Design, synthesis and biological evaluation of novel 5α, 8α-endoperoxide steroidal derivatives with hybrid side chain as anticancer agents

Article abstract of DOI:10.1016/j.steroids.2019.108471

A series of novel 5α, 8α-endoperoxide steroidal hybrid derivatives containing isatin or indole substituents on the C-17 side chain were synthesized and characterized. The preliminary anti-proliferative activity of the compounds against HepG2, MCF-7, HT-29 and HeLa cell lines were investigated. Compounds 7g and 7l displayed significant anti-proliferative activity in vitro against HepG2 and Hela cells, with IC₅₀ values lower than 8 μM. Furthermore, the biological functions of 7g were examined by flow cytometry and colony analysis. The results showed that 7g could induce HepG2 cell apoptosis, inhibited cell cycle progression, and colony growth. The studies indicated that structural modification at C-17 position could be a promising launch point for design steroidal anticancer agents.

Full text of DOI:10.1016/j.steroids.2019.108471

Journal Pre-Proof
Design, synthesis and biological evaluation of novel 5α, 8α-endoperoxide ster-
oidal derivatives with hybrid side chain as anticancer agents
Hongling Li, Haijun Wang, Jing Wang, Yu Lin, Yukun Ma, Ming Bu
PII:
S0039-128X(19)30161-8
DOI:
https://doi.org/10.1016/j.steroids.2019.108471
STE 108471
Reference:
Steroids
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20 April 2019
30 July 2019
2 August 2019
Please cite this article as: Li, H., Wang, H., Wang, J., Lin, Y., Ma, Y., Bu, M., Design, synthesis and biological
evaluation of novel 5α, 8α-endoperoxide steroidal derivatives with hybrid side chain as anticancer agents,
Steroids (2019), doi: https://doi.org/10.1016/j.steroids.2019.108471
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Design, synthesis and biological evaluation of novel 5α,
8α-endoperoxide steroidal derivatives with hybrid side chain as
anticancer agents
Hongling Lia, Haijun Wanga, Jing Wanga, Yu Lina, Yukun Mab and Ming Bua*
a College of Pharmacy, Qiqihar Medical University, Qiqihar 161006, China
b Research Institute of Medicine & Pharmacy, Qiqihar Medical University, Qiqihar 161006,
China
* Corresponding author: Tel.: +86 045 22663881
E-mail addresses: buming@qmu.edu.cn (M. Bu)
Abstract
A series of novel 5α, 8α-endoperoxide steroidal hybrid derivatives containing isatin or
indole substituents on the C-17 side chain were synthesized and characterized. The
preliminary anti-proliferative activity of the compounds against HepG2, MCF-7,
HT-29 and HeLa cell lines were investigated. Compounds 7g and 7l displayed
significant anti-proliferative activity in vitro against HepG2 and Hela cells, with IC50
values lower than 8 μM. Furthermore, the biological functions of 7g were examined by
flow cytometry and colony analysis. The results showed that 7g could induce HepG2
cell apoptosis, inhibited cell cycle progression, and colony growth. The studies
indicated that structural modification at C-17 position could be a promising launch
point for design steroidal anticancer agents.
Keywords: ergosterol peroxide, isatin, indole, hybrids, antiproliferative activity.

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1. Introduction
Nowadays, natural products have attracted extensive attention in disease
treatment and human health promotion. Natural product-based drug discovery is a
major route leading to developing potent drugs for diseases [1]. Natural
endoperoxides are cyclic organic compounds, such as artemisinin, talaperoxides B,
schinalactone A and gracilioethers A, which all have a peroxide bridge in their
structures (Figure 1) [2, 3]. A plenty of natural endoperoxides have been proved with
a range of bioactivities, such as anticancer, antifungal and antiviral activity [4-7].
Figure 1. Structures of natural endoperoxides with bioactivities.
Ergosterol peroxide (5α,8α-epidioxyergosta-6,22-diene-3β-ol, EP), is one
metabolite of series natural sterol endoperoxide derivatives, has been continuously
isolated from many kinds of medicinal fungi (Figure 2) [8, 9]. A plenty of literatures
have been reported that EP can inhibit cancer cells growth by cytotoxicity [10-13]. In
our previous study, we proved that EP could induce Foxo3a activity by inhibiting
pAKT to induce cancer cell death [14].
Figure 2. Structure of ergosterol peroxide (EP).
Isatin is well known as an important natural product, and which is also a

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common scaffold in various drugs, agrochemicals and dyes [15-17]. Many isatin
derivatives exhibit various pharmacological activities. It is noteworthy that, some
isatin derivatives have been successfully developed and used as anticancer drugs such
as semaxanib, intedanib and sunitinib (Figure 3). Isatin moiety has been proved as an
attractive pharmacophore in the design and development of new drugs [18-20]. In
addition, the indole structure is also found in plenty of natural products, and which is
also considered to be a potential scaffold for new drugs design and development [21].
Figure 3. Structures for some anticancer drugs derived from isatin/indole.
Steroidal compounds have always been widespread application in medicine field,
such as anti-inflammatory, contraceptive, diuretic, and anticancer agents [22-24]. The
special skeleton features of steroidal provide additional fascination for us to design and
synthesis new entities, which with the potential and prospect to become new drugs [25,
26].
In addition, hydrazine and its analogues have also been demonstrated to exhibit
wide activities including antibacterial, antivirus and antitumor for a long time [27].
Especially, steroidal hydrazones have attracted the great attention of many researchers
in recent years, and some novel steroidal hydrazones analogues (Figure 4) also
presented obviously pharmaceutical activities such as antibacterial, antivirous, and
cytotoxicity activities [17, 28].

