Design, syntheses, and characterization of pharmacophore based chemokine receptor CCR5 antagonists as anti prostate cancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.09.004

Accumulating evidence has shown multiple roles that chemokine receptor CCR5 may play to promote the progression of several types of cancer. The mechanism of such promotion is believed to involve chronic inflammation that creates a microenvironment which e

Full text of DOI:10.1016/j.ejmech.2013.09.004

European Journal of Medicinal Chemistry 69 (2013) 647e658
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, syntheses, and characterization of pharmacophore based
chemokine receptor CCR5 antagonists as anti prostate cancer agents
Christopher K. Arnatt a, Saheem A. Zaidi a, Zhu Zhang b, Guo Li a, Amanda C. Richardson c,
,
, d,
*
Joy L. Ware c d, Yan Zhang a
a Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, 800 East Leigh Street, Richmond, VA 23298, USA
b Department of Chemistry, College of Pharmacy, Tianjin Medical University, Tianjin 300070, China
c Department of Pathology, Virginia Commonwealth University, Richmond, VA 23298, USA
d Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Accumulating evidence has shown multiple roles that chemokine receptor CCR5 may play to promote the
progression of several types of cancer. The mechanism of such promotion is believed to involve chronic
inflammation that creates a microenvironment which enhances tumor survival. Therefore, blocking CCR5
function with an antagonist may provide a novel treatment of cancers such as prostate cancer. Currently,
several CCR5 antagonists are available, but all have been optimized for their inhibitory activity on HIV-1
cellular membrane invasion process rather than inhibition on cytoplasmic signaling pathways. Thus,
there is need to develop CCR5 antagonists focusing on blockage of CCR5 downstream signaling and
inhibition of CCR5 related prostate cancer proliferation and progression. In this report, a pharmacophore
analysis was conducted based on docking studies of several known CCR5 antagonists in a CCR5 homology
model. A unique structural skeleton for CCR5 antagonist was constructed and functionalized, resulting in
a new series of small molecules to be synthesized and characterized. A combination of CCR5 calcium flux
inhibition, anti prostate cancer cell proliferation, basal cytotoxicity, and in vivo animal model studies
were applied to screen the newly synthesized compounds. Results from this study provided a potential
lead compound for future CCR5 antagonist development focusing on prostate cancer therapy.
Published by Elsevier Masson SAS.
Received 22 July 2013
Received in revised form
28 August 2013
Accepted 1 September 2013
Available online 20 September 2013
Keywords:
Prostate cancer
CCR5 antagonist
Pharmacophore based molecular design
1. Introduction
invasion and proliferation can be inhibited by the CCR5 antagonist
TAK-779 (Fig. 1) [9]. Therefore, this mechanism of prostate cancer
Chemokine receptor CCR5, a G protein-coupled receptor (GPCR),
has been shown to be a viable target in drug discovery due to its
involvement in HIV entry and cancer [1]. In HIV pathogenesis, CCR5
acts as an essential co-receptor for HIV invasion into host cells;
whereas in cancer, it provides a pro-inflammatory environment
promoting cell invasion and proliferation in several cancers [2e10].
Within the immune system, CCR5 primarily functions through
interaction with endogenous small cytokines (chemokines) which
progression and inhibition represents a novel cancer therapy
target.
Currently, prostate cancer is the most common non-cutaneous
solid cancer in men in the U.S.; in all, approximately one sixth of
U.S. men will develop prostate cancer [16]. Several therapies exist
for prostate cancer, but are beneficially limited to early stages of the
disease. Upon the onset of prostate cancer metastasis no signifi-
cantly effective therapies exist [17e21]. Therefore, exploiting
RANTES-induced cancer cell invasion and proliferation could be a
useful therapy to stop the progression of prostate cancer at later
stages. In order to do so, CCR5 antagonists specifically targeting
prostate cell proliferation and invasion need to be developed.
Several small molecule CCR5 antagonists have been developed
as HIV-1 entry inhibitors. All of them have shown high efficacy
inhibiting CCR5 mediated virus entry [22e28]. However, there has
not been much success in getting them through clinical trials due to
toxicity, cardiac side effects, lack of efficacy and bioavailability
[1,24e26,29]. In fact, only one of them has been approved by the
include CCL3 (MIP-1a), CCL4 (MIP-1b) and CCL5 (RANTES) [11,12].
Of those chemokines, RANTES expression has been correlated to
the progression of several cancers [9,13e15]. Within those cancers,
prostate cancer specimens have also been shown to overexpress
CCR5 [15]. Importantly, the RANTES induced prostate cancer cell
* Corresponding author. Massey Cancer Center, Virginia Commonwealth Uni-
versity, Richmond, VA 23298, USA. Tel.: þ1 804 828 0021; fax: þ1 804 828 7625.
E-mail address: yzhang2@vcu.edu (Y. Zhang).
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2013.09.004

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C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
F
F
H
N
O
HN
O
N+
N
N
N
N
O
Maraviroc
TAK-779
O
O
O
OH
N
N
N
N
O
F3C
NH
N
N
HO
N
O
O
Vicriviroc
Aplaviroc
Fig. 1. The chemical structures of several known CCR5 antagonists.
FDA for the treatment of HIV in 2007 (Maraviroc, Fig. 1) [27,28].
Therefore, there is still a need to continue looking for new chemical
structures in order to curtail the negative side effects of those
compounds. Herein, we designed and synthesized a set of novel
CCR5 antagonists based on a shared pharmacophore model of
several known CCR5 antagonists obtained through molecular
modeling, and tested them for their activity on inhibition of
RANTES signaling and cancer cell proliferative, basal cytotoxicity,
and tumor growth inhibitory potency.
Ile198 (TM5), Phe109 (TM3), Tyr108 (TM3), and Phe112 (TM3); and
all four had an aromatic group binding within an aromatic binding
pocket formed by Tyr89 (TM2), Trp86 (TM2), and Trp248 (TM6).
Such results further supported the identification of the general
pharmacophore of these ligands.
Based upon the shared scaffold of these known CCR5 antago-
nists and the analysis of their binding modes in the homology
model of CCR5, a new series of ligands was proposed as a proof-of-
concept to test our observation (Fig. 2). The skeleton chosen was
based upon a 1,2,4-trisubstituted benzene ring with its 4-position
attached to an N-substituted piperidine ring and provides three
different R-group attachment sites, allowing for a library of diverse
ligands to be easily synthesized. In detail, R1 group was mainly
different sizes of hydrophobic moieties to test the bulkiness influ-
ence to the binding pocket; R2 group was introduced as two
different aromatic systems; and R3 group was applied to test va-
riety of electronic effects on the hydrophobic moiety.
2. Results and discussion
2.1. Molecular design
To identify the common pharmacophore of known CCR5 an-
tagonists, a conformational analysis of several CCR5 antagonists
was first performed to find the lowest energy conformation for
each of them. Superimposing the lowest energy conformations of
these ligands yielded a general molecular scaffold that each of the
structures shared. In all, in the center of the scaffold a secondary or
tertiary amino group was connected to an amide moiety at a dis-
2.2. Chemistry
A total of 24 compounds with varying substituents were syn-
thesized by applying the route in Fig. 4. The first step involved a
Grignard reaction between 1-benzyl-4-piperidone and 1-bromo-
4(R2-substituted)benzene to synthesize 1. After hydrogenation to
ꢀ
tance of 5e7 A, on the right of the scaffold an aromatic moiety is
ꢀ
attached to the amide at a distance of 3.5e5.5 A, and on the left of
the scaffold a hydrophobic moiety is linked to the secondary or
ꢀ
tertiary amine at a distance of 4.5e6 A. When looking at the
o
o
o
3.5 - 5.5 A
5 - 7 A
4.5 - 6 A
overlaid conformations three-dimensionally the whole scaffold
was somehow bent, rather than linear (Fig. 2). Such conformation
provided us a starting point of analyzing the possible binding mode
of the ligands in the protein.
H
N
H
N
All the ligands in their lowest energy conformation were docked
into a CCR5 homology model constructed based on the crystal
structure of CXCR4 [30]. After that, energy minimization and dy-
namics simulation were conducted to optimize the ligand binding
mode. Analyses of the docking modes of four known CCR5 antag-
onists in the CCR5 homology model (Fig. 3) indicated only a slight
change of their final docking mode compared to their original
lowest energy conformation, and revealed several commonalities
shared among the compounds. All four compounds carried a sec-
ondary or tertiary amino group which formed a putative salt bridge
with Glu283 (TM7). Additional common interacted included: a
hydrophobic moiety fitting into a hydrophobic pocket formed by
O
O
Aromatic
Group
Hydrophobic group
with polar moiety
R2
O
NH
R1
R3
N
Fig. 2. Proposed pharmacophore of known CCR5 antagonists and molecular scaffold
designed to meet its requirements.

C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
649
Fig. 3. Docked poses for maraviroc, vicriviroc, SCHC, and TAK779. Ligands are shown in orange balls and sticks, possible interacting residues in cyan sticks.
deprotect the amino group and reduce the alkene group, com-
pound 2 was obtained. Trifluoroacetyl protection of the amino
group on the piperidine ring to afford 3 was achieved under typical
acetylation conditions. In order to mono-nitrate the ortho-position
to synthesize 4, very mild nitration conditions at low temperature
(ꢀ60 ꢁC) were applied. The nitro group was then reduced through
palladium/carbon catalyzed hydrogenation to give 5, and com-
pound 5 was coupled to either of the two R3 substituted carboxylic
acids using standard amide coupling conditions to afford 6. Sub-
sequently, the piperidine amine group was deprotected under basic
conditions to yield intermediate 7, and with another amide
coupling reaction and following modification operation the
4-substituted R1 groups were added to obtain the final target
compounds 8 to 31. All of the final compounds were obtained in
reasonable yields and characterized with NMR, IR, MS, and HPLC.
Overall, the designed synthetic route allowed a divergent synthesis
that was capable of introducing a variety of different substitutions
efficiently (Fig. 5).
2.3. Biological screenings
2.3.1. Inhibition of CCR5 agonist RANTES stimulated calcium flux
Inhibition of RANTES induced CCR5 calcium ion mobilization
was first applied to test the compounds’ ability to bind and inhibit
R1
R1
R2
R1
O
O
O
O
NO2
O
N
Br
O
1. Mg, I2, THF
2. NH4Cl, H2O
Ac2O, HNO3
-60 0
Pd/C, H2
(CF3CO)2O
Py, CH2Cl2
MeOH, H2O
C
R1
N
H
N
N
N
O
CF3
O
CF3
R1=CH3,
CH2CH3,
or i-Pr
1
2 (a, b, c)
3 (a, b, c)
4 (a, b, c)
1a, R1=CH3
1b, R1=CH2CH3
1c, R1=i-Pr
R1
R1
H
R1
O
O
O
H
N
R2
R2
NH2
N
O
K2CO3
MeOH, H2O
O
R2COOH
6a, R1=CH3, R2=phenyl
Pd/C, H2
MeOH
6b, R1=CH3, R2=pyrazinyl
6c, R1=CH2CH3, R2=phenyl
6d, R1=CH2CH3, R2=pyrazinyl
6e, R1=i-Pr, R2=phenyl
HOBT, EDCI, TEA
DMF
N
H
R2 = -phenyl, -pyrazinyl
N
N
6f, R1=i-Pr, R2=pyrazinyl
O
CF3
O
CF3
7 (a-f)
5 (a, b, c)
6
Fig. 4. Synthetic route for the key intermediate compound 7.

