Synthesis and structure–activity relationship of N-(piperidin-4-yl)benzamide derivatives as activators of hypoxia-inducible factor 1 pathways

Article abstract of DOI:10.1007/s12272-018-1050-2

Guided by bioisosterism and pharmacokinetic parameters, we designed and synthesized a series of novel benzamide derivatives. Preliminary in vitro studies indicated that compounds 10b and 10j show significant inhibitory bioactivity in HepG2 cells (IC₅₀ values of 0.12 and 0.13?μM, respectively). Compounds 10b and 10j induced the expression of HIF-1α protein and downstream target gene p21, and upregulated the expression of cleaved caspase-3 to promote tumor cells apoptosis.

Full text of DOI:10.1007/s12272-018-1050-2

Arch. Pharm. Res.
Online ISSN 1976-3786
Print ISSN 0253-6269
https://doi.org/10.1007/s12272-018-1050-2
RESEARCH ARTICLE
Synthesis and structure–activity relationship of N-(piperidin-4-
yl)benzamide derivatives as activators of hypoxia-inducible factor
1 pathways
Zhi-Ning Huang1 Han Liang1 Hong Qiao2 Bao-Rui Wang1 Ning Qu2
•
•
•
•
•
Hua Li3 Run-Run Zhou1 Li-Juan Wang1 Shan-Hua Li2 Fu-Nan Li1
•
•
•
•
Received: 18 January 2018 / Accepted: 25 June 2018
Ó The Pharmaceutical Society of Korea 2018
Abstract Guided by bioisosterism and pharmacokinetic
parameters, we designed and synthesized a series of novel
benzamide derivatives. Preliminary in vitro studies indi-
cated that compounds 10b and 10j show significant inhi-
bitory bioactivity in HepG2 cells (IC50 values of 0.12 and
0.13 lM, respectively). Compounds 10b and 10j induced
the expression of HIF-1a protein and downstream target
gene p21, and upregulated the expression of cleaved cas-
pase-3 to promote tumor cells apoptosis.
constitutively expressed in cells, whereas the unique sub-
unit of HIF-1, HIF-1a, is only found in the hypoxic nucleus
and determines the HIF-1 activity (Majmundar et al. 2010).
HIF-1a performs conflicting functions in tumor cells,
especially in hepaticliver cancer cells, which are always in
state of hypoxia. HIF-1a promotes transcription and
expression of target genes to accelerate glycolysis in tumor
cells and upregulates a variety of growth factors, allowing
cancer cells to proliferate, differentiate, and adapt to
hypoxic microenvironment (Guo et al. 2016). In contrast,
HIF-1a overexpression in hypoxic cancer cells was sug-
gested to inhibit cell division by inducing p21, p27, or p53
and to promote apoptosis by inducing p53, Nip3, Noxa, or
HGTD-P. Apoptosis factor p21 is a general cell cycle
inhibitory protein, inhibiting many protein kinases. Acti-
vation of p21 expression or p53–p21 signaling pathway
promotes cell cycle arrest and apoptosis. HIF-1a induces
p21 expression by dissociating c-Myc (Lina and Brown
2006), a protein blocking p21 promoter, whereas phos-
phorylated HIF-1a interacts with p53, promoting p53-me-
diated apoptosis (Sendoel et al. 2010).
Keywords Diaryl ethers Á HIF-1a Á p21 Á Cleaved caspase-
3 Á Antitumor activity
Introduction
Hypoxia-inducible factor 1 (HIF-1) is an important tran-
scription factor that targets a series of adaptation genes
under hypoxic conditions. HIF-1 is a heterodimeric protein
composed of HIF-1a and HIF-1b subunits. HIF-1b is
However, there is a difference between scholars’ opin-
ions about whether HIF can promote or inhibit the growth
and proliferation of hepatocellular carcinoma cells in the
course of the treatment. Some scholars think that the
increase of HIF expression in a hypoxic environment
around the tumor tissue is beneficial to the formation of
hepatic vessels, and invasion and metastasis, thereby pro-
moting the development of tumors (Brahimi-Horn et al.
2007). Some scholars believe that inducing HIF gene
overexpression can promote the expression of apoptosis-
related proteins such as Bax and Bad, while inhibiting the
target gene TFDP3, in turn inhibiting tumor growth and
development (Sun et al. 2013). However, there is no doubt
that using HIF as a breakthrough holds great potential for
Zhi-Ning Huang and Han Liang have equally contributed.
& Shan-Hua Li
lish@xmu.edu.cn
& Fu-Nan Li
fnlee5@xmu.edu.cn
1
School of Pharmaceutical Sciences, Xiamen University,
Xiamen 361102, People’s Republic of China
2
Department of Basic Medical Sciences of Medical College,
Xiamen University, Xiamen 361102, People’s Republic of
China
3
Department of Gastroenterology, First Affiliated Hospital of
Xiamen University, Xiamen 361003, People’s Republic of
China
123

Z.-N. Huang et al.
General synthetic procedure for (5a–j;
10a–r and 15a–d)
the development of targeted chemosynthetic antitumor
drugs.