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Figure 4. Structure of some steroidal hydrazones derivatives.
As an important active lead compound, EP is well known for its 5α,
8α-peroxybridge. In our previous studies, we have synthesized series of sterol 5α,
8α-endoperoxides from available natural sterols and found that several compounds
displayed significantly cytotoxic activity against kinds of human tumor cells [29, 30].
As we known, pharmacophores hybridization of natural products is the most efficient
approach for discovery of novel bioactive agents in the field of medicinal chemistry. In
the present study, we integrated the structural features of ergosterol peroxide,
hydrazone, and isatin/indole unit to design and synthesize a new series of steroidal
hybrids as shown in Figure 5, wishing to find novel functional steroidal molecules with
significant inhibited activity to tumor cells.
Figure 5. Design strategy of novel derivatives.
2. Experimental section
2.1. Chemistry
2.1.1. General
1
Reagents were used without further purification. H NMR (400 MHz) and 13C
NMR (100 MHz) spectra were measured on a Bruker Avance DRX400 spectrometer
with tetramethylsilane (TMS) as internal standards. Chemical shifts were given as δ
ppm values. Melting points were determined on an MP120 auto point apparatus. Mass
spectra of all compounds were tested on an Esquire 6000 mass spectrometer in ESI

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mode. Flash chromatography in the experiments was performed using silica gel (400
mesh).
2.1.2. Synthesis of 3β-acetoxyandrosta-5-en-17-one (2)
To a suspension of DHEA (1) (14.4 g, 0.05 mol) in CH2Cl2-pyridine 4︰1 (80
mL) was added Ac2O (6 mL, 0.07 mol) over 15 min. The mixture was stirred for 8 hat
room temperature, and then water (50 mL) was added. The resulting mixture extracted
with EtOAc (2 × 80 mL). The combined organic phase was washed with NaHCO3
saturated aqueous (2 × 50 mL) and brine (2 × 50 mL) and dried over anhydrous
Na2SO4. Evaporation of solvent gave crude 2 as a white solid (16 g, 97.6% yield). mp:
1
168-170 °C. H NMR (CDCl3, 400 MHz) δ 5.40 (1H, d, J = 5.0 Hz), 4.65-4.54 (1H,
m), 2.45 (1H, m), 2.34 (2H, d, J = 7.8 Hz), 2.11 (2H, m, 2H), 2.05 (3H, s), 1.93 (1H, d,
J = 6.2 Hz), 1.90 (1H, d, J = 3.2 Hz), 1.87-1.81 (2H, m), 1.71-1.62 (4H, m), 1.55 (1H,
m), 1.49 (1H, m), 1.34-1.25 (2H, m), 1.19-1.09 (1H, m), 1.03(3H, s), 1.00 (1H, d, J =
3.8 Hz), 0.90 (3H, s). MS m/z: 353.9 [M+Na]+.
2.1.3. Synthesis of 3β-acetoxyandrosta-5,7-diene-17-one (3)
To a solution of intermediate 2 (16 g, 0.05 mol) in cyclohexane (70 mL) was
heated to 70 °C for 20 min, and then was added NBS (12.5 g, 0.07 mol). The mixture
was refluxed for 1.5 h. The mixture was diluted with 150 mL water. The precipitate
was collected and washed with water. Then the solid was dissolved in DCM and
washed with brine, dried over anhydrous Na2SO4. Evaporationof solvent gave a light
brown solid (17.5 g, 85%). The mixture of the crude bromide (17.5 g, 0.043 mol)
prepared above was added into tetrabutylammonium fluoride (45 mL 1.0 M solution
in THF, 0.07 mol, 1.5 equiv). The resulting brown solution was stirred for 10 h, and
then followed by rotary evaporation under 45 °C to a yellow solid. The solid was
diluted with DCM (100 mL) and washed with water (2 × 50 mL), dried over
anhydrous Na2SO4.Purified by chromatographic column to give a pale yellow solid as
1
compound 3 (5.8 g). Yield: 35%, mp: 112-115 °C; H NMR (CDCl3, 400 MHz) δ
5.62 (1H, d, J = 3.0 Hz), 5.58 (1H, d, J = 3.2 Hz), 4.70 (1H, m), 2.56-2.49 (2H, m),
2.38 (1H, d, J = 12.5 Hz), 2.24-2.17 (2H, m), 2.07 (3H, s), 1.97-1.92 (2H, m), 1.90
(1H, d, J = 3.8 Hz), 1.74 (2H, d, J = 4.8 Hz), 1.70(1H, d, J = 5.0 Hz), 1.58 (2H, s),
1.40-1.34 (2H, m), 1.27 (1H, s), 0.99 (3H, s), 0.83 (3H, s). MS m/z 351.7 [M+Na]+.

