17-Oximino-5-androsten-3β-yl esters: Synthesis, antiproliferative activity, acute toxicity, and effect on serum androgen level

Article abstract of DOI:10.1007/s00044-010-9393-3

The 17-oximino-5-androsten-3β-yl esters (10a- 10j) were synthesized from commercially available (25R)- 5-Spirosten-3β-ol (Diosgenin) (4) as starting material. The synthesized compounds were evaluated for their antiproliferative activity against prostate specific cancer cell line DU-145, acute toxicity, and effect on serum androgen level and were compared with Finasteride used as positive control. Some of the compounds exhibited better cytotoxicity and antiandrogenic activity than the reference control. The detailed synthesis, spectroscopic data, and biological evaluation for the synthesized compounds are reported. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9393-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:817–825
DOI 10.1007/s00044-010-9393-3
ORIGINAL RESEARCH
17-Oximino-5-androsten-3b-yl esters: synthesis, antiproliferative
activity, acute toxicity, and effect on serum androgen level
•
Neelima Dhingra Tilak Raj Bhardwaj Neeraj Mehta
•
•
•
Tapas Mukhopadhyay Ashok Kumar Manoj Kumar
•
Received: 11 September 2009 / Accepted: 20 July 2010 / Published online: 4 August 2010
Ó Springer Science+Business Media, LLC 2010
Abstract The 17-oximino-5-androsten-3b-yl esters (10a–
10j) were synthesized from commercially available (25R)-
5-Spirosten-3b-ol (Diosgenin) (4) as starting material. The
synthesized compounds were evaluated for their antiprolif-
erative activity against prostate specific cancer cell line
DU-145, acute toxicity, and effect on serum androgen level
and were compared with Finasteride used as positive con-
trol. Some of the compounds exhibited better cytotoxicity
and antiandrogenic activity than the reference control. The
detailed synthesis, spectroscopic data, and biological eval-
uation for the synthesized compounds are reported.
MTT
[3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide]
DMEM Dulbecco’s modified eagle medium
Introduction
Benign prostatic hyperplasia is the nonmalignant enlarge-
ment of the prostate gland with increase in numbers of both
epithelial and stromal cells within the periurethral transi-
tion zone of the prostate, resulting in the constriction of
prostatic urethra (Bullock and Andriole, 2006). The prev-
alence increases to 50% by the age of 60 years and to 90%
by the age of 85 years (Berry et al., 1984).
Keywords Dihydrotestosterone ꢀ 5-Alpha reductase
enzyme ꢀ Benign prostatic hyperplasia ꢀ Steroids ꢀ
Androgen
Abnormal increase in the number of cells in prostate
may result not only from increased cell proliferation but
also from decreased level in programmed cell death
(apoptosis) (Isaacs and Coffey, 1989). Number of available
cytotoxic agents are able to induce apoptosis, and thus, can
cause significant decrease in proliferation rate and are
useful for the treatment of disease that involves abnormal
or uncontrolled cell proliferation (Perez-Stable, 2006;
Jakobsen et al., 2001; Brady et al., 2002; Garsky et al.,
2001; Gediya et al., 2005; Gududuru et al., 2005). Treat-
ments with standard cytotoxic agents do provide some
palliative relief, but are associated with system toxicity.
On the other hand, excessive production of dihydrotes-
tosterone has been implicated in this pathological condition.
Steroidal 5a-reductase is an NADPH-dependent enzyme that
catalyzes the irreversible conversion of 4-en-3-oxosteroid,
i.e., testosterone (T) to the corresponding 5a-H-3-oxoster-
oid, i.e., dihydrotestosterone (DHT) (Fig. 1) (Bruchoksky
et al., 1996). Two isozymes of 5a-reductase have been
Abbreviations
BPH
T
Benign prostatic hyperplasia
Testosterone
DHT
DCC
Dihydrotestosterone
Dicyclohexylcarbodiimide
DU-145 Prostate cancer cell line
N. Dhingra ꢀ T. R. Bhardwaj ꢀ N. Mehta ꢀ M. Kumar (&)
University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh 160-014, India
e-mail: manoj_uips@pu.ac.in
T. R. Bhardwaj
I.S.F. College of Pharmacy, Ferozepur Road,
Moga 142001, India
T. Mukhopadhyay ꢀ A. Kumar
National Centre for Human Genome Studies and Research,
Panjab University, Chandigarh 160-014, India
123

818
Med Chem Res (2011) 20:817–825
Fig. 1 Mechanism of 5a-reductase enzyme
Fig. 3 Structures of some potent progesterone esters
cloned, expressed, and characterized based on difference in
chromosomal localization, tissue expression pattern, and
biochemical properties (Bruchoksky et al., 1996; Andersson
and Russell, 1990). Therefore, 5a-reductase inhibitors rep-
resent one of the mainstay interventions in the treatment of
benign prostatic hyperplasia. During the past two decades a
number of non-steroidal (Occhiato et al., 2004) and steroidal
compounds (Chen Li et al., 1994; Kenny et al., 1997) have
been prepared as competitive or non-competitive inhibitors
of 5a-reductase.
literature has not mentioned much about the evaluation of
cytotoxicity along with 5a-reductase inhibitory activity.
