Synthesis and antiproliferative evaluation of novel steroid-benzisoselenazolone hybrids

Article abstract of DOI:10.1016/j.steroids.2019.108502

The two different types of steroidal benzisoselenazolone hybrids were synthesized by incorporating benzisoselenazolone scaffold into dehydroepiandrosterone and B-norcholesterol. The antiproliferative activity of the synthesized compounds against some carcinoma cell lines were investigated. The results showed that some of these compounds have better inhibitory activity than abiraterone on the proliferation of tumor cells associated with human growth hormone, and have less cytotoxicity on normal human cells. In particular, the IC₅₀ values of the compound 8a and 8f are 5.4 and 6.5 μmol/L against human ovarian carcinoma (SKOV3) cell line, and possess SI values of 13.9 and 10.5, respectively. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.

Full text of DOI:10.1016/j.steroids.2019.108502

Journal Pre-proofs
Synthesis and Antiproliferative Evaluation of Novel Steroid-Benzisoselenazo-
lone Hybrids
Jianguo Cui, Meizhen Wei, Liping Pang, Chunfang Gan, Junan Xiao, Haixin
Shi, Junyan Zhan, Zhiping Liu, Yanmin Huang
PII:
S0039-128X(19)30192-8
DOI:
https://doi.org/10.1016/j.steroids.2019.108502
STE 108502
Reference:
Steroids
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Revised Date:
Accepted Date:
7 June 2019
31 August 2019
17 September 2019
Please cite this article as: Cui, J., Wei, M., Pang, L., Gan, C., Xiao, J., Shi, H., Zhan, J., Liu, Z., Huang, Y., Synthesis
and Antiproliferative Evaluation of Novel Steroid-Benzisoselenazolone Hybrids, Steroids (2019), doi: https://
doi.org/10.1016/j.steroids.2019.108502
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Synthesis and Antiproliferative Evaluation of Novel
Steroid-Benzisoselenazolone Hybrids
Jianguo Cuia,b*
Meizhen Weia
Liping Pang a
Chunfang Gana
Junan Xiaoa
Haixin Shib
Junyan Zhana
Zhiping Liua
Yanmin Huanga*
a. Guangxi Key Laboratory of Natural Polymer Chemistry and Physics, Nanning Normal University, Nanning, 530001, PR
China;
b. Guangxi Colleges and University Key Laboratory of Beibu Gulf Oil and Natural Gas Resource Effective Utilization, Beibuwan
University, China.
*Corresponding author at: College of Chemistry and Material Science, Nanning Normal University, Nanning 530001, China.
E-mail address: Huangyanmin828@163.com (YM. Huang); cuijg1954@126.com (Jianguo Cui).
Abstract: The two different types of steroidal benzisoselenazolone hybrids were synthesized by
incorporating benzisoselenazolone scaffold into dehydroepiandrosterone and B-norcholesterol.
The antiproliferative activity of the synthesized compounds against some carcinoma cell lines
were investigated. The results showed that some of these compounds had better inhibitory activity
than abiraterone on the proliferation of tumor cells associated with human growth hormone, and
had less cytotoxicity on normal human cells. In particular, the IC50 values of the compound 8a and
8f are 5.4 and 6.5 µmol/L against human ovarian carcinoma (SKOV3) cell line, and possess SI
values of 13.9 and 10.5, respectively. The information obtained from the studies may be useful for
the design of novel chemotherapeutic drugs.
Keywords: Selenosteroids; Steroid-benzisoselenazolone hybrids; Benzisoselenazolone;
dehydroepiandrosterone; B-norcholesterol.
1. Introduction
Selenium is a necessary trace element for the human body and some animals to sustain life.
Compared with inorganoselenium compounds, organoselenium compounds have some excellent
characteristics, such as higher bioavailability, stronger bioactivity, lower toxicity, smaller
environmental pollution, and anti-oxidation, anti-inflammatory and anti-cancer, etc. [1-2]. Karam et
al. found that synthetic organoselenium compounds have a stronger antitumor activity and lower
toxicity than inorganoselenium compounds [3]. At present, some organoselenium compounds have

[5-7]
been used in anti-tumor clinical studies and achieved some good results [4], such as ebselen
ethaselen [8-11] and selenazofurin [12-13], etc.
,
O
N
Se
O
N
O
N
O
N
NH2
O
HO
OH
Se
Se
Se
HO
Ebselen
Ethaselen
Selenazofurin
It can be seen from the structures of ebselen and ethaselen that there is an organoselenium
heterocyclic structure of the benzisoselenazolone in both structures, which is the major
pharmacophore in the compounds. Some researchers had further introduced the
[15-16]
benzisoselenazolone into some different compounds, such as resveratrol [14], sugar
,
1,3,4-thiadiazoles [17-18], 1,2,4-triazole [19], and some of the obtained compounds possessed good
anti-tumor activity.
Steroids are an important class of essential physiologically active substances in the human
life process, which have high permeability to cells and binding ability to the cell nucleus and cell
membrane. Steroidal compounds have been widely used in medicine as anti-inflammatory,
anabolic and contraceptive drugs. Changing the steroidal side chain or substitution of the steroidal
skeleton, introducing heteroatom or replacing one or more carbon atoms by a heteroatom in
steroidal molecule may result in the alteration of its biological activity [20-25]
.
We have been carrying out the structural modification of steroidal compounds and the
research on steroidal compounds as anti-tumor agents. Our research results showed that when
different heterocyclic functional groups were introduced into the 17-position of steroid [26-29] or the
6-position of B-norcholesterol [30-31], the compounds obtained had a good inhibitory effect on the
proliferation of some tumor cells.
Recently, a few of reports about selenium-containing steroidal compounds have appeared.
[33]
[34]
Some scholars introduced selenocyano [32], selenourea groups
or selenide
into steroids
respectively, and the compounds produced displayed excellent antiproliferative activity on the
tested tumor cells. O. Lopez et al. synthesized benzisoselenazolone derivatives of diosgenin,
hecogenin and smilagenin, and these compounds behaved as moderate antiproliferative agents,
with GI50 values ranging from 27 to >100 μM [35]. We recently introduced the selenadiazole into
pregnenolone, and the products obtained showed moderate cytotoxicity [36]
.

However, although some steroidal selenium compounds have been reported recently, the
synthesis of organoselenium steroids by introducing Se atoms or selenium-containing groups into
steroids and studying their bioactivity, especially anti-tumor activity or antibacterial activity,
much work remains to be done in this field [37]
.
In view of above facts, we report herein the synthesis of some selenium-containing steroidal
hybrids by incorporating benzisoselenazolone scaffold into dehydroepiandrosterone or
B-norcholesterol, and their antiproliferative activities in vitro were evaluated against four types of
human hormone-related cancer cell lines, including human ovarian carcinoma (SKOV3), human
prostate carcinoma (PC-3), human breast infiltrating duct carcinoma (T47D), human breast
adenocarcinoma (MCF-7) and a normal human kidney epithelial cell lines (HEK293T).
2. Result and discussion
2.1 Chemistry
2.1.1 Synthesis of dehydroepiandrosterone-benzisoselenazolone hybrids
First, the key intermediates 4a-4k with various substituent groups on benzene ring were
synthesized according to the method of the reference [38] (Scheme 1). The 4a-4k were not
purified and went straight to the next step reaction.
COOH
NH2
COOH
N2
NaNO2
HCl
R
R
COOH
COCl
SeCl
Cl
SOCl2
80oC
R
R
1a-1k
2a-2k
Se
2
KBH4
Ar
4a-4k
Se
K2Se2
3a-3k
R =
H
a
3-Cl 4-Cl 6-Cl 3-F 4-F
b
c
d
e
f
5-F 5-CH3 4-OCH3 5-OCH3 4-NO2
g
h
i
j
k
Scheme 1 Synthesis of the intermediates 4a-4k
[39]
Dehydroepiandrosterone is a substance with a wide range of physiological activities
.
Here, we synthesized the hybrids 8a-8j of dehydroepiandrosterone by the reaction of compound 7
with 4a-4j. Scheme 2 outlines the synthetic procedure of compounds 8a-8j. First,
dehydroepiandrosterone (5) was transformed to oxime
6
by the reaction of

dehydroepiandrosterone with hydroxylamine hydrochloride. Then, compound 6 was reduced to
compound 7 using sodium cyanoborohydride as reductant in the presence of molybdenum
pentachloride and sodium hydrogen sulfate [40]. Last, the reaction of compound 7 with the
intermediates 4a-4j afforded corresponding dehydroepiandrosterone-benzisoselenazolone hybrids
8a-8j.
The structure of all synthesized compounds had been confirmed based on analytical and
spectral data. In the NMR spectrum of compound 8a, resonances signals of Ar-H at δ 7.41, 7.73,
8.04ppm and Ar-C at δ 121.07-141.12ppm demonstrate the presence of a benzisoselenazolone
group. The triplet of 4.61 ppm (J = 9.4) in the 1H NMR spectrum is the chemical shift of 17-H. It
can be seen from the NOESY spectrum of compound 8i that the 18-methyl group is not correlated
with 17-H, so 17-H should be in the α-configuration, that is, the 17-substituent of compounds 8 is
in the β-configuration [41]. Moreover, the HREI mass spectrum of compound 8a exhibits a
molecular ion peak ([M+Na]+) at m/z 494.1574 (calcd. for C26H33NO2SeNa 494.1574). All spectra
confirm the structure of compound 8a. Similarly, the structure of compounds 8b-8j had been
determined by their spectral data.
NH2
NOH
O
NH2OH.HCl
NaBH3CN
MoCl5, NaHSO4
HO
HO
HO
6
(70%)
7
5
R
Se
N
COCl
SeCl
O
R
4a-4j
(yield: 17%-46%)
8a-8j
HO
Scheme 2 Synthesis of dehydroepiandrosterone-benzisoselenazolone hybrids 8a-8j
2.1.2 Synthesis of B-norcholestane-3β,5β-diol-benzisoselenazolone hybrids
In previous studies, we found that when various functional groups were introduced into the
6-position of B-norcholesterol, the compounds obtained displayed distinct antiproliferative
activity. Therefore, we further introduced the benzisoselenazolone pharmacophore into the
6-position of B-norcholesterol and synthesized compounds 14a-14k (Scheme 3). Meanwhile, the
anti-tumor activity of the compounds in vitro was investigated.

