Synthesis, antiproliferative, acute toxicity and assessment of antiandrogenic activities of some newly synthesized steroidal lactams

Article abstract of DOI:10.1016/j.ejmech.2010.01.064

The 17-oxo-17a-aza-d-homo-5-androsten-3β-yl esters (13-22) were synthesized from commercially available (25R)-5-spirosten-3β-ol (Diosgenin) (6) as starting material. The synthesized compounds were evaluated for their antiproliferative activity, acute toxicity and effect on serum androgen level and were compared with Finasteride as positive controls. Some of the compounds exhibited better cytotoxicity and antiandrogenic activity than the reference control. The detailed synthesis, spectroscopic data and pharmacological screening for the synthesized compounds were reported.

Full text of DOI:10.1016/j.ejmech.2010.01.064

European Journal of Medicinal Chemistry 45 (2010) 2229–2236
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative, acute toxicity and assessment of antiandrogenic
activities of some newly synthesized steroidal lactams
,
Neelima Dhingra a, T.R. Bhardwaj a b, Neeraj Mehta a, Tapas Mukhopadhyayc, Ashok Kumar c,
,
Manoj Kumar a
*
a University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160 014, India
b I.S.F. College of Pharmacy, Ferozepur Road, Moga 142 001, India
c National Centre for Human Genome Studies and Research, Panjab University, Chandigarh 160 014, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The 17-oxo-17a-aza-
D-homo-5-androsten-3b-yl esters (13–22) were synthesized from commercially
Received 13 June 2009
Received in revised form
23 January 2010
Accepted 25 January 2010
Available online 2 February 2010
available (25R)-5-spirosten-3
b
-ol (Diosgenin) (6) as starting material. The synthesized compounds were
evaluated for their antiproliferative activity, acute toxicity and effect on serum androgen level and were
compared with Finasteride as positive controls. Some of the compounds exhibited better cytotoxicity and
antiandrogenic activity than the reference control. The detailed synthesis, spectroscopic data and
pharmacological screening for the synthesized compounds were reported.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Dihydrotestosterone
Five alpha reductase enzyme
Benign prostatic hyperplasia
17a-Aza steroids
Androgen
1. Introduction
cause significant decrease in proliferation rate and are useful for
the treatment of disease that involve abnormal or uncontrolled
Benign prostatic hyperplasia (BPH) is the nonmalignant
enlargement of the prostate gland with increase in numbers of both
epithelial and stromal cells within the periurethal transition zone
of the prostate, resulting in the constriction of prostatic urethra [1].
The prevalence increases to 50% by the age of 60 years and to 90%
by the age of 85 years [2].
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) [3]. Cells
die in response to developmental signals and the process is
characterized by number of biochemical changes. Any influence
between the physiological process of cell proliferation and cell
death may lead to change in prostate size with the subsequent
development of abnormalities in the gland [4]. So it is reasonable
to assume that cytotoxic agents are able to induce apoptosis,
cell proliferation.
Nature treasure has an abundant source of cytotoxic agents
obtained from various plant sources like Paclitaxel [5], Thapsia
garganica [6] and extract of Vitex agnus–castus fruit [7]. Number of
semi-synthetic derivatives like vinblastine [8], doxorubicin A [9],
fluoroindolo carbazoles [10], certain derivatives of quinoline [11]
have also been reported as therapeutic agents for the treatment
of symptomatic BPH. Various synthetic derivatives of sub-
eroylanilide hydroxamic acid [12], 2-arylthiazolidine-4-carboxylic
acids [13] etc. has been reported to possess significant cytotoxic
property. Though treatment with standard cytotoxic agents does
provide some palliative relief but are associated with system
toxicity.
The management of BPH has undergone a rapid evolution over
the past decade to aid men with lower urinary tract symptoms
attributed to bladder outlet obstruction. Although not fully defined,
the sources of symptoms in patient with BPH appear to be both
static and dynamic component [14,15]. Treatment of clinical BPH
aims to improve symptoms, prevent urinary tract infections, avoid
renal insult, relief obstruction and improve bladder emptying.
Great strides in the development of antiandrogen have fueled this
evolution.
Abbreviations: Benign prostatic hyperplasia, BPH; Testosterone, T; Dihy-
drotestosterone, DHT; Dicylcohexylcarbodiimide, DCC; Prostate cancer cell lines,
DU-145; [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium-bromide], MTT;
Dulbecco’s modified eagle medium, DMEM.
* Corresponding author. Tel.: þ91 172 2534115(office); fax: þ91 172 2541142.
E-mail address: manoj_uips@pu.ac.in (M. Kumar).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.064

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N. Dhingra et al. / European Journal of Medicinal Chemistry 45 (2010) 2229–2236
OH
OH
5 α - reductase type 1 and 2
Prostatic Growth
O
O
H
NADP
NADPH
Dihydrotestosterone
Testosterone
Fig. 1. Androgen dependent growth of prostate.
The biological basis of androgen ablation therapy lies in the
observation that the embryonic development of the prostate is
4-azasteroids were also studied for 5a-reductase inhibitory activity.
This led to the development of Dutasteride (2), by Glaxo Smith
Kline in 2002, a new dual inhibitor able to reduce the DHT level by
85% [26].
dependent on the androgen [16]. Steroidal 5
dependent enzyme that catalyzes the irreversible conversion of
4-ene-3-oxosteroid i.e. testosterone (T) to the corresponding 5 -H-
3-oxosteroid i.e. dihydrotestosterone (DHT) (Fig. 1) [17]. Two
isozymes of 5 -reductase have been cloned, expressed and char-
a-reductase is a NADPH
a
The 5a-reductase inhibitory activity of these azasteroids is
considered to be attributed by the lactam in ring A of the steroidal
nucleus that mimics intermediate transition state [27]. Other
inhibitors which look promising are the steroidal SK& F 105 687 (3)
[27] and turosteride (4) [28] and progesterone ester (5) [29].
