Design, synthesis, and biological evaluation of novel 2-methylpiperazine derivatives as potent CCR5 antagonists

Article abstract of DOI:10.1016/j.bmc.2014.12.052

Three series of novel 2-methylpiperazine derivatives were designed and synthesized using a fragment-assembly strategy. Among them, six compounds (13, 16, 18, 22, 33, and 36) showed potent activity against CCR5 comparable to that of the positive control, maraviroc, in calcium mobilization assay. Moreover, some compounds were selected and further tested for their antiviral activity in HIV-1 single cycle assay. As a result, four compounds (13, 16, 33, and 36) showed antiviral activity at the nanomolar level. Additionally, the potent four compounds showed no cytotoxicity at a concentration of 10 μM.

Full text of DOI:10.1016/j.bmc.2014.12.052

Accepted Manuscript
Design, Synthesis, and Biological Evaluation of Novel 2-methylpiperazine de-
rivatives as Potent CCR5 Antagonists
Suwen Hu, Zhilong Wang, Tingjun Hou, Xiaodong Ma, Jing Li, Tao Liu, Xin
Xie, Yongzhou Hu
PII:
S0968-0896(14)00910-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.052
BMC 11984
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 October 2014
19 December 2014
20 December 2014
Please cite this article as: Hu, S., Wang, Z., Hou, T., Ma, X., Li, J., Liu, T., Xie, X., Hu, Y., Design, Synthesis, and
Biological Evaluation of Novel 2-methylpiperazine derivatives as Potent CCR5 Antagonists, Bioorganic &
Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.12.052
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Design, Synthesis, and Biological Evaluation of Novel 2-methylpiperazine derivatives as Potent
CCR5 Antagonists
Suwen Hua†, Zhilong Wangb†, Tingjun Houa,c, Xiaodong Maa, Jing Lib, Tao Liua*, Xin Xieb* ,Yongzhou
Hua*,
a. ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, China
b. State Key Laboratory of Drug Research, National Center for Drug Screening, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
c. Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou, Jiangsu
215123, China.
† These authors contributed equally to this work
*Corresponding author: Tao Liu: Tel: +86-571-88208458 Fax: +86-571-88981051
Email: Lt601@zju.edu.cn
Xin Xie: Tel. & Fax: +86-21-50801313 E-mail: xxie@mail.shcnc.ac.cn
Yongzhou Hu: Tel. & Fax: +86-571-88208460 E-mail: huyz@zju.edu.cn
Abstract
Three series of novel 2-methylpiperazine derivatives were designed and synthesized using a
fragment-assembly strategy. Among them, six compounds (13, 16, 18, 22, 33, and 36) showed potent
activity against CCR5 comparable to that of the positive control, maraviroc, in calcium mobilization
assay. Moreover, some compounds were selected and further tested for their antiviral activity in HIV-1
single cycle assay. As a result, four compounds (13, 16, 33, and 36) showed antiviral activity at the
nanomolar level. Additionally, the potent four compounds showed no cytotoxicity at a concentration of
10 µM.
Keywords: CCR5 antagonist; Anti-HIV-1 agent; 2-Methylpiperazine derivatives

1. Introduction
Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV),
is one of the fatal diseases threatening human. Although highly active antiretroviral therapy (HAART),
a combination of drugs targeting HIV-1 protease and reverse transcriptase, has been successful in
reducing HIV-1-associated mortality and morbidity, many patients are still suffered from long term
toxicity, incomplete efficacy, and the eventual emergence of resistance. 1 Thereby, it seems urgent to
develop novel classes of antiretroviral agents with superior potency and less toxicity.
The chemokine receptor 5 (CCR5), a member of the seven-transmembrane G-protein coupled
receptor family, serves a critical role for the entrance of R5-tropic HIV-1 into host cells.2As such, the
close association between CCR5 and HIV-1 that has been observed provides a foundation for novel
methods aimed at controlling HIV-1 infection.3 This, in turn,has stimulated a great deal of effort in the
identification of new CCR5 antagonists. As a result, several novel CCR5 antagonistshave become
available. For example, maraviroc (1, Fig. 1) was the first marketed small molecular CCR5 antagonist4
andTAK-220 (2, Fig. 1), which was developed by the Takeda Pharmaceutical Co. Ltd., has potent
anti-HIV-1 activity at nanomolar concentrations.5
In our previous work, we designed a series of novel piperazinederivatives.6Among themwas a
CCR5 antagonist containing a 4-cyanophenylpiperazin moiety with an IC50 value of 6.29 µM(3,
Fig.1).Furtherresearch resulted in the identification of two additionalpiperazine derivatives with CCR5
antagonist activity, 4 and 5(see Fig. 2).These two additional compounds were designed using a
fragment-assembly strategy(Fig.2) combining the structural features of TAK-220and compound 3(Part
A, 2; Part B,3). Interestingly, evaluation of the CCR5 antagonist activity in a calcium mobilization
assay revealed that the 2-methylpiperazin derivative 5(IC50 = 163.8 nM) exhibited better CCR5
inhibitor activity thanderivative 4 (IC50 = 239.5 nM), which displayed only moderateactivity.
In order to further explore the structure–activity relationship between 2-methylpiperazin
derivatives and potential CCR5 antagonists that may havetherapeutic potential, compound 5 was
chosen as our lead compound; we subsequently designed three series of target compounds. First, we
replaced the substituent (R1) at the 4-position of the 2-methylpiperazine ring in compound 5 with a
variety of aromatic rings. Then, we focused on modifyingthe terminal group tethered to the amide
moiety (R4). Finally, the 2-methylpiperazine moiety was replaced with aheptatomic ring and bridged
ring. Herein, we report the synthesis and biological evaluation of these novel 2-methylpiperazin

derivatives.
2. Results and discussion
2.1. Chemistry
The required N-substituted piperazines7a-k and 9 were synthesized as depicted in scheme 1.
Specifically, N-alkylation of the 2-methylpiperazine 6a with a substituted benzyl chloride in the
presence of potassium carbonate afforded the 1-benzyl-3-methylpiperazine analogues 7a-f. In contrast,
treatment of2-methylpiperazine 6a with 4-fluorobenzoyl chloride or 4-fluorobenzenesulfonyl chloride
resulted in the generation ofthe benzamide7g and sulfonamide 7h, respectively. Moreover, reacting
4-fluorobenzaldehyde with homopiperazine6b, (R)-2-methylpiperazine 6c, (S)-2-methylpiperazine 6d
or(1S,4S)-2,5-diazabicyclo[2.2.1]heptanes8 in the presence of sodium triacetoxyborohydride led to the
formation of compounds7i, 7j, 7k, and 9, respectively.
The preparation of target compounds 13-22, 26-29 and 32-36is outlined in schemes2 and 3.
N-alkylation of anilines 10a-c with 1-bromo-3-chloropropane in the presence of potassium iodide
under microwave irradiation provided the corresponding substituted secondary amines 11a-c.
Subsequently, acylation of 11a-c with 1-acetylpiperidine-4-carbonyl chloride led to formation ofthe
amides 12a-c. Furthermore, we used theN-substituted 2-methylpiperazine analogues 7a-hto react with
the amides 12a-c in the presence of potassium iodide and potassium carbonate in orderto produce the
target compounds 13-22(scheme 2).
Coupling of 3-chloro-4-methylaniline 10a with different carboxylic acids gave amides 23a-e.
Afterward, N-alkylation of 23a-e with 2-(2-bromoethyl)-1, 3-dioxolane in the presence of sodium
hydride afforded the dioxolane derivatives 24a-e, which were subsequently treated with hydrochloric
acid to produce the aldehydes 25a-e. Finally, treatment of the aldehydes 25a-d with
1-(4-fluorobenzyl)-3-methylpiperazine 7d in the presence of sodium triacetoxyborohydride generated
target compounds 26-29.
The acid chloride 307 was reacted with 3-chloro-N-(3-chloropropyl)-4-methylaniline 11a to
provide 31, then the reaction of compound 31 with 1-(4-fluorobenzyl)-3-methylpiperazine 7d yielded
compound 32.Compounds 33-36 were prepared from aldehydes25e and 9 or 7i-k with the same
synthetic route as compounds 26-29 (scheme 3).
2.2. Calcium mobilization assay

All compounds were evaluated for antagonistic activity of RANTES induced CCR5 calcium ion
mobilization using maraviroc as the positive control.No CCR5 agonist activitywas observed for any of
the test compounds.Furthermore, we tested all compounds for purity, ensuring that all compounds were
greater than 95% pure. The results are summarized in Table 1, 2, and 3.
As shown in Table 1, most of the tested compounds displayed potent CCR5antagonism in
thisseries. Among them, compounds 13-18, which contained a benzyl moiety at the 4-position of the
2-methylpiperazine ring, exhibited improved activities compared to the reference structure 5 (IC50
=
163.8 nM). In particular, compound 16(IC50 = 6.6 nM) with a 4-fluorobenzyl group had the highest
CCR5 binding affinity of all the compounds tested. Compound 13 (IC50 = 8.3 nM) with a
4-cyanobenzyl group showed similar potency compared to 16. However, when a benzyl or
4-methylbenzyl group was placed in the R1position, the potency was reduced compared to compound
16. This is demonstrated by the activity of compound 14(IC50 = 15.1 nM) and15(IC50 = 25.1 nM),
respectively, and may indicate that an appropriate orientation of the electron-withdrawing group might
be required for high potency. Changing the position of the fluorine atomon the phenyl ring (17, o-F:
IC50 = 30.1 nM and18, m-F: IC50 = 11.7 nM) also resulted in a loss of potency compared to 16.
Furthermore, replacement of the 4-fluorobenzyl group in 16 with a 4-fluorobenzoyl group (21: IC50 =
30.0 nM) led to a 4-fold decrease in potency, while the alteration of the 4-fluorobenzyl group to a
4-fluorobenzenesulfonyl group (22: IC50 = 10.9 nM) only had a slight loss of potency. This suggests
that compounds with a 4-fluorobenzyl group might be more effective to enhance CCR5 activity.
The substituent found at the central phenyl moiety (R2, R3)of the target compounds alsoaffected
the antagonist activity for CCR5. For example, compound 19, which lacks asubstituent on the central
phenyl ring (IC50 = 319.9 nM), showed a 48-fold decrease in potency compared tocompound16.
Furthermore, compound 20 (IC50 = 25.1 nM),which contains only achlorine atom at the 3-position of
the central phenyl ring, displayed an approximate 3-fold loss in potency compared to that of compound
16. These results suggest that a 3-chloro-4-methylphenyl group might be beneficial forretaining high
potency for CCR5.
Table 2 summarizes the activity of compounds obtained via alteration of the terminal group
tethered to the amide moiety (R4). Compounds 26-29 and 32 in this series showed lower activity than
that of compound 16, which contains a 1-acetylpiperidine-4-yl moiety at the R4 position. Compound 26
(IC50 = 2158 nM), which contains a methyl group at the R4 position, displayed a drastic loss in potency

