Synthesis and biological evaluation of novel unsaturated carboxysteroids as human 5α-reductase inhibitors: A legitimate approach

Article abstract of DOI:10.1016/j.ejmech.2012.06.026

In the present study, novel steroidal 17a-substituted 3-cyano-17a-aza-D- homo-3,5-androstadien-17-ones (12-19) and 17a-substituted 17-oxo-17a-aza-D-homo- 3,5-androstadien-3-oic acids (20-26) were synthesized from dehydroepiandrosterone acetate (6) along with 17-oxo-19-nor-3,5-androstadien-3- oic acid (30) through a multistep synthesis. Compounds were evaluated for their in vitro 5α-reductase inhibitory activity by measuring the conversion of [³H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity was also determined using rat prostate weighing method. Compounds 21-23 and 25 showed potent inhibition of 5α-reductase II enzyme with IC₅₀ values of 54.1 ± 9.5, 22.1 ± 2.4, 72.8 ± 2.3 and 26.5 ± 4.4 nM respectively as compared to Finasteride (30.3 nM) along with a significant (p < 0.05) reduction in rat prostate weight.

Full text of DOI:10.1016/j.ejmech.2012.06.026

European Journal of Medicinal Chemistry 54 (2012) 728e739
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel unsaturated carboxysteroids
as human 5a-reductase inhibitors: A legitimate approach
,
Saurabh Aggarwal a, Suresh Thareja a, T.R. Bhardwaj a b, Jörg Haupenthal c, R.W. Hartmann d,
,
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 142001, India
c Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2_3, 66123 Saarbrücken, Germany
d Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2_3, 66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, novel steroidal 17a-substituted 3-cyano-17a-aza-D-homo-3,5-androstadien-17-
ones (12e19) and 17a-substituted 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acids (20e26) were
synthesized from dehydroepiandrosterone acetate (6) along with 17-oxo-19-nor-3,5-androstadien-3-oic
Received 12 December 2011
Received in revised form
10 June 2012
Accepted 13 June 2012
Available online 21 June 2012
acid (30) through a multistep synthesis. Compounds were evaluated for their in vitro 5
inhibitory activity by measuring the conversion of [3H] androstenedione in human embryonic kidney
(HEK) cells. In vivo 5 -reductase inhibitory activity was also determined using rat prostate weighing
method. Compounds 21e23 and 25 showed potent inhibition of 5 -reductase II enzyme with IC50 values
a-reductase
a
a
Keywords:
of 54.1 ꢀ 9.5, 22.1 ꢀ 2.4, 72.8 ꢀ 2.3 and 26.5 ꢀ 4.4 nM respectively as compared to Finasteride (30.3 nM)
along with a significant (p < 0.05) reduction in rat prostate weight.
Ó 2012 Elsevier Masson SAS. All rights reserved.
17a-Aza steroids
Benign prostatic hyperplasia
Carboxysteroids
Testosterone
Epristeride
Finasteride
5a-Reductase
1. Introduction
-Reductase inhibition has emerged as a key target for the
prostate, testosterone (T) is enzymatically converted to an active
metabolite, 5a-dihydrotestosterone (DHT), by steroid 5a-reductase
enzyme (3-oxo-steroid-4-ene dehydrogenase {E.C. 1.3.99.5}) which
is a system of two membrane bound nicotinamide dinucleotide
5a
management of benign prostatic hyperplasia (BPH) as abnormally
high activity of the enzyme in human’s results in excessive dihy-
drotestosterone levels in peripheral tissues leading to hyperplasia.
BPH is a noncancerous growth of the prostate gland resulting due to
the over-proliferation of the stromal and glandular elements of the
prostate [1]. About 60% of men aged over 50 years have histological
evidence of BPH and, after the age of 70, the proportion increases to
80% [2]. It is a chronic, progressive and highly prevalent disease
which clinically manifests as lower urinary tract symptoms (LUTS)
which includes frequency, hesitancy, urgency, nocturia, slow
urinary stream and incomplete emptying thereby causing socio-
economic burden to the patients [3,4].
phosphate (NADPH) dependent enzymes. Thus
5a-reductase
dictates the cellular availability of DHT to prostatic epithelial cells
and consequently modulates its growth. Once formed, DHT can
bind reversibly to the androgen receptor to regulate prostatic
cellular proliferation and survival. The presence of DHT and its
binding to androgen receptors can directly up-regulate expression
of prostate-specific differentiation markers such as prostate-
specific antigen (PSA) synthesized by the human prostatic epithe-
lial cells and locally active growth factors e the andromedins which
stimulates the proliferation of so-called transit amplifying (TA) cells
within the prostate [8,9]. This has led to the development of
Normal growth, development and maintenance of the prostate
are dependent on the testicular androgens [5e7]. Within the
steroidal and non-steroidal 5
the conversion of T (1) to DHT (2) as shown in Fig. 1 [10e12].
There are mainly two types of 5 -reductase enzymes: The type I
a-reductase inhibitors as they inhibit
a
enzyme which is present mainly in the hair follicles and peripheral
skin and is not the major species expressed in the prostate while
* Corresponding author. Tel.: þ91 172 2541142, þ91 0172 2534115 (O); fax: þ91
172 2541142.
E-mail addresses: aggarwalsau@gmail.com (S. Aggarwal), mkgw4pu@
yahoo.com, manoj_uips@pu.ac.in (M. Kumar).
type II 5
a-reductase is the major isozyme in genital tissues and a
deletion in the gene leads to male pseudohermaphroditism [13,14].
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.026

S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
729
Fig. 1. Site of action of 5a-reductase inhibitors.
The type I isozymes is active at pH 6.0e8.5 while type II is active at
pH 5.0e5.5. More recently with the development of genome-wide
gene expression profile analyses a third type of 5a-reductase
enzyme (type III) has been identified in hormone-refractory pros-
tate cancer cells (HRPC) [15]. This enzyme also converts T (1) to DHT
(2) in HRPC cells in a similar way to type I enzyme and was found to
be active at pH 6.9 [16].
reductase I. It has been shown to be an uncompetitive inhibitor
against both T and NADPH. Because of presumably favourable
electrostatic interaction between the carboxylate and the positively
charged oxidized cofactor, the acrylate preferentially binds in
a ternary complex with enzyme and NADPþ, which leads to the
uncompetitive kinetic mechanism [22e24].
17a-aza-D-Homosteroids belongs to another category of
Finasteride (MK-906) was the first
approved in U.S. for the treatment of BPH [17,18]. Finasteride (3) is
a potent inhibitor of 5 -reductase type II with weak in vitro activity
for 5 -reductase type I having IC50 value of 9.4 and 410 nM,
respectively. At clinical dose, 5 mg/day, it caused 65e80% lowering
of plasma DHT levels [19]. Dutasteride (GG745) (4) is another
related drug approved by United States food and drug adminis-
tration (U.S. FDA) in 2002 for the symptomatic treatment of BPH.
Unlike Finasteride (3), Dutasteride (4) is a competitive inhibitor of
5
a
-reductase inhibitor
compounds which have shown 5a-reductase inhibitory activity. The
most interesting aspect concerning them is the possibility of
“inverted action” or “back binding” as proposed by McDonald et al.
Their proposition was based on the fact that the steroids have the
potential to bind in two orientations at the active site of various
a
a
metabolizing enzymes. Research on 5a-reductase inhibitors has
shown that steroids without side chains can bind to enzymes with
the A-ring of the substance simulating the D-ring of the substrate,
whiletheD-ringemulates the A-ring. Thiscouldlead this compounds
exhibiting same mechanism of action as 4-azasteroids [25].
both 5a-reductase type I and type II isozymes and it reduces DHT
levels >90% following one year of oral administration [20]. It is
a time dependent inhibitor as Finasteride (3) and forms a stable
complex with a slow rate of dissociation constant and does not bind
to the androgen receptor [21]. Epristeride (SK&F 105657) (5),
It is evident from the clinically used 5a-reductase inhibitors in
the management of BPH that planarity is required in ring A of the
steroidal nucleus so as to enter the active site of the enzyme.
Planarity is provided by the presence of double bond between C-1
and C-2 in case of Finasteride (3) and Dutasteride (4), and conjugate
double bonds at C-3 and C-5 in Epristeride (5). Therefore it was
envisaged to synthesize Epristeride (5) related analogues having
a novel 5
a-reductase inhibitor, is an interesting drug in the treat-
ment of BPH. It belongs to class of carboxysteroids (Fig. 2). It is
a potent inhibitor of 5
a-reductase II while a weak inhibitor of 5a-
O
CF3
O
NHC
H
N
F3C
O
N
H
O
N
H
H
H
Finasteride (3)
Dutasteride (4)
O
NHC
HOOC
Epristeride (5)
Fig. 2. Structures of active molecules.

