Synthesis and evaluation of some 17-acetamidoandrostane and N,N-dimethyl-7-deoxycholic amide derivatives as cytotoxic agents: Structure/activity studies

Article abstract of DOI:10.3390/molecules18077436

Using pregnenolone and 7-deoxycholic acid as starting materials, some 17-acetamidoandrostane and N,N-dimethyl-7-deoxycholic amide derivatives were synthesized. The cytotoxicity of the synthesized compounds was tested in vitro against two tumor cell lines: SGC 7901 (human gastric carcinoma) and Bel 7404 (human liver carcinoma). The result showed that the blockage of the interaction of the amide group with outside groups might cause a decrease of the cytotoxicity, and an O-benzyloximino group at the 3-position of N,N-dimethyl-7-deoxycholic amide could enhance the cytotoxic activity of the compound. The information obtained from the studies provides the structure-activity relationship for these compounds and may be useful for the design of novel chemotherapeutic drugs.

Full text of DOI:10.3390/molecules18077436

Molecules 2013, 18, 7436-7447; doi:10.3390/molecules18077436
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Evaluation of Some 17-Acetamidoandrostane and
N,N-Dimethyl-7-deoxycholic Amide Derivatives as Cytotoxic
Agents: Structure/Activity Studies
Yanmin Huang 1, Jianguo Cui 1,*, Linyi Jia 2, Chunfang Gan 1, Huacan Song 3, Chun Zeng 4 and
Aimin Zhou 4,*
1
College of Chemistry and Life Science, Guangxi Teachers Education University,
Nanning 530001, China
2
No. 37 Middel School in Nanning, Nanning 530001, China
3
School of Chemistry and Chemical Engineering, SUN YAT-SEN University,
Guangzhou 510275, China
4
Clinical Chemistry Program, Department of Chemistry, SI 424, Cleveland State University,
Cleveland, OH 44115, USA
* Authors to whom correspondence should be addressed; E-Mails: cuijg1954@126.com (J.C.);
a.zhou@csuohio.edu (A.Z.); Tel.: +86-771-553-0019 (J.C.); +1-216-687-2416 (A.Z.).
Received: 9 May 2013; in revised form: 13 June 2013 / Accepted: 18 June 2013 /
Published: 26 June 2013
Abstract: Using pregnenolone and 7-deoxycholic acid as starting materials, some
17-acetamidoandrostane and N,N-dimethyl-7-deoxycholic amide derivatives were
synthesized. The cytotoxicity of the synthesized compounds was tested in vitro against two
tumor cell lines: SGC 7901 (human gastric carcinoma) and Bel 7404 (human liver
carcinoma). The result showed that the blockage of the interaction of the amide group with
outside groups might cause a decrease of the cytotoxicity, and an O-benzyloximino group at
the 3-position of N,N-dimethyl-7-deoxycholic amide could enhance the cytotoxic activity of
the compound. The information obtained from the studies provides the structure-activity
relationship for these compounds and may be useful for the design of novel
chemotherapeutic drugs.
Keywords: pregnenolone; 17-acetamidoandrostane; N,N-dimethyl-7-deoxycholic amide;
antiproliferative activity

Molecules 2013, 18
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1. Introduction
Steroidal compounds display a variety of biological functions and play a very important role in
life [1–3]. Azahomosteroids are also a class of steroid compounds which were synthesized and
modified in order to increase the biological activity of steroids. These compounds have been tested
successfully as anti-cancer drugs against several types of cancer cells [4–8]. Some researchers
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 [9,10].
Several new steroidal lactams with the introduction of NHCO groups on the A-ring, C-ring or
D-ring of the steroidal nucleus were synthesized and assayed in our group [11–14]. We found that
these compounds displayed distinct cytotoxicity against different cancer cells, for instance, the
steroidal lactam compounds I and II (Figure 1) displayed distinct cytotoxicity against some cancer
cells through effective induction of cancer cell apoptosis by activation of the intrinsic pathway [15].
Figure 1. The structure of compounds I–IV.
O
O
HN
HN
NOH
OH
I
II
O
OH
CH3
CH3
CH3
N
CH3
HO
HO
OH
Ⅲ
IV
A survey of the literature suggested steroidal compounds with a NHCO group in the side chain
exhibit some valuable biological activities, such as cytotoxicity and antibacterial properties. For
example, finasteride, dutasteride and PNU-157706 [16,17], used as clinical benign prostatic
hyperplasia drugs, have a NHCO group in their branch chain. In order to further evaluate the
antiproliferative activity of new steroidal derivatives and study their structure-activity relationships,
some 17-acetamidoandrostane (Scheme 1) and N,N-dimethyl-7-deoxycholic amide derivatives
(Scheme 2) with a NHCO group in their side chain were designed and synthesized. Their
antiproliferative activities were tested in vitro against two tumor cell lines: SGC 7901 (human gastric
carcinoma) and Bel 7404 (human liver carcinoma).
