Discovery of a novel CCR5 antagonist lead compound through fragment assembly

Article abstract of DOI:10.3390/molecules13102426

CCR5, as the major co-receptor for HIV-1 entry, is an attractive novel target for the pharmaceutical industry in the HIV-1 therapeutic area. In this study, based on the structures of maraviroc and 1,4-bis(4-(7-chloroquinolin-4- yl)piperazin-1-yl)butane-1,4-dione (1), which was identified using structure-based virtual screening in conjunction with a calcium mobilization assay, a series of novel small molecule CCR5 antagonists have been designed and synthesized through fragment assembly. Preliminary SARs were obtained, which are in good agreement with the molecular binding model and should prove helpful for future antagonist design. The novel scaffold presented here might also be useful in the development of maraviroc-derived second generation CCR5 antagonists.

Full text of DOI:10.3390/molecules13102426

Molecules 2008, 13, 2426-2441; DOI: 10.3390/molecules13102426
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.org/molecules
Article
Discovery of a Novel CCR5 Antagonist Lead Compound
Through Fragment Assembly
Yanqing Liu 1,†, Enkun Zhou 2,3,†, Kunqian Yu 4,†, Jin Zhu 1, Yu Zhang 1, Xin Xie 2,*, Jian Li 1,*
and Hualiang Jiang 1,4
1
School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road,
Shanghai 200237, P.R. China; E-mails: yanqing_liu@163.com (Y. L.), jinzh@mail.ecust.edu.cn (J.
Z.), yuloveyou8133@sina.com (Y. Z.)
2
The National Center for Drug Screening, Chinese Academy of Sciences, Shanghai 201203, P.R.
China; E-mail: nickey838@sina.com (E. Z.)
3
School of Biotechnology, Southwest University, Chongqing 400715, P.R. China
4
Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P.R. China; E-mail: kqyu@mail.shcnc.ac.cn
(K-Q. Y.), hljiang@mail.shcnc.ac.cn (H-L. J.)
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-mail: jianli@ecust.edu.cn (J. L.);
xxie@mail.shcnc.ac.cn (X. X.).
Received: 3 September 2008; in revised form: 18 September 2008 / Accepted: 2008 / Published: 1
October 2008
Abstract: CCR5, as the major co-receptor for HIV-1 entry, is an attractive novel target for
the pharmaceutical industry in the HIV-1 therapeutic area. In this study, based on the
structures of maraviroc and 1,4-bis(4-(7-chloroquinolin-4-yl)piperazin-1-yl)butane-1,4-
dione (1), which was identified using structure-based virtual screening in conjunction with
a calcium mobilization assay, a series of novel small molecule CCR5 antagonists have
been designed and synthesized through fragment assembly. Preliminary SARs were
obtained, which are in good agreement with the molecular binding model and should prove
helpful for future antagonist design. The novel scaffold presented here might also be useful
in the development of maraviroc-derived second generation CCR5 antagonists.

Molecules 2008, 13
Keywords: CCR5 antagonist; fragment assembly, HIV-1; molecular modeling
2427
Introduction
Almost a decade ago now, the chemokine receptor CCR5 was identified as the major co-receptor
for HIV-1 entry, besides the cellular CD4 receptor [1-5]. CCR5 plays an integral role in the R5-tropic
HIV-1 entry process by serving as a critical co-receptor for the viral envelope protein gp120 [6-7]. The
natural ligands of CCR5 (RANTES, MIP-1α, MIP-1β) [8] and their derivatives [9-10], as well as some
specific monoclonal antibodies against certain epitopes of CCR5 [11] possess anti-HIV-1 activity, and
homozygous individuals with a 32-base pair deletion in the gene encoding CCR5 do not express the
functional receptor and are ultimately resistant to R5-tropic HIV-1 infection [12]. These facts have
made CCR5 an attractive novel target for the pharmaceutical industry in the HIV-1 therapeutic area.
In the last decade, numerous small molecule CCR5 antagonists have been reported. The discovery
and development of CCR5 antagonists have been systmatically reviewed by Palani [13]. They include
anilide-, oximino-piperidino-piperidine-, chiral piperazine-, tropane-, spirodiketopiperazine-, acyclic
and cyclic scaffold-based compounds. These efforts have resulted recently in the FDA approval of the
first small molecule CCR5 antagonist, maraviroc (Selzentry®, Figure 1) [14] for the treatment of HIV-
1 infection. But there are still various challenges and unknowns associated with CCR5 antagonists
such as drug resistance, viral tropism and possible long term adverse events, so development of second
generation CCR5 antagonists with improved properties is still much needed.
Figure 1. Design of novel scaffold 2 through fragment assembly.
O
N
F
F
NH
N
O
N
Cl
N
Cl
N
N
N
N
O
N
N
Maraviroc
Hit 1
Fragment Assembly
O
F
NH
F
Cl
N
N
N
Compound 2
The fragment assembly method is a simply, effective structural modification strategy applied in
medicinal chemistry field. These fragments are usually from active compounds or launched drugs, and
possess comparatively good properties, such as high affinity, excellent metabolic stability, and low
toxicity. Our CCR5 program was initiated from compound 1 (Figure 1) featuring the symmetric bis-4-

