Synthesis and biological evaluation of 3-tetrazolo steroidal analogs: Novel class of 5α-reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.12.048

In the present study, a series of steroidal tetrazole derivatives of androstane and pregnane have been prepared in which the tetrazole moiety was appended at C-3 and 17a-aza locations. 3-Tetrazolo-3,5-androstadien-17-one (6), 3-tetrazolo-19-nor-3,5-androstadien-17-one (10), 3-tetrazolo-3,5-pregnadien-20-one (14), 17a-substituted 3-tetrazolo-17a-aza-d-homo-3,5-androstadien-17-one (26-31) and 3-(2-acetyltetrazolo)-17a-aza-d-homo-3,5-androstadien-17-one (32) were synthesized from dehydroepiandrosterone acetate (1) through multiple synthetic steps. Some of the synthesized compounds were evaluated for their in vitro 5α-reductase (5AR) inhibitory activity by measuring the conversion of [³H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity also showed a significant reduction (p <0.05) in rat prostate weight. The most potent compound 14 showed 5AR-2 inhibition with IC₅₀ being 15.6 nM as compared to clinically used drug finasteride (40 nM). There was also a significant inhibition of 5AR-1 with IC₅₀ 547 nM compared to finasteride (453 nM).

Full text of DOI:10.1016/j.bmc.2015.12.048

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 3-tetrazolo steroidal analogs:
Novel class of 5a-reductase inhibitors
Saurabh Aggarwal a, Manoj Kumar Mahapatra a, Rajnish Kumar a, Tilak R. Bhardwaj a,b, Rolf W. Hartmann c,d
,
Jörg Haupenthal c, Manoj Kumar a,
⇑
a University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India
b School of Pharmacy & Emerging Sciences, Baddi University of Emerging Sciences & Technology, Baddi, Himachal Pradesh, India
c Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus 81, D-66123 Saarbrücken, Germany
d Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, 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, a series of steroidal tetrazole derivatives of androstane and pregnane have been
prepared in which the tetrazole moiety was appended at C-3 and 17a-aza locations. 3-Tetrazolo-3,5-
androstadien-17-one (6), 3-tetrazolo-19-nor-3,5-androstadien-17-one (10), 3-tetrazolo-3,5-pregnadien-
Received 19 November 2015
Revised 28 December 2015
Accepted 29 December 2015
Available online xxxx
20-one (14), 17a-substituted 3-tetrazolo-17a-aza-
3-(2-acetyltetrazolo)-17a-aza- -homo-3,5-androstadien-17-one
dehydroepiandrosterone acetate (1) through multiple synthetic steps. Some of the synthesized
compounds were evaluated for their in vitro 5 -reductase (5AR) inhibitory activity by measuring the
conversion of [3H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5
-reductase
D
-homo-3,5-androstadien-17-one (26–31) and
D
(32) were synthesized from
Keywords:
Tetrazole
5AR
a
a
inhibitory activity also showed a significant reduction (p <0.05) in rat prostate weight. The most potent
compound 14 showed 5AR-2 inhibition with IC50 being 15.6 nM as compared to clinically used drug
finasteride (40 nM). There was also a significant inhibition of 5AR-1 with IC50 547 nM compared to
finasteride (453 nM).
HEK
Finasteride
Testosterone
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
a different tissue distribution pattern and with distinct biochemi-
cal and pharmacological properties.3 A third type of isozyme has
Androgens plays a vital role in benign prostatic hyperplasia
(BPH) and cancerous growth in prostate by acting through a
member of steroid nuclear receptor super family, that is, androgen
receptor.1 It directs the transcription of genes responsible for
growth and maintenance of prostate, for which androgenic path-
way has emerged as a target of therapeutic intervention in both
been detected and described in hormone refractory prostate cancer
(HRPC) and is ubiquitous in mammals, being found also in
non-androgenic tissues such as pancreas and brain.4,5 The most
commonly used 5AR inhibitor in BPH treatment is finasteride, a
steroidal compound inhibiting mainly 5AR-2. Finasteride was the
first 5AR inhibitor approved in U.S. for the treatment of BPH.6,7
The long term pharmacotherapy with finasteride has generally
been well tolerated.8 However, its limited activity and side effects
which are related with sexual function (libido, impotence, and
ejaculatory disorder) have prompted to look for newer 5AR
inhibitors.9 Epristeride, a carboxy steroid, is also a potent inhibitor
of 5AR-2 while a weak inhibitor of 5AR-1. Earlier we have also
reported a series unsaturated carboxysteroids (17a-substituted
benign and cancerous diseases. 5a-Reductase (5AR), a NADPH-
dependent and membrane bound enzyme, catalyzes the conver-
sion of testosterone (T) to more potent dihydrotestosterone
(DHT) (Fig. 1).
BPH is the most common benign tumor affecting over 40% of
men above the age of 70.2 DHT stimulates several growth factors
that drive cellular proliferation in the human prostate. Therefore,
inhibition of 5AR has been considered as a legitimate therapeutic
target and there is a greater thrust for development of newer
inhibitors. There are two main isozymes (5AR-1 and 5AR-2) with
17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acids) and their
5AR inhibitory potency.10 The mechanism of ‘inverted action’ or
‘back binding’, as proposed by McDonald et al. has been observed
in 17a-Aza-D-Homo-steroids which have also exhibited significant
5AR inhibitory activity. Thus it can be considered that 17a-aza ster-
oids might be exhibiting same type of mechanism of action as 4-
azasteroids.11
⇑
Corresponding author. Mobile: +91 9417455011.
E-mail address: manoj_uips@pu.ac.in (M. Kumar).
http://dx.doi.org/10.1016/j.bmc.2015.12.048
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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2
S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
lactam and substituting hydrogen with bulky groups. Therefore it
was envisioned to synthesize epristeride related analogs having
3-tetrazolo-3,5-diene moiety in androstane. In addition com-
pounds would have 17-oxo-17a-aza-
instead of ring A as in finasteride.
D-homo lactam in ring D
Guarna et al. synthesized a novel class of compounds 19-nor-
10-azasteroids which were found to be better inhibitors of the
5AR enzyme.20,21 Therefore, it was also envisaged to synthesize
molecule not having 19-methyl group and having rings A and B
mimicking that of epristeride. The present paper describes the syn-
thesis of a novel class of steroidal compounds which were evalu-
ated in vitro for 5AR inhibition against both 5AR-1 and 5AR-2,
DU-145 cancer cell lines and in vivo by prostate weighing method.
Figure 1. Formation of dihydrotestosterone by 5a-reductase.
