Design and synthesis of novel dehydroepiandrosterone analogues as potent antiproliferative agents

Article abstract of DOI:10.3390/molecules23092243

The aim of the present study was to determine the cytotoxic effects of a series of novel dehydroepiandrosterone derivatives containing triazole at the C₁₆ position on human cancer cells. The cancer cells used in the present study were A549, Hel

Full text of DOI:10.3390/molecules23092243

molecules
Article
Design and Synthesis of Novel
Dehydroepiandrosterone Analogues as Potent
Antiproliferative Agents
Xing Huang, Qing-Kun Shen, Hong-Jian Zhang, Jia-Li Li, Yu-Shun Tian * and Zhe-Shan Quan *
Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry
of Education, College of Pharmacy, Yanbian University, Yanji 133002, China; 2016010676@ybu.edu.cn (X.H.);
2016001065@ybu.edu.cn (Q.-K.S.); zhjzhishixiang@163.com (H.-J.Z.); 2016010667@ybu.edu.cn (J.-L.L.)
* Corresponding authors: ystian@ybu.edu.cn (Y.-S.T.); zsquan@ybu.edu.cn (Z.-S.Q.); Tel.: +86-433-243-6028
(Y.-S.T.); Fax: +86-433-243-5026 (Y.-S.T.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 August 2018; Accepted: 31 August 2018; Published: 3 September 2018
Abstract: The aim of the present study was to determine the cytotoxic effects of a series of novel
dehydroepiandrosterone derivatives containing triazole at the C16 position on human cancer
cells. The cancer cells used in the present study were A549, Hela, HepG-2, BEL7402, MCF-7, and
HCT116. Several of the synthesised compounds exhibited potent antiproliferative effects. The most
promising compound was (E)-3-hydroxy-16-((1-(4-iodophenyl)-1H-1,2,3-triazole-4-yl)methylene)-
10,13-dimet-hyl-1,3,4,7,8,9,10,11,12,13,15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14)-one
(compound 2n), which showed considerably high antiproliferative activity in the HepG-2 cell line,
with an IC50 value of 9.10
µM, and considerably high activity against the MCF-7 cell line, with an IC50
value of 9.18 M. Flow cytometry assays demonstrated that compound 2n exerted antiproliferative
µ
effects by arresting cells in the G2 phase of the cell cycle and inducing apoptosis.
Keywords: dehydroepiandrosterone; 1,2,3-Triazoles; antiproliferative; synthesis
1. Introduction
Cancer is one of the leading causes of death worldwide [
1]. Because of its severity, cancer is
considered one of the greatest social and economic concerns for the public health-care system [
2].
Over the years, several anticancer drugs have been developed with excellent cytotoxicity such as
paclitaxel and cisplatin [2].
Many anticancer drugs are nonselective, with paclitaxel and cisplatin being no exception.
Therefore, their use is limited because of their inability to differentiate between normal cells and
cancerous cells. Despite several advances made towards the diagnosis, prevention, and cure of cancer,
it remains one of the major causes of human morbidity and mortality worldwide. Therefore, there
is a requirement for safer and more effective anticancer drugs, which is a challenge for medicinal
chemists [3].
More than 60% of all clinically used anticancer drugs originate from natural sources [4]. To develop
selective drugs, many natural products are modified. Steroids are important physiological and
pharmacological regulators of cell growth and survival. There are several examples in the literature
that indicate the use of natural and synthetic steroids for the treatment of sex-hormone-dependent
cancer tumours, including breast cancer [
These hormone-dependent tumour cells can control tumour growth through hormone receptor
antagonists [5–7] Different types of steroids have been modified as cytotoxic and cytostatic
5], endometrial cancer [6], and cervical cancer [7].
.
(antiproliferative) anticancer agents. The majority of steroidal anticancer drugs have been developed
as enzyme inhibitors and cytotoxic drugs.
Molecules 2018, 23, 2243; doi:10.3390/molecules23092243
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2243
2 of 14
Recent years have seen an extensive focus on the modification of steroids. The rational
modifications of the perhydrocyclopentanophenanthrene nucleus of steroids have yielded several
important antiproliferative lead molecules. Exemestane, 2-methoxyestradiol, abiraterone, and
fulvestrant are some of the successful leads that have emerged from steroidal pharmacophores [2].
In order to maintain preselected characteristics of the lead compounds, the molecular
hybridization was a preferred choice for medicinal chemists [8–10]. Dehydroepiandrosterone (DHEA)
(Figure 1) is secreted by the adrenal glands [11]. Other DHEA derivatives have also been reported
as having antiproliferative properties in ovarian cancer cells (ES-2), human lung cells (A549), and
liver carcinoma cell (HepG-2) lines [12]. Heterocyclic compounds containing 1,2,3-triazoles display a
broad spectrum of biological activities, including antiproliferative [13–15]. Additionally, 1,2,3-triazole
fragment is widely applied in organic, medicinal, and material sciences [14].
O
HO
O
O
N
O
DHEA
O
O
N
N
N
N
Ν
Ο
Ο
O
Molecular
F3C
Hybridization
Ο
N
N
Compound A
A549 IC50<0.1μM
Compound B
AGS IC50=7.5μM
HO
N
2a-2w
R
ΟΗ
O
ΗΟ
OH
Ο
Cl
Ο
HO
Compound C
HCT116 IC50<0.1μM
Compound D
KB IC50=0.6μM
Figure 1. Design of target compound 2a–2w.
The 2-(substituted benzylidene) derivatives of C-3 oxidized ursolic acid (compound C) and
16-(substituted benzylidene) derivatives of DHEA (compound D) can be utilised as potent anticancer
agents [16 17]. Chemically, compound C and compound D are open-chain precursors of flavonoids
and isoflavonoids in which two aromatic rings are linked by a three-carbon unsaturated carbonyl
,
α-β
system [16]. The cytotoxic potential of the described compounds is mainly attributed to the position
and nature of the substituted group on the benzylidene pendant [17].
Therefore, α-β unsaturated androsterone derivatives containing the exocyclic double bond at the
C16 position could be utilised as potentially potent chemotherapeutic agents. The 1,2,3-triazoles can
also increase the antitumour activity of the compound. In the present study, we utilised the molecular
hybridization principle and designed, synthesised, and evaluated the antitumour biological activity
of a series of new compounds that contained a 1,2,3-triazole between the
α,β-unsaturated carbonyl
group and phenyl, and then evaluated any antitumour biological activities.

Molecules 2018, 23, 2243
3 of 14
2. Results and Discussion
2.1. Chemistry
Compounds 1a
to 61%, based on procedures described in references [18
aniline diazotisation generated substituted 1-azidobenzene, and in the second step the substituted
1-azidobenzene with propargyl alcohol and CuSO4 5H2O and L-ascorbic acid sodium salt generated
substituted phenyltriazole. The last step involved phenyltriazole oxidation by MnO2. From these three
steps, compounds 1a 1w were obtained. Compounds 2a 2w were obtained by treating DHEA with
–
1w were obtained in three synthetic steps, with overall yields from 40%
–
21]. In the first step, the substituted
·
–
–
different compounds 1a–1w under the catalysis of KOH in anhydrous C2H5OH, with 90–98% yield.
2.2. In Vitro Antiproliferative Activity
All synthesised compounds were evaluated for their antiproliferative activities in vitro against
six human cancer cell lines (A549, Hela, HepG-2, BEL7402, MCF-7 and HCT116) and compared with
those of DHEA and 5-fluorouracil (5-FU). Cells were allowed to proliferate in the presence of the tested
compounds for 48 h and the results are presented as the inhibition rate (Table 1) or IC50 values (Table 2).
Some of the synthesised compounds showed highly significant antiproliferative effects. Among the
compounds tested, compound 2n exhibited better bioactivities against HepG-2 in vitro than 5-FU.
Compound 2n exhibited potent activity against HepG-2, HCT116, and MCF-7, with IC50 values of 9.10,
31.04, and 9.18
µM, respectively. Compound 2h exhibited potent activity against A549, Hela, BEL7402,
HCT116, and MCF-7, with IC50 values of 17.46, 15.11, 15.96, 11.86, and 14.93 µM, respectively.
Table 1. The antiproliferative activity of compounds 2a–2w (% Growth Inhibition at 100 µM). DHEA:
Dehydroepiandrosterone, NA: antiproliferative activity <5%.
% Growth Inhibition at 100 µM
Compound
R
A549
Hela
HepG-2
BEL7402
HCT116
MCF-7
L02
DHEA
2a
2b
2
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
-
H
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
3,4-Cl2
2-Br
24.8
30.3
NA
60.5
68.8
NA
11.9
69.8
70.7
NA
NA
47.8
NA
NA
46.8
NA
63.1
7.6
15.7
NA
NA
55.2
78.9
32.8
9.2
62.8
68.7
17.0
20.5
30.7
NA
NA
17.3
12.7
64.6
NA
NA
14.9
NA
60.5
NA
44.1
10.9
10.2
NA
78.8
65.6
NA
51.7
32.5
48.2
NA
10.8
66.4
NA
NA
83.0
12.8
76.0
NA
NA
42.3
NA
59.1
12.4
48.3
46.5
12.9
NA
66.7
60.4
10.5
36.5
66.9
70.1
NA
37.6
NA
NA
14.1
NA
30.5
23.7
10.0
NA
38.4
NA
51.2
13.2
62.2
47.3
11.1
NA
78.3
84.9
23.0
41.9
78.7
74.4
20.2
42.0
NA
NA
7.6
65.5
40.9
76.5
14.3
NA
70.7
NA
59.2
10.2
75.0
17.1
NA
NA
31.2
83.0
37.3
41.0
39.9
80.0
NA
42.1
45.5
NA
NA
81.3
11.2
21.7
34.6
NA
58.1
NA
36.3
NA
41.3
44.6
22.3
NA
79.3
80.3
12.4
60.2
40.9
67.9
NA
43.8
11.1
NA
NA
34.2
40.1
67.1
NA
16.9
58.5
9.4
3-Br
4-Br
2-I
3-I
4-I
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
3,4-(OCH3)2
3,4,5-(OCH3)3
4-NO2
4-CF3
NA
44.0
NA
40.3
NA
75.2
2s
2t
2u
2v
2w
36.4
5.5
57.6

