Synthesis and bioevaluation of novel steroidal isatin conjugates derived from epiandrosterone/androsterone

Article abstract of DOI:10.1080/14756366.2019.1659790

Steroids are classes of natural products widely distributed in nature, which have been demonstrated to exhibit broad biological functions, and have also attracted increasing interest from bioorganic and pharmaceutical researches. In order to develop novel chemical entities as potential cytotoxic agents, a series of steroidal isatin conjugations derived from epiandrosterone and androsterone were efficiently prepared and characterized, and all these obtained compounds were screened for their potential cytotoxic activities. The preliminary bioassay indicated that most of the newly synthesized compounds exhibited good cytotoxic activities against human gastric cancer (SGC-7901), melanoma (A875), and hepatocellular liver carcinoma (HepG2) cell lines compared with 5-fluorouracil (5-FU), which might be considered as promising scaffold for further development of potential anticancer agents.

Full text of DOI:10.1080/14756366.2019.1659790

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and bioevaluation of novel steroidal
isatin conjugates derived from epiandrosterone/
androsterone
Shaoyong Ke, Zhigang Zhang, Manli Liu, Wei Fang, Daye Huang, Zhongyi
Wan, Ronghua Zhou, Kaimei Wang & Liqiao Shi
To cite this article: Shaoyong Ke, Zhigang Zhang, Manli Liu, Wei Fang, Daye Huang, Zhongyi
Wan, Ronghua Zhou, Kaimei Wang & Liqiao Shi (2019) Synthesis and bioevaluation of novel
steroidal isatin conjugates derived from epiandrosterone/androsterone, Journal of Enzyme
Inhibition and Medicinal Chemistry, 34:1, 1607-1614, DOI: 10.1080/14756366.2019.1659790
To link to this article: https://doi.org/10.1080/14756366.2019.1659790
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Group.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1607–1614
https://doi.org/10.1080/14756366.2019.1659790
RESEARCH PAPER
Synthesis and bioevaluation of novel steroidal isatin conjugates derived from
epiandrosterone/androsterone
Shaoyong Ke, Zhigang Zhang, Manli Liu, Wei Fang, Daye Huang, Zhongyi Wan, Ronghua Zhou, Kaimei Wang and
Liqiao Shi
National Biopesticide Engineering Research Centre, Hubei Biopesticide Engineering Research Centre, Hubei Academy of Agricultural Sciences,
Wuhan, China
ABSTRACT
ARTICLE HISTORY
Received 10 June 2019
Revised 5 August 2019
Accepted 16 August 2019
Steroids are classes of natural products widely distributed in nature, which have been demonstrated to
exhibit broad biological functions, and have also attracted increasing interest from bioorganic and
pharmaceutical researches. In order to develop novel chemical entities as potential cytotoxic agents, a ser-
ies of steroidal isatin conjugations derived from epiandrosterone and androsterone were efficiently pre-
pared and characterized, and all these obtained compounds were screened for their potential cytotoxic
activities. The preliminary bioassay indicated that most of the newly synthesized compounds exhibited
good cytotoxic activities against human gastric cancer (SGC-7901), melanoma (A875), and hepatocellular
liver carcinoma (HepG2) cell lines compared with 5-fluorouracil (5-FU), which might be considered as
promising scaffold for further development of potential anticancer agents.
KEYWORDS
Epiandrosterone; androster-
one; isatin; conjugate;
synthesis; cytotoxic activity
Introduction
explore the potential structure-activity relationship, we plan to
replace the dehydroepiandrosterone unit with epiandrosterone
and androsterone to construct two series of novel isatin deriva-
tives derived from epiandrosterone/androsterone as shown in
Figure 1, wishing to identify novel functional molecules with
potent antiproliferative effects on tumor cells. The results may
provide useful information for design of novel chemotherapeu-
tic drugs.
We utilized epiandrosterone/androsterone as a basic scaffold
and planned for the hybridization of the active pharmacophore to
the core structure as shown in Figure 1. Therefore, a series of
novel steroid-isatin conjugates 3a–h and 6a–h were designed and
synthesized as shown in Scheme 1, and their potential inhibition
on three cell lines (SGC-7901, A875, HepG2) were evaluated by
MTT (3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium brom-
ide) colorimetric method, and the possible structure-activity rela-
tionships have also been summarized and discussed.
Cancer is the second cause of death in the world, which denotes
a real crisis for public health in the worldwide with the ecological
changes and environmental deterioration1–3
. Although many
efforts have been made to control the cancer in the past decades,
the outlook is still not optimistic. Among all treatment methods,
chemotherapy is still the most common options for cancer
patients2,4–6. Therefore, searching for and developing neotype
chemical entities with special structure features as potential anti-
cancer agents are an important challenge in the field of
drug discovery.
Natural products play important roles in the development of
novel drugs7–11 because of their unique structures and broad bio-
activities, which always offer chemists a range of uncharted che-
motypes for the discovery of novel drugs. Steroids are classes of
natural products widely distributed in plants, beverages, well-
cooked foods and tobacco smoke and with special four-ring struc-
tural features, which have been demonstrated to exhibit broad
biological functions, and have also attracted increasing interest
from bioorganic and pharmaceutical researches12–14. On the other
hand, isatin is also a natural indole alkaloid consisted in a number
of plants, which has also been found to be a common scaffold in
Results and discussion
Synthesis
In the present study, a series of novel steroidal derivatives derived
from epiandrosterone and androsterone were efficiently prepared
and characterized using a similar method in the reference20. The
general synthetic route for the synthesis of these steroidal deriva-
tives 3a–h and 6a–h is outlined in Scheme 1.
various drugs, agrochemicals, and dyes15–18
.