Previously, we introduced amide groups as ureido
bioisosteres in the structure of anti-cancer drug sorafenib
and optimized the compounds to include the hydrophilic
pyridyl group (Yang et al. 2015), obtaining a series of N-
(piperidin-4-yl)benzamide derivatives. Preliminary bioac-
tivity screening indicated that most compounds featuring
pyridine and 2,6-difluorobenzyl fragments in the C region
of the structure exhibit promising biological potential and
inhibitory activity, with compound HJ-47 showing greatest
activity (Hou et al. 2015). This study extends our previous
work, retaining N-(1-(2,6-difluorobenzyl)-4-piperidinyl)
benzamide core and optimizing diaryl ether substituents in
the A region of the derivatives, with the goal of comparing
the importance of diaryl ether moiety and substituents,
further clarifying structure–activity relationships, and
exploring potential anti-cancer activity (Fig. 1).
To a stirred solution of phenol 6a or 2-chloropyridine 11a
(1.1 mmol), ethyl 4-fluorobenzoate 7 of ethyl 4-hydroxy-
benzoate 12 (1.0 mmol) in N, N-dimethylformamide was
added K2CO3 or CsCO3 (2.0 mmol) at 130 °C for12 h. The
reaction mixture was extracted with EtOAc. The organic
layers dried over anhydrous MgSO4 and concentrated.
Then the residue was dissolved in ethanol, a little NaOH
(1.5 mmol) was added. The reaction mixture was stirred at
80 °C for 1 h and then concentrated in vacuo to give of the
crude acid 8a–r and 13a–d, which was directly used for the
next step (48–89% for 2 steps).
To a solution of acids 3a–j, 8a–r and 13a–d (0.8 mmol)
and tert-butyl 4-aminopiperidine-1-carboxylate (1.0 mmol)
in dichloromethane (3 mL) at 0 °C were added 1-hydrox-
ybenzotriazole HOBt (0.9 mmol), 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide
hydrochloride
EDCI
(0.9 mmol). The reaction mixture was stirred at 0 °C and at
room temperature for a further 12 h. Then washed with
10% aqueous HCl, 5% aqueous NaOH, H2O and brine,
dried over MgSO4, and concentrated in vacuo. The residue
was dissolved in AcOEt and 4 N HCl in dichloromethane
was added dropwise. The reaction was stirred at rt for 2 h
and then evaporated to dryness. The resdiue was diluted
with dichloromethane and washed with 2 N NaOH. The
organic layer was dried over MgSO4, and concentrated in
vacuo. The residue was purified by crystallization from
dichloromethane to afford as a white solid (66–90% for 2
steps).
Materials and methods
Chemistry
All reagents were purchased and used without further
purification unless otherwise noted. Reactions were moni-
tored by thin-layer chromatography (TLC) on YanTai slica
gel GF-254 thin-layer plates. 1H NMR and 13C NMR
spectra were determined with a Bruker Avance III
600 MHz spectrometer (600 MHz for 1H and 150 MHz for
13C). Chemical shifts are expressed in d values (ppm),
using tetramethylsilane (TMS) as the internal standard;
coupling constants (J) are given in Hz. Signal multiplicities
are characterized as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), brs (broad signal). High-resolution
mass spectral (HRMS) were acquired on a Q-Exac-
tiveseries MS instrument with UV deterction at 254 nm in
low-resonance electrospray mode (ESI).
To a solution of intermediate 4a–j, 9a–r and 14a–
d (0.3 mmol) and 2-(bromomethyl)-1,3-difluorobenzene
(0.3 mmol) in DMF (3 mL) was added. K2CO3
(0.6 mmol). The reaction was stirred at rt for 20 h was then
added some water. The aqueous mixture was extracted with
AcOEt. The organic phase was combined and washed with
brine, dried over anhydrous MgSO4, filtered, and concen-
trated in vacuo. The residue was purified by column
chromatography (ethyl acetate/hexane) to provide the title
compound (54–91%).
Fig. 1 Designs of N-(Piperidin-4-yl)benzamide analogues
123

Synthesis and structure–activity relationship of N-(piperidin-4-yl)benzamide derivatives as…
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)benzamide (5a)
131.8, 129.4, 128.5, 126.1, 111.2, 51.4, 48.7, 46.7, 32.1;
LCMS (ESI): m/z 409, 411 [M ? H?].
1
White solid; yield: 84%; H NMR (600 MHz, CDCl3):d
7.65 (dd, J = 8.4, 1.7 Hz, 2H), 7.41 (tt, J = 7.5, 2.0 Hz,
1H), 7.34 (t, J = 7.9 Hz, 2H), 7.18 (q, J = 7.3 Hz, 1H),
6.83 (q, J = 7.5 Hz, 2H), 5.89 (d, J = 7.5 Hz, 1H), 3.84 -
3.92 (m, 1H), 3.65 (s, 2H), 2.85 (d, J = 11.7 Hz, 2H), 2.22
(t, J = 11.2 Hz, 2H), 1.96 (d, J = 13.0 Hz, 2H), 1.51 (qd,
J = 11.7, 3.9 Hz, 2H); 13C NMR (150 MHz, CDCl3): d
166.8, 162.9, 161.2, 134.8, 131.4, 129.4, 128.6, 126.8,
112.8, 111.2, 51.5, 48.7, 46.7, 32.2; HRMS (ESI): m/z
calcd for C19H21F2N2O [M ? H?] 331.1622, found
331.1627.