Bioisosteres are those chemical substituents or groups with
analogous physical or chemical properties which imparts broadly
similar biological properties to the chemical compound. Tetrazoles
are the most commonly used metabolism-resistant bioisostere of
the carboxylate moiety, for which medicinal chemists paid a speci-
fic attention toward tetrazole group.12 Tetrazoles, after getting ion-
ized at physiological pH, shows a planar structure like their
carboxylate anion. Tetrazole anions are 10 times more lipophilic
than carboxylate anions but the interesting fact is both of them
have similar acidity (pKa 4.5–4.9 and 4.2–4.4 for tetrazole and car-
boxylate anion, respectively) which accounts for high membrane
permeability of tetrazoles.13 Moreover, tetrazoles are resistant to
many biological metabolic degradation pathways which causes
increased half life. Matta and co-workers computational study
has given evidence that anions of tetrazole and carboxyl group
shows bioisosterism, due to similarity in the geometrical arrange-
ment of four local minima in their respective electrostatic
potentials (a quartet of minima).14
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of 3-tetrazolo-3,5-androstadien-17-one (6)
17-Oxo-5-androsten-3b-yl acetate (dehydroepiandrosterone)
(1) was hydrolyzed by refluxing with methanolic potassium
hydroxide to obtain 3b-hydroxy-5-androsten-17-one (2). Oppe-
nauer oxidation of 2 gave 4-androstene-3,17-dione (3). Compound
3 on reaction with phosphorous tribromide in glacial acetic acid
yielded 3-bromo-3,5-androstadien-17-one (4). The bromo com-
pound (4) was refluxed with cuprous cyanide in dimethylfor-
mamide to yield 3-cyano-3,5-androstadien-17-one (5). The cyano
compound (5) when treated with sodium azide in presence of tri-
ethylamine hydrochloride gave the desired 3-tetrazolo-3,5-
androstadien-17-one (6) (Scheme 1).
Planarity in certain part of steroidal ring A seems to be a prereq-
uisite for the steroidal nucleus to enter into the active site of the
enzyme, as suggested by different literatures.15–18 Planarity has
been provided to certain extent by sp2 hybridized carbon of C-1
and C-2 in case of Finasteride, Dutasteride, and conjugated double
bonds at C-3 and C-5 in Epristeride (Fig. 2).
QSAR studies from our laboratory also indicated that a bulky
group at 17 position will lead to favorable activity.19 A tetrazole
is an isostere of carboxylic group, hence it was envisaged to
synthesize molecules having tetrazole group at C-3 mimicking
the charge replacement of carboxylate ion and having various alkyl
substituents at 17a-nitrogen of D-ring lactam. This can also be
accomplished by enlarging 5 membered D-ring to 6 membered
2.1.2. Synthesis of 3-tetrazolo-19-nor-3,5-androstadien-17-one
(10)
19-Nor-4-androsten-3,17-dione (7) was converted to its bromo
derivative (8) by treating with phosphorous tribromide in glacial
acetic acid. The bromo derivative on refluxing with cuprous cya-
nide in dimethylformamide gave 3-cyano-19-nor-3,5-androsta-
dien-17-one (9). The cyano compound (9) on reaction with
sodium azide in presence of triethylamine hydrochloride gave 3-
tetrazolo-19-nor-3,5-androstadien-17-one (10) (Scheme 2).
2.1.3. Synthesis of 3-tetrazolo-3,5-pregnadien-20-one (14)
4-Pregnene-3,20-dione (11) was treated with phosphorous tri-
bromide in glacial acetic acid to yield 3-bromo-3,5-pregnadien-
20-one (12). Compound 12 was converted to its cyano derivative
(13) by refluxing with cuprous cyanide in dimethylformamide.
The cyano derivative was treated with sodium azide and triethy-
lamine hydrochloride to yield 3-tetrazolo-3,5-pregnadien-20-one
(14) (Scheme 3).
2.1.4. Synthesis of 3-cyano-17a-aza-D-homo-3,5-androstadien-
17-one (20)
17-Oxo-5-androsten-3b-yl acetate(1) wasconverted toits oxime
(15) by refluxing in ethanol with hydroxylamine hydrochloride and
sodium acetate trihydrate. 17-Oximino-5-androsten-3b-yl acetate
(15) on Beckmann’s rearrangement with thionyl chloride gave 17-
oxo-17a-aza-
(16) was hydrolyzed by refluxing with methanolic potassium
hydroxide to obtain 3b-hydroxy-17a-aza- -homo-5-androsten-17-
one (17). Oppenaeur oxidation of 17 gave 17a-aza- -homo-4-
androstene-3,17-dione (18) which on reacting with phosphorous
tribromide in glacial acetic acid gave 3-bromo-17-a-aza- -homo-
D-homo-5-androsten-3b-yl acetate (16). The lactam
D
D
D
3,5-androstadien-17-one (19). The bromo derivative was refluxed
with cuprous cyanide in dimethylformamide to yield 3-cyano-17a-
aza-D-homo-3,5-androstadien-17-one (20) (Scheme 4).
Figure 2. Structures of active 5a-reductase inhibitors.
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S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Scheme 1. Synthesis of 3-tetrazolo-3,5-androstadien-17-one.
Scheme 2. Synthesis of 3-tetrazolo-19-nor-3,5-androstadien-17-one.
2.1.5. Synthesis of N-substituted 3-tetrazolo-17a-aza-
3,5-androstadien-17-one derivatives (26–31)
The cyano compound (20) was treated with sodium azide in
presence of triethylamine hydrochloride to form 3-tetrazolo-17a-
D
-homo-
to get 17a-methyl-3-cyano-17a-aza-
(21), 17a-ethyl-3-cyano-17a-aza-
(22), 17a-allyl-3-cyano-17a-aza-
one (23), 17a-benzyl-3-cyano-17a-aza-
17-one (24) and 17a-cyanoethyl-3-cyano-17a-aza-
5-androstadien-17-one (25) respectively. Compounds 21–25 on
reaction with sodium azide gave 17a-methyl-3-tetrazolo-17a-aza-
homo-3,5-androstadien-17-one (27), 17a-ethyl-3-tetrazolo-17a-
D
-homo-3,5-androstadien-17-one
D
-homo-3,5-androstadien-17-one
-homo-3,5-androstadien-17-
-homo-3,5-androstadien-
-homo-3,
D
D
aza-
D-homo-3,5-androstadien-17-one (26). 3-Cyano-17a-aza-
D
-
D
homo-3,5-androstadien-17-one (20) was also treated with methyl
iodide, ethyl bromide, allyl bromide, benzyl chloride and acryloni-
trile respectively in presence of sodium hydride in tetrahydrofuran
D-
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S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Scheme 3. Synthesis of 3-tetrazolo-3,5-pregnadien-20-one.
aza-
D
-homo-3,5-androstadien-17-one (28), 17a-allyl-3-tetrazolo-
-homo-3,5-androstadien-17-one (29), 17a-benzyl-3-
tetrazolo-17a-aza- -homo-3,5-androstadien-17-one (30) and
17a-tetrazoloethyl-3-tetrazolo-17a-aza-
-homo-3,5-androstadien-17-one (31), respectively (Scheme 5).
