Evaluation of Novel N-(piperidine-4-yl)benzamide Derivatives as Potential Cell Cycle Inhibitors in HepG2 Cells

Article abstract of DOI:10.1111/cbdd.12484

In this study, a series of novel N-(piperidine-4-yl)benzamide derivatives was designed, synthesized, and evaluated for antitumor activity. Some compounds were found to have potent antitumor activity. In particular, compound 47 showed the most potent biological activity against HepG2 cells, with an IC₅₀ value of 0.25 μm. Western blot analysis demonstrated that compound 47 inhibited the expression of cyclin B1 and p-Rb and enhanced the expression of p21, p53, Rb, and phospho-adenosine monophosphate-activated protein kinase (p-AMPK). Further, cell cycle arrest was observed by flow cytometry (FCM). In summary, compound 47 was screened to have potential activity for the treatment of hepatocarcinoma via the induction of cell cycle arrest by a p53/p21-dependent pathway.

Full text of DOI:10.1111/cbdd.12484

Chem Biol Drug Des 2015; 86: 223–231
Research Article
Evaluation of Novel N-(piperidine-4-yl)benzamide
Derivatives as Potential Cell Cycle Inhibitors in HepG2
Cells
Jin Hou1, Wei Zhao1, Zhi-Ning Huang1, Shao-Mei
(CDKs), and cyclin-dependent kinase inhibitors (CDKIs).
CDKs are an important class of serine/threonine protein
kinases, and their activity is regulated by INK4 and the
Cip/Kip family of proteins. p21, a Cip/Kip family protein,
Yang1, Li-Juan Wang1, Yu Jiang2, Zhong-Shi
Zhou3, Man-Yi Zheng1, Ji-Li Jiang1, Shan-Hua
Li3,* and Fu-Nan Li1,*
can alter the conformation of the CDK catalytic subunit,
1School of Pharmaceutical Sciences, Xiamen University,
thereby blocking its activity and inhibiting the formation of
cyclin-CDK complexes in the G1 phase (3). p21 is also a
downstream regulator of p53, which is the most common
target for cancer gene therapy treatments. Mutations in
p53 and abnormal expression of p21 contribute to tumor
development. Thus, cell cycle progression is regulated
through a balance in the level and activity of cyclin-CDK
complexes, CDKIs, and other growth inhibitory proteins,
such as the p53 tumor suppressor.
Xiang’an South Road, Xiamen 361102, China
2Department of Outpatient, Air Force of Chengdu Military
Command, 87 New South Road, Chengdu 610041,
China
3Department of Basic Medical Sciences of Medical
College, Xiamen University, Xiang’an South Road, Xiamen
361102, China
*Corresponding authors: Fu-Nan Li, fnlee5@xmu.edu.cn;
Shan-Hua Li, lish@xmu.edu.cn
Adenosine monophosphate-activated protein kinase
(AMPK) is a master regulator of cellular energy homeosta-
sis. The activation of AMPK has toxic effects on most
human tumor cells, including lung, prostate, gastric, and
breast cancer cells, through the regulation of p53. Studies
indicate that activation of AMPK can induce phosphoryla-
tion of p53, block the binding of phosphorylated p53
to MDM2, and eventually increase intracellular p53
levels (4,5). AMPK, via a p53-p21-dependent pathway or
a p53-, p21-, and p27-independent pathway, restricts
the synthesis of fatty acids, proteins, and cholesterol
in cells and changes the cell metabolism pathway
to induce cell cycle arrest, inhibit tumor biosynthesis,
and cause tumor cell apoptosis and autophagy (6–9).
Therefore, p53 and AMPK can mutually coregulate cell
growth.
In this study, a series of novel N-(piperidine-4-yl)ben-
zamide derivatives was designed, synthesized, and
evaluated for antitumor activity. Some compounds
were found to have potent antitumor activity. In partic-
ular, compound 47 showed the most potent biological
activity against HepG2 cells, with an IC50 value of
0.25 lM. Western blot analysis demonstrated that com-
pound 47 inhibited the expression of cyclin B1 and
p-Rb and enhanced the expression of p21, p53, Rb,
and phospho-adenosine monophosphate-activated
protein kinase (p-AMPK). Further, cell cycle arrest was
observed by flow cytometry (FCM). In summary,
compound 47 was screened to have potential
activity for the treatment of hepatocarcinoma via the
induction of cell cycle arrest by a p53/p21-dependent
pathway.