O3, CH2Cl2/MeOH (4:1)
-78 oC, then Me2S
NH2OH.HCl
65oC
neutral Al2O3
benzene, rt
HO
HO
O
CHO
HO
CHO
OH
11
9
10
COCl
SeCl
R
NaBH3CN
MoCl5, NaHSO4
HO
O
N
HO
NH2
4a-4k
OH
OH
12 (85%)
R
HO
N
OH
H
OH
Se
13 (80%)
14a-14k (28%-70%)
Scheme 3 Synthesis of B-norcholestane-3β,5β-diol-benzisoselenazolone hybrids 14a-14k
The configuration of compound 11 at C-6 had been described in reference [42-43] and the
synthesis of compound 12 referred to reference [44]. After compound 12 was reduced into 13
using the method in Scheme 2, the 4a-4k were further incorporated into 13 to give target products
14a-14k. The structure of the synthesized compounds had been determined by their analysis and
spectra data.
2.2 The evaluation of the antiproliferative activity in vitro
All synthesized compounds had been evaluated for their antiproliferative activities in vitro
against SKOV3, PC-3, T47D, MCF-7 and HEK293T cell lines using MTT assay. Abiraterone, a
CYP17 inhibitor with a similar structure to compounds 8a-8j, has been clinically used in the
treatment of prostate cancer，using as a positive control. The results were summarized as IC50
values in µM in Table 1.
Table 1 In vitro antiproliferative activities (IC50 in μM) of compounds 8a-8j and 14a-14k
Compd.
SKOV-3
PC-3
T47D
MCF-7
HEK-293T
7
>100
5.40±3.61
>100
>100
40.75±0.83
>100
>100
44.37±1.18
>100
>100
40.48±0.66
>100
76.58±1.27
75.16±1.32
>100
8a
8b
8c
8d
8e
10.41±0.37
>100
16.78±1.04
78.60±6.99
>100
26.80±1.72
84.42±4.27
>100
23.39±1.69
15.64±1.54
>100
70.44±0.16
>100
>100
>100

8f
6.49±0.59
14.26±0.77
35.39±1.35
33.00±2.01
68.19±0.09
8g
8h
>100
14.13±0.29
22.95±1.28
36.52±0.64
15.14±0.56
>100
>100
30.19±0.48
29.53±0.19
35.16±0.12
ND
>100
>100
>100
60.03±0.28
51.36±0.60
74.10±1.31
17.75±1.12
>100
36.46±2.35
33.86±0.20
36.01±0.31
ND
23.27±1.53
34.24±1.84
41.58±2.51
24.36±1.70
>100
8i
8j
14a
14b
14c
>100
>100
21.29±0.39
>100
19.60±1.65
61.04±4.13
>100
16.68±0.70
84.60±1.69
>100
24.63±0.06
75.90±1.10
>100
>100
14d
14e
>100
>100
>100
14f
10.01±0.05
17.28±0.92
8.72±0.21
6.50±0.50
>100
14.45±1.05
14.59±0.33
7.01±0.14
9.07±0.06
>100
16.96±0.59
10.25±0.34
10.70±0.63
11.27±0.66
61.56±4.14
45.45±3.54
34.66±1.69
19.89±0.54
22.52±0.24
9.59±1.04
11.47±0.83
>100
40.72±1.19
38.03±0.29
53.49±1.09
42.03±0.18
>100
14g
14h
14i
14j
14k
Abiraterone
39.94±10.12
51.51±15.98
>100
27.73±0.06
44.70±0.67
>100
37.61±0.12
>100
ND: not determined.
It can be seen from the Table 1 that the synthesized compounds display a different level of
inhibitory activity against the proliferation of MCF-7, PC-3, SKOV-3 and T47D cancer cells. For
compounds 8a-8j, there is an important relationship between the antiproliferative activity of
compounds and substituent on the benzene ring of benzisoselenazolone. When there is no
substituent or there is an electron-withdrawing substituent, such as halogen, on the 6-position of
the benzisoselenazolone group, the compounds show an excellent antitumor activity, such as 8a,
8c and 8f, which possess IC50 value of less than 10µM to SKOV-3 cell. However, for the same
type of substituent, the compound has little cytotoxicity to the tumor cells tested when the
substituent is located elsewhere (8b, 8d, 8e, 8g). Additionally, when the substituent is the
electron-donating group, the compounds show a moderate cytotoxicity (8h, 8i, 8j). Furthermore,
the cytotoxicity of compounds 8a-8j are higher than that of its precursor compound 7.
For compounds 14a-14k, the cytotoxicity of the compounds has nothing to do with the
electronic factors of the substituent, and the main determinant factor is the substituting position of
functional groups. Similar to compounds 8a-8j, when the substituent is at the 6-position of the
benzisoselenazolone group, the compound has good cytotoxicity (14f, 14i). In addition, compound

14h with 5-methyl substitution also shows good antiproliferative activity to the tested tumor cells.
At the same time, it can be seen from Table 1 that the most of these compounds show higher
cytotoxicity to these hormone-related tumor cells than the positive control abiraterone.
The Selectivity Index (SI) is defined as the ratio of the cytotoxicity of a compound with
respect to normal cells (IC50 HEK293T) versus cancer cells and used to determine the criterion of
effectiveness of the compounds. It determines the damage degree of a compound to human normal
cells. The higher SI value is, the less damage the compound will do to normal cells. The calculated
SI values of compound 8a and 8f for SKOV-3 cells are 13.9 and 10.5, respectively. So,
comparison of the cytotoxicity of the compounds with the SI values suggests that the compound
8a and 8f may be potent anticancer agents against human ovarian carcinoma (SKOV-3).
3. Conclusion
In
this
paper,
dehydroepiandrosterone-benzisoselenazolone
and
B-norcholestane-3β,5β-diol-benzisoselenazolone hybrids were synthesized and their antitumor
activity in vitro was assayed. The results show that some of these compounds have better
inhibitory activity than abiraterone on the proliferation of tumor cells associated with human
growth hormone, and have less cytotoxicity on normal human cells. Compounds 8a and 8f have a
lower IC50 value and a higher SI value to human ovarian carcinoma SKOV-3 cells, so they are
expected to be candidates for such anti-tumor drugs, which deserve further study.
4. EXPERIMENTAL
4.1 Chemistry
4.1.1 Reagents and Instruments
The cholesterol, dehydroepiandrosterone and various substituted aminobenzoic acid were
purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. All chemicals and
solvents were analytical grade. Melting points were measured on an X6 apparatus (Beijing Tech
Instrument Co. Ltd., Beijing, China), uncorrected. Infrared spectra were determined with a
Thermo Scientific Nicolet IS-10 Spectrophotometer (Thermo Scientific, America); The 1H and 13C
NMR spectra were recorded on a Bruker AV-600 spectrometer at working frequencies 600 and
150 MHz and a Bruker AV-300 spectrometer at working frequencies 300 and 75 MHz. Chemical
shifts are expressed in parts per million (δ) values and coupling constants (J) in Hertz. HREIMS