Molecules 2008, 13
2428
(piperazin-1-yl)quinoline framework, and identified using a docking based virtual screening approach
in conjunction with a calcium mobilization assay (IC50 = 2.00 μM). Mono-4-(piperazin-1-yl)quinoline
derivatives have previously been reported as CCR5 antagonists [15]. By combining the attractive
features of maraviroc and the 4-(piperazin-1-yl)quinoline moiety from hit 1, and with the goal of
identifying novel compounds that could be optimized to be more potent than compound 1, compound 2
was identified via a calcium mobilization assay as a modest starting point (IC50 = 692 nM) for further
optimization. Twelve derivatives 2a-l of compound 2 were designed and synthesized, and their
antagonistic activities against CCR5 were tested.
Results and Discussion
Identification of Compound 1 by Virtual Screening and Calcium Mobilization Assay
Targeting the optimized 3D model of CCR5 [16] which was constructed based on the bovine
rhodopsin crystal structure (PDB entry 1F88) [17], a total of 80,000 compounds [18] in the SPECS
database (http://www.specs.net/) were subsequently docked and ranked according to the scoring
functions of DOCK 4.0 [19], the top-2,000 molecules were selected and re-scored by the scoring
function of AutoDock 3.0 [20]. The 150 molecules with the highest score were selected from the
SPECS database. Finally, 95 commercially available molecules were purchased and submitted to
biological evaluation. Applying a calcium mobilization assay, compound 1 was identified among the
above 95 compounds as a CCR5 antagonist (IC50 = 2.00 μM), so compound 1 was designated as a hit
for further structural optimization.
Scheme 1a. The synthetic route to compounds 2 and 2a-j.
H
H
N
N
a
R1 Cl
+
N
N
H
R1
3
f
O
Boc
Boc
NH2
4
O
NH2
5
O
NH O
NH
O
c
e
b
d
O
O
O
6
7
O
NH2
R2
NH
Boc
NH
g
h
N
N
N
N
N
R1
R1
N
R1
2 and 2a-j
9
8
a
Reagents and conditions: (a) K2CO3, DMF, N2, 45 °C; (b) (i) NH4OAc, EtOH, 45 °C, (ii)
CH2(COOH)2, 60 °C, (iii) MeOH, SOCl2, 25 °C; (c) L(+)-tartaric acid, -20 °C; (d) (BOC)2O,
Na2CO3, THF/H2O, 25 °C; (e) DIBAl-H, DCM, -78 °C; (f) NaBH(OAc)3, HOAc, DCM, 25 °C; (g)
Saturated HCl in MeOH, 65 °C; (h) R2COOH, EDCI, HOBt, DIPEA, DCM, 25 °C or R2COCl,
pyridine, 25 ºC. R1 and R2 are as in Table 1.

Molecules 2008, 13
2429
Design and Synthesis of Compounds 2 and 2a-l
To discover novel compounds that could be optimized to be more potent than compound 1, we
designed novel chemical scaffold 2 through fragment assembly (Figure 1). Based on the structure of 2,
first, compounds 2a-f were designed to prove if the 4,4-difluorocyclohexanecarbonyl unit is essential
for antagonistic activity. Next, we changed the 7-chloroquinolin-4-yl of 2 for different heterocyclic
aryl moieties (compounds 2g-j) to examine if the heterocyclic aryl moiety would affect antagonistic
activity. Lastly, compounds 2k-l were prepared to investigate the importance of the chiral center and
the (1-phenyl)propylamine spacer of 2.
Scheme 2a. The synthetic route of compound 2k.
Boc
NH
Boc
NH2 O
Boc
NH O
NH O
10
Cl
a
b
c
N
O
O
N
N
4
11
12
O
F
NH2
NH
Cl
e
N
F
d
Cl
N
N
N
N
N
13
2k
a Reagents and conditions: (a) (BOC)2O, Na2CO3, THF/H2O, 25 °C; (b) DIBAL-H, DCM, -78 °C;
(c) NaBH(OAc)3, HOAc, DCM, 25 °C; (d) Saturated HCl in MeOH, 65 °C; (e) 4,4-
Difluorocyclohexanecarboxylic acid, EDCI, HOBt, DIPEA, DCM, 25 °C.
Scheme 3a. The synthetic route to compound 2l.
H
N
O
Cl
H
N
F
N
Cl
N
a
b
F
+
N
N
H
N
Cl
N
Cl
N
2l
3a
a Reagents and conditions: (a) K2CO3, DMF, N2, 135 °C; (b) 4,4-Difluorocyclohexanecarboxylic
acid, EDCI, HOBt, DIPEA, DCM, 25°C.
Compounds 2, and 2a-l were synthesized through the routes outlined in Schemes 1-3, and the
details for synthetic procedures and structural characterizations are described in the Experimental
section.

