Synthesis and antiproliferative activity of some steroidal thiosemicarbazones, semicarbazones and hydrozones

Article abstract of DOI:10.1016/j.steroids.2014.05.026

Steroidal thiosemicarbazones, semicarbazones and hydrazones have received extensive attention of scientists recently because they exhibit some biological activities such as antibacterial, antiviral and anticancer. Using different steroids as starting mate

Full text of DOI:10.1016/j.steroids.2014.05.026

Steroids 87 (2014) 99–107
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antiproliferative activity of some steroidal
thiosemicarbazones, semicarbazones and hydrozones
⇑
⇑
Chunfang Gan, Jianguo Cui , Shaoyang Su, Qifu Lin, Linyi Jia, Lianghua Fan, Yanmin Huang
College of Chemistry and Life Science, Guangxi Teachers Education University, Nanning 530001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Steroidal thiosemicarbazones, semicarbazones and hydrazones have received extensive attention of
scientists recently because they exhibit some biological activities such as antibacterial, antiviral and anti-
cancer. Using different steroids as starting materials, through different chemical methods, 24 steroidal
compounds with thiosemicarbazone, semicarbazone or hydrazone groups in their structures, were syn-
thesized, characterized by IR, NMR and MS. The antiproliferative activity of the compounds was evaluated
against human gastric cancer (SGC-7901) and human liver cancer (Bel-7404) cells. The structure–activity
relationship of these compounds was discussed. The results showed that compound 3 and 12a–12c
exhibited significant inhibitory activity to Bel-7404 cells, and IC50 values of them were 4.2, 11.0, 7.4
Received 2 February 2014
Received in revised form 24 May 2014
Accepted 29 May 2014
Available online 11 June 2014
Keywords:
Cholesterol
Stigmasterol
Steroidal thiosemicarbazones
Steroidal semicarbazones
Antiproliferative activity
and 15.0 lM respectively (Cisplatin, IC50: 11.6 lM).
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
the 3-, 6- or 22-position of steroidal nucleus respectively, and
other potency groups such as hydroxyl, oxime or carbonyl groups
were introduced into another position. Using some natural sterols
with different side chains, such as cholesterol and stigmasterol as
starting materials, a total of 24 steroidal hydrazone derivatives
with different structural characteristics were designed and synthe-
sized through different methods. The antiproliferative activity of
synthesized compounds was evaluated.
Recently, numerous compounds with biological activities were
separated from marine organism [1–6]. Many compounds with ste-
roidal skeletons showed significant antibacterial, antiviral, antitu-
mor or other biological activities [7–11]. Hydrazone compounds
and their derivatives are a class of substances displaying the above
[12,13] activities. A variety of steroidal hydrazone derivatives with
unique structures had been synthesized with their bioactivities
assayed [14–17]. Especially, steroids with thiosemicarbazone
structure were known to possess antiamoebic, antinociceptive,
antiangiogeni, antibacterial, antifungal and anti-inflammatory
properties [18–22]. However, steroidal thiosemicarbazone with
antiproliferative activity was rarely reported.
2. Results and discussion
2.1. Chemistry
Compounds 1, 4a–4c and 10 were prepared according to the
method of literature [25].
In previous researches, a series of steroidal oxime compounds
had been synthesized by our group, and the in vitro antiprolifera-
tive activity of these compounds had been investigated [23–25].
The results indicated that some steroidal compounds with
3,6-dioxime groups in the steroidal nucleus showed a good cyto-
toxicity against some cancer cells. In this paper, some steroidal
hydrazone derivatives with 3,6-disubstituted structure and
different side chains were synthesized. In these compounds, a
semicarbazone or thiosemicarbazone group was introduced into
First, compounds 2 and 3 were obtained by the reaction of
compound 1 with semicarbazide or thiosemicarbazide (Scheme 1).
The structures of 2 and 3 were confirmed by NMR spectra. In the
13C NMR of 2 and 3, the chemical shift of 6-carbonyl in the com-
pound 1 disappeared, and resonances of C-6 that appeared at
155.5 ppm for compound 2 and at 158.6 ppm for compound 3
showed that the 6-carbonyl of 1 had been transformed into the
6-C@N group of 2 or 3. Moreover, in the 1H NMR, the chemical
shift of 2 at 5.433 (NH2), 6.116 (NH2), 8.356 (ANHA) and 3 at
6.326 (NH2), 7.249 (NH2), 8.749 (ANHA) ppm demonstrated
further formation of 6-semicarbazone and 6-thiosemicarbazone
groups.
⇑
Corresponding authors. Tel.: +86 771 3908065; fax: +86 771 3908308 (J. Cui).
Tel.: +86 13977159868 (Y. Huang).
E-mail addresses: cuijg1954@126.com (J. Cui), huangyanmin828@163.com
(Y. Huang).
http://dx.doi.org/10.1016/j.steroids.2014.05.026
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

100
C. Gan et al. / Steroids 87 (2014) 99–107
group and resonances which showed at 179.0 and at 178.9 ppm
were belonged to the C@S chemical shift of (E)- and (Z)-5 respec-
tively. The chemical shift of 6-carbonyl at 209.9 and 209.5 ppm
demonstrated further that compound 5 was the mixture of (E)-
and (Z)-configuration isomer. Next, the 6-carbonyl of 5 was con-
verted to 6-hydroxyl of compound 6 by the reduction of NaBH4,
but the 3-thiosemicarbazone group was still kept in compound 6.
From the 13C NMR spectra of compound 6, the characteristic signal
of the 6-carbonyl of 5 was disappeared at 209 ppm, instead of at
70.9 ppm which was the chemical shift of 6-hydroxyl’s carbon.
The presence of multiple peaks at 3.88 ppm to 3.80 ppm in C6AH
of 6 illustrated further that the 6-carbonyl group of 5 had been
converted to 6-hydroxyl, where product 6 was generated.
To investigate the effect of substituted group to biological activ-
ity in steroidal thiosemicarbazones, several new phenylthiosemic-
arbazonesteroid derivatives were prepared and evaluated for their
cytotoxic activity (Scheme 3). The reaction of compounds 4a–4c
with phenylthiosemicarbazide gave 3-(40-phenyl)-thiosemicarbaz-
onesteroids 7a–7c with different branched chain. 7a–7c were also
composed of the mixture of (3E)- and (3Z)-isomer with 1:1 propor-
tion, and their structures were confirmed by their spectral data.
Interestingly, when 7a–7c were reduced by sodium borohydride,
differing from compound 5, compounds 8a–8c and 9a–9c were
obtained in which both 6-carbonyl and 3-(40-phenyl)thiosemicar-
bazone groups were reduced simultaneously. From the NMR spec-
trum of 8a and 9a, it was obviously seen that 13C chemical shift of
a
HO
HO
H
N
N
N
NH2
2
O
S
HO
O
1
b
H
N
NH2
3
Scheme 1. Reagents and conditions: (a) NH2NHCONH2/95%EtOH, CH3COONaꢀ3H2O,
70 °C; and (b) NH2NHCSNH2/EtOH, 70 °C.
Next, starting from compound 4a, compound 5 with 3-thiosem-
icarbazone group was yielded by controlling an appropriate molar
ratio of 4a and thiosemicarbazide because 3-carbonyl group was
more active than 6-carbonyl, and thiosemicarbazide was selec-
tively reacted with 3-carbonyl (see Scheme 2). Compound 5 was
a mixture of (3E)- and (3Z)-isomer (1:1, from the 1H NMR). In 13C
NMR spectrum of compound 5, the characteristic peak at
154.9 ppm indicated that the presence of 3-thiosemicarbazone
a
b
H
H
H2N
N
H2N
N
N
O
N
OH
O
O
S
S
5
6
4a
Scheme 2. Reagents and conditions: (a) NH2CSNHNH2/CH3CH2OH, 80 °C; and (b) NaBH4, CH3OH.
R
R
R
R
I
II
H
N
H
N
H
N
H
N
H
N
H
N
+
N
H
N
H
O
Ph
Ph
N
Ph
OH
OH
O
S
S
O
S
9
8
7
4
c
R =
a
b
Scheme 3. Reagents and conditions: (I) PhNHCSNHNH2/CH3CH2OH, 80 °C; and (II) NaBH4, CH3OH.
R
R
R
NaBH3
H
CH3O
H
H
H
N
N
N
N
N
N
N
N
H
Ph
N
Ph
Ph
O
O
S
O
SH
SH
A
7
B
R
R
H
N
H
N
H
N
H
N
+
N
H
N
H
Ph
Ph
OH
OH
S
S
9
8
Scheme 4. The formation mechanism of compounds 8a–8c and 9a–9c.

C. Gan et al. / Steroids 87 (2014) 99–107
101
R
R
I-III
HO
N
HO
N
X
O
10
11a-c, 12a-c, 13a-c
11a-11c: X=NNHCSNH2
12a-12c: X=NNHCONH2
13a-13c: X=NNH2
c
R =
a
b
Scheme 5. Reagents and conditions: (I) NH2NHCSNH2/CH3COOH/CH3CH2OH; (II) NH2NHCONH2ꢀHCl/95%EtOH; and (III): N2H4ꢀH2O (80%)/CH3COOH/CH3CH2OH.
H
H
H
N
NH2
O
N
S
I
II
O
O
O
O
O
O
4b
14
15
H
H
N
NH2
N
S
III
HO
N
O
16
Scheme 6. Reagents and conditions: (I) O3, CH2Cl2, Me2S, ꢂ78 °C; (II) NH2NHCSNH2/C2H5OH/CH3COOH, pH = 4; and (III) NH2OH.HCl/95%EtOH/NaOAcꢀ3H2O.