compared to compound16. Although the 4-fluorophenyl group found at the R4 position of compound
27(IC50 = 204.1nM) partly compensated for the loss of CCR5 antagonism observed for compound 26, it
was still considerably less potent than 16--indicating that the 1-acetylpiperidine moiety might be
important for retaining potency. We also examined structural alterations at the piperidine ring to
determine the optimum replacement of alternate moieties. Replacing the 1-acetylpiperidine-4-yl moiety
on 16 with a 1-acetylpiperidine-3-yl moiety (28, IC50 = 259.7 nM) or 1-(methylsulfonyl)piperidine
moiety (29, IC50 = 17.1nM) led to a decrease in potency compared to compound 16. In addition,
introduction of a benzoylpiperidine-4-yl moiety to replace the 1-acetylpiperidine-4-yl moiety on 16
resulted in a one-fold reduction in potency (32, IC50 = 33.2 nM), suggesting that a bulky group at the
para-position reduces the activity for CCR5 as seen in compound32compared to compound16. These
data provide strong evidence that the 1-acetyl piperidine moiety is vital for maintaining high activity.
The results observed due to an alteration of the 2-methylpiperazine moiety are summarized in
Table 3. Compound 33 (IC50 = 8.8 nM), containing a (1S, 4S)-2, 5-diazabicyclo [2.2.1]heptane group
showed a slight loss in activity compared to 16. Additionally, the introduction of a homopiperazine
moiety (34, IC50 = 906.0 nM) to replace the 2-methylpiperazine group of compound16 resulted in a
137-fold decrease in potency. This indicates that the 2-methylpiperazine moiety might be used as an
appropriate basic center. Subsequently, we investigated the chiral center on the 2-methylpiperazine
moiety. It has been demonstrated that the S-enantiomer (36: IC50 = 3.0 nM) exhibits much higher
activity than the R-enantiomer (35: IC50 = 49.9 nM). The difference in activity between the two
compounds (35 and 36) might stem from the conformational restriction of the methyl group of the
2-methylpiperazine moiety.
2.3. Viral infectivity assays
In an attempt to assess the antagonistic activities of the target compounds against a viral infection,
the most potent compounds 13, 16, 33, and 36 were selected for further analysis using the anti-HIV-1
single cycle antiviral assay. As shown in Table 4, all of the tested compounds showed
nanomolarantagonistic activities. The S-Enantiomer 36 showed
a
one-fold increase
inantagonisticactivity compared to its raceme 16, an observation thatis consistent with that of the CCR5
antagonism results.
2.4. Cell Cytotoxicity Assays
In order to evaluate cytotoxicity of the most potent compounds 13, 16, 33, and 36, we treated

human macrophage cells with 10 µM of each compound and then utilized the CCK-8 assay to measure
inhibition rates(data and methodsareincludedin supplementarymaterials).
3. Conclusions
In summary, three series of novel 2-methylpiperazine derivatives were designed and
synthesizedby using a fragment-assembly strategy. The CCR5 antagonistic activities of the target
compounds were evaluated based on a calcium mobilization assay.Several compounds showed
promising activities, with IC50 values ranging from 3.0 to 11.7 nM. Among them, six compounds (13,
16, 18,22,33,and36) showed better activity in comparison with that of the positive control, maraviroc.
The most potent compounds, as identified by the calcium mobilization assay, were selected for further
antiviral evaluation. As a result, four compounds showedpotent efficacy at the nanomolar level (13:
IC50 = 47.2 nM, 16: IC50 = 140.6 nM,33: IC50 = 31.4 nM and 36: IC50 = 75.1 nM, respectively).
Additionally, the four compounds chosen for additional study (13, 16, 33, and 36) showed no
cytotoxicityat a concentration of 10 µM by the CCK-8 assay. We believe that the results of this research
will provide a foundation that will prove useful in the further design and optimization of novel small
molecular CCR5 antagonists.
4. Experimental protocols
4.1. Chemistry
4.1.1 General procedures
Melting points were obtained on a B-540 Buchi melting point apparatus which were uncorrected.
1H- NMR and 13C-NMR spectra were recorded on a Bruker Acance III instrument at 500 MHz
(chemical shifts were expressed as δ values relative to TMS as internal standard). Mass spectra (MS)
and ESI (positive) were recorded on an Esquire-LC-00075 spectrometer. High-resolution mass spectra
(HRMS) were measured by Agilent 6224 LC/MS.
4.1.2 Synthesis of 1, 3-disubstituted piperazine precursors (7a-f): general procedure
A mixture of 2-methylpiperazine (5.00 mmol), substituted benzyl chlorides (5.00 mmol) and K2CO3
(7.50 mmol) in DMF (10.00 ml) was stirred at reflux for 12h. The mixture was concentrated in vacuo,
diluted with water (20.00 ml) and extracted with EtOAc (15.00 ml×3). The organic layer was dried
(MgSO4), filtered and concentrated in vacuo. The residue was purified by column chromatography on

silica gel (EtOAc/PE 1/2 to 1/0) to afford corresponding compounds 7a-f.
4.1.2.1 ( )-4-((3-methylpiperazin-1-yl) methyl)benzonitrile (7a).
According to the general
procedure, the reaction of ( )-2-methylpiperazine with 4-(chloromethyl)benzonitrile produced
1
compound 7a (0.88g, 82%) as yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.60-7.59 (d, J =8Hz, 2H,
Ar-H), 7.45-7.43 (d, J =8Hz, 2H, Ar-H), 3.52 (brs, 2H, CH2), 2.98-2.85 (m, 3H), 2.70-2.68 (d, J =11Hz,
2H), 2.09-2.03 (m, 1H), 1.89-1.71 (m, 2H), 1.03-1.02 (m, 3H, CH3). ESI-MS m/z: 216 [M+H]+.
4.1.2.2 ( )-1-benzyl-3-methylpiperazine (7b). According to the general procedure, the reaction of
( )-2-methylpiperazine with (chloromethyl)benzene produced compound 7b (0.80g, 85%) as yellow oil.
1H-NMR (CDCl3, 500 MHz) δ: 7.50-7.32 (m, 5H, Ar-H), 3.51 (s, 2H), 2.97-2.85 (m, 3H), 2.71-2.67 (m,
2H), 2.09-2.05 (m,1H), 1.89-1.71 (m, 2H), 1.03-1.02 (m, 3H). ESI-MS m/z: 191 [M+H]+.
4.1.2.3 ( )-3-methyl-1-(4-methylbenzyl)piperazine (7c). According to the general procedure, the
reaction of ( )-2-methylpiperazine with 1-(chloromethyl)-4-methylbenzene produced compound 7c
(0.87g, 86%) as yellow oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.19-7.17 (d, J =8Hz, 2H, Ar-H), 7.10-7.09
(d, J =8Hz, 2H, Ar-H), 3.42 (s, 2H), 2.90-2.81 (m, 3H), 2.75-2.71 (m, 2H), 2.31 (s, 3H, CH3), 1.96-1.93
(m, 1H), 1.64-1.62 (m, 2H), 0.99-0.95 (m, 3H). ESI-MS m/z: 205 [M+H]+.
4.1.2.4 ( )-1-(4-fluorobenzyl)-3-methylpiperazine (7d). According to the general procedure, the
reaction of ( )-2-methylpiperazine with 1-(chloromethyl)-4-fluorobenzene produced compound 7d
1
(0.81 g, 78%) as white oil. H-NMR (CDCl3, 500 MHz) δ: 7.27-7.24 (d, J =8Hz, 2H, Ar-H), 6.99-6.96
(d, J =8Hz, 2H, Ar-H), 3.42 (s, 2H, CH2), 2.94-2.81 (m, 3H), 2.72-2.70 (d, J =11Hz, 2H), 2.00-1.95 (m,
1H), 1.65-1.55 (m, 2H), 0.99-0.98 (m, 3H, CH3). ESI-MS m/z: 209 [M+H]+.
4.1.2.5 ( )-1-(2-fluorobenzyl)-3-methylpiperazine (7e). According to the general procedure, the
reaction of ( )-2-methylpiperazine with 1-(chloromethyl)-2-fluorobenzene produced compound
1
7e(0.87 g, 84%) as white oil. H-NMR (CDCl3, 500 MHz) δ: 7.46-7.44 (m, 1H, Ar-H), 7.34-7.32 (m,
1H, Ar-H), 7.23-7.15 (m, 2H, Ar-H), 3.58 (s, 2H), 2.97-2.86 (m, 3H), 2.79-2.76 (m, 2H), 1.96 (brs, 2H),
1.02-0.99 (m, 3H). ESI-MS m/z: 209 [M+H]+.
4.1.2.6 ( )-1-(3-fluorobenzyl)-3-methylpiperazine (7f). According to the general procedure, the
reaction of ( )-2-methylpiperazine with 1-(chloromethyl)-2-fluorobenzene produced compound 7f
(0.81 g, 78%) as white oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.46-7.44 (m, 1H, Ar-H), 7.33-7.32 (m, 1H,
Ar-H), 7.23-7.14 (m, 2H, Ar-H), 3.58 (s, 2H, CH2), 2.94-2.85 (m, 3H), 2.79-2.75 (m, 2H), 2.13-2.08
(m,1H), 1.85-1.76 (m, 2H), 1.02-1.00 (m, 3H). ESI-MS m/z: 209[M+H]+.

