Synthesis and anticancer activity of 4-azasteroidal-20-oxime derivatives

Article abstract of DOI:10.3184/174751915X14401666837860

A new series of 4-azasteroidal-20-oxime derivatives have been synthesised from progesterone. All the new compounds have been characterised by analysis and spectroscopic data and subsequently evaluated for their anticancer activity in vitro against T24 cell. The studies show that the methyl and ethyl oxime-ethers exhibited higher activity than the aryl substituted oxime-esters.

Full text of DOI:10.3184/174751915X14401666837860

JOURNAL OF CHEMICAL RESEARCH 2015
VOL. 39 SEPTEMBER, 527–530
RESEARCH PAPER 527
Synthesis and anticancer activity of 4-azasteroidal-20-oxime derivatives
Shao-Rui Chena*, Feng-Juan Shena, Guo-Liang Fenga and Rui-Xian Yuanb
aCollege of Science, Hebei University of Science and Technology, Shijiazhuang 050018, P.R. China
bAnalysis and Testing Research Center in Hebei Province, Hebei University of Science and Technology, Shijiazhuang 050018, P.R. China
A new series of 4-azasteroidal-20-oxime derivatives have been synthesised from progesterone. All the new compounds have been
characterised by analysis and spectroscopic data and subsequently evaluated for their anticancer activity in vitro against T24 cell. The
studies show that the methyl and ethyl oxime-ethers exhibited higher activity than the aryl substituted oxime-esters.
Keywords: progesterone, synthesis, configuration, anticancer activity
Cancer is a major cause of death in both developing and
developed countries according to the WHO.1 A number of
natural products and their semi synthetic analogues have been
discovered and evaluated for their role in the treatment and
cure of cancer and other fatal diseases.2,3 Steroids and their
derivatives elicit diverse biological effects though various
functional groups located around the periphery of their rigid
tetracyclic core.4,5 It has been reported that 4-azasteroidal
analogues may lead to compounds having anticancer activity or
5α-reductase inhibitor activity.6-8 In several studies, derivatives
of steroidal oximes have been tested for a variety of anticancer,
antimicrobial and antioxidant activities.9,10 The advantage
of employing hydrophobic steroid units with an oxime group
enhances their ability to interact with cell membranes and thus
paves the way for biological activity of such hybrid molecules.
There are few reports of the synthesis of oximes of 4-azasteroids
and their biological screening. We have synthesised novel
4-azasteroidal-20-oxime derivatives from the commercially
available progesterone and evaluated their anticancer activities
against the T24 cell.
The synthesis of 3-oxo-4-aza-5-pregnene-20-oxime derivatives
are shown in Scheme 1. Starting from progesterone (1), ring
A was oxidatively cleared by treatment withsodium periodate
and potassium permanganate to give 5,20-dioxo-A-nor-3,5-
secopregnane-3-oic acid (2) in a yield of 92%.11,12 Ozonolysis of
progesterone followed by oxidative work-up(H2O2–NaOH) also
produced compound 2, but the yield was 80% and the procedure
was complex.13 This compound was treated with ammonium
formate to afford 4-aza-5-pregnene-3,20-dione (3) in 91.5%
yield,14 and this was converted into its 20-oxime analogue 4 in
6a R1=R2=CH3; 6b R1=R2=Et; 6c R1=H, R2=Et; 7a R1=R2=CH3; 7b R1=R2=Et; 7c R1=H, R2=Et;
8a R=CH3; 8b R=Ph; 8c R=p-CH3-Ph; 8d R=p-OCH3-Ph; 8e R=p-Cl-Ph.
Scheme 1 Synthesis of 3-oxo-4-substituted-4-aza-5-pregnene-20-oxime derivatives.
* Correspondent. E-mail: sjz_wgq@126.com

528 JOURNAL OF CHEMICAL RESEARCH 2015
67% yield using hydroxylamine in the presence of pyridine.15
In the IR spectrum of 4, the characteristic oxime bands were
observed at 3402 cm-1(OH) and 1,654 cm-1 (C=N), whilst
the 20-C=O carbon signal (209.4 ppm) of compound 3 was
not observed in 13C NMR spectrum of compound 4. Instead
the 20-C=NOH carbon signal was identified at 155.5ppm.