Molecules 2008, 13
2430
Calcium Mobilization Assay
For the primary assay, the percent inhibitory rates of the compounds 1, 2 and 2a-l at 1 μM were
measured. Except compounds 2a and 2c, the other twelve compounds were remarkably antagonist of
CCR5 activity. Therefore, we determined their IC50 values (Table 1). From the data in Table 1, we can
see that antagonistic activity of compound 2 remarkably decreased 3-fold compared to that of
compound 1 (IC50 from 2.00 μM down to 0.692 μM, respectively, Table 1).
Table 1. Chemical Structures of Compounds 1, 2 and 2a-l and Their Antagonistic
Activities against CCR5.
Compound
Structure
IC50 (μM)
N
N
O
N
Cl
1
2.00
Cl
N
O
N
N
O
F
NH
F
Cl
N
2
0.692
N
N
O
F
NH
Cl
N
2a
2b
2c
2d
--
N
N
O
NH
Cl
N
2.66
N
N
O
NH
Cl
N
--
N
N
O
O
NH
Cl
N
> 1.00
N
N

Molecules 2008, 13
2431
Table 2. Cont.
O
NH
Cl
2e
N
> 10.0
> 10.0
> 1.00
> 10.0
> 10.0
0.233
N
N
O
O
S
NH
Cl
N
2f
2g
2h
2i
N
N
O
F
NH
F
N
N
N
N
O
F
NH
F
N
N
NH
N
N
N
O
F
NH
F
N
N
N
O
F
NH
F
N
2j
N
N
O
F
NH
F
Cl
N
2k
2l
0.669
N
N
O
F
Cl
N
F
> 1.00
N
N

Molecules 2008, 13
2432
Table 2. Cont.
O
F
F
NH
Maraviroc
0.00261
N
N
N
N
Structure and Activity Relationship Correction with the Binding Models
According to the above results, we can draw some SAR conclusions: (1) the 4,4-difluorocyclo-
hexanecarbonyl of 2 (Table 1) is favorable for maintaining the antagonistic activity, as its replacement
with various substituted carbonyls (compounds 2a-e) or substituted sulfonyl (compound 2f) resulted in
decrease or loss of potency; (2) changing the 7-chloroquinolin-4-yl of 2 for some other heterocyclic
aryl group (compounds 2g-j) proved to be quite beneficial, resulting in a 3-fold increase in potency in
the case of 2j (IC50 from 0.692 μM down to 0.233 μM, respectively); (3) racemization of 2 (compound
2k) has no effect on potency; (4) removing the (1-phenyl)propylamine spacer from 2 nearly leads to a
loss of potency (compound 2l). Although the best compound 2j is 90-fold less potent than the control
compound (maraviroc), these preliminary structure-activity relationships provide some valuable clues
for structural optimization which could lead to development of useful second generation maraviroc-
derived CCR5 antagonists.
To illustrate these SARs and gain structural information for further structural optimization, we
compared the 3D binding models of the control compound maraviroc to CCR5 with that of the most
potent antagonist 2j to CCR5 generated based on the docking simulation (Figures 2a-b). Figure 2a
shows that one fluorine of the cyclohexane ring and the amide N atom of maraviroc form two H-bond
interactions with the Gly202 and Gly286 residues, respectively. Simultaneously, two excellent
hydrophobic groups, the tropane moiety and the isopropyl group of maraviroc, form potent
hydrophobic interactions with the Leu107, Tyr108, and Trp86 residues, respectively. Compound 2j
also forms two similar H-bond interactions with the Gly202 and Gly286 residues, whereas the
piperazinyl group and quinoline ring of compound 2j form weak hydrophobic interactions with the
Leu107, Tyr108, and Trp86 residues, respectively. The above difference in strength of hydrophobic
interactions between maraviroc and 2j might partly affect the antagonistic activity. Considering that
antagonistic activity of 2j is ten times less potent than maraviroc, the definite interaction mechanism
between antagonists and CCR5 needs further experimental verification. Inspired by molecular
modeling, introduction some hydrophobic substituents to piperazinyl group and/or quinoline ring of
compound 2j would be likely to produce some additional hydrophobic interactions and be useful to
improve activity.

Molecules 2008, 13
Figure 2. Detailed interactions of maraviroc (a) and 2j (b) with the binding sites of CCR5.
2433
Compounds maraviroc and 2j are indicated by yellow and blue thick sticks, respectively.
Key residues of the binding pocket are shown as thin sticks. Hydrogen bonds are shown as
yellow dotted lines with distance between donor and acceptor atoms. These pictures were
prepared using ViewerPro (http://www.accelrys.com/).
(a)
(b)
Conclusions
In this study, we have discovered a novel lead (2j) by using structure-based virtual screening
approach in conjunction with chemical synthesis and bioassay. The preliminary SARs were obtained,
which show the 4,4-difluorocyclohexanecarbonyl of compound 2 is necessary to maintain the
antagonistic activity, and appropriate heterocyclic aryl of compound 2 could remarkably improve the
antagonistic activity. These primary SARs are in good agreement with the molecular binding models
and helpful for future antagonists design, and the novel scaffold presented here also provides potential
application in development of maraviroc-derived second generation CCR5 antagonists.