Here, the N,N-dimethyl-7-deoxycholic amide (III) has a similar atom rank of the side chain of as
24-methylenecholest-4-en-3β,6β-diol (IV), which is a polyhydroxylsterol obtained from the soft coral
Alcyonium patagonicum that showed potent cytotoxicity to murine leukemia cell with an IC50 value of
1 µg/mL [18], except for different atoms substituted in the 25- and 28-positions of the side chain.

Molecules 2013, 18
Scheme 1. Synthetic procedures of 17-acetamido-androst-5-en derivatives.
7438
HON
O
O
18
21
NH
20
12
19 11
10
17
13
O
16
iii
1
i
ii
14
2
6
9
15
5
8
3
4
HO
7
AcO
AcO
AcO
1
3
4
2
NH
NH
NH
O
O
O
iv
v
vi
HO
HO
O
O
OH
7
6
5
vii
NH
O
HO
O
8
Reagents and conditions: (i) Ac2O/Pyridine/r.t., 24 h; (ii) NH2OH.HCl/NaOAc.3H2O/95%
CH3CH2OH, 60 °C, 2h; (iii) SOCl2/THF, r.t., 2 h; (iv) 13% K2CO3/CH3OH/reflux, 4 h; (v) Jones’
reagent/acetone/r.t., 2 h; (vi) NaBH4/CH3OH, r.t., 30 min; (vii) NiCl2·6H2O/NaBH4.
Scheme 2. Synthetic procedures of N,N-dimethyl-7-deoxycholic amide derivatives.
O
COOH
COOH
OH
O
CH3
CH3
O
N
a
b
HO
O
O
9
10
11
O
CH3
O
N
CH3
d
O
AcO
H2N
CH3
CH3
O
N
13
c
NH
O
N
HO
S
CH3
N
12
CH3
e
HO
14
Reagents and conditions: (a) Jones’ reagent, (CH3)2CO, 0 °C; (b) Toluene, (CO)2Cl2, Py/(CH3)2NH
(33%); (c) NaBH4/CH3OH; (d) Ac2O/Pyridine/r.t., 24 h; (e) NH2NHCSNH2/EtOH, 80 °C.

Molecules 2013, 18
7439
2. Results and Discussion
2.1. Chemistry
Scheme 1 outlines the synthetic procedures for compounds 4–8. First, the 3β-hydroxy group of
pregnenolone (1) was protected by acetylation, then oximation of 3β-acetoxypregnenolone (2) was
performed using NH2OH·HCl and NaOAc·3H2O to give compound 3. Compound 4 was generated by
Beckmann rearrangement of 3 with SOCl2/THF. Afterwards, compound 5 was obtained via the
deacetylation of 4 with alcoholic K2CO3. Compound 6 was prepared by the oxidation of 5 with Jones’
reagent and multiple extraction because of better water solubility of compound 6. Lastly, compound 6
was converted to compounds 7 and 8 by selective reduction using NaBH4 under different conditions.
Synthetic procedures to obtain N,N-dimethyl-7-deoxycholic amide derivatives 11–14 are outlined in
Scheme 2. First, 3,12-dioxo-7-deoxycholic acid (10) was obtained by oxidation of 7-deoxycholic acid (9)
with Jones’ reagent. Then, the reaction of 10 with (CO)2Cl2 and (CH3)2NH in toluene gave
N,N-dimethyl-3,12-dioxo-7-deoxycholic amide (11). The structure of compound 11 had been confirmed
by its IR, NMR and MS spectra. In the IR spectrum, the disappearance of the broad absorption peak at
3,200–2,500 cm−1 showed that the COOH functional group had been converted to another group. The
1
chemical shifts of 2.923 and 2.985 ppm (single peak) in the H-NMR confirmed the existence of the
CON(CH3)2 moiety. Next, the functional groups on the 3-position were modified in order to compare the
relative antiproliferative activities of different groups. Compound 11 was converted to
N,N-dimethyl-3-hydroxy-12-oxo-7-deoxycholic amide (12) by selective reduction using NaBH4.
Finally, compound 13 was produced by the esterification of the 3-hydroxy group of 12, and compound
14 was obtained by the reaction of 12 with thiosemicarbazide.
To further determine the effect of the 3-substituent groups of N,N-dimethyl-7-deoxycholic
amide derivatives on the cytotoxic activity, compounds 15–18 with different 3-substitutent groups
were prepared. Similarly, the reaction of compound 11 with hydroxylamine hydrochloride,
thiosemicarbazide, CH3ONH2·HCl or PhCH2ONH2·HCl afforded the corresponding products 15, 16, 17
or 18, respectively (Scheme 3).
Scheme 3. Synthetic procedures of N,N-dimethyl-7-deoxycholic amide derivatives.
