Synthesis and antiproliferative activity of 17-[1′,2′,3′]-selenadiazolylpregnenolone compounds

Article abstract of DOI:10.1016/j.steroids.2018.10.004

Using pregnenolone as a starting material, some 3-substituted 17-[1′,2′,3′]-selenadiazolylpregnenolone derivatives were synthesized, and their structures were characterized by IR, NMR and HRMS. The in vitro antitumor activity of the compounds was assayed

Full text of DOI:10.1016/j.steroids.2018.10.004

Accepted Manuscript
Synthesis and AntiproliferativeActivity of 17-[1’,2’,3’]-selenadiazolylpregne-
nolone compounds
Jianguo Cui, Liping Pang, Meizhen Wei, Chunfang Gan, Dandan Liu, Haiyan
Yuan, Yanmin Huang
PII:
S0039-128X(18)30191-0
https://doi.org/10.1016/j.steroids.2018.10.004
STE 8326
DOI:
Reference:
Steroids
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Revised Date:
Accepted Date:
13 September 2018
25 September 2018
1 October 2018
Please cite this article as: Cui, J., Pang, L., Wei, M., Gan, C., Liu, D., Yuan, H., Huang, Y., Synthesis and
AntiproliferativeActivity of 17-[1’,2’,3’]-selenadiazolylpregnenolone compounds, Steroids (2018), doi: https://
doi.org/10.1016/j.steroids.2018.10.004
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Synthesis and AntiproliferativeActivity of
17-[1ʹ,2ʹ,3ʹ]-selenadiazolylpregnenolone compounds
Jianguo Cui Liping Pang MeizhenWei Chunfang Gan
Dandan Liu Haiyan Yuan Yanmin Huang*
College of Chemistry and Material Science, Guangxi Teachers Education University, Nanning, 530001, PR China
*Corresponding author at: College of Chemistry and Material Science, Nanning Normal University, Nanning.
530001, China. E-mail address: Huangyanmin828@163.com (YM. Huang).
ABSTRACT
Using pregnenolone as a starting material, some 3-substituted
17-[1ʹ,2ʹ,3ʹ]-selenadiazolylpregnenolone derivatives were synthesized, and their structures were
characterized by IR, NMR and HRMS. The in vitro antitumor activity of the compounds was
assayed against PC-3、SKOV3、T47D、MCF-7 and HEK293T cell lines. The results show that
some compounds display selective antiproliferative activity against PC-3 and SKOV3 cells lines
and are almost inactive to normal kidney epithelial cells (HEK293T). The IC50 value are much
better than that of abiraterone (positive control).
KEYWORDS
17-[1ʹ,2ʹ,3ʹ]-selenadiazolylpregnenolone; antiproliferative activity; [1ʹ,2ʹ,3ʹ]-selenadiazole;
Pregnenolone; Organoselenium compounds.
1. Introduction
Selenium is a key component of severalmajor metabolic pathways in human, including
thyroidhormone metabolism, antioxidant defense system, andimmune function[1]. Organoselenium
compounds have attracted special attention over the last years mainly because of their diversified
biological activity. Compared with inorganoselenium compound, organoselenium compound has
some excellent characteristics, such as higher bioavailability, stronger bioactivity, lower toxicity,
smaller environmental pollution, and anti-oxidation, anti-inflammatory and anti-cancer, etc. [2-3]
.
Karam et al. found that synthetic organoselenium compounds had a stronger antitumor activity
and lower toxicity than inorganoselenium compounds [4]. At present, some organoselenium
compounds have been used in anti-tumor clinical studies and achieved some good results, such as

ebselen [5-6], ethaselen [7-9] and selenazofurin [10-11], etc.
Selenazoles are interesting five-membered heterocycles which are being reported for their
potential pharmacological features [12-15]. In addition, some selenadiazole derivatives have
triggered intense research interest due to their excellent anticancer biological activities[16-20]
.
[1,2,3]-selenadiazole compounds are a type of compounds in which a selenium atom is directly
connected with two nitrogen atoms. Because of its simple preparation [21-22] and good bioactivity
[23-25], it has received great attention. Recently, Tibor Pasinszki synthesized a
4,5-dihydrobenzoferroceno[1,2-d][1,2,3]selenadiazole and its benzo-fused analogue, and found
that the antiproliferative effect of both compounds were active against cervical HeLa, ovarian
A2780 and breast MDA-MB-231 cell lines[26]
.
Steroidal compounds are a kind of essential physiological active substances in the human life
process, which have high permeability to cells and binding ability to the cell nucleus and cell
membrane. Small changes in steroids often lead to widespread changes in their biological activity,
and may be used to treat different diseases in humans. In recent years, pharmacologist and chemist
have been committed to the structure transformation of steroid compounds through different ways,
such as introducing different hetero atoms and functional groups or heterocyclic groups into the
steroid, or changing the structure of steroid nucleus or branched chain, and then further explore
their biological activity and clinical application [27].However, 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 wasn’t carried out in
this field [28-31]
.
Recently, Fernández-Herrera M. A. et al. introduced a selenocyano group into
22-oxo-26-hydroxycholestane and synthesized two novel 22-oxo-26-selenocyanocholestanic
steroids [32]. Romero-Hernández L. L. et al. introduced a selenourea into the diosgenin to prepare a
selenourea-steroid compound [33]. Fuentes-Aguilar A. et al. introduced abselen into diosgenin,
hecogenin and smilagenin, and prepared three steroidal ebselen analogues [34]. The result of
bioactivity tests showed that all the steroidal selenium compounds obtained above had a good
antiproliferative activity in vitro.
In previous studies, we synthesized some novel heterosteroids[35-38], and the results of
antiproliferative test showed that some B-norcholesteryl benzimidazoles, thiazoles and

17-hydrazone aromatic steroidal heterocycle derivatives exhibited an excellent antiproliferative
activity. In continuation of our work on the synthesis and anticancer activity evaluation of various
heterosteroids, herein, we designed and synthesized a series of novel heterosteroids possessing
[1,2,3]selenadiazole ring as the side chain by using pregnenolone as a starting material. The
antiproliferative activity of compounds had been evaluated against SKOV3, PC-3, T47D and
MCF-7 cancer cell lines.
2. Result and discussion
2.1 Chemistry
2.1.1 Synthesis of pregnenolone-17-[1ʹ,2ʹ,3ʹ] selenadiazole
First, pregnenolone was transformed to compound 2 by the reaction with semicarbazide and
CH3CH2ONa. Then, the reaction of compound 2 with selenium dioxide and acetic acid afforded
corresponding pregnenolone-17-[1ʹ,2ʹ,3ʹ]selenadiazole (3) (Scheme 1). The structure of compound
3 had been confirmed on analytical and spectral data. In the NMR spectrum of compound 3,
resonances signals of Ar-H at δ 8.86 ppm and Ar-C at δ 137.79, 164.49ppm demonstrate the
presence of a selenadiazole ring. The HREI mass spectrum of compound 3 exhibits a molecular
ion peak ([M+H]+) at m/z 407.1604. All spectra confirm the structure of compound 3.
Scheme 1 The synthesis of compound pregnenolone-17-[1ʹ,2ʹ,3ʹ] selenadiazole
2.1.2 Synthesis of pregnenolone-17-[1ʹ,2ʹ,3ʹ]selenadiazole derivatives
To investigate the effect of different 3-substituted group in compound 3 to antiproliferative
acitivity, pregnenolone-17-[1ʹ,2ʹ,3ʹ]selenadiazole derivatives 6a-6i were prepared (Scheme 2) and
evaluated for their antiproliferative activity.