Furthermore, its structure was confirmed by ESI mass spectra.
With ethanol as the solvent, a different oxime 5 was obtained in
82% yield.8 The configuration of the 20-oxime was determined
using the 2D-NOESY spectrum. The NOESY spectrum of
compound 5 did not exhibit correlation between the peak of the
activity against T24 cells, with the IC50 values of 1.94μM.
However, compounds 6b and 6c bearing the same substituent at
C20-position exhibited low inhibitory activity against the tested
cell. Compound 5 exhibited no inhibitory activity of these cells,
while its oxime-ether derivatives 7b and 7c showed significant
influence on the cytotoxic activity with the IC50 values of 1.99
and 2.62 μM respectively, suggesting that substituent at the
4N-position of the steroidal moiety also affected the anticancer
activity.
In conclusion, we have reported the synthesis and anticancer
activity against T24 cell of 4-azasteroidal-20-oxime derivatives.
The results from MTS-assays showed that some compounds
had excellent activity against T24 (compound 4, 6a, 7b, 7c,
8a). It was evident that introduction of aryl groups resulted in a
marked decrease in potency (compounds 8b–e).
21-CH3 (1.73ppm) and the proton 20-oxime group (10.34ppm)
16
while compound 4 showed this NOE effect.
Therefore
compound 5 was identified as Z-isomer. In the formation of the
ethers, compound 4 reacts faster than 5 because of the influence
of steric hindrance. This indirectly confirmed its configuration
as the E-isomer.
Experimental
The oxime was treated with the alkyl iodide under basic
conditions to afford 3-oxo-4-substituted-4-aza-5-pregnene-
20-oxime ether derivatives. The ESI mass spectra of 6a–c and
All the reagents were obtained commercially and used without further
purification. Melting points were measured on an X4 apparatus and
uncorrected. IR spectra were determined as KBr pellets on a FTS-135
spectrophotometer. 1H NMR spectra were measured with a Varian
INOVA 500 spectrometer at 500 MHz in CDCl3 as the solvent with
TMS as internal standard. ESI-MS were performed on LCQ Advantage
MAX spectrometer. Elementary analyses were performed by a Carlo-
Erba CHNO-S analyser.
5,20-Dioxo-A-nor-3,5-secopregnane-3-oic acid (2): A mixture of
progesterone (15.0 g, 47.6 mmol) in tert-butanol (450 mL), anhydrous
sodium carbonate (6.1 g, 57.6 mmol) and H2O (20 mL) was heated to
reflux, and a solution of sodium periodate (61.2 g, 286.0 mmol) and
potassium permanganate (0.45 g, 2.9 mmol) in hot water (300 mL)
was added dropwise to a refluxing mixture. This was then refluxed for
another 3 h. All inorganic material was filtered off, and washed with
water and methylene chloride. The filtrate was acidified with dilute
hydrochloric acid and extracted with dichloromethane (150 mL×3).
The organic layer was washed with brine, dried over anhydrous sodium
sulfate, and evaporated under reduced pressure. Compound 2 (14.7 g,
92.1%) was obtained as pale yellow solid, which was used directly
without further purification; m.p. 176–178 °C (lit.6 177–179 °C). IR
(KBr), ν/cm−1: 3148(OH), 1734(CO), 1698(3-CO); ESI-MS m/z:
333(M-H)-; 1H NMR (CDCl3, 500 MHz), δ 2.13 (3H, s, 21-CH3), 1.13
(3H, s, 19-CH3), 0.69 (3H, s, 18-CH3).
