Design, synthesis and biological profiling of aryl piperazine based scaffolds for the management of androgen sensitive prostatic disorders

Article abstract of DOI:10.1039/c6md00426a

In the quest for novel scaffolds for the management of androgen sensitive prostatic disorders like prostate cancer and benign prostatic hyperplasia, a series of twenty-six aryl/heteroaryl piperazine derivatives have been described. Three compounds, 8a, 8c

Full text of DOI:10.1039/c6md00426a

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Gupta, D. Pandey, D. Mandalapu, V. Bala, V. Sharma, M. Shukla, S. K. Yadav, N. Singh, S. Jaiswal, J. P.
Maikhuri, J. Lal, M. I. Siddiqi and G. Gupta, Med. Chem. Commun., 2016, DOI: 10.1039/C6MD00426A.
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Design, Synthesis and Biological Profiling of Aryl Piperazine Based Scaffold for the
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Management of Androgen Sensitive Prostatic Disorders#
Sonal Gupta,a,e Deepti Pandey,b Dhanaraju Mandalapu,a Veenu Bala,a,e Vikas Sharma,b Mahendra
Shukla,c,e Santosh K.Yadav,b Nidhi Singh,d Swati Jaiswal,c,e Jagdamba P. Maikhuri,b Jawahar Lal,c,e
Mohammad I. Siddiqi,d Gopal Guptab and Vishnu L. Sharmaa,e,*
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aMedicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
bEndocrinology Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
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cPharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
dMolecular & Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow-226031 (India)
eAcademy of Scientific and Innovative Research (AcSIR), New Delhi-110001 (India)
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*Corresponding author. Dr. V. L. Sharma
Medicinal and Process Chemistry Division,
CSIRꢀCentral Drug Research Institute, Sector 10, Jankipuram ext.,
Lucknow, Uttar Pradesh 226031, India.
Tel.: 91ꢀ522ꢀ2772450; Ext. 4671; Fax: 91ꢀ522ꢀ2771941
Eꢀmail address: vl_sharma@cdri.res.in; vlscdri@gmail.com
#The authors declare no competing interests.
CDRI Manuscript No. 276/2015/VLS
Communication No. 9294
1

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ABSTRACT
In the quest of novel scaffolds for the management of androgen sensitive prostatic disorders
like prostate cancer and benign prostatic hyperplasia, a series of twentyꢀsix aryl/heteroaryl
piperazine derivatives have been described. Three compounds 8a, 8c and 9a exhibited good
activity profile against androgen sensitive prostate cancer cell line (LNCaP) with EC50 values
of 9.8, 7.6 and 11.2 µM, respectively. These compounds caused a decrease in luciferase
activity and decline in the PSA and Ca2+ levels, which are indicative of its antiꢀandrogenic
and α1Aꢀadrenergic receptor blocking activities, respectively. Compound 9a reduced prostatic
weights of rat (47%) and in pharmacokinetic analysis at 10 mg/kg it demonstrated an MRT of
~14 h post dose, exhibiting high levels in prostate. Compound 9a docked in a similar
orientation as hydroxyflutamide on androgen receptor and showed strong πꢀπ interactions.
These findings reveal that compound 9a is a promising candidate for management of
prostatic disorders with antiꢀandrogenic and α1Aꢀblocking activities.
Keywords: Prostate Cancer, Benign Prostatic Hyperplasia, Piperazine, Antiꢀandrogenic,
Alpha Blocker
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1. INTRODUCTION
Malignant or benign proliferation of prostatic cells in peripheral and transition zones
respectively is common in aged men. While prostatic cancer (PCa) is a leading cause of
cancerꢀrelated death in men over 60, ageꢀassociated voiding dysfunction exclusively in
elderly males primarily occurs due to benign prostatic hyperplasia (BPH). Both significantly
lead to the erosion of quality of life. 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 2008.1 The risk of prostate cancer increases with
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age and by the age of sixty a significant increase in risk is noticeable 2
. On the other hand,
nonꢀmalignant unregulated proliferation of the cellular elements of prostatic transition zone
leads to BPH. The prevalence of BPH increases gradually from the age of 50 and by the age
70, the average size of prostate gets doubled. The consequent lower urinary tract symptoms
(LUTS) affect about 70% of men aged 80 years, and more than half of them seek medical
consultation.3 By the year 2020, the number of persons over 60 years of age has been
expected to increase4 by 200% and recently prostate cancer (PCa)5 as well as LUTS caused
by BPH4 has been associated with depressive symptoms. In the U.S., the total estimated
expenditure on prostate cancer was 9.862 billion US dollars ($) in 2006,6 while BPH
accounted for the seventh highest annual diseaseꢀspecific medical cost as about 1 in 5 men
have significant clinical event.7 Therefore, in the upcoming future the socioꢀeconomic burden
is likely to increase with the growing incidence of these diseases.