17a-aza-
D
D
D
2.1.6. Synthesis of 3-(2-acetyltetrazolo)-17a-aza-
androstadien-17-one (32)
D-homo-3,5-
3-Tetrazolo-17a-aza-
treated with acetic anhydride in pyridine to obtain 3-(2-acetyltetra-
zolo)-17a-aza- -homo-3,5-androstadien-17-one (32) (Scheme 6).
D-homo-3,5-androstadien-17-one (26) was
D
2.2. In vitro human 5AR inhibitory activity (HEK 293 cells
method)
Human embryonic kidney cell line (HEK293) lacking endoge-
nous 5a-reductase activity has replaced the conventional BPH
microsomal assay and the DU-145 cell free assay. The whole cell
system seemed to be more close to the in vivo situation and offers
faster and selective results as compared to the use of microsomal
enzymes. The main disadvantages of the earlier assay methods
were reported in our previous report.10
Previously we have reported a series of unsaturated 3-carboxys-
teroids which were active against both 5AR-1 and 5AR-2. Since
tetrazoles are isosteres of the carboxylic acids, some 3-tetrazolo
steroidal derivatives were prepared from their cyano precursors
and some of them were evaluated for their inhibitory potency
against 5AR-1 and 5AR-2 at 10
pound 6 was found to be a potent dual inhibitor of 5
showing 100% inhibition for 5AR-2 isozyme at both 10
M concentration with IC50 being 83.8 nM. It showed around
81.1% inhibition for 5AR-1 isozyme at 10 M. Similarly, compound
10 has exhibited around 80% inhibition for both 5AR-1 and 5AR-2
isozyme at 10 M concentration, while it also showed about 60%
inhibition at 2 M concentration against 5AR-2 isozyme. Com-
pound 14 showed 100% inhibition for 5AR-2 isozyme at 10
and 2 M concentration with IC50 being 15.6 nM indicating that
the compound is twice more potent than finasteride. It has shown
around 95% inhibition for 5AR-1 isozyme at 10 M with IC50 being
547 nM. Compound 27 showed 91.2% inhibition at 10 M and
l
M and 2
l
M concentrations. Com-
-reductase
M and
a
l
2
l
l
l
l
Scheme 4. Synthesis of 3-cyano-17a-aza-D-homo-3,5-androstadien-17-one.
lM
l
around 84.3% inhibition against 5AR-2 at 2
IC50 of 273.8 nM. Compound 29 showed 100% inhibition at 10
and 95% inhibition at 2 M concentration against 5AR-2 with IC50
lM concentration with
l
M
l
l
l
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S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Scheme 5. Synthesis of compounds 26–31.
is, the Prostate Cancer Prevention Trial (PCPT). Therefore, 5AR-1
inhibitors can be considered as a treatment strategy against pro-
static cancer whereas inhibition of 5AR-2 may not be an appropri-
ate therapeutic modality for the treatment of prostatic cancer.
Some of the synthesized compounds, based on their in vitro and
in vivo activity, were evaluated for their antiproliferative potential
on DU-145 cells as described by Mosamann.25 All the compounds
were tested at seven different concentrations in the culture med-
ium and finasteride was used as reference drug. Linear regression
line was drawn to calculate the concentration required to cause
50% inhibition in cell growth (IC50) and the results for DU-145 have
been reported in Table 2.
Some of the synthesized compounds (10, 27, 29) were found to
be weakly active against DU-145 cell line probably due to the fact
that the compounds were 5AR-2 active, that is, they inhibit synthe-
sis of DHT and hence were unable to show effect on androgen inde-
pendent cell lines. 3-Tetrazolo-19-nor-3,5-androstadien-17-one
(10) exhibited nearly same activity as that of Finasteride on DU-
145 cell line while there was no response to cell lines in case of
3-tetrazolo-3,5-androstadien-17-one (6), 3-tetrazolo-3,5-pregna-
dien-20-one (14) and 17a-aza-3-tetrazolo-D-homo-3,5-androsta-
Scheme 6. Synthesis of 3-(2-acetyltetrazolo)-17a-aza-D-homo-3,5-androstadien-
17-one.
dien-17-one (26). N-Methyl derivative (27) and N-allyl derivative
(29) however showed anti-proliferative activity on cell lines
similar to that of Finasteride (Table 2).
being 157 nM. Finasteride, a clinically used drug, was used as the
standard in the assay. It showed IC50 value of 453.0 nM for 5AR-1
enzyme while for 5AR-2 enzyme the value was 40 nM in the pre-
sent assay. Compounds 28, 30–32 could not be evaluated because
of the stability challenges for these molecules. The in vitro 5AR
inhibition data have been summarized in Table 1.
2.4. In vivo inhibitory activity (change in rat prostate weight in
mature male rats)
Despite of the histological and biochemical differences, rat
prostate is commonly used as the prostate growth model because
of its similar growth pattern with that of humans. Only active com-
2.3. In Vitro evaluation against DU-145 prostate cancer cell line
pounds (based on their in vitro 5a-reductase inhibitory activity)
along with standard drug finasteride were selected for in vivo
activity.26–28 The results of the activity have been given in Table 3.
The percentage decrease in the organ weights (ventral prostate,
dorsal prostate, vas deferens and epididymis) has been reported
in Table 4. In other organs the decrease was not significant. None
of the tested compounds induced any mortality.
Animals treated (2 weeks) with Finasteride or a 5AR inhibitor
were expected to have prostate weights significantly smaller than
those of control animals. Consistent with this expectation, Finas-
teride produced a statistically significant decrease in ventral and
dorsal prostate where it reduced the weight to around 44% as com-
pared to control.
Human prostate carcinoma cell lines have been available since
1977 and represent a good experimental model to assess new hor-
monal therapies. There are many prostate cancer cell lines avail-
able out of which DU145 is androgen-independent cell line.22
Finasteride, a selective 5AR-2 inhibitor, has been reported to
reduce the proliferation rate in vitro of DU145 prostate cancer cell
line although they being ‘hormone-independent’ suggesting that
the local formation of even small amounts of DHT in prostate neo-
plastic tissues may play some role also in the pathogenesis of
androgen-dependent prostate cancer.23 There are also some
reports that DU145 cancer cell line expresses mRNA for and
expresses functional 5AR-1 activity.24 Finasteride has been evalu-
ated for reducing the risk of prostate cancer in a clinical trial, that
3-Tetrazolo-3,5-androstadien-17-one (6) significantly reduced
the weight of ventral prostate (35.20%), vas deferens (59.11%)
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6
S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
In vitro activity data of synthesized compounds against 5AR-1 and 5AR-2
Compound
code
Activity
code
5AR-1% inhibition at
10
5AR-1% inhibition at
5AR-2% inhibition at
10
5AR-2% inhibition at
2 lM
IC50 of 5AR-2
(nM)
l
M
2
lM
l
M
6
10
14
SAR-43
SAR-47
SAR-49
81.1 2.5
79.7 5.0
95.0 7.1
38.7 5.8
36.2 6.6
82.6 0.0
(IC50-547.0 nM)
n.d.
n.d.
n.d.