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-
hydroxybenzamide (5e)
White solid; yield: 67%; 1H NMR (600 MHz, DMSO-d):
d 9.89–10.17 (m, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.69 (d,
J = 8.8 Hz, 2H), 7.41 (q, J = 7.7 Hz, 1H), 7.10 (t,
J = 7.8 Hz, 2H), 6.77 (d, J = 8.6 Hz, 2H), 3.64–3.73 (m,
1H), 3.57 (s, 2H), 2.81 (d, J = 11.6 Hz, 2H), 2.08 (t,
J = 11.2 Hz, 2H), 1.73 (d, J = 10.3 Hz, 2H), 1.52 (qd,
J = 11.9, 3.7 Hz, 2H); 13C NMR (150 MHz, DMSO-d): d
165.7, 162.7, 161.1, 160.4, 130.4, 129.6, 125.8, 115.1,
113.6, 111.8, 52.3, 48.8, 46.9, 31.9; LCMS (ESI): m/z 347
[M ? H?].
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-
fluorobenzamide (5b)
1
White solid; yield: 86%; H NMR (600 MHz, CDCl3): d
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-
methylbenzamide (5f)
7.74 (td, J = 6.6, 2.0 Hz, 2H), 7.22 - 7.28 (m, 1H), 7.09 (t,
J = 8.6 Hz, 2H), 6.90 (t, J = 7.9 Hz, 2H), 5.89 (d,
J = 7.3 Hz, 1H), 3.88–3.98 (m, 1H), 3.71 (s, 2H), 2.91 (d,
J = 11.7 Hz, 2H), 2.28 (t, J = 11.2 Hz, 2H), 2.01 (d,
J = 11.6 Hz, 2H), 1.55 (qd, J = 11.6, 3.9 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 165.7, 165.5, 163.8, 162.9,
161.3, 130.9, 129.3, 129.2, 115.6, 115.5, 113.0, 111.1,
51.5, 48.7, 46.9, 32.3; HRMS (ESI): m/z calcd for C19-
H20F3N2O [M ? H?] 349.1528, found 349.1533.
1
White solid; yield: 76%; H NMR (600 MHz, CDCl3): d
7.55 (d, J = 8.3 Hz, 2H), 7.15 (q, J = 7.5 Hz, 1H), 7.14 (m,
J = 8.1 Hz, 2H), 6.82 (q, J = 6.8 Hz, 2H), 5.83 (d,
J = 7.7 Hz, 1H), 3.83–3.91 (m, 1H), 3.64 (s, 2H), 2.84 (d,
J = 11.7 Hz, 2H), 2.31 (s, 3H), 2.22 (t, J = 11.1 Hz, 2H),
1.94 (d, J = 12.8 Hz, 2H), 1.49 (qd, J = 11.6, 3.8 Hz, 2H);
13C NMR (150 MHz, CDCl3): d 166.7, 162.9, 161.3,
141.8, 131.9, 129.3, 129.2, 126.8, 111.1, 51.5, 48.7, 46.6,
32.3, 21.4; LCMS (ESI): m/z 345 [M ? H?].
4-Chloro-N-(1-(2,6-difluorobenzyl)piperidin-4-
yl)benzamide (5c)
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-
methoxybenzamide (5g)
1
White solid; yield: 73%; H NMR (600 MHz, CDCl3): d
7.66 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 7.25 (q,
J = 7.1 Hz, 1H), 6.90 (t, J = 7.9 Hz, 2H), 5.89 (d,
J = 7.5 Hz, 1H), 3.89–3.97 (m, 1H), 3.71 (s, 2H), 2.91 (d,
J = 11.9 Hz, 2H), 2.28 (t, J = 11.2 Hz, 2H), 2.01 (d,
J = 11.9 Hz, 2H), 1.55 (qd, J = 11.9, 3.5 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 165.7, 162.9, 161.2, 137.6,
133.1, 129.3, 128.8, 128.3, 112.9, 111.2, 51.5, 48.7, 46.9,
32.2; HRMS (ESI): m/z calcd for C19H20ClF2N2O
[M ? H?] 365.1232, found 365.1235.
1
White solid; yield: 83%; H NMR (600 MHz, CDCl3): d
7.62 (dt, J = 8.6, 2.6 Hz, 2H), 7.18 (q, J = 7.5 Hz, 1H),
6.84 (d, J = 9.4 Hz, 2H), 6.82 (t, J = 9.0 Hz, 2H), 5.78 (d,
J = 7.7 Hz, 1H), 3.83–3.90 (m, 1H), 3.77 (s, 3H), 3.64 (s,
2H), 2.84 (d, J = 11.6 Hz, 2H), 2.22 (t, J = 11.2 Hz, 2H),
1.93 (d, J = 12.5 Hz, 2H), 1.49 (qd, J = 11.7, 3.1 Hz, 2H);
13C NMR (150 MHz, CDCl3): d 166.3, 162.9, 162.1,
161.3, 129.3, 128.6, 127.1, 113.7, 111.2, 55.4, 51.5, 48.7,
46.6, 32.3; HRMS (ESI): m/z calcd for C20H23F2N2O2
[M ? H?] 361.1728, found 361.1731.