Recently, during the course of our research for highly active
compounds based on the scaffold of natural steroids19–23, a series
of isatin-dehydroepiandrosterone conjugates have been demon-
strated to exhibit significant inhibition on the proliferation of
According to the above aforementioned, in order to explore
human tumor cells20, which suggest that this natural four-ring the possible structure-activity relationship, the available diasterom-
scaffold might contribute to the cytotoxic activity. In order to ers (epiandrosterone and androsterone) were selected to construct
CONTACT Shaoyong Ke
keshaoyong@163.com; Liqiao Shi
liqiao.shi@nberc.com
National Biopesticide Engineering Research Centre, Hubei Biopesticide
Engineering Research Centre, Hubei Academy of Agricultural Sciences, Wuhan, 430064, China
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1608
S. KE ET AL.
Figure 1. Design strategy of novel steroid-isatin conjugates.
Scheme 1. Synthesis of novel steroid-isatin conjugates. Reagents and conditions: (a) NH2NH2ꢀH2O, NH2NH2ꢀH2SO4, EtOH/H2O, rt; (b) Substituted isatin, EtOH, 30–45 ꢁC.
two series of novel steroidal isatin derivatives (3a–h and 6a–h). derivatives revealed a distinct molecular peak in positive ion
Firstly, the corresponding epiandrosterone or androsterone was mode, the value of [M þ H]þ absorption signal was consistent with
treated with hydrazine hydrate under the catalytic condition of the calculated value of the molecular weight.
hydrazine sulfate to obtain the intermediates 2 and 5. Then the
compounds 2 or 5 was condensed with various substituted isatin
Cytotoxic activity
to the target steroidal isatin conjugates 3a–h or 6a–h. All the tar-
get derivatives were characterized by 1H NMR, 13C NMR, and ESI-
The in vitro cytotoxic activity of target derivatives 3a–h and 6a–h
All obtained compounds 3a–h and 6a–h were screened for their
MS spectroscopic analyses.
in vitro cytotoxic activity against SGC-7901 (Human gastric cancer),
A875 (human melanoma), and HepG2 (human hepatocellular liver
Spectroscopy
carcinoma) cell lines using the standard MTT assay24–26 compared
All the structures of target derivatives 3a–h and 6a–h were con-
firmed by their 1H NMR, 13C NMR, and ESI-MS analyses. For 1H
NMR spectrum of all epiandrosterone or androsterone-isatin deriv-
atives 3a–h and 6a–h, the signals at 11.50–10.67 ppm were attrib-
uted to NH protons as indicated in molecular structures, and the
signals at lower fields in the range of 8.65–6.70 ppm were
with 5-FU (5-Fluorouracil). The preliminary cytotoxic screening
results were summarized in Figure 3.
According to the results shown in Figure 3, most of these ster-
oidal isatin derivatives 3a–h and 6a–h present good cytotoxic
activities (75–85%) against the tested cell lines compared with the
control at the concentration of 20 mg/mL. Notably, the androster-
one-isatin conjugates 6a–h exhibited better inhibitory activities
against all tested cell lines than that of the parent compound
androsterone. Meanwhile, the epiandrosterone-isatin hybrids 3a–h
also indicated better inhibitory actiities than that of the parent
epiandrosterone especially for SGC7901 and A875 cell lines. The
preliminary results demonstrated that the strategy combined ster-
oid and isatin was effective, and these novel steroidal isatin
hybrids might be used as a potential active scaffold for optimiza-
tion of cytotoxic agents.
1
assigned to the aromatic protons of isatin unit. All H NMR spectra
of target derivatives indicated distinctive signals for protons of
hydroxyl group in the part of steroidal ring were resonated as a
singlet between d 4.46–4.18 ppm, and the signals of methine pro-
ton attached to hydroxyl group were indicated at about
1
3.30–3.80 ppm. The several set of signals that appeared in their H
NMR spectra at higher fields were attributed to the other protons
of steroidal scaffold. All the characteristic peaks observed within
1
the H NMR spectra for title compounds are given in Experimental
Section, and the representative 1H NMR spectra analysis of target
In addition, the IC50 values for all derivatives were also eval-
uated, and the cytotoxic activities expressed as IC50 values are
compound 3c is shown in Figure 2.
For the 13C NMR spectrum, the signals at 180.21–109.04 ppm presented in the following Table 1. From these data, we can find
were assigned to the carbon of the aromatic ring, and the other most of the derivatives exhibited higher inhibition activity than
peaks appearing at the range of 84.87–11.60 ppm were the signals the commercial 5-FU, and the activity of epiandrosterone-isatin
of steroidal carbons. All mass spectra were obtained on a Waters derivatives 3a–h are slightly better than that of androsterone-isa-
R
R
ACQUITY UPLCV H-CLASS PDA (WatersV) instrument. According to tin hybrids 6a–h. Especially, the compounds with 5-I group have
the experiments displayed, the ESI-MS spectrum of the target poor activities (Entries 5 and 13), and the two parent compounds

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1609
1
Figure 2. Representative H NMR spectra analysis of compound 3c.