In its primary stage prostate cancer is androgen sensitive and can be managed effectively by
androgen deprivation therapy using potent antiꢀandrogens. Hence, androgen ablation remains
the first line treatment strategy for prostate cancer.
Besides androgen sensitivity, primary prostate cancer and LNCaP cells also express α1Aꢀ
adrenergic receptors and are prone to proliferation by αꢀagonists and inhibition by αꢀblockers
like prazosin.8 Similarly, BPH also has essentially two active components, static and
dynamic. The static component is androgenꢀsensitive and caused by proliferation of cells in
the transition zone whereas the dynamic component is due to increased adrenergicꢀα1A
receptor activity, hence sensitive to α1ꢀadrenergic blockers (e.g. prazosin, tamsulosin).
Currently, androgen antagonists display the potential of an alternative therapy for the
treatment of the androgen sensitive prostatic disorders. The steroidal androgen antagonists
(Cyproterone acetate9,10 Figure 1), due to its multiꢀtargeting nature, has a battery of side
effects such as cardiovascular problems, hepatotoxicity,11 thrombosis, fluid retention, and loss
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of libido.12 On the contrary, nonꢀsteroidal androgen antagonists do not show steroidal side
effects as they do not bind with the targets of specific steroidal action. The well known
examples of nonꢀsteroidal androgen antagonists includes flutamide,13ꢀ15 nilutamide,16 and
bicalutamide17ꢀ21 (Figure 1), and literature reveals that some of the compounds have reached
the clinical stage.22 To cater the future medical needs of ageing population (due to increased
life expectancy), the development of novel, potent, nonꢀsteroidal androgen antagonists with
profound ARꢀantagonist activity and fewer side effects is highly desirable. Complementary
α1ꢀadrenergic blocking activity would be highly desirable to potentiate their action against the
diseased prostate. Currently such molecules are not available for clinical use.
Figure 1. Structure of androgen antagonists
The piperazine scaffold has gained much attention of the scientific community due to its
diverse pharmacological potential across various therapeutic areas such as antipsychotic,23
antidepressant,24 antimicrobial,25,26 anticancer,27 antioxidant28 and antimalarial leads29 etc.
Recently several novel nonsteroidal androgen antagonists have been identified that contain
piperazine motif.30,31 On the other hand, terazosin, and doxazosin (the wellꢀknown marketed
alpha blockers) comprise of the piperazine structure (Figure 2), and piperazine moiety is also
present in many of the other reported scaffolds with alpha blocking activity.32ꢀ35
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Figure 2. Structure of alpha blockers
Our previous work revealed that piperazine containing scaffolds are potent androgen
antagonists against BPH,36 therefore with an aim to incorporate α1Aꢀadrenergic blocking
properties in androgen antagonists for simultaneously targeting both the AR and α1A receptors
of PCa and BPH it was envisaged to synthesize compounds with two piperazine moieties in
same scaffold. The synthesis, biology, pharmacokinetics and docking of aryl piperazine
derivatives containing two piperazine cores (Figure 3) have been described.
Ar1
Ar2
Ar1
N
N
N
N
Ar2
N
N
X
X
Ar1, Ar2 = aryl, heteroaryl
X = CO, CH2
Ar1, Ar2 = aryl, heteroaryl
X = CO,COCH2
Present Work
Previous Work
Figure 3. General structure of synthesized compounds
The mode of action of these type of compounds was already discussed in our previous lab
work which was found to be as androgen antagonist36 so based on the fact the present
compounds were docked with androgen receptor.