4-Bromo-N-(1-(2,6-difluorobenzyl)piperidin-4-
yl)benzamide (5d)
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-
(trifluoromethoxy)benzamide (5h)
1
White solid; yield: 68%; H NMR (600 MHz, CDCl3): d
7.52 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.19 (q,
J = 7.2 Hz, 1H), 6.83 (t, J = 7.9 Hz, 2H), 5.83 (d,
J = 7.5 Hz, 1H), 3.82–3.90 (m, 1H), 3.66 (s, 2H), 2.86 (d,
J = 11.7 Hz, 2H), 2.23 (t, J = 11.3 Hz, 2H), 1.94 (d,
J = 10.5 Hz, 2H), 1.51 (qd, J = 11.6, 3.7 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 165.8, 162.9, 161.2, 133.6,
1
White solid; yield: 76%; H NMR (600 MHz, CDCl3): d
7.85 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.1 Hz, 2H), 7.28 (q,
J = 7.5 Hz, 1H), 6.92 (t, J = 7.7 Hz, 2H), 6.01 (d,
J = 7.5 Hz, 1H), 3.94–4.01 (m, 1H), 3.74 (s, 2H), 2.95 (d,
J = 11.6 Hz, 2H), 2.31 (t, J = 11.3 Hz, 2H), 2.05 (d,
J = 10.5 Hz, 2H), 1.61 (qd, J = 11.6, 3.8 Hz, 2H); 13C
123

Z.-N. Huang et al.
NMR (150 MHz, CDCl3): d 165.5, 162.9, 161.3, 138.0,
133.1, 129.4, 127.3, 125.6, 124.6, 122.7, 112.8, 111.2,
51.4, 48.7, 47.0, 32.1; LCMS (ESI): m/z 415 [M ? H?].
7.16–7.22 (m, 2H), 7.14 (t, J = 8.8 Hz, 1H), 7.11 (t,
J = 7.7 Hz, 1H), 6.96 (d, J = 8.4 Hz, 2H), 6.89 (t,
J = 7.6 Hz, 2H), 5.88 (d, J = 7.7 Hz, 1H), 3.90 - 3.97 (m,
1H), 3.70 (s, 2H), 2.90 (d, J = 11.4 Hz, 2H), 2.27 (t,
J = 11.3 Hz, 2H), 2.00 (d, J = 11.6 Hz, 2H), 1.55 (qd,
J = 11.4, 3.1 Hz, 2H); 13C NMR (150 MHz, CDCl3): d
166.1, 162.9, 161.2, 160.2, 155.3, 153.7, 142.5, 129.3,
129.2, 128.8, 125.8, 124.9, 122.7, 117.3, 116.4, 115.5,
113.0, 111.1, 58.4, 51.5, 48.7, 46.7, 32.3, 18.4; HRMS
(ESI): m/z calcd for C25H24F3N2O2 [M ? H?] 441.1790,
found 441.1790.
2-Chloro-N-(1-(2,6-difluorobenzyl)piperidin-4-yl)-4-
fluorobenzamide (5i)
1
White solid; yield: 84%; H NMR (600 MHz, CDCl3): d
7.61 (dd, J = 8.7, 6.1 Hz, 1H), 7.18 (q, J = 7.7 Hz, 1H),
7.05 (dd, J = 8.4, 2.6 Hz, 1H), 6.96 (td, J = 8.6, 2.8 Hz,
1H), 6.83 (q, J = 7.5 Hz, 2H), 6.00 (d, J = 7.2 Hz, 1H),
3.87–3.94 (m, 1H), 3.65 (s, 2H), 2.82 (d, J = 11.2 Hz, 2H),
2.25 (t, J = 10.9 Hz, 2H), 1.96 (dd, J = 13.4, 2.9 Hz, 2H),
1.54 (qd, J = 12.1, 3.1 Hz, 2H); 13C NMR (150 MHz,
CDCl3): d 164.7, 164.0, 162.9, 162.3, 161.3, 132.1, 131.8,
131.3, 129.4, 117.6, 117.5, 114.7, 114.5, 111.2, 51.2, 48.7,
47.0, 31.8; LCMS (ESI): m/z 383 [M ? H?].
4-(2-Chlorophenoxy)-N-(1-(2,6-difluorobenzyl)piperidin-4-
yl)benzamide (10c)
1
White solid; yield: 85%; H NMR (600 MHz, CDCl3): d
7.70 (d, J = 8.8 Hz, 2H), 7.48 (dd, J = 8.0, 1.4 Hz, 1H),
7.28 (d, J = 7.7 Hz, 1H), 7.25 (q, J = 7.2 Hz, 1H), 7.16 (td,
J = 7.7, 0.9 Hz, 1H), 7.06 (dd, J = 8.1, 1.3 Hz, 1H), 6.92
(d, J = 8.8 Hz, 2H), 6.89 (t, J = 7.7 Hz, 2H), 5.88 (d,
J = 7.7 Hz, 1H), 3.89–3.98 (m, 1H), 3.70 (s, 2H), 2.90 (d,
J = 11.4 Hz, 2H), 2.28 (t, J = 11.1 Hz, 2H), 2.01 (d,
J = 10.8 Hz, 2H), 1.54 (qd, J = 11.6, 2.8 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 166.1, 165.8, 163.8, 162.9,
161.3, 159.8, 151.2, 131.0, 129.4, 129.2, 128.8, 128.2,
126.6, 125.8, 122.1, 116.8, 115.5, 111.1, 51.5, 48.7, 46.7,
32.3; LCMS (ESI): m/z 457 [M ? H?].