O
O
O
O
CH3
CH3
CH3
N
N
CH3
a, b, c, d
R
O
11
15-18
15: R = NOH, 16: R = NNHCSNH2
17: R = NOCH3, 18: R = NOCH2C6H5
Reagents
and
Conditions:
(a)
NH2OH·HCl/95%EtOH,
CH3COONa,
70
°C;
(b) NH2NHCSNH2/EtOH, 80 °C; (c) CH3ONH2·HCl/95%EtOH, CH3COONa, 70 °C;
(d) PhCH2ONH2·HCl/95% EtOH, CH3COONa, 70 °C.

Molecules 2013, 18
7440
2.2. In Vitro Evaluation of the Antiproliferative Activity
2.2.1. Cell Culture and Assay for Cell Viability
SGC 7901 (human gastric carcinoma) and Bel 7404 (human liver carcinoma) cell lines were obtained
from Guangxi Traditional Chinese Medical University. Cells were grown in RPMI-1640 supplemented
with 10% cosmic calf serum (Hyclone, Beijing, China) and antibiotics in a humidified atmosphere of 5%
CO2 at 37 °C. The viability of these cells was determined using the colorimetric CellTiter 96 aqueous
Cell Proliferation Assay (MTT) according to the instructions provided by the manufacturer (Promega,
Madison, WI, USA). Briefly, cells (1~3 × 104 cells per well) were seeded in 96 wells plates. An equal
amount of DMSO was added to the cells used as negative controls. All were treated in triplicate. One day
after seeding, the cells were treated with or without different concentration of each compound and
reincubated for 72 h. After the cells were washed with sterile phosphate buffer saline (PBS), 190 µL of
RPMI-1640 and 10 µL of the tetrazolium dye (MTT) (5 mg/mL) solution were added to each well, and
the cells were incubated for an additional 4 h. The medium was discarded; 200 µL of DMSO was added
to dissolve the purple formazan crystals formed. The absorbance (A) at 492 nm was measured using a
MULTISCAN MK3 analysis spectrometer (Thermo Scientific, Shanghai, China). The results were
summarized as IC50 values in µ mol/L in Table 1.
Table 1. In vitro antiproliferative activities (IC50 in μmol/L) of the compounds 3–18.
Componuds
SGC 7901
Bel 7404
3
4
5
6
7
8
11
>100
>100
Cisplatin
6.7
12
23.4
51.2
13
65.8
>100
14
63.4
>100
15
49.0
80.1
16
58.4
>100
17
61.5
>100
18
>100
>100
Compounds
SGC 7901
Bel 7404
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
15.0
14.4
23.2
2.2.2. Structure-Activity Relationships
As shown in Table 1, compound 3 with a 20-hydroximino group displayed distinct cytotoxicity
against the two studied cancer cell lines. However, after the 20-hydroximino group of 3 was converted
into an amide group (compounds 4–8), the antiproliferative activity of compounds was remarkably
decreased. The result could be ascribed to the blockage of the interaction of the amide group with outside
groups, which may be a main reason causing a decrease of the cytotoxicity because of the steric
hindrance of the steroidal nucleus [14]. Therefore, changing the hydroximino group to the amide group
at position-20 did not increase the cytotoxic activities of these 17-acetamidoandrost-5-ene derivatives.
Comparing the result with the earlier studies [12,13], we can see that the cytotoxicity of the steroidal
amide compounds is not only dependent on the structure of the side chain, but also the position of the
CONH functional group. When the compounds had a cholesteric side chain and A-ring or B-ring was
modified into an A-lactam [13] or a B-lactam [19], it would display distinctive cytotoxicity against the
cancer cells.
To investigate the effect of the 24-amide group on the biological function of the compounds, we
synthesized some N,N-dimethyl-7-deoxycholic amide derivatives with various 3-substitutent groups

Molecules 2013, 18
7441
(compounds 11–18). Here, compounds 11–17 were almost inactive against the cancer cells. Once again,
the reason may be the steric hindrance of N,N-dimethyl group inhibiting the affinity of the amide group
with outside groups. Nevertheless, a 3-O-benzyloxime group played an important role in the
cytotoxicity of compound 18 against these cancer cells. Compound 18 showed almost the same
cytotoxicity as cisplatin to SGC 7901 and Bel 7404 cells (18: 15.0 and 14.4; cisplatin: 6.7 and 23.2 µmol/L),
indicating that the presence of the 3-O-benzyloxime group on the compound 18 confers a positive
effect on the cytotoxicity against these cancer cells, which provides clues the seeking more powerful
anti-cancer drug candidates based on O-benzyloxime groups via structural optimization.