Key words: cell cycle, HepG2, N-(piperidine-4-yl) benzamide
In this study, we designed a series of N-(piperidine-4-yl)
benzamide derivatives. Screening of these derivatives in a
series of human cancer cell lines showed that some of
these compounds exhibited stronger activity than sorafenib
against HepG2 cells. Compound 47, N-(1-(2, 6-difluorob-
enzyl)-piperidine-4-yl)-4-phenoxybenzamide, was found to
be the effective against HepG2 cells, and more selective
for HepG2 cells than A549 and MCF-7. Further, Com-
pound 47 exerted its effects through regulation of the cell
cycle. Thus, we hypothesize that compound 47 might
offer a promising therapeutic strategy for the treatment of
HCC.
derivatives, p53/p21-dependent pathway
Received 20 September 2014, revised 11 November 2014
and accepted for publication 20 November 2014
Hepatocellular carcinoma (HCC) is a common cancer with
a high mortality rate. Cancer is characterized by uncon-
trolled cell growth and proliferation, most commonly due
to dysregulation of the cell cycle (1,2). Studies have shown
that the cell cycle is regulated by three classes of proteins:
cell cycle proteins (cyclins), cyclin-dependent kinases
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12484
[The copyright line for this article was changed on 29th October, 2015 after original online publication]
223

Hou et al.
diluted with AcOEt, washed with 2 M HCl (29), brine, dried
over anhydrous MgSO4, and concentrated. KOH (44.8 mg,
0.8 mmol) in water (0.2 M) was added to the residue in
EtOH at room temperature. The reaction mixture was
stirred at 100 °C for 1–6 h and monitored by TLC analy-
sis. EtOH was evaporated from the reaction mixture upon
completion; the resultant solution was cooled to 0 °C
and acidified to pH 2.0 with 2M HCl. Then, we got com-
pound 5 white solid with the yield of 85%. 1H NMR
(600 MHz, CDCl3) d ppm: 12.24 (br s, 1H), 7.98 (dd,
J = 8.8, 1.8 Hz, 2H), 7.35 (t, J = 8.3 Hz, 2H), 7.19 (d,
J = 7.5 Hz, 2H), 7.09 (t, J = 7.3 Hz, 1H), 7.00 (d,
J = 8.6 Hz, 2H), 6.08 (br s, 1H). LC-MS (ESI): m/z 214.1
[M + H+].
Methods and Materials
Chemicals
All reagents were purchased and used without further puri-
fication unless otherwise indicated. Reactions were mag-
netically stirred and monitored by thin-layer chromatography
(TLC) on Merck silica gel 60F-254 by fluorescence
quenching under UV light. All of the final compounds were
purified by column chromatography. 1H NMR and 13C
NMR spectra were recorded on Bruker AV2 400 or AV2
600 MHz. Chemical shifts were given in parts per million
(ppm) relative to tetramethylsilane (TMS) as an internal
standard. Multiplicities were abbreviated as follows: single
(s), doublet (d), doublet-doublet (dd), doublet-triplet (dt),
triplet (t), triplet-triplet (tt), triplet-doublet (td), quartet (q),
quartet-doublet (qd), multiplet (m), and broad signal (br s).
Elemental analysis was determined on a Vario EL III ele-
mental analysis instrument (Elementar Analysen Syetem
GmbH, Hanau, Germany). Low-resolution and high-resolu-
tion mass spectral (MS) data were acquired on a Q Exac-
tive LC-MS/MS instrument with UV detection at 254 nm in
low-resonance electrospray mode (ESI). The molecular
masses of compounds were calculated by CHEMDRAW ULTRA
12.0 Software (PerkinElmer Inc., Waltham, MA, USA).
The compounds (6–8) were synthesized following the gen-
eral procedure as described above with the yield of 80–87%.
4-Phenoxy-N-(piperidine-4-yl)benzamide (9)