Recently our laboratory has reported 3D-QSAR SOMFA studies
focused on refining the molecular architecture of new steroidal
a
acterized based on difference in chromosomal localization, tissue
expression pattern and biochemical properties [18,19]. Therefore,
5a-reductase inhibitors represent one of the mainstay interven-
tions in the treatment of BPH. During the last two decades
a number of non-steroidal [20] and steroidal compounds [21,22]
have been prepared as competitive or non-competitive inhibitors
inhibitors of human 5
[30,31].
a-reductase for the management of BPH
of 5
comparatively high inhibitory activity as exemplified by Finasteride
(MK-906) (1) (Fig. 2) [23]. Finasteride was the first 5 -reductase
inhibitor clinically approved in 1992 in U.S. for the treatment of
BPH. It has demonstrated its biochemical efficacy with an 80%
reduction of intraprostatic DHT with and a 28% reduction in pros-
tate size in patients with BPH and this compound is currently used
for the treatment of BPH [24,25]. A series of other 17–substituted
a
-reductase. Of these, 4-aza steroids were found to possess
Thus aiming at the discovery of potent, selective anti-
proliferative agent with reduced toxicity and active 5 -reductase
inhibitor, steroid molecules were utilized as biological vector for
chemotherapeutic agents. Given the significance of lactam in some
of clinically proved drugs (1,2) and ester group in 5 with increased
antiandrogenic activity, we reasoned to synthesize compounds
a
a
with general structure having lactam in ring
D and various esters at
3b
position of androsten nucleus. The synthesized compounds
CF3
CF3
CH3
CH3
O
H
O
NH
N
C
C
CH3
CH3
C
CH3
CH3
CH3
H
CH3
H
O
N
H
O
N
H
(1)
(2)
CH(CH3)2
CH(CH3)2
H
O
O
H
N
N
N
C
C
C
CH3
CH3
CH3
CH3
O
CH3
H
CH3
O
N
HOOC
(3)
CH3
(4)
CH3
C
O
R
CH3
CH3
O
R=OCOCH3
R=OCO-C6H5
Cl
(5)
Fig. 2. Structures of some reported potent compounds.

N. Dhingra et al. / European Journal of Medicinal Chemistry 45 (2010) 2229–2236
2231
were evaluated for antiproliferative activity and effect on serum
androgen level.
were tested at five different concentrations in the culture medium
and Finasteride (1) was used as reference drug. The percentage
viable cells and percentage growth inhibition value are presented in
Fig. 4 and Table 1. Linear regressed line was drawn to calculate the
2. Results and discussion
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).
2.1. Chemistry
In comparison to Finasteride, 17a-aza-
compounds reported in this paper revealed a general reduction in
the level of cellular cytotoxicity. 17-Oxo-17a-aza- -homo-5-
androsten-3 -yl 4-nitrobenzoate (15) displayed a better cytotox-
icity comparable with that of Finasteride in DU-145 cells.
Compound 16 with amino group at para position instead of nitro
exhibited similar activity as 15. More in the terms of p-substitution,
groups like -OH (17), –Cl (19) and –CH3 (20) have been found to
increase the potency, suggesting that para-position of phenyl ring
can tolerate wide variety of groups. At low concentrations,
compound 17, 19 and 20 showed strong activities and maintained
D-homo-17-one based
For the syntheses of compounds 13–22, 3b-hydroxy-17a-aza-D-
homo-5-androsten-17-one (12) was used as starting material. The
12 was synthesized from commercial available (25R)-5-spirosten-
D
b
3
b
-ol (Diosgenin) (6) according to the literature as shown in Fig. 3
[32–34]. Esters 13–22 of 3 -hydroxy-17a-aza- -homo-5-andros-
ten-17-one (12) were prepared by treating 3 -hydroxyl function
b
D
b
with various acids in dichloromethane in presence of dicyclohex-
ylcarbodiimide (DCC) [35].
2.2. In vitro antiproliferative activity using cell lines DU-145
a relatively high activity upto 2.0
teride but there was no further significant increase in growth
mg/mL as comparable to Finas-
Compounds were tested for antiproliferative activity using
DU-145 cell line as described by Mosamann [36]. All the compounds
Fig. 3. Synthesis of 17-oxo-17a-aza-D-homo-5-androsten-3b-yl esters (13–22).

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N. Dhingra et al. / European Journal of Medicinal Chemistry 45 (2010) 2229–2236
obtained allowed us to determine toxicity index (LC50). The
summarized data is presented in Table 3.
Data from this study clearly indicated that compounds 13, 17
and 19 with high LC50 values were non toxic to mouse macro-
phages. We found that low doses of 17 upon 24 h of exposure
induced more than 20% of cell mortality and further there was not
significant decrease in the number of viable cell over the range of
five concentrations, whereas the viability of cells were hardly
reduced to 6% with 5
mg/mL of 13 and 19. The prepared analogue 14
was less toxic to mouse macrophages showing LC50 of 43.65
m
m.
Acute toxicity of the compounds 15, 16, 20 and 21 was compa-
rable to Finasteride while the toxicity of compounds 18 and 22 was
about 1.5–4 times higher than that of reference drug.
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).
2.4. In vivo effect on steroid androgen level
Enzyme involved in the biosynthesis and metabolism of
testosterone are attractive target for designing and development of
the drugs to be useful in treatment of BPH as indicated in 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 effect on serum androgen level, as judged by the their ability to
attenuate the conversion of T into DHT (Fig. 2). ELISA for T was
found to be suitable for determination in serum of rats since the
cross reactive DHT levels were extra low in male. The procedure
measure T equally well and method met all the requirements of
precision, accuracy, sensitivity and selectivity [38].
inhibition. This loss in potency may be due to saturation of cells.