2. RESULT AND DISCUSSION
2.1. Chemistry
Aryl/hetroaryl piperazine derivatives (9aꢀn) were synthesized according to scheme 1 in a
simple two step reaction procedure. In the first step substituted aryl/heteroaryl piperazine (7aꢀ
e) were allowed to react with chloroacetyl chloride under basic conditions at low temperature
to furnish the respective intermediate (8aꢀe). These intermediates were further on treatment
with differently substituted aryl/heteroarylpiperazines delivered the final piperazine
derivatives (9aꢀn).
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Scheme 1. Synthesis of compounds 9aꢀn
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NꢀArylpiperazinecarboxamide derivatives (12aꢀg) were prepared by simple chemical
transformation (Scheme 2). Arylpiperazines (10aꢀb) were stirred with aryl chloroformate in
presence of triethylamine to form aryl piperazinecarboxylate (11aꢀb) which were further
heated with various arylpiperazines in DMF to offer final products (12aꢀg).
Scheme 2. Synthesis of compounds 12aꢀg
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2.2. Biology
2.2.1. In vitro Evaluation on LNCaP Cell Line
As human prostate cancer LNCaP cellꢀline is sensitive to both androgens and α1A agonists,
hence, initially all the synthesized twenty six compounds (8aꢀe, 9aꢀn, 12aꢀg) were screened
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against LNCaP cell lines via MTT assay to evaluate their inhibitory activity using flutamide
(EC50 = 7.0 µM) as standard. The twenty two compounds exhibited EC50 in the range of 7.6ꢀ
79.8 µM except four compounds (9b, 12a, 12c, 12g) which were found to have EC50 >80 µM.
Compounds having EC50 (µM) values comparable to the standard drug were subjected to
further analysis. The structure of synthesized scaffolds and their EC50 (µM) values derived
from MTT assay on LNCaP cell lines have been shown in Table 1 & 2.
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Table 1. In vitro activity in LNCaP cell line of 8aꢀe and 9aꢀn
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LNCaP
Compd
LNCaP
EC50
Compd
no.
Ar1
Ar2
EC50
Ar1
Ar2
no.
(µM)
(µM)
ꢀ
ꢀ
ꢀ
48.2
47.1
50
8a
8b
8c
9.8
71.3
7.6
9f
9g
9h
ꢀ
ꢀ
53.3
63.1
8d
9i
41.1
11.2
>80
52.2
45.9
48.2
8e
9a
9b
9j
9k
9l
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46.6
9c
9m
55.2
67.9
48.2
7.0
9d
9e
9n
Flutamide
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Table 2. In vitro activity in LNCaP cell line of 12aꢀg
LNCaP
Compd no.
12a
Ar1
Ar2
EC50 (µM)
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>80
79.8
12b
>80
69.5
72.3
12c
12d
12e
76.1
>80
12f
12g
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The study comprised of three categories of piperazine derivatives, viz., 1ꢀarylꢀ4ꢀ
chloroacetylpiperazines (8aꢀe), 1ꢀarylꢀ4ꢀ(4ꢀarylpiperazino)acetylpiperazines (9aꢀn) and
diarylpiperazinylmethanones (12aꢀg). Among 8aꢀe (Table 1), aryl substitutions were
preferred over pyrimidyl group whereas in aryl moieties 2ꢀOCH3 and 4ꢀNO2 gave much better
activity than diꢀsubstituted with CF3 and NO2 groups. 2ꢀchloroꢀ1ꢀ(4ꢀ(2ꢀ
methoxyphenyl)piperazinꢀ1ꢀyl)ethanone (8c) was most active (EC50 = 7.6 µM).
The activity data of 1ꢀarylꢀ4ꢀ(4ꢀarylpiperazino)acetylpiperazines (9aꢀn, Table 1) suggested
that among the compounds with 4ꢀnitrophenyl group at Ar1 (9aꢀd) the more preferred
substituent at Ar2 position was found to be 4ꢀnitrophenyl (9a) over 2ꢀmethoxyphenyl (9b),
pyrimidyl (9c) and 2ꢀpyridyl (9d). With Ar1 as pyrimidyl (9eꢀh), 2ꢀmethoxyphenyl (9iꢀl) and
2ꢀCF3ꢀ4ꢀNO2phenyl (9mꢀn) displayed comparable LNCaP inhibition ranging between EC50
45.9 to 67.9 µM.
