Novel aryl piperazines for alleviation of ‘andropause’ associated prostatic disorders and depression

Article abstract of DOI:10.1016/j.ejmech.2017.03.036

A series of seventeen piperazine derivatives have been synthesized and biologically evaluated for the management of andropause-associated prostatic disorders and depression. Five compounds 16, 19, 20, 21 and 22 significantly inhibited proliferation of and

Full text of DOI:10.1016/j.ejmech.2017.03.036

Accepted Manuscript
Novel aryl piperazines for alleviation of ‘andropause’ associated prostatic disorders
and depression
Sonal Gupta, Deepti Pandey, Dhanaraju Mandalapu, Vikas Sharma, Mahendra
Shukla, Seema Singh, Nidhi Singh, Santosh Kumar Yadav, Dilip Kumar Tanpula,
Surabhi Singh, Jagdamba P. Maikhuri, Shubha Shukla, Jawahar Lal, Mohammad I.
Siddiqi, Gopal Gupta, Vishnu L. Sharma
PII:
S0223-5234(17)30193-9
10.1016/j.ejmech.2017.03.036
EJMECH 9296
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 January 2017
Revised Date: 17 March 2017
Accepted Date: 19 March 2017
Please cite this article as: S. Gupta, D. Pandey, D. Mandalapu, V. Sharma, M. Shukla, S. Singh, N.
Singh, S.K. Yadav, D.K. Tanpula, S. Singh, J.P. Maikhuri, S. Shukla, J. Lal, M.I. Siddiqi, G. Gupta,
V.L. Sharma, Novel aryl piperazines for alleviation of ‘andropause’ associated prostatic disorders and
depression, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.036.
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Table of Content

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Novel Aryl Piperazines for alleviation of ‘Andropause’ associated Prostatic Disorders and
Depression#
Sonal Gupta,†,$ Deepti Pandey,‡ Dhanaraju Mandalapu,† Vikas Sharma,‡ Mahendra Shukla,§,$ Seema
Singh,£,$ Nidhi Singh,# Santosh Kumar Yadav,‡ Dilip Kumar Tanpula,§,┴ Surabhi Singh,£,χ Jagdamba P.
$
Maikhuri,‡ Shubha Shukla,£,$ Jawahar Lal,§,$ Mohammad I. Siddiqi,# Gopal Gupta,‡ and Vishnu L.
Sharma*,†,$
†Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
‡Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
§Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
£Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
#Molecular & Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
┴Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Raebareli-
229010, India
χDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research
(NIPER), Raebareli-229010, India
$Academy of Scientific and Innovative Research (AcSIR), New Delhi-110001 (India)
*Corresponding author. Dr. V. L. Sharma
Medicinal and Process Chemistry Division,
CSIR-Central Drug Research Institute, Sector 10, Jankipuram ext.,
Lucknow, Uttar Pradesh 226031, India.
Tel.: 91-522-2772450; Ext. 4671; Fax: 91-522-2771941
E-mail address: vl_sharma@cdri.res.in; vlscdri@gmail.com
#CDRI Manuscript No. 09/2016/VLS

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ABSTRACT
A series of seventeen piperazine derivatives have been synthesized and biologically evaluated for
the management of andropause-associated prostatic disorders and depression. Five compounds
16, 19, 20, 21 and 22 significantly inhibited proliferation of androgen-sensitive LNCaP prostatic
cell line with EC50 values of 12.4 µM, 15.6 µM, 11.8 µM, 10.4 µM, 12.2 µM respectively and
decreased Ca2+ entry through adrenergic-receptor α1A blocking activity. Anti-androgenic
behaviour of compound 19 and 22 was evident by decreased luciferase activity. The high EC50
value in AR-negative cells PC3 and DU145 suggested that the cytotoxicity of compounds was
due to AR down regulation. Compound 19 reduced the prostate weight of rats by 53.8%. Further,
forced-swimming and tail-suspension tests revealed antidepressant-like activity of compound 19,
lacking effects on neuromuscular co-ordination. In silico ADMET predictions revealed that the
compound 19 had good oral absorption, aqueous solubility, non-hepatotoxic and good affinity for plasma
protein binding. Pharmacokinetic and tissue uptake of 19 at 10 mg/kg demonstrated an oral
bioavailability of 35.4%. In silico docking studies predicted similar binding pattern of compound
19 on androgen receptor as hydroxyflutamide. Compound 19 appears to be a unique scaffold
with promising activities against androgen associated prostatic disorders in males like prostate
cancer and BPH and associated depression.
Keywords: Piperazine derivatives; MTT assay; α1A blocking activity, Pharmacokinetics

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INTRODUCTION
Testosterone (T) and dihydrotestosterone (DHT) are the two androgenic hormones governing the
normal tissue growth and differentiation of the prostate gland. These two male hormones are
potent activators of androgen receptor (AR), the member of nuclear hormone receptor
superfamily, which operates as a transcription factor and regulates downstream gene expressions
[1]. AR signaling shows an array of pharmacological functions such as development and
maintenance of male sexual characteristics and musculoskeletal tissues [2]. Androgen antagonists
bind with the androgen receptors and display antagonist actions on prostate. Due to a range of
side effects of steroidal androgen antagonists such as hepatotoxicity [3], loss of libido [4] etc. the
non-steroidal template came into existence such as flutamide [5], nilutamide [6], and
bicalutamide [7] (Figure 1) which are used for the treatment of proliferative androgen dependent
prostatic disorders such as benign prostatic hyperplasia (BPH) and prostate cancer. Further, the
tissue selective action of these androgen antagonists is yet ambiguous and needs clarification [8].
BPH and prostate cancer are age associated disorders and the risk of the incidence increases with
age [9,10]. Globally, prostate cancer is the second most frequently diagnosed cancer in men
(899,000 new cases, 13.6 % of the total) and the fifth most common cancer overall, leading to
258,000 deaths in 2008 [11]. On the other hand, BPH is increasingly becoming a major old-age
disorder in human males causing morbidity due to troublesome Lower Urinary Tract Symptoms
(LUTS). According to the global data it has been anticipated that the market value for treatment
of BPH may rise from $2 billion in 2014 to approximately $4.9 billion by 2024 [12]. Similarly,
the total estimated expenditure on prostate cancer in US was ~12 billion dollars ($) in 2010, and
based on the growing aged population it is project to go up by 27%, by year 2020 [13].
ABBREVIATIONS
BPH, Benign prostatic hyperplasia; T, testosterone; DHT, dihydrotestosterone; SARM, selective androgen receptor
modulator; PSA, prostate specific antigen; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetra-zolium bromide;
DMF, dimethylsulphoxide; RFU, relative fluorescence unit; FST, forced swim test; TST, tail suspension test;
AUClast, area under the serum/tissue concentration-time curve up to last observation; Cmax, peak serum/tissue
concentration; tmax, time to Cmax; Vd/F, volume of distribution; Cl/F, clearance; F, fraction of dose absorbed; HF,
hydroxyflutamide; AR, androgen receptor; ET3N, triethylamine; DCM, dichloromethane; ETOAc, ethyl acetate;
DMSO, dimethyl sulfoxide; OD, optical density; SDS, sodium dodecyl sulphate; TBS, tri buffered saline; FBS, fetal
bovine serum; UFLC, ultra fast liquid chromatography.