Figure 3. Antitumor activities of compounds 3a–h and 6a–h at 20 mg/mL. SGC-7901: Human gastric cancer cell line; A875: Human melanoma cell line; HepG2: Human
hepatocellular liver carcinoma cell line; 5-FU: 5-Fluorouracil, used as a positive control.
epiandrosterone (Entry 17) and androsterone (Entry 18) also indi- some selective cytotoxicity between cancer cells (IC50
cated poor cytotoxic activity at the same conditions. These results 3.85–6.40 lM) and normal cells (HL-7702, IC50 ¼ 11.23 lM). In add-
can further confirm the cytotoxic activity of these steroidal isatin ition, this class of steroidal isatin derivatives 3a–h and 6a–h exhib-
derivatives are highly potential scaffold to discovery of potential ited slightly better inhibitory activities than that of the steroidal
anti-cancer agents. Meanwhile, the selectivity of the target deriva- derivatives derived from dehydroepiandrosterone19,20
,
which
tives on tumor cell lines and normal cells have been investigated
and the non-cancer cells HL-7702 have been used in our experi-
ment, and the results in Table 1 indicated that most of the
deserved to carry out further structural optimization and in-
depth study.
The Figure 4 described the dose-response curve of cell growth
obtained compounds exhibited cytotoxic activity against HL-7702 inhibition activities for high potential compounds 3b, 3c, 6c and
cell lines, and the cytotoxic activities of compound 6g exhibited 6g, which indicated that these steroidal isatin derivatives

1610
S. KE ET AL.
Table 1. Cytotoxic activity of the steroid-isatin hybrids.
a
In vitro cytotoxicity IC50 (lM)
Entry
Compd. No.
Substituents (R)
SGC-7901
A875
HepG2
HL-7702
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
6a
6b
6c
6d
6e
6f
6g
H
5-F
5-Cl
5-Br
5-I
5-CH3
5-NO2
6-F
7.69 2.54
6.67 1.77
4.92 1.13
8.43 2.23
21.67 3.86
7.06 1.27
11.88 2.45
7.22 1.17
13.82 2.65
9.51 0.49
6.83 2.01
7.14 1.41
17.11 3.68
9.12 1.61
6.40 1.74
9.20 1.68
69.77 8.47
>100
9.51 1.34
8.58 1.64
7.92 1.95
11.58 2.84
23.75 4.39
6.01 1.95
26.20 4.59
6.85 1.99
21.72 4.82
42.21 8.19
8.78 0.64
8.08 0.51
19.72 1.89
8.25 1.97
3.85 1.44
7.71 2.13
63.57 13.22
>100
6.72 1.59
6.12 1.79
2.39 0.36
8.29 2.41
18.62 4.79
6.91 0.58
19.44 3.37
4.85 0.84
20.36 6.69
32.31 4.21
4.84 1.05
6.10 2.09
8.99 3.13
4.85 1.07
4.31 0.92
5.41 1.44
36.67 10.61
>100
5.65 1.59
5.49 1.33
5.01 1.37
14.06 3.79
13.95 2.83
6.08 0.54
33.39 6.15
7.87 2.04
8.29 2.24
7.09 1.57
6.36 1.37
4.40 0.68
18.49 1.00
12.50 4.36
11.23 3.05
12.76 2.79
91.24 12.29
>100
9
H
5-F
10
11
12
13
14
15
16
17
18
19
5-Cl
5-Br
5-I
5-CH3
5-NO2
6-F
–
–
–
6h
Epiandrosterone
Androsterone
5-FUb
62.03 8.38
65.26 7.30
63.18 12.61
86.02 9.61
Abbreviations: SGC-7901: Human gastric cancer cell line; A875: Human melanoma cell line; HepG2: Human hepatocellular liver carcinoma cell
line; HL-7702: Human normal liver cells.
aIC50: Compound concentration required to inhibit tumor cell proliferation by 50%.
b5-Fluorouracil, used as a positive control.
Figure 4. Dose-response analysis of cell growth inhibition activity for representative compounds 3b, 3c, 6c and 6g against SGC-7901 (left), A875 (middle) and HepG2
(right) cell lines.
exhibited cytotoxic effects on SGC-7901, A875 and HepG2 cell speculated it would have been more interesting to test the further
lines in a dose-dependent manner.
activity of the nitro-group derivatives against other cell lines, and
the special properties of different configuration will be helpful to
investigate the real potent.
The structure and activity relationships (SARs)
According to the in vitro bioassay results presented in the
Figure 3 and Table 1, the possible structure-activity relationships
for these steroidal isatin derivatives can be obtained.
Conclusion
Two series of steroidal isatin derivatives were synthesized, charac-
terized and evaluated for their in vitro cytotoxicity against three
cancer cell lines (SGC-7901, A875, HepG2). The preliminary bio-
assay indicated that most of the newly synthesized compounds
exhibited good cytotoxic activities against tested cell lines com-
pared with 5-fluorouracil (5-FU), especially, the two compounds
containing a nitro-group (3g and 6g) present significant activity
difference. These findings will help to design and discover novel
anticancer agents.