3. Experimental
Sterols and NaBH4 were purchased from Merck Co. (Shanghai, China) 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
Thermo Scientific IS-10 Spectrophotometer. The 1H and 13C-NMR spectra were recorded in CDCl3 on a
Bruker AV-300 spectrometer at working frequencies 300 and 75 MHz. Chemical shifts are expressed in
parts per million (δ) values and coupling constants (J) in Hertz. LREIMS were recorded on a
Thermo-DSQ instrument, while HREIMS were measured on a Agilent 6210 TOFMS instrument.
3-Acetoxy-17-acetamidoandrost-5-ene (4) and 3,12-dioxydeoxycholic acid (10) were prepared
according to the literature [6] and [20], respectively.
Synthesis of 3-hydroxy-17-acetamidoandrost-5-en (5). K2CO3 solution (13%, 20 mL) was added to a
solution of compound 4 (438 mg, 3.32 mmol) in CH3OH (200 mL). The reaction mixture was heated
under reflux for 1 h. After completion of the reaction as indicated by TLC, the solvent was removed
under vacuum. CH2Cl2 (200 mL) was added to dissolve the solid and the resulting solution was
washed with cold water and saturated brine. 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 methanol/dichloromethane (30:1) as eluent to give 709 mg (76%) of 5 as a white
solid. θ
196–197 °C; IR (KBr) ν/cm−1: 3354, 2962, 2937, 1646, 1556, 1433, 1372, 1315;
mp
1H-NMR (CDCl3, 300 MHz) δ: 0.721 (3H, s, 18-CH3), 1.028 (3H, s, 19-CH3), 2.002 (3H, s, N-COCH3),
2.36–2.24 (2H, m, C4-H), 3.58–3.51 (1H, m, C3-H), 3.908 (1H, q, J = 9.0, C17-H), 5.302 (1H, brd, J = 9.9,
13
N-H), 5.367(1H, dd, J = 3.6, 1.8, C6-H); C-NMR (CDCl3, 75 MHz) δ: 169.9 (-NHCOCH3), 140.9
(5-C), 121.3 (6-C), 71.7 (3-C), 58.9 (17-C), 52.8 (9-C), 50.1 (13-C), 42.7 (4-C), 42.2 (14-C), 37.2
(10-C), 36.8(1-C), 36.7 (12-C), 32.0 (8-C), 31.6 (7-C), 31.5 (16-C), 28.7 (2-C), 27.7 (15-C), 23.6
(21-C), 20.6 (11-C), 19.4 (19-C), 11.9 (18-C); ESI-MS m/z: 332 (M+1)+.
Synthesis of 17-acetamidoandrost-4-en-3,6-dione (6). Jones’ reagent (0.7 mL, 0.267 mol/L) was added
dropwise to the solution of 5 (709 mg) in acetone (30 mL) in 20 min. The mixture was stirred at room
temperature for 1 h and then neutralized with 10% K2CO3 solution. The suspension was poured over a
silica gel column and eluted with ethyl acetate. The solvent was removed under reduced pressure. The
residue was purified by chromatography on silica gel using methanol/dichloromethane (20:1) as
elution to give 590 mg (80%) of 6 as white solid. θ mp 198–199 °C; IR (KBr) ν/cm−1: 3321, 2941, 2864,
1
1691, 1654, 1556, 1442, 1384; H-NMR (CDCl3, 300 MHz) δ: 0.728 (3H, s, 18-CH3), 1.150 (3H, s,

Molecules 2013, 18
7442
19-CH3), 1.976 (3H, s, N-COCH3), 2.17–2.10 (2H, m, C7-βH and C2-αH), 2.495 (1H, dd, J = 13.8, 5.1,
C7-αH), 2.662 (1H, dd, J = 15.9, 3.9, C2-βH), 3.937 (1H, q, J = 9.0, C17-H), 5.582 (1H, d, J = 9.0,
N-H), 6.149 (1H, s, C4-H); 13C-NMR (CDCl3, 300 MHz) δ: 201.8 (6-C), 199.3 (3-C), 170.1
(-NHCOCH3), 160.7 (5-C), 125.6 (4-C), 58.5 (14-C), 52.7 (17-C), 50.9 (9-C), 46.3 (13-C), 43.0 (10-C),
39.7 (1-C), 36.3 (12-C), 35.5 (7-C), 34.3 (8-C), 33.9 (16-C), 28.2 (2-C), 23.4 (15-C), 23.2 (21-C), 20.5
(11-C), 17.6 (19-C), 11.9 (18-C); ESI-MS m/z: 344 (M+1)+.
Synthesis of 3,6-dihydroxy-17-acetamidoandrost-4-en (7). NaBH4 (42 mg, 1.1 mmol) was added at
room temperature to a solution of 6 (124 mg, 0.36 mmol) in CH3OH (50 mL) in one portion. After 30
min, the reaction was stopped. The solution was neutralized with 1 M HCl. After evaporation of the
majority of MeOH under reduced pressure, the residue was extracted with ethyl acetate (15 mL × 3).