IC50-453 nM
100.0 0.0
79.7 5.0
100.0 0.0
100.0 0.0
60.6 2.1
100.0 0.0
83.8 17.4
n.d.
15.6 4.7
26
27
29
SAR-44
SAR-45
SAR-46
—
9.6 10.0
6.4 9.0
31.7 0.9
—
100.0 0.0
91.2 2.8
100.0 0.0
—
90.6 8.4
84.3 1.6
94.7 7.6
—
220.1 66.2
273.8 45.2
157.0 27.6
40 nM
Finasteride
n.d.—not determined; activity of compounds 28 and 30–32 couldn’t be performed due to stability challenges.
Table 2
nor-3,5-androstadien-17-ones (10), 3-tetrazolo-3,5-pregnadien-
20-one (14), 17a substituted 3-tetrazolo-17a-aza- -homo-3,5-
androstadien-17-one (26–31) and 3-(2-acetyltetrazolo)-17a-aza-
-homo-3,5-androstadien-17-one (32) have been synthesized as a
novel class of 5ARIs through multistep synthesis. 3-Tetrazolo-3,5-
pregnadien-20-one (14) emerged as a potent dual inhibitor for
5AR-1 and 5AR-2 isozymes as it showed 5AR-2 inhibition with
IC50 being 15.6 nM as compared to finasteride (40 nM) and a signif-
icant 5AR-1 inhibition with IC50 547 nM as compared to finasteride
being 453 nM. These results provide support to the hypothesis that
having tetrazole group at C-3 mimicks the charge replacement of
carboxylate ion and may provide a potential lead for design and
development of newer class of 5AR inhibitors.
In vitro activity of synthesized compounds against DU-145 cell line
D
Compound code
Activity code
IC50
N.I.
174.40
N.I.
(
l
g/ml)
IC50
N.I.
536.34
N.I.
(lM)
D
6
10
14
26
27
29
SAR-43
SAR-47
SAR-49
SAR-44
SAR-45
SAR-46
—
N.I.
N.I.
143.20
195.10
204.20
389.12
495.50
548.00
Finasteride
N.I.—no siginificant inhibition; activity of compounds 28 and 30–32 couldn’t be
performed due to stability challenges.
and epididymis (36.25%) thereby exhibiting strong inhibition of
4. Experimental
4.1. Chemistry
5a-reductase as displayed in vitro activity. It also reduced the
weights of dorsal prostate (15.25%) and seminal vesicles but the
reduction was not significant. However, its 19-nor counterpart
(10) displayed weak inhibition in vivo as the reduction in the
weights of organs, that is, ventral and dorsal prostate, epididymis
and vas deferens were not significant. This indicated that loss of
methyl group has lead to a reduction in activity in vivo. Other
organs like liver, adrenal and testis have miniscule effect on weight
which was not statistically significant.
3-Tetrazolo-3,5-pregnadien-20-one (14) however, emerged as
one of the potent 5AR inhibitor in vitro as well as in vivo. It showed
statistically significant reduction of ventral prostate weight
(35.40%), epididymis (31.76%), vas deferens (57.10%) and seminal
vesicles at a dose of 1 mg/kg. Dorsal prostate weight was also
reduced (17.78%) but was not significant. Hence, substitution of
tetrazole moiety at 3rd position led to the formation of more
The synthetic procedure of different intermediates 3-cyano-3,5-
androstadien-17-one (5), 3-cyano-19-nor-3,5-androstadien-17-one (9),
3-cyano-3,5-pregnadien-20-one (13), 3-cyano-17a-aza-
D
-homo-3,
5-androstadien-17-one (20), 17a-methyl-3-cyano-17a-aza-
D
-homo-3,
5-androstadien-17-one (21), 17a-ethyl-3-cyano-17a-aza-
3,5-androstadien-17-one (22), 17a-allyl-3-cyano-17a-aza- -homo-3,
5-androstadien-17-one (23), 17a-benzyl-3-cyano-17a-aza- -homo-
3,5-androstadien-17-one (24) and 17a-cyanoethyl-3-cyano-17a-
D-homo-
D
D
aza-D-homo-3,5-androstadien-17-one (25) were described in detail
in our previous report.10 The synthesis of the novel compounds are
described in the following subsections.
The preparation of the tetrazole compounds from their respec-
tive cyano derivatives followed a similar procedure. The cyano
compound (5 mmol) and sodium azide (0.84 g, 13.0 mmol) were
dissolved in toluene (10 ml) and added to the solution of freshly
prepared triethylamine hydrochloride formed by passing dry
hydrogen chloride gas in triethylamine (2 ml) in toluene. The mix-
ture was heated at 110–120 °C for 20–42 h with continuous stir-
ring. After completion of the reaction, the mixture was cooled
and extracted with water (3 ꢀ 20 ml). To the aqueous extract, conc.
hydrochloric acid was added drop wise to precipitate the tetrazole.
Any differences from the standard procedure have been noted
below in the individual compound synthesis.
potent 5a-reductase inhibitor. Probably pregnane type moiety
along with the tetrazole one affords better binding of the com-
pounds at the active site of the enzyme. Effects on other organ
weights like liver, adrenal and testis were not significant.
The unsubstituted compound (26) although caused reduction in
the weights of ventral prostate, dorsal prostate, epididymis but the
effect was significant only on vas deferens (37.09%). On N-alkyla-
tion the methyl (27) and allyl (29) derivatives formed, caused sig-
nificant reduction in the weight of ventral prostate (29.94% and
33.61%, respectively) while the N-allyl derivative also showed sig-
nificant reduction in the weight of vas deferens (37.07%). But these
compounds didn’t show any significant effect on other organs such
as liver, adrenal, testis and seminal vesicles. Not much effect on the
body weight was seen. Hence, it can be concluded that
N-alkylation leads to the potent 5AR inhibitors in vitro as well as
good inhibitory activity in vivo.