Thus, in this study, we prepared a series of 17-oximino-5-
androsten-3b-yl ester steroids, and all the synthesized
compounds were evaluated for antiproliferative activity,
acute toxicity, and their effect on serum androgen levels.
Results and discussion
Hartmann synthesized number of pregnenolone (1, 2a–
2e) (Fig. 2)-based steroids bearing an oxime group con-
nected directly or via a spacer to the steroidal D ring, where
the oxime group is capable to form a coordinate bond with
heme iron of enzyme (Hartmann et al., 2000). On the other
hand, progesterone esters (3a, 3b) (Fig. 3) synthesized in
Mexico laboratories exhibited high antiandrogenic activity
(Cabeza et al., 2001).
Chemistry
For the syntheses of compounds (10a–10j), 17-oximino-5-
androsten-3b-ol (9) was used as starting material. The 9 was
synthesized from commercially available (25R)-5-spirosten-
3b-ol (Diosgenin) (4) according to the literature (Scheme 1)
(Mason and Kepler, 1945; Hershberg, 1948; Regan and
Hayes, 1956). Representative esters (10a–10j) were pre-
pared by treating 3b-hydroxyl function with various acids in
dichloromethane in the presence of dicyclohexylcarbodi-
imide (DCC). In the esterification reaction, DCC acts as
dehydrating agent which forms an O-acylurea called acid–
DCC complex, similar to an acid anhydride or acyl halide.
This is followed by attack of alcohol on carboxylic carbon of
acid–DCC complex, as a nucleophilic catalyst to give esters
and dicyclohexyl urea as side product (March, 2001).
Taking into consideration the 5a-reductase inhibitory
activity of the reported oximes (1, 2a–2e) and importance
of the ester group in 3a and 3b, it was considered of
interest to introduce both the oxime and ester functional-
ities in the steroidal androstane skeleton. It is expected that
such molecules will suitably bind with the enzyme. The
Biological evaluation
Antiproliferative activity on DU-145
Newly synthesized compounds were evaluated for their
antiproliferative activity in comparison to reference drug
Finasteride at five different concentrations using prostate
specific cancer cell line DU-145 (Mosmann, 1983). The
percentage of viable cells and percentage growth inhibition
values are presented in Fig. 4 and Table 1. Linear
Fig. 2 Structures of some potent pregnenolone oximes
123

Med Chem Res (2011) 20:817–825
819
Scheme 1
regressed line was drawn to calculate the concentration
required to cause 50% inhibition in cell growth (IC50)
(Table 2). The conclusions summarized in the following
paragraph are based on the significance (P \ 0.001).
All the compounds (10a–10j) evaluated at National
Centre for Human Genome Studies and Research, Panjab
University, Chandigarh for antiproliferative activity against
DU-145 cell line demonstrated a general reduction in the
level of cellular cytotoxicity. Antiproliferative activity was
optimal with unsubstituted analog (phenyl ring) and its
para-substituted analogs. Compounds 10b–10e, 10g, 10h,
and 10j were found to be more active with over 80%
123

820
Med Chem Res (2011) 20:817–825
Fig. 4 Log dose–response
relationship with regard to
cytotoxicity of the compounds
on the number of living cells
(DU-145) relative to the control.