To a solution of compound (1) (200 mg, 0.94 mmol) and
4-amino-1-Boc-piperidine (224.4 mg, 1.1 mmol) in CH2Cl2
was added EDCI (198.2 mg, 1.3 mmol) and HOBT
(140 mg, 1.3 mmol) at 0 °C and then kept overnight at
room temperature. The reaction was diluted with CH2Cl2,
washed with 5% HCl (39), 5% NaOH (19), water (19),
and brine (19). Then, the organic layer was dried over
anhydrous MgSO4 and concentrated. To a solution of the
residue in AcOEt (10 mL) was added 4 M HCl solution
(5 mL) and stirred at room temperature for 4 h. Then, the
solvent was evaporated. The residue was diluted with
CH2Cl2 and water, alkalized to pH 9.0 with 2M NaOH,
and extracted with CH2Cl2 (39). The combined organic
layer was washed with water and brine, dried over anhy-
drous MgSO4, and concentrated. The residue was purified
by column chromatography to get white solid with the
yield of 83%. 1H NMR (400 MHz, DMSO-d6) d ppm: 8.82
(br s, 1H), 8.45 (d, J = 7.3 Hz, 1H), 7.91 (d, J = 8.8 Hz,
2H), 7.44 (td, J = 7.5, 2.0 Hz, 2H), 7.21 (t, J = 7.3 Hz,
1H), 7.08 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H),
4.05 (m, 1H), 3.28-3.32 (m, 2H), 3.00 (td, J = 12.3,
2.5 Hz, 2H), 1.96 (d, J = 10.8 Hz, 2H), 1.78 (qd, J = 12.0,
3.5 Hz, 2H). LC-MS (ESI): m/z 297.2 [M+H+].
4-Phenoxybenzoic acid (1)
To
a solution of ethyl 4-hydroxybenzoate (100 mg,
0.6 mmol) and fluoro benzene (67.2 mg, 0.7 mmol) in
DMF was added K2CO3 (248.4 mg, 1.8 mmol) and stirred
at 100 °C. After 12 h, the reaction was diluted with water
and extracted with AcOEt. The combined organic layer
was washed with brine, dried over anhydrous MgSO4, and
concentrated. Dissolve it in THF and methanol (4:1), added
LiOH (63 mg, 1.5 mmol), and followed by stirring for
30 min at room temperature and monitored by TLC analy-
sis. Then the residue was purified by column chromatogra-
phy to get white solid with the yield of 90%. 1H NMR
(600 MHz, CDCl3) d ppm: 12.18 (br s, 1H), 8.08 (dd,
J = 9.0, 2.2 Hz, 2H), 7.41 (td, J = 7.5, 2.0 Hz, 2H), 7.21
(t, J = 7.4 Hz, 1H), 7.09 (d, J = 7.7 Hz, 2H), 7.01 (dd,
J = 9.0, 2.0 Hz, 2H). LC-MS (ESI): m/z 215.1 [M + H+].
The compounds (2–4) were synthesized following the gen-
eral procedure as described above with the yield of 82–
90%.
The compounds (10–16) were synthesized following the
general procedure as described above with the yield of
76–92%.
4-(Phenylamino)benzoic acid (5)
N-(1-(2,6-Difluorobenzyl)piperidine-4-yl)-4-
To
a
solution of ethyl 4-bromobenzoate (100 mg,
phenoxybenzamide (47)
0.4 mmol) and benzenamine (49.3 mg, 0.5 mmol) in DMF
was added Cs2CO3 (202 mg, 0.6 mmol), BINAP (21.9 mg,
0.03 mmol), Pd(AcO)2 (7.9 mg, 0.03 mmol) at room tem-
perature. The reaction mixture was stirred at 120 °C for
4–48 h. Once the reaction appeared to be complete by
consumption of the 4-bromobenzoate by TLC analysis,
the mixture was allowed to cool to room temperature,
To a solution of compound (9) (50 mg, 0.2 mmol) and 2,6-
Difluorobenzyl bromide (35 mg, 0.2 mmol) in DMF was
added K2CO3 (70 mg, 0.5 mmol) and kept for 20 h at
room temperature. Then, the reaction was diluted with
water and extracted with AcOEt (39). The combined
organic layer was washed with brine, dried over anhydrous
224
Chem Biol Drug Des 2015; 86: 223–231

Cell Cycle Inhibitors by p53/p21-Dependent Pathway in HepG2
MgSO4, and concentrated. The residue was purified by
column chromatography to get white solid with the yield of
65%. 1H NMR (600 MHz, CDCl3) d ppm: 7.73 (dd,
J = 8.9, 2.0 Hz, 2H), 7.39 (td, J = 7.6, 2.0 Hz, 2H), 7.24-
7.29 (m, 1H), 7.19 (tt, J = 7.6, 1.0 Hz, 1H), 7.06 (d,
J = 8.8 Hz, 2H), 7.02 (dd, J = 8.9, 2.0 Hz, 2H), 6.92 (t,
J = 7.9 Hz, 2H), 5.90 (d, J = 7.6 Hz, 1H), 3.97 (m, 1H),
3.73 (s, 2H), 2.93 (d, J = 11.9 Hz, 2H), 2.31 (t,
J = 11.2 Hz, 2H), 2.04 (d, J = 9.6 Hz, 2H), 1.58 (qd,
J = 11.2, 3.6 Hz, 2H). 13C NMR (150 MHz, CDCl3) d ppm:
166.1, 162.9, 161.2, 156.1, 130.0, 129.3, 129.2, 128.7,
124.2, 119.7, 117.9, 113.0, 111.2, 111.1, 51.5, 48.7,
46.7, 32.3. HRMS (ESI): m/z calcd for C25H25F2N2O2
[M + H+] 423.1884, found 423.1893. Anal. Calcd for
C25H24F2N2O2: C, 71.07, H, 5.73, N, 6.63. Found: C,
71.21, H, 5.59, N, 6.71.
incubated at 37 °C for 4 h. The supernatant was removed,
and cells were dissolved in 150 lL DMSO. Next, the
plates were shaken for 5 min on a plate shaker to ensure
adequate solubility. Finally, the light absorption (OD) of the
dissolved cells was measured at 490 nm using a Spectra
MAX M5 microplate spectrophotometer (Molecular
Devices, Shanghai, China). All experiments were per-
formed in triplicate. The IC50 values were calculated using
Graph Pad Prism 5.0 software (Dragon Springs, Beijing,
China).
