Synthesis and cytotoxicity of A-homo-lactam derivatives of cholic acid and 7-deoxycholic acid

Article abstract of DOI:10.1016/j.steroids.2011.03.009

Using cholic acid and deoxycholic acid as starting materials, a series of 3-aza-A-homo-4-one bile acid and 7-deoxycholic acid derivatives were synthesized by the esterification, oxidation, reduction, oximation and Beckman rearrangement etc. The cytotoxicity of the synthesized compounds against MGC 7901 (human ventriculi carcinoma cell line), hela (human cervical carcinoma cell line), SMMC 7404 (human liver carcinoma cell line) were investigated. The results showed that bile acid and 7-deoxycholic-acid derivatives with 3-aza-A-homo-4-one configuration bearing a 6-hydroximino or 12-hydroximino group displayed a distinct cytotoxicity to Hela tumor cell line. In particular, the IC₅₀ values of the compounds 6 and 13 were 14.3 and 24.3 μmol/L against Hela human tumor cell line respectively. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.

Full text of DOI:10.1016/j.steroids.2011.03.009

Steroids 76 (2011) 690–694
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and cytotoxicity of A-homo-lactam derivatives of cholic acid and
7
-deoxycholic acid
Yanmin Huanga,b, Sijing Chen , Jianguo Cui , Chunfang Gan , Zhiping Liu ,
b
b,∗
b
b
c
a,∗∗
Yingliang Wei , Huachan Song
a
School of Chemistry and Chemical Engineering, SUN YAT-SEN University, Guangzhou 510275, PR China
College of Chemistry and Life Science, Guangxi Teachers Education University, Mingxiu Road 175, Nanning 530001, PR China
Guangxi Analysis Testing Center, Nanning 530022, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Using cholic acid and deoxycholic acid as starting materials, a series of 3-aza-A-homo-4-one bile acid and
Received 1 January 2011
Received in revised form 16 March 2011
Accepted 17 March 2011
Available online 2 April 2011
7
-deoxycholic acid derivatives were synthesized by the esterification, oxidation, reduction, oximation
and Beckman rearrangement etc. The cytotoxicity of the synthesized compounds against MGC 7901
human ventriculi carcinoma cell line), hela (human cervical carcinoma cell line), SMMC 7404 (human
(
liver carcinoma cell line) were investigated. The results showed that bile acid and 7-deoxycholic-acid
derivatives with 3-aza-A-homo-4-one configuration bearing a 6-hydroximino or 12-hydroximino group
displayed a distinct cytotoxicity to Hela tumor cell line. In particular, the IC50 values of the compounds
Keywords:
Cholic acid lactam
Deoxycholic acid lactam
Synthesis
6
and 13 were 14.3 and 24.3 ␮mol/L against Hela human tumor cell line respectively. The information
obtained from the studies may be useful for the design of novel chemotherapeutic drugs.
© 2011 Elsevier Inc. All rights reserved.
Cytotoxicity
1
. Introduction
chain carboxyl or hydroxyl on position-3. Compounds modified on
the steroidal nucleus of cholic acid or deoxycholic acid have rarely
been reported. In our previous work, we found that a polyhydrox-
ysterol or a steroidal oxime has an excellent cytotoxicity when it
has the cholesteric side chain and the 3- or 6-position on steroidal
nucleus is substituted by the hydroxyl or hydroximino [15–17].
In order to evaluate the antitumor activity of new steroidal
derivatives, we designed and synthesized some new steroidal com-
pounds with the introduction of N atom on A-ring using cholic acid
and 7-deoxycholic acid as starting materials. The cytotoxicity of
synthesized compounds was tested in vitro against three tumor
cell lines: MGC 7901 (human ventriculi carcinoma cell line), Hela
(human cervical carcinoma cell line) and SMMC 7404 (human liver
carcinoma cell line). The results reveal that the compounds have a
better cytotoxicity against Hela human tumor cell line.