Each point represents a
mean SEM of 3 independent
experiments. Linear regressed
line was drawn to calculate the
IC50. ANOVA followed by
Tukey’s was applied. Data
significantly different from the
reference drug (P \ 0.001)
Table 1 Antiproliferative activity of the compounds 9, 10a–10j
% Growth inhibition (mean SEM)a
Compound
0.01 lg/ml
0.5 lg/ml
1.0 lg/ml
2.0 lg/ml
5.0 lg/ml
Finasteride
9
5.00 1.58
Nib
8.83 8.40
Nib
16.48 2.49
Nib
36.0 8.95
Nib
78.51 4.63
Nib
10a
10b
10c
10d
10e
10f
53.0 0.48
47.02 1.67
41.50 0.71
71.82 4.24
62.65 2.46
30.68 3.52
43.41 0.77
42.60 2.21
Nib
55 4.23
48.86 1.66
60.51 4.90
62.10 11.39
76.89 5.94
51.43 3.22
57.1 5.96
62.99 4.78
Nib
56 1.22
56.0 0.75
85.87 4.08
74.03 0.77
83.89 6.70
91.39 1.50
65.56 0.51
69.17 7.48
89.55 0.96
Nib
59.23 2.43
91.10 2.10
81.53 1.79
91.02 1.25
91.54 0.19
69.46 3.76
84.47 0.74
92.94 0.13
Nib
67.62 1.62
67.26 3.70
79.54 7.76
83.08 0.75
59.23 2.77
64.90 7.40
84.99 3.99
Nib
10g
10h
10i
10j
33.59 7.66
24.25 6.37
36.19 9.85
76.52 5.58
87.81 1.11
a
Antiproliferative effect is expressed as a percentage of control and is mean SEM of triplicate measurements
b
Ni no significant inhibition
growth inhibition at concentration of 5.0 lg/ml relative to
activity up to 2.0 lg/ml as comparable to Finasteride, but
further there was no significant increase in growth
inhibition.
Finasteride (78% at 5.0 g/ml), whereas compounds 10a
(4.8 lm) and 10i (6.5 lm) showed somewhat lower
activity than that of reference Finasteride. Structure activ-
ity relationship from present antiproliferative study dem-
onstrated that unsubstituted phenyl analog 10b and
compounds 10d, 10e, and 10h with an electron donating
moiety at para position found to be more potent than ref-
erence Finasteride. On the other hand, analogs 10c, 10g
with electron withdrawing substituents demonstrated rela-
tively less cytotoxicity. However, compounds 10a and
10i were mainly inactive. 17-Oximino-5-androsten-3b-yl
4-methoxybenzoate (10f) showed and maintained strong
In vitro cytotoxicity using mouse macrophages
(acute toxicity)
In vitro cytotoxicity test using cancer cell lines in the
preliminary evaluation of cytotoxic agents enables us to
select most potent compound, but cytotoxic agents, how-
ever, frequently exhibit unspecific toxicity. Nevertheless,
the ability to selectively kill the target cell remains a highly
desirable property of potential new therapeutic cytotoxic
123

Med Chem Res (2011) 20:817–825
821
Table 2 Inhibitory
concentration of the
investigated compounds
Table 3 Acute toxicity of the
investigated compounds
Compound
IC50 (lm)
Compound
LC50 (lm)
Finasteride
9
3.9
NfaNdb
Finasteride
9
28.2
Nda
28.0
89.4
19.5
29.4
22.0
14
10a
4.8
10a
10b
10c
3.8
10b
10c
3.07
3.7
10d
10e
10d
10e
2.9
10f
5.4
10f
10g
10h
10i
3.4
10g
10h
10i
24.0
22
2.3
6.5
11.8
89
a
Ni not significant inhibition
10j
6.3
10j
b
a
Nd not determined
Nd not determined
agents (Cholody et al., 2005). In vitro toxicity of newly
synthesized compounds was tested with Red dye uptake
(MTT) assay (Valasinas et al., 2001). The assay quantifies
the viable cells, after 24 h incubation of cells with five
different concentrations. Figure 5 demonstrated a direct
and proportional relation between cell number and con-
centration. The results obtained from MTT assay were
statistically significant (P \ 0.001) and linear equation
obtained allowed us to determine toxicity index (LC50).
The summarized data have been presented in Table 3.
Concerning the toxicity of the compounds toward mouse
macrophages, the results of our study clearly indicated that
compounds 10b and 10j with high LC50 values were non-
toxic to mouse macrophages. Acute toxicity of the com-
pounds 10a, 10d, 10e, 10g, and 10h was comparable to
Finasteride while the toxicity of compounds 10c, 10i, and
10f was about 1.5 times higher than that of reference drug.
In vivo (effect on serum androgen level)
Enzymes involved in the biosynthesis and metabolism of
testosterone are attractive target for designing and develop-
ment of the drugs to be useful in treatment of benign prostatic
hyperplasia (BPH) as indicated Fig. 1. Intact male rats
(Sprague–Dawley, 200–250 g) were used in the designed
study in which various compounds were compared for in
vivo 5a-reductase inhibitory potency, as judged by their
ability to attenuate the conversion of testosterone into
dihydrotestosterone (DHT) (Gerhard, 2002; Hartmann et al.,
2000). ELISA for T were found to be suitable for determi-
nation in the serum of rats since the cross-reactive DHT
levels were quite low in males. The procedure measures T
equally well, and method met all the requirements of preci-
sion, accuracy, sensitivity, and selectivity (Stahl et al., 1984)
The results of the various administrated compounds on the
serum concentration level of testosterone have been pre-
sented in Fig. 6. All the compounds except for 10e and 10f
have shown significant increase in serum testosterone level
as compared to the control. Ester derivatives 10d and 10h
with electron releasing group at p-position of phenyl ring
have been found to possess increased activity. While the
presence of electron withdrawing moiety at this position
causes the loss of activity (10c and 10g). Significant decrease
in activity has been found in the compound 10a and 10j with
an extra methylene group.