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C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
O
O
O
Cl
R1
HN
R2
R1
HN
R2
R1
HN
R2
NO2
Pd/C/H2
NO2
NH2
K2CO3, KI, DMF
NH
N
N
O
O
O
7
9, 13, 17, 21, 25, 29
10, 14, 18, 22, 26, 30
i) benzoic acid
p-toluenesulfonic acid
pyridine
Ac2O
OHC
N(CH2CH3)2
ii) NaBH4, ethanol
O
R1
HN
R2
O
NHCOCH3
R1
HN
R2
N(CH2CH3)2
N
O
N
O
8, 12, 16, 20, 24, 28
11, 15, 19, 23, 27, 31
Fig. 5. Syntheses of final compounds 8e31.
CCR5 signaling since it has been shown to correlate well with
radioligand binding and functional assays [31,32]. Briefly, CCR5
expressing MOLT-4 cells [33] were transfected with a chimeric G-
protein, Gqi5 [34], and stimulated with RANTES with and without
the presence of the newly synthesized compounds, the results were
shown in Table 1. First of all, no agonism was observed for any of the
new compounds. Second, they all followed a dose-dependent in-
hibition and acted CCR5 antagonists with moderate inhibitory ac-
tivity in the calcium flux assay. Several trends were seen: R1 group
bulkiness change is not significant enough to influence the CCR5
antagonism; For R2 group, it seemed pyrazinyl substitution in
general was more favorable to the inhibitory effect; For R3 groups,
it seemed overall nitro group would be better suited. In all, several
compounds showed promising CCR5 inhibition below 40
which supported our original molecular design.
mM,
2.3.2. Inhibition of prostate cancer cell proliferation assay
Two prostate cancer cell lines, PC-3 and M12, which express both
RANTES and CCR5, were chosen asmodel prostate cancer cell systems
[35e37] inorder to assesstheanti-proliferativeactivityof theseseries
of compounds. Using a protocol previously described, each com-
pound showed dose-dependent anti-proliferative activity in both cell
lines [38]. The results in Table 2 suggested that several of the com-
poundswere capable ofinhibiting proliferation inbothof theprostate
cancer cell lines, including compound 8,18,19, 26, and 27. For several
other compounds they only showed significant inhibitory effect on
one cancer cell line, e.g. compound 12, 16, 20, and 24.
Table 1
Synthesized derivatives and their respective CCR5 calcium mobilization inhibition.
O
R1
R3
HN
N
O
R2
2.3.3. Basal cytotoxicity studies
To ensure the above results were not due the toxicity of the com-
pounds, a basal cytotoxicity assay was conducted. The NIH-3T3 cells
used for the assay are mouse fibroblasts that have been used exten-
sivelyalongwith neutralred uptake(NRU) to assess compounds’ basal
cytotoxicity levels [39,40]. In all, several of the compounds that
showed high anti-proliferative activity in both M12 and PC-3 were
also cytotoxic in NIH-3T3 cells which indicates their observed anti-
proliferative activity may be related to their basal cytotoxicity from
off-target protein interactions. For example, compound 8 showed an
Compound
R1
R2
R3
CCR5 inhibition
IC50 M)
(m
8
9
eCH3
eCH3
eCH3
eCH3
eCH3
eCH3
eCH3
eCH3
ePhenyl
ePhenyl
ePhenyl
ePhenyl
ePyrazinyl
ePyrazinyl
ePyrazinyl
ePyrazinyl
ePhenyl
ePhenyl
ePhenyl
ePhenyl
ePyrazinyl
ePyrazinyl
ePyrazinyl
ePyrazinyl
ePhenyl
ePhenyl
ePhenyl
ePhenyl
ePyrazinyl
ePyrazinyl
ePyrazinyl
ePyrazinyl
eN(CH2CH3)2
eNO2
eNH2
eNCOCH3
eN(CH2CH3)2
eNO2
90 ꢂ 22
28.7 ꢂ 7.9
77 ꢂ 18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
70.8 ꢂ 8.8
56 ꢂ 11
73 ꢂ 29
eNH2
38.8 ꢂ 4.0
59 ꢂ 34
eNCOCH3
eN(CH2CH3)2
eNO2
eCH2CH3
eCH2CH3
-CH2CH3
64 ꢂ 25
ED50 of 12.5 ꢂ1.6 and 37.2 ꢂ 7.6
m
M in M12 and PC-3 respectively, but
28.0 ꢂ 3.1
63 ꢂ 26
it showed basal cytotoxicity in NIH-3T3 with a TC50 of 28.1 ꢂ 0.6
mM,
eNH2
similar cases for compound 26 and 27. Therefore, these compounds
would not be a good lead due to their similar effects in both cancerous
and noncancerous cells. However, two compounds in the series have
anti-proliferative activity while displaying low cytotoxicity. Com-
-CH2CH3
eNCOCH3
eN(CH2CH3)2
eNO2
115 ꢂ 48
51.8 ꢂ 4.3
61 ꢂ 11
eCH2CH3
eCH2CH3
eCH2CH3
eCH2CH3
eCH(CH3)2
eCH(CH3)2
eCH(CH3)2
eCH(CH3)2
eCH(CH3)2
eCH(CH3)2
eCH(CH3)2
eCH(CH3)2
eNH2
43.5 ꢂ 5.1
47 ꢂ 11
eNCOCH3
eN(CH2CH3)2
eNO2
pound 18 showed an ED50 of 31.9 ꢂ 0.42 and 43.0 ꢂ 2.3
m
M for M12
138 ꢂ 34
45 ꢂ 15
and PC-3 respectively and displayed a TC50 of greater than 80
mM.
eNH2
66.2 ꢂ 1.8
92.2 ꢂ 4.9
61 ꢂ 13
Similar results were seen for 19, but due to solubility issues it was not
further pursued for the following in vivo studies.
eNCOCH3
eN(CH2CH3)2
eNO2
eNH2
eNCOCH3
65.7 ꢂ 1.1
77.8 ꢂ 1.4
88.1 ꢂ 6.6
2.3.4. In vivo tumor growth inhibition studies
To assess the effect of the potential lead compound 18 on
prostate tumor growth in vivo, we injected mice subcutaneously
with M12 prostate cancer cells M12, followed by treatment with
the compound at a dose of 0.3 mg/kg (or saline) once every four
Values shown are mean ꢂ S.E.M. from at least three separate experiments per-
formed in triplicate. The calculated IC50 values for CCR5 calcium inhibition were
calculated using GraphPad Prism.

C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
651
Table 2
modeling studies of the CCR5 binding pocket. In CCR5 calcium
mobilization inhibition assays all of the compounds acted as antag-
onists. By using a combination of anti-proliferation assays and basal
cytotoxicityassays compounds having the most desirable therapeutic
Proliferation inhibition in prostate cancer cell lines.
Compound
M12 anti-proliferation
ED50 M)
PC-3 anti-proliferation
ED50 M)
NIH-3T3
cytotoxicity
TC50 (mM)
(m
(m
potential were determined. With an ED50 of 32
and PC-3 prostate cancer cells and no observable cytotoxicity up to
80 M, and more impressively an in vivo tumor growth inhibition
mM and 43 mM in M12
8
9
12.5 ꢂ 1.6
ND
67.6 ꢂ 9.9
79.9 ꢂ 1.8
84.8 ꢂ 5.7
ND
73.7 ꢂ 8.4
89.2 ꢂ 2.3
15.9 ꢂ 0.7
122 ꢂ 12
31.9 ꢂ 0.42
34.5 ꢂ 1.9
18.7 ꢂ 2.9
ND
37.2 ꢂ 7.6
104 ꢂ 3
28.1 ꢂ 0.6
>100
96 ꢂ 2.2
92.2 ꢂ 0.3
63 ꢂ 10
>100
m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
53.1 ꢂ 7.5
60 ꢂ 14
effects observed, compound 18 was defined as our new lead com-
pound. Further modification of this lead will be pursued based upon
in depth molecular modeling study and more extensive structure
modification on the substitutions of the basic skeleton.
21.6 ꢂ 3.9
ND
72.3 ꢂ 4.8
99.2 ꢂ 2.8
86.6 ꢂ 0.8
151 ꢂ 34
43.0 ꢂ 2.3
34.7 ꢂ 0.7
77 ꢂ 11
>100
95.9 ꢂ 1.5
49.5 ꢂ 1.7
>100
84 ꢂ 11
87.5 ꢂ 5.9
46.9 ꢂ 4.7
>100
4. Experimental sections
4.1. Chemical syntheses
109 ꢂ 4
Melting points were obtained with a Fisher scientific micro
melting point apparatus and were uncorrected. Proton (300 MHz)
and carbon-13 (75 MHz) nuclear magnetic resonance (NMR)
spectra were recorded on a Varian Gemini-300 “Tesla” spectrom-
eter (300 MHz), with tetramethylsilane as the internal standard. All
IR spectra were recorded on a Nicolet Avatar 360 FT-IR Instruments.
TLC analyses were carried out on Analtech Uniplate F254 plates.
Chromatographic purification was carried out on silica gel columns
(160 meshes). Yields were not maximized. For representative
spectra, please refer to the Supplementary information.
52.4 ꢂ 8.0
61.7 ꢂ 4.4
50 ꢂ 19
76 ꢂ 14
58 ꢂ 13
>100
28.7 ꢂ 0.6
83.6 ꢂ 8.4
23.9 ꢂ 3.2
35.6 ꢂ 4.2
67 ꢂ 15
70 ꢂ 16
26.5 ꢂ 3.8
>100
71.2 ꢂ 6.3
25.1 ꢂ 4.7
41 ꢂ 12
27.3 ꢂ 0.87
42.0 ꢂ 6.6
45.3 ꢂ 4.6
56.9 ꢂ 9.4
>100
75 ꢂ 13
35.1 ꢂ 1.3
32.7 ꢂ 2.6
35.0 ꢂ 1.3
57.2 ꢂ 1.9
ND
57.2 ꢂ 1.9
94 ꢂ 15
ND denotes that the compound was not tested due to solubility issues. Values
shown are mean ꢂ S.E.M. from at least three separate experiments performed in
triplicate. The calculated EC50 and TC50 values for CCR5 calcium inhibition were
calculated using GraphPad Prism.
4.1.1. 1-Bromo-4-isopropoxybenzene
The solution of 4-bromophenol (15.0 g, 86.7 mmol) and isopropyl
bromide (16.0 g, 130 mmol) in DMF (50 mL) was added potassium
carbonate (14.4 g, 104 mmol). The reaction mixture was heated to
reflux overnight. After cooling down, the suspension was filtered, the
filtrate was concentrated under vacuum to remove DMF. The residue
was partitioned between water (50 mL) and dichloromethane
(100 mL). The organic layer was washed with brine, dried over so-
dium sulfate, concentrated to give 14 g colorless oil, in 75% yield.
After dried, the crude product was used for next step without further
days for total of sixteen days before data analysis. No apparent
toxicity of the compounds to the animal, e.g., significant weight
loss, was observed during the experiment period. The experiment
was terminated when the control group mice were lost due to their
fully developed tumors. The results (Fig. 6) showed that compound
18 reduced the subcutaneous growth of M12 tumors in athymic
nude mice by over 50% at this dose (P value < 0.05). These results
establish compound 18 as our new lead for future molecular design
and development of a novel anti prostate cancer agent.
purification. 1HNMR (CDCl3, 300 MHz):
d
7.35 (d, J ¼ 7.8 Hz, 2H), 6.76
(d, J ¼ 7.8 Hz, 2H), 4.49 (m, 1H), 1.31 (d, J ¼ 5.7 Hz, 6H).
3. Conclusions
4.1.2. 1-Benzyl-4-(4-methoxyphenyl)-1,2,3,6-tetrahydropyridine
(1a)
In summary, a series of ligands was designed and synthesized as
potentialCCR5antagonists targeting the treatmentofprostate cancer.