Scheme 2 The synthesis of pregnenolone-17-[1ʹ,2ʹ,3ʹ]selenadiazole derivatives
Firstly, the reaction of compound 1 with various acyl chlorides afforded corresponding
carboxylic pregnenoloneesters 4a-4i. After that, compounds 5a-5i were obtained by the reaction of
compounds 4a-4i with semicarbazide. Last, the reaction of compounds 5a-5i with selenium
dioxide gave the target compounds 6a-6i. The structure of all synthesized compounds had been
confirmed on analytical and spectral data.
2.2 The evaluation of the antiproliferative activityin vitro
All synthesized compounds were evaluated for their antiproliferative activities in vitro
against PC-3 (human prostate carcinoma), SKOV3 (human ovarian carcinoma), T47D (human
breast infiltrating duct carcinoma), MCF-7(human breast adenocarcinoma) and HEK293T (normal
kidney epithelial cells) cell lines using a MTT assay. The results were summarized as IC50 values
in µmol/L in Table 1.
Tab.1 In vitro antiproliferative activities (IC50 inµM) of pregnenolone selenadiazole compounds
compd
3
SKOV3
PC-3
T47D
MCF-7
>100
>100
>100
>100
>100
>100
>100
>100
HEK-293T
>100
57.59±0.86
42.35±0.66
36.60±1.05
55.31±11.02
35.79±0.47
>100
37.35±0.06
13.24±0.41
31.74±0.32
56.31±2.01
12.48±0.44
>100
71.28±1.31
40.65±0.10
41.65±0.24
>100
>100
6a
>100
6b
6c
>100
>100
>100
6d
6e
>100
>100
>100
59.85±5.48
>100
>100
>100
6f
>100
>100
>100
6g

33.88±0.13
18.26±0.49
51.51±15.98
18.56±0.82
21.40±0.37
37.61±0.12
37.39±0.60
46.67±0.19
34.66±1.69
>100
>100
>100
>100
6h
>100
6i
44.70±0.67
abiraterone
As showed in Table 1, all pregnenolone selenadiazole compounds do not exhibit obvious
inhibitory activity against MCF-7 and T47D breast cancer cell lines, but show selective
antiproliferative activity against PC-3 and SKOV3 cell lines. From the perspective of SKOV3
cells, compounds (6a, 6b and 6c) display obvious inhibitory activity when the substituted group R
is the alkyl groups. When the substituent R is phenyl or substituted phenyls (6e, 6f, 6g),
compounds show no obvious anticancer activity to SKOV3 cells. However, when R is the
thiophenyl or furanyl of 5-member heterocycle, the compounds manifest good antiproliferative
activity. For example, IC50 of compound 7i is 18.26 μmol/L against SKOV3 cells which is vastly
superior to the 51.51μmol/L of the positive control abiraterone.
When the substituent R is propyl, cyclopropyl or thiophenyl and furanyl, the compounds (6a,
6d and 7h, 7i) display very good inhibitive effect on the proliferation of PC-3 cells with IC50 value
of 13.24, 12.48, 18.56 and 21.40 μmol/L, respectively. The result is much better than the
37.61μmol/L of abiraterone.
Furthermore, all tested compounds are almost inactive to normal kidney epithelial cells
(HEK293T).
3. Conclusion
Using pregnenolone as a starting material, some 3-substituted
17-[1ʹ,2ʹ,3ʹ]-selenadiazolylpregnenolone compounds were synthesized, and the structures of all
investigated compounds were characterized by IR, NMR and HRMS. The in vitro antitumor
activity of the compounds was assayed against PC-3、SKOV3、T47D、MCF-7 and HEK293T cell
lines. The results show that some compounds display selective inhibitory activity against PC-3 and
SKOV3 cells when the substituted group R is the alkyl groups, thiophenyl or furanyl, and the IC50
value are much better than that of abiraterone (positive control). Moreover, all compounds are
almost inactive to normal kidney epithelial cells (HEK293T). The information obtained from the
studies is valuable for the design of novel steroidal chemotherapeutic drugs.
4. EXPERIMENT
4.1 Chemistry
4.1.1 Reagent and Instrument

The sterols were purchased from Sinopharm Chemical Reagent Co., Ltd, Shanghai, China.
All chemicals and solvents are analytical grade. Melting points were determined on an X4
apparatus (Beijing Tech Instrument Co. Ltd., Beijing, China) and were uncorrected. Infrared
spectra were measured with a Thermo Scientific Nicolet IS-10 Spectrophotometer (Thermo
Scientific, America). The 1H and 13C NMR spectra were recorded in CDCl3 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, respectively. 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 a MTT method using 96-well plates on a MLLTISKAN MK3 analysis spectrometer (Thermo
Scientific, Shanghai, China).
4.1.2 General procedure for the synthesis of compounds 4a-4i.
600μL of acyl chloride was injected slowly to a solution of pregnenolone (500mg,1.6mol）) in
15 mL of pyridine. The mixture was stirred at 45oC until no starting material was observed (the
progress of the reaction was monitored by TLC). 2 mL of ice water was added to terminate the
reaction. Then 30 mL of 1M hydrochloric acid and 50 mL of ethyl acetate were added, organic
phase was separated and water phase was extracted with ethyl acetate. The combined organic
phase was washed twice with 1 M hydrochloric acid and one time with 10mL solution of saturated
copper sulfate to ensure residual pyridine removed. Then the organic phase was washed in turn
with saturated NaHCO3, water and saturated brine, dried with anhydrous sodium sulfate. After
solvent was removed under reduced pressure, the crude product was purified by the column
chromatography on silica gel using CH3COOEt/petroleum ether (1:20) as the eluent to give the
target product.
3β-Propionyloxypregnenolone (4a)
White solid, Yield: 57.52%, m.p. 121-122oC; 1H NMR (300 MHz, CDCl3) δ: 0.58 (3H, s,
18-CH3), 0.97 (3H, s, 19-CH3)，1.08 (3H, t, J = 7.5, 3′-CH3), 2.07 (3H, s, 21-CH3), 2.26 (2H, q, J
= 7.5, 2′-CH2), 2.49 (1H, t, J = 8.7, C4-βH), 4.50-4.61 (1H, m, C3-αH), 5.31 (1H, d, J = 4.8,
C6-H); 13C NMR (75MHz, CDCl3) δ: 9.14 (3′-C), 13.16 (18-C), 19.26 (19-C), 20.98 (11-C), 22.75
(15-C), 24.43 (16-C), 27.69 (2′-C), 27.82 (2-C), 31.49 (21-C), 31.71 (8-C), 31.76 (7-C), 36.54

(10-C), 36.95 (1-C), 38.04 (4-C), 38.72 (12-C), 43.90 (13-C), 49.81 (9-C), 56.76 (14-C), 63.58
(17-C), 73.51 (3-C), 122.19 (6-C), 139.64 (5-C), 173.80 (-C=O), 209.34 (20-C); IR(KBr) ν/cm-1:
3728.81, 3628.14, 3068.31, 2963.20, 2939.87, 2899.06, 2874.33, 2825.44, 1728.49, 1469.90,
1383.11, 1364.35, 1310.29, 1260.18, 1188.78, 1136.16, 1088.55, 1044.45, 1015.68, 1000.50,
957.92, 900.90, 803.86, 669.88, 603.80; HREIMS (m/z): 395.2564 [M+Na]+ (calcd. For
C24H36NaO3 395.2562).
3β-Butanoyloxypregnenolone (4b)
White solid, Yield: 67.81%, m.p. 88-91 oC; 1H NMR (300 MHz, CDCl3) δ: 0.60 (3H, s,
18-CH3), 0.91 (3H, t, J = 7.5, 4′-CH3), 0.99 (3H, s, 19-CH3), 1.61 (2H, q, J = 7.5, 3′-CH3), 2.09
(2H, s, 21-CH3), 2.22 (2H, t, J = 7.5, 2′-CH2), 2.51 (1H, t, J = 9.0, C4-βH), 4.53-4.64 (1H, m,
C3-αH), 5.34 (1H, d, J = 4.5, C6-H); 13C NMR (75 MHz, CDCl3) δ: 13.18 (18-C), 13.61 (4′-C),
18.42 (3′-C), 19.28 (19-C), 20.99 (11-C), 22.77 (15-C), 24.44 (16-C), 27.73 (2-C), 31.51 (21-C),
31.72 (8-C), 31.77 (7-C), 36.30 (2′-C), 36.50 (10-C), 36.56 (1-C), 38.07 (4-C), 38.74 (12-C),
43.92 (13-C), 49.83 (9-C), 56.78 (14-C), 63.60 (17-C), 73.46 (3-C), 122.21 (6-C), 139.65 (5-C),
173.03 (1′-C), 209.40 (20-C); IR(KBr) ν/cm-1: 2943.08, 2872.36, 2849.21, 1734.72, 1708.76,
1467.82, 1386.16, 1356.89, 1300.75, 1226.88, 1189.11, 1135.60, 1111.55, 1013.52, 985.43, 955.69,
922.28, 797.96, 592.42; HREIMS (m/z): 409.2716 [M+Na]+ (calcd. For C25H38NaO3 409.2719).
3-Trichloroacetoxypregnenolone (4c)
White solid, Yield: 86.05%, m.p. 177-180 oC; 1H NMR (300 MHz, CDCl3) δ: 0.61 (3H, s,
18-CH3), 1.03 (3H, s, 19-CH3), 2.10 (3H, s, 21-CH3), 2.52 (1H, t, J = 9.0, C4-βH), 4.68-4.77 (1H,
m, C3-αH), 5.42 (1H, d, J = 4.8, C6-H); 13C NMR (75MHz, CDCl3) δ: 13.20 (18-C), 18.42 (19-C),
19.28 (11-C), 21.02 (15-C), 22.79 (16-C), 24.45 (2-C), 27.04 (21-C), 31.54 (8-C), 31.73 (7-C),
36.53 (10-C), 36.73 (1-C), 37.23 (4-C), 38.69 (12-C), 43.91 (13-C), 49.75 (9-C), 56.73 (14-C),
63.57 (17-C), 79.57 (3-C), 90.18 (CCl3), 123.32 (6-C), 138.60 (5-C), 161.25 (-C=O), 209.37
(20-C); IR(KBr) ν/cm-1: 3123.78, 2967.14, 2933.09, 2860.77, 2829.75, 1758.20, 1705.66, 1450.45,
1379.93, 1358.97, 1319.66, 1256.33, 1175.34, 1155.51, 1137.14, 1089.24, 1007.24, 980.66,
943.15, 927.38, 858.25, 827.67, 754.80, 680.41, 639.39, 597.26, 577.54; HREIMS (m/z):
483.1239 [M+Na]+ (calcd. For C23H31Cl3NaO3 483.1236).
3-Cyclopropylformyloxypregnenolone (4d)
White solid, Yield: 49.75%, m.p. 217-219 oC; 1H NMR (300 MHz, CDCl3) δ: 0.54 (3H, s,