4.1.3 Synthesis of ( )-(4-fluorophenyl)(3-methylpiperazin-1-yl)methanone (7g). To an ice-cooled
stirred suspension of ( )-2-methylpiperazine (0.32 g, 3.20 mmol) in DCM (10.00 mL) was added Et3N
(0.41 mL, 3.20 mmol) dropped with 4-fluorobenzoyl chloride (0.47 g, 3.00 mmol) in DCM (5.00 mL),
and the mixture was stirred at room temperature for 3 h. Then it was concentrated in vacuo and
water(20.00 mL) was added to it. The residue was extracted by DCM (20 ml×3), the organic layer was
combined, then washed with brine (10.00 mL), dried (MgSO4), filtered and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (EtOAc /PE 3/1) to afford the title
1
compound 7g (0.56 g, 84%) as a light yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.40-7.38 (m, 2H,
Ar-H), 7.10-7.06 (m, 2H, Ar-H), 3.60 (s, 2H), 3.00 -2.98 (m, 3H), 2.30-2.28 (m, 1H), 1.89-1.86 (m,1H),
1.49 (brs, 1H), 1.01-0.99 (m, 3H). ESI-MS m/z: 223 [M+H]+.
4.1.4 Synthesis of ( )- 1-(4-fluorophenylsulfonyl)-3-methylpiperazine (7h). To an ice-cooled
stirred suspension of ( )-2-methylpiperazine (0.32 g, 3.20 mmol) in DCM (10.00 mL) was added Et3N
(0.41 mL, 3.20 mmol) dropped with 4-fluorobenzene-1-sulfonyl chloride (0.58 g, 3.00 mmol) in DCM
(5.00 mL), and the mixture was stirred at room temperature for 3 h. Then it was concentrated in vacuo
and water (20.00 mL) was added to it. The residue was extracted by DCM (20 ml×3), the organic layer
was combined, then washed with brine (10.00 mL), dried (MgSO4), filtered and concentrated in vacuo.
The residue was purified by column chromatography on silica gel (EtOAc /PE 1/1) to afford the title
compound 7h. 1H-NMR (CDCl3, 500 MHz) δ: 7.75-7.74 (m, 2H, Ar-H), 7.20-7.17 (m, 2H, Ar-H), 3.57
(s, 2H), 2.97-2.88 (m, 3H), 2.26-2.25 (m, 1H), 1.88-1.84 (m,1H), 1.46 (brs, 1H), 1.01-0.99 (m, 3H).
ESI-MS m/z: 259 [M+H]+.
4.1.5 1-(4-fluorobenzyl)-1, 4-diazepane (7i).
To a stirred mixture of homopiperazine (0. 60g,
6.00mmol), 4-fluorobenzaldehyde (0. 60g, 5.00mmol) in DCM (15ml) was added AcOH (0.08 ml, 0.
50mmol) and the mixture was stirred at room temperature for 0.5 h. Then Sodium
triacetoxyborohydride (1.60g, 7.50mmol) was added in portions. The reaction was stirred at the same
temperature for 6h. Finally, the mixture was diluted with saturated aqueous NaHCO3 (15.00ml)
followed by water (15.00ml) and extracted with EtOAc (3×15.00ml). The organic layer was dried
(MgSO4), filtered, and concentrated in vacuo. The residue was purified by column chromatography on
1
silica gel (EtOAc/PE 5/1) to afford yellow oil (0.90g, yield 77%). H-NMR (CDCl3, 500 MHz) δ:
7.25-7.22 (m, 2H, Ar-H), 6.95-6.92 (m, 2H, Ar-H), 5.15 (brs, 1H), 4.82 (brs, 1H), 3.55-3.53 (m, 2H),
3.48-3.45 (m, 1H), 3.02-3.00 (m,1H), 2.95-2.93 (m,1H), 2.65-2.53 (m, 4H), 1.79-1.76 (m, 2H). ESI-MS

m/z: 209 [M+H]+.
4.1.6 (R)-1-(4-fluorobenzyl)-3-methylpiperazine (7j). According to the procedure of synthesis of
1-(4-fluorobenzyl)-1, 4-diazepane 7i, the reaction of (R)-2-methylpiperazine (0. 60g, 6mmol) with
1
4-fluorobenzaldehyde (0.60g, 5mmol) produced compound 8j (1.10g, 86%) as yellow oil. H-NMR
(CDCl3, 500 MHz) δ: 7.26-7.23 (m, 2H, Ar-H), 6.98-6.94 (m, 2H, Ar-H), 3.41 (s, 2H), 2.94-2.85 (m,
2H), 2.84-2.82 (m, 1H), 2.71-2.69 (m,2H), 2.21 (brs, 1H), 2.01-1.95 (m, 1H), 1.67-1.62 (m, 1H),
1.00-0.98 (d, J=6.5Hz, 3H). ESI-MS m/z: 209 [M+H]+.
4.1.7 (S)-1-(4-fluorobenzyl)-3-methylpiperazine (7k).
According to the procedure of synthesis of
1-(4-fluorobenzyl)-1, 4-diazepane 7i, the reaction of (S)-2-methylpiperazine (0.60g, 6mmol) with
4-fluorobenzaldehyde (0. 60g, 5mmol) produced compound 7k (1.00g, yield 84%) as yellow oil.
1H-NMR (CDCl3, 500 MHz) δ: 7.26-7.23 (m, 2H, Ar-H), 6.98-6.94 (m, 2H, Ar-H), 3.41 (s, 2H),
2.93-2.87 (m, 2H), 2.85-2.80 (m, 1H), 2.71-2.68 (m, 2H), 1.99-1.94 (m, 1H), 1.76 (brs, 1H), 1.64-1.60
(m, 1H), 0.98-0.97 (d, J=6.5Hz, 3H). ESI-MS m/z: 209 [M+H]+.
4.1.8 (1S,4S)-2-(4-fluorobenzyl)-2, 5-diazabicyclo [2.2.1] heptanes (9). According to the procedure
of synthesis of 1-(4-fluorobenzyl)-1, 4-diazepane 7i, the reaction of (1S,4S)-2,5-diazabicyclo
[2.2.1]heptanes 8 (0. 6g, 6mmol) with 4-fluorobenzaldehyde (0. 60g, 5.00mmol) produced yellow oil
(0.70g, yield 69%). 1H-NMR (CDCl3, 500 MHz) δ: 7.31-7.28 (m, 2H, Ar-H), 7.00-6.97 (m, 2H, Ar-H),
4.23 (s, 2H, CH2), 3.69-3.68 (m, 2H), 3.49-3.41 (m, 2H), 3.17-3.13 (m,1H), 2.89-2.82 (m,1H),
1.86-1.83 (m, 1H), 1.72-1.64 (m, 1H). ESI-MS m/z: 207 [M+H]+.
4.1.9 Synthesis of 3,4-disubstituted-N-(3-chloropropyl)aniline (11a-c): general grocedure
A mixture of anilines 10a-c (9.00 mmol), 1-bromo-3-chloropropane (0.49 g, 3.00 mmol), and KI
o
(0.05 g, 0.30 mmol) in MeCN (5.00 ml) was reacted in microwave reactor at 110 C for 15 minutes.
The solvent was evaporated in vacuo, diluted with water (30 ml), and extracted with EtOAc (30 ml×3).
The combined organic layer was dried (MgSO4) and filtered, then the solvent was evaporated in vacuo.
The residue was purified by column chromatography on silica gel to yield corresponding compounds
11a-c.
4.1.9.1 3-chloro-N-(3-chloropropyl)-4-methylaniline (11a). Yellow oil (0.49g, 75%). 1H-NMR
(CDCl3, 500 MHz) δ: 7.52 (d, J = 2.3 Hz, 1H), 7.50 (d,J = 8.3 Hz, 1H), 7.38 (m, 1H), 3.73 (m, 2H),
3.53 (2H, m), 2.40 (s, 3H, CH3), 2.23 (m, 2H,). ESI-MS m/z: 219 [M+H]+.
4.1.9.2 N-(3-chloropropyl) aniline (11b). Yellow oil (0.34g, 67%).1H-NMR (CDCl3, 500 MHz)δ:

7.36-7.18 (m, 2H, Ar-H), 7.16-6.99 (m, 3H, Ar-H), 3.68-3.65 (m, 2H, CH2), 3.60-3.56 (m, 2H, CH2),
3.03-2.99 (m, 2H, CH2). ESI-MS m/z: 170 [M+H]+.
1
4.1.9.3 3-chloro-N-(3-chloropropyl)aniline (11c). Yellow oil (0.36g, 60%). H-NMR (CDCl3,
500 MHz) δ: 7.34-7.20 (m, 2H, Ar-H), 7.18-7.00 (m, 2H, Ar-H), 3.67-3.64 (m, 2H, CH2), 3.57-3.53 (m,
2H, CH2), 3.05-2.98 (m, 2H, CH2). ESI-MS m/z: 204 [M+H]+.
4.1.10 1-acetyl-N-(3,4-disubstitutedphenyl)-N-(3-chloropropyl)piperidine-4-carboxamide (12a-c):
general grocedure.
To an ice-cooled stirred suspension of anilines 11a-c (1.00 mmol) in DCM (3.00 mL) was added
Et3N (0.40 mL, 3.00 mmol) followed by 1-acetylpiperidine-4-carbonyl chloride (0.23g, 1.20 mmol),
and the mixture was stirred at room temperature for 5 h. Then it was poured to a saturated solution of
sodium bicarbonate (10.00 mL). The residue was extracted by EtOAc (20.00 ml×3), the organic layer
was combined, then washed with water (15.00 ml×3) and brine (10.00 mL), dried (MgSO4), filtered
and concentrated in vacuo. The residue was purified by column chromatography on silica gel to afford
the title compound.
4.1.10.1 1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-chloropropyl)piperidine-4-carboxamide (12a).
White solid (88%), mp: 112-114 °C.1H NMR (CDCl3, 500 MHz) δ: 7.30 (d, J = 7.7 Hz, 1H), 7.16 (d,
J=2.2 Hz, 1H), 6.98 (m, 1H), 4.53 (m, 1H), 3.77 (t, J = 7.1Hz, 2H), 3.65- 3.85 (m, 1H), 3.53 (t, J = 6.6
Hz, 2H), 2.76-2.97 (m, 1H), 2.43 (s, 3H), 2.26-2.49 (m, 2H), 2.03 (s, 3H), 1.52-2.12 (m, 6H). ESI-MS
m/z: 372 [M+H]+.
4.1.10.2 1-acetyl-N-(3-chloropropyl)-N-phenylpiperidine-4-carboxamide (12b). White solid
1
(80%); mp: 110-112oC. H-NMR (CDCl3, 500 MHz) δ: 7.48-7.39 (m, 3H, Ar-H), 7.417-7.15 (m, 2H,
Ar-H), 4.50-4.48 (m, 1H), 3.80-3.75 (m, 3H), 3.54-3.52 (t, J = 6.5 Hz, 2H, CH2), 2.79 (brs, 1H),
2.37-2.29 (m, 2H), 2.04 (s, 3H, CH3), 2.02-1.98 (m, 2H), 1.75-1.61 (m, 4H). ESI-MS m/z: 323 [M+H]+.
4.1.10.3 1-acetyl-N-(3-chlorophenyl)-N-(3-chloropropyl) piperidine-4-carboxamide (12c). Brown
1
solid (88%); mp: 115-117 oC. H-NMR (CDCl3, 500 MHz) δ: 7.31-6.98 (m, 4H, Ar-H), 4.55-4.50 (m,
1H), 3.83-3.80 (m, 1H), 3.76 (t, J = 7.0 Hz, 2H, CH2), 3.54 (t, J = 7.0 Hz, 2H, CH2), 2.94-2.81 (m, 1H),
2.44-2.34 (m, 2H), 2.06 (s, 3H, CH3), 2.10-2.06 (m, 2H, CH2), 1.87-1.62 (m, 4H). ESI-MS m/z: 357
[M+H]+.
4.1.11 Synthesis of ( )-1-acetyl-N-(3, 4-disubstituted phenyl)-N-(3-substituted-2-substituted
piperazin- 1-yl)propyl)piperidine-4-carboxamide (13-22): general procedure

