Arylpiperazines for management of benign prostatic hyperplasia: Design, synthesis, quantitative structure-activity relationships, and pharmacokinetic studies

Article abstract of DOI:10.1021/jm101163m

A series of 27 aryl/heteroaryl/aralkyl/aroyl piperazines were synthesized, and most of these compounds reduced prostate weight of mature rats by 15-47%. Three compounds, 10, 12, and 18, had better activity profile (reduced prostate weight by 47%, 43%, and 39%, respectively) than the standard drug flutamide (24% reduction). QSAR suggested structures with more cyclic and branched moieties, increased topological separation of O and N therein, and reduced solvation connectivity index for better activity. Pharmacokinetic study with compound 10 at an oral dose of 10.0 mg/kg indicated good absorption, negligible extrahepatic elimination, and rapid distribution to the target organ (prostate) but restricted entry through the blood-brain barrier. A 10-fold decrease in PSA and 15-fold increase in ER-β gene expressions of human prostate cancer cells (LNCaP) by compound 10 in vitro indicated AR and ER-β mediated actions. The findings may stimulate further explorations of identified lead for the management of benign prostatic hyperplasia.

Full text of DOI:10.1021/jm101163m

302 J. Med. Chem. 2011, 54, 302–311
DOI: 10.1021/jm101163m
Arylpiperazines for Management of Benign Prostatic Hyperplasia: Design, Synthesis, Quantitative
Structure-Activity Relationships, and Pharmacokinetic Studies†
Amit Sarswat,‡ Rajeev Kumar,§ Lalit Kumar,‡ Nand Lal,‡ Smriti Sharma,‡ Yenamandra S. Prabhakar,‡
Shailendra K. Pandey, Jawahar Lal, Vikas Verma,§ Ashish Jain,§ Jagdamba P. Maikhuri,§ Diwakar Dalela,^
Kirti,^ Gopal Gupta,§ and Vishnu L. Sharma*,‡
‡Medicinal & Process Chemistry Division, §Endocrinology Division, and Pharmacokinetics & Metabolism Division,
Central Drug Research Institute (CSIR), Lucknow-226001, India, and ^Department of Urology, CSM Medical University,
Lucknow-226001, India
Received September 8, 2010
A series of 27 aryl/heteroaryl/aralkyl/aroyl piperazines were synthesized, and most of these compounds
reduced prostate weight of mature rats by 15-47%. Three compounds, 10, 12, and 18, had better
activity profile (reduced prostate weight by 47%, 43%, and 39%, respectively) than the standard drug
flutamide (24% reduction). QSAR suggested structures with more cyclic and branched moieties,
increased topological separation of O and N therein, and reduced solvation connectivity index for
better activity. Pharmacokinetic study with compound 10 at an oral dose of 10.0 mg/kg indicated good
absorption, negligible extrahepatic elimination, and rapid distribution to the target organ (prostate) but
restricted entry through the blood-brain barrier. A 10-fold decrease in PSA and 15-fold increase in ER-
β gene expressions of human prostate cancer cells (LNCaP) by compound 10 in vitro indicated AR and
ER-β mediated actions. The findings may stimulate further explorations of identified lead for the
management of benign prostatic hyperplasia.
Introduction
volume and hence the risk of acute urinary retention.6 Potential
cardiovascular consequences and sexual dysfunction are the
major issues associated with therapies involving R-adrenergic
blockers and 5R-reductase inhibitors, respectively. Tamsulosin
has fewer cardiovascular side effects due to its selectivity for R1a-
adrenergic receptors which are concentrated mainly in the
prostate but may cause floppy iris syndrome and sulfa drug-
type adverse effects.7 Steroidal AR antagonists, such as
cyproterone acetate8 (Figure 1), block androgen action and
also have progestational and glucocorticoid activities. Their
overlapping effects with other hormonal systems cause a
range of unpleasant side effects including thrombosis, fluid
retention, and loss of libido, which hinder their use as agents
for management of BPH.9 A number of nonsteroidal AR
antagonists, including flutamide10-12 (Figure 1), nilutamide13
(Figure 1), and bicalutamide14-18 (Figure 1), have been
reported in the literature.19-29
Nonsteroidal AR antagonists selectively block androgen
action without affecting other hormonal systems, and side
effects such as loss of libido and impotence are therefore less
profound. However, they do inhibit the binding of testoster-
one to ARs in the central nervous system (CNS) which, in
turn, interrupts the negative feedback of testosterone on
gonadotropin secretion, causing an increased serum testoster-
one level.30 This increase in serum testosterone impairs the
antiandrogenic activityof ARantagonists and alsocauses side
effects including gynecomastia and breast tenderness. There-
fore, potent AR antagonists with fewer adverse effects are
highly desirable.
Benign prostatic hyperplasia (BPH)a is an ubiquitous1 con-
dition in aging males, such that the incidence of BPH detected at
autopsy increases from approximately 30% at age 50 to >80%
at age 80. Unfortunately, the urinary symptoms attributed to
BPH lead to significant erosion in the quality of life for affected
men.2 The available treatments for BPH are either highly
invasive (surgical) or partially effective with unwanted side
effects and put a significant burden on employees and their
employers through direct medical costs and lost work time.
Direct and indirect private sector costs related to BPH treatment
are an estimated $3.9 billion.3 It is well established that andro-
gens, such as testosterone and 5R-dihydrotestosterone (DHT),
play an essential role in stimulating hyperplasia and carcinoma
of hormone-sensitive tissue such as that of the prostate.4 The
two medical treatments currently used for BPH are R-adrenergic
blockers (e.g., terazosin, tamsulosin) and 5R-reductase inhibi-
tors (e.g., finasteride). While the former are more effective
in relieving BPH symptoms,5 only the latter reduce prostate
†CDRI Communication No. 7892.
*To whom correspondence should be addressed. Phone: 91-522-
2612411 ext 4320. Fax: 91-522-2623405. E-mail: vlscdri1@rediffmail.
com; vlscdri@gmail.com.
a Abbreviations: BPH, benign prostatic hyperplasia; QSAR, quanti-
tative structure-activity relationship; PSA, prostate-specific antigen;
ER, estrogen receptor; AR, androgen receptor; DHT, 5R-dihydrotes-
tosterone; SARMs, selective androgen receptor modulators; PCR,
polymerase chain reaction; MRT, mean residence time; BR, biological
response; CP-MLR, combinatorial protocol in multiple linear regres-
sion; LOO, leave-one-out; SPI, superpendentic index; TEA, triethyla-
mine; DCM, dichloromethane; TURP, trans-urethral resection of
prostate; FCS, fetal calf serum; dNTP, deoxyribonucleoside tripho-
sphate; HPLC, high-performance liquid chromatography.
Recently, arylpiperazine derivatives have been reported as
potent nonsteroidal AR antagonists,31,32 and some of them
r
pubs.acs.org/jmc
Published on Web 12/03/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 303
Figure 1. Structure of SARMs and general structure of compounds synthesized.
Scheme 1a
have exhibited better activity profile than bicalutamide
(Figure 1) both in vitro and in the testosterone propionate
treated castrated rat model.33 However, they exhibited lower
activity in the mature intact rat model which is a more
appropriate model for BPH. Thus, it was thought worthwhile
to synthesize aryl/heteroaryl/aralkyl/aroyl piperazine deriva-
tives (Figure 1) as selective androgen receptor modulators
(SARMs) that can achieve AR blockade without causing
increased testosterone levels and to evaluate their in vivo effect
on the prostate of mature intact rat model that simulates the
clinical situation more appropriately.
The synthesis, quantitative structure-activity relationship
(QSAR), and in vivo activity in mature rats of these piperazine
derivatives (Figure 1) have been described in this report. The
pharmacokinetics in rats and in vitro effect on prostate-
specific antigen (PSA) and steroid hormone receptor expres-
sions in human prostate cancer cells of the most promising
structure are also presented.
a Reagents and conditions: (a) (Et)3N, neat, 80-100 ꢀC, 5-10 h; (b)
(Et)3N, dry DCM, -5 to 0 ꢀC, 0.5-2 h; (c) (Et)3N, neat, 80-100 ꢀC, 2-4 h.
and two compounds (6 and 7) had no effect at all. Nine
compounds (9-12, 14-16, 18, 32) were more active than
flutamide (nonsteroidal SARM, 24% reduction at 10.0 mg/
kg) while five compounds (17, 25, 29-31) were comparable
to flutamide in activity. Three compounds (10, 12, 18) were
almost twice as active as flutamide. The data have been
presented in Table 1.