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The LNCaP inhibition data of diarylpiperazinylmethanones (12aꢀg, Table 2) revealed that
the scaffolds containing 2ꢀpyridyl (12aꢀd) and 4ꢀnitrophenyl (12eꢀg) at Ar1 and 2ꢀCF3ꢀ4ꢀNO2
phenyl (12a, 12f), 3ꢀCF3ꢀ4ꢀNO2 phenyl (12b), 4ꢀCF3ꢀ2ꢀNO2 phenyl (12g), 2ꢀpyridyl (12c)
and 4ꢀnitrophenyl (12d, 12e) at Ar2 were found to exhibit moderate activity profile with EC50
72.3 to >80 µM.
Among mono piperazine derivatives 8aꢀe with 4ꢀchloroacetyl substituent it was found that
the presence of 4ꢀNO2 phenyl substituent on the Ar1 of piperazine 8a (EC50 = 9.8 µM)
showed a remarkable activity towards LNCaP cell line. On replacing the 4ꢀNO2 group with
the 2ꢀOCH3 8c (EC50 = 7.6 µM) caused the increase in activity. However when the NO2
substituent was accompanied by CF3 over the phenyl ring as in compound 8d (EC50 = 41.1
µM) there occurred a significant decline in the inhibitory profile. Whereas the activity
decreased notably when the phenyl ring gets replaced with the heterocyclic pyrimidyl ring
(8b, EC50 = 71.3 µM).
Further, piperazine derivatives 9aꢀn and 12aꢀg were screened to explore the effect of the
presence of linkers COCH2 and CO respectively. SAR studies revealed that 9a (EC50 = 11.2
µM) having a 4ꢀNO2 phenyl on both the A and B rings of piperazine with COCH2 linker
possessed good activity comparable to standard flutamide. With 4ꢀNO2 phenyl at Ar1 in ring
A (9aꢀd) and 2ꢀOCH3 (9b), 2ꢀpyrimidyl (9c) and 2ꢀpyridyl (9d) at Ar2 in Bꢀring the activity
decreased several folds. On the other hand, with pyrimidyl moiety at Ar1 in ring A (9eꢀh), the
increasing order of activity was found to be 4ꢀNO2 phenyl (9e), 2ꢀpyridyl (9h), 2ꢀOCH3
phenyl (9g), 2ꢀpyrimidyl (9f) at Ar2 in ring B. However with 2ꢀOCH3 phenyl at Ar1 the
activity pattern followed was 2ꢀpyridyl (9k) > 2ꢀpyrimidyl (9l) > 2ꢀOCH3 phenyl (9j) > 4ꢀ
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NO2 phenyl (9i) at Ar2. As compared to 9a the inclusion of CF3 group in either Ar1 or Ar2
(9mꢀn) decreased activity by about 4 times. A carbonyl linker between two piperazine rings
(12aꢀg) resulted in the compounds with moderate activity.
The activity profile in LNCaP cell line of the compounds 1ꢀarylꢀ4ꢀchloroacetylpiperazines
(8aꢀe) revealed that the 4ꢀNO2 phenyl (8a) and 2ꢀOCH3 phenyl (8c) substituent’s at the Ar1
position were the most desirable scaffolds however among 1ꢀarylꢀ4ꢀ(4ꢀ
arylpiperazino)acetylpiperazines (9aꢀn), derivative 9a with 4ꢀNO2 phenyl substituent at both
Ar1 and Ar2 position was found to display maximum inhibitory effect. It seems that 4ꢀNO2
phenyl substituent on either or both the piperazine rings (8a and 9a) were most desirable for
activity. Data also revealed that the COCH2 linker was preferential over the CO linker
scaffolds so the two carbon distance between the two fragments was much desired condition
for affinity activity. The three compounds 8a, 8c and 9a demonstrated a prominent reduction
of cell proliferation in androgen dependent LNCaP cell lines with 9.8 µM, 7.6 µM and 11.2
µM respectively, comparable to flutamide.
