Synthesis and antitumor activities of steroidal 5α,8α-endoperoxide derivatives with side chain of 17-hydrazone aromatic heterocycle

Article abstract of DOI:10.3987/COM-17-13658

Here a series of novel 5α,8α-epidioxyandrostan-3β-ol-17-hydrazone derivatives possessing various aromatic heterocycle structures in 17-side chain of their steroidal nucleus were synthesized and characterized. The antiproliferative activity of synthesized compounds against some cancer cells was investigated. The results have demonstrated that compound 8b with quinoline and 8c with indole structure in side chain display excellent anti-proliferative activity in vitro against tested cancer cell lines.

Full text of DOI:10.3987/COM-17-13658

HETEROCYCLES, Vol. 94, No. 4, 2017
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HETEROCYCLES, Vol. 94, No. 4, 2017, pp. 691 - 701. © 2017 The Japan Institute of Heterocyclic Chemistry
Received, 22nd January, 2017, Accepted, 9th March, 2017, Published online, 23rd March, 2017
DOI: 10.3987/COM-17-13658
SYNTHESIS AND ANTITUMOR ACTIVITIES OF STEROIDAL
5α,8α-ENDOPEROXIDE DERIVATIVES WITH SIDE CHAIN OF
17-HYDRAZONE AROMATIC HETEROCYCLE
Ming Bu,1,# Yujie Wang,1,# Tingting Cao,1 Hongxia Li,1 Mingzhou Guo,3 Yue
Zhou,1 Na Zhang,1 Chengchu Zeng,1 and Liming Hu1,2,*
1
Department of Biomedical Engineering, College of Life Science and
Bioengineering, Beijing University of Technology, Beijing, 100124, China.
2 Beijing Key Laboratory of Environmental and Viral Oncology, College of Life
Science and Bioengineering, Beijing University of Technology, Beijing, 100124,
3
#
China. Chinese PLA General Hospital, Beijing, 100853, China. These authors
contributed equally to this work. *E-mail: huliming@bjut.edu.cn
Abstract – Here a series of novel 5α,8α-epidioxyandrostan-3β-ol-17-hydrazone
derivatives possessing various aromatic heterocycle structures in 17-side chain of
their steroidal nucleus were synthesized and characterized. The antiproliferative
activity of synthesized compounds against some cancer cells was investigated.
The results have demonstrated that compound 8b with quinoline and 8c with
indole structure in side chain display excellent anti-proliferative activity in vitro
against tested cancer cell lines.
INTRODUCTION
It is well known that N, O, S-heterocycles are important structural units present in many drugs, natural
and synthetic products with potent bioactivity profile.1 They possess hydrogen bond donors and acceptors
in a rigid framework, and they can therefore effectively interact with target enzymes and receptors via
hydrogen bond interactions.2 They can enhance binding affinity and improve in vitro potency, thus
providing desired pharmacokinetic properties and pharmaceutical properties.3 Studies have found that
introducing some heterocycles into the steroids, changing the steroidal side chain or substitution of the
steroidal skeleton, introducing heteroatom or replacing one or more carbon atoms in steroidal molecule
with heteroatom may result in change of its biological activities.4 So far, the steroids containing
heterocycles had been widely researched and reported.5

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Sterol endoperoxides are the important active lead compounds in drug discovery, which are well known
for their 5α,8α-peroxy moiety. Among sterol endoperoxides, ergosterol peroxide (5α,8α-
epidioxyergosta-6,22-dien-3β-ol, EP) (Figure 1), is a member of a class of fungal secondary metabolites
of sterol 5α,8α-endoperoxide derivatives.6 In our previous study, we found that EP purified from
Ganoderma lucidum, induced cell death and inhibited cell migration, cell cycle progression, and colony
growth of human hepatocellular carcinoma cells. We further examined the mechanism associated with
this effect and found that treatment with EP increased expression of Foxo3a mRNA and protein in HepG2
cells. The levels of Puma and Bax, pro-apoptotic proteins, were effectively enhanced. Our results suggest
that ergosterol peroxide stimulated Foxo3 activity by inhibiting pAKT and c-Myc and activating
pro-apoptotic protein Puma and Bax to induce cancer cell death.7
Figure 1. Molecular structures of ergosterol peroxide, DHEA, and designed compound
In order to obtain biologically potent compounds with diverse structures, therefore in this paper, a series
of steroidal compounds containing heterocycle attached to the steroidal 17-side chain by a hydrazone
structure were designed and synthesized using dehydroepiandrosterone as staring materials (Figure 1).