The synthesis of some aza-homosteroid compounds with
unusual and interesting structures have been reported recently
1–5]. These compounds exhibit valuable biological activities
[
such as cytotoxicity, antibacterium and antileukemic activity.
Researches of azahomosteroids indicated that the presence of the
characteristic group (–NH–CO–) in the aza-homosteroid molecule
had been proven to be important in lowering the acute toxicity and
improving antitumour activity of the compound in cancer research
[
6,7]. In other way, bile acids have been considered very useful
in the preparation of new pharmaceutical drugs because of their
inherent chemical and biological properties [8,9]. They are phar-
macological interesting as potential carriers of liver-specific drugs,
absorption enhancers and cholesterol lowering agent. The facial
amphiphilic structure of bile acids is the potential to be antibi-
otic and polymyxin [10–12]. Recently, researches found that bile
acid conjugates synthesized or obtained from nature have exhibit
activity in the respect of antibiotics, ionophores and molecular car-
riers [13,14]. However, the investigations of this kind of compounds
mainly focus on bile acids conjugates which were modified at side
2. Experimental
2.1. Chemistry
The sterols and NaBH4 were purchased from the Merck Co.
Compounds 4 and 10 were prepared according to the procedures
in the literature [18]. All chemicals and solvents were analytical
grade and solvents were purified by general methods before being
used. Melting points were determined on an X4 apparatus and
were uncorrected. Infrared spectra were measured with a Nico-
let FT-360 Spectrophotometer. The 1H and C NMR spectra were
∗
Corresponding author. Tel.: +86 771 3908065; fax: +86 771 3908308.
Corresponding author. Tel.: +86 20 84110918; fax: +86 20 84112245.
E-mail addresses: cuijg1954@126.com (J. Cui),
∗
∗
13
yihxhc@mail.sysu.edu.cn (H. Song).
0
039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.03.009

Y. Huang et al. / Steroids 76 (2011) 690–694
691
recorded in CDCl on a Bruker AV-600 spectrometer at working fre-
2.1.3. The synthesis of methyl
3
quencies 600 and 150 MHz and a Bruker AV-300 spectrometer at
4,12-dioxy-3-aza-A-homo-7-deoxycholicate (11)
working frequencies 300 and 75 MHz, respectively. Chemical shifts
are expressed in ppm (ı) values and coupling constants (J) in Hz.
LRESIMS were recorded on a Thermo-DSQ instrument. The cell pro-
liferation assay was undertaken by a MTT method using 96-well
plates on Biocell ELIASA analysis spectrometer.
Freshly distilled thionyl chloride (1.4 mL) in 4 mL dry THF
was added dropwise to a solution of methyl (Z)-3-hydroxyimino-
12-oxo-deoxycholicate (10, 300 mg, 0.72 mmol) in 12 mL dry
◦
◦
THF cooled to 0 C, while maintaining the temperature at 0 C.
◦
After this addition, the reaction mixture was stirred at 0–10 C
for 90 min under a argon atmosphere. The mixture was then
poured into ice–water, neutralized with a solution of NH , and
extracted with DCM. The organic layer was washed with water
3
2
4
.1.1. The synthesis of methyl
,7,12-trioxo-3-aza-A-homo-cholicate (5)
To a solution of oxime 4 (300 mg, 0.74 mmol) in dry THF (12 mL)
and saturated brine, dried over anhydrous Na SO . Evapora-
2
4
tion of the solvent under reduced pressure gave a solid residue,
which was chromatographed over silica gel using petroleum
the solution of thionyl chloride (1.4 mL) in 4 mL dry THF was added
under argon. The solution was stirred under anhydrous conditions
for 30 min at 0 C. Then the reaction was terminated and water was
added to the solution. The solution was neutralized with ammo-
nia and the product was extracted with CH Cl . The combined
extract was washed with water and saturated brine, dried over
anhydrous Na SO , and evaporated under reduced pressure to give
a crude product which was chromatographed on silica gel (elution:
Vmethanol:Vdichloromethane = 1:50) to give 180 mg of 5 as a white solid.
