Synthesis and evaluation of some steroidal oximes as cytotoxic agents: Structure/activity studies (I)

Article abstract of DOI:10.1016/j.steroids.2008.09.003

The side chain of a compound plays an important role in its biological function. In our studies, we have found that hydroximinosteroid derivatives with different side chains and position of hydroximino on ring A and B displayed remarkable distinct cytotoxicities against a diversity of cancer cell types. Presence of an oxime group on ring B and a hydroxy on ring A or B resulted in a higher cytotoxicity than other structural motifs. In addition, a cholesterol-type side chain at position 17 was required for the biological activity. Our findings provide new evidence showing the relationship between the chemical structure and biological function. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.

Full text of DOI:10.1016/j.steroids.2008.09.003

steroids 7 4 ( 2 0 0 9 ) 62–72
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Synthesis and evaluation of some steroidal oximes as
cytotoxic agents: Structure/activity studies (I)
a,∗
a
a
b
b,∗∗
Jian-Guo Cui , Lei Fan , Li-Liang Huang , Hong-Li Liu , Ai-Min Zhou
a
Department of Chemistry, Guangxi Teachers Education University, Nanning 530001, China
Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The side chain of a compound plays an important role in its biological function. In our
studies, we have found that hydroximinosteroid derivatives with different side chains and
position of hydroximino on ring A and B displayed remarkable distinct cytotoxicities against
a diversity of cancer cell types. Presence of an oxime group on ring B and a hydroxy on
ring A or B resulted in a higher cytotoxicity than other structural motifs. In addition, a
cholesterol-type side chain at position 17 was required for the biological activity. Our find-
ings provide new evidence showing the relationship between the chemical structure and
biological function. The information obtained from the studies may be useful for the design
of novel chemotherapeutic drugs.
Received 13 July 2008
Received in revised form
3
September 2008
Accepted 5 September 2008
Published on line 16 September 2008
Keywords:
Steroidal oximes
Synthesis
© 2008 Elsevier Inc. All rights reserved.
Antiproliferative activity
Cytotoxicity
1
.
Introduction
leukemia), A-549 (human lung carcinoma), HT-29 (human
colorectal adenocarcinoma) and MEL-28 (human myeloma)
A variety of steroids with unusual and interesting struc-
tures have been isolated from marine sponges recently
tumor cells [6].
In recent years, several 6-hydroximinosteroid analogues
have been synthesized and evaluated for their cytotoxicity
[7–9]. Interestingly, studies have revealed that the cytotoxicity
of these compounds against cancer cells is dependent on the
location of the hydroximino group on the steroidal nucleus.
The parental steroids with a hydroximino group located at a
different position show a remarkable difference in their cyto-
toxicities, suggesting the importance of a side chain location
on a steroid compound in its biological functions. In this report
we present more evidence that the cytotoxicity of steroidal
oximes we synthesized is not only dependent on the location
of a hydroximino group but also the type of a side chain at
position 17 on the parental steroid. Our results may provide
useful information for the design of chemotherapeutic drugs.
[
1–3]. Among these steroidal compounds, marine steroids
with oxime groups have been reported rarely. Two steroidal
oximes, (6E)-hydroximino-24-ethylcholest-4-en-3-one and
(
6E)-hydroximinocholest-4-en-3-one, were isolated from
Cinachyrella alloclada and C. apion [4] in 1997, and another
steroidal oxime, (3E)-hydroximinocholest-4-en-6-one, was
isolated from marine sponge Cinachyrella australiensis of South
China Sea [5] in 2005. The structures of the three compounds
are shown in Fig. 1. Studies have revealed that these steroids
exert interesting biological activities. For example, the com-
pound 3 displays antiviral function to hepatitis virus (Hep
G2) in vitro [5] and the compound 2 exerts cytotoxic activities
against several types of cancer cells such as P-388 (murine
∗
Corresponding author. Tel.: +86 771 3908065; fax: +86 771 3908308.
∗
∗
Corresponding author. Tel.: +1 216 687 2416; fax: +1 216 687 9298.
E-mail addresses: cuijg1954@126.com (J.-G. Cui), a.zhou@csuohio.edu (A.-M. Zhou).
039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.09.003
0

steroids 7 4 ( 2 0 0 9 ) 62–72
63
Fig. 1 – Natural steroidal oximes. (1) (6E)-hydroximino-24-ethylcholest-4-en-3-one; (2) (6E)-hydroximinocholest-4-en-3-one;
3) (3E)-hydroximinocholest-4-en-6-one.
(
2
.1.2. Synthesis of analogues of
2
.
Results and discussion
(
6E)-hydroximino-4-en-3-one steroids (Scheme 2)
Seven steps were needed to synthesize compound 1 as
reported in Ref. [4]. Here, we introduce a new synthetic method
for the steroidal oxime compounds 1, 2 and 10 with higher
overall yields and fewer synthetic steps [10].
2
.1.
Chemistry
To determine the effect of the type of a side chain and the
hydroximino position on the biological role of a steroidal com-
pound, we have synthesized several analogues of steroidal
oximes with the variation of the side chain at position 17
and the hydroximino group on the A ring or B ring by using
cholesterol, stigmasterol and ␤-sitosterol.
The cholest-4-en-3,6-dione (5a) was converted to 8a by
selective reduction using NaBH4 in the presence of CoCl2
according to the synthetic method we developed. The struc-
ture of 8a was confirmed by comparing IR and 1H NMR
spectra with those of the analogous compound that was ana-
lyzed previously. The oxime 9a was generated by the reaction
of 8a with hydroxylamine hydrochloride in ethanol in the
presence of NaOAc. At the same time, Z-isomer of 9a was
yielded in the reaction with a lower yield (3%). The oxida-
tion of 9a with a Jones’ reagent in acetone produced the
compound 2.
2
.1.1. Synthesis of analogues of
(
3E)-hydroximino-4-en-6-one steroids (Scheme 1)
The steroidal oxime 3 and its analogues, with a hydroxymino
group on the A ring, were synthesized in two steps accord-
ing to the sequence shown in Scheme 1. First, the compound
4
a was converted to the corresponding 4-en-3,6-dione (5a) via
oxidation with pyridinium chlorochromate (PCC) in CH2Cl2.
Next, the oxime 3 was produced in a yield of 87% by the reac-
tion of 5a with hydroxylamine hydrochloride in ethanol in
the presence of NaOAc. At the same time, the compound 2
was obtained as a byproduct in 3% yield. The structure of 3
and 2 was confirmed by analysis of the proton and carbon
NMR chemical shifts at C-2 and C-7. Resonances showing H-2
at 3.095 ppm (dd, J = 18.0 and 3.8 Hz) and C-3 at 155.808 ppm
demonstrated a position of 3-hydroxymino in 3, while the
chemical shifts found for 7-␤H and C-6 at 3.437 ppm (1H, dd,
J = 15.9 and 4.6 Hz) and 149.173 ppm, respectively were indica-
tive of the E-configuration of 6-hydroxymino in 2.
2
.1.3. Synthesis of analogues of
(
7E)-hydroximino-5-en-3-ol steroids (Scheme 3)
We designed and synthesized a series of analogues of (7E)-
hydroximino-5-en-3-ol steroids. These compounds have a
hydroxyimino group at C-7 on the B ring. The following steps
were used to synthesize these compounds. First, the 3␤-
hydroxy group of 4a was protected by forming the acetic ester
(
11a), which was then converted to a 5-ene-7-one 12a by oxi-
dation with CrO3 in pyridine and dichloromethane for 25 h at
ambient temperature. The yield of the product was about 71%.
The hydrolysis of 12a with alcoholic K2CO3 obtained the com-
pound 13a in a yield of 73%. Final oximination of 12a and 13a
Scheme 1 – Reagents: (a) PCC; CH2Cl2 (4a: 84%); (b) NH2OH·HCl, AcONa (3: 87%, 2: 3%).