Furthermore, the antiproliferative activity of the compounds against some cancer cells was evaluated in
vitro.
RESULTS AND DISCUSSION
Chemistry. The synthetic route of new steroidal derivatives with side chain of hydrazone aromatic
heterocycle 7a-h is shown in Scheme 1. First, acetylize of 1 to get compound 2, then bromination of 2 and
O
O
O
O
Ac2O, DCM,
pyridine
Bu4NF, THF
rt, 12 h
NBS, cyclohexane
reflux, 1 h
rt, 12 h
AcO
Br
HO
AcO
AcO
DHEA (1)
2
98%
4 35%
3
86%
O
N
NH2
EtOH, AcOH
N
R
N
NH2NH2-H2O
NaOMe, MeOH
reflux, 1 h
different aldehydes
EtOH, 45 oC, 2 h
rt, 2~4 h
HO
HO
HO
7a-h
5
96%
6
70%
Scheme 1. Synthetic route of the target compounds 7a-h

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693
dehydrobromination of 3 to afforded 5,7-diene acetate 4,8 which was then converted to 5 via solvolysis in
30% overall yield from 1. Then, 5 was converted to the corresponding steroidal hydrazone 6 via reaction
with hydrazine hydrate in anhydrous ethanol.9 After crystallized, the reaction of pure steroidal hydrazone
with appropriate aldehydes gave steroidal hydrazone derivatives 7a-h (Table 1).
Table 1. Synthesis of the intermediates 7a-h
Entry
7a
R
Yielda
78%
Entry
7d
R
Yielda
82%
Entry
7g
R
Yielda
80%
86%
73%
77%
80%
91%
7b
7c
7e
7f
7h
a Isolated yields.
Then treatment of 7a-h with eosin Y (EY) as photosensitizer in pyridine, irradiated by iodine tungsten
lamp and kept bubbling oxygen for 0.5 h to get target compounds 8a-h (Scheme 2).10 The synthesis of
compounds 8a-h were achieved by the α-π-facial stereoselectivity of the addition reaction of singlet
oxygen with Δ5,7-sterol 7a-h. Reactions were monitored by thin layer chromatography (TLC) plates
and/or by staining with H2SO4-EtOH solution (5 mL concentrated H2SO4 + 95 mL EtOH). The chemical
structures of the compounds synthesized were elucidated on the basis of 1H NMR, 13C NMR, and LC-MS.
1
The detailed physical and analytical data are given in experimental part. In the H NMR spectrum, the
downfield chemical shifts of C6-H at 6.50 ppm (d) and C7-H at 6.35 ppm (d) demonstrate a formation of
5,8-peroxy bond and 6-CH=CH-7 bond in compound 8a (Figure 2). In addition, the resonances showing
of C22-H at 8.25 ppm (s) and 22-C at 155.3 ppm demonstrate a formation of 17=N̾N=C̾Ar bond in 8a.
N
N
R
R
N
O2, pyridine
hv, 0 oC, 0.5 h
N
O
O
HO
HO
7a-h
8a-h
Scheme 2. Synthetic route of the target compounds 8a-h
Biology. The newly synthesized compounds 8a-h and EP were evaluated for their antiproliferative
activities against human cancer cell lines derived from various human cancer types, including human
hepatocellular carcinoma cell lines (HepG2, SK-Hep1) and human breast cervical cancer cell lines
(MDA-MB-231, MCF-7). In vitro cell-based evaluation of the antiproliferative activities of the

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synthesized compounds was carried out using MTT assay. Cisplatin was employed as the positive control.
The anticancer potency of these compounds were indicated by IC50 values. The results were summarized
in Table 2. As compared to the parent EP molecule, most of compounds 8a-h possess distinct
antiproliferative activity against all the four tested adherent cancer cell lines. The screening results
suggested a rough structure-activity relationships: among the steroidal 5α,8α-endoperoxide derivatives
with side chain of 17-hydrazone aromatic heterocycle, compound 8b and 8c with quinoline and
indolyl-group in 17-side chain displayed better antiproliferative activity.