Molecules 2008, 13
Experimental
General
2434
The chemicals used were purchased from Alfa, Acros and Shanghai Chemical Reagent Company,
and used without further purification. Analytical thin-layer chromatography (TLC) was done on HSGF
254 plates (150-200 µm thickness, Yantai Huiyou Company, P.R. China). Yields were not optimized.
Melting points were measured in capillary tubes on a SGW X-4 melting point apparatus without
correction. Nuclear magnetic resonance (NMR) spectra were given on a Brucker AVANCE 500 NMR
(TMS as IS). Chemical shifts were reported in parts per million (ppm, δ) downfield from
tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). Low- and high-resolution mass spectra (LRMS and HRMS) were
given with electric ionization (EI) produced by a Finnigan MAT-95 instrument.
Virtual Screening by Molecular Docking
The 3D molecular model was previously described [16]. Briefly, a homology model of the human
CCR5 receptor was constructed based on the bovine rhodopsin crystal structure (PDB entry 1F88) [17]
using the program Insight II (version 2000.1, Accelrys Inc., San Diego, CA, U.S.A.), and optimized
using molecular mechanics method with the following parameters: a distance-dependent dielectric
constant of 5.0; non-bonded cutoff of 8Å, Amber force field and Kollman all-atom charges [21]; and
conjugate gradient minimization. The minimized structure was validated using the PROCHECK [22]
program. The initial structures of the synthesized compounds (2 and 2a-l) and maraviroc were
constructed with Insight II (version 2000.1, Accelrys Inc., San Diego, CA) and energetically
minimized using Tripos force field with Gasteiger–Hückel charges [23]. The N-protonated forms of
the molecules, which are the prevalent species at physiological pH, were used in the calculations.
The optimized 3D model of CCR5 was used as the target for virtual screening on database SPECS
(http://www.specs.net/). The program DOCK4.0 [19] was employed for the primary screening.
Residues around the catalytic center at radius of 6 Å was isolated for constructing the grids of docking
screening, and the pocket composed by these residues was larger enough to include residues of the
binding pocket. During the docking calculations, Kollman-all-atom charges [21] were assigned to the
protein, and Geisterger-Hückel charges [23] were assigned to the small molecules in the SPECS
databases. The conformational flexibility of the compounds from the database was considered in the
docking searching.
The orientation of a ligand is evaluated with a shape scoring function and/or a function
approximating the ligand-receptor binding energy. After the initial orientation and scoring evaluation,
a grid-based rigid body minimization is carried out for the ligand to locate the nearest local energy
minimum within the receptor binding site. The position and conformation of each docked molecule
were optimized using single anchor search and torsion minimization method of DOCK4.0 [19].
Thirty configurations per ligand building a cycle and 50 maximum anchor orientations were used in
the anchor-first docking algorithm. All docked configurations were energy minimized using 100
maximum iterations and 1 minimization cycle. Next, the top-2000 molecules were selected for further

