (m, 3H), 1.28–1.16 (m,
2H), 1.14–1.07 (m, 2H), 0.97 (s, 3H), 0.60 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ 209.8, 166.8, 157.5, 153.5, 141.2, 139.7, 130.4, 122.4, 120.0,
105.4, 105.1, 74.8, 63.8, 61.1, 56.9, 56.2, 50.0, 44.1, 40.8, 38.9, 38.7,
38.6, 37.1, 36.6, 31.9, 31.8, 31.7, 28.2, 24.6, 22.9, 21.1, 19.4, 13.3. ESI-
+
+Na]+. HR-ESIMS: [M+H]+ calcd for C35H49N2O6 593.3591, found
593.3595.
+
MS m/z: 623.3 [M+H]+. HR-ESIMS: [M+H]+ calcd for C36H51N2O7
2.1.1.12. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
623.3696, found 623.3687.
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(2,5-dimethoxybenzamido)ethylcarbamate (5l). White
solid (31 mg, 35%). 1H NMR (600 MHz, CDCl3) δ 7.83 (d, J = 15.8 Hz,
1H), 6.99 (d, J = 2.9 Hz, 1H), 6.85 (dd, J = 9.0, 2.9 Hz, 1H), 6.81 (d,
J = 9.0 Hz, 1H), 6.50 (d, J = 15.8 Hz, 1H), 6.47 (s, –NH), 5.32 (s, 1H),
5.29 (s, –NH), 4.47 (m, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.51 (m, 2H),
3.37 (m, 2H), 2.52 (t, J = 8.9 Hz, 1H), 2.33 (d, J = 12.4 Hz, 1H), 2.25
(t, J = 11.8 Hz, 1H), 2.17 (m, 1H), 2.11 (s, 3H), 2.03 (m, 1H), 1.95 (m,
1H), 1.86 (m, 1H), 1.82 (m, 1H), 1.70–1.61 (m, 2H), 1.61–1.49 (m, 3H),
1.48–1.39 (m, 3H), 1.27–1.17 (m, 2H), 1.16 – 1.06 (m, 2H), 0.97 (s,
3H), 0.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 209.8, 167.3, 157.2,
153.5, 152.8, 139.9, 136.4, 124.5, 122.3, 121.7, 116.2, 113.7, 112.4,
74.8, 63.8, 56.9, 56.1, 55.9, 49.9, 44.1, 41.1, 40.7, 38.9, 38.6, 37.1,
2.1.1.15. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(2,4,5-trimethoxybenzamido)ethylcarbamate (5o). White
solid (19 mg, 20%). 1H NMR (600 MHz, CDCl3) δ 7.81 (d, J = 15.8 Hz,
1H), 6.94 (s, 1H), 6.54 (s, –NH), 6.45 (s, 1H), 6.38 (d, J = 15.8 Hz, 1H),
5.41 (d, J = 5.4 Hz, 1H), 5.28 (s, 1H), 4.44 (m, 1H), 3.88 (s, 3H), 3.81 (s,
6H), 3.49 (t, J = 4.9 Hz, 2H), 3.35 (t, J = 4.9 Hz, 2H), 2.50 (t, J = 8.8 Hz,
1H), 2.30 (m, 1H), 2.23 (m, 1H), 2.18–2.13 (m, 1H), 2.09 (s, 3H), 2.00 (m,
1H), 1.93 (m, 1H), 1.84 (m, 1H), 1.79 (m, 1H), 1.66–1.59 (m, 2H),
1.58–1.47 (m, 3H), 1.45–1.38 (m, 3H), 1.23 (s, 1H), 1.18 (m, 1H),
1.13–1.04 (m, 2H), 0.95 (s, 3H), 0.59 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ 209.7, 167.8, 157.2, 153.7, 151.5, 143.2, 139.8, 136.1, 122.2, 118.0,
115.3, 111.3, 97.0, 74.5, 63.7, 56.9, 56.5, 56.4, 56.1, 49.9, 44.0, 41.1,
38.8, 38.7, 38.6, 37.0, 36.6, 31.9, 31.6, 29.3, 28.2, 24.5, 22.9, 21.1, 19.4,
13.3. ESI-MS m/z: 645.3 [M+Na]+. HR-ESIMS: [M+H]+ calcd for
36.6, 31.9, 31.8, 31.7, 28.2, 24.6, 22.9, 21.2, 19.4, 13.4. ESI-MS m/z:
+
615.3 [M+Na]+
.