As a proof-of-concept, the general skeleton of the molecules was
constructed based on known CCR5 antagonists and molecular
A solution of methoxybromobenzene (3.74 g, 20 mmol) in THF
(20 mL) was added dropwise to a stirred mixture of Magnesium
(442 mg, 18.2 mmol) and iodine (catalytic amount) in THF (30 mL)
over a 30 min period, then heated to reflux for 30 min. After cooled
down,1-benyl-4-piperidinone (3.78g, 20mmol)wasadded dropwise
to the stirred mixture at room temperature over 20 min, and then
refluxed for 30 min. After cooling down to room temperature, sat. aq.
NH4Cl (53 mL) and H2O (25 mL) were added to the mixture, then
allowed to stand for a short time. The mixture was added 6 N HCl
(53 mL), then refluxed for 3 h. After cooling down, the mixture was
concentrated in vacuum to remove THF, the compound was precipi-
tatedout fromwaterlayer. Filtration gave 3.7 gofcompound1ain60%
Compound 18 anti proliferation in vivo
assay results
400
350
300
250
yield. 1HNMR (CDCl3, 300 MHz):
d 12.9 (br, 1H), 7.69 (m, 2H), 7.46 (m,
200
3H), 7.31 (d, J ¼ 8.1 Hz, 2H), 6.88(d, J ¼ 8.1 Hz, 2H), 5.88(s,1H), 4.25(m,
2H), 3.98 (m,1H), 3.80 (s, 3H), 3.58 (m, 2H), 3.19 (m, 2H), 2.65 (m,1H).
Under a similar procedure, the following two compounds (R1 is
ethoxyl, or isoproxyl group) were prepared in 55e70% yield.
Compd 18
150
100
50
Control
4.1.3. 1-Benzyl-4-(4-ethoxyphenyl)-1,2,3,6-tetrahydropyridine (1b)
0
1HNMR (CDCl3, 300 MHz):
d 12.45 (br, 1H), 7.72 (m, 2H), 7.46 (m.
0
4
8
12
16
3H), 7.30 (d, J ¼ 8.4 Hz, 2H), 6.86 (d, J ¼ 8.4 Hz, 2H), 5.88 (s, 1H), 4.28
(m, 2H), 4.03 (q, J ¼ 6.9 Hz, 2H), 3.94 (m, 1H), 3.57 (m, 2H), 3.20 (m,
2H), 2.68 (m, 1H), 1.42 (t, J ¼ 6.9 Hz, 3H).
Tumor Growth During Injection Period over 16 days
Fig. 6. In vivo studies of prostate tumor growth inhibition of the lead compound 18.

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C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
4.1.4. 1-Benzyl-4-(4-isopropoxyphenyl)-1,2,3,6-tetrahydropyrine
extracted with CH2Cl2 (50 mL ꢃ 3), washed with brine, dried, and
concentrated to yield 1.7 g of compound 4a (in 98% yield). 1HNMR
(1c)
1HNMR (CDCl3, 300 MHz):
d
12.49 (br, 1H), 7.71 (m, 2H), 7.46 (m.
(CDCl3, 300 MHz):
d
7.38 (d, J ¼ 9.0 Hz, 1H), 7.38 (d, J ¼ 9.0 Hz, 1H),
3H), 7.29 (d, J ¼ 12.0 Hz, 2H), 6.85 (d, J ¼ 12.0 Hz, 2H), 5.87 (s, 1H),
4.56 (m, 1H), 4.28 (m, 2H), 3.96 (d, J ¼ 13.2 Hz, 1H), 3.62 (m, 1H),
3.50 (d, J ¼ 16.2 Hz, 1H), 3.20 (m, 2H), 2.67 (m, 1H), 1.29 (d,
J ¼ 6.3 Hz, 6H).
7.03 (d, J ¼ 9.0 Hz, 1H), 4.72 (d, J ¼ 14.1 Hz, 1H), 4.15 (d, J ¼ 14.1 Hz,
1H), 3.95 (s, 3H), 3.25 (m, 1H), 2.86 (m, 2H), 2.00 (d, J ¼ 12.9 Hz, 2H),
1.69 (m, 2H).
Under a similar procedure, the following two compounds (R1 is
ethoxyl, or isoproxyl group) were prepared in comparable yields.
4.1.5. 4-(4-Methoxyphenyl)piperidine (2a)
A solution of compound 1a (2.3 g, 8.2 mmol) in methanol (100 mL)
was hydrogenated in the presence of 5% Pd/C (230 mg) under a H2
atmosphere (57 psi) at room temperature for 24 h. The mixture was
filtered, and the filtrate was concentrated to give compound 2a (1.8 g,
4.1.12. 2,2,2-Trifluoro-1-(4-(4-ethoxy-3-nitrophenyl)piperidin-1-yl)
ethanone (4b)
1HNMR (CDCl3, 300 MHz):
d
7.66 (d, J ¼ 2.4 Hz, 1H), 7.34 (dd,
J ¼ 2.4, 8.4 Hz, 1H), 7.03 (d, J ¼ 8.4 Hz, 1H), 4.70 (m. 2H), 4.17 (q,
J ¼ 6.6 Hz, 3H), 3.24 (m, 1H), 2.83 (m, 2H), 2.02 (d, J ¼ 13.8 Hz, 2H),
1.67 (m, 2H), 1.47 (t, J ¼ 6.6 Hz, 3H).
96% yield).1HNMR (CDCl3, 300 MHz):
d 9.65 (br,1H), 9.43 (br,1H), 7.16
(d, J ¼ 8.1 Hz, 2H), 6.86 (d, J ¼ 8.1 Hz, 2H), 3.79 (s, 3H), 3.62 (m, 2H),
3.01 (m, 2H), 2.73 (m, 1H), 2.21 (m, 2H), 2.02 (m, 2H).
Under a similar procedure, the following two compounds (R1 is
ethoxyl, or isoproxyl group) were prepared in 95e100% yield.
4.1.13. 2,2,2-Trifluoro-1-(4-(4-isopropoxy-3-nitrophenyl)piperidin-
1-yl)ethanone (4c)
1HNMR (CDCl3, 300 MHz):
d
7.60 (d, J ¼ 2.1 Hz, 1H), 7.30 (dd,
4.1.6. 4-(4-Ethoxyphenyl)piperidine (2b)
J ¼ 2.4 Hz, 8.4 Hz, 1H), 7.03 (d, J ¼ 8.4 Hz, 1H), 4.70 (m, 1H), 4.64 (q,
J ¼ 6.0 Hz, 1H), 4.15 (m, 1H), 3.24 (m, 1H), 2.83 (m, 2H), 1.99 (m, 2H),
1.70 (m, 2H), 1.38 (d, J ¼ 6.3 Hz, 6H).
1HNMR (CDCl3, 300 MHz):
6.84 (d, J ¼ 7.5 Hz, 2H), 4.01 (q, J ¼ 6.9 Hz, 2H), 3.63 (m, 2H), 3.05 (m,
2H), 2.72 (m, 1H), 2.16 (m, 1H), 2.04 (m, 3H), 1.40 (t, J ¼ 6.9 Hz, 3H).
d
9.65 (br, 1H), 7.14 (d, J ¼ 7.5 Hz, 2H),
4.1.14. 1-(4-(3-Amino-4-methoxyphenyl)piperidin-1-yl)-2,2,2-
trifluoroethanone (5a)
4.1.7. 4-(4-Isopropoxyphenyl)piperidine (2c)
1HNMR (CDCl3, 300 MHz):
d
9.27 (br, 1H), 9.07 (br, 1H), 7.13 (d,
A solution of compound 4a (1.0 g, 3 mmol) in methanol (50 mL)
was hydrogenated in the presence of 5% Pd/C (100 mg) under a H2
atmosphere (57 psi) at room temperature for 24 h. The mixture was
filtered, and the filtrate was concentrated to give compound 5a as
J ¼ 8.4 Hz, 2H), 6.84 (d, J ¼ 8.4 Hz, 2H), 4.52 (m, 1H), 3.66 (d,
J ¼ 11.7 Hz, 2H), 3.02 (m, 2H), 2.72 (m, 1H), 2.23 (m, 2H), 2.04 (d,
J ¼ 13.5 Hz, 2H), 1.32 (m, 6H).
gray foam (0.9 g, 99% yield). 1HNMR (CDCl3, 300 MHz):
d 6.72 (d,
4.1.8. 2,2,2-Trifluoro-1-(4-(4-methoxyphenyl)piperidin-1-yl)
ethanone (3a)
J ¼ 6.9 Hz, 1H), 6.55 (m, 2H), 4.66 (d, J ¼ 12.0 Hz, 1H), 4.10 (d,
J ¼ 13.5 Hz, 1H), 3.83 (s, 3H), 3.79 (m, 2H), 3.20 (t, J ¼ 12.9 Hz, 1H),
2.82 (t, J ¼ 12.6 Hz, 1H), 2.68 (m, 1H), 1.93 (t, J ¼ 13.5 Hz, 2H), 1.64
(m, 2H).
Under a similar procedure, the following two compounds (R1 is
ethoxyl, or isoproxyl group) were prepared in comparable yields.
To a solution of compound 2a (1.8 g, 8 mmol) in dichloro-
methane (100 mL) and pyridine (1.4 g, 17.6 mmol), trifluoroacetic
anhydride (1.85 g, 8.8 mmol) was added dropwise. After stirred at
room temperature overnight, the resulting suspension was washed
with 1 N HCl (25 mL ꢃ 2) and brine, dried with Na2SO4. The CH2Cl2
solution was filtered, the filtrate was concentrated to give com-
4.1.15. 1-(4-(3-Amino-4-ethoxyphenyl)piperidin-1-yl)-2,2,2-
pound 3a (2.07 g, 90% yield). 1HNMR (CDCl3, 300 MHz):
d
7.11 (d,
trifluoroethanone (5b)
J ¼ 8.7 Hz, 2H), 6.86 (d, J ¼ 8.7 Hz, 2H), 4.68 (d, J ¼ 12.0 Hz, 1H), 4.12
(d, J ¼ 12.0 Hz, 1H), 3.80 (s, 3H), 3.23 (m, 1H), 2.80 (m, 2H), 1.95 (d,
J ¼ 12.3 Hz, 2H), 1.69 (m, 2H).
1HNMR (CDCl3, 300 MHz):
d 10.4 (br, 1H), 7.52 (s, 1H), 7.16 (d,
J ¼ 10.9 Hz, 1H), 6.92 (q, J ¼ 8.1 Hz, 1H), 4.68 (d, J ¼ 12.0 Hz, 1H), 4.13
(q, J ¼ 7.2 Hz, 1H), 3.21 (d, J ¼ 13.5 Hz, 1H), 2.82 (m, 2H), 1.95 (d,
J ¼ 12.9 Hz, 2H), 1.68 (m, 2H), 1.46 (t, J ¼ 7.2 Hz, 3H).
Under a similar procedure, the following two compounds (R1 is
ethoxyl, or isoproxyl group) were prepared in comparable yield.
4.1.16. 1-(4-(3-Amino-4-isopropoxyphenyl)piperidin-1-yl)-2,2,2-
4.1.9. 2,2,2-Trifluoro-1-(4-(4-ethoxyphenyl)piperidin-1-yl)
ethanone (3b)
trifluoroethanone (5c)
1HNMR (CDCl3, 300 MHz):
d
7.55 (d, J ¼ 2.1 Hz, 1H), 7.13 (dd,
1HNMR (CDCl3, 300 MHz):
d
7.09 (d, J ¼ 8.7 Hz, 2H), 6.84 (d,
J ¼ 2.1 Hz, 8.7 Hz, 1H), 6.92 (d, J ¼ 8.4 Hz, 1H), 4.64 (m, 2H), 4.12 (d,
J ¼ 12.6 Hz, 1H), 3.20 (m, 1H), 2.80 (m, 2H), 1.95 (d, J ¼ 13.2 Hz, 2H),
1.65 (m, 2H), 1.39 (d, J ¼ 5.7 Hz, 6H).