4.1.1. 3-Tetrazolo-3,5-androstadien-17-one (6)
3-Cyano-3,5-androstadien-17-one (5) (1.47 g, 5.0 mmol) and
sodium azide (0.84 g, 13.0 mmol) were dissolved in toluene
(10 ml) and added to the solution of freshly prepared triethylamine
hydrochloride formed by passing dry hydrogen chloride gas in tri-
ethylamine (2 ml) in toluene. The mixture was heated at 110–
120 °C for 20 h with continuous stirring. After completion of the
reaction the mixture was cooled and extracted with water
(3 ꢀ 20 ml). To the aqueous extract concentrated hydrochloric acid
3. Conclusion
In the present study a series ten steroidal compounds consisting
of 3-tetrazolo-3,5-androstadien-17-ones (6), 3-tetrazolo-19-
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S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
was added drop wise to precipitate the tetrazole. The product was
filtered, washed and dried under vacuum to afford 3-tetrazolo-3,5-
androstadien-17-one (6) (1.20 g, 71.4%): mp 230–232 °C; UVmax
(MeOH): 274.0 nm (loge
4.40); IR (KBr, cmꢁ1): 2946, 2862, 1733,
1625 and 1547; 1H NMR (DMSO-d6): d 0.89 (s, 3H, 18-CH3), 1.00
(s, 3H, 19-CH3), 5.79 (br s, 1H, 6-vinylic), 6.98 (br s, 1H, 4-vinylic)
and 15.8 ppm (br s, NH); 13C NMR (DMSO-d6): d 219.26 (C-17),
155.38 (C-N), 32.57 (C-1), 22.42 (C-2), 118.47 (C-3), 132.00 (C-4),
140.33 (C-5), 128.64 (C-6), 30.49 (C-7), 30.73 (C-8), 51.05 (C-9),
34.37 (C-10), 19.83 (C-11), 35.22 (C-12), 46.97 (C-13), 47.57
(C-14), 21.27 (C-15), 30.97 (C-16), 13.25 (C-18) and 18.68 (C-19);
Mass (APCI): 339.60 [M+1]+.
4.1.2. 3-Tetrazolo-19-nor-3,5-androstadien-17-one (10)
A
solution of 3-cyano-19-nor-3,5-androstadien-17-one (9)
(1.40 g, 5.0 mmol) and sodium azide (0.84 g, 13.0 mmol) in toluene
(10 ml) and added to the freshly prepared triethylamine
hydrochloride prepared by passing dry hydrogen chloride gas in
triethylamine (2 ml) in toluene. The reaction mixture was heated
at 110–120 °C for 24 h with continuous stirring. After completion
of the reaction the mixture was cooled and extracted with water
(3 ꢀ 20 ml). Tetrazole was precipitated by adding concentrated
hydrochloric acid drop wise to the aqueous layer. The solid product
was filtered and dried under vacuum to get 3-tetrazolo-19-nor-
3,5-androstadien-17-one (10) (1.10 g, 68.3%); mp 265–267 °C;
UVmax (MeOH): 272.8 nm (loge
4.45); IR (KBr, cmꢁ1): 3116, 2875,
1734, 1628 and 1543; 1H NMR (DMSO-d6): d 0.92 (s, 3H, 18-
CH3), 5.88 (br s, 1H, 6-vinylic), 7.05 (br s, 1H, 4-vinylic) and
15.84 ppm (br s, NH); 13C NMR (DMSO-d6): d 219.26 (C-17),
155.45 (C-N), 35.20 (C-1), 25.64 (C-2), 120.36 (C-3), 132.40 (C-4),
135.67 (C-5), 128.85 (C-6), 26.18 (C-7), 35.59 (C-8), 50.21 (C-9),
40.20 (C-10), 21.16 (C-11), 30.09 (C-12), 47.05 (C-13), 43.18 (C-
14), 25.21 (C-15), 30.97 (C-16), and 13.22 (C-18); Mass (APCI):
325.33 [M+1]+.
4.1.3. 3-Tetrazolo-3,5-pregnadien-20-one (14)
3-Cyano-3,5-pregnadien-20-one (13) (1.61 g, 5.0 mmol) and
sodium azide (0.84 g, 13.0 mmol) were dissolved in toluene
(10 ml) and added to the freshly prepared triethylamine
hydrochloride prepared by passing dry hydrogen chloride gas in
triethylamine (2 ml) in toluene. The reaction mixture was heated
at 110–120 °C for 24 h with continuous stirring. After completion
of the reaction the mixture was cooled and extracted with water
(3 ꢀ 20 ml). Tetrazole was precipitated by adding concentrated
hydrochloric acid dropwise to the aqueous layer. The solid product
was filtered and dried under vacuum to get 3-tetrazolo-3,5-preg-
nadien-20-one (14) (0.89 g, 66.9%); mp 240–242 °C; UVmax
(MeOH): 270.4 nm (loge
4.32); IR (KBr, cmꢁ1): 3424, 2938, 1698,
1631 and 1546; 1H NMR (DMSO-d6): d 0.61 (s, 3H, 18-CH3), 0.96
(s, 3H, 19-CH3), 2.09 (s, 3H, 21-CH3), 5.76 (br s, 1H, 6-vinylic)
and 6.95 (br s, 1H, 4-vinylic); 13C NMR (DMSO-d6): d 208.02 (C-
20), 155.57 (C-N), 62.64 (C-17), 32.63 (C-1), 20.51 (C-2), 118.49
(C-3), 140.23 (C-4), 131.98 (C-5), 129.18 (C-6), 32.39 (C-7), 31.50
(C-8), 56.15 (C-9), 34.25 (C-10), 22.47 (C-11), 37.97 (C-12), 43.37
(C-13), 47.29 (C-14), 22.20 (C-15), 23.83 (C-16), 12.95 (C-18),
18.69 (C-19) and 31.10 (C-21); Mass (APCI): 339.60 [M+1]+.
4.1.4. 3-Tetrazolo-17a-aza-D-homo-3,5-androstadien-17-one
(26)
3-Cyano-3,5-pregnadien-17a-aza-D-homo-3.5-androstadien-17-
one (20) (1.55 g, 5.0 mmol) and sodium azide (0.84 g, 13.0 mmol)
were dissolved in toluene (5 ml) and added to the freshly prepared
triethylamine hydrochloride prepared by passing dry hydrogen
chloride gas in triethylamine (2 ml) in toluene. The reaction mix-
ture was heated at 110–120 °C for 35 h with continuous stirring.
After completion of the reaction the mixture was cooled and
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8
S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 4
get 17a-ethyl-3-tetrazolo-17a-aza-
one (28) (1.30 g, 68.4%); mp 220–222 °C; UVmax (MeOH):
272.0 nm (log
4.42); IR (KBr, cmꢁ1): 2942, 2741, 2677, 1598 and
D-homo-3,5-androstadien-17-
Percentage decrease in organ weight after treatment with synthesized compounds
e
Compound
code
Ventral
prostate
Dorsal
prostate
Vas
deferens
Epididymis
1462; 1H NMR (DMSO-d6): d 0.97 (s, 3H, 19-CH3), 1.18 (s, 3H, 18-
CH3), 1.13 (t, 3H, CH3-CH2-N), 3.16 (dq, 1H, CH3CHH-N), 3.67 (dq,
1H, CH3CHH-N), 5.79 (br s, 1H, 6-vinylic) and 7.02 ppm (br s, 1H,
4-vinylic); 13C NMR (DMSO-d6): 169.34 (C-17), 129.51 (C-3),
132.11 (C-4), 140.09 (C-5), 128.86 (C-6), 32.56 (C-1), 22.61 (C-2),
31.40 (C-7), 32.32 (C-8), 48.93 (C-9), 34.60 (C-10), 19.28 (C-11),
37.08 (C-12), 59.29 (C-13), 46.40 (C-14), 21.02 (C-15), 31.74
(C-16), 15.42 (C-18), 18.65 (C-19), 35.95 (N-CH2-CH3) and 19.04
(N-CH2-CH3); Mass (APCI): 382.24 [M+1]+.