HR-ESIMS: [M+H]+ calcd for C35H49N2O6
593.3591, found 593.3591.
+
C36H51N2O7 623.3696, found 623.3687.
2.1.1.13. (3S,8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yl 2-(4-hydroxy-3,5-dimethoxybenzamido)ethylcarbamate
(5m). White solid (36 mg, 39%). 1H NMR (600 MHz, MeOD) δ 7.43
(d, J = 15.6 Hz, 1H), 6.84 (s, 2H), 6.45 (d, J = 15.6 Hz, 1H), 5.29 (s,
1H), 4.37 (m, 1H), 3.86 (s, 6H), 3.43–3.38 (m, 2H), 3.30–3.23 (m, 2H),
2.60 (t, J = 8.8 Hz, 1H), 2.38–2.21 (m, 2H), 2.11 (s, 3H), 2.03 (m, 1H),
1.93 (m, 1H), 1.84 (m, 2H), 1.70–1.62 (m, 2H), 1.61–1.51 (m, 3H),
1.49–1.41 (m, 3H), 1.26–1.04 (m, 4H), 0.98 (s, 3H), 0.94 (m, 1H), 0.59
(s, 3H). 13C NMR (150 MHz, MeOD) δ 212.3, 169.4, 158.9, 149.4,
142.5, 141.1, 138.9, 127.1, 123.3, 119.1, 106.4, 75.7, 64.6, 57.9, 56.8,
51.3, 45.0, 41.4, 40.6, 39.8, 39.7, 38.2, 37.7, 33.1, 32.8, 31.7, 29.2,
25.4, 23.8, 22.1, 19.7, 13.6. ESI-MS m/z: 631.3 [M+Na]+. HR-ESIMS:
2.2. Biological evaluation
2.2.1. Cell line and culture condition
All of the cell lines were purchased from Cell Bank of Chinese Academy
of Sciences, Shanghai Branch (Shanghai, China). A549, H157, HepG2,
MCF-7, and HL-60 cells were cultured in specified medium supplemented
with 10% fetal bovine serum (Gibco), 100 IU/mL penicillin and 100 µg/
mL streptomycin (both from Thermo Fisher Scientific, Inc., Waltham, MA,
USA) in a humidified atmosphere containing 5% CO2 at 37 °C.
2.2.2. Cell viability assay
The cells were cultured in 96-well plates, and each well was seeded
with 1 × 104 cells. After incubation for 16 h, the medium was removed
[M+H]+ calcd for C35H49N2O7 609.3540, found 609.3542.
+
4

Y.-X. Ge, et al.
Steroids152(2019)108499
Table 1
a
In vitro antiproliferative activity of 5a-5o presented as IC50 (μM).
Compounds
R
MCF-7
> 50b
A549
> 50
H157
> 50
HepG2
> 50
HL-60
> 50
5a
5b
> 50
> 50
3.72
0.32
2.62
5.46
0.35
0.42
> 50
> 50
5c
5d
4.16
8.85
1.12
1.23
> 50
30.98
24.50
21.33
6.55
2.11
2.36
3.33
22.59
1.66
15.68
> 50
1.54
5e
> 50
> 50
24.54
3.81
0.23
5f
4.91
6.46
0.13
0.35
3.22
5.16
0.42
0.61
4.28
3.77
0.11
0.21
1.26
3.45
8.86
0.23
0.67
6.87
3.51
1.11
0.22
3.22
5g
5h
5i
6.11
0.47
> 50
> 50
17.98
> 50
5.22
0.33
25.64
> 50
> 50
> 50
5j
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
10.19
> 50
0.14
5k
5l
> 50
> 50
> 50
5.5
> 50
5m
5n
5o
6.55
0.54
36.20
> 50
> 50
1.65
30.67
> 50
> 50
2.75
> 50
> 50
3.10
11.26
> 50
> 50
1.33
> 50
> 50
0.51
0.01
Doxorubicin
a
1.51
0.02
0.31
0.01
0.48
0.02
1.82
0.91
0.03
IC50 values are taken as a mean from three experiments.
b
Compounds with inhibition ratio less than 50% at 50 μM were not tested further.