was measured on an Agilent 6210 TOFMS instrument (Agilent Technologies, America). The cell
proliferation assay was undertaken by MTT method using 96-well plates on a MLLTISKAN MK3
analysis spectrometer (Thermo Scientific, Shanghai, China).
4.1.2 Synthesis of compound 2 (2a-2k)
10mL of HCl solution (concentrated HCl : H2O = 1 : 1) was added into 4.15 mmol
substituted o-aminobenzoic acid, stirred for 30 min in ice bath, then 290 mg (4.2mmol) of sodium
nitrite (dissolved in 1.5 mL water) was slowly added. The reaction was continued for 30 min to
obtain compound 2, set aside.
4.1.3 Synthesis of K2Se2
198 mg (2.5 mmol) of selenium powder was added to a reaction flask, and 3-5 mL of distilled
water was added. Another 440 mg (8.02 mmol) of potassium borohydride was added to a constant
pressure funnel, and 5 mL of water was added to dissolve it. Under the protection of argon, the
potassium borohydride solution was slowly added to the selenium powder in about 10 minutes.
After the selenium powder was completely dissolved, 396 mg (5 mmol) of selenium powder was
quickly added and stirred at room temperature for 30 min. Then, 4 mL of a 6 M potassium
hydroxide solution was added to obtain a K2Se2 solution.
4.1.4 Synthesis of compound 3 (3a-3k)
The K2Se2 solution obtained above was transferred to an ice bath, and compound 2 was
added slowly under constant agitation. The reaction was continued for 5 min under the ice bath,
o
and then stirred for 2 h at 70 C and 3 h at room temperature. Last, the pH value of the solution
was tested.
1) If the pH was less than 7, directly filtered, added the filter cake to 1M sodium carbonate
solution, dissolved it until the solution was alkaline, heated until the solution boiled, cooled and
filtered, the filtrate was acidified with 1M hydrochloric acid until the pH of solution was less than
1, precipitate appeared. After filtration, solid was dried to give compound 3.
2) If the pH was greater than 7, directly filtered, the filtrate was acidified with 1M
hydrochloric acid until the pH of the solution was less than 1, heated until the solution boiled,
cooled and filtered, and the resulting precipitate was added to 1 M sodium carbonate solution to
dissolve until the solution was alkaline. The solution was heated to micro-boiling, after cooling,
filtered, and the filtrate was acidified with a hydrochloric acid solution until the pH of the solution
was less than 1, at which time the precipitate occurred, filtered, and solid was dried to obtain
compound 3.
4.1.5 Synthesis of compounds 4a-4k
200 mg of compound 3 (3a-3k) and 20 mL of SOCl2 were added into the reactive flask and

refluxed for 3 h at 80oC until no starting material was observed. The reaction was stopped and
SOCl2 was evaporated under reduced pressure to give an oily compound 4 (4a-4k). Compound 4
was not further purified and was directly used in the next reaction.
4.1.6 Synthesis of compound 7 and 13
0.27 mmol of compound 6 was dissolved in 40 mL of anhydrous ethanol, and 1.08 mmol of
NaBH3CN, 0.80 mmol NaHSO4, and 0.27 mmol MoCl5 were added. After addition, the solution
turned brown and large number of bubbles were produced. The reaction mixture was stirred at
room temperature for 2 h until no starting material was observed (the progress of the reaction was
monitored by TLC). Then the reaction was stopped and a silica gel short column was used for
filtration, and methanol was used for washing until there was no target product. The solvent was
evaporated under reduced pressure. The residue was purified by flash chromatography on silica
gel with Vdichloromethane : Vmethanol : Vammonia water= 10 : 1 : 1 as mobile phase to afford the
corresponding target product 7 as a white solid. Yield: 70%, m.p.154-156oC. IR(KBr) ν/cm-1:
1
3404, 2930, 1651, 1581, 1434, 1369, 1068, 953, 796; H NMR (CDCl3, 300MHz) δ: 0.64 (3H, s,
18-CH3), 1.00 (3H, s, 19-CH3), 2.64 (1H, t, J = 9.0, C17-H), 2.26-2.21 (2H, m), 3.54-3.41 (1H, m,
C3-H), 5.36 (1H, d, J = 5.1, C6-H); 13C NMR (75MHz, CDCl3): 141.0 (5-C), 121.3 (6-C), 71.4
(3-C), 62.7 (17-C), 53.3 (14-C), 50.3 (9-C), 42.4 (13-C), 42.3 (4-C), 37.3 (1-C), 36.6 (12-C), 32.2
(10-C), 31.6 (7-C), 31.5 (8-C), 31.1 (2-C), 29.7 (16-C), 23.7 (15-C), 20.7 (11-C), 19.4 (19-C),
11.0 (18-C); HREIMS m/z: 290.2505 [M+H]+ (calcd. for C19H32NO 290.2486)。
The synthesis of compound 13 was similar with that of compound 7:
1
White solid, Yield: 80%, m.p. 121-123oC; H NMR (300 MHz, CDCl3) δ: 0.64 (3H, s,
18-CH3), 0.86 (3H, d, J = 6.6, 26-CH3 or 27-CH3), 0.87 (3H, d, J = 6.6, 26-CH3 or 27-CH3), 0.91
(3H, s, 19-CH3), 0.92 (3H, d, J = 6.3, 21-CH3), 2.78-2.87 (2H, m, C6-H), 3.92 (1H, br s, C3-αH),
3.99 (1H, s, -OH); 13C NMR (75MHz, CDCl3) δ: 12.44 (18-C), 18.72 (19-C), 18.78 (21-C), 21.28
(11-C), 22.50 (26-C or 27-C), 22.76 (26-C or 27-C), 23.76 (23-C), 24.55 (16-C), 27.23 (15-C),
27.91 (25-C), 28.08 (1-C), 28.56 (8-C), 35.57 (2-C), 36.18 (20-C), 39.43 (22-C), 39.81 (6-C),
42.15 (12-C), 44.19 (24-C), 44.53 (13-C), 44.83 (4-C), 46.28 (7-C), 49.92 (10-C), 53.10 (9-C),
55.47 (14-C), 56.52 (17-C), 67.11 (3-C), 82.17 (5-C); IR(KBr) ν/cm-1: 3785, 3695, 3404, 2927,
2868, 2319, 1733, 1594, 1546, 1382, 1075, 716; HREIMS m/z: 420.3845 [M+H]+ (calcd. for
C27H50NO2, 420.3842).
4.1.7 General procedure for the synthesis of compounds 8a-8j or 14a-14k

The solution of 0.47 mmol compound 7 (or compound 13) in 20 mL of CH2Cl2 was added
slowly to the different compound 4 (4a-4k) in about 15 minutes, and then 1.5 mL of
trimethylamine was added. The reaction mixture was stirred for 3 h at room temperature until no
starting material was observed (the progress of the reaction was monitored by TLC). Then the
reaction was stopped and 20 mL of CH2Cl2 was added. The organic phase was washed with
saturated NaHCO3 solution, water and saturated brine, dried with anhydrous sodium sulfate, and
evaporated under reduced pressure. The residue was purified by flash chromatography on silica
gel with Vethyl acetate ：Vpetroleum ether = 1 : 2 to afford the corresponding target products 8a-8j (or
14a-14k) as a faint yellow solid.
17β-[3'-oxobenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8a)
Faint yellow solid. Yield: 45.84%, m.p. 242-245oC; 1H NMR (300 MHz, CDCl3) δ: 0.86 (3H,
s, 18-CH3), 1.01 (3H, s, 19-CH3), 3.54 (1H, ddd, J =10.8, 5.6, 5.0, C3-αH), 4.61 (1H, t, J = 9.4,
C17-H), 5.37 (1H, d, J = 5.1, C6-H), 7.41 (1H, s, J = 7.4, 3′-ph-H), 7.73-7.52 (2H, m, 3′,4′-ph-H),
8.04 (1H, d, J = 7.8, 6′-ph-H); 13C NMR (75MHz, CDCl3) δ: 13.01 (18-C), 19.44 (19-C), 20.51
(11-C), 23.78 (15-C), 29.37 (16-C), 31.70 (2-C), 31.43 (8-C), 31.93 (7-C), 32.09 (12-C), 36.56
(1-C), 37.27 (10-C), 42.20 (4-C), 45.17 (13-C), 50.11 (9-C), 52.44 (14-C), 64.21 (17-C), 71.60
(3-C), 121.07 (2′-Ph-C), 123.34 (6-C), 125.98 (6′-Ph-C), 128.22 (5′-Ph-C), 128.65 (3′-Ph-C),
131.70 (4′-Ph-C), 138.35 (1′-Ph-C), 141.12 (5-C), 168.11 (-C=O); IR(KBr) ν/cm-1: 3407.65,
2924.87, 2853.65, 1732.29, 1619.32, 1568.03, 1448.63, 1374.02, 1321.33, 1246.95, 1069.12,
1028.70, 953.91, 737.87, 672.53, 586.69; HREIMS (m/z): 494.1574 [M+Na]+ (calcd. for
C26H33NO2SeNa 494.1574).
17β-[3'-Oxo-7'-chlorobenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8b)
Faint yellow solid. Yield: 32.79%, m.p. 221-222oC; 1H NMR (600 MHz, CDCl3) δ: 0.87 (3H,
s, 18-CH3), 1.01 (3H, s, 19-CH3), 2.22-2.32 (2H, m, C4-H), 3.51-3.56 (1H, m, C3-αH), 4.61 (1H,
t, J = 8.4, C17-H), 5.37 (1H, d, J = 5.4, C6-H), 7.41 (1H, t, J = 7.8, 5′-Ph-H), 7.54 (1H, d, J = 7.8,
13
4′-Ph-H), 7.94 (1H, d, J = 7.8, 6′-Ph-H)； C NMR (75MHz, CDCl3) δ: 13.15 (18-C), 19.56 (19-C),
20.63 (11-C), 23.91 (15-C), 29.82 (16-C), 29.86 (2-C), 31.58 (8-C), 31.73 (7-C), 32.21 (12-C),
36.69 (1-C), 37.34 (10-C), 42.35 (4-C), 45.33 (13-C), 50.19 (9-C), 52.57 (14-C), 64.49 (17-C),