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O
NH
O2N
F3C
F3C
O2N
N
O
O
N
H
Nilutamide 2
Flutamide 1
HO
O
O
O
H
F3C
NC
N
S
F
Bicalutamide 3
Figure 1. Structure of SARMs
It is well known that the progression of static component of the BPH [14] and early stages of
prostate cancer [15] are androgen dependent. Besides being androgen sensitive, prostate cancer
[16] and BPH [17] display increased adrenergic-α1A receptor activity, hence sensitive to α1-
adrenergic blockers (e.g. prazosin, tamsulosin). Currently androgen antagonists are the potential
option for management of androgen sensitive prostatic disorders in ageing males. Thus the
discovery of novel androgen antagonists with α1A-adrenergic blocking activity would be highly
desirable to complement the activity of scaffolds. Different androgen antagonists have been
reported in literature [18,19] based on different scaffolds.
It has been reported that prostatic disorders [20,21] and depression are closely associated with
each other [10,22]. Depressive symptoms are generally diagnosed while experiencing
complications due to prostatic problems and for a longer duration in elderly males [21,23].
Further, the presence of LUTS also increases the occurrence of depression in males [24,25]. BPH
derived depression may be attributed to the fact that androgen imbalance (andropause, a hormone
imbalance caused by lower levels of male hormone testosterone) [26] and depression are
interlinked [27,28]. Hence it would be worthwhile to develop a scaffold with inbuilt anti-
depressant activity.
Therapeutic potential and versatility of piperazines are well documented in literature as
anticancer [29], anticonvulsant [30], antimalarial [31], antidepressant [32], etc. making it a
privileged moiety in the field of medicinal chemistry. Additionally, piperazine bearing scaffolds
hold a significant position in the area of non-steroidal androgen antagonists [19], as well as α1
adrenoceptor antagonists [33] as well as dual activity towards α1 adrenoceptor and 5-HT1A
receptors [34,35,36]. Our previous lab work explains that the piperazine containing scaffolds
display an antagonistic profile against prostate, acting as androgen antagonists [19]. Piperazine

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provides the molecular backbone to the molecule and the basicity of nitrogen atoms help in
improving the potency of the scaffolds. Our endeavours of further exploring with piperazine
motif containing scaffolds with both androgen antagonists and α1A-adrenergic blocking activity
led to the designing and development of new compounds containing two piperazines joined with
variable number of methylene linkers. This report explains the synthesis, biological evaluation,
pharmacokinetic studies and docking studies of synthesized piperazine based motifs (Figure 2).
Ar1
Ar2
Ar1
N
N
N
N
Ar2
N
N
X
X
Ar1, Ar2 = aryl, heteroaryl
X = (CH2)n
Ar1, Ar2 = aryl, heteroaryl
X = CO, CH2
Previous Work
Present Work
Figure 2. General structure of synthesized compounds
RESULT AND DISCUSSION
Chemistry
Aryl/hetroaryl piperazine derivatives may be synthesized by the following Scheme 1. In a single
step reaction the substituted aryl/heteroaryl piperazine (4-10) were allowed to react with
dihaloalkanes in basic conditions under heating in acetonitrile to furnish the respective final
differently substituted aryl/heteroaryl piperazines derivatives (11-23).
Scheme 1. Synthesis of compounds 11-23

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Scheme 2 further defines the synthesis of more derivatives (28-31). Substituted piperazine (5-6)
on reaction with bromoethanol and subsequently with thionyl chloride gives the respective
intermediates (26-27). Further 26-27 on refluxing with substituted piperazines in basic conditions
gives final compounds (28-31).
Scheme 2. Synthesis of compounds 28-31
Biology
In vitro evaluation
Studies have shown that LNCaP cell are androgen dependent and also expresses α1A [37].
Accordingly, the initial screening of seventeen synthesized compounds (11-23, 28-31) has been
done against the androgen dependent LNCaP cells via MTT assay taking flutamide (EC50 = 7.0
µM) as standard. The motive of this experiment was to obtain preliminary data on the inhibitory
potential of compounds through a convenient screening method. The EC50 value of new
compounds varied between 10.4-78.7 µM. Based on the results of MTT assay, compounds 16,
19, 20, 21 and 22 exhibited good EC50 (µM) values in the range of 10 to 15 µM, which was
comparable to the standard drug. Therefore, these five compounds were selected for further
biological profiling. The structure of synthesized scaffolds and their EC50 (µM) values have been
given in table 1.

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Table 1. In vitro activity in LNCaP cell line (EC50 µM) of 11-23 and 28-31
EC50 (µM) in
LNCaP
Compound
number
n
Ar1
Ar2
2
40
11
2
2
25.2
71.2
12
13
2
40
14
2
2
2
72.2
12.4
63.2
15
16
17
3
3
3
49.2
15.6
11.8
18
19
20
3
3
3
21
22
23
10.4
12.2
55.5

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2
2
2
46.5
77.9
78.7
28
29
30
2
68
31
Detailed study of the data revealed the importance of methoxy, nitro, fluoro, trifluoromethyl
substituent at the phenyl ring of piperazine and the heterocyclic pyridyl nucleus over the
piperazine core in the synthesized scaffolds. The compounds 11-23 were symmetric having
similar substituent on both the piperazines with 2 and 3 methylene spacer unit i.e. ethylene and
propylene spacer while 28-31 were asymmetric with different substitution over both piperazines
separated with 2 methylene spacers. It was found that in compounds 11-17 containing 2
methylene spacer units, compound 11 (EC50 = 40 µM) with 2-OCH3 phenyl substituent on the
N4-position of both the piperazine showed moderate inhibition in the androgen dependent cell
lines. Replacement of 2-OCH3 with the 4-NO2 group 12 (EC50 = 25.2 µM) increased the activity
by 1.6 folds suggesting that the 4-NO2 phenyl substituent was more favorable over 2-OCH3
phenyl group. Further in scaffold 15 (EC50 = 72.2 µM), when 4-NO2 group is accompanied by 2-
CF3 on the phenyl ring of both the piperazines, showed a considerable decrease in potency
exhibiting that both the groups were not complementary to each other. On the other hand when
the 4-NO2 and 2-CF3 were interchanged as in 17 (EC50 = 63.2 µM) having the 2-NO2 and 4-CF3
groups over the phenyl ring of both piperazine displayed a slight improvement in activity.
Replacement of 4-F by 4-NO2 group as in compound 13 (EC50 = 71.2 µM) resulted in decreased
activity. The EC50 values of 13 and 15 and 17 confirm that the presence of 4-F, 2-CF3 and 4-CF3
respectively over the phenyl rings decrease the antagonistic behavior of the scaffolds. While the
reshuffling of the fluoro substituent from the position-4 to position-2 of phenyl ring (compound
14, EC50 = 40 µM) leads to the improvement of the activity of scaffold, showing that 2-F is more
favourable than 4-F group. The switch over of the phenyl ring by the heterocyclic 2-pyridyl

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nucleus (compound 16, EC50 = 12.4 µM) causes a remarkable increase in the effectiveness of the
scaffold as an antagonist.
Scaffolds 18-23, had propylene as a linker between the differently substituted piperazine
fragments. Compound 18 (EC50 = 49.2 µM) with 4-F phenyl core exhibited moderate inhibition.
Moving this fluoro substituent from position-4 to position-2 (compound 21, EC50 = 10.4 µM)
caused five-fold enhancement of activity in comparison to 18. Presence of 4-NO2 phenyl
(compound 20, EC50 = 11.8 µM) led to a significant increase in the antagonistic behavior of the
motif and additional 2-CF3 group (compound 23, EC50 = 55.5 µM) showed a diminished activity.
However, interchanging the positions of these two groups (compound 22, EC50 = 12.2 µM)
enhanced the activity by about 4.5 folds. Adding the heterocyclic 2-pyridyl component to the
nucleus (compound 19, EC50 = 15.6 µM) also retained the good activity. The piperazine
scaffolds 28-31 with dissimilar aryl substituent showed moderate activity (EC50 = 46.6-77.9
µM).
SAR of the scaffolds showed that with ethylene linker with the 2-pyridyl substitution (16) was
the most privileged showing maximum inhibition followed by 4-NO2 phenyl (12), 2-OCH3
phenyl (11) = 2-F phenyl (14), 2-NO2-4-CF3 phenyl (17), 4-F phenyl (13) and 4-NO2-2-CF3
phenyl (15). While with propylene linker the order of activity was found to be 2-F phenyl (21) >
4-NO2 phenyl (20) > 2-NO2-4-CF3 phenyl (22) > 2-pyridyl (19) > 4-F phenyl (18) > 4-NO2-2-
CF3 phenyl (23).
The five scaffolds 16, 19, 20, 21 and 22 with EC50 values of 12.4 µM, 15.6 µM, 11.8 µM, 10.4
µM, 12.2 µM respectively, and comparable with flutamide, were subjected to time dependent
analysis of Ca2+ mobilization in α1A expressing Chem-1 cells and in vivo evaluation in rats.
AR-antagonist nature of test compounds was exhibited by luciferase reporter gene assay. An AR
negative Cos7 cell line was transiently transfected with AR and ARE- LUC plasmids and treated
with compounds 19 and 22 at a concentration of 7.5 µM. Bicalutamide (3µM), was used as
positive control. Test compounds (19 and 22, P<0.01) showed a significant decrease in luciferase
activity as compared to control at 24 h, which displayed their anti-androgenic nature (Figure 3).