37 °C. In this step, 100 µL of DMSO was added to dissolve the formazan
crystals. The optical density (OD) was measured at an absorbance wave-
length of 490 nm while using a Microplate Reader (Tecan, Switzerland).
Table 2
Cytotoxicity of 5a-5o against non-tumor cells presented as inhibition ratio (%)
at 50 μM.
Compounds
BEAS2B
MCF-10A
Compounds
BEAS2B
MCF-10A
2.2.3. Hoechst 33258 staining assay
5a
5b
5c
5d
5e
5f
41.10
24.68
9.55
45.89
22.52
8.99
5i
8.44
10.44
12.59
21.72
16.02
3.70
A549 cells (1 × 105 cells/well) were seeded in confocal dishes.
After attachment, cells were treated with the concentration of 2.5, 5,
10 μM 5f for 24 h. After treatment, A549 cells were incubated with
Hoechst 33258 (2 μg/ml, Sigma-Aldrich, USA) away from light for
15 min at 37 °C. Stained cells were washed twice with PBS and then
viewed under a Leica DMI8 (Germany) fluorescence microscope.
5j
15.26
23.15
19.72
2.18
5k
5l
16.82
22.71
15.76
4.37
16.25
21.32
28.55
2.53
5m
5n
5o
17.68
10.54
20.32
10.79
5g
5h
28.59
26.90
2.2.4. Flow cytometric analysis
and replaced with 100 µL medium containing the indicated concentrations
of compounds and further incubated for 24 h. Then, the viability of cells
was measured by the MTT method [20]. Briefly, 0.02 mL of MTT solution
(5 mg/mL in PBS) was added to each well and then incubated for 4 h at
A549 cells were plated in six-well plates treated with tested com-
pounds or DMSO (control). Cells were harvested at 24 h and measured
using Annexin V–FITC Apoptosis Detection Kit (Vazyme Biotech) ac-
cording to the manufacturer’s instructions for cell apoptosis analysis.
5

Y.-X. Ge, et al.
Steroids152(2019)108499
Fig. 2. Compound 5f blocked A549 cell cycle in G1 phase. (A) Compound 5f induced A549 cells cycle arrest. Cells were treated with 2.5, 5.0 and 10 µM of 5f for 24 h.
Propidium iodide (PI) staining was done to determine the DNA content; (B) Percentage of A549 cells in different phases of cell cycle.
Fig. 3. Fluorescent micrographs of 5f-treated and untreated A549 cells after Hoechst 33258 staining.
For cell cycle analysis, the harvested cells were re-suspended in 70%
ethanol overnight at 4 °C for fixation. Then samples were washed with
PBS and incubated with propidium iodide/rnase staining buffer (BD
Pharmingen) for 0.5 h at room temperature.
inhibitor cocktails (Roche, South San Francisco, CA, USA). Protein con-
centrations were determined while using BCA protein assay kit
(Beyotime Institute of Biotechnology, Haimen, Jiansu, China. Equivalent
quantities of total protein were electrophoretically separated on a 10%
sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The total pro-
tein were then transferred onto polyvinylidene fluoride (PVDF) mem-
branes and blocked for 60 min with 3% bovine serum albumin (BSA)
solution prepared in PBS at room temperature. A 1:1000 dilution of the
2.2.5. Western blotting analysis
A549 cells were treated with different doses of 5f (2.5, 5, 10 μM) for
24 h. The cells were lysed in 1 × RIPA buffer containing protease
6

Y.-X. Ge, et al.
Steroids152(2019)108499
Fig. 4. Flow cytometry analysis of 5f-treated A549 cells death by double staining with Annexin V-FITC/PI.
primary antibodies (Bax, Bcl-2, and β-actin) were incubated overnight at
4 °C. The membranes were washed three times with TBST buffer (0.05%
Tris-buffered saline and Tween 20). The appropriate peroxidase-con-
jugated secondary antibody was incubated for 2 h at room temperature.