Chemistry
The aryl/heteroaryl/aralkyl/aroyl piperazines (6-32) were
synthesized according to Scheme 1. Aryl/heteroaryl pipera-
zines (1-5) were treated with appropriate aryl halide to
provide diaryl piperazines (6-19) whereas the reaction of
1-5 with suitable aroyl chloride furnished 1-aryl-4-aroylpi-
perazines (20-28). Similarly, appropriate benzyl chlorides
gave 1-aryl-4-arylmethylpiperazines (29-32). The attempts
havebeenmade tosynthesize piperazineswithtrifluoromethyl
(CF3) and nitro (NO2) group in aryl ring keeping an analogy
with the structures of flutamide (Figure 1), nilutamide
(Figure 1), and bicalutamide (Figure 1).
This study included two types of aryl piperazines, com-
pounds having an aryl group directly attached to N4-position
(6-19) and compounds in which the N4-aryl substituent was
separated by a one-carbon linker (a methylene or a carbonyl
group, 20-32). The aryl substituents at N1-position were
3-chlorophenyl, 3-(trifluoromethyl)phenyl, 4-fluorophenyl,
2-pyridyl, and 2-pyrimidyl, while the aryl group at N4-
position was either nitro substituted or nitro and trifluoro-
methyl disubstituted. A broad analysis of the structure
-activity relationship indicated that a 4-nitrophenyl (9, 10,
12, 14, 16, 18) or a 4-nitrobenzyl (29-32) group at N4-
position of piperazines generally resulted in potent activity.
Addition of a carbonyl linker (20-28) imparted better
activity, and addition of a methylene linker (29-32) reduced
activity. For example, introducing the carbonyl linker in
compounds 7 and 8 augmented the activity (24, 25), while
placing the methylene linker in compound 9 decreased the
activity (31). Nevertheless, the active compound (32) with linker
had a 4-nitrophenylmethyl group at N4-position. On the other
hand, structure-activity comparisons of compounds without
Result and Discussion
In Vivo Effect on Prostate Weight. Adult mature rats
(Sprague-Dawley strain, n = 5) were given test compounds
6-32 as water/gum acacia suspension at an oral dose of 10.0
mg/kg, daily for 21 days. Parallel control animals received
vehicle while positive control animals received flutamide
(10.0 mg/kg) for similar duration. At autopsy (24 h after
the last treatment), the prostates were dissected out and
weighed (Table 1). Twenty-one of these piperazine deriva-
tives (8-19, 21, 24-27, 29-32) decreased the size of prostate
appreciably by 15-47% whereas four compounds (20, 22,
23, 28) decreased the prostatic size moderately by up to 10%

304 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Table 1. Effect of Compounds 6-32 on Prostate of Rata
Sarswat et al.
a * = prostate weight (per 100 mg of control); a = test set compounds.
linker (10-19) indicated that molecules having nitro group at
position 4 and trifloromethyl group at position 2 (10, 12, 14, 16,
and18) in thearyl ring at N4-positionof piperazine were more
potent than those having nitro group at position 2 and
trifloromethyl group at position 4 (11, 13, 15, 17, and 19).
Among the former (10, 12, 14, 16, and 18), the activity profile
with respect to the substituent at N1-position (R1) of piper-
azine was of the following order: 3-(trifluoromethyl)phenyl
(10, 47.70%) > pyridyl (12, 43.40%) > 4-fluorophenyl (18,
39.50%) > 3-chlorophenyl (16, 35.61%) > 2-pyrimidyl (14,
32.30%). However, in the entire study, the most active com-
pound (10) had a 3-(trifluoromethyl)phenyl group at N1-
position and a (4-nitro-2-trifloromethyl)phenyl group directly
attached at N4-position of piperazine.
With a view to assess the potential of the most active
compound 10 as a candidate for BPH management, it
was evaluated for the expression of prostate-specific
antigen (PSA) gene expression (Table 2) and estrogen
receptors (Figure 2) in human BPH-derived prostatic
stromal cells and prostate cancer derived epithelial cell

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 305
line, as well as the pharmacokinetic parameters (Table 3,
Figure 3).
Pharmacokinetic Studies. The pharmacokinetic studies
revealed that the animals tolerated the treatment as no
peculiarities in the animals’ behavior were observed. In a
comparative study of tissue distribution and pharmacokinetics,
the absorption of the compound was rapid with a peak con-
centration (Cmax 1) at 0.5 h and could be monitored up to 24 h
(Figure 3, Table 3). The compound exhibited a double-peak
phenomenon, and the Cmax 2 (139.31 ( 56.64 ng/mL) occurred
after 3.0 h postdose. Because of the occurrence of a double-peak
Expression of Prostate-Specific Antigen (PSA) in Human
Prostate Cancer Epithelial Cells (LNCaP). Prostatic cells
secrete a serine protease called PSA which is used as an early
marker for prostatic hyperplasia, especially cancer. The
regulation of PSA gene is strictly androgen dependent and
regulated by AR at transcriptional level.34 Consequently,
PSA mRNA expression has been used for antiandrogenic
assays.35 We established the SARM nature of new com-
pounds by establishing the effect of the most promising
compound 10 on expression of PSA gene in androgen-
dependent human prostate cancer epithelial cells (LNCaP)
by real time PCR. Flutamide was used as positive control.
Compound 10 (10.0 μmol) reduced PSA secretion of LNCaP
cells by ∼90%, which established its antagonistic activity
against AR. Flutamide (10.0 μmol) reduced PSA secretion of
LNCaP cells by 99% (Table 2).
Compound 10 stimulated the expressions of ER-R and
ER-β by approximately 3- and 6-fold in human BPH stromal
cells and 11- and 16-fold in LNCaP cells, respectively.
Flutamide stimulated the expressions of ER-R and ER-β
by approximately 11- and 14-fold in LNCaP cells. Both
flutamide and compound 10 shifted ER-β/ER-R ratio in
favor of ER-β, though compound 10 was more effective
(1.37) than flutamide (1.32). Hence, compound 10 is likely to
promote prostatic regression through elevated ER-β
mediated signaling in stromal and epithelial cells. Human
prostatic stromal cells are highly responsive to androgens
due to the high expression of AR and the highly favorable
AR:ER-β ratio, which further increases 3-fold in BPH
stromal cells.36 Hence, upregulation of ER-β expression is
highly desirable to control aberrant growth.
Table 3. Pharmacokinetic Parameters of Compound 10 after Single
Administration at 10 mg/kg (per Oral) in Male Sprague-Dawley Rats a
parameter
serum
prostate
hypothalamus
C
max (ng/mL or μg/g)
1
2
1
2
106.01 ( 2.47 442.19 ( 18.82
139.31 ( 2.47 364.73 ( 39.94
99.47
123.82
1.0
t
max (h)
0.5
2.0
712
1.5
6.0
2.0
1999
AUC0-t ((ng h)/mL
5653
3
or (μg h)/g)
ratio to prostate
3
0.13
6.97
0.010
1.00
10.98
0.001
0.35
11.57
0.001
MRT (h)
CLoral (L h-1 kg-1
)
a Each value represents the average of three rats dosed at 10 mg/kg po.
Serum and prostate concentrations are mean ( SEM from three rats.
Hypothalamus concentrations were the mean of replicates from the
pooled tissue of three rats.
Table 2. Fold Change in PSA Expression of LNCaP (Androgen-
Dependent Human Prostate Cancer) Cells after 48 h of Treatment with
Compound 10 and Flutamide
treatment
control
fold change
1
Figure 3. Concentration-time profile of compound 10 and testos-
terone after a single 10 mg/kg oral dose in male Sprague-Dawley
rats. Bar represents SEM.
flutamide (10 μmol)
compound 10 (10 μmol)
0.01 ( 0.23
0.106 ( 0.16
Figure 2. Changes in ER-R and ER-β gene expressions by compound 10 (10.0 μmol) and flutamide (10.0 μmol) in human prostate cancer
epithelial cells (LNCaP) and BPH-derived stromal cells (ST) in vitro (mean ( SE of three values).