18-CH3), 0.73-0.76 (2H, m, 3′-CH2), 0.84-0.89 (2H, m, 4′-CH2), 0.94 (3H, s, 19-CH3), 2.03 (3H, s,
21-CH3), 2.45 (1H, t, J = 8.7, C4-βH), 4.44-4.55 (1H, m, C3-αH), 5.28 (1H, d, J = 3.3, C6-H); 13C
NMR (75MHz, CDCl3) δ: 8.25 (3′,4′,-C), 13.01 (18,-C), 13.13 (2′-C), 19.24 (19-C), 20.93 (11-C),
22.72 (15-C), 24.41 (16-C), 27.69 (2-C), 31.44 (21-C), 31.69 (8-C), 31.72 (7-C), 36.50 (10-C),
36.94 (1-C), 38.04 (4-C), 38.68 (12-C), 43.84 (13-C), 49.78 (9-C), 56.71 (14-C), 63.51 (17-C),
73.59 (3-C), 122.14 (6-C), 139.61 (5-C), 174.05 (1′-C=O), 209.14 (20-C); IR(KBr) ν/cm-1:
3448.45, 2939.46, 2852.15, 1699.24, 1469.21, 1450.99, 1384.04, 1356.75, 1291.05, 1229.42,
1196.29, 1133.57, 979.01, 927.14, 882.35, 854.98, 839.47, 784.52, 764.10, 722.21, 592.69;
HREIMS (m/z): 407.2566 [M+Na]+ (calcd. For C25H36NaO3 407.2562).
3-Benzoyloxypregnenolone (4e)
White solid, Yield: 78.41%, m.p. 203-204 oC; 1H NMR (300 MHz, CDCl3) δ: 0.63 (3H, s,
18-CH3), 1.06 (3H, s, 19-CH3), 2.13 (3H, s, 21-CH3), 2.54 (1H, t, J = 9.0, C4-βH), 4.81-4.91 (1H,
m, C3-αH), 5.42 (1H, d, J = 4.2, C6-H), 7.42 (2H, t, J = 7.5, 3′, 5′-Ph-H), 7.54 (1H, t, J = 6.0,
4′-Ph-H), 8.04 (2H, d, J = 6.9, 2′,6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.22 (18-C), 19.37
(19-C), 21.05 (11-C), 22.81 (15-C), 24.48 (16-C), 27.84 (2-C), 31.56 (21-C), 31.78 (8-C), 31.81
(7-C), 36.64 (10-C), 37.02 (1-C), 38.16 (4-C), 38.76 (12-C), 44.00 (13-C), 49.86 (9-C), 56.80
(14-C), 63.65 (17-C), 74.45 (3-C), 122.46 (6-C), 128.27 (3′- or 5′-Ph-C), 128.41 (3′- or 5′-Ph-C),
129.52 (2′- or 6′-Ph-C), 130.09 (2′- or 6′-Ph-C), 130.71 (1′-Ph-C), 132.77 (4′-Ph-C), 139.61 (5-C),
166.01 (-C=O), 209.75 (20-C); IR(KBr) ν/cm-1: 3387.33, 2945.99, 2899.52, 2882.82, 2834.51,
1714.35, 1701.92, 1450.62, 1382.87, 1350.71, 1312.01, 1271.81, 1253.84, 1225.41, 1178.60,
1163.08, 1111.36, 1066.60, 1026.07, 994.46, 980.21, 946.75, 837.23, 711.89, 683.58, 592.83;
HREIMS (m/z): 443.2561 [M+Na]+ (calcd. For C28H36NaO3 443.2562).
3-(2′-Methylbenzoyloxy)pregnenolone (4f)
White solid, Yield: 77.48%, m.p. 187-189 oC; 1H NMR (300MHz, CDCl3) δ: 0.64 (3H, s,
18-CH3), 1.06 (3H, s, 19-CH3), 2.13 (3H, s, 21-CH3), 2.54 (1H, t, J = 9.0, C4-βH), 2.60 (3H, s,
Ph-CH3), 4.80-4.91 (1H, m, C3-αH), 5.43 (1H, d, J = 4.2, C6-H), 7.26-7.21 (2H, m, 3′,5′-Ph-H),
7.38 (1H, t, J = 6.3, 4′-Ph-H), 7.91 (1H, d, J = 8.4, 6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.23
(18-C), 19.37 (19-C), 21.05 (11-C), 21.78 (Ph-CH3), 22.82 (15-C), 24.49 (16-C), 27.91 (2-C),
31.56 (21-C), 31.78 (8-C), 31.82 (7-C), 36.66 (10-C), 37.07 (1-C), 38.23 (4-C), 38.77 (12-C),
43.98 (13-C), 49.88 (9-C), 56.80 (14-C), 63.65 (17-C), 74.30 (3-C), 122.44 (6-C), 125.66

(5′-Ph-C), 130.23 (6′-Ph-C), 130.42 (1′-Ph-C), 131.61 (3′-Ph-C), 131.75 (4′-Ph-C), 139.64
(2′-Ph-C), 139.82 (5-C), 167.12 (-C=O), 209.60 (20-C); IR(KBr) ν/cm-1: 3060.45, 2967.81,
2935.60, 2852.84, 1700.45, 1601.26, 1438.21, 1383.57, 1352.77, 1292.48, 1258.04, 1192.13,
1166.55, 1131.30, 1087.40, 1068.81, 1051.83, 1025.85, 1011.38, 978.45, 947.51, 838.15, 801.19,
742.23, 592.72; HREIMS (m/z): 457.2718 [M+Na]+ (calcd. For C29H38NaO3 457.2719).
3-(4′-Chlorobenzoyloxy)pregnenolone (4g)
White solid, Yield: 73.94%, m.p. 185-187 oC; 1H NMR (300 MHz, CDCl3) δ: 0.61 (3H, s,
18-CH3), 1.03 (3H, s, 19-CH3), 2.10 (3H, s, 21-CH3), 2.45 (2H, d, J = 7.8, C7-H), 2.51 (1H, t, J =
9.0, C4-βH), 4.77-4.88 (1H, m, C3-αH), 5.39 (1H, d, J = 4.5, C6-H), 7.38 (2H, d, J = 8.4,
3′,5′-Ph-H), 7.95 (2H, d, J = 8.4, 2′,6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.20 (18-C), 19.33
(19-C), 21.03 (11-C), 22.80 (15-C), 24.46 (16-C), 27.79 (2-C), 31.53 (21-C), 31.77 (8-C), 31.78
(7-C), 36.61 (10-C), 36.98 (1-C), 38.10 (4-C), 38.14 (12-C), 43.9 (13-C), 49.84 (9-C), 56.77
(14-C), 63.61 (17-C), 74.72 (3-C), 122.55 (6-C), 128.57 (1′-Ph-C), 129.17 (3′,5′-Ph-C), 130.92
(2′,6′-Ph-C), 139.12 (4′-Ph-C), 139.45 (5-C), 165.00 (-C=O), 209.40 (20-C); IR(KBr) ν/cm-1:
2942.67, 2883.00, 2834.21, 1787.23, 1716.31, 1701.31, 1592.60, 1486.42, 1448.21, 1399.50,
1382.55, 1354.13, 1321.34, 1274.44, 1190.53, 1167.36, 1114.81, 1089.94, 1067.79, 1013.97,
993.63, 979.10, 946.03, 850.83, 760.35, 682.79, 593.66; HREIMS (m/z): 477.2169 [M+Na]+
(calcd. For C28H35ClNaO3 477.2172).
3-(2′-Thiophenoyloxy)pregnenolone (4h)
White solid, Yield: 87.16%, m.p. 225-226 oC; 1H NMR (300 MHz, CDCl3) δ: 0.64 (3H, s,
18-CH3), 1.06 (3H, s, 19-CH3), 2.14 (3H, s, 21-CH3), 2.46 (2H, d, J = 7.8, C7-H), 2.55 (1H, t, J =
9.0, C4-βH), 4.77-4.88 (1H, m, C3-αH), 5.42 (1H, d, J = 3.9, C6-H), 7.09 (1H, dd, J = 5.1, 3.9,
4′-thiophene-H), 7.54 (1H, dd, J = 5.1, 1.5, 5′-thiophene-H), 7.79 (1H, dd, J = 3.9, 1.5,
3′-thiophene-H); 13C NMR (75MHz, CDCl3) δ: 13.23 (18-C), 19.36 (19-C), 21.05 (11-C), 22.82
(15-C), 24.48 (16-C), 27.81 (2-C), 31.56 (21-C), 31.77 (8-C), 31.81 (7-C), 36.62 (10-C), 36.99
(1-C), 38.12 (4-C), 38.76 (12-C), 44.03 (13-C), 49.85 (9-C), 56.81 (14-C), 63.68 (17-C), 74.79
(3-C), 122.54 (6-C), 128.03 (4′-thiophene-C), 132.19 (5′-thiophene-C), 133.23 (3′-thiophene-C),
134.77 (2′-thiophene-C), 139.54 (5-C), 161.76 (-C=O), 209.95(20-C); IR(KBr) ν/cm-1: 3377.77,
3097.14, 2967.31, 2943.69, 2895.70, 2853.16, 1697.32, 1526.21, 1434.16, 1416.09, 1382.74,
1355.99, 1281.41, 1258.55, 1235.69, 1191.14, 1168.75, 1131.47, 1099.14, 1080.54, 1036.82,