Subsequently, the results were visualized with enhanced chemilumines-
cence reagent (Merck Millipore, Darmstadt, Germany) while using ima-
ging system (Chemi-Doc XRS imager, Bio-Rad, Hercules, CA, USA.
using MTT assay. The compounds with inhibition ratios > 50% at 50 μM
were further tested for IC50 values. Table 1 gave the cancer cell inhibitory
data and IC50 value for each compound. By comparison of their IC50
values, it is clear that two compounds 5f and 5g showed the best broad-
spectrum antiproliferative activity against these cancer cell lines with
IC50 values less than 10 μM. It is worth noting that compound 5e has
selective antiproliferative activity towards HepG2 compared to other
cancer cells. The comparison of activities of 5b-5g indicated that 3-OH or
4-OH was beneficial to increase compounds’ potency, while methylation
of hydroxyl or changing the position of hydroxyl would significantly
decrease the activity of compound or even make the activity completely
lose towards certain cancer cell lines, such as A549 and HL-60. More-
over, introduction of another methoxyl (5h) or dialkylation of hydroxyl
(5i-5l) led to decreased antiproliferative activity. This result highlighted
that the 3-OH and 4-OH on benzene ring were necessary for its anti-
proliferative activity. This deduction was also supported by the ob-
servation that compound 5m exhibited improved activity compared with
5n and 5o. Thus, the 3-OH and 4-OH of the benzene group probably
played an important role in these bioactive compound’s binding to target
protein. To prove the efficacy of this hybridization, the activity of the
synthetic precursors, including pregnenolone and cinnamic acid deriva-
tives, towards these cancer cells were then tested. The result shown in
Table S1 (ESI†) revealed that all these precursors were inactive towards
MCF-7, A549, H157, HepG2 and HL-60 at the testing concentration of
25, 50 and 100 μM, with inhibition ratio less than 50%. This result
clearly indicated that the hybridization of pregnenolone with cinnamic
acids led to the new antiproliferative prototype.
3. Results and discussion
3.1. Design and synthesis
As introduced above, cinnamic acid and pregnenolone are privi-
leged framework used in the design of anticancer drugs, thus the cin-
namic acid-pregenolone hybrids I were designed for the first time on
the basis of molecular hybridization strategy [21,22]. In this new
chemotype, the ethanediamine (–N–CH2–CH2–N–) linker was chosen
due to the fact that a large number of bioactive compounds or drugs
contain this moiety [23]. Here, a four-step concise synthetic route for
target compounds 5a-5o is depicted in Scheme 1. Briefly, acylation of
pregnenolone (2) using p-nitrophenyl chloroformate in the presence of
pyridine provided intermediate 3, which was then reacted with N-Boc-
ethylenediamine to yield compound 4. The N-Boc group of 4 was re-
moved by treating with trifluoroacetic acid, and the following coupling
with substituted cinnamic acids in the presence of HATU yielded cor-
responding 5a-5o. The 1H, 13C NMR and HRMS spectra of target
compounds were provided in Supplementary material.
To further verify the selectivity of these hybrids towards cancer
cells, the cytotoxicity of 5a-5o against two non-tumor cell lines, in-
cluding BEAS2B and MCF-10A, was evaluated at the initial concentra-
tion of 50 μM. Interestingly, as shown in Table 2 all of them exhibited
inhibition ratio less than 50% at 50 μM, which were considered to be
3.2. Antiproliferative activity
All cinnamic acid-pregenolone hybrids 5a-5o were evaluated for
their in vitro antiproliferative activity against five human cancer cell lines
7

Y.-X. Ge, et al.