Figure 2. The partial 1H NMR spectrums of compound 7a and 8a
Compounds 8a-h and EP were selected for further cytotoxic activity evaluation in vitro using a
non-cancer cell line 293T as a control. As shown in Table 2, only compound 8b showed little cytotoxicity
(CC50 <50 μM). It is apparent from the results depicted in Table 2 that most of compounds exhibit
obvious selectivity between cancer cell line and non-cancer cell line.
In summary, we have prepared a series of steroidal 5α,8α-epidioxyandrost-6-en-3β-ol-17-hydrazone
derivatives possessing various aromatic heterocycle structures. The antiproliferative activity of the
synthesized compounds against human hepatocellular carcinoma cell lines HepG2, SK-Hep1 and human
breast cervical cancer cell lines MDA-MB-231, MCF-7 were investigated. The results had demonstrated
that some steroidal endoperoxide-17-hydrazone heterocycle derivatives showed distinct antiproliferative
activity. Preliminary SARs were put forward based on the biological results. Compound 8b and 8c with
quinoline and indole structure in 17-side chain displayed better antiproliferative activity in vitro against
tested cells. In an overall view, the steroidal 5α,8α-endoperoxide-17-hydrazone derivatives will provide
better insight into the effect of the C-17 site on anticancer activity for designing potential steroidal

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endoperoxide anticancer agents. Future work will also focus on the synthesis of additional candidate
structures with different side-chain to address specific cancer cell lines.
Table 2. In vitro antiproliferative activity and cytotoxic activity data of compounds
IC50 (μM)a
CC50 (μM)a
293T
>80
Entry
HepG2
35.40
8.74
SK-Hep1
MCF-7
31.60
14.30
15.78
24.25
47.62
49.34
17.76
>60
MDA-MB-231
26.53
17.54
10.66
35.07
38.22
>60
27.76
13.99
9.38
8a
8b
44.10
54.52
62.60
>80
10.63
19.54
40.46
>60
8c
16.34
34.73
43.54
20.14
>60
8d
8e
>60
8f
23.22
48.62
16.80
0.65
15.63
>60
54.40
>80
8g
8h
17.25
2.39
23.31
6.63
19.17
5.50
56.82
24.22
EP
Cisplatin
a Data represent the mean values of three independent determinations.
EXPERIMENTAL
General methods. All commercially available reagents were used without further purification. Melting
points (uncorrected) were determined on a MP120 auto point apparatus (Jinan Hanon Instruments Co.
1
13
Ltd). H NMR and C NMR spectra were measured on a Bruker Avance DRX400 spectrometer with
1
TMS and solvent signals allotted as internal standards. The chemical shifts of the H NMR and 13C NMR
were expressed in ppm (δ). MS-ESI mass spectra were obtained on an Esquire 6000 mass spectrometer.

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LC-MS data were measured using an Agilent 6120 mass spectrometer. Silica gel (300-400 mesh) was
used for analytical and flash chromatography. HRMS data were measured using a Bruker APEX IV
Fourier transform ion cyclotron resonance mass spectrometer.
Synthesis of 3β-acetoxyandrost-5-en-17-one (2). To a suspension of DHEA (1) (28.8 g, 0.10 mol) in
CH2Cl2 (40 mL) and pyridine (10 mL) was added Ac2O (12 mL, 0.13 mol) over 20 min. After the mixture
was stirred in room temperature for 6 h, water (30 mL) was added, and then extracted with EtOAc. The
combined organic layers were washed with saturated aqueous NaHCO3 and brine. The organic extracts
were dried with anhydrous MgSO4 and concentrated to get crude product 2 as a white solid (32 g). Yield:
1
98%, mp 167-170 °C. H NMR (CDCl3, 400 MHz) δ 5.41 (d, J = 5.1 Hz, 1H), 4.65-4.54 (m, 1H), 2.46
(dd, J = 19.3, 8.6 Hz, 1H), 2.33 (t, J = 7.9 Hz, 2H), 2.10 (dd, J = 18.8, 9.4 Hz, 2H), 2.04 (s, 3H), 1.94 (d,
J = 5.7 Hz, 1H), 1.89 (d, J = 2.9 Hz, 1H), 1.87-1.81 (m, 2H), 1.71-1.61 (m, 4H), 1.56 (dd, J = 10.4, 6.4
Hz, 1H), 1.48 (dd, J = 13.3, 4.4 Hz, 1H), 1.33-1.26 (m, 2H), 1.19-1.11 (m, 1H), 1.04 (s, 3H), 0.99 (d, J =