Molecules 2008, 13
2435
analyses. These molecules were re-scored by the scoring function of AutoDock3.0 [20]. Based on the
second scoring results, 150 molecules were selected from the database. We purchased 95 available
molecules for further bioassay.
Calcium Mobilization Assay
CHO cells stably expressing CCR5 and Gα16 were loaded with 2 μmol/L Fluo-4 AM in Hanks
balanced salt solution (HBSS, containing KCl 5.4 mM, Na2HPO4 0.3 mM, KH2PO4 0.4 mM, NaHCO3
4.2 mM, CaCl2 1.3 mM, MgCl2 0.5 mM, Mg2SO4 0.6 mM, NaCl 137 mM, BSA 5 g/L, Glucose 5.6
mM, Sulfinpyrazone 250 μM, pH 7.4) at 37 °C for 45 minutes. After the cells being rinsed with the
reaction buffer, 50 μL HBSS containing known antagonists (positive control), compounds of interest
or DMSO (negative control, final concentration 1%) were added. After incubation at room temperature
for 10 minutes, 25 μL RANTES (final concentration 30 nM) was dispensed into the well using a
FlexStation II micro-plate reader (Molecular Devices, Sunnyvale, CA, USA) and intracellular calcium
change was recorded with an excitation wavelength of 485 nm and emission wavelength of 525 nm.
The half maximal inhibitory concentrations (IC50s) of compounds were determined with GraphPad
Prism software by constructing their dose-response curves.
N-((S)-3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexanecarbox-
amide (2). To a solution of (S)-3-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropan-1-amine (9)
(0.11 g, 0.29 mmol) and 4,4-difluorocyclohexanecarboxylic acid (0.06 g, 0.37 mmol) in DCM (5 mL)
was added EDCI (0.07 g, 0.36 mmol), HOBt (0.05 g, 0.37 mmol) and DIPEA (0.15 mL, 0.85 mmol).
The mixture was stirred for 4 h. The reaction mixture washed with brine and dried, then concentrated
in vacuo. The residue was purified by flash column chromatography on silica gel, eluted with a
1
mixture of MeOH/DCM (1:12, v/v), to afford 2 (0.12 g, 76%) as a yellow foam: mp 177-179 °C; H-
NMR (CDCl3) δ 1.65-1.90 (m, 4H), 1.90-2.10 (m, 3H), 2.12-2.30 (m, 4H), 2.40-2.60 (m, 2H), 2.70-
2.85 (m, 4H), 3.20-3.40 (m, 4H), 5.15 (q, 1H), 6.87 (d, 1H), 7.25-7.30 (m, 2H), 7.33-7.39 (m, 2H),
7.42-7.49 (m, 2H), 7.92 (d, 1H), 8.08 (d, 1H), 8.78 (d, 1H); EI-MS m/z 526 (M+), 260 (100%); HRMS
(EI) m/z calcd. C29H33ClF2N4O (M+) 526.2311, found 526.2313.
N-((S)-3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropyl)-4-fluorobenzamide (2a). Prepared
in the same manner as described for 2 by replacing 4,4-difluorocyclohexanecarboxylic acid with 4-
1
fluorobenzoic acid. Yellow foam: mp 77-79 °C; H-NMR(CDCl3) δ 2.12-2.30 (m, 2H), 2.50-2.62 (m,
2H), 2.72-2.90 (m, 4H), 3.15-3.30 (m, 4H), 5.38 (q, 1H), 6.81(d, 1H), 7.10 (t, 2H), 7.29 (m, 1H), 7.38
(m, 4H), 7.45 (dd, 1H), 7.90 (m, 3H), 8.08 (d, 1H), 8.78 (d, 1H); EI-MS 502 (M+), 274 (100%); HRMS
(EI) m/z calcd. C29H28ClFN4O (M+) 502.1936, found 502.1932.
N-((S)-3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropyl)cyclohexanecarboxamide
(2b).
1
Prepared as described for 2 using cyclohexanecarboxylic acid. Yellow solid: mp 153-158 °C; H-
NMR (CDCl3,) δ 1.20-1.35 (m, 4H), 1.40-1.50 (m, 2H), 1.63-1.72 (m, 4H), 1.85-2.04 (m, 3H), 2.41-
2.58 (m, 2H), 2.71-2.87 (m, 4H), 3.20-3.39 (m, 4H), 5.15 (q, 1H), 6.85 (d, 1H), 7.18-7.38 (m, 5H),

