Synthesis and antiproliferative activity of some androstene oximes and their O-Alkylated derivatives

Article abstract of DOI:10.1002/ardp.201300216

In order to study the structure-activity relationship with respect to the cytotoxicity of steroidal oximes, several 6E-hydroximino-4-ene steroids and their O-alkylated derivatives were synthesized. The oxime ethers were solidified and purified by preparing their corresponding oxalate salts. The new derivatives as well as some previously synthesized ones were evaluated for in vitro antineoplastic activity against a panel of 60 cancer cell lines at 10 μM. The oximes and oxime ethers were found to have moderate to good antiproliferative activity against various leukemia, colon, melanoma, and renal cancer cell lines. Several 6E-hydroximino-4-ene steroids and their O-alkylated derivatives were synthesized. Their structure-activity relationship with respect to the cytotoxicity of steroidal oximes was studied. The oximes and oxime ethers were found to have moderate-to-good antiproliferative activity against various leukemia, colon, melanoma, and renal cancer cell lines.

Full text of DOI:10.1002/ardp.201300216

Arch. Pharm. Chem. Life Sci. 2014, 347, 193–199
193
Full Paper
Synthesis and Antiproliferative Activity of Some Androstene
Oximes and Their O-Alkylated Derivatives
Pratap Chandra Acharya and Ranju Bansal
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India
In order to study the structure–activity relationship with respect to the cytotoxicity of steroidal oximes,
several 6E-hydroximino-4-ene steroids and their O-alkylated derivatives were synthesized. The oxime
ethers were solidified and purified by preparing their corresponding oxalate salts. The new derivatives
as well as some previously synthesized ones were evaluated for in vitro antineoplastic activity against a
panel of 60 cancer cell lines at 10 mM. The oximes and oxime ethers were found to have moderate to
good antiproliferative activity against various leukemia, colon, melanoma, and renal cancer cell lines.
Keywords: Antileukemic activity / Cytotoxic steroids / Oxime ethers / Steroidal oximes
Received: June 7, 2013; Revised: September 20, 2013; Accepted: September 23, 2013
DOI 10.1002/ardp.201300216
Introduction
androsterone skeleton leads to remarkable changes in the
antineoplastic activity [10]. Steroidal oximes of natural origin
have also emerged as a new perspective of cytotoxic agents in
the recent past. Two steroidal oximes, (6E)-hydroximino-
cholest-4-en-3-one and its 24-ethyl analog (Fig. 1), isolated from
marine sponges Cinachyrella alloclada and C. apion [12], have
shown good antiproliferative activity against several types of
cancer cells [13]. Several endocrine active antitumor steroidal
oximes like aromatase or 5a-reductase inhibitors are also
reported in the literature [14, 15].
In continuation with our earlier similar studies [10, 11],
wherein synthesis of a variety of oximes and their etheral
derivatives have been reported as potent cytotoxic agents, it
was envisaged to synthesize ether derivatives of 6E-hydrox-
imino steroid. Impact of substituting 2-alkylaminoethyl side
chain on antiproliferative activity of the parent 6E-hydroxi-
minoandrostene framework was also observed. To study the
effect of the location of the oxime on the steroid skeleton and
establish the structure–activity relationship of steroidal
oximes, the hydroximino androstene derivatives with oxime
positioned at C3 (13) and C7 (14) of steroid (Fig. 2) were also
evaluated for antineoplastic activity.
The fact that the available cytotoxic cancer chemotherapeutic
agents worsen the quality of life has driven the quest to find a
safer and target-oriented anticancer agent [1]. However, most
of the FDA-approved drugs for the clinical use are either
analogs of the older cytotoxic drugs or reintroduced after the
patent expiry, indicating their importance either alone or in
combination with newer innovative agents for cancer
therapy [1]. Several steroids, which are in clinical use to
treat the complications associated with malignancy, include
prednisone, cyproterone acetate, and fluoxymesterone [2–4].
Inhibition of specific enzymes like aromatase and 5a-
reductase are the frontline treatment approaches for breast
and prostate carcinoma [5, 6]. Extensive chemical modifica-
tions of the steroid skeleton have been carried out to
synthesize many potent antineoplastic agents, which could
target hormone-dependent cancers like prostate (e.g., finaste-
ride, dutasteride), breast (e.g., exemestane), and uterine
carcinomas [7–9].