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2.1.4. Synthesis of 3β-hydroxyandrosta-5,7-diene-17-one (4)
To a suspension of intermediate 3 (11 g, 0.034 mol) in MeOH (100 mL) was
added 25% (wt) NaOMe in MeOH (10 mL). The mixture was reflux for 1 h. 100 mL
of water was added into the mixture over 0.5 h, and the mixture was continually
stirred for 1 h. The precipitate was collected and washed with water (50 mL), and
dried under high vacuum at 40 °C to get a brown solid crude product. Purified by
chromatographic column to give a pale solid as intermediate 4 (9.3 g). Yield: 96%,
mp: 157-159 °C; 1H NMR (400 MHz, CDCl3) δ 6.00 (1H, d, J = 9.8 Hz), 5.70 (1H, d,
J = 9.8 Hz), 4.30 (1H, t, J = 7.9 Hz), 3.76-3.60 (1H, m), 2.67-2.41 (2H, m), 1.03 (3H,
s), 0.96 (3H, s). MS (ESI) m/z 309.9 [M+Na]+.
2.1.5. Synthesis of 5α,8α-cyclicobioxygen-6-vinyl-3β-DHEA (5)
To a solution of intermediate 4 (140 mg) in pyridine (20 mL) was added eosin (1
mg) in a quartz tube. The mixture kept in a water-cooled bath and vigorously stirred
by bubbling into the oxygen. At the same time, the mixture was irradiated with an
iodine tungsten lamp (220 V 500 W) for 0.5 h. The solution was poured into ice-water
(20 mL) and extracted with ethyl acetate (2 × 50 mL). The combined organic phase
was washed with brine (2 × 50 mL) and dried over anhydrous Na2SO4. Evaporated of
solvent and then purified by chromatographic column (40% EA in PE) to give 5 as
1
white needles (101.4 mg, 63%), m.p. 167.0-168.1 °C. H NMR (400 MHz, CDCl3) δ
6.53 (1H, d, J = 8.5 Hz), 6.35 (1H, d, J = 8.5 Hz), 4.01 (1H, m), 2.58-2.47 (1H, m),
2.24-2.12 (2H,m), 2.08-2.00 (1H, m), 1.98-1.94 (1H, m), 1.88 (1H, m), 1.86-1.81 (2H,
m), 1.72 (1H, m), 1.67-1.62 (1H, m), 1.58-1.54 (4H, m), 1.54-1.49 (1H, m), 1.38-1.22
(2H, m), 1.00 (3H, s), 0.92 (3H, s). 13C NMR (100 MHz, CDCl3) δ 218.3, 136.5, 129.8,
82.5, 78.9, 66.3, 51.5, 48.9, 47.7, 37.0, 36.8, 35.6, 34.7, 31.6, 29.9, 22.7, 19.0, 18.3,
15.0. MS (ESI) m/z 319.19 [M+H]+.
2.1.6. Synthesis of 5α,8α-epidioxy-17-hydrazonoandrostan-3β-ol (6)
To a solution of intermediate 5 (2.0 g, 6.7mmol) in EtOH (50 mL) was added 85%
hydrazine hydrate (3 mL). The mixture was heated to 45 °C and stirred for 2 h. The
white solid was collected by filtrating. Intermediate 6 was obtained as white solid (2.1
g) after recrystallizing in MeOH. LC-MS (ESI) m/z: 333.1 [M+H]+.
2.1.7. Synthesis of novel derivatives 7a-n