6
4
steroids 7 4 ( 2 0 0 9 ) 62–72
Scheme 2 – Reagents: (a) NaBH4/CH3OH, CoCl2·6H2O (8a: 88%); (b) NaAc·3H2O, 95% C2H5OH, H2OH·HCl (9a: 75%); (c) Jones’
reagent, acetone (2: 61%).
generated analogue 14a and 15a. The downfield chemical shift
of H-6 at 6.568 ppm (5.706 ppm for 13a) for 15a confirmed the
Z configuration of the oxime group because of the influence of
hydroxy in the hydroxyimino group.
2.2.
Biological evaluation
To evaluate the effect of the location of the hydroximino
group(s) and the type of a side chain at position 17 on the bio-
logical functions of steroidal analogues, we determined the
cytotoxicity of these compounds to a variety of cancer cell
types such as Sk-Hep-1 (human liver carcinoma cell line), H-
292 (human lung carcinoma cell line), PC-3 (human prostate
carcinoma cell line) and Hey-1B (human ovarian carcinoma
cell line) cells. Interestingly, we found that the biological activ-
ity of a steroidal oxime was significantly dependent on the
location of the hydroximino group(s) and the type of a side
chain at position 17 on the parental steroid. The results,
expressed as IC50 values in ␮g, are summarized in Table 1.
Apparently the structure of a side chain at position 17 on
the steroidal oxime plays an important role in its cytotoxic-
ity against cancer cells. An increased antineoplastic activity
among these analogueues was observed along with the order
of the side chain attached at position 17: cholesterol-type
side chain (2, 15a, 19a) >stigmasterol-type side chain (7, 15b,
19b) >sitosterol-type side chain (1, 19c). The presence of a
cholesterol-type side chain appears to be necessary for the
biological activity. The analogues 2, 7, 1, with an oxime group
at C-6, showed a remarkable increase in their cytotoxic activ-
ity in comparison with the analogues 3, 6b, 6c, which have an
oxime group at C-3. The compound 18a–c without any sub-
stitute group on ring B, were found no obvious cytotoxicity
against these cancer cells.
2
.1.4. Synthesis of analogues of (3E)-hydroximino-4-ene
steroids (Scheme 4)
The compounds 18a–c lacking of a substituted group on B ring
were synthesized from the compound 4a which was oxidized
to 5-ene-3-one (16a) with Jones’ reagent in acetone and sub-
sequent treatment with oxalic acid gave 4-ene-3-one (17a) in
8
3% yield. Oximination of 17a with hydroxylamine hydrochlo-
ride produced the hydroximinosteroid analogues (18a) in 73%
yield.
2
.1.5. Synthesis of analogues of
(
3E,6E)-dihydroximino-4-ene steroids (Scheme 5)
We synthesized steroid analogues with two hydroxyimino
groups on the steroidal rings. Two hydroxyimino groups were
introduced by oximination of 5a and b in the presence of
superfluous hydroxylamine hydrochloride to generate the
compound 19a and b.
2
.1.6. 3E-Hydroximinocholest-4-en-6-ol
The compound 20 as shown in Scheme 6 was produced by the
reduction of the compound 3. In the presence of CeCl3·7H2O
as an additive in the reaction, the compound 20 with 6␤-OH
was obtained as a major product.
Scheme 3 – Reagents: (a) Ac2O/Py (11a: 97%); (b) CrO3/Py, CH2Cl2 (12a: 71%); (c) K2CO3, CH3OH, reflux (13a: 73%); (d)
NH2OH·HCl, AcONa, EtOH (14a: 95%); (e) NH2OH·HCl, NaOH, EtOH (15a: 99%).

steroids 7 4 ( 2 0 0 9 ) 62–72
65
Scheme 4 – Reagents: (a) Jones’ reagent, acetone (16a: 83%); (b) oxalic acid, EtOH (17a: 89%); (c) NH2OH·HCl, AcONa, EtOH
(
18a: 73%).
Scheme 5 – Reagents: (a) NH2OH·HCl, AcONa, EtOH (19a: 96%).
The conversion of a hydroxyl group to a keto group at C3
activity of the compounds was markedly decreased (the IC50
values of 15a/14a, 15b/14b in Table 1).
resulted in a dramatic loss of cytotoxic activity, suggesting the
importance of the hydroxyl group in the biological function of
a steroidal oxime (comparing the IC50 values of 9a/2, 9b/7, 9c/1
and 20/3 in Table 1). This result is different from the conclusion
obtained by Rodriguez and co-workers [6]. The reason for that
is under investigation.
Compounds 2 and 7, with a hydroximino group at C-6,
showed a slight increase in their cytotoxic activity when com-
pared to compound 15a and 15b with the same group at C-7
Compound 19a, with a cholesterol-type side chain and two
oxime groups at C-3 and C-6, showed a slight increase in its
cytotoxicity against Sk-Hep-1 and Hey-1B cells in comparison
of compound 2 with the same side chain at position 17, a
keto at C-3 and a oxime group at C-6. However, 19b with a
stigmasterol-type side chain and a similar steroidal nucleus
was less active than analogues 7 of compound 2. Furthermore,
conversion of the oxime group at C-6 (19a, 19b) to a keto (3,
6b) caused a loss of activity indicating that an oxime on ring
B (analogues 2, 7, 15a, 15b, 19a and 19b) or a hydroxy group
(
except of Sk-Hep-1 and Hey-1B cell lines for 2/15a). However,
after the 3-hydroxy on 15a or 15b was acetylated, the cytotoxic
Scheme 6 – Reagents: (a) NaBH4/CH3OH, CeCl3·7H2O (20: 82%).

6
6
steroids 7 4 ( 2 0 0 9 ) 62–72
Table 1 – In vitro antitumor activities (IC50 in ␮g/mL) of the synthetic hydroximinosteroid analogues.
Compound
Structurea
Sk-Hep-1
H-292
PC-3
Hey-1B
>100
1
>100
>100
>100
2
3
33
32.6
35
54
>100
>100
>100
>100
6
6
b
c
>100
>100
>100
>100
>100
>100
>100
>100
7
43
59.5
44
49
9
9
a
b
20.1
26.2
32.5
26.3
37
37
40.5
41.5
45
9
1
c
45
62.5
53
4a
>100
>100
>100
>100

steroids 7 4 ( 2 0 0 9 ) 62–72
67
Table 1 – (Continued )
Compound
Structurea
Sk-Hep-1
>100
H-292
>100
PC-3
>100
Hey-1B
1
4b
>100
1
5a
5b
25
46
70
76
38
78
1
76.8
>90
1
1
9a
9b
24
57
33
76
36
66
37
51
1
2
9c
>100
>100
>100
>100
0
34.5
53
52
45
a
.
on ring B (20) plays a key role in enhancing the cytotoxicity of
this type of compounds.
and Hey-1B (human ovarian carcinoma cell line) cells was
investigated. The results have demonstrated that the pres-
ence of a cholesterol-type side chain is very important in
determining the biological activity of these compounds. We
have found that presence of a hydroximino on the B ring and
a hydroxy on the A ring or B ring resulted in an increase
of cytotoxic activity for the compounds against tumor cells.
Our findings provide new evidence showing the relation-
ship between the chemical structure and biological function.
The information obtained from the studies may be useful
for the design of novel chemotherapeutic drugs for can-
cer.
3
.
Conclusions
We have prepared a series of hydroximinosteroid derivatives
with different substituted groups and the position of a hydrox-
imino on the ring A and B, and different side chains. The
cytotoxicity of the synthesized compounds against sk-Hep-1
(
human liver carcinoma cell line), H-292 (human lung carci-
noma cell line), PC-3 (human prostate carcinoma cell line)

6
4
8
.
steroids 7 4 ( 2 0 0 9 ) 62–72
J = 6.5, 26- or 27-CH3), 1.036(d, 3H, J = 7.0, 21-CH3), 1.169(s, 3H,
Experimental
19-CH3), 5.040(dd, 1H, J = 9.0, 15.2, 22-CH), 5.150(dd, 1H, J = 8.5,
15.2, 23-CH), 6.171(s, 1H, 4-CH).
4
.1.