Unsubstituted benzoyloxy ester 14 was found to be 19 fold less
active and the concentration required to cause 50% growth inhi-
bition will be more than reference drug. Introduction of spacer like
–CH2– and –OCH2– separating the phenyl ring and carboxylic
carbon furnished the compounds 21 and 22, but no significant
increase in the percentage growth inhibition was observed.
However, 18 has not shown any growth inhibition in prostate cells,
further suggesting that unsubstituted (benzoate) and p-substituted
Serum T level were increased in Finasteride treated rats
(1.26 ꢀ 0.02 ng/mL compared with 0.742 ꢀ 0.07 ng/mL control rats).
benzoate at 3
b of 17a-aza-D-homo-5-androsten-17-one plays an
important role in providing potency and selectivity.
It is apparent from Fig. 6 that almost all the ester derivatives of 3b-
hydroxy-17a-aza-D-homo-5-androsten-17-one have increased the
2.3. In vitro cytotoxicity using mouse macrophages (acute toxicity)
serum T level as compared to control. The effect on potency of
different substitutents at para-position of phenyl ring is in
approximate order of –NO2 (15) > –NH2 (16) > –CH3 (20) > –OH
(17) > –Cl (19) except for –OCH3 (18) where no significant change in
concentration of serum T has been found. Analogues 13 and 18 with
poor antiproliferative activity showed satisfactory increased level
of T upto 0.82 ꢀ 0.029 and 1.05 ꢀ 0.068 ng/mL. It is worth
In vitro cytotoxicity using DU-145 cells in the preliminary eval-
uation of anti-cancer drugs enable us to select most potent
compound, but cytotoxic agents however frequently exhibit-
unspecific toxicity. Nevertheless the ability to selectively kill the
target cell remains a highly desirable property of potential new
therapeutic cytotoxic agents. In this study, we have demonstrated
the applicability of red dye uptake (MTT) assay using mouse
macrophages (Balb C) for in vitro toxicity testing of newly synthe-
sized compounds [37]. The assay quantifies the viable cells after
24 h incubation of cells with five different concentrations. Fig. 5
demonstrated a direct and proportional relation between cell
number and concentration. The results obtained from MTT assay
were statistically significant (P < 0.001) and linear equation
mentioning that 17a-aza-
substituted at 3 with benzoate or p-substituted benzoate offer the
optimal ring substitution pattern for good antiandrogenic
activity. This increased biological activity of 17a-aza- -homo-5-
D-homo-5-androsten-17-one steroids
b
A
D
androsten-17-one steroids may be because of formation of product
like transition state. These results are consistent with earlier
observations of increased activity of 4-azasteroids as 5
inhibitor [39].
a-reductase
Table 1
Antiproliferative activity of compounds 13–22.
Compound
% Growth inhibition (mean ꢀ SEM)a
0.01
m
g/mL
0.5
m
g/mL
1.0
m
g/mL
2.0
mg/mL
5.0 mg/mL
Finasteride
5.00 ꢀ 1.58
8, 83 ꢀ 8.40
16.48 ꢀ 2.49
36.0 ꢀ 8.95
78.51 ꢀ 4.63
12
13
14
15
16
17
18
19
20
21
22
Nib
Nib
Nib
Nib
Nib
Nib
Nib
Nib
Nib
Nib
28.11 ꢀ 1.18
33.92 ꢀ 2.52
30.54 ꢀ 2.49
30.68 ꢀ 3.52
17.37 ꢀ 0.07
18.98 ꢀ 0.34
21.63 ꢀ 1.22
Nib
35, 69 ꢀ 3.33
41.43 ꢀ 0.70
43.41 ꢀ 1.10
51.43 ꢀ 3.22
25.17 ꢀ 3.19
27.88 ꢀ 2.19
29.51 ꢀ 0.19
Nib
38.78 ꢀ 2.73
57.91 ꢀ 6.51
45.03 ꢀ 5.12
59.23 ꢀ 2.7
21.34 ꢀ 4.55
42.24 ꢀ 2.19
40.69 ꢀ 5.59
Nib
45.03 ꢀ 4.20
73.51 ꢀ 2.14
48.05 ꢀ 2.83
65.56 ꢀ 0.51
33.92 ꢀ 1.70
49.37 ꢀ 0.85
68.06 ꢀ 2.05
48.27 ꢀ 4.07
Nib
63.65 ꢀ 1.06
88.89 ꢀ 1.51
82.41 ꢀ 2.69
69.46 ꢀ 3.76
43.05 ꢀ 5.32
67.62 ꢀ 2.58
76.97 þ 2.83
73.14 ꢀ 14.28
Nib
Nib
Nib
Nib
a
Antiproliferative effect is expressed as a percentage of control and is mean ꢀ SEM of triplicate measurements.
Ni ¼ not significant inhibition.
b

N. Dhingra et al. / European Journal of Medicinal Chemistry 45 (2010) 2229–2236
2233
Table 2
Inhibitory concentrations of the investigated compounds.
Compound
IC50
3.9
(mm).
Finasteride
12
13
14
15
16
17
18
19
20
21
22
NiaNdb
NiaNdb
9.5
5.2
7.1
5.7
15.1
8.3
7.5
7.1
NiaNdb
a
Ni ¼ not significant inhibition.
b
Nd ¼ not determined.
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 Finas-
teride according to the one-way ANOVA followed by Tukey’s test.
3. Conclusions
In this paper various 3
b esters of androsten having lactam in
ring are prepared and evaluated for antiproliferative effect on well
D
established prostate cancer cells, acute toxicity on mouse macro-
phages and in vivo effect on serum androgen level.