Molecules 2018, 23, 2243
4 of 14
Table 2. IC50 values (µM) of some active compounds.
IC50 Values (µM) a
Compound
R
A549
Hela
HepG-2
BEL7402
HCT116
MCF-7
L02
2c
2d
2g
2h
2k
2n
2p
2s
2w
5-FU
3-F
4-F
4-Cl
3,4-Cl2
4-Br
4-I
4-CH3
4-OCH3
4-CF3
51.64
15.80
37.38
17.46
>100
>100
56.72
>100
72.55
23.65
a
83.52
13.21
57.3
15.11
>100
>100
62.42
>100
>100
34.61
11.07
36.19
>100
>100
37.11
9.10
18.94
>100
>100
10.59
68.86
40.41
58.22
15.96
>100
>100
>100
>100
94.03
21.30
26.32
33.25
23.37
11.86
>100
31.04
44.07
56.91
22.65
24.80
>100
17.75
>100
14.93
>100
9.18
>100
69.4
>100
28.11
83.37
27.75
>100
26.36
>100
>100
62.23
81.08
93.69
19.12
IC50: concentration that inhibits 50% of cell growth.
2.3. Structure Activity Relationship Studies
Based on an overall comparison, the compounds derived from structures with
electron-withdrawing substituents on the 1,2,3-triazole ring exhibited potent activity, whereas
those with electron-donating substituents on the 1,2,3-triazole ring displayed no apparent activity
against the six cancer cell lines. For the 3-substituent compounds, only 3-F substitution exhibited
potent activity, and all 2-substituent compounds showed no significant activity. The special 3,4-Cl2
replacement exhibited potent activity.
Compound 2n displayed the highest activity against HepG-2 and MCF-7; however, in other
cancer cells, this compound exhibited lower activity and was not toxic in human normal liver (L02)
cells with good selectivity. Compound 2k displayed activity only against HepG-2, and for other cancer
cells, this compound exhibited weaker activity. Compound 2h displayed activity against all cancer
cells except for HepG-2.
2.4. Selective Inhibition of Cancer Cell Growth by Compounds 2c, 2d and 2n
Lack of selective cytotoxicity is the main factor that restricts the dose of most conventional
chemotherapeutic agents [22]. We compared the toxicities of compounds 2c, 2d, and 2n with 5-FU in
L02 cells. Selectivity indexes between cancer cells and L02 cells were calculated. As shown in Table 3,
compound 2n exhibited more than a 10.99-fold and more than a 10.89-fold higher selectivity for
HepG-2 and MCF-7 cells, respectively, than that for L02 cells; this selectivity displayed by compound
2n was significantly higher than that displayed by 5-FU.
Table 3. In vitro antiproliferative activities of compounds 2c
,
2d, and 2n against normal cell line (L02).
(IC50, µM)
Selectivity Index a
Compounds
R
L02
Hela
HepG-2
BEL7402
HCT116
MCF-7
A549
b
2c
2d
2n
3-F
4-F
4-I
-
83.37
27.75
>100
19.12
0.99
2.10
-
7.53
0.77
>10.99
1.80
1.21
0.69
-
3.17
0.83
>3.22
0.65
1.61
1.76
-
-
1.56
>10.89
1.47
5-FU
0.55
0.90
0.81
a
b
SI: selective index (IC50 on normal cells/IC50 on tumour cells). not calculated.
2.5. Compound 2n Induces HepG-2 Cell Cycle Arrest
Numerous cytotoxic compounds exert their antiproliferative effect by inducing cell cycle arrest
(at a particular cell cycle checkpoint), apoptosis, or both [23]. These mechanisms are considered to
be effective anticancer strategies [24]. In the present study, flow cytometry was used to determine

Molecules 2018, 23, 2243
5 of 14
whether compound 2n mediated the inhibition of growth and whether proliferation was associated
with apoptosis. HepG-2 cell lines were treated with compound 2n at concentrations of 5.0, 10.0, and
20.0
µM for 48 h, and the results are shown in Figure 2. The proportion of cells in the G2 phase
decreased from 11.47% (control) to 11.14% (5.0
µ
M), 0.41% (10.0 M), and 0.0% (20.0 M). The rate of
µ
µ
change in G2 phases showed a significant difference with the increasing concentration of compound
2n. This finding suggests that compound 2n significantly reduces the proportion of cells in the mitotic
G2 phase.
Figure 2. Flow cytometry analyses of cell cycle distribution of HepG-2 cancer cell after treatment of
compound 2n (5.0 Mm (B), 10.0 µM (C), 20.0 µM (D), and no treatment (Ctrl) (A) as a reference control
for 48 h).
2.6. Compound 2n Induces HepG-2 Cell Apoptosis
Cell apoptosis analysis was performed to determine whether compound 2n induced apoptosis
of cells. As shown in Figure 3, the percentage of total apoptotic cells (right quadrants)
increased from 13.65% (control) to 22.24% (5.0 µM), 27.24% (10.0 µM), and 47.86% (20.0 µM). This
suggests that compound 2n induced apoptosis and caused a marked increase in apoptosis in a
concentration-dependent manner.

Molecules 2018, 23, 2243
6 of 14
Figure 3. Apoptosis induction in HepG-2 cancer cell after treatment of compound 2n (5.0 µM (B),
10.0 µM (C), 20.0 Mm (D), and no treatment (Ctrl) (A) as a reference control for 48 h).
3. Experimental Section
3.1. General Information
Petroleum ether (PE), ethyl acetate (EA), ethanol (EtOH), and other reagents were obtained
commercially and were used without further purification. Solvents were dried according to standard
procedures. Reactions were monitored by thin-layer chromatography (TLC) on silica gel plates.
1H-NMR and 13C-NMR spectra were measured on an AV-300 (Bruker, Flawil, Switzerland), and all
chemical shifts were given in ppm relative to TMS. Mass spectra were measured on an HP1100LC
(Agilent Technologies, Palo Alto, CA, USA). High resolution mass spectra were measured on a
MALDI-TOF/TOF mass spectrometer (Bruker Daltonik, Bremen, Germany).
3.2. Chemistry
3.2.1. General Procedure for Preparation of 2a–2w
A mixture of dehydroepiandrosterone (DHEA) (2.0 mmol), aromatic aldehydes (2.1 mmol), and
KOH (2.0 mmol) in EtOH (20 mL) was heated under reflux for about 1 h. After completion of the
reaction as evident from TLC, the reaction solution was condensed under reduced pressure and the
solid residue was transferred to 15 mL of water with 15 mL EA. The EA layer was washed with
water (5 mL
×
3) and saline (5 mL
×
3) and dried using anhydrous MgSO4. The mixture was then
purified by normal-phase column chromatography (PE:EA = 5:2) to obtain the target compounds
2a–2w (Scheme 1).