1013.02, 976.72, 943.93, 914.14, 862.53, 820.88, 796.39, 753.71, 738.98, 652.33, 591.80;
HREIMS (m/z): 449.2127 [M+Na]+ (calcd. For C26H34SNaO3 449.2126).
3-(2′-Furoyloxy)pregnenolone (4i)
White solid, Yield: 41.97%, m.p. 209-211 oC; 1H NMR (300MHz, CDCl3) δ: 0.60 (3H, s,
18-CH3), 1.02 (3H, s, 19-CH3), 2.11 (3H, s, 21-CH3), 2.43 (2H, d, J = 7.8, C7-H), 2.55 (1H, t,
C17-H), 4.78-4.85 (1H, m, C3-αH), 5.38 (1H, d, C6-H), 6.47-6.79 (1H, m, 3′-furan-H), 7.40 (1H,
d, 5′-furan-H), 7.69 (1H, d, 4′-furan-H); 13C NMR (75MHz, CDCl3) δ: 13.22 (18-C), 19.33 (19-C),
21.04 (11-C), 22.82 (15-C), 24.48 (16-C), 27.79 (2-C), 31.56 (21-C), 31.78 (8-C), 31.82 (7-C),
36.63 (10-C), 37.00 (1-C), 38.10 (4-C), 38.78 (12-C), 43.98 (13-C), 49.89 (9-C), 56.83 (14-C),
63.68 (17-C), 74.56 (3-C), 111.76 (3′-Ph-C), 117.69 (4′-Ph-C), 122.61 (6-C), 139.48 (5-C), 145.07
(2′-C), 146.09 (2′-furan-C), 158.21 (-C=O), 209.54 (20-C); IR(KBr) ν/cm-1: 3136.84, 3107.95,
2945.57, 2894.70, 2871.60, 2852.11, 2835.41, 1715.29, 1699.61, 1569.01, 1470.03, 1451.96,
1437.14, 1393.25, 1380.34, 1355.51, 1321.00, 1294.77, 1230.99, 1172.66, 1122.00, 1079.55,
1015.47, 983.44, 950.14, 884.00, 772.54, 597.97; HREIMS (m/z): 433.2353 [M+Na]+ (calcd. For
C26H34NaO4 433.2355).
4.1.3 General procedure for the synthesis of congpound 2 and compounds 5a-5i.
5.5 mmol of semicarbazide hydrochloride and 6.8 mmol of sodium acetate trihydrate were
added into 30 mL of acetonitrile and stirred for 2 h at 80 oC. During the process of the reaction,
some white precipitation appeared in the solution. After the solid was filtrated, 1.0 mmol of
compound 1 or compounds 4a-4i was added into the filter liquor. After the solution was heated to
80 oC, the mixture was refluxed and stirred at the temperature for 8 h (the progress of the reaction
was monitored by TLC). Then, the reaction was terminated and solvent was removed under
reduced pressure. 400mL of distilled water and 10mL of ethanol was added to the solid, and the
product was sonicated for 10 min under ultrasonication, filtered and dried to obtain the target
product.
Pregnenolon-20-semicarbazone (2)
White solid, Yield: 91.32%, m.p. 206-209℃; 1H NMR (300 MHz, CDCl3) δ: 0.61(3H, s,
18-CH3), 1.03 (3H, s, 18-CH3), 1.82 (3H, s, 21-CH3), 3.55 (1H, br s, C3-αH), 5.37 (1H, s, C6-H),
7.44 (1H, s, -CONH); 13C NMR (75 MHz, CDCl3) δ: 13.25 (18-C), 16.79 (21-C), 19.44 (19-C),

21.09 (11-C), 23.24 (15-C), 24.15 (16-C), 31.61 (8-C), 31.74 (2-C), 32.06 (7-C), 36.53 (10-C),
37.25 (1-C), 38.92 (12-C), 42.25 (4-C), 43.97 (13-C), 50.09 (9-C), 56.4(17-C), 58.80 (14-C),
71.97 (3-C), 121.45 (6-C), 140.77 (5-C), 150.06 (20-C), 157.55 (-CONH2); IR(KBr) ν/cm-1:
3468.21, 3263.71, 3201.53, 2931.29, 2846.33, 1688.58, 1569.41, 1435.61, 1374.25, 1241.27,
1155.73, 1108.67, 1080.69, 1060.09, 955.60, 834.54, 800.59, 767.16, 742.52, 671.47, 616.98,
574.60; HREIMS (m/z): 396.2627 [M+Na]+ (calcd. For C22H35N3NaO2 396.2627).
3β-Propionyloxypregnenolon-20-semicarbazone (5a)
White solid, Yield: 76.15%, m.p. 238-240℃; 1H NMR (300 MHz, CDCl3) δ: 0.59 (3H, s,
18-CH3), 1.03 (3H, s, 19-CH3), 1.14 (3H, t, J = 7.5, 3′-CH3), 1.84 (3H, s, 21-CH3), 2.31 (2H, t, J =
7.5, 2′-CH2), 4.57-4.67 (1H, m, C3-αH), 5.38 (1H, d, J = 4.5, C6-H), 8.13 (1H, s, -CONH);
13CNMR (75MHz, CDCl3) δ: 9.18 (3′-C), 13.20 (18-C), 17.16 (19-C), 19.34 (11-C), 21.03 (21-C),
23.29 (15-C), 24.14 (2-C ), 27.75 (2′-C), 27.91 (17-C), 31.73 (8-C), 32.01 (7-C), 36.61 (16-C),
37.00 (1-C), 38.10 (10-C), 38.84 (4-C), 43.91 (12-C ), 50.00 (13-C ), 56.34 (9-C), 58.79 (14-C),
73.62 (3-C), 122.30 (6-C), 139.73 (5-C), 150.33 (20-C), 158.08 (-CONH2), 173.96 (-COO);
IR(KBr) ν/cm-1: 3467.35, 3190.50, 2967.64, 2941.32, 2848.50, 1733.60, 1695.75, 1579.42,
1439.71, 1376.55, 1237.86, 1195.66, 1110.65, 1082.62, 1026.40, 769.26, 670.06, 577.19;
HREIMS (m/z): 452.2890 [M+Na]+ (calcd. For C25H39NaN3O3 452.2889).
3β-Butanoyloxypregnenolon-20-semicarbazone (5b)
White solid, Yield: 82.32%, m.p. 224-226 ℃; 1H NMR (300 MHz, CDCl3) δ: 0.60 (3H, S,
18-CH3)，0.93 (3H, t, J = 7.5, 4′-CH3), 1.03 (3H, s, 19-CH3), 1.66 (2H, q, J = 7.5, 3′-CH3), 1.84
(3H, s, 21-CH3), 2.27 (2H, t, J = 7.2, 2′-CH2), 2.33 (1H, d, J = 6.9, C4-H), 4.58-4.68 (1H, m,
C3-αH), 5.39 (1H, d, J = 4.8, C6-H), 7.95 (1H, s, -CONH); 13C NMR (75MHz, CDCl3) δ: 13.21
(18-C), 13.64 (4′-C), 17.06 (19-C), 18.54 (3′-C), 19.35 (11-C), 21.03 (21-C), 23.28 (15-C), 24.14
(2-C), 27.77 (17-C), 31.77 (8-C), 32.02 (7-C), 36.57 (2′-C), 36.61 (16-C), 37.00 (1-C), 38.12
(10-C), 38.85 (4-C), 43.92 (12-C), 50.00 (13-C), 56.34 (9-C), 58.79 (14-C), 73.55 (3-C), 122.30
(6-C), 139.73 (5-C), 150.28 (20-C), 157.88 (-CONH2), 173.17 (-COO); IR(KBr) ν/cm-1: 3745.89,
3471.00, 3194.10, 2964.65, 2940.57, 1732.40, 1696.79, 1668.28, 1569.73, 1447.94, 1374.75,
1256.52, 1188.64, 1106.14, 999.82, 957.04, 768.19; HREIMS (m/z): 466.3044 [M+Na]+ (calcd.
For C26H41NaN3O3 466.3046).
3-Trichloroacetoxypregnenolon-20-semicarbazone (5c)