Chemistry
4
5
.1.4. (3E)-Hydroximinocholest-4-en-6-one (3)
The sterol and NaBH4 were purchased from the Merck Co. All
chemicals and solvents were analytical grade and solvents
were purified by general methods before being used. Melt-
ing points were determined on an X4 apparatus and were
uncorrected. Infrared spectra were measured with a Nicolet
FT-360 Spectrophotometer. The H and 13C NMR spectra were
recorded in CDCl3 on a Bruker AV-500 spectrometer at working
frequencies 500 and 125 MHz, respectively. Chemical shifts are
expressed in ppm (ı) values and coupling constants (J) in Hz.
The cell proliferation assay was performed by a MTS method
using 96-well plates in Beckman coulter LD400 AD/LD analysis
spectrometer.
a (84 mg, 0.20 mmol) was dissolved in 10 mL 95% CH3CH2OH.
◦
After the mixture was heated to 60 C CH3COONa·3H2O(33 mg,
0.24 mmol) and NH2OH·HCl (22.0 mg, 0.32 mmol) were added
◦
to the solution. The mixture was stirred for 1 h at 60 C. Then
the reaction was terminated and the majority of solvent was
evaporated under reduced pressure. Proper water was added
into the reaction mixture, and the product was extracted
with ethyl acetate (3× 20 mL). The combined extracts were
washed with saturated brine, dried with anhydrous sodium
sulfate, and evaporated under reduced pressure. The residue
was subjected to chromatography to give 76 mg of 3 (87.4%)
1
◦
−1
as pale yellow crystals, ꢀmp 136–138 C; IR(KBr) ꢁ (cm ): 3264,
Compounds 1, 2 and 7 were prepared according to Ref. [10].
2
941, 2868, 1683, 1658, 1585, 1462, 1384, 1245, 1176, 1025, 984;
1
H NMR(CDCl3) ı: 0.735(s, 3H, 18-CH3), 0.889(d, 3H, J = 6.5 Hz,
26 or 27-CH ), 0.892(d, 3H, J = 6.5, 26 or 27-CH ), 0.948(d, 3H,
4
.1.1. 24-Ethylcholest-4-en-3,6-dione (5a)
3
3
Pyridinium chlorochromate (PCC) (2.564 g, 2.0 mmol) was
added to a solution of sitosterol (4c) (0.852 g, 0.50 mmol) in
dried CH2Cl2 (40 mL) in one portion at room temperature.
The reaction was completed in 26 h. To the mixture was then
added 30 mL of CH2Cl2, and the suspension was poured over
a silica gel column and eluted with CH2Cl2. The resulting
solution was washed with cold water and saturated brines.
After drying over anhydrous sodium sulfate, the solvent was
removed under reduced pressure, and the resulting crude
product was purified by chromatography on silica gel using
3 3
J = 6.5, 21-CH ), 1.064(s, 3H, 19-CH ), 2.039–1.976(m, 1H, 7-C␣H),
2.272(ddd, 1H, J = 18.0, 13.5, 5.4, 2-C H), 2.661(dd, 1H, J = 16.4,
␤
3.7, 7-C H), 3.095(dd, 1H, J = 18.0, 3.8, 2-C␣H), 6.776(s, 1H, 4-CH),
␤
8.667(brs, 1H, N–OH); 13C NMR(CDCl ) ı: 200.9(6-C), 155.8(3-C),
3
149.2(5-C), 126.3(4-C), 56.7(14-C), 56.05(17-C), 50.0(9-C), 46.0(7-
C), 42.5(13-C), 39.5(24-C), 39.4(12-C), 38.9(10-C), 36.1(22-C),
35.7(20-C), 33.5(1-C), 33.4(8-C), 28.1(16-C), 28.0(25-C), 24.0(23-
C), 23.8(15-C), 22.8(26-C), 22.5(27-C), 21.3(11-C), 18.9(19-C),
18.7(2-C), 18.6(21-C), 11.9(18-C).
In the reaction, the compound 2 was obtained as a byprod-
uct in 3.4% yield.
◦
petroleum ether (60–90 C)/EtOAc (5:1) as eluent to give 0.75 g
◦
(
86%) of 5a as pale yellow crystals, ꢀmp 172–174 C. IR(KBr) ꢁ:
−
2
1
959, 1683, 1601, 1581, 1461, 1377, 1246, 1124, 948, 871 cm 1
;
4.1.5. (3E)-Hydroximino-24-ethylcholest-4,22-dien-
6-one (6b)
H NMR(CDCl3): 0.724(s, 3H, 18-CH3), 0.816(d, 3H, J = 7.0, 26-
or 27-CH3), 0.841(d, 3H, J = 7.0, 26- or 27-CH3), 0.848(t, 3H,
J = 8.0, 29-CH3), 0.935(d, 3H, J = 6.5, 21-CH3), 1.167(s, 3H, 19-CH3),
The preparing method is similar to 3, yield 70.6%; ꢀ
215–216 ◦C; IR(KBr) ꢁ (cm−1): 3289, 3040, 2954, 2864, 1675, 1581,
mp
2
2
.13–2.17(m, 1H, 2-C␣H), 2.44–2.58(m, 2H, 7-C␤H and 2-C␤H),
.682(dd, 1H, J = 4.5, 15.5, 7-C␣H), 6.170(s, 1H, 4-CH).
1454, 1238, 972; 1H NMR(CDCl ) ı: 0.759(s, 3H, 18-CH ), 0.824(d,
3
3
3H, J = 7.1, 26-CH3 or 27-CH ), 0.840(d, 3H, J = 7.7, 26-CH or
3
3
27-CH3), 0.833(t, 3H, J = 7.5, 29-CH3), 0.874(d, 3H, J = 6.3, 21-
4
.1.2. Cholest-4-en-3,6-dione (5b)
PCC (2.564 g, 2.4 mmol) was added to a solution of cholesterol
0.924 g, 2.2 mmol) in dried CH2Cl2 (40 mL) in one portion at
CH ), 1.069(s, 3H, 19-CH ), 2.271(ddd, 1H, J = 18.5, 14.0, 5.5,
3
3
2-C H), 2.657(dd, 1H, J = 16.5, 3.5, 7-C H), 3.097(dd, 1H, J = 18.3,
␤
␤
(
3.5, 2-C␣H), 5.063(dd, 1H, J = 15.1, 8.6, 22-CH), 5.179(dd, 1H,
J = 15.2, 8.6, 23-CH), 6.775(s, 1H, 4-CH), 8.386(brs, N–OH). 13C
room temperature. The reaction was completed in 28 h. The
workup similar to 5a provided 0.795 g (83.5%) of 5b as pale
yellow crystals, ꢀmp 90–91 C; IR(KBr) ꢁ: 2953, 2865, 1693, 1600,
NMR(CDCl ) ı: 200.9(6-C), 155.8(3-C), 149.2(5-C), 137.9(22-C),
129.7(23-C), 126.3(4-C), 56.8(14-C), 55.9(17-C), 51.3(9-C), 50.1(24-
C), 46.0(13-C), 42.4(10-C), 40.4(20-C), 39.3(7-C), 38.9(12-C),
33.6(8-C), 33.4(25-C), 31.9(1-C), 28.7(16-C), 25.4(2-C), 24.1(28-C),
21.3(15-C), 21.2(11-C), 21.1(21-C), 19.0(26-C), 19.0(27-C), 18.7(19-
C), 12.2(18-C), 12.1(29-C).
3
◦
−
1
1
2
2
486, 1249, 1221, 1117, 942 cm 1. 1H NMR(CDCl3): 0.746(s, 3H,
8-CH3), 0.886(d, 3H, J = 6.4, 26 or 27-CH3), 0.899(d, 3H, J = 6.4,
6 or 27-CH3), 0.952(d, 3H, J = 6.5, 21-CH3), 1.172(s, 3H, 19-CH3),
.546(dd, 1H, J = 5.2, 14.6, 2-C␤H), 2.706(dd, 1H, J = 4.0, 16.0, 7-
C␣H), 6.196(s, 1H, 4-CH).
4.1.6. (3E)-Hydroximino-24-ethylcholest-4-en-6-one (6c)
4
5
.1.3. Stigmast-4,22-dien-3,6-dione (5c)
c was prepared similarly according to the procedure of 5a.