71.80 (3-C), 121.26 (6-C), 126.82 (6′-C), 127.82 (5′-Ph-C), 128.89 (4′-Ph-C), 130.35 (3′-Ph-C),
130.86 (1′-Ph-C), 138.96 (2′-Ph-C), 141.13 (5-C), 168.00 (C=O); IR(KBr) ν/cm-1: 3727.82,
3627.07, 3594.52, 3384.52, 2925.41, 2855.17, 1626.66, 1589.80, 1561.04, 1455.64, 1412.55,
1371.76, 1321.21, 1300.45, 1204.73, 1139.85, 1089.60, 1055.32, 1029.03, 954.12, 908.13, 841.36,
804.01, 742.70, 669.74；HREIMS (m/z): 506.1376 [M+H]+ (calcd. for C26H33ClNO2Se 506.1365).
17β-[3'-Oxo-6'-chlorobenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8c)
Faint yellow solid. Yield: 17.12%, m.p. 218-221oC; 1H NMR (300 MHz, CDCl3) δ: 0.86 (3H,
s, 18-CH3), 1.02 (3H, s, 19-CH3), 2.23-2.31 (2H, m, C4-H), 3.50-3.60 (1H, m, C3-αH), 4.62 (1H,
t, J = 9.3, C17-H), 5.38 (1H, d, J = 5.1, C6-H), 7.38 (1H, dd, J = 8.4, 1.8, 5′-Ph-H), 7.64 (1H, d, J
= 1.8, 3′-Ph-H), 7.95 (1H, d,J = 8.4, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.03 (18-C), 19.43
(19-C), 20.50 (11-C), 23.77 (15-C), 29.60 (16-C), 31.45 (2-C), 31.60 (8-C), 32.08 (7-C), 36.56
(12-C), 37.16 (1-C), 37.24 (10-C), 42.20 (4-C), 45.19 (13-C), 50.07 (9-C), 52.41 (14-C), 64.41
(17-C), 71.63 (3-C), 121.07 (6-C), 123.19 (3′-Ph-C), 126.70 (6′-Ph-C), 126.82 (5′-Ph-C), 129.58
(1′-Ph-C), 138.34 (4′-Ph-C), 139.48 (2′-Ph-C), 141.07 (5-C), 167.23 (-C=O); IR(KBr) ν/cm-1:
3726.72, 3704.38, 3626.24, 3597.83, 3395.08, 2927.72, 2851.64, 1723.12, 1611.58, 1584.71,
1548.04, 1453.43, 1396.54, 1374.04, 1306.68, 1274.16, 1189.51, 1125.07, 1090.86, 1046.65,
953.97, 872.92, 827.26, 760.00, 669.87, 653.72, 617.43, 574.26; HREIMS(m/z): 506.1367
[M+H]+ (calcd. for C26H33ClNO2Se 506.1365).
17β-[3'-Oxo-4'-chlorobenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8d)
Faint yellow solid. Yield: 35.17%, m.p. 142-144oC; 1H NMR (300 MHz, CDCl3) δ: 0.81 (3H,
s, 18-CH3), 0.97 (3H, s, 19-CH3), 2.19-2.29 (2H, m, C4-H), 2.60 (1H, br s, OH), 3.49-3.59 (1H, m,
C3-αH), 4.57 (1H, t, J = 9.3, C17-H), 5.31 (1H, s, C6-H), 7.32-7.41 (2H, m, 3′,4′-Ph-H), 7.52 (1H,
dd, J = 7.5, 1.2, 5′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.02 (18-C), 19.40 (19-C), 20.46 (11-C),
23.77 (15-C), 29.68 (16-C), 31.39 (2-C), 31.57 (8-C), 32.02 (7-C), 36.50 (12-C), 37.32 (1-C),
37.39 (10-C), 42.02 (4-C), 45.07 (13-C), 50.14 (9-C), 52.48 (14-C), 64.12 (17-C), 71.42 (3-C),
120.88 (6-C), 122.21 (3′-Ph-C), 123.21 (5′-Ph-C), 128.23 (6′-Ph-C), 131.46 (4′-Ph-C), 136.07
(1′-Ph-C), 141.27 (2′-Ph-C), 141.31 (5-C), 165.95 (-C=O); IR(KBr) ν/cm-1: 3726.56, 3704.80,
3414.50, 2926.82, 2852.16, 2239.85, 1711.13, 1632.72, 1578.35, 1555.32, 1443.36, 1373.68,

1295.18, 1175.02, 1088.87, 1052.14, 1029.67, 953.42, 908.66, 783.32, 731.21, 669.73, 649.03,
586.07, 555.38; HREIMS(m/z): 506.1376 [M+H]+ (calcd. for C26H33ClNO2Se 506.1365).
17β-[3'-Oxo-7'-fluorobenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8e)
Faint yellow solid. Yield: 41.21%, m.p. 236-238oC; 1H NMR (600 MHz, CDCl3) δ: 0.83 (3H,
s, 18-CH3), 0.97 (3H, s, 19-CH3), 2.19-2.24 (2H, m, C4-H), 2.56 (1H, br s, OH), 3.48-3.54 (1H, m,
C3-αH), 4.54 (1H, t, J = 9.6, C17-H), 5.31 (1H, d, J = 5.4, C6-H), 7.24 (1H, t, J = 8.4, 4′-Ph-H),
7.36-7.39 (1H, m, 5′-Ph-H), 7.80 (1H, d, J = 7.8, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.11
(18-C), 19.55 (19-C), 20.61 (11-C), 23.86 (15-C), 29.81 (16-C), 31.54 (2-C), 31.69 (8-C), 32.17
(7-C), 36.66 (12-C), 37.29 (1-C), 37.41 (10-C), 42.26 (4-C), 45.29 (13-C), 50.23 (9-C), 52.56
(14-C), 64.58 (17-C), 71.61 (3-C), 117.31 (2JC-F=9.75, 4′-Ph-C), 121.05 (6-C), 124.35 (6′-Ph-C),
128.10 (2′-Ph-C), 130.00 (5′-Ph-C), 141.35 (5-C), 156.65 (1′-Ph-C), 158.28 (3′-Ph-C), 167.52
(-C=O); IR(KBr) ν/cm-1: 3425.82, 2933.34, 2849.82, 1641.97, 1574.20, 1471.10, 1365.74,
1325.72, 1300.86, 1247.87, 1200.19, 1087.84, 1059.44, 1030.28, 981.49, 955.10, 799.80, 737.64,
669.59, 648.92; HREIMS (m/z): 490.1654 [M+H]+ (calcd. for C26H33FNO2Se 490.1661).
17β-[3'-Oxo-6'-fluorobenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8f)
Faint yellow solid. Yield: 46.12%, m.p. 273-275oC; 1H NMR (300 MHz, CDCl3) δ: 0.87 (3H,
s, 18-CH3), 1.02 (3H, s, 19-CH3), 2.24-2.32 (2H, m, C4-H), 3.51-3.59 (1H, m, C3-αH), 4.59 (1H,
t, J = 9.3, C17-H), 5.38 (1H, d, J = 5.1, C6-H), 7.10-7.17 (1H, m, 3′-Ph-H), 7.33 (1H, dd, J = 7.8,
2.1, 5′-Ph-H), 8.02 (1H, dd, J = 8.7, 5.1, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.04 (18-C),
19.44 (19-C), 20.48 (11-C), 23.77 (15-C), 29.71 (16-C), 31.45 (2-C), 31.59 (8-C), 32.07 (7-C),
36.56 (12-C), 37.12 (1-C), 37.21 (10-C), 42.21 (4-C), 45.15 (13-C), 50.04 (9-C), 52.35 (14-C),
64.37 (17-C), 71.66 (3-C), 110.03 (5′-Ph-C), 110.73 (3′-Ph-C), 121.14 (6-C), 124.57 (6′-Ph-C),
130.50 (1′-Ph-C), 130.63 (2′-Ph-C), 139.75 (5-C), 141.01 (4′-Ph-C), 167.17 (-C=O); IR(KBr)
ν/cm-1: 3727.48, 3448.34, 2924.38, 2852.20, 1605.63, 1470.33, 1409.92, 1375.95, 1309.37,
1245.47, 1215.96, 1174.70, 1089.97, 1061.13, 1027.90, 950.27, 888.46, 859.92, 834.32, 796.51,
762.14, 720.86, 670.73, 611.15, 586.34; HREIMS (m/z): 490.1658 [M+H]+ (calcd. For
C26H33FNO2Se 490.1661).
17β-[3'-Oxo-5'-fluorobenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8g)