A mixture of 1-acetyl-N-(3, 4-disubstitutedphenyl)-N-(3-chloropropyl)piperidine-4-carboxamide
12a-c (0.50 mmol), compounds 7a-h (0.50 mmol), KI (0.05 mmol) and K2CO3 (0.75 mmol) in DMF
(5.00 ml) was stirred at reflux for 16h. The mixture was concentrated in vacuo, diluted with water (20.0
ml) and extracted with EtOAc (15.00 ml×3). The organic layer was dried (MgSO4), filtered and
concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc/PE
1/1 to 1/0) to afford corresponding target compounds 13-22.
4.1.11.1 ( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-cyanobenzyl)-2-methylpiperazin-1-yl)
propyl)piperidine-4-carboxamide (13). The reaction of 7a with 12a produced compound 13 (31.6
mg, 23%) as light yellow oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.58-7.56 (d, J= 8.5Hz, 2H, Ar-H),
7.41-7.40 (d, J= 8.5Hz, 2H, Ar-H), 7.28-7.26 (m, 1H, Ar-H ), 7.15 (m, 1H, Ar-H), 6.95-6.93 (m, 1H,
Ar-H), 4.50-4.48 (m, 1H), 3.75-3.72 (m, 1H), 3.63-3.56 (m, 2H), 3.47 (s, 2H), 2.84-2.75 (m, 2H),
2.70-2.53 (m, 4H), 2.40 (s, 3H, CH3), 2.35-2.30 (m, 2H), 2.22-2.18 (m, 3H), 2.02 (s, 3H, CH3),
1.92-1.89 (m, 1H), 1.79-1.54 (m, 6H), 1.02-0.94 (m, 3H, CH3). 13C-NMR (CDCl3, 125 MHz) δ: 173.81,
168.69, 144.18, 140.89, 136.41, 135.16, 131.99, 131.87, 129.38, 129.26, 128.41, 127.28, 126.27,
118.87, 110.73, 62.21, 60.51, 57.62, 54.83, 53.29, 50.54, 47.96, 45.43, 40.59, 39.26, 29.95, 28.76,
28.26, 24.32, 21.27, 19.69. HRMS (ESI+) m/z calculated for C31H41ClN5O2 [M + H]+,550.2959; found,
550.2955.
4.1.11.2 ( )-1-acetyl-N-(3-(4-benzyl-2-methylpiperazin-1-yl)propyl)-N-(3-chloro-4-methylphenyl)
piperidine -4-carboxamide (14). The reaction of 7b with 12a produced compound 14 (63.00 mg,
1
24%) as light yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.31-7.29 (m, 5H, Ar-H), 7.25-7.23 (m, 1H,
Ar-H), 7.16 (brs, 1H, Ar-H), 6.95-6.93 (m, 1H, Ar-H), 4.51-4.49 (m, 1H), 3.76-3.74 (m, 1H), 3.64-3.56
(m, 2H), 3.44 (brs, 2H), 2.84-2.76 (m, 2H), 2.75-2.60 (m, 4H), 2.41 (s, 3H, CH3), 2.36-2.16 (m, 5H),
2.03 (s, 3H, CH3), 1.92-1.90 (m, 1H), 1.80-1.55 (m, 6H),1.02-0.95 (m, 3H, CH3). 13C-NMR (CDCl3,
125 MHz) δ: 173.76, 168.55, 140.73, 135.43, 133.44, 131.92, 130.72, 128.23, 128.23, 127.32, 126.39,
126.24, 124.62, 61.72, 60.52, 56.66, 54.53, 52.85, 50.31, 47.67, 45.47, 40.43, 39.22, 31.39, 28.68,
28.23, 24.21, 21.24, 19.61. HRMS (ESI+) m/z calculated for C30H42ClN4O2 [M + H]+,525.2996; found,
525.3001.
4.1.11.3
( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(2-methyl-4-(4-methylbenzyl)piperazin
-1-yl)propyl)piperidine-4-carboxamide (15). The reaction of 7b with 12a produced compound 15
1
(70.00 mg, 26%) as light yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.26-7.21 (m, 4H, Ar-H),

7.18-7.17 (m, 1H, Ar-H), 7.00-6.96 (m, 2H, Ar-H), 4.51-4.49 (m, 1H), 3.76-3.73 (m, 1H), 3.67-3.58 (m,
2H), 3.47(s, 2H), 2.84-2.72 (m, 2H), 2.71 (s, 3H, CH3), 2.68-2.52 (m, 4H), 2.40 (s, 3H, CH3), 2.36-2.17
(m, 5H), 2.03 (s, 3H, CH3), 1.91-1.90 (m, 1H), 1.81-1.64 (m, 6H), 1.00-0.94 (m, 3H, CH3). 13C-NMR
(CDCl3, 125 MHz) δ: 173.76, 168.65, 140.73, 136.38, 135.01, 133.54, 131.73, 130.41, 130.34, 129.13,
128.27, 126.32, 61.83, 60.22, 56.66, 54.85, 52.93, 50.56, 47.81, 45.37, 40.57, 39.21, 31.26, 28.65,
28.23, 24.21, 21.30, 21.17, 19.56. HRMS (ESI+) m/z calculated for C31H44ClN4O2 [M + H]+,539.3153;
found, 539.3155.
4.1.11.4 ( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-1-
yl) propyl)piperidine-4-carboxamide (16). The reaction of 7d with 12a produced compound 16
1
(75.80 mg, 28%) as light yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.27-7.22 (m, 3H, Ar-H),
7.17-7.16 (m, 1H, Ar-H), 6.98-6.93(m, 3H, Ar-H), 4.50-4.47 (m, 1H), 3.76-3.72 (m, 1H), 3.65-3.62 (m,
2H), 3.43 (s, 2H), 2.91-2.57 (m, 6H), 2.40 (s, 3H, CH3), 2.38-2.36 (m, 2H), 2.32-2.29 (m, 3H), 2.02 (s,
3H, CH3), 1.93-1.90 (m, 1H), 1.77-1.55 (m, 6H), 1.01-0.95 (m, 3H, CH3). 13C-NMR (CDCl3, 125 MHz)
δ: 173.77, 168.65, 162.50, 160.06, 140.81, 136.39, 135.11, 133.53, 131.76, 130.44, 130.37, 129.33,
126.20, 114.92, 114.71, 61.85, 60.25, 56.60, 54.51, 52.89, 50.49, 47.80, 45.37, 40.54, 39.18, 31.35,
28.61, 28.14, 24.10, 21.10, 19.65. HRMS (ESI+) m/z calculated for C30H41ClFN4O2 [M + H]+,543.2903;
found, 543.2898.
4.1.11.5 ( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(2-fluorobenzyl)-2-methylpiperazin-1-
yl) propyl)piperidine-4-carboxamide (17). The reaction of 7e with 12a produced compound 17
(71.10 mg, 26%) as yellow oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.44-7.42 (m, 1H, Ar-H), 7.31-7.25 (m,
2H, Ar-H), 7.20-7.18 (m, 1H, Ar-H), 7.15 (m, 2H, Ar-H), 6.94-6.92 (m, 1H, Ar-H), 4.49-4.46 (m, 1H),
3.74-3.71 (m, 1H), 3.64-3.60 (m, 2H), 3.53 (s, 2H), 2.91-2.81(m, 2H), 2.78-2.73 (m, 2H), 2.67-2.66 (m,
2H), 2.38 (s, 3H, CH3), 2.34-2.19 (m, 5H), 2.01 (s, 3H, CH3), 1.98-1.92 (m, 1H), 1.78-1.74 (m, 1H),
1.65-1.54 (m, 5H), 1.00-0.94 (m, 3H, CH3). HRMS (ESI+) m/z calculated for C30H41ClFN4O2 [M +
H]+,543.2903; found, 543.2909.
4.1.11.6 ( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(3-fluorobenzyl)-2-methylpiperazin-1-
yl) propyl)piperidine-4-carboxamide (18). The reaction of 7f with 12a produced compound 18
(57.00 mg, 21%) as yellow oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.28-7.23 (m, 3H, Ar-H), 7.16 (brs, 1H,
Ar-H), 7.07-7.06 (m, 2H, Ar-H), 6.94-6.92 (m, 1H, Ar-H), 4.52-4.49 (m, 1H), 3.76-3.73 (m, 1H),
3.66-3.58 (m, 2H), 3.43 (s, 2H), 2.89-2.85 (m, 3H), 2.68-2.58 (m, 3H), 2.41 (s, 3H, CH3), 2.36-2.31 (m,