6-carbonyl at 210 ppm disappeared, replaced by 13C chemical shift
of 6-hydroxyl at 71.8 ppm. In addition, the characteristic signal of
3-C@N at 154.7 ppm for 7a disappeared and was replaced by the
signals of 3-CANHA at 60.4 for 8a and 55.7 ppm for 9a. It showed
clearly that 3-(40-phenyl)thiosemicarbazone group of 7a had been
reduced. In their 1H NMR, the chemical shift of C3AH at
chemical shift of NH2 for 11a appears in 6.758, 7.232–7.248 ppm.
A resonance signal is found within 3.273–3.322 ppm, which is
chemical shift of C2AbH and moved to the downfield due to the
deshielding influence of the hydroxyl in (3E)-hydroximino group.
In the 13C NMR of 11a, the chemical shift of carbonyl carbon is
not found, the 13C chemical shift of two C@N appear at 159.5 and
155.0 ppm respectively. The chemical shift at 179.0 ppm is indica-
tive of carbon in C@S bond of 6-thiosemicarbazone group.
Using 4b as starting material, another nitrogenous steroidal com-
pound 16 with 3-hydroximino and 22-thiosemicarbazone groups in
its structure was synthesized (Scheme 6). The ozonolysis of 4b was
performed in a mixture of CH2Cl2 and MeOH (CH2Cl2:MeOH = 4:1) at
ꢂ78 °C. After bubbling O2 to expel the excess O3 and adding Me2S to
decompose the produced ozonide, compound 14 was obtained.
Utilizing the difference between the activity of 22-aldehyde and
3,6-dicarbonyl group in 14, compound 15 with 22-thiosemicarbarz-
one group in the structure was obtained by a selective reaction of
22-aldehyde with thiosemicarbarzide. Furthermore, a selective oxi-
mation of 3-carbonyl group in compound 15 produced compound
2.95–2.80 ppm for 8a (3a-configuration, CAH connected to
amino-group) and 3.34–3.27 ppm for 9a (3b-configuration) illus-
trated further that the C@N double bond of 3-thiosemicabazone
had been transformed into CAN single bond. Here, the chemical
shift at 2.95–2.80 ppm in upfield for 8a was assigned to 3-aH,
and 3.34–3.27 ppm in downfield for 9a assigned to 3-bH, respec-
tively. Compound 8a (8a:9a ꢁ 5:1) was a main product because
3-(4’-phenylthiosemicarbamide) group located at e-bond is more
stable than that at a-bond.
Under the same reaction conditions, why only 6-carbonyl in
compound
5
was reduced, but both 6-carbonyl and 3-(40-
phenyl)thiosemicarbazone group in compound 7 were reduced?
A proposed mechanism for the formation of 8 and 9 is shown in
Scheme 4.
In the reaction, an intermediate A with enol-configuration in
compound 7 was formed due to the conjugated effect of 40-phenyl.
Sodium borohydride attacking the N@N double bond of A pro-
duced an intermediate B. The B obtaining a proton from CH3OH
produced compounds 8 and 9. Within this reaction, compound 8
with 3b-substituent was the main product.
In order to study the effect of various nitrogenous substituent
on the substrate to the antiproliferative activity, compounds
11a–11c, 12a–12c and 13a–13c possessing 3-oxime group and dif-
ferent side chains in their structures were synthesized by com-
pounds 10a–10c reacted with different nitrogenous reagents
(Scheme 5). Their structures were confirmed by spectrum analysis.
Comparing the 1H NMR of compound 11a with 10a, the proton
Table 1
In vitro antiproliferative activities of target compounds (lM).
Compounds
2
3
6
7a
7b
7c
8a
8b
SGC-7901
Bel-7404
47.8
28.3
8c
76.3
4.2
9a
>100 30.8
>100 >100 32.8
9b 9c 11a
67.4
>100 >100 >100
89.5
11b
>100 >100
11c
12a
SGC-7901
Bel-7404
>100 >100 >100 >100 17.2
>100 32.4 >100 >100 27.8
13a 13b 13c
>100 15.9
>100 11.0
15
13.2
11.0
Cisplatin
12b
12c
16
SGC-7901
Bel-7404
32.3
7.4
26.2
15.0
>100 >100 >100 19.1
>100 >100 >100 43.1
>100 6.7
78.7 23.2

102
C. Gan et al. / Steroids 87 (2014) 99–107
16. The structures of all synthesized compounds were confirmed by
their spectral data. Compounds 15 and 16 have similar atom rank
with the side chain of cholesterol, except that the carbon atoms in
branch chain of 15 and 16 were replaced by nitrogen atoms and a
sulfur atom.
4. Experimental
4.1. Chemistry
4.1.1. Reagent and Instrument
The synthetic reagents were analytically pure, and the solvents
were purified by general methods before being used. Infrared spec-
tra were measured with a Nicolet FT-360 Spectrophotometer
(Thermo Scientific, America); Melting points were determined by
X6 microscopic melting point meter and was uncorrected; 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 Bru-
ker AV-300 spectrometer at working frequencies 300 and 75 MHz,
respectively. Chemical shifts are expressed in parts per million (d)
values and coupling constants (J) in Hertz. LRESIMS were recorded
on a Thermo-DSQ instrument, while HRESIMS were measured on a
Agilent 6210 TOFMS instrument.
2.2. In vitro evaluation of the antiproliferative activity
All synthesized compounds were evaluated for their antiprolif-
erative activities in vitro against SGC 7901 (human gastric carci-
noma) and Bel 7404 (human liver carcinoma) cell lines using a
MTT assay. The results were summarized as IC50 values in
Table 1.
lM in
Comparing compound 3 with 2, compound 3 with 3-hydroxyl
and 6-thiosemicarbazone groups had better antiproliferative activ-
ity than compound 2 with 3-hydroxyl and 6-semicarbazone groups
against Bel 7404 cells. Compound 3 showed significant increase in
its cytotoxicity against these cells in comparison of compound 6
with same 3,6-disubstituted groups. However, the structure of 6
possesses an opposite arrangement of the two functional groups.
The results showed that thiosemicarbazone group in 6-position
favored the increase of the compound’s cytotoxicity.
In addition, substituting hydrogen atom in thiosemicarbazone
group with phenyl group increased the antiproliferative activity
of these compounds (compare 7a–7c with 6). However, com-
pounds 8a–8c and 9a–9c showed an obvious decrease in their
cytotoxicity against these cancer cells after 3-(40-phenyl)thiosemi-
carbazone group of 7a–7c was reduced.
The results showed that the compounds’ cytotoxicity increased
when 6-substituted group was thiosemicarbazone or semicarba-
zone for the compounds with 3-hydroximino group (see 11a–11c
and 12a–12c), but it was almost inactive when 6-substituted group
was hydrazone (13a–13c). For compounds possessing 6-thiosemi-
carbazone group in the structure, the presence of double bond in
the side chain resulted in a remarkable decrease of the cytotoxicity
of compounds (comparing 11b and 11a, 11c). Though no obvious
difference was found in their cytotoxicity for compounds 12a–
12c bearing 6-semicarbazone group which displayed a better anti-
proliferative activity than cisplatin did (a positive control) against
Bel-7404 cells. Among them, compound 12b with 22,23-double
bond in its structure showed an excellent antiproliferative activity
4.1.2. 3b-hydroxycholestan-6-semicarbazone (2)
CH3COONaꢀ3H2O (34 mg, 0.25 mmol) and semicarbazide hydro-
chloride (34 mg, 0.3 mmol) were added to the solution of 100 mg
(0.25 mmol) compound 1 in 20 mL 95% ethanol. After the solution
was heated to 70 °C, the mixture was stirred at the temperature
for 10 h (the progress of the reaction was monitored by TLC,
Vdichloromethane:Vmethanol = 20:1). Then, the reaction was terminated
and majority of solvent was evaporated under reduced pressure.
Suitable amount of water was added to the reaction mixture, and
the product was extracted with CH2Cl2 (15 mL ꢃ 3). The combined
extract was washed with saturated NaHCO3 solution, water and
saturated brine, dried with anhydrous sodium sulfate, and evapo-
rated under reduced pressure. The crude product was separated
by
the
column
chromatography
using
CH2Cl2/CH3OH
(eluent:Vdichloromethane:Vmethanol = 40:1) to give 65 mg of 2 (56%) as
white solid. m.p. 234–236 °C. IR (KBr)
m
/cmꢂ1: 3468, 2929, 2864,
1687, 1662, 1580, 1466, 1384, 1066; 1H NMR (CDCl3, 300 MHz) d:
0.645 (s, 3H, 18-CH3), 0.703 (s, 3H, 19-CH3), 0.867 (d, 3H, J = 6.6,
26-CH3 or 27-CH3), 0.871 (d, 3H, J = 6.6, 26-CH3 or 27-CH3), 0.907
(d, 3H, J = 6.3, 21-CH3), 2.687 (dd, 1H, J = 13.2, 3.0, C5AH), 3.67–
3.52 (m, 1H, C3AaH), 5.433 (br s, 1H, ANH2), 6.116 (br s, 1H,
ANH2), 8.356 (br s, 1H, ANHA); 13C NMR (CDCl3, 75 MHz) d:
158.6 (6-C), 152.7 (AC@O), 71.0 (3-C), 56.5 (14-C), 56.2 (17-C),
54.3 (9-C), 50.9 (5-C), 42.9 (13-C), 39.6 (10-C), 39.5 (24-C), 39.2
(12-C), 36.3 (1-C), 36.1 (22-C), 35.7 (20-C), 31.7 (4-C), 31.4 (7-C),
30.9 (8-C), 29.7 (2-C), 28.2 (16-C), 28.0 (25-C), 24.1 (19-C), 23.8
(15-C), 22.8 (26-C), 22.6 (27-C), 21.4 (23-C), 18.6 (11-C), 12.7 (21-
C), 12.1 (18-C); HRESI-MS, m/z: 460.3919 [M + H]+, (calcd for
with IC50 value of 7.4 lM.