Faint yellow solid. Yield: 26.00%, m.p. 236-238oC; 1H NMR (600MHz, CDCl3) δ: 0.84 (3H,
s, 18-CH3), 0.99 (3H, s, 19-CH3), 2.19-2.25 (2H, m, C4-H), 3.50-3.55 (1H, m, C3-αH), 4.56 (1H,
t, J = 9.0, C17-H), 5.35 (1H, d, J = 4.2, C6-H), 7.30-7.33 (1H, m, 4′-ph-H), 7.56 (1H, dd, J = 9.0,
4.8, 3′-ph-H), 7.71 (1H, dd, J = 8.4, 2.4, 6′-ph-H); 13C NMR (75MHz, CDCl3) δ: 13.10 (18-C),
19.54 (19-C), 20.61 (11-C), 23.88 (15-C), 29.81 (16-C), 31.56 (2-C), 31.56 (8-C), 32.20 (7-C),
36.67 (12-C), 37.30 (1-C), 37.36 (10-C), 42.32 (4-C), 45.34 (13-C), 50.19 (9-C), 52.55 (14-C),
64.63 (17-C), 71.73 (3-C), 114.90 (2JC-F=11.25, 6′-Ph-C), 120.26 (2JC-F=12.00, 4′-Ph-C), 121.19
(6-Ph-C), 124.92 (3JC-F=3.75, 3′-Ph-C), 130.02 (3JC-F=3.75, 1′-Ph-C ), 132.95 (2′-Ph-C), 141.22
(5-C), 161.86 (1JC-F=122.25, 5′-Ph-C), 167.31 (-C=O); IR(KBr) ν/cm-1: 3377.38, 2925.88,
2852.73, 1714.80, 1613.80, 1576.88, 1461.13, 1331.20, 1272.55, 1219.15, 1143.89, 1107.39,
1090.76, 1057.26, 1029.83, 953.25, 930.84, 879.75, 812.26, 764.18, 730.38, 669.83, 646.36,
600.67, 536.00; HREIMS(m/z): 490.1662 [M+H]+ (calcd. for C26H33FNO2Se 490.1661).
17β-[3'-Oxo-5'-methylbenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8h)
Faint yellow solid. Yield: 23.65%, m.p. 263-265oC; 1H NMR (600 MHz, CDCl3) δ: 0.82 (3H,
s, 18-CH3), 0.98 (3H, s, 19-CH3), 2.42 (3H, s, ph-CH3), 3.49-3.53 (1H, m, C3-αH), 4.56 (1H, t, J =
9.0, C17-H), 5.32 (1H, s, C6-H), 7.36 (1H, t, J = 6.6, 4′-ph-H), 7.47 (1H, t, J = 7.2, 5′-ph-H), 7.81
(1H, d, J = 3.6, 6′-ph-H); 13C NMR (75MHz, CDCl3): 13.09 (18-C), 19.54 (19-C), 20.61 (11-C),
21.12 (5′-Ph-CH3), 23.85 (15-C), 29.65 (16-C), 31.55 (2-C), 31.70 (8-C), 32.16 (7-C), 36.65
(12-C), 37.31 (1-C), 37.40 (10-C), 42.29 (4-C), 45.26 (13-C), 50.24 (9-C), 52.55 (14-C), 64.29
(17-C), 71.63 (3-C), 121.10 (6-C), 123.19 (6′-Ph-C), 128.32 (3′-Ph-C), 128.73 (1′-Ph-C), 133.19
(4′-Ph-C), 135.11 (5′-Ph-C), 136.16 (2′-Ph-C), 141.30 (5-C), 168.23 (-C=O); IR (KBr) ν/cm-1:
3726.79, 3381.71, 2927.72, 2853.08, 1618.63, 1537.51, 1465.03, 1335.11, 1290.91, 1251.42,
1220.53, 1146.81, 1060.50, 953.41, 906.84, 808.14, 767.67, 732.16, 669.59, 649.92, 602.27,
513.20; HREIMS (m/z): 486.1907 [M+H]+ (calcd. for C27H36NO2Se 486.1911)
17β-[3'-Oxo-6'-methoxylbenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8i)
o
Faint yellow solid. Yield: 11.58%, m.p. 243-246 C；1H NMR (300 MHz, CDCl3) δ: 0.85
(3H, s, 18-CH3), 1.01 (3H, s, 19-CH3), 2.20-2.31 (2H, m, C4-H), 3.49-3.57 (1H, m, C3-αH), 3.89
(3H, s, ph-OCH3), 4.57 (1H, t, J = 9.3, C17-H), 5.37 (1H, d, J = 4.8, C6-H), 6.96 (1H, dd, J = 8.7,

2.4, 5′-ph-H), 7.08 (1H, d, J = 2.4, 3′-ph-H), 7.92 (1H, d, J = 8.7, 6′-ph-H); 13C NMR (75MHz,
CDCl3) δ: 13.03 (18-C), 19.45 (19-C), 20.50 (11-C), 23.75 (15-C), 29.70 (16-C), 31.46 (2-C),
31.60 (8-C), 32.07 (7-C), 36.56 (12-C), 37.14 (1-C), 37.25 (10-C), 42.19 (4-C), 45.06 (13-C),
50.09 (9-C), 52.35 (14-C), 55.73 (4-Ph-OCH3), 64.10 (17-C), 71.62 (3-C), 106.78 (3′-Ph-C),
114.21 (5′-Ph-C), 121.13 (6-C), 121.32 (1′-Ph-C), 129.68 (6′-Ph-C), 140.16 (5-C), 141.11
(2′-Ph-C), 162.74 (4′-Ph-C), 167.91(-C=O); IR(KBr) ν/cm-1: 3380.80, 2929.85, 2852.82, 1596.24,
1480.34, 1437.72, 1416.98, 1364.04, 1317.13, 1256.64, 1187.70, 1043.30, 954.36, 907.38, 880.14,
846.75, 763.98, 731.36, 680.28; HREIMS (m/z): 502.1866 [M+H]+ (calcd. for C27H36NO3Se
502.1860).
17β-[3'-Oxo-5'-methoxylbenzisoselenazol-2'-(3H)-yl]androst-5-en-3β-ol (8j)
Faint yellow solid. Yield: 8.15%, m.p. 238-241oC；1H NMR (300 MHz, CDCl3) δ: 0.87 (3H,
s, 18-CH3), 1.03 (3H, s, 19-CH3), 2.21-2.32 (2H, m, C4-H), 3.50-3.60 (1H, m, C3-αH), 3.89 (3H,
s, ph-OCH3), 4.61 (1H, t, J = 9.3, C17-H), 5.38 (1H, d, J = 5.1, C6-H), 7.22 (1H, dd, J = 8.7, 2.7,
4′-ph-H), 7.49 (1H, d, J = 8.7, 3′-ph-H), 7.55 (1H, d, J = 2.7, 6′-ph-H); 13C NMR (75MHz, CDCl3)
δ: 12.99 (18-C), 19.44 (19-C), 20.52 (11-C), 23.77 (15-C), 29.70 (16-C), 31.47 (2-C), 31.61 (8-C),
32.09 (7-C), 36.57 (12-C), 37.18 (1-C), 37.23 (10-C), 42.22 (4-C), 45.23 (13-C), 50.06 (9-C),
52.41 (14-C), 55.68 (5-Ph-OCH3), 64.34 (17-C), 71.65 (3-C), 110.31 (6′-Ph-C), 121.18 (4′-Ph-C),
121.95 (6-C), 124.09 (3′-Ph-C), 129.21 (1′-Ph-C), 139.58 (2′-Ph-C), 141.03 (5-C), 158.75
(5′-Ph-C), 168.00 (-C=O); IR(KBr) ν/cm-1: 3369.77, 2926.41, 2852.94, 1609.03, 1568.82,
1469.44, 1436.58, 1341.20, 1289.76, 1274.50, 1230.54, 1188.71, 1153.22, 1123.39, 1049.84,
1025.64, 955.11, 809.48, 764.25, 729.98, 669.52, 647.90, 605.43; HREIMS (m/z): 502.1864
[M+H]+ (calcd. for C27H36NO3Se 502.1860).
6β-[3'-Oxobenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14a)
Faint yellow solid. Yield: 43.75%, m.p. 222-223oC; 1H NMR (300MHz, CDCl3) δ: 0.71 (3H,
s, 18-CH3), 0.87 (3H, s, 19-CH3), 0.89 (6H, d, J = 6.5, 26- and 27-CH3), 0.93 (3H, d, J = 6.5,
21-CH3), 3.93-3.86 (2H, m, C6-H), 4.02 (1H, br s, C3-αH), 4.78(1H, s, -OH), 7.47-7.31 (1H, m,
3′-Ph-H), 7.78-7.47 (2H, m, 4′,5′-Ph-H), 8.02 (1H, d, J = 7.7, 6′-Ph-H); 13C NMR (75MHz,
CDCl3)δ: 12.60 (18-C), 18.72 (19-C), 18.83 (21-C), 21.41 (11-C), 22.56 (26- or 27-C), 22.83 (26-