J ¼ 8.7 Hz, 2H), 4.68 (d, J ¼ 13.2 Hz, 1H), 4.11 (d, J ¼ 14.1 Hz, 1H), 4.01
(q, J ¼ 7.2 Hz, 2H), 3.22 (m, 1H), 2.77 (m, 2H), 1.95 (d, J ¼ 14.1 Hz,
2H), 1.70 (m, 2H), 1.40 (t, J ¼ 7.2 Hz, 3H).
4.1.17. N-(2-Methoxy-5-(1-(2,2,2-trifluoroacetyl)piperidin-4-yl)
phenyl)benzamide (6a)
On an ice-water bath, a solution of benzoic acid (650 mg,
5.5 mmol) (or 2-pyrazinecarboxylic acid) in DMF (2 mL), was added
4.1.10. 2,2,2-Trifluoro-1-(4-(4-isopropoxyphenyl)piperidin-1-yl)-
ethanone (3c)
1HNMR (CDCl3, 300 MHz):
d
7.09 (d, J ¼ 8.7 Hz, 2H), 6.84 (d,
ꢀ
J ¼ 8.7 Hz, 2H), 4.67 (m, 1H), 4.53 (m, 1H), 4.13 (d, J ¼ 13.8 Hz, 1H),
3.23 (m, 1H), 2.77 (m, 2H), 1.96 (d, J ¼ 12.9 Hz, 2H), 1.67 (qd,
J ¼ 4.2 Hz, 13.2 Hz, 2H), 1.33 (m, 6H).
EDC (750 mg, 4 mmol), HOBt (535 mg, 4 mmol), 4 A molecular
sieves, and TEA (800 mg, 8 mmol) under N2 protection. After
20 min, a solution of compound 5a (800 mg, 2.65 mmol) in DMF
(2 mL) was added dropwise. After stirring at r.t. overnight, the
resulting mixture was filtered through celite. The filtrate was
concentrated in vacuum to remove DMF. The residue was added
brine, taken up with CH2Cl2 (50 mL ꢃ 3). The organic layer was
washed with brine, dried, and concentrated. The residue was pu-
rified with chromatography (Hexane/EtOAc: 5/1) to give 800 mg
4.1.11. 2,2,2-Trifluoro-1-(4-(4-methoxy-3-nitrophenyl)piperidin-1-
yl)ethanone(4a)
To a solution of compound 3a (1.5 g, 5.22 mmol) in Ac2O (40 mL),
on a dry ice-acetone bath, nitric acid (4.75 g, 69%, 52 mmol) was
added dropwise. The reaction mixture was stirred at ꢀ30 ꢁC for 6 h.
Then the mixture was poured into ice (50 mL). The water phase was
compound 6a in 74% yield. 1HNMR (CDCl3, 300 MHz):
d 8.59 (s, 1H),

C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
653
8.48 (s, 1H), 7.90 (d, J ¼ 7.2 Hz, 2H), 7.52 (m, 2H), 6.89 (d, J ¼ 8.1 Hz,
1H), 6.86 (d, J ¼ 8.1 Hz, 1H), 4.68 (d, J ¼ 13.2 Hz, 1H), 4.13 (d,
J ¼ 13.8 Hz, 1H), 3.91 (s, 3H), 3.23 (t, J ¼ 12.9 Hz, 1H), 2.84 (m, 2H),
2.01 (d, J ¼ 13.2 Hz, 2H), 1.73 (m, 2H).
J ¼ 2.1 Hz, 1H), 6.98 (dd, J ¼ 2.1, 8.4 Hz, 1H), 6.89 (d, J ¼ 8.4 Hz, 1H),
3.95 (s, 3H), 3.18 (m, 2H), 2.70 (m, 3H), 1.87 (d, J ¼ 12.6 Hz, 2H), 1.65
(m, 2H).
Under a similar procedure, the following compounds (R1 is methyl,
ethoxyl, or isoproxyl group; R2 is phenyl, or pyrazinyl group) were
prepared in 74e85% yield.
4.1.25. N-(2-Ethoxy-5-(piperidin-4-yl)phenyl)benzamide (7c)
1HNMR (CDCl3, 300 MHz):
d
8.64 (br, 1H), 8.48 (d, J ¼ 2.1 Hz, 1H),
7.89 (d, J ¼ 7.8 Hz, 2H), 7.53 (m, 3H), 6.99 (d, J ¼ 8.7 Hz, 1H), 6.85 (d,
J ¼ 8.7 Hz, 1H), 4.13 (q, J ¼ 7.2 Hz, 2H), 3.18 (d, J ¼ 12.3 Hz, 2H), 2.70
(m, 3H), 1.86 (d, J ¼ 12.0 Hz, 2H), 1.66 (td, J ¼ 3.9 Hz, 12.0 Hz, 2H),
1.48 (t, J ¼ 7.2 Hz, 2H).
4.1.18. N-(2-Methoxy-5-(1-(2,2,2-trifluoroacetyl)piperidin-4-yl)
phenyl)pyrazine-2-carboxamide (6b)
1HNMR (CDCl3, 300 MHz):
d 10.29 (br, 1H), 9.50 (s. 1H), 8.81 (d,
J ¼ 2.4 Hz, 1H), 8.63 (dd, J ¼ 1.5, 2.4 Hz, 1H), 8.52 (d, J ¼ 2.4 Hz, 1H),
6.93 (m, 2H), 4.70 (m, 1H), 4.14 (d, J ¼ 12.6 Hz, 1H), 3.96 (s, 3H), 3.26
(m, 1H), 2.85 (m, 2H), 2.02 (d, J ¼ 13.5 Hz, 2H), 1.73 (m, 2H).
4.1.26. N-(2-Ethoxy-5-(piperidin-4-yl)phenyl)pyrazine-2-
carboxamide (7d)
1HNMR (CDCl3, 300 MHz):
d 10.34 (s, 1H), 9.50 (s, 1H), 8.78 (m,
1H), 8.61 (dd, J ¼ 1.5 Hz, 2.4 Hz,1H), 8.51 (d, J ¼ 2.1 Hz,1H), 6.95 (dd,
J ¼ 2.1 Hz, 8.1 Hz, 1H), 6.88 (d, J ¼ 8.1 Hz, 1H), 4.16 (q, J ¼ 7.2 Hz, 2H),
3.19 (d, J ¼ 12.3 Hz, 2H), 2.75 (td, J ¼ 2.1 Hz, 12.0 Hz, 2H), 2.65 (m,
1H), 1.87 (d, J ¼ 14.6 Hz, 2H), 1.65 (m, 2H), 1.51 (t, J ¼ 7.2 Hz, 3H).
4.1.19. N-(2-Ethoxy-5-(1-(2,2,2-trifluoroacetyl)piperidin-4-yl)
phenyl)benzamide (6c)
1HNMR (CDCl3, 300 MHz):
d 8.66 (br, 1H), 8.49 (s, 1H), 7.89 (d,
J ¼ 7.2 Hz, 2H), 7.54 (m, 3H), 6.87 (m, 2H), 4.69 (d, J ¼ 13.2 Hz, 1H),
4.13 (m, 3H), 3.23 (t, J ¼ 12.9 Hz, 2H), 2.85 (m, 2H), 2.00 (d,
J ¼ 13.5 Hz, 2H), 1.73 (m, 2H), 1.49 (t, J ¼ 6.6 Hz, 3H).
4.1.27. N-(2-Isopropoxy-5-(piperidin-4-yl)phenyl)benzamide (7e)
1HNMR (CDCl3, 300 MHz):
d 8.67 (s, 1H), 8.49 (s, 1H), 7.90 (m,
2H), 7.53 (m, 3H), 6.91 (m, 2H), 4.61 (q, J ¼ 6.3 Hz, 1H), 3.21 (d,
J ¼ 12.9 Hz, 2H), 2.76 (t, J ¼ 12.3 Hz, 2H), 2.65 (m, 1H), 1.75 (m, 2H),
1.66 (m, 2H), 1.39 (d, J ¼ 6.3 Hz, 6H).
4.1.20. N-(2-Ethoxy-5-(1-(2,2,2-trifluoroacetyl)piperidin-4-yl)
phenyl)pyrazine-2-carboxamide (6d)
1HNMR (CDCl3, 300 MHz):
d
10.36 (br, 1H), 9.50 (d, J ¼ 1.5 Hz,
1H), 8.79 (d, J ¼ 2.4 Hz,1H), 8.62 (m,1H), 8.51 (d, J ¼ 1.5 Hz,1H), 6.91
(m, 2H), 4.69 (m, 1H), 4.18 (q, J ¼ 6.9 Hz, 2H), 4.13 (m, 1H), 3.25 (td,
J ¼ 3.3 Hz, 14.4 Hz, 1H), 2.84 (m, 2H), 2.02 (d, J ¼ 13.2 Hz, 2H), 1.72
(m, 2H), 1.54 (t, J ¼ 6.9 Hz, 3H).
4.1.28. N-(2-Isopropoxy-5-(piperidin-4-yl)phenyl)pyrazine-2-
carboxamide (7f)
1HNMR (CDCl3, 300 MHz):
d
10.39 (br, 1H), 9.50 (d, J ¼ 1.5 Hz,
1H), 8.79 (d, J ¼ 2.1 Hz, 1H), 8.63 (m, 2H), 6.96 (dd, J ¼ 2.1, 8.4 Hz,
1H), 6.91 (d, J ¼ 8.4 Hz, 1H), 4.59 (q, J ¼ 6.3 Hz, 1H), 3.22 (d,
J ¼ 11.7 Hz, 2H), 2.76 (t, J ¼ 12.0 Hz, 2H), 2.66 (m, 1H), 1.90 (m, 2H),
1.70 (m, 2H), 1.42 (dd, J ¼ 1.8, 6.3 Hz, 6H).
4.1.21. N-(2-Isopropoxy-5-(1-(2,2,2-trifluoroacetyl)piperidin-4-yl)
phenyl)benzamide (6e)
1HNMR (CDCl3, 300 MHz):
d 8.69 (br, 1H), 8.50 (s, 1H), 7.88 (m,
2H), 7.54 (m, 3H), 6.88 (s, 2H), 4.72 (d, J ¼ 12.3 Hz, 1H), 4.64 (m, 1H),
4.14 (d, J ¼ 13.5 Hz,1H), 3.23 (m,1H), 2.83 (m, 2H), 2.01 (m, 2H),1.75
(m, 2H), 1.40 (d, J ¼ 6.0 Hz, 6H).
4.2. Preparation of the final compounds
4.2.1. Procedure A
4.2.1.1. N-(5-(1-(4-(Diethylamino)benzyl)piperidin-4-yl)-2-
methoxyphenyl)benzamide (8). A mixture of compound 7a (180 mg,
0.58 mmol), benzoic acid (206 mg, 1.16 mmol), 4-(diethylamino)-
benzaldehyde (142 mg, 1.16 mmol), and a trace of p-toluenesulfonic
acid in toluene (40 mL), ethanol (40 mL) was refluxed for 24 h,
using a DeaneStark trap for removal of water. The solution was
concentrate to 15 mL at 1 atm. Anhydrous ethanol (40 mL), NaBH4
4.1.22. N-(2-Isopropoxy-5-(1-(2,2,2-trifluoroacetyl)piperidin-4-yl)
phenyl)pyrazine-2-carboxamide (6f)
1HNMR (DMSO-d6, 300 MHz):
d
10.41 (br, 1H), 9.50 (d, J ¼ 1.2 Hz,
1H), 8.80 (dd, J ¼ 1.2, 2.4 Hz, 1H), 8.63 (dd, J ¼ 1.2, 2.4 Hz, 1H), 8.52
(s, 1H), 6.91 (s, 2H), 4.70 (d, J ¼ 13.2 Hz, 1H), 4.60 (q, J ¼ 6.0 Hz, 1H),
4.14 (d, J ¼ 13.2 Hz, 1H), 3.25 (t, J ¼ 11.4 Hz, 1H), 2.84 (m, 2H), 2.02
(d, J ¼ 13.8 Hz, 2H), 1.77 (m, 2H), 1.43 (dd, J ¼ 1.2, 6.9 Hz, 6H).