Literature reports indicate prominent cytotoxicity of
steroidal oximes and their O-alkylated derivatives on various
cancer cell lines [10, 11]. It has been observed that varying
the position of the hydroximino group on the parental Results and discussion
Chemistry
Correspondence: Dr. Ranju Bansal, University Institute of Pharmaceuti-
cal Sciences, Panjab University, Chandigarh 160014, India.
E-mail: ranju29in@yahoo.co.in
Fax: þ91 172 2543101
Synthesis of the steroidal oxime 8, 6E-hydroximino-androst-4-
ene-3,17-dione, starting from dehydroepiandrosterone acetate
was achieved based on the methodology available in the
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P. C. Acharya and R. Bansal
Arch. Pharm. Chem. Life Sci. 2014, 347, 193–199
To obtain compounds with higher degree of oxidation in
ring A, diacetoxy 6-oxime 6 was hydrolyzed using methanolic
KOH to yield 3b,17b-dihydroxy-6E-hydroximino-androst-4-ene
(7), which was further oxidized with CrO3/pyridine complex to
obtain 3,17-dioxo-6E-hydroximino-androst-4-ene (8) [16].
O-Alkylation of 6E-hydroximino-androst-4-en-3,17-dione (8)
with hydrochlorides of 2-chloroethylmorpholine and 2-
diethylaminoethyl chloride in the presence of K2CO3 and
KOH in ethylmethyl ketone afforded a sticky mass of 9 and 11,
respectively (Scheme 2). Attempts to crystallize the dried mass
using various solvents remained unsuccessful. Hence, it was
decided to prepare the salt of the steroidal oxime ether to
obtain solid product. Oxalate salt was prepared by adding a
saturated ethereal solution of oxalic acid to a solution of the
steroidal oxime ethers in dry solvent ether to obtain the
corresponding solid product of 6E-(2-morpholinoethoxy-
imino)-androsta-4-ene-3,17-dione oxalate dihydrate (10) and
6E-(2-(diethylamino)ethoxyimino)-androst-4-ene-3,17-dione ox-
Figure 1. Structures of (6E)-hydroximino cholestene derivatives
isolated from marine sponges [12, 13].
alate dihydrate (12).
ꢀ
The broad singlet for –N–(CH2)2– of morpholine and
ꢀ
multiplet of –CH2N< appeared downfield at d 2.98 and
3.16 ppm, respectively, in the 1H NMR spectrum of 10 due to
presence of quaternary nitrogen. Similarly, the multiplets for
Figure 2. Structures of C3 and C7 hydroximino androstene
derivatives [10, 11].
protons adjacent to the quaternary nitrogen of the oxalate
ꢀ
salt 12 appeared downfield at d 3.08 (–N–(CH2CH3)2) and
ꢀ
literature [13, 16, 17] with some suitable modifications as
shown in Scheme 1. The NaBH4 reduction of the C17 keto
functionality of dehydroepiandrosterone acetate led to the
formation of reduced steroid 3-acetoxy-17-hydroxy-androst-5-
ene. However, this also resulted in simultaneous hydrolysis of
the C3 acetate group leading to the formation of an 80:20
mixture of 3-acetoxy-17-hydroxy and 3,17-dihydroxy steroid
represented by 1 as observed from 1H NMR data. Splitted
3.31 ppm (–CH2–N) in comparison to its tertiary nitrogen
steroidal substrate 11 in the proton NMR spectrum. Similarly,
the broad singlet for the –OCH2– of the aminoethyl side chain
and singlet for 4-CH vinylic proton were observed downfield at
d ꢁ4.39 and ꢁ5.99 ppm, respectively, for the oxalate dihydrate
salts 10 and 12. The quaternization of the oxime ether
nitrogen and formation of the oxalate dihydrate salt was
further confirmed by the appearance of the molecular ion
[Mþ] peaks at 555.4 and 541.3 in the mass spectra of the
oxalate salts 10 and 12.
1
multiplets were observed for 3a-H in the H NMR spectrum
at d 4.55–4.64 and 3.49–3.54 ppm, which together (area
ratio 80:20) integrated for one proton. The reduced mixture
of steroids was subjected to acetylation using acetic
anhydride and pyridine [17], which yielded 3b,17b-diacetyl
derivative 2.