2.2.2. Western Blotting Analysis
On the basis of these results the three most active compounds (8a, 8c and 9a) were further
subjected for the western blotting analysis to validate the antiꢀandrogenic effect on LNCaP.
Prostate specific antigen (PSA, androgenꢀsensitive prostatic marker protein) expression was
found to be increased in LNCaP cells by the treatment of various concentration of
testosterone (10 nM, 20 nM, 40 nM; Figure 4a) and decreased by treating with antiandrogen
drug flutamide (10 µM, 20 µM, 40 µM; Figure 4b). The results were also confirmed by
treating with testosterone (20 nM) and subsequently with flutamide (10 µM, 20 µM, 40 µM;
Figure 4c). Western blots of the three active compounds (8a, 8c and 9a) prominently showed
declined PSA level in LNCaP cell line at 10 µM concentration (Figure 4c). Data suggested
that at 10 µM the compound 8a reduces the PSA expression to the maximum extent in
comparison to the 8c and 9a.
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Figure 4. PSA expression in androgen sensitive LNCaP human prostatic cell line treated with
(a) testosterone (T); (b) nonꢀsteroidal antiandrogen flutamide (F) ; and (c) testosterone (T=20
nM) along with flutamide (20 µM) or compound 8a, 8c or 9a (10 µM) for 24 h. Control
(C=vehicle). [20 µg of protein loaded in each lane].
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2.2.3. Antiꢀandrogenic property of 8a and 9a
Luciferase reporter gene assay was performed to assess the ARꢀantagonist nature of
compounds more precisely. An AR negative Cos7 cell line was transiently transfected with
AR and AREꢀ LUC plasmids and treated with compounds 8a and 9a at a concentration of 7.5
µM. A potent antiꢀandrogen, bicalutamide (3µM), was used as positive control. Significant
decrease in luciferase activity in the presence of test compounds (8a, 9a, P<0.01), as
compared to control, after 24 h supports their antiꢀandrogenic nature (Figure 5).
Figure 5. Transactivation of AREꢀLUC by AR in presence of test compounds was measured
by dualꢀluciferase reporter assay in terms of RLU (Control=100%). 8a (7.5 µM), 9a (7.5
µM) and bicalutamide (3 µM). Significant difference from control is indicated as **P<0.01
and ***P<0.001).
2.2.4. Cell Viability Test
To conclude whether the test compounds exhibited antiproliferative activity due to AR
downregulation, cell viability test was performed in ARꢀnegative PC3 and DU145 cells lines.
All the three compounds tested (8a, 8c, 9a) exhibited antiꢀandrogenic activity as depicted by
their specific action against AR positive LNCaP cells (Table 3). These compounds showed
EC50 of 9.8 µM (8a), 7.6 µM (8c) and 11.2 µM (9a) against LNCaP. On the other hand, the
EC50 values were 65 µM for 8a and >80 µM for 8c and 9a against PCꢀ3 and 70.6 µM for 8a
and >80 µM for 8c and 9a against DU145. The high EC50 value in ARꢀnegative cells PC3
and DU145 suggested that the cytotoxicity of compounds was specifically mediated through
AR.