4-Aza-5-pregnene-3,20-dione (3): The mixture of 5,20-dioxo-A-
nor-3, 5-secopregnane-3-oic acid (5.0 g, 15.0 mmol), and ammonium
acetate (3.5g, 45.1 mmol) in acetic acid (50 mL) was heated to reflux
for 4 h. After removal of the acetic acid under reduced pressure, the
residue was poured into water. The precipitate was filtered and washed
with water to give 3(4.3g, 91.5%) as pale yellow solid; m.p. 273–275°C
(274–276°C12). IR(KBr), σ/cm-1: 2938(–NH), 1702(20-C=O), 1667(3-
C=O); ESI-MS (m/z): 316.4(M+H)+; 1H NMR (CDCl3, 500 MHz)
δ 0.65 (s, 3H, 18-CH3), 1.08 (s, 3H, 19-CH3), 2.15 (s, 3H, 21-CH3), 4.85
(m, 1H, 6-H), 7.76 (s, 1H, 4-H); 13C NMR (125 MHz, DMSO) δ 209.4,
169.3, 140.0, 103.0, 63.5, 56.6, 47.8, 44.0, 38.5, 34.3, 31.6, 31.5, 31.4,
29.6, 28.5, 24.4, 22.8, 21.0, 18.7, 13.3.
1
7a–c were characterised by their molecular ion peaks. The H
NMR spectrum of 6c displayed the signals at 4.06–4.10, which
were assigned to the proton of –N–O–CH2, while the –NH
signal appeared at 7.22 ppm. The expected singles for 18-CH3 (δ
= 0.65 ppm), 19-CH3(δ = 1.09 ppm), 21-CH3(δ = 1.82 ppm) and
double bond (δ = 4.81ppm) were observed. Under these reaction
conditions, we concluded that the 20E-oxime was more reactive
than the 20Z-oxime. A reaction using KOH to obtain the ethyl
ester of 3-oxo-4-substituted-4-aza-5-pregnene-20Z-oxime did
not succeed. Consequently, potassium t-butoxide was used to
afford the target products. However, using iodopropane and
other reactants, the reaction was so complex that the products
could not be isolated. Because of steric hindrance, only
compound 4 was treated with the acyl chlorides in a mixture
of pyridine and dichloromethane as the solvent to afford the
4-acyl-3-oxo-4-aza-5-pregnene-20E-oxime ester derivatives.16
The novel compounds were evaluated for their anticancer
activity in vitro against T24 (human bladder carcinoma) cell.
The inhibition of test compounds was determined using the
MTS assay. The anticancer activity was indicated in terms of
IC50 (μM) value and the results were presented in Table 1.
From the data shown in Table 1, it is evident that the oxime-
ether derivatives have a higher activity against T24 than the
oxime-ester derivatives. In the oxime-ester derivatives, only
compound 8a which contained the acetyl group had significant
influence on the cytotoxicity. In contrast, for compounds
8b–e the introduction of aryl group resulted in a marked
decrease in potency compared to 8a. Compounds in which
the configuration of oxime was E displayed strong cell growth
inhibitory activity against T24, with IC50 values of 2.41μM. It is
interesting that compound 6a bearing methyl groups at the 4N
and 20C=N–O– positions demonstrated the best anticancer
3-Oxo-4-aza-5-pregnene-20E-oxime (4): Hydroxylamine hydrochloride
(3.7 g, 48.6 mmol) was added to a stirred soluion of 4-aza-5-pregnene-
3,20-dione (5.0g , 15.8 mmol) in pyridine (120 mL). The mixture was
stirred for 12 h at 80–90°C, and then poured into water (150 mL), extracted
with dichloromethane (50 mL×3). The organic layer was washed with
brine, dried over anhydrous sodium sulfate, and evaporated under reduced
pressure. The residue was purified by column chromatography on silica
gel and eluted with petroleum ether/ethyl acetate to afford compound 4
(3.2 g, 60.7%) as a white solid; m.p. 230–231 °C; IR(KBr), σ/cm-1: 3350.18,
2928.18, 1654.09, 1368.00, 1217.6, 930.51; ESI-MS (m/z)：331.4(M+H)+;
1H NMR (500 MHz, DMSO) δ 0.58 (s, 3H, 18-CH3), 1.02 (s, 3H, 19-CH3),
1.74 (s, 3H, 21-CH3), 5.31 (s, 1H, 6-H), 9.71 (s, 1H, –NH), 10.35 (s, 1H,
–OH); 13C NMR (125 MHz, DMSO) δ 162.3, 155.5, 142.3, 101.3, 56.6,
55.8, 48.5, 43.6, 38.4, 36.2, 31.3, 31.2, 29.8, 28.9, 24.2, 23.1, 21.1, 19.3,
Table 1 The in vitro anticancer activity of 4-azasteroidal-20-oxime
derivatives
Compound
IC50/μM
Compound
IC50/μM
4
5
2.41
>100
1.94
50.45
>100
>100
1.99
7c
8a
8b
8c
8d
8e
2.62
1.90
80.89
91.34
>100
>100
6a
6b
6c
7a
7b

JOURNAL OF CHEMICAL RESEARCH 2015 529
15.7, 13.6. Anal. calcd for C20H30N2O2: C, 72.69; H, 9.15; N, 8.48;
found: C, 72.73; H, 9.34; N, 8.60%.