306 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Sarswat et al.
phenomenon, the data sets could not be fitted to an appropriate
compartmental pharmacokinetic model. Hence, the phar-
macokinetic parameters were determined by a noncompart-
mental analysis. The mean residence time (MRT) and
leave-one-out (LOO) and leave group of three out, respec-
tively, s is the standard error of the estimate, F is the F-ratio
between the variances of calculated and observed activities,
and r2
is the average squared correlation coefficient
Yrand
AUC0-t were 6.97 h and 712 (ng h)/mL, respectively. The
3
from randomization study involving 100 simulations with its
maximum (max). In the equation, the values given in the
parentheses are the standard errors of the regression coeffi-
cients. The model is validated with an external test set of five
compounds. The predictions of the test set compounds are
found to be satisfactory as reflected in the test set r2.
Equation 2 is a derivative of eq 1, derived by using the scaled
X (XS) in place of X as shown.
clearance (0.01 L h-1 kg-1) was smaller than the hepatic
blood flow (2.9 L h-1 kg-1 37
of the rat, suggesting an
)
insignificant amount of extrahepatic elimination of this
compound. However, the prostate levels achieved the Cmax
(Cmax 1, 442.19 ( 18.82 ng/g, and Cmax 2, 364.73 ( 39.94 ng/g)
occurred after 1.5 and 6.0 h postdose. Whereas the hypothala-
mus showed the Cmax (Cmax 1, 99.47 ng/g, and Cmax 2, 123.82 ng/g)
which occurred after 1.0 and 2.0 h post dose. The compound
levels were higher in prostate (target tissues for main effects)
than in hypothalamus (target tissues for side effects such as
increased serum testosterone concentrations), probably because
of poor penetration across the blood-brain barrier (Figure 3,
Table 3). However, since some amount of compound crosses the
blood-brain barrier, its interference with the negative feedback
mechanism resulted in increased serum testosterone levels dur-
ing 0.5-6.0 h postdose. Similar rise in serum testosterone levels
has been reported for bicalutamide.33 Moreover, the flip-flop
serum testosterone levels correspond to the levels of compound
10 in serum, prostate, and hypothalamus (Figure 3). Never-
theless, significantly higher levels of compound 10 in prostate
than in brain (hypothalamus) kept the prostate weight sup-
pressed.
QSAR Study. A QSAR study has been carried out on the
27 piperazine derivatives shown in the Table 1 to offer
rationale for the activity. Adopting the standard procedure,
the structures were generated in the ChemDraw.38 These
structures were used in Dragon software to compute the
topological features of the compounds.39 This has resulted in
222 descriptors as explanatory variables of the molecular
structures (Table 1). The biological response (BR) of these
compounds (effect on prostate) was considered as dependent
variable after logarithmic transformation and expressed as
log BR. For the purpose of QSAR model validation, these
27 compounds are randomly divided into training (22
compounds) and test sets (5 compounds). Only the training
set compounds are used for the model development, and test
set compounds are used for the external validation of the
developed model. The QSAR analysis has been carried out
using combinatorial protocol in multiple linear regression
(CP-MLR) procedure.40 Among several models the follow-
ing equation has optimally explained the activity:
X - Xmin
Xmax - Xmin
XS
¼
ð3Þ
where Xmin and Xmax are the training set feature X’s mini-
mum and maximum values. This transforms the descriptor
values between “0” and “1” and provides an opportunity for
direct comparison of the regression coefficients within the
equation. The scaled descriptors are identified with subscript
“S” suffixed to the abbreviated names.
In the equation the coefficient of solvation connectivity
index of order 5 (X5sol) suggested in favor of smaller
connectivity values for better activity. Its regression coeffi-
cient is marginally less significant when compared to coeffi-
cients of other descriptors. However, this and the other three
descriptors are statistically significant in the regression equa-
tion of full data set. The regression coefficients of SPI
(superpendentic index), VAR2 (average Randic-type eigen-
vector-based index from adjacency matrix), and T(N O)
3 3 3
(sum of topological distances between nitrogen and oxygen)
suggested that a larger value of these features is favorable for
the activity. The index SPI accounts for the branches and/or
loops present in the molecules. The equation suggests in
favor of increased cyclic and branched analogues for the
activity. Also, this regression equation suggests that an
increased separation of O and N is favorable for the activity.
This in turn suggests in favor of increased and/or distantly
placed amino, nitro, and hydroxyl groups on the template
molecule for better activity.
Conclusion
The new series of designed SARMs consisting of aryl/
heteroaryl/aralkyl/aroyl piperazines have yielded some very
interesting structures that reduced prostatic weight in mature
rats to the extent of 47%, which was twice more than that
reduced by flutamide (24%). The QSAR study revealed that
cyclic and branched moieties with topological separation of O
and N andreduced solvation connectivityindex showed better
activity profile. The most promising compound 10 was well
absorbed andrapidlydistributed tothe targetorgan(prostate)
but restricted by blood-brain barrier from reaching hypotha-
lamus (the target organ for major side effect). Compound 10
inhibited PSA expression in prostatic cells by 90%, which is a
marker for both prostatic hyperplasia and AR activation. On
the other hand, upregulation of ER-β in prostatic (especially
BPH) cells indicated an ER-β mediated mechanism of action.
Some of these structures are worth further optimization for
activity against prostatic hyperplasia.
log BR ¼ 1:918 - 0:000016ð0:0000043ÞSPI
þ 0:045ð0:029ÞX5sol - 0:025ð0:008ÞVRA2
- 0:001ð0:0005ÞTðN OÞ
3 3 3
n ¼ 22
r ¼ 0:861
Q2 ¼ 0:516
s ¼ 0:044
r2 ¼ 0:544
t
Q2 ¼ 0:498
ð1Þ
ð2Þ
G3
F ¼ 12:242
r2YrandðmaxÞ ¼ 0:290ð0:376Þ
log BR ¼ 1:969 - 0:139SPIS þ 0:046X5solS
- 0:092VRA2S - 0:127TðN OÞS
3 3 3
Experimental Section
In this regression equation, n is the number of compounds,
r is the multiple correlation coefficient of regression, r2 is the
Chemistry. In general, all reagents and solvents were commercial
quality and were used without further purification. Melting points
t
test set r2 value, Q2 and Q2 are cross-validated r2 from
G3

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 307
were determined in open capillary tubes on an electrically heated
block and are uncorrected. IR spectra (νmax in cm-1) of the
compounds were recorded on Perkin-Elmer’s FT-IR RX1 PC
spectrophotometer. 1H NMR and 13C NMR spectra were recorded
on Bruker Supercon Magnet Avance DPX-200/DRX-300 spectrom-
eters (operating at 200 and 300 MHz, respectively, for 1H and 50
and 75 MHz, respectively, for 13C) in deuterated solvents with
TMS as internal reference (chemical shifts δ in ppm, J in Hz).
Electrospray ionization mass spectra (ESI-MS) were recorded
on Thermo Lcq Advantage Max-IT, and HR-DART MS were
recorded on JEOL, JMS T100LC Accu TOF. Elemental ana-
lyses were performed on Carlo Erba EA-1108 micro analyzer/
Vario EL-III C H N S analyzer. All compounds were analyzed
for C, H, and N, and the results obtained were within (0.4% of
calculated values. The reaction progress was routinely monitored by
thin-layer chromatography (TLC) on precoated alumina/silica gel
plates (Aldrich). Column chromatography was performed over
Merck silica gel (60-120 mesh). All compounds were characterized
by TLC, 1H and 13C NMR, MS, and HRMS. Elemental analyses
data meet the criteria of g95% purity. All chemicals and solvents
were procured from Sigma-Aldrich/Merck India Ltd.
1-(3-Chlorophenyl)-4-(2-nitrophenyl)piperazine (6). To the mix-
ture of 1-(3-chlorophenyl)piperazine (1, 1.7 g, 9.0 mmol) and
triethylamine (TEA, 2.5 mL, 18.1 mmol) was added 1-chloro-2-
nitrobenzene (2.1 g, 13.5 mmol) and stirred at 100 ꢀC for 10 h.