730
S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
sp2-hybridized 3,5-diene-3-oic acid moiety in androstane. In
addition compounds would have 17-oxo-17a-aza-D-homo lactam
in ring D instead of ring A as in Finasteride (3). Results of our
previously published studies have indicated that the bulk around
17 position has to be increased and carboxylic group at position-3
as in Epristeride (5) has to be less bulky and unsubstituted one
acetate) (6) as the starting material by standard procedures. It was
converted to its oxime (7) by refluxing in ethanol with hydroxylamine
hydrochloride and sodium acetate trihydrate. 17-Oximino-5-
androsten-3
onyl chloride gave 17-oxo-17a-aza-D-homo-5-androsten-3
(8). The lactam (8) was hydrolyzed by refluxing with methanolic
potassium hydroxide to obtain -hydroxy-17a-aza-D-homo-5-
b-yl acetate (7) on Beckmann’s rearrangement with thi-
b-yl acetate
for better 5
a
-reductase inhibition [26]. This can be done by
3b
replacing a 5 membered D-ring with a 6 membered D-ring and
substituting the lactam hydrogen with bulky groups.
androsten-17-one (9). Oppenaeur oxidation of compound 9 was
carried out in a mixture of cyclohexanone and toluene to yield 17a-aza-
D-homo-4-androstene-3,17-dione (10).
Guarna et al. synthesized a novel class of compounds 19-nor-10-
azasteroids which were found to be better inhibitors of the 5a-
reductase enzyme [27,28]. Therefore, it was also envisaged to
synthesize some molecules not having 19-methyl group and having
rings A and B mimicking that of Epristeride (5).
2.1.2. Synthesis of 17a-substituted 3-cyano-17a-aza-D-homo-3,5-
androstadien-17-ones
17a-Aza-D-homo-4-androstene-3,17-dione (10) on treatment
with phosphorus tribromide in glacial acetic acid gave 3-bromo-
17a-aza-D-homo-3,5-androstadien-17-one (11) in 85.0% yield. The
bromo compound (11) was refluxed with copper cyanide in dime-
thylformamide to yield 3-cyano-17a-aza-D-homo-3,5-androsta
dien-17-one (12). The cyano compound (12) was treated with
methyl iodide in the presence of sodium hydride in tetrahydrofuran
to form 17a-methyl-3-cyano-17a-aza-D-homo-3,5-androstadien-
17-one (13) (Fig. 4a).
The present paper describes the synthesis of novel steroidal
compounds which were evaluated for 5
a-reductase inhibitory
activity in vitro against 5 -reductase type I and II and in vivo by
a
prostate weighing method.
2. Results and discussion
2.1. Chemistry
3-Cyano-17a-aza-D-homo-3,5-androstadien-17-one (12) was
also treated with ethyl bromide, allyl bromide, benzyl chloride,
acrylonitrile and acetyl chloride respectively to get 17a-ethyl-3-
cyano-17a-aza-D-homo-3,5-androstadien-17-one (14), 17a-allyl-3-
2.1.1. Synthesis of 17a-aza-D-homo-4-androstene-3,17-dione (10)
For the synthesis of proposed compounds, 17a-aza-D-homo-4-
androstene-3,17-dione (10), was synthesized first as depicted in Fig. 3
using 17-oxo-5-androsten-3b-yl acetate (dehydroepiandrosterone
cyano-17a-aza-D-homo-3,5-androstadien-17-one
(15),
17a-
O
NOH
NH2OH.HCl,
C2H5OH
H3COCO
H3COCO
SOCl2,
C6H6
(6)
(7)
H
N
H
N
O
O
KOH,
CH3OH
H3COCO
HO
(8)
(9)
Cyclohexanone,
C6H5CH3
H
N
O
O
(10)
Fig. 3. Synthesis of 17a-aza-D-homo-4-androstene-3,17-dione (10).

S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
731
benzyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-one (16),
17a-cyanoethyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (17) and 17a-acetyl-3-cyano-17a-aza-D-homo-3,5-andros
tadien-17-one (18), 17a-benzoyl-3-cyano-17a-aza-D-homo-3,5-
androstadien-17-one (19) respectively (Fig. 4b).
2.1.4. Synthesis of 17-oxo-19-nor-3,5-androstadien-3-oic acid (30)
4-Androstene-19-nor-dione (27) was converted to 3-bromo-19-
nor-3,5-androstadien-17-one (28) by treating with phosphorus
tribromide in glacial acetic acid. The bromo compound was refluxed
with copper cyanide in dimethylformamide to get 3-cyano-19-nor-
3,5-androstadien-17-one (29). The cyano compound (29) was
hydrolyzed in the presence of ethanolic sodium hydroxide to obtain
17-oxo-19-nor-3,5-androstadien-3-oic acid (30) (Fig. 6).
2.1.3. Synthesis of 17a-substituted 17-oxo-17a-aza-D-homo-3,5-
androstadien-3-oic acids
3-Cyano-17a-aza-D-homo-3,5-androstadien-17-one (12) was
hydrolyzed in the presence of ethanolic sodium hydroxide to
obtain 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acid (20).
Compounds 14e18 were also hydrolyzed to obtain N-methyl (21),
N-ethyl (22), N-allyl (23), N-benzyl (24) and N-17a-(2-propionoxy
ethyl) (25) respectively. 17a-Acetyl-17-oxo-17a-aza-D-homo-3,5-
androstadien-3-oic acid (26) was prepared by reacting 17-oxo-
17a-aza-D-homo-3,5-androstadien-3-oic acid (20) directly with
acetic anhydride in pyridine (Fig. 5) but the hydrolysis of 17a-
benzoyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-one (19)
was not successful because of the N-benzoyl side chain breakdown
in strong basic conditions.
2.2. In vitro human 5
a-reductase inhibitory activity against type I
and type II 5 -reductase enzyme (HEK 293 cells method)
a
Compounds were evaluated in vitro for their inhibitory activity
against human 5 -reductase enzyme I and II. For this purpose
human embryonic kidney cell line (HEK293) which lacks endoge-
nous -reductase activity was transfected with the cDNAs
encoding for both 5 -reductase type I and type II by inserting them
into pRcCMV (Cytomegalovirus promoter of the eukaryotic
expression vector). Single cell clones with substantially high
enzymatic activity were selected and established as permanent cell
a
5
a
a
a
H
H
N
O
N
O
PBr3,
CH3COOH
O
Br
CuCN,
DMF
(10)
(11)
CH3
N
H
N
O
O
NaH,
THF, CH3I
NC
NC
(12)
(13)
R
b
H
N
O
N
O
NaH, THF
Alkylating
agents
NC
NC
(12)
(14) R= -C2H5
(15) R= -CH2CH=CH2
(16) R= -CH2C6H5
(17) R= -CH2CH2CN
(18) R= -COCH3
(19) R= -COC6H5
Fig. 4. (a and b) Synthesis of 17a-substituted 3-cyano-17a-aza-D-homo-3,5-androsta-dien-17-ones.

732
S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
R
R
N
N
O
O
NaOH,
C2H5OH
NC
HOOC
(12) R= -H
(20) R= -H
(13) R= -CH3
(14) R= -C2H5
(21) R= -CH3
(22) R= -C2H5
(15) R= -CH2CH=CH2
(16) R= -CH2C6H5
(23) R= -CH2CH=CH2
(24) R= -CH2C6H5
(17) R= -CH2CH2CN
(25) R= -CH2CH2COOC2H5
COCH3
H
N
O
N
O
(CH3CO)2O, C5H5N
HOOC
HOOC
(20)
(26)
Fig. 5. Synthesis of 17a-substituted 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acids.
lines. The cell lines were used to test selected synthesized
compounds as well as the clinically used steroidal inhibitor Finas-
teride (3) by measuring the conversion of [3H] androstenedione
[29,30]. Reaction products were quantified by a HPLC reversed
phase technique. By the development of this assay method the
conventional BPH microsomal assay and the DU-145 cell free assay
were replaced. Compared to use of microsomal enzymes, whole cell
systems are closer to the in vivo situation. The main disadvantages
of the earlier methods were that in those methods the substrates
used were different i.e. in BPH microsomal assay the substrate used
was T, in DU-145 cell free assay it was androstenedione. In the
present assay, androstenedione is used as substrate for both types
of isozymes. Since source of enzyme is same, better comparable
inhibition values for both isozymes can be obtained in the new
O
O
H
H
PBr3,
CH3COOH
O
Br
(27)
(28)
CuCN,
DMF
O
O
NaOH,
C2H5OH
H
H
NC
HOOC
(29)
(30)
Fig. 6. Synthesis of 17-oxo-19-nor-3,5-androstadien-3-oic acid (30).