2H), 2.26-2.18 (m, 3H), 2.03 (s, 3H, CH3), 1.90 (brs, 1H), 1.77-1.55 (m, 6H), 1.02-0.95(m, 3H, CH3).
HRMS (ESI+) m/z calculated for C30H41ClFN4O2 [M + H]+,543.2903; found, 543.2907.
4.1.11.7 ( )-1-acetyl-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-1-yl)propyl)-N-phenylpiperidine-
4 -carboxamide (19). The reaction of 7d with 12b produced compound 19 (71.60 mg, 29%) as
1
yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.42-7.25 (m, 7H, Ar-H), 7.18-7.17 (m, 2H, Ar-H),
4.52-4.49 (m, 1H), 3.75-3.73 (m, 1H), 3.64-3.59 (m, 2H), 3.44 (s, 2H), 2.91-2.78 (m, 2H), 2.73-2.59 (m,
4H), 2.41-2.15 (m, 5H), 2.03 (s, 3H, CH3), 1.92-1.91 (m, 1H), 1.83-1.56 (m, 6H), 1.03-0.97 (m, 3H,
CH3). 13C-NMR (CDCl3, 125 MHz) δ: 173.71, 168.63, 162.47,160.03, 140.72, 135.13, 133.52, 131.74,
130.44, 129.21, 126.29, 114.81, 114.59, 61.85, 60.23, 56.62, 54.80, 52.89, 50.21, 47.82, 45.32, 40.57,
39.28, 31.31, 28.62, 28.17, 24.30, 21.22. HRMS (ESI+) m/z calculated for C29H40ClFN4O2 [M +
H]+,495.3135; found, 495.3138.
4.1.11.8 ( )-1-acetyl-N-(3-chlorophenyl)-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-1-yl)propyl)
piperidine-4-carboxamide (20). The reaction of 7d with 12c produced compound 20 (66.10 mg,
25%) as yellow oil.1H-NMR (CDCl3, 500 MHz) δ: 7.39-7.37 (m, 2H, Ar-H), 7.24-7.23 (m, 1H, Ar-H),
7.18 (s, 1H, Ar-H), 7.05 (brs, 2H, Ar-H), 7.00-6.95 (m, 2H, Ar-H), 4.52-4.49 (m, 1H), 3.77-3.74 (m,
1H), 3.70-3.62 (m, 2H), 3.40 (brs, 2H), 2.98-2.57 (m, 6H), 2.35-2.30 (m, 2H), 2.26-2.16 (m, 3H), 2.03
(s, 3H, CH3), 1.90-1.56 (m, 7H), 1.03-0.95 (m, 3H, CH3). 13C-NMR (CDCl3, 125 MHz) δ: 173.72,
168.63, 162.47, 160.03, 140.71, 136.31, 135.13, 133.57, 131.34, 130.62, 130.43, 129.13, 126.31,
114.82, 114.60, 61.83, 60.32, 56.61, 54.48, 52.91, 50.31, 47.83, 45.39, 40.52, 39.28, 31.35, 28.51,
28.14, 24.29, 21.17. HRMS (ESI+) m/z calculated for C29H39ClFN4O2 [M + H]+,529.2745; found,
529.2751.
4.1.11.9 ( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzoyl)-2-methylpiperazin-1-
yl) propyl) piperidine-4-carboxamide (21). The reaction of 7g with 12a produced compound 21
(0.064 g, 23%) as yellow oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.46-7.28 (m, 4H, Ar-H), 7.18-7.17 (m,
1H, Ar-H), 7.11-6.98 (m, 2H, Ar-H), 4.52-4.49 (m, 1H), 3.76-3.73 (m, 1H), 3.68-3.62 (m, 2H),
3.19-3.12 (m, 2H), 2.86-2.80 (m, 2H), 2.63-2.59 (m, 2H), 2.41 (s, 3H, CH3), 2.37-2.06 (m, 5H), 2.03 (s,
3H, CH3), 1.96-1.50 (m, 7H), 1.02-0.94 (m, 3H, CH3). HRMS (ESI+) m/z calculated for
C30H39ClFN4O3 [M + H]+,557.2694; found, 557.2698.
4.1.11.10
( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorophenylsulfonyl)-2-
The reaction of 7h with 12a
methylpiperazin-1-yl)propyl)piperidine-4-carboxamide (22).

produced compound 22 (0.077 g, 26%) as yellow oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.54-7.46 (m, 4H,
Ar-H), 7.20-7.18 (m, 1H, Ar-H), 7.15-6.99 (m, 2H, Ar-H), 4.51-4.49 (m, 1H), 3.76-3.74 (m, 1H),
3.70-3.69 (m, 2H), 3.20-3.12 (m, 2H), 2.88-2.58 (m, 4H), 2.40 (s, 3H, CH3), 2.39-2.05 (m, 5H), 2.03 (s,
3H, CH3), 2.00-1.93 (m, 1H), 1.85-1.59 (m, 6H), 1.02-0.95 (m, 3H, CH3). HRMS (ESI+) m/z calculated
for C29H39ClFN4O2 [M + H]+,529.2745; found, 529.2751.
4.1.12 Synthesis of ( )-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-
1-yl) propyl)acetamide (26)
4.1.12.1 N-(3-chloro-4-methylphenyl)acetamide (23a). To a solution of 3-chloro-4-methylaniline
10a (0.85g, 6.00 mmol) in DCM (15.00 mL), EDC·HCl (1.72g, 9.00 mmol) and acetic acid (0.34ml,
6.00 mmol) were added successively at room temperature, and the resulting reaction mixture was
stirred for 12h at the same temperature. Then the reaction mixture was concentrated under reduced
pressure. The residue was purified by silica gel column chromatography (EtOAc/PE 1/4) to furnish the
title compound 23a (0.84g, yield 76%). 1H-NMR (CDCl3, 500 MHz) δ: 7.92 (s, 1H, NH), 7.57 (brs, 1H,
Ar-H), 7.26-7.23 (m, 1H, Ar-H), 7.11-7.09 (m, 1H, Ar-H), 2.29 (s, 3H, CH3), 2.14 (s, 3H, CH3).
ESI-MS m/z: 184 [M+H]+.
4.1.12.2 N-(2-(1,3-dioxolan-2-yl)ethyl)-N-(3-chloro-4-methylphenyl)acetamide (24a).
To an
ice-cooled stirred solution of N-(3-chloro-4-methylphenyl)acetamide 23a (0.65 g, 4.00mmol) in DMF
(8.00ml) was added NaH (60% in oil, 160mg, 4.00mmol). After 1 h the mixture was treated with
2-(2-bromoethyl)-1, 3-dioxolane (0.47ml, 4mmol) and stirred at 80 °C for 12 h. The mixture was
concentrated in vacuo, diluted with water (20ml), and extracted with DCM (3×20ml). The organic layer
was dried (MgSO4), filtered, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/PE 1/1) to give the product (0.81 g, yield 71%) as oil.1H-NMR
(CDCl3, 500 MHz) δ: 7.25-7.23 (d, J=8Hz, 1H, Ar-H), 7.18 (brs, 1H, Ar-H), 6.98-6.96 (dd, J=2Hz, 8Hz,
1H, Ar-H), 4.88-4.86 (m, 1H), 3.91-3.88 (m, 2H), 3.80-3.77(m, 4H), 2.37 (s, 3H, CH3), 1.88-1.84 (m,
2H), 1.81 (s, 3H, CH3). ESI-MS m/z: 284 [M+H]+.
4.1.12.3
N-(3-chloro-4-methylphenyl)-N-(3-oxopropyl)acetamide
(25a).
N-(2-(1,3-dioxolan-2-yl)ethyl)-N-(3-chloro-4-methylphenyl)acetamide 24a (0.79 g, 2.80mmol) was
dissolved in1N HCl (10.00ml), and the mixture was stirred at room temperature for 18 h. The mixture
was extracted with DCM (3×15.00ml), and the organic layer was dried (MgSO4), filtered, and
concentrated in vacuo to give 25a (0.65g, yield 97%) as an oil.1H-NMR (CDCl3, 500 MHz) δ: 9.74(s,

1H), 7.26(s, 1H), 7.17(brs, 1H, Ar-H), 6.97-6.95(m, 1H, Ar-H), 4.00-3.98 (m, 2H), 2.70-2.66 (m, 2H),
2.39 (s, 3H, CH3), 1.83 (s, 3H, CH3). ESI-MS m/z: 240 [M+H]+.
4.1.12.4
propyl)acetamide
N-(3-chloro-4-methylphenyl)-N-(3-oxopropyl)acetamide
( )-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-1-yl)
(26).
To
a
stirred
mixture
of
25a
(0.12g,
0.5mmol),
( )-1-(4-fluorobenzyl)-3-methylpiperazine 7d (0.10g, 0.5mmol) in DCM (8ml) was added AcOH
(0.008 ml, 0.05mmol) and the mixture was stirred at room temperature for 0.5 h. Then Sodium
triacetoxyborohydride (0.16g, 0.75mmol) was added in portions. The reaction was stirred at the same
temperature for 6h. Finally, the mixture was diluted with saturated aqueous NaHCO3 (10ml) followed
by water (10ml) and extracted with EtOAc (3×10ml). The organic layer was dried (MgSO4), filtered,
and concentrated in vacuo. The residue was purified by column chromatography on silica gel
1
(EtOAc/PE 3/1) to afford yellow oil (0.071g, yield 33%). H-NMR (CDCl3, 500 MHz) δ: 7.25-7.22(m,
3H, Ar-H), 7.15(brs, 1H, Ar-H), 6.96-6.94(m, 3H, Ar-H), 3.71-3.58(m, 2H), 3.40(brs, 2H), 2.79-2.57
(m, 4H), 2.37 (s, 3H, CH3), 2.31-2.21 (m, 4H), 1.95-1.89 (m, 1H), 1.81 (s, 3H, CH3), 1.69-1.66 (m, 2H),
0.98-0.96(m, 3H, CH3). HRMS (ESI+) m/z calculated for C24H32ClFN3O [M + H]+,432.2218; found,
432.2223.
4.1.13
Synthesis
of
( )-N-(3-chloro-4-methylphenyl)-4-fluoro-N-(3-(4-(4-fluorobenzyl)-
2-methylpiperazin -1-yl) propyl)benzamide (27)
4.1.13.1 N-(3-chloro-4-methylphenyl)-4-fluorobenzamide (23b). According to the method of
synthesis of N-(3-chloro-4-methylphenyl)acetamide 23a, the reaction of 10a (0.85g, 6.00 mmol) with
4-fluorobenzoic acid (0.84g, 6.00 mmol) produced compound 23b as a white oil (1.38g, yield 87%).
1H-NMR (CDCl3, 500 MHz) δ: 8.05(s, 1H), 7.85-7.82(m, 2H), 7.67(brs, 1H, Ar-H), 7.37-7.35(m, 1H,
Ar-H), 7.15-7.14(m, 1H, Ar-H), 7.11-7.07(m, 2H, Ar-H), 2.33 (s, 3H, CH3). ESI-MS m/z: 264 [M+H]+.
4.1.13.2 N-(2-(1, 3-dioxolan-2-yl) ethyl)-N-(3-chloro-4-methylphenyl)-4-fluorobenzamide (24b).
According to the method of synthesis of N-(2-(1, 3-dioxolan-2-yl) ethyl)-N-(3-chloro-4-methylphenyl)
acetamide 24a, the reaction of 23b (1.05g, 4.00mmol) with 2-(2-bromoethyl)-1,3-dioxolane (0.47ml,
1
4mmol) produced compound 24b as a white oil (1.38g, yield 77%). H-NMR (CDCl3, 500 MHz) δ:
7.30-7.27(m, 2H), 7.09 (brs, 1H, Ar-H), 7.05-7.03(m, 1H, Ar-H), 6.87-6.84(m, 2H, Ar-H),
6.78-6.76(m, 1H, Ar-H), 4.97-4.95(m, 1H), 4.02-3.99(m, 2H), 3.95-3.92(m, 2H), 3.84-3.82(m, 2H),
2.28 (s, 3H, CH3), 2.02-1.98(m, 2H). ESI-MS m/z: 365 [M+H]+.