Apparently, for compounds possessing 22-thiosemicarbazone,
compound 15 with 3-carbonyl had better cytotoxicity than com-
pound 16 with 3-hydroximino group.
C28H50N3O2, 460.3903).
3. Conclusion
4.1.3. 3b-hydroxycholestan-6-thiosemicarbazone (3)
Using various steroids as starting materials, through different
chemical methods, 24 steroidal compounds with the character-
istic thiosemicarbazone, semicarbazone or hydrazone groups in
their structures were synthesized, and their structures were
characterized by IR, NMR and MS. The in vitro antitumor activ-
ity of these compounds was assayed against human gastric can-
cer (SGC-7901) and human liver cancer (Bel-7404) cells. The
results showed that compounds with the 6-thiosemicarbazone
or 6-semicarbazone group in the structures exhibited significant
antiproliferative activity in vitro. Moreover, compounds 3 and
Compound 1 (100 mg, 0.25 mmol) was dissolved in 20 mL of
CH3CH2OH, and some glacial acetic acid was dripped into the solu-
tion to adjust pH value of solution to 3–5 after the solid was com-
pletely dissolved. The mixture was heated to 70 °C, and 27 mg
(0.3 mmol) thiosemicarbazide was added into the solution after
10 min. The mixture was stirred for 6 h until no starting material
was observed (the progress of the reaction was monitored by
TLC, petroleum ether: ethyl acetate = 1:1). The reaction was termi-
nated and majority of solvent was evaporated under reduced pres-
sure. The mixture was extracted with ethyl acetate (10 mL ꢃ 3).
The combined extract was washed with saturated NaHCO3 solu-
tion, water and saturated brine, dried with anhydrous sodium sul-
fate. After solvent was removed under reduced pressure, the
residue was purified by flash chromatography on silica gel using
petroleum ether/ethyl acetate (2:1) as the eluent. Compound 3
was obtained as white solid (78 mg, 65%), m.p. 259–261 °C.
12a–12c, with IC50 value of 4.2, 11.0, 7.4 and 15.0 lM, dis-
played better antiproliferative activity than cisplatin did (a posi-
tive control) against Bel-7404 cells. The presence of 22-double
bond in the side chain resulted in a remarkable decrease of
the cytotoxicity for compounds possessing 6-thiosemicarbazone
group.

C. Gan et al. / Steroids 87 (2014) 99–107
103
IR (KBr)
m
/cmꢂ1: 3432, 2929, 2864, 1662, 1580, 1466, 1384, 1066;
4.1.6. 6-Oxo-cholestan-3-(4’-phenyl)thiosemicarbazone (7a)
1H NMR (CDCl3, 300 MHz) d: 0.669 (s, 3H, 18-CH3), 0.717 (s, 3H, 19-
CH3), 0.892 (d, 6H, J = 6.3, 26-CH3 or 27-CH3), 1.151 (d, 3H, J = 6.3,
21-CH3), 2.646 (br d, 1H, J = 12.9, C5AH), 3.73–3.56 (m, 1H,
Light yellow solid, yield 49.3%, m.p. 203–205 °C. 7a was the
mixture of (3E)- and (3Z)-isomer (ratio: E:Z = 1:1). IR (KBr)
m
/cmꢂ1: 3415, 3260, 2949, 2868, 1711, 1597, 1527, 1466, 1442,
C3A
a
H), 6.326 (br s, 1H, ANH2), 7.249 (br s, 1H, ANH2), 8.749 (s,
1380, 1278, 1176; 1H NMR (CDCl3, 300 MHz) d: 0.668 (s, 3H, 18-
CH3), 0.855 (s, 3H, 19-CH3), 0.866 (d, 6H, J = 6.9, 26-CH3 and 27-
CH3), 0.915 (d, 3H, J = 6.0, 21-CH3), 2.002–2.091 (m, 2H, 7-CH2),
2.747 (d, 1H, J = 14.7, C5AH), 7.189 (t, 1H, J = 7.2, p-PhAH), 7.351
(t, 2H, J = 7.5, m-PhAH), 7.633 (d, 2H, J = 7.8, o-PhAH), 9.017 (s,
0.5H, ACNHAN, Z-), 9.089 (s, 0.5H, ACNHAN, E-), 9.284 (s, 0.5H,
APhANHAC, Z-), 9.344 (s, 0.5H, APhANHAC, E-); 13C NMR (CDCl3,
75 MHz) d: 210.0 (6-C, E), 209.8 (6-C, Z), 176.1 (C@S, E), 176.0 (C@S,
Z-), 154.7 (3-C), [138.1,128.7,128.7,125.8,124.1,124.1] (C6H5), 57.8
(14-C), 56.7 (5-C, Z-), 56.6 (5-C, E-), 56.1 (17-C), 53.4 (13-C, Z-),
53.3 (13-C, E-), 46.6 (10-C, E-), 46.5 (10-C, Z-), 42.9 (7-C), 41.7 (9-
C), 41.6 (12-C), 39.5 (24-C), 39.3 (22-C), 38.2 (4-C, Z-), 37.8 (20-
C), 37.1 (4-C, E-), 36.1 (8-C), 35.7 (25-C), 30.5 (2-C, E-), 30.1 (2-C,
Z-), 28.0 (16-C), 23.9 (1-C), 23.8 (15-C), 22.9 (27-C), 22.6 (26-C),
21.5 (23-C), 18.7 (11-C), 12.6 (21-C), 12.4 (19-C), 12.0 (18-C);
HRESI-MS: 550.3827 [M+H]+ (calcd for C34H52N3OS, 550.3831).
1H, ANHA); 13C NMR (CDCl3, 75 MHz) d: 179.1 (C@S), 155.5 (6-
C), 71.0 (3-C), 56.4 (14-C), 56.1 (17-C), 54.3 (9-C), 51.1 (5-C), 43.1
(13-C), 39.6 (24-C), 39.5 (12-C), 39.4 (10-C), 36.5 (1-C), 36.3 (22-
C), 36.1 (20-C), 35.7 (8-C), 31.6 (4-C), 31.5 (7-C), 31.0 (2-C), 28.1
(16-C), 28.0 (25-C), 24.2 (19-C), 23.8 (15-C), 22.8 (26-C), 22.6
(27-C), 21.5 (23-C), 18.6 (11-C), 12.7 (21-C), 12.1 (18-C); HRESI-
MS: m/z: 476.3670 [M+H]+ (calcd for C28H50N3OS, 476.3675).
4.1.4. Cholestan-3,6-dione 3-thiosemicarbazone (5)
Compound 5 was prepared similarly as the procedure for the
synthesis of 3, but from compound 4a.
White solid, yield: 60%. m.p. 189–191 °C. Compound 5 was the
mixture of (3E)- and (3Z)-isomer (ratio: E:Z = 1:1, 1H NMR integral
area determined). IR (KBr) m
/cmꢂ1: 3403, 3256, 2929, 2851, 1699,
1593, 1409, 1364, 1245; 1H NMR (CDCl3, 300 MHz) d: 0.683 (s,
3H, 18-CH3), 0.861 (s, 3H, 19-CH3), 0.872 (d, 6H, J = 6.3, 26-CH3
and 27-CH3), 0.922 (d, 3H, J = 6.3, 21-CH3), 2.43–2.34 (m, 4H,
C4AH and C7AH), 2.686 (d, 1H, J = 12.3, 3.6, C5AH), 6.414 (br s,
1H, ANH2), 7.234 (br s, 1H, ANH2), 8.823 (s, 0.5H, ANHA, Z-),
8.881 (s, 0.5H, ANHA, E-); 13C NMR (CDCl3, 75 MHz) d: 209.9
(6-C, E-), 209.5 (6-C, Z-), 179.0 (C@S, E-), 178.9 (C@S, Z-), 154.9
(3-C), 57.8 (5-C), 56.6 (14-C), 56.1 (17-C), 53.6 (13-C, E-), 53.4
(13-C, Z-), 46.6 (10-C, E-), 46.5 (10-C, Z-), 42.9 (9-C), 41.7 (7-C, E-),
41.6 (7-C, Z-), 39.5 (12-C), 39.3 (24-C), 38.1 (22-C), 37.8 (8-C),
37.1 (20-C), 36.1 (2-C), 35.6 (16-C), 30.0 (25-C), 28.0 (4-C), 24.0
(1-C), 23.8 (23-C), 22.8 (26-C), 22.6 (27-C), 22.5 (15-C), 21.5 (11-
C), 18.6 (21-C), 12.5 (19-C), 12.0 (18-C); HRESI-MS: 474.3532
[M+H]+ (calcd for C28H48N3OS, 474.3518).