S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
733
Table 1
In vitro 5
(12e19).
assay as processing method is similar. In case of earlier BPH
microsomal assay homogenate was prepared from the prostate
tissue of BPH patient. It was cumbersome to seek approval
from patient every time and also individual fluctuations among
the patients were there which in new HEK-II assay has been
replaced. Therefore, this whole cell system gives better and faster
results.
a-reductase inhibitory activities of bromo (11) and cyano compounds
Compound code
Activity
code
Type-I 5
percentage inhibition
at 10
a
-reductase
Type-II 5
percentage inhibition
at 10
a-Reductase
m
M
mM
11
12
13
SAR-16
SAR-1
SAR-2
34.5 ꢀ 1.7
27.6 ꢀ 9.3
93.0 3.1
(64.0 10.1 at 2
56.1 ꢀ 3.7
20.8 ꢀ 3.9
0.0 ꢀ 0.0
45.6 ꢀ 5.2
9.9 ꢀ 5.9
13.5 ꢀ 7.1
Another reason for the use of new assay was that in prostate
gland both types of isozymes of 5
fore, homogenate prepared from prostatic tissue of BPH patient
contained not only 5 -reductase II but also smaller quantity of 5
reductase I due to which inhibition values of the selective inhibitors
could be easily falsified. In HEK-II cells, it is only active 5 -reductase
a-reductase are present; there-
m
M)
14
15
16
17
18
19
SAR-5
SAR-7
SAR-9
SAR-10
SAR-12
SAR-13
48.9 ꢀ 2.7
3.3 ꢀ 5.7
a
a-
34.7 ꢀ 8.6
9.9 ꢀ 10.8
23.2 ꢀ 9.8
11.4 ꢀ 3.9
30.3 nM (IC50)
0.0 ꢀ 0.0
a
2.2 ꢀ 3.0
0.0 ꢀ 0.0
II which is in form of an enzyme preparation from these defined
cells in high quantity. Similarly, in DU-145 cells apart from the
3 (Finasteride)
453.0 nM (IC50
)
5
17
a
b
-reductase I there is also some amount of 5
-hydroxysteroid dehydrogenase which can convert T and
androstenedione. In addition, the 5 -reductase I activity is much
a-reductase II and
Bold values represents to signify the important results.
a
cyano derivatives were not active against the enzyme probably
due to the lack of charge replacement of the enolate oxyanion at the
active site as all other features such as sp2-hybridized centre at C-3,
C-4 and bulk in ring D were present in the molecules as those
present in active molecule Epristeride (5).
With the aim of improving potency and providing charge
replacement of the enolate oxyanion at the active site the above
compounds were hydrolyzed and a carboxylic acid group was
smaller in DU145 cells than in HEK-I cells, so that for one HEK-I
homogenate assay much less cells for the same enzyme yield
would be required which in turn finally save time and costs. For
example in order to obtain an androstenedione conversion about
25% cells must be incubated for 5 h in previous assay while in HEK-I
cells time is 30 min. Hence new assay method provides better,
faster and selective results.
Lysates are obtained after harvesting and resuspending 80%
confluent cells in homogenate buffer (containing 300 mM saccha-
rose, 5 mM TriseHCl and 0.1 mM EDTA) followed by homogeni-
zation using ultrasonication. Suspensions of both cell lines were
used for all following assays. In the inhibitor assays, the compounds
dissolved in DMSO were mixed with androstenedione (test
concentration containing 3H androstenedione: 500 nM), which
served as a substrate, NADPH regenerating system (containing
NADP, glucose-6-phosphate and glucose-6-phosphate dehydroge-
nase) and tris buffer. To start the incubation one volume of the cell
introduced as in Epristeride (5). The results of 5
inhibitory activity of compounds 20e26 at 10 and 2
both type of isozymes along with IC50 against type II enzyme is
reported in Table 2. Out of the series four compounds were found to
a
m
-reductase
M against
be potent inhibitors of 5a-reductase showing inhibition around or
better than the clinically used drug Finasteride (3). 17-oxo-17a-aza-
D-homo-3,5-androstadien-3-oic acid (20) obtained from
compound 12 showed better inhibition than the latter one towards
5a
-reductase II enzyme. Increase in chain length to methyl (21)
leads to increase in potency with strong inhibition of 5 -reductase
a
suspension was added to a total volume of 500
m
l. After an incu-
II with an IC50 value of 54.1 nM which was near to that of the
standard drug Finasteride (3) (30.3 nM).
Further increasing the chain length to ethyl (22) led to the
increase in the activity as the compound 22 exhibited an IC50 value
bation of 30 min at 37 ꢁC, the reaction was stopped by the addition
of ether. The steroids were extracted, dried and resuspended in
methanol followed by radioactivity HPLC based detection. The
amount of converted tritiated androstenedione was measured for
each sample which served to determine the inhibitory activity of
the compounds [29,30].
of 22.1 nM against
5a-reductase II which was better than
the standard drug Finasteride (3) (IC50 ¼ 30.3 nM). It emerged as
the most potent compound of the series. Further substitution with
Finasteride (3), a clinically used drug, was used as the standard in
an allyl group (23) led to the retention of 5a-reductase II activity
the assay. It showed IC50 value of 453.0 nM for 5
enzyme while for 5 -reductase II enzyme the value was 30.3 nM in
the present assay. Epristeride (5) although not tested in assay has
a literature IC50 value of 58e60 nM [31]. The results of 5 -reductase
inhibitoryactivity of our synthesized compounds 11e19 at 10 M are
a
-reductase I
(IC50 value ¼ 72.8 nM). Finally, 17a-(2-propionoxy ethyl)-17-oxo-
a
17a-aza-D-homo-3,5-androstadien-3-oic acid (25) derivative
also showed potent inhibition against
5a-reductase II (IC50
a
value ¼ 26.5 nM) while two synthesized derivatives benzyl (24)
and acetyl (26) were not evaluated because of being unstable on
storage. All the compounds showed very less inhibition against
m
reported in Table 1. Out of the series 17a-methyl-3-cyano-17a-aza-
D-homo-3,5-androstadien-17-one (13) emerged as the most potent
5a-reductase I enzyme suggesting that the carboxylic group
selective inhibitor of 5
a
-reductase type I enzyme. It possesses an
at position-3 provides the selective inhibition towards 5
tase II.
a-reduc-
additional 17a-methyl group as compared to the unsubstituted
compound (12) which also possesses 3-cyano-3,5-diene and D-
homo system. The observation is in concordance with the previously
reported SAR studies [32,33] concluding that an NeCH3 group is
Among 19-nor compounds, the carboxy derivative (30) proved
to be potent 5 -reductase II inhibitor having IC50 in nanomolar
range (212.9 nM) showing that removal of 19-methyl group doesn’t
have a drastic effect on the 5 -reductase inhibitory activity
although the cyano compound (29) was found to be inactive
showing only 1% inhibition against type II enzyme. Results of 5
a
required to have higher potency against 5
to tighter binding at the active site but the compound showed less
potency towards 5 -reductase II inhibition. Further increase in
chain length from methyl to ethyl (14) resulted in reduction of 5
reductase type I inhibition although there was an improvement in
-reductase II inhibition. Substitutions like allyl (15), benzyl (16),
cyanoethyl (17), acetyl (18) and benzoyl (19) resulted in decrease of
inhibition against 5 -reductase type I as well as 5 -reductase type II.
In conclusion, substitution beyond methyl group leads to loss of
activity against both types of 5 -reductase isozymes. In general 3-
a
-reductase I enzyme due
a
a
a-
a
-
reductase inhibitory activity of compounds 29e30 are reported in
Table 3.
5
a
The synthesized compounds are expected to be uncompetitive
inhibitors as Epristeride (5) due to presence of the acrylate in A and
B ring which could lead to formation of ternary complex with the
positively charged cofactor, enzyme and NADPþ thereby possibly
demonstrating uncompetitive mechanism.
a
a
a

734
S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
Table 2
In vitro 5a-reductase inhibitory activities of carboxy compounds (20e26).
Compound code
Activity code
Type-I 5
inhibition at 10
a
-reductase percentage
Type-II 5
inhibition at 10
a
-reductase percentage
Type-II 5
inhibition at 2 mM
a
-reductase percentage
IC50 of type-II
enzyme (nM)
mM
m
M
20
21
22
23
SAR-4
SAR-3
SAR-6
SAR-8
e
0.0 ꢀ 0.0
18.9 ꢀ 1.1
32.3 ꢀ 1.5
0.4 ꢀ 0.5
n.d.
62.9 2.2
100.0 0.0
100.0 0.0
100.0 0.0
n.d.
n.d.
n.d.
99.0 2.1
100.0 0.0
99.7 0.5
n.d.
54.1 9.5
22.1 2.4
72.8 2.3
n.d.
24
25
26
SAR-11
e
8.5 ꢀ 3.0
100.0 0.0
n.d.
100.0 0.0
n.d.
26.5 4.4
n.d.
n.d.
3 (Finasteride)
453.0 nM (IC50
)
e
e
30.3 nM
n.d. ¼ not determined.
Bold values represents to signify the important results.
2.3. In vivo 5
weight in mature male rats)
a
-reductase inhibitory activity (change in rat prostate
partially dependent on DHT for their homeostasis but the reduction
was less significant. There were no significant differences between
treatment groups in adrenal weight, body weight gain, liver weight,
or testicular weights of rats at the conclusion of these studies. The
suppression of prostate and other organs growth in the animals
treated with Finasteride (3) can be explained as a consequence of
an inhibition of DHT formation in these organs.
5a-Reductase enzyme plays a crucial role in human prostate
growth which is an age-related and androgen dependent process
being mediated by DHT [34]. As there are many similarities
between human and rat prostate growth, rat prostate is commonly
used as the growth model although histological and biochemical
17a-Methyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
differences still exist. 5
a
-Reductase enzyme has been isolated and
one (13) was found to be 5
likely to reduce weights of organs where there is presence of 5
reductase I and have lower effect on prostate where predominantly
-reductase II enzyme is present. The results of the study are in
conformation with the same as the compound has miniscule effect
on the reduction of prostate weight. Although it did reduce the
weight of vas deferens and epididymis but the reduction was not
significant.
In case of 17a-substituted-17-oxo-17a-aza-D-homo-3,5-
androstadien-3-oic acids which were found to be potent inhibi-
tors of 5a-reductase II enzyme in vitro; there was a statistically
significant reduction in the weight of ventral prostate and the
magnitude of the reduction of these organ weights were almost
equal with those of the standard drugs.
Although there was reduction in the weight of ventral prostate
as in the case of 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic
acid (20) but it was smaller as compared to the N-substituted
compounds and was not statistically significant. Hence substitution
at 17a-position leads to better inhibitory compounds with signifi-
cant inhibition of enzyme in the ventral prostate. In case of 17a-
methyl-17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acid (21)
presence of N-methyl group increased the reduction (39.50%) over
unsubstituted one (27.33%). N-ethyl (22), N-allyl (23), and N-(2-
propionoxy ethyl) (25) derivatives lead to a statistically significant
reduction of 39.35%, 35.34%, and 33.86%, respectively in the weights
of ventral prostate. In all the compounds there was a reduction in
the weights of dorsal prostate, vas deferens, epididymis and
seminal vesicles on the expected lines. N-methyl (21) derivative
showed statistically significant reduction (31.75%) in weight of
a-reductase I active in vitro and hence is
shown to be expressed in the ventral prostate of the rats [35,36].
Although the rat enzyme has showed similarities to human
enzymes in amino acid sequences (approximately 60% homology)
and activities but in some cases different pharmacological
responses were also shown. For each inhibitor to be evaluated,
mature male wistar rats were domesticated and were given once
daily dose of 1 mg/kg of compounds under test for 14 days and the
inhibitory effects on the growth of prostate and other androgen-
target organs was examined on 15th day after 24 h of last dose
when they were sacrificed and weighed. The following organs were
removed, free of adherent tissue and weighed: ventral prostate,
dorsal prostate, testis (both), vas deferens, epididymis, liver,
adrenal and seminal vesicles. Values are reported as mean ꢀ S.E.M.
for actual weights and percentage of vehicle control and were
obtained by using the analysis of variance feature [37,38].
a-
5a
Among the synthesized compounds, active compounds based
on their in vitro 5a-reductase inhibitory activity along with stan-
dard drug Finasteride (3) were selected for activity. One group
received only vehicle (0.50% CMC) and served as control. The results
of the activity are given in Table 4. The percentage decrease in the
organ weights (ventral prostate, dorsal prostate, vas deferens and
epididymis) of importance is reported in Table 5. In other organs
the decrease was not significant. Treatment with any compound
did not induce any mortality.
Animals treated (2 weeks) with Finasteride (3) or a 5a-reductase
inhibitor were expected to have prostate weights significantly
smaller than those of control animals. Consistent with this expec-
tation, Finasteride (3) produced a statistically significant decrease
in ventral and dorsal prostate where it reduces the weight to
around 44% as compared to control.
epididymis suggesting greater reduction of epididymal 5a-reduc-
tase enzyme while N-(2-propionoxy ethyl) (25) and 19-nor carboxy
compound (30) showed significant reduction in the weight of vas
deferens (37.55% and 34.80% respectively).
Finasteride (3) also reduced the weights of seminal vesicle, vas
deferens and epididymis on expected lines as these organs are also
Table 3
In vitro 5a-reductase inhibitory activities of 19-nor compounds (29e30).
Compound code
Activity code
Type-I 5
inhibition at 10
a
-reductase percentage
Type-II 5
inhibition at 10
a
-reductase percentage
Type-II 5
inhibition at 2 mM
a
-reductase percentage
IC50 of type-II
enzyme (nM)
mM
m
M
29
30
SAR-17
SAR-19
0.0 ꢀ 0.0
1.2 ꢀ 2.1
n.d.
89.5 ꢀ 7.6
n.d.
212.9 28.9
82.1 ꢀ 10.0
100.0 ꢀ 0.0
Bold values represents to signify the important results.