Conclusion
Antiandrogenic activity of the newly synthesized esters of
17-oximino-5-androsten-3b-ol, together with acute toxicity
and cytotoxicity, supports the fact that esterification of the
hydroxy group at position 3 with p-substituted aromatic
acid gives compounds better antiproliferative and antian-
drogenic activity.
Fig. 5 Toxicity of the compounds to mouse macrophages (Balb C).
Cell viability was determined based on the MTT assay. Each point
represents a mean value and SEM of 3 independent experiments.
* P \ 0.001 are significantly different compared to Finasteride
according to the one-way ANOVA followed by Tukey’s test
123

822
Med Chem Res (2011) 20:817–825
stirred for 48 h at room temperature. Disappearance of the
starting material and completion of the reaction were
confirmed by TLC. The precipitated dicyclohexylurea
(DCU) was filtered, and solvent was removed under vac-
uum. The resulting residue was crystallized from ethyl
acetate: petroleum ether (60:80).
17-Oximino-5-androsten-3b-yl chloroacetate (10a) 17-
Oximino-5-androsten-3b-yl chloroacetate (10a) (0.24 g,
48.0%), was prepared by method as described above using
chloroacetic acid (0.16 g, 1.6 mmol): mp 96–100°C; IR
(KBr): 3490, 2940, 2820, 1750, 1700, and 1200 cm-1; 1H-
NMR (400 MHz, CDCl3): d 1.01 (s, 3H, 18-CH3), 1.05 (s,
3H 19-CH3), 4.03 (s, ClCH2COO), 4.70 (m, 1H, 3a-H), and
5.40 ppm (br, 1H, 6-vinylic); Anal. Calcd for C21H30NO3Cl:
N, 3.69. Found: N, 3.52.
Fig. 6 Effect of compounds on serum level of testosterone. Results
are mean SEM of five experiments. * P \ 0.05 significant as
compared to control, a P \ 0.05 significant as compared to Finasteride
17-Oximino-5-androsten-3b-yl benzoate (10b) The com-
pound 10b (0.32 g, 64.0%) was prepared using benzoic
acid (0.2 g, 1.6 mmol) by above described method: mp
166–170°C; IR (KBr): 3300, 2930, 1700, 1650, and
Experimental section
Chemistry
1
1235 cm-1; H-NMR (400 MHz, CDCl3): d 0.89 (s, 3H,
The melting points were determined on Veego melting
point apparatus and are uncorrected. 1H-NMR spectra were
obtained using Brucker AC-300F, 300 MHz and Brucker
AC-400F, 400 MHz spectrometer for solutions in CDCl3,
DMSO-d6 and are reported in parts per million (ppm),
downfield from tetramethylsilane (TMS) as internal stan-
dard. The spin multiplicities are indicated by the symbols, s
(singlet), d (doublet), t (triplet), q (quartlet), m (multiplet),
and br (broad). Infrared (IR) spectra were obtained with
Perkin-Elmer 882 Spectrum and RXI, FT-IR model using
potassium bromide pellets (in cm-1). The ultraviolet
spectra were recorded on Perkin-Elmer, Lambda 15 spec-
trophotometer. Elemental analyses were carried out on a
Perkin-Elmer 2400 CHN elemental analyzer. The reactions
were monitored, and the homogeneity of the products was
checked by TLC. Plates for thin layer chromatography
(TLC) were prepared with silica gel G and activated at
110° for 30 min. Silica gel G60 F aluminum sheets were
used for final monitoring. The plates were developed by
exposure to iodine vapor. Anhydrous sodium sulfate was
utilized as drying agents. All the solvents were freshly
distilled and dried prior to use according to standard
procedure.
18-CH3), 1.1 (s, 3H 19-CH3), 4.07 (m, 1H, 3a-H), 5.40 (br,
1H, 6-vinylic), and 6.84–7.28 (m, 5H, aromatic); Anal.
Calcd for C26H33NO3: N, 3.44. Found: N, 3.74.