ꢀ
(66 mg, 1.74 mmol) and 4 A molecular sieves was added, and the
resulting solution was stirred under N2 for 8 h. The mixture was
diluted with MeOH (40 mL) and filtered. The residue obtained after
removal of solvent was partitioned between CH2Cl2 and NH4OH.
The combined CH2Cl2 extracts was washed with brine, dried, and
concentrated. The residue was purified with chromatography
(CH2Cl2/MeOH: 50/1) to give 80 mg product 8 in 37% yield. M.p.:
4.1.23. N-(2-Methoxy-5-(piperidin-4-yl)phenyl)benzamide(7a)
To the solution of compound 6a (0.70 g, 1.72 mmol) in CH3OH
(40 mL) and H2O (2.4 mL) was added potassium carbonate (1.23 g,
8.94 mmol). The reaction mixture was heated to reflux for 2 h. After
the solvent was evaporated, water was added to the residue. The
water layer was basified with NH4OH (20 mL), then extracted with
CHCl3 (40 mL ꢃ 4). The organic phase was washed with brine, dried,
and concentrated to give the product (510 mg oil, in 96% yield).
177e179 ꢁC. IR (KBr, cmꢀ1
1HNMR (CDCl3, 300 MHz):
)
nmax: 3447, 2932, 1670, 1536, 1025, 699.
8.54 (s, 1H), 8.45 (s, 1H), 7.88 (d,
d
J ¼ 6.9 Hz, 2H), 7.50 (m, 3H), 7.18 (d, J ¼ 8.7 Hz, 2H), 6.95 (d,
J ¼ 8.7 Hz, 1H), 6.85 (d, J ¼ 8.7 Hz, 1H), 6.64 (d, J ¼ 8.7 Hz, 2H), 3.90
(s, 3H), 3.48 (s, 2H), 3.34 (q, J ¼ 6.8 Hz, 4H), 3.05 (d, J ¼ 11.4 Hz, 2H),
2.51 (m, 1H), 2.05 (m, 2H), 1.83 (m, 4H), 1.16 (t, J ¼ 6.9 Hz, 6H).
1HNMR (CDCl3, 300 MHz):
d
8.56 (s, 1H), 8.47 (d, J ¼ 2.1 Hz,1H), 7.90
(m, 2H), 7.53 (m, 2H), 6.95 (dd, J ¼ 2.1, 8.4 Hz,1H), 6.86 (d, J ¼ 8.4 Hz,
1H), 3.92 (s, 3H), 3.21 (d, J ¼ 11,7 Hz, 2H), 2.76 (t, J ¼ 12,0 Hz, 2H),
2.66 (m, 1H), 1.88 (d, J ¼ 15.6 Hz, 2H), 1.68 (m, 2H).
13CNMR (100 MHz, CD3OD)
d: 168.27, 150.93, 137.18, 136.02, 134.51,
Under a similar procedure, the following compounds (R1 is methyl,
ethoxyl, or isoproxyl group; R2 is phenyl, or pyrazinyl group) were
prepared in comparable yields.
134.51, 133.13, 133.13, 129.87, 129.86, 129.85, 128.40, 128.39, 128.38,
128.35, 124.63, 121.94, 112.26, 112.25, 40.35, 33.33, 31.87, 31.86,
11.82. MS (ESI) m/z: 472.3 (M þ H)þ.
Procedure A was applied to prepare the following compounds,
12, 16, 20, 24, 28.
4.1.24. N-(2-Methoxy-5-(piperidin-4-yl)phenyl)pyrazine-2-
carboxamide (7b)
1HNMR (CDCl3, 300 MHz):
1H), 8.79 (d, J ¼ 2.1 Hz, 1H), 8.62 (dd, J ¼ 1.2, 2.1 Hz, 1H), 8.52 (d,
d
10.27 (br, 1H), 9.50 (d, J ¼ 1.2 Hz,
4.2.1.2. N-(5-(1-(4-(Diethylamino)benzyl)piperidin-4-yl)-2-
methoxyphenyl)pyrazine-2-carboxamide (12). M.p.: 200e202 ꢁC. IR

654
C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
(KBr, cmꢀ1
)
nmax: 3354, 2945, 1670, 1545, 1202, 691. 1HNMR
1.28 mmol) in DMF (12 mL), potassium carbonate (265 mg,
(300 MHz, CD3OD):
d
8.90 (br, 1H), 8.42 (m, 3H), 7.92 (d, J ¼ 6.9 Hz,
1.92 mmol), a trace amount of potassium iodine, 4-nitrobenzyl
chloride (264 mg, 1.54 mmol) was added. Then the mixture was
stirred overnight at r.t. After removal of DMF in vacuum, the residue
was purified with chromatography (CH2Cl2/MeOH: 100/1) to afford
2H), 7.83 (d, J ¼ 6.9 Hz, 2H), 7.05 (m, 2H), 4.51 (s, 3H), 3.34 (m, 2H),
2.93 (m, 2H), 2.14 (m, 4H), 1.90 (m, 6H). 13CNMR (100 MHz, DMSO)
d: 160.29, 148.17, 148.16, 147.29, 144.00, 143.55, 143.40, 136.58,
133.03, 133.00, 133.00, 126.39, 126.38, 123.70, 122.52, 117.69, 111.18,
58.20, 56.40, 56.17, 56.15, 51.53, 51.53, 38.40, 29.77, 29.76, 10.10,
10.09. MS (ESI) m/z: 446.3 (M þ H)þ.
550 mg product 9 in 96% yield. M.p.: 218e220 ꢁC. IR (KBr, cmꢀ1
)
nmax: 3400, 2900, 1665, 1534, 1348, 1026, 701. 1HNMR (CDCl3,
300 MHz):
d
8.57 (br, 1H), 8.50 (d, J ¼ 2.1 Hz, 1H), 8.19 (d, J ¼ 9.0 Hz,
2H), 7.90 (d, J ¼ 6.9 Hz, 2H), 7.53 (m, 5H), 6.95 (dd, J ¼ 2.1, 8.4 Hz,
1H), 6.86 (d, J ¼ 8.4 Hz, 1H), 3.91 (s, 3H), 3.62 (s, 2H), 2.96 (d,
J ¼ 13.2 Hz, 2H), 2.54 (m, 1H), 2.13 (m, 3H), 1.85 (m, 3H). 13CNMR
4.2.1.3. N-(5-(1-(4-(Diethylamino)benzyl)piperidin-4-yl)-2-
ethoxyphenyl)benzamide (16). M.p.: 182e184 ꢁC. IR (KBr, cmꢀ1
)
nmax: 3454, 2976, 1672, 1536, 1266, 697. 1HNMR (CDCl3, 300 MHz):
(75 MHz, DMSO-d6) d: 164.90,150.06,148.09,137.23,136.02, 134.45,
d
8.62 (s,1H), 8.48 (s,1H), 7.89 (d, J ¼ 6.9 Hz, 2H), 7.52 (m, 3H), 7.17 (d,
132.91, 132.43, 131.60, 128.47, 128.46, 127.38, 127.36, 126.81, 123.61,
123.60, 123.40, 122.38, 111.49, 57.72, 55.85, 55.84, 51.79, 38.10,
29.74, 29.73. MS (ESI) m/z: 446.3 (M þ H)þ.
J ¼ 8.1 Hz, 2H), 6.92 (d, J ¼ 8.1 Hz, 1H), 6.82 (d, J ¼ 8.1 Hz,1H), 6.64 (d,
J ¼ 8.1 Hz, 2H), 4.11 (q, J ¼ 7.2 Hz, 2H), 3.44 (s, 2H), 3.30 (q, J ¼ 6.9 Hz,
4H), 3.02 (d, J ¼ 11.1 Hz, 2H), 2.49 (m, 1H), 2.03 (m, 2H), 1.81 (m, 4H),
1.46 (t, J ¼ 6.9 Hz, 3H), 1.15 (t, J ¼ 6.9 Hz, 6H). 13CNMR (100 MHz,
Procedure B was applied to prepare compounds 13, 17, 21, 25, 29
with similar yields.
CDCl3) d: 165.20,146.23,135.99,135.22,134.50,133.51,131.84,129.00,
128.89, 128.87, 128.85, 127.75, 127.10, 126.94, 126.93, 126.90, 121.05,
119.17, 114.42, 64.51, 53.50, 53.48, 50.00, 46.10, 46.09, 40.05, 34.90,
30.27, 30.27, 14.91, 14.91. MS (ESI) m/z: 486.4 (M þ H)þ.
4.2.2.2. N-(5-(1-(4-Nitrobenzyl)piperidin-4-yl)-2-methoxyphenꢀy1l)
nmax: 3355, 2987, 1680, 1351, 1020, 696. 1HNMR (CDCl3, 300 MHz):
pyrazine-2-carboxamide (13). M.p.: 238e240 ꢁC. IR (KBr, cm
)
d
10.28 (br, 1H), 9.50 (d, J ¼ 1.2 Hz, 1H), 8.80 (d, J ¼ 2.1 Hz, 1H), 8.62
4.2.1.4. N-(5-(1-(4-(Diethylamino)benzyl)piperidin-4-yl)-2-
ethoxyphenyl)pyrazine-2-carboxamide (20). M.p.: 192e194 ꢁC. IR
(dd, J ¼ 1.5, 2.1 Hz, 1H), 8.54 (d, J ¼ 1.8 Hz, 1H), 8.19 (dd, J ¼ 2.1,
12.0 Hz, 2H), 7.55 (d, J ¼ 12.0 Hz, 2H), 6.98 (dd, J ¼ 2.4, 8.4 Hz, 1H),
6.88 (d, J ¼ 8.4 Hz, 1H), 3.95 (s, 3H), 3.62 (s, 2H), 2.96 (m, 2H), 2.55
(KBr, cmꢀ1
300 MHz):
)
nmax: 3430, 2950, 1687, 1535, 1018, 701. 1HNMR (CDCl3,
d
10.33 (s, 1H), 9.49 (d, J ¼ 1.2 Hz, 1H), 8.77 (d, J ¼ 2.4 Hz,
(m, 1H), 2.15 (m, 2H), 1.85 (m, 4H). 13CNMR (75 MHz, DMSO-d6)
d:
1H), 8.60 (dd, J ¼ 1.5, 2.4 Hz, 1H), 8.50 (d, J ¼ 2.1 Hz, 1H), 7.17 (d,
J ¼ 8.4 Hz, 2H), 6.99 (dd, J ¼ 2.1 Hz, 8.4 Hz, 1H), 6.85 (d, J ¼ 8.4 Hz,
1H), 6.64 (d, J ¼ 8.4 Hz, 2H), 4.14 (q, J ¼ 7.2 Hz, 2H), 3.45 (s, 2H), 3.34
(q, J ¼ 7.2 Hz, 4H), 3.04 (d, J ¼ 8.1 Hz, 2H), 2.52 (m, 1H), 2.04 (m, 2H),
1.83 (m, 4H), 1.48 (t, J ¼ 6.9 Hz, 3H), 1.16 (t, J ¼ 6.9 Hz, 6H). 13CNMR
163.30, 148.18, 147.29, 147.22, 143.88, 143.54, 143.39, 137.09, 136.44,
132.84, 126.44, 126.35, 123.67, 133.63, 117.49, 117.28, 111.12, 57.88,
56.12, 56.11, 51.89, 51.87, 38.27, 29.88, 29.87. MS (ESI) m/z: 448.2
(M þ H)þ.