S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
735
Table 5
Percentage change in organ weights after treatment with compounds.
Compound code
Ventral
Dorsal
Vas
Epididymis
prostate
prostate
deferens
Finasteride (3)
13; SAR-2
20; SAR-4
21; SAR-3
22; SAR-6
23; SAR-8
25; SAR-11
30; SAR-19
44.76
6.72
45.32
ꢃ8.07
46.50
39.96
28.37
31.25
28.75
15.62
14.45
14.58
26.33
29.36
19.32
27.89
37.55
34.80
14.40
10.11
13.98
31.75
17.25
24.75
25.41
29.88
27.33
39.50
39.35
35.34
33.86
22.56
Positive values indicate percentage decrease; negative values indicate percentage
increase.
3. Conclusion
In the present study a series of eight novel 17a-substituted 3-
cyano-17a-aza-D-homo-3,5-androstadien-17-ones (12e19), seven
17a-substituted 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic
acids (20e26) and 17-oxo-19-nor-3,5-androstadien-3-oic acid (30)
have been synthesized as novel human 5a-reductase inhibitors
through multistep synthesis. In vitro biological evaluation against
HEK cells revealed that most of the synthesized compounds showed
potent inhibition when compared to standard drug Finasteride (3).
The most potent compounds 21e23, and 25 showed 5a-reductase II
inhibition with IC50 being 54.1, 22.1, 72.8, and 26.5 nM respectively
as compared to Finasteride (30.3 nM) with significant (p < 0.05)
reduction in rat prostate weights. Also compound 13 was found to
be potent inhibitor of 5a-reductase I. The study concludes that D-
homo ring is well tolerated at the enzyme site and substitution of
17a-aza position with alkyl groups enhances the activity. The
results may prove as potential lead for the development of
compounds to be used in the treatment of BPH.
4. Experimental
4.1. Chemistry
For the synthesis of 17-oximino-5-androsten-3
b
-yl acetate (7),
-hydroxy-
17-oxo-17a-aza-D-homo-5-androsten-3 -yl acetate (8), 3
b
b
17a-aza-D-homo-5-androsten-17-one (9) and 17a-aza-D-homo-4-
androstene-3,17-dione (10) refer to supplementary material.
4.1.1. 3-Bromo-17a-aza-D-homo-3,5-androstadien-17-one (11)
17a-Aza-D-homo-4-androstene-3,17-dione (10) (3.33 g, 9.16 mmol)
was dissolved in glacial acetic acid (25.0 ml) and 1.61 ml of phosphorus
tribromide (4.60 g, 17.0 mmol) was added drop wise to the mixture at
room temperature. The dark solution was kept at 8e10 ꢁC for 24 h
during which the product got precipitated from the solution. The solid
product was filtered and washed with cold glacial acetic acid, followed
by cold water (3 ꢂ 50.0 ml). The product was dried under vacuum to
afford
3-bromo-17a-aza-D-homo-3,5-androstadien-17-one
(11)
(3.42 g, 85.0%): mp 239e241 ꢁC; UVmax (MeOH): 241.4 nm (log & 4.28);
IR (KBr, cmꢃ1): 3184, 2946 and 1645; 1H NMR (400 MHz, DMSO-d6):
d
0.95 (s, 3H, 19-CH3), 1.24 (s, 3H, 18-CH3), 5.41 (br s, 1H, 6-vinylic), 6.28
(br s,1H, 4-vinylic) and 7.61 ppm (1H, NH); 13C NMR (400 MHz, DMSO-
d6): 167.52 (C-17),116.18 (C-3),135.86 (C-4),125.71 (C-5),118.60 (C-6),
d
29.57 (C-1), 25.19 (C-2), 26.03 (C-7), 27.94 (C-8), 42.83 (C-9), 30.14 (C-
10), 14.57 (C-11), 33.97 (C-12), 49.68 (C-13), 42.26 (C-14), 15.95 (C-15),
27.04 (C-16), 16.88 (C-18) and 13.79 (C-19) ppm; Mass (APCI): 364.47
[M]þ, 366.20 [Mþ2]þ.
4.1.2. 3-Cyano-17a-aza-D-homo-3,5-androstadien-17-one (12)
3-Bromo-17a-aza-D-homo-3,5-androstadien-17-one (11) (1.08 g,
3.0 mmol) was dissolved in dimethylformamide (25.0 ml) and to this