4.6 Hz, 1H), 0.88 (s, 3H). MS (ESI) m/z: 353.9 [M+Na]+, 369.7 [M+K]+.
Synthesis of 7α-bromo-3β-(acetyloxy)androst-5-en-17-one (3). A mixture of 2 (32 g, 0.1 mol) and
cyclohexane (80 mL) was warmed to 65 °C to obtain a clear solution, then NBS (23.3 g, 0.15 mol) was
added and the mixture was heated to reflux for 1 h. The mixture was cooled to room temperature and
diluted with 200 mL water. After being stirred for 1 h, the precipitate was collected by filtration and
washed with water. Then the solid was dissolved in CH2Cl2, the solution was washed with brine, dried
with anhydrous MgSO4, and concentrated to get crude 7α-bromo-3β-(acetyloxy)androst-5-en-17-one 3 as
a light brown solid (35 g, 86%). The crude product was used for the next step.
Synthesis of 3β-acetoxyandrosta-5,7-dien-17-one (4). A mixture of the crude bromide 3 (35 g, 0.085
mol) prepared above and 1.0 M Bu4NF in THF (88 mL, 0.127 mol, 1.5 equiv) was stirred overnight. Then,
the mixture was diluted with cyclohexane, washed with water, dried with anhydrous MgSO4, and
concentrated to get crude product 4 as a brown solid. The product was purified by chromatographic
1
column to give a pale yellow solid as compound 4 (11.5 g). Yield: 35%, mp 112-115 °C; H NMR
(CDCl3, 400 MHz) δ 5.6 (1H, s), 5.57 (1H, d, J = 2.6 Hz), 4.7 (1H, m), 2.56-2.49 (2H, m), 2.38 (1H, d, J
= 12.5 Hz), 2.24-2.17 (2H, m), 2.06 (3H, s), 1.97-1.92 (2H, m), 1.91 (1H, d, J = 3.7 Hz), 1.74 (2H, d, J =
4.8 Hz), 1.70 (1H, d, J = 5.2 Hz), 1.58 (2H, s), 1.40-1.34 (2H, m), 1.27 (1H, s), 0.98 (3H, s), 0.82 (3H, s).
MS (ESI) m/z: 351.7 [M+Na]+.
Synthesis of 3β-hydroxyandrosta-5,7-dien-17-one (5). To a suspension of intermediate 4 (11 g, 0.034
mol) in 100 mL of MeOH was added 25% (wt) NaOMe in MeOH (10 mL). After the mixture was reflux
for 1 h, 100 mL of water was added dropwise over 1 h. Then, the mixture was stirred for 2 h, the
precipitate was collected by filtration, washed with MeOH-H2O (1:2), and dried at 40 °C under high
vacuum to get crude product as a brown solid. The product was purified by chromatographic column to

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1
give a pale yellow solid as intermediate 5 (9.3 g). Yield: 96%, mp 157-159 °C; H NMR (400 MHz,
CDCl3) δ 6.00 (1H, d, J = 9.8 Hz, C6-H), 5.69 (1H, d, J = 9.8 Hz, C7-H), 4.29 (1H, t, J = 7.9 Hz, C3-αH),
3.75-3.58 (1H, m, C3-OH), 2.66-2.40 (2H, m, C16-H), 1.03 (3H, s, C19-CH3), 0.95 (3H, s, C18-CH3).
MS (ESI) m/z: 309.8 [M+Na]+, 325.8 [M+K]+.
Synthesis of 17-hydrazonoandrosta-5,7-dien-3β-ol (6). Intermediate 5 (3.0 g, 10 mmol) was dissolved
in 40 mL of EtOH. The mixture was heated to 45 °C, and 4 mL of 85% hydrazine hydrate was added. The
solution was stirred for 2 h until no starting material was observed. The reaction was terminated and some
white solid was separated out. The solid was filtrated and recrystallized using MeOH as solvent.