Molecules 2018, 23, 2243
7 of 14
OH
O
Ν3
ΝΗ2
a
b
R
N
R
R
N
N
O
c
R
N
N
N
1a-1w
N
N
O
d
HO
N
2a-2w
R
HO
DHEA
Scheme 1. Reagents and conditions: (
) propargyl alcohol, CuSO4, sodium ascorbate, H2O: t-butanol=1:1, 24 h, rt; (
(d) KOH/C2H5OH, overnight, reflux.
a
) (i) NaNO2, HCl, 0 ◦C, 30 min; (ii) NaN3, H2O, 0 ◦C, 2–4 h;
) MnO2/EtOAc, 1 h, rt;
(
b
c
(E)-3-hydroxy-10,13-dimethyl-16-((1-phenyl-1H-1,2,3-triazol-4-yl)methylene)-1,3,4,7,8,9,10,11,12,13,15,16-
1
dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2a) Yield 90%. H-NMR (300 MHz, CDCl3)
δ 8.11
(s, 1H, triazole-H), 7.80 (s, 1H,-CO-C=CH), 7.77 (s, 1H, Ar-H), 7.58 (t, J = 6 Hz, 2H, Ar-H), 7.50 (t, J =
6 Hz, 2H, Ar-H), 5.44 (s, 1H, C6-H), 3.57 (s, 1H, -OH), 3.22 (dd, J1 = 15 MHz, J2 = 6 Hz, 1H, C3-H),
2.57–2.47 (m, 4H), 2.06–1.41 (m, 12H), 1.18–1.10 (m, 4H), 1.01 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ
213.80, 149.37, 146.34, 142.43, 141.38, 134.59, 133.81, 127.47, 125.33, 125.04, 124.98, 75.49, 55.05, 54.07,
52.36, 47.05, 41.95, 41.47, 36.27, 35.91, 35.67, 34.29, 25.09, 34.23, 19.05; ESI-HR MS (m/z): calcd. for
C28H34N3O2+ [M + H]+: 444.2646; found: 444.2651. Purity: 98.066% by HPLC (A: H2O; B: methanol,
graded: 60–100%), tR 15.873 min, λ: 292 nm.
(E)-16-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenan-thren-17(14H)-one (2b) Yield 98%. H-NMR (300 MHz, CDCl3)
δ
8.71 (s, 1H, triazole-H), 8.08 (s, 1H,-CO-C=CH), 7.86 (t, J = 6 Hz, 2H, Ar-H), 7.61–7.39 (m, 3H, Ar-H),
7.50 (t, J = 6 Hz, 2H, Ar-H), 5.53 (s, 1H, C6-H), 4,53 (s, 1H, -OH), 3.07 (dd, J1 = 15 Hz, J2 = 6 Hz, 1H,
C3-H), 2.42–2.17 (m, 4H), 1.90–1.36 (m, 12H), 1.04 (s, 4H), 0.94 (m, 3H); 13C-NMR (75 MHz, DMSO-d6)
δ
208.50, 143.89, 141.87, 137.99, 128.10, 128.05, 126.79, 126.05, 126.01, 120.47, 120.03, 117.74, 117.49, 70.43,
50.25, 48.97, 47.54, 42.69, 37.31, 36.77, 31.88, 31.63, 31.13, 30.78, 29.53, 20.47, 19.66, 14.43; ESI-HR MS
+
(m/z): calcd. for C28H33FN3O2 [M + H]+: 462.2551; found: 462.2558. Purity: 100% by HPLC (A: H2O;
B: methanol, graded: 80–100%), tR 5.820 min, λ: 292 nm.
(E)-16-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,12,13,
15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2c) Yield 98%. 1H-NMR (300 MHz,
DMSO-d6) δ 8.95 (s, 1H, triazole-H), 7.86 (t, J = 6 Hz, 1H, -CO-C=CH), 7.69–7.56 (m, 2H, Ar-H), 7.47
(t, J = 6 Hz, 2H, Ar-H), 7.34 (s, 1H, Ar-H), 5.33 (s, 1H, C6-H), 4.61 (d, J = 3 Hz, 1H, -OH), 4.08 (dd, J1
= 9 Hz, J2 = 6 Hz, 1H, C3-H), 3.17 (d, J = 3 Hz, 2H), 3.07–2.99 (m, 1H), 2.20–2.12 (m, 3H), 1.83–1.66
(m, 6H), 1.53–1.30 (m, 4H), 1.01 (s, 4H), 0.89 (s, 3H); 13C-NMR (75 MHz, DMSO-d6)
δ 213.37, 169.30,
149.37, 146.45, 142.61, 136.36, 128.27, 124.97, 120.86, 120.58, 120.27, 112.99, 112.64, 75.29, 55.03, 53.97,
52.30, 47.22, 41.98, 41.50, 36.40, 36.31, 35.95, 35.72, 34.26, 25.11, 24.30, 19.09; ESI-HR MS (m/z): calcd. for
+
C28H33FN3O2 [M + H]+: 462.2551; found: 462.2560. Purity: 100% by HPLC (A: H2O; B: methanol,
graded: 60–100%), tR 17.187 min, λ: 292 nm.