The preparing method is similar to 3, yield 79.6% ꢀmp
◦
−1
190–192 C; IR(KBr) ꢁ (cm ): 3252, 3035, 2933, 2868, 1658,
1585, 1462, 1376, 1249, 984; 1H NMR(CDCl3) ı: 0.741(s, 3H,
18-CH3), 0.843(d, 3H, J = 6.8, 26-CH3 or 27-CH3), 0.865(d,
3H, J = 6.8, 26-CH3 or 27-CH3), 0.874(t, 3H, J = 7.8, 29-CH3),
0.959(d, 3H, J = 6.4, 21-CH3), 1.067(s, 3H, 19-CH3), 2.271(ddd,
1H, J = 19.5, 14, 5.5, 2-C␤H), 2.663(dd, 1H, J = 16.0, 3.5, 7-
C␤H), 3.097(dd, 1H, J = 18.3, 4.0, 2-C␣H), 6.777(s, 1H, 4-CH),
8.507(brs, N–OH). 13C NMR(CDCl3) ı: 200.9(6-C), 155.9(3-C),
PCC (1.30 g, 6.0 mmol) was added to a solution of stigmas-
terol (0.50 g, 1.2 mmol) in dried CH2Cl2 (10 mL) in one portion
at room temperature. The reaction was completed in 27 h.
The workup similar to 5a gives 0.42 g (83%) of 5c as pale yel-
◦
low crystals, ꢀmp 134–135 C; IR(KBr) ꢁ: 2959, 1714, 1686, 1609,
9
69, 864 cm 1; 1H NMR(CDCl3): 0.743(s, 3H, 18-CH3), 0.805(t, 3H,
−
J = 7.0, 29-CH3), 0.798(d, 3H, J = 6.5, 26- or 27-CH3), 0.849(d, 3H,

steroids 7 4 ( 2 0 0 9 ) 62–72
69
1
49.2(5-C), 126.3(4-C), 56.7(14-C), 56.0(17-C), 50.1(9-C), 46.0(24-
4.1.11. (7Z)-Hydroximinocholest-5-en-3-ol acetate (14a)
C), 45.9(13-C), 42.5(10-C), 39.4(7-C), 38.9(12-C), 36.1(20-C),
Compound 12a (75 mg) was dissolved in 10 mL 95%
3
2
3.9(8-C), 33.6(22-C), 33.4(1-C), 29.2(25-C), 28.1(16-C), 26.2(2-C),
4.0(23-C), 23.1(15-C), 21.3(28-C), 19.8(11-C), 19.1(19-C), 19.0(21-
CH CH OH. After the mixture was stirred for 5 min,
3
2
CH COONa·3H O (23 mg) and NH OH·HCl (18 mg) were
3
2
2
C), 18.7(26-C), 18.7(27-C), 12.0(18-C), 11.9(29-C).
added to the solution. The mixture was stirred for 3.5 h at
room temperature. After removal of the majority of solvent,
proper water was added into the reaction mixture, and
the product was extracted with ethyl acetate (3× 15 mL).
The combined extracts were washed with saturated brine,
dried with anhydrous sodium sulfate, and evaporated under
reduced pressure. The residue was subjected to column
4
4
6
.1.7. 3-Hydroxycholest-5-en-7-one acetate (12a)
.400 g CrO3 was dissolved in a mixture of 8.8 mL pyridine and
0 mL CH2Cl2. After stirring for 10 min, a sulution of 13 (0.640 g)
in 20 mL CH2Cl2 was added slowly. The mixture was stirred
at room temperature for 25 h. The reaction mixture was fil-
tered and filtrate was neutralized with 5% HCl, washed (NaCl,
NaHCO3, and water), dried with anhydrous sodium sulfate,
and evaporated under reduced pressure. The residue was sub-
chromatography (silica gel, ethyl acetate/petroleum ether
◦
(
ꢀ
60–90 C) 1:6) to afford 73 mg of 14a (95%) as white solid,
◦
−1
mp 136–137 C; IR(KBr) ␯ (cm ): 3469, 2954, 2872, 1719, 1670,
1466, 1389, 1262, 1037, 956; H NMR(CDCl ) ı: 0.717(s, 3H,
1
◦
jected to chromatography (petroleum ether (60–90 C)/ether
3
1
8-CH3), 0.877(d, 6H, J = 1.6, 26-CH3 or 27-CH3), 0.890(d, 6H,
4
1
1
3
2
2
:1) to give 480 mg of 12a (70.9%) as a white solid, ꢀmp
56–158 C. IR(KBr) ꢁ (cm ): 2969, 1731, 1668, 1467, 1374,
190, 965, 624; 1H NMR(CDCl3) ı: 0.701(s, 3H, 18-CH3), 0.877(d,
H, J = 2.4, 26-CH3 or 27-CH3), 0.890(d, 3H, J = 2.4, 26-CH3 or
7-CH3), 0.941(d, 3H, J = 6.5, 21-CH3), 1.231(s, 3H, 19-CH3),
.077(s, 3H, CH3CO), 4.769–4.705(m, 1H, 3-C␣H), 5.724(d, 1H,
◦
−1
J = 1.6, 26-CH3 or 27-CH ), 0.951(d, 3H, J = 6.5, 21-CH ), 1.154(s,
3H, 19-CH ), 2.064(s, 3H, CH CO), 4.769–4.676(m, 1H, 3-C␣H),
3
3
3
3
6
.591(s, 1H, 6-CH), 6.970(s, 1H, N–OH). 13C NMR(CDCl3)
ı: 170.3(–C O), 163.8(7-C), 152.0(5-C), 113.8(6-C), 73.0(3-C),
4.7(17-C), 50.2(14-C), 49.6(13-C), 42.9(9-C), 39.5(8-C), 38.7(10-
C), 38.3(24-C), 38.1(12-C), 38.0(4-C), 37.8(1-C), 36.2(22-C),
5.7(20-C), 28.3(16-C), 28.0(25-C), 27.5(2-C), 27.2(15-C), 23.9(23-
C), 22.8(26-C), 22.6(27-C), 20.8(11-C), 18.9(21-C), 17.9(19-C),
2.2(18-C).
5
J = 1.6, 6-CH).
3
4
.1.8. 3-Hydroxy-24-ethylcholest-5,22-dien-7-one
acetate (12b)
Yield 72%, ꢀmp 170–172 C; IR(KBr) ꢁ (cm ): 2962, 2872, 1728,
1
◦
−1
4
.1.12. (7Z)-Hydroximino-24-ethylcholest-5, 22-dien-3-ol
acetate (14b)
Yield 93%, ꢀmp 140–142 C; IR(KBr) ꢁ (cm ): 3407, 2958, 2872,
1
1
675, 1458, 1377, 1254, 1037; 1H NMR(CDCl3) ı: 0.713(s, 3H,
8-CH3), 0.819(d, 3H, J = 6.4, 26-CH3 or 27-CH3), 0.825(t, 3H,
◦
−1
J = 7.0, 29-CH3), 0.863(d, 3H, J = 6.4, 26-CH3 or 27-CH3), 1.044(d,
1
0
0
2
2
1
6
1
1
732, 1662, 1467, 1368, 1246, 1037, 972; 1H NMR(CDCl3) ı:
.737(s, 3H, 18-CH3), 0.820(d, 3H, J = 7.0, 26-CH3 or 27-CH3),
.827(t, 3H, J = 5.5, 29-CH3), 0.835(d, 3H, J = 7.0, 26-CH3 or
7-CH3), 0.871(d, 3H, J = 5.5, 21-CH3), 1.159(s, 3H, 19-CH3),
.067(s, 3H, CH3COO–), 4.749–4.700(m, 1H, 3-C␣H), 5.043(dd,
H, J = 15.0, 8.5, 22-CH), 5.201(dd, 1H, J = 15.0, 9.0, 23-CH),
.589(s, 1H, 6-CH), 6.881(s, 1H, N–OH). 13C NMR(CDCl3) ı:
70.3(–C O), 163.8(7-C), 152.1(5-C), 138.2(22-C), 129.5(23-C),
13.7(6-C), 73.0(3-C), 54.7(17-C), 51.2(14-C), 50.1(24-C), 49.7(13-
3H, J = 6.5, 21-CH3), 1.228(s, 3H, 19-CH3), 2.078(s, 3H, CH3CO),
4.735(m, 1H, 3-CH), 5.035(dd, 1H, J = 15.1, 8.7, 22-CH), 5.185(dd,
1H, J = 15.1, 8.7, 23-CH), 5.714(s, 1H, 6-CH).