The C3 and C7 hydroximino androstene derivatives 13
and 14 were synthesized according to the literature
methods [10, 11].
The diacetyl product 2 on epoxidation with m-chloroper-
benzoic acid in ice-cold chloroform afforded a mixture of a/b-
isomers of 3b,17b-diacetoxy-5,6-epoxy-androstane (3) consis-
tent with literature reports [16, 18]. The isomeric mixture 3 on
oxidation with CrO3 was converted to 3b,17b-diacetoxy-5a-
hydroxy-androstan-6-one (4), which, on further treatment
with SOCl2 and pyridine, dehydrated to 3b,17b-diacetoxy-
androst-4-en-6-one (5) [16, 19]. Oximation of compound 5 with
NH2OH · HCl in the presence of CH3COONa yielded 3b,17b-
diacetoxy-6E-hydroximino-androst-4-ene (6). The 7b-H proton
resonated downfield as a double doublet at d 3.34 ppm
(Jtrans ¼ 15.16 Hz, Jgem ¼ 4.64 Hz), confirming the steroidal
oxime 6 being the E-isomer consistent with earlier reports [13].
Antineoplastic activity
The newly synthesized 6E-oxime steroidal derivatives 6–8, 10,
and 12 and previously reported steroidal oximes 13 and 14
were submitted to NCI, Bethesda, USA, for anticancer
screening. Out of these, compounds 6–8 and 12–14 were
selected for the preliminary in vitro anticancer screening. The
screening begins with the evaluation of selected compounds
at a single dose of 10 mM against the 60 cell lines, which
include cells from eight melanomas, six leukemias, eight
breast cancers, two prostate, nine lung, seven colon, six ovary,
eight kidney, and six central nervous system cancers. A 48-h
continuous drug exposure protocol was used, and
sulforhodamine B (SRB) protein assay was used to estimate
a
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Arch. Pharm. Chem. Life Sci. 2014, 347, 193–199
Antiproliferative Androstene Oximes
195
Scheme 1. Reagents and reaction conditions: (a) NaBH4/MeOH, stirring, rt, 4 h, (b) Ac2O/pyridine, 100°C, 2 h, (c) MCPBA, 0°C, stirring,
12 h, (d) CrO3/H2O/ethylmethyl ketone, stirring, 0°C, 1 h, (e) SOCl2/pyridine, stirring, 0°C, 1 h, (f) NH2OH · HCl/CH3COONa, stirring, rt,
overnight, (g) KOH/MeOH, reflux, 1 h, and (h) CrO3/pyridine, stirring, 1 h.
the cell viability or growth [20]. The percentage growth of
some selected cancer cell lines treated with various steroidal
derivatives at 10 mM is summarized in Table 1.
effect on these cell lines. This is also in sharp contrast
to earlier reports indicating the good antiproliferative
activity of (6E)-hydroximino cholestane analogs, isolated
from marine sponges, against several types of cancer
cells [13].
Although steroidal 6E-oximes of the current androstene
series possess all important structural features such as
elevated oxidation in ring A and presence of double bond
at C4,5 required for the biological activity [13], the compounds
are almost inactive, suggesting that cholesterol type steroidal
skeleton not only is playing the major role, but might be an
essential factor for biological activity of 6E-oximes.
C3-substituted (13) and C7-substituted (14) androstene
oximes displayed considerable inhibitory effect on the
entire panel of 60-cell lines at 10 mM, being more pronounced
on leukemia cell lines. They exhibited significant growth
reduction against five leukemia cell lines at 10 mM with
mean growth percentage of 53.52 and 57.41, respectively,
but C6-substituted androstene oximes 6–8 produced negligi-
ble antiproliferative effects in comparison to the standard
drug adriyamycin, which has
a pronounced cytotoxic
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P. C. Acharya and R. Bansal
Arch. Pharm. Chem. Life Sci. 2014, 347, 193–199
Scheme 2. Reagents and reaction conditions: (a) K2CO3/KOH/ethylmethyl ketone, (b) 2-chloroethylmorpholine · HCl, reflux, 18 h,
(c) 2-diethylaminoethyl chloride · HCl, reflux, 18 h, and (d) dry ether, oxalic acid, stirring.