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Figure 3. Transactivation of ARE-LUC by AR in presence of test compounds was measured by
dual-luciferase reporter assay in terms of RLU (Control=100%). 19 (7.5 µM), 22 (7.5 µM) and
bicalutamide (3 µM). Significant difference from control is indicated as **P<0.01 and
***P<0.001).
Cell viability test was performed in AR-negative PC3 and DU145 cells lines, which exhibited
that the anti proliferative activity of test compounds 16, 19, 20, 21 and 22 was due to AR down
regulation. EC50 values of the compounds in AR-negative cells PC3, DU145 and AR-positive
cells LNCaP cells was shown in table 2. The high EC50 value in AR-negative cells PC3 and
DU145 suggested that the cytotoxicity of compounds was specifically mediated through AR.
Table 2. Cell Viability Test in PC3 and DU145 of compounds 16, 19, 20, 21 and 22
Compound EC50 for PC3 (µM) EC50 for DU145 (µM) EC50 for LNCaP (µM)
>80
>80
>80
>80
>80
>80
>80
>80
>80
76.5
12.4
15.6
11.8
10.4
12.2
16
19
20
21
22
Alpha blocker (α1A-adrenoceptor antagonist) alleviates the symptom score of the disease by
binding with the α1A-adrenoceptor present in the smooth muscle of the prostate. The five
synthesized piperazine derivatives 16, 19, 20, 21 and 22 were analyzed for the alpha blocking
activity via time dependent analysis of Ca2+ mobilization in α1a expressing Chem-1 cells. Assay
was done with dose responses for compounds 16, 19, 20, 21 and 22 at 1 µM, agonist epinephrine

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at 20 µM and standard antagonist tamsulosin at 100 nM and 1 µM. The values of relative
fluorescence unit (RFU) correspond to the Ca2+ released in the cells. It was elaborated from the
results that the epinephrine, an agonist, increased the Ca2+ level significantly about 2.5 times in
comparison to the untreated cell. Contrary to this the standard tamsulosin (1 µM), an antagonist
causes the diminution of the Ca2+ level about 1.6 times of the epinephrine treated cells. The five
tested scaffold exhibited the alpha blocking potency at concentration of 1 µM. Relative
fluorescence unit (RFU) data reveals that the cells treated with compound 19 and 22 experience
Ca2+ level reduction 1.6 times of epinephrine treated cells which is approximately same as that of
tamsulosin. On the other side the next three scaffolds 16, 20 and 21 showed a drop of 1.5 times
which very close to standard drug tamsulosin (Figure 4). Hence, these scaffolds displayed good
potency as α1A-adrenoceptor antagonist.
Figure 4. Time dependent analysis of Ca2+ mobilization in α1A expressing Chem-1 cells with
different concentration of compounds 16 (1 µM), 19 (1 µM), 20 (1 µM), 21 (1 µM), 22 (1 µM),
agonist epinephrine (20 µM) and antagonist tamsulosin (1 µM)
In vivo Experiment on Sprague–Dawley Rats
Compounds 16, 19, 20, 21 and 22 were subjected to in vivo experiment performed on the adult
male Sprague–Dawley rats (weighing 250–280 g, n = 5). 29 days study included the induction of
disease (prostatic hyperplasia) by initial 8 days oral dosing with citral (100 mg/kg) followed by
giving compounds 16, 19, 20, 21, 22 and flutamide at 10 mg/kg for rest of 21 days parallel along
with citral. Table 3 shows the results of experiment after dissection and weighing of the prostate
24 h of last treatment.

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Table 3. Effect of compound 16, 19, 20, 21 and 22 on prostate
Prostate wt.
per 100 g of body
% Reduction of
prostate weight
Compd no.
% Prostate wt.*
weight
0.16
0.13
0.21
0.18
0.14
0.12
0.29
0.11
56.0
46.2
71.0
63.0
48.3
41.2
100
44.0
53.8
29.0
37.0
51.7
58.8
0
16
19
20
21
22
Flutamide
Citral
Control
37.9
-
* Prostate weight of citral treated rats was taken as 100%.
Citral being a disease inducer caused the increase in the prostatic weight up to 290 mg/100 g of
body weight which is significantly higher than that of control 110 mg/100 g of body weight.
Experiment displayed the prominent reduction of prostatic weight by treatment with all the five
derivatives. Compound 19 and 22 exhibited reduction of 54% and 52% respectively which is
comparable with flutamide. Interestingly, compounds did not affect the weights of seminal
vesicle, testis and epididymis (Figure 5).
Figure 5. Effect of citral (10 mg/Kg) compound flutamide, 16, 19, 20, 21 and 22 (10 mg/Kg) on
prostate, testis, epididymis, seminal vesicle
Histological investigations of prostatic tissues were also carried out for all five compounds.
Compound 19 was found to show the maximum (54%) reduction of the prostatic weight than
other compounds so was also subjected for pharmacokinetic studies.

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Histology
The histological investigations of periurethral prostatic tissues obtained from the in vivo
experiments were done microscopically and presented in figure 6. Pictures clearly demonstrated
that the BPH generated tissue have very little stromal differentiation as compared to control
tissue having clear stroma. Treatment with compounds 16, 19, 20, 21, 22 and flutamide in
parallel with citral increased the distinction between the stromal and acinar compartments. The
results obtained from this study support the in vitro and in vivo results.
Figure 6. Representative histological architecture of the prostate of rats given vehicle control,
citral (100 mg/Kg), citral + flutamide (10 mg/Kg), (A) citral + 16 (10 mg/Kg), (B) citral + 19 (10
mg/Kg), (C) citral + 20 (10 mg/Kg), (D) citral + 21 (10 mg/Kg) and (E) citral + 22 (10 mg/Kg)
orally once daily for 21 days
Evaluation of antidepressant-like activity of BPH active compounds in different mice
models
Forced Swim Test (FST)
Five compounds 16, 19, 20, 21, 22 active in in vivo studies for BPH were also studied for
antidepressant like activity and tested in FST, a primary model of depression. Active compounds
at dose of 20 mg/kg were administered orally to the mice and after 60 min of administration
subjected to FST and immobility time was monitored. The reduction in the immobility time was
found to be highly significant (p<0.001) after the administration of compound 19 (69.40%) and
20 (69.74%) as compared to control mice (Figure 7). The two compounds 19 and 20 also resulted

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in significant reduction of immobility time as compared to Imipramine (IMI) (60 mg/kg)
whereas was comparable with Flouxetine (FXT) (20mg/kg) (Table 4).
FST
***
**
250
200
150
100
50
***
***
0
Figure 7. Force Swimming Test. Bar diagram shows immobility time duration (in seconds)
during 5 minutes of test. The compounds were given orally at dose of 20 mg/kg.i.p. ***P<0.001;
**P<0.01 vs control. Data was expressed as mean ± SEM; N=7-9 and analyzed by unpaired t-test
Table 4. Percentage decrease of immobility time (seconds) of compounds in FST. ***P < 0.001
vs control
% decrease of
Significance
level
Compound
Mean
194.10
SEM
14.86
immobility
-
Ns
P < 0.001
P < 0.001
Ns
Ns
P < 0.01
P < 0.001
-
Control
16 (29)
19 (32)
20 (33)
21 (34)
161.2
59.39
58.72
153.60
144.20
115.70
47.84
18.57
12.28
9.55
23.35
36.47
17.02
17.66
16.95
69.40
69.74
20.86
25.71
40.39
75.35
22 (35)
Imipramine (IMI)
Fluoxetine (FXT)
Tail Suspension Test (TST)
Further compounds 19 and 20 were also tested (at same dose) in tail suspension test, which is
another model of antidepressant like activity to further confirm this activity. Treated mice were

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suspended with tail in a horizontal rod and immobility time was recorded. Both the compounds
showed significant reduction in immobility time and their effect was found comparable to
Imipramine (60 mg/kg) in TST (Figure 8).
TST
250
200
150
100
50
***
***
***
0
Figure 8. Tail Suspension Test. Diagram represents immobility time duration in TST when
suspended in rod. ***P<0.001 as compared to control. Data was expressed as mean ± SEM; N=5
FST and TST experiments confirmed the antidepressant like activity of compounds 19 and 20.
Thereafter, both these compounds were further evaluated for their effect on motor activity.
Locomotor Activity and Rotarod Test
Locomotor activity and rotarod test (n = 6) were also performed for compounds 19 and 20,
which were found active in both FST and TST. There was no significant difference in locomotor
activity when compared to control animals. This indicated that the compounds were not causing
any alteration in locomotor activity (Figure 9). Similarly, effect of compounds on rotarod test for
motor co-ordination showed that the latency to fall was not affected after the administration of
compounds and stayed for 120 sec on the rotating rod. This suggests that the neuromuscular co-
ordination of the animals was not impaired by treatment with the compounds 19 and 20.