White solid, Yield: 74.14%, m.p. 240-242 ℃; 1H NMR (300 MHz, CDCl3) δ: 0.61 (3H, S,
18-CH3), 1.07 (3H, s, 19-CH3), 1.84 (3H, s, 21-CH3), 2.48 (1H, d, J = 9.0, C4-βH), 4.73-4.84 (1H,
m, C3-αH), 5.46 (1H, d, J = 4.8, C6-H), 7.85 (1H, s, -CONH); 13C NMR (75MHz, CDCl3) δ:
13.22 (18-C), 17.02 (18-C), 19.33 (11-C), 21.04 (21-C), 23.27 (15-C), 24.14 (2-C), 27.07 (17-C),
31.73 (8-C), 31.97 (7-C), 36.57 (16-C), 36.76 (1-C), 37.27 (10-C), 38.80 (4-C), 43.92 (12-C),
49.92 (13-C), 56.29 (9-C), 58.77 (14-C), 79.65 (3-C), 90.19 (2′-C), 123.40 (6-C), 138.66 (5-C),
150.16 (20-C), 157.72 (-CONH2), 161.37 (-COO); IR(KBr) ν/cm-1: 3465.29, 3204.36, 2939.84,
2845.71, 1761.22, 1693.94, 1570.32, 1439.72, 1370.45, 1243.20, 1107.43, 1084.43, 984.41,
925.83, 860.43, 828.33, 768.26, 681.19; HREIMS (m/z): 540.1569 [M+Na]+ (calcd. For
C24H34Cl3NaN3O3 540.1563).
3-Cyclopropylformyloxypregnenolon-20-semicarbazone (5d)
White solid, Yield: 74.31%, m.p.231-233℃; 1H NMR (300 MHz, CDCl3) δ: 0.60 (3H, s,
18-CH3), 0.86 (2H, d, J = 7.8, 3′-CH2), 0.98 (2H, d, J = 7.8, 4′-CH2), 1.04 (3H, s, 19-CH3), 1.83
(3H, s, 21-CH3), 2.35 (2H, d, J = 9.0, C4-H), 4.57-4.68 (1H, m, C3-αH), 5.39 (1H, d, J = 4.5,
C6-H), 7.78 (1H, s, -CONH); 13C NMR (75MHz, CDCl3) δ: 8.36 (3′,4′-C), 13.15 (18-C), 13.22
(2′-C), 16.97 (19-C), 19.35 (11-C), 21.03 (21-C), 23.27 (15-C), 24.14 (2-C), 27.78 (17-C), 31.73
(8-C), 32.02 (7-C), 36.61 (16-C), 37.00 (1-C), 38.13 (10-C), 38.86 (4-C), 43.94 (12-C), 50.00
(13-C), 56.34 (9-C), 58.79 (14-C), 73.76 (3-C), 122.29 (6-C), 139.76 (5-C), 150.21 (20-C), 157.65
(-CONH2), 174.34 (-COO); IR(KBr) ν/cm-1: 3466.51, 3238.33, 2935.45, 2849.37, 1717.99,
1694.07, 1579.59, 1474.95, 1439.46, 1399.47, 1372.66, 1300.98, 1233.12, 1186.43, 1118.99,
1084.19, 1014.28, 951.86, 756.55, 669.92, 611.09; HREIMS (m/z): 464.2887 [M+Na]+ (calcd. For
C26H39NaN3O3 464.2889).
3-Benzoyloxypregnenolon-20-semicarbazone (5e)
White solid, Yield: 80.42%, m.p.221-223 ℃; 1H NMR (300 MHz, CDCl3) δ: 0.62 (3H, s,
18-CH3), 1.09 (3H, s, 19-CH3), 1.84 (3H, s, 21-CH3)，2.50 (1H, d, J = 7.5, C4-H), 4.82-4.92 (1H,
m, C3-αH), 5.45 (1H, d, J = 5.4, C6-H), 6.09 (1H, br s, -NH), 7.45 (2H, t, J = 7.5, 3′,5′-Ph-H),
7.56 (1H, s, 4′-Ph-H), 8.09 (2H, d, J = 7.2, 2′,6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.25
(18-C), 16.87 (19-C), 19.42 (11-C), 21.06 (21-C), 23.25 (15-C), 24.15 (2-C), 27.85 (17-C), 31.76
(8-C), 32.04 (7-C), 36.68 (16-C), 37.04 (1-C), 38.18 (10-C), 38.87 (4-C), 43.97 (12-C), 50.02
(3-C), 56.34 (9-C), 58.79 (14-C), 74.45 (3-C), 122.51 (6-C), 128.28 (3′,5′-Ph-C), 129.53

(2′,6′-Ph-C), 130.74 (1′-Ph-C), 132.77 (4′-Ph-C), 139.68 (5-C), 150.14 (20-C), 157.41 (-CONH2),
166.03 (-COO); IR(KBr) ν/cm-1: 3466.75, 3237.49, 2934.47, 2849.05, 1712.17, 1582.67, 1450.81,
1373.42, 1317.23, 1277.37, 1180.07, 1116.03, 1085.67, 1069.70, 1028.84, 952.40, 769.88;
HREIMS (m/z): 478.3073 [M+H]+ (calcd. For C29H40N3O3 478.3070).
3-(2′-Methylbenzoyloxy)pregnenolon-20-semicarbazone (5f)
White solid, Yield: 67.47%, m.p.226-228 ℃; 1H NMR (300 MHz, CDCl3) δ: 0.62 (3H, s,
18-CH3), 1.08 (3H, s, 19-CH3), 1.84 (3H, s, 21-CH3), 2.49 (2H, d, J = 6.9, C17-H), 2.61 (3H, s,
Ph-CH3), 4.81-4.92 (1H, m, C3-αH), 5.45 (1H, d, J = 5.2, C6-H), 7.25 (1H, d, J = 7.2, 3′-Ph-H),
7.40 (1H, t, J = 7.5, 5′-Ph-H), 7.79 (1H, s, 4′-Ph-H), 7.91 (1H, d, J = 8.4, 6′-Ph-H); 13C NMR
(75MHz ,CDCl3) δ: 13.24 (18-C), 16.99 (19-C), 19.41 (11-C), 21.06 (21-C), 21.75 (2′-Ph-CH3),
23.28 (15-C), 24.16 (2-C), 27.79 (17-C), 31.76 (8-C), 32.04 (7-C), 36.03 (16-C), 36.69 (1-C),
37.08 (10-C), 38.24 (4-C), 43.96 (12-C), 50.03 (13-C), 56.35 (9-C), 58.80 (14-C), 74.33 (3-C),
122.48 (6-C), 125.66 (5′-Ph-C), 130.28 (6′-Ph-C), 130.40 (1′-Ph-C), 131.61 (3′-Ph-C), 131.74
(4′-Ph-C), 139.70 (2′-Ph-C), 139.81 (5-C), 150.22 (20-C), 157.66 (-CONH2), 167.19 (-COO);
IR(KBr) ν/cm-1: 3474.03, 3196.31, 2940.47, 2851.65, 1696.34, 1571.98, 1437.22, 1372.65,
1293.22, 1257.71, 1132.87, 1079.89, 1050.66, 995.80, 767.82, 739.26; HREIMS (m/z): 514.3046
[M+Na]+ (calcd. For C30H41NaN3O3 514.3046).
3-(4′-Chlorobenzoyloxy)pregnenolon-20-semicarbazone (5g)
White solid, Yield: 80.42%, m.p. 241-243℃; 1H NMR (300 MHz, CDCl3) δ: 0.62 (3H, s,
18-CH3), 1.09 (3H, s, 19-CH3), 1.86 (3H, s, 21-CH3)，2.48 (2H, d, J = 7.8, C4-H), 4.81-4.88 (1H,
m, C3-αH), 5.45 (1H, d, J = 4.5, C6-H), 6.12 (1H, br s, -NH), 7.42 (2H, d, J = 8.4, 3′,5′-Ph-H),
7.99 (2H, d, J = 8.4, 2′,6′-Ph-H); 13C NMR (75MHz, CDCl3) δ: 13.23 (18-C), 17.03 (19-C), 19.40
(11-C), 21.05 (21-C), 23.29 (15-C), 24.15 (2-C), 27.81 (17-C), 31.75 (8-C), 32.04 (7-C), 36.67
(16-C), 37.01 (1-C), 38.13 (10-C), 38.86 (4-C), 43.97 (12-C), 50.01 (13-C), 56.35 (9-C), 58.80
(14-C), 74.79 (3-C), 122.63 (6-C), 128.63 (3′,5′-Ph-C), 130.94 (1′-Ph-C), 131.35 (2′,6′-Ph-C),
139.19 (4′-Ph-C), 139.54 (5-C), 150.72 (20-C), 158.00 (-CONH2), 165.17 (-COO); IR(KBr) ν/cm-1:
3456.96, 3126.77, 2936.34, 2847.62, 1720.42, 1691.47, 1575.77, 1440.20, 1399.81, 1374.13,
1274.88, 1237.22, 1168.53, 1114.21, 1086.07, 1014.32, 949.13, 843.57, 758.93; HREIMS (m/z):
512.2680 [M+H]+ (calcd. For C29H39ClN3O3 512.2680).
3-(2′-Thiophenoyloxy)pregnenolon-20-semicarbazone (5h)