Western blot analysis
After treatment with a series of concentrations of com-
pound 47 for 12, 24, and 36 h at 37 °C, HepG2 cells
were harvested, washed with ice-cold physiological saline,
and lysed with NP40 lysis buffer (Beyotime, Shanghai,
China) including 1% phosphatase inhibitor cocktail (Sigma-
Aldrich, St. Louis, MO, USA). Total protein was separated
by SDS-PAGE and transferred to a PVDF membrane. The
membrane was blocked for 1 h in TBST(10 mM Tris–HCl,
pH 7.5, 150 mM NaCl, Tween-20) solution containing 5%
skimmed milk, then probed with primary antibody at 4 °C
overnight, washed 3 9 10 min in TBST, and probed with
corresponding secondary antibody at room temperature
for 1 h. After washed with TBST (3 9 10 min), autoradiog-
raphy was conducted with ECL chemiluminescence
regents. The relative expression of the target protein was
valuated with the gray value ratio of target protein content
to b-actin (target protein/b-actin) content. The primary anti-
bodies were cyclinD1 (H-295; Santa Cruz Biotechnology,
Dallas, TX, USA), p53 (DO-1; Santa Cruz Biotechnology),
Rb (C-15; Santa Cruz Biotechnology), p-Rb (F-4; Santa
Cruz Biotechnology), cyclinB1 [#4138; Cell Signaling Tech-
nology (CST), Danvers, MA, USA], cyclinE1 (HE-12; CST),
CDK2 (78B2; CST), CDK4 (DCS156; CST),p21 (Waf1/
Cip1; 12D1;CST), p-AMPK (t172; CST).
The other target compounds except compound 34 were
synthesized following the general procedure as described
above with the yield of 51–82%.
4-((4-(4-Phenoxybenzamido)piperidine-1-yl)methyl)
benzoic acid (34)
Procedure: Compound 33 (50 mg, 0.1 mmol) was dis-
solved in 10 mL of EtOH; to the stirred solution, 3 mL of
2M NaOH solution was added drop by drop and kept for
1 h at room temperature. Then, the EtOH was evaporated,
the resultant solution was cooled to 0 °C, acidified to pH
2.0 with 2 M HCl, and collected the precipitate to get white
1
solid with the yield of 58%. H NMR (600 MHz, DMSO-d6)
d ppm: 10.93 (br s, 1 H), 8.51 (br s, 1 H), 8.14–8.17 (m, 2
H), 7.85 (d, J = 8.3 Hz, 2 H), 7.69 (d, J = 8.3 Hz, 2 H),
7.43 (t, J = 8.0 Hz, 2 H), 7.20–7.24 (m, 1 H), 7.07 (dd,
J = 7.7, 1.1 Hz, 2 H), 7.00–7.03 (m, 2 H), 4.44 (s, 2 H),
4.15–4.20 (m, 1 H), 3.55 (d, J = 11.9 Hz, 2 H), 3.23–3.27
(m, 2 H), 2.24 (d, J = 13.8 Hz, 2 H), 1.95–2.04 (m, 2 H).
13C NMR (150 MHz, CDCl3) d ppm: 172.1, 170.1, 164.6,
160.9, 136.8, 135.5, 134.7, 134.2, 129.5, 127.6, 124.6,
122.5, 119.9, 117.6, 63.51, 56.0, 50.4, 33.7. HRMS (ESI):
m/z calcd for C26H27N2O4 [M + H+] 431.1971, found
431.1992.
A flow cytometry assay
The effect of compound 47 on cell cycle of HepG2 cells
was investigated with flow cytometry. Then, the cells were
detached by trypsin, washed twice in PBS, and fixed in
70% cold ethanol overnight at 4 °C. The next day, the cells
were centrifuged in 3000 9 g for 5 min, washed twice in
PBS, and incubated with DNase A solution (150 lg/mL) for
20 min at 37 °C. Then, the cells were incubated in PI solu-
tion (100 lg/mL) at room temperature for 30 min. The cell
cycle was detected by flow cytometry (Beckman Coulter,
Inc., Brea, CA, USA). The experiment was performed in
triplicate.