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1000
800
600
400
200
0
Control
19
20
Time points (mins)
Figure 9. Locomotor activity of compound 19 and 20 treated and control mice (n=5)
In silico ADMET Prediction of Selected Compounds
The in silico ADMET predictions using BIOVIA Discovery Studio 4.1, for the compounds 16, 19,
20, 21 and 22 are summarized in the table 5. The ADMET absorption ratio of 0 predicted for most of the
compounds indicated good absorption. The ADMET solubility level of 2 indicated that although the
compound is having some aqueous solubility but it is low, whereas ADMET solubility level of 3
indicated good aqueous solubility. The ADMET BBB level of 0, 1 and 4 indicated very high, high and
undefined respective blood brain barrier penetration levels of compounds. The compounds with ADMET
EXT CYP2D6 PRED as true indicated CYP2D6 inhibition potential and false indicated no inhibition
potential. The compounds with false predicted ADMET hepatotoxicity are categorized into nontoxic. The
ADMET EXT PPB PRED indicated good affinity of all the compounds for plasma protein binding [38].
Overall, the compound 19 was predicted to have good oral absorption, good aqueous solubility, non-
hepatotoxic with CYP2D6 inhibition potential and good affinity for plasma protein binding. The
confirmation of these predictions is provided by the in vivo pharmacokinetic studies of the compound 19
in Sprague Dawley rats.
Table 5. In silico ADMET Prediction of Selected Compounds
ADMET
Comp. solubility
level
ADMET
BBB_level
ADMET EXT
CYP2D6 PRED
ADMET EXT
hepatotoxic pred
ADMET EXT
PPB PRED
ADMET
absorption
3
1
true
false
true
0
16

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3
2
2
2
1
4
0
4
true
false
true
false
true
true
false
true
true
true
true
0
0
0
2
19
20
21
22
false
Pharmacokinetic Studies
The animals tolerated the treatment as no peculiarities in the animals’ behaviour were observed.
Serum levels of the compound 19 were monitored up to 24 h. Following 10 mg/kg oral dose, it
showed a peak serum concentration (Cmax, 328.6 ± 45.9 ng/mL) at 1 h indicating rapid absorption
(Figure 9). Moreover, it was monitored up to 48 h in hypothalamus and prostate with Cmax at 1
and 4 h post dose, respectively. The concentration-time profile was analyzed using both non-
compartmental and compartmental approach. The pharmacokinetic models were compared
according to maximal correlation between observed and predicted concentration, minimal sum of
squared residuals, Akaike’s Information Criterion (AIC) and Schwarz Bayesian Criterion (SBC)
[39,40]. The concentration-time data was best described by a two-compartmental open model
and the calculated pharmacokinetic parameters are shown in table 6. The volume of distribution
(28.7 ± 3.8 L/kg) is larger than the total blood volume of rat (0.054 L/kg [41]) and systemic
clearance (4.1 ± 0.3 L/h/kg) is higher than the total hepatic blood flow in rats (2.9 L/h/kg [41])
indicating extravascular distribution along with the extrahepatic elimination of compound. The
oral bioavailability was found to be 35.4%. Compound 19 displayed higher tissue to serum
concentration ratio in prostate. However, hypothalamus showed lower tissue to serum ratio
(Figure 10, table 6).

ACCEPTED MANUSCRIPT
Figure 10. Mean concentration-time profile of 19 in serum, prostate and hypothalamus after
single 10 mg/kg oral and intravenous administration in male Sprague Dawley rats. Bar represents
SEM (n = 3).
Table 6. Pharmacokinetic parameters of 19 after single 10 mg/kg oral and intravenous
administration in male Sprague Dawley ratsa
Parameters
Intravenous
Serum
520.0 ± 37.7
-
Oral
Prostate
573.3 ± 10.2
4.0
Serum
328.6 ± 45.9
1.0
Hypothalamus
Cmax (ng/mL or ng/g)
tmax (h)
461.0
1.0
AUClast (µg h/mL or
µg h/g)
2.5 ± 0.2
0.9
10.7
4.2
T1/2 (h)
5.2 ± 0.7
4.1 ± 0.3
28.3 ± 3.8
5.4
3.9
23.7
1.0
155.5
0.3
3.6
11.8
-
128.8
0.8
6.6
4.6
-
Cl (L/h/kg or g/h/kg)
Vss (L/kg or g/kg)
Tissue to serum ratio
Bioavailability (%)
-
-
35.4
aEach value represent the average of three rats. Serum and prostate concentration are mean ± SEM from
three rats. Hypothalamus concentrations are the mean of the pooled tissue of three rats.
Docking Studies
Previous lab work showed that the piperazine containing scaffolds possibly act via androgen
receptor modulation [19] therefore the present compounds were docked with androgen receptor.
The binding mode of compounds 16, 19, 20, 21 and 22 has been compared to Hydroxyflutamide
(HF) which is known antagonist of androgen receptor (AR). The crystal structure of androgen
receptor in complex with HF has been reported (PDB accession code: 2AX6). It has been shown
that T877A mutation converts the HF to agonist [42]. In another study it has been proposed that
repositioning of helix-12 of AR causes the change in activity of HF [43]. Therefore, docking
studies of HF in the wild type AR was done and the reproduced binding pose was used as
reference for the reported active compounds (Figure 11, table 7). The predicted binding pattern
of compounds 16, 19, 20, 21 and 22 was similar to binding mode of HF. The protein-ligand
interactions were dominated by strong pi-pi stacking interactions with residues Phe 764 and Trp
741. The residues Met 749, Met 745 and Leu 707 are facilitating hydrophobic contacts. The
backbone of residue Met 742 is involved in hydrogen bond formation in case of compound 16,
19 and 22 while residues Gln 711 and Arg 752 showed to form hydrogen bond in case of

ACCEPTED MANUSCRIPT
compound 20. Compound 19 with propylene linker showed strong pi-pi stacking in comparison
to compound 16 with ethylene linker. Compound 21 with 2-fluoro phenyl substituent over
piperazine showed good pi-pi stacking but devoid of hydrogen bonding character. However,
compound 20 and 22 showed similar docking properties.
Figure 11. (A) Hydroxyflutamide binding mode to WT AR (B) Predicted binding mode of
compound 19 (C) compound 22 (D) compound 16 (E) compound 21 (F) compound 20 in AR
binding site. Protein is shown in mixed representation (ribbon and stick). Hydrogen bonds are
shown as black line
Table 7. Docking studies and the residues involved in interaction
Compound No.
Hydrogen bond
Met 742
Pi-pi stacking interactions
Phe 764, Trp 741
16
19
20
21
Met 742
Phe 764, Trp 741, His 874
Phe 764, Trp 741
Arg 752/Gln711
-
Phe 764, Trp 741, His 874