4.1.13.3 N-(3-chloro-4-methylphenyl)-4-fluoro-N-(3-oxopropyl)benzamide (25b). According to
the method of synthesis of N-(3-chloro-4-methylphenyl)-N-(3-oxopropyl)acetamide 25a, the reaction
of 24b(1.02g, 2.80mmol) with 1N HCl produced compound 25b as a white oil (0.86g, yield 96%).
1H-NMR (CDCl3, 500 MHz) δ: 9.79(s, 1H), 7.30-7.27(m, 2H), 7.09 (brs, 1H, Ar-H), 7.05-7.03(m, 1H,
Ar-H), 6.87-6.83(m, 2H, Ar-H), 6.78-6.74(m, 1H, Ar-H), 4.01-3.98(m, 2H), 3.93-3.92(m, 2H), 2.28 (s,
3H, CH3). ESI-MS m/z: 320 [M+H]+.
4.1.13.4 ( )-N-(3-chloro-4-methylphenyl)-4-fluoro-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-1-
yl)
propyl)benzamide
(27).
According
to
the
method
of
synthesis
of
( )-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)- 2-methylpiperazin-1-yl) propyl)acetamide
26, the reaction of 25 (0.16g, 0.50mmol)with ( )-1-(4-fluorobenzyl)-3-methylpiperazine 7d (0.10g,
0.50mmol) produced compound 27 (0.11g, yield 45%)as a yellow oil. 1H-NMR (CDCl3, 500 MHz) δ:
7.29-7.27(m, 2H), 7.26-7.23(m, 2H),7.08 (brs, 1H, Ar-H), 7.29-7.23 (m, 4H, Ar-H), 7.08-6.95 (m, 4H,
Ar-H), 6.88-6.84 (m, 2H, Ar-H), 6.74-6.72 (m, 1H, Ar-H), 3.93-3.83(m, 2H), 3.41 (s, 2H), 2.80-2.41(m,
5H), 2.28 (s, 3H, CH3), 2.25-1.70 (m, 6H), 1.02-0.97 (m, 3H, CH3). 13C-NMR (CDCl3, 125 MHz) δ:
169.14, 164.47, 163.17, 161.98, 160.74, 142.12, 134.75, 134.66, 133.77, 133.74, 131.91, 131.88,
131.18, 130.94, 130.85, 130.57, 130.49, 127.68, 126.10, 115.05, 114.83, 62.03, 60.38, 54.90, 53.15,
50.91, 50.58, 49.01, 29.66, 24.10, 19.53. HRMS (ESI+) m/z calculated for C29H33ClF2N3O [M +
H]+,512.2280; found, 512.2285.
4.1.14
Synthesis
of
( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-
2-methylpiperazin-1-yl) propyl) piperidine-3-carboxamide (28)
4.1.14.1 1-acetyl-N-(3-chloro-4-methylphenyl) piperidine-3-carboxamide (23c). According to the
method of synthesis of N-(3-chloro-4-methylphenyl)acetamide 23a, the reaction of 10a (0.85g, 6.00
mmol) with 1-acetylpiperidine-3-carboxylic acid (1.03g, 6.00 mmol) produced compound 23c (1.49g,
1
yield 84%) as a yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.69 (brs, 1H, Ar-H), 7.33-7.29 (m, 1H,
Ar-H), 7.09-7.06 (m, 1H, Ar-H), 4.44-4.41 (m, 1H), 4.15-4.11 (m, 2H), 3.58-3.55 (m, 1H), 3.47-3.24
(m,2H), 2.50-2.45 (m, 1H), 2.26 (s, 3H, CH3), 2.08 (s, 3H, CH3), 1.94-1.68 (m, 2H), 1.49-1.32 (m, 1H).
ESI-MS m/z: 295.7 [M+H]+.
4.1.14.2
carboxamide
3-dioxolan-2-yl)ethyl)-N-(3-chloro-4- methylphenyl)acetamide 24a, the reaction of 23c (1.18g,
N-(2-(1,
3-dioxolan-2-yl)ethyl)-1-acetyl-N-(3-chloro-4-methylphenyl)piperidine-3-
(24c).
According to the method of synthesis of N-(2-(1,

4.00mmol) with 2-(2-bromoethyl)-1,3- dioxolane (0.47ml, 4mmol) produced compound 24c(1.25g,
1
yield 79%) as a yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.74 (brs, 1H, Ar-H), 7.33-7.30 (m, 1H,
Ar-H), 7.13-7.11 (m, 1H, Ar-H), 4.90-4.88 (m, 2H), 4.55-4.46 (m, 2H), 3.69-3.63 (m, 2H), 3.48-3.46
(m,1H), 2.56 (brs, 1H), 2.26-2.22 (m,2H), 2.12 (s, 3H, CH3), 2.03 (s, 3H, CH3), 1.78-1.74 (m, 4H),
1.73-1.56 (m, 4H). ESI-MS m/z: 396 [M+H]+.
4.1.14.3 1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-oxopropyl) piperidine-3-carboxamide (25c).
According to the method of synthesis of N-(3-chloro-4-methylphenyl)-N-(3-oxopropyl)acetamide 25a,
the reaction of 24c (1.11g, 2.80mmol) with 1N HCl produced compound 25c (0.92g, yield 94%) as a
1
yellow oil. H-NMR (CDCl3, 500 MHz) δ: 9.10-9.09(m, 1H), 7.69(brs, 1H, Ar-H), 7.31-7.29(m, 1H,
Ar-H), 7.08-7.06 (m, 1H, Ar-H), 4.44-4.41 (m, 1H), 4.15-4.01 (m, 2H), 3.58-3.55 (m, 1H), 3.47-3.24
(m,2H), 2.49-2.45 (m, 1H), 2.26 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.05-1.68 (m, 5H), 1.54-1.44 (m, 1H).
ESI-MS m/z: 351 [M+H]+.
4.1.14.4
( )-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-
According to the method of synthesis of
-1-yl) propyl) piperidine-3-carboxamide (28).
( )-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl) -2-methylpiperazin-1-yl) propyl)acetamide
26, the reaction of 25c(0.18g, 0.50mmol) with ( )-1-(4-fluorobenzyl)- 3-methylpiperazine 7d (0.10g,
1
0.50mmol) produced compound 28(0.11g, yield 39%) as a yellow oil. H-NMR (CDCl3, 500 MHz) δ:
7.73 (brs, 1H, Ar-H), 7.33-7.31 (m, 1H, Ar-H), 7.27-7.24 (m, 2H, Ar-H), 7.11-7.09 (m, 1H, Ar-H),
6.99-6.96 (m, 2H, Ar-H), 4.60-4.45 (m, 1H), 3.97-3.95 (m, 1H), 3.72-3.65 (m, 1H), 3.54-3.50 (m, 1H),
3.42 (brs, 2H, CH2), 2.95-2.82 (m, 4H), 2.72-2.70 (m, 2H), 2.54-2.40 (m, 2H), 2.29 (s, 3H, CH3),
2.23-2.16 (m, 1H), 2.11 (s, 3H, CH3), 2.07-1.86 (m, 5H), 1.82-1.50 (m, 4H), 1.00-0.94 (m, 3H, CH3).
HRMS (ESI+) m/z calculated for C30H41ClFN4O2 [M + H]+,543.2902; found, 543.2908.
4.1.15 Synthesis of ( )-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-
1-yl) propyl)-1-(methylsulfonyl)piperidine-4-carboxamide (29)
4.1.15.1
1-methylsulfonyl-N-(3-chloro-4-methylphenyl)
piperidine-4-carboxamide
(23d).
According to the method of synthesis of N-(3-chloro-4-methylphenyl)acetamide 23a , the reaction of
10a (0.85g, 6.00 mmol) with 1-(methylsulfonyl)piperidine-4-carboxylic acid (1.24g, 6.00 mmol)
1
produced compound 23d (1.63g, yield 82%) as a yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.73(s,
1H), 7.61(brs, 1H, Ar-H), 7.30-7.28 (m, 1H, Ar-H), 7.14-7.13 (m, 1H, Ar-H), 4.61-4.58 (d, J =13Hz,
1H), 3.91-3.88 (d, J =13Hz, 1H), 3.14-3.09 (m, 1H), 2.71-2.68 (m, 2H), 2.51-2.46 (m, 1H), 2.31 (s,

3H, CH3), 2.10 (s, 3H, CH3), 1.93 (brs, 2H), 1.83-1.69 (m, 2H). ESI-MS m/z: 332 [M+H]+.
4.1.15.2
N-(2-(1,3-dioxolan-2-yl)
ethyl)-N-(3-chloro-4-methylphenyl)-1-(methylsulfonyl)
piperidine -4-carboxamide (24d).
According to the method of synthesis of N-(2-(1,
3-dioxolan-2-yl)ethyl)-N-(3-chloro- 4-methylphenyl) acetamide 24a, the reaction of 23d (1.32g,
4.00mmol) with 2-(2-bromoethyl)-1,3- dioxolane produced (0.47ml, 4mmol) compound 24d (1.29g,
1
yield 72%) as a yellow oil. H-NMR (CDCl3, 500 MHz) δ: 7.28-7.27 (d, J=8Hz, 1H, Ar-H), 7.20 (brs,
1H, Ar-H), 7.00-6.98 (m, 1H, Ar-H), 4.90-4.88 (m, 1H), 3.93-3.90 (m, 2H), 3.82-3.76 (m, 4H),
3.71-3.68 (m, 2H), 2.72 (s, 3H, CH3), 2.60-2.54 (m,2H), 2.40 (s, 3H, CH3), 2.28-2.22 (m, 1H),
1.88-1.84 (m, 4H), 1.69-1.66 (m, 2H). ESI-MS m/z: 432 [M+H]+.
4.1.15.3
N-(3-chloro-4-methylphenyl)-1-(methylsulfonyl)-N-(3-oxopropyl)piperidine-4-
(25d). According to the method of synthesis of
carboxamide
N-(3-chloro-4-methylphenyl)-N-(3-oxopropyl)acetamide 25a, the reaction of 24d (1.20g, 2.80mmol)
1
with 1N HCl produced compound 25d (1.03g, yield 92%) as a yellow oil. H-NMR (CDCl3, 500 MHz)
δ: 9.73 (s, 1H), 7.31-7.29 (d, J=8Hz, 1H, Ar-H), 7.18 (brs, 1H, Ar-H), 6.99 -6.96 (m, 1H, Ar-H),
3.98-3.95 (m, 1H), 3.71-3.67 (m, 2H), 2.72 (s, 3H, CH3), 2.68-2.65 (m, 2H), 2.58-2.53 (m, 2H), 2.41 (s,
3H, CH3), 2.28-2.22 (m, 1H), 1.88-1.80 (m, 4H), 1.68-1.64 (m, 2H). ESI-MS m/z: 387 [M+H]+.
4.1.15.4
( )-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-1-yl)
propyl)- -1-(methylsulfonyl)piperidine-4-carboxamide (29). According to the method of synthesis
of
propyl)acetamide
(methylsulfonyl)-N-(3-oxopropyl)piperidine-4-carboxamide
( )-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-
fluorobenzyl)-
N-(3-chloro-4-
25d (0.19g,
2-methylpiperazin-1-yl)
26, the reaction
of
methylphenyl)-1-
0.50mmol)
with
( )-1-(4-fluorobenzyl)- 3-methylpiperazine 7d (0.10g, 0.5mmol) produced compound 29 (0.12g, yield
43%) as yellow oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.28-7.22 (m, 3H, Ar-H), 7.16-7.15 (m, 1H, Ar-H),
6.99-6.93 (m, 3H, Ar-H), 3.71-3.68 (m, 2H), 3.64-3.59 (m, 2H), 3.39 (m, 2H), 2.77-2.75 (m, 1H), 2.71
(s, 3H, CH3), 2.68-2.63 (m, 2H), 2.58-2.52 (m, 3H), 2.40 (s, 3H, CH3), 2.37 (brs, 1H), 2.27-2.15 (m,
4H), 1.90-1.81 (m, 3H), 1.67-1.64 (m, 4H), 1.02-0.95(m, 3H, CH3). 13C-NMR (CDCl3, 125 MHz) δ:
173.86, 162.65,160.22, 140.73, 136.41, 135.07, 133.49, 131.71, 130.45, 130.39, 128.32, 126.21, 114.97,
114.76, 61.71, 60.53, 56.80, 54.89, 52.92, 50.23, 47.61, 45.69, 40.58, 39.18, 31.38, 28.65, 28.28, 26.21,
21.16, 19.77. HRMS (ESI+) m/z calculated for C29H41ClFN4O3S [M + H]+,579.2572; found, 579.2579.
4.1.16
Synthesis
of
( )-1-benzoyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-