Cell lines and cell culture
All of the cell lines were obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA) and grown
in DMEM culture medium containing 10% fetal bovine
serum (v/v) in 5% CO2 at 37 °C.
Cell antitumor activities assays
We used the MTT assay to test the cell viability. The tumor
cells in good state were collected and seeded in 96-well
plates (5–6 9 103 cells/well) and incubated at 37 °C for
24 h. Different concentrations of compounds were added
to the cells and incubated at 37 °C for 48 h. Then, 20 lL
of 5 mg/mL MTT reagent was added to each well and
Colony formation assay
HepG2 cells were seeded in 6-well plate (500 cells/well).
After stabilized for 24 h, treated with different concentra-
tions of compound 47 in 0.5% serum medium for 6 days
Chem Biol Drug Des 2015; 86: 223–231
225

Hou et al.
and fixed with 4% paraformaldehyde. Colonies were
stained with 0.1% crystal violet.
nucleophilic substitution reaction on the N atom of piperidi-
nyl was used to obtain compounds 17–61 with the yield of
51–82% (Scheme 1).
Results and Discussion
Tumor cell growth inhibition
Chemistry
An overview of the in vitro antitumor activity screen using
the N-(piperidine-4-yl)benzamide derivatives (17–61) is
reported in Tables 1–3.
Based on the structure of sorafenib, a multitargeted diaryl
urea antitumor agent with high efficacy (10,11), we
retained the diaryl ether and optimized a functional amide
moiety to further improve its antihepatocarcinoma activity.
We replaced the urea moiety, which regulates dipolar
interactions, with an amide moiety. We then attempted to
introduce a piperidine to hydrophobically interact with
another aromatic ring to obtain the N-(piperidine-4-yl)ben-
zamide class of compounds (Figure 1).
Tables 1 and 2 show the bioactivities of a series of N-
(1-benzylpiperidine-4-yl)-4- phenoxybenzamide derivatives
that contain varied B-ring substituents. The growth inhib-
itory potency of N-(1-benzylpiperidine-4-yl)-4-phenoxyb-
enzamide derivatives in HepG2 cells is shown in
Table 1. Derivative potency was strongly influenced by
B-ring substituents and displayed strong positional
effects.
To explore the structure–activity relationship (SAR) of the
N-(piperidine-4-yl)benzamide class of compounds, a series
of derivatives was obtained using the synthetic pathway
shown in Scheme 1 (12–15). All of the target compounds
were obtained via a three-step reaction. First, a Buchwald–
Hartwig C-N or C-O coupling followed by an ester hydroly-
sis reaction was used to obtain compounds 1–8 with the
yield of 80–90%. Next, EDCI (1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide) and HOBT (1-hydroxybenzotriazole
hydrate) were used to catalyze amide formation, and the
protecting group (-Boc) was removed to produce
compounds 9–16 with the yield of 76–92%. Finally, a
Compounds with halogen, methyl, nitro, or carboxyl
groups at the different positions of ring B showed stronger
against HepG2 than compound 17, which had no substit-
uents on ring B. Some compounds showed higher anti-
proliferative activity against HepG2 cells than the reference
drug, sorafenib (IC50 16.30 lM) (16), including compounds
18–23, 25, 26, 28, which had IC50 values of 3.41, 1.03,
7.84, 15.40, 9.35, 6.26, 3.69, 3.32 and 4.68 lM, respec-
tively. In particular, compound 19 was the most potent
agent, with an IC50 15-fold higher than sorafenib.
Figure 1: Structure design of derivative 2 based on sorafenib.
Scheme 1: Synthetic route to obtain target compounds. Reagents and conditions: (a) K2CO3, DMF, 12 h, 100 °C; LiOH, THF/MeOH,
0 °C to rt, 30 min, 82–90% for 2 steps; (b) Cs2CO3, BINAP, Pd(OAc)2, DMF, 120 °C, 4–48 h; KOH, H2O/EtOH, 100 to 0 °C, 1–6 h,
80–87% for two steps; (c) 4-amino-1-Boc-piperidine, EDCI, HOBT, CH2Cl2, 0 °C to rt, overnight; 2 M HCl, 2 h, 76–92% for two steps;
(d) substituted benzylbromide, K2CO3, DMF, rt, 20 h, 51–82%.
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Cell Cycle Inhibitors by p53/p21-Dependent Pathway in HepG2
Table 1: Inhibitory potency of compounds 17–35 against HepG2
cells
decreased potency, as shown in Table 1 (compound 28
versus 32). Additionally, compounds 29–33 and 35 exhib-
ited much lower antiproliferative activity than the other
mono-substituted derivatives. Ultimately, the ortho-Cl
substituted analog (compound 19) was the most potent
inhibitor among the mono-substitute analogs on ring B.