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Met 742
Phe 764, Trp 741
22
CONCLUSION
A series of seventeen alkylene bis(4-aryl/heteroarylpiperazine) derivatives (11-23, 28-31) were
synthesized and evaluated for proliferation inhibitory activity against androgen dependent
LNCaP cell lines via MTT assay using flutamide as standard. Compounds 16, 19, 20, 21 and 22,
which exhibited activity comparable to flutamide and were supplemented with α1A blocking
activity, were chosen for in vivo experiments, histology and docking studies. Anti-androgenic
behavior of the compounds was assessed by luciferase reporter gene assay. Further the anti
proliferative activity of the test compounds was due to AR down regulation. In vivo experiments
revealed that all the five compounds 16, 19, 20, 21 and 22 displayed a significant decrease in the
prostatic weight. Compound 19 showed 54% reduction of prostatic weight, which is similar to
flutamide (58%). Histological experiment also supported the results of in vivo studies. Further
compound 19 and 20 were also found to possess significant antidepressant like behavior without
affecting the neuromuscular co-ordination. In silico ADMET predictions revealed that the
compound 19 had good oral absorption, good aqueous solubility, non-hepatotoxic with CYP2D6
inhibition potential and good affinity for plasma protein binding. The compound 19 exhibited a higher
tissue to serum concentration ratio in prostate showing an oral bioavailability of 35.4%. In
docking studies it displayed similar binding mode as that of hydroxyflutamide. Conclusively, 1,
2-bis(4-(pyridin-2-yl)piperazin-1-yl)propane (19) might act as a potent lead candidate for
management of androgen sensitive prostatic disorders and associated depression in aged men.
EXPERIMENTAL
Chemistry
General Information
All reagents and solvents were commercial available and were used without further purification.
Analytical thin layer chromatography (TLC) was performed on pre-coated silica gel plates 60
GF254 (Aldrich) to monitor the progress of the reaction. Electrospray ionization mass spectra
(ESI-MS) were recorded on Ion Ttrap LCQ Advantage Max-IT (Thermo Electron Corporation).
High-resolution mass spectra (HRMS) were recorded on a 6520 Agilent Q Tof LC MS/MS
13
(accurate mass).1H and C NMR spectra were recorded on Bruker Supercon Magnet Avance

ACCEPTED MANUSCRIPT
DPX-200/DRX-300 spectrometers operating at 400 and 100 MHz respectively in deuterated
solvents with TMS as internal reference (chemical shifts
δ in ppm, J in Hz.). IR spectra (νmax in
cm-1) of the compounds were recorded on Perkin Elmer’s FT-IR RX1 PC spectrophotometer.
Melting points were determined in open capillary tubes on the melting point apparatus.
Elemental analyses were performed on Carlo Erba EA-1108 micro analyzer/Vario ELIII C H N S
analyzer. All compounds were analyzed for C, H, N and the results obtained were within ±0.4%
of calculated values. All compounds were characterized by TLC, 1H and 13C NMR, MS, and
HRMS. Elemental analyses data meet the criteria of >95% purity.
1, 2-Bis(4-(2-methoxyphenyl)piperazin-1-yl)ethane (11)
A mixture of 1-bromo-2-chloroethane (0.43 mL, 5.2 mmol), K2CO3 ( 0.71 g, 5.2 mmol) in ACN
(10mL) was stirred at 50 °C for 15 min and then 1-(2-methoxyphenyl)piperazine (4, 0.5 g, 2.6
mmol) in ACN (5 mL), was added dropwise within 1 h duration. Reaction mixture was further
stirred for 4 h. Then the mixture was concentrated under reduced pressure and residue was
dissolved in EtOAc (15 mL) and the EtOAc layer was then washed with water (10 mL × 3). The
combined organic layer was dried (anhyd. Na2SO4) and concentrated under reduced pressure.
The resultant was washed with distilled hexane and again dried under high vaccum. The pure
offwhite crystals were obtained by recrystallization using EtOAc/Hexane (yield: 69%); mp: 137-
1
140 °C; IR (KBr) ν (cm-1): 3019, 2834, 2400, 1594, 1499, 1451, 1411; H NMR (400 MHz,
13
CDCl3):
δ
7.04-6.87 (8H, m), 3.88 (6H, s), 3.15 (8H, bs), 2.78-2.72 (12H, m); C NMR (75.4
MHz, CDCl3):
δ
152.3, 141.3, 122.9, 121.0, 118.2, 111.2, 55.9, 55.3, 53.9, 50.6; HRMS (ESI
positive) m/z calcd. for C24H34N4O2 [M+H]+: 411.2760, found: 411.2761; Anal calcd. for
C24H34N4O2: C, 70.21; H, 8.35; N, 13.65, found: C, 70.09; H, 8.21; N, 13.43.
Further compounds 12-23 was synthesized by using the procedure similar to compound 11.
1, 2-Bis(4-(4-nitrophenyl)piperazin-1-yl)ethane (12)
The title compound was synthesized using 1-(4-nitrophenyl)piperazine (5) and 1-bromo-2-
chloroethane as a starting material and yellow colour crystals were obtained as a product (yield
77%); mp: 189-193 °C; IR (KBr) ν (cm-1); 3021, 2955, 2401, 1595, 1502, 1397, 1327; 1H NMR
(400 MHz, CDCl3):
δ
8.06-8.04 (4H, m), 6.76-6.74 (4H, m), 3.36 (8H, t, J = 5.0 Hz), 2.59 (8H, t,
13
J = 5.1 Hz), 2.5 (4H, s); C NMR (100 MHz, CDCl3):
δ 154.8, 138.5, 125.9, 112.6, 55.7, 53.1,
47.0; HRMS (ESI positive) m/z calcd. for C22H28N6O4 [M+H]+: 441.2250, found: 441.2247;
Anal calcd. for C22H28N6O4: C, 59.99; H, 6.41; N, 19.08, found: C, 59.72; H, 6.31; N, 19.01.

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1, 2-Bis(4-(4-fluorophenyl)piperazin-1-yl)ethane (13)
The title compound was synthesized from 1-(4-fluorophenyl)piperazine (6) and 1-bromo-2-
chloroethane as white solid (yield 70%); mp: 183-185 °C; IR (KBr) ν (cm-1): 3019, 2954, 2827,
1
2400, 1600, 1509, 1452, 1420, 1386; H NMR (400 MHz, CDCl3):
δ
6.97-6.93 (4H, m), 6.88-
13
6.85 (4H, m), 3.12 (8H, t, J = 4.8 Hz), 2.67 (8H, t, J = 5.0 Hz), 2.62 (4H, s); C NMR (100
MHz, CDCl3):
δ 158.3, 156.0, 147.9, 117.8, 117.7, 115.6, 115.4, 55.8, 53.6, 50.1; HRMS (ESI
positive) m/z calcd. for C22H28F2N4 [M+H]+: 387.2360, found: 387.2349; Anal calcd. for
C22H28F2N4: C, 68.37; H, 7.30; N, 14.50, found: C, 68.12; H, 7.21; N, 14.35.
1, 2-Bis(4-(2-fluorophenyl)piperazin-1-yl)ethane (14)
The title compound was synthesized from 1-(2-fluorophenyl)piperazine (7) and 1-bromo-2-
chloroethane to give pure compound as white solid (yield 59%); mp: 149-153 °C; IR (KBr) ν
(cm-1) 3020, 2951, 2829, 2401, 1613, 1501, 1450; 1H NMR (400 MHz, CDCl3)
m), 3.05 (8H, t, J = 4.6 Hz), 2.63 (8H, t, J = 4.7 Hz), 2.56 (4H, s); 13C NMR (100 MHz, CDCl3)
156.9, 154.5, 140.1, 140.0, 124.4, 124.4, 122.4, 122.4, 118.9, 118.9, 116.2, 115.9, 55.9, 53.7,
δ 7.00-6.82 (8H,
δ
50.5, 50.4; HRMS (ESI positive) m/z calcd. for C22H28F2N4 [M+H]+: 387.2360, found:
387.2359; Anal calcd. for C22H28F2N4: C, 68.37; H, 7.30; N, 14.50; found: C, 68.24; H, 7.01; N,
14.29.
1, 2-Bis(4-nitro-2-(trifluoromethyl)phenyl)piperazin-1-yl)ethane (15)
The title compound was synthesized from 1-(4-nitro-2-(trifluoromethyl)phenyl)piperazine (8)
and 1-bromo-2-chloroethane to give pure compound as yellow solid (yield 70%); mp: 138-143
°C; IR (KBr) ν (cm-1): 1638, 1386; 1H NMR (400 MHz, CDCl3):
δ 8.43 (2H, d, J = 2.6 Hz), 8.25
(2H, dd, J = 2.4, 8.9 Hz), 7.20-7.19 (2H, m), 3.10 (8H, t, J = 4.5 Hz), 2.63-2.62 (8H, m) 2.57
13
(4H, s); C NMR (100 MHz, CDCl3):
δ 156.9, 142.1, 127.8, 124.6, 124.6, 122.2, 122.0, 55.7,
53.4, 52.7, 51.2; HRMS (ESI positive) m/z calcd. for C24H26F6N6O4 [M+H]+: 577.1998, found:
577.1995; Anal calcd. for C24H26F6N6O4: C, 50.00; H, 4.55; N, 14.58, found: C, 49.85; H, 4.41;
N, 14.46.
1, 2-Bis(4-(pyridin-2-yl)piperazin-1-yl)ethane (16)
The title compound was synthesized from 1-(pyridine-2-yl)piperazine (9) and 1-bromo-2-
chloroethane as yellow solid (yield 74%); mp: 129-131 °C; IR (KBr) ν (cm-1): 3399, 2953, 1602,
1
1482, 1386; H NMR (400 MHz, CDCl3):
δ 8.20 (2H, dd, J = 1.4, 4.8 Hz), 7.50-7.46 (2H, m),