Yield: 60.0%, ꢀmp 221–223 C. IR(KBr) ␯/cm : 3382, 2949, 2872,
◦
◦
ether (60–90 C)/EtOAc (1:3) as the eluent to give 220 mg
◦
−1
:
(
73%) of 11 as a white solid, m.p. 213–215 C; IR(KBr) ␯/cm
3
1
1
322, 2937, 2864, 1744, 1708, 1671, 1446, 1380, 1335, 1266,
2
2
192; 1H NMR(CDCl , 600 MHz) ı: 0.875(3H, d, J = 6.6, 21-CH ),
3
3
.058(3H, s, 19-CH ), 1.105(3H, s, 18-CH ), 2.26-2.22(1H, m, C23-
3
3
2
4
H), 2.294(1H, dd, J = 9.6, 6.6, C11-␣H), 2.416(1H, ddd, J = 15.6,
.6, 5.4, C23-H), 2.551(1H, dd, J = 15.0, 9.6, C11-␤H), 2.583(1H,
9
◦
−1
dd, J = 12.0, 5.4, C4a-␣H), 2.981(1H, dd, J = 15.0, 12.0, C4a-␤H),
3.074(1H, ddd, J = 15.0, 7.8, 7.2, C2-␤H), 3.256(1H, ddd, J = 15.0,
732, 1703, 1654, 1142, 1380, 1331; 1H NMR(CDCl , 600 MHz)
1
3
9
.6, 5.4, C2-␣H), 3.686(3H, s, –OCH ), 5.878(1H, brs, –NH);
3
ı: 0.861(3H, d, J = 6.6, 21-CH ), 1.069(3H, s, 19-CH ), 1.389(3H, s,
1
2
C11-␣H), 2.415(1H, ddd, J = 15.6, 9.6, 5.4, C23-H), 2.614(1H, dd,
J = 15.6, 12.6, C4a-␣H), 2.788(1H, t, J = 12.6, C -H), 2.838(1H, t,
J = 11.4, C5-H), 2.990(1H, dd, J = 12.6, 5.4, C4a-␤H), 3.059–3.107(1H,
m, C -␤H), 3.237(1H, ddd, J = 15.6, 10.2, 5.4, C -␣H), 3.686(3H, s,
3
3
1
3
C NMR(CDCl , 75 MHz,) ı: 214.2(12-C), 177.2(4-C), 174.6(24-
3
8-CH ), 2.088(1H, dd, J = 12.6, 5.4, C -␣H), 2.159(1H, dd, J = 12.6,
3
9
C), 58.3(9-C), 58.2(17-C), 57.5(14-C), 51.5(O–CH ), 46.5(13-C),
3
.4, C -␤H), 2.27-2.22(1H, m, C23-H), 2.293(1H, dd, J = 9.0, 7.2,
6
4
3
2
0.1(5-C), 39.3(1-C), 38.6(4a-C), 38.5(2-C), 37.9(11-C), 36.7(8-C),
5.7(10-C), 35.6(20-C), 31.3(23-C), 30.5(22-C), 29.8(7-C), 27.4(6-C),
8
5.7(16-C), 24.2(15-C), 22.6(21-C), 18.6(19-C), 11.7(18-C); LRES-
+
IMS(m/z): 418(M +1).
2
2
1
3
–
OCH ), 5.979(1H, brs, –NH); C NMR(CDCl , 75 MHz) ı: 212.1(12-
3
3
2
1
.1.4. The synthesis of menthyl
2-hydroxy-4-oxy-3-aza-A-homo-deoxycholicate (12)
C), 208.5(7-C), 175.6(4-C), 174.5(24-C), 56.8(8-C), 51.59(17-C),
1.51(O–CH ), 48.9(14-C), 47.5(9-C), 46.7(13-C), 45.6(6-C), 43.2(1-
5
3
To the stirred solution of 11 (200 mg, 0.48 mmol) in CH OH
3
C), 39.4(5-C), 38.64(4a-C), 38.60(11-C), 38.4(2-C), 36.3(10-C),
3
C), 18.6(18-C), 11.8(19-C); LRESIMS(m/z): 432(M +1).