EtOAc (15.0 mL) was added in reaction mixture, and solid triethy-
lamine hydrochloride salt was filtered off. Filtrate was washed with
water (2 ꢀ 5.0 mL), and organic layer was separated. Combined
organic phases were dried over sodium sulfate and concentrated
under reduced pressure. Solid product was washed with hexane
(10.0 mL) and recrystallized from EtOAc/hexane to give the title
compound (2.4 g, 85%) as yellow solid: mp 91-93 ꢀC; IR (KBr) ν
(cm-1) 2941, 2928, 1596, 1483, 1231; 1H NMR (200 MHz, CDCl3 þ
CCl4) δ 7.76 (1H, d, J = 7.5 Hz), 7.45-7.53 (1H, m), 7.04 -7.19
(3H, m), 6.75 -6.87 (3H, m), 3.29 -3.31 (4H, m), 3.20 (4H, br s);
13C NMR (75 MHz, CDCl3 þ CCl4) δ 152.3 (s), 145.9 (s), 144.1 (s),
135.2 (s), 133.5 (d), 130.3 (d), 126.0 (d), 122.5 (d), 121.3 (d), 119.8
(d), 116.2 (d), 114.2 (d), 51.8 (t), 49.0 (t); ESI-MS m/z 318 (MHþ
with 35Cl), 320 (MHþ with 37Cl). Anal. Calcd for C16H16ClN3O2:C,
60.47; H, 5.08; N, 13.22. Found: C, 60.51; H, 5.25; N, 13.29.
The following compounds 7-19 were prepared using a
procedure similar to that described for compound 6 from the
corresponding substituted piperazines and aryl halides.
1-(2-Nitrophenyl)-4-(3-(trifluoromethyl)phenyl)piperazine (7).
The title compound was synthesized from 1-(3-(trifluoromethyl)-
phenyl)piperazine (2) and 1-chloro-2-nitrobenzene in 88% yield as
yellow solid: mp 66-68 ꢀC; IR (KBr) ν (cm-1) 2982, 2857, 1597,
1540, 1280; 1H NMR (300 MHz, CDCl3 þ CCl4) δ 7.77 (1H, dd,
J = 8.1, 1.4 Hz), 7.47-7.52 (1H, m), 7.32-7.37 (1H, m), 7.19 (1H,
d, J = 8.2 Hz), 7.08-7.13 (4H, m), 3.38-3.41 (4H, m), 3.21-3.25
(4H, m); 13C NMR (75 MHz, CDCl3) δ 151.4 (s), 145.9 (s), 143.8
(s), 133.8 (d), 130.9-132.1 (CF3), 129.8 (d), 126.0 (d), 122.7 (s),
122.6 (d), 121.3 (d), 119.12-119.13 (d), 116.2-116.4 (d), 112.4-
112.6 (d), 51.8 (t), 48.9 (t); HRMS m/z calcd for C17H17F3N3O2
(MHþ) 352.1273, found 352.1258. Anal. Calcd for C17H16F3N3O2:
C, 58.12; H, 4.59; N, 11.96. Found: C, 57.91; H, 4.58; N, 11.79.
1-(2-Nitrophenyl)-4-(pyridin-2-yl)piperazine (8). The title com-
pound was synthesized from 1-(pyridin-2-yl)piperazine (3) and
1-chloro-2-nitrobenzene in 80% yield as yellow solid: mp 118-
120 ꢀC; IR (KBr) ν (cm-1) 2888, 2831, 1598, 1498, 1229; 1H NMR
(300 MHz, CDCl3 þ CCl4) δ 8.17 (1H, d, J = 3.9 Hz), 7.77 (1H,
dd, J = 8.1, 1.1 Hz), 7.44-7.50 (2H, m), 7.17 (1H, d, J = 8.20 Hz),
7.04-7.09 (1H, m), 6.60-6.65 (2H, m), 3.69-3.72 (4H, m),
3.17-3.20 (4H, m); 13C NMR (75 MHz, CDCl3 þ CCl4) δ 159.5
(s), 148.3 (d), 146.2 (s), 144.1 (s), 137.6 (d), 133.5 (d), 126.1 (d),
122.3 (d), 121.3 (d), 113.7 (d), 107.2 (d), 51.8 (t), 45.5 (t); ESI-MS
m/z 285 (MHþ). Anal. Calcd for C15H16N4O2: C, 63.37; H, 5.67;
N, 19.71. Found: C, 63.51; H, 5.73; N, 19.53.
82% yield as yellow solid: mp 165-167 ꢀC; IR (KBr) ν (cm-1
)
2926, 2848, 1594, 1535, 1280; 1H NMR (300 MHz, CDCl3 þ CCl4)
δ 8.20-8.23 (1H, m), 8.14 (2H, d, J = 9.4 Hz), 7.50-7.56 (1H, m),
6.84 (2H, d, J = 9.4 Hz), 6.66-6.70 (2H, m), 3.73-3.77 (4H, m),
3.57-3.60 (4H, m); 13C NMR (75 MHz, CDCl3 þ CCl4) δ 159.0
(s), 154.8 (s), 148.2 (d), 138.7 (s), 137.9 (d), 126.2 (d), 114.1 (d),
112.6 (d), 107.2 (d), 46.8 (t), 44.7 (t); ESI-MS m/z 285 (MHþ).
Anal. Calcd for C15H16N4O2: C, 63.37; H, 5.67; N, 19.71. Found:
C, 63.52; H, 5.71; N, 19.97.
1-(4-Nitro-2-(trifluoromethyl)phenyl)-4-(3-(trifluoromethyl)-
phenyl)piperazine (10). The title compound was prepared from
2 and 1-chloro-4-nitro-2-(trifluoromethyl)benzene in 95%
yield as dark brown solid: 110-112 ꢀC; IR (KBr) ν (cm-1
)
2841, 1612, 1591, 1216; 1H NMR (300 MHz, CDCl3 þ CCl4) δ
8.53 (1H, d, J = 2.4 Hz), 8.35 (1H, dd, J = 8.9, 2.4 Hz),
7.34-7.40 (2H, m), 7.10-7.16 (3H, m), 3.40-3.42 (4H, m),
3.29-3.32 (4H, m); 13C NMR (75 MHz, CDCl3) δ 157.1 (s),
151.4 (s), 142.9 (s), 131.1-132.4 (CF3), 129.9 (d), 128.2 (d),
125.1 (s), 124.7-124.8 (CF3, s), 122.9 (d), 119.3 (d),
116.6-116.7 (d), 112.6-112.7 (d), 52.9 (t), 49.1 (t); HRMS m/
z calcd for C18H16F6N3O2 (MHþ) 420.1147, found 420.1130.
Anal. Calcd for C18H15F6N3O2: C, 51.56; H, 3.55; N, 10.02.
Found: C, 51.32; H, 3.66; N, 10.11.
1-(2-Nitro-4-(trifluoromethyl)phenyl)-4-(3-(trifluoromethyl)-
phenyl)piperazine (11). The title compound was prepared from
2 and 1-chloro-2-nitro-4-(trifluoromethyl)benzene in 95%
yield as dark yellow solid: mp 76-78 ꢀC; IR (KBr) ν (cm-1
)
1
2926, 2848, 1622, 1526, 1275; H NMR (300 MHz, CDCl3 þ
CCl4) δ 8.06 (1H, d, J = 1.4 Hz), 7.70 (1H, dd, J = 8.7, 1.7 Hz),
7.33-7.39 (1H, m), 7.20 (1H, d, J = 8.7 Hz), 7.12-7.13 (2H,
m), 7.06 (1H, d, J = 8.3 Hz), 3.39-3.43 (4H, m), 3.31-3.34
(4H, m); 13C NMR (75 MHz, CDCl3) δ 151.1 (s), 148.0 (s),
140.9 (s), 131.8 (d), 131.4 (d), 130.4-130.5 (CF3), 129.9 (d),
126.3 (s), 124.3-124.4 (CF3), 123.1 (s), 120.8 (d), 119.1 (d),
116.5-116.6 (d), 112.4-112.5 (d), 50.6 (t), 48.6 (t); ESI-MS m/z
420 (MHþ). Anal. Calcd for C18H15F6N3O2: C, 51.56; H, 3.61;
N, 10.02. Found: C, 51.70; H, 3.67; N, 10.10.