Structure activity relationship from antiproliferative study
revealed that 17a-aza-D-homo-5-androsten-17-one steroids
dimethylsulfoxide and are reported in parts per million (ppm),
downfield from tetramethylsilane (TMS) as internal standard. 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 Spec-
trum and RXI, FT-IR model using a potassium bromide pellets (in
cmꢁ1). The ultraviolet spectra were recorded on Perkin–Elmer,
Lambda 15 spectrophotometer. Elemental analyses were carried
out on a Perkin–Elmer 2400 CHN elemental analyzer. 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 110ꢂ for 30 min. Silica gel G60 F
aluminum sheets plates were used for final monitoring. The plates
were developed by exposure to iodine vapour. Anhydrous sodium
sulphate was used as drying agents. All the solvents were dried and
freshly distilled prior to use according to standard procedure.
possess significant antiproliferative activity. With respect to
substitution, p-substituted benzoyloxy (15,16) were by far the most
effective antiproliferative agents. However, the exact mechanism of
action these compounds remain to be elucidated. The present study
indicates that compounds possess a potential for development into
anti-cancer drugs that may prove to be effective against prostate
cells.
Among the compounds tested for acute toxicity using mouse
macrophages, none of the compound has been found to be toxic to
the normal cells even at high concentration except few of them.
Thus preliminary evaluation in vitro cytotoxicity and toxicity
studies enabled us to screen or select potent compounds with
reduced toxicity such as 13, 15, 16, 17 and 20.
As the compounds were potent in vitro on the prostate cancer
cells it was worthwhile to pursue with investigation of in vivo effect
on serum androgen level by measuring the serum T level in rat
model. Some of the selected compounds (15, 16, 17 and 20) showed
potent antiandrogenic activity. It is noteworthy that steroid struc-
tural requirement for good antiandrogenic activity and anti-
proliferative activity are similar.
In vitro and in vivo experiments have been found to be very
encouraging and that may prove as potential lead for the devel-
opment of compounds to be used in the treatment of BPH with
their dual action by controlling the growth of prostate with their
cytotoxicity activity towards prostate cell and by decreasing the
DHT level without having negative effect on normal cells.
4.1.1. General procedure for preparation of 13–22
To a stirred solution of 3b-hydroxy-17a-aza-D-homo-5-andros-
ten-17-one (12) (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 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 removed under
vacuum. The resulting residue was crystallized from ethyl acetate:
petroleum ether (60:80).
Table 3
Acute toxicity of the investigated compounds.
4. Experimental section
Compound
LC50 (mm).
Finasteride
28.2
Nda
4.1. Chemistry
12
13
14
15
16
17
18
19
20
21
22
94.07
43.65
27.79
25.10
75
The melting points were determined on Veego melting point
apparatus and are uncorrected. Proton nuclear magnetic resonance
(1H NMR) spectra were obtained using Brucker AC-300F, 300 MHz
and Brucker AC-400F, 400 MHz spectrometer for solutions in
deuteriochloroform, deuterated dimethylsulfoxide and are repor-
ted in parts per million (ppm), downfield from tetramethylsilane
(TMS) as internal standard. Carbon nuclear magnetic resonance
8.4
100.5
22.79
26
11.7
(
13C NMR) spectra were obtained using Brucker AC-400F, 400 MHz
spectrometer for solutions in deuteron chloroform, deuterated
a
Nd ¼ not determined.

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N. Dhingra et al. / European Journal of Medicinal Chemistry 45 (2010) 2229–2236
5.95 (br, 1H, 6-vinylic), 6.62 (d, J ¼ 8.3, 2H, 3-CH and 5-CH aromatic)
and 7.42 ppm (d, J[8.9, 2H, 2-CH and 6-CH aromatic); 13C NMR
(400 MHz, CDCl3): 171.67 (NHCO), 167.23 (COO), 154.47 (ArC-4),
d
141.19 (C-5), 130.04 (2ArCH), 120.77 (ArC-1), 115.60 (2ArCH), 120.04
(C-6), 70.50 (C-3), 24.68 (C-19) and 20.45 ppm (C-18). Anal. Calcd. for
C26H34N2O3 (%): N, 6.63. Found: N, 6.10.
4.1.1.5. 17-Oxo-17a-aza-D-homo-5-androsten-3b-yl 4-hydroxybenzoate
(17). The compound 17 (0.29 g, 58.0%) was prepared using
4-hydroxybenzoic acid (0.23 g, 1.6 mmol) by above described method:
mp 162–165 ꢂC; IR (KBr, cmꢁI): 3310, 2935, 1720, 1680, 1240; 1H NMR
(400 MHz, CDCl3):
(m, 1H, 3
-H), 5.80 (br, 1H, 6-vinylic), 6.73 (d, J ¼ 8.1, 2H, 3-CH and
5-CH aromatic), 6.86 (1H, NH) and 7.55 ppm (d, J ¼ 8.4, 2H, 2-CH and
6-CH aromatic); 13C NMR (400 MHz, CDCl3):
171.89 (NHCO), 166.37
d 1.18 (s, 3H, 18-CH3), 1.40 (s, 3H, 19-CH3), 4.12
a
d
(COO), 157.56 (ArC-4), 141.73 (C-5), 131.19 (2ArCH), 123.21 (ArC-1),
114.83 (2ArCH), 120.27 (C-6), 70.53 (C-3), 24.39 (C-19) and 20.53 ppm
(C-18). Anal. Calcd. for C26H33 NO4 (%): N, 3.31. Found: N, 2.91.
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, ap < 0.05 significant as
compared to Finasteride.