(50 mL) was added NaBH4 (63 mg, 1.7 mmol) in 10 min at room
5.5(20-C), 31.3(23-C), 30.4(22-C), 27.6(15-C), 25.0(16-C), 22.9(21-
+
temperature. After 20 min, the reaction was stopped. The solution
was neutralized with 1 M HCl. After evaporation of the major-
ity of MeOH under reduced pressure, the residue was extracted
with ethyl acetate. The organic layer was washed with cold
water and saturated brines. After drying over anhydrous sodium
sulfate, the solvent was removed under reduced pressure to
obtain a crude product, which was chromatographed on sil-
ica gel (elution: Vmethanol:Vdichloromethane = 1:40) to give 104 mg
2
.1.2. The synthesis of methyl 4,12-dioxo-7-hydroxyimino-3-
aza-A-homo-cholicate(6)
CH COONa·3H O (31.5 mg, 0.23 mmol) and NH OH.HCl
3
2
2
(
16.1 mg, 0.23 mmol) were added to the solution of 5 (100 mg,
0
.23 mmol) in 30 mL 95% ethanol. After the solution was heated
◦
to 60 C, the mixture was stirred at the temperature for 40 min.
Then the reaction was terminated and the majority of solvent was
evaporated under reduced pressure. Distilled water was added into
the reaction mixture, and the product was extracted with ethyl
acetate. The combined extracts were washed with saturated brine,
dried, and evaporated under reduced pressure. The residue was
subjected to chromatography using methanol/dichloromethane
◦
−1
of 12 (52%) as a white solid. m.p 105–107 C; IR(KBr) ␯/cm :
3
1
432, 3215, 2945, 2868, 2353, 2320, 1732, 1663, 1437, 1360,
245, 1168, 1012, 788; 1H NMR(CDCl , 600 MHz) ı: 0.761(3H,
3
s, 18-CH ), 1.028(3H, d, J = 6.6, 21-CH ), 1.029(3H, s, 19-CH ),
3
3
3
2
7
.213(1H, dd, J = 15.6, 9.0, C17-H), 2.289(1H, ddd, J = 15.6, 9.0,
.2, C23-H), 2.407(1H, ddd, J = 15.6, 9.6, 5.4, C23-H), 2.551(1H,
dd, J = 15.6, 12.0, C4a-␣H), 3.031(1H, dd, J = 15.0, 12.0, C4a-␤H),
(
1:40) as the eluent to give 83 mg of 6 (80%) as a white solid,
3
.09-3.04(1H, m, C -␤H), 3.245(1H, ddd, J = 15.0, 9.6, 4.8, C -
2
2
◦
−1
ꢀ
mp 198–200 C. IR(KBr) ␯/cm : 3395, 2925, 2872, 1736, 1707,
␣
H), 3.487(1H, dd, J = 15.6, 4.8, C12-␣H), 3.684(3H, s, -OCH ),
3
1
662, 1433, 1388, 1164, 976; 1H NMR(CDCl , 600 MHz) ı: 0.841
13
3
4.034(1H, brs, –OH), 5.972(1H, brs, –NH);
C NMR(CDCl3,
(
3H, d, J = 6.6, 21-CH ), 1.068(3H, s, 19-CH ), 1.233(3H, s, 18-
3
3
75 MHz,) ı: 177.8(4-C), 174.7(24-C), 73.1(12-C), 51.5(O–CH ),
3
CH ), 2.10-2.16(2H, m, C11-␤H, C4a-␤H), 2.274(1H, dd, J = 9.6,
6
brd, J = 15.6, C -␤H), 2.395(1H, ddd, J = 15.6, 10.2, 5.4, C23-H),
2
3
47.9(17-C), 47.3(14-C), 46.5(13-C), 45.5(9-C), 40.2(1-C), 39.7(5-C),
38.7(4a-C), 36.9(8-C), 36.7(2-C), 36.0(8-C), 35.1(20-C), 33.8(10-C),
31.1(22-C), 30.9(23-C), 29.7(11-C), 29.0(7-C), 27.4(6-C), 25.7(16-
C), 23.5(15-C), 17.3(21-C), 12.8(19-C), 12.7(18-C); LRESIMS(m/z):
.6, C11-␣H), 2.260(1H, ddd, J = 15.6, 9.6, 6.6, C23-H), 2.350(1H,
6
.592(1H, t, J = 11.4, C -H), 2.659(1H, dd, J = 15.6, 12.0, C4a-␣H),
8
.693(1H, t, J = 12.6, C5-H), 3.056-3.006(1H, m, C -␤H), 3.283(1H,
2
+
2
420(M +1).