White solid, Yield: 88.50%, m.p.227-228 ℃; 1H NMR (300 MHz, CDCl3) δ: 0.61 (3H, s,
18-CH3), 1.08 (3H, s, 19-CH3), 1.84 (3H, s, 21-CH3), 2.48 (1H, d, J = 7.5, C4-H), 4.78-4.89 (1H,
m, C3-αH), 5.44 (1H, d, J = 2.1, C6-H), 6.11 (1H, br s, -NH), 7.11 (1H, dd, J = 4.8, 3.9,
4′-thiophene-H), 7.56 (1H, dd, J = 4.8, 0.9, 5′-thiophene-H), 7.81 (1H, s, 3′-thiophene-H); 13C
NMR (75 MHz, CDCl3) δ: 13.23 (18-C), 16.98 (19-C), 19.40 (11-C), 21.05 (21-C), 23.28 (15-C),
24.15 (2-C), 27.82 (17-C), 31.76 (8-C), 32.04 (7-C), 36.65 (16-C), 37.01 (1-C), 38.15 (10-C),
38.87 (4-C), 43.95 (12-C), 50.02 (13-C), 56.35 (9-C), 58.50 (14-C), 74.78 (3-C), 122.56 (6-C),
127.65 (4′-thiophene-C), 132.11 (5′-thiophene-C), 133.17 (3′-thiophene-C), 134.47
(2′-thiophene-C), 139.60 (5-C), 150.20 (20-C), 157.67 (-CONH2), 161.72 (-COO); IR(KBr) ν/cm-1:
3459.67, 3252.06, 3188.23, 2937.12, 2880.93, 2847.83, 1690.35, 1569.84, 1527.64, 1420.08,
1360.08, 1259.80, 1237.29, 1224.00, 1100.09, 1075.82, 1035.18, 768.75, 750.34, 707.04;
HREIMS (m/z): 506.2456 [M+Na]+ (calcd. For C27H37NaSN3O3 506.2453).
3-(2′-Furoyloxy)pregnenolon-20-semicarbazone (5i)
White solid, Yield: 65.71%, m.p.244-246 ℃; 1H NMR (300 MHz, CDCl3) δ: 0.61 (3H, s,
18-CH3), 1.07 (3H, s, 19-CH3), 1.84 (3H, s, 21-CH3), 2.47 (1H, d, J = 7.5, C4-H), 4.82-4.93 (1H,
m, C3-αH), 5.44 (1H, d, J = 4.5, C6-H), 6.52 (1H, d, J = 3.3, 4′-furan-H), 7.19 (1H, d, J = 3.3,
3′-furan-H), 7.59 (1H, s, NH), 7.67 (1H, s, 5′-furan-H); 13C NMR (75MHz, CDCl3) δ: 13.23
(18-C), 16.90 (19-C), 19.37 (11-C), 21.04 (21-C), 23.27 (15-C), 24.14 (2-C), 27.80 (17-C), 31.74
(8-C), 32.03 (7-C), 36.64 (16-C), 37.00 (1-C), 38.12 (10-C), 38.86 (4-C), 43.95 (12-C), 50.01
(13-C), 56.33 (9-C), 58.79 (14-C), 74.57 (3-C), 111.78 (4′-furan-C), 117.70 (3′-furan-C), 122.65
(6-C), 139.50 (5-C), 145.06 (2′-furan-C), 146.10 (5′-furan-C), 150.16 (20-C), 157.53 (-CONH2),
158.23 (-COO); IR(KBr) ν/cm-1: 3465.68, 2933.02, 2848.60, 1717.76, 1693.73, 1580.81, 1476.97,
1440.35, 1400.30, 1371.72, 1305.18, 1232.61, 1189.29, 1118.85, 1085.62, 1015.43, 755.60;
HREIMS (m/z): 490.2680 [M+Na]+ (calcd. For C27H37NaN3O4 490.2682).
4.1.4 General procedure for the synthesis of congpound 3 and compounds 6a-6i.
0.33mmol of compound 2 or compounds 5a-5i was dissolved in 30mL of glacial acetic acid
with vigorous stirring and gentle heating to 45-50 ºC. Selenium dioxide powder (3.0mmol) was
added into the solution and the mixture was kept under vigorous stirring. After about 0.5hr, the
color of the mixture became red color gradually. The reaction mixture was stirred until no starting

material was observed (the progress of the reaction was monitored by TLC, eluent: Vethyl
acetate:Vpetroleum ether=1:4). Then the reaction was stopped and the roduct was extracted with 30 mL of
ethyl acetate. The solution was washed with water (30 mL×2), saturated NaHCO3 (20 mL×4) and
saturated brine, dried with anhydrous sodium sulfate, and evaporated under reduced pressure. The
crude product was separated by the column chromatography using ethyl acetate/petroleum ether
(eluent: Vethyl acetate:Vpetroleum ether=1:20) to give the target product.
17-[1ʹ,2ʹ,3ʹ-Selenadiazol-4ʹ-yl]pregnenolon (3)
Faint red solid, Yield: 57.83%; m.p.158-159℃。1H NMR (300MHz, CDCl3) δ: 0.55 (3H, s,
18-CH3), 1.20 (3H, s, 19-CH3), 3.33 (1H, t, J = 9.6, C4-βH), 3.56-3.65 (1H, m, C3-αH), 4.18 (1H,
s, -OH), 5.73 (1H, d, J = 3.3, C6-H), 8.86 (1H, s, 21-H); 13C NMR (75MHz, CDCl3) δ: 13.00
(18-C), 20.24 (19-C), 21.05 (11-C), 24.67 (16-C), 25.34 (15-C), 27.42 (8-C), 32.06 (2-C), 32.26
(7-C), 36.12 (10-C), 36.96 (1-C), 37.76 (12-C), 44.62 (4-C), 50.33 (13-C), 51.38 (9-C), 56.21
(14-C), 63.64 (17-C), 72.45 (3-C), 128.43 (6-C), 137.79 (21-CSe), 142.88 (5-C), 164.49 (20-C);
IR(KBr) ν/cm-1: 3475.47, 3071.65, 2964.16, 2934.15, 2896.62, 2854.90, 2822.57, 1663.90,
1472.13, 1450.35, 1424.11, 1364.90, 1337.61, 1312.27, 1256.84, 1200.62, 1177.42, 1116.22,
1069.53, 1050.80, 1029.66, 985.50, 967.67, 902.92, 866.29, 823.17, 801.68, 758.34, 644.60;
HREIMS (m/z): 407.1604 [M+H]+ (calcd. For C21H31N2OSe 407.1601).
3β-Propionyloxy-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6a)
Faint red solid, Yield: 34.98%, m.p. 148-150℃; 1H NMR (300MHz, CDCl3) δ: 0.54 (3H, S,
18-CH3), 1.04 (3H, s, 19-CH3), 1.14 (3H, t, J = 7.5, 3′-CH3), 2.32 (2H, q, J = 7.5, 2′-CH2), 3.32
(1H, t, J = 7.5, C17-H), 4.59-4.70 (1H, m, C3-αH), 5.43 (1H, d, J = 4.8, C6-H), 8.86 (1H, s,
C21-H); 13C NMR (75MHz, CDCl3) δ: 9.20 (3′-C), 13.01 (18-C), 19.36 (19-C), 20.71 (11-C),
24.71 (16-C), 27.43 (15-C), 27.76 (2′-C), 27.92 (2-C), 31.88 (8-C), 32.29 (7-C), 36.72 (1-C),
37.03 (10-C), 37.78 (4-C), 38.14 (12-C), 44.62 (13-C), 50.15 (9-C), 51.43 (14-C), 55.98 (17-C),
73.66 (3-C), 122.29 (6-C), 137.82 (21-CSe), 139.88 (5-C), 164.54 (20-C), 173.96 (-COO); IR(KBr)
ν/cm-1: 2967.09, 2941.72, 2904.77, 2888.75, 2854.57, 2826.25, 1732.73, 1457.86, 1382.56,
1362.60, 1330.74, 1310.73, 1196.02, 1135.89, 1082.29, 1023.95, 957.92, 797.53; HREIMS (m/z):
463.1860 [M+H]+ (calcd. For C24H35N2O2Se 463.1864).
3β-Butanoyloxy-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6b)
Brownish red solid, Yield: 37.01%，m.p. 154-157℃; 1H NMR (300MHz, CDCl3) δ: 0.54 (3H,