First, we can observe that most of these conjugates exhibited
higher activity than those of parent compounds, and the com-
pounds from the epiandrosterone series 3a–h were slightly better
than those from androsterone series 6a–h. For the epiandroster-
one series, the 5-I or 5-NO2 group decrease the cytotoxicity of the
corresponding compounds (3e or 3g), which may be due to the
large size of these groups is unfavorable for activity. The com-
pound 3c with 5-Cl substituent is the highest potent against
tested cell lines. For the androsterone series, the 5-I group also
decrease the potential cytotoxicity of the corresponding com-
pound (6e), and the range of IC50 value is 8–20 mM. Especially, the
compound bearing 5-F substituent 6b present a wide IC50 range,
which exhibit selective inhibition against SGC-7901 cell lines (IC50
9.51 0.49 mM). In particular, it’s interesting that the two com-
pounds containing a nitro-group (3g and 6g) present significant
Experimental section
Instrumentation and chemicals
All starting materials and reagents commercially available were
used without further purification, unless otherwise specified. 1H
activity difference, which testified the configuration might also NMR and 13C NMR spectra were recorded on a Bruker Avance III
affect the activity. According to these findings, it can be 600 MHz FT-NMR spectrometer using DMSO-d6 as the solvent and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1611
tetramethylsilane (TMS) as the internal standard. Chemical shifts 5-Chloro-3-(((3S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyldode-
are reported in d (parts per million) values, and coupling constants
cahydro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hy-
drazono)indolin-2-one 3c
nJ are reported in Hz. Mass spectra were performed on a Waters
R
R
ACQUITY UPLCV H-CLASS PDA (WatersV) instrument. Analytical
thin-layer chromatography was carried out on precoated silica gel
plates GF254 (Qindao Haiyang Chemical, China), and spots were
visualized with ultraviolet light.
This compound was obtained following the above method as
bright yellow powder, yield 72%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 10.93 (s, 1H), 7.81 (d, J ¼ 2.2 Hz, 1H), 7.43 (dd, J ¼ 8.3, 2.2 Hz,
1H), 6.90 (d, J ¼ 8.4 Hz, 1H), 4.44 (d, J ¼ 4.7 Hz, 1H), 3.41–3.34 (m,
1H), 2.45–2.27 (m, 2H), 2.01 (d, J ¼ 12 Hz, 1H), 1.80–1.60 (m, 5H),
1.55–1.33 (m, 5H), 1.31–1.11 (m, 5H), 1.11–1.02 (m, 1H), 1.00–0.88
(m, 5H), 0.80 (s, 3H), 0.72 (t, J ¼ 11.2 Hz, 1H); 13C NMR (150 MHz,
DMSO-d6): d ¼ 179.58, 164.42, 146.30, 143.66, 132.93, 127.99,
126.02, 118.24, 112.71, 69.74, 54.42, 53.04, 45.37, 44.84, 38.61,
37.07, 35.77, 35.00, 34.24, 31.83, 31.49, 28.70, 28.11, 23.31, 20.93,
16.99, 12.56; MS (ESI) m/z 468.4 (M þ H)þ, calcd. for C27H34ClN3O2
m/z ¼ 467.2; HRMS: calcd for C27H34ClN3O2 ([M þ H]þ), 469.0308;
found, 468.5869.
General synthetic procedure for target compounds
The key intermediates (Compd. 2 and 5) and target steroidal
derivatives 3a–h and 6a–h were conveniently synthesized accord-
ing to the modified procedures20. The general process for prepar-
ation of these steroidal isatin derivatives is shown as following: To
a solution of intermediates 2 or 5 (2 mmol) in ethanol (20 ml) was
added the equal amounts of substituted isatin (2 mmol), and then
the mixture was stirred at 30–45 ꢁC for additional hours. After
that, the solution was concentrated, and the condensation prod-
uct separated out on cooling (0–4 ꢁC) and was recrystallized from
5-Bromo-3-(((3S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyldode-
cahydro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hy-
ethanol. All the compounds were characterized by ESI-MS, 1H drazono)indolin-2-one 3d
NMR and 13C NMR spectroscopic analyses. Their physico-chemical
properties and spectra data are as follows:
This compound was obtained following the above method as yel-
1
low powder, yield 81%. H NMR (600 MHz, DMSO-d6): d ¼ 10.93 (s,
1H), 7.95 (d, J ¼ 1.9 Hz, 1H), 7.55 (dd, J ¼ 8.3, 2.1 Hz, 1H), 6.85 (d,
J ¼ 8.3 Hz, 1H), 4.44 (d, J ¼ 4.6 Hz, 1H), 3.46–3.43 (m, 1H), 2.46–2.38
(m, 1H), 2.34–2.25 (m, 1H), 2.00 (d, J ¼ 9.6 Hz, 1H), 1.81–1.62 (m,
5H), 1.53–1.35 (m, 5H), 1.28–1.05 (m, 6H), 1.00–0.90 (m, 5H), 0.81
(s, 3H), 0.77 (s, 1H); 13C NMR (150 MHz, DMSO-d6): d ¼ 179.18,
164.50, 145.10, 143.96, 135.89, 130.32, 125.50, 118.91, 113.60,
69.90, 54.58, 53.14, 45.55, 44.90, 38.62, 37.20, 35.75, 35.08, 34.31,
32.95, 31.55, 28.81, 28.02, 23.44, 20.95, 17.03, 12.59; MS (ESI) m/z
512.3 (M þ H)þ, calcd. for C27H34BrN3O2 m/z ¼ 511.2; HRMS: calcd
for C27H34BrN3O2 ([M þ H]þ), 512.1834; found, 512.5112.