38.18, 37.09, 31.16, 29.43, 29.05, 24.09, 23.10, 22.80, 19.89, 18.95, 18.18,
16.99, 13.37. Anal. calcd for C24H34N2O4: C, 69.54; H, 8.27; N, 6.76;
found: C, 69.65; H, 8.37; N, 6.78%.
3-Oxo-4-aza-5-pregnene-20Z-oxime (5): Hydroxylamine hydrochloride
(1.0 g, 14.1 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added
to a stirred solution of 4-aza-5-pregnene-3,20-dione (3.3 g, 10.0 mmol)
in ethanol (100 mL). Then the mixture was heated to reflux for 12 h,
cooled to room temperature, poured into water (80 mL), extracted with
dichloromethane (50 mL×3). The organic layer was washed with brine,
dried over anhydrous sodium sulfate, and evaporated under reduced
pressure. The residue was purified by column chromatography on silica
gel and eluted with petroleum ether/ethyl acetate to afford compound
5 (2.7 g, 81.8%) as white solid; m.p. 243–245°C; IR (KBr), σ/cm-1:
3316.16, 2928.18, 1664.98, 1368.00, 1056.48; ESI-MS (m/z)：347.4
(M+NH3)+; 1H NMR (500 MHz, DMSO) δ 0.58 (s, 3H, 18-CH3), 0.99 (s,
3H, 19-CH3), 1.73 (s, 3H, 21-CH3), 4.84 (s, 1H, 6-H), 9.27 (s, 1H, –NH),
10.34 (s, 1H, –OH); 13C NMR (125 MHz, DMSO) δ 168.3, 155.5, 141.3,
101.6, 56.6, 55.9, 48.2, 43.6, 38.4, 34.0, 31.3, 31.8, 31.7, 29.7, 28.7, 24.3,
23.1, 21.0, 19.0, 15.7, 13.6. Anal. calcd for C20H30N2O2: C, 72.69; H, 9.15;
N, 8.48; found: C, 72.89; H, 9.02; N, 8.50%.
4-Benzoyl-3-oxo-4-aza-5-pregnene-20E-oxime-N–O-benzoate ester
(8b): Yield 54.6%; m.p. 260–262 °C; ESI-MS (m/z): 477.9 (M + K)+;
1H NMR (500 MHz, CDCl3) δ 0.79 (s, 3H, 18-CH3), 1.58 (s, 3H, 19-
CH3), 2.10 (s, 3H, 21-CH3), 5.25 (s, 1H, 6-H), 7.45–7.51 (m, 4H, Ph),
7.58–7.66 (m, 2H, Ph), 8.06–8.08 (m, 2H, Ph), 8.13–8.14 (m, 2H, Ph);
13C NMR (125 MHz, CDCl3) δ 167.60, 163.83, 163.03, 162.24, 141.57,
134.20, 133.11, 130.12, 129.52, 128.75, 128.51, 101.44, 56.84, 55.89,
48.35, 48.33, 44.24, 38.24, 37.30, 37.28, 31.22, 31.20, 29.47, 29.27,
29.25, 24.17, 23.23, 20.86, 19.04, 17.14, 13.89, 13.49. Anal. calcd for
C34H38N2O4: C, 75.81; H, 7.11; N, 5.20; found: C, 75.89; H, 7.21; N,
5.29%.