17-Oximino-5-androsten-3b-yl 4-nitrobenzoate (10c) 4-
Nitrobenzoic acid (0.27 g, 1.6 mmol) was used to prepare
17-oximino-5-androsten-3b-yl 4-nitrobenzoate compound
(10c) (0.34 g, 68.0%) by above described method: mp
185–188°C; IR (KBr): 3300, 2930, 1700, 1650, and
1
1234 cm-1, H-NMR (400 MHz, CDCl3): d 0.89 (s, 3H,
18-CH3), 1.03 (s, 3H 19-CH3), 3.85 (m, 1H, 3a-H), 5.4 (br,
1H, 6-vinylic), 6.65 (d, J = 6.8, 2H, 3-CH, and 5-CH aro-
matic), 7.87 (d, J = 7.0, 2H, 2-CH, and 6-CH aromatic);
Anal. Calcd for C26H32N2O5: N, 6.19. Found: N, 5.64.
17-Oximino-5-androsten-3b-yl 4-aminobenzoate (10d) 17-
Oximino-5-androsten-3b-yl 4-aminobenzoate (10d) (0.28 g,
56.0%) was prepared by method described above using
4-aminobenzoic acid (0.22 g, 1.6 mmol): mp 165–168°C;
IR (KBr): 3330, 2930, 1700, 1630, and 1240 cm-1; 1H-NMR
(400 MHz, CDCl3): d 0.93 (s, 3H, 18-CH3), 1.05 (s, 3H
19-CH3), 2.17 (1H, NOH), 4.08 (m, 1H, 3a-H), 6.20 (br, 1H,
6-vinylic), 7.22 (d, J = 8.3, 2H, 3-CH, and 5-CH aromatic),
and 8.26 ppm (d, J = 8.4, 2H, 2-CH, and 6-CH aromatic);
Anal. Calcd for C26H34 N2O3: N, 6.63. Found: N, 6.79.
General procedure for preparation of compounds
10a–10j
17-Oximino-5-androsten-3b-yl 4-hydroxybenzoate (10e)
The compound 10e (0.31 g, 62.0%) was prepared using
4-hydroxybenzoic acid (0.23 g, 1.6 mmol) by above
described method: mp 179–182°C; IR (KBr): 3310, 2930,
1710, 1680, and 1235 cm-1; 1H-NMR (400 MHz, CDCl3):
To a stirred solution of 17-oximino-5-androsten-3-ol (9)
(0.5 g, 1.6 mmol) and dicyclohexylcarbodiimide (DCC)
(0.34 g, 1.6 mmol) in anhydrous dichloromethane
(30.0 ml) was added acid (1.6 mmol) and the mixture was
123

Med Chem Res (2011) 20:817–825
823
d 0.89 (s, 3H, 18-CH3), 1.05 (s, 3H 19-CH3), 2.17 (1H,
NOH), 4.18 (m, 1H, 3a-H), 6.35 (br, 1H, 6-vinylic), 6.83
(d, J = 8.1, 2H, 3-CH, and 5-CH aromatic), and 7.46 ppm
(d, J = 8.4, 2H, 2-CH, and 6-CH aromatic); Anal. Calcd
for C26H33 NO4: N, 3.31. Found: N, 3.32.
mentioned method to get the 17-Oxo-17a-aza-D-homo-5-
androsten-3b-yl phenoxyacetate (10j) (0.27 g, 54.0%): mp
118–122°C; IR (KBr): 3200, 2940, 1750, 1650, and
1
1220 cm-1; H-NMR (400 MHz, CDCl3): d 0.88 (s, 3H,
18-CH3), 1.02 (s, 3H, 19-CH3), 4.60 (s, 2H, –OCH2), 4.7 (m,
1H, 3a-H), 5.40 (br, 1H, 6-vinylic), and 7.29 (m, 5H, aro-
matic); Anal. Calcd for C27H35 NO4: N, 3.20. Found: N, 3.60.
17-Oximino-5-androsten-3b-yl 4-methoxybenzoate (10f) 4-
Methoxybenzoic acid (p-anisic acid) (0.24 g, 1.6 mmol) was
used to prepare 17-oximino-5-androsten-3b-yl 4-methoxy-
benzoate compound (10f) (0.31 g, 62.0%) by above descri-
bed method: mp 122–125°C; IR (KBr): 3340, 2935, 1735,
1670, and 1250 cm-1; 1H-NMR (400 MHz, CDCl3): d 0.77
(s, 3H, 18-CH3), 0.84 (s, 3H 19-CH3), 3.78 (s, 3H, –OCH3),
4.06 (m, 1H, 3a-H), 5.97 (br, 1H, 6-vinylic), 6.83 (d, J = 7.9,
2H, 3-CH, and 5-CH aromatic), and 7.46 ppm (d, J = 8.0,
2H, 2-CH, and 6-CH aromatic); Anal. Calcd for C27H35 NO4:
N, 3.20. Found: N, 3.07.