1-(4-Nitro-2-(trifluoromethyl)phenyl)-4-(pyridin-2-yl)piperazine
(12). The title compound was prepared from 3 and 1-chloro-4-
nitro-2-(trifluoromethyl)benzene in 82% yield as dark brown
solid: mp 62-64 ꢀC; IR (KBr) ν (cm-1) 2964, 2847, 1593, 1570,
1
1283; H NMR (300 MHz, CDCl3 þ CCl4) δ 8.52 (1H, d, J =
2.6 Hz), 8.33 (1H, dd, J = 8.9, 2.6 Hz), 8.17-8.20 (1H, m),
7.46-7.52 (1H, m), 7.32 (1H, d, J = 8.9 Hz), 6.63-6.67 (2H, m),
3.70-3.74 (4H, m), 3.22-3.25 (4H, m); 13C NMR (75 MHz,
CDCl3) δ 159.2 (s), 148.2 (d), 148.2 (s), 140.6 (s), 137.9 (d),
130.3-130.5 (d), 124.5-124.6 (CF3), 120.5 (d), 114.1 (d), 107.3
(d), 50.7 (t), 45.0 (t); ESI-MS m/z 353 (MHþ). Anal. Calcd for
C16H15F3N4O2: C, 54.55; H, 4.29; N, 15.90. Found: C, 54.63; H,
4.45; N, 15.87.
1-(2-Nitro-4-(trifluoromethyl)phenyl)-4-(pyridin-2-yl)piperazine
(13). The title compound was prepared from 3 and 1-chloro-2-
nitro-4-(trifluoromethyl)benzene in 87% yield as oil: IR (neat)
1
ν (cm-1) 2968, 2850, 1598, 1570, 1290; H NMR (300 MHz,
CDCl3 þ CCl4) δ 8.16-8.18 (1H, m), 8.05 (1H, d, J = 1.4 Hz),
7.66 (1H, dd, J = 8.7, 1.8 Hz), 7.45-7.51 (1H, m), 7.19 (1H, d,
J = 8.7 Hz), 6.61-6.66 (2H, m), 3.71-3.74 (4H, m), 3.27-3.30
(4H, m); 13C NMR (75 MHz, CDCl3) δ 159.5 (s), 157.2 (s), 148.2
(d), 142.6 (s), 137.9 (d), 128.1 (d), 125.2 (s), 124.3-124.9 (d, CF3),
122.7 (d), 114.1 (d), 107.5 (d), 52.8 (t), 45.6 (t); HRMS m/z calcd
for C16H16F3N4O2 (MHþ) 353.1225, found 353.1216. Anal.
Calcd for C16H15F3N4O2: C, 54.55; H, 4.29; N, 15.90. Found:
C, 54.78; H, 4.37; N, 15.87.
2-(4-(4-Nitro-2-(trifluoromethyl)phenyl)piperazin-1-yl)pyrimidine
(14). The title compound was prepared from 2-(piperazin-1-yl)-
pyrimidine (4) and 1-chloro-4-nitro-2-(trifluoromethyl)benzene in
75% yield as brown solid: mp 95-97 ꢀC; IR (KBr) ν (cm-1) 2922,
2854, 1587, 1496, 1226; 1H NMR (300 MHz, CDCl3 þ CCl4) δ 8.53
(1H, d, J = 2.6 Hz), 8.30-8.36 (3H, m), 7.31 (1H, d, J =
8.9 Hz), 6.53 (1H, t, J = 4.7 Hz), 3.99-4.02 (4H, m), 3.16-3.19
1-(4-Nitrophenyl)-4-(pyridin-2-yl)piperazine (9). The title com-
pound was synthesized from 3 and 1-chloro-4-nitrobenzene in

308 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Sarswat et al.
(4H, m); 13C NMR (75 MHz, CDCl3) δ 161.8 (s), 157.9 (d), 157.3
(s), 142.7 (s), 128.1 (d), 125.3 (s), 124.6-124.9 (d, CF3), 122.8 (d),
110.6 (d), 52.9 (t), 43.9 (t); HRMS m/z calcd for C15H15F3N5O2
(MHþ) 354.1178, found 354.1172. Anal. Calcd for C15H14F3N5O2:
C, 50.99; H, 3.99; N, 19.82. Found: C, 51.02; H, 3.45; N, 19.88.
2-(4-(2-Nitro-4-(trifluoromethyl)phenyl)piperazin-1-yl)pyrimidine
(15). The title compound was prepared from 4 and 1-chloro-2-
nitro-4-(trifluoromethyl)benzene in 78% yield as yellow solid: mp
66-68 ꢀC. IR (KBr) ν (cm-1) 3020, 2856, 1624, 1586, 1501, 1218;
1H NMR (300 MHz, CDCl3) δ 8.34 (2H, d, J = 4.8 Hz), 8.10 (1H,
d, J = 1.4 Hz), 7.69 (1H, dd, J = 8.8, 1.8 Hz), 7.20 (1H, d, J = 8.7
Hz), 6.55 (1H, t, J = 4.7 Hz), 3.99-4.03 (4H, m), 3.24-3.28 (4H,
m); 13C NMR (75 MHz, CDCl3) δ 161.7 (s), 157.9 (d), 148.3 (s),
141.0 (s), 130.1-130.2 (d), 124.3-124.5 (CF3), 122.9 (s), 120.7 (d),
110.6 (d), 50.9 (t), 43.5 (t); HRMS m/z calcd for C15H15F3N5O2
(MHþ) 354.1178, found 354.1158. Anal. Calcd for C15H14F3N5O2:
C, 50.99; H, 3.99; N, 19.82. Found: C, 50.86; H, 4.09; N, 19.98.
1-(3-Chlorophenyl)-4-(4-nitro-2-(trifluoromethyl)phenyl)piperazine
(16). The title compound was prepared from 1 and 1-chloro-4-nitro-
2-(trifluoromethyl)benzene in 93% yield as dark brown solid: mp
separated, followed by water washing (2 ꢀ 5.0 mL). Combined
organic phases were dried over sodium sulfate and concentrated
under reduced pressure. Solid product was recrystallized from
EtOAc/hexane to give the title compound (0.97 g, 97%) as white
solid: mp 68-70 ꢀC; IR (KBr) ν (cm-1) 2910, 2857, 1629, 1595; 1H
NMR (300 MHz, CDCl3 þ CCl4) δ 8.20 (1H, d, J = 3.6 Hz),
7.43-7.53 (6H, m), 6.65-6.68 (2H, m), 3.89 (2H, br s), 3.57 (6H, br s);
13C NMR (50 MHz, CDCl3) δ 170.7 (s), 159.3 (s), 148.2 (d), 137.9
(d), 135.8 (s), 130.1 (d), 128.7 (d), 127.3 (d), 114.2 (d), 107.5 (d),
45.6 (t); ESI-MS m/z 268 (MHþ). Anal. Calcd for C16H17N3O: C,
71.89; H, 6.41; N, 15.72. Found: C, 71.79; H, 6.59; N, 15.56.
The following compounds 21-28 were prepared using a
procedure similar to that described for compound 20 from the
corresponding substituted piperazines and benzoyl chloride.
Phenyl(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methanone
(21). The title compound was prepared from 2 and benzoyl chloride
in 95% yield as white solid: mp 75-77 ꢀC; IR (KBr) ν (cm-1) 2907,
2839, 1715, 1636, 1598; 1H NMR (300 MHz, CDCl3 þ CCl4) δ 7.42
(5H, br s), 7.33-7.38 (1H, m), 7.05-7.14 (3H, m), 3.77 (4H, br s),
3.25 (4H, br s); 13C NMR (75 MHz, CDCl3 þ CCl4) δ 170.3 (s),
151.3 (s), 135.6 (s), 131.2-132.5 (CF3), 130.1 (d), 129.8 (d), 128.7
(d), 127.4(d), 122.5(s), 119.6(d), 116.9-117.0 (d), 112.9-113.1 (d),
49.4 (t); ESI-MS m/z335 (MHþ). Anal. Calcd for C18H17F3N2O: C,
64.66; H, 5.13; N, 8.38. Found: C, 64.86; H, 5.25; N, 8.67.
(4-(3-Chlorophenyl)piperazin-1-yl)(phenyl)methanone (22).