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To a suspension of intermediate 6 (0.2 mmol) in anhydrous EtOH (30 mL) was
added different substituent (0.2 mmol). The mixture was continually stirred under
50 °C for 2~3 h until no starting material. Then the solvent was evaporated by rotary
evaporation. The residue was purified by flash chromatography to afford compounds
7a-n.
2.1.7.1. 5α,8α-Epidioxy-17-(4-fluoro-benzylidene)hydrazonoand-rost-3β-ol (7a).
White powder, yield 70%, mp 72.3-74.8 °C, 1H NMR (400 MHz, CDCl3) δ 8.27 (1H,
s), 7.78-7.74 (2H, dd, J = 8.5, 5.5 Hz), 7.13-7.09 (2H, t, J = 8.6 Hz), 6.55 (1H, d, J =
8.2 Hz), 6.33 (1H, d, J = 8.0 Hz), 4.01 (1H, m), 2.78-2.63 (2H, m), 2.15 (1H, m),
2.05-2.04 (1H, m), 1.96-1.93 (2H, m), 1.89-1.86 (2H, m), 1.77-1.63 (4H, m),
13
1.63-1.56 (2H, m), 1.36-1.27 (2H, m), 1.13 (3H, m), 1.05 (1H, s), 0.94 (3H, s). C
NMR (100 MHz, CDCl3) δ 181.2, 165.5, 160.8, 156.7, 136.1, 130.1, 115.8, 115.7,
82.3, 79.1, 66.2, 51.5, 49.1, 45.9, 37.1, 36.9, 34.7, 33.9, 30.1, 29.7, 27.8, 23.0, 20.0,
18.1, 16.7. MS (ESI) m/z: 439.2 [M+H]+.
2.1.7.2. 5α,8α-Epidioxy-17-(benzylidene)hydrazonoandrost-3β-ol (7b). White powder,
1
yield 72%, mp 75.4-76.7 °C, H NMR (400 MHz, CDCl3) δ 8.28 (1H, s), 7.74 (1H,
m), 7.54 (2H, d, J = 5.7 Hz), 7.42 (2H, m), 6.54 (1H, d, J = 8.4 Hz), 6.32 (1H, d, J =
8.2 Hz), 3.98 (1H, dd, J = 11.3, 5.6 Hz, C3-αH), 2.83-2.60 (2H, m), 1.11 (3H, m),
13
0.93-0.88 (3H, m). C NMR (100 MHz, CDCl3) δ 182.0, 155.7, 138.5, 136.2, 132.4,
129.4, 128.4, 118.4, 113.8, 71.6, 53.7, 50.4, 44.3, 42.2, 37.2, 36.8, 33.9, 31.6, 31.5,
31.3, 28.0, 23.3, 20.6, 19.5, 16.5. MS (ESI) m/z: 421.2 [M+H]+.
2.1.7.3. 5α,8α-Epidioxy-17-(3-trifluoromethyl-benzylidene)hydra-zonoandrost-3β-ol
(7c). White powder, yield 78%, mp 80.1-82.2 °C, 1H NMR (400 MHz, CDCl3) δ 8.32
(1H, s), 8.03 (1H, s), 7.93 (1H, d, J = 7.5 Hz), 7.69 (1H, d, J = 7.6 Hz), 7.55 (1H, t, J
= 7.7 Hz), 6.56-6.54 (1H, d, J = 8.4 Hz), 6.35-6.33 (1H, d, J = 8.4 Hz), 4.05-3.99 (1H,
m), 2.81-2.65 (2H, m), 2.18-2.14 (1H, m), 2.08-2.05 (1H, m), 1.99-1.91 (2H, m),
1.89-1.82 (2H, m), 1.77-1.69 (4H, m), 1.67-1.56 (2H, m), 1.37-1.26 (2H, m), 1.15 (3H,
s), 1.06 (1H, s), 0.95 (3H, s). 13C NMR (100 MHz, CDCl3) δ 181.7, 161.0, 156.6,
136.2, 131.8, 131.2, 130.0, 129.4, 129.2, 124.6, 124.5, 82.3, 79.1, 66.3, 51.5, 49.1,
46.0, 37.2, 36.8, 34.7, 33.9, 30.1, 27.8, 23.0, 20.3, 18.3, 18.0. MS (ESI) m/z: 489.2
[M+H]+.

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2.1.7.4.
5α,8α-Epidioxy-17-(3-methoxy-4-hydroxy-benzylidene)hydrazonoandrost-3β-ol (7d).
White powder, yield 84%, mp 95.3-96.2 °C; 1H NMR (400 MHz, CDCl3) δ 8.20 (1H,
s), 7.43 (1H, d, J = 1.4 Hz), 7.28 (1H, s), 7.14 (1H, dd, J = 8.1, 1.5 Hz), 6.94 (1H, d, J
= 8.1 Hz), 6.54 (1H, d, J = 8.5 Hz), 6.32 (1H, d, J = 8.5 Hz), 6.06 (1H, s), 4.00 (1H,
m), 3.95 (3H, m), 2.76-2.71 (2H, m), 2.04 (1H, m), 1.92-1.86 (1H, m), 1.80-1.72 (4H,
m), 1.63-1.52 (3H, m), 1.36-1.27 (2H, m), 1.14-1.11 (3H, m), 1.04 (1H, s), 0.94 (3H,
13
s). C NMR (100 MHz, CDCl3) δ 180.2, 157.7, 148.5, 147.0, 136.1, 130.1, 129.7,
123.9, 114.4, 108.4, 82.3, 79.1, 66.2, 55.3, 51.5, 49.2, 45.9, 37.2, 36.8, 34.5, 34.0,
30.1, 27.8, 23.0, 20.3, 18.3, 18.0. MS (ESI) m/z: 451.2 [M+H]+.
2.1.7.5. 5α,8α-Epidioxy-17-(isatin)hydrazonoandrost-3β-ol (7e). Bright yellow
powder, yield 74%, mp 198-200 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.78 (1H, s),
7.77 (1H, d, J = 7.0 Hz), 7.37 (1H, d, J = 7.2 Hz), 7.01 (1H, d, J = 7.1 Hz), 6.88 (1H,
d, J = 7.4 Hz), 6.53 (1H, d, J = 8.5 Hz), 6.31 (1H, d, J = 7.6 Hz), 4.59 (1H, m),
2.41-2.39 (2H, m), 2.01-1.86 (5H, m), 1.76-1.23 (10H, m), 1.08 (3H, s), 0.88 (3H, s).
13C NMR (100 MHz, DMSO-d6) δ 177.0, 164.7, 147.2, 145.1, 136.5, 133.6, 130.2,
128.6, 122.5, 117.0, 111.1, 82.2, 80.9, 78.7, 65.1, 55.3, 51.5, 48.9, 46.9, 46.1, 37.1,
36.0, 30.4, 22.8, 20.3, 18.4, 18.2. MS (ESI) m/z: 462.3 [M+H]+.
2.1.7.6. 5α,8α-Epidioxy-17-(5-fluoro-isatin)hydrazonoandrost-3β-ol (7f). Yellow
powder, yield 75%, mp 169-170 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.82 (1H, s),
7.60-7.50 (1H, dd, J = 2.4, 2.0 Hz), 7.28-7.24 (1H, t, J = 7.9 Hz), 6.89 (1H, dd, J =
8.5, 4.2 Hz), 7.26 (1H, m), 6.89 (1H, dd, J = 8.5, 4.3 Hz), 6.54 (1H, d, J = 8.5 Hz),
6.31 (1H, d, J = 8.5 Hz), 2.48-2.36 (2H, m), 2.04-1.23 (16, m), 1.11 (3H, s), 0.85 (3H,
s). 13C NMR (100 MHz, DMSO-d6) δ 179.1, 164.7, 156.6, 147.2, 141.4, 136.6, 130.2,
120.5, 117.5, 115.3, 112.2, 82.2, 80.9, 78.7, 78.6, 65.1, 51.9, 48.8, 46.7, 37.1, 35.9,
34.8, 30.4, 22.8, 20.3, 18.4, 18.2. MS (ESI) m/z: 480.2 [M+H]+.
2.1.7.7. 5α,8α-Epidioxy-17-(5-chloro-isatin)hydrazonoandrost-3β-ol (7g). Yellow
powder, yield 69%, mp 171-172 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.93 (1H, s),
7.79 (1H, s), 7.43 (1H, d, J = 8.3 Hz), 6.90 (1H, d, J = 8.4 Hz), 6.53 (1H, d, J = 8.5
Hz), 6.30 (1H, d, J = 8.4 Hz), 4.58 (1H, m), 2.40-2.32 (2H, m), 2.04-1.85 (5H, m),
1.73-1.15 (10H, m), 1.06 (3H, s), 0.86 (3H, s). 13C NMR (100 MHz, DMSO-d6) δ
178.8, 164.4, 146.9, 143.8, 136.6, 133.0, 130.1, 128.2, 126.1, 118.2, 112.7, 82.2, 80.9,