The oxime ether 12 at 10 mM has shown moderate activity
against HT29 (colon cancer) and UO-31 (renal cancer) cell lines
with percentage growth inhibition of 57.41% and 64.48%,
respectively. The oxime ethers 10 and 12 (as these were not
screened by NCI against leukemia cell lines) were tested for
cytotoxicity against the K562 leukemia cell line at the
Advanced Centre for Treatment, Research and Education in
Cancer (ACTREC), Navi Mumbai, India. The typical SRB assay
Table 1. Percentage growth of some selected cancer cell lines treated with various steroidal derivatives at 10 mM.
Percentage growth
Cell lines
6
7
8
12
13
14
Adriyamycin
Leukemia
HL-60 (TB)
K562
MOLT-4
RPMI-8226
SR
Colon cancer
HT29
Melanoma
MDA-MB-435
99.17
87.33
92.52
99.23
92.76
–
–
–
–
–
93.47
85.26
91.99
101.67
80.61
–
–
–
–
–
23.05
45.90
72.61
89.35
36.72
67.11
10.07
50.55
75.01
29.88
ꢂ21.8
2.1
ꢂ12.0
ꢂ17.4
ꢂ5
102.89
107.86
98.04
90.72
99.80
57.41
88.48
76.04
20.33
88.60
46.36
4.7
102.07
ꢂ83.9
Renal cancer
A498
UO-31
53.29
73.86
–
73.14
76.30
–
53.11
65.11
72.87
72.59
–
66.19
64.48
4.0
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Arch. Pharm. Chem. Life Sci. 2014, 347, 193–199
Antiproliferative Androstene Oximes
197
various steroidal oximes results in increased antiproliferative
activity; however, the location of the oximino moiety on the
steroid skeleton is very important. C3 and C17 positions at
the far end of the steroidal core are the preferred sites,
whereas C6 and C7 positions well within the nucleus are less
preferred ones for such substitutions.
Conclusions
A
new series of hydroximino-4-ene steroids and their
O-alkylated derivatives were synthesized and studied for
antiproliferative potential. It is concluded that steroidal
oximes and their ethereal derivatives display potent cytotoxic
effects, O-alkylation generally results in increased antiproli-
ferative activity. However, the location of the oximino moiety
on the steroid skeleton is very significant for these effects.
Figure 3. Dose–response curves of compounds 10, 12, and
adriamycin depicting percentage growth of K562 human leukemia
cell line at various concentrations.
protocol was used for the in vitro testing of each compound
at four dose levels (10ꢂ7, 10ꢂ6, 10ꢂ5, and 10ꢂ4 M). Each Experimental
experiment was repeated thrice along with appropriate
negative controls and the standard drug adriamycin. Dose–
response curve of compounds 10 and 12 depicting percentage
growth of K562 human leukemia cell line at various
concentrations is shown in Fig. 3. Various dose–response
parameters such as GI50 (drug concentration resulting in a 50%
reduction in the net protein increase), total growth inhibition
(drug concentration of TGI), and LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the end
of the drug treatment as compared to that at the beginning) of
compounds 10 and 12 against K562 cell line are summarized in
Table 2. The compounds 10 and 12 were found to be moderately
active with GI50 ¼ 65.0 and 35.7mM, respectively, in compari-
son to the standard drug adriyamycin (GI50 ꢃ 1 mM).
Chemistry
Melting points were determined on a Veego melting point
apparatus and are uncorrected. Infrared (wavenumbers in cmꢂ1
)
spectra were recorded on Perkin-Elmer RX1 FTIR spectrophotom-
eter model using potassium bromide pellets. 1H NMR spectra
were obtained on a Bruker Avance II 400 MHz spectrometer using
deuterated-chloroform (CDCl3) or deuterated dimethylsulfoxide
(DMSO-d6) as solvents containing tetramethylsilane as internal
standard (chemical shifts in d, ppm). Mass spectra were obtained
on an Applied Biosystems API 2000TM mass spectrophotometer.
Elemental analyses were carried out on a Perkin Elmer-2400
model CHN analyzer. Plates for thin layer chromatography (TLC)
were prepared with silica gel G according to method of Stahl (E.
Merck) using ethyl acetate as solvent and activated at 110°C for
30 min. Iodine was used to develop the TLC plates. Anhydrous
sodium sulfate was utilized as drying agent. All solvents were
distilled prior to use according to standard procedures.