4-p-Methylbenzoyl-3-oxo-4-aza-5-pregnene-20E-oxime-N–O-p-
methylbenzoate ester (8c): Yield 51.9%; m.p. 188–189 °C; ESI-MS
1
(m/z): 605.5 (M + K)+; H NMR (500 MHz, CDCl3) δ 0.78 (s, 3H,
18-CH3), 1.58 (s, 3H, 19-CH3), 2.09 (s, 3H, 21-CH3), 2.43 (s, 6H, 2Ph-
CH3), 5.24 (s, 1H, 6-H), 7.25–7.30 (m, 4H, Ph), 7.95–7.96 (m, 2H, Ph),
8.01–8.03 (m, 2H, Ph); 13C NMR (125 MHz, CDCl3) δ 167.39, 163.91,
163.08, 162.24, 145.15, 143.81, 141.60, 140.53, 130.16, 129.55, 129.44,
129.20, 126.72, 101.40, 56.83, 55.90, 48.36, 48.35, 44.23, 38.24, 37.28,
37.27, 31.22, 31.20, 29.48, 29.46, 29.26, 29.25, 24.17, 23.22, 21.83,
21.70, 20.85, 19.02, 17.09, 13.49. Anal. calcd for C36H42N2O4: C, 76.29;
H, 7.47; N, 4.94; found: C, 76.39; H, 7.57; N, 4.88.
Synthesis of 3-oxo-4-substituted-4-aza-5-pregnene-20E-oxime-ether
derivatives (6a–c); general procedure
A
mixture of compound 4 (0.21 g, 0.64 mmol) in N,N-
dimethylformamide (DMF, 30mL), containing potassium hydroxide
(KOH, 0.1g, 1.78 mmol) was stirred for 0.5h at room temperature,
then iodomethane or iodoethane (0.2 mL) was added dropwise. The
mixture was heated to 110–115°C for 5 h, poured into water (10 mL),
and extracted with dichloromethane (20 mL×3). The organic layer was
washed with water (50mL×3), dried over anhydrous sodium sulfate,
andevaporated under reduced pressure. The crude was purified by
column chromatography on silica gel and eluted with petroleum ether/
ethyl acetate to afford compounds 6a–c.
3-Oxo-4-methyl-4-aza-5-pregnene-20E-oxime-N–O-methyl ether
(6a): Yield 66.4%; m.p. 177–179 °C; ESI-MS (m/z): 375.4 (M+NH3)+;
1H NMR (500 MHz, CDCl3) δ 0.65 (s, 3H, 18-CH3), 1.09 (s, 3H, 19-
CH3), 1.85 (s, 3H, 21-CH3), 3.72 (s, 3H, 4-NCH3), 3.84 (s, 3H, –N–O–
CH3), 5.39 (s, 1H, 6-H). Anal. calcd for C22H34N2O2: C, 73.70; H, 9.56;
N, 7.81; found: C, 73.64; H, 9.47; N, 7.62%.
4-p-Methoxybenzoyl-3-oxo-4-aza-5-pregnene-20E-oxime-N–O-
p-methoxybenzoate ester (8d): Yield 48.7%; m.p. 186–188 °C;
1
ESI-MS (m/z): 637.4 (M + K)+; H NMR (500 MHz, CDCl3) δ 0.78
(s, 3H, 18-CH3), 1.55 (s, 3H, 19-CH3), 2.08 (s, 3H, 21-CH3), 3.87 (s,
3H, Ph-OCH3), 3.88 (s, 3H, Ph-OCH3), 5.24 (s, 1H, 6-H), 6.93–6.97
(m, 4H, Ph), 8.2–8.04 (m, 2H, Ph), 8.08–8.10 (m, 2H, Ph); 13C NMR
(125 MHz, CDCl3) δ 167.19, 164.35, 163.60, 163.49, 162.70, 162.33,
141.62, 140.57, 132.30, 131.56, 121.75, 114.07, 113.79, 101.39, 56.83,
55.89, 55.56, 55.47, 48.36, 48.12, 44.22, 38.23, 37.04, 37.03, 31.22,
31.20, 29.49, 29.48, 29.26, 24.17, 23.22, 21.69, 20.84, 19.02, 17.07,
13.45. Anal. calcd for C36H42N2O6: C, 72.22; H, 7.07; N, 4.68; found: C,
72.32; H, 7.17; N, 4.78%.