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78.6, 65.1, 51.6, 48.9, 46.8, 46.3, 37.1, 35.9, 34.8, 28.4, 22.8, 20.4, 18.3, 18.2. MS
(ESI) m/z: 496.2 [M+H]+.
2.1.7.8. 5α,8α-Epidioxy-17-(5-bromo-isatin)hydrazonoandrost-3β-ol (7h). Bright
1
yellow powder, yield 74%, mp 156-158 °C; H NMR (400 MHz, DMSO-d6) δ 10.94
(1H, s), 7.94 (1H, s), 7.56 (1H, dd, J = 8.3, 1.9 Hz), 6.86 (1H, d, J = 8.3 Hz), 6.53 (1H,
d, J = 8.5 Hz), 6.31 (1H, d, J = 8.5 Hz), 4.58 (1H, m), 2.41 (1H, m), 1.98 (1H, m),
1.86-1.75 (2H, m), 1.71-1.20 (13H, m), 1.11 (3H, s), 0.85 (3H, s). 13C NMR (100
MHz, DMSO-d6) δ 178.5, 164.2, 146.6, 144.1, 136.6, 135.8, 130.9, 130.1, 118.6,
113.6, 82.2, 80.9, 78.6, 65.1, 51.6, 48.8, 46.7, 37.3, 37.1, 34.0, 30.4, 28.0, 26.8, 22.9,
20.4, 18.4, 18.2. MS (ESI) m/z: 540.2 [M+H]+.
2.1.7.9. 5α,8α-Epidioxy-17-(5-nitro-isatin)hydrazonoandrost-3β-ol (7i). Yellow
powder, yield 72%, mp 177-179 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.49 (1H, s),
8.62 (1H, s), 8.29 (1H, d, J = 8.7 Hz), 7.06 (1H, d, J = 8.6 Hz), 6.54 (1H, d, J = 8.4
Hz), 6.32 (1H, d, J = 8.3 Hz), 4.63 (1H, s), 2.43 (2H, m), 2.05 (1H, m), 1.86-1.75 (5H,
13
m), 1.72-1.25 (9H, m), 1.15 (3H, s), 0.87 (3H, s). C NMR (100 MHz, DMSO-d6) δ
178.6, 164.8, 150.4, 145.9, 142.4, 136.6, 130.1, 129.7, 123.8, 116.8, 111.3, 82.2, 78.7,
65.1, 51.6, 48.8, 46.8, 37.3, 34.9, 33.9, 30.4, 28.1, 26.8, 22.9, 20.4, 18.4, 18.2. MS
(ESI) m/z: 507.3 [M+H]+.
2.1.7.10. 5α,8α-Epidioxy-17-(5-methoxy-isatin)hydrazonoandro-st-3β-ol (7j). Yellow
powder, yield 71%, mp 156-159 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.59 (1H, s),
7.41 (1H, d, J = 2.0 Hz), 6.98 (1H, dd, J = 8.4, 2.3 Hz), 6.80 (1H, d, J = 8.5 Hz), 6.53
(1H, d, J = 8.4 Hz), 6.31 (1H, d, J = 8.5 Hz), 4.63 (1H, s), 3.70 (3H, s), 2.44-2.33 (1H,
m), 1.99 (1H, m), 1.95-1.50 (10H, m), 1.48-1.22 (5H, m), 1.11 (3H, s), 0.85 (3H, s).
13C NMR (100 MHz, DMSO-d6) δ 177.5, 164.8, 155.0, 147.1, 138.7, 136.6, 130.2,
119.3, 117.5, 114.1, 111.8, 82.2, 78.7, 65.1, 56.0, 51.6, 48.9, 46.6, 37.3, 37.1, 34.9,
34.1, 30.4, 28.0, 22.9, 20.4, 18.4, 18.2. MS (ESI) m/z: 492.2 [M+H]+.
2.1.7.11. 5α,8α-Epidioxy-17-(5-trifluoromethoxy-isatin)hydrazo-noandrost-3β-ol (7k).
Yellow powder, yield 70%, mp 148-150 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.99
(1H, s), 7.77 (1H, s), 7.40 (2H, d, J = 7.5 Hz), 6.97 (2H, d, J = 8.5 Hz), 6.52 (1H, d, J
= 8.5 Hz), 6.30 (1H, d, J = 8.4 Hz), 3.66-3.49 (1H, m), 2.35 (1H, m), 2.03-1.50 (10H,
13
m), 1.48-1.22 (6H, m), 1.07 (3H, s), 0.86 (3H, s). C NMR (100 MHz, DMSO-d6) δ