ACCEPTED MANUSCRIPT
13
6.66-6.61 (4H, m), 3.56 (8H, t, J = 4.9 Hz), 2.63 (12H, t, J = 4.5 Hz); C NMR (100 MHz,
CDCl3): 159.5, 147.9, 137.4, 113.2, 107.7, 56.0, 53.5, 45.1; HRMS (ESI positive) m/z calcd.
δ
for C20H28N6 [M+H]+: 353.2454, found: 353.2449; Anal calcd. for C20H28N6: C, 68.15; H, 8.01;
N, 23.84, found: C, 68.04; H, 7.90; N, 23.62.
1, 2-Bis(2-nitro-4-(trifluoromethyl)phenyl)piperazin-1-yl)ethane (17)
The title compound was synthesized from 1-(2-nitro-4-(trifluoromethyl)phenyl)piperazine (10)
and 1-bromo-2-chloroethane as yellow crystals (yield 78%); mp: 150-153 °C; IR (KBr) ν (cm-1):
1
3019, 2400, 1625, 1534, 1385, 1326; H NMR (400 MHz, CDCl3):
δ 7.95 (2H, d, J = 1.6 Hz),
7.57 (2H, dd, J = 1.9, 8.8 Hz), 7.07 (2H, d, J = 8.7 Hz), 3.09 (8H, t, J = 4.7 Hz), 2.58 (8H, t, J =
13
4.8 Hz), 2.53 (4H, s); C NMR (100 MHz, CDCl3):
δ 146.9, 139.5, 129.1, 129.1, 123.2, 123.1,
119.5, 54.6, 52.0, 49.8; HRMS (ESI positive) m/z calcd. for C24H26F6N6O4 [M+H]+: 577.1998,
found: 577.2023; Anal calcd. for C24H26F6N6O4: C, 50.00; H, 4.55; N, 14.58, found: C, 49.82; H,
4.33; N, 14.36.
1, 2-Bis(4-(4-fluorophenyl)piperazin-1-yl)propane (18)
The title compound was synthesized from 1-(4-fluorophenyl)piperazine (6) and 1-bromo-3-
chloropropane when pure compound was obtained as white solid (yield 55%); mp: 176-179 °C;
IR (KBr) ν (cm-1): 3019, 1654, 1627, 1509, 1404; 1H NMR (400 MHz, CDCl3):
δ 6.97-6.93 (4H,
m), 6.89-6.85 (4H, m), 3.13 (8H, t, J = 4.8 Hz), 2.61 (8H, t, J = 5.0 Hz), 2.45 (4H, t, J = 7.5 Hz),
13
1.80-1.75 (2H, m) ; C NMR (100 MHz, CDCl3):
δ 158.3, 155.9, 147.9, 147.9, 117.8, 117.7,
115.6, 115.3, 56.6, 53.2, 50.1, 24.3; HRMS (ESI positive) m/z calcd. for C23H30F2N4 [M+H]+:
401.2517, found: 401.2512; Anal calcd. for C23H30F2N4: C, 68.97; H, 7.55; N, 13.99, found: C,
68.75; H, 7.36; N, 13.74.
1, 2-Bis(4-(pyridin-2-yl)piperazin-1-yl)propane (19)
The title compound was synthesized from 1-(pyridine-2-yl)piperazine (9) and 1-bromo-3-
chloropropane as offwhite crystals (yield 66%); mp: 166-168 °C; IR (KBr) ν (cm-1): 3019, 2823,
1
1636, 1595, 1481, 1437, 1402, 1385, 1313; H NMR (400 MHz, CDCl3):
δ 8.19-8.18 (2H, m),
7.49-7.45 (2H, m), 6.65-6.60 (4H, m), 3.55 (8H, t, J = 5.0 Hz), 2.57 (8H, t, J = 5.1 Hz), 2.44 (4H,
t, J = 7.4 Hz), 1.82-1.75 (2H, m); 13C NMR (100 MHz, CDCl3):
159.5, 147.9, 137.4, 113.2,
δ
107.0, 56.7, 53.0, 45.1, 24.2; HRMS (ESI positive) m/z calcd. for C21H30N6 [M+H]+: 367.2610,
found, 367.2606; Anal calcd. for C21H30N6: C, 68.82; H, 8.25; N, 22.93, found: C, 68.90; H,
8.14; N, 22.72.

ACCEPTED MANUSCRIPT
1, 2-Bis(4-(4-nitrophenyl)piperazin-1-yl)propane (20)
The title compound was synthesized from 1-(4-nitrophenyl)piperazine (5) and 1-bromo-2-
chloropropane as pure yellow colour crystals (yield 80%); mp: 144-149 °C; IR (KBr) ν (cm-1):
1
3019, 1626, 1598, 1458, 1403, 1385, 1328; H NMR (400 MHz, CDCl3):
δ 8.05 (4H, d, J = 9.4
Hz), 6.75 (4H, d, J = 9.4 Hz), 3.36 (8H, t, J = 5.0 Hz), 2.53 (8H, t, J = 5.1 Hz), 2.39 (4H, t, J =
13
7.4 Hz), 1.71-1.63 (2H, m); C NMR (100 MHz, CDCl3):
δ 154.8, 138.4, 125.9, 112.6, 56.4,
52.7, 47.0, 24.3; HRMS (ESI positive) m/z calcd. for C23H30N6O4 [M+H]+: 455.2407, found,
455.2400; Anal calcd. for C23H30N6O4: C, 60.78; H, 6.65; N, 18.49, found: C, 60.61; H, 6.42; N,
18.25.
1, 2-Bis(4-(2-fluorophenyl)piperazin-1-yl)propane (21)
The title compound was synthesized from 1-(2-fluorophenyl)piperazine (7) and 1-bromo-2-
chloropropane to give pure compound as white solid (yield 68%); mp: 136-138 °C; IR (KBr) ν
1
(cm-1): 3016, 2915, 1654, 1457, 1403, 1385; H NMR (400 MHz, CDCl3):
δ 7.00-6.84 (8H, m),
3.05 (8H, t, J = 4.7 Hz), 2.58 (8H, t, J = 4.7 Hz), 2.40 (4H, t, J = 7.5 Hz), 1.74-1.69 (2H, m); 13C
NMR (100 MHz, CDCl3): 156.9, 154.5, 140.1, 140.0, 124.4, 124.4, 122.4, 122.3, 118.9, 118.9,
δ
116.1, 115.9, 56.6, 53.3, 50.5, 50.4, 24.2; HRMS (ESI positive) m/z calcd. for C23H30F2N4
[M+H]+: 401.2517, found, 401.2511; Anal calcd. for C23H30F2N4: C, 68.97; H, 7.55; N, 13.99,
found: C, 68.79; H, 7.34; N, 13.72.
1, 2-Bis(2-nitro-4-(trifluoromethyl)phenyl)piperazin-1-yl)propane (22)
The title compound was synthesized from 1-(2-nitro-4-(trifluoromethyl)phenyl)piperazine (10)
and 1-bromo-2-chloropropane to give pure compound as yellow solid (yield 80%); mp: 131-135
°C; IR (KBr) ν (cm-1): 3020, 1626, 1403, 1326; 1H NMR (400 MHz, CDCl3):
δ 8.04 (2H, d, J =
1.5 Hz), 7.66 (2H, dd, J = 2.0, 8.7 Hz), 7.17 (2H, d, J = 8.7 Hz), 3.18 (8H, t, J = 4.7 Hz), 2.61
13
(8H, t, J = 4.8 Hz), 2.47 (4H, t, J = 7.3 Hz), 1.79-1.70 (2H, m); C NMR (100 MHz, CDCl3):
δ
147.9, 140.6, 130.1, 130.0, 124.1 124.1, 120.5, 56.3, 52.7, 50.8, 24.1; HRMS (ESI positive) m/z
calcd. for C25H28F6N6O4 [M+H]+: 591.2154, found, 591.2151; Anal calcd. for C25H28F6N6O4: C,
50.85; H, 4.78; N, 14.23, found: C, 50.62; H, 4.52; N, 14.02.
1, 2-Bis(4-nitro-2-(trifluoromethyl)phenyl)piperazin-1-yl)propane (23)
The title compound was synthesized from 1-(4-nitro-2-(trifluoromethyl)phenyl)piperazine (8)
and 1-bromo-2-chloropropane as yellow solid (yield 63%); mp: 169-172 °C; IR (KBr) ν (cm-1)
1
3429, 1626, 1459, 1402, 1385, 1333; H NMR (400 MHz, CDCl3):
δ 8.42 (2H, d, J = 2.5 Hz),