4.1.1.6. 17-Oxo-17a-aza-
(18). 4-Methoxybenzoic acid (p-anisic acid) (0.24 g, 1.6 mmol)
was used to prepare 17-oxo-17a-aza- -homo-5-androsten-3 -yl
D-homo-5-androsten-3b-yl 4-methoxybenzoate
D
b
4-methoxybenzoate compound (18) (0.22 g, 44.0%) by above
4.1.1.1. 17-Oxo-17a-aza-
(13). 17-Oxo-17a-aza-
D
-homo-5-androsten-3
b
-yl chloro acetate
-yl chloroacetate
described method: mp 180–183 ꢂC; IR (KBr, cmꢁI): 3440, 2940, 1730,
D
-homo-5-androsten-3
b
1680, 1245; 1H NMR (400 MHz, CDCl3):
d
1.07 (s, 3H, 18-CH3), 1.19
(13), (0.24 g, 48.0%), was prepared by method as described above
using chloroacetic acid (0.15 g, 1.6 mmol): mp 165–170 ꢂC; IR
(KBr, cmꢁI): 3290, 2890, 1720, 1620, 1210; 1H NMR (400 MHz,
(s, 3H, 19-CH3), 3.83 (s, 3H, CH3O–), 4.12 (m, 1H, 3 -H), 6.12 (br, 1H,
a
6-vinylic), 6.89 (d, J ¼ 7.9, 2H, 3-CH and 5-CH aromatic) and 7.54 ppm
(d, J ¼ 8.0, 2H, 2-CH and 6-CH aromatic); 13C NMR (400 MHz, CDCl3):
CDCl3):
-H), 4.06 (s, 2H, CH2COO), 5.36 (br, 1H, 6-vinylic) and 6.96 ppm
(1H, NH); 13C NMR (400 MHz, CDCl3):
171.82 (NHCO), 168.50
d
0.99 (s, 3H, 18-CH3), 1.18 (s, 3H, 19-CH3), 3.54 (m, 1H,
d 171.45 (NHCO), 169.75 (COO), 154.87 (ArC-4), 141.06 (C-5), 129.48
3
a
(2ArCH), 120.96 (ArC-1), 113.37 (2ArCH), 120.32 (C-6), 70.77 (C-3),
53.63 (CH3O–), 24.54 (C-19) and 20.38 ppm (C-18). Anal. Calcd. for
C27H35 NO4 (%): N, 3.20. Found: N, 3.03.
d
(COO), 140.42 (C-5), 119.66 (C-6), 70.10 (C-3), 48.77(–CH2COO–),
24.27 (C-19) and 20.24 ppm (C-18). Anal. Calcd. for C21H30NO3Cl
(%): C, 66.39; H, 7.96; N, 3.69. Found: C, 65.81; H, 8.23; N, 3.16.
4.1.1.7. 17-Oxo-17a-aza-D-homo-5-androsten-3b-yl 4-chlorobenzoate
(19). 4-Chlorobenzoic acid (0.26 g, 1.6 mmol) was used to obtain
4.1.1.2. 17-Oxo-17a-aza-
D
-homo-5-androsten-3
b
-yl benzoate (14). The
17-oxo-17a-aza-D-homo-5-androsten-3b-yl 4-chlorobenzoate (19)
compound 12 (0.25 g, 50.0%) was prepared using benzoic acid (0.2 g,
(0.3 g, 60.0%) by above described method: mp 172–175 ꢂC; IR (KBr,
1.6 mmol) by above described method: mp 155–158 ꢂC; IR (KBr,
cmꢁI): 3320, 2930, 1722, 1680, 1240; 1H NMR (400 MHz, CDCl3):
d 1.26 (s, 3H, 18-CH3),1.45 (s, 3H, 19-CH3), 4.08 (m, 1H, 3a-H), 6.14 (br,
cmꢁI): 3320, 2960, 1710, 1680,1240; 1H NMR (400 MHz, CDCl3):
d
0.99
-H), 5.37 (br, 1H, 6-
vinylic) and 7.42 ppm (m, 5H, aromatic); 13C NMR (400 MHz, CDCl3):
171.40 (NHCO), 167.87 (COO), 141.09 (C-5), 137.03 (ArC-4), 130.73
(s, 3H, 18-CH3), 1.20 (s, 3H, 19-CH3), 3.53 (m, 1H, 3
a
1H, 6-vinylic), 7.36 (1H, NH), 7.50 (d, J ¼ 6.2, 2H, 3-CH and 5-CH
aromatic) and 8.07 ppm (d, J ¼ 7.2, 2H, 2-CH and 6-CH aromatic); 13C
d
NMR (400 MHz, CDCl3): d 171.23 (NHCO), 168.41 (COO), 156.31 (ArC-
(ArC-1), 128.56 (2ArCH), 126.65 (2ArCH), 120.47 (C-6), 70.73 (C-3),
24.51 (C-19) and 19.82 ppm (C-18). Anal. Calcd. for C26H33 NO2 (%): N,
3.44. Found: N, 4.0.
4), 141.09 (C-5), 131.91 (2ArCH), 128.83 (2ArCH), 126.96(ArC-1),
120.30 (C-6), 70.70 (C-3), 25.69 (C-19) and 20.32 ppm (C-18). Anal.
Calcd. for C26H32NO3Cl (%): N, 3.17. Found: N, 3.52.