Biological evaluation
Chemicals and biochemicals
Reagent grade chemicals were used without purification.
Dulbecco’s modified eagle medium (DMEM), fetal bovine
serum, sodium dihydrogen phosphate, disodium hydrogen
phosphate, and dimethyl sulfoxide were purchased from Hi
Media (Bombay). Finasteride was obtained as a gift sample
from Cipla, Bombay (India) and was of analytical grade
(assay 99.9%). MTT for assay was purchased from Sigma-
Aldrich Chemicals.
17-Oximino-5-androsten-3b-yl 4-chlorobenzoate (10g) 4-
Chlorobenzoic acid (0.26 g, 1.6 mmol) was used to obtain
17-oximino-5-androsten-3b-yl 4-chlorobenzoate (10g)
(0.3 g, 60.0%) by above described method: mp 167–170°C;
IR (KBr): 3310, 2930, 1740, 1650, and 1235 cm-1; 1H-NMR
(400 MHz, CDCl3): d 1.03 (s, 3H, 18-CH3), 1.04 (s, 3H
19-CH3), 2.17 (1H, NOH), 4.15 (m, 1H, 3a-H), 5.40 (br, 1H,
6-vinylic), 7.50 (d, J = 7.0, 2H, 3-CH, and 5-CH aromatic),
and 7.97 ppm (d, J = 7.2, 2H, 2-CH, and 6-CH aromatic);
Anal. Calcd for C26H32NO3Cl: N, 3.17. Found: N, 3.25.
Cell culture and animals
Human prostate cancer cell line, DU-145, was procured
from National Centre for Cell Science (Pune, India), and
cells were grown in DMEM supplemented with 10% heat
inactivated fetal bovine serum, 100 lg/ml streptomycin,
and 100 lg/ml penicillin in a highly humidified 5% CO2 at
37°C in NUAIRE incubator.
17-Oximino-5-androsten-3b-yl 4-methylbenzoate (10h)
The compound 10h (0.29 g, 58.0%),was prepared using
4-methylbenzoic acid (p-toluic acid) (0.22 g, 1.6 mmol) by
above described method: mp 135–139°C; IR (KBr): 3310,
Albino mice (laca strain) weighing 20–25 g of either sex
and Sprague–Dawley rats were procured from Central
Animal House, Panjab University, Chandigarh. Animals
were housed under standard conditions and allowed to free
access to both food and water available ad libitum until
used.
1
2930, 1700, 1650, and 1235 cm-1; H-NMR (400 MHz,
CDCl3): d 0.89 (s, 3H, 18-CH3), 1.03 (s, 3H 19-CH3), 2.41
(1H, NOH), 4.08 (m, 1H, 3a-H), 6.25 (br, 1H, 6-vinylic),
7.20 (d, J = 7.9, 2H, 3-CH, and 5-CH aromatic), and
7.43 ppm (d, J = 8.0, 2H, 2-CH, and 6-CH aromatic);
Anal. Calcd for C27H35NO3: N, 3.32. Found: N, 3.77.
Samples
All steroids were dissolved in ethanol and diluted to
appropriate concentration: 0.01, 0.5, 1.0, 2.0, and 5.0 lg/ml
from the two stock solutions of 1 mg/ml and 0.001 lg/ml.
Stocks were maintained at room temperature.
17-Oximino-5-androsten-3b-yl 4-phenylacetate (10i) 17-
Oximino-5-androsten-3b-yl phenylacetate (10i) (0.29 g,
58.0%) was prepared by method as described above using
phenylacetic acid (0.22 g, 1.6 mmol): oily residue; IR
In vitro antiproliferative activity on DU-145
(MTT assay)
(KBr): 3320, 2935, 1730, and 1250 cm-1
;
1H-NMR
(400 MHz, CDCl3): d 0.91 (s, 3H, 18-CH3), 1.05 (s, 3H 19-
CH3), 3.65 (s, 2H, –CH2), 4.65 (m, 1H, 3a-H), 5.45 (br, 1H,
6-vinylic), and 7.32 (m, 5H, aromatic); Anal. Calcd for
C27H35 NO3: N, 3.37. Found: N, 3.39.
Newly synthesized compounds were evaluated for their
growth inhibitory activity using MTT assay. This assay
quantifies the viable cells by observing the reduction of
tetrazolium salt, MTT, to formazan crystals by the live
cells. Based on the absorbance of the cell sample after the
test is carried out, cell viable can be measured.
17-Oximino-5-androsten-3b-yl phenoxyacetate (10j) Phe-
noxyacetic acid (0.24 g, 1.6 mmol) was used in above-
123

824
Med Chem Res (2011) 20:817–825
For this purpose DU-145 cell line was used, and cells
ethanol (95:5) and administered once intraperitoneally
equimolar to 40 mg/kg body weight of Finasteride. Control
animals were given corresponding amount of vehicle only.