or 27-C), 23.88 (23-C), 24.89 ( 16-C), 27.39 (15-C), 28.02 (25-C), 28.10 (1-C), 28.83 (8-C), 35.67
(2-C), 36.25 (20-C), 39.48 (22-C), 39.87 (6-C), 43.35 (12-C), 44.66 (24-C), 44.81 (13-C), 45.01
(4-C), 46.33 (7-C), 49.96 (10-C), 55.29 (9-C), 55.56 ( 14-C), 56.68 (17-C), 67.38 (3-C), 80.60
(5-C), 123.66 (2′-ph-C), 125.97 (6′-ph-C), 127.37 (5′-ph-C), 128.58 (3′-ph-C), 131.85 (4′-ph-C),
138.43 (1′-ph-C), 167.84 (-C=O); IR (KBr) ν/cm-1: 3364.18、2927.15、2862.08、1619.20、
1564.57、1445.99、1378.98、1352.75、1171.23、1078.73、1020.57、956.54、738.28、675.55;
HREIMS (m/z): 624.2939 [M+Na]+ (calcd. for C34H51NO3SeNa 624.2932).
6β-[3'-Oxo-7'-chlorobenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14b)
Faint yellow solid. Yield: 31.48%,m.p. 198-201oC; 1H NMR (600 MHz, CDCl3) δ: 0.69 (3H,
s, 18-CH3), 0.86 (3H, d, J = 6.6, 26 or 27-CH3), 0.87 (3H, d, J = 6.6, 26 or 27-CH3), 0.90 (3H, s,
19-CH3), 0.91 (3H, d, J = 6.6, 21-CH3), 1.97 (1H, d, J = 15.0, C4-H), 2.04 (1H, d, J = 12.6,
C17-H), 2.87 (1H, s, -OH), 3.77 (1H, dd, J = 13.8, 9.6, C6-H), 4.06 (1H, br s, C3-αH), 4.07 (1H,
d, J = 13.8, C6-H), 4.39 (1H, s, -OH), 7.37 (1H, t, J = 7.2, 5′-Ph-H), 7.51 (1H, d, J = 7.8, 4′-Ph-H),
7.91 (1H, d, J = 7.2, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 12.71 (18-C), 18.95 (19-C), 19.01
(21-C), 21.54 (11-C), 22.67 (26 or 27-C), 22.94 (26 or 27-C), 24.00 (23-C), 24.92 (16-C), 27.19
(15-C), 28.14 (25-C), 28.6 (1-C), 29.82 (8-C), 35.80 (2-C), 36.36 (20-C), 39.59 (22-C), 39.99
(6-C), 43.51 (12-C), 44.77 (24-C), 44.93 (13-C), 45.24 (4-C), 46.58 (7-C), 49.86 (10-C), 55.20
(9-C), 55.70 (14-C), 56.81 (17-C), 67.64 (3-C), 80.62 (5-C), 126.68 (6′-ph-C), 127.69 (5′-ph-C),
129.13 (4′-ph-C), 129.68 (3′-ph-C), 130.94 (1′-ph-C), 140.17 (2′-ph-C), 167.64 (-C=O); IR(KBr)
ν/cm-1: 3726.67, 3627.70, 3298.78, 2927.35, 2861.18, 1628.56, 1559.99, 1456.37, 1424.71,
1379.21, 1348.46, 1273.59, 1206.07, 1172.55, 1147.43, 1078.69, 1016.46, 969.80, 870.18, 802.61,
762.56, 744.85, 669.72; HREIMS (m/z): 636.2722 [M+H]+ (calcd. for C34H51ClNO3Se 636.2723).
6β-[3'-Oxo-6'-chlorobenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14c)
o
1
Faint yellow solid. Yield: 39.61%, m.p. 201-204 C; H NMR (600 MHz, CDCl3) δ: 0.67
(3H, s, 18-CH3), 0.85 (3H, d, J = 6.6, 26 or 27-CH3), 0.86 (3H, d, J = 6.6, 26 or 27-CH3), 0.87
(3H, s, 19-CH3), 0.91 (3H, d, J = 6.6, 21-CH3), 1.94-1.87 (2H, m, C4-H), 2.03 (1H, d, J = 13.6,
C17-H), 3.30 (1H, s, OH), 3.74 (1H, dd, J = 13.8, 9.6, C6-H), 4.01 (1H, d, J = 14.4, C6-H), 4.03
(1H, br s, C3-αH), 4.52 (1H, s, OH), 7.33 (1H, dd, J = 12.6, 2.7, 5′-Ph-H), 7.59 (1H, d, J = 2.7,

3′-Ph-H), 7.89 (1H, d, J = 12.6, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 12.70 (18-C), 18.91
(19-C), 18.94 (21-C), 21.53 (11-C), 22.68 (26 or 27-C), 22.94 (26 or 27-C), 24.01 (23-C), 24.96
(16-C), 27.44 (15-C), 28.16 (25-C), 28.94 (1-C), 29.82 (8-C), 35.79 (2-C), 36.36 (20-C), 39.59
(22-C), 39.96 (6-C), 43.38 (12-C), 44.67 (24-C), 44.91 (13-C), 45.21 (4-C), 46.49 (7-C), 50.10
(10-C), 55.21 (9-C), 55.68 (14-C), 56.76 (17-C), 67.57 (3-C), 80.68 (5-C), 123.58 (3′-ph-C),
126.12 (6′-ph-C), 126.79 (5′-ph-C), 129.50 (1′-ph-C), 138.48 (4′-ph-C), 141.12 (2′-ph-C), 167.06
(-C=O); IR(KBr) ν/cm-1: 3384.08, 3096.66, 2927.18, 2864.60, 1602.53, 1586.39, 1442.70,
1405.91, 1380.02, 1306.97, 1237.91, 1165.77, 1126.33, 1077.60, 1016.10, 951.07, 869.58, 830.21,
762.41, 669.04, 577.26; HREIMS (m/z): 636.2719 [M+H]+ (calcd. for C34H51ClNO3Se 636.2723).
6β-[3'-Oxo-4'-chlorobenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14d)
Faint yellow solid. Yield: 62.46%, m.p. 196-198oC; 1H NMR (600 MHz, CDCl3) δ: 0.66 (3H,
s, 18-CH3), 0.84 (3H, d, J = 6.6, 26- or 27-CH3), 0.85 (3H, d, J = 6.6, 26- or 27-CH3), 0.86 (3H, s,
19-CH3), 0.89 (3H, d, J = 6.6, 21-CH3), 1.97-1.87 (2H, m, C4-H), 2.03 (1H, d, J = 12.6, C17-H),
3.63 (1H, dd, J = 14.4, 10.2, C6-H), 4.02 (1H, br s, C3-αH), 4.07 (1H, dd, J = 14.4, 1.8, C6-H),
4.32 (1H, s, OH), 7.29 (1H, d, J = 7.8, 3′-Ph-H), 7.35 (1H, t, J = 8.4, 4′-Ph-H), 7.45 (1H, d, J =
7.8, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 12.67 (18-C), 18.82 (19-C), 18.93 (21-C), 21.52
(11-C), 22.67 (26 or 27-C), 22.93 (26 or 27-C), 23.99 (23-C), 25.06 (16-C), 27.71 (15-C), 28.10
(25-C), 28.96 (1-C), 29.79 (8-C), 35.78 (2-C), 36.34 (20-C), 39.57 (22-C), 39.94 (6-C), 43.19
(12-C), 44.51 (24-C), 44.86 (13-C), 45.22 (4-C), 46.46 (7-C), 50.39 (10-C), 54.89 (9-C), 55.66
(14-C), 56.60 (17-C), 67.50 (3-C), 80.77 (5-C), 122.41 (3′-ph-C), 122.69 (5′-ph-C), 128.69
(6′-ph-C), 131.50 (4′-ph-C), 136.04 (1′-ph-C), 142.93 (2′-ph-C), 165.91 (-C=O); IR(KBr) ν/cm-1:
3382.70, 2926.88, 2865.45, 1580.98, 1556.46, 1444.29, 1379.16, 1334.54, 1309.42, 1259.76,
1169.26, 1131.05, 1075.13, 1018.44, 955.26, 867.54, 778.81, 668.28, 556.98; HREIMS (m/z):
636.2720 [M+H]+ (calcd. For C34H51ClNO3Se 636.2723).
6β-[3'-Oxo-7'-fluorobenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14e)
Faint yellow solid. Yield: 41.76%, m.p. 219-221oC; 1H NMR (600 MHz, CDCl3) δ: 0.69 (3H,
s, 18-CH3), 0.85 (3H, d, J = 6.6, 26- or 27-CH3), 0.86 (3H, d, J = 6.6, 26- or 27-CH3), 0.89 (3H, s,
19-CH3), 0.91 (3H, d, J = 6.6, 21-CH3), 1.98-2.05 (2H, m, C4-H), 3.03 (1H, s, OH), 3.69 (1H, dd,