3-(((3S,8R,9S,10S,13S,14S)-3-Hydroxy-10,13-dimethyldodecahydro-
1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hydrazo-
no)indolin-2-one 3a
This compound was obtained following the above method as
bright yellow powder, yield 73%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 10.77 (s, 1H), 7.79 (d, J ¼ 7.6 Hz, 1H), 7.37–7.35 (m, 1H), 7.01 (t,
J ¼ 7.6 Hz, 1H), 6.87 (d, J ¼ 7.8 Hz, 1H), 4.44 (d, J ¼ 4.8 Hz, 1H),
3.39–3.34 (m, 1H), 2.39 (dd, J ¼ 19.3, 8.4 Hz, 1H), 2.32–2.21 (m, 1H),
2.03 (d, J ¼ 12.4 Hz, 1H), 1.84–1.73 (m, 1H), 1.71–1.59 (m, 4H),
1.55–1.40 (m, 3H), 1.39–1.31 (m, 2H), 1.19 (s, 4H), 1.15 (s, 1H), 1.06
(d, J ¼ 12.3 Hz, 1H), 1.01–0.89 (m, 5H), 0.80 (s, 3H), 0.77–0.70 (m,
1H); 13C NMR (150 MHz, DMSO-d6): d ¼ 178.12, 164.73, 146.78,
144.91, 133.54, 128.53, 122.54, 117.11, 111.11, 69.75, 54.38, 53.10,
45.20, 44.85, 38.63, 37.07, 35.78, 35.03, 34.21, 31.84, 31.51, 28.71,
27.96, 23.31, 20.89, 16.97, 12.60; MS (ESI) m/z 434.5 (M þ H)þ,
3-(((3S,8R,9S,10S,13S,14S)-3-Hydroxy-10,13-dimethyldodecahydro-
1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hydrazono)-
5-iodoindolin-2-one 3e
This compound was obtained following the above method as yel-
1
low powder, yield 74%. H NMR (600 MHz, DMSO-d6): d ¼ 10.99 (s,
1H), 8.15 (d, J ¼ 1.7 Hz, 1H), 7.68 (dd, J ¼ 8.2, 1.8 Hz, 1H), 6.76 (d,
calcd. for C27H35N3O2 m/z ¼ 433.3; HRMS: calcd for C27H35N3O2 J ¼ 8.2 Hz, 1H), 4.46 (d, J ¼ 4.7 Hz, 1H), 3.38–3.32 (m, 1H), 2.42–2.21
([M þ H]þ), 434.5857; found, 434.6535.
(m, 2H), 2.00 (d, J ¼ 12.3 Hz, 1H), 1.82–1.73 (m, 1H), 1.73–1.60 (m,
4H), 1.55–1.34 (m, 5H), 1.23 (m, 4H), 1.18–1.12 (m, 1H), 1.05 (t,
J ¼ 5.7 Hz, 1H), 1.01–0.89 (m, 5H), 0.80 (s, 3H), 0.77–0.71 (m, 1H);
13C NMR (150 MHz, DMSO-d6): d ¼ 178.15, 164.02, 145.66, 144.37,
141.27, 136.48, 119.14, 113.60, 84.87, 69.72, 54.48, 53.06, 45.37,
44.84, 38.61, 37.11, 35.77, 34.99, 34.25, 31.84, 31.50, 28.70, 27.96,
23.31, 20.99, 16.94, 12.56; MS (ESI) m/z 560.3 (M þ H)þ, calcd. for
C27H34IN3O2 m/z ¼ 559.2.
5-Fluoro-3-(((3S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyldode-
cahydro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hy-
drazono)indolin-2-one 3b
This compound was obtained following the above method as
brown yellow powder, yield 65%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 10.82 (s, 1H), 7.56 (dd, J ¼ 8.4, 2.7 Hz, 1H), 7.27–7.23 (m, 1H),
6.88 (dd, J ¼ 8.6, 4.3 Hz, 1H), 4.44 (d, J ¼ 4.7 Hz, 1H), 3.41–3.33 (m,
1H), 2.45 (dd, J ¼ 19.4, 8.6 Hz, 1H), 2.37–2.26 (m, 1H), 2.02 (d,
J ¼ 12.2 Hz, 1H), 1.81–1.74 (m, 1H), 1.74–1.60 (m, 4H), 1.55–1.32 (m,
5H), 1.31–1.13 (m, 5H), 1.09–1.03 (m, 1H), 0.99–0.88 (m, 5H), 0.80
(s, 3H), 0.76–0.69 (m, 1H); 13C NMR (150 MHz, DMSO-d6):
d ¼ 180.14, 164.74, 156.98, 146.98, 141.32, 120.01, 119.85, 117.50,
115.35, 112.18, 69.74, 54.38, 53.06, 45.36, 44.84, 38.62, 37.06,
35.77, 35.01, 34.21, 31.84, 31.50, 28.70, 28.15, 23.30, 20.89, 17.00,
12.58; MS (ESI) m/z 452.5 (M þ H)þ, calcd. for C27H34FN3O2 m/
z ¼ 451.3; HRMS: calcd for C27H34FN3O2 ([M þ H]þ), 452.5762;
found, 452.6553.