4.1.1.3. 17-Oxo-17a-aza-
D
-homo-5-androsten-3
b
-yl 4-nitrobenzoate
4.1.1.8. 17-Oxo-17a-aza-D-homo-5-androsten-3b-yl 4-methylbenzoate
(15). 4-Nitrobenzoic acid (0.27 g, 1.6 mmol) was used to prepare
(20). The compound 20 (0.32 g, 64.0%) was prepared using
4-methylbenzoic acid (p-toluic acid) (0.22 g, 1.6 mmol) by above
described method: mp 165–172 ꢂC; IR (KBr, cmꢁI): 3320, 2930, 1720,
17-oxo-17a-aza-D-homo-5-androsten-3b-yl 4-nitrobenzoate (15)
(0.33 g, 66.0%) by above described method: mp 290–292 ꢂC; IR (KBr,
cmꢁI): 3180, 2970, 1740, 1670, 1250; 1H NMR (400 MHz, CDCl3) :
1660, 1240; 1H NMR (400 MHz, CDCl3):
d
1.07 (s, 3H, 18-CH3), 1.19
d
1.07 (s, 3H, 18-CH3), 1.19 (s, 3H, 19-CH3), 4.90 (m, 1H, 3
a-H), 5.45
(s, 3H, 19-CH3), 2.37 (s, 3H, CH3), 4.06 (m, 1H, 3 -H), 6.26 (br, 1H,
a
(br, 1H, 6-vinylic), 5.99 (1H, NH), 8.20 (d, J ¼ 7.0, 2H, 3-CH and 5-CH
6-vinylic), 7.19 (d, J ¼ 7.9, 2H, 3-CH and 5-CH aromatic), 7.26 (1H, NH)
aromatic) and 8.28 ppm (d, J ¼ 6.8, 2H, 2-CH and 6-CH aromatic);
and 7.43 ppm (d, J ¼ 8.0, 2H, 2-CH and 6-CH aromatic); 13C NMR
13C NMR (400 MHz, CDCl3):
d
171.47 (NHCO), 166.43 (COO), 150.19
(400 MHz, CDCl3): d 171.80 (NHCO), 170.44 (COO), 144.10 (ArC-4),
(ArC-4), 141.05 (C-5), 136.80 (ArC-1), 130.83 (2ArCH), 123.47
(2ArCH), 120.07 (C-6), 70.64 (C-3), 24.91 (C-19) and 20.07 ppm (C-
18). Anal. Calcd. for C26H32 N2O5 (%): N, 6.19. Found: N, 5.84.
141.19 (C-5), 134.10 (2ArCH), 129.13 (2ArCH), 127.13 (ArC-1), 120.87
(C-6), 70.75 (C-3), 24.86 (C-19), 21.73(4 CH3–) and 21.47 ppm (C-18).
Anal. Calcd. for C27H35 NO3 (%) : N, 3.32. Found: N, 3.20.
4.1.1.4. 17-Oxo-17a-aza-D-homo-5-androsten-3b-yl 4-aminobenzoate
4.1.1.9. 17-Oxo-17a-aza- -yl phenylacetate
D
-homo-5-androsten-3
b
(16). 17-Oxo-17a-aza- -homo-5-androsten-3
D
b-yl 4-aminobenzoate
(21). 17-Oxo-17a-aza- -homo-5-androsten-3b-yl phenylacetate
D
(16) (0.26 g, 52.0%) was prepared by method described above using
4-aminobenzoic acid (0.22 g, 1.6 mmol): mp 177–182 ꢂC; IR (KBr,
cmꢁI): 3460, 3220, 2970, 1730, 1640, 1240; 1H NMR (400 MHz,
(21) (0.27 g, 54.0%) was prepared by method as described above
using phenylacetic acid (0.22 g, 1.6 mmol): mp 195–197 ꢂC; IR (KBr,
cmꢁI): 3200, 2960, 1730, 1690, 1240; 1H NMR (400 MHz, CDCl3):
CDCl3):
d
1.18 (s, 3H, 18-CH3), 1.40 (s, 3H, 19-CH3), 4.15 (m, 1H, 3
a-H),
d 0.99 (s, 3H, 18-CH3), 1.16 (s, 3H, 19-CH3), 3.59 (s, 2H, CH2COO), 4.62

N. Dhingra et al. / European Journal of Medicinal Chemistry 45 (2010) 2229–2236
2235
(m, 1H, 3
a
-H), 5.36 (br, 1H, 6-vinylic), 6.09 (1H, NH) and 7.30 ppm
vigorously mixed to dissolve the reacted dye. 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 ꢃ 100.
(m, 5H, aromatic); 13C NMR (400 MHz,CDCl3):
d
170.54 (NHCO),
168.89 (COO), 141.02 (C-5), 131.47 (ArC-1), 129.40 (2ArCH), 128.77
(2ArCH), 122.16 (ArC-4), 120.96 (C-6), 71.62 (C-3), 38.14(CH2COO–),
24.54 (C-19) and 20.38 ppm (C-18). Anal. Calcd. for C27H35NO3 (%):
N, 3.32. Found: N, 3.48.
4.1.1.10. 17-Oxo-17a-aza-
(22). Phenoxyacetic acid (0.24 g, 1.6 mmol) was used in above
mentioned method to get the 17-oxo-17a-aza- -homo-5-androsten-
-yl phenoxyacetate (22), (0.25 g, 50.0%): mp 192–195 ꢂC; IR (KBr,
cmꢁI): 3270, 2930,1735,1700,1240; 1H NMR (400 MHz, CDCl3):
0.88
(s, 3H, 18-CH3), 1.04 (s, 3H, 19-CH3), 3.71 (m, 1H, 3 -H), 4.60(s, 2H,
OCH2), 5.41 (br, 1H, 6-vinylic) and 6.97 ppm (m, 5H, aromatic); 13C
NMR (400 MHz, CDCl3): 170.67 (NHCO), 168.12 (COO), 151.65 (ArC-
D-homo-5-androsten-3b-yl phenoxyacetate
% Growth Inhibition ¼ ½ODꢄcontrolꢁ½ODꢄ test=½ODꢄcontrolꢃ100
D
3b
[ OD]test ¼ absorbance test sample
d
[ OD]control ¼ absorbance control sample
a
d
4.2.2. In vitro cytotoxicity using mouse macrophages (acute
toxicity)
1), 139.75 (C-5), 129.30 (2ArCH), 121.23 (ArC-4), 120.47 (C-6), 114.33
(2ArCH),72.57 (C-3), 64.78 (OCH2COO–), 24.59 (C-19) and 20.57 ppm
(C-18). Anal. Calcd. for C27H35 NO4 (%): N, 3.20. Found: N, 3.04.