The title compound was prepared from 1 and benzoyl chloride in
91% yield as oil: IR (neat) ν(cm-1) 2910, 2857, 1639, 1595, 1570; 1H
NMR (300 MHz, CDCl3 þ CCl4) δ 7.30 (5H, br s), 7.02-7.07 (1H,
m), 6.71-6.75 (2H, m), 6.66 (1H, d, J = 8.3 Hz), 3.45-3.76 (4H, br
m), 3.03 (4H, br s); 13C NMR (75 MHz, CDCl3) δ 170.3 (s), 152.0
(s), 135.5 (s), 135.0 (s), 130.2 (d), 129.9 (d), 128.6 (d), 127.1 (d), 120.1
(d), 116.4 (d), 114.5 (d), 49.1 (t); ESI-MS m/z 301 (MHþ). Anal.
Calcd for C17H17ClN2O: C, 67.88; H, 5.70; N, 9.31. Found: C,
67.94; H, 5.94; N, 9.21.
1
55-57 ꢀC; IR (KBr) ν (cm-1) 2857, 1625, 1586, 1270; H NMR
(300 MHz, CDCl3 þ CCl4) δ8.52 (1H, d, J= 2.3 Hz), 8.34 (1H, dd,
J = 8.9, 2.3 Hz), 7.34 (1H, d, J = 8.9 Hz), 7.16-7.22 (1H, m), 6.92
(1H, s), 6.82-6.85 (2H, m), 3.34-3.36 (4H, m), 3.27-3.29 (4H, m);
13C NMR (75 MHz, CDCl3) δ 157.0 (s), 152.2 (s), 142.8 (s), 135.2
(s), 130.3 (d) 128.1 (d), 125.4 (s), 124.7-125.1 (CF3), 122.8 (d), 120.1
(d), 116.4 (d), 114.4 (d), 52.9 (t), 49.1 (t); HRMS m/z calcd for
C17H16ClF3N3O2 (MHþ) 386.0883, found 386.0910. Anal. Calcd
for C17H15ClF3N3O2: C, 52.93; H, 3.92; N, 10.89. Found: C, 52.79;
H, 3.73; N, 10.97.
1-(3-Chlorophenyl)-4-(2-nitro-4-(trifluoromethyl)phenyl)piperazine
(17). The title compound was prepared from 1 and 1-chloro-2-nitro-
4-(trifluoromethyl)benzene in 90% yield as light brown solid: mp
126-128 ꢀC; IR (KBr) ν (cm-1) 2965, 2841, 1625, 1593, 1282; 1H
NMR (300 MHz, CDCl3 þ CCl4) δ 8.06 (1H, br s), 7.69 (1H, dd,
J = 8.7, 1.8 Hz), 7.15-7.21 (2H, m), 6.83-6.88 (2H, m), 6.78 (1H,
dd, J = 8.3, 2.1 Hz), 3.35-3.37 (4H, m), 3.30-3.32 (4H, m); 13C
NMR (75 MHz, CDCl3) δ 152.1 (s), 148.1 (s), 141.1 (s), 135.3 (s),
130.4 (d), 125.3 (d) 124.4-124.5 (CF3), 122.8 (d), 121.7 (d), 120.8
(d), 120.2 (d), 116.3 (d), 114.3 (d), 51.0 (t), 48.8 (t); ESI-MS m/z 386
(MHþ). Anal. Calcd for C17H15ClF3N3O2: C, 52.93; H, 3.92; N,
10.89. Found: C, 52.72; H, 3.60; N, 10.67.
1-(4-Fluorophenyl)-4-(4-nitro-2-(trifluoromethyl)phenyl)piperazine
(18). The title compound was prepared from 1-(4-fluorophe-
nyl)piperazine (5) and 1-chloro-4-nitro-2-(trifluoromethyl)benzene
in 90% yield as brown solid: mp 80-82 ꢀC;IR(KBr) ν(cm-1) 2932,
2858, 1587, 1498, 1230; 1H NMR (300 MHz, CDCl3) δ 8.52 (1H, d,
J=2.4Hz),8.34(1H,dd,J= 8.9, 2.4 Hz), 7.35 (1H, d, J=9.0Hz),
6.87-6.99 (4H, m), 3.27 (8H, s); 13C NMR (50 MHz, CDCl3 þ
CCl4) δ 162.20 (s), 157.1 (s), 155.4 (s), 147.8 (d), 147.8 (d), 143.0 (s),
128.1 (d), 124.6-124.9 (CF3), 122.7 (d), 118.7 (d), 118.5 (d), 116.2
(d), 115.7 (d), 53.2 (t), 50.7 (t); ESI-MS m/z369 (MHþ). Anal. Calcd
for C17H15F4N3O2: C, 55.29; H, 4.09; N, 11.38. Found: C, 55.42; H,
4.2; N, 11.58.
Phenyl(4-(pyrimidin-2-yl)piperazin-1-yl)methanone (23). The
title compound was prepared from 4 and benzoyl chloride in
88% yield as white solid: mp 113-115 ꢀC; IR (KBr) ν (cm-1
)
1
2924, 2855, 1628, 1585, 1550; H NMR (300 MHz, CDCl3 þ
CCl4) δ 8.29 (2H, d, J = 4.7 Hz), 7.42 (5H, s), 6.51 (1H, t, J = 4.7
Hz), 3.70-3.86 (8H, m); 13C NMR (50 MHz, CDCl3 þ CCl4) δ
170.5 (s), 161.7 (s), 157.9 (d), 135.9 (s), 130.0 (d), 128.7 (d), 127.4
(d), 110.6 (d), 44.1 (t); ESI-MS m/z 269 (MHþ). Anal. Calcd for
C15H16N4O: C, 67.15; H, 6.01; N, 20.88. Found: C, 67.29; H,
6.27; N, 20.98.
(2-Nitrophenyl)(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)-
methanone (24). The title compound was prepared from 2 and
2-nitrobenzoyl chloride in 95% yield as pale yellow solid: mp
62-64 ꢀC; IR (KBr) ν (cm-1) 2936, 2858, 1700, 1630, 1594, 1278;
1H NMR (300 MHz, CDCl3 þ CCl4) δ 8.21 (1H, d, J = 8.1 Hz),
7.72-7.77 (1H, m), 7.58-7.62 (1H, m), 7.43 (1H, d, J = 6.7 Hz),
7.34-7.40 (1H, m), 7.06-7.14 (3H, m), 3.95-4.05 (2H, m),
3.39-3.41 (4H, m), 3.17 (2H, br s); 13C NMR (75 MHz, CDCl3)
δ 166.8 (s), 151.3 (s), 145.7 (s), 134.8 (s), 131.5-132.7 (CF3), 131.1
(d), 130.2 (d), 129.9 (d), 128.3 (d), 125.1 (d), 122.6 (s), 119.7-119.8
(d), 116.9-117.1 (d), 113.0-113.2 (d), 49.0 (t), 48.9 (t), 46.7 (t),
41.8 (t); HRMS m/z calcd for C18H17F3N3O3 (MHþ) 380.1222,
found 380.1195. Anal. Calcd for C18H16F3N3O3: C, 56.99; H, 4.25;
N, 11.08. Found: C, 56.69; H, 4.53; N, 11.04.
1-(4-Fluorophenyl)-4-(2-nitro-4-(trifluoromethyl)phenyl)piperazine
(19). The title compound was prepared from 5 and 1-chloro-2-
nitro-4-(trifluoromethyl)benzene in 90% yield as brown solid: mp
1
70-72 ꢀC; IR (KBr) ν (cm-1) 2935, 2858, 1580, 1496, 1237; H
NMR (300 MHz, CDCl3) δ 8.05 (1H, s), 7.69 (1H, dd, J = 8.6, 1.4
Hz), 7.20 (1H, d, J = 8.7 Hz), 6.86-6.99 (4H, m), 3.25-3.31 (8H,
m); 13C NMR δ (75 MHz, CDCl3) δ 159.1 (s), 155.9 (s), 147.9 (s),
147.5 (d), 147.5 (d), 130.2-130.3 (s), 124.1-124.3 (CF3), 120.7 (d),
118.3 (d), 118.2 (d), 115.8 (d), 115.5 (d), 51.0 (t), 50.0 (t); ESI-MS
m/z 369 (MHþ). Anal. Calcd for C17H15F4N3O2: C, 55.29; H, 4.09;
N, 11.38. Found: C, 55.46; H, 4.22; N, 11.48.