The results imply that the decreased activity of 6E-oximes in
androstene series may be attributed to missing cholesteryl
side chain. The O-alkylation of 6E-androstene oxime 8
(Scheme 1) marginally improved the antiproliferative
potential but was insignificant in comparison to 3-substituted
The
compounds
3b-acetyloxy-17b-hydroxy-androst-5-ene/
3b,17b-dihydroxy-androst-5-ene (1), 3b,17b-diacetoxy-androst-5-
ene (2), 3b,17b-diacetoxy-5a/b,6a/b-epoxy-androstane (3), 3b,17b-
diacetoxy-5a-hydroxy-androstan-6-one (4), and 3b,17b-diacetoxy-
androst-4-en-6-one (5) were synthesized according to reported
methods [16–19].
(mean GI50 ¼ ꢁ5 mM) and 17-substituted (mean GI50
¼
ꢁ2.5 mM) oxime ethers that we reported previously [10].
However, the results obtained in 6E-oxime ether series
(Scheme 2) are similar to those obtained in 7-substituted
steroidal oxime ethers [11]. It seems that O-alkylation of
3b,17b-Diacetoxy-6E-hydroximino-androst-4-ene (6)
A
solution of 3b,17b-diacetoxy-androst-4-en-6-one (5; 1.4 g,
3.6 mmol) in methanol (20 mL) was treated with a solution of
NH2OH · HCl (1.1 g, 15.7 mmol) and anhydrous CH3COONa (1.1 g,
13.09 mmol) in 30% aqueous methanol (20 mL). The reaction
mixture was stirred at room temperature overnight and
concentrated under vacuum, and cold water was added.
The precipitate obtained was filtered, washed, dried, and
Table 2. Dose–response parameters (GI50, TGI, and LC50) of
compounds 10 and 12 against the K562 cell line.
Dose–response parameters (mM)
recrystallized from methanol to give compound
6 (1.2 g,
82.75%), m.p. 115–117°C. FT-IR: 3371, 2938, 1713, 1648, 1453,
1256, 1058, 949. 1H NMR (CDCl3): d 0.80 (s, 3H, 18-CH3), 1.00
(s, 3H, 19-CH3), 2.04 (s, 3H, 17b –OCOCH3), 2.17 (s, 3H, 3b-OCOCH3),
3.34 (dd, 1H, 7b-H, Jtrans ¼ 15.16 Hz, Jgem ¼ 4.64 Hz), 4.62
(t, 1H, 17a-H), 5.28 (m, 1H, 3a-H), 5.70 (s, 1H, 4-CH), 8.2 (br,
–
Compound
GI50
LC50
TGI
10
12
65.0
35.7
<1
>100
>100
>100
>100
>100
Adriamycin
79.1
1H, N–OH).
–
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Arch. Pharm. Chem. Life Sci. 2014, 347, 193–199
3b,17b-Dihydroxy-6E-hydroximino-androst-4-ene (7)
A solution of 3b,17b-diacetoxy-6E-hydroximino-androst-4-ene (6;
0.5 g, 1.2 mmol) and KOH (0.2 g, 3.5 mmol) in methanol (30 mL)
was heated under reflux for 1 h. The reaction mixture was
concentrated under vacuum, cold water added, and then
neutralized with glacial CH3COOH. The precipitate obtained
was filtered, washed, dried, and recrystallized with aqueous
ethanol to yield the compound 7 (0.4 g, 50.63%), m.p. 225–227°C
(lit [16] 227–229°C).
oily mass, which was washed several times with cold water to
remove unreacted amine and dried in a desiccator over activated
silica for 2 days to obtain a sticky mass of 11. 1H NMR (CDCl3):
d
0.82 (s, 3H, 18-CH3), 1.11 (s, 3H, 19-CH3), 1.18 (t, 6H,
–N–(CH2CH3)2), 2.65 (m, 4H, –N–(CH2CH3)2), 2.98 (m, 2H,
–CH2–N–), 4.48 (s, 2H, –OCH2–), 5.83 (s, 1H, 4-CH).
6E-(2-(Diethylamino)ethoxyimino)-androst-4-ene-3,17-
dione oxalate dihydrate (12)
A saturated solution of oxalic acid (0.2 g) in dry ether (10 mL) was
added to the solution of compound 11 in dry ether (5 mL). The
oxalate salt formed immediately and was kept aside overnight.
The yellowish solid obtained was filtered and washed well with
dry ether to yield oxalate salt 12 (0.19 g, 55%), m.p. 148–150°C.