Molecules 2018, 23, 2243
8 of 14
(E)-16-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2d) Yield 96%. H-NMR (300 MHz, CDCl3)
δ
8.06 (s, 1H, triazole-H), 7.78–7.74 (m, 2H, Ar-H), 7.47 (s, 1H, Ar-H), 7.29 (d, J = 6 Hz, 2H, Ar-H), 5.44
(s, 1H, C6-H), 3.57 (s, 1H, -OH), 3.22 (dd, J1 = 18 Hz, J2 = 6 Hz, 1H, C3-H), 2.57–2.46 (m, 1H), 2.40–2.29
(m, 3H), 2.06–1.40 (m, 12H), 1.30–1.15 (m, 1H), 1.10 (m, 4H), 1.01 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ
209.32, 144.98, 141.09, 138.21, 122.69, 122.66, 122.57, 120.93, 119.56, 117.05, 116.74, 71.59, 50.37, 49.41,
47.71, 42.24, 37.17, 26.73, 31.59, 31.51, 31.21, 30.98, 29.70, 20.43, 19.47, 14.29; ESI-HR MS (m/z): calcd. for
+
C28H33FN3O2 [M + H]+: 462.2551; found: 462.2547. Purity: 97.271% by HPLC (A: H2O; B: methanol,
graded: 80–100%), tR 5.987 min, λ: 292 nm.
(E)-16-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-di-methyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenan-thren-17(14H)-one (2e) Yield 92%. H-NMR (300 MHz, CDCl3)
δ
8.14 (s, 1H, triazole-H), 7.68–1,62 (m, 2H, Ar-H), 7.54–7.47(m, 3H, Ar-H), 5.43 (s, 1H, C6-H), 3.55 (s,
1H, -OH), 3.22 (dd, J1 = 18 Hz, J2 = 6 Hz, 1H, C3-H), 2.57–2.46 (m, 1H), 2.39–2.24 (m, 3H), 2.06–1.99 (m,
1H), 1.92–1.74 (m, 4H), 1.64–1.56 (m, 5H), 1.51–1.41 (m, 3H), 1.14–1.10 (m, 3H), 1.01 (s, 3H); 13C-NMR
(75 MHz, CDCl3) δ 209.31, 144.08, 141.06, 138.04, 131.03, 130.88, 128.63, 128.06, 127.68, 126.55, 120.95,
119.75, 71.59, 50.38, 49.43, 47.72, 42.23, 37.17, 36.73, 31.58, 31.22, 30.97, 29.68, 20.43, 19.46, 14.29; ESI-HR
MS (m/z): calcd. for C28H33ClN3O2+ [M + H]+: 478.2256; found: 478.2663. Purity: 98.081% by HPLC
(A: H2O; B: methanol, graded: 80–100%), tR 4.933 min, λ: 292 nm.
(E)-16-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2f) Yield 90%. H-NMR (300 MHz, CDCl3)
δ
8.07 (s, 1H, triazole-H), 7.80 (s, 1H,-CO-C=CH), 7.68 (d, J = 3 Hz, 1H, Ar-H), 7.50–7.45 (m, 3H, Ar-H),
5.42 (s, 1H, C6-H), 3.55 (s, 1H, -OH), 3.20 (dd, J1 = 12 Hz, J2 = 3 Hz, 1H, C3-H), 2.53–2.47 (m, 1H),
2.37–2.25 (m, 3H), 2.04–1.98 (m, 2H), 1.90–1.82 (m, 4H), 1.75–1.70 (m, 2H), 1.52–1.44 (m, 4H), 1.15–1.08
(m, 4H), 0.99 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ 209.37, 145.06, 141.04, 137.45, 135.70, 130.98, 129.15,
122.39, 120.90, 120.78, 119.53, 119.50, 118.98, 71.56, 50.34, 49.38, 47.73, 42.20, 37.15, 36.70, 31.53, 31.20,
+
31.17, 30.97, 29.70, 20.42, 19.46, 14.29; ESI-HR MS (m/z): calcd. for C28H33ClN3O2 [M + H]+: 478.2256;
found: 478.2680. Purity: 98.406% by HPLC (A: H2O; B: methanol, graded: 60–100%), tR 19.453 min,
292 nm.
λ:
(E)-16-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2g) Yield 97%. H-NMR (300 MHz, CDCl3)
δ
8.09 (s, 1H, triazole-H), 7.75 (s, 1H,-CO-C=CH), 7.72 (s, 1H, Ar-H), 7.55 (d, J = 6 Hz, 2H, Ar-H), 7.46
(s, 1H, Ar-H), 5.43 (s, 1H, C6-H), 3.21 (dd, J1 = 15 Hz, J2 = 6 Hz, 1H, C3-H), 2.56–2.46 (m, 1H), 2.35–2.24
(m, 3H), 2.02–1.40 (m, 12H), 1.09 (m, 5H), 1.00 (s, 3H); 13C-NMR (75 MHz, CDCl3)
213.39, 149.39,
δ
146.44, 142.52, 140.09, 138.92, 134.66, 128.13, 126.65, 124.96, 75.31, 55.04, 53.99, 52.30, 47.20, 41.98, 41.50,
36.32, 35.94, 35.70, 34.25, 25.12, 24.29, 23.56, 19.09; ESI-HR MS (m/z): calcd. for C28H33ClN3O2+ [M +
H]+: 478.2256; found: 478.2563. Purity: 100% by HPLC (A: H2O; B: methanol, graded: 95–100%), tR
2.860 min, λ: 292 nm.
(E)-16-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,12,
13,15,16-dodecahydro-2H-cyclopenta[a]phenan-thren-17(14H)-one (2h) Yield 98%. 1H-NMR (300 MHz,
CDCl3)
δ 8.08 (s, 1H, triazole-H), 7.94 (s, 1H,-CO-C=CH), 7.66 (s, 2H, Ar-H), 7.45 (s, 1H, Ar-H), 5.44 (s,
1H, C6-H), 3.57 (s, 1H, -OH), 3.21 (dd, J1 = 15 Hz, J2 = 6 Hz, 1H, C3-H), 2.57–2.46 (m, 1H), 2.38–2.25
(m, 3H), 2.06–2.02 (m, 1H), 1.92–1.69 (m, 5H), 1.64–1.55 (m, 4H), 1.50–1.40 (m, 2H), 1.18–1.10 (m, 4H),
1.00 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ 209.19, 145.29, 141.08, 138.72, 135.65, 134.17, 133.30, 131.62,
122.29, 122.13, 120.91, 119.52, 119.17, 71.61, 50.38, 49.38, 47.72, 42.23, 37.17, 36.73, 31.59, 31.51, 31.22,
+
30.99, 29.71, 20.43, 19.46, 14.28; ESI-HR MS (m/z): calcd. for C28H32Cl2N3O2 [M + H]+: 512.1866;
found: 512.1859. Purity: 98.857% by HPLC (A: H2O; B: methanol, graded: 60–100%), tR 22.315 min,
292 nm.
λ:

Molecules 2018, 23, 2243
9 of 14
(E)-16-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenan-thren-17(14H)-one (2i) Yield 98%. H-NMR (300 MHz, CDCl3)
δ
8.27 (s, 1H, triazole-H), 7.80 (d, J = 6 Hz, 1H, -CO-C=CH), 7.61–7.45 (m, 4H, Ar-H), 5.43 (s, 1H, C6-H),
3.22 (dd, J1 = 15 Hz, J2 = 6 Hz, 1H, C3-H), 3.25–3.18 (m, 1H), 2.55–2,46 (m, 1H), 2.35–2.24 (m, 3H),
1.93–1.88 (m, 3H), 1.82–1.58 (m, 6H), 1.51–1.45 (m, 3H), 1.09 (m, 4H), 1.01 (m, 3H); 13C-NMR (75 MHz,
CDCl3)
δ 209.34, 143.94, 141.14, 137.96, 136.11, 134.01, 131.48, 128.66, 128.10, 126.71, 120.85, 119.87,
118.57, 71.47, 50.34, 49.38, 47.71, 42.19, 37.17, 36.72, 31.53, 31.18, 30.95, 29.66, 20.42, 19.48, 14.30; ESI-HR
MS (m/z): calcd. for C28H33BrN3O2+ [M + H]+: 522.1751; found: 522.1743. Purity: 99.701% by HPLC
(A: H2O; B: methanol, graded: 60–100%), tR 15.587 min, λ: 292 nm.
(E)-16-((1-(3-bromophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenan- thren-17(14H)-one (2j) Yield 98%. H-NMR (300 MHz, CDCl3)
δ
8.09 (s, 1H, triazole-H), 7.97 (d, J = 6 Hz, 1H, -CO-C=CH), 7.75 (dd, J1 = 6 Hz, J2 = 3 Hz, 1H, Ar-H),
7.63 (t, J = 3 Hz, 1H, Ar-H), 7.48–7.43 (m, 2H, Ar-H), 5.43 (s, 1H, C6-H), 3.57 (s, 1H, -OH), 3.22 (dd,
J1 = 18 Hz, J2 = 6 Hz, 1H, C3-H), 2.57–2.46 (m, 1H), 2.38–2.25 (m, 3H), 2.06–1.86 (m, 9H), 1.52–1.40
(m, 3H), 1.15–1.06 (m, 4H), 1.01 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ 209.31, 145.07, 141.06, 138.42,
137.55, 132.10, 131.22, 123.61, 123.42, 122.35, 120.91, 119.48, 119.10, 71.58, 50.35, 49.37, 47.72, 42.21, 37.16,
36.72, 31.56, 31.20, 30.98, 29.69, 20.42, 19.47, 14.29; ESI-HR MS (m/z): calcd. for C28H33BrN3O2+ [M +
H]+: 522.1751; found: 522.1740. Purity: 98.860% by HPLC (A: H2O; B: methanol, graded: 60–100%), tR
20.073 min, λ: 292 nm.
(E)-16-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-di-methyl-1,3,4,7,8,9,10,11,12,13,
15,16-dodecahydro-2H-cyclopenta[a]phenan- thren-17(14H)-one (2k) Yield 95%. 1H-NMR (300 MHz,
CDCl3)
Ar-H), 5.44 (s, 1H, C6-H), 3.60–3.52 (m, 1H), 3.25–3.17 (m, 1H, C3-H), 2.56–2.45 (m, 1H), 2.38–2.24
(m, 3H), 2.02–1.39 (m, 12H), 1.14–1.1.09 (m, 4H), 1.00 (s, 3H); 13C-NMR (75 MHz, CDCl3)
208.48,
δ 8.09 (s, 1H, triazole-H), 7.72–7.65 (m, 4H, Ar-H), 7.46–7.44 (dd, J1 = 6 Hz, J2 = 3 Hz, 1H,
δ
144.40, 141.96, 138.06, 135.98, 135.26, 124.45, 122.79, 122.18, 120.43, 120.12, 70.43, 50.24, 49.06, 48.93,
47.54, 42.70, 37.28, 36.77, 31.89, 31.60, 31.16, 30.87, 29.48, 19.67, 14.42; ESI-HR MS (m/z): calcd. for
+
C28H33BrN3O2 [M + H]+: 522.1751; found: 522.1743. Purity: 99.001% by HPLC (A: H2O; B: methanol,
graded: 60–100%), tR 19.640 min, λ: 292 nm.
(E)-3-hydroxy-16-((1-(2-iodophenyl)-1H-1,2,3-triazol-4-yl)methylene)-10,13-dim-ethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2l) Yield 91%. H-NMR (300 MHz, CDCl3)
δ
8.06 (s, 1H, triazole-H), 8.03 (d, J = 3 MHz, 1H, -CO-C=CH), 7.59 (m, 3H, Ar-H), 7.32 (s, 1H, Ar-H),
5.43 (s, 1H, C6-H), 3.56 (s, 1H, -OH), 3.23 (dd, J1 = 15 Hz, J2 = 6 Hz, 1H, C3-H), 2.56–2.43 (m, 1H),
2.35–2.24 (m, 3H), 2.03–1.73 (m, 5H), 1.56–1.42 (m, 4H), 1.18–1.10 (m, 4H), 1.01 (m, 3H); 13C-NMR
(75 MHz, CDCl3) δ 209.28, 144.05, 141.11, 140.35, 139.65, 137.98, 133.47, 131.92, 129.38, 127.36, 126.54,
124.42, 120.88, 119.82, 117.47, 71.51, 50.37, 49.81, 49.41, 48.82, 47.71, 42.21, 37.17, 36.72, 31.55, 31.51,
+
31.20, 30.96, 30.89, 29.69, 20.41, 19.45, 14.29; ESI-HR MS (m/z): calcd. for C28H33IN3O2 [M + H]+:
570.1612; found: 570.1595. Purity: 98.823% by HPLC (A: H2O; B: methanol, graded: 60–100%), tR
15.273 min, λ: 292 nm.
(E)-3-hydroxy-16-((1-(3-iodophenyl)-1H-1,2,3-triazol-4-yl)methylene)-10,13-dim-ethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2m) Yield 96%. H-NMR (300 MHz, CDCl3)
δ
8.14 (s, 1H, triazole-H), 8.08 (s, 1H,-CO-C=CH), 7.87–7.77 (m, 2H, Ar-H), 7.33 (s, 1H, Ar-H), 5.44 (s,
1H, C6-H), 3.58 (s, 1H, -OH), 3.21 (dd, J1 = 15 Hz, J2 = 6 Hz, 1H, C3-H), 2.56–2.47 (m, 1H), 2.34–2.25 (m,
3H), 2.08–1.74 (m, 7H), 1.54–1.27 (m, 5H), 1.10 (m, 4H), 1.01 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ
209.30, 145.06, 141.05, 138.40, 138.05, 137.41, 131.28, 129.31, 122.32, 120.94, 119.82, 119.49, 71.54, 50.44,
49.32, 47.67, 42.20, 37.23, 36.74, 31.58, 31.51, 31.28, 31.17, 31.06, 29.73, 20.45, 19.53, 14.36; ESI-HR MS
+
(m/z): calcd. for C28H33IN3O2 [M + H]+: 570.1612; found: 570.1604. Purity: 98.003% by HPLC (A:
H2O; B: methanol, graded: 60–100%), tR 20.673 min, λ: 292 nm.