Phenyl(4-(pyridin-2-yl)piperazin-1-yl)methanone (20). To a solu-
tion of 3 (0.6 mL, 3.9 mmol) and TEA (1.0 mL, 7.8 mmol) in
dichloromethane (DCM, 15.0 mL) was added benzoyl chloride
(0.5 mL, 4.7 mmol) and stirred at -5 to 0 ꢀC for 2 h. Reaction
mixture was treated with ice (2.0 g), and organic layer was
(2-Nitrophenyl)(4-(pyridin-2-yl)piperazin-1-yl)methanone (25).
The title compound was prepared from 3 and 2-nitrobenzoyl
chloride in 92% yield as yellow solid: mp 110-112 ꢀC; IR (KBr)
ν (cm-1) 2923, 2853, 1718, 1635, 1592, 1235; 1H NMR (300 MHz,
CDCl3 þ CCl4) δ8.19-8.24 (2H, m), 7.71-7.76 (1H, m) 7.58-7.63
(1H, m) 7.49-7.54 (1H, m), 7.43 (1H, d, J = 7.5 Hz), 6.66-6.68
(2H, m), 3.96 (2H, br s), 3.70 (2H, br s), 3.56 (2H, br s), 3.33-3.36
(2H, m); 13C NMR (75 MHz, CDCl3) δ 166.7 (s), 159.1 (s), 148.1
(d), 145.5 (s), 137.8 (d), 134.7 (d), 132.8 (s), 130.1 (d), 128.2 (d),
124.9 (d), 114.1 (d), 107.5 (d), 46.6 (t), 45.1 (t), 45.0 (t), 41.7 (t); ESI-
MS m/z 313 (MHþ). Anal. Calcd for C16H16N4O3: C, 61.53; H,
5.16; N, 17.94. Found: C, 61.39; H, 5.28; N, 17.74.

Article
(4-(Pyridin-2-yl)piperazin-1-yl)(4-(trifluoromethyl)phenyl)-
methanone (26). The title compound was prepared from 3 and
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 309
δ 8.19 (2H, d, J = 8.7 Hz), 8.14-8.16 (1H, m), 7.55 (2H, d, J = 8.6
Hz), 7.41-7.47 (1H, m), 6.58-6.62 (2H, m), 3.64 (2H, s), 3.54-3.57
(4H, m), 2.55-2.58 (4H, m); 13C NMR (50 MHz, CDCl3) δ 159.6
(s), 148.1 (d), 147.4 (s), 146.3 (s), 137.6 (d), 129.7 (d), 123.7 (d), 113.5
(d), 107.2 (d), 62.3 (t), 53.2 (t), 45.4 (t); ESI-MS m/z 299 (MHþ).
Anal. Calcd for C16H18N4O2: C, 64.41; H, 6.08; N, 18.78. Found: C,
64.50; H, 6.27; N, 18.57.
4-(trifluoromethyl)benzoyl chloride in 92% yield as white solid:
1
mp 80-82 ꢀC; IR (KBr) ν (cm-1) 2923, 2851, 1638, 1596; H
NMR (300 MHz, CDCl3 þ CCl4) δ 8.18 (1H, d, J = 3.8 Hz),
7.70 (2H, d, J = 8.0 Hz), 7.46-7.56 (3H, m), 6.62-6.69 (2H, m),
3.59-3.89 (8H, m); 13C NMR (50 MHz, CDCl3) δ 169.2 (s),
159.1 (s), 148.1 (s), 139.3 (s), 137.9 (d), 131.6 (s), 127.7 (d),
125.7-125.9 (CF3), 114.3 (d), 107.6 (d), 45.6 (t); ESI-MS m/z
336 (MHþ). Anal. Calcd for C17H16F3N3O: C, 60.89; H, 4.81; N,
12.53. Found: C, 61.18; H, 4.98; N, 12.34.
2-(4-(4-Nitrobenzyl)piperazin-1-yl)pyrimidine (32). The title
compound was prepared from 4 and 1-(bromomethyl)-4-nitro-
benzene in 76% yield as pale yellow solid: mp 130-132 ꢀC; IR
1
(KBr) ν (cm-1) 2925, 2838, 1594, 1544, 1255; H NMR (300
(4-(Trifiuoromethyl)phenyl)(4-(3-(trifluoromethyl)phenyl)-
piperazin-1-yl)methanone (27). The title compound was prepared
from 2 and 4-(trifluoromethyl)benzoyl chloride in 90% yield as
white solid: mp 65-66 ꢀC; IR (KBr) ν (cm-1) 2927, 2857, 1656,
1598; 1H NMR (300 MHz, CDCl3 þ CCl4) δ 7.71 (2H, d, J = 8.1
Hz), 7.55 (2H, d, J = 8.0 Hz), 7.34-7.39 (1H, m), 7.04-7.15 (3H,
m), 3.46-3.90 (4H, m), 3.25 (4H, br s); 13CNMR(50MHz,CDCl3)
δ 168.9 (s), 151.3 (s), 139.3 (s), 131.9-132.6 (CF3), 130.0 (d), 127.9
(d), 126.2 (d), 125.9-126.0 (CF3), 122.6 (s), 122.1 (s), 119.9 (d),
117.4-117.5 (d), 113.3-113.4 (d), 49.7 (t); HRMS m/z calcd for
C19H17F6N2O (MHþ) 403.1245, found 403.1228. Anal. Calcd for
C19H16F6N2O: C, 56.72; H, 4.01; N, 6.96. Found: C, 56.81; H, 4.20;
N, 7.04.
(4-(3-Chlorophenyl)piperazin-1-yl)(4-(trifluoromethyl)phenyl)-
methanone (28). The title compound was prepared from 1 and
4-(trifluoromethyl)benzoyl chloride in 88% yield as oil; IR (neat)
ν (cm-1) 2925, 2846, 1689, 1594; 1H NMR (300 MHz, CDCl3 þ
CCl4) δ 7.70 (2H, d, J = 8.1 Hz), 7.54 (2H, d, J = 8.0 Hz),
7.14-7.20 (1H, m), 6.85-6.87 (2H, m), 6.77 (1H, dd, J = 8.2, 1.8
Hz), 3.56-3.90 (4H, m), 3.20 (4H, br s); ESI-MS m/z 369 (MHþ).
Anal. Calcd for C18H16ClF3N2O: C, 58.62; H, 4.37; N, 7.60.
Found: C, 58.92; H, 4.40; N, 7.51.