Compounds
R
HepG2 (IC50, lM)a
It has been reported that increased halogen-induced lipo-
philicity has an additive effect (21). Thus, we designed and
synthesized compounds with the addition of two or three
halogen substituents on the ring B. However, the results
indicated that additional halogen atoms did not improve
the effect. We hypothesize that the addition of halogen
substituents on the ring B may influence the absorption
and steric effect of compounds, due to their high molecu-
lar weight and large volume, with the exception of the fluo-
rine atom. The fluorine atom, which has the lowest
molecular weight and strongest electron-withdrawing
effect among all the halogen atoms, exhibited an additive
effect without affecting the absorption and steric effect
(21,22). Compared to mono-substituted compounds, di-
substitution of the compounds with fluorine enhanced
lipophilicity, permeability, stability, and bioavailability (21).
When comparing compounds 37, 43, 47, 49, and 52 di-
substituted with fluorine, only compound 47 exhibited
excellent activity. This phenomenon could occur due to
the electronic effect of fluorine, which is also reflected in
the fluorine atom of receptors involved in the formation of
weak hydrogen bonds, which could improve the struc-
tural stability and biological activity (17). The two adjacent
fluorine substituents may be more conducive to form
hydrogen bonds with the nitrogen atom of piperidine,
which make its structure more stable than others. In con-
trast, tri-substituted compound 54 presented low antipro-
liferative activity against HepG2 cells, indicating that two
fluorine substituents on ring B were sufficient to mediate
the inhibitory effect. Additionally, compound 42 (2-CH3,
4-F) showed a better inhibitory activity than compound
25 (4-F) which may be caused by the sterics of the
methyl group. Therefore, compound 47 (2-F, 6-F) exhib-
ited the best inhibitory activity with an IC50 value of
0.25 lM, which represented an 65-fold increase in
potency compared to sorafenib and a fourfold increase
compared with compound 19.
17
18
H
2-F
2-Cl
2-Br
2-NO2
3-F
3-Cl
3-Br
4-F
30.70
3.41
1.03
7.84
15.40
9.35
6.26
23.64
3.69
3.32
23.41
4.68
19
20
21
22
23
24
25
26
4-Cl
4-Br
27
28
4-CH3
4-CN
4-SO2CH3
4-CF3
4-t-Bu
4-COOCH3
4-COOH
4-(2-naphthalene)
–
29
30
>100
>100
>100
>100
>100
21.42
35.14
16.30
31
32
33
34
35
Sorfenib
aEach compound was tested in triplicate.
When comparing compounds containing halogen substitu-
ents, the position of the substituents was found to effect
the inhibitory potency for the growth of HepG2 cell
(ortho > para > meta), such as compounds 18, 25, and
22 had IC50 values of 3.41, 3.69, and 9.35 lM, respec-
tively. Further, the specific halogen also affected potency
(-Cl > -F > -Br), such as compounds 19, 18, and 20 had
IC50 values of 1.03, 3.41, and 7.84 lM, respectively.
Meantime, we found that compounds with halogen groups
at the different positions on ring B were more potent
inhibitors of HepG2 cell growth than compound 17, which
had no substituents on ring B. Perhaps this is due to the
nature of halogen atoms. First, their strong electron-with-
drawing effect can change the acid–base properties of the
compounds, increasing the stability and receptor affinity of
the compounds (17,18). Second, it is known that C-H
bond cleavage by oxidation is a common metabolic path-
way. Because the C-halogen bond has higher bond
energy than the C-H bond, it is harder to oxidize C-halo-
gen bonds than C-H bonds. Thus, compounds with halo-
gen groups could block metabolism (19). Third, the
introduction of halogen groups could increase lipophilicity,
thereby enhancing the permeability and improving biologi-
cal activity (20). Therefore, compounds with halogen
groups on ring B exhibited good activity.
As our compounds were designed for the treatment of he-
patocarcinoma, HepG2, MCF-7, and A549 which are
common cancer cell lines were chosen to see whether
compound 47 had selectivity toward HepG2 cells. As
shown in Table 2, compound 47 exhibited better inhibition
in HepG2 cells than in A549 (IC50 > 100 lM) and MCF-7
(IC50 > 100 lM) cells, indicating that compound 47 was a
potent and specific inhibitor of HepG2 cells. The excep-
tional ability of compound 47 to inhibit HepG2 cell growth
prompted us to examine its effect in a colony formation
assay. Treatment of HepG2 cells with compound 47 for
6 days completely inhibited the colony-forming activity of
HepG2 cells shown in Figure 2.