Finasteride
6; SAR-43
44.76
35.20
5.66
35.40
24.79
29.94
33.61
45.32
15.25
7.21
17.78
37.31
11.39
16.94
14.45
59.11
12.40
57.10
37.09
11.56
37.07
14.40
36.25
3.44
31.76
11.09
9.03
10; SAR-47
14; SAR-49
26; SAR-44
27; SAR-45
29; SAR-46
9.30
4.1.7. 17a-Allyl-3-tetrazolo-17a-aza-
17-one (29)
D-homo-3,5-androstadien-
extracted with water (3 ꢀ 20 ml). Tetrazole was precipitated by
adding concentrated hydrochloric acid dropwise to the aqueous
layer. The solid product was filtered and dried under vacuum to
17a-Allyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-one
(23) (1.75 g, 5.0 mmol) and sodium azide (0.84 g, 13.0 mmol) were
dissolved in toluene (10.0 ml) and added to the freshly prepared
triethylamine hydrochloride prepared by passing dry hydrogen
chloride gas in triethylamine (2 ml) in toluene. The reaction mix-
ture was heated at 110–120 °C for 30 h with continuous stirring.
After completion of the reaction the mixture was cooled and
extracted with water (3 ꢀ 20 ml). Tetrazole was precipitated by
adding concentrated hydrochloric acid dropwise to the aqueous
layer. The solid product was filtered and dried under vacuum to
get 3-tetrazolo-17a-aza-
(0.70 g, 61.4%); mp 265–267 °C; UVmax (MeOH): 269.2 nm (log
D-homo-3,5-androstadien-17-one (26)
e
4.34); IR (KBr, cmꢁ1): 3172, 2942, 2770, 1660, 1631, 1550 and
1237; 1H NMR (DMSO-d6): d 0.92 (s, 3H, 19-CH3), 1.16 (s, 3H, 18-
CH3), 5.78 (br s, 1H, 6-vinylic), 6.98 (br s, 1H, 4-vinylic) and
7.51 ppm (1H, NH); 13C NMR (DMSO-d6): 170.82 (C-17), 155.84
(C-N), 119.10 (C-3), 132.24 (C-4), 140.41 (C-5), 129.16 (C-6),
32.80 (C-1), 22.88 (C-2), 31.03 (C-7), 32.05 (C-8), 47.95 (C-9),
34.91 (C-10), 20.05 (C-11), 39.07 (C-12), 53.77 (C-13), 47.35 (C-
14), 20.92 (C-15), 31.48 (C-16), 19.00 (C-18) and 22.09 (C-19);
Mass (APCI): 354.27 [M+1]+.
get 17a-allyl-3-tetrazolo-17a-aza-
one (29) (0.95 g, 51.1%); mp 255–257 °C; UVmax (MeOH):
270.4 nm (log
3.97); IR (KBr, cmꢁ1): 2933, 1622 and 1457; 1H
D-homo-3,5-androstadien-17-
e
NMR (DMSO-d6): d 0.94 (s, 3H, 19-CH3), 1.17 (s, 3H, 18-CH3),
3.74 (dd, 1H, N-HHC), 4.19 (dd, 1H, N-HHC), 5.03 (dd, 1H,
N-CH2-CH@CHH), 5.09 (dd, 1H, N-CH2-CH@CHH), 5.83 (ddq, 1H,
N-CH2-CH@CHH), 5.76 (br s, 1H, 6-vinylic) and 6.98 ppm (br s,
1H, 4-vinylic); 13C NMR (DMSO-d6): 169.26 (C-17), 118.48 (C-3),
131.87 (C-4), 139.76 (C-5), 128.64 (C-6), 36.76 (C-1), 22.36 (C-2),
31.03 (C-7), 32.11 (C-8), 48.52 (C-9), 34.24 (C-10), 19.07 (C-11),
42.97 (C-12), 59.23 (C-13), 46.03 (C-14), 20.71 (C-15), 32.25
(C-16), 18.37 (C-18), 18.93 (C-19), 135.79 (N-CH2CH@CH2),
114.89 (N-CH2CH@CH2) and 45.53 (N-CH2CH@CH2); Mass (APCI):
394.40 [M+1]+.
4.1.5. 17a-Methyl-3-tetrazolo-17a-aza-
androstadien-17-one (27)
D-homo-3,5-
17a-Methyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-one
(21) (1.62 g, 5.0 mmol) and sodium azide (0.84 g, 13.0 mmol) were
dissolved in toluene (5 ml) and added to the freshly prepared
triethylamine hydrochloride prepared by passing dry hydrogen
chloride gas in triethylamine (2 ml) in toluene. The reaction mix-
ture was heated at 110–120 °C for 42 h with continuous stirring.
After completion of the reaction the mixture was cooled and
extracted with water (3 ꢀ 20 ml). Tetrazole was precipitated by
adding concentrated hydrochloric acid dropwise to the aqueous
layer. The solid product was filtered and dried under vacuum
4.1.8. 17a-Benzyl-3-tetrazolo-17a-aza-
androstadien-17-one (30)
D-homo-3,5-
to get 17a-methyl-3-tetrazolo-17a-aza-
17-one (27) (1.19 g, 65.0%); mp 260–262 °C; UVmax (MeOH):
270.4 nm (log
4.46); IR (KBr, cmꢁ1): 3098, 2945, 2755, 1606 and
D-homo-3,5-androstadien-
17a-Benzyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-one
e
1539; 1H NMR (DMSO-d6): d 0.95 (s, 3H, 19-CH3), 1.20 (s, 3H, 18-
CH3), 2.85 (s, 3H, N-CH3), 5.78 (br s, 1H, 6-vinylic), 6.98 (br s, 1H,
4-vinylic) and 16.26 ppm (br s, 1H, NH); 13C NMR (DMSO-d6):
168.91 (C-17), 154.79 (C-N), 118.50 (C-3), 131.87 (C-4),
139.82 (C-5), 128.83 (C-6), 32.27 (C-1), 22.37 (C-2), 31.05 (C-7),
31.98 (C-8), 48.36 (C-9), 34.30 (C-10), 19.02 (C-11), 36.58
(C-12), 58.10 (C-13), 46.08 (C-14), 20.80 (C-15), 31.34 (C-16),
17.48 (C-18), 18.41 (C-19) and 26.11 (N-CH3); Mass (APCI):
368.27 [M+1]+.