s, 18-CH3), 0.96 (3H, t, J = 7.5, 4′-CH3), 1.04 (3H, s, 19-CH3), 1.66 (2H, q, J = 7.5, 3′-CH2), 2.27
(2H, t, J = 7.5, 2′-CH2), 3.32 (1H, t, J = 9.6, C17-H), 4.59-4.70 (1H, m, C3-αH), 5.43 (1H, d, J =
5.1, C6-H), 8.86 (1H, s, 21-C); 13C NMR (75MHz, CDCl3) δ: 13.01 (18-C), 13.67 (4′-C), 18.55
(3′-C), 19.37 (19-C), 20.71 (11-C), 24.71 (16-C), 27.43 (15-C), 27.78 (2-C), 31.88 (8-C), 32.28
(7-C), 36.58 (2′-C), 36.72 (1-C), 37.03 (10-C), 37.77 (4-C), 38.15 (12-C), 44.62 (13-C), 50.13
(9-C), 51.43 (14-C), 55.97 (17-C), 73.58 (3-C), 122.29 (6-C), 137.83 (21-CSe), 139.87 (5-C),
164.55 (20-C), 173.17 (-C=O); IR(KBr) ν/cm-1: 3728.81, 3628.14, 3068.31, 2963.20, 2939.87,
2899.06, 2874.33, 2825.44, 1728.49, 1469.90, 1383.11, 1364.35, 1310.29, 1260.18, 1188.78,
1136.16, 1088.55, 1044.45, 1015.68, 1000.50, 957.92, 900.90, 803.86, 669.88, 603.80; HREIMS
(m/z): 477.2018 [M+H]+ (calcd. For C25H37N2O2Se 407.2020).
3-Trichloroacetoxy-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6c)
Faint yellow solid, Yield: 35.03%, m.p. 162-163℃; 1H NMR (300 MHz, CDCl3) δ: 0.55 (3H,
S, 18-CH3), 1.08 (3H, s, 19-CH3), 2.49 (2H, d, J = 6.9, C4-H), 3.33 (1H, t, J = 9.6, C17-H),
4.74-4.83 (1H, m, C3-αH), 5.48 (1H, d, J = 5.1, C6-H), 8.86 (1H, s, C21-H); 13C NMR (75MHz,
CDCl3) δ: 13.00 (18-C), 19.33 (19-C), 20.74 (11-C), 24.69 (16-C), 27.09 (15-C), 27.42 (2-C),
31.89 (8-C), 32.25 (7-C), 36.68 (1-C), 36.80 (10-C), 37.30 (4-C), 37.76 (12-C), 44.61 (13-C),
50.09 (9-C), 51.42 (14-C), 55.95 (17-C), 79.69 (3-C), 90.22 (2′-C), 123.36 (6-C), 137.80 (-CSe),
138.82 (5-C), 161.35 (20-C), 164.47 (1′-C=O ); IR(KBr) ν/cm-1: 3097.89, 2938.54, 2892.05,
1762.51, 1472.02, 1447.25, 1368.02, 1311.57, 1248.91, 1199.86, 1140.69, 982.42, 926.69, 899.03,
859.32, 836.62, 821.70, 753.84, 675.50, 618.45; HREIMS (m/z): 551.0524 [M+H]+ (calcd. For
C23H30Cl3N2O2Se 551.0538).
3-Cyclopropylformyloxy-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6d)
Faint yellow solid, Yield: 37.85%, m.p.153-154℃; 1H NMR (300MHz, CDCl3) δ: 0.54 (3H,
S, 18-CH3), 0.85 (2H, d, J = 7.8, 3′-CH2), 0.99 (2H, d, J = 7.8, 4′-CH2), 1.04 (3H, s, 19-CH3), 2.36
(2H, d, J = 7.2, C4-H), 3.32 (1H, t, J = 9.6, C17-H), 4.58-4.65 (1H, m, C3-αH), 5.42 (1H, d, J =
5.1, C6-H), 8.85 (1H, s, C21-H); 13C NMR (75MHz, CDCl3) δ: 8.34 (3′,4′-C), 13.00 (18-C), 13.15
(2′-C), 19.36 (19-C), 20.72 (11-C), 24.70 (16-C), 27.43 (15-C), 27.79 (2-C), 31.88 (8-C), 32.29
(7-C), 36.71 (1-C), 37.04 (10-C), 37.79 (4-C), 38.17 (12-C), 44.62 (13-C), 50.15 (9-C), 51.44
(14-C), 55.98 (17-C), 73.79 (3-C), 122.27 (6-C), 137.79 (21-CSe), 139.90 (5-C), 164.53 (20-C),
174.31 (-C=O); IR(KBr) ν/cm-1: 3077.48, 2931.14, 2896.45, 2862.32, 1727.71, 1469.46, 1445.11,

1390.90, 1308.05, 1196.37, 1170.78, 1141.49, 1095.09, 1063.46, 998.67, 959.31, 921.17, 894.80,
834.91, 805.51, 745.96, 668.71, 618.83; HREIMS (m/z): 475.1866 [M+H]+ (calcd. For
C25H35N2O2Se 475.1864).
3-Benzoyloxy-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6e)
White solid, Yield: 56.58%, m.p.168-169℃; 1H NMR (300MHz, CDCl3) δ: 0.56 (3H, s,
18-CH3), 1.10 (3H, s, 19-CH3), 2.51 (2H, d, J = 7.2, C4-H), 3.34 (1H, t, J = 9.9, C17-H),
4.85-4.95 (1H, m, C3-αH), 5.48 (1H, d, J = 4.8, C6-H), 7.45 (2H, t, J = 7.5, 3′,5′-Ph-H), 7.56 (1H,
t, J = 7.5, 4′-Ph-H), 8.07 (2H, d, J = 7.5, 2′,6′-Ph-H), 8.87 (1H, s, 21-CH); 13C NMR (75MHz,
CDCl3) δ: 13.02 (18-C), 19.43 (19-C), 20.75 (11-C), 24.72 (16-C), 27.45 (15-C), 27.86 (2-C),
31.92 (8-C), 32.32 (7-C), 36.79 (1-C), 37.08 (10-C), 37.81 (4-C), 38.22 (12-C), 44.64 (13-C),
50.19 (9-C), 51.45 (14-C), 56.00 (17-C), 74.49 (3-C), 122.49 (6-C), 128.27 (21-CSe), 129.54
(3′,5′-Ph-C), 130.79 (2′,6′-Ph-C), 132.74 (1′-Ph-C), 137.81 (4′-Ph-C), 139.83 (5-C), 164.54
(-C=O), 166.00 (20-C); IR(KBr) ν/cm-1: 3394.60, 3184.26, 3076.10, 2922.24, 2851.39, 1714.90,
1646.54, 1469.14, 1450.42, 1379.91, 1310.99, 1272.33, 1203.33, 1175.38, 1111.23, 1066.34,
1025.28, 974.69, 953.80, 900.73, 903.46, 737.82, 711.47, 684.00; HREIMS (m/z): 533.1683
[M+Na]+ (calcd. For C25H36N2O2SeNa 533.1683).
3-(2′-MethylBenzoyloxy)-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6f)
Faint red solid, Yield: 33.17%, m.p. 162-164℃; 1H NMR (300 MHz, CDCl3) δ: 0.56 (3H, s,
18-CH3), 1.09 (3H, s, 19-CH3), 2.51 (2H, d, J = 5.1, C4-H), 2.62 (3H, s, ph-CH3), 3.33 (1H, t, J =
9.6, C17-H), 4.83-4.94 (1H, m, C3-αH), 5.48 (1H, d, J = 4.8, C6-H), 7.25 (1H, m, 3′,5′-Ph-H),
7.40 (1H, t, J = 7.2, 4′-Ph-H), 7.91 (1H, d, J = 7.2, 6′-Ph-H), 8.86 (1H, s, 21-C); 13C NMR
(75MHz, CDCl3) δ: 13.02 (18-C), 19.43 (19-C), 20.75 (11-C), 21.76 (2′-Ph-CH3), 24.72 (16-C),
27.45 (15-C), 27.93 (2-C), 31.92 (8-C), 32.32 (7-C), 36.80 (1-C), 37.12 (10-C), 37.80 (4-C), 38.28
(12-C), 44.64 (13-C), 50.18 (9-C), 51.45 (14-C), 55.99 (17-C), 74.37 (3-C), 122.47 (6-C), 125.65
(5′-Ph-C), 130.31 (21-CSe), 130.41 (6′-Ph-C), 131.60 (1′-Ph-C), 131.72 (3′-Ph-C), 137.82
(4′-Ph-C), 139.82 (2′-Ph-C), 139.86 (5-C), 164.55 (-C=O), 167.19 (20-C); IR(KBr) ν/cm-1:
3068.39, 2940.90, 2898.57, 2854.51, 2825.16, 1715.04, 1489.84, 1469.67, 1453.32, 1367.56,
1310.96, 1255.70, 1200.59, 1142.24, 1082.85, 1014.02, 957.32, 900.13, 802.70, 731.74, 669.41;
HREIMS (m/z): 563.1577 [M+K]+ (calcd. For C29H36N2O2SeK 563.1579).
3-(4′-Chlorobenzoyloxy)-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6g)