(100 MHz, CD3OD)
d: 169.00, 149.65, 148.00, 144.91, 144.00, 137.42,
4.2.2.3. N-(5-(1-(4-Nitrobenzyl)piperidin-4-yl)-2-ethoxyphenyl)ben-
136.10, 135.05, 135.03, 128.26, 124.53, 124.51, 124.50, 119.41, 119.40,
113.09, 113.00, 60.80, 56.82, 56.80, 54.61, 54.38, 54.36, 40.44, 31.92,
31.90, 30.00, 10.84, 10.83. MS (ESI) m/z: 488.3 (M þ H)þ.
zamide (17). M.p.: 212e215 ꢁC. IR (KBr, cmꢀ1
)
nmax: 2950, 1676,
1523, 1351, 697. 1HNMR (CDCl3, 300 MHz):
d 8.64 (s, 1H), 8.51 (d,
J ¼ 2.1 Hz, 1H), 8.19 (d, J ¼ 7.8 Hz, 2H), 7.89 (m, 2H), 7.53 (m, 5H),
6.94 (dd, J ¼ 2.1 Hz, 8.4 Hz, 1H), 6.85 (d, J ¼ 8.4 Hz, 1H), 4.14 (q,
J ¼ 6.9 Hz, 2H), 3.62 (s, 2H), 2.95 (d, J ¼ 10.8 Hz, 2H), 2.55 (m, 1H),
2.15 (m, 2H), 1.85 (m, 4H), 1.48 (t, J ¼ 6.9 Hz, 3H). 13CNMR (100 MHz,
4.2.1.5. N-((5-(1-(4-(Diethylamino)benzyl)piperidin-4-yl)-2-
isopropoxy)phenyl)benzamide (24). M.p.: 168e170 ꢁC. IR (KBr, cmꢀ1
)
nmax: 3430, 2977, 1670, 1533, 1111, 702. 1HNMR (CDCl3, 300 MHz):
CDCl3) d: 165.23, 149.08, 146.55, 135.52, 135.47, 135.23, 132.87,
d
8.65 (s,1H), 8.47 (s,1H), 7.88 (m, 2H), 7.51 (m, 3H), 7.17 (d, J ¼ 8.1 Hz,
131.88, 128.93, 128.92, 127.97, 126.95, 126.94, 124.29, 124.25, 120.99,
119.14, 114.37, 111.57, 64.63, 60.07, 53.56, 53.55, 40.03, 30.23, 30.22,
14.90. MS (ESI) m/z: 460.3 (M þ H)þ.
2H), 6.89 (d, J ¼ 8.4 Hz,1H), 6.85 (d, J ¼ 8.4 Hz,1H), 6.64 (d, J ¼ 8.1 Hz,
2H), 4.58 (m, 1H), 3.44 (s, 2H), 3.36 (q, J ¼ 7.2 Hz, 4H), 3.03 (d,
J ¼ 10.8 Hz, 2H), 2.51 (m, 1H), 2.04 (m, 2H), 1.82 (m, 4H), 1.39 (d,
J ¼ 5.7 Hz, 6H), 1.16 (t, J ¼ 6.9 Hz, 6H). 13CNMR (100 MHz, CDCl3)
d:
4.2.2.4. N-(5-(1-(4-Nitrobenzyl)piperidin-4-yl)-2-ethoxyphenyl)pyr-
165.10, 145.43, 139.20, 135.89, 134.14, 134.13, 131.85, 131.84, 128.96,
128.90, 128.88, 128.82, 126.91, 126.90, 123.84, 121.17, 120.01, 119.13,
113.20, 71.82, 59.73, 53.60, 53.59, 53.32, 53.30, 39.93, 30.33, 22.33,
22.30, 20.29, 10.51, 10.50. MS (ESI) m/z: 500.4 (M þ H)þ.
azine-2-carboxamide (21). M.p.: 236e238 ꢁC. IR (KBr, cmꢀ1
)
nmax
:
3362, 2934, 1681, 1535, 1347, 1018, 688. 1HNMR (CDCl3, 300 MHz):
d
10.35 (br, 1H), 9.50 (d, J ¼ 1.2 Hz, 1H), 8.79 (d, J ¼ 2.7 Hz, 1H), 8.61
(dd, J ¼ 1.5, 2.7 Hz, 1H), 8.53 (d, J ¼ 2.1 Hz, 1H), 8.19 (dd, J ¼ 2.1,
6.9 Hz, 2H), 7.54 (m, 2H), 6.96 (d, J ¼ 8.4 Hz, 1H), 6.87 (d, J ¼ 8.4 Hz,
1H), 4.16 (q, J ¼ 6.9 Hz, 2H), 3.63 (s, 2H), 3.00 (m, 2H), 2.53 (m, 1H),
2.17 (m, 2H), 1.86 (dd, J ¼ 3.0, 8.1 Hz, 4H), 1.51 (t, J ¼ 6.9 Hz, 3H).
4.2.1.6. N-(5-(1-(4-(Diethylamino)benzyl)piperidin-4-yl)-2-
isopropoxyphenyl)pyrazine-2-carboxamide (28). M.p.: 156e158 ꢁC.
IR (KBr, cmꢀ1
)
nmax: 3567, 3349, 2977, 1685, 1253, 1019, 820. 1HNMR
13CNMR (100 MHz, DMSO-d6)
d: 160.18, 148.13, 146.21, 143.97,
(DMSO, 300 MHz):
d
10.30 (s, 1H), 9.34 (d, J ¼ 1.2 Hz, 1H), 8.98 (m,
143.53, 143.45, 132.90, 130.00, 126.71,123.50,123.49,122.51, 122.50,
117.47, 117.46, 112.22, 112.20, 79.12, 64.39, 64.38, 52.00, 38.29,
29.80, 29.79, 14.61. MS (ESI) m/z: 461.2 (M þ H)þ.
1H), 8.84 (dd, J ¼ 1.2, 2.4 Hz, 1H), 8.40 (s, 1H), 7.86 (m, 2H), 7.53 (m,
2H), 7.14 (d, J ¼ 8.4 Hz, 1H), 6.99 (d, J ¼ 8.4 Hz, 1H), 4.68 (q,
J ¼ 5.7 Hz, 1H), 4.08 (s, 2H), 3.95 (m, 4H), 3.43 (m, 2H), 3.06 (m, 2H),
2.78 (m, 1H), 1.99 (m, 4H), 1.36 (d, J ¼ 5.7 Hz, 6H) 1.08 (t, J ¼ 6.9 Hz,
4.2.2.5. N-(5-(1-(4-Nitrobenzyl)piperidin-4-yl)-2-isopropoxyphenyl)
6H). 13CNMR (100 MHz, DMSO)
d: 160.14, 148.16, 145.28, 144.10,
benzamide (25). M.p.: 124e125 ꢁC. IR (KBr, cmꢀ1
)
nmax: 3421, 2975,
143.96, 143.52, 143.50, 136.85, 136.20, 132.99, 132.98, 129.97, 127.78,
122.90, 122.41, 117.48, 114.32, 71.69, 52.25, 52.23, 51.47, 51.45, 38.47,
29.75, 29.73, 21.93, 21.77, 9.70, 9.68. MS (ESI) m/z: 502.4 (M þ H)þ.
1670, 1523, 1328, 1109, 699. 1HNMR (DMSO-d6, 300 MHz):
d
10.95
(br, 1H), 9.26 (s, 1H), 8.34 (d, J ¼ 8.7 Hz, 2H), 7.95 (d, J ¼ 8.7 Hz, 2H),
7.91 (d, J ¼ 7.8 Hz, 2H), 7.58 (m, 3H), 7.06 (d, J ¼ 8.4 Hz, 1H), 6.99 (br
of d, J ¼ 8.4 Hz, 1H), 4.60 (q, J ¼ 6.3 Hz, 1H), 4.49 (d, J ¼ 3.9 Hz, 2H),
3.45 (m, 2H), 3.10 (m, 2H), 2.77 (m, 1H), 1.98 (m, 4H), 1.30 (d,
4.2.2. Procedure B
4.2.2.1. N-(5-(1-(4-Nitrobenzyl)piperidin-4-yl)-2-methoxyphenyl)
benzamide (9). To the solution of compound 7a (400 mg,
J ¼ 6.3 Hz, 6H). 13CNMR (100 MHz, DMSO-d6)
d: 164.71, 148.09,
147.65, 137.23, 136.18, 134.60, 132.92, 132.43, 131.63, 128.63, 128.62,

C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
655
128.61, 127.17, 127.15, 123.61, 123.06, 123.05, 121.60, 114.28, 71.64,
57.73, 51.78, 51.77, 38.18, 29.72, 29.70, 21.88, 21.86. MS (ESI) m/z:
474.3 (M þ H)þ.
J ¼ 9.0 Hz, 2H), 4.14 (q, J ¼ 6.9 Hz, 2H), 3.63 (br, 1H), 3.46 (s, 2H),
3.02 (d, J ¼ 10.8 Hz, 2H), 2.50 (m,1H), 2.07 (m, 2H),1.82 (m, 4H),1.50
(t, J ¼ 6.9 Hz, 3H). 13CNMR (100 MHz, CD3OD)
d: 165.00, 148.68,
144.90, 137.35, 136.30, 134.55, 134.54, 134.12, 131.48, 128.38, 125.90,
125.00, 125.00, 124.05, 121.80, 119.15, 113.09, 65.89, 60.82, 54.17,
54.16, 40.44, 31.85, 31.84, 15.13. MS (ESI) m/z: 432.3 (M þ H)þ.
4.2.2.6. N-(5-(1-(4-Nitrobenzyl)piperidin-4-yl)-2-isopropoxyphenyl)
pyrazine-2-carboxamide (29). M.p.: 215e218 ꢁC. IR (KBr, cmꢀ1
)
nmax: 3378, 2981, 1685, 1539, 1344, 1117, 856. 1HNMR (DMSO-d6,
300 MHz):
d
10.68 (br, 1H), 9.33 (d, J ¼ 1.5 Hz, 1H), 8.97 (dd, J ¼ 1.5,
4.2.3.5. N-((5-(1-(4-Aminobenzyl)piperidin-4-yl)-2-isopropoxy)
2.4 Hz, 1H), 8.84 (dd, J ¼ 1.5, 2.4 Hz, 1H), 8.41 (s, 1H), 8. 36 (d,
J ¼ 7.5 Hz, 2H), 7.94 (d, J ¼ 7.5 Hz, 2H), 7.13 (d, J ¼ 7.8 Hz, 1H), 6.98
(br of d, J ¼ 7.8 Hz, 1H), 4.68 (q, J ¼ 5.4 Hz, 1H), 4.51 (d, J ¼ 4.8 Hz,
2H), 3.47 (m, 2H), 3.15 (m, 2H), 2.80 (m, 1H), 2.03 (m, 4H), 1.36 (m,
phenyl)benzamide (26). M.p.: 165e167 ꢁC. IR (KBr, cmꢀ1
)
nmax: 3418,
10.63 (br,
2929,1662,1473,1109, 710.1HNMR (DMSO-d6, 300 MHz):
d
1H), 9.26 (s,1H), 7.91 (d, J ¼ 6.9 Hz, 2H), 7.82 (s,1H), 7.57 (m, 5H), 7.20
(d, J ¼ 7.5, 2H), 7.06 (d, J ¼ 8.7 Hz, 1H), 6.98 (br of d, J ¼ 7.2 Hz, 1H),
4.58 (q, J ¼ 5.4 Hz,1H), 4.26 (s, 2H), 3.39 (br of d, J ¼ 10.8 Hz, 2H), 3.03
(m, 2H), 2.75 (m, 1H), 2.00 (m, 4H), 1.28 (d, J ¼ 6.0 Hz, 6H). 13CNMR
6H). 13CNMR (100 MHz, DMSO-d6)
d: 160.15, 148.16, 148.10, 145.29,
143.95, 143.53, 143.50, 137.20, 136.78, 132.92, 132.90, 127.79, 123.63,
123.31, 122.41, 117.45, 114.31, 71.69, 57.75, 51.78, 51.77, 38.37, 29.73,
29.71, 21.92, 21.90. MS (ESI) m/z: 476.2 (M þ H)þ.