3-(((3S,8R,9S,10S,13S,14S)-3-Hydroxy-10,13-dimethyldodecahydro-
1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hydrazono)-
5-methylindolin-2-one 3f
This compound was obtained following the above method as yel-
1
low powder, yield 68%. H NMR (600 MHz, DMSO-d6): d ¼ 10.67 (s,
1H), 7.62 (s, 1H), 7.18 (d, J ¼ 8.7 Hz, 1H), 6.77 (d, J ¼ 7.9 Hz, 1H),
4.44 (d, J ¼ 4.7 Hz, 1H), 3.41–3.35 (m, 1H), 2.40–2.24 (m, 2H), 2.22
(s, 3H), 2.04 (d, J ¼ 9.6 Hz, 1H), 1.75–1.40 (m, 10H), 1.30–1.11 (m,
5H), 1.10–1.03 (m, 1H), 1.03–0.88 (m, 5H), 0.80 (s, 3H), 0.77–0.70
(m, 1H); 13C NMR (150 MHz, DMSO-d6): d ¼ 177.12, 164.78, 146.60,
142.57, 133.77, 131.22, 128.99, 117.15, 110.88, 69.75, 54.45, 53.10,

1612
S. KE ET AL.
45.18, 44.85, 38.62, 37.10, 35.78, 35.01, 34.26, 31.84, 31.51, 28.71, 2.48–2.29 (m, 2H), 2.03 (d, J ¼ 12.4 Hz, 1H), 1.82–1.65 (m, 3H),
27.88, 23.33, 21.14, 20.96, 16.92, 12.58; MS (ESI) m/z 448.4
(M þ H)þ, calcd. for C28H37N3O2 m/z ¼ 447.3.
1.60–1.42 (m, 5H), 1.40–1.30 (m, 5H), 1.28–1.13 (m, 4H), 1.07–0.89
(m, 4H), 0.87–0.74 (m, 4H); 13C NMR (150 MHz, DMSO-d6):
d ¼ 180.21, 164.75, 158.55, 156.98, 146.98, 141.30, 119.84, 117.53,
112.18, 64.53, 54.56, 53.12, 45.37, 39.03, 36.30, 36.20, 35.03, 34.23,
32.40, 31.62, 29.11, 28.58, 28.14, 23.27, 20.46, 17.01, 11.62; MS (ESI)
m/z 452.5 (M þ H)þ, calcd. for C27H34FN3O2 m/z ¼ 451.3; HRMS:
calcd for C27H34FN3O2 ([M þ H]þ), 452.5762; found, 452.6553.
3-(((3S,8R,9S,10S,13S,14S)-3-Hydroxy-10,13-dimethyldodecahydro-
1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hydrazono)-
5-nitroindolin-2-one 3g
This compound was obtained following the above method as
bright yellow powder, yield 78%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 11.50 (s, 1H), 8.65 (d, J ¼ 2.4 Hz, 1H), 8.30 (dd, J ¼ 8.7, 2.4 Hz,
1H), 7.07 (d, J ¼ 8.7 Hz, 1H), 4.44 (d, J ¼ 4.7 Hz, 1H), 3.38–3.34 (m,
5-Chloro-3-(((3R,5S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyl-
1H), 2.47–2.27 (m, 2H), 2.06 (d, J ¼ 12.6 Hz, 1H), 1.80–1.35 (m, 10H), dodecahydro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylide-
1.32–1.12 (m, 5H), 1.06 (d, J ¼ 7.0 Hz, 1H), 1.03–0.88 (m, 5H), 0.82
ne)hydrazono)indolin-2-one 6c
This compound was obtained following the above method as
(s, 3H), 0.75 (t, J ¼ 11.4 Hz, 1H); 13C NMR (150 MHz, DMSO-d6):
deep yellow powder, yield 75%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 10.93 (s, 1H), 7.82 (d, J ¼ 2.2 Hz, 1H), 7.43 (dd, J ¼ 8.4, 2.3 Hz,
1H), 6.90 (d, J ¼ 8.4 Hz, 1H), 4.19 (d, J ¼ 3.0 Hz, 1H), 3.82 (d,
J ¼ 2.6 Hz, 1H), 2.45–2.23 (m, 2H), 2.02 (d, J ¼ 12.4 Hz, 1H),
1.82–1.66 (m, 3H), 1.61–1.44 (m, 5H), 1.43–1.28 (m, 5H), 1.26–1.10
(m, 4H), 1.03–0.92 (m, 4H), 0.85–0.75 (m, 4H); 13C NMR (150 MHz,
DMSO-d6): d ¼ 179.48, 164.42, 146.24, 143.65, 132.91, 127.98,
126.02, 118.24, 112.69, 64.53, 54.61, 53.12, 45.37, 39.02, 36.29,
36.19, 35.01, 34.25, 32.41, 31.62, 29.11, 28.58, 28.09, 23.28, 20.50,
16.99, 11.60; MS (ESI) m/z 468.4 (M þ H)þ, calcd. for C27H34ClN3O2
m/z ¼ 467.2; HRMS: calcd for C27H34ClN3O2 ([M þ H]þ), 469.0308;
found, 468.6769.
d ¼ 179.37, 164.91, 150.35, 145.44, 142.44, 129.69, 123.68, 116.90,
111.39, 69.74, 54.45, 53.04, 45.44, 44.86, 38.61, 37.11, 35.78, 35.01,
34.20, 31.85, 31.50, 28.69, 28.16, 23.32, 20.99, 16.96, 12.58; MS (ESI)
m/z 479.4 (M þ H)þ, calcd. for C27H34N4O4 m/z ¼ 478.3.