The organic layer was washed with cold water and saturated brines. After drying over anhydrous
sodium sulfate, the solvent was removed under reduced pressure and a crude product was obtained.
After recrystallization from methanol, 95 mg (78%) of compound 7 was obtained as a white solid. θ mp
1
198–200 °C; IR (KBr) ν/cm−1: 3331, 2941, 2866, 1654, 1442, 1384, 1217, 1131; H-NMR (CDCl3,
300 MHz) δ: 0.703 (3H, s, 18-CH3), 1.008 (3H, s, 19-CH3), 1.988 (3H, s, N-COCH3), 3.57–3.47 (1H,
m, C3-H), 3.93–3.80 (1H, m, C6-H), 4.13 (1H, dd, J = 7.0, 1.5, C17-H), 5.36 (1H, d, J = 2.1, C4-H),
13
5.40 (1H, br s, N-H); C-NMR (CDCl3, 75 MHz) δ: 170.0 (-NHCOCH3), 140.9 (5-C), 121.3 (4-C),
71.6 (6-C), 68.8 (3-C), 58.9 (17-C), 52.8 (14-C), 50.1 (9-C), 42.7 (13-C), 42.2 (7-C), 37.2 (10-C), 36.8
(1-C), 36.5 (12-C), 32.1 (8-C), 31.6 (2-C), 31.5 (16-C), 28.7 (15-C), 23.6 (21-C), 20.6 (11-C), 19.4
(19-C), 12.0 (18-C); ESI-MS m/z: 348 (M+1)+.
Synthesis of 3-hydroxy-17-acetamidoandrost-4-en-6-one (8). NaBH4 (42 mg, 1.1 mmol) was added to
a solution of 6 (124 mg, 0.36 mmol) and NiCl2·6H2O (87 mg, 0.37 mmol) in CH3OH (15 mL) in the
interval of 5 min at room temperature. After 5 min, the reaction was stopped. The solution was
neutralized with 1 M HCl. After evaporation of the majority of MeOH under reduced pressure, water
of 15 mL was added. Then the residue was extracted with ethyl acetate (15 × 3 mL). The resulting
solution was washed with cold water and saturated brine. After drying over anhydrous sodium sulfate,
the solvent was removed under reduced pressure, and the resulting crude product was purified by flash
chromatography on silica gel using methanol/dichloromethane (20:1) as the eluent. Compound 8 was
obtained as white solid (75 mg, 60%), θ mp 187–188 °C; IR(KBr) ν/cm–1: 3330, 2942, 2865, 1675,
1442, 1385, 1218; 1H-NMR (CDCl3, 300 MHz) δ: 0.747 (3H, s, 18-CH3), 1.196 (3H, s, 19-CH3), 2.004
(3H, s, N-COCH3), 2.22–2.14 (2H, m, C16-H), 2.418 (1H, dd, J = 12.9, 4.5, C7-αH), 3.929 (1H, q,
J = 9.0, C17-H), 4.338 (1H, ddd, J = 11.4, 4.2, 1.2, C3-βH), 5.330 (1H, br d, J = 7.5, N-H), 6.196 (1H, d,
J = 1.2, C4-H); 13C-NMR (CDCl3, 75 MHz) δ: 199.3 (6-C), 170.0 (-NHCO), 169.9 (5-C), 119.9 (4-C),
68.5 (3-C), 58.6 (17-C), 53.7 (9-C), 51.7 (14-C), 42.9 (13-C), 40.9 (7-C), 39.0 (10-C), 36.5 (8-C), 36.3
(1-C), 34.3 (12-C), 33.7 (2-C), 28.6 (16-C), 23.6 (15-C), 23.5 (21-C), 20.6 (11-C), 18.3 (19-C), 12.1
(18-C); ESI-MS m/z: 346 (M+1)+.