Molecules 2018, 23, 2243
10 of 14
(E)-3-hydroxy-16-((1-(4-iodophenyl)-1H-1,2,3-triazol-4-yl)methylene)-10,13-dimethyl-1,3,4,7,8,9,10,11,12,13,
1
15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2n) Yield 92%. H-NMR (300 MHz, CDCl3)
δ
8.08 (s, 1H, triazole-H), 7.91 (d, J = 6 Hz, 1H, Ar-H), 7.53 (m, 3H, Ar-H), 5.44 (s, 1H, C6-H), 3.56 (s,
1H, -OH), 3.21 (dd, J1 = 15 Hz, J2 = 6 Hz, 1H, C3-H), 2.59–2.46 (m, 1H), 2.39–2.25 (m, 3H), 2.12–1.99
(m, 2H), 1.92–1.71 (m, 6H), 1.62–1.46 (m, 5H), 1.10 (s, 4H), 1.00 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ
209.32, 145.12, 141.05, 139.02, 138.39, 136.27, 122.18, 122.07, 120.96, 119.45, 94.04, 71.62, 50.37, 49.40,
47.72, 42.21, 37.17, 36.73, 31.57, 31.51, 31.21, 30.98, 29.71, 20.43, 19.41, 14.30; ESI-HR MS (m/z): calcd. for
+
C28H33IN3O2 [M + H]+: 570.1612; found: 570.1601. Purity: 100% by HPLC (A: H2O; B: methanol,
graded: 60–100%), tR 20.460 min, λ: 292 nm.
(E)-3-hydroxy-10,13-dimethyl-16-((1-m-tolyl-1H-1,2,3-triazol-4-yl)methylene)-1,3,4,7,8,9,10,11,12,13,15,16-
1
dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2o) Yield 90%. H-NMR (300 MHz, CDCl3)
δ 8.08
(s, 1H, triazole-H), 7.60 (s, 1H,-CO-C=CH), 7.55 (d, J = 6 Hz, 1H, Ar-H), 7.50–1.42 (m, 2H, Ar-H), 7.31 (s,
1H, Ar-H), 5.43 (s, 1H, C6-H), 3.57 (s, 1H, -OH), 3.22 (dd, J1 = 18 Hz, J2 = 6 Hz, 1H, C3-H), 2.56–2.47
(m, 4H), 2.35–2.25 (m, 3H), 2.06–1.87 (m, 5H), 1.83–1.74 (m, 3H), 1.61–1.40 (m, 8H), 1.13–1.06 (m, 4H),
1.01 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ 209.40, 144.72, 141.09, 140.16, 137.90, 136.59, 129.87, 129.65,
122.62, 121.24, 120.92, 119.89, 117.70, 71.56, 50.38, 49.42, 47.71, 42.23, 37.17, 36.72, 31.56, 31.53, 31.20,
30.98, 29.68, 21.43, 20.43, 19.46, 14.29; ESI-HR MS (m/z): calcd. for C29H36N3O2+ [M + H]+: 458.2802;
found: 458.2804. Purity: 100% by HPLC (A: H2O; B: methanol, graded: 680–100%), tR 6.500 min,
292 nm.
λ:
(E)-3-hydroxy-10,13-dimethyl-16-((1-p-tolyl-1H-1,2,3-triazol-4-yl)methylene)-1,3,4,7,8,9,10,11,12,13,15,16-
1
dodecahydro-2H-cyclopenta[a]phenanthren-17(14H)-one (2p) Yield 96%. H-NMR (300 MHz, CDCl3)
δ 8.06
(s, 1H, triazole-H), 7.66 (s, 1H,-CO-C=CH), 7.63 (s, 1H, Ar-H), 7.48 (s, 1H, Ar-H), 7.35 (t, J = 6 Hz, 2H,
Ar-H), 5.44 (s, 1H, C6-H), 3.57 (s, 1H, -OH), 3.22 (dd, J1 = 15 MHz, J2 = 6 Hz, 1H, C3-H), 2.56–2.46 (m,
4H), 2.41–2.25 (m, 3H), 2.06–1.44 (m, 12H), 1.14–1.10 (m, 4H), 1.00 (s, 3H); 13C-NMR (75 MHz, CDCl3)
δ 209.44, 144.69, 141.05, 139.33, 137.85, 134.34, 130.37, 122.57, 120.96, 120.52, 119.91, 71.60, 50.35, 49.40,
47.71, 42.20, 31.17, 36.72, 31.50, 31.19, 30.97, 29.68, 21.15, 20.43, 19.47, 14.30; ESI-HR MS (m/z): calcd. for
C29H36N3O2+ [M + H]+: 458.2802; found: 458.2811. Purity: 97.937% by HPLC (A: H2O; B: methanol,
graded: 60–100%), tR 18.167 min, λ: 292 nm.
(E)-3-hydroxy-16-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)-10,13-dimethyl-1,3,4,7,8,9,10,11,12,
13,15,16-dodecahydro-2H-cyclopenta[a]phenan-thren-17(14H)-one (2q) Yield 96%. 1H-NMR (300 MHz,
CDCl3)
δ 8.27 (s, 1H, triazole-H), 7.82 (d, J = 6 Hz, 1H,-CO-C=CH), 7.47 (t, J = 9 Hz, 2H, Ar-H), 7.15 (t, J
= 9 Hz, 2H, Ar-H), 5.44 (s, 1H, C6-H), 3.93 (s, 3H, -OCH3), 3.23 (dd, J1 = 18 Hz, J2 = 3 Hz, 1H, C3-H),
2.55–2.47 (m, 1H), 2.33–2.24 (m, 3H), 2.06–1.88 (m, 3H), 1.82–1.62 (m, 5H), 1.58–1.28 (m, 5H), 1.10–1.01
(m, 8H); 13C-NMR (75 MHz, DMSO-d6)
δ 209.55, 151.06, 143.74, 141.08, 137.31, 130.39, 126.93, 125.90,
125.31, 121.35, 120.98, 120.28, 112.36, 71.57, 56.07, 50.38, 49.47, 47.70, 42.23, 37.17, 36.73, 31.57, 31.21,
30.96, 29.66, 20.44, 19.46, 14.31; ESI-HR MS (m/z): calcd. for C29H36N3O3+ [M + H]+: 474.2751; found:
474.2762. Purity: 98.888% by HPLC (A: H2O; B: methanol, graded: 60–100%), tR 15.440 min, λ: 292 nm.
(E)-3-hydroxy-16-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)-10,13-dimethyl-1,3,4,7,8,9,10,11,12,
13,15,16-dodecahydro-2H-cyclopenta[a]phenan-thren-17(14H)-one (2r) Yield 95%. 1H-NMR (300 MHz,
CDCl3)
δ 8.09 (s, 1H, triazole-H), 7.49 (s, 1H,-CO-C=CH), 7.45 (d, J = 3 Hz, 1H, Ar-H), 7.38 (d, J = 3
Hz, 1H, Ar-H), 7.32 (s, 1H, Ar-H), 7.02 (dd, J1 = 9 Hz, J2 = 3 Hz, 1H, Ar-H), 5.45 (s, 1H, C6-H), 3.92
(s, 3H, -OCH3), 3.57 (s, 1H, -OH), 3.21 (dd, J1 = 15 MHz, J2 = 6 MHz, 1H, C3-H), 2.57–2.46 (m, 1H),
2.36–2.25 (m, 3H), 2.07–1.40 (m, 13H), 1.18–1.10 (m, 3H), 1.01 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ
209.35, 160.67, 144.79, 141.07, 138.03, 137.64, 130.67, 122.59, 120.94, 119.76, 114.76, 112.42, 106.56, 71.57,
55.67, 50.37, 49.48, 47.71, 42.23, 37.17, 36.72, 31.58, 31.51, 31.20, 30.97, 29.68, 20.43, 19.47, 14.30; ESI-HR
+
MS (m/z): calcd. for C29H36N3O3 [M + H]+: 474.2751; found: 474.2754. Purity: 100% by HPLC (A:
H2O; B: methanol, graded: 60–100%), tR 16.440 min, λ: 292 nm.