Compound 6 was obtained as white solid (2.1 g) after drying. Yield: 70%, mp 190-192 °C. 1H NMR (400
MHz, DMSO-d6) δ 5.51 (1H, d, J = 3.7 Hz, C6-H), 5.45 (1H, d, J = 2.7 Hz, C7-H), 5.36 (2H, d, J = 10.9
Hz, NH2), 4.69 (1H, m, C3-αH), 3.17 (1H, m, C3-OH), 2.29 (2H, m, C16-H), 0.88 (3H, s, C19-CH3),
0.69 (3H, s, C18-CH3). LC-MS (ESI) m/z: 301.2 [M+H]+, 323.2 [M+Na]+.
General procedure for the synthesis of compounds 7a-h. After 0.2 mmol of compound 6 was dissolved
in 25 mL of anhydrous EtOH, 0.2 mmol of aldehyde was added. Then the mixture was heated to 50 °C
and stirred continually at the temperature until no starting material. Then the reaction was terminated and
solvent was evaporated under reduced pressure. The residue was purified by recrystallization or flash
chromatography on silica gel to afford the corresponding compounds 7a-h.
General procedure for the synthesis of compounds 8a-h. For the synthesis of the title compound 8a-h,
a mixture of 7a-h (100 mg, 0.23 mmol) and eosin Y (1 mg) in pyridine (10 mL) in a quartz tube kept in a
water-cooled bath. Then the tube was irradiated with an iodine tungsten lamp. The oxygen was kept
bubbling during the irradiation for 0.5 h. The mixture was then poured into cold water and extracted with
EtOAc. The combined organic fractions were dried over anhydrous MgSO4 and the solvent was removed
under reduced pressure. The product was purified by chromatographic column to give the corresponding
compounds 8a-h.
5α,8α-Epidioxy-17-(pyridin-4-ylmethylene)hydrazonoandrostan-3β-ol (8a). Faint yellow crystals,
yield: 62%, mp 197.8-200.2 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 8.21 (1H, s, C22-H), 7.71 (1H, dd,
J = 21.4, 18.0 Hz, 3′-Ar-H), 7.60 (2H, d, J = 4.8 Hz, 2′,5′-Ar-H), 7.55-7.46 (1H, m, 4′-Ar-H), 6.53 (1H, d,
J = 8.5 Hz, C6-H), 6.32(1H, d, J = 8.5 Hz, C7-H), 4.03-3.92 (1H, m, C3-αH), 2.82-2.56 (2H, m, C16-H),
13
1.12 (3H, s, C19-CH3), 0.92 (3H, s, C18-CH3). C NMR (100 MHz, CDCl3) δ 181.9 (17-C), 155.3
(22-C), 150.1 (4′-Ar-C), 141.5 (3′-Ar-C), 136.3(7-C), 130.9 (2′-Ar-C), 129.9 (6-C), 128.8 (6′-Ar-C),
121.1 (5′-Ar-C), 82.4 (8-C), 79.0 (5-C), 71.8 (3-C), 66.1 (14-C), 51.5 (9-C), 49.1 (4-C), 46.1 (13-C), 37.2
(12-C), 34.7 (10-C), 33.6 (1-C), 30.1 (2-C), 27.9 (16-C), 23.0 (15-C), 20.3 (11-C), 19.1 (18-C), 18.1
(19-C). LC-MS (ESI) m/z: 422.2 [M+H]+. HRMS (ESI) m/z calcd for C25H31N3O3 [M+H]+ 422.2444,
found 422.2439.