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Table 3. Cell Viability Test in PC3, DU145 and LNCaP cells of compounds 8a, 8c and 9a
EC50 for PC3
EC50 for DU145
EC50 for LNCaP
Compound
(µM)
65
(µM)
>70.6
>80
(µM)
9.8
8a
8c
9a
>80
>80
7.6
>80
11.2
2.2.5. Time Dependent Analysis of Ca2+ Mobilization in α1A Expressing Chemꢀ1 Cells
As mentioned earlier many of the piperazine containing scaffolds acts as alpha blocker (α1Aꢀ
adrenoceptor antagonist)32ꢀ35 acting on the dynamic component so as to relieve the symptom
score due to the disease. Taking this into consideration the three motifs 8a, 8c and 9a were
subjected to time dependent analysis of Ca2+ mobilization in α1A expressing Chemꢀ1 cells to
evaluate the alpha blocking behavior of these compounds. Ca2+ mobilization assay was done
with dose responses for compounds 8a (10 ꢁM), 8c (10 ꢁM), 9a (10 ꢁM), α1A agonist
epinephrine (20 ꢁM) and antagonist tamsulosin (1 ꢁM) in α1A adrenergic receptor GPCR
frozen cells. The agonist epinephrine increased the calcium level by ~2.5 times to that of
control while tamsulosin being an antagonist significantly decreased Ca2+ which diminished
the relative fluorescence unit (RFU) value by ~1.6 times of that of epinephrine. It was found
that 8a (10 ꢁM), 8c (10 ꢁM) and 9a (10 ꢁM) showed comparable decrease as that of standard
tamsulosin at 1 ꢁM of Ca2+ level, about 1.6 times of the epinephrine (Figure 6). It may be
inferred that these compounds may also have promising potentials of an alphaꢀblocker and
androgen antagonist, which could improve symptoms as well as act on the prostatic volume.
Figure 6. Time dependent analysis of Ca2+ mobilization in α1A expressing Chemꢀ1 cells with
different concentration of compounds 8a (10 µM), 8c (1 µM), 9a (10 µM), agonist
epinephrine (20 µM) and antagonist tamsulosin (1 µM)
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The compounds 8a, 8c and 9a showed comparable activity towards androgen dependent
LNCaP cell line, western blotting analysis and time dependent analysis of Ca2+ mobilization
in α1A expressing Chemꢀ1 cells. Further in vivo experiments were carried out to assess the
effect of compound 8a and 9a on the volume of prostate gland in BPH induced male
Sprague–Dawley rats.
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2.2.6. In vivo Studies Adult Male Sprague–Dawley Rats
Compounds 8a and 9a were taken up for in vivo experiments, which were done on BPH
induced adult male sprague–dawley rats (weighing 250–280 g, n = 5). BPH was induced by
giving citral (100 mg/kg) daily for 30 days. After 8 days of treatment, compounds 8a and 9a
at 10 mg/kg were given orally for rest of 21 days simultaneously with citral in experimental
animals. Parallel control animals received vehicle and while the positive control animals were
given flutamide (10 mg/kg) for same the regimen. The prostates were dissected and weighed
after 24 h of last treatment (Table 4). The study was approved by the Institutional Animal
Ethics Committee (IAEC) of CSIRꢀCDRI and the experiments were performed in compliance
with the IAEC guidelines.
Table 4. Effect of compound 8a and 9a on prostate
Prostate wt.
% Reduction of
prostate weight
Compd no.
per 100 g of body
weight
0.191
% Prostate wt.*
68.4
53.8
48.2
100
31.6
46.2
51.8
0
8a
9a
0.151
0.135
0.280
Flutamide
Citral
Control
0.162
57.7
ꢀ
380
381
382
383
384
385
386
387
388
389
* Prostate weight of citral treated rats was taken as 100%.
Prostate weight of citral treated rats was found to be significantly higher about 280 mg/100g
of body weight in comparison to experimental control 162 mg/100g of body weight.
However, diseased model treated with aryl piperazine derivatives 8a and 9a showed a
prominent reduction in the prostate weight of about 191 mg/100 g and 151 mg/100 g
respectively in comparison to standard drug flutamide 135 mg/100 g. The study showed that
the compound 9a reduces the weight of prostate by 46.2 % which is comparable to flutamide
(51.8 %). Further it was found that lower reduction of 32 % by 8a was also considerable. In
the view of in vivo results it can be inferred that the presence of 4ꢀNO2 phenyl over both the
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piperazine rings was more desirable for the activity. The seminal vesicle, testis and
epididymis were least affected by the compound (Figure 7). The histological investigations
were also carried out on the prostatic tissues dissected out in this in vivo experiment to
validate the results.