FT-IR nmax (KBr): 3429, 2927, 1736, 1663, 1459, 1227, 1023, 951.
1H NMR (DMSO-d6): d 0.82 (s, 3H, 18-CH3), 1.09 (s, 3H, 19-CH3), 1.15
(t, 6H,ꢀ–N–(CH2CH3)2), 3.08 (m, 4H, –N–(CH2CH3)2), 3.31 (m, 2H,
–CH2–N–), 4.40 (s, 2H, –OCH2–), 5.98 (s, 1H, 4-CH). ESI-MS m/z: 541.3
[Mþ]. Anal. calcd. for C25H38N2O3 · C2H2O4 · 2H2O: C, 59.98; H, 8.20;
N, 5.18. Found: C, 60.01; H, 8.18; N, 5.09.
6E-Hydroximino-androst-4-ene-3,17-dione (8)
A solution of compound 7 (0.12 g, 0.37 mmol) in pyridine (2 mL)
was added to CrO3–pyridine complex (0.33 g CrO3 in 3.5 mL
pyridine). The reaction mixture was stirred at room temperature
for 1 h. Ethyl acetate was added and the precipitate obtained was
filtered. Filtrate was washed successively with 10% HCl and 10%
NaHCO3, dried, evaporated, and residue recrystallized from
methanol to yield compound 8 (0.08 g, 67.79%), m.p. 135–137°C
(lit [16] m.p. 138–140°C).
ꢀ
ꢀ
6E-(2-Morpholinoethoxyimino)-androst-4-ene-3,17-
Antineoplastic activity
dione (9)
The human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum
and 2 mM L-glutamine. For a typical screening experiment, cells
were inoculated into 96-well microtiter plates in 100 mL at plating
densities ranging from 5000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37°C, 5% CO2, 95% air, and
100% relative humidity for 24 h prior to addition of experimental
drugs.
A mixture of 6E-hydroximino-androst-4-ene-3,17-dione (8; 0.2 g,
0.63 mmol) and 2-chloroethylmorpholine hydrochloride (0.3 g,
2.2 mmol) in ethylmethyl ketone (50 mL) in presence of K2CO3 (2 g)
and KOH (0.25 g) was heated under reflux for 18 h. The reaction
mixture was filtered hot, the residue was washed with fresh
ethylmethyl ketone and washings combined with the filtrate. The
combined filtrate was vacuum evaporated to get an oily mass,
which was washed several times with cold water to remove
unreacted amine and dried in a desiccator over activated silica for
2 days to obtain a sticky mass of 9. 1H NMR (CDCl3): d 0.82 (s, 3H,
18-CH3), 1.17 (s, 3H, 19-CH3), 2.53 (s (br), 4H, –N–(CH2)2–,
morpholine), 2.73 (m, 2H, –CH2N), 3.73 (s (br), 4H, –O–(CH2)2–,
morpholine), 4.30 (t, 2H, –OCH2–), 6.17 (s, 1H, 4-CH).
After 24 h, two plates of each cell line were fixed in situ with
TCA, to represent a measurement of the cell population for each
cell line at the time of drug addition (Tz). Experimental drugs
were solubilized in dimethyl sulfoxide at 400-fold the desired
final maximum test concentration and stored frozen prior to use.
At the time of drug addition, an aliquot of frozen concentrate was
thawed and diluted to twice the desired final maximum test
concentration with complete medium containing 50 mg/mL
6E-(2-Morpholinoethoxyimino)-androst-4-ene-3,17-dione
oxalate dihydrate (10)
A saturated solution of oxalic acid (0.2 g) in dry ether (10 mL) was
added to the solution of compound 9 in dry ether (5 mL). The
oxalate salt formed immediately and was kept aside overnight.
The yellowish solid obtained was filtered and washed well with
dry ether to yield oxalate salt 10 (0.18 g, 52%), m.p. 152–156°C.