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5α,8α-Epidioxy-17-(quinolin-4-ylmethylene)hydrazonoandrostan-3β-ol (8b). White crystals, yield:
1
64%, mp 207.5-209.6 °C; H NMR (400 MHz, CDCl3) δ (ppm) 8.98 (1H, d, J = 4.3 Hz, 2′-Ar-H), 8.89
(1H, s, C22-H), 8.73 (1H, d, J = 8.5 Hz, 5′-Ar-H), 8.18 (1H, d, J = 8.4 Hz, 8′-Ar-H), 7.78 (1H d, J = 4.8,
3′-Ar-H), 7.76 (1H, m, 6′-Ar-H),7.64 (1H, t, J = 7.6 Hz, 7′-Ar-H), 6.55 (1H, d, J = 8.4 Hz, C6-H), 6.33
(1H, d, J = 8.4 Hz, C7-H), 4.04-3.92 (1H, m, C3-αH), 2.90-2.63 (2H, m, C16-H), 1.16 (3H, s, C19-CH3),
0.94 (3H, s, C18-CH3). 13C NMR (100 MHz, CDCl3) δ (ppm) 182.5 (17-C), 155.6 (22-C), 150.1 (2′-Ar-C),
148.9(4′-Ar-C), 137.6 (10′-Ar-C), 136.3 (7-C), 130.1 (8′-Ar-C), 129.6 (6-C), 128.8 (7′-Ar-C), 127.6
(6′-Ar-C), 125.6 (5′-Ar-C), 124.5 (9′-Ar-C), 121.1 (3′-Ar-C), 82.4 (8-C), 79.0 (5-C), 71.7 (3-C), 66.14
(14-C), 51.5 (9-C), 49.1 (4-C), 46.1 (13-C), 37.0 (12-C), 34.7 (10-C), 34.3(1-C), 30.1 (2-C), 28.3 (16-C),
23.0 (15-C), 20.3 (11-C), 19.1 (18-C), 18.1 (19-C). LC-MS (ESI) m/z: 472.2 [M+H]+. HRMS (ESI) m/z
calcd for C29H33N3O3 [M+H]+ 472.2600, found 472.2589.
5α,8α-Epidioxy-17-(indol-3-ylmethylene)hydrazonoandrostan-3β-ol (8c). White crystals, yield: 57%,
1
mp 187.4-189.3 °C; H NMR (400 MHz, CDCl3) δ (ppm): 8.65 (1H, s, NH), 8.62 (1H, s, 1′-Ar-H), 8.34
(1H, d, J = 7.3 Hz, C22-H), 7.72 (1H d, J = 3.6 Hz, 4′-Ar-H), 7.53 (1H, d, J = 4.8 Hz, 7′-Ar-H), 7.50 (1H,
s, 5′-Ar-H), 7.39 (1H, d, J = 7.4 Hz, 6′-Ar-H), 6.56 (1H, d, J = 8.5 Hz, C6-H), 6.31 (1H, d, J = 8.4 Hz,
C7-H), 4.03-3.94 (1H, m, C3-αH), 2.99-2.75 (2H, m, C16-H), 0.99 (3H, s, C19-CH3), 0.93(3H, s,
13
C18-CH3). C NMR (100 MHz, CDCl3) δ (ppm): 180.3 (17-C), 154.4 (22-C), 136.9 (9′-Ar-C), 136.0
(7-C), 130.9 (2′-Ar-C), 129.6 (6-C), 125.1 (8′-Ar-C), 123.5 (4′-Ar-C), 122.7 (6′-Ar-C), 121.6 (5′-Ar-C),
113.8 (3′-Ar-C), 111.3 (7′-Ar-C), 82.4 (8-C), 79.2 (5-C), 71.8 (3-C), 66.3 (14-C), 51.5 (9-C), 49.2 (4-C),
45.8 (13-C), 37.2 (12-C), 34.7 (10-C), 34.1 (1-C), 30.1 (2-C), 28.2 (16-C), 27.8 (15-C), 20.3 (11-C), 19.2
(18-C), 18.1 (19-C). LC-MS (ESI) m/z: 460.3 [M+H]+. HRMS (ESI) m/z calcd for C28H33N3O3 [M+H]+:
460.2600, found 460.2596.