Molecules 2008, 13
2436
7.42 (dd, 1H), 7.91 (d, 1H), 8.06 (d, 1H), 8.72 (d, 1H); EI-MS 490 (M+), 274 (100%); HRMS (EI) m/z
calcd. C29H35ClN4O (M+) 490.2499, found 490.2500.
N-((S)-3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-henylpropyl)-4-tert-butylcyclohexanecarbox-
amide (2c). Prepared as described for 2 using 4-tert-butylcyclohexanecarboxylic acid. Yellow solid:
mp 79-82 °C; 1H-NMR (CDCl3) δ 0.78 (9H, d), 1.00 (m, 2H), 1.21 (m, 1H), 1.45-1.65 (m, 4H), 1.80-
2.00 (m, 2H), 2.10 (m, 1H), 2.15-2.25 (m, 2H), 2.45-2.65 (m, 2H), 2.75-2.90 (m, 4H), 3.28-3.40 (m,
4H), 5.15 (q, 1H), 6.87 (d, 1H), 7.23-7.38 (m, 5H), 7.47 (dd, 1H), 7.92 (d, 1H), 8.07 (d, 1H), 8.75 (d,
1H); EI-MS 546 (M+), 274 (100%); HRMS (EI) m/z calcd. C33H43ClN4O (M+) 546.3125, found
546.3127.
N-((S)-3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropyl)-4-oxocyclohexanecarboxamide
(2d). Prepared as described for 2 by replacing the 4,4-difluorocyclohexanecarboxylic acid with 4-oxo-
cyclohexanecarboxylic acid. Yellow solid: mp 56-59 °C; 1H-NMR (CDCl3) δ 1.90-2.22 (m, 7H), 2.25-
2.35 (m, 2H), 2.40-2.60 (m, 4H), 2.75-2.90 (m, 4H), 3.20-3.32 (m, 4H), 5.12 (q, 1H), 6.87 (d, 1H),
7.28-7.40 (m, 5H), 7.45 (dd, 2H), 7.89 (d, 1H), 8.06 (d, 1H), 8.75 (d, 1H); EI-MS 504 (M+), 260
(100%); HRMS (EI) m/z calcd. C29H33ClN4O2 (M+) 504.2292, found 504.2293.
N-((S)-3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropyl)-2-phenylacetamide (2e). To a
solution of (S)-3-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropan-1-amine (9, 0.09 g, 0.24
mmol) in DCM (5 mL) was added 2-phenylacetyl chloride (0.04 g, 0.26 mmol) and pyridine (2 drops),
respectively. The mixture was stirred overnight at 25 °C and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel, eluted with a mixture of MeOH/DCM (1:12,
v/v), to afford 2e (0.11 g, 92%) as a brown solid: mp 69-72 °C; 1H-NMR (CDCl3) δ 1.80-1.94 (m, 2H),
2.35-2.55 (m, 2H), 2.91-3.17 (m, 4H), 3.55-3.65 (m, 6H), 5.05 (q, 1H), 6.94 (d, 1H), 7.22-7.35 (m, 8H),
7.38-7.43 (m, 2H), 7.50 (dd, 1H), 7.79 (d, 1H), 8.18 (d, 1H), 8.75 (d, 1H); EI-MS 498 (M+), 274
(100%); HRMS (EI) m/z calcd. C30H31ClN4O (M+) 498.2186, found 498.2183.
N-((S)-3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropyl)-4-methylbenzenesulfonamide (2f).
Prepared in the same manner as described for 2e by replacing the 2-phenylacetyl chloride with
p-toluenesulfonyl chloride. Yellow solid: mp 132-136 °C; 1H-NMR (CDCl3) δ 2.08-2.15 (m, 2H), 2.32
(s, 3H), 2.80-3.10 (m, 6H), 3.51-3.63 (m, 4H), 4.53 (q, 1H), 6.90 (d, 1H), 7.05-7.19 (m, 7H), 7.45-7.55
(m, 3H), 7.88 (d, 1H), 8.18 (d, 1H), 8.74 (d, 1H); EI-MS 534 (M+), 260 (100%); HRMS (EI) m/z
calcd. C29H31ClN4O2S (M+) 534.1856, found 534.1858.
N-((S)-3-(4-(Pyrimidin-2-yl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexanecarboxamide
(2g). Replacing 4,7-dichloroquinoline with 4-chloropyrimidine, in the same manner as described for 2,
1
2g was prepared as a yellow oil; H-NMR(CDCl3) δ 1.69-2.10 (m, 7H), 2.10-2.30 (m, 4H), 2.40-2.50
(m, 2H), 2.60-2.75 (m, 4H), 3.80-3.97 (m, 4H), 5.10 (q, 1H), 6.51 (t, 1H), 7.25-7.35 (m, 5H), 8.31 (d,
2H); EI-MS 443 (M+), 177 (100%); HRMS (EI) m/z calcd. C24H31F2N5O (M+) 443.2497, found
443.2498.