Steroids152(2019)108499
Fig. 5. Effect of 5f on expression of apoptosis-related proteins in A549 cells. (A) The protein level of Bax and Bcl-2 was examined by western blot assay, and β-actin
was used as a loading control; (B) Protein expression of Bax/β-action and Bcl-2/β-action ratio; (C) Ratio of Bax/Bcl-2. *p < 0.05 and **p < 0.01 versus control.
inactive towards BEAS2B and MCF-20A. This result suggested that these
analogs exhibited selective cytotoxicity towards above cancer cell lines
compared to the normal human BEAS2B and MCF-10A cell lines.
cells exhibited obvious apoptosis characteristics, including nuclear
fragmentation, nuclear condensation, and membrane integrity loss.
Thus, compound 5f could induce the apoptosis of A549 cells.
To understand the potential apoptosis inducing mechanism of this
series of cinnamic acid-pregenolone hybrids, different concentrations of
model compound 5f was tested in A549 cells. Annexin V-FITC/PI fluor-
escence assay was performed to evaluate necrotic cells as well as early
stage and late stage apoptotic cells. The percentage of cells in different
phases of the cell cycle was analyzed by flow cytometry. As shown in
Fig. 4, a significant accumulation in late stage of apoptotic cells of A549
cells treated by 5f (0, 1, 10, 20 μM) could be observed. These results
suggested that the effect of 5f on the growth inhibition of A549 cells was
closely related with its enhanced apoptosis in A549 cells.
3.3. Compound 5f arrested A549 cells in G1 phase
Anticancer therapies such as anti-proliferation and inducing apop-
tosis of cancer cells are linked to cell cycle [24,25]. Among these hy-
brids, compound 5f served as the most broad-spectrum antiproliferative
agent, thus it was chosen for preliminary mechanism of action study on
A549 cell model. To verify the blockade of cell cycle at a specific
checkpoint by 5f, its effect on distribution of A549 cell cycle phase was
studied by cytometric analysis. The cell cycle results showed that 5f
arrested the A549 cells progression in G1 phase (Fig. 2). As shown in
Fig. 2B, the ratio of cell in G1 phase was increased in a dose-dependent
way, from 58.85% in the control to 63.04% at 2.5 μM, 69.40% at
5.0 μM, and 76.42% at 10 μM. Clearly, the effect of G1 arrest con-
tributed to the growth inhibition of A549 cells caused by 5f.
3.5. Compound 5f induced A549 cells apoptosis by up-regulating expression
of Bax and down-regulating expression of Bcl-2.
Apoptosis is significantly regulated by the Bcl-2 family proteins,
such as Bax as an activator and Bcl-2 as an inhibitor [26]. The ratio of
Bax/Bcl-2 determines the survival or death of cells following an apop-
totic stimulus [27]. Thus, to better figure out the molecular mechanism
involved in apoptosis inducement, the effect of 5f on the expression of
Bax and Bcl-2 in A549 cells was evaluated by western blot. When the
cells were treated with 0, 2.5, 5.0 and 10 μM of 5f for 24 h, the up-
regulation of apoptosis-induced Bax and down-regulation of anti-
3.4. Compound 5f induced apoptosis of A549 cells
To investigate the effect of 5f on the proliferation and apoptosis of
cancer cells, a Hoechst 33258 staining assays was performed. This re-
sult shown in Fig. 3 indicated that 5f-treated A549 cells were under-
going apoptosis. As the concentration of 5f increased, the treated A549
8

Y.-X. Ge, et al.
Steroids152(2019)108499
apoptotic Bcl-2 protein were observed (Fig. 5A,B). Fig. 5C showed
significantly (P less than 0.01) increased ratio of Bax/Bcl-2 under
treatment of 5f in a dose-dependent manner. These obtained results
revealed that the expression of Bax/Bcl-2 regulated by 5f contributed to
the apoptosis of A549.
cinnamic acids, Anticancer Agents Med. Chem. 15 (2015) 980–987.
[6] M. Mielecki, B. Lesyng, Cinnamic acid derivatives as inhibitors of oncogenic protein
kinases-structure, mechanisms and biomedical effects, Curr. Med. Chem. 23 (2016)
954–982.