(100 MHz, DMSO-d6) d: 164.71, 147.70, 147.69, 136.25, 134.80, 134.60,
132.78, 132.76, 131.63, 128.63, 128.60, 128.05, 127.17, 127.16, 123.11,
122.49, 122.48, 121.69, 114.30, 71.04, 58.25, 51.47, 51.46, 38.26, 29.71,
29.70, 21.89, 21.88. MS (ESI) m/z: 444.3 (M þ H)þ.
4.2.3. Procedure C
4.2.3.1. N-((5-(1-(4-Aminobenzyl)piperidin-4-yl)-2-methoxy)phenyl)
benzamide (10). A solution of compound 9 (70 mg, 0.16 mmol) in
methanol (50 mL) was hydrogenated in the presence of 5% Pd/C
(7 mg) under a H2 atmosphere (57 psi) at room temperature for
24 h. The mixture was filtered, and the filtrate was concentrated to
4 . 2 . 3 . 6 . N - ( 5 - ( 1 - ( 4 - A m i n o b e n z yl ) p i p e r i d i n - 4 - yl ) - 2 -
isopropoxyphenyl)pyrazine-2-carboxamide (30). M.p.: 168e169 ꢁC.
IR (KBr, cmꢀ1 nmax: 3355, 2929, 1685, 1541, 1110, 821. 1HNMR
)
(DMSO-d6, 300 MHz): d 10.65 (s, 1H), 10.30 (s, 1H), 9.33 (s, 1H), 8.98
give foam (65 mg, 99% yield). M.p.: 210e211 ꢁC. IR (KBr, cmꢀ1
)
nmax
:
(s, 1H), 8.83 (s, 1H), 8.40 (s, 1H), 7.62 (d, J ¼ 7.8 Hz, 2H), 7.56 (m, 3H),
7.38 (d, J ¼ 7.8 Hz, 2H), 7.13 (d, J ¼ 8.4 Hz,1H), 6.97 (d, J ¼ 8.4 Hz,1H),
4.68 (q, J ¼ 6.0 Hz, 1H), 4.30 (s, 2H), 3.42 (m, 2H), 3.20 (m, 2H), 2.90
(m, 1H), 2.04 (m, 4H), 1.13 (d, J ¼ 6.0 Hz, 6H). 13CNMR (100 MHz,
3418, 2839, 1655, 1024, 712. 1HNMR (CDCl3, 300 MHz):
d 8.54 (s,
1H), 8.47 (s, 1H), 7.89 (d, J ¼ 7.8 Hz, 2H), 7.52 (m, 3H), 7.13 (d,
J ¼ 7.5 Hz, 2H), 6.95 (d, J ¼ 8.4 Hz, 1H), 6.85 (d, J ¼ 7.5 Hz, 1H), 6.66
(d, J ¼ 6.9 Hz, 2H), 3.90 (s, 3H), 3.62 (s, 2H), 3.44 (s, 2H), 3.00 (d,
J ¼ 10.3 Hz, 2H), 2.49 (m, 1H), 2.02 (m, 2H), 1.82 (m, 4H). 13CNMR
DMSO-d6) d: 164.14, 148.16, 145.29, 143.96, 143.52, 143.50, 136.84,
135.10, 132.79, 132.78, 132.70, 128.00, 127.78, 122.44, 122.00, 117.45,
114.32, 71.70, 64.86, 58.31, 51.47, 38.47, 29.73, 29.70, 21.92, 21.90.
MS (ESI) m/z: 446.3(M þ H)þ.
(75 MHz, DMSO-d6) d: 164.63, 147.92, 135.60, 134.47, 132.50, 132.46,
132.45, 131.26, 128.56, 128.55, 128.29, 127.80, 127.14, 126.80, 126.80,
124.91, 122.01, 119.41, 110.37, 55.66, 55.55, 51.73, 51.70, 38.71, 29.86,
29.00. MS (ESI) m/z: 416.3 (M þ H)þ.
4.2.4. Procedure D
Procedure C was applied to prepare compounds 14, 18, 22,
26, 30.
4.2.4.1. N-(5-(1-(4-Acetamidobenzyl)piperidin-4-yl)-2-
methoxyphenyl)benzamide (11). To a solution of compound 10
(300 mg, 0.67 mmol) in dichloromethane (30 mL) and pyridine
(116 mg, 2.35 mmol), acetic anhydride (102 mg, 1.01 mmol) was
added dropwise. After stirred at room temperature overnight, the
resulting suspension was washed with brine, dried with NaSO4. The
CH2Cl2 solution was filtered, then the filtrate was concentrated. The
residue was purified with chromatography (CH2Cl2/MeOH: 100/1) to
4.2.3.2. N-(5-(1-(4-Aminobenzyl)piperidin-4-yl)-2-methoxyphenꢀy1l)
pyrazine-2-carboxamide (14). M.p.: 209e211 ꢁC. IR (KBr, cm
)
nmax: 3306, 2836, 1672, 1541, 1020, 808. 1HNMR (CDCl3, 300 MHz):
d
10.25 (br, 1H), 9.49 (d, J ¼ 1.5 Hz, 1H), 8.78 (d, J ¼ 2.7 Hz, 1H), 8.62
(dd, J ¼ 1.5, 2.4 Hz, 1H), 8.51 (d, J ¼ 2.1 Hz, 1H), 7.14 (d, J ¼ 8.4 Hz,
2H), 6.98 (dd, J ¼ 2.4, 8.7 Hz, 1H), 6.87 (d, J ¼ 8.4 Hz,1H), 6.67 (d,
J ¼ 8.1 Hz, 2H), 3.95 (s, 3H), 3.45 (s, 2H), 3.02 (m, 2H), 2.51 (m, 1H),
give GL-2-49 190 mg in 62% yield. M.p.: 168e170 ꢁC. IR (KBr, cmꢀ1
nmax: 3415, 2938, 1670, 1519, 1536, 1024, 761. 1HNMR (CDCl3,
300 MHz):
)
2.04 (m, 2H), 1.82 (m, 4H). 13CNMR (75 MHz, CD3OD)
d: 165.00,
d
8.54 (s, 1H), 8.47 (s, 1H), 7.89 (d, J ¼ 7.8 Hz, 2H), 7.54 (m,
149.60, 148.91, 144.83, 142.50, 137.40, 134.54, 134.53, 134.40, 131.23,
128.15, 124.85, 124.84, 124.12, 119.40, 112.06, 112.05, 60.93, 56.79,
54.24, 54.23, 40.43, 31.83, 31.82. MS (ESI) m/z: 418.3 (M þ H)þ.
5H), 7.30 (d, J ¼ 8.4 Hz, 2H), 7.20 (br, 1H), 6.95 (dd, J ¼ 1.8 Hz, 8.4 Hz,
1H), 6.85 (d, J ¼ 8.4 Hz, 1H), 3.90 (s, 3H), 3.50 (s, 2H), 2.98 (d,
J ¼ 13.2 Hz, 2H), 2.51 (m, 1H), 2.17 (s, 3H), 2.07 (m, 2H), 1.84 (m, 4H).
13C NMR (100 MHz, CDCl3)
d: 168.00, 165.45, 147.63, 144.00, 140.00,
4.2.3.3. N-((5-(1-(4-Aminobenzyl)piperidin-4-yl)-2-ethoxy)phenyl)
135.96, 135.26, 132.27, 131.98, 128.90, 128.89, 127.77, 127.10, 127.09,
125.00, 124.10, 121.38, 120.37, 119.27, 110.61, 56.20, 53.20, 40.01,
30.37, 29.71, 24.50, 22.50, 14.90. MS (ESI) m/z: 458.2 (M þ H)þ.
Procedure D was applied to prepare compounds 15, 19, 23,
27, 31.
benzamide (18). M.p.: 165e167 ꢁC. IR (KBr, cmꢀ1
)
d
nmax: 3417, 2926,
8.61 (s, 1H), 8.47
1688, 1535, 1141, 711. 1HNMR (CDCl3, 300 MHz):
(d, J ¼ 2.1 Hz, 1H), 7.88 (d, J ¼ 6.9 Hz, 2H), 7.53 (m, 3H), 7.13 (d,
J ¼ 8.1 Hz, 2 Hz, 2H), 3.61 (s, 2H), 3.44 (s, 2H), 3.00 (d, J ¼ 11.1 Hz,
2H), 2.48 (m, 1H), 2.02 (m, 2H), 1.83 (m, 4H). 13CNMR (100 MHz,
DMSO-d6)
d
: 164.81, 149.00, 136.09, 134.56, 132.75, 132.74, 131.61,
4.2.4.2. N-(5-(1-(4-Acetamidobenzyl)piperidin-4-yl)-2-
128.56, 128.55, 128.50, 127.26, 127.25, 127.21, 127.09, 123.27, 121.94,
121.68, 121.67, 112.54, 64.08, 58.35, 51.45, 51.40, 38.22, 29.75, 29.74,
14.62. MS (ESI) m/z: 430.3 (M þ H)þ.
methoxyphenyl)pyrazine-2-carboxamide (15). M.p.: 181e183 ꢁC. IR
(KBr, cmꢀ1
)
nmax: 3421, 2950, 1670, 1654, 1540, 1261, 1021, 709.
1HNMR (CDCl3, 300 MHz):
d
8.54 (s,1H), 8.46 (d, J ¼ 1.8 Hz,1H), 7.89
(m, 2H), 7.50 (m, 4H), 7.35 (d, J ¼ 8.7 Hz, 2H), 6.95 (dd, J ¼ 2.4 Hz,
8.4 Hz, 1H), 6.85 (d, J ¼ 8.7 Hz, 1H), 3.90 (s, 3H), 3.58 (s, 2H), 3.03 (d,
J ¼ 13.2 Hz, 2H), 2.54 (m, 1H), 2.18 (s, 3H), 2.18 (m, 2H), 1.86 (m, 4H).
4.2.3.4. N-(5-(1-(4-Aminobenzyl)piperidin-4-yl)-2-ethoxyphenꢀy1l)
pyrazine-2-carboxamide (22). M.p.: 180e182 ꢁC. IR (KBr, cm
)
nmax: 3421, 2930, 1678, 1401, 1251, 809. 1HNMR (CDCl3, 300 MHz):
13CNMR (100 MHz, CD3OD)
d: 151.10, 146.30, 134.27, 134.26, 133.19,
d
10.33 (br, 1H), 9.49 (d, J ¼ 1.5 Hz, 1H), 8.78 (d, J ¼ 2.4 Hz, 1H), 8.61
133.11, 129.90, 129.89, 128.40, 128.39, 124.70, 122.90, 121.88, 121.75,
121.46, 113.10, 112.30, 112.15, 61.19, 56.62, 54.02, 53.91, 40.37, 31.89,
31.88, 19.60. MS (ESI) m/z: 460.2 (M þ H)þ.
(dd, J ¼ 1.5, 2.4 Hz, 1H), 8.50 (d, J ¼ 1.8 Hz, 1H), 7.14 (d, J ¼ 9.0 Hz,
2H), 6.95 (dd, J ¼ 2.4, 8.4 Hz, 1H), 6.86 (d, J ¼ 8.4 Hz, 1H), 6.66 (d,

656
C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
4.2.4.3. N-(5-(1-(4-Acetamidobenzyl)piperidin-4-yl)-2-
ethoxyphenyl)benzamide (19). M.p.: 231e234 ꢁC. IR (KBr, cmꢀ1
nmax: 3089, 1733, 1700, 1535, 1252, 708. 1HNMR (CDCl3, 300 MHz):
most GPCRs and used for model construction for both the inactive
and active models. The comparative modeling software, MODELLER
9v8, was used to generate 100 homology models for each state
using the default parameters.