Molecules 2018, 23, 2243
11 of 14
(E)-3-hydroxy-16-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)-10,13-dimethyl-1,3,4,7,8,9,10,11,12,
13,15,16-dodecahydro-2H-cyclopenta[a]phenan-thren-17(14H)-one (2s) Yield 97%. 1H-NMR (300 MHz,
CDCl3)
δ 8.01 (s, 1H, triazole-H), 7.67 (s, 1H, -CO-C=CH), 7.64 (s, 1H, Ar-H), 7.47 (s, 1H, Ar-H), 7.05 (d,
J = 6 Hz, 2H, Ar-H), 5.42 (s, 1H, C6-H), 3.88 (s, 3H, -OCH3), 3.55 (s, 1H, -OH), 3.19 (dd, J1 = 18 Hz,
J2 = 6 Hz, 1H, C3-H), 2.54–2.23 (m, 4H), 2.05–1.44 (m, 12H), 1.08 (s, 4H), 0.99 (s, 3H); 13C-NMR (75
MHz, CDCl3)
δ 213.78, 164.71, 149.14, 146.34, 142.15, 134.76, 127.85, 126.97, 125.16, 125.03, 119.59, 75.46,
60.43, 55.05, 54.07, 52.35, 47.07, 41.96, 41.47, 36.29, 35.90, 35.67, 34.26, 25.06, 24.24, 19.06; ESI-HR MS
+
(m/z): calcd. for C29H36N3O3 [M + H]+: 474.2751; found: 474.2759. Purity: 100% by HPLC (A: H2O; B:
methanol, graded: 95–100%), tR 2.527 min, λ: 292 nm.
(E)-16-((1-(3,4-dimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)-3-hydroxy-10,13-dimethyl-1,3,4,7,8,9,10,11,
1
12,13,15,16-dodecahydro-2H-cyclopenta[a]phenan-thren-17(14H)-one (2t) Yield 93%. H-NMR (300 MHz,
CDCl3) δ 8.04 (s, 1H, triazole-H), 7.47 (s, 1H,-CO-C=CH), 7.39 (d, J = 3 Hz, 1H, Ar-H), 7.20 (dd, J1 = 9
Hz, J2 = 3 Hz, 1H, Ar-H), 7.00 (d, J = 3 Hz, 1H, Ar-H), 5.44 (s, 1H, C6-H), 3.99 (s, 3H, -OCH3), 3.97(s, 3H,
-OCH3), 3.21 (dd, J1 = 15 Hz, J2 = 6 Hz, 1H, C3-H), 2.56–2.25 (m, 5H), 2.05–1.98 (m, 2H), 1.91–1.74 (m,
5H), 1.62–1.44 (m, 4H), 1.17–1.00 (m, 8H); 13C-NMR (75 MHz, CDCl3)
δ 209.49, 149.89, 144.68, 141.08,
137.88, 130.19, 122.76, 120.94, 119.85, 112.55, 111.25, 105.06, 71.59, 56.27, 50.36, 49.41, 47.71, 42.21, 37.16,
36.72, 31.56, 31.20, 30.97, 29.67, 20.42, 19.46, 14.30; ESI-HR MS (m/z): calcd. for C30H38N3O4 [M + H]+:
+
504.2857; found: 504.2863. Purity: 98.680% by HPLC (A: H2O; B: methanol, graded: 95–100%), tR 2.427
min, λ: 292 nm.
(E)-3-hydroxy-10,13-dimethyl-16-((1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)-1,3,4,7,8,9,10,
1
11,12,13,15,16-dodecahydro-2H-cyclopenta[a]phenanthren17(14H)-one (2u) Yield, 90%. H-NMR (300 MHz,
CDCl3)
δ 8.05 (s, 1H, triazole-H), 7.45 (s, 1H,-CO-C=CH), 6.97 (s, 2H, Ar-H), 5.44 (s, 1H, C6-H), 3.96 (s,
6H, -OCH3), 3.91 (s, 3H, -OCH3), 3.24 (dd, J1 = 18 Hz, J2 = 3 Hz, 1H, C3-H), 2.57–2.47 (m, 1H), 2.35–2.23
(m, 3H), 2.05–1.99 (m, 1H), 1.90–1.83 (m, 1H), 1.78–1.63 (m, 5H), 1.49–1.39 (m, 3H), 1.09–1.00 (m, 8H);
13C-NMR (75 MHz, CDCl3)
δ 209.57, 153.96, 144.72, 141.12, 138.09, 132.47, 123.32, 122.93, 121.88, 121.61,
98.62, 71.55, 61.06, 56.49, 51.33, 49.56, 47.70, 42.20, 37.15, 36.70, 31.55, 31.17, 30.95, 29.68, 20.39, 19.45,
+
14.28; ESI-HR MS (m/z): calcd. for C31H40N3O5 [M + H]+: 534.2962; found: 534.2955. Purity: 100% by
HPLC (A: H2O; B: methanol, graded: 80–100%), tR 15.187 min, λ: 292 nm.
(E)-3-hydroxy-10,13-dimethyl-16-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methylene)1,3,4,7,8,9,10,11,12,13,
15,16-dodecahydro-2H-cyclopenta[
DMSO-d6) 9.19 (s, 1H, triazole-H), 8.46 (s, 1H,-CO-C=CH), 8.43 (s, 1H, Ar-H), 8.25 (d, J = 6 Hz, 2H,
α
]phenan- thren-17(14H)-one (2v) Yield 95%. 1H-NMR (300 MHz,
δ
Ar-H), 7.24 (s, 1H, Ar-H), 5.31 (s, 1H, C6-H), 4.60 (s, 1H, -OH), 2.99 (dd, J1 = 12 Hz, J2 = 3 Hz, 1H,
C3-H), 2.42–2.15 (m, 4H), 1.77–1.66 (m, 7H), 1.49–1.34 (m, 4H), 0.98 (m, 5H), 0.86 (s, 3H); 13C-NMR (75
MHz, DMSO-d6) δ 208.29, 147.28, 144.76, 141.96, 140.95, 138.44, 125.94, 124.55, 121.26, 120.32, 119.74,
70.40, 50.24, 48.90, 47.50, 42.68, 40.30, 40.02, 39.74, 39.47, 39.19, 37.29.36.75, 31.86, 31.58, 31.14, 29.48,
+
20.42, 19.63, 14.37; ESI-HR MS (m/z): calcd. for C28H33N4O4 [M + H]+: 489.2496; found: 489.2503.
Purity: 98.048% by HPLC (A: H2O; B: methanol, graded: 80–100%), tR 5.413 min, λ: 292 nm.
(E)-3-hydroxy-10,13-dimethyl-16-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol4-y)methylene)-1,3,4,7,8,9,10,
11,12,13,15,16-dodecahydro-2H-cyclopenta[
MHz, CDCl3) 8.13 (s, 1H, triazole-H), 7.94 (s, 1H,-CO-C=CH), 7.92 (s, 1H, Ar-H), 7.84 (t, J = 3 Hz, 2H,
α
] phenanthren-17(14H)-one (2w) Yield 93%. 1H-NMR (300
δ
Ar-H), 7.45 (s, 1H, Ar-H), 5.42 (s, 1H, C6-H), 3.55 (s, 1H, -OH), 3.22 (dd, J1 = 9 Hz, J2 = 3 Hz, 1H, C3-H),
2.51 (t, J = 9 Hz, 2H, Ar-H), 2.36–2.25 (m, 3H), 2.01–1.69 (m, 7H), 1.52–1.44 (m, 3H), 1.15–1.03 (m, 4H),
0.99 (m, 3H), 0.86 (s, 2H); 13C-NMR (75 MHz, CDCl3)
δ 209.22, 145.32, 141.08, 139.04, 138.74, 131.29,
127.28, 127.23, 122.21, 120.92, 120.54, 119.16, 71.60, 50.36, 49.38, 47.73, 42.23, 37.17, 36.73, 31.59, 31.50,
+
31.22, 30.98, 29.73, 20.43, 19.47, 14.29; ESI-HR MS (m/z): calcd. for C29H33F3N3O2 [M + H]+: 512.2519;
found: 512.2509. Purity: 99.465% by HPLC (A: H2O; B: methanol, graded: 60–100%), tR 19.600 min,
292 nm.
λ:

Molecules 2018, 23, 2243
12 of 14
3.3. Biological Evaluation
3.3.1. Cell Cultures and Antiproliferative Assays
The antiproliferative activity of the title compounds against the panel of six different human
cancer cell lines, viz. lung (A549), cervix (HeLa), liver (HepG-2), liver (BEL7402), colorectal (HCT116),
and breast (MCF-7) cell lines, was evaluated using a standard MTT-based colorimetric assay. All cell
lines were obtained from the Key Laboratory of Natural Resources and Functional Molecules of the
Changbai Mountain (Yanbian University) and maintained in Dulbecco’s modified Eagle’s medium
(DMEM) and RPMI Media 1640 (RPMI1640), supplemented with 10% foetal bovine serum (FBS) at
37 ◦C in a humidified atmosphere containing 5% CO2.
Cells were plated in 96-well plates at appropriate densities to ensure exponential growth
throughout the experimental period (9
h. Cells were then treated for 48 h with each compound. After 48 h of incubation, 10
solution were added to each well to a final concentration of 2 mg
mL−1. Plates were then incubated
for a further 4 h. After incubation, the MTT solution was removed and 150 L of DMSO were added to
each well for coloration. The plates were shaken vigorously for 10 min at room temperature to ensure
complete solubilisation. The optometric density (OD) was read on a microplate reader (EL 800,
×
103 cells per well), and then allowed to adhere for 24
µL of MTT
·
µ
×
BioTek, Highland Park, Winooski, VT, USA) at 492 nm, and the data were subsequently analysed. The
percentage of cell growth inhibition was calculated from the following equation:
Inhibitory rate (%) = [1 − (ODtreated − ODblank)/(ODcontrol − ODblank)] × 100.
To obtain the antiproliferative activity of compounds 2a–2w, the compounds were selected in the
same way with four serial concentrations (1, 10, 50 and 100
density (OD) reading was then used to calculate the IC50.
µM) of those compounds. The optometric
3.3.2. Analysis for Cell Cycle by Flow Cytometry
◦
HepG-2 cells were plated in 96-well plates (5.0
×
105 cells per well) and incubated at 37 C for 12 h.
Exponentially growing cells were then incubated with compound 2n at different concentrations (5, 10
and 20 M). After 48 h, untreated cells (control) or cells treated with compound 2n were centrifuged at
1000 rpm (177 g) for 10 min, and then fixed in 70% ethanol at
20 ◦C for at least 24 h. The cells were
subsequently resuspended in phosphate-buffered saline (PBS) containing 0.1mg mL−1 RNase A and
mL−1 propidium iodide (PI). The cellular DNA content for the cell cycle distribution analysis
was measured by flow cytometry using a FACS Calibur flow cytometer with Cell Quest software
(Becton-Dickinson, Franklin Lakes, NJ, USA), plotting at least 30,000 events per sample. The percentage
of cells in the G1, S and G2 phases of the cell cycle were determined using the ModFit LT V4.0 software
package (Verity Software, Topsham, ME, USA).
µ
−
5
µg
·
3.3.3. Analysis for Apoptosis by Flow Cytometry
Apoptosis was detected using an Apoptosis Detection Kit (Invitrogen, Eugene, OR, USA). In brief,
cells were cultured in 96-well plates (5.0
with exponential growth were then incubated with compound 2n at different concentrations (5, 10 and
20 M). Following 48 h of incubation, the cells were collected, washed twice with PBS and once with
1binding buffer, and then stained with 5
×
105 cells per well) and incubated at 37 ◦C for 12 h. Cells
µ
µ
M of annexin V-FITC and 2.5
µ
M of PI (5 mg
·
mL−1) in 1
×
binding buffer for 30 min at 20 ◦C in the dark. Apoptotic cells were enumerated using a FACSCalibur
flow cytometer with Cell Quest software (Becton–Dickinso, Franklin Lakes, NJ, USA).
4. Conclusions
In summary, we designed and synthesised one series of DHEA derivatives (compounds 2a
–2w)
and evaluated their antiproliferative effects against six cancer cell lines (A549, Hela, HepG-2, BEL7402,