2-methylpiperazin- -1-yl)propyl)piperidine-4-carboxamide (32)
4.1.16.1 1-benzoyl-N-(3-chloro-4-methylphenyl)-N-(3-chloropropyl) piperidine-4-carboxamide
(31). To an ice-cooled stirred suspension of 3-chloro-N-(3-chloropropyl)-4-methylaniline 10a (0.22g,
1.00 mmol) in DCM (3.00 mL) was added Et3N (0.40 mL, 3.00 mmol) followed by
1-benzoylpiperidine-4-carbonyl chloride 30 (0.30 g, 1.20 mmol), and the mixture was stirred at room
temperature for 5 h. Then it was poured to a saturated solution of sodium bicarbonate (10.00 mL). The
residue was extracted by EtOAc (20 ml×3), the organic layer was combined, then washed with water
(15 ml×3) and brine (10.00 mL), dried (MgSO4), filtered and concentrated in vacuo. The residue was
1
purified by column chromatography on silica gel to afford yellow oil (0.32g, yield 75%). H-NMR
(CDCl3, 500 MHz) δ: 7.38-7.35 (m, 5H, Ar-H), 7.31-7.29 (m, 1H, Ar-H), 7.17 (brs, 1H, Ar-H),
6.98-6.96 (m, 1H, Ar-H), 4.66-4.56 (m, 1H), 3.78-3.75 (m, 2H), 3.54-3.52 (m, 2H), 3.29-3.26 (m, 2H),
2.84-2.53 (m, 2H), 2.42 (s, 3H, CH3), 2.01-1.99 (m, 2H), 1.79-1.52 (m, 4H). ESI-MS m/z: 434 [M+H]+.
4.1.16.2 ( )-1-benzoyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-methylpiperazin-
-1-yl)propyl)piperidine-4-carboxamide
(32).
A
mixture
of
1-benzoyl-N-(3-chloro-4-methylphenyl)-N- (3-chloropropyl)piperidine-4- carboxamide 31 (0.22g, 0.50
mmol), ( )-1-(4-fluorobenzyl)-3- methylpiperazine 7d (0.10g, 0.50 mmol), KI (0.008, 0.05 mmol) and
K2CO3 (0.10g, 0.75 mmol) in DMF (5.00 ml) was stirred at reflux for 16h. The mixture was
concentrated in vacuo, diluted with water (20.00 ml) and extracted with EtOAc (15 ml×3). The organic
layer was dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/PE 1/2) to afford yellow oil (0.08g, yield 29%). 1H-NMR (CDCl3,
500 MHz) δ: 7.37-7.27 (m, 5H, Ar-H), 7.24-7.14 (m, 2H, Ar-H), 6.99-6.93 (m,3H, Ar-H), 6.60 (brs,1H,
Ar-H), 6.44-6.42 (m,1H, Ar-H), 4.62-4.60 (m, 1H), 3.76-3.73 (m, 1H), 3.66-3.63 (m, 2H), 3.42 (s, 2H),
2.82-2.76 (m, 2H), 2.69-2.57 (m, 4H), 2.40 (s, 3H, CH3), 2.34-2.18 (m, 5H), 1.91-1.90 (m, 1H),
1.80-1.53 (m, 6H), 1.01-0.95 (m, 3H, CH3). HRMS (ESI+) m/z calculated for C35H43ClFN4O2 [M +
H]+,605.3058; found, 605.3060.
4.1.17 Synthesis of 1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-((1S,4S)-5-(4-fluorobenzyl)-2,5-
diazabicyclo [2.2.1]heptan-2-yl)propyl)piperidine-4-carboxamide (33).
To a mixture of
1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-oxopropyl) piperidine-4-carboxamide 25e (0.18g, 0.5mmol)
and (1S,4S)-2-(4-fluorobenzyl)-2, 5-diazabicyclo [2.2.1] heptanes 9 (0.10g, 0.50mmol) in DCM
(8.00ml) was added AcOH (0.008 ml, 0.05mmol) and the mixture was stirred at room temperature for

0.5 h. Then Sodium triacetoxyborohydride(0.16g, 0.75mmol) was added in portions. The reaction was
stirred at the same temperature for 6h. Finally, the mixture was diluted with saturated aqueous NaHCO3
(10.00ml) followed by water (10ml) and extracted with EtOAc (3×10.00ml). The organic layer was
dried (MgSO4), filtered, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/PE 1/1) to afford yellow oil (0.11g, yield 41%). 1H-NMR (CDCl3,
500 MHz) δ: 7.29-7.27 (m, 3H, Ar-H), 7.18-7.17 (m, 1H, Ar-H), 6.98-6.95 (m, 3H, Ar-H), 4.52-4.49 (m,
1H), 3.76-3.73 (m, 1H), 3.71-3.58(m, 4H), 3.27-3.22 (m, 2H), 2.86-2.80 (m, 1H), 2.71-2.58 (m, 5H),
2.45-2.44 (m, 1H), 2.41 (s, 3H, CH3), 2.38-2.31 (m, 2H), 2.03 (s, 3H, CH3), 1.77-1.56 (m, 8H).
13C-NMR (CDCl3, 125 MHz) δ: 173.82, 168.72, 162.94, 160.51, 141.02, 136.36, 135.53, 135.13,
131.85, 129.82, 129.75, 128.46, 126.31, 115.00, 114.79, 61.96, 61.24, 57.44, 56.43, 55.71, 51.46, 47.93,
45.46, 40.63, 39.30, 33.59, 28.77, 28.26, 27.46, 21.31, 19.72. ESI-MS m/z: HRMS (ESI+) m/z
calculated for C30H39ClFN4O2 [M + H]+,541.2745; found, 541.2749.
4.1.18 Synthesis of 1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-1,4-diazepan-1-
yl) propyl)piperidine-4-carboxamide (34).
According to the method of synthesis of
(4- fluorobenzyl)-2,5- diazabicyclo
1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-((1S,4S)-5-
[2.2.1]heptan-2-yl)propyl)piperidine-4-carboxamide (33), the reaction of 1-acetyl-N-(3-chloro-
4-methylphenyl)-N-(3-oxopropyl) piperidine-4-carboxamide 25e (0.18g, 0.50mmol) with
1-(4-fluorobenzyl)-1,4-diazepane 7i (0.10g, 0.50mmol) produced compound 34 (0.12g, 46%) as yellow
oil. 1H-NMR (CDCl3, 500 MHz) δ: 7.30-7.26 (m, 3H, Ar-H), 7.18 (brs, 1H, Ar-H), 6.99-6.97(m, 3H,
Ar-H), 4.53-4.50 (m, 1H), 4.08 (brs, 2H), 3.76-3.74 (m, 3H), 3.55-3.50 (m, 4H), 3.41(brs, 2H),
2.86-2.80 (m, 1H), 2.69-2.55 (m, 4H), 2.41 (s, 3H, CH3), 2.36-2.30 (m, 2H), 2.04 (s, 3H, CH3),
1.85-1.57 (m, 8H). 13C-NMR (CDCl3, 125 MHz) δ: 174.01, 168.75, 162.85, 160.42, 140.94, 136.53,
135.30, 131.97, 130.22, 129.01, 128.43, 127.90, 126.38, 115.15, 114.94, 62.48, 61.52, 57.70, 55.62,
54.52, 53.39, 50.71, 46.62, 45.49, 40.65, 39.34, 28.83, 28.28, 27.51, 21.34, 19.76. HRMS (ESI+) m/z
calculated for C30H41ClFN4O2 [M + H]+,543.2904; found, 543.2903.
4.1.19
Synthesis
of
(R)-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-
According to the method of
1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-((1S,4S)-5- (4- fluorobenzyl)-2,5-
[2.2.1]heptan-2-yl)propyl)piperidine-4-carboxamide (33), the reaction of
methylpiperazin-1-yl)propyl)piperidine-4-carboxamide (35).
synthesis
of
diazabicyclo
1-acetyl-N-(3-chloro- 4-methylphenyl)-N-(3-oxopropyl) piperidine-4-carboxamide 25e (0.18g,