(24) (1.00 g, 2.5 mmol) and sodium azide (0.41 g, 6.5 mmol) were
dissolved in toluene (5.0 ml) and added to the freshly prepared tri-
ethylamine hydrochloride prepared by passing dry hydrogen chlo-
ride gas in triethylamine (1 ml) in toluene. The reaction mixture
was heated at 110–120 °C for 30 h with continuous stirring. After
completion of the reaction the mixture was cooled and extracted
with water (3 ꢀ 20 ml). Tetrazole was precipitated by adding
concentrated hydrochloric acid dropwise to the aqueous layer.
The solid product was filtered and dried under vacuum to get
17a-benzyl-3-tetrazolo-17a-aza-
(30) (0.58 g, 52.2%); mp 260–262 °C; UVmax (MeOH): 274.4 nm
(log
4.32); IR (KBr, cmꢁ1): 3401, 2942, 1606 and 1449; 1H NMR
D-homo-3,5-androstadien-17-one
4.1.6. 17a-Ethyl-3-tetrazolo-17a-aza-D-homo-3,5-androstadien-
e
17-one (28)
(DMSO-d6): d 0.91 (s, 3H, 19-CH3), 1.23 (s, 3H, 18-CH3), 5.00 (dd,
1H, PhCHH-N), 4.26 (dd, 1H, PhCHH-N), 5.78 (br s, 1H, 6-vinylic),
6.95 (br s, 1H, 4-vinylic) and 7.22 ppm (m, 5H, aromatic protons);
13C NMR (DMSO-d6): 169.72 (C-17), 129.02 (C-3), 131.67
(C-4), 139.77 (C-5), 129.60 (C-6), 32.20 (C-1), 22.35 (C-2), 31.05
(C-7), 31.06 (C-8), 48.57 (C-9), 34.25 (C-10), 19.34 (C-11), 37.02
(C-12), 59.50 (C-13), 45.94 (C-14), 20.67 (C-15), 32.25 (C-16),
18.39 (C-18), 18.79 (C-19), 125.91, 126.27, 127.82, 139.94
(aromatic carbons) and 43.71 (N-CH2C6H5); Mass (APCI): 444.30
[M+1]+.
17a-Ethyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-one
(22) (1.69 g, 5.0 mmol) and sodium azide (0.84 g, 13.0 mmol) were
dissolved in toluene (10.0 ml) and added to the freshly prepared
triethylamine hydrochloride prepared by passing dry hydrogen
chloride gas in triethylamine (2 ml) in toluene. The reaction mix-
ture was heated at 110–120 °C for 35 h with continuous stirring.
After completion of the reaction the mixture was cooled and
extracted with water (3 ꢀ 20 ml). Tetrazole was precipitated by
adding concentrated hydrochloric acid dropwise to the aqueous
layer. The solid product was filtered and dried under vacuum to
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S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
4.1.9. 17a-Tetrazoloethyl-3-tetrazolo-17a-aza-
androstadien-17-one (31)
D
-homo-3,5-
For transfection experiments cells were used at passage number
8–10. Tissue culture reagents were from c.c.pro (Neustadt/W.,
Germany), except Geneticin selective antibiotic (G418 sulfate),
which was from Calbiochem (Bad Soden, Germany).28
17a-Cyanoethyl-3-cyano-17a-aza-D-homo-3,5-androstadien-17-
one (25) (0.91 g, 2.5 mmol) and sodium azide (0.41 g, 6.5 mmol)
were dissolved in toluene (10.0 ml) and added to the freshly
prepared triethylamine hydrochloride prepared by passing dry
hydrogen chloride gas in triethylamine (1 ml) in toluene. The reac-
tion mixture was heated at 110–120 °C for 30 h with continuous
stirring. After completion of the reaction the mixture was cooled
and extracted with water (3 ꢀ 10 ml). Tetrazole was precipitated
by adding concentrated hydrochloric acid dropwise to the aqueous
layer. The solid product was filtered and dried under vacuum to get
4.2.3. Construction of 5AR expression plasmids
The Not I-insert of the plasmid ph5a45 is a full length human
cDNA encoding the 5AR-1 isoenzyme. It was inserted downstream
of the Cytomegalovirus (CMV) promoter of the eucaryotic expres-
sion vector pRcCMV (Invitrogen, Groningen, Netherlands). This
vector carries an additional neomycin resistance. The new con-
struct which encoded human 5AR-1 was named pRcCMV-I and
used for transfection.
The Sal I/Not I-insert of the plasmid pBS-76-1 correspond to the
full length human cDNA encoding the 5AR-2 isoenzyme. It was first
inserted by the SalI/NotI-sites into pUC21-vector and recleaved by
Hind III and XbaI. By this strategy a 50-Hind III-and a 30-XbaI-site
was added to the 5AR-2 encoding DNA fragment, by which it
was inserted into the expression vector pRcCMV. The resulting
plasmid (pRcCMV-II) was used for transfecting HEK293 cells.28
17a-tetrazoloethyl-3-tetrazolo-17a-aza-
17-one (31) (0.63 g, 62.3%); mp 250–255 °C; UVmax (MeOH):
274.0 nm (log
4.45); IR (KBr, cmꢁ1): 3400, 2929, 1624, 1456 and
D-homo-3,5-androstadien-
e
1259; 1H NMR (DMSO-d6): d 0.95 (s, 3H, 19-CH3), 1.18 (s, 3H, 18-
CH3), 3.41 (m, 1H, CH2-CHH-N), 3.87 (m, 1H, CH2-CHH-N), 2.97
(m, 1H, CHH-CH2-N), 2.71 (m, 1H, CHH-CH2-N), 5.77 (br s, 1H, 6-
vinylic) and 6.98 ppm (br s, 1H, 4-vinylic); 13C NMR (DMSO-d6):
170.19 (C-17), 156.04 (C-N), 154.18 (N-CH2CH2-C-N–), 119.40
(C-3), 131.92 (C-4), 140.32 (C-5), 128.97 (C-6), 32.78 (C-1), 23.99
(C-2), 31.57 (C-7), 32.49 (C-8), 48.90 (C-9), 34.81 (C-10), 21.24
(C-11), 37.00 (C-12), 59.91 (C-13), 46.59 (C-14), 22.90 (C-15),
31.98 (C-16), 18.89 (C-18), 19.07 (C-19), 46.35 (N-CH2CH2C-N–)
and 19.43 (N-CH2CH2C-N–); Mass (APCI): 430.35 [Mꢁ18]+.
4.2.4. Transfection procedure
One day before the transfection experiment 1 ꢀ 107 HEK293
cells were seeded in 100 mm culture dishes. By this procedure
the culture is approximately 70% confluent on the day of transfec-
tion. The liposomal transfecting reagent RotiÒ-Fect was used for
transfecting cells either with pRcCMV-I or pRcCMV-II following
the manufacturers recommendations. The optimal DNA/reagent
4.1.10. 3-(2-Acetyltetrazolo)-17a-aza-D-homo-3,5-
androstadien-17-one (32)
3-Tetrazolo-17a-aza-
D
-homo-3,5-androstadien-17-one
(26)
ratio was 10
lg plasmid and 20 l
l RotiÒ-Fect reagent.