4
.1.9. 3-Hydroxycholest-5-en-7-one (13a)
K2CO3 solution (13%) of 15 mL was added to a solution of 12a
0.500 g) in CH3OH (30 mL) at room temperature. The reaction
(
mixture was heated under reflux for 4 h. Then the reaction was
terminated and the majority of solvent was evaporated under
reduced pressure. CH2Cl2 of 60 mL was added to dissolve a
solid and the resulting solution was washed with cold water
and saturated brines. After drying over anhydrous sodium sul-
fate, the solvent was removed under reduced pressure, and the
resulting crude product was purified by chromatography on
silica gel using petroleum ether (60–90 C)/EtOAc (2:1) as elu-
ent to give 0.330 g (73%) of 13a as white solid, ꢀmp 171–172 C.
IR(KBr) ꢁ (cm 1): 3434, 2929, 2866, 1670, 1464, 1263, 1060,
C), 43.0(20-C), 42.8(9-C), 40.3(8-C), 40.2(10-C), 38.6(12-C),
8.1(4-C), 37.8(1-C), 32.0(25-C), 29.0(16-C), 27.5(2-C), 27.3(28-
C), 25.4(15-C), 21.5(11-C), 21.3(CH3–C O), 21.1(21-C), 20.8(26-C),
9.0(27-C), 17.9(19-C), 12.4(29-C), 12.3(18-C).
3
1
4
.1.13. (7Z)-Hydroximinocholest-5-en-3-ol (15a)
◦
NaOH solution (0.25 mol/L) of 1.3 mL was added to a solution
of 13a (83 mg) in 95% CH3CH2OH (15 mL) at room tempera-
ture. After the mixture was stirred for 10 min, NH2OH·HCl
◦
−
9
49, 799; 1H NMR(CDCl3) ı: 0.701(s, 3H, 18-CH3), 0.875(d, 3H,
J = 2.4, 26-CH3 or 27-CH3), 0.888(d, 3H, J = 2.4, 26-CH3 or 27-CH3),
(
40 mg) were added to the solution, and the mixture was
◦
heated at 78 C for 11 h. After removal of the majority of
solvent, proper water was added into the reaction mixture,
and the product was extracted with ethyl acetate (3× 15 mL).
The combined extracts were washed with saturated brine,
dried with anhydrous sodium sulfate, and evaporated under
reduced pressure. The residue was recrystallized and 84 mg
0
1
.939(d, 3H, J = 6.6, 21-CH3), 1.216(s, 3H, 19-CH3), 3.714–3.669(m,
H, 3-C␣H), 5.707(d, 1H, J = 1.1, 6-CH).
4
.1.10. 24-Ethylcholest-5,22-dien-3-hydroxy-7-one (13b)
◦
−1
Yield 95%, ꢀmp 153–155 C; IR(KBr) ꢁ (cm ): 2962, 2872, 1728,
1
1
675, 1458, 1377, 1254, 1037; H NMR(CDCl3) ı: 0.722(s, 3H, 18-
◦
of 15a (99%) was obtained as a white crystal, ꢀmp 235–236 C;
IR(KBr) ꢁ (cm ): 3378, 2937, 2864, 1711, 1466, 1384, 1262, 1172,
CH3), 0.819(d, 3H, J = 6.4, 26-CH3 or 27-CH3), 0.826(t, 3H, J = 7.0,
9-CH3), 0.871(d, 3H, J = 6.4, 26-CH3 or 27-CH3), 1.051(d, 3H,
J = 6.7, 21-CH3), 1.224(s, 3H, 19-CH3), 3.731–3.677(m, 1H, 3-C␣H),
.043(dd, 1H, J = 15.0, 8.6, 22-CH), 5.191(dd, 1H, J = 15.0, 8.6, 23-
CH), 5.714(d, 1H, J = 1.5, 6-CH).
−1
2
021, 959, 796; 1H NMR(CDCl3) ı: 0.723(s, 3H, 18-CH3), 0.882(d,
1
3
2
3
H, J = 1.6, 26-CH3 or 27-CH3), 0.895(d, 3H, J = 1.6, 26-CH3 or
7-CH3), 0.955(d, 3H, J = 6.5, 21-CH3), 1.149(s, 3H, 19-CH3),
.708–3.628(m, 1H, 3-C␣H), 6.568(s, 1H, 6-CH), 7.119(s, 1H, 7
5

7
0
steroids 7 4 ( 2 0 0 9 ) 62–72
N–OH); 13C NMR(CDCl3) ı: 158.2(7-C), 153.5(5-C), 112.8(6-C),
26-CH3 or 27-CH3), 0.930(d, 3H, J = 7.5, 21-CH3), 1.120(s, 3H, 19-
7
3
1.2(3-C), 54.8(17-C), 50.3(14-C), 49.8(13-C), 42.9(4-C), 42.2(9-C),
9.5(8-C), 38.6(10-C), 38.4(24-C), 38.0(12-C), 36.7(1-C), 36.2(22-
CH3), 6.486(s, 1H, 4-CH).
C), 35.6(20-C), 31.3(2-C), 28.3(16-C), 28.0(25-C), 27.2(15-C),
3.8(23-C), 22.8(26-C), 22.6(27-C), 20.8(11-C), 19.0(21-C), 18.0(19-
C), 12.2(18-C).
4.1.18. (3E)-Hydroximinocholest-4-en (18a)
Compound 17a (60 mg, 0.156 mmol) was dissolved in 10 mL
95% CH3CH2OH. After the mixture was heated to 60 C,
2
◦
CH3COONa·3H2O (25 mg, 0.18 mmol) and NH2OH·HCl (15 mg,
4
(
.1.14. (7Z)-Hydroximino-24-ethylcholest-5, 22-dien-3-ol
15b)
Yield 99%, ꢀmp 232–233 C; IR(KBr) ꢁ (cm ): 3378, 2937, 2864,
711, 1466, 1384, 1262, 1172, 1021, 959, 796; 1H NMR(CDCl3)
0.21 mmol) were added into the solution. The mixture was
◦
stirred for 1 h at 60 C. Then the reaction was terminated
◦
−1
and the majority of solvent was evaporated under reduced
pressure. Proper water was added into the reaction mix-
ture, and the product was extracted with ethyl acetate (3×
20 mL). The combined extracts were washed with saturated
brine, dried with anhydrous sodium sulfate, and evaporated
under reduced pressure. The residue was subjected to chro-
matography to produce 46 mg of 18a (73%) as pale yellow
1
ı: 0.739(s, 3H, 18-CH3), 0.821(d, 3H, J = 6.5, 26-CH3 or 27-
CH3), 0.828(t, 3H, J = 7.5, 29-CH3), 0.870(d, 3H, J = 6.5, 26-CH3
or 27-CH3), 1.060(d, 3H, J = 6.6, 21-CH3), 1.149(s, 3H, 19-CH3),
3
5
.702-3.635(m, 1H, 3-C␣H), 5.046(dd, 1H, J = 15.0, 8.5, 22-CH),
.200(dd, 1H, J = 15.0, 9.0, 23-CH), 6.566(s, 1H, 6-CH), 7.093(s, 1H,
N–OH); 13C NMR(CDCl3) ı: 158.1(7-C), 153.4(5-C), 138.2(22-C),
29.4(23-C), 112.8(6-C), 71.2(3-C), 54.7(17-C), 51.2(14-C), 50.4(24-
◦
−1
crystals, ꢀmp 158–159 C; IR(KBr) ꢁ (cm ): 3276, 3066, 2933,
1
2864, 1629, 1466, 1376, 1291, 1237, 1200, 1134, 997, 967, 930,
1
C), 49.8(13-C), 42.8(20-C), 42.2(4-C), 40.1(9-C), 38.5(10-C),
857; H NMR(CDCl3) ı: 0.722(s, 3H, 18-CH3), 0.884(d, 3H, J = 2.0,
3
2
8.4(12-C), 38.0(1-C), 36.7(8-C), 31.9(25-C), 31.4(2-C), 28.8(16-C),
7.3(28-C), 25.4(15-C), 21.5(11-C), 21.0(21-C), 20.8(26-C), 19.0(27-
26-CH3 or 27-CH3), 0.897(d, 3H, J = 2.0, 26-CH3 or 27-CH3),
0.934(d, 3H, J = 6.7, 21-CH3), 1.084(s, 3H, 19-CH3), 2.137(ddd,
1H, J = 17.0, 14.0, 5.2, 6-C␤H), 2.232(ddd, 1H, J = 14.0, 4.0, 2.0,
C), 18.0(19-C), 12.4(29-C), 12.2(18-C).