brd, J = 10.2, C -␣H), 3.206(1H, ddd, J = 15.0, 9.6, 4.8, C -␣H),
6
2
3
.669(3H, s, –OCH ), 6.078(1H, brs, –NH), 7.927(1H, brs, NOH);
3
2.1.5. The synthesis of methyl
4-oxy-12-hydroxyimino-3-aza-A-homo-deoxycholicate (13)
Compound 11 (80 mg, 0.19 mmol) was dissolved in 30 mL
13
C NMR(CDCl , 75 MHz) ı: 213.9(12-C), 177.0(4-C), 174.7(24-
3
C), 157.2(7-C), 57.1(17-C), 52.5(O–CH ), 51.5(13-C), 48.5(14-C),
3
4
3
5.8(9-C), 41.7(1-C), 41.6(8-C), 39.5(4a-C), 39.0(10-C), 38.4(11-C),
7.4(5-C), 36.5(2-C), 35.6(20-C), 31.3(23-C), 30.4(22-C), 29.3(6-
◦
of 95% CH CH OH. After the mixture was heated to 70 C,
3
2
CH COONa·3H O (77 mg, 0.57 mmol) and NH OH·HCl (40 mg,
3
2
2
C), 27.6(15-C), 25.6(16-C), 22.9(21-C), 18.6(19-C), 11.7(18-C);
LRESIMS(m/z): 447(M +1).
0
.57 mmol) were added into the solution in 10 min. The mix-
+
◦
ture was stirred for 3 h at 70 C. Then the reaction was

6
92
Y. Huang et al. / Steroids 76 (2011) 690–694
OH
COOH
OH
COOMe
O
COOMe
a
b
HO
OH
HO
O
OH
O
2
1
3
O
COOMe
COOMe
O
O
COOMe
c
d
e
HN
O
HN
N
O
O
N
O
OH
4
HO
5
6
Scheme 1. Reagents and conditions: (a) CH3OH/HCl; (b) PCC/CH2Cl2, 9 h; (c) NH2OH·HCl/95% CH3CH2OH, 60 ◦C; (d) THF/SOCl2; (e) NH2OH·HCl/95% CH3CH2OH.
terminated and the majority of solvent was evaporated under
reduced pressure. Distilled water was added into the reac-
tion mixture, and the product was extracted with ethyl
acetate. The combined extracts were washed with water and
saturated brine, dried with anhydrous sodium sulfate, and evap-
orated under reduced pressure. The residue was subjected to
chromatography (elution: Vmethanol:Vdichloromethane = 1:40) to pro-
2.2. Antiproliferative activity
2.2.1. Material and methods
Stock solutions of the compounds 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 streptomycin sulfate.