J = 14.4, 10.6, C6-H), 4.08 (1H, br s, C3-αH), 4.12 (1H, d, J = 14.4, C6-H), 4.39 (1H, s, OH), 7.23
(1H, t, J = 8.4, 4′-Ph-H), 7.35-7.39 (1H, m, 5′-Ph-H), 7.80 (1H, d, J = 7.8, 6′-Ph-H); 13C NMR
(75MHz, CDCl3) δ: 12.73 (18-C), 18.97 (19-C), 19.07 (21-C), 21.57 (11-C), 22.70 (26- or 27-C),
22.97 (26- or 27-C), 24.92 (23-C), 27.20 (16-C), 28.16 (15-C), 28.18 (25-C), 28.98 (1-C), 29.85
(8-C), 35.82 (2-C), 36.38 (20-C), 39.61 (22-C), 40.02 (6-C), 43.49 (12-C), 44.67 (24-C), 44.93
(13-C), 45.28 (4-C), 46.60 (7-C), 49.89 (10-C), 55.14 (9-C), 55.73 (14-C), 56.82 (17-C), 67.66
(3-C), 80.63 (5-C), 117.29 (2JC-F =18.9, 4′-ph-C), 124.21 (6′-ph-C), 126.24 (2JC-F =24.6, 2′-ph-C),
127.90 (5′-ph-C), 130.55 (1′-ph-C), 157.76 (1JC-F =244.5, 3′-ph-C), 167.23 (-C=O); IR(KBr)
ν/cm-1: 3371.13, 2923.92, 2866.17, 1632.23, 1575.66, 1473.46, 1429.71, 1379.06, 1352.68,
1272.64, 1247.00, 1204.10, 1169.75, 1132.87, 1075.70, 996.87, 867.55, 800.58, 737.61, 669.36,
615.40; HREIMS (m/z): 620.3018 [M+H]+ (calcd. for C34H51FNO3Se 620.3018).
6β-[3'-Oxo-6'-fluorobenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14f)
o
1
Faint yellow solid. Yield: 27.68%, m.p. 194-196 C; H NMR (600 MHz, CDCl3) δ: 0.68
(3H, s, 18-CH3), 0.85 (3H, d, J = 6.6, 26- or 27-CH3), 0.86 (3H, d, J = 6.6, 26- or 27-CH3), 0.87
(3H, s, 19-CH3), 0.91 (3H, d, J = 6.6, 21-CH3), 1.93-2.04 (2H, m, C4-H), 3.29 (1H, s, -OH), 3.75
(1H, dd, J = 14.4, 9.6, C6-H), 3.99 (1H, d, J = 14.4, C6-H), 4.03 (1H, br s, C3-αH), 4.56 (1H, s,
-OH), 7.08 (1H, td, J = 8.4, 1.8, 5′-Ph-H), 7.30 (1H, dd, J = 7.8, 1.9, 3′-Ph-H), 7.96 (1H, dd, J =
8.4, 4.8, 6′-Ph-H); 13C NMR (75MHz, CDCl3)δ: 12.68 (18-C), 18.89 (19-C), 18.92 (21-C), 21.51
(11-C), 22.63 (26 or 27-C), 22.92 (26 or 27-C), 23.99 (23-C), 24.94 (16-C), 27.39 (15-C), 28.17
(25-C), 28.91 (1-C), 29.79 (8-C), 35.77 (2-C), 36.34 (20-C), 39.57 (22-C), 39.95 (6-C), 43.37
(12-C), 44.68 (24-C), 44.89 (13-C), 45.18 (4-C), 46.47 (7-C), 50.03 (10-C), 55.24 (9-C), 55.66
(14-C), 56.76 (17-C), 67.55 (3-C), 80.65 (5-C), 110.57 (2JC-F=25.4, 5′-ph-C), 114.53 (2JC-F=23.4,
3′-ph-C), 123.95 (1′-ph-C), 130.46 (3JC-F=9.0, 6′-ph-C), 141.50 (3JC-F=9.0, 2′-ph-C), 165.15
(1JC-F=252.0, 4′-ph-C), 167.03 (-C=O); IR(KBr) ν/cm-1: 3727.06, 3704.22, 3627.01, 3599.97,
3375.24, 2922.88, 2864.18, 1599.65, 1468.74, 1441.94, 1417.27, 1378.65, 1313.59, 1242.15,
1216.14, 1166.58, 1126.59, 1077.81, 1016.07, 951.65, 877.40, 828.87, 763.55, 670.55,
654.71;HREIMS(m/z): 620.3019 [M+H]+ (calcd. for C34H51FNO3Se 620.3018).
6β-[3'-Oxo-5'-fluorobenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14g)

Faint yellow solid. Yield: 61.99%, m.p. 212-215 oC; 1H NMR (600MHz, CDCl3) δ: 0.67 (3H,
s, 18-CH3), 0.85 (3H, d, J = 6.6, 26- or 27-CH3), 0.86 (3H, d, J = 6.6, 26- or 27-CH3), 0.87 (3H, s,
19-CH3), 0.90 (3H, d, J = 6.6, 21-CH3), 1.93-2.08 (2H, m, C4-H), 3.41 (1H, s, OH), 3.77 (1H, dd,
J = 14.4, 9.6, C6-H), 3.99 (1H, d, J = 14.4, C6-H), 4.03 (1H, br s, C3-αH), 4.57 (1H, s, OH),
7.29-7.31 (1H, td, J = 8.4, 2.4, 4′-Ph-H), 7.55 (1H, dd, J = 8.4, 4.2, 3′-Ph-H), 7.68 (1H, dd, J = 8.4,
2.4, 6′-Ph-H); 13C NMR (75MHz, CDCl3)δ: 12.19 (18-C), 18.41 (19-C), 18.43 (21-C), 21.02
(11-C), 22.17 (26 or 27-C), 22.44 (26 or 27-C), 24.43 (23-C), 26.88 (16-C), 27.62 (15-C), 27.67
(25-C), 28.43 (1-C), 29.31 (8-C), 35.28 (2-C), 35.85 (20-C), 39.08 (22-C), 39.46 (6-C), 42.96
(12-C), 44.16 (24-C), 44.41 (13-C), 44.8 (4-C), 46.15 (7-C), 49.51 (10-C), 54.72 (9-C), 55.17
(14-C), 56.27 (17-C), 67.03 (3-C), 80.15 (5-C), 114.18 (2JC-F=22.5, 6′-ph-C), 119.77 (2JC-F=24.0,
4′-ph-C), 124.68-124.73 (3′-ph-C), 128.71 (1′-ph-C), 133.89 (2′-ph-C), 161.27 (1JC-F=244.5,
5′-ph-C), 166.51 (-C=O); IR(KBr) ν/cm-1: 3726.86, 3704.25, 3291.87, 2924.73, 2865.52, 1873.02,
1623.65, 1577.74, 1464.02, 1438.15, 1379.27, 1269.01, 1170.63, 1138.74, 1113.31, 1076.13,
961.10, 883.18, 865.36, 814.56, 762.94, 669.64, 654.15, 619.63, 539.61; HREIMS (m/z):
620.3024 [M+H]+ (calcd. for C34H51FNO3Se 620.3018).
6β-[3'-Oxo-5'-methylbenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14h)
Faint yellow solid. Yield: 64.75%, m.p. 165-166 oC; 1H NMR (600MHz, CDCl3) δ: 0.68 (3H,
s, 18-CH3), 0.85 (3H, d, J = 6.6, 26- or 27-CH3), 0.86 (3H, d, J = 6.6, 26- or 27-CH3), 0.87 (3H, s,
19-CH3), 0.91 (3H, d, J = 6.6, 21-CH3), 2.43 (3H, s, Ph-CH3), 3.49 (1H, s, OH), 3.80 (1H, d, J =
14.4, C6-H), 3.92 (1H, dd, J = 14.4, 8.4, C6-H), 3.97 (1H, br s, C3-αH), 4.89 (1H, s, OH), 7.37
(1H, d, J = 7.8, 4′-Ph-H), 7.46 (1H, d, J = 7.8, 3′-Ph-H), 7.81 (1H, s, 6′-Ph-H); 13C NMR (75MHz,
CDCl3) δ: 12.69 (18-C), 14.29 (5-Ph-CH3), 18.77 (19-C), 18.92 (21-C), 21.10 (11-C), 21.50 (26-
or 27-C), 22.92 (26- or 27-C), 24.99 (23-C), 27.54 (16-C), 28.11 (15-C), 28.25 (25-C), 28.92
(1-C), 29.79 (8-C), 35.76 (2-C), 36.35 (20-C), 39.59 (22-C), 39.97 (6-C), 43.44 (12-C), 44.81
(24-C), 44.92 (13-C), 45.07 (4-C), 46.41 (7-C), 50.08 (10-C), 55.51 (9-C), 55.65 (14-C), 56.80
(17-C), 67.44 (3-C), 80.69 (5-C), 123.47 (6′-ph-C), 127.44 (3′-ph-C), 128.79 (1′-ph-C), 133.40
(4′-ph-C), 135.97 (5′-ph-C), 136.20 (2′-ph-C), 167.96 (-C=O); IR(KBr) ν/cm-1: 3374.12, 2927.54,
2863.30, 1616.62, 1562.35, 1465.74, 1378.87, 1277.09, 1237.90, 1167.51, 1129.58, 1078.50,
958.51, 921.51, 865.63, 824.13, 767.76, 730.90, 669.89, 618.26, 538.0; HREIMS (m/z): 616.3279