4-Chloro-N-(1-(2,6-difluorobenzyl)piperidin-4- yl)-2-
fluorobenzamide (5j)
1
White solid; yield: 79%; H NMR (600 MHz, CDCl3): d
7.95 (t, J = 8.5 Hz, 1H), 7.17 (dd, J = 8.3, 2.0 Hz, 1H),
7.18 (q, J = 7.3 Hz, 1H), 7.07 (dd, J = 11.6, 1.8 Hz, 1H),
6.82 (q, J = 7.6 Hz, 2H), 6.43 (dd, J = 11.3, 8.2 Hz, 1H),
3.87–3.95 (m, 1H), 3.64 (s, 2H), 2.82 (d, J = 11.0 Hz, 2H),
2.23 (t, J = 11.0 Hz, 2H), 1.95 (dd, J = 13.2, 2.9 Hz, 2H),
1.52 (qd, J = 11.7, 3.7 Hz, 2H); 13C NMR (150 MHz,
CDCl3): d 162.9, 161.5, 161.3, 161.0, 159.4, 138.3, 133.1,
129.3, 125.4, 119.8, 116.8, 116.6, 111.2, 51.4, 48.7, 46.7,
32.0; LCMS (ESI): m/z 383 [M ? H?].
4-(2-Bromophenoxy)-N-(1-(2,6-difluorobenzyl)piperidin-4-
yl)benzamide (10d)
1
White solid; yield: 81%; H NMR (600 MHz, CDCl3): d
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-phenoxy
benzamide (10a)
7.64 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 7.7 Hz, 1H), 7.24 (t,
J = 7.2 Hz, 1H), 7.18 (d, J = 9.9 Hz, 1H), 7.01 (d,
J = 5.3 Hz, 1H), 6.96 (d, J = 7.9 Hz, 1H), 6.85 (d,
J = 8.6 Hz, 2H), 6.81 (d, J = 12.8 Hz, 2H), 5.86 (d,
J = 6.8 Hz, 1H), 3.81–3.91 (m, 1H), 3.63 (br s, 2H), 2.83
(d, J = 8.4 Hz, 2H), 2.20 (t, J = 10.5 Hz, 2H), 1.94 (d,
J = 9.4 Hz, 2H), 1.48 (d, J = 11.0 Hz, 2H); 13C NMR
(150 MHz, CDCl3): d 166.1, 165.8, 162.9, 161.3, 159.8,
152.5, 134.1, 129.4, 129.2, 128.9, 128.8, 126.1, 121.9,
117.0, 115.6, 115.5, 112.9, 111.2, 51.5, 48.7, 46.7, 32.3;
LCMS (ESI): m/z 501, 503 [M ? H?].
1
White solid; yield: 65%; H NMR (600 MHz, CDCl3): d
7.73 (dd, J = 8.9, 2.0 Hz, 2H), 7.39 (td, J = 7.6, 2.0 Hz,
2H), 7.26 (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
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.
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(2-
(trifluoromethyl)phenoxy)benzamide (10e)
1
White solid; yield: 66%; H NMR (600 MHz, CDCl3): d
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(2-
fluorophenoxy)benzamide (10b)
7.66 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 7.7 Hz, 1H), 7.43 (t,
J = 7.7 Hz, 1H), 7.17 (t, J = 8.1 Hz, 1H), 7.18 (q,
J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 2H), 6.91 (d,
J = 8.3 Hz, 1H), 6.83 (t, J = 7.7 Hz, 2H), 5.85 (d,
J = 7.7 Hz, 1H), 3.83–3.91 (m, 1H), 3.65 (s, 2H), 2.86 (d,
1
White solid; yield: 63%; H NMR (600 MHz, CDCl3): d
7.70 (d, J = 8.8 Hz, 2H), 7.25 (q, J = 7.0 Hz, 1H),
123

Synthesis and structure–activity relationship of N-(piperidin-4-yl)benzamide derivatives as…
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(3-
J = 11.4 Hz, 2H), 2.23 (t, J = 11.2 Hz, 2H), 1.96 (d,
J = 11.4 Hz, 2H), 1.52 (qd, J = 12.1, 3.1 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 166.0, 162.9, 161.3, 159.5,
154.1, 133.4, 130.1, 129.4, 128.9, 127.4, 124.1, 123.8,
122.3, 120.4, 118.4, 111.2, 51.5, 48.7, 46.7, 32.1, 29.7;
HRMS (ESI): m/z calcd for C26H24F5N2O2 [M ? H?]
491.1758, found 491.1761.