4.1.7. 6-oxo-stigmastan-3-(40-phenyl)thiosemicarbazone (7b)
From compound 4b. Light yellow solid, yield: 67.7%. m.p. 193–
195 °C. 7b was the mixture of (3E)- and (3Z)-isomer (ratio:
E:Z = 1:1). IR (KBr) m
/cmꢂ1: 3411, 3284, 2945, 2864, 1711, 1593,
1531, 1437, 1388, 1270, 1184, 1074; 1H NMR (CDCl3, 300 MHz)
d: 0.708 (s, 3H, 18-CH3), 0.804 (d, 3H, J = 6.6, 26-CH3), 0.815 (t,
3H, J = 6.3, 29-CH3), 0.856 (d, 3H, J = 6.6, 27-CH3), 0.878 (s, 3H,
19-CH3), 1.037 (d, 3H, J = 6.6, 21-CH3), 2.52–2.34 (m, 4H, C4AH,
C5AH and C7AH), 2.730 (dd, 1H, J = 12.9, 2.4, C7AH), 5.033 (dd,
1H, J = 15.0, 8.4, C22AH), 5.155 (dd, 1H, J = 15.3, 8.4, C23AH),
7.218 (t, 1H, J = 7.2, p-PhAH), 7.379 (t, 2H, J = 7.8, m-PhAH),
7.652 (d, 2H, J = 8.1, o-PhAH), 8.878 (s, 0.48H, ACNHAN, Z-),
8.917 (s, 0.5H, ACNHAN, E-), 9.280 (s, 0.48H, APhANHAC, Z-),
9.326 (s, 0.48H, APhANHAC, E-); 13C NMR (75 MHz, CDCl3) d:
210.0 (6-C, E-), 209.6 (6-C, Z-), 176.2 (C@S, Z-), 176.1 (C@S, E-),
154.2 (3-C), [138.0, 128.7, 128.7, 125.9, 124.1, 124.1] (C6H5),
137.9 (22-C), 129.6 (23-C), 57.8 (14-C), 56.7 (5-C), 55.8 (17-C),
53.5 (24-C), 53.4 (13-C), 51.2 (9-C), 46.6 (10-C, E-), 46.5 (10-C, Z-),
42.8 (20-C), 41.8 (7-C, Z-), 41.6 (7-C, E-), 40.5 (9-C), 39.2 (12-C),
38.1 (8-C, E-), 37.9 (25-C), 37.1 (8-C, Z-), 31.9 (16-C), 30.5 (4-C,
E-), 30.1 (4-C, Z-), 28.7 (2-C), 25.4 (1-C), 24.0 (15-C), 22.7 (28-C),
22.5 (11-C), 21.2 (26-C), 21.1 (27-C), 21.2 (21-C), 19.0 (C-19),
12.5 (18-C), 12.2 (29-C); HRESI-MS: 576.3989 [M+H]+ (calcd for
4.1.5. 6-Hydroxycholestan-3-thiosemicarbazone (6)
NaBH4 (20 mg, 0.5 mmol) was added in batches to a solution of
5 (120 mg, 0.25 mmol) in anhydrous methanol (20 mL) at room
temperature in 10 min. The mixture was stirred at room tempera-
ture until no raw material was observed (by TLC, eluent:Vpetroleum
ether:Vethyl acetate = 1:1). Then the solution was neutralized with
1 mol.Lꢂ1 HCl. After evaporation of the majority of MeOH under
reduced pressure, a small amount of water was added and
extracted with ethyl acetate (15 mL ꢃ 3). The combined extract
was washed with cold water, saturated NaHCO3 and brine. After
drying over anhydrous sodium sulfate, the solvent was removed
under reduced pressure and the resulting crude product was puri-
fied by flash chromatography on silica gel using petroleum ether/
ethyl acetate (1:1) as the eluent to give 60 mg of white solid 6,
yield: 50%. m.p. 221–223 °C. Product 6 was the mixture of (3E)-
C36H54N3OS, 576.3988).
4.1.8. 6-Oxo-sitostan-3-(40-phenyl)thiosemicarbazone (7c)
From compound 4c. Light yellow solid, yield: 40.9%. m.p. 203–
206 °C. 7c was the mixture of (3E)- and (3Z)-isomer (ratio:
and (3Z)-isomer (ratio: E:Z = 1:1). IR (KBr)
m
/cmꢂ1: 3460, 1686,
E:Z = 1:1). IR (KBr) m
/cmꢂ1: 3427, 3246, 2941, 2868, 1703, 1589,
1666, 1564, 1466, 1384; 1H NMR (CDCl3, 300 MHz) d: 0.723 (s,
3H, 18-CH3), 0.880 (d, 6H, J = 6.3, 26-CH3 and 27-CH3), 0.924 (d,
3H, J = 6.3, 21-CH3), 1.125 (s, 3H, 19-CH3), 2.46–2.34 (m, 3H,
C4AH and C7AH), 3.88–3.80 (m, 1H, C6AH), 6.24 (br s, 1H,
ANH2), 7.25 (br s, 1H, ANH2), 8.694 (s, 0.5H, ANHA, Z-), 8.937 (s,
0.5H, ANHA, E-); 13C NMR (CDCl3, 75 MHz) d: 178.9 (C@S), 157.1
(3-C, E-), 156.6 (3-C, Z-), 70.9 (6-C), 56.3 (14-C), 55.9 (17-C), 53.8
(9-C), 49.1 (5-C), 47.9 (13-C), 42.7 (10-C), 39.8 (7-C), 39.5 (12-C),
36.1 (24-C), 36.0 (22-C), 35.7 (20-C), 30.2 (8-C), 29.7 (2-C), 28.2
(4-C), 28.0 (16-C), 27.4 (25-C), 24.2 (1-C), 23.8 (23-C), 22.8 (26-
C), 22.5 (27-C), 21.1 (15-C), 21.0 (11-C), 18.7 (21-C), 15.0 (19-C),
12.5 (18-C); HRESI-MS: 476.3667 [M+H]+ (calcd for C28H50N3OS,
476.3675).
1527, 1442, 1380, 1266, 1188; 1H NMR (CDCl3, 300 MHz) d:
0.695 (s, 3H, 18-CH3), 0.793 (d, 3H, J = 6.9, 26-CH3), 0.826 (d, 3H,
J = 6.9, 27-CH3), 0.860 (t, 3H, J = 6.6, 29-CH3), 0.883 (s, 3H, 19-
CH3), 0.943 (d, 3H, J = 4.8, 21-CH3), 2.53–2.37 (m, 4H, C4AH,
C5AH and C7AH), 2.722 (br d, 1H, J = 9.3, C7AH), 7.225 (t, 1H,
J = 7.2, p-PhAH), 7.386 (t, 2H, J = 7.2, m-PhAH), 7.657 (d, 2H,
J = 7.5, o-PhAH), 8.802 (br s, 1H, ACSNHAN), 9.280 (s, 0.5H,
APhANHAC, Z-), 9.321 (s, 0.5H, APhANHAC, E-); 13C NMR (CDCl3,
75 MHz) d: 209.9 (6-C, E-), 209.6 (6-C, Z-), 176.2 (C@S, E-), 176.1
(C@S, Z-), 154.2 (3-C), [138.0, 128.7, 128.7, 125.8, 124.1, 124.1]
(C6H5), 57.8 (14-C), 56.7 (5-C, Z-), 56.6 (5-C, E-), 56.0 (17-C), 53.5
(24-C), 53.4 (9-C), 46.6 (13-C, E-), 46.5 (13-C, Z-), 45.8 (7-C), 43.0
(12-C), 41.7 (10-C, E-), 41.6 (10-C, Z-), 39.4 (20-C), 39.0 (8-C, Z-),
38.8 (8-C, Z-), 38.1 (4-C, Z-), 37.8 (4-C, E-), 37.1 (22-C), 36.0 (16-
C), 35.8 (23-C), 33.8 (2-C, E-), 33.6 (2-C, Z-), 32.4 (1-C, E-), 31.4
(1-C, Z-), 30.5 (25-C), 24.0 (15-C), 23.0 (11-C), 21.5 (26-C), 21.4
Compounds 7a–7c were prepared similarly as the procedure for
the synthesis of 5, but 4-phenyl-3-thiosemicarbazide was used as
an attack reagent instead of thiosemicarbazide.

104
C. Gan et al. / Steroids 87 (2014) 99–107
(27-C), 20.2 (21-C), 19.0 (19-C), 18.8 (28-C), 12.4 (18-C), 12.0 (29-
C); HRESI-MS: 578.4139 [M+H]+ (calcd for C36H56N3OS, 578.4144).
C6AbH), 5.019 (dd, 1H, J = 15.3, 8.3, C22AH), 5.157 (dd, 1H,
J = 15.3, 8.3, C23AH), 7.213 (t, 1H, J = 7.5, p-PhAH), 7.380 (t, 2H,
J = 7.5, m-PhAH), 7.633 (d, 2H, J = 7.8, o-PhAH), 8.131 (br s, 1H,
CSNH), 9.351 (br s, 1H, PhANHA); 13C NMR (CDCl3, 75 MHz) d:
180.2 (C = S), 138.3 (22-C), [138.0, 128.7, 128.7, 125.7, 123.9,
123.9] (AC6H5), 129.3 (23-C), 71.8 (6-C), 60.3 (3-C), 56.3 (14-C),
56.0 (17-C), 54.2 (9-C), 51.3 (24-C), 47.4 (5-C), 42.5 (20-C), 40.6
(13-C), 39.8 (12-C), 39.7 (7-C), 38.4 (10-C), 35.8 (1-C), 31.9 (8-C),
30.8 (25-C), 30.3 (4-C), 28.9 (16-C), 26.9 (2-C), 25.4 (28-C), 24.3
(15-C), 21.2 (11-C), 21.1 (26-C), 20.9 (27-C), 19.0 (21-C), 15.9
(18-C), 12.3 (19-C), 12.2 (29-C); ESI-MS, m/z: 580.5 (M + H)+; HRES-
I-MS: 580.4316 [M+H]+ (calcd for C36H56N3OS, 580.4301).
4.1.9. 6b-Hydroxycholestan-3-(40-phenyl)thiosemicarbamides (8a,
9a)
NaBH4 (28 mg, 0.72 mmol) was added in batches to a solution of
7a (200 mg, 0.36 mmol) in 10 mL of methanol (add 2 mL of CH2Cl2
to help dissolving) at room temperature in 10 min. The mixture
was stirred for 20 min at room temperature until no raw material.