(1.06 g, 3.0 mmol) was dissolved in pyridine (10.0 ml) and acetic
anhydride (5.0 ml) was added to it. The solution was refluxed for
5 h. The excess of acetic anhydride was treated with methanol after
cooling to room temperature was poured into crushed ice. The
resulting precipitate was filtered, washed thoroughly with water
4.2.5. Selection of stable clones
Initially the concentration at which G418 sulfate inhibits the
growth of untransfected HEK293 cells was determined. Therefore,
varying concentrations of G418 sulfate (50 lg/ml-1000 lg/ml)
and dried to yield 3-(2-acetyltetrazolo)-17a-aza-
androstadien-17-one (32) (0.75 g, 63.0%); mp 220–222 °C; UVmax
(MeOH): 283.6 nm (log
4.45); IR (KBr, cmꢁ1): 3423, 2933, 1685
D
-homo-3,5-
were added to adherent HEK293 cells seeded in 24-well tissue cul-
ture plates at a density of 200,000 cells/well. After 6 days incubation
at 37 °C, viable cells were determined using the trypan blue exclu-
e
and 1631; 1H NMR (DMSO-d6): d 0.88 (s, 3H, 19-CH3), 1.09 (s,
3H, 18-CH3), 2.45 (s, 3H, N-COCH3), 5.75 (br s, 1H, 6-vinylic),
6.77 (br s, 1H, 4-vinylic) and 7.34 ppm (br s, 1H, NH); 13C NMR
(DMSO-d6): 170.29 (C-17), 162.41 (C-N), 118.34 (C-3), 132.05
(C-4), 139.84 (C-5), 129.60 (C-6), 32.15 (C-1), 21.29 (C-2), 30.52
(C-7), 31.53 (C-8), 47.46 (C-9), 34.44 (C-10), 19.56 (C-11), 38.58
(C-12), 53.26 (C-13), 46.82 (C-14), 20.41 (C-15), 31.06 (C-16),
18.51 (C-18), 21.59 (C-19), 10.54 (N-CO-CH3) and 164.76 (N-CO-
CH3); Mass (APCI): 410.31 [M+15]+.
sion test. At a dose of 400
killed. Two days after transfection the growth medium was replaced
by medium containing 500 g/ml G418 sulfate. During the follow-
lg/ml G418 sulfate 50% of the cells were
l
ing 3 weeks of incubation untransfected cells subsequently died.
To remove cell debris the medium was replaced every four days.
Stable cell clones could be identified by phase contrast microscopy
at the end of the second week. Single cell clones were picked and
transferred into 60 rnm culture plates for further analysis.
Using these cell lines, selected synthesized compounds as well
as the clinically used drug finasteride were tested, by measuring
the conversion of [3H] androstenedione.25,26 HPLC reversed phase
technique was used to quantify the reaction products. The details
regarding preparation of solutions and HPLC analysis have been
discussed in our previous report.10
4.2. Biological activity
4.2.1. In vitro human 5a-reductase inhibitory activity
HEK-I and HEK-II served cell lines were used as a source of 5AR
enzyme. Human embryonic kidney cell line (HEK293) lacking
endogenous 5AR activity was transfected with the cDNAs encoding
for both 5AR-1 and 5AR-2 by inserting them into pRcCMV (Cyto-
megalovirus promoter of the eukaryotic expression vector). Single
cell clones were selected having substantially high enzymatic
activity and were established as permanent cell lines.
4.2.6. Inhibition assay
Lysates were obtained after harvesting and resuspending 80%
confluent cells in homogenate buffer (300 mM saccharose + 5 mM
Tris–HCl + 0.1 mM EDTA) followed by homogenization by using
ultrasonication. Suspensions of both cell lines (HEK-I and HEK-II)
were used for all the assays. A master mix of 124.7
50 l androstenedione solution (500 nM) and 50 l regenerating
system (NADP/glucose/glucose-6-P-dehydrogenase = 1:2:1) was
made and mixed with 0.3 l of
3H labeled androstenedione. 25
the compound solution (in 2% DMSO) to be tested was added to
this solution to get a final test volume of 250 l. 250 l of the cell
suspension was added to 250 l of the master mix to make a total
volume of 500 l and incubated at 37 °C for 30 min. The reaction
was stopped by addition of 750 l of ether. The steroids were
ll tris buffer,
4.2.2. Cell culture
l
l
The adherent fibroblastoid HEK293 cell line was obtained from
DSMZ, Braunsch-weig, Germany (DSM ACC 305) and maintained in
Dulbecco’s modified Eagle medium (DMEM) with 10% fetal calf
serum (FCS), 0.25% sodium hydrogen carbonate, 100 units peni-
ll
l
l
l
cillin/ml, and 100
l
g streptomycin/ml. The cells were grown in a
l
humidified 95% O2–5% CO2 atmosphere at 37 °C in 175 cm2 tissue
l
culture flasks. Every 3–4 days they were split at a ratio of 1:6.
l
Please cite this article in press as: Aggarwal, S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2015.12.048

10
S. Aggarwal et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
extracted, dried and resuspended in 35 ll of methanol followed by
Supplementary data
radioactivity HPLC based detection. The amount of converted triti-
ated androstenedione was measured for each synthesized com-
pound to determine the inhibitory activity of the compounds.29,30
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.12.048.
4.2.7. In vivo 5AR inhibitory activity
References and notes
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 facil-
ities 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, 1–1.5% of each mineral
and vitamin mix. The control diet was obtained from Ashirwad
Industries (Chandigarh, India). Finasteride was procured from Dr
Reddy’s Laboratories, Hyderabad as gift sample. Mature male Wis-
tar 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
suspended 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
identified, removed and after removal of adhering fat and connec-
tive tissue weighed: ventral prostate, dorsal prostate, seminal vesi-
cles, testes, epididymis, vas deferens, liver and adrenal glands.
Organ weights were recalculated (mg/100 g body weight), that is,
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 Shimadzu 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
receiving 5 ml/kg Finasteride) and synthesized compounds (ani-
mals receiving 5 ml/kg compound). Results are expressed as
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test.31,32 For the statistical determination, statistical computerized
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Acknowledgements
The authors gratefully acknowledge UGC (University Grant
Commission), New Delhi, India for providing UGC-RFSMS fellow-
ship to Saurabh Aggarwal. We thank Mr. Avtar Singh (SAIF, Panjab
University, Chandigarh, India) for doing the NMR study/character-
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India for generous gift of Finasteride and Cipla Limited, Mumbai,
India for dehydroepiandrosterone as gift sample.
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Please cite this article in press as: Aggarwal, S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2015.12.048