Figure 7. Effect of citral (100 mg/kg), flutamide compound, 8a and 9a (10 mg/kg) on
prostate, testis, epididymis and seminal vesicle
2.2.7. Histological Investigations
Periurethral prostatic tissues were obtained from the sprague–dawley rats after successive
treatment with citral (100 mg/kg) for 8 days and followed by citral plus compound 8a, 9a and
flutamide at 10 mg/kg treatment for 21 days. The histological investigations were made
microscopically and represented in figure 8. With some variation between animals, it was
quite apparent from pictures of stained sections that control tissue (Figure 8A) had clear
stroma while the BPH tissue (Figure 8B) displayed a very little stromal differentiation, which
signifies the growth of disease in the rat model. Treatment with compound 8a, 9a and
flutamide in parallel with citral reduced the stromal component and differentiated the tissues
into stromal and acinar compartments (Figure 8CꢀE). It is clear from the pictures that in
comparison to compound 8a, compound 9a was more potent and is somewhat comparable
with flutamide.
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Figure 8. Representative histological architecture of the prostate of rats given (A) vehicle
control; (B) citral (100 mg/kg); (C) citral (100 mg/kg) + 8a (10 mg/kg); (D) citral (100
mg/kg) + 9a (10 mg/kg) and (E) citral (100 mg/kg) + flutamide (10 mg/kg) orally once daily
for 21 days
2.3. Pharmacokinetic Studies
The animals tolerated the treatment as no changes in the behaviour were observed.
Compound 9a was monitored up to 48 h with peak serum concentration (Cmax, 1.1 ± 0.1
µg/mL) at 10 h indicating slow absorption (Figure 9). Moreover, it was monitored up to 48 h
in hypothalamus and up to 72 h in prostate with Cmax at 10 h post dose. The serum/tissue
concentrationꢀtime profile was analyzed using nonꢀcompartmental and compartmental
approaches. 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).37,38 The concentrationꢀ
time data was best described by the oneꢀcompartmental open model and the calculated
pharmacokinetic parameters are given in table 5. The volume of distribution (4.2L/kg) is
larger than the total blood volume of rat (0.054 L/kg)39 and systemic clearance (0.4 L/h/kg) is
lower than the total hepatic blood flow in rats (2.9 L/h/kg)39 indicating extravascular
distribution with negligible extrahepatic elimination. The compound 9a has higher tissue to
serum concentration ratio in prostate (target tissues). However, hypothalamus (tissues for side
effects) showed lower tissue to serum ratio indicating poor penetration across the bloodꢀbrain
barrier (Figure 9, Table 5).
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Figure 9. Mean concentrationꢀtime profile of 9a in serum, prostate and hypothalamus after
single 10 mg/kg oral dose in male Sprague Dawley rats. Bar represents SD (n = 3)
Table 5. Pharmacokinetic parameters of 9a after single 10 mg/kg per oral dose in male
Sprague Dawley ratsa.
Parameters
Cmax (µg/mL or µg/g)
tmax (h)
Serum
Prostate Hypothalamus
1.1 ± 0.1 2.4 ± 0.2
1.2
10.0
10.0
24.1
1.0
10.0
37.3
26.1
AUClast (µg h/mL or µg h/g)
Tissue to serum ratio
Cl/F (L/h/kg or g/h/kg)
Vd/F (L/kg or g/kg)
1.5
1.1
0.4
268.2
2311.9
383.2
3313.7
4.2
461
462
463
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470
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472
473
474
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479
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481
aEach value represents the average of three rats dosed at 10 mg/kg per oral. Serum and
prostate concentration are mean ± SD from three rats. Hypothalamus concentrations are the
mean of the pooled tissue of three rats.
2.4. Docking Studies
Molecular docking studies were performed to predict the binding mode and interaction
pattern of compounds 8a, 8c and 9a and were compared to hydroxyflutamide (HF) which is a
known antagonist of androgen receptor (AR). The crystal structure of HF has been solved as
T877A mutant (PDB: 2AX6). In previous studies it has been reported that T877A mutation
affects the activity of HF and converts it to agonist.40 In another study the probable effect of
T877A mutation on repositioning of helixꢀ12 of AR and subsequent change in the activity has
been proposed.41 Therefore docking studies of HF in the wild type AR (2AXA) was done and
the probable binding pose of reported active compounds has been compared to it (Figure 10).