ACCEPTED MANUSCRIPT
8.24 (2H, dd, J = 2.4, 8.9 Hz), 7.21-7.18 (2H, m), 3.11 (8H, bs), 2.63-2.57 (8H, m) 2.42 (4H, t, J
= 7.4 Hz), 1.73-1.66 (2H, m); 13C NMR (100 MHz, CDCl3):
δ 156.9, 142.0, 127.8, 124.6, 124.6,
122.2, 56.4, 53.0, 52.8, 52.7, 51.2, 24.1; ESI-MS m/z calcd for C25H28F6N6O4 [M+H]+: 591; Anal
calcd. for C25H28F6N6O4: C, 50.85; H, 4.78; N, 14.23; found: C, 50.71; H, 4.65; N, 14.12.
2-(4-(4-Nitrophenyl)piperazin-1-yl)ethanol (24)
A mixture of 1-(4-nitrophenyl)piperazine (5, 2.5 g, 12 mmol), 2-bromoethanol (1.28 mL, 18
mmol) and Et3N (1.6 mL, 18 mmol) were heated at 50 °C in CHCl3 (15 mL) for 30-35 h.
Reaction mixture was then concentrated under reduced pressure and residue was dissolved in
EtOAc (15 mL). The organic layer was washed with water (10 mL × 3). The combined organic
layer was then dried (anhyd. Na2SO4) and was concentrated under reduced pressure. The solid
separated out was washed with distilled hexane and again dried under high vacuum. The pure
yellow crystals were recrystallized using EtOAc/Hexane in excellent yield (yield 97%); mp: 180-
1
184 °C; IR (KBr) ν (cm-1): 3019, 2399, 2344, 1597, 1535, 1507, 1476, 1350, 1330; H NMR
(400 MHz, CDCl3):
δ
8.12-8.09 (2H, m), 6.83-6.81 (2H, m), 3.69 (2H, t, J = 5.2 Hz), 3.47-3.43
13
(4H, m), 2.67 (4H, t, J = 5.1 Hz), 2.62 (2H, t, J = 5.4 Hz), 2.33 (1H, bs); C NMR (100 MHz,
CDCl3):
δ 154.7, 138.5, 125.9, 112.7, 59.3, 57.9, 52.4, 47.0; HRMS (ESI positive) m/z calcd. for
C12H17N3O3 [M+H]+: 252.1348, found, 252.1347; Anal calcd. for C12H17N3O3; C, 57.36; H, 6.82;
N, 16.72, found: C, 57.13; H, 6.64; N, 16.60.
Similar procedure was used to synthesize compound 25.
2-(4-(4-Fluorophenyl)piperazin-1-yl)ethanol (25)
The title compound was synthesized from 1-(4-fluorophenyl)piperazine (6) and 2-bromoethanol
as offwhite crystals (yield 89%); mp: 136-139 °C; IR (KBr) ν (cm-1) 3019, 2400, 1638, 1510,
1405; 1H NMR (400 MHz, CDCl3):
Hz), 3.31 (1H, bs), 3.15 (4H, t, J = 4.8 Hz), 2.72 (4H, t, J = 5.0 Hz), 2.64 (2H, t, J = 5.3 Hz); 13C
NMR (100 MHz, CDCl3): 158.4, 147.7, 118.0, 117.9, 115.6, 155.4, 59.4, 57.7, 52.9, 50.0;
δ 6.97-6.93 (2H, m), 6.88-6.85 (2H, m), 3.69 (2H, t, J = 5.2
δ
HRMS (ESI positive) m/z calcd. for C12H17FN2O [M+H]+: 225.1403, found, 225.1400; Anal
calcd. for C12H17FN2O: C, 64.26; H, 7.64; N, 12.49, found: C, 64.01; H, 7.40; N, 12.23.
1-(2-Chloroethyl)-4-(4-nitrophenyl)piperazine (26)
The mixture of 24 (1 g, 0.39 mmol), Et3N (1 mL, 0.78 mmol) in dry DMF (5mL) was stirred at
room temperature for 15 min under dry condition and then thionyl chloride (0.28 mL, 0.39
mmol) in dry DMF (2 mL) was added dropwise at 0 to 5 °C within 1 h duration. Reaction

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mixture was further stirred at room temperature for 3 h. The resultant mixture was concentrated
under reduced pressure, dissolved in CHCl3 (10mL) and washed with cold distilled water (10mL
x 5). The combined organic layer was dried (anhyd. Na2SO4) and concentrated under reduced
pressure and recrystallization was done using EtOAc/Hexane. The title compound was obtained
as yellow solid (yield 62%); mp: 140-143 °C; IR (KBr) ν (cm-1): 3019, 2963, 2400, 1637, 1599,
1509, 1475, 1419, 1331, 1261; 1H NMR (400 MHz, CDCl3):
δ 8.14-8.12 (2H, m), 6.84-6.82 (2H,
m), 3.69-3.63 (2H, m), 3.49-3.45 (4H, m), 2.84-2.80 (2H, m), 2.71-2.68 (4H, m); ESI-MS m/z
calcd. for C12H16ClN3O2 [M+H]+: 270; Anal calcd. for C12H16ClN3O2; C, 53.43; H, 5.98; N,
15.58, found: C, 53.59; H, 6.12; N, 15.69.
Compound 27 was also synthesized using the procedure similar to compound 26.
1-(2-Chloroethyl)-4-(4-fluorophenyl)piperazine (27)
The title compound was synthesized from 25 and thionyl chloride as pure white crystals (yield
1
60%); mp: 152-155 °C; IR (KBr) ν (cm-1): 3019, 2400, 1638, 1510, 1403; H NMR (400 MHz,
CDCl3):
δ 7.01-6.96 (2H, m), 6.92-6.89 (2H, m), 4.14-4.03 (4H, m), 3.55 (1H, bs), 3.51-3.46
(3H, m), 3.38-3.29 (4H, m); ESI-MS m/z calcd. for C12H16ClFN2 [M+H]+: 243; Anal calcd. for
C12H16ClFN2: C, 59.38; H, 6.64; N, 11.54, found: C, 59.52; H, 6.82; N, 11.73.
Further compounds 28-31 was synthesized by using the procedure similar to compound 11.
1-(2-Methoxyphenyl)-4-(2-(4-(4-nitrophenyl)piperazin-1-yl)ethyl)piperazine (28)
The title compound was synthesized from 26 and 1-(2-methoxyphenyl)piperazine (4) to give
pure yellow crystals (yield 72%); mp: 181-183 °C; IR (KBr) ν (cm-1) 3019, 2400, 1637, 1599,
1419, 1330; 1H NMR (400 MHz, CDCl3):
3.69 (3H, bs), 3.47-3.44 (2H, m), 3.16-3.13 (5H, m), 2.76-2.67 (4H, m), 2.35 (6H, bs), 2.03 (1H,
s); 13C NMR (100 MHz, CDCl3):
154.8, 152.2, 141.1, 138.4, 125.9, 123.4, 123.0, 121.0, 118.4,
δ 8.20-8.13 (2H, m), 7.04-6.83 (6H, m), 3.88 (2H, bs),
δ
118.2, 112.3, 111.3, 55.4, 55.3, 53.8, 53.1, 50.4, 50.2, 46.9, 46.1, 45.0; HRMS (ESI positive) m/z
calcd. for C23H31N5O3 [M+H]+: 426.2505, found, 426.2505; Anal calcd. for C23H31N5O3: C,
64.92; H, 7.34; N, 16.46, found: C, 64.81; H, 7.20; N, 16.32.
1-(4-Fluorophenyl)-4-(2-(4-(4-nitrophenyl)piperazin-1-yl)ethyl)piperazine (29)
The title compound was synthesized from 26 and 1-(4-fluorophenyl)piperazine (6) as pure
yellow crystals (yield 57%); mp: 142-144 °C; IR (KBr) ν (cm-1): 3019, 2400, 2346, 1636, 1599,
1
1510, 1476, 1420, 1330; H NMR (400 MHz, CDCl3):
δ 8.15-8.12 (4H, m), 6.85-6.82 (4H, m),
3.46-3.39 (8H, m), 3.16-3.13 (1H, m), 3.05-3.03 (7H, m), 2.69-2.64 (4H, m); 13C NMR (100