Molecules 2008, 13
2437
N-((S)-3-(4-(9H-Purin-6-yl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexanecarboxamide (2h).
Replacing 4,7-dichloroquinoline with 6-chloro-9H-purine, in the same manner as described for 2, 2h
1
was prepared as a white soild; H-NMR (CDCl3) δ 1.70-2.20 (m, 11H), 2.40-2.52 (m, 2H), 2.70-2.85
(m, 4H), 4.30-4.50 (m, 4H), 5.10 (q, 1H), 7.25-7.37 (m, 5H), 7.91 (s, 1H), 8.34 (s, 1H); EI-MS 483
(M+), 231 (100%); HRMS (EI) m/z calcd. C25H31F2N7O (M+) 483.2558, found 483.2559.
N-((S)-3-(4-(pyridin-4-yl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexanecarboxamide (2i).
Replacing 4,7-dichloroquinoline with 4-chloropyridine, in the same manner as described for 2, 2i was
prepared as a brown oil; 1H-NMR (CDCl3) δ 1.65-2.20 (m, 9H), 2.25-2.36 (m, 4H), 2.40-2.40 (m, 4H),
3.23-3.42 (m, 4H), 5.12 (q, 1H), 6.87 (d, 2H), 7.24-7.30 (m, 2H), 7.38-7.43 (m, 3H), 8.78 (d, 2H); EI-
MS 442 (M+), 106 (100%); HRMS (EI) m/z calcd. C25H32F2N4O (M+) 442.2544, found 442.2540.
N-((S)-3-(4-(Quinolin-2-yl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexanecarboxamide (2j).
Replacing 4,7-dichloroquinoline with 2-chloroquinoline, in the same manner as described for 2, 2j was
1
prepared as a brown soild: mp 50-53 °C; H-NMR (CDCl3) δ 1.65-2.40 (m, 11H), 2.45-2.63 (m, 2H),
2.70-2.94 (m, 4H), 3.80-3.94 (m, 4H), 5.08 (q, 1H), 6.98 (d, 1H), 7.25-7.38 (m, 6H), 7.58 (t, 1H), 7.63
(d, 1H), 7.73 (d, 1H), 7.95 (d, 1H); EI-MS 492 (M+), 157 (100%); HRMS (EI) m/z calcd. C29H34F2N4O
(M+) 492.2701, found 492.2700.
N-(3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexanecarboxamide
(2k). Replacing (S)-methyl 3-amino-3-phenylpropanoate (5) with (±)-methyl 3-amino-3-
phenylpropanoate (4), in the same manner as described for 2, 2k was prepared as a brown solid: mp
1
177-120 °C; H-NMR (CDCl3) δ 1.67-1.89 (m, 4H), 1.90-2.33 (m, 7H), 2.59-2.65 (m, 2H), 2.80-2.95
(m, 4H), 3.30-3.40 (m, 4H), 5.15 (q, 1H), 6.88 (d, 1H), 7.25-7.30 (m, 2H), 7.33-7.39 (m, 3H), 7.46 (dd,
1H), 7.92 (d, 1H), 8.08 (d, 1H), 8.78 (d, 1H); EI-MS 526 (M+), 260 (100%); HRMS (EI) m/z calcd.
C29H33ClF2N4O (M+) 526.2311, found 526.2310.
(4-(7-chloroquinolin-4-yl)piperazin-1-yl)(4,4-difluorocyclohexyl)methanone (2l). Replacing (S)-3-(4-
(7-chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropan-1-amine (9) with 7-chloro-4-(piperazin-1-
1
yl)quinoline (3a), in the same manner as described for 2, 2l was prepared as a yellow oil; H- NMR
(CDCl3,) δ 1.65-2.02 (m, 5H), 2.16-2.32 (m, 4H), 3.16-3.30 (m, 4H), 3.70-3.90 (m, 4H), 6.87 (d, 1H),
7.50 (dd, 1H), 7.98 (d, 1H), 8.12 (d, 1H), 8.76 (d, 1H); EI-MS 393 (M+), 217 (100%); HRMS (EI) m/z
calcd. C20H22ClF2N3O (M+) 393.1419, found 393.1417.
7-Chloro-4-(piperazin-1-yl)quinoline (3a). A mixture of 4,7-dichloroquinoline (0.5 g, 2.5 mmol),
piperazine (2.5 g, 12.6 mmol), K2CO3 (0.1 g, 0.72 mmol), triethylamine (0.1 mL, 0.71 mmol) in N-
methyl-2-pyrrolidinone (10 mL) was stirred under nitrogen at 135 °C for 4 h. After cooling to room
temperature, the mixture was diluted with CH2Cl2 (20mL) and washed with brine. The organic layer
was separated, dried, filtered and condensed. The residue was purified by flash column
chromatography on silica gel, eluted with a mixture of MeOH/EtOAc (1:4, v/v), to afford 3a (0.33 g,
54%) as a white solid: mp 113-114 °C ; 1H-NMR (CDCl3): δ 3.08 (m, 8H), 6.85 (d, 1H), 7.40 (dd, 1H),
7.91 (d, 1H), 8.09 (d, 1H), 8.70 (d, 1H).