(trifluoromethyl)phenoxy)benzamide (10i)
1
White solid; yield: 62%; H NMR (600 MHz, CDCl3): d
7.68 (d, J = 8.6 Hz, 2H), 7.40 (t, J = 8.0 Hz, 1H), 7.33 (d,
J = 7.9 Hz, 1H), 7.21 (q, J = 6.9 Hz, 1H), 7.19 (d,
J = 5.1 Hz, 1H), 7.12 (dd, J = 8.2, 1.9 Hz, 1H), 6.96 (dt,
J = 8.6, 1.8 Hz, 2H), 6.84 (t, J = 7.7 Hz, 2H), 5.92 (d,
J = 7.7 Hz, 1H), 3.85–3.93 (m, 1H), 3.68 (s, 2H), 2.89 (d,
J = 11.6 Hz, 2H), 2.25 (t, J = 11.1 Hz, 2H), 1.96 (dd,
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(3-
fluorophenoxy)benzamide (10f)
J = 12.7, 3.1 Hz, 2H), 1.56 (qd, J = 11.4, 2.4 Hz, 2H); 13
C
1
NMR (150 MHz, CDCl3): d 166.0, 162.9, 161.2, 159.2,
156.7, 130.5, 130.2, 129.6, 129.0, 122.4, 120.6, 118.6,
116.1, 111.2, 58.4, 51.5, 48.6, 46.6, 32.0, 18.4; HRMS
(ESI): m/z calcd for C26H24F5N2O2 [M ? H?] 491.1758,
found 491.1763.
White solid; yield: 78%; H NMR (600 MHz, CDCl3): d
7.73 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.3 Hz, 1H), 7.26 (q,
J = 6.1 Hz, 1H), 7.03 (d, J = 8.1 Hz, 2H), 6.89 (t,
J = 6.6 Hz, 2H), 6.85 (t, J = 7.9 Hz, 1H), 6.80 (d,
J = 8.1 Hz, 1H), 6.73 (d, J = 9.7 Hz, 1H), 5.96 (d,
J = 13.9 Hz, 1H), 3.90–3.98 (m, 1H), 3.70 (br s, 2H), 2.91
(d, J = 10.3 Hz, 2H), 2.28 (t, J = 10.9 Hz, 2H), 2.01 (d,
J = 11.7 Hz, 2H), 1.56 (qd, J = 10.5, 3.5 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 166.0, 164.3, 162.9, 162.7,
161.2, 159.3, 157.6, 130.7, 130.1, 129.3, 128.9, 118.6,
114.8, 113.0, 111.1, 110.9, 110.8, 106.9, 51.5, 48.8, 46.8,
32.3; LCMS (ESI): m/z 441 [M ? H?].
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(4-
fluorophenoxy)benzamide (10j)
1
White solid; yield: 67%; H NMR (600 MHz, CDCl3): d
7.63 (d, J = 8.8 Hz, 2H), 7.16–7.21 (m, 1H), 6.99 (t,
J = 8.1 Hz, 2H), 6.91–6.95 (m, 2H), 6.88 (d, J = 8.8 Hz,
2H), 6.83 (t, J = 7.7 Hz, 2H), 5.83 (d, J = 7.7 Hz, 1H),
3.83–3.91 (m, 1H), 3.66 (s, 2H), 2.85 (d, J = 11.7 Hz, 2H),
2.22 (t, J = 11.1 Hz, 2H), 1.94 (d, J = 9.9 Hz, 2H), 1.51
(qd, J = 11.9, 3.9 Hz, 2H); 13C NMR (150 MHz, CDCl3):
d 166.1, 162.9, 161.2, 160.6, 160.1, 158.5, 151.7, 129.4,
129.2, 128.8, 121.4, 121.3, 117.3, 116.7, 116.5, 111.2,
51.5, 48.7, 46.7, 32.2, 29.7, 18.4; HRMS (ESI): m/z calcd
for C25H24F3N2O2 [M ? H?] 441.1790, found 441.1788.
4-(3-Chlorophenoxy)-N-(1-(2,6-difluorobenzyl)piperidin-4-
yl)benzamide (10g)
1
White solid; yield: 91%; H NMR (600 MHz, CDCl3): d
7.73 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 8.1 Hz, 1H), 7.28 (q,
J = 7.7 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 7.01 (d,
J = 8.1 Hz, 2H), 7.01 (t, J = 2.4 Hz, 1H), 6.91 (dd,
J = 7.0, 2.2 Hz, 1H), 6.90 (t, J = 7.9 Hz, 2H), 5.89 (d,
J = 7.9 Hz, 1H), 3.90–3.98 (m, 1H), 3.71 (s, 2H), 2.91 (d,
J = 11.6 Hz, 2H), 2.28 (t, J = 11.2 Hz, 2H), 2.03 (d,
J = 11.2 Hz, 2H), 1.56 (qd, J = 11.6, 2.9 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 166.0, 162.9, 161.2, 159.3,
157.1, 135.2, 130.7, 130.1, 129.3, 128.9, 124.2, 119.6,
118.5, 117.5, 115.6, 113.0, 111.1, 51.5, 48.7, 46.8, 32.3,
29.7; LCMS (ESI): m/z 457 [M ? H?].
4-(4-Chlorophenoxy)-N-(1-(2,6-difluorobenzyl)piperidin-4-
yl)benzamide (10k)
1
White solid; yield: 75%; H NMR (600 MHz, CDCl3): d
7.64 (d, J = 8.8 Hz, 2H), 7.25 (d, J = 9.0 Hz, 2H),
7.16–7.21 (m, 1H), 6.90 (d, J = 8.2 Hz, 2H), 6.89 (d,
J = 8.8 Hz, 2H), 6.83 (t, J = 7.9 Hz, 2H), 5.84 (d,
J = 7.7 Hz, 1H), 3.83–3.92 (m, 1H), 3.66 (s, 2H), 2.86 (d,
J = 11.6 Hz, 2H), 2.23 (t, J = 11.2 Hz, 2H), 1.94 (d,
J = 10.5 Hz, 2H), 1.52 (qd, J = 11.2, 3.5 Hz, 2H); 13C
NMR (CDCl3): d 166.0, 162.9, 161.2, 159.9, 154.7, 130.0,
129.6, 129.4, 129.3, 128.9, 120.9, 118.0, 111.2, 51.5, 48.7,
46.7, 32.2, 29.7; HRMS (ESI): m/z calcd for C25H24ClF2-
N2O2 [M ? H?] 457.1494, found 457.1499.