4-p-Chlorobenzoyl-3-oxo-4-aza-5-pregnene-20E-oxime-N–O-
p-chlorobenzoate ester (8e): Yield 49.7%; m.p. 170–172 °C; ESI-MS
(m/z): 645.4 (M + K)+; 1H NMR (500 MHz, CDCl3) δ 0.78 (s, 3H, 18-
CH3), 1.55 (s, 3H, 19-CH3), 2.08 (s, 3H, 21-CH3), 5.21 (s, 1H, 6-H),
7.43–7.45 (m, 4H, Ph), 7.99–8.01 (m, 4H, Ph); 13C NMR (125 MHz,
CDCl3) δ 167.93, 163.13, 162.40, 162.31, 141.68, 140.96, 139.71, 131.02,
129.29, 129.01, 128.07, 101.58, 56.95, 56.01, 48.44, 48.43, 44.38, 38.34,
37.45, 37.36, 31.34, 31.32, 29.57, 29.55, 29.36, 29.35, 24.28, 23.36,
20.98, 19.16, 17.26, 13.62. Anal. calcd for C34H36Cl2N2O4: C, 67.21; H,
5.97; N, 4.61; found: C, 67.35; H, 5.89; N, 4.58%.
3-Oxo-4-methyl-4-aza-5-pregnene-20Z-oxime-N–O-methyl
ether (7a): A mixture of compound 5(0.11 g, 0.31 mmol) in N,N-
Dimethylformamide(DMF, 15mL), containing potassium hydroxide
(KOH, 0.1 g, 1.78 mmol) was stirred for 0.5h at room temperature,
and then iodomethane (0.1mL) was added dropwise. The mixture was
heated to 95–100°C for 12 h, poured into water (15 mL), and extracted
with dichloromethane (30 mL×3). The organic layer was washed with
water (50 mL×3), dried over anhydrous sodium sulfate, and evaporated
under reduced pressure. The crude product was purified by column
chromatography on silica gel eluted with petroleum ether/ethyl acetate
to afford compound 7a; yield 66.4%; m.p. 197–199°C; ESI-MS (m/z):
375.4(M+NH3)+; 1H NMR (500 MHz, CDCl3) δ 0.66 (s, 3H, 18-CH3),
1.10 (s, 3H, 19-CH3), 1.87 (s, 3H, 21-CH3), 3.12 (s, 3H, 4-NCH3), 3.84
(s, 3H, –N–O–CH3), 5.05 (s, 1H, 6-H). Anal. calcd for C22H34N2O2: C,
73.70; H, 9.56; N, 7.81; found: C, 73.65; H, 9.49; N, 7.72%.
3-Oxo-4-ethyl-4-aza-5-pregnene-20E-oxime-N–O-ethyl ether (6b):
Yield 16.9%; ESI-MS (m/z): 385.4(M-H)-; 1H NMR (500 MHz, CDCl3)
δ 0.67 (s, 3H, 18-CH3), 1.09 (s, 3H, 19-CH3), 1.83 (s, 3H, 21-CH3),
3.87–3.99 (m, 2H, 4–N–CH2), 4.03–4.10 (m, 2H, –N–O–CH2), 5.39
(s, 1H, 6-H). Anal. calcd for C24H38N2O2: C, 74.57; H, 9.91; N, 7.25;
found: C, 74.64; H, 9.97; N, 7.12%.
3-Oxo-4-aza-5-pregnene-20E-oxime-N–O-ethyl ether (6c): Yield
46.1%; m.p. 178–180°C; ESI-MS (m/z): 381.3(M+Na)+; 1H NMR (500
MHz, CDCl3) δ 0.65 (s, 3H, 18-CH3), 1.09 (s, 3H, 19-CH3), 1.82 (s,
3H, 21-CH3), 4.06–4.10 (m, 2H, –N–O–CH2), 4.81 (s, 1H, 6-H), 7.22
(s, 1H, –NH). Anal. calcd for C22H34N2O2: C, 73.70; H, 9.56; N, 7.81;
found: C, 73.89; H, 9.57; N, 7.88%.