Animals were divided into 3 groups: vehicle (control),
Finasteride (standard), and treated (test sample), and each
group consists of 5 animals. Sprague–Dawley rats were
treated with Finasteride and equimolar dose of compounds.
After 6 h of treatment, blood was withdrawn by cardiac
puncture under diethyl ether anesthesia and serum was
separated from cells by centrifugation. Plasma testosterone
values were obtained by ELISA plate reader at 450 nm and
are given in ng/ml [38, 39].
were grown as described above. Cells were cultured at a
density of 5 9 103 cells/well in 96-well plates at 37°C in
5.0% CO2 atmosphere and were allowed to attach for 24 h.
The cells were treated in triplicate with graded concen-
tration of sample and reference drug Finasteride at 37°C for
48 h. A 20 ll aliquot of MTT solution was added directly
to all the appropriate wells. Following 4 h of incubation at
37 ll, the media were removed and formazan crystals,
which results from the reduction of MTT by active cell
were dissolved in 100 ll DMSO and vigorously mixed to
dissolve the reacted dye. The absorbance of each well was
read on Elisa plate reader (Merck) at 570 nm. The spec-
trometer was calibrated to zero absorbance using culture
medium without cells. The relative cell viability (%) rela-
ted to control well containing cell culture medium without
drug was calculated by [A]test/[A]control 9 100.
Elisa
The aliquots of 50 ll of each of standards, control, and
unknown (serum samples) were added to testosterone
antibody-coated wells. 100 ll of HRP–testosterone conju-
gate was added to all the wells, and the plates were shaken
gently on a shaker for proper mixing of the reagents. Fol-
lowing 4 h of incubation at 37°C, the incubation mixture
was removed. The wells were washed with phosphate buffer
for 5–6 times (200 ll each time), followed by addition of
100 ll of H2O2 substrate in each of the wells. The plates
were further incubated at 37°C for 20 min. At the end of
incubation, reaction was stopped by using 100 ll of 0.5 M
H2SO4 as stopping reagent. The absorbance of each well
was read on Elisa plate reader at 450 nm [40, 41].
% Growth inhibition
¼ ½ODꢁcontrol ꢂ ½ODꢁtest=½ODꢁcontrol ꢃ 100
½ODꢁtest ¼ absorbance test sample
½ODꢁcontrol ¼ absorbance control sample
In vitro cytotoxicity using mouse macrophages
[acute toxicity (MTT assay)]
Cells (mouse macrophages) were used as normal cells and
plated at a density of 5 9 103 cells/well in 96 plates at 37°C
in 5% CO2. Cells were exposed in graded concentration of
compounds at designated various concentration. Each
concentration was tested in triplicate wells. After 48 h,
fresh MTT 20 ll (1 mg/ml) was added directly to all the
wells and culture was incubated for 4 h at 37°C. During this
incubation, MTT was converted into a water insoluble
formazan complex by metabolic activity of viable cells.
Formazan crystals were taken and dissolved in 100 ll of
DMSO, which gives light pink color. The absorbance of
each well was read on Elisa plate reader (Merck) at 570 nm.
The spectrometer was calibrated to zero absorbance using
culture medium without cells. The relative cell viability (%)
related to control well containing cell culture medium
without drug was calculated by [A]test/[A]control 9 100.
Acknowledgments The grants received from the University Grants
Commission, New Delhi and Council of Scientific and Industrial
Research, New Delhi are gratefully acknowledged. We are thankful to
Mr. Dewank, Archer Chem. Mumbai, for providing free sample of
finasteride.
References
Andersson S, Russell DW (1990) Structural and biochemical
properties of cloned and expressed human and rat steroid 5
alpha-reductase. Proc Natl Acad Sci USA 87:3640–3644
Berry SJ, Coffey DS, Walsh PC, Ewing LL (1984) The development
of human benign prostatic hyperplasia with age.