A methyl group para to ring B improved potency; however,
increasing the size of the alkyl substituents gradually
Chem Biol Drug Des 2015; 86: 223–231
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Hou et al.
Table 2: Inhibition of cell lines (HepG2, A549, and MCF-7 cells) by N-(1-benzylpiperidine-4-yl)-4-phenoxybenzamide derivatives with
modified B rings
Compounds
R
HepG2 (IC50, lM)a
A549 (IC50, lM)a
MCF-7 (IC50, lM)a
36
37
2-F, 3-Cl
2-F, 4-F
2-F, 4-Cl
2-F, 4-Br
2-Cl, 4-F
2-Br, 4-CN
2-CH3,4-F
2-F, 5-F
2-F, 5-Cl
2-F, 5-Br
2-Br, 5-F
2-F, 6-F
2-Cl, 6-Cl
3-F, 4-F
3-Cl, 4-Cl
3-F, 4-Br
3-F, 5-F
3-Br, 5-Br
2-F, 4-F, 6-F
–
5.99
>100
65.26
19.29
>100
>100
3.24
28.32
>100
>100
22.22
0.25
>100
>100
8.03
>100
>100
>100
>100
16.30
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
>100
ndb
ndb
ndb
ndb
ndb
ndb
ndb
13.15
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
ndb
>100
ndb
ndb
ndb
ndb
ndb
ndb
ndb
>100
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Sorfenib
aEach compound was tested in triplicate.
bNot determined.
Table 3: Effect of ring A substituents and the 4-position linker
atom (X) of N-(piperidine-4-yl)benzamide derivatives on growth
inhibition in HepG2 cells
derivatives were more potent than their corresponding
N-(1-benzyl-piperidine-4-yl)-4-(phenyl-amino)benzamide der-
ivatives. Moreover, among the N-(1-benzylpiperidine-4-yl)-
4-phenoxybenzamide derivatives, compounds 55 and 56,
which had bromine and methoxy at the para-position of
ring A, exhibited excellent inhibitory activity, but were less
potent than compounds 47 and 57. However, because of
the similar inhibitory potency of compounds 47 and 57,
the influence of ring A moieties on N-(1-benzylpiperidine-
4-yl)-4-phenoxybenzamide derivatives needs further study.
Here, we used compound 47, which has an oxygen (–O)
atom at the 4-position linker site and no substituents at
ring A, for further biological activity studies.
Compounds
R
X
HepG2 (IC50, lM)a
47
55
56
57
58
59
60
61
H
Br
OCH3
CF3
H
Br
OCH3
CF3
O
O
O
O
NH
NH
NH
NH
0.25
2.29
2.29
0.34
>100
>100
29.03
>100
Signaling inhibition in HepG2 cells
The present study sought to determine how compound 47
inhibits HepG2 cell growth. Microscopic analysis indicated
that the number of HepG2 cells decreased after com-
pound 47 treatment, as compared to the control group.
Therefore, we hypothesized that the inhibitory activity of
compound 47 may be associated with regulation of the
cell cycle. Flow cytometry was performed to examine cell
cycle inhibition after 24 or 48 h of compound 47 treatment
(10 lM, Figure 3). The results revealed a slight accumula-
tion of cells in the S phase at 24 h and significant G2/M
phase arrest at 48 h.
aEach compound was tested in triplicate.
According to the principles of bioisostere, we designed
and synthesized two series of N-(piperidine-4-yl)benzamide
derivatives, N-(1-benzylpiperidine-4-yl)-4-phenoxy benza-
mide and N-(1-benzyl-piperidine-4-yl)-4-(phenyl-amino)
benzamide, which contained a secondary amine (-NH-) or
oxygen atom at the linker site, respectively. As shown in
Table 3, N-(1-benzylpiperidine-4-yl)-4-phenoxybenzamide
228
Chem Biol Drug Des 2015; 86: 223–231

Cell Cycle Inhibitors by p53/p21-Dependent Pathway in HepG2
Figure 2: Compound 47 inhibits the
colony-forming activity of HepG2 cells. Cells
were grown in 6-well plates for 6 days and
treated with compound 47 (0.02, 0.25, and
1 lM).
A
B
Figure 3: A flow cytometry assay was
performed to examine cell cycle arrest.
HepG2 cells were harvested after treatment
with 10 lM compound 47 for 24 and 48 h.
(A) Treatment for 24 h. (B) Treatment for
48 h. (a) Control group. (b) Treated with
compound 47.
A
B
Figure 4: Western blot analysis was used
to examine the expression of cell cycle-
regulated proteins. (A, B, D) Expression was
evaluated in a dose-dependent manner in
HepG2 cells after incubation with compound
47 for 24 h. (C) Expression was determined
after 12, 24, and 36 h in HepG2 cells after
treatment with 10 lM compound 47.