MHz, CDCl3 þ CCl4) δ 8.20 (2H, d, J = 4.7 Hz), 8.13 (2H, d,
J = 8.7 Hz), 7.47 (2H, d, J = 8.6 Hz), 6.39 (1H, t, J = 4.7 Hz),
3.75-3.78 (4H, m), 3.56 (2H, s), 2.42-2.45 (4H, m); 13C NMR
(50 MHz, CDCl3) δ 161.8 (s), 157.8 (d), 147.3 (s), 146.3 (s), 129.6
(d), 123.7 (d), 110.1 (d), 62.3 (t), 53.2 (t), 43.8 (t), ESI-MS m/z
300 (MHþ). Anal. Calcd for C15H17N5O2: C, 60.19; H, 5.72; N,
23.40. Found: C, 60.23; H, 5.68; N, 23.20.
Biology
Expression of Estrogen Receptors in Human BPH Stromal
and Cancer Epithelial (LNCaP) Cells. Estrogen receptors
(ER-R, ER-β) are ligand-dependent transcription factors
belonging to the superfamily of nuclear receptors. Recent
studies have shown that estrogen signaling plays an impor-
tant role in pathogenesis of prostatic hyperplasia. Compell-
ing evidence is available in literature which supports that
ER-β signaling mediates antiproliferative and proapoptotic
effects while ER-R mediates proliferative effects (aberrant
growth) in prostate.41 ER-R is chiefly located in the stroma,
which is the site of BPH initiation, and regulates epithelial
growth through paracrine signaling. ER-β is present in both
stromal and epithelial compartments, with higher prevalence
in the latter. The mRNA expression of these receptors in
human BPH-derived stromal cells and cancer epitheial cells
(LNCaP) in response to treatment with compound 10 was
studied by real time PCR. Human BPH-derived stromal cells
[isolated from prostatic tissue of patients undergoing trans-
urethral resection of prostate (TURP) for BPH at the CSM
Medical University, Lucknow] were maintained in primary
culture by following standard method.42 Briefly, viable
tissues were washed three times in medium A [a mixture of
DMEM/Ham’s F-12 and Waymouth MD 752/1 (1:1 v/v)
supplemented with antibiotic-antimycotic solution (Sigma
Aldrich)], minced, and digested with collagenase (5.0 mg/
mL) in medium A enriched with 2% fetal calf serum (FCS) at
37 ꢀC for 4 h. Subsequently, the tissues were passed 6-7
times through an 18G needle and centrifuged at 1200 rpm for
3 min. The pellets were washed and resuspended in medium
A with 10% FCS. The resulting cells were vimentin positive
and grown in DMEM/Ham’s F-12 medium supplemented
with 10% FCS. Cells from passages 4-6 were used in these
studies. LNCaP cells were grown in RPMI-1640 medium
supplemented with 10% FCS, 100 units/mL penicillin G
sodium, and 100 μg/mL streptomycin sulfate using gelatin-
coated plates. Prostatic cells grown to 80% confluence
were treated with either 10.0 μmol of compound 10 or
flutamide for 24 h. Later, total RNA of cells was extracted
using the Trireagent (Invitrogen) by following the manufac-
turer’s instructions. cDNA was prepared from 1.0-2.0 μg of
total RNA using Revert Aid H-Minus first strand cDNA
synthesis kit (Fermentas Life Sciences, USA). Synthesis was
performed for 1 h at 42 ꢀC (for reverse transcription), and the
thermocycling for each reaction was done in a final volume
of 25.0 μL containing 1.0 μL of cDNA sample, 0.5 μmol of
1-(4-Nitrobenzyl)-4-(3-(trifluoromethyl)phenyl)piperazine (29).
Tothe mixture of 2 (0.7 g, 3.3mmol) andTEA (0.9 mL, 6.7mmol)
was added 1-(bromomethyl)-4-nitrobenzene (1.0 g, 5.0 mmol) and
heated at 100 ꢀC for 4 h. EtOAc (15.0 mL) was added in reaction
mixture and washed with water (2 ꢀ 5.0 mL), and organic layer was
separated. Combined organic phases were dried over sodium sulfate
and concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (EtOAc/hexane =1/5) to give
the title compound in 83% yield as yellow oil: IR (neat) ν (cm-1
)
2925, 2838, 1582, 1544, 1255; 1H NMR (300 MHz, CDCl3 þ CCl4)
δ8.19 (2H, d, J = 8.6 Hz), 7.54 (2H, d, J= 8.6 Hz), 7.29-7.35 (1H,
m), 67.00-7.07 (3H, m), 3.66 (2H, s), 3.24-3.27 (4H, m), 2.61-2.64
(4H, m); 13C NMR (50 MHz, CDCl3) δ 151.5 (s), 147.3 (s), 146.2
(s), 130.4-132.3 (CF3), 129.7 (d), 129.6 (d), 127.2 (s), 123.6 (d),
118.8 (d), 115.7-115.9 (d), 112.0-112.2 (d), 62.0 (t), 53.0 (t), 48.7
(t); ESI-MS m/z 366 (MHþ). Anal. Calcd for C18H18F3N3O2: C,
59.17; H, 4.97; N, 11.50. Found: C, 59.28; H, 5.10; N, 11.48.
The following compounds 30-32 were prepared using a
procedure similar to that described for compound 29 from the
corresponding substituted piperazines and 1-(bromomethyl)-
4-nitrobenzene.
1-(3-Chlorophenyl)-4-(4-nitrobenzyl)piperazine (30). The title
compound was prepared from 1 and 1-(bromomethyl)-4-nitro-
benzene in 85% yield as pale yellow solid: mp 108-110 ꢀC; IR
1
(KBr) ν (cm-1) 2942, 2878, 1592, 1564, 1230; H NMR (300
MHz, CDCl3 þ CCl4) δ 8.18 (2H, d, J = 8.7 Hz), 7.54 (2H, d, J =
8.7 Hz), 7.13-7.18 (1H, m), 6.85-6.87 (1H, m), 6.76-6.81 (2H,
m), 3.64 (2H, s), 3.19-3.22 (4H, m), 2.58-2.61 (4H, m); 13C
NMR (75 MHz, CDCl3) δ 152.4 (s), 147.3 (s), 146.2 (s), 135.0 (s),
130.2 (d), 129.6 (d), 123.7 (d), 119.4 (d), 115.8 (d), 114.0 (d), 62.1
(t), 53.1(t), 48.7(t); ESI-MSm/z 332 (MHþ with 35Cl), 334 (MHþ
with 37Cl). Anal. Calcd for C17H18ClN3O2: C, 61.54; H, 5.47; N,
12.66. Found: C, 61.29; H, 5.61; N, 12.81.
1-(4-Nitrobenzyl)-4-(pyridin-2-yl)piperazine (31). The title com-
pound was prepared from 3and 1-(bromomethyl)-4-nitrobenzene in
80% yield as yellow solid: mp 136-138 ꢀC;IR (KBr) ν(cm-1) 2941,
2847, 1685, 1594, 1560, 1248; 1H NMR (300 MHz, CDCl3 þ CCl4)

310 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Sarswat et al.
each primer, 2ꢀ ready-to-use reaction mixture (ABi SYBR
green master mixture) including Taq DNA polymerase, reac-
tion buffer, and deoxyribonucleoside triphosphate (dNTP)
mixture. After 10 s of initial denaturation at 95 ꢀC, the
following cycling conditions (45 cycles) were used: denatura-
tion at 95 ꢀC for 20 s, annealing at 58 ꢀC for 30 s, and
elongation at 72 ꢀC for 30 s. The detection of the PCR reaction
based on fluorescence monitoring (Light Cycler 480; Roche)
was employed. Quantitative results were obtained by the cycle
threshold (CT) value where a signal rose above background
level. Expression of the investigated genes was compared to the
steady expression of GAPDH. The primer sequences used were
as follows. ER-R: forward, 50-CCACCAACCAGTGCAC-
CATT-30; reverse, 50-GGTCTTTTCGTATCCCACCTTTC-
30. ER-β: forward, 50-AAGAAGATTCCCGGCTTTGT-30;
reverse, 50-CTTCTACGCATTTCCCCTCA-30.
Pharmacokinetic Evaluation. The pharmacokinetic studies
of compound 10 were carried out in young and healthy male
Sprague-Dawley rats weighing 250 ( 25 g, obtained from
Laboratory Animal Division, CDRI, Lucknow, India. The
animals were housed in plastic cages under standard labora-
tory conditions with a regular 12 h day-night cycle. Stan-
dard pelleted laboratory chow (Goldmohar Laboratory
Animal Feed, Lipton India Ltd., Chandigarh, India) and
water were allowed ad libitum. The rats were acclimatized to
this environment for at least 2 days before conducting the
experiments. In all of the studies mentioned below the dose
was administered after overnight fasting (12-16 h). The
study was conducted in n = 3 per time point. In all experi-
ments, euthanasia and disposal of carcasses were carried out
as per the guidelines of Local Ethics Committee for animal
experimentation.
models using WinNonlin program, version 1.5 (Scientific Con-
sulting Inc.).
Acknowledgment. The authors gratefully acknowledge the
technical assistance received from Mrs. Tara Rawat and
Research Fellowships from the Council of Scientific and
Industrial Research (A.S.), University Grants Commission
(L.K., N.L., A.J.), and Indian Council of Medical Research
(R.K., V.V.). Staff members of SAIF Division, CDRI, are
acknowledged for providing the spectral data. This study was
partly supported by a grant from the Ministry of Health and
Family Welfare, Government of India.
Supporting Information Available: 1H NMR and 13C NMR
spectral data of compounds 6-32, HRMS data of compounds 7,
10, 13, 14, 15, 16, 24, and 27, and representative chromatogram
of compound 10. This material is available free of charge via the
Internet at http://pubs.acs.org.
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