5α,8α-Epidioxy-17-(thiophene-3-ylmethylene)hydrazonoandrostan-3β-ol (8d). Faint yellow crystals,
yield: 59%, mp 173.8-175.2 °C; 1H NMR (400 MHz, CDCl3) δ 8.30 (1H, s, C22-H), 7.61 (1H, d, J = 1.6
Hz, 2′-Ar-H), 7.54 (1H, d, J = 4.9 Hz, 5′-Ar-H), 7.34 (1H, d, J = 2.8 Hz, 4′-Ar-H), 6.53 (1H, d, J = 8.4 Hz,
C6-H), 6.31 (1H, d, J = 8.4 Hz, C7-H), 4.04-3.89 (1H, m, C3-αH), 2.90-2.51 (2H, m, C16-H), 1.09 (3H, s,
13
C19-CH3), 0.92 (1H, s, C18-CH3). C NMR (100 MHz, CDCl3) δ (ppm): 180.8 (17-C), 152.6 (22-C),
136.1 (7-C), 131.0 (3′-Ar-C), 130.1 (6-C), 128.8 (2′-Ar-C), 126.6 (5′-Ar-C), 125.5 (4′-Ar-C), 82.4 (8-C),
79.1 (5-C), 71.8 (3-C), 66.2 (14-C), 51.5 (9-C), 49.1 (4-C), 45.9 (13-C), 37.1 (12-C), 34.7 (10-C), 34.0
(1-C), 30.1 (2-C), 27.8 (16-C), 23.0 (15-C), 20.3 (11-C), 19.2 (18-C), 18.0 (19-C). LC-MS (ESI) m/z:
427.2 [M+H]+. HRMS (ESI) m/z calcd for C24H30N2O3S [M+H]+ 427.2055, found 427.2045.
5α,8α-Epidioxy-17-(furan-3-ylmethylene)hydrazonoandrostan-3β-ol (8e). Faint yellow crystals, yield:
55%, mp 165.7-168.2 °C; 1H NMR (400 MHz, CDCl3) δ 8.16 (1H, s, C22-H), 7.81-7.63 (1H, m, 2′-Ar-H),
6.81 (1H, d, J = 3.3 Hz, 4′-Ar-H), 6.52 (1H, s, 3′-Ar-H), 6.51 (1H, d, J = 3.1 Hz, C6-H), 6.32 (1H, d, J =

HETEROCYCLES, Vol. 94, No. 4, 2017
699
8.4 Hz, C7-H), 4.03-3.90 (1H, m, C3-αH), 2.72 (2H, m, C16-H), 1.09 (3H, s, C19-CH3), 0.93 (3H, s,
C18-CH3). 13C NMR (100 MHz, CDCl3) δ 181.2 (17-C), 149.9 (22-C), 147.3 (2′-Ar-C), 145.2 (4′-Ar-C),
136.1 (7-C), 130.1 (6-C), 114.9 (3′-Ar-C), 111.9 (5′-Ar-C), 82.3 (8-C), 79.1 (5-C), 71.8 (3-C), 66.3
(14-C), 51.5 (9-C), 49.1 (4-C), 46.0 (13-C), 37.1 (12-C), 34.7 (10-C), 34.0 (1-C), 30.1 (2-C), 27.8 (16-C),
23.0 (15-C), 20.3 (11-C), 19.2 (18-C), 18.0 (19-C). LC-MS (ESI) m/z: 411.2 [M+H]+. HRMS (ESI) m/z
calcd for C24H30N2O4 [M+H]+ 411.2284, found 411.2276.
5α,8α-Epidioxy-17-(benzylidene)hydrazonoandrostan-3β-ol (8f). White solid, yield: 61%, mp 72.5-
74.4 °C. 1H NMR (400 MHz, CDCl3) δ 8.28 (1H, s, C22-H), 7.74 (1H, m, Ar-H), 7.54 (2H, d, J = 5.7 Hz,
Ar-H), 7.42 (2H, m, Ar-H), 6.54 (1H, d, J = 8.4 Hz, C6-H), 6.32 (1H, d, J = 8.4 Hz, C7-H), 3.98 (1H, dd,
J = 11.2, 5.5 Hz, C3-αH), 2.83-2.60 (2H, m, C16-H), 1.11 (3H, m, C18-CH3), 0.93-0.88 (3H, m,
13
C18-CH3). C NMR (100 MHz, CDCl3) δ 183.1 (17-C), 162.2 (22-C), 141.1 (5-C), 134.6 (1′-Ar-C),
130.6 (4′-Ar-C), 128.8 (3′-Ar-C), 128.7 (5′-Ar-C), 128.5 (2′-Ar-C), 128.1 (6′-Ar-C), 121.0 (6-C), 71.6
(3-C), 53.6 (14-C), 50.4 (9-C), 44.3 (4-C), 42.2 (13-C), 37.2 (10-C), 36.7 (1-C), 33.9 (12-C), 31.6 (7-C),
31.4 (2-C), 31.3 (8-C), 28.0 (16-C), 23.3 (15-C), 20.7 (11-C), 19.5 (18-C), 16.5 (19-C). LC-MS (ESI)
m/z: 421.2 [M+H]+. HRMS (ESI) m/z calcd for C26H32N2O3 [M+H]+ 421.2491, found 421.2479.