(by TLC, elution:Vpetroleum:Vethyl acetat = 4:1). Then the solution was
neutralized with 1 mol Lꢂ1 HCl. After most of methanol was evap-
orated under reduced pressure, a small amount of water was added
and extracted with ethyl acetate (20 mL ꢃ 3). The extract was
washed with cold water, saturated NaHCO3 and brine. After drying
over anhydrous sodium sulfate, the solvent was removed under
reduced pressure. The residue was purified by flash chromatogra-
phy on silica gel using petroleum ether/ethyl acetate (4:1) as the
eluent to give 59 mg of 8a (light yellow solid), yield 30%. m.p.
6b-Hydroxystigmastan-3
a
-(40-phenyl)thiosemicarbamide (9b):
/cmꢂ1
light yellow solid, yield: 12.0%. m.p. 165–167 °C. IR (KBr)
m
:
3432, 2941, 2868, 1625, 1544, 1442, 1376, 1266, 1192, 1086; 1H
NMR (CDCl3, 300 MHz) d: 0.715 (s, 3H, 18-CH3), 0.810 (d, 3H,
J = 6.3, 26-CH3 or 27-CH3), 0.819 (t, 3H, J = 7.2, 29-CH3), 0.862 (d,
3H, J = 6.3, 26-CH3 or 27-CH3), 1.028 (d, 3H, J = 6.3 Hz, 21-CH3),
1.029 (s, 3H, 19-CH3), 2.06–1.97 (m, 2H, C4AH), 3.37–3.29 (m,
1H, C3AbH), 3.702 (br s, 1H, C6AbH), 3.798 (br s, 1H, 3-CANHA),
5.023 (dd, 1H, J = 15.0, 8.4, C22AH), 5.158 (dd, 1H, J = 15.3, 8.4,
C23AH), 7.231 (t, 1H, J = 7.5, p-PhAH), 7.400 (t, 2H, J = 7.5, m-
PhAH), 7.608 (d, 2H, J = 7.8, o-PhAH), 7.858 (br s, 1H, CSNH),
9.248 (br s, 1H, PhANHA); 13C NMR (CDCl3, 75 MHz) d: 180.3
(C@S), 138.3 (22-C), [138.0, 128.8, 128.8, 125.7, 123.7, 123.7]
(AC6H5), 129.3 (23-C), 71.6 (6-C), 56.3 (14-C), 56.0 (17-C), 55.6
(3-C), 54.6 (9-C), 51.3 (24-C), 43.4 (5-C), 42.5 (13-C), 40.6 (20-C),
39.8 (12-C), 39.7 (7-C), 36.3 (1-C), 35.0 (10-C), 31.9 (8-C), 30.3
(25-C), 29.7 (4-C), 28.9 (16-C), 28.3 (2-C), 25.4 (28-C), 24.4 (15-
C), 24.2 (21-C), 21.2 (26-C), 21.1 (27-C), 20.6 (11-C), 19.0 (18-C),
15.1 (19-C), 12.3 (29-C); ESI-MS, m/z: 580.5 (M+H)+; HRESI-MS:
580.4266 [M+H]+ (calcd for C36H56N3OS, 580.4301).
200–203 °C. IR (KBr)
m
/cmꢂ1: 3432, 2921, 2851, 1589, 1544,
1495, 1454, 1384, 1270, 1200, 1082, 1041, 739, 686; 1H NMR
(CDCl3, 300 MHz) d: 0.688 (s, 3H, 18-CH3), 0.876 (d, 3H, J = 6.6,
26-CH3 or 27-CH3), 0.881 (d, 3H, J = 6.6, 26-CH3 or 27-CH3), 0.917
(d, 3H, J = 6.3, 21-CH3), 1.010 (s, 3H, 19-CH3), 1.998 (br d, 1H,
J = 12.6, C4AbH), 2.95–2.80 (m, 1H, C3AaH), 3.782 (br s, 1H, 3-
CANHA), 3.948 (br s, 1H, C6AbH), 7.212 (t, 1H, J = 7.5, p-PhAH),
7.381 (t, 2H, J = 7.8, m-PhAH), 7.637 (d, 2H, J = 7.8, o-PhAH),
8.122 (br s, 1H, CSNH), 9.349 (br s, 1H, PhANHA); 13C NMR (CDCl3,
75 MHz) d: 180.4 (C@S), [138.0, 128.7, 128.7, 125.7, 123.9, 123.9]
(AC6H5), 71.8 (6-C), 60.4 (3-C), 56.3 (14-C), 56.2 (17-C), 54.2 (9-
C), 47.4 (5-C), 42.7 (13-C), 39.9 (7-C), 39.7 (12-C), 39.5 (24-C),
38.4 (10-C), 36.2 (1-C), 35.8 (22-C), 35.7 (20-C), 30.8 (8-C), 30.3
(4-C), 28.2 (16-C), 28.0 (25-C), 26.9 (2-C), 24.2 (15-C), 23.9 (23-
C), 22.8 (26-C), 22.6 (27-C), 21.0 (11-C), 18.7 (21-C), 15.9 (19-C),
12.1 (18-C); ESI-MS, m/z: 554.4 (M+H)+; HRESI-MS: 554.4152
[M+H]+(calcd for C34H56N3OS, 554.4144).
4.1.11. 6b-hydroxysitostan-3-(40-phenyl)thiosemicarbamides (8c, 9c)
Compounds 8c and 9c were prepared similarly as the procedure
for the synthesis of 8a and 9a, but from compound 7c.
Isomer 9a of 8a, 6b-hydroxycholestan-3
micarbamide, was obtained as a byproduct (13 mg, yield 6.5%).
m.p. 107–108 °C. IR (KBr)
/cmꢂ1: 3440, 2937, 2365, 2336, 1642,
a
-(40-phenyl)thiose-
6b-hydroxysitostan-3b-(40-phenyl)thiosemicarbamide
light yellow solid, yield: 33.3%. m.p. 214–215 °C. IR (KBr)
(8c):
m
m
/cmꢂ1
:
1540, 1380, 1078; 1H NMR (CDCl3, 300 MHz) d: 0.699 (s, 3H, 18-
CH3), 0.878 (d, 3H, J = 6.6, 26-CH3 or 27-CH3), 0.882 (d, 3H,
J = 6.6, 26-CH3 or 27-CH3), 0.921 (d, 3H, J = 6.3, 21-CH3), 1.037 (s,
3H, 19-CH3), 2.016 (br d, 2H, J = 12.3, C4AH), 3.34–3.27 (m, 1H,
C3AbH), 3.713 (br s, 1H, C6AbH), 3.751 (br s, 1H, 3-CANHA),
7.234 (t, 1H, J = 7.5, p-PhAH), 7.404 (t, 2H, J = 7.5, m-PhAH),
7.485 (br s, 1H, CSNH), 7.614 (d, 2H, J = 7.8, o-PhAH), 9.237 (br s,
1H, PhANHA); 13C NMR (CDCl3, 75 MHz) d: 180.5 (C@S), [138.0,
128.8, 128.8, 125.7, 123.6, 123.6] (AC6H5), 71.7 (6-C), 56.3 (14-
C), 56.2 (17-C), 55.7 (3-C), 54.6 (9-C), 43.4 (5-C), 42.7 (13-C), 39.9
(12-C), 39.5 (24-C), 36.3 (1-C), 36.2 (7-C), 35.8 (22-C), 35.1 (20-
C), 30.3 (4-C), 30.0 (8-C), 28.2 (16-C), 28.0 (25-C), 24.4 (2-C), 24.2
(15-C), 23.8 (23-C), 22.8 (26-C), 22.7 (27-C), 20.6 (11-C), 18.7
(21-C), 15.1 (19-C), 12.1 (18-C); ESI-MS, m/z: 554.4 (M + H)+; HRES-
I-MS: 554.4151 [M+H]+(calcd for C34H56N3OS, 554.4144).
3436, 2953, 2361, 2328, 1634, 1552, 1503, 1413; 1H NMR (CDCl3,
300 MHz) d: 0.696 (s, 3H, 18-CH3), 0.789 (d, 3H, J = 6.6, 26-CH3 or
27-CH3), 0.817 (d, 3H, J = 6.6, 26-CH3 or 27-CH3), 0.860 (t, 3H,
J = 6.6, 29-CH3), 0.926 (d, 3H, J = 6.3, 21-CH3), 1.022 (s, 3H, 19-
CH3), 2.004 (br d, 1H, J = 12.6, C4AbH), 2.94–2.82 (m, 1H,
C3AaH), 3.800 (br s, 1H, 3-CANHA), 3.886 (d, 1H, J = 3.6,
C6AbH), 7.219 (t, 1H, J = 7.5, p-PhAH), 7.388 (t, 2H, J = 7.5,
m-PhAH), 7.643 (d, 2H, J = 7.8, o-PhAH), 7.853 (br s, 1H, CSNHA),
9.340 (br s, 1H, PhANHA); 13C NMR (CDCl3, 75 MHz) d: 180.3
(C@S), [138.0, 128.7, 128.7, 125.7, 123.9, 123.9] (AC6H5), 71.8 (6-
C), 60.4 (3-C), 56.3 (14-C), 56.1 (17-C), 54.1 (9-C), 47.4 (5-C), 45.8
(24-C), 42.6 (13-C), 39.8 (7-C), 39.7 (12-C), 38.8 (10-C), 38.4 (1-
C), 36.2 (20-C), 35.8 (8-C), 33.8 (22-C), 30.8 (4-C), 30.3 (25-C),
29.1 (2-C), 28.2 (16-C), 26.9 (23-C), 24.2 (15-C), 23.0 (28-C), 20.9
(11-C), 20.2 (26-C), 19.9 (27-C), 18.7 (21-C), 15.9 (19-C), 12.1
(18-C), 12.0 (29-C); HRESI-MS: 582.4451 [M + H]+ (calcd for
4.1.10. 6b-Hydroxystigmastan-3-(40-phenyl)thiosemicarbamides (8b,
9b)
C
36H56N3OS, 582.4457).