[M+H]+ (calcd. for C35H54NO3Se 616.3269).
6β-[3'-Oxo-6'-methoxylbenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14i)
Faint yellow solid. Yield: 62.86%, m.p. 235-236 oC; 1H NMR (300MHz, CDCl3) δ: 0.71 (3H,
s, 18-CH3), 0.88 (6H, d, J = 6.6, 26- and 27-CH3), 0.89 (3H, s, 19-CH3), 0.94 (3H, d, J = 6.3,
21-CH3), 3.31 (1H, s, OH), 3.80-3.94 (2H, m, C6-H), 3.89 (3H, s, Ph-OCH3), 4.01 (1H, br s,
C3-αH), 4.88 (1H, s, OH), 6.96 (1H, dd, J = 8.7, 2.1, 5′-Ph-H), 7.06 (1H, d, J = 2.1, 3′-Ph-H), 7.91
(1H, d, J = 8.7, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 12.60 (18-C), 18.65 (19-C), 18.82 (21-C),
21.41 (11-C), 22.56 (26 or 27-C), 22.83 (26 or 27-C), 23.88 (23-C), 27.41 (16-C), 28.03 (15-C),
28.17 (25-C), 28.84 (1-C), 29.69 (8-C), 35.68 (2-C), 36.24 (20-C), 39.48 (22-C), 39.86 (6-C),
43.22 (12-C), 44.81 (24-C), 44.96 (13-C), 45.85 (4-C), 46.27 (7-C), 49.97 (10-C), 55.39 (9-C),
55.72 (14-C), 56.68 (17-C), 60.42 (4-Ph-OCH3), 67.43 (3-C), 80.63 (5-C), 107.14 (3′-ph-C),
114.23 (5′-ph-C), 120.38 (1′-ph-C), 129.70 (6′-ph-C), 141.07 (2′-ph-C), 162.93 (4′-ph-C),
167.67(-C=O); IR(KBr) ν/cm-1: 3726.39, 3261.70, 2932.91, 2864.96, 1872.93, 1781.03, 1598.13,
1481.35, 1462.22, 1438.30, 1379.77, 1337.55, 1257.55, 1173.43, 1129.43, 1078.23, 1043.84,
957.38, 865.49, 835.10, 764.57, 732.68, 679.61; HREIMS (m/z): 654.3040 [M+Na]+ (calcd. for
C35H53NO3SeNa 654.3037).
6β-[3'-Oxo-5'-methoxylbenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14j)
Faint yellow solid. Yield: 67.97%, m.p. 209-210 oC; 1H NMR (300MHz, CDCl3) δ: 0.69 (3H,
s, 18-CH3), 0.86 (6H, d, J = 6.3, 26- and 27-CH3), 0.87 (3H, s, 19-CH3), 0.92 (3H, d, J = 6.3,
21-CH3), 3.66 (1H, s, -OH), 3.81-3.88 (2H, m, C6-H), 3.84 (3H, d, Ph-OCH3), 4.00 (1H, br s,
C3-αH), 4.87 (1H, s, -OH), 7.18 (1H, dd, J = 8.7, 2.7, 4′-Ph-H), 7.47 (1H, d, J = 8.7, 3′-Ph-H),
7.49 (1H, d, J = 2.7, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 12.59 (18-C), 18.74 (19-C), 18.84
(21-C), 21.39 (11-C), 22.57 (26- or 27-C), 22.84 (26- or 27-C), 23.88 (23-C), 24.87 ( 16-C), 27.39
(15-C), 28.01 (25-C), 28.10 (1-C), 28.84 (8-C), 35.67 (2-C), 36.24 (20-C), 39.48 (22-C), 39.85
(6-C), 43.34 (12-C), 44.67 (24-C), 44.80 (13-C), 44.98 (4-C), 46.44 (7-C), 49.91 (10-C), 55.35
(9-C), 55.53 ( 14-C), 55.64 (17-C), 56.68 (5-Ph-OCH3), 67.32 (3-C), 80.55 (5-C), 110.21
(6′-ph-C), 121.93 (4′-ph-C), 124.47 (3′-ph-C), 128.30 (1′-ph-C), 130.41 (2′-ph-C), 158.72
(5′-ph-C), 167.70 (-C=O); IR(KBr) ν/cm-1: 3269.67, 2935.69, 2862.10, 1616.41, 1568.18,

1470.64, 1427.48, 1352.40, 1276.45, 1231.53, 1170.31, 1150.74, 1128.47, 1077.13, 1021.74,
977.83, 959.87, 921.86, 866.09, 813.82, 764.44, 732.28, 634.11, 550.86, 506; HREIMS (m/z):
654.3028 [M+Na]+ (calcd. for C35H53NO3SeNa 654.3037).
6β-[3'-Oxo-6'-nitrobenzisoselenazol-2'-(3H)-ylmethyl]-B-norcholestane-3β,5β-diol (14k)
o
1
Faint yellow solid. Yield: 47.59%, m.p. 217-220 C; H NMR (600 MHz, CDCl3) δ: 0.70
(3H, s, 18-CH3), 0.865 (3H, d, J = 6.6, 26- or 27-CH3), 0.870 (3H, d, J = 6.6, 26- or 27-CH3), 0.88
(3H, s, 19-CH3), 0.91 (3H, d, J = 6.6, 21-CH3), 1.98-2.07 (2H, m, C4-H), 2.35 (1H, OH),
3.66-3.72 (1H, m, C3-αH), 4.13 (1H, d, J = 13.8, C6-H), 4.15 (1H, OH), 4.25 (1H, d, J = 13.8,
C6-H), 8.14 (1H, d, J = 8.4, 6′-Ph-H), 8.21 (1H, d, J = 8.4, 5′-Ph-H), 8.50 (1H, s, 3′-Ph-H);
13CNMR(75MHz,CDCl3) δ: 12.68 (18-C), 18.92 (19-C), 19.03 (21-C), 21.53 (11-C), 22.64 (26 or
27-C), 22.91 (26 or 27-C), 24.00 (23-C), 27.14 (16-C), 28.12 (15-C), 28.20 (25-C), 28.91 (1-C),
29.79 (8-C), 35.78 (2-C), 36.34 (20-C), 39.57 (22-C), 39.95 (6-C), 43.39 (12-C), 44.54 (24-C),
44.91 (13-C), 45.33 (4-C), 46.89 (7-C), 49.96 (10-C), 54.93 (9-C), 55.71 (14-C), 56.75 (17-C),
67.85 (3-C), 80.58 (5-C), 119.57 (3′-ph-C), 120.88 (5′-ph-C), 129.44 (6′-ph-C), 132.22 (1′-ph-C),
140.91 (2′-ph-C), 150.00 (4′-ph-C), 166.00 (-C=O); IR(KBr) ν/cm-1:670.18、729.59、1078.91、
1164.35、1346.29、1401.15、1496.31、1527.42、1616.28、1653.77、2953.70、3133.78、
3429.25; HREIMS (m/z): 649.2956 [M+H]+ (calcd. for C34H51N2O5Se 647.2963).
4.2 Biological Studies
Using 96-well plates on a MLLTISKAN MK3 analysis spectrometer, the cell proliferation
was tested with MTT method. SKOV3 (ovarian carcinoma), PC-3 (prostatic carcinoma), T47D
(breast carcinoma) and MCF-7 (breast carcinoma) cell were obtained by Guangxi Medical
University (China).
Materials
The compounds were dissolved by sterile dimethyl sulfoxide (DMSO) (Sigma) at initial
concentration of 10 mg/mL. The stock solution was diluted with complete nutrient medium
(RPMI-1640) supplemented with 10% heat inactivated fetal bovine serum and 0.1 g/L penicillin G
+ 0.1 g/L streptomycin sulfate.

Cell culture
All cells were grown in the medium (RPMI-1640) supplemented with 10% heat inactivated
fetal bovine serum, 0.1 g/L penicillin G + 0.1 g/L streptomycin sulfate. The cell culture was
incubated in a humidified atmosphere of 5% CO2 at 37 oC.
Assay for cell viability
Cells (1~2×104 cells/mL) were seeded into 96-wells plates for 24 h. The medium contained
test compound with different concentrations was added to the cells. After 72 h incubation, 20 µL
of the tetrazolium dye (MTT) (5 mg/mL) solution were added to each well. After additional 4 h
incubation, the medium was discarded, and 200 µL of DMSO were added to dissolve the purple
formazan. The untreated cells were used as controls. Triplicate wells were prepared for each
individual dose. The absorbance values (A) at 492 nm were determined using a MLLTISKAN
MK3 analysis spectrometer (Thermo Scientific Co.). The IC50 values were calculated as the
concentration of drug yielding 50% cell survival.
Acknowledgments
The authors acknowledge the financial support of “The National Natural Science Foundation
of China, China (No: 21762008; No: 21462009)”, “the Guangxi Natural Science Foundation of
China (No: 2018JJA120087)” and Universities Key Laboratory Funds of Synthetic and Natural
Functional Molecular Chemistry.
Conflict of Interest
The authors declare no conflict of interest.
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Highlights
1. Two novel steroid-benzisoselenazolone hybrids were synthesized.
2. Hybrids were obtained by condensing of 2-chloroselanylbenzoyl
chloride with amino-steroids.
3. The antiproliferative activity of the hybrids was investigated.
4. Some hybrids had excellent inhibitory activity on SKOV3 cells.
Graphical Abstract
Synthesis and Antiproliferative Evaluation of
Novel Steroid-Benzisoselenazolone Hybrids
Leave this area blank for abstract info.
Jianguo Cui*, Meizhen Wei, Liping Pang, Chunfang Gan, Haixin Shi, Junan Xiao, Junyan Zhan, Zhiping Liu
Yanmin Huang*
R
Se
O
N
O
O
HO
HO
N
HO
OH
R
Se
HO