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MHz, CDCl3):
δ
155.2, 154.8, 147.9, 138.3, 126.0, 125.9, 117.8, 117.7, 115.6, 115.4, 112.5,
112.2, 55.8, 53.6, 53.1, 50.1, 48.1, 47.0, 45.7; HRMS (ESI positive) m/z calcd. for C22H28FN5O2
[M+H]+: 414.2305, found, 414.2301; Anal calcd. for C22H28FN5O2; C, 63.90; H, 6.83; N, 16.94,
found: C, 63.75; H, 6.61; N, 16.73.
1-(4-Fluorophenyl)-4-(2-(4-(4-methoxyphenyl)piperazin-1-yl)ethyl)piperazine (30)
The title compound was synthesized from 27 and 1-(2-methoxyphenyl)piperazine (4) as pure
yellow crystals (yield 82%); mp: 133-136 °C; IR (KBr) ν (cm-1) 3019, 2399, 1638, 1533, 1500,
1
1419, 1350, 1301; H NMR (400 MHz, CDCl3):
δ
6.93-6.76 (8H, m), 3.77 (4H, s), 3.05-2.95
158.3, 155.9, 152.2,
(12H, m), 2.60-2.55 (4H, m), 2.39 (3H, bs); 13C NMR (100 MHz, CDCl3):
δ
147.9, 141.7, 122.9, 120.9, 118.2, 117.7, 115.6, 115.3, 111.2, 59.6, 57.9, 55.9, 55.3, 53.9, 53.6,
53.0, 51.9, 50.6, 50.1, 46.1; HRMS (ESI positive) m/z calcd. for C23H31FN4O [M+H]+:
399.2560, found, 399.2550; Anal calcd. for C23H31FN4O; C, 69.32; H, 7.84; N, 14.06, found: C,
69.49; H, 7.96; N, 14.35.
1-(2-Fluorophenyl)-4-(2-(4-(4-fluorophenyl)piperazin-1-yl)ethyl)piperazine (31)
The title compound was synthesized from 27 and 1-(2-fluorophenyl)piperazine (7) to obtain
offwhite crystals (yield 71%); mp: 171-174 °C; IR (KBr) ν (cm-1) 3019, 2952, 2829, 2400, 1613,
1
1535, 1509, 1453, 1406, 1385, 1331, 1302; H NMR (400 MHz, CDCl3):
δ
7.00-6.78 (8H, m),
13
3.07-3.04 (4H, m), 2.99 (6H, s), 2.64-2.59 (5H, m), 2.56-2.52 (2H, m), 2.29 (3H, s); C NMR
(100 MHz, CDCl3): δ 158.4, 157.0, 154.5, 147.9, 140.5, 124.4, 124.4, 122.4, 117.8, 117.8, 117.7,
115.6, 59.3, 57.8, 53.7, 53.6, 52.9, 51.6, 50.1, 46.0; HRMS (ESI positive) m/z calcd. for
C22H28F2N4 [M+H]+: 387.2360, found, 387.2357; Anal calcd. for C22H28F2N4; C, 68.37; H, 7.30;
N, 14.50, found: C, 68.63; H, 7.50; N, 14.68.
Biology
Cell Cultures
The PC3 and DU145, monkey kidney Cos-7 and LNCaP prostate cancer cells were procured
from National Centre for Cell sciences (NCCS, Pune). Human prostate cancer cells, PC3 and
DU145, were maintained in DMEM/ HAM’S F-12 medium (Sigma–Aldrich, St. Louis, MO),
and Cos-7 cells were grown in DMEM medium (Sigma–Aldrich, St. Louis, MO) supplemented
with 10% FBS and 0.01% antibiotic/antimycotic solutions. These cells were grown in Roswell
Park Memorial Institute (RPMI) medium supplemented with 12.5% fetal bovine serum (charcoal

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stripped, Life Technologies Inc), 100 units/mL penicillin G sodium and 100 µg/mL streptomycin
sulfate (Sigma-Aldrich, USA) in an atmosphere of 5% CO2/95% air at 37 °C.
Ready-to-Assay α1a Adrenergic Receptor GPCR frozen cells were used for rapid calcium assays
and procured from Millipore (cat no. HTS087RTA).
Cell Proliferation Assay
Prostate cancer cells and normal cells were seeded in 96-well plates at a density of 2×104
cells/well and allowed 24 h for attachment. The treatments were proceed in triplicate with
flutamide (Stock 10mM; Sigma-Aldrich, USA) and with compounds (Stock 10mM) at
concentrations ranging from 2.5 µM to 80 µM in 2% charcoal stripped serum for 24 h at 37 °C
in 5% CO2 atmosphere. The stock solution of flutamide (10mM) was dissolved in molecular
grade dimethyl sulfoxide (DMSO) and diluted with culture medium to different concentrations
before adding to the cells. Final concentration of DMSO was not more than 0.05%. Controls
were treated with DMSO (0.05% in culture medium). After 24 h of incubation in CO2 incubator,
5 µl of 5mg/ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide)] was
added to the cells. After a further incubation of 3 h, the formazan crystals formed in viable cells
were dissolved in DMSO with gentle shaking on a plate shaker for 5 min. The absorbance was
measured at 540 nm using a microplate reader (Microquant, Bio-Tek, USA). The percent
viability was calculated according to formula given below:
% viability of cells = {(OD of treatment)/OD of Vehicle control} x 100
Reporter Gene Assay by Luciferase Expression
AR-negative Cos7 cells were seeded at a density of 5x104 cells/well into 48-well plates on the
day prior to transfection with 500ng of luciferase reporter gene construct pARE-eElB-LUC along
with the expression vector for AR, pCR 3.1.AR, using DharmaFECT transfection reagent
according to manufacturer’s protocol. At the end of 8 h transfection period, cells were switched
to complete medium and thereafter, treated with test compounds and Bicalutamide (AR-
antagonist, 3µM) was used as positive control for 24 h. Luciferase activity was determined with
luciferase assay systems (Promega, Madison, WI) following the manufacturer’s protocol to
detect the AR mediated transcriptional activity. Luciferase activity was normalized for
transfection efficiency using pRL-SV40-luc as an internal control.
Calcium Mobilization Assay