4.2.2.1. MTT assay. Cells (mouse macrophages) were used as
normal cells and plated at a density of 5 ꢃ 103 cells/well in 96
plates at 37 ꢂC in 5% CO2. Cells were exposed in graded concen-
tration of compounds at designated various concentration. Each
concentration was tested in triplicate wells. After 48 h fresh MTT
4.2. Biological evaluation
4.2.1. In vitro antiproliferative activity using cell lines DU-145
4.2.1.1. Chemical and biochemicals. All the chemicals were of
reagent grade and 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 (Mumbai).
Finasteride was obtained as a gift sample from Cipla, Mumbai
(India) and was of analytical grade (assay 99.9%). MTT for assay was
obtained from Sigma Aldrich Chemicals.
20 ml (1 mg/mL) was added directly to all the wells and culture was
incubated for 4 h at 37 ꢂC. During this incubation, MTT was con-
verted into a water insoluble formazan complex by metabolic
activity of viable cells. Formazan crystal were taken and dissolved
in 100 ml of DMSO, which give light pink color. The absorbance of
each well was read on ELISA plate reader at 570 nm. The spec-
trometer 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 ꢃ 100.
4.2.1.2. Cell culture and animals. Human prostate cancer cell line,
DU-145 was procured from National Center for Cell Science
(Pune, India) and cell line were grown in DMEM media sup-
% Cell Viability ¼ ½Aꢄtest=½Aꢄcontrol ꢃ 100
plemented with 10% heat inactivated fetal bovine serum, 100
m
g/
mL streptomycin and 100
mg/mL penicillin in a highly humidi-
fied atmosphere of 95% air with 5% CO2 at 37 ꢂC in NUAIRE
incubator.
[ A]test ¼ absorbance test sample
[ A]control ¼ absorbance control sample
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 stan-
dard conditions and allowed to free access to both food and water
available ad.libitium until used.
4.2.3. In vivo effect on steroid androgen level
In order to measure the serum androgen level, all the
compounds were suspended in mixture of olive oil and 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),
treated (test sample) and each group consist of 5 animals.
Sprague Dawley rats were treated with Finasteride and equi-
molar 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 [40,41].
4.2.1.3. Samples. All synthesized steroidal compounds were
dissolved in ethanol and diluted to appropriate concentration:
0.01, 0.5, 1.0, 2.0, 5.0
m
g/mL from the two stock solutions of
1 mg/mL and 0.001
temperature.
mg/mL. Stocks were maintained at room
4.2.1.4. MTT assay. 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, viable
cells can be measured.
DU-145 cell line was used and cells were grown as described
above. Cells were cultured at a density of 5 ꢃ 103 cells/well in 96
well plates at 37 ꢂC in 5.0% CO2 atmosphere and were allowed to
attach for 24 h. The cells were treated in triplicate with graded
concentration of sample and reference drug Finasteride at 37 ꢂC for
4.2.3.1. ELISA. The aliquots of 50
and unknown (serum samples) were added to T-antibody coated
wells. 100 l of HRP-T conjugate was added to all the wells and the
ml of each of standards, control
m
plates were shaken gently on a shaker for proper mixing of the
reagents. The mixture was incubated for 4 h at 37 ꢂC. The wells
were washed with phosphate buffer for 5–6 times (200
time), followed by addition of 100 l of H2O2 substrate in each of
the wells. The plates were further incubated at 37 ꢂC for 20 min. At
the end of incubation, reaction was quenched by using 100 l of
ml each
m
48 h. A 20
appropriate wells. Following 4 h of incubation at 37
was removed and formazan crystals, which results from the
reduction of MTT by active cell were dissolved in 100 l DMSO and
mL aliquot of MTT solution was added directly to all the
ml, the media
m
0.5 M H2SO4. The absorbance of each well was read on ELISA plate
reader at 450 nm [42,43].
m

2236
N. Dhingra et al. / European Journal of Medicinal Chemistry 45 (2010) 2229–2236
[18] N. Bruchoksky, M.D. Sadar, K. Akakura, S.L. Goldenberg, K. Matsuoka,
Acknowledgment
P.S. Rennie, J. Steroid. Biochem. Mol. Chem. 59 (1996) 397.
[19] S. Andersson, D.W. Russell, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 3640.
[20] E.G. Occhiato, A. Guarna, G. Danza, M. Serio, J. Steroid Biochem. Mol. Biol. 88
(2004) 1.
[21] X. Li, C. Chen, S.M. Singh, F. Labrie, J. Med. Chem. 60 (1995) 430.
[22] B. Kenny, S. Ballard, J. Blagg, D. Fox, J. Med. Chem. 40 (1997) 1293.
[23] H. Weisser, S. Tunn, M. Debus, M. Krieg, Steroids 59 (1994) 616.
[24] J.R. Brooks, C. Berman, R.L. Primka, G.F. Reynolds, G.H. Rasmsson, Steroids 47
(1986) 1.
The grants received from University Grants Commission, New
Delhi and Council of Scientific and Industrial Research, New Delhi is
gratefully acknowledged. We are thankful to Mr Dewank, Archer
Chem, Mumbai, for generous gift of Finasteride.
[25] E. Bratoeff, E. Ramirez, E. Murillo, G. Flores, M. Cabeza, Curr. Med. Chem. 6
(1999) 1107.