736
S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
solution was added cuprous cyanide (0.27 g, 3.0 mmol). The reaction
mixture was refluxed for 5 h and allowed to cool to around 100 ꢁC and
quenched with stirring into a solution of 20.0 ml of concentrated
aqueous ammonia and 40.0ml of water. The resultingsuspensionwas
extracted twice with 100.0 ml of dichloromethane and organic phase
was washed with three 50.0 ml portions of 50/50 v/v concentrated
aqueous ammonia/water followed by water. The organic phase was
concentrated under vacuum and crystallised from absolute ethanol.
The solid product was filtered and dried under vacuum to afford 3-
cyano-17a-aza-D-homo-3,5-androstadien-17-one (12) (0.52 g,
56.5%): mp 288e290 ꢁC; UVmax (MeOH): 262.2 nm (log & 4.42); IR
(KBr, cmꢃ1): 3189, 2945, 2203 and 1662; 1H NMR (400 MHz, CDCl3):
4.1.5. 17a-Allyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (15)
3-Cyano-17a-aza-D-homo-3,5-androstadien-17-one (12) (0.34
g, 1.0 mmol) and allyl bromide (1.5 ml) were reacted using
procedure as mentioned above and crystallization from ethanol
yielded 17a-allyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (15) (0.23 g, 60.5%) : mp 179e180 ꢁC; UVmax (MeOH):
258.4 nm (log & 4.17); IR (KBr, cmꢃ1): 2945, 2877, 2209, 1630 and
1613; 1H NMR (400 MHz, CDCl3): 0.90 (s, 3H, 19-CH3), 1.21 (s, 3H,
18-CH3), 3.74 (dd, 1H, NeHHC), 4.33 (dd, 1H, NeHHC), 5.07 (dd, 1H,
NeCH2CH]CHH), 5.10 (dd, 1H, NeCH2CH]CHH), 5.87 (ddq, 1H,
NeCH2CH]CHH), 5.79 (br s, 1H, 6-vinylic) and 6.67 (br s, 1H, 4-
d
0.91 (s, 3H,19-CH3),1.22 (s, 3H,18-CH3), 5.79(brs,1H, 6-vinylic), 6.67
(br s, 1H, 4-vinylic) and 7.07 ppm (1H, NH, disappeared on D2O
exchange); 13C NMR (400 MHz, CDCl3):
171.94 (C-17), 120.14 (CN),
vinylic) ppm; 13C NMR (400 MHz, CDCl3):
d 170.06 (C-17), 120.14
(CN), 107.07 (C-3), 142.75 (C-4), 139.36 (C-5), 131.71 (C-6), 32.34 (C-
1), 24.23 (C-2), 31.51 (C-7), 32.56 (C-8), 49.11 (C-9), 34.46 (C-10),
19.65 (C-11), 37.30 (C-12), 59.63 (C-13), 46.37 (C-14), 21.11 (C-15),
31.59 (C-16), 19.42 (C-18) 18.70 (C-19), 135.90 (NeCH2CH]CH2),
115.51 (NeCH2CH]CH2) and 43.74 (NeCH2CH]CH2) ppm; Mass
(APCI): 351.33 [Mþ1]þ.
d
107.01 (C-3),142.79 (C-4),139.45 (C-5),131.64 (C-6), 32.32 (C-1), 24.21
(C-2), 30.64 (C-7), 31.92 (C-8), 47.88 (C-9), 34.57 (C-10), 19.88 (C-11),
39.16 (C-12), 54.03 (C-13), 47.12 (C-14), 20.71 (C-15), 31.43 (C-16),
22.02 (C-18) and 18.79 (C-19)ppm; Mass (APCI): 311.40 [Mþ1]þ.
Similarly,
17a-benzyl-3-cyano-17a-aza-D-homo-3,5-androsta-
4.1.3. 17a-Methyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (13)
dien-17-one (16) and 17a-cyanoethyl-3-cyano-17a-aza-D-homo-3,5-
androstadien-17-one (17) were synthesized. The spectral data is
reported in the supplementary material.
3-Cyano-17a-aza-D-homo-3,5-androstadien-17-one (12) (0.31
g, 1.0 mmol) was dissolved in anhydrous tetrahydrofuran (65.0 ml)
and stirred with sodium hydride (60% dispersion in mineral oil;
0.64 g, 1.74 mmol) which was previously washed three times under
nitrogen atmosphere with hexane to remove paraffin oil. The
reaction mixture was allowed to stir for 10 min, 1.2 ml of methyl
iodide was added drop wise and the mixture was kept on stirring at
room temperature for 24 h. The progress of reaction was monitored
with the help of TLC. After the completion of the reaction excess of
hydride was neutralized with methanol. The resulting solution was
concentrated, acidified with dilute hydrochloric acid, poured into
water and extracted with chloroform (3 ꢂ 50.0 ml). The combined
organic layer was washed with water, dried and solvent removed
under reduced pressure to get a residue which was crystallised
from methanol to give 17a-methyl-3-cyano-17a-aza-D-homo-3,5-
androstadien-17-one (13) (0.29 g, 90.6%): mp 233e235 ꢁC; UVmax
(MeOH): 262.0 nm (log & 4.41); IR (KBr, cmꢃ1): 2949, 2916, 2205
4.1.6. 17a-Acetyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (18)
3-Cyano-17a-aza-D-homo-3,5-androstadien-17-one (12) (1.02
g, 3.0 mmol) was dissolved in pyridine (10.0 ml) and acetic anhy-
dride (5.0 ml) was added to the mixture. The solution was refluxed
for 5 h at about 120 ꢁC. The excess of acetic anhydride was
destroyed with methanol after cooling to room temperature and
poured into crushed ice. The resulting precipitate was filtered,
washed thoroughly with water and dried to yield 17a-acetyl-3-
cyano-17a-aza-D-homo-3,5-androstadien-17-one (18) (0.95 g,
93.1%): mp 191e193 ꢁC; UVmax (MeOH): 261.8 nm (log & 4.26); IR
(KBr, cmꢃ1): 2942, 2855, 2203, 1727 and 1659; 1H NMR (400 MHz,
CDCl3):
COCH3), 5.78 (br s, 1H, 6-vinylic) and 6.67 (br s, 1H, 4-vinylic) ppm;
13C NMR (400 MHz, CDCl3):
173.43 (C-17), 120.12 (CN), 107.26 (C-
d 0.91 (s, 3H, 19-CH3), 1.45 (s, 3H, 18-CH3), 2.34 (s, 3H, N-
d
and 1631; 1H NMR (400 MHz, CDCl3):
3H, 18-CH3), 2.91 (s, 3H, N-CH3), 5.79 (br s, 1H, 6-vinylic) and 6.67
(br s, 1H, 4-vinylic) ppm; 13C NMR (400 MHz, CDCl3):
170.13 (C-
d
0.91 (s, 3H, 19-CH3), 1.23 (s,
3), 142.66 (C-4), 139.47 (C-5), 131.36 (C-6), 32.84 (C-1), 24.22 (C-2),
31.63 (C-7), 32.41 (C-8), 49.00 (C-9), 34.45 (C-10), 19.83 (C-11),
35.46 (C-12), 62.10 (C-13), 46.51 (C-14), 21.09 (C-15), 32.33 (C-16),
20.04 (C-18), 18.68 (C-19), 29.46 (NeCOeCH3) and 179.04
(NeCOeCH3); Mass (APCI): 353.20 [Mþ1]þ, 311.33 [M ꢃ 42]þ.
d
17),120.14 (CN),107.12 (C-3),142.74 (C-4),139.40 (C-5),131.71 (C-6),
32.42 (C-1), 24.25 (C-2), 31.59 (C-7), 32.37 (C-8), 48.94 (C-9), 34.50
(C-10), 19.49 (C-11), 37.08 (C-12), 58.66 (C-13), 46.44 (C-14), 21.19
(C-15), 31.67 (C-16), 18.73 (C-18), 17.95 (C-19) and 26.81 (NeCH3)
ppm; Mass (APCI): 325.33 [Mþ1]þ.
4.1.7. 17a-Benzoyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (19)
3-Cyano-17a-aza-D-homo-3,5-androstadien-17-one (12) (0.34
g, 1.0 mmol) was dissolved in 10.0 ml of dry benzene and benzoyl
chloride (0.35 ml; 3.0 mmol) was added to it. The reaction mixture
was refluxed under anhydrous conditions for 18 h. Then, the
solvent was removed under vacuum, the residue was diluted with
diethyl ether and filtered to give 17a-benzoyl-3-cyano-17a-aza-D-
homo-3,5-androstadien-17-one (19) (0.32 g, 71.1%): mp
240e241 ꢁC; UVmax (MeOH): 259.2 nm (log & 4.39); IR (KBr, cmꢃ1):
4.1.4. 17a-Ethyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (14)
Following similar procedure as mentioned above using 3-cyano-
17a-aza-D-homo-3,5-androstadien-17-one (12) (0.31 g, 1.0 mmol)
and bromoethane (1.5 ml) with crystallization from methanol
yielded 17a-ethyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (14) (0.19 g, 55.9%): mp 208e210 ꢁC; UVmax (MeOH):
261.6 nm (log & 3.87); IR (KBr, cmꢃ1): 2943, 2891, 2203 and 1634;
2954, 2198, 1710 and 1644; 1H NMR (400 MHz, CDCl3):
d 0.91 (s, 3H,
1H NMR (400 MHz, CDCl3):
d
0.91 (s, 3H, 19-CH3), 1.15 (s, 3H, 18-
19-CH3), 1.59 (s, 3H, 18-CH3), 7.8 (d, 2H), 7.52 (t, 1H), 7.41 (t, 2H)
CH3), 1.18 (t, 3H, CH3CH2Ne), 3.08 (dq, 1H, CH3CHHeN), 3.72 (dq,
1H, CH3CHHeN), 5.80 (br s, 1H, 6-vinylic) and 6.67 (br s, 1H, 4-
(aromatic hydrogens), 5.81 (br s, 1H, 6-vinylic) and 6.68 (br s, 1H, 4-
vinylic) ppm; 13C NMR (400 MHz, CDCl3):
d173.08 (C-17), 120.12
vinylic) ppm; 13C NMR (400 MHz, CDCl3):
d
169.78 (C-17), 120.14
(CN), 107.28 (C-3), 142.69 (C-4), 139.50 (C-5), 131.37 (C-6), 32.49 (C-
1), 24.24 (C-2), 31.61 (C-7), 32.35 (C-8), 48.54 (C-9), 34.50 (C-10),
20.26 (C-11), 35.65 (C-12), 61.44 (C-13), 46.57 (C-14), 20.86 (C-15),
31.92 (C-16), 21.13 (C-18), 18.75 (C-19) 128.68, 128.98, 133.02,
135.86 (aromatic carbons) and 176.28 (NeCOC6H5); Mass (APCI):
415.20 [Mþ1]þ, 311.47 [MꢃCOC6H5]þ.
(CN), 107.05 (C-3), 142.76 (C-4), 139.37 (C-5), 131.78 (C-6), 32.47 (C-
1), 24.25 (C-2), 31.63 (C-7), 32.36 (C-8), 49.17 (C-9), 34.50 (C-10),
19.54 (C-11), 37.29 (C-12), 59.39 (C-13), 46.47 (C-14), 21.14 (C-15),
31.85 (C-16), 18.71 (C-18), 15.54 (C-19), 36.34 (NeCH2CH3) and
19.22 (NeCH2CH3) ppm; Mass (APCI): 339.53 [Mþ1]þ.