Synthesis of N,N-dimethyl-3,12-dioxo-7-deoxycholic amide (11). Oxalyl chloride (200 µL, 2.23 mmol)
was added to a solution of 10 (535 mg, 1.38 mmol) and pyridine (150 µL, 1.80 mmol) in toluene (25
mL) at ice bath temperature. The mixture was stirred for 2 h. After completion of the reaction as

Molecules 2013, 18
7443
indicated by TLC, a solution of dimethyl amine (540 µL) was added dropwise. The solution was
stirred for 10 min at 0 °C and 10 mL ice water was added to the solution. The solution was extracted
with dichloromethane (30 × 3 mL). The organic solution was washed with cold water and saturated
brine. 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 petroleum/ethyl
acetate (1:2) as the eluent. Compound 11 was obtained as a white solid (480 mg, 71%), θ
mp
271–272 °C; IR(KBr) ν/cm–1: 2929, 2859, 1695, 1634; H-NMR (CDCl3, 300 MHz) δ: 0.797 (3H, d,
1
J = 5.7, 21-CH3), 1.000 (3H, s, 18-CH3), 1.050 (3H, s, 19-CH3), 2.13–2.04 (2H, m), 2.37–2.18 (3H, m),
2.58–2.48 (2H, m, C11-H), 2.872 (3H, s, NCH3), 2.955 (3H, s, NCH3); 13C-NMR (CDCl3, 75 MHz) δ:
214.2 (12-C), 212.1 (3-C), 173.4 (24-C), 58.5 (14-C), 57.5 (17-C), 46.5 (9-C), 44.2 (13-C), 43.6 (5-C),
42.1 (4-C), 38.3 (8-C), 37.3 (11-C), 36.8 (1-C), 36.7 (2-C), 35.6 (10-C), 35.5 (N-C), 35.4 (N-C), 35.3
(20-C), 30.6 (7-C), 30.2 (23-C), 27.4 (22-C), 26.5 (15-C), 25.4 (6-C), 24.3 (16-C), 22.1 (21-C), 18.7
(19-C), 11.7 (18-C); ESI-MS m/z: 416 (M+1)+.
Synthesis of N,N-dimethyl-3-hydroxy-12-oxo-7-deoxycholic amide (12). Compound 12 was prepared
according to the procedure of 7. Yield 30%, θ mp 279–280 °C; IR(KBr) ν/cm–1: 3465, 2925, 2859, 1707,
1
1621; H-NMR (CDCl3, 300 MHz) δ: 0.843 (3H, d, J = 6.3, 21-CH3), 0.986 (3H, s, 19-CH3), 0.995
(3H, s, 18-CH3), 2.26–2.17 (3H, m), 2.49-2.32 (2H, m, C11-H), 2.912 (3H, s, NCH3), 2.994 (3H, s,
NCH3), 3.63-3.53 (1H, m, C3-H); 13C-NMR (CDCl3, 75 MHz) δ: 215.2 (12-C), 173.7 (24-C), 71.2
(3-C), 58.7 (14-C), 57.5 (17-C), 46.4 (13-C), 44.1 (5-C), 41.5 (9-C), 38.1 (8-C), 37.3 (11-C), 36.3
(1-C), 35.6 (4-C), 35.4 (10-C), 35.3 (N-C), 35.3 (N-C), 35.2 (20-C), 30.6 (2-C), 30.3 (23-C), 30.2
(22-C), 27.5 (6-C), 27.1 (15-C), 26.1 (16-C), 24.3 (7-C), 22.8 (21-C), 18.8 (18-C), 11.7 (19-C);
ESI-MS m/z: 418 (M+1)+.
Synthesis of N,N-dimethyl-3-acetoxy-12-oxo-7-deoxycholic amide (13). Compound 13 was prepared by
the esterification of 12 using acetic anhydride in pyridine. Yield: 78%. θ mp 153–154 °C; IR(KBr)
1
ν/cm–1: 3440, 2953, 1730, 1700, 1656, 1465, 1447; H-NMR (CDCl3, 300 MHz) δ: 0.885 (3H, d,
J = 6.3, 21-CH3), 1.035 (6H, br s, 18-CH3 and 19-CH3), 2.025 (3H, s, CH3CO-), 2.951 (3H, s, NCH3),
3.025 (3H, s, NCH3), 4.659–4.763 (1H, m, C3-H); 13C-NMR (CDCl3, 75 MHz) δ: 214.9 (12-C), 173.6
(24-C), 170.6 (CH3CO-), 73.7 (3-C), 58.7 (14-C), 57.6 (17-C), 46.5 (13-C), 44.1 (5-C), 41.4 (9-C),
38.1 (8-C). 37.3 (11-C), 35.7 (1-C), 35.4 (4-C), 35.3 (N-C), 34.9 (N-C), 32.1 (20-C), 30.7 (2-C), 30.3
(23-C), 27.5 (10-C), 26.9 (22-C), 26.4 (6-C), 26.4 (15-C), 24.4 (16-C), 22.7 (7-C), 21.4 (21-C), 18.8
(18-C), 11.7 (19-C); ESI-MS m/z: 460 (M+1)+.