The predicted binding pattern of compound 9a is similar to binding of HF as evident by
hydrogen bond formation with residue Gln 711 and Arg 752. The rings attached to both nitro
groups at the ends showed piꢀpi stacking with the residues Phe 764 and Trp 741 (Figure
10B). The docked pose of compound 8a showed the hydrogen bond formation with residue
Arg 752 and piꢀpi stacking interaction between ring attached to nitro group and residue Phe
764 (Figure 10C). The predicted binding pose of compound 8c shows piꢀpi stacking
interaction with residue Phe 764 and hydrogen bond formation with residue Thr 877, the ring
at other terminal shows hydrophobic interaction with residues Met 745 (Figure 10D). The
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strong piꢀpi interaction and formation of hydrogen bonds can be plausible explanation for the
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484
better activity of compound 9a than 8a and 8c.
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Figure 10. (A) Hydroxyflutamide binding mode to Wild Type AR (B) Predicted binding
mode of compound 9a (C) compound 8a (D) compound 8c in AR binding site. Protein is
shown in mixed representation (ribbon and stick). Hydrogen bonds are shown as black line
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3. CONCLUSION
A series of novel piperazine derivatives as androgen antagonists have been synthesized and
subjected to MTT assay on androgen sensitive LNCaP prostate cell line. The three most
potent compounds 8a, 8c and 9a had activity comparable with flutamide and were chosen for
further evaluation i.e. western blotting for PSA, time dependent analysis of Ca2+ mobilization
in α1Aexpressing Chemꢀ1 cells and cell viability test. Further the results of Luciferase reporter
gene assay support the antiꢀandrogenic nature of the compounds 8a and 9a. Of these, two
compounds 8a and 9a were selected for in vivo studies in BPH induced diseased adult male
rats. Compounds 8a and 9a showed significant prostatic reduction of 32% and 46%
respectively, which was also validated by histological investigations. Since 9a possessed
comparable in vivo activity with standard flutamide it was taken up for pharmacokinetic
studies, which revealed that compound 9a targeted the prostate as it exhibited higher levels
than that in serum and hypothalamus. The molecular docking studies revealed the probable
binding mode of compound 9a similar to hydroxy flutamide. It may be concluded from the
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study that 1,2ꢀbis(4ꢀ(4ꢀ4ꢀnitrophenyl)piperazinꢀ1ꢀyl)ethanone (9a) might be a promising lead
candidate for the management of androgen sensitive prostatic disorders.
SUPPORTING INFORMATION AVAILABLE
1
1
Experimental details of chemistry and biology, H NMR, 13C NMR spectral data, H NMR,
13C NMR spectra, HRMS of compounds 8aꢀe, 9aꢀn, 11aꢀb and 12aꢀg and representative
chromatogram of compound 9a.
ABBREVIATIONS
PCa, Prostate cancer; BPH, Benign prostatic hyperplasia; androgen antagonist, selective
androgen receptor modulator; PSA, prostate specific antigen; MTT, 3ꢀ(4, 5ꢀdimethylthiazolꢀ
2ꢀyl)ꢀ2, 5ꢀdiphenyl tetraꢀzolium bromide; DMF, dimethylsulphoxide; RFU, relative
fluorescence unit; AUClast = area under the serum/tissue concentrationꢀtime 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; UFLC, ultra fast liquid chromatography.
CONFLICT OF INTEREST
Authors confirm that this article has no conflict of interest
ACKNOWLEDGEMENT
Research fellowships from Council of Scientific and Industrial Research (D. M., M. S., N. S.,
S. J.), University Grants Commission (V. S.) and Indian Council of Medical Research (S. G.,
V.B., D. P.) are sincerely acknowledged. Staff members of SAIF Division, CDRI, are
acknowledged for providing the spectral data. The authors gratefully acknowledge Dr. Nancy
L. Weigel, Baylor College of Medicine, Houston, TX, USA, for kindly providing the
plasmids for gene reporter assay. This study was supported by a grant from Indian Council of
Medical Research, New Delhi, Government of India (GAP 0120).
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534
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Table of Content
Twenty-six piperazine derivatives were synthesized and findings revealed that compound 9a
is promising candidate for management of prostatic disorders.