4-Acyl-3-oxo-4-aza-5-pregnene-20E-oxime-ester derivatives (8a–e);
general procedure
A stirred solution of compound 4 (0.21 g, 0.64 mmol) in a mixture of
pyridine (3 mL) and dichloromethane (3 mL) was treated with the acyl
chloride (1.92 mmol) at 0 °C. The mixture was then stirred for 0.5 h
at room temperature, poured into water (10 mL), and extracted with
dichloromethane (20 mL × 3). The organic layer was washed with
dilute hydrochloric acid, saturated sodium carbonate solution, brine,
dried over anhydrous sodium sulfate, and evaporated under reduced
pressure. The crude product was purified by column chromatography
on silica gel. Elution with petroleum ether/ethyl acetate afforded
compounds 8a–e as white solids.
4-Acetyl-3-keto-4-aza-5-pregnene-20E-oxime-N–O-acetate ester
(8a): Yield 64.1%; m.p. 175–176 °C; ESI-MS (m/z): 437.3 (M + Na)+;
1H NMR (500 MHz, CDCl3) δ 0.61 (s, 3H, 18-CH3), 1.18 (s, 3H, 19-
CH3), 1.89 (s, 3H, 21-CH3), 2.11 (s, 3H, OCO–CH3), 2.21 (s, 3H, 4-N–
CO–CH3), 5.08 (s, 1H, 6-H); 13C NMR (125 MHz, CDCl3) δ 169.24,
166.75, 166.10, 162.05, 141.39, 101.43, 60.09, 56.67, 55.84, 48.26, 44.05,
3-Oxo-4-substituted-4-aza-5-pregnene-20Z-oxime-ether derivatives
(7b–c); general procedure
A
mixture of compound 5(0.81 g, 2.40 mmol) in N,N-
Dimethylformamide(DMF, 85mL), containing potassium t-butoxide
(from t-BuOH, 0.82 g, 6 mmol) was stirred for 0.5h at room

530 JOURNAL OF CHEMICAL RESEARCH 2015
temperature, and then iodoethane (2.0mL) was added dropwise. The
mixture was heated to 100-110°C for 10 h, poured into water (100 mL),
and extracted with dichloromethane (100 mL×3). The organic layer
was washed with water (100 mL×3), dried over anhydrous sodium
sulfate, and evaporated under reduced pressure. The crude was
purified by column chromatography on silica gel eluted with petroleum
ether/ethyl acetate to afford compounds 7b–c.
measured at 570 nm with an automatic microplate reader. All the data
of the experiment were expressed as the IC50 (μM) values.
The authors appreciate the financial support from Natural
Science Foundation of Hebei Province (B2012208036,
B2012208051, B2013208016), XiaoLi Foundation of Hebei
University of Science and Technology (YY14).
3-Oxo-4-ethyl-4-aza-5-pregnene-20Z-oxime-N–O-ethyl ether (7b):
1
Yield 15.2%; m.p. 97–99°C; ESI-MS (m/z): 409.0(M+Na)+; H NMR
Received 3 July 2015; accepted 7 August 2015
Paper 1503465 doi: 10.3184/174751915X14401666837860
Published online: 1 September 2015
(500 MHz, CDCl3) δ 0.66 (s, 3H, 18-CH3), 1.10 (s, 3H, 19-CH3), 1.86
(s, 3H, 21-CH3), 3.83–3.91 (m, 2H, 4–N–CH2), 3.98–4.08 (m, 2H,
–N–O–CH2), 5.03 (s, 1H, 6-H). Anal. calcd for C24H38N2O2: C, 74.57;
H, 9.91; N, 7.25; found: C, 74.66; H, 9.95; N, 7.46%.
3-Oxo-4-aza-5-pregnene-20Z-oxime-N–O-ethyl ether (7c): Yield
39.7%; m.p. 113–115°C; ESI-MS (m/z): 359.3(M+H)+; 1H NMR
(500 MHz, CDCl3) δ 0.66 (s, 3H, 18-CH3), 1.10 (s, 3H, 19-CH3), 1.87
(s, 3H, 21-CH3), 4.05–4.10 (m, 2H, –N–O–CH2), 4.83 (s, 1H, 6-H),
7.54 (s, 1H, –NH). Anal. calcd for C22H34N2O2: C, 73.70; H, 9.56; N,
7.81; found: C, 73.99; H, 9.59; N, 7.68%.
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