Faint yellow solid, Yield: 40.58%, m.p.163.165oC; 1H NMR (300 MHz, CDCl3) δ: 0.56 (3H,
s, 18-CH3), 1.09 (3H, s, 19-CH3), 2.50 (2H, d, J = 7.5, C4-H), 3.34 (1H, t, J = 9.6, C17-H),
4.83-4.94 (1H, m, C3-αH), 5.47 (1H, d, J = 4.5, C6-H), 7.43 (2H, d, J = 8.7, 3′,5′-Ph-H), 8.00 (2H,
d, J = 8.7, 2′,6′-Ph-H), 8.86 (1H, s, 21-C); 13C NMR (75MHz, CDCl3) δ: 13.01 (18-C), 19.41
(19-C), 20.74 (11-C), 24.71 (16-C), 27.44 (15-C), 27.83 (2-C), 31.91 (8-C), 32.31 (7-C), 36.77
(1-C), 37.05 (10-C), 37.80 (4-C), 38.17 (12-C), 44.64 (13-C), 50.17 (9-C), 51.45 (14-C), 55.99
(17-C), 74.82 (3-C), 122.61 (6-C), 128.61 (1′-Ph-C), 129.23 (21-CSe), 130.94 (3′,5′-Ph-C), 137.80
(2′,6′-Ph-C), 139.16 (4′-Ph-C), 139.69 (5-C), 164.54 (-COO), 165.14 (20-C); IR(KBr) ν/cm-1:
3418.26, 3088.98, 2939.09, 2848.20, 1716.17, 1593.42, 1485.33, 1447.58, 1399.30, 1367.96,
1312.52, 1275.25, 1199.78, 1169.91, 1120.41, 1088.93, 1065.64, 1014.08, 977.18, 850.29, 798.76,
758.97, 732.34, 683.97; HREIMS (m/z): 545.1476 [M+H]+ (calcd. for C28H34ClN2O2Se
545.1474).
3-(2′-Thiophenoyloxy)-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6h)
White solid, Yield: 13.39%, m.p. 217-220oC; 1H NMR (300 MHz, CDCl3) δ: 0.56 (3H, s,
18-CH3), 1.09 (3H, s, 19-CH3), 2.49 (2H, d, J = 7.5, C4-H), 3.33 (1H, t, J = 9.6, C17-H),
4.80-4.91 (1H, m, C3-αH), 5.46 (1H, d, J = 4.8, C6-H), 7.11 (1H, t, J = 4.5, 4′-thiophene-H), 7.56
(1H, d, J = 4.8, 5′-thiophene-H), 7.82 (1H, d, J = 3.9, 3′-thiophene-H), 8.86 (1H, s, 21-C); 13C
NMR (75MHz, CDCl3) δ: 13.02 (18-C), 19.42 (19-C), 20.75 (11-C), 24.72 (16-C), 27.44 (15-C),
27.83 (2-C), 31.92 (8-C), 32.31 (7-C), 36.76 (1-C), 37.05 (10-C), 37.80 (4-C), 38.10 (12-C), 44.64
(13-C), 50.17 (9-C), 51.45 (14-C), 55.99 (17-C), 74.83 (3-C), 122.55 (6-C), 127.65
(4′-thiophene-C), 132.10 (21-CSe), 133.16 (5′-thiophene-C), 134.51 (3′-thiophene-C), 137.80
(2′-thiophene-C), 139.74 (5-C), 161.71 (-COO), 164.54 (20-C); IR(KBr) ν/cm-1: 3077.07, 2938.21,
2853.12, 1705.98, 1523.95, 1448.70, 1417.80, 1360.71, 1312.36, 1277.37, 1256.20, 1224.58,
1202.45, 1131.62, 1097.72, 1077.92, 1064.37, 1036.43, 1014.49, 989.92, 972.86, 952.82, 899.65,
860.46, 800.13, 751.55, 737.11, 620.29; HREIMS(m/z): 539.1243 [M+Na]+ (calcd. for
C26H33N2O2SeNaS 539.1247).
3-(2′-Furoyloxy)-17-[1ʹ,2ʹ,3ʹ-selenadiazol-4ʹ-yl]pregnenolon (6i)
Faint yellow solid, Yield: 24.60%, m.p.154-156oC; 1H NMR (300 MHz, CDCl3) δ: 0.55 (3H,
s, 18-CH3), 1.07 (3H, s, 19-CH3), 2.48 (2H, d, J = 7.2, C4-H)，3.32 (1H, t, J = 9.6, C17-H),
4.83-4.91 (1H, m, C3-αH), 5.46 (1H, d, J = 4.5, C6-H), 6.51 (1H, dd, J = 3.6, 1.8, 4′-furan-H),

7.19 (1H, d, J = 4.2, 3′-furan-H), 7.58 (1H, d, J = 1.5, 5′-furan-H), 8.86 (1H, s, 21-C); 13C NMR
(75MHz, CDCl3): 13.01 (18-C), 19.38 (19-C), 20.73 (11-C), 24.71 (16-C), 27.43 (15-C), 27.80
(2-C), 31.90 (8-C), 32.29 (7-C), 36.74 (1-C), 37.03 (10-C), 37.78 (4-C), 38.14 (12-C), 44.63
(13-C), 50.15 (9-C), 51.43 (14-C), 55.97 (17-C), 74.61 (3-C), 111.78 (4′-furan-C), 117.70
(3′-furan-C), 122.64 (6-C), 137.83 (21-CSe), 139.63 (5-C), 145.07 (2′-furan-C), 146.09
(5′-furan-C), 158.22 (-COO), 164.53 (20-C); IR(KBr) ν/cm-1: 2942.40, 2907.12, 2852.97, 2829.04,
1717.34, 1475.95, 1399.41, 1303.91, 1234.21, 1191.46, 1119.98, 1013.77, 952.87, 797.77, 753.80;
HREIMS (m/z): 501.1662 [M+H]+ (calcd. for C26H33N2O3Se 501.1656).
4.2 Biological studies
Using 96-well plates on a MLLTISKAN MK3 analysis spectrometer, the cell proliferation
was tested with MTT method. PC-3 (human prostate carcinoma), SKOV3 (human
ovarian carcinoma), T47D (human breast infiltrating duct carcinoma), MCF-7 (human breast
adenocarcinoma) and HEK293T (normal kidney epithelial cells) cell lines were obtained by
Guangxi Medical University (China).
4.2.1 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.
4.2.2 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.
4.2.3 Assay for cell viability
Cells (1～2×104 cells/mL) were seeded into 96-wells plates for 24 hr. The medium contained
test compound with different concentrations was added to the cells. After 72 hr incubation, 20 µL
of the tetrazolium dye (MTT) (5 mg/mL) solution were added to each well. After additional 4 hr
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.: 21462009; No: 21562007), the Nanning technology development project.
of China（20171125-5）and Universities Key Laboratory Funds of Synthetic and Natural
Functional Molecular Chemistry.
Conflict of Interest
The authors declare no conflict of interest.
[5] Lesyk R, Zimenkovsky B, Atamanyuk D, Jensen F, Kononowicz KK, Gzella A. Anticancer
thiopyrano[2,3-d][1,3]thiazol-2-ones with norbornane moiety. Synthesis, cytotoxicity, physico-
chemical properties, and computational studies. Bioorg. Med. Chem. 2006;14:5230-40.
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Graphical Abstract:

Research Highlights
1. 3-substituted 17-[1ʹ,2ʹ,3ʹ]-selenadiazolylpregnenolone derivatives
were synthesized.
2. The structures of all synthesized compounds had been were
characterized.
3. The antiproliferative activity of synthesized compounds was assayed.
4. Some compounds show a distinct antiproliferative activity.