References
[26] T.H. Tarter, E.D. Vaughan, Curr. Pharm. Design 12 (2006) 775.
[27] S.H. Georgianna, J.W. Kozarich, Curr. Opin. Chem. Biol. 1 (1997) 254.
[28] Di Salle, E.G. Briatico, D. Giudici, G. Ornati, A. Panzeri, J. Steroid Biochem. Mol.
Biol. 48 (1994) 241.
[29] M. Cabeza, I. Heuze, E. Bratoeff, E. Murillo, E. Ramirez, A. Lira, Chem. Pharm.
Bull. (Tokyo) 49 (2001) 1081.
[30] S. Thareja, S. Aggarwal, T.R. Bhardwaj, M. Kumar, Eur. J. Med. Chem. 44 (2009)
4920.
[31] S. Aggarwal, S. Thareja, T.R. Bhardwaj, M. Kumar, Eur. J. Med. Chem. 45 (2010)
476.
[32] H.L. Mason, E.J. Kepler, J. Biol. Chem. (1945) 235.
[33] E.B. Hershberg, J. Org. Chem. 13 (1948) 542.
[34] E.M. Regan, F.N. Hayes, J. Am. Chem. Soc. 78 (1956) 639.
[35] J. March, Advanced Organic Chemistry, third ed. John Wiley and Sons, New
York, 2001, p. 349.
[36] T.J. Mosmann, J. Immunol. Methods 65 (1983) 55.
[37] A. Valasinas, A. Sarkar, V.K. Reddy, L.J. Marton, H.S. Basu, B. Frydman, J. Med.
Chem. 44 (2001) 390.
[38] F. Stahl, F. Gotz, G. Dorner, Exp. Clin. Endorinol 84 (1984) 277.
[39] R.K. Bakshi, G.F. Patel, G.H. Rasmusson, W.F. Baginsky, G. Cimis, K. Ellsworth,
B. Chang, H. Bull, R.L. Tolman, G.S. Harris, J. Med. Chem. 37 (1994) 3871.
[40] H. Gerhard, Drug Discovery and Evaluation: Pharmacological Assays, second
ed. Springer-Verlag, Berlin Heidelberg, 2002, p. 1178.
[41] R.W. Hartmann, M. Hector, S. Haidar, P.B. Ehmer, W. Reichert, J. Jose, J. Med.
Chem. 43 (2000) 4266.
[1] T.L. Bullock, G.L. Andriole, Expert Opin. Emerg. Drugs 11 (2006) 111.
[2] S.J. Berry, D.S. Coffey, P.C. Walsh, L.L. Ewing, J. Urol. 132 (1984) 474.
[3] J.T. Isaacs, D.S. Coffey, Prostate(Suppl. 2) (1989) 33.
[4] B. Djavan, M. Remzi, B. Erne, M. Marberger, Drugs of Today 38 (2002) 867.
[5] C. Perez-Stable, Cancer Lett. 231 (2006) 49.
[6] C.M. Jakobsen, S.R. Denmeade, J.T. Isaacs, A. Gady, C.E. Olsen, S.B. Christensen,
J. Med. Chem. 44 (2001) 4696.
[7] M. Weisskop, W. Schaffner, G. Jundt, T. Sulser, S. Wyler, H. Tullberg-Reinert,
Planta Med. 71 (2005) 910.
[8] S.F. Brady, J.M. Pawluczyk, P.K. Lumma, D.M. Feng, J.M. Wai, R. Jones, D. DeFeo-
Jones, B.K. Wong, C. Miller-Stein, J.H. Lin, A. Oliff, R.M. Freidinger, V.M. Garsky,
J. Med. Chem. 45 (2002) 4706.
[9] V.N. Garsky, P.K. Lumma, D.M. Feng, A. Oliff, R.E. Jones, R.M. Frendinger, J. Med.
Chem. 44 (2001) 4216.
[10] B.N. Balasubramanian, D.R. St Laurent, M.G. Saulnier, B.H. Long, C. Bachand,
F. Beaulieu, W. Clarke, M. Deshpande, J. Eummer, C.R. Fairchild,
D.B. Frennesson, R. Kramer, F.Y. Lee, M. Mahler, A. Martel, B.N. Naidu,
W.C. Rose, J. Russell, E. Ruediger, C. Solomon, K.M. Stoffan, H. Wong, J.J. Wright,
K. Zimmermann, D.M. Vyas, J. Med. Chem. 47 (2004) 1609.
[11] I. Gomez-Monterrey, P. Campiglia, P. Grieco, M.V. Diurno, A. Bolognese, P. La
Colla, E. Novellino, Bioorg. Med. Chem. 11 (2003) 3769.
[12] L.K. Gediya, P. Chopra, P. Purushottamachar, N. Maheshwari, V.C. Njar, J. Med.
Chem. 48 (2005) 5047.
[13] V. Gududuru, E. Hurh, J.T. Dalton, D.D. Miller, J. Med. Chem. 48 (2005) 2584.
[14] T. Sraddha, H. Kourals, J. Pharm. Tech 21 (2005) 330.
[15] M. Jonler, M. Riehmann, R.C. Bruskewitz, Drugs 47 (1994) 66.
[16] N. Bruchovsky, J.D. Wilson, J. Biol. Chem. 243 (1968) 2012.
[17] S. Aggarwal, S. Thareja, A. Verma, T.R. Bhardwaj, M. Kumar, Streoids 75 (2010)
109.
[42] M.J. Rassaie, G.L. Kumari, P.K. Pandey, N. Gupta, N. Kochupillai, P.K. Grover,
Steroids 57 (1992) 288.
[43] H. Hosoda, H. Yoshida, Y. Sakai, S. Miyairi, T. Nambara, Chem. Pharm. Bull. 28
(1980) 3035.