6-C␣H), 2.324(ddd, 1H, J = 14.0, 5.0, 2.0, 2-C␤H), 3.061(ddd, 1H,
J = 18.5, 5.0, 2.5, 2-C␣H), 5.777(s, 1H, 4-CH); 13C NMR(CDCl3) ı:
156.9(3-C), 155.7(5-C), 117.2(4-C), 56.2(14-C), 56.1(17-C), 53.8(9-
C), 42.4(13-C), 39.9(10-C), 39.6(12-C), 38.0(24-C), 36.2(22-C),
35.9(8-C), 35.8(20-C), 34.7(7-C), 32.5(1-C), 32.2(6-C), 28.2(2-C),
28.0(25-C), 24.3(16-C), 23.9(15-C), 22.9(23-C), 22.6(26-C), 21.4(27-
C), 18.9(11-C), 18.7(19-C), 17.8(21-C), 12.0(18-C).
4
.1.15. Cholest-4-en-3-one (17a)
The Jones’ reagent of 1 mL (0.267 mol/L) was gradually added
into the solution of 4a (386 mg, 1 mmol) in 50 mL of acetone
in 10 min. The reaction mixture was stirred at 0 C for 15 min
◦
and then neutralized with 10% K2CO3 solution. The majority of
solvent was evaporated under reduced pressure and then the
product was extracted with ethyl acetate (3× 20 mL). The com-
bined extracts were washed with saturated brine, dried with
anhydrous sodium sulfate, and evaporated under reduced
pressure. The crude product was recrystallized in CH3OH to
obtain 16a as a white crystal. The white crystal was dissolved
in 5 mL 95% CH3CH2OH, and subsequent treatment with oxalic
acid gave cholest-4-en-3-one 17a as pale yellow crystals in 89%
In the reaction, the 3Z-isomer of 18a was obtained
◦
−1
in 24% yield, ꢀmp 97–98 C; IR(KBr) ꢁ (cm ): 3276, 3060,
1
2933, 2855, 1634, 1462, 1376, 963, 841; H NMR(CDCl3) ı:
0.722(s, 3H, 18-CH3), 0.883(d, 3H, J = 2.0, 26-CH3 or 27-CH3),
0.896(d, 3H, J = 2.0, 26-CH3 or 27-CH3), 0.931(d, 3H, J = 6.5,
21-CH3), 1.125(s, 3H, 19-CH3), 2.030(dd, 1H, J = 13.0, 3.5, 6-
C␤H), 2.288–2.246(m, 1H, 6-C␣H), 2.395–2.314(m, 1H, 2-CH),
6.484(s, 1H, 4-CH).
◦
−1
yield. ꢀmp 84–85 C; IR(KBr) ꢁ (cm ): 3019, 2945, 2864, 1670,
1
1
609, 1462, 1376, 1333, 1266, 1225, 1192, 1026, 951, 922, 865; H
NMR(CDCl3) ı: 0.737(s, 3H, 18-CH3), 0.886(d, 3H, J = 2.5, 26-CH3
or 27-CH3), 0.899(d, 3H, J = 2.5, 26-CH3 or 27-CH3), 0.938(d, 3H,
J = 6.5, 21-CH3), 1.207(s, 3H, 19-CH3), 2.428(dd, 1H, J = 14.5, 5.3,
4.1.19. (3E)-Hydroximino-24-ethylcholest-4,22-dien (18b)
◦
−1
Yield 70%, ꢀmp 168–169 C; IR(KBr) ꢁ (cm ): 3285, 3046, 2951,
2883, 1629, 1466, 1437, 1372, 1295, 1237, 1218, 1126, 995,
930, 873; 1H NMR(CDCl3) ı: 0.740(s, 3H, 18-CH3), 0.821(d, 3H,
J = 6.5, 26-CH3 or 27-CH3), 0.829(t, 3H, J = 8.0, 29-CH3), 0.871(d,
3H, J = 6.5, 26-CH3 or 27-CH3), 1.039(d, 3H, J = 6.6, 21-CH3),
1.085(s, 3H, 19-CH3), 2.136(ddd, 1H, J = 17.0, 14.0, 5.0, 6-C␤H),
2.230(ddd, 1H, J = 14.0, 4.0, 2.5, 6-C␣H), 2.323(ddd, 1H, J = 14.0,
4.5, 2.0, 2-C␤H), 3.065(ddd, 1H, J = 17.0, 4.5, 3.0, 2-C␣H), 5.040(dd,
1H J = 15.2, 9.0, 22-CH), 5.170(dd, 1H, J = 15.2, 8.5, 23-CH),
5.778(s, 1H, 4-CH); 13C NMR(CDCl3) ı: 157.2(3-C), 155.9(5-
C), 138.2(22-C), 129.4(23-C), 117.1(4-C), 56.2(14-C), 56.0(17-C),
53.8(9-C), 51.3(24-C), 42.3(13-C), 40.5(10-C), 39.8(20-C), 38.0(12-
C), 35.9(8-C), 34.7(7-C), 32.6(1-C), 32.3(25-C), 31.9(6-C), 28.9(2-C),
25.4(28-C), 24.3(16-C), 21.4(15-C), 21.2(11-C), 21.1(21-C), 19.0(27-
C), 18.7(19-C), 17.8(26-C), 12.3(18-C), 12.2(29-C).
2
-C␤H), 2.461(dd, 1H, J = 15.0, 5.3, 2-C␣H), 5.747(s, 1H, 4-CH).
4
.1.16. 24-Ethylcholest-4,22-dien-3-one (17b)
◦
−1
Yield 85%, ꢀmp 121–122 C; IR(KBr) ꢁ (cm ): 2969, 2937,
2871, 1679, 1619, 1462, 1446, 1435, 1384, 1270, 1229, 994,
9
61, 868; 1H NMR(CDCl3) ı: 0.748(s, 3H, 18-CH3), 0.816(d,
3H, J = 6.5, 26-CH3 or 27-CH3), 0.824(t, 3H, J = 7.3, 29-CH3),
0.866(d, 3H, J = 6.5, 26-CH3 or 27-CH3), 1.037(d, 3H, J = 6.6, 21-
CH3), 1.203(s, 3H, 19-CH3), 2.282(ddd, 1H, J = 14.5, 4.0, 2.5,
6
5
-C␣H), 2.356(dt, 1H, J = 16.5, 4.0, 6-C␤H), 2.426(dd, 1H, J = 14.5,
.2, 2-C␤H), 2.459(dd, 1H, J = 15.0, 5.2, 2-C␣H), 5.040(dd, 1H
J = 15.1, 8.7, 22-CH), 5.165(dd, 1H, J = 15.1, 8.7, 23-CH), 5.743
s, 1H, 4-CH).