S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
737
4.1.8. 17-Oxo-17a-aza-D-homo-3,5-androstadien-3-oic acid (20)
4.1.11. 17a-Allyl-17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic
acid (23)
3-Cyano-17a-aza-D-homo-3,5-androstadien-17-one (12) (0.31
g, 1.0 mmol) was dissolved in 20.0 ml of absolute alcohol, to the
solution 50% aqueous sodium hydroxide (2.0 ml) was added and
allowed to reflux for 24 h. The reaction was monitored with the
help of TLC, after completion of reaction it was cooled to 50 ꢁC
and to this was added with stirring mixture of 50% hydrochloric
acid (5.0 ml) and dichloromethane (50.0 ml). The aqueous phase
pH was kept between pH 1.5e2.0. The aqueous layer was
extracted with dichloromethane (3 ꢂ 50.0 ml), combined organic
layers were washed with water and dried. The dichloromethane
was removed under vacuum and ethyl acetate (30.0 ml) was
added. This was refluxed for 2 h, and then kept at 0e5 ꢁC. The
precipitated compound was filtered and dried under vacuum to
afford 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acid (20)
(0.20 g, 60.6%): mp 264e265 ꢁC; UVmax (MeOH): 268.0 nm (log &
4.48); IR (KBr, cmꢃ1): 3464, 3216, 2939, 1723, 1687, 1635, 1608 and
17a-Allyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-one (15)
(0.35 g, 1.0 mmol) was hydrolyzed to obtain 17a-allyl-17-oxo-17a-aza-
D-homo-3,5-androstadien-3-oic acid (23) (0.25 g, 67.6%) via similar
procedure: mp 194e196 ꢁC; UVmax (MeOH): 266.2 nm (log & 4.11); IR
(KBr, cmꢃ1): 3397, 2948, 1702, 1611 and 1259; 1H NMR (400 MHz,
CDCl3):
d 0.81 (s, 3H, 19-CH3), 1.15 (s, 3H, 18-CH3), 3.69 (dd, 1H,
NeHHC), 4.24 (dd, 1H, NeHHC), 5.03 (dd, 1H, NeCH2CH]CHH), 5.07
(dd, 1H, NeCH2CH]CHH), 5.82 (ddq, 1H, NeCH2CH]CHH), 5.79 (br s,
1H, 6-vinylic) and 7.07 (br s, 1H, 4-vinylic) ppm; 13C NMR (400 MHz,
CDCl3):
d 172.00 (COOH), 170.85 (C-17), 125.52 (C-3), 139.57 (C-4),
140.79 (C-5),131.63 (C-6), 33.00 (C-1), 21.41 (C-2), 31.27 (C-7), 32.71 (C-
8), 49.09 (C-9), 34.75 (C-10), 21.29 (C-11), 37.31 (C-12), 60.12 (C-13),
46.58 (C-14), 21.09 (C-15), 31.80 (C-16), 19.46 (C-18), 18.76 (C-19),
135.48 (NeCH2CH]CH2), 115.92 (NeCH2CH]CH2) and 44.02
(NeCH2CH]CH2) ppm; Mass (APCI): 370.60 [Mþ1]þ.
1184; 1H NMR (400 MHz, DMSO-d6):
d
0.89 (s, 3H, 19-CH3), 1.19 (s,
For synthesis and spectral data of 17a-benzyl-17-oxo-17a-aza-D-
homo-3,5-androstadien-3-oic acid (24)refertosupplementary material.
3H, 18-CH3), 5.79 (br s, 1H, 6-vinylic), 6.99 (br s, 1H, 4-vinylic) and
7.04 (1H, NH, disappeared on D2O exchange) ppm; 13C NMR
(400 MHz, DMSO-d6):
d
171.23 (COOH), 169.43 (C-17), 126.46 (C-
4.1.12. 17a-(2-Propionoxyethyl)-17-oxo-17a-aza-D-homo-3,5-
androstadien-3-oic acid (25)
3), 137.65 (C-4), 140.86 (C-5), 130.39 (C-6), 33.09 (C-1), 21.69 (C-2),
30.85 (C-7), 32.05 (C-8), 47.98 (C-9), 34.83 (C-10), 19.96 (C-11),
39.21 (C-12), 53.95 (C-13), 47.37 (C-14), 20.92 (C-15), 31.56 (C-16),
22.05 (C-18), 18.81 (C-19); Mass (APCI Mode): 330.40 [Mþ1]þ,
344.33 [Mþ15].þ
17a-Cyanoethyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (17) (0.36 g, 1.0 mmol) was hydrolyzed by ethanolic sodium
hydroxide using similar procedure to obtain 17a-(2-propionoxy ethyl)-
17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acid (25) (0.21 g,
50.0%): mp 200e205 ꢁC; UVmax (MeOH): 267.2 nm (log & 4.44); IR
(KBr, cmꢃ1): 3424, 2951, 1730, 1610 and 1260; 1H NMR (400 MHz,
4.1.9. 17a-Methyl-17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic
acid (21)
CDCl3):
d 0.89 (s, 3H, 19-CH3), 1.22 (s, 3H, 18-CH3), 3.36 (m,
17a-Methyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (13) (0.32 g, 1.0 mmol) was dissolved in 20.0 ml of absolute
alcohol, to the solution 50% aqueous sodium hydroxide (2.0 ml)
was added and allowed to reflux for 24 h. Following similar
procedure as mentioned above and crystallization from methanol
yielded 17a-methyl-17-oxo-17a-aza-D-homo-3,5-androstadien-3-
oic acid (21) (0.19 g, 55.9%): mp 238e240 ꢁC; UVmax (MeOH):
267.0 nm (log & 4.27); IR (KBr, cmꢃ1): 3501, 2955, 1690, 1594 and
1H, CH3CH2COOCH2CHHeN), 3.90 (m, 1H, CH3CH2COOCH2
CHHeN), 2.84 (m, 1H, CH3CH2COOCHHCHHeN), 2.56 (m, 1H,
CH3CH2COOCHHCHHeN), 5.86 (br s, 1H, 6-vinylic), 7.14 (br s, 1H, 4-
vinylic), 4.14 (q, 2H, CH3CH2COOCH2CHHeN), 1.24 (t, 3H,
CH3CH2COOCH2 CH2eN) ppm; 13C NMR (400 MHz, CDCl3):
d 172.04
(COOH), 171.93 (C-17), 172.46 (NeCH2CH2COOC2H5), 125.43 (C-3),
139.66 (C-4),140.83 (C-5),131.67 (C-6), 34.30 (C-1), 21.39 (C-2), 31.86 (C-
7), 32.56 (C-8), 49.27 (C-9), 34.80 (C-10),19.41 (C-11), 37.28 (C-12), 59.92
(C-13), 46.72 (C-14), 21.29 (C-15), 32.56 (C-16), 19.04 (C-18), 18.75 (C-
19), 60.49 (NeCH2CH2 COOCH2CH3), 37.55 (NeCH2CH2COOCH2CH3),
14.22 (NeCH2 CH2COOCH2CH3); Mass (APCI): 430.27 [Mþ1]þ.
1235; 1H NMR (400 MHz, CDCl3):
d 0.89 (s, 3H, 19-CH3), 1.20 (s,
3H, 18-CH3), 2.92 (s, 3H, N-CH3), 5.84 (br s, 1H, 6-vinylic), 7.14 (br
s, 1H, 4-vinylic) and 7.8 (br s, 1H, COOH) ppm; 13C NMR (400 MHz,
CDCl3):
d 172.04 (COOH), 170.71 (C-17), 125.70 (C-3), 139.38 (C-4),
140.82 (C-5), 131.51 (C-6), 33.01 (C-1), 21.42 (C-2), 31.56 (C-7),
32.52 (C-8), 48.92 (C-9), 34.76 (C-10), 19.37 (C-11), 37.10 (C-12),
58.90 (C-13), 46.63 (C-14), 21.33 (C-15), 31.78 (C-16), 18.76
(C-18), 17.98 (C-19) and 26.96 (NeCH3); Mass (APCI Mode):
344.40 [Mþ1]þ.
4.1.13. 17a-Acetyl-17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic
acid (26)
17-Oxo-17a-aza-D-homo-3,5-androstadien-3-oic
acid
(20)
(0.32 g, 1.0 mmol) was dissolved in pyridine (10.0 ml) and acetic
anhydride (5.0 ml) was added to the mixture. The solution was
refluxed for 5 h at about 120 ꢁC. The excess of acetic anhydride was
destroyed with methanol after cooling to room temperature and
poured into crushed ice. The resulting precipitate was filtered,
washed thoroughly with water and dried to yield 17a-acetyl-17-oxo-
17a-aza-D-homo-3,5-androstadien-3-oic acid (26) (0.26 g, 81.2%):
mp 198e200 ꢁC; UVmax (MeOH): 265.2 nm (log & 4.32); IR (KBr,
cmꢃ1): 3422, 2937, 1701, 1697, 1635 and 1200; 1H NMR (400 MHz,
4.1.10. 17a-Ethyl-17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic
acid (22)
17a-Ethyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-one
(14) (0.34 g, 1.0 mmol) was hydrolyzed using similar procedure
and after crystallization from methanol 17a-ethyl-17-oxo-17a-
aza-D-homo-3,5-androstadien-3-oic acid (22) (0.20 g, 55.5%) was
obtained: mp 249e250 ꢁC; UVmax (MeOH): 267.2 nm (log &
4.34); IR (KBr, cmꢃ1): 3430, 2941, 1688, 1638, 1584 and 1241; 1H
DMSO-d6):
COCH3), 5.80 (br s, 1H, 6-vinylic) and 6.95 (br s, 1H, 4-vinylic) ppm;
13C NMR (400 MHz, DMSO-d6):
172.74 (COOH),168.51 (C-17),126.18
d 0.85 (s, 3H, 19-CH3), 1.40 (s, 3H, 18-CH3), 2.34 (s, 3H, N-
NMR (400 MHz, CDCl3):
d
0.82 (s, 3H, 19-CH3), 1.13 (s, 3H, 18-
d
CH3), 1.18 (t, 3H, CH3CH2N-), 3.03 (dq, 1H, CH3CHHeN), 3.67
(dq, 1H, CH3CHHeN), 5.78 (br s, 1H, 6-vinylic) and 7.08 (br s, 1H,
(C-3),136.85 (C-4),140.18 (C-5),129.93 (C-6), 32.57 (C-1), 21.26 (C-2),
31.13 (C-7), 31.93 (C-8), 48.10 (C-9), 34.15 (C-10), 20.73 (C-11), 34.97
(C-12), 61.47 (C-13), 46.12 (C-14),19.31 (C-15), 32.25 (C-16),19.76 (C-
18),18.26 (C-19), 28.98 (NeCOeCH3) and 178.75 (NeCOeCH3); Mass
(APCI): 372.05 [Mþ1]þ, 330.26 [M ꢃ 42]þ.
4-vinylic) ppm; 13C NMR (400 MHz, CDCl3):
d 172.08 (COOH),
170.73 (C-17), 125.66 (C-3), 139.43 (C-4), 140.81 (C-5), 131.53 (C-
6), 33.04 (C-1), 21.42 (C-2), 31.52 (C-7), 32.62 (C-8), 49.12 (C-9),
34.79 (C-10), 19.30 (C-11), 37.25 (C-12), 59.92 (C-13), 46.67 (C-
14), 21.31 (C-15), 31.82 (C-16), 18.75 (C-18), 15.40 (C-19), 36.74
(NeCH2CH3) and 19.26 (NeCH2CH3) ppm; Mass (APCI Mode):
358.53 [Mþ1]þ.
4.1.14. 3-Bromo-19-nor-3,5-androstadien-17-one (28)
Following a similar procedure as for the synthesis of compound
11 and using 19-nor-4-androstene-3,17-dione (27) (2.49 g,