5α,8α-Epidioxy-17-(4-bromo-benzylidene)hydrazonoandrostan-3β-ol (8g). White crystals, yield: 60%,
1
mp 76.4-78.7 °C; H NMR (400 MHz, CDCl3) δ 8.23 (1H, s, C22-H), 7.62 (2H, d, J = 8.1 Hz, Ar-H),
7.54 (2H, d, J = 7.8 Hz, Ar-H), 6.53 (1H, d, J = 8.4 Hz, C6-H), 6.31 (1H, d, J = 8.4 Hz, C7-H), 4.03-3.89
13
(1H, m, C3-αH), 2.82-2.56 (2H, m, C16-H), 1.10 (3H, s, C19-CH3), 0.93 (3H, s, C18-CH3). C NMR
(100 MHz, CDCl3) δ 181.3, 156.7, 136.1, 131.9, 130.9, 130.0, 129.9, 129.5, 128.8, 82.4, 79.0, 71.8, 66.3,
51.5, 49.1, 46.0, 37.2, 36.8, 34.6, 33.9, 30.1, 27.8, 23.0, 20.3, 19.2, 18.1. LC-MS (ESI) m/z: 500.2, 502.2
[M+H]+. HRMS (ESI) m/z calcd for C26H31BrN2O3 [M+H]+ 501.1596, found 501.1570.
5α,8α-Epidioxy-17-(6-methoxynaphthalen-2-ylmethylene)hydrazonoandrostan-3β-ol (8h). White
1
crystals, yield: 55%, mp 112.1-114.3 °C; H NMR (400 MHz, CDCl3) δ 8.41 (1H, s, C22-H), 8.05-7.91
(2H, m, 4′,5′-Ar-H), 7.76 (2H, dd, J = 14.0, 8.7 Hz, 7′-Ar-H), 7.16 (2H, d, J = 9.3 Hz, Ar-H), 6.55 (1H, d,
J = 8.4 Hz, C6-H), 6.32 (1H, d, J = 8.5 Hz, C7-H), 3.96 (3H m, OCH3), 3.72 (1H, m, C3-αH), 2.83-2.71
(2H, m, C16-H), 1.12 (3H, s, C19-CH3), 0.94 (3H, s, C18-CH3). LC-MS (ESI) m/z: 501.3 [M+H]+.
HRMS (ESI) m/z calcd for C31H36N2O4 [M+H]+ 501.2753, found 501.2742.
Biological assays. The effect of derivatives on cell proliferation was evaluated by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Human hepatic carcinoma cells
(HepG2, SK-Hep1) and human breast cancer cells (MCF-7, MDA-MB-231) were used in the study. The
cells were cultured in DMEM/RPMI1640 supplemented with 10% FBS, 100 U/mL penicillin/
streptomycin at 37 °C in an incubator containing 5% CO2. Cells (1 × 104 cells/mL) were seeded into
96-well plates and were incubated at 37 °C overnight in a humidified incubator containing 5% CO2. Cells

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HETEROCYCLES, Vol. 94, No. 4, 2017
were dosed with compounds at final concentrations ranging from 5 μM to 60 μM in each well of the
plates. The cells were incubated for various periods and analyzed by MTT assay to analyze rates of cell
proliferation as described. Cell survival was determined by measuring the absorbance at 490 nm using a
microplate reader. A calibration curve was prepared using the SPSS to determine the IC50 of the target
compounds. Cytotoxicity as IC50 for each cell line is the concentration of compound which reduced by
50% the optical density of treated cells (48 h) with respect to untreated cells using the MTT assay.
ACKNOWLEDGEMENTS
The authors would like to acknowledge financial support from the Chinese Natural Science Foundation
Project (21272020), Beijing Key Laboratory of Environmental and Viral Oncology and Beijing Key
Laboratory for Green Catalysis and Separation.
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