◦
duce 68 mg of 13 (82%) as a white solid, m.p. 212–213 C;
−
1
IR(KBr) ␯/cm : 3310, 3239, 2937, 2868, 1740, 1663, 1645,
1
1
0
433, 1380, 1168, 923, 735;
.945(3H, s, 19-CH ), 0.947(3H, d, J = 6.0, 21-CH ), 1.083(3H,
H NMR(CDCl3, 600 MHz) ı:
2.2.2. Cell culture
3
3
s, 18-CH ), 2.120(1H, dd, J = 18.6, 9.0, C4a-␤H), 2.28-2.24(1H,
m, C4a-␣H), 2.271(1H, ddd, J = 15.6, 9.6, 6.6, C23-H), 2.408(1H,
SMMC 7404, Hela and MGC 7901 cells were cultured in the
medium(RPMI-1640) supplemented with 10% fetal bovine serum
and 0.1 g/L penicillin G + 0.1 g/L streptomycin sulfate in a humidi-
3
ddd, J = 15.6, 10.2, 4.8, C23-H), 3.029(1H, dd, J = 15.0, 12.0, C11
H), 3.102(1H, ddd, J = 15.0, 7.8, 6.0, C2-␤H), 3.271(1H, dd,
J = 15.6, 9.0, C11-␣H), 3.325(1H, ddd, J = 15.0, 9.6, 4.8, C -␣H),
-
◦
␤
fied atmosphere of 5% CO2 at 37 C.
2
3
.685(3H, s, –OCH ), 6.066(1H, brs, –NH), 7.789(1H, brs, N–OH);
3
13
C NMR(CDCl , 75 MHz,) ı: 177.8(4-C), 174.8(24-C), 165.3(12-
2.2.3. Treatment of cancer cells
3
4
C), 59.2(14-C), 51.5(-OCH ), 49.8(17-C), 47.0(13-C), 45.2(9-C),
Cancer cells (1–2 × 10 cells/mL, 200 ␮L) were seeded into each
3
4
3
2
3.9(1-C), 40.4(5-C), 39.6(4a-C), 38.6(10-C), 37.7(8-C), 36.7(2-C),
5.9(20-C), 31.5(23-C), 30.7(22-C), 29.7(7-C), 28.0(6-C), 25.8(15-C),
4.1(16-C), 22.7(11-C), 20.4(21-C), 19.1(19-C), 12.1(18-C); LRES-
well of a 96-well microtiterplate. After incubation for 24 h, the com-
pounds with a series of concentrations (range 1–80 ␮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 triplicate.
+
IMS(m/z): 433(M +1).
COOH
COOMe
COOMe
OH
OH
O
a
b
c
HO
HO
O
7
8
9
OH
COOMe
COOMe
O
O
COOMe
HN
e
f
O
d
12
HN
N
OH
HO
N
COOMe
O
1
0
11
HN
O
1
3
Scheme 2. Reagents and conditions: (a) CH3OH/HCl; (b) PCC/CH2Cl2; (c) NH2OH·HCl/95% C2H5OH, 60 ◦C; (d) THF/SOCl2; (e) NaBH4/anhydrous CH3OH; (f) NH2OH·HCl/95%
◦
C2H5OH, 70 C.

Y. Huang et al. / Steroids 76 (2011) 690–694
693
Table 1
In vitro antitumor activities (IC50 in ␮mol/L) of the bile acids’ A-lactam derivatives.
Compounds
Structure
7901
>200
Hela
>200
7404
>200
O
COOMe
4
N
O
OH
O
COOMe
COOMe
5
6
>200
48.7 ± 4.1
>200
>200
HN
O
O
O
150 ± 10
14.3 ± 1.2
HN
O
N
HO
COOMe
O
1
0
>200
>200
>200
>200
N
OH
O
COOMe
11
12
13
96 ± 7.5
68.7 ± 5.3
44.8 ± 3.6
HN
O
OH
COOMe
52.0 ± 4.8
50.9 ± 6
HN
O
HO
N
COOMe
56.2 ± 5.1
13.7 ± 1.2
24.3 ± 1.5
20.6 ± 1.8
74.0 ± 4.6
11.6 ± 0.9
HN
O
NH
Cl
3
Pt
Cisplatin
NH
3
Cl
2
.2.4. Determination of cell viability
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
crystals formed. The absorbance (A) at 550 nm was measured
using a Biocell ELIASA analysis spectrometer. The IC50 value was
calculated as the concentration of drug yielding 50% cell survival.