6-Fluoro-3-(((3S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyldode-
cahydro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hy-
drazono)indolin-2-one 3h
This compound was obtained following the above method as
brown yellow powder, yield 62%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 10.94 (s, 1H), 7.84 (dd, J ¼ 8.4, 5.9 Hz, 1H), 6.87–6.82 (m, 1H),
6.70 (dd, J ¼ 9.1, 2.3 Hz, 1H), 4.44 (d, J ¼ 4.7 Hz, 1H), 3.38–3.35 (m,
1H), 2.45–2.26 (m, 2H), 2.01 (d, J ¼ 9.4 Hz, 1H), 1.80–1.73 (m, 1H),
1.70–1.60 (m, 4H), 1.54–1.34 (m, 5H), 1.30–1.25 (m, 4H), 1.18–1.12
(m, 1H), 1.06 (t, J ¼ 7.0 Hz, 1H), 0.98–0.90 (m, 5H), 0.80 (s, 3H), 0.73
(d, J ¼ 9.3 Hz, 1H); 13C NMR (150 MHz, DMSO-d6): d ¼ 179.51,
165.05, 147.23, 146.04, 130.85, 130.77, 113.91, 109.20, 109.04,
69.75, 54.37, 53.06, 45.26, 44.85, 38.62, 37.06, 35.77, 35.03, 34.17,
31.83, 31.51, 28.70, 28.05, 23.30, 20.88, 16.99, 12.59; MS (ESI) m/z
452.4 (M þ H)þ, calcd. for C27H34FN3O2 m/z ¼ 451.3.
5-Bromo-3-(((3R,5S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyl-
dodecahydro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylide-
ne)hydrazono)indolin-2-one 6d
This compound was obtained following the above method as
yellowish powder, yield 84%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 10.93 (s, 1H), 7.96 (d, J ¼ 2.0 Hz, 1H), 7.55 (dd, J ¼ 8.3, 2.1 Hz,
1H), 6.85 (d, J ¼ 8.3 Hz, 1H), 4.21 (d, J ¼ 8.8 Hz, 1H), 3.86–3.78 (m,
1H), 2.45–2.24 (m, 2H), 2.01 (d, J ¼ 12.3 Hz, 1H), 1.82–1.66 (m, 3H),
1.61–1.43 (m, 5H), 1.40–1.25 (m, 7H), 1.21–1.12 (m, 2H), 1.03–0.91
(m, 4H), 0.82–0.74 (m, 4H); 13C NMR (150 MHz, DMSO-d6):
d ¼ 178.96, 164.27, 145.94, 143.99, 135.67, 130.74, 118.70, 113.60,
113.18, 64.53, 54.64, 53.14, 45.38, 39.03, 36.30, 36.19, 35.01, 34.27,
32.43, 31.63, 29.12, 28.58, 28.05, 23.28, 20.52, 16.99, 11.62; MS (ESI)
m/z 512.4 (M þ H)þ, calcd. for C27H34BrN3O2 m/z ¼ 511.2; HRMS:
calcd for C27H34BrN3O2 ([M þ H]þ), 512.1834; found, 512.5112.
3-(((3R,5S,8R,9S,10S,13S,14S)-3-Hydroxy-10,13-dimethyldodecahy-
dro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hydra-
zono)indolin-2-one 6a
This compound was obtained following the above method as
bright yellow powder, yield 77%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 10.77 (s, 1H), 7.80 (d, J ¼ 7.2 Hz, 1H), 7.38–7.35 (m, 1H),
7.03–7.00 (m, 1H), 6.87 (d, J ¼ 7.8 Hz, 1H), 4.18 (d, J ¼ 3.0 Hz, 1H),
3.82 (d, J ¼ 2.6 Hz, 1H), 2.42–2.22 (m, 2H), 2.03 (dd, J ¼ 9.5, 3.0 Hz,
1H), 1.81–1.65 (m, 3H), 1.63–1.44 (m, 5H), 1.43–1.28 (m, 5H),
1.25–1.12 (m, 4H), 1.01–0.92 (m, 4H), 0.86–0.75 (m, 4H); 13C NMR
(150 MHz, DMSO-d6): d ¼ 178.18, 164.74, 146.78, 144.91, 133.53,
128.54, 122.54, 117.11, 111.11, 64.53, 54.56, 53.18, 45.21, 39.03,
36.30, 36.20, 35.04, 34.23, 32.40, 31.63, 29.12, 28.59, 27.96, 23.28,
20.46, 16.98, 11.64; MS (ESI) m/z 434.5 (M þ H)þ, calcd. for
C27H35N3O2 m/z ¼ 433.3; HRMS: calcd for C27H35N3O2 ([M þ H]þ),
434.5857; found, 434.5635.