132:474–479
J Urol
Brady SF, Pawluczyk JM, Lumma PK, Feng DM, Wai JM, Jones R,
DeFeo-Jones D, Wong BK, Miller-Stein C, Lin JH, Oliff A,
Freidinger RM, Garsky VM (2002) Design and synthesis of a
prodrug of vinblastine targeted at treatment of prostate cancer
with enhanced efficacy and reduced systematic toxicity. J Med
Chem 45:4706–4715
Bruchoksky N, Sadar MD, Akakura K, Goldenberg SL, Matsuoka K,
Rennie PS (1996) Characterization of 5 alpha-reductase gene
expression in stroma and epithelium of human prostate. J Steroid
Biochem Mol Biol 59:397–404
% Cell viability ¼ ½Aꢁtest=½Aꢁcontrol ꢃ 100
½Aꢁtest ¼ absorbance test sample
½Aꢁcontrol ¼ absorbance control sample
In vivo (effect on serum androgen levels)
Bullock TL, Andriole GL (2006) Emerging drug therapies for
benign prostatic hyperplasia. Expert Opin Emerg Drugs
11:111–123
In order to measure the serum androgen level, all the
compounds were suspended in mixture of olive oil and
123

Med Chem Res (2011) 20:817–825
825
Cabeza M, Heuze I, Bratoeff E, Murillo E, Ramirez E, Lira A (2001)
New progesterone esters as 5a-reductases inhibitors. Chem
Pharm Bull (Tokyo) 49:1081–1084
Chen Li X, Singh C, Labrie SM (1994) The enzyme and inhibitors of
4-ene-3-oxosteroids 5-alpha. Steroids 60:430–441
Jakobsen CM, Denmeade SR, Isaacs JT, Gady A, Olsen CE,
Christensen SB (2001) Design, synthesis and pharmacological
evaluation of thapsigargin analogues for targeting apoptosis to
prostatic cancer cells. J Med Chem 44:4696–4703
Kenny B, Blagg SJ, Fox D (1997) Options in the treatment of benign
prostatic hyperplasia. J Med Chem 40:1293–1312
March J (2001) Advanced organic chemistry, 5th edn. Wiley, New
York
Mason HL, Kepler EJ (1945) Isolation of steroids from the urine of
patients with adrenal cortical tumors and adrenal cortical
hyperplasia: a new 17-ketosteroid, androstane-3(a),11-diol-17-
one. J Biol Chem 161:235–237
Mosmann TJ (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
Immunol Methods 65:55–63
Occhiato EG, Guarna A, Danza G, Serio M (2004) Selective non-
steroidal inhibitors of 5a-reductase type-1. J Steroid Biochem
Mol Biol 88:1–16
Cholody WM, Cholody TK, Hollingshead MG, Haripraksha HK,
Michejda CJ (2005) A new synthetic agent with potent but
selective cytotoxic activity against cancer.
48:4474–4481
J Med Chem
Garsky VN, Lumma P, Feng DM, Wai J, Ramjit HG, Sardana MK,
Oliff A, Jones RE, Frendinger RM (2001) The synthesis of a
prodrug of doxorubicin designed to provide reduced systematic
toxicity and greater target efficacy. J Med Chem 44:4216–4224
Gediya LK, Chopra P, Purushottamachar P, Maheshwari N, Njar VC
(2005) A new simple and high yield synthesis of suberoylanilide
hydroxamic acid and its inhibitory effect alone or in combination
with retinoids on proliferation of human prostate cancer cells.
J Med Chem 48:5047–5057
Gerhard VH (2002) Drug discovery and evaluation: pharmacological
assays, 2nd edn. Springer-Verlag, New York
Perez-Stable C (2006) 2-Methoxyestradiol and paclitaxel have similar
effects on the cell cycle and induction of apoptosis in prostate
cancer cells. Cancer Lett 231:49–64
Regan EM, Hayes FN (1956) 17- and 17a-Aza-D-homosteroids. J Am
Chem Soc 78:639–643
Gududuru V, Hurh E, Dalton JT, Miller DD (2005) Discovery of 2-
arylthiazolidine-4-carboxylic acid amides as a new class of
cytotoxic agents for prostate cancer. J Med Chem 48:2584–2588
Hartmann RW, Hector M, Haidar S, Ehmer PB, Reichert W, Jose J
(2000) Synthesis and evaluation of novel steroidal oxime
inhibitors of P450 17 (17a-hydroxylase/C17–20-lyase) and 5a-
reductase types 1 and 2. J Med Chem 43:4266–4277
Stahl F, Gotz F, Dorner G (1984) Plasma testosterone levels in rats
under various conditions. Exp Clin Endorcinol 84:277–284
Valasinas A, Sarkar A, Reddy VK, Marton LJ, Basu HS, Frydman B
1
(2001) Conformationally restricted analogues of N, 14N-biseth-
Hershberg EB (1948) Regeneration of steroid ketones from their
semicarbazones with pyruvic acid. J Org Chem 13:542–546
Isaacs JT, Coffey DS (1989) Etiology and disease process of benign
prostatic hyperplasia. Prostate Suppl 2:33–50
ylhomospermine (BE-4 -4 -4 -): synthesis and growth inhibitory
effects on human prostate cancer cells. J Med Chem 44:390–403
123