bromide] dye reduction assay was used to determine the cell
viability. The cancer cells at approximately 80% confluence (i.e.
logarithmically growing cells) were selected for trypsinization, and
stained with trypan blue and their numbers were recorded. The
cells (200 ␮L) were seeded in 96-well microtiterplates after the cell
3. Result and discussion
3.1. Chemistry
4
concentrations were adjusted to 1–2 × 10 cells/mL RPMI-1640
◦
culture medium, and incubated at 37 C and 5% CO for 24 h. The
Schemes 1 and 2 outline the synthetic procedures of compounds
5–6 and 11–13 respectively. First, cholic acid (1) is transformed
into the corresponding methyl 4,7,12-trioxycholicate (3) via ester-
ification in methanol and oxidation with PCC in CH Cl . Next, the
2
cells were treated with the compounds dissolved in DMSO in
different concentrations (range 1.0–80 ␮g/mL) and reincubated
for 72 h. After the cells were washed with sterile phosphate buffer
saline (PBS), 190 ␮L of RPMI-1640 and 10 ␮L of the tetrazolium dye
2
2
compound 4 was synthesized by the oximation of 3 with ration of
(
MTT) (5 mg/mL) solution were added to each well, and the cells
NH OH·HCl = 1:1 [18]. Compound 5 was generated by Beckmann
2
◦
were incubated for an additional 4 h. The medium was discarded
and 200 ␮L of DMSO was added to dissolve the purple formazan
rearrangement of 4 with SOCl /THF at 0 C in 60% yield. The struc-
2
1
13
ture of 5 was confirmed by analysis of the H and C NMR chemical

6
94
Y. Huang et al. / Steroids 76 (2011) 690–694
shifts. In the NMR spectrum, the resonances showing of N–H at
.979 ppm(brs) and C-4 at 175.6 ppm demonstrated a formation
In conclusion, we have prepared a new series of A-homo-
lactam derivatives of cholic acid and 7-deoxycholic acid which
were proved to be potent antitumor activities against Hela tumor
cell line. Compounds 6, 12, 13 were found to be better potent
compounds, especially the compound 6, therefore, the further
structural modification of 6 and antitumor activity study of the
compounds in vivo would be in progress. Our findings could pro-
vide new evidence showing the relationship between the chemical
structure and biological activity and may be useful for the design
of novel chemotherapeutic drugs.
5
of lactam in 5. Resonances showing of C -␣H at 3.237 ppm (ddd,
2
J = 15.6, 10.2, 5.4) demonstrated a position of 3-NH in the compound
5
(If there is a 4-NH in 5, a dd-peak would been appeared on C4a-H).
The compound 6 was obtained by the treatment of 5 with hydrox-
ylamine hydrochloride (1:1.1) in ethanol in the presence of NaOAc
in 80% yield. The structure of 6 was confirmed by analysis of its IR
and NMR. The downfield chemical shift of 18-CH3 at 1.233 ppm for
6
confirmed which 12-carbonyl group was held.
Compound 11 was prepared similarly according to the pro-
cedure of 5 using 7-deoxycholic acid as a starting material. The
structure of 11 was confirmed by comparing IR and NMR spectra in
the similar method of compound 5 analyzed. The compound 12 was
obtained by a reduction of the carbonyl group at position 12 in 11
using NaBH as a reductive agent in CH OH in 52% yield. The struc-
Acknowledgment
The authors acknowledge the financial supports of the Nat-
ural Science Foundation of Guangxi Province of China (Nos:
2010GXNSFD013019 and 0832106).
4
3
ture of compound 12 was deduced from its analytical and spectral
1
data. In the H NMR spectrum, the resonances showing of C12-␣H
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