738
S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
9.16 mmol) and 1.61 ml of phosphorus tribromide (4.60 g,
17.0 mmol) afforded 3-bromo-19-nor-3,5-androstadien-17-one
(28) (2.70 g, 88.23%): mp 174e176 ꢁC; UVmax (MeOH): 234.4 nm
(log & 4.14); IR (KBr, cmꢃ1): 2927, 1732, 1608 and 1435; 1H NMR
used is 5 mM. In case of glucose-6-phosphate dehydrogenase 5 ml of
the sample obtained from Sigma (G8404) was directly used and it
was diluted in 1 ml tris buffer. A 1:2:1 mixture of this regenerating
system (NADP: glucose: glucose-6-P-dehydrogenase) was stored in
(400 MHz, CDCl3):
and 6.36 ppm (br s, 1H, 4-vinylic) ppm; 13C NMR (400 MHz, CDCl3):
221.06 (C-17), 36.02 (C-1), 28.89 (C-2), 122.92 (C-3), 136.50 (C-4),
d
0.90 (s, 3H, 18-CH3), 5.51 (br s, 1H, 6-vinylic)
the fridge and 50 ml of it was used in the end assay.
Androstenedione: A concentrated stock solution of 1 mM in
methanol was used for the assay. For the test from the stock solu-
d
131.61 (C-5), 123.80 (C-6), 30.13 (C-7), 36.21 (C-8), 50.97 (C-9),
40.00 (C-10), 21.70 (C-11), 31.32 (C-12), 47.81 (C-13), 43.72 (C-14),
25.89 (C-15), 35.82 (C-16) and 13.67 (C-18); Mass (APCI): 335.27
[M]þ and 337.20 [Mþ2]þ.
tion a 5
mM solution is prepared by first diluting it 1:1 in methanol
and then 1 ml of this solution is diluted to 100 ml in tris buffer. 50
of it is used in the inhibitory assay i.e. an end concentration of
500 nM.
Master mix: A master mix of 124.7
tenedione solution and 50 l regenerating system was made and
l [3H] androstenedione. 25
mixed with 0.3 l of the compound
solution to be tested was added to the solution prepared so as to get
a final test volume of 250 l.
Standard: Finasteride, a clinically used drug, was used as
standard.
ml
ml tris buffer, 50 ml andros-
4.1.15. 3-Cyano-19-nor-3,5-androstadien-17-one (29)
m
Following similar procedure as for compound 12 but using 3-
bromo-19-nor-3,5-androstadien-17-one (28) (1.04 g, 3.0 mmol)
and cuprous cyanide (0.27 g, 3.0 mmol) afforded 3-cyano-19-nor-
3,5-androstadien-17-one (29) (0.54 g, 64.3%): mp 230e232 ꢁC;
UVmax (MeOH): 262.2 nm (log & 3.87); IR (KBr, cmꢃ1): 2925,
m
m
m
2202, 1730 and 1630 cmꢃ1; 1H NMR (400 MHz, CDCl3):
d
0.91 (s, 3H,
18-CH3), 5.89 (br s, 1H, 6-vinylic) and 6.72 ppm (br s, 1H, 4-vinylic)
ppm; 13C NMR (400 MHz, CDCl3):
120.25 (CN), 220.67 (C-17),
Reversed phase HPLC: HPLC analyses were performed by the use
of a high pressure solvent delivery pump (Waters M6000A, Milford,
USA), a radioactivity detector (LB506C, Berthold, Wildbad,
d
35.74 (C-1), 26.23 (C-2), 108.46 (C-3), 143.53 (C-4), 135.22 (C-5),
132.20 (C-6), 27.38 (C-7), 35.99 (C-8), 50.85 (C-9), 40.06 (C-10),
21.63 (C-11), 30.61 (C-12), 47.74 (C-13), 43.48 (C-14), 25.65 (C-15),
31.20 (C-16) and 13.63 (C-18); Mass (APCI): 300.33 [M þ 18]þ.
Germany) and an autosampler system (851-AS, Jasco, Tokyo, Japan).
Nucleosil 120-3-C8 was applied as stationary phase using pre-
packed columns (125 ꢂ 4 mm; MachereyeNagel, Duren, Germany).
Following parameters were used for analysis: Flow rate: 0.325 ml/
min; Run time: 11 min; Additive flow for Scintillator: 1.2 ml from
3.5 to 10 min, Solvent System: 28% MeOH: 72% water.
4.1.16. 17-Oxo-19-nor-3,5-androstadien-3-oic acid (30)
Following similar procedure as for compound 20 and hydro-
lyzing 3-cyano-19-nor-3,5-androstadien-17-one (29) (0.84 g,
3.0 mmol) by 50% aqueous sodium hydroxide solution (2.0 ml)
afforded 17-oxo-19-nor-3,5-androstadien-3-oic acid (30) (0.60 g,
71.4%): mp 250e252 ꢁC; UVmax (MeOH): 269.6 nm (log & 4.45); IR
(KBr, cmꢃ1): 3450, 2931, 1732, 1675, 1627, 1279 and 1081 cmꢃ1; 1H
Inhibition assay: Lysates were obtained after harvesting and
resuspending 80% confluent cells in homogenate buffer (containing
300 mM saccharose, 5 mM TriseHCl and 0.1 mM EDTA) followed by
homogenization using ultrasonication. Suspensions of both cell
lines were used for all following assays. In the inhibitor assay to
250
was added so as to make a total volume of 500
bation of 30 min at 37 ꢁC, the reaction was stopped by the addition
of 750 l of ether. The steroids were extracted by shaking the
ml of master mix prepared above 250
ml of the cell suspension
NMR (400 MHz, CDCl3):
vinylic), 7.20 ppm (br s, 1H, 4-vinylic); 13C NMR (400 MHz,
CDCl3): 172.61 (COOH), 221.03 (C-17), 126.88 (C-3), 140.73 (C-4),
d
0.92 (s, 3H, 18-CH3), 5.98 (br s, 1H, 6-
ml. After an incu-
d
m
136.59 (C-5), 132.49 (C-6), 35.79 (C-1), 25.80 (C-2), 26.81 (C-7),
36.14 (C-8), 50.95 (C-9), 40.64 (C-10), 21.66 (C-11), 30.86 (C-12),
47.81 (C-13), 43.70 (C-14), 24.56 (C-15), 31.27 (C-16) and 13.64 (C-
18); Mass (APCI): 300.53 [Mþ].
reaction mixture for 10 min, then centrifuging it for further 10 min.
and finally freezing the aqueous phase (in an ethanol-dry ice bath)
to collect the ether phase containing the steroids. The latter phase
is dried in a speed vacuum, resuspended again in 35 ml of methanol
and then transferred to HPLC vials for radioactivity HPLC based
detection. The amount of converted tritiated androstenedione was
measured for each sample which served to determine the inhibi-
tory activity of the compounds.
4.2. Biological activity
4.2.1. In vitro human 5
and type II 5 -reductase enzyme
HEK-I and HEK-II served cell lines were used as a source of 5
reductase enzyme. HEK 293 cells are human embryonic kidney cells
which were made cultivatable for 5 -reductase I and II when
cDNAs encoding 5 -reductase type I and type II were inserted into
a-reductase inhibitory activity against type I
a
a
-
4.2.2. In vivo 5a-reductase inhibitory activity
Male wistar rats were used in the study in accordance with
a protocol (6598/PS dated 21.08.2010) approved by the Institutional
Animal Ethical Committee (IAEC) at the central animal house
facilities of Panjab University, Chandigarh, India. The experiments
were conducted as per CPCSEA (Committee for Prevention, Control
and Supervision of Experimental Animals) guidelines. All rats were
housed under standard conditions with free access to food and
water. The control diet for the rats contained 50.0% nitrogen free
extract, 20% protein, 8% fat, 4% cellulose, 1e1.5% of each mineral and
vitamin mix. The control diet was obtained from Ashirwad Indus-
tries (Chandigarh, India). Finasteride was procured from Dr Reddy’s
Laboratories, Hyderabad as gift sample. Mature male wistar rats
were domesticated for a month and oral administration was started
after 9 weeks. Following this varying amounts of drug suspension
at a dose of 5 ml/kg was administered orally once daily for 14
consecutive days. All the test and reference compounds were sus-
pended in 0.50% carboxymethylcellulose sodium salt (Himedia)
solution. Rats were weighed and sacrificed by ether anaesthesia on
the 15th day after 24 h of last dosing. The following organs were
a
a
pRcCMV vector and expressed in them.
4.2.1.1. Preparation of solutions used in inhibitory assay. Inhibitor
preparation: A stock solution of 10 mM in DMSO was prepared.
Before the test it was diluted to a concentration which is 50 times
more concentrated than the concentration in the test. In the next
step one volume of this solution was diluted with 2.5 volumes of
tris buffer (so that it’s now 20 times more concentrated than that
would be used in the test). This results in a test concentration of 2%
DMSO. As controls 2% DMSO (without inhibitor) was used. From
this 20 times compound solution, 25
of 500 l.
ml is used in a total test volume
m
NADPH regenerating system: NADP was dissolved in tris buffer to
a concentration of 22 mM while in the test the concentration used is
0.55 mM. Also glucose-6-phosphate was dissolved in tris buffer to
a concentration of 100 mM while in the test the end concentration