6b-Hydroxysitostan-3
a
-(40-phenyl)thiosemicarbamide
(9c):
/cmꢂ1
:
Compounds 8b and 9b were prepared similarly as the proce-
dure for the synthesis of 8a and 9a, but from compound 7b.
6b-Hydroxystigmastan-3b-(40-phenyl)thiosemicarbamide (8b):
light yellow solid, yield: 11.7%. m.p. 148–150 °C. IR (KBr)
m
3452, 2953, 2353, 2332, 1629, 1556, 1503, 1413; 1H NMR (CDCl3,
300 MHz) d: 0.698 (s, 3H, 18-CH3), 0.788 (d, 3H, J = 6.3, 26-CH3 or
27-CH3), 0.817 (d, 3H, J = 6.3, 26-CH3 or 27-CH3), 0.838 (t, 3H,
J = 6.6, 29-CH3), 0.928 (d, 3H, J = 6.6, 21-CH3), 1.030 (s, 3H,
19-CH3), 2.06–1.96 (m, 2H, C4AH), 3.35–3.29 (m, 1H, C3AbH),
3.707 (br s, 1H, C6AbH), 3.781 (br s, 1H, 3-CANHA), 7.232 (t, 1H,
J = 7.5, p-PhAH), 7.401 (t, 2H, J = 7.5, m-PhAH), 7.607 (d, 2H,
J = 7.8, o-PhAH), 7.729 (br s, 1H, CSNH), 9.243 (br s, 1H, PhANHA);
13C NMR (CDCl3, 75 MHz) d: 180.4 (C@S), [138.0, 128.8, 128.8,
light yellow solid, yield: 37%. m.p. 145–146 °C. IR (KBr) m :
/cmꢂ1
3411, 2925, 2868, 1613, 1544, 1503, 1446, 1380, 1266, 1078,
972; 1H NMR (CDCl3, 300 MHz) d: 0.708 (s, 3H, 18-CH3), 0.809 (d,
3H, J = 6.3, 26-CH3 or 27-CH3), 0.818 (t, 3H, J = 7.2, 29-CH3), 0.861
(d, 3H, J = 6.3, 26-CH3 or 27-CH3), 1.013 (s, 3H, 19-CH3), 1.025 (d,
3H, J = 6.9, 21-CH3), 2.08–1.96 (m, 2H, C4AH), 2.95–2.80 (m, 1H,
C3AaH), 3.780 (br s, 1H, 3-CANHA), 3.960 (br d, 1H, J = 3.3,

C. Gan et al. / Steroids 87 (2014) 99–107
105
125.7, 123.6, 123.6] (AC6H5), 71.7 (6-C), 56.2 (14-C), 55.7 (17-C),
4.1.15. (3E)-hydroximinocholestan-6-semicarbazone (12a)
White solid, yield: 42.4%. m.p. 258–260 °C. IR (KBr)
3447, 3370, 3219, 3149, 2941, 2863, 1653, 1580, 1474, 1380,
1286, 954; 1H NMR (CDCl3, 300 MHz) d: 0.679 (s, 3H, 18-CH3),
0.832 (s, 3H, 19-CH3), 0.882 (d, 3H, J = 6.6, 26-CH3 or 27-CH3),
0.886 (d, 3H, J = 6.6, 26-CH3 or 27-CH3), 0.925 (d, 3H, J = 6.3, 21-
CH3), 2.41–2.37 (m, 1H, C7AbH), 2.639 (dd, 1H, J = 13.5, 3.6,
55.6 (3-C), 54.6 (9-C), 45.8 (24-C), 43.4 (5-C), 42.6 (13-C), 39.9
(7-C), 39.8 (12-C), 38.8 (10-C), 36.3 (1-C), 36.1 (20-C), 35.1 (8-C),
33.9 (22-C), 30.3 (4-C), 29.7 (25-C), 29.1 (2-C), 28.2 (16-C), 26.1
(23-C), 24.2 (15-C), 23.1 (28-C), 20.6 (11-C), 20.2 (26-C), 19.8
(27-C), 18.7 (21-C), 15.1 (19-C), 12.1 (18-C), 12.0 (29-C); ESI-MS,
m/z: 582.7 (M+H)+; HRESI-MS: 582.4453 [M+H]+(calcd for C36H56-
N3OS, 582.4457).
m :
/cmꢂ1
C7AaH), 3.38–3.27 (m, 1H, C2AbH), 5.044 (br s, 1H, ANH2),
Compounds 11a–11c were prepared similarly as the procedure
for the synthesis of 3, but from 10a–10c.
6.063 (br s, 1H, ANH2), 7.935 (s, 1H, ANHA); 13C NMR (CDCl3,
75 MHz) d: 160.1 (3-C), 158.0 (6-C), 152.0 (C@O), 56.1 (14-C),
54.0 (17-C), 52.1 (9-C), 51.0 (5-C), 42.9 (10-C), 40.1 (13-C), 39.9
(24-C), 39.5 (12-C), 36.5 (22-C), 36.1 (20-C), 35.7 (7-C), 31.2 (8-
C), 29.7 (16-C), 28.6 (25-C), 28.1 (2-C), 28.0 (1-C), 24.1 (4-C), 23.8
(15-C), 22.8 (26-C), 22.6 (27-C), 21.4 (23-C), 19.9 (11-C), 18.6
(21-C), 12.0 (19-C), 11.9 (18-C); HRESI-MS, m/z: 473.3837 [M+H]+
(calcd for C28H49N4O2, 473.3856).
4.1.12. (3E)-hydroximinocholestan-6-thiosemicarbazone (11a)
White solid, yield: 35.4%. m.p. 165–168 °C. IR (KBr)
m :
/cmꢂ1
3390, 3308, 3223, 2924, 2851,1727, 1600, 1510, 1465, 1384,
1281, 1069, 954; 1H NMR (CDCl3, 300 MHz) d: 0.683 (s, 3H, 18-
CH3), 0.820 (s, 3H, 19-CH3), 0.872 (d, 3H, J = 6.6, 26-CH3), 0.893
(d, 3H, J = 6.6, 27-CH3), 0.924 (d, 3H, J = 6.3, 21-CH3), 2.391 (dd,
4.1.16. (3E)-hydroximinostigmastan-6-semicarbazone (12b)
White solid, yield: 76%. m.p 290–291 °C. IR (KBr) m
/cmꢂ1: 3472,
1H, J = 8.1, 2.4, C7AbH), 2.689 (dd, 1H, J = 11.7, 2.4, C7AaH),
3244, 2953, 2868, 1691, 1572, 1458, 1380, 972; 1H NMR (CDCl3,
300 MHz) d: 0.695 (s, 3H, 18-CH3), 0.807 (s, 3H, 19-CH3), 0.823 (t,
3H, J = 6.3, 29-CH3), 0.859 (d, 3H, J = 6.3, 26-CH3), 0.869 (d, 3H,
J = 6.3, 27-CH3), 1.031 (d, 3H, J = 6.3, 21-CH3), 2.81–2.70 (m, 1H,
3.323 (td, 1H, J = 12.9, 5.2, C2AbH), 6.754 (br s, 1H, ANH2), 7.194
(d, 1H, J = 4.8, ANH2), 8.843 (s, 1H, ANH); 13C NMR(CDCl3,
75 MHz) d: 179.0 (C@S), 159.5 (3-C), 155.0 (6-C), 56.4 (14-C),
56.1 (17-C), 53.9 (9-C), 52.3 (5-C), 43.0 (13-C), 40.5 (10-C), 40.3
(24-C), 39.5 (12-C), 36.4 (22-C), 36.3 (20-C), 36.1 (8-C), 35.7 (7-
C), 31.7 (16-C), 29.7 (25-C), 28.6 (2-C), 28.0 (1-C), 24.2 (4-C), 23.8
(15-C), 22.8 (27-C), 22.6 (26-C), 21.4 (23-C), 19.9 (11-C), 18.6
(21-C), 12.0 (19-C), 11.9 (18-C); HRESI-MS, m/z: 489.3601 [M+H]+
(calcd for C28H49N4OS, 489.3627).
C7AaH), 2.996 (br d, 1H, J = 13.4, C2AbH), 5.039 (dd, 1H, J = 15.0,
8.4, C22AH), 5.166 (dd, 1H, J = 15.3, 8.4, C23AH), 6.096 (br s, 1H,
ANH2), 8.838 (br s, 1H, ANH2), 9.831 (s, 1H, ANHA); 13C NMR
(CDCl3, 75 MHz) d: 159.2 (3-C), 153.5 (6-C), 151.3 (C@O), 138.0
(22-C), 129.5 (23-C), 56.6 (14-C), 55.9 (17-C), 53.8 (9-C), 51.2
(24-C), 50.6 (5-C), 42.8 (10-C), 40.4 (13-C), 39.7 (20-C), 39.2 (12-
C), 37.3 (25-C), 35.6 (7-C), 31.9 (8-C), 31.2 (16-C), 30.3 (2-C), 29.7
(28-C), 25.4 (1-C), 24.3 (4-C), 22.7 (15-C), 21.3 (11-C), 21.2 (26-
C), 21.1 (27-C), 19.1 (21-C), 12.3 (19-C), 11.9 (18-C), 11.7 (29-C);
HRESI-MS, m/z: 499.3994 [M+H]+ (calcd for C30H51N4O2, 499.4012).