(
The 3Z-isomer of 18b was obtained in 29% yield, ꢀmp
◦
−1
4
.1.17. 24-Ethylcholest-4-en-3-one (17c)
166–168 C; IR(KBr) ꢁ (cm ): 3281, 3055, 2933, 2862, 1630,
1462, 1377, 995, 868; 1HNMR(CDCl3) ı: 0.737(s, 3H, 18-CH3),
0.818(d, 3H, J = 6.0, 26-CH3 or 27-CH3), 0.826(t, 3H, J = 7.5,
29-CH3), 0.868(d, 3H, J = 6.0, 26-CH3 or 27-CH3), 1.034(d, 3H,
J = 6.6, 21-CH3), 1.123(s, 3H, 19-CH3), 2.305-2.335(m, 1H, 2-C␣H),
◦
−1
Yield 85%, ꢀmp161–163 C; IR(KBr) ꢁ (cm ): 2957, 2039, 2867,
2
852, 1681, 1620, 1466, 1438, 1384, 1367, 1271, 1120, 1030, 867;
1
H NMR(CDCl3) ı: 0.719(s, 3H, 18-CH3), 0.836(d, 3H, J = 6.5, 26-
CH3 or 27-CH3), 0.867(t, 3H, J = 7.5, 29-CH3), 0.859(d, 3H, J = 6.5,

steroids 7 4 ( 2 0 0 9 ) 62–72
71
4
.146(N–OH), 5.036(dd, 1H, J = 15.1, 8.7, 22-CH), 5.165(dd, 1H,
2.5, C2-␣H), 3.369(ddd, 1H, J = 17.5, 15.0, 4.5, 7-C␤H), 5.057(dd,
1H, J = 15.1, 8.7, 22-CH), 5.179(dd, 1H, J = 15.1, 8.6, 23-CH),
6
J = 15.1, 8.7, 23-CH), 6.482(s, 1H, 4-CH).
.528(s, 1H, 4-CH), 6.961(–OH); 13C NMR(CDCl3) ı: 157.2(6-
4
.1.20. (3E)-Hydroximino-24-ethylcholest-4-en (18c)
C), 156.6(3-C), 147.6(5-C), 138.1(22-C), 129.6(23-C), 119.3(4-C),
56.8 (14-C), 55.9(17-C), 51.4(9-C), 51.3(24-C), 42.5(10-C), 40.4(13-
C), 39.4(20-C), 38.4(12-C), 33.7(25-C), 33.1(8-C), 31.9(1-C),
29.7(16-C), 28.8(2-C), 25.4(7-C), 24.2(28-C), 21.3(15-C), 21.2 (21-
C), 21.1(11-C), 19.1(27-C), 18.6(19-C), 17.6(26-C), 12.3(18-C),
12.2(29-C).
◦
−1
Yield 70%, ꢀmp 174–175 C; IR(KBr) ꢁ (cm ): 3195, 2958, 2929,
2864, 1660, 1629, 1454, 1397, 1295, 1237, 1200, 1130, 1022,
9
67, 930, 868; 1H NMR(CDCl3) ı: 0.720(s, 3H, 18-CH3), 0.836(d,
3H, J = 6.8, 26-CH3 or 27-CH3), 0.859(d, 3H, J = 6.8, 26-CH3 or
27-CH3), 0.868(t, 3H, J = 7.5, 29-CH3), 0.938(d, 3H, J = 6.5, 21-
CH3), 1.081(s, 3H, 19-CH3), 2.136(ddd, 1H, J = 17.0, 14.0, 5.0,
6
-C␤H), 2.250–2.200(m, 1H, 6-C␣H), 2.357–2.289(m, 1H, 2-C␤H),
4.1.23. (3E,6E)-Dihydroximino-24-ethylcholest-4-en (19c)
◦
−1
3
.063(ddd, 1H, J = 17.3, 4.5, 3.0, 2-C␣H), 5.776(s, 1H, 4-CH);
Yield 93%, ꢀmp 207–208 C; IR(KBr) ꢁ (cm ): 3542, 3340, 3061,
2964, 2861, 1642, 1455, 1377, 1307, 1275, 1175, 1005, 956; 1H
NMR(CDCl3) ı: 0.721(s, 3H, 18-CH3), 0.846(d, 3H, J = 7.0, 26-CH3
or 27-CH3), 0.867(d, 3H, J = 7.0, 26-CH3 or 27-CH3), 0.877(t, 3H,
J = 8.0, 29-CH3), 0.951(d, 3H, J = 6.0, 21-CH3), 1.034(s, 3H, 19-CH3),
2.227–2.152(m, 1H, 7-C␣H), 2.436–2.402(m, 1H, 2-C␤H), 3.111(dd,
1H, J = 16.5, 2.0, 2-C␣H), 3.382(ddd, 1H, J = 17.0, 15.0, 4.5, 7-C␤H),
6.532(s, 1H, 4-CH), 6.963(N–OH); 13C NMR(CDCl3) ı: 157.3(C-6),
156.7(3-C), 147.7(5-C), 119.3(4-C), 56.7(14-C), 56.1(17-C), 51.4(9-
C), 45.9(24-C), 42.6(10-C), 39.5(13-C), 38.4(12-C), 36.1(20-C),
34.0(22-C), 33.7(8-C), 33.1(1-C), 29.6(25-C), 29.3(16-C), 28.1(2-C),
26.2(7-C), 24.1(23-C), 23.1(15-C), 21.3(28-C), 19.8(11-C), 19.1(27-
C), 18.7(19-C), 18.5(26-C), 17.6(21-C), 12.0(29-C), 11.9(18-C).
1
3C NMR(CDCl3) ı: 157.3(3-C), 155.9(5-C), 117.1(4-C), 56.1(14-
C), 56.0(17-C), 53.8(9-C), 45.9(24-C), 42.4(13-C), 39.9(10-C),
3
3
C), 21.4(28-C), 19.8(11-C), 19.1(27-C), 18.8(19-C), 18.7(26-C),
1
8.0(12-C), 36.2(20-C), 35.9(8-C), 34.7(22-C), 34.0(7-C), 32.6(1-C),
2.3(6-C), 29.2(25-C), 28.2(2-C), 26.2(23-C), 24.3(16-C), 23.1(15-
7.8(21-C), 12.0(29-C), 12.0(18-C).
The 3Z-isomer of 18c was obtained in 28% yield, ꢀmp
◦
−1
162–163 C; IR(KBr) ꢁ (cm ): 3456, 2966, 2934, 2867, 1629, 1456,
1
400, 1291, 1012, 973, 935, 864; 1H NMR(CDCl3) ı: 0.721(s, 3H,
18-CH3), 0.837(d, 3H, J = 6.8, 26-CH3 or 27-CH3), 0.859(d, 3H,
J = 6.8, 26-CH3 or 27-CH3), 0.869(t, 3H, J = 7.5, 29-CH3), 0.936(d,
H, J = 6.5, 21-CH3), 1.124(s, 3H, 19-CH3), 2.284-2.243(m, 1H, 6-
CH), 2.383–2.328(m, 2H, 2-CH), 6.482(s, 1H, 4-CH).
3
4.1.24. (3E)-Hydroximinocholest-4-en-6˛-ol (20)
4
.1.21. (3E,6E)-Dihydroximinocholest-4-ene (19a)
NaBH4 (19 mg, 0.28 mmol) was added to a solution of 3 (140 mg,
0.339 mmol) and CeCl3·7H2O (126 mg, 0.339 mmol) in CH3OH
(20 mL) in the interval of 5 min at room temperature. After
30 min, the reaction was terminated. The solution was neu-
tralized with 1 M HCl. After evaporation of the majority of
the MeOH under reduced pressure, ethyl acetate (30 mL) was
added to the residue. The resulting solution was washed
with cold water and saturated brines. After drying over anhy-
drous sodium sulfate, the solvent was removed under reduced
pressure, and the resulting crude product was purified by
chromatography. The 20 was obtained as pale yellow crys-
tals (115 mg, 82%), ꢀmp 172–173 C; IR(KBr) ꢁ (cm ): 3374,
3043, 2933, 2905, 2872, 1629, 1462, 1376, 1315, 1287, 1154,
1123, 1074, 972, 886, 849; 1H NMR(CDCl3) ı: 0.704(s, 3H, 18-
CH3), 0.877(d, 3H, J = 2.0, 26 or 27-CH3), 0.890(d, 3H, J = 2.0,
26 or 27-CH3), 0.923(d, 3H, J = 6.5, 21-CH3), 1.044(s, 3H, 19-
CH3), 2.153–2.089(m, 1H, 2-C␤H), 3.026(ddd, 1H, J = 13.6, 6.8,
3.5, 2-C␣H), 4.243(dd, 1H, J = 14.5, 3.5, 6-C␤H), 6.233(s, 1H,
4-CH); 13C NMR(CDCl3) ı: 157.0(5-C), 156.9(3-C), 113.8(4-C),
68.9(6-C), 56.3(17-C), 55.8(14-C), 53.6(9-C), 42.5(13-C), 41.2(10-
C), 39.7(24-C), 39.5(12-C), 38.5(7-C), 36.2 (22-C), 35.8(20-C),
35.2(1-C), 34.3(8-C), 28.2(2-C), 28.1(25-C), 28.0(16-C), 24.3(15-C),
23.9(23-C), 22.8(26-C), 22.6(27-C), 21.4(11-C), 18.7(19-C), 18.6(21-
C), 12.0(18-C).