S. Aggarwal et al. / European Journal of Medicinal Chemistry 54 (2012) 728e739
[6] G. Bartsch, R.S. Rittmaster, H. Klocker, World J. Urol. 19 (2002) 413e425.
739
identified, removed and after removal of adhering fat and
connective tissue weighed: ventral prostate, dorsal prostate,
seminal vesicles, testes, epididymis, vas deferens, liver and adrenal
glands. Organ weights were recalculated (mg/100 g body weight)
i.e. dividing the weight of the tissue by the body weight so as to
remove variation due to the body weights among the groups. All
weighing of the organs were made on Shimazdu AW 220 balance.
Animals were divided into groups of 5 animals each. The following
groups were taken in the study: Control (animals receiving only
vehicle i.e. 0.50% CMC), others were being Finasteride (animals
[7] C. Carson, R. Rittmaster, Urology 61 (2003) 2e7.
[8] A. Morales, Eur. Urol. 41 (2002) 113e120.
[9] S. Basaria, A.S. Dobs, Drug Aging 15 (1999) 131e142.
[10] S. Aggarwal, S. Thareja, A. Verma, T.R. Bhardwaj, M. Kumar, Steroids 75 (2010)
109e153.
[11] Y.J. Abul-Hajj, Steroids 20 (1972) 215e222.
[12] D.W. Russell, J.D. Wilson, Annu. Rev. Biochem. 63 (1994) 25e61.
[13] J.D. Wilson, J.E. Griffin, D.W. Russell, Endocr. Rev. 14 (1993) 577e593.
[14] S. Andersson, D.M. Berman, E.P. Jenkins, D.W. Russell, Nature 354 (1991)
159e161.
[15] K. Tamura, M. Furihata, T. Tsunoda, S. Ashida, R. Takata, W. Obara, H. Yoshioka,
Y. Daigo, Y. Nasu, H. Kumon, H. Konaka, M. Namiki, K. Tozawa, K. Kohri,
N. Tanji, M. Yokoyama, T. Shimazui, H. Akaza, Y. Mizutani, T. Miki, T. Fujioka,
T. Shuin, Y. Nakamura, H. Nakagawa, Cancer Res. 67 (2007) 5117e5125.
[16] M. Uemura, K. Tamura, S. Chung, S. Honma, A. Okuyama, Y. Nakamura,
H. Nakagawa, Cancer Sci. 99 (2008) 81e86.
[17] D. Vaughan, J. Imperato-McGinley, J. McConnell, A.M. Matsumoto, B. Bracken,
J. Roy, M. Sullivan, F. Pappas, T. Cook, C. Daurio, A. Meehan, E. Stoner,
J. Waldstreicher, Urology 60 (2002) 1040e1044.
[18] H.G. Bull, M. Garcia-Calvo, S. Andersson, W.F. Baginsky, H.K. Chan,
D.E. Ellsworth, R.R. Miller, R.A. Stearns, R.K. Bakshi, G.H. Rasmusson,
R.L. Tolman, R.W. Myers, J.W. Kozarich, G.S. Harris, J. Am. Chem. Soc. 118
(1996) 2359e2365.
[19] B. Faller, D. Farley, H. Nick, Biochemistry 32 (1993) 5705e5710.
[20] R.V. Clark, D.J. Hermann, G.R. Cunningham, T.H. Wilson, B.B. Morrill, S. Hobbs,
J. Clin. Endocrinol. Metab. 89 (2004) 2179e2184.
[21] C. Schulman, P. Pommerville, K. Höfner, B. Wachs, BJU Int. 97 (2006) 73e80.
[22] M.A. Levy, M. Brandt, J.R. Heys, D.A. Holt, B.W. Metcalf, Biochemistry 29 (1990)
2815e2824.
[23] B.W. Metcalf, D.A. Holt, M.A. Levy, J.M. Erb, J.I. Heaslip, M. Brandt, H.J. Oh,
Bioorg. Chem. 17 (1989) 372e378.
[24] D.A. Holt, M.A. Levy, H.J. Oh, J.M. Erb, J.I. Heaslip, M. Brandt, H.Y. Lan-Hargest,
B.W. Metcalf, J. Med. Chem. 33 (1990) 943e950.
receiving
5 ml/kg Finasteride) and synthesized compounds
(animals receiving 5 ml/kg compound). Results are expressed as
mean ꢀ standard error of mean (SEM) of 5 animals per group.
Statistical significance of differences between groups was deter-
mined by one-way analysis of variance (ANOVA) followed by
Dunnett’s test. For the statistical determination, statistical
computerized software SIGMASTAT 3.5 was used. A probability (P)
value of less than 0.05 indicates a statistically significant difference
between the treatment groups.
Acknowledgements
The authors gratefully acknowledge UGC (University Grant
Commission), New Delhi, India for providing UGC-RFSMS fellow-
ship to the one of the author (Saurabh Aggarwal) along with Dr.
Vaibhav Gaur, Dr. Ruchita V. Kumar, Ms. Abhilasha Verma, and Mr.
Rohit Shukla for their help in carrying out studies. We thank Mr.
Avtar Singh (SAIF, Panjab University, Chandigarh, India) for doing
the NMR study/characterization. We are also thankful to techni-
cians Victoria Kraemer and Martina Jankowski of Saarland
University, Germany for their technical support. The authors report
no conflict of interest. We are also thankful to Dr Reddy Laborato-
ries, Hyderabad, India for generous gift of Finasteride and Cipla
Limited, Mumbai, India for dehydroepiandrosterone as gift sample.
[25] I.A. McDonald, P.L. Nyce, D.M. Muench, C.A. Gates, T.R. Blohm, M.E. Laughlin,
P.M. Weintraub, Bioorg. Med. Chem. Lett. 4 (1994) 847e851.
[26] S. Thareja, S. Aggarwal, T.R. Bhardwaj, M. Kumar, Eur. J. Med. Chem. 44 (2009)
4920e4925.
[27] A. Guarna, C. Belle, F. Machetti, E.G. Occhiato, A.H. Payne, C. Cassiani,
A. Comerci, G. Danza, A. De Bellis, S. Dini, A. Marrucci, M. Serio, J. Med. Chem.
40 (1997) 1112e1129.
[28] A. Guarna, E.G. Occhiato, F. Machetti, A. Marrucci, G. Danza, M. Serio, P. Paoli,
J. Med. Chem. 40 (1997) 3466e3477.
[29] W. Reichert, R.W. Hartmann, J. Jose, J. Enzyme Inhib. Med. Chem. 16 (2001)
47e53.
[30] B.U. Panter, J. Jose, R.W. Hartmann, Mol. Cell. Biochem. 270 (2005) 201e208.
[31] Z.Y. Sun, H.Y. Wu, M.Y. Wang, Z.H. Tu, Eur. J. Pharmacol. 371 (1999) 227e233.
[32] B. Kenny, S. Ballard, J. Blagg, D. Fox, J. Med. Chem. 40 (1997) 1293e1315.
[33] G.H. Rasmusson, G.F. Reynolds, N.G. Steinberg, E. Walton, G.F. Patel, T. Liang,
M.A. Cascieri, A.H. Cheung, J.R. Brooks, C. Berman, J. Med. Chem. 29 (1986)
2298e2315.
[34] J. Imperato-McGinley, L. Guerrero, T. Gautier, R.E. Peterson, Science 186
(1974) 1213e1215.
[35] S. Andersson, R.W. Bishop, D.W. Russell, J. Biol. Chem. 264 (1989)
16249e16255.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.06.026.
References
[36] K. Normington, D.W. Russell, J. Biol. Chem. 267 (1992) 19548e19554.
[37] J. Hirosumi, O. Nakayama, N. Chida, M. Inami, T. Fagan, K. Sawada,
S. Shigematsu, H. Kojo, Y. Notsu, M. Okuhara, J. Steroid Biochem. Mol. Biol. 52
(1995) 365e373.
[38] H.N. Bramson, D. Hermann, K.W. Batchelor, F.W. Lee, M.K. James, S.V. Frye,
J. Pharmacol. Exp. Ther. 282 (1997) 1496e1502.
[1] A. Tiwari, N.S. Krishna, K. Nanda, A. Chugh, Expert Opin. Invest. Drugs 14
(2005) 1359e1372.
[2] S.J. Berry, D.S. Coffey, P.C. Walsh, L.L. Ewing, J. Urol. 132 (1984) 474e479.
[3] J. Barkin, Can. Med. Assoc. J. 156 (1997) 978e979.
[4] L.S. Marks, C.G. Roehrborn, G.L. Andriole, J. Urol. 176 (2006) 1299e1306.
[5] J.D. McConnell, Br. J. Urol. 76 (1995) 5e10.