3-(((3R,5S,8R,9S,10S,13S,14S)-3-Hydroxy-10,13-dimethyldodecahy-
dro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hydra-
zono)-5-iodoindolin-2-one 6e
This compound was obtained following the above method as yel-
1
low powder, yield 79%. H NMR (600 MHz, DMSO-d6): d ¼ 10.99 (s,
1H), 8.15 (d, J ¼ 6.0 Hz, 1H), 7.69 (dd, J ¼ 6.0 Hz, 1H), 6.76 (d,
J ¼ 8.4 Hz, 1H), 4.22 (d, J ¼ 6.0 Hz, 1H), 3.85–3.79 (m, 1H), 2.41–2.23
5-Fluoro-3-(((3R,5S,8R,9S,10S,13S,14S)-3-hydroxy-10,13-dimethyldo- (m, 2H), 2.02–1.98 (m, 1H), 1.80–1.75 (m, 1H), 1.72–1.70 (m, 2H),
1.59–1.45 (m, 5H), 1.39–1.30 (m, 4H), 1.28–1.22 (m, 5H), 1.19–1.12
(m, 2H), 0.99 (s, 3H), 0.79 (s, 3H); 13C NMR (150 MHz, DMSO-d6):
d ¼ 177.88, 164.01, 144.37, 141.26, 136.42, 129.17, 123.47, 119.14,
113.61, 64.52, 54.67, 53.17, 45.37, 43.67, 39.04, 36.31, 36.19, 35.00,
34.26, 32.44, 31.63, 28.58, 27.95, 23.28, 20.55, 16.95, 11.62; MS (ESI)
decahydro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)-
hydrazono)indolin-2-one 6b
This compound was obtained following the above method as yel-
1
low powder, yield 71%. H NMR (600 MHz, DMSO-d6): d ¼ 10.82 (s,
1H), 7.57 (dd, J ¼ 8.4, 2.7 Hz, 1H), 7.27–7.24 (m, 1H), 6.89 (dd,
J ¼ 8.6, 4.3 Hz, 1H), 4.19 (d, J ¼ 3.0 Hz, 1H), 3.82 (d, J ¼ 2.6 Hz, 1H), m/z 560.2 (M þ H)þ, calcd. for C27H34IN3O2 m/z ¼ 559.2.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1613
3-(((3R,5S,8R,9S,10S,13S,14S)-3-Hydroxy-10,13-dimethyldodecahy-
dro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hydra-
zono)-5-methylindolin-2-one 6f
data were analyzed with SPSS 16.0 software, and the 50% inhibi-
tory concentrations (IC50) of each compound for the different cell
lines were determined. Assays were performed in triplicate on
three independent experiments.
This compound was obtained following the above method as yel-
1
low powder, yield 74%. H NMR (600 MHz, DMSO-d6): d ¼ 10.67 (s,
1H), 7.63 (s, 1H), 7.19 (dd, J ¼ 6.0 Hz, 1H), 6.77 (d, J ¼ 7.8 Hz, 1H),
3.82–3.80 (m, 2H), 2.39–2.26 (m, 2H), 2.23 (s, 3H), 1.72–1.69 (m,
2H), 1.53–1.45 (m, 5H), 1.38–1.32 (m, 6H), 1.28–1.22 (m, 4H),
1.18–1.12 (m, 3H), 0.98 (s, 3H), 0.79 (s, 3H); 13C NMR (150 MHz,
DMSO-d6): d ¼ 177.01, 164.78, 146.53, 142.57, 133.75, 131.22,
128.98, 117.15, 110.88, 64.53, 54.64, 53.19, 45.18, 39.03, 36.31,
36.20, 35.02, 34.28, 32.44, 31.64, 29.12, 28.60, 27.86, 23.30, 21.14,
20.52, 16.93, 11.62; MS (ESI) m/z 448.4 (M þ H)þ, calcd. for
C28H37N3O2 m/z ¼ 447.3.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
We gratefully acknowledge the support of this work by the
National Key R&D Program of China (2017YFD0200502) and the
Program for Leading Talents of Hubei Academy of Agricultural
Sciences, and the authors also gratefully acknowledge the partial
support from Hubei Agricultural Science Innovation Center
(2019–620-000–001-27).
3-(((3R,5S,8R,9S,10S,13S,14S)-3-Hydroxy-10,13-dimethyldodecahy-
dro-1H-cyclopenta[a]phenanthren-17(2H,10H,14H)-ylidene)hydra-
zono)-5-nitroindolin-2-one 6g
This compound was obtained following the above method as
bright yellow powder, yield 78%. 1H NMR (600 MHz, DMSO-d6):
d ¼ 11.49 (s, 1H), 8.65 (d, J ¼ 2.0 Hz, 1H), 8.30 (dd, J ¼ 8.6, 2.1 Hz,
1H), 7.06 (d, J ¼ 8.7 Hz, 1H), 4.19 (d, J ¼ 1.9 Hz, 1H), 3.82 (s, 1H),
2.48–2.24 (m, 2H), 2.06 (d, J ¼ 12.2 Hz, 1H), 1.85–1.65 (m, 3H),
1.66–1.46 (m, 5H), 1.42–1.13 (m, 9H), 1.08–0.93 (m, 4H), 0.90–0.66
(m, 4H); 13C NMR (150 MHz, DMSO-d6): d ¼ 179.37, 164.90, 150.33,
145.40, 142.43, 129.66, 123.67, 116.89, 111.37, 64.53, 54.61, 53.11,
45.44, 39.03, 36.30, 36.20, 35.02, 34.21, 32.43, 31.62, 29.13, 28.58,
28.14, 23.29, 20.56, 16.96, 11.61; MS (ESI) m/z 479.4 (M þ H)þ,
calcd. for C27H34N4O4 m/z ¼ 478.3.
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