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179.4, 164.6, 147.2, 144.0, 143.2, 136.6, 130.2, 126.7, 121.2, 117.5, 112.3, 82.2, 78.7,
65.1, 51.6, 48.9, 46.7, 37.3, 37.1, 34.9, 33.9, 30.4, 28.0, 26.8, 22.9, 20.3, 18.4, 18.1.
MS (ESI) m/z: 546.2 [M+H]+.
2.1.7.12. 5α,8α-Epidioxy-17-(5-fluoro-indole)hydrazonoandrost-3β-ol (7l). Yellow
powder, yield 68%, mp 151-153 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.98 (1H, s),
8.56 (1H, d, J = 6.2 Hz), 7.83 (2H, m), 7.48 (1H, dd, J = 8.7, 4.5 Hz), 7.07 (1H, d, J =
9.1 Hz), 6.55 (1H, d, J = 6.0 Hz), 6.18 (1H, d, J = 6.0 Hz), 3.78 (1H, m), 2.83-2.61
13
(2H, m), 191-1.52 (11H, m), 1.48-1.24 (4H, m), 1.01 (3H, s), 0.93 (3H, s). C NMR
(100 MHz, DMSO-d6) δ 180.4, 158.9, 156.6, 154.2, 133.7, 133.5, 125.1, 124.9, 113.1,
112.1, 110.7, 106.8, 75.4, 72.9, 65.2, 57.3, 49.2, 45.0, 43.6, 33.8, 32.3, 30.9, 27.5,
26.7, 22.0, 20.0, 19.7, 16.6. MS (ESI) m/z: 478.2 [M+H]+.
2.1.7.13. 5α,8α-Epidioxy-17-(5-chloro-indole)hydrazonoandrost-3β-ol (7m). Yellow
powder, yield 70%, mp 143-145 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.98 (1H, s),
8.56 (1H, s), 8.19 (1H, s), 7.93 (1H, d, J = 7.0 Hz), 7.50 (1H, d, J = 6.7 Hz), 7.24 (1H,
d, J = 7.6 Hz), 6.56 (1H, d, J = 7.2 Hz), 6.18 (1H, d, J = 7.4 Hz), 4.31 (1H, s), 2.74
(1H, m), 2.04-1.78 (2H, s), 1.76-1.50 (10H, m), 1.49-1.25 (4H, m), 1.01 (3H, s), 0.94
13
(3H, s). C NMR (100 MHz, DMSO-d6) δ 180.1, 154.5, 153.5, 137.7, 132.1, 128.3,
127.5, 127.2, 125.2, 115.1, 111.8, 105.3, 75.4, 72.9, 65.2, 59.7, 57.3, 49.3, 45.0, 43.6,
33.8, 32.3, 30.9, 27.4, 26.7, 22.0, 20.0, 19.7, 16.6, 14.1. MS (ESI) m/z: 494.2 [M+H]+.
2.1.7.14.
5α,8α-Epidioxy-17-(5-methoxy-indole)hydrazonoandro-st-3β-ol
(7n).
Yellow powder, yield 71%, mp 153-155 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.56
(1H, s), 8.56 (1H, s), 7.77 (2H, d, J = 11.6 Hz), 7.35 (1H, d, J = 8.8 Hz), 6.84 (1H, d,
J = 8.8 Hz), 6.55 (1H, s), 6.14 (1H, s), 3.90-3.68 (3H, m), 2.88-2.56 (2H, m),
1.94-1.91 (2H, m), 1.79-1.50 (11H, m), 1.48-1.25 (4H, m), 1.01 (3H, s), 0.93 (3H, s).
13C NMR (100 MHz, DMSO-d6) δ 180.1, 154.6, 154.5, 132.1 131.9, 125.3, 112.5,
112.3, 111.9, 103.9, 75.4, 72.9, 65.2, 61.3, 57.4, 55.0, 49.3, 45.0, 43.5, 33.9, 32.3,
30.9, 27.4, 26.7, 22.1, 20.1, 19.7, 16.6, 15.1. MS (ESI) m/z: 490.2 [M+H]+.
2.2. Biological Evaluation
2.2.1. MTT Cytotoxicity Assay.
Cytotoxicity activities of all synthesized compounds were tested in the human
HepG2, HT-28, MCF-7 and HeLa cancer cell lines by MTT assay. Compounds were