0.50mmol) with (R)-1-(4-fluorobenzyl)-3-methylpiperazine 7j (0.11g, 0.50mmol) produced compound
35 as yellow oil (0.068g, yield 26%). 1H-NMR (CDCl3, 500 MHz) δ: 7.28-7.27(m, 2H, Ar-H), 7.17 (m,
1H, Ar-H), 7.00-6.97 (m, 2H, Ar-H), 6.60 (brs, 1H, Ar-H), 6.42-6.40 (m, 1H, Ar-H), 4.45-4.43 (m, 1H),
3.80-3.77 (m, 1H), 3.71-3.60 (m, 2H), 3.44(s, 2H), 2.96-2.81 (m, 6H), 2.41 (s, 3H, CH3), 2.37-2.29 (m,
2H), 2.24-2.21 (m, 3H), 2.09 (s, 3H, CH3), 1.91-1.0 (m, 1H), 1.70-1.59 (m, 6H), 1.01-1.00 (d, J =6.5Hz,
3H, CH3). 13C-NMR (CDCl3, 125 MHz) δ: 173.71, 168.63, 162.47, 160.04, 140.75, 136.34, 135.08,
133.53, 131.81, 130.45, 130.37, 128.30, 126.20, 114.91, 114.70, 61.88, 60.23, 56.52, 53.41, 52.93,
50.16, 47.29, 45.58, 40.52, 39.13, 31.41, 28.63, 28.16, 24.24, 21.20, 19.19. HRMS (ESI+) m/z
calculated for C30H41ClFN4O2 [M + H]+,543.2902; found, 543.2906.
4.1.20
methylpiperazin1-yl)propyl)piperidine-4-carboxamide (36). According to the method of synthesis
of 1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-((1S,4S)-5- (4- fluorobenzyl)-2,5-diazabicyclo
Synthesis
of
(S)-1-acetyl-N-(3-chloro-4-methylphenyl)-N-(3-(4-(4-fluorobenzyl)-2-
[2.2.1]heptan-2-yl)propyl)piperidine-4-carboxamide (33), the reaction of 1-acetyl-N-(3-chloro-
4-methylphenyl)-N-(3-oxopropyl) piperidine-4-carboxamide 25e (0.18g, 0.50mmol) with
(S)-1-(4-fluorobenzyl)-3-methylpiperazine 7k (0.11g, 0.50mmol) produced compound 36 as yellow oil
(0.08g, yield 31%). 1H-NMR (CDCl3, 500 MHz) δ: 7.28-7.22 (m, 3H, Ar-H), 7.16 (s, 1H, Ar-H),
7.02-6.94(m, 3H, Ar-H), 4.51-4.48 (m, 1H), 3.76-3.73 (m, 1H), 3.64-3.62(m, 2H), 3.40 (s, 2H),
2.86-2.57 (m, 6H), 2.40 (s, 3H, CH3), 2.36-2.31 (m, 2H), 2.27-2.17 (m, 2H), 2.03 (s, 3H, CH3),
1.90-1.87 (m, 1H), 1.77-1.58 (m, 6H), 0.97-0.95(d, J =6.0Hz, 3H, CH3). 13C-NMR (CDCl3, 125 MHz)
δ: 173.71, 168.63, 162.49, 160.06, 140.76, 136.33, 135.08, 133.53, 131.76, 130.45, 130.37, 128.33,
126.19, 114.91, 114.70, 61.89, 60.24, 56.60, 54.81, 52.98, 50.51, 47.85, 45.34, 40.53, 39.17, 31.32,
28.65, 28.16, 24.14, 21.19, 19.61. HRMS (ESI+) m/z calculated for C30H41ClFN4O2 [M + H]+,543.2902;
found, 543.2908.
4.2. Biological assay methods
4.2.1. Calcium mobilization assay
CHO cells stably expressing CCR5 and Gα16 were loaded with 2.00 µmol/L Fluo-4 AM in Hanks
balanced salt solution (HBSS, containing KCl 5.40 mmol/L, Na2HPO4 0.30 mmol/L, KH2PO4 0.4
mmol/L, NaHCO3 4.20 mmol/L, CaCl2 1.30 mmol/L, MgCl2 0.50 mmol/L, Mg2SO4 0.60 mmol/L,
NaCl 137.00 mmol/L, BSA 5.00 g/L, 2.5 mmol/L probenacid，Glucose 5.60 mmol/L, Sulfinpyrazone

250 µmol/L, 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 3nmol/L) was dispensed into the well using a FlexStation II
microplate 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 (IC50) of compounds were determined with GraphPad Prism
software by constructing their dose-response curves. All experiments were repeated a total of three
times. 8
4.2.2. Viral infectivity assays
Plasmids: HIV-1 proviral indicator construct pNL-Luc-E- contains a full-length HIV-1 genome, in
which env was replaced by firefly Luciferase coding sequence. pENV-Ad8 expresses R5-tropic
envelope (AD8).
Viral infectivity assays: Single-cycle HIV-1 replication assays were performed as described
previously. In brief, 4×105 293T cells were co-transfected with 0.4 µg of pNL-Luc-E- and 0.4 µg of
pENV-R5. After 48h, the supernatant containing pseudovirion was harvested by filtration through a
0.45 µm filter and the amount of viral capsid protein was measured by p24 antigen capture ELISA
(Biomerieux). The resultant supernatant (10 µL) was used to infect SupT1 cells (1×105) in 96-well
plates in the presence of testing compound at the concentration indicated. The SupT1 cells were lysed
48h post-infection and firefly luciferase activities were determined using a firefly Luciferase Assay
System (Promega). Values were normalized to the control group treated with DMSO and represented
relative infectivity of each sample testing. 8
Acknowledgements
The authors are grateful to the support of the National Natural Science Foundation of China
(81072515), the Ministry of Health-medical Science Critical Technological Program of Zhejiang
Province (WKJ20122024), the Program for Zhejiang Leading Team of S&T Innovation (2011R50014),
National Basic Research Program of China (973 Program) (2014CB541906), the National Natural
Science Foundation of China (81202341). The authors also thank Jianyang Pan (Insititute of
Pharmaceutical Informatics, Zhejiang University, China) for performing NMR spectrometry and Mass

spectrometry for structure elucidation.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
Rererences and notes
1. Yeni, P. G.; Hammer, S. M.; Carpenter, C. C. J.; Cooper, D. A.; Fischl, M. A.; Gatell, J. M.; Gazzard,
B. G.; Hirsch, M. S.; Jacobsen, D. M.; Katzenstein, D. A.; Montaner, J. S. G.; Richman, D. D.; Saag,
M. S.; Schechter, M.; Schooley, R. T.; Thompson, M. A.; Vella, S.; Volberding, P. A. JAMA 2002,
288, 222.
2. Lederman, M.M.; Penn-Nicholson, A.; Cho M.; Mosier, D. JAMA 2006, 296, 815.
3. Berger, E. A.; Murphy, P. M.; Farber, J. M. Annu. Rev. Immunol. 1999, 17, 657.
4. Liu, T.; Weng, Z.; Dong, X.; Hu, Y. Mini-Rev. Med. Chem. 2010, 10, 1277.
5. Takashima, K.; Miyake, H.; Kanzaki, N.; Tagawa, Y.; Wang, X.; Sugihara, Y.; Iizawa, Y.; Baba, M.
Antimicrob. Agents Chemother. 2005, 49, 3474.
6. Liu, T.; Weng, Z.; Dong, X.; Chen, L.; Ma, L.; Cen, S.; Zhou, N.; Hu, Y. PLoS ONE 2013, 8, e53636.
7. Rossi, C.; Porcelloni, M.; D'Andrea, P.; Fincham. C. I.; Ettorre, A.; Mauro, S.; Squarcia, A.; Bigioni,
M.; Parlani, M.; Nardelli, F.; Binaschi, M.; Maggi, C. A.; Fattori, A D. Bioorg. Med. Chem. Lett.
2011, 21, 2305 – 2308.
8. Hu, S., Gu, Q.; Wang, Z.; Weng, Z.; Cai, Y.; Dong, X.; Hu, Y.; Liu, T.; Xie, X. Eur J. Med. Chem.
2014, 71, 259-266.

Fig. 1. Structures of Maraviroc, TAK-220, and piperazine derivative 3.
Fig. 2. Design of target compounds.
Scheme 1. Synthesis of N-substituted piperazines. Reagents and conditions: (a) substituted benzyl
chloride, K2CO3, DMF, reflux, 12h; (b) 4-fluorobenzaldehyde, NaBH(OAc)3, DCM, rt, 6h; (c)
4-fluorobenzoyl chloride or 4-fluorobenzeneulfonyl chloride , Et3N, DCM, rt, 4h.
Scheme 2. Synthesis of target compounds 13-22. Reagents and conditions: (a) 1-bromo-3-
chloropropane, K2CO3, KI, CH3CN, 110°C, 0.5h; (b) 1-acetylpiperidine-4-carbonyl chloride, pyridine,
DCM, rt, 4h; c) K2CO3, KI, DMF, reflux, 12h.
Scheme 3. Synthesis of target compounds 26-29, 32-36. Reagents and conditions: (a) EDC, R4COOH,
DCM, rt, 12h; (b)2-(2-bromoethyl)-1,3-dioxolane, NaH, DMF, 80°C, 12h; (c) 1N HCl, 18h; (d)
NaBH(OAc)3, rt, 6h, (e) Et3N, DCM, rt, 4h; (f) K2CO3, KI, DMF, reflux, 12h.

Fig. 1.
O
O
N
F
F
N
N
N
N
NH2
H
N
N
N
O
O
Cl
TAK-220, 2
Maraviroc, 1
CN
N
H
N
N
O
3

Fig. 2.
O
O
N
N
NH2
O
R
N
N
N
O
TAK-220, 2
Cl
N
N
O
Part A
Cl
4: R= H
CN
CN
5: R= CH3
N
H
N
O
N
R4
N
N
Part B
O
N
R1
3
R2
R3
Target Compounds

Scheme 1.
7a: R1= 4-cyanobenzyl, R5=CH3(racemic), n=1
7b
: R1= benzyl, R5=CH3(racemic), n=1
7c: R1= 4-methylbenzyl, R5=CH3(racemic), n=1
R5
HN
7d
: R1= 4-fluorobenzyl, R5=CH3(racemic), n=1
: R1= 2-fluorobenzyl, R5=CH3(racemic), n=1
7f: R1= 3-fluorobenzyl, R5=CH3(racemic), n=1
N R1
n
7e
7i
: R1= 4-fluorobenzyl, R5=H, n=2
7j: R1= 4-fluorobenzyl, R5=CH3(R), n=1
7k
: R1= 4-fluorobenzyl, R5=CH3(S), n=1
7a-f, 7i-k
R5
O
c
HN
NH
N
n
HN
c
6a-d
F
7g
N
O
O
S
HN
F
7h
F
b
HN
HN
N
NH
8
9

Scheme 2.
O
NH2
HN
Cl
N
Cl
a
b
N
c
R2
7a-h
R2
O
R2
R3
R3
R3
10a-c
11a-c
12a-c
O
N
N
N
N
R1
O
R2
R3
13-22

Scheme 3.
O
O
O
O
O
NH2
O
R4
NH
R4
N
R4
N
a
b
c
Cl
Cl
Cl
Cl
10a
23a-e
24a-e
25a-e
O
N
O
O
R4
N
F
R4
N
d
N
7d
Cl
Cl
25a-d
O
26-29
O
Cl
N
Cl
e
f
N
N
7d
11a
O
O
Cl
30
31
O
F
N
N
N
N
O
Cl
32
O
N
O
O
X
N
d
N
N
F
9, 7i-k
O
O
Cl
33-36
Cl
25e