4-(3-Bromophenoxy)-N-(1-(2,6-difluorobenzyl)piperidin-4-
yl)benzamide (10h)
1
White solid; yield: 81%; H NMR (600 MHz, CDCl3): d
7.66 (d, J = 8.6 Hz, 2H), 7.12–7.24 (m, 5H), 6.94 (d,
J = 8.4 Hz, 2H), 6.89 (d, J = 8.1 Hz, 2H), 5.85 (d,
J = 5.9 Hz, 1H), 3.82–3.94 (m, 1H), 3.65 (br s, 2H), 2.85
(d, J = 8.6 Hz, 2H), 2.22 (t, J = 10.0 Hz, 2H), 1.94 (d,
J = 10.1 Hz, 2H), 1.50 (d, J = 10.5 Hz, 2H); 13C NMR
(150 MHz, CDCl3): d 166.0, 162.9, 161.2, 159.3, 157.2,
131.0, 130.1, 129.4, 128.9, 127.1, 123.0, 122.5, 118.5,
118.0, 112.8, 111.2, 51.5, 48.7, 46.8, 32.2, 31.0, 29.7;
LCMS (ESI): m/z 501, 503 [M ? H?].
N-(1-(2,6-difluorobenzyl)piperidin-4-yl)-4-(4-
bromophenoxy)benzamide (10l)
1
White solid; yield: 79%; H NMR (600 MHz, CDCl3): d
7.73 (dd, J = 8.6, 1.8 Hz, 2H), 7.47 (dd, J = 8.8, 2.0 Hz,
2H), 7.13 (m, 1H), 6.96-7.01 (m, 3H), 6.89-6.93 (m, 3H),
123

Z.-N. Huang et al.
5.93 (d, J = 7.9 Hz, 1H), 4.01 (m, 1H), 3.56 (s, 2H), 2.86
(d, J = 11.7 Hz, 2H), 2.26 (t, J = 10.6 Hz, 2H), 2.03 (d,
J = 11.0 Hz, 2H), 1.57 (qd, J = 12.5, 3.9 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 166.0, 159.8, 159.5, 157.9,
156.4, 155.3, 133.0, 129.7, 128.9, 121.3, 118.1, 117.1,
116.8, 116.2, 114.9, 54.9, 52.2, 46.9, 32.4; HRMS (ESI):
m/z calcd for C25H23BrF2N2O2 501.0989 [(M ? H?) for
79Br] and 503.0969 [(M ? H?) for 81Br], found 501.0986
[(M ? H?) for 79Br] and 503.0963 [(M ? H?) for 81Br].
128.7, 128.5, 121.4, 116.8, 115.0, 111.2, 55.7, 51.5, 48.7,
46.8. 32.2; HRMS (ESI): m/z calcd for C26H27F2N2O3
[M ? H?] 453.1990, found 453.1991.
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(3,4-
difluorophenoxy)benzamide (10p)
White solid; yield: 76%; 1H NMR (600 MHz, DMSO-d6):
d 8.18 (d, J = 7.7 Hz, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.50
(q, J = 10.5 Hz, 1H), 7.42 (q, J = 7.5 Hz, 1H), 7.26–7.32
(m, 1H), 7.10 (t, J = 7.8 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H),
6.92 (dt, J = 8.6, 2.8 Hz, 1H), 3.68–3.75 (m, 1H), 3.57 (s,
2H), 2.82 (d, J = 11.6 Hz, 2H), 2.09 (t, J = 11.0 Hz, 2H),
1.74 (dd, J = 11.6, 2.6 Hz, 2H), 1.54 (qd, J = 11.9, 2.9 Hz,
2H); 13C NMR (150 MHz, DMSO-d6): d 165.2, 162.7,
161.0, 159.2, 152.5, 151.1, 149.5, 147.6, 145.9, 130.4,
130.0, 118.8, 117.9, 116.3, 113.6, 111.9, 109.9, 52.2, 48.8,
47.1, 31.8, 31.2; LCMS (ESI): m/z 459 [M ? H?].
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(4-
nitrophenoxy)benzamide (10m)
1
White solid; yield: 65%; H NMR (600 MHz, CDCl3): d
8.15 (d, J = 9.0 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.19 (q,
J = 7.4 Hz, 1H), 7.04 (d, J = 8.6 Hz, 2H), 6.98 (d,
J = 9.0 Hz, 2H), 6.83 (q, J = 7.0 Hz, 2H), 5.91 (d,
J = 7.7 Hz, 1H), 3.84–3.93 (m, 1H), 3.66 (s, 2H), 2.87 (d,
J = 11.4 Hz, 2H), 2.23 (t, J = 11.2 Hz, 2H), 1.96 (dd,
J = 12.8, 2.4 Hz, 2H), 1.53 (qd, J = 11.7, 2.8 Hz, 2H); 13
C
4-(4-Chloro-3-(trifluoromethyl)phenoxy)-N-(1-(2,6-
difluorobenzyl)piperidin-4-yl)benzamide (10q)
NMR (150 MHz, CDCl3): d 165.7, 162.9, 162.3, 161.2,
157.5, 143.2, 131.6, 129.4, 129.3, 126.0, 120.0, 117.9,
112.7, 111.2, 51.5, 48.7, 46.9, 32.2, 29.7, 18.4; HRMS
(ESI): m/z calcd for C25H24F2N3O4 [M ? H?] 468.1735,
found 468.1742.