Compound 5a (80 mg, 0.2 mmol) was dissolved in 10 mL of
◦
5% CH3CH2OH. After the mixture was heated to 60 C,
9
CH3COONa·3H2O (54 mg, 0.4 mmol) and NH2OH·HCl (42 mg,
0
6
.6 mmol) were added. The mixture was stirred for 1 h at
0 C. Then the reaction was terminated and the majority
◦
of solvent was evaporated under reduced pressure. Proper
water was added into the reaction mixture, and the product
was extracted with ethyl acetate (3× 20 mL). The combined
extracts were washed with saturated brine, dried with anhy-
drous sodium sulfate, and evaporated under reduced pressure.
The residue was subjected to recrystallize in methanol
to give 82 mg of 19a (96.4%) as pale yellow crystals, ꢀmp
◦
−1
◦
−1
1
1
3
3
1
1
41–142 C. IR(KBr) ꢁ (cm ): 3554, 3178, 3039, 2941, 2864,
634, 1458, 1376, 1307, 1000, 792; 1H NMR (CDCl3) ı: 0.709(s,
H, 18-CH3), 0.885(d, 3H, J = 6.5, 26-CH3 or 27-CH3), 0.889(d,
H, J = 6.5, 26-CH3 or 27-CH3), 0.936(d, 3H, J = 6.4, 21-CH3),
.051(s, 3H, 19-CH3), 3.103(d, 1H, J = 17.6, C2-␣H), 3.353(dd,
H, J = 15.6, 4.3, C7-␤H), 6.544(s, 1H, 4-CH), 6.780(s, 1H, 6-
NOH), 6.948(s, 1H, 3-NOH); 13C NMR(CDCl3) ı: 157.3(6-C),
56.8(3-C), 147.8(5-C), 119.3(4-C), 56.7(14-C), 56.1(17-C), 51.4(9-
C), 42.6(13-C), 39.5(7-C), 39.4(24-C), 38.3(12-C), 36.1(22-C), 35.7
20-C), 33.6(1-C), 33.0(10-C), 29.6(8-C), 28.1(16-C), 28.0(25-C),
4.1(23-C), 23.8(15-C), 22.8(26-C), 22.5(27-C), 21.3(11-C), 18.5(21-
C), 17.6(2-C), 17.5(19-C), 11.9(18-C).
1
(
2
4
.2.
Antiproliferative activity
4
4
.1.22. (3E,6E)-Dihydroximino-24-ethylcholest-
,22-dien (19b)
4.2.1. Materials and methods
◦
−1
Yield 98%, ꢀmp 140–141 C; IR(KBr) ꢁ (cm ): 3317, 2953, 2864,
Stock solutions of compounds 1, 2 and 4, were prepared in
sterile dimethyl sulfoxide (DMSO) (Sigma) at a concentration
of 10 mg/mL and afterwards 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 strepto-
mycin sulfate.
1
3
3
1
1
629, 1454, 1376, 1298, 1176, 959; 1H NMR(CDCl3) ı: 0.731(s,
H, 18-CH3), 0.826(d, 3H, J = 6.3, 26-CH3 or 27-CH3), 0.876(d,
H, J = 6.3, 26-CH3 or 27-CH3), 0.833(t, 3H, J = 7.0, 29-CH3),
.026(s, 3H, 19-CH3), 1.047(d, 3H, J = 6.0, 21-CH3), 2.222-2.148(m,
H, 7-C␣H), 2.436–2.407(m, 1H, 2-C␤H), 3.101(dd, 1H, J = 14.9,

7
2
steroids 7 4 ( 2 0 0 9 ) 62–72
4
.2.2. Cell culture
the Natural Science Foundation of Guangxi Province (Guike:
Sk-Hep-1, H-292, PC-3 (ATCC) and Hey-1B (A gift from Dr.
Yan Xu, University of Indiana) cells were cultured in a proper
medium supplemented with 10% fetal bovine serum in a
057554).
r e f e r e n c e s
◦
humidified atmosphere of 5% CO2 at 37 C.
4
.2.3. Treatment of cancer cells
[
1] Blunt JW, Copp BR, Hu WP, Munro MHG, Northcote PT,
Prinsep MP. Marine natural products. Nat Prod Rep
3
Cancer cells (4 × 10 cells/200 ␮L) were seeded into each well
of a 96-well microtiter plate. After incuation for 24 h, the com-
pounds with a series of concentrations (range 20–80 ␮g/mL)
were added to the cells. An equal amount of DMSO was added
to the cells used as negative controls. All were treated in trip-
licate.
2007;24:31–86.
[2] Blunt JW, Copp BR, Munro MHG, Northcote PT, Prinsep MP.
Marine natural products. Nat Prod Rep 2005;22:15–61.
[
3] Faulkner DJ. Marine natural product. Nat Prod Rep
001;18:1–49.
2
[
4] Jaime R, Lucia N, Solange P, Carlos J. Isolation and synthesis
of the first natural 6-hydroximino-4-en-3-one steroids from
the sponges cinachyrella spp. Tetrahedron Lett 1997;38:
4
.2.4. Determination of cell viability
MT stetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium)
1833.
[
5] Xiao DJ, Peng XD, Deng SZ, Ma WZ, Wu HM. Structure
elucidation of (3E)-cholest-4-en-3,6-dione-3-oxime in
marine sponge Cinachyrella australiensis from the south china
sea. Chin J Org Chem 2005;25(12):1606–9.
(
CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay,
Cat.# G5421, Promega Corporation) dye reduction assay was
used. The assay is dependent on the MTS being reduced to
an aqueous, soluble formazan by dehydrogenase enzymes
found in metabolically active cells. The quantity of formazan
product as measured by the amount of 490 nm absorbance is
directly proportional to the number of living cells in culture.
Briefly, after treatment (see Section 4.2.3) for 72 h, the medium
was removed and the cells were incubated with 100 ␮L of
fresh medium plus 20 ␮L of MTS solution according to the
instruction provided by the manufacturer for additional
[
6] Deive N, Rodrı ˇı guez J, Jime ˇı nez C. Synthesis of cytotoxic
6E-hydroximino-4-ene steroids: structure/activity studies. J
Med Chem 2001;44:2612–8.
[
7] Jindal DP, Chattopadhaya R, Guleria S, Gupta R. Synthesis
and antineoplastic activity of 2-alkylaminoethyl derivatives
of various steroidal oximes. Euro J Med Chem
2003;38:1025–34.
8] Krsti c´ a NM, Bjelakovi c´ MS, Zi zˇ akb Z, Pavlovic MD, Juraníc
ZD, Pavlovíc VD. Synthesis of some steroidal oximes,
lactams, thiolactams and their antitumor activities. Steroids
[
[
4
h. The absorbance (A) at 490 nm was measured using an
2007;72:406–14.
Beckman coulter LD400 AD/LD analysis spectrometer. IC50
concentration was defined as the concentration of an agent
inhibiting cell survival by 50%, compared to a control.
9] Poza J, Rega M, Paz V, Alonso B, Rodríguez J, Salvador N, et al.
Synthesis and evaluation of new 6-hydroximinosteroid
analogues as cytotoxic agents. Bioorg Med Chem
2007;15:4722–40.
[
10] JianGuo Cui, Liliang Huang, Lei Fan, Aimin Zhou. A facile
and efficient synthesis of some
Acknowledgments
(
6E)-hydroximino-4-en-3-one steroids, steroidal oximes
The authors acknowledge the financial support of the National
Natural Science Foundation of China (Project: 20562001),
from Cinachyrella spp. Sponges. Steroids 2008;73(3):
252–6.