4.1.13. (3E)-hydroximinostigmastan-6-thiosemicarbazone (11b)
White solid, yield: 72%. m.p. 203–205 °C; IR (KBr) m
/cmꢂ1: 3423,
3374, 3260, 2953, 2868, 1593, 1486, 1384, 1282, 1074, 968; 1H
NMR (CDCl3, 300 MHz) d: 0.703 (s, 3H, 18-CH3), 0.825 (d, 3H,
J = 6.3, 26-CH3), 0.821 (s, 3H, 19-CH3), 0.823 (t, 3H, J = 7.2, 29-
CH3), 0.873 (d, 3H, J = 6.3, 27-CH3), 1.033 (d, 3H, J = 6.5, 21-CH3),
2.392 (dd, 1H, J = 8.1, 3.0, C7AbH), 2.677 (dd, 1H, J = 13.1, 3.0,
4.1.17. (3E)-hydroximinositostan-6-semicarbazone (12c)
White solid, yield: 72%. m.p. 246–248 °C. IR (KBr) m
/cmꢂ1: 3447,
3370, 3210, 2953, 2867, 1674, 1584, 1461, 1380, 1077; 1H NMR
(CDCl3, 300 MHz) d: 0.678 (s, 3H, 18-CH3), 0.807 (s, 3H, 19-CH3),
0.832 (d, 3H, J = 6.6, 26-CH3), 0.854 (d, 3H, J = 6.6, 27-CH3), 0.865
(t, 3H, J = 6.6, 29-CH3), 0.930 (d, 3H, J = 6.0, 21-CH3), 2.84–2.79
C7AaH), 3.306 (dd, 1H, J = 14.5, 3.9, C2AbH), 5.049 (dd, 1H,
J = 15.2, 8.3, C22AH), 5.163 (dd, 1H, J = 15.2, 8.2, C23AH), 6.736
(br d, 1H, J = 4.2, ANH2), 7.194 (br d, 1H, J = 4.2, ANH2), 8.130 (br
s, 1H, ANOH), 8.829 (s, 1H, ANH); 13C NMR (CDCl3, 75 MHz) d:
179.1 (C@S), 159.5 (3-C), 154.9 (6-C), 137.9 (22-C), 129.7 (23-C),
56.5 (14-C), 55.9 (17-C), 53.9 (9-C), 52.3 (24-C), 51.2 (5-C), 42.9
(13-C), 40.5 (10-C), 40.4 (20-C), 40.3 (12-C), 39.3 (8-C), 36.5 (25-
C), 36.3 (7-C), 31.9 (16-C), 31.7 (2-C), 28.7 (28-C), 28.6 (1-C), 25.3
(4-C), 24.3 (15-C), 21.4 (11-C), 21.2 (26-C), 21.1 (27-C), 19.0 (21-
C), 12.3 (19-C), 12.2 (18-C), 11.9 (29-C); HRESI-MS, m/z:
513.3623 [MꢂH]ꢂ (calcd for C30H49N4OS, 513.3627).
(m, 1H, C7AaH), 2.964 (br d, 1H, J = 14.1, C2AbH), 6.031 (br s,
1H, ANH2), 8.924 (br s, 1H, ANH2), 9.733 (s, 1H, ANHA); 13C
NMR (CDCl3, 75 MHz) d: 159.0 (3-C), 153.6 (6-C), 151.3 (C@O),
56.5 (14-C), 56.1 (17-C), 53.4 (9-C), 50.6 (5-C), 45.8 (24-C), 42.9
(10-C), 39.6 (13-C), 39.3 (12-C), 37.3 (20-C), 36.1 (22-C), 35.6 (7-
C), 33.9 (8-C), 31.3 (25-C), 30.3 (16-C), 29.7 (2-C), 29.1 (23-C),
28.2 (1-C), 26.1 (4-C), 24.1 (15-C), 23.1 (28-C), 21.3 (11-C), 19.8
(26-C), 19.0 (27-C), 18.7 (21-C), 12.1 (19-C), 12.0 (18-C), 11.9
(29-C); HRESI-MS, m/z: 501.4151 [M + H]+ (calcd for C30H53N4O2,
501.4169).
4.1.14. (3E)-hydroximinositostan-6-thiosemicarbazone (11c)
White solid, yield: 69%. m.p 197–199 °C. IR (KBr) m
/cmꢂ1: 3407,
2916, 2851, 1711, 1461, 1384, 1277, 1122, 938; 1H NMR (CDCl3,
4.1.18. (3E)-hydroximinocholestan-6-hydrozone (13a)
300 MHz) d: 0.688 (s, 3H, 18-CH3), 0.821–0.938 (m, 15H, 5CH3),
Light yellow solid, yield: 73.5%. m.p. 173–175 °C. IR (KBr) m/
2.706 (d, 1H, J = 12.9, C7AaH), 3.281ꢄ3.368 (m, 1H, C2AbH),
cmꢂ1: 3452, 2945, 2863, 1641, 1465, 1380, 1241, 1171, 1020; 1H
NMR (CDCl3, 300 MHz) d: 0.658 (s, 3H, 18-CH3), 0.878 (d, 3H,
J = 6.6, 26-CH3), 0.881 (d, 3H, J = 6.6, 27-CH3), 0.924 (d, 3H, J = 6.6,
21-CH3), 0.938 (s, 3H, 19-CH3), 2.258 (t, 1H, J = 13.8, C7AbH),
6.934 (br s, 1H, ANH2), 7.209 (br s, 1H, ANH2), 8.899 (br s, 1H,
ANH); 13C NMR (CDCl3, 75 MHz) d: 178.9 (C@S), 159.5 (3-C),
155.2 (6-C), 56.4 (14-C), 56.0 (17-C), 53.8 (9-C), 52.3 (5-C), 51.1
(24-C), 45.8 (13-C), 43.0 (10-C), 40.4 (12-C), 39.5 (20-C), 37.7 (8-
C), 36.5 (22-C), 36.0 (7-C), 33.9 (25-C), 31.8 (16-C), 29.2 (2-C),
28.6 (23-C), 28.1 (1-C), 26.1 (4-C), 24.2 (15-C), 23.1 (28-C), 21.4
(11-C), 19.8 (26-C), 19.0 (27-C), 18.7 (21-C), 12.1 (19-C), 12.0
(18-C), 11.9 (29-C); HRESI-MS, m/z: 517.3951 [M+H]+ (calcd for
2.416 (dd, 1H, J = 12.3, 5.4, C2A
aH), 3.311 (dd, 1H, J = 13.8, 4.2,
C7Aa
H), 3.392 (br d, 1H, J = 13.2, C2AbH); 13C NMR (CDCl3,
75 MHz) d: 169.7 (6-C), 165.7 (3-C), 57.0 (14-C), 56.2 (17-C), 54.5
(9-C), 52.6 (5-C), 43.0 (10-C), 40.0 (13-C), 39.8 (24-C), 39.5 (12-
C), 39.0 (22-C), 36.1 (20-C), 35.7 (8-C), 35.6 (7-C), 32.8 (16-C),
30.7 (25-C), 28.1 (2-C), 28.0 (1-C), 25.6 (4-C), 24.1 (23-C), 23.9
(15-C), 22.8 (26-C), 22.6 (27-C), 21.6 (11-C), 18.7 (21-C), 12.2
(19-C), 12.1 (18-C); HRESI-MS, m/z: 430.3796 [M+H]+ (calcd for
C30H53N4OS, 517.3940).
Compounds 12a–12c, 13a–13c were prepared similarly as the
procedure for the synthesis of 2, but from 10a–10c, and semicarb-
azide hydrochloride and N2H4ꢀH2O were used as attack reagents.
C
27H48N3O, 430.3797).

106
C. Gan et al. / Steroids 87 (2014) 99–107
4.1.19. (3E)-hydroximinostigmastan-6-hydrozone (13b)
C), 208.8 (6-C), 178.2 (C@S), 152.5 (22-C), 57.5 (17-C), 56.2 (14-
Light yellow solid, yield: 83%. m.p. 197–198 °C; IR (KBr)
m
/cmꢂ1
:
C), 53.7 (5-C), 53.4 (13-C), 46.5 (9-C), 43.4 (7-C), 41.2 (10-C), 39.5
(4-C), 39.1 (12-C), 38.0 (2-C), 37.9 (1-C), 37.4 (8-C), 37.0 (20-C),
27.5 (16-C), 24.1 (15-C), 21.6 (11-C), 17.4 (21-C), 12.6 (19-C),
12.3 (18-C).
3448, 2953, 2868, 1642, 1462, 1380, 975; 1H NMR (CDCl3,
300 MHz) d: 0.693 (s, 3H, 18-CH3), 0.821 (t, 3H, J = 6.0, 29-CH3),
0.833 (d, 3H, J = 6.6, 26 or 27-CH3), 0.861 (d, 3H, J = 6.6, 26 or 27-
CH3), 0.934 (s, 3H, 19-CH3), 1.031 (d, 3H, J = 6.3, 21-CH3), 2.246
(t, 1H, J = 14.7, C5-H), 2.40 (br s, 2H, ANH2), 3.42–3.29 (m, 2H,
4.1.23. 6-Oxo-3-hydroximino-22,23-secostigmastan-22-
thiosemicarbazone (16)
C7AaH and C2AaH), 5.036 (dd, 1H, J = 15.0, 8.4, C22AH), 5.158
(dd, 1H, J = 15.0, 8.4, C23AH); 13C NMR (CDCl3, 75 MHz) d: 169.3
(6-C), 165.4 (3-C), 138.1 (22-C), 129.5 (23-C), 57.1 (14-C), 56.0
(17-C), 54.5 (9-C), 52.6 (24-C), 51.3 (5-C), 42.8 (10-C), 40.4 (13-
C), 40.0 (20-C), 39.7 (12-C), 39.0 (8-C), 35.7 (25-C), 32.7 (7-C),
31.9 (16-C), 30.8 (2-C), 28.9 (28-C), 25.5 (1-C), 25.4 (4-C), 24.1
(15-C), 21.5 (11-C), 21.2 (26-C), 21.1 (27-C), 19.0 (21-C), 12.2
(19-C), 12.1 (18-C), 11.9 (29-C); HRESI-MS, m/z: 456.3952 [M+H]+
(calcd for C29H50N3O, 456.3954).