Synthesis of N,N-dimethyl-3-hydroxy-12-thiosemicarbazone-7-deoxycholic amide (14). The compound
12 (100 mg, 0.24 mmol) was dissolved in 15 mL of CH3CH2OH and the solution was adjusted to
pH ≈ 3–5 by adding a few drops of glacial acetic acid. After the solution was heated to 80 °C, and
thiosemicarbazide (32 mg, 0.36 mmol) was added. The mixture was stirred for 10 h at 80 °C until no
starting material (the progress of the reaction was monitored by TLC). Then the reaction was
terminated and the majority of solvent was evaporated under reduced pressure. Water (10 mL) was
added to the mixture which was then extracted with dichloromethane. The organic layer was washed
with water and saturated brine. After drying over anhydrous sodium sulfate, the solvent was removed

Molecules 2013, 18
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under reduced pressure, and the resulting crude product was purified by flash chromatography on silica
gel using dichloromethane/methanol (40:1) as the eluent to give 56 mg of 14 as a white solid (yield:
49%), θ mp 294–295 °C; IR (KBr) ν/cm–1: 3427, 2921, 1634, 1503, 1401; 1H-NMR (CDCl3, 300 MHz):
0.843 (3H, d, J = 6.3, 21-CH3), 0.986 (3H, s, 18-CH3), 0.995 (3H, s, 19-CH3), 2.912 (3H, s, N-CH3),
2.994 (3H, s, N-CH3), 3.577 (1H, m, C3-H), 6.576 (1H, br s, -NH2), 7.234 (1H, br s, -NH2), 9.284 (1H,
s, -NH-); 13C-NMR (CDCl3, 75 MHz): 178.8 (C=S), 173.6 (24-C), 162.6 (12-C), 71.1 (3-C), 58.7
(14-C), 57.5 (17-C), 46.4 (13-C), 44.1 (5-C), 41.6 (9-C), 38.1 (8-C), 37.4 (4-C), 36.3 (11-C), 35.7 (10-C),
35.6 (1-C), 35.4 (N-C), 35.3 (20-C), 35.1 (N-C), 30.7 (2-C), 30.3 (22-C), 30.2 (23-C), 27.5 (6-C), 27.1
(15-C), 26.1 (16-C), 24.4 (7-C), 22.8 (21-C), 18.8 (18-C), 11.7 (19-C); ESI-MS m/z: 491(M+1)+.
Synthesis of N,N-dimethyl-3-hydroximino-12-oxo-7-deoxycholic amide (15). Compound 11 (91 mg,
0.22 mmol) was dissolved in 95% CH3CH2OH (15 mL). After the mixture was heated to 70 °C,
CH3COONa·3H2O (30 mg, 0.22 mmol) and NH2OH·HCl (18 mg, 0.26 mmol) were added. The
mixture was stirred for 3 h at 70 °C. 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 dichloromethane. The combined extracts were washed with water and
saturated brine, dried with anhydrous sodium sulfate, and evaporated under reduced pressure. The
residue was purified by flash chromatography on silica gel using dichloromethane/methanol (40:1) as
the eluent. Compound 15 was obtained as a white solid (56 mg, 60%). θmp 232–233 °C; IR (KBr)
1
ν/cm–1: 3440, 1691, 1601, 1437, 1392; H-NMR (CDCl3, 300 MHz): 0.853 (3H, d, J = 6.3, 21-CH3),
1.025 (3H, s, 18-CH3), 1.035 (3H, s, 19-CH3), 2.534 (2H, m, C11-H), 2.923 (3H, s, N-CH3), 2.999 (3H,
13
s, N-CH3), 3.070 (1H, dd, J = 14.4, 3.9, C2-βH); C-NMR (CDCl3, 75 MHz): 214.9 (12-C), 173.7
(24-C), 160.2 (3-C), 58.6 (14-C), 57.6 (17-C), 46.4 (9-C), 44.2 (5-C), 44.0 (13-C), 43.3 (10-C), 41.9
(8-C), 38.3 (11-C), 37.4 (N-C), 36.9 (N-C), 36.1 (20-C), 35.6 (7-C), 35.5 (4-C), 30.6 (23-C), 30.3
(6-C), 27.4 (22-C), 26.6 (2-C), 26.5 (1-C), 25.6 (15-C), 24.8 (16-C), 24.3 (21-C), 22.6 (19-C), 18.8
(18-C); ESI-MS m/z: 431(M+1)+.
Synthesis of N,N-dimethyl-3-thiosemicarbazone-12-oxo-7-deoxycholic amide (16). Compound 16 was
prepared similarly to 14, but from the compound 11. Yield: 69%, θmp 282–283 °C. IR (KBr) ν/cm–1: 3432,
1
2929, 2868, 1703, 1638, 1499; H-NMR (CDCl3, 300 MHz): 0.856 (3H, d, J = 5.7, 21-CH3), 1.034
(3H, s, 18-CH3), 1.060 (3H, s, 19-CH3), 2.539 (2H, m, C11-H), 2.926 (3H, s, N-CH3), 3.002 (3H, s,
N-CH3), 6.487 (1H, br s, N-H), 7.213 (H, br s, N-H), 8.873 (1H, s, -NH-); 13C-NMR (CDCl3, 75
MHz,): 214.5 (12-C), 178.7 (S=C), 173.6 (24-C), 156.8 (3-C), 58.5 (14-C), 57.5 (17-C), 46.5 (9-C),
44.4 (5-C), 44.1 (13-C), 43.7 (10-C), 42.3 (8-C), 38.3 (11-C), 37.3 (N-C), 36.1 (N-C), 36.0 (20-C),
35.6 (4-C), 35.4 (7-C), 30.6 (23-C), 30.3 (6-C), 29.9 (2-C), 27.4 (22-C), 26.7 (1-C), 25.6 (15-C), 24.3
(16-C), 22.5 (21-C), 18.8 (19-C), 11.8 (18-C); ESI-MS m/z: 489 (M+1)+.