Molecules 2008, 13
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(±)-Methyl 3-amino-3-phenylpropanoate (4). Benzaldehyde (5 g, 47.2 mmol) and NH4OAc (7.3 g,
94.8 mmol) were dissolved in EtOH (50 mL). The solution was stirred at 45 °C for 12 h and then was
added to a solution of malonic acid (4.9 g, 47.1 mmol) in EtOH (25 mL). The mixture was stirred
overnight at 60 °C and then at reflux for 6 h. The reaction mixture was cooled to 5 °C. The resulting
precipitate was collected by filtration and washed with ice-cold EtOH. The white solid was dried in
vacuo, to give 3-amino-3-phenylpropionic acid. The acid was dissolved in MeOH (25 mL) and cooled
to 5 °C. To this mixture was added dropwise SOCl2 (2.5 mL) and the mixture was stirred overnight at
r.t. The solution was refluxed for 2h and subsequently concentrated to dryness. To the white residue
was added 2 M Na2CO3 (20 mL) and the mixture was extracted with EtOAc (3 x 20 mL). The
combined organic layers were dried over Na2SO4 and concentrated in vacuo, to give 4 (3.88 g, 46%) as
white oil. 1H-NMR (CDCl3) δ 1.82 (s, 2H), 2.70 (d, 2H), 3.70 (s, 3H), 4.45 (t, 1H), 7.28 (m, 1H), 7.38
(m, 4H).
(S)-Methyl 3-amino-3-phenylpropanoate (5). A solution of 4 (1 g, 5.6 mmol) in MeOH (5 mL) was
added to a refluxing soln. of (+)-L-tartaric acid (0.84 g, 5.6 mmol) in MeOH (6 mL). The product
crystallized overnight at -20 °C and was filtered, to give tartrate of 5 as a white powder, mp 174-176
°C (Lit. [24] 170.6-171.6 °C). The tartrate was dissolved in 1 N aq. NaOH, then was extracted with
CHCl3 (3 x 10 mL). The organic layer was dried with Na2SO4 and concentrated, to give 5 (0.30 g, 60%)
as a colorless oil. [α]28D = -15.6 (c = 1.13 in CH2Cl2) (Lit. [24] [α]22D = -20.3 (c = 1.53 in CHCl3)); 1H-
NMR (CDCl3) δ 2.85 (m, 1H), 2.95 (m, 1H), 3.68 (s, 3H), 4. 52 (dd, 1H), 7.16-7.48 (m, 5H).
tert-Butyl (S)-2-(methoxycarbonyl)-1-phenylethylcarbamate (6). To a cold (0 °C) mixture of Na2CO3
(2.8 g, 26.5 mmol) in THF/H2O (5 mL, 2:1, v/v) was added 5 in one portion (0.85 g, 4.7 mmol). A
solution of (BOC)2O (1.2 g, 5.5 mmol) in THF (4 mL) was added in one portion and the reaction
stirred at 0 °C for 1 h, then at 25 °C for 2 h, extracted with EtOAc (3 x 20 mL). The combined organic
extracts were dried and concentrated in vacuo. The crude white solid product 6 was used in the next
step without further purification.
tert-Butyl (S)-2-formyl-1-phenylethylcarbamate (7). To a soln. of 6 (1.5 g, 5.4 mmol) in DCM (10 mL)
cooled to -78 °C was added DIBAL-H (8.7 mL of 25% W/W in hexane, 12.0 mmol) dropwise at a rate
that maintained the temperature below -70 °C. After 2 h the reaction was quenched by the addition of
MeOH (1.7 mL) and H2O (1 mL) at -78 °C, then allowed to warm to 25 °C. The mixture was filtered.
The filtrate was dried and concentrated in vacuo. The crude colorless oil 7 (0.76 g, 56%) was used in
the next step without further purification. 1H-NMR (CDCl3) δ 1.50 (s, 9H), 2.85-3.05 (m, 2H), 5.10 (s,
1H), 7.25-7.40 (m, 5H), 9.80 (s, 1H).
Tert-butyl (S)-3-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropylcarbamate (8). To a solution
of 7 (0.25 g, 1 mmol), 3a (0.25 g, 1 mmol) and HOAc (0.14 mL) in DCM (10 mL) was added
NaBH(OAc)3 (0.26 g, 1.2 mmol) in one portion and the mixture was stirred for 5 h. The reaction was
quenched by adding 10% K2CO3 soln. (20 mL) and stirred for an additional 30 min, extracted with
DCM (3 x 20 mL). The combined organic layer was dried and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel, eluted with a mixture of MeOH/DCM (1:12,

Molecules 2008, 13
2439
v/v), to afford 8 (0.32 g, 67%) as a yellow foam. 1H-NMR (CDCl3) δ 1.35 (s, 9H), 2.15 (m, 2H), 2.61
(m, 2H), 2.91 (m, 4H), 3.35 (s, 4H), 4.69 (m, 1H), 6.80 (d, 1H), 7.22-7.32 (m, 5H), 7.40 (dd, 1H), 7.81
(d, 1H), 8.08 (d, 1H), 8.65 (d, 1H).
(S)-3-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-1-phenylpropan-1-amine (9). A solution of 8 (0.19 g,
0.40 mmol) in saturated HCl in MeOH (6 mL) heated at 65 °C for 2 h. The MeOH was evaporated
under reduced pressure and the residue cautiously partitioned between DCM (20 mL) and 20% K2CO3
solution (20 mL). The aqueous layer was re-extracted with DCM (3 x 20mL). The combined organic
layer was dried and concentrated in vacuo, to afford 9 (0.14 g, 93%) as a straw yellow oil which was
used in the next step without further purification. 1H NMR (CDCl3) δ 1.98-2.12 (m, 2H), 2.58 (m, 2H),
2.81 (m, 4H), 3.25 (m, 4H), 4.18 (m, 1H), 6.81 (d, 1H), 7.20 (t, 1H), 7.32 (t, 2H), 7.35-7.44 (m, 3H),
7.90 (d, 1H), 8.05 (d, 1H), 8.68 (d, 1H).
Acknowledgements
We gratefully acknowledge the financial support from the 863 Hi-Tech Program of China (Grants
2006AA020404, 2006AA01A124), and the Shanghai Rising-Star Program (A type) of Shanghai
Ministry of Science and Technology (Grant 07QA14013). We also thank Shanghai Supercomputing
Center (http://www.ssc.net.cn/) for providing the computational resources.
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© 2008 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).