[7] K. Swiatek-Machado, J. Mieczkowski, A. Ellert-Miklaszewska, P. Swierk, I. Fokt,
S. Szymanski, S. Skora, W. Szeja, G. Grynkiewicz, B. Lesyng, W. Priebe,
B. Kaminska, Novel small molecular inhibitors disrupt the JAK/STAT3 and FAK
signaling pathways and exhibit a potent antitumor activity in glioma cells, Cancer
Biol. Ther. 13 (2012) 657–670.
4. Conclusions
[8] Y. Qian, H.J. Zhang, H. Zhang, C. Xu, J. Zhao, H.L. Zhu, Synthesis, molecular
modeling, and biological evaluation of cinnamic acid metronidazole ester deriva-
tives as novel anticancer agents, Bioorg. Med. Chem. 18 (2010) 4991–4996.
[9] H. Zou, H. Wu, X. Zhang, Y. Zhao, J. Stöckigt, Y. Lou, Y.Yu. Synthesis, biological
evaluation, and structure-activity relationship study of novel cytotoxic aza-caffeic
acid derivatives, Bioorg. Med. Chem. 18 (2010) 6351–6359.
In summary, a series of new cinnamic acid-pregenolone hybrids (5a-
5o) was prepared and evaluated for their in vitro antiproliferative ac-
tivity against a panel of human cancer cell line, including A549, H157,
HepG2, MCF-7, and HL-60. From the bioassay results, 5f and 5g showed
the most promising potency in suppressing the growth of all these
tested cancer cells. The preliminary SAR study highlighted that the 3-
OH and 4-OH on the benzene ring were necessary for antiproliferative
activity. In addition, all these antiproliferative compounds have se-
lectivity towards above cancer cell lines compared to the normal human
BEAS2B and MCF-10A cell lines. The data obtained from Flow cyto-
metry experiment indicated that 5f could arrest A549 cells in G1 phase
and induce apoptosis of A549 cells. In addition, the effect of 5f on A549
cells apoptosis was associated with up-regulation of pro-apoptotic
protein Bax and down-regulation of anti-apoptotic protein Bcl-2. These
findings established the cinnamic acid-pregenolone hybrids as a new
chemotype for development of antiproliferative agents.
[10] A. Gupta, B.S. Kumar, A.S. Negi, Current status on development of steroids as an-
ticancer agents, J. Steroid Biochem. Mol. Biol. 137 (2013) 242–270.
[11] A.H. Banday, S.M. Akram, S.A. Shameem, Benzylidine pregnenolones and their
oximes as potential anticancer agents: synthesis and biological evaluation, Steroids
84 (2014) 64–69.
[12] V.A. D'yakonov, R.A. Tuktarova, L.U. Dzhemileva, S.R. Ishmukhametova,
M.M. Yunusbaeva, U.M. Dzhemilev, Catalytic cyclometallation in steroid chemistry
V: synthesis of hybrid molecules based on steroid oximes and (5Z,9Z)-tetradeca-5,9-
dienedioic acid as potential anticancer agents, Steroids 138 (2018) 14–20.
[13] A.H. Banday, S.A. Shameem, J.A. Banday, B.A. Ganaie, Synthesis, 17α-hydroxylase-
C17,20-lyase inhibitory and 5AR reductase activity of novel pregnenolone deriva-
tives, Anticancer Agents Med. Chem. 18 (2018) 1919–1926.
[14] L.A. Shervington, N. Smith, E. Norman, T. Ward, R. Phillips, A. Shervington, To
determine the cytotoxicity of chlorambucil and one of its nitro-derivatives, con-
jugated to prasterone and pregnenolone, towards eight human cancer cell-lines,
Eur. J. Med. Chem. 44 (2009) 2944–2951.
[15] L.H. Shan, H.M. Liu, K.X. Huang, G.F. Dai, C. Cao, R.J. Dong, Synthesis of
3β,7α,11α-trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives and their cy-
totoxic activities, Bioorg. Med. Chem. Lett. 19 (2009) 6637–6639.