Compounds 17 and 18 were prepared similarly according to the procedure of 15, but CH3ONH2·HCl
and PhCH2ONH2·HCl were used as reagents instead of NH2OH·HCl.
N,N-dimethyl-3-O-methyloximino-12-oxo-7-deoxycholic amide (17). Yield: 70%, θmp 188–189 °C. IR
(KBr) ν/cm–1: 3436, 2855, 1703, 1654, 1446, 1041; 1H-NMR (CDCl3, 300 MHz): 0.875 (3H, d, J = 6.3,
21-CH3), 1.043 (3H, s, 18-CH3), 1.054 (3H, s, 19-CH3), 2.55–2.40 (2H, m, C11-H), 2.940 (3H, s,

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N-CH3), 3.014 (3H, s, N-CH3), 3.804 (3H, s, O-CH3); 13C-NMR (CDCl3, 75MHz): 214.7 (12-C), 173.5
(24-C), 160.0 (3-C), 61.0 (-OCH3), 58.6 (14-C), 57.6 (17-C), 46.5 (9-C), 44.2 (13-C), 44.0 (10-C),
43.5 (5-C), 42.0 (8-C), 38.3 (11-C), 37.3 (N-C), 36.1 (N-C), 36.0 (20-C), 35.6 (4-C), 35.4 (7-C), 30.6
(23-C), 30.2 (6-C), 27.4 (22-C), 26.7 (2-C), 26.6 (1-C), 25.6 (15-C), 24.3 (16-C), 22.6 (21-C), 18.8
(19-C), 11.7 (18-C); ESI-MS m/z: 445 (M+1)+.
N,N-dimethyl-3-O-benzyloximino-12-oxo-7-deoxycholic amide (18). Yield: 72%, θmp 148–149 °C. IR
(KBr) ν/cm–1: 2970, 2868, 1707, 1625, 1450, 1397; 1H-NMR (CDCl3, 300 MHz): 0.882 (3H, d, J = 6.3,
21-CH3), 1.048 (3H, s, 18-CH3), 1.056 (3H, s, 19-CH3), 2.35–2.45 (1H, m, C11-βH), 2.553 (1H, t,
J = 12.6, C11-αH), 2.947 (3H, s, N-CH3), 3.019 (3H, s, N-CH3), 5.052 (2H, s, O-CH2-Ph), 7.36–7.29
13
(5H, m, -C6H5); C-NMR (CDCl3, 75 MHz): 214.7 (12-C), 173.5 (24-C), 160.5 (3-C), [138.2, 128.3,
128.3, 127.9, 127.8, 127.6 (-C6H5)], 75.2 (O-C), 58.6 (14-C), 57.5 (17-C), 46.5 (9-C), 44.3 (13-C),
44.0 (5-C), 43.5 (10-C), 41.9 (8-C), 38.3 (11-C), 37.3 (N-C), 36.1 (N-C), 36.0 (20-C), 35.6 (2-C), 35.4
(7-C), 30.6 (23-C), 30.3 (6-C), 27.4 (1-C), 26.6 (22-C), 25.8 (4-C), 25.6 (15-C), 24.3 (16-C), 22.6
(21-C), 18.8 (19-C), 11.8 (18-C); ESI-MS m/z: 521 (M+1)+.
4. Conclusions
We have prepared some 17-acetamidoandrostane and N,N-dimethyl-7-deoxycholic amide derivatives
with different substitutent groups on different positions of the steroidal nucleus. The antiproliferative
activity of the synthesized compounds against the SGC 7901 and Bel 7404 cell lines was assayed. The
results showed that the blockage of the interaction of the amide group with outside groups might
cause a decrease of the cytotoxicity, and an O-benzyloxime group on the 3-position of
N,N-dimethyl-7-deoxycholic amide could enhance the cytotoxic activity of the compound. Our result
revealed some structure-activity relationships of the compounds and although some displayed
indistinctive cytotoxicity against these cancer cells, this informstion may be useful for the design of
novel chemotherapeutic drugs with better activity.
Acknowledgments
The authors acknowledge the financial support of the Natural Science Foundation of Guangxi
Province (No. 2010GXNSFA013019, No. 2010GXNSFD013052) and Natural Science Fund of
Education Department of Guangxi Province (No. 201202ZD059).
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compound 4, 5, 11, 12 are available from the authors.
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