Molecules 2018, 23, 2243
13 of 14
MCF-7, and HCT116). Several of the target compounds exhibited potent inhibitory activity in vitro
and the antiproliferative activity of these compounds was screened via the MTT assay. In particular,
compound 2n exhibited excellent inhibitory activity against HepG-2 cells, with an IC50 value of
9.10 µM. Moreover, it showed high inhibitory activities against MCF-7, with IC50 values of 9.18 µM.
The results of experiments on cell cycle arrest and apoptosis induced by compound 2n suggested that
it induced apoptosis in HepG-2 cells. Therefore, the modifications to the C16 position of DHEA in the
present study were helpful in improving its antiproliferative activity.
Supplementary Materials: The Supplementary materials are available online, 1H and 13C-NMR spectra of these
compounds are available in the supplementary materials.
Author Contributions: The authors X.H., Q.-K.S., H.-J.Z., J.-L.L., Y.-S.T., and Z.-S.Q. designed and accomplished
research. Also, they analyzed data and wrote the paper together. Finally, all authors read and approved the
final manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (No. 21662036).
Conflicts of Interest: We declare that we have no conflict of interest with respect to this study.
References
1.
2.
3.
Mahapatra, D.K.; Bharti, S.K.; Asat, V. Anti-cancer chalcones: Structural and molecular target perspectives.
Eur. J. Med. Chem. 2015, 98, 69–114. [CrossRef] [PubMed]
Atul, G.B.; Sathish, K.; Arvind, S.N. Current status on development of steroids as anticancer agents. J. Steroid
Biochem. Mol. Biol. 2013, 137, 242–270.
Huang, X.C.; Jin, L.; Wang, M.; Liang, D.; Chen, Z.F.; Zhang, Y.; Pan, Y.M.; Wang, H.S. Design, synthesis
and in vitro evaluation of novel dehydroabietic acid derivatives containing a dipeptide moiety as potential
anticancer agents. Eur. J. Med. Chem. 2015, 89, 370–385. [CrossRef] [PubMed]
4.
5.
6.
7.
Cragg, G.M.; Newman, D.J. Nature: A vital source of leads for anticancer drug development. Phytochem. Rev.
2009, 8, 313–331. [CrossRef]
Pearce, S.T.; Jordan, V.C. The biological role of estrogen receptors alpha and beta in cancer. Crit. Rev. Oncol.
Hematol. 2004, 50, 3–22. [CrossRef] [PubMed]
Nishiyama, T. Androgen deprivation therapy in combination with radiotherapy for high-risk clinically
localized prostate cancer. J. Steroid Bioch. Mol. Biol. 2012, 129, 179–190. [CrossRef] [PubMed]
Minorics, R.; Bózsity, N.; Wölfling, J.; Mernyák, E.; Schneider, G.; Márki, A.; Falkay, G.; Ocsovszki, I.;
Zupkó, I. Antiproliferative effect of normal and 13-epi-dhomoestrone and their 3-methyl ethers on human
reproductive cancer cell lines. J. Steroid Bioch. Mol. Biol. 2012, 132, 168–175. [CrossRef] [PubMed]
Colín-Lozano, B.; León-Rivera, I.; Chan-Bacab, M.J.; Ortega-Morales, B.O.; Moo-Puc, R.; López-Guerrero, V.;
Hernández-Núñez, E.; Argüello-Garcia, R.; Scior, T.; Barbosa-Cabrera, E.; et al. Synthesis, in vitro and in vivo
giardicidal activity of nitrothiazole-NSAID chimeras displaying broad antiprotozoal spectrum. Bioorg. Med.
Chem. Lett. 2017, 27, 3490–3494. [CrossRef] [PubMed]
Nava-Zuazo, C.; Estrada-Soto, S.; Guerrero-Alvarez, J.; León-Rivera, I.; Molina-Salinas, G.M.;
Said-Fernández, S.; Chan-Bacab, M.J.; Cedillo-Rivera, R.; Moo-Puc, R.; Mirón-López, G.; et al.
Design, synthesis, and in vitro antiprotozoal, antimycobacterial activities of N-{2-[(7-chloroquinolin-4-yl)
amino]ethyl}ureas. Bioorg. Med. Chem. 2010, 18, 6398–6403. [CrossRef] [PubMed]
8.
9.
10. Viegas-Junior, C.; Danuello, A.; da Silva Bolzani, V.; Barreiro, E.J.; Fraga, C.A.M. Molecular hybridization:
A useful tool in the design of new drug prototypes. Curr. Med. Chem. 2007, 14, 1829–1852. [CrossRef]
[PubMed]
11. Kikuchi, A.; Furutani, T.; Azami, H.; Watanabe, K.; Niimi, T.; Kamiyama, Y.; Kuromitsu, S.; Baskin-Bey, E.;
Heeringa, M.; Ouatas, T.; et al. In vitro and in vivo characterisation of ASP9521: A novel, selective, orally
bioavailable inhibitor of 17b-hydroxysteroid dehydrogenase type 5 (17bHSD5; AKR1C3). Invest New Drugs.
2014, 32, 860–870. [CrossRef] [PubMed]
12. Liu, X.K.; Ye, B.J.; Wu, Y.; Nan, J.X.; Lin, Z.H.; Piao, H.R. Synthesis and antitumor activity of
dehydroepiandrosterone derivatives on AS-2, A549, and HepG2 cells in vitro. Chem. Bio. Drug Des. 2012, 79,
523–529. [CrossRef] [PubMed]

Molecules 2018, 23, 2243
14 of 14
13. Elmegeed, G.A.; Khalil, W.K.B.; Mohareb, R.M.; Ahmed, H.H.; Abd-Elhalim, M.M.; Elsayed, G.H.
Cytotoxicity and gene expression profiles of novel synthesized steroid derivatives as chemotherapeutic
anti-breast cancer agents. Bioorg. Med. Chem. 2011, 19, 6860–6872. [CrossRef] [PubMed]
14. Shen, Q.K.; Liu, C.F.; Zhang, H.J.; Tian, Y.S.; Quan, Z.S. Design and synthesis of new triazoles linked to
xanthotoxin for potent and highly selective anti-gastric cancer agents. Bioorg. Med. Chem. Lett. 2017, 27,
4871–4875. [CrossRef] [PubMed]
15. Rashid, S.; Dar, B.A.; Majeed, R.; Hamid, A.; Bhat, B.A. Synthesis and biological evaluation of ursolic
acid-triazolyl derivatives as potential anti-cancer agents. Eur. J. Med. Chem. 2013, 66, 238–245. [CrossRef]
[PubMed]
16. Dar, B.A.; Lone, A.M.; Shah, W.A.; Qurishi, M.A. Synthesis and screening of ursolic acid-benzylidine
derivatives as potential anti-cancer agents. Eur. J. Med. Chem. 2016, 111, 26–32. [CrossRef] [PubMed]
17. Mohsen, V.; Hoda, Y.; Kouros, D.; Shamsa, H.; Kheirollahi, A.; Safavi, M.; Ardestani, S.K.; Sadeghi-Neshat, S.;
Mohammadhosseini, N.; Edraki, N.; et al. Synthesis and In vitro cytotoxic activity evaluation of
(E)-16-(substituted benzylidene) derivatives of dehydroepiandrosterone. J. Pharm. Sci. 2013, 21, 21–34.
18. Dai, C.Z.; Chen, Y.F.; Zhang, M.; Li, S.K.; Yang, T.T.; Shen, L.; Wang, J.X.; Qian, S.S.; Zhu, H.L.; Ye, Y.H.
Synthesis and antifungal activity of 1,2,3-triazole phenylhydrazone derivatives. Org. Biomol. Chem. 2015, 13,
477–486. [CrossRef] [PubMed]
19. Gonzaga, D.; Senger, M.R.; Silva, F.C.; Ferreira, V.F.; Silva, F.P., Jr. 1-Phenyl-1H-2-phenyl-2H-1,2,3-triazol
derivatives: Design, synthesis and inhibitory effect on alpha-glycosidases. Eur. J. Med. Chem. 2014, 74,
461–476. [CrossRef] [PubMed]
20. Boechat, N.; Ferreira, V.F.; Ferreira, S.B.; Ferreira, M.L.G.; Silva, F.C.; Bastos, M.M.; Costa, M.S.;
Lourenco, M.C.S.; Pinto, A.C.; Krettli, A.U.; et al. Novel 1,2,3-Triazole Derivatives for Use against
Mycobacterium tuberculosis H37Rv (ATCC 27294) Strain. J. Med. Chem. 2011, 54, 5988–5999. [CrossRef]
[PubMed]
21. Costa, M.S.; Boechat, N.; Rangel, E.A.C. Synthesis, tuberculosis inhibitory activity, and SAR study of
N-substituted-phenyl-1,2,3-triazole derivatives. Bioorg. Med. Chem. 2006, 14, 8644–8653. [CrossRef]
[PubMed]
22. Zhou, H.Y.; Dong, F.Q.; Du, X.L.; Zhou, Z.K.; Huo, H.R.; Wang, W.H.; Zhan, H.D.; Dai, Y.F.; Jing, M.; Sui, Y.P.;
et al. Antitumor activities of biscoumarin and dihydropyran derivatives. Bioorg. Med. Chem. Lett. 2016, 26,
3876–3880. [CrossRef] [PubMed]
23. Hileman, E.O.; Liu, J.; Albitar, M.; Keating, M.J.; Huang, P. Intrinsic oxidative stress in cancer cells:
A biochemical basis for therapeutic selectivity. Cancer Chemother Pharmacol. 2004, 53, 209–219. [CrossRef]
[PubMed]
24. Luo, G.S.; Muyaba, M.; Lyu, W.; Tang, Z.C.; Zhao, R.H.; Xu, Q.; You, Q.D.; Xiang, H. Design, synthesis and
biological evaluation of novel 3-substituted 4-anilino-coumarin derivatives as antitumor agents. Bioorg. Med.
Chem. Lett. 2017, 27, 867–874. [CrossRef] [PubMed]
Sample Availability: Not available.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).