Compound 15 (150 mg, 0.36 mmol) and CH3COONaꢀ3H2O
(49 mg, 0.36 mmol) were dissolved in 25 mL of 95% CH3CH2OH.
After the mixture was heated to 60 °C, NH2OHꢀHCl (26 mg,
0.37 mmol) was added. The mixture was stirred for 1 h at 65 °C
and monitored by TLC (Vethyl acetate:Vpetroleum ether = 1:1). Then the
reaction was terminated and majority of solvent was evaporated
under reduced pressure. Distilled water was added and white solid
generated, and the product was extracted with ethyl acetate
(20 ꢃ 3 mL). The combined extract was washed with water and
saturated brine, dried with anhydrous sodium sulfate, and evapo-
rated under reduced pressure. The residue was purified by flash
chromatography (Vethyl acetate:Vpetroleum ether = 1:1) to give 48 mg
of 16 as light yellow solid. Yield: 30.9%. m.p. 182–184 °C. IR (KBr)
4.1.20. (3E)-hydroximinositostan-6-hydrozone (13c)
Light yellow solid, yield: 81%. m.p 189–191 °C; IR (KBr) m :
/cmꢂ1
3423, 2953, 2867, 1637, 1461, 1380, 1249; 1H NMR (CDCl3,
300 MHz) d: 0.672 (s, 3H, 18-CH3), 0.825 (d, 3H, J = 6.6, 26 or 27-
CH3), 0.847 (d, 3H, J = 6.6, 26 or 27-CH3), 0.858 (t, 3H, J = 6.9, 29-
CH3),, 0.924 (d, 3H, J = 6.3, 21-CH3), 0.932 (s, 3H, 19-CH3), 2.252
(t, 1H, J = 14.7, C5AH), 2.419 (br s, 2H, ANH2), 3.299 (dd, 1H,
m
/cmꢂ1: 3432, 3256, 3150, 2945, 2859, 1707, 1597, 1540, 1376,
1111; 1H NMR (DMSO, 300 MHz) d: 0.663 (s, 3H, 18-CH3), 0.750
(s, 3H, 19-CH3), 1.057 (d, 3H, J = 6.6, 21-CH3), 2.51–2.38 (m, 4H,
C4 and C7AH), 3,10–2.96 (m, 1H, C20AH), 7.279 (d, 1H, J = 6.6,
C22AH), 7.424 (br s, 1H, ANH), 7.944 (br s, 1H, ANH), 10.26 (br
s, NAOH), 10.949 (br s, 1H, @NNHA); 13C NMR (DMSO, 75 MHz)
d: 210.4 (6-C), 178.0 (C@S), 156.7 (3-C), 152.1 (22-C), 57.1 (5-C),
56.0 (17-C), 53.8 (14-C), 52.6 (13-C), 46.2 (9-C), 43.2 (7-C), 41.7
(10-C), 37.6 (12-C), 36.7 (8-C), 30.0 (20-C), 27.5 (4-C), 27.1 (2-C),
26.9 (1-C), 24.1 (15-C), 21.4 (11-C), 19.7 (21-C), 17.8 (19-C), 12.4
(18-C); ESI-MS, m/z: 433.4 [M + H]+ (calcd for C23H37N4O2S, 433.3).
J = 14.1, 4.2, C7AaH), 3.381 (br d, 1H, J = 13.2, C2Aa
H); 13C NMR
(CDCl3, 75 MHz) d: 169.7 (6-C), 165.7 (3-C), 57.0 (14-C), 56.2 (17-
C), 54.5 (9-C), 52.6 (5-C), 45.8 (24-C), 43.0 (10-C), 40.0 (13-C),
39.8 (12-C), 39.0 (20-C), 36.1 (22-C), 35.7 (8-C), 33.9 (7-C), 32.8
(25-C), 30.7 (16-C), 29.1 (2-C), 28.2 (23-C), 26.1 (1-C), 25.6 (15-
C), 24.1 (4-C), 23.1 (28-C), 21.6 (11-C), 19.8 (26-C), 19.0 (27-C),
18.7 (21-C), 12.2 (19-C), 12.1 (18-C), 11.9 (29-C); HRESI-MS, m/z:
458.4124 [M + H]+ (calcd for C29H50N3O, 458.4110).
4.1.21. 3,6-Dioxo-22,23-secostigmastan-22-aldehyde (14)
A flow of O3 in O2 was bubbled through a solution of 4b
(500 mg, 1.17 mmol) in a mixture of CH2Cl2 (16 mL) and MeOH
(4 mL) at ꢂ78 °C until the solution turned pale blue. The reaction
was monitored by TLC (Vethyl acetate:Vpetroleum ether = 4:1). Then the
mixture was purged with O2 for 20 min, and Me2S (2 mL) was
added. The mixture was allowed to warm to room temperature
and was stirred overnight. After removing solvent and dimethyl-
sulfide under reduced pressure, the residue was dissolved with
60 mL CH2Cl2. The organic layer was washed with distilled water
and saturated salt water, dried with anhydrous sodium sulfate,
and evaporated under reduced pressure. The residue was separated
by column chromatography (Vpetroleum ether:Vethyl acetate = 4:1) to
afford compound 14 (242 mg, 70%) as white solid. m.p. 168–
4.2. Antiproliferative activity
4.2.1. Materials
Stock solutions of the compounds were prepared in sterile
dimethyl sulfoxide (DMSO) (Sigma) at a concentration of 10 mg/
mL and afterward 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
SGC 7901 and Bel 7404 cancer cells were grown in the medium
(RPMI-1640) supplemented with 10% heat inactivated fetal bovine
serum and 0.1 g/L penicillin G + 0.1 g/L streptomycin sulfate in a
humidified atmosphere of 5% CO2 at 37 °C.
169 °C. IR (KBr)
m
/cmꢂ1: 2931, 2871, 2734, 1709, 1463, 1386,
1265, 1227, 920; 1H NMR (DMSO, 600 MHz) d: 0.689 (3H, s, 18-
CH3), 0.869 (3H, s, 19-CH3), 1.045 (3H, d, J = 6.5, 21-CH3), 2.782
(1H, dd, J = 13.5, 4.0, C5AH), 9.538 (1H, d, J = 6.5, C22AH); 13C
NMR (CDCl3, 150 MHz) d: 209.4 (3-C), 209.3 (6-C), 205.7 (22-C),
56.5 (14-C), 55.5 (5-C), 52.4 (17-C), 51.0 (20-C), 49.0 (13-C), 46.2
(9-C), 43.5 (7-C), 41.0 (10-C), 39.0 (4-C), 37.7 (12-C), 37.6 (2-C),
37.4 (1-C), 37.2 (8-C), 26.9 (16-C), 24.4 (15-C), 21.6 (11-C), 13.6
(19-C), 12.6 (18-C), 12.5 (21-C).
4.2.3. Assay for cell viability
The cell proliferation assay was undertaken by a MTT method
using 96-well plates. Using cisplatin as a positive control, the anti-
proliferative activity of the compounds was determined. Briefly,
cells (3 ꢃ 104 cells per well) were seeded in 96-wells plates. One
day after seeding, cells in the wells were respectively treated with
target compounds at various concentrations. An equal amount of
DMSO was added to the cells used as negative controls. All were
treated in triplicate. After reincubated for 72 h, 20 lL of the tetra-
zolium dye (MTT) (5 mg/mL) solution were added to each well, and
the cells were incubated for an additional 4 h. After the superna-
tant was discarded, 200 lL of DMSO were added to dissolve the
4.1.22. 3,6-Dioxo-22,23-secostigmastan-22-thiosemicarbazone (15)
Compound 15 was prepared similarly as the procedure for the
synthesis of 3, but from compound 14. White solid, yield: 20.0%.
m.p. 210–212 °C. IR (KBr) m
/cmꢂ1: 3436, 3256, 3141, 2937, 2847,
1711, 1605, 1540, 1375, 1236, 1102; 1H NMR (CDCl3, 300 MHz)
d: 0.745 (s, 3H, 18-CH3), 0.971 (s, 3H, 19-CH3), 1.148 (d, 3H,
J = 6.6, 21-CH3), 2.65–2.54 (m, 2H, C4AH and C5AH), 6.401 (br s,
1H, ANH), 7.054 (br s, 1H, ANH), 7.170 (d, 1H, J = 6.6, C22AH),
9.702 (s, 1H, @NANHA); 13C NMR (CDCl3, 75 MHz) d: 211.2 (3-
purple formazan crystals formed. The absorbance values (A) at
492 nm were determined using a MLLTISKAN MK3 analysis spec-
trometer (Thermo Scientific Co.). The IC50 values were calculated
as the concentration of drug yielding 50% cell survival.

C. Gan et al. / Steroids 87 (2014) 99–107
107
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Science Foundation of Guangxi Province (No. 2010GXNSFD013019),
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