FT-IR nmax (KBr): 3408, 1732, 1632, 1455, 1379, 1223, 1133, 1036,
1
gentamicin. Additional four, 10-fold or 2 log serial dilutions
are made to provide a total of five drug concentrations plus
control. Aliquots of 100 mL of these different drug dilutions were
added to the appropriate microtiter wells already containing
100 mL of medium, resulting in the required final drug
concentrations. Following drug addition, the plates were
incubated for an additional 48 h at 37°C, 5% CO2, 95% air, and
100% relative humidity. For adherent cells, the assays were
terminated by the addition of cold TCA. Cells were fixed in situ
by the gentle addition of 50 mL of cold 50% w/v TCA (final
concentration, 10% TCA) and incubated for 60 min at 4°C. The
supernatant was discarded, and the plates were washed five times
with tap water and air dried. SRB solution (100 mL) at 0.4% w/v in
1% acetic acid was added to each well, and plates were incubated
for 10 min at room temperature. After staining, unbound dye was
removed by washing five times with 1% acetic acid and the plates
were air dried. Bound stain was subsequently solubilized with
10 mM Trizma base, and the absorbance was read on an
914, 701. 1H NMR (DMSO-d6): d 0.83 (s, 3H, 18-CH3), 1.10 (s, 3H,
ꢀ
19-CH3), 2.98 (s (br), 4H, –N–(CH2)2–, morpholine), 3.16 (m, 2H,
ꢀ
–CH2–N), 3.72 (s (br), 4H, –O–(CH2)2–, morpholine), 4.38 (s (br), 2H,
–OCH2–), 5.99 (s, 1H, 4-CH). ESI-MS m/z: 555.4 [Mþ]. Anal. calcd. for
C25H36N2O4 · C2H2O4 · 2H2O: C, 58.47; H, 7.63; N, 5.05. Found: C,
58.39; H, 7.70; N, 4.49.
6E-(2-(Diethylamino)ethoxyimino)-androst-4-ene-3,17-
dione (11)
A mixture of 6E-hydroximino-androst-4-ene-3,17-dione (10; 0.2 g,
0.63 mmol) and 2-diethylaminoethyl chloride hydrochloride
(0.3 g, 1.7 mmol) in ethylmethyl ketone (50 mL) in presence of
K2CO3 (2 g) and KOH (0.25 g) was heated under reflux for 18 h. The
reaction mixture was filtered hot, the residue was washed with
fresh ethylmethyl ketone, and washings combined with the
filtrate. The combined filtrate was vacuum evaporated to get an
automated plate reader at
a wavelength of 515 nm. For
suspension cells, the methodology was the same except that
the assay was terminated by fixing settled cells at the bottom of
the wells by gently adding 50 mL of 80% TCA (final concentration,
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Arch. Pharm. Chem. Life Sci. 2014, 347, 193–199
Antiproliferative Androstene Oximes
199
16% TCA). Using the seven absorbance measurements [time zero References
(Tz), control growth (C), and test growth in the presence of drug at
the five concentration levels (Ti)], the percentage growth was
calculated at each of the drug concentrations levels. Percentage
growth inhibition was calculated as:
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ꢀ
ꢀ
ꢁ
ꢁ
ðTi ꢂ TzÞ
ðC ꢂ TzÞ
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ꢄ 100 for concentrations for which Ti ꢅ Tz
ꢄ 100 for concentrations for which Ti < Tz
ðTi ꢂ TzÞ
Tz
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Three dose–response parameters were calculated for each
experimental agent. Growth inhibition of 50% (GI50 was
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calculated from [(Ti ꢂ Tz)/(C ꢂ Tz)] ꢄ 100 ¼ 50, which was the
drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells
during the drug incubation. The drug concentration resulting in
TGI is calculated from Ti ¼ Tz. The LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the end of
the drug treatment as compared to that at the beginning)
indicating a net loss of cells following treatment is calculated
from [(Ti ꢂ Tz)/Tz] ꢄ 100 ¼ ꢂ50. Values were calculated for each of
these three parameters if the level of activity is reached; however,
if the effect is not reached or is exceeded, the value for that
parameter is expressed as greater or less than the maximum or
minimum concentration tested. Two standard drugs, meaning
that their activities against the cell lines are well documented,
were tested against each cell line: NSC 19893 (5-fluorouracil) and
NSC 123127 (adriamycin).
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The financial support provided by the UGC, New Delhi, India for this
project is gratefully acknowledged. The authors express appreciation to
National Cancer Institute, Bethesda, Maryland, USA for evaluation of
compounds for antineoplastic activity and Cipla Ltd., Bombay, India
for the generous supply of steroids.
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The authors have declared no conflict of interest.
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www.archpharm.com