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solubilized in DMSO at gradient concentrations from 5 μM to 60 μM. Cells were
inoculated into 96-well plates for 24 h. The cells were treated with gradient
concentrations of compounds for 48 h and then were added with 10 μL of MTT for 2
h. The formazan dye product was measured by the absorbance at 490 nm on a Spectra
Max 340 microplate reader. The IC50 values of compounds were derived by SPSS
nonlinear regression analysis.
2.2.2. Flow Cytometry Analysis.
Cell cycle analysis was detected by a cell cycle staining (PI) kit purchased from
BD Pharmingen according to the instructions. HepG2 cells were inoculated into
6-well plates at a concentration of 8 × 104 cells/mL to attach overnight. The cells were
treated with different concentrations of compound 7g for 24 h. The cells were
collected, washed twice with PBS, and centrifuged (300 g, 5 min). To detect cell cycle
analysis, the collected pellets were incubated (106 cells/mL) with propidium iodide
staining solution (50 µg/mL PI) at room temperature for 1 h. The cells were analyzed
by flow cytometry (BD Biosciences).
2.2.3. Colony Analysis.
HepG2 cells were inoculated into 6-well plates at a concentration of 500
cells/well. Then 15 µM of compound 7g were added into each plate and mixed with
low melting (0.25%) agarose gel. The 6-well plates were maintained in incubator for
14 days. The formation of colonies were counted, stained, and analyzed under a
microscope.
3. Results and Discussion
3.1 Chemistry.
The general procedure for the synthesis of steroidal hybrids derivatives is shown in
Scheme 1. Dehydroepiandrosterone (DHEA, 1) as starting material, was acetylize with
acetic anhydride to give compound 2, which then underwent bromination and
debromination with NBS, n-Bu4NBr and n-Bu4NF to afford 5,7-diene acetate 3.
△
Subsequently, compound 3 was converted to compound 4 via deacetylation reaction
with potassium hydroxide in 30% overall yield from 1. The key compound 4 was
converted to endoperoxide 5 through photooxidation reaction with eosin as

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photosensitizer. Then, the reactions of compound 5 with hydrazine hydrate yielded the
corresponding steroidal hydrazine 6. At last, the reaction of steroidal hydrazine 6 with
different substituent gave our target compounds 7a-n.The structures were characterized
by MS, 1H NMR and 13C NMR spectrum.
Scheme 1. Synthesis of compounds 7a-n. Reagents and conditions: (i) Ac2O, pyridine, DCM, rt; (ii)
Cyclohexane, NBS, reflux, 1 h; (iii) NaOMe, MeOH, reflux, 1 h; Bu4NF, THF, rt, 12 h; (iv)
pyridine, O2, light, 0 °C, 0.5 h; (v) NH2NH2-H2O, EtOH, 45 °C, 1 h; (vi) EtOH, AcOH, appropriate
substituents, rt, 1~2 h.
3.2. Biological evaluation
3.2.1. In vitro anti-proliferative activity assays of 7a-n
The newly synthesized compounds 7a-n and EP were investigated for their
anti-proliferative activities against human hepatocellular carcinomacells (HepG2),
human breast carcinoma cells (MCF-7), human colon carcinoma cells (HT-29), and
human cervical carcinoma cells (HeLa) by the MTT assay. Cisplatin was used as
positive control drug. The anti-proliferative activity data of compounds were
summarized as IC50 values (μM) in Table 1.
Results of the MTT assay indicated that most of synthesized compounds showed
anti-proliferative activity against all the tested adherent cancer cell lines. Compounds