)
d
8.62 (s, 1H), 8.47 (d, J ¼ 2.1 Hz, 1H), 7.88 (m, 2H), 7.50 (m, 5H), 7.29
(d, J ¼ 8.1 Hz, 2H), 6.92 (dd, J ¼ 2.1 Hz, 8.1 Hz,1H), 6.83 (d, J ¼ 8.1 Hz,
1H), 4.12 (q, J ¼ 6.6 Hz, 2H), 3.50 (s, 2H), 2.98 (d, J ¼ 10.8 Hz, 2H),
2.50 (m, 1H), 2.17 (s, 3H), 2.07 (m, 2H), 1.81 (m, 4H), 1.47 (t,
Model screening was performed by using the genetic-algorithm
docking program GOLD 5.1 (Cambridge Crystallographic Data
Centre, Cambridge, UK) to dock maraviroc into the CCR5 homology
models using GOLD score as the fitness function. Once receptor
model was chosen based upon the discrete optimized protein en-
ergy (DOPE) scores, fitness function values, and the electronic and
steric interactions between the ligands and receptor, further model
refinement was done by using molecular mechanics based energy
minimization in Sybyl-X 2.0. Briefly, the model was minimized
using a Tripos Force Field with GasteigereHückel charges, a non-
J ¼ 7.2 Hz, 3H). 13CNMR (100 MHz, CDCl3)
d: 169.77, 165.22, 146.57,
140.88, 135.85, 135.30, 132.13, 131.86, 130.00, 128.93, 128.92, 127.93,
126.98, 126.97, 123.00, 121.25, 120.37, 120.36, 119.11, 111.55, 64.63,
61.05, 53.09, 40.03, 30.33, 30.32, 25.88, 24.66, 14.92. MS (ESI) m/z:
472.2(M þ H)þ.
4.2.4.4. N-(5-(1-(4-Acetamidobenzyl)piperidin-4-yl)-2-
ethoxyphenyl)pyrazine-2-carboxamide (23). M.p.: 195e198 ꢁC. IR
bonded interaction cutoff of 8 A with a distance-dependent
ꢀ
(KBr, cmꢀ1
)
nmax: 3366, 2929, 1674, 1596, 1499, 1020, 671. 1HNMR
dielectric constant of ε ¼ 4 being terminated at 0.05 kcal/(mol A).
ꢀ
(CDCl3, 300 MHz):
d
9.40 (s, 1H), 8.89 (s, 1H), 8.77 (s, 1H), 8.47 (s,
The minimized models were then analyzed using PROCHECK and
ProTable within SYBYL-X 2.0 to ensure the overall quality of the
models (i.e. acceptable torsion angles, steric clashes, bond lengths,
etc.).
1H), 7.74 (d, J ¼ 9.0 Hz, 2H), 7.54 (d, J ¼ 9.0 Hz, 2H), 7.06 (m, 2H),
4.37 (s, 2H), 4.22 (q, J ¼ 7.2 Hz, 2H), 3.64 (m, 2H), 3.35 (m, 2H), 3.20
(m, 2H), 2.90 (m, 2H), 2.54 (m, 2H), 2.17 (s, 3H), 1.52 (t, J ¼ 7.2 Hz,
3H). 13CNMR (100 MHz, CD3OD)
d: 171.99, 162.31, 148.91, 148.61,
Four CCR5 antagonists (including maraviroc) were docked into
the CCR5 homology model using GOLD 4.1. The compounds were
docked twenty times each with standard GOLD parameters and the
default GOLD scoring was used. For all the ligands the best GOLD-
docked solutions were selected and merged into the CCR5re-
ceptor. The combined receptoreligand structures were energy-
minimized following the method described above. The minimiza-
tion step optimized the interaction between ligands and the CCR5
homology model by removing clashes and lowering strain energy.
146.10, 144.82, 141.80, 139.50, 137.40, 133.10, 133.09, 128.32, 126.10,
125.49, 124.07, 121.41, 119.06, 113.06, 65.88, 61.48, 53.85, 53.84,
40.48, 31.87, 31.86, 23.93, 15.12. MS (ESI) m/z: 474.3 (M þ H)þ.
4.2.4.5. N-(5-(1-(4-Acetamidobenzyl)piperidin-4-yl)-2-
isopropoxyphenyl)benzamide (27). M.p.: 226e228 ꢁC. IR (KBr, cmꢀ1
)
nmax: 3335, 2977, 1684, 1660, 1538, 1251, 710. 1HNMR (DMSO-d6,
300 MHz): 10.16 (s, 1H), 9.24 (s, 1H), 7.90 (m, 3H), 7.68 (d,
d
J ¼ 8.4 Hz, 2H), 7.56 (m, 3H), 7.48 (d, J ¼ 8.4 Hz, 2H), 7.06 (d,
J ¼ 9.0 Hz, 1H), 6.97 (d, J ¼ 6.6 Hz, 1H), 4.59 (q, J ¼ 6.0 Hz, 1H), 4.25
(d, J ¼ 5.1 Hz, 2H), 3.40 (m, 2H), 3.03 (m, 2H), 2.73 (m, 1H), 1.95 (m,
4.4. Biological screening protocols
4H), 1.28 (d, J ¼ 6.0 Hz, 6H). 13CNMR (100 MHz, DMSO-d6)
d: 168.55,
4.4.1. CCR5 calcium inhibition assays
164.70, 147.61, 140.35, 136.26, 134.61, 131.97, 131.96, 131.63, 128.63,
128.60, 128.08, 127.16, 127.15, 123.87, 123.08, 121.52, 118.83, 118.82,
114.26, 71.04, 58.65, 51.35, 51.34, 38.29, 29.79, 29.78, 23.99, 23.98,
21.88, 21.87. MS (ESI) m/z: 486.3 (M þ H)þ.
CCR5-MOLT-4 cells (Obtained through the AIDS Research and
Reference Reagent Program, NIAID, NIH, from Dr. Masanori Baba,
Dr. Hiroshi Miyake, Dr. Yuji Iizawa) were transfected with Gqi5
pcDNA1 using Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s recommended procedure and maintained in RPMI
1640 supplemented with 10% fetal bovine serum, 100 u/mL peni-
4.2.4.6. N-(5-(1-(4-Acetamidobenzyl)piperidin-4-yl)-2-
isopropoxyphenyl)pyrazine-2-carboxamide (31). M.p.: 162e164 ꢁC.
cillin, 100 m
g/mL streptomycin, and 1 mg/mL G418 at 37 ꢁC and 5%
CO2. 48 h after transfection a total of 2,500,000 cells were spun
down and brought back up in 8 mL of 50:1 HBSS:HEPES assay
buffer. Cells were then plated at 25,000 cells per well into a clear
IR (KBr, cmꢀ1
)
nmax: 3350, 2929, 1681, 1594, 1540, 1110, 702. 1HNMR
(DMSO-d6, 300 MHz): d 10.31 (s, 1H), 10.21 (s, 1H), 9.34 (s, 1H), 8.97
(s, 1H), 8.84 (s, 1H), 8.40 (s, 1H), 7.68 (d, J ¼ 8.4 Hz, 2H), 7.52 (d,
J ¼ 8.4 Hz, 2H), 7.13 (d, J ¼ 8.4 Hz, 1H), 6.97 (d, J ¼ 9.0 Hz, 1H), 4.70
(q, J ¼ 5.7 Hz, 1H), 4.25 (s, 2H), 3.43 (m, 2H), 3.03 (m, 2H), 2.78 (m,
1H), 2.08 (s, 3H), 1.99 (m, 4H), 1.35 (d, J ¼ 5.7 Hz, 6H). 13CNMR
bottom, black 96-well plate (Greiner Bio-one) and 50
loading buffer (40 L 2 M Fluo4-AM (Invitrogen), 100
probenacid, in 5 mL assay buffer) was added to bring the volume up
to 130 L. After incubating for 45 min, 50 L of varying concen-
m
L of Fluo4
m
m
mL 2.5 mM
(100 MHz, DMSO-d6)
d: 168.56, 160.15, 148.16, 145.21, 143.97,
m
m
143.52, 143.51, 136.87, 131.97, 131.96, 127.80, 127.79, 123.86, 122.46,
118.83, 118.82, 117.41, 114.31, 71.70, 64.86, 58.67, 51.33, 51.32, 38.49,
29.79, 29.78, 23.99, 21.93. MS (ESI) m/z: 488.3 (M þ H)þ.
trations of ligands and controls were added and the plate was
incubated for an additional 15 min. Plates were then read on a
FlexStation3 microplate reader (Molecular Devices) at 494/516 ex/
em for a total of 120 s. After 16 s of reading, 20
(Biosource) in assay buffer, or assay buffer alone, was added to the
wells to bring the total volume up to 200
L. The changes in Ca2þ
mL of 200 nM RANTES
4.3. Molecular modeling protocols
m
All ligands used in the docking studies were built with standard
bond lengths and angles using the molecular modeling package
SYBYL-X 2.0. The small molecules were assigned GasteigereHuckel
charges and energy minimized with the Tripos Force Field.
mobilization were monitored and peak height values were ob-
tained using SoftMaxPro software (Molecular Devices). Non-linear
regression curves and IC50’s were generated using GraphPad Prism.
All experiments were repeated a total of three times.
All molecular modeling was collected using the SYBYL-X 2.0
molecular modeling package (Tripos LP, St. Louis, MO) on dual-core
AMD Opteron(tm) 2.4 GHz processors. The amino acid sequence of
chemokine receptor CCR5 was obtained from UniProtKB/Swiss-Prot
(P51681). Within ClustalX a multiple alignment was performed
with a gap opening penalty of 15 using the BLOSUM protein weight
matrix series. Sequence alignment between CCR5 and CXCR4 was
further optimized based on the most conserved residues among
4.4.2. Anti-proliferation assays
All cell lines, PC-3 and M12, were incubated at 37 ꢁC in the
presence of 5% CO2. RPMI 1640 serum free media (GIBCO Invi-
trogen) containing 1%
L
-glutamine, 0.1% ITS (insulin, 5
mg/ml;
transferrin, 5 g/ml; and selenium, 5
m
mg/ml; Collaborative Research,
Bedford) and 0.1% gentamicin was used to cultivate all cells. M12
cells were first incubated in media with 5% fetal bovine serum

C.K. Arnatt et al. / European Journal of Medicinal Chemistry 69 (2013) 647e658
657
(FBS); after 24 h serum free media was added with 0.01% epidermal
growth factor (EGF). DU-145 and PC-3 cell lines were incubated in
media containing 10% FBS at all times.
Prostate cancer tumor cells (PC-3, and M12) were plated into 96
well plates (BD Falcon, VWR) at a concentration of 1000 cells per
well. Each cell line was plated in its respective serum containing
and does not necessarily represent the official views of the US Army
Prostate Cancer Research Program. We thank the NIH AIDS
Research and Reference Reagent Program for providing the MOLT-
4/CCR5 cell line.
Appendix A. Supplementary data
media for a total concentration of 100
various concentrations of drugs in a 50 L PBS solution were added
to the cells. Control cells were given 50 L of PBS. Seventy-two
hours after incubation with drug, the serum containing media
was replaced with 100 L of a 9:1 solution of serum free media and
mL per well. After 24 h,
m
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.004.
m
m
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We are grateful for the partial funding support from US Army
Prostate Cancer Research Program PC073739 and A.D. Williams
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