[16] J. Zhang, W. Tavares de Sousa Júnior, V.C. Mello da Silva, M.C. Rodrigues,
J.A. Vasconcelos Morais, J.L. Song, Z.Q. Cheng, J.P.F. Longo, R. Bentes Azevedo,
C.S. Jiang, L. Alexandre Muehlmann, H. Zhang, Synthesis and evaluation of new
potential benzo[a]phenoxazinium photosensitizers for anticancer photodynamic
therapy, Molecules 23 (2018) pii: E1436.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[17] J.L. Song, J. Zhang, C.L. Liu, C. Liu, K.K. Zhu, F.F. Yang, X.G. Liu, J.P. Figueiró
Longo, L. Alexandre Muehlmann, R.B. Azevedo, Y.Y. Zhang, Y.W. Guo, C.S. Jiang,
H. Zhang, Design and synthesis of pregnenolone/2-cyanoacryloyl conjugates with
dual NF-κB inhibitory and anti-proliferative activities, Bioorg. Med. Chem. Lett. 27
(2017) 4682–4686.
Acknowledgements
This research work was financially supported by the Natural Science
Foundation of China (No. 21672082), Natural Science Foundation of
Shandong Province (Nos. ZR2019YQ31, JQ201721, ZR2018ZC0944),
the Young Taishan Scholars Program (No. tsqn20161037), and
Shandong Talents Team Cultivation Plan of University Preponderant
Discipline (No. 10027).
[18] C.S. Jiang, C.G. Huang, B. Feng, J. Li, J.X. Gong, T. Kurtán, Y.W. Guo, Synthesis and
antitumor evaluation of methyl spongoate analogs, Steroids 75 (2010) 1153–1163.
[19] C.S. Jiang, T. Ru, X.-J. Huan, Z.-H. Miao, Y.W. Guo, New cytotoxic ergostane sterols
from the Chinese soft coral Sinularia sp. Steroids 149 (2019) 108425.
[20] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63.
[21] C. Viegas-Junior, A. Danuello, V. da Silva Bolzani, E.J. Barreiro, C.A. Fraga,
Molecular hybridization: a useful tool in the design of new drug prototypes, Curr.
Med. Chem. 14 (2007) 1829–1852.
Appendix A. Supplementary data
[22] G. Bérubé, An overview of molecular hybrids in drug discovery, Expert Opin. Drug
Discov. 11 (2016) 281–305.
[23] S.R. Saibabu Kotti, C. Timmons, G. Li, Vicinal diamino functionalities as privileged
structural elements in biologically active compounds and exploitation of their
synthetic chemistry, Chem. Biol. Drug Des. 67 (2006) 101–114.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.steroids.2019.108499.
[24] N. Shankaraiah, S. Nekkanti, U.R. Brahma, N. Praveen Kumar, N. Deshpande,
D. Prasanna, K.R. Senwar, U. Jaya Lakshmi, Synthesis of different heterocycles-
linked chalcone conjugates as cytotoxic agents and tubulin polymerization in-
hibitors, Bioorg. Med. Chem. 25 (2017) 4805–4816.
References
[1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer sta-
tistics, CA Cancer J. Clin. 61 (2011) 69–90.
[25] J. Zhang, L. Ma, Z.F. Wu, S.L. Yu, L. Wang, W.C. Ye, Q.W. Zhang, Z.Q. Yin,
Cytotoxic and apoptosis-inducing activity of C21 steroids from the roots of
Cynanchum atratum, Steroids 122 (2017) 1–8.
[2] https://www.who.int/news-room/fact-sheets/detail/cancer (accessed at 26/06/
2019).
[26] Y. Tsujimoto, S. Shimizu, Bcl-2 family: life-or-death switch, FEBS Lett. 466 (2000)
6–10.
[3] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs from 1981 to
2014, J. Nat. Prod. 79 (2016) (1981) 629–661.
[27] Z.N. Oltval, C.L. Milliman, S.J. Korsmeyer, Bcl-2 heterodimerizes in vivo with a
conserved homolog, Bax, that accelerates programed cell death, Cell 74 (1993)
609–619.
[4] T. Takahashi, M. Miyazawa, Tyrosinase inhibitory activities of cinnamic acid ana-
logues, Pharmazie 65 (2010) 913–918.
[5] P. Su, Y. Shi, J. Wang, X. Shen, J. Zhang, Anticancer agents derived from natural
9