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7g and 7l exhibited the most prominent inhibitory activity against the tested cell lines
with IC50 values of 7.97 μM and 6.46 μM for HepG2 cells, 5.69 μM and 7.43 μM for
Hela cells, respectively. The results suggested the following rough structure-activity
relationships (SAR) considerations. Compound 5, which has only one carbonyl group
at the C-17 position, showed hardly any inhibitory activity (IC50 values more than 60
μM). On the contrary, most of compounds with hybrid side-chain exhibited inhibitory
activity against the tested cell lines. The results indicated that the introduction of
hybrids to the C-17 position resulted in increased inhibitory activity. In addition,
compared to EP, some of compounds exhibited better antitumor activity. It appeared
that chemcial structural modification to the C-17 position could be an opportunity for
development of ergosterol peroxide analogues with more remarkable bioactivity.
Besides, it is notable that the type of substituent at C-17 position provided
opportunity to further increase the inhibitory activity. Compounds 7e-n with isatin or
indole substituents on the side chain exhibited more obvious cytotoxic activity as
compared to compounds 7a-d that with aromatic substituents. Especially, most of
hybrids 7e-n displayed significant cytotoxic activities against HepG2 and HeLa cell
lines. The IC50 values for compound 7g bearing a 5-Cl-isatin substituent is up to 7.97
and 5.69 μM against HepG2 and Hela cell lines, respectively. In addition, compound 7l
bearing a 5-F-indole substituent is up to 6.46 and 7.43 μM against HepG2 and HeLa
cell lines, respectively. The preliminary results accounted for the strategy that
combined isatin/indole and peroxide steroidal was effective, and the novel hybrids
might be used as an active scaffold for further optimization of potential anticancer
agents.
Table 1. In vitro anti-proliferativeactivities of compounds.
IC50 (μM)a
Compd
HepG2
HT-29
MCF-7
HeLa
>60
44.30
>60
>60
32.21
>60
>60
37.40
>60
>60
>60
>60
5
7a
7b
32.50
>60
>60
36.47
7c
23.02
13.23
10.04
7.97
>60
37.20
15.10
14.35
9.05
16.83
24.50
15.90
23.02
10.80
>60
27.62
12.58
11.46
5.69
13.90
16.43
15.54
12.72
7.43
7d
7e
7f
7g
7h
7i
7j
7k
7l
25.30
19.60
20.42
19.65
41.10
31.93
40.05
17.50
22.28
>60
9.22
21.42
13.12
17.11
6.46
9.27
15.45
10.64
19.42
7m
7n
26.33

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EP
Cisplatin
23.35
3.04
26.42
4.52
18.00
6.56
19.54
5.68
a Data represent the mean values of three independent determinations.
Among the compounds under study, most of compounds showed more obvious
anti-proliferative activity against HepG2 cell lines. Compound 7g and HepG2 cell lines
were further chosen for more biological functions experiments.
3.2.2. Effect of 7g on the cell cycle progression.
The effect of synthesized compound 7g on tumor cell cycle progression was
examined by flow cytometry. The HepG2 cells were treated with 7g at a very low
concentration (3 μM) for 24 h. As shown in Figure 6A, the number of cells in the G1
phase increased significantly, suggesting a distinct inhibitory effect of 7g on cell cycle
progression. On the other hand, as shown in Figure 6B, we also detected that the
number of HepG2 cells decreased in S-G2 phases when the cells were treated with 7g.
Figure 6. Compound 7g inhibited HepG2 cell cycle progression. (A) Typical
distribution of cell cycles treated with or without 7g. (B) Statistical analysis of cells
distributed in each phase of cell cycles when the cells were treated with or without
7g.
3.2.3. Effect of 7g on the cell apoptosis.
Furthermore, to test the induction of apoptosis, HepG2 cells treated with 7g for 24
h was doubly stained with annexin V/PI. As shown in Figure 7, compound 7g could

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effectively induce apoptosis of HepG2 cells. At the 8 μM concentration 7g causes
35.81% late apoptosis annexin V-positive and PI-positive cells, suggesting compound
7g is a potent apoptotic inducer in HepG2 cells.
Figure 7. Compound 7g induced HepG2 cell apoptosis. Typical distribution of cell
apoptosis treated with or without 7g analyzed by annexin V/PI staining.
3.2.4. Effect of 7g on the cell colony.
As we known, HepG2 cell is a non-tumorigenic cell line, which would be difficult
for us to test the inhibitory effect of compound 7g on tumor growth in vivo. Hence, we
evaluated the effect of compound 7g on cancer colony formation in vitro. The HepG2
cells were treated with 7g at a concentration of 8 μM for 20 days. Then the colony
formation was examined under light microscopy. As shown in Figure 8A and 8B, the
formed number of colonies of HepG2 cells treated with 7g distinctly lower than the
untreated cells.

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Figure 8. Compound 7g inhibited cell colony formation. (A) Representative
images of HepG2 cells colonies after treatment with 7g. The plates were stained
with Coomassie Blue. (B) Data are presented as the mean ± SD of three
experiments. *p<0.01.
4. Conclusions
In summary, we have successfully prepared
a
series of 5α,
8α-endoperoxidesteroidal hybrid derivatives possessing various substituents on the
C-17 side chain. The in vitro anti-proliferative activities of all synthesized compounds
were investigated. Some of the synthesized compounds exhibited significant anticancer
activities against all the four tested cell lines. In particular, compound 7g and 7l were
the most promising derivatives, with IC50 values lower than 8 μM against HepG2 and
HeLa cell lines. Furthermore, the biological experiments showed that compound 7g
inhibited cycle progression of HepG2 cells, induced cell apoptosis, and colony growth.
It appeared that chemical structural modification of this type to the C-17 position could
be an opportunity for development of novel steroidal anticancer agents.
Supplementary data
Supplementary data: the 1H NMR and 13C NMR spectra of new compounds associated
with this article can be found, in the online version, at
Supplementary data

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The authors are grateful to Administration of Traditional Chinese Medicine of
Heilongjiang Province (ZHY18-164).
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Graphical Abstract