C
D
It is well known that the G1/S transition is mediated by
cyclin-CDK complex-induced phosphorylation of Rb,
which promotes the activation of the transcription factor
E2F, thereby regulating the expression of various genes
that mediate cell cycle progression (23). And the com-
plexes of cyclin D-CDK4/6 and cyclin E-CDK2 play an
important role in G1/S transition by phosphorylating Rb
(24,25). Our results showed that the expression of cyclin
D1-CDK4 and cyclin E1-CDK2 complexes was not
affected by compound 47 (Figure 4A). Thus, there may be
other mechanisms contributing to S phase arrest in this
study. CDKIs can negatively regulate CDKs by binding to
cyclin-CDK complexes (26), while p53, an essential cell
cycle regulator, can transcriptionally activate a series of
genes involved in both G1/S and G2/M transitions (27).
Thus, we examined the expression of CDKIs and p53.
Compound 47 upregulated p21 and p53 in a dose-depen-
dent manner suggests that the activity of CDKs may be
inhibited by p21 and p53 (Figure 4B). Furthermore, com-
pound 47 suppressed the expression of p-Rb, which may
promote the interaction between Rb and E2F, resulting in
S phase arrest (Figure 4C). Taken together, the data
Chem Biol Drug Des 2015; 86: 223–231
229

Hou et al.
indicated that compound 47 may block the S phase by
regulation of p21, p53, and p-Rb.
derivatives as potential anticancer agents. The synthetic
method was relatively simple; the compounds were easily
purified and produced in high yields. Some compounds
possessed greater than sorafenib with regard to inhibition of
HepG2 cells. Compound 47 showed significant inhibitory
activity against HepG2 cells, with an IC50 value of 0.25 lM,
as well as excellent selectivity toward HepG2 over A549
and MCF-7 cells. Western blot analysis and flow cytometry
assay demonstrated that compound 47 could regulate
AMPK phosphorylation, activate downstream signaling pro-
teins, and arrest the cell cycle in a p53/p21-dependent
manner. However, determining its roles in preventing cancer
still requires further intensive study.
The cell cycle analysis revealed that a significant G2/M
phase arrest occurred at 48 h after treatment with com-
pound 47. G2/M transition is partially governed by the
activity of CDK1, which is positively regulated by cyclin B
(28). p21 also inhibits the CDK1/cyclin B complex causing
G2 arrest (29). The increase in p53 activity and p21 levels
may lead G2 arrest in the event that DNA damage has
been reported (30,31). Thus, the downregulation of cyclin
B1 (Figure 4C) associated with the upregulation of p21
and p53 suggests that the p53-p21 pathway may have
contributed to the G2/M phase arrest in HepG2 cells trea-
ted with compound 47.
Acknowledgments
The significant changes in p53 and p21 expression sug-
gested that components of the p53/p21-dependent path-
way, such as the AMPK signaling pathway, might be
involved in cell cycle arrest (6). Further, the effect of Rb
may be mediated through AMPK activation, leading to p53/
p21-dependent cellular senescence (30). We therefore
evaluated whether compound 47 inhibited the cell cycle by
activating the upstream protein kinase AMPK. Intriguingly,
upregulation of p-AMPK and Rb was observed (Figure 4C
and D). Taken together, our data suggest that the upregu-
lation of p53, likely induced by the activation of p-AMPK,
may promote the synthesis of p21. p21 could then com-
bine with cyclin-CDK complexes to form a trimer, thereby
negatively regulating cyclin-CDK. Inactivation of cyclin-CDK
complexes limits the cell restriction points, thereby prevent-
ing the transition and conversion between each phase of
the cell cycle. p21 also inhibits the phosphorylation of Rb.
Non-phosphorylated Rb could bind E2F, resulting in the
inactivation of E2F and inhibition of the cell cycle. Thus, we
concluded that compound 47, which inhibited the cell cycle
through a p53/p21-dependent pathway, might be a poten-
tial agent for HCC therapy.
This work was supported by the National Natural Science
Foundation of China (NSFC-81101924 and NSFC-
81472230) and the Scientific Research Foundation for the
Returned Overseas Chinese Scholars, State Education
Ministry.
Conflict of Interest
None.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. 1H and 13C NMR and MS spectra of com-
pound 47.
Figure S2. Western blot analysis was used to examine
the expression of p53 and p21.
1
Table S1. H and 13C NMR and MS spectra of the chemi-
cal intermediate and N-(piperidine-4-yl)benzamide deriva-
ties.
Chem Biol Drug Des 2015; 86: 223–231
231