1
White solid; yield: 63%; H NMR (600 MHz, CDCl3): d
7.69 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.6 Hz, 1H), 7.27 (d,
J = 2.6 Hz, 1H), 7.18 (q, J = 7.3 Hz, 1H), 7.03 (dd,
J = 8.7, 2.7 Hz, 1H), 6.95 (d, J = 8.6 Hz, 2H), 6.83 (t,
J = 7.6 Hz, 2H), 5.87 (d, J = 7.7 Hz, 1H), 3.82–3.93 (m,
1H), 3.64 (s, 2H), 2.86 (d, J = 11.2 Hz, 2H), 2.22 (t,
J = 11.2 Hz, 2H), 1.95 (d, J = 11.0 Hz, 2H), 1.51 (qd,
J = 11.2, 2.2 Hz, 2H). 13C NMR (150 MHz, CDCl3): d
165.9, 162.9, 162.9, 161.3, 161.2, 158.7, 155.1, 132.9,
130.6, 129.9, 129.7, 129.4, 129.1, 126.7, 123.1, 118.6,
118.4, 112.7, 111.2, 51.5, 48.7, 46.8, 32.2, 18.4; HRMS
(ESI): m/z calcd for C26H23ClF5N2O2 [M ? H?] 525.1368,
found 525.1374.
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(4-
(trifluoromethyl)phenoxy)-benzamid (10n)
1
White solid; yield: 61%; H NMR (600 MHz, CDCl3): d
7.73 (dd, J = 8.8, 1.8 Hz, 2H), 7.37 (td, J = 7.3, 2.0 Hz,
2H), 7.17 (t, J = 7.4 Hz, 1H), 7.04 (d, J = 8.6 Hz, 2H),
7.01 (dd, J = 8.8, 2.2 Hz, 2H), 6.87–6.89 (m, 2H), 6.69 (tt,
J = 8.9, 2.4 Hz, 1H), 5.92 (d, J = 7.7 Hz, 1H), 4.01 (m,
1H), 2.83 (d, J = 11.6 Hz, 2H), 2.20 (t, J = 10.7 Hz, 2H),
2.01–2.06 (m, 2H), 1.57 (qd, J = 11.6, 3.9 Hz, 3H); 13C
NMR (150 MHz, CDCl3): d 165.9, 162.9, 161.2, 159.4,
158.7, 130.6, 129.3, 129.1, 127.3, 125.9, 124.9, 123.1,
119.1, 118.7, 112.9, 111.2, 51.5, 48.7, 46.8, 32.2; HRMS
(ESI): m/z calcd for C26H24F5N2O2 [M ? H?] 491.1758,
found 491.1766.
4-(2-Bromo-5-fluorophenoxy)-N-(1-(2,6-
difluorobenzyl)piperidin-4-yl)benzamide (10r)
1
White solid; yield: 82%; H NMR (600 MHz, CDCl3): d
7.67 (d, J = 5.1 Hz, 2H), 7.52 (t, J = 7.2 Hz, 1H), 7.19 (d,
J = 8.8 Hz, 1H), 6.90 (d, J = 5.1 Hz, 2H), 6.82 (t,
J = 7.0 Hz, 2H), 6.75 (t, J = 7.7 Hz, 1H), 6.64 (d,
J = 5.9 Hz, 1H), 5.86 (s, 1H), 3.81–3.93 (m, 1H), 3.64 (br
s, 2H), 2.84 (d, J = 6.0 Hz, 2H), 2.21 (t, J = 10.0 Hz, 2H),
1.95 (d, J = 9.7 Hz, 2H), 1.50 (d, J = 8.6 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 165.9, 163.2, 162.9, 161.5,
161.2, 158.8, 153.7, 134.4, 130.3, 129.3, 129.0, 117.8,
112.9, 111.2, 109.5, 108.8, 51.5, 48.7, 46.8, 32.2, 14.2;
LCMS (ESI): m/z 519, 521 [M ? H?].
N-(1-(2, 6-Difluorobenzyl) piperidin-4-yl)-4-(4-
methoxyphenoxy)benzamide (10o)
1
White solid; yield: 58%; H NMR (600 MHz, CDCl3): d
7.72 (dd, J = 8.8, 2.2 Hz, 2H), 7.47 (td, J = 8.0, 7.2 Hz,
1H), 7.37 (td, J = 7.7, 2.2 Hz, 2H), 7.14–7.18 (m, 2H),
7.03 (dd, J = 7.9, 2.2 Hz, 2H), 6.97–7.01 (m, 3H), 5.94 (d,
J = 7.9 Hz, 1H), 4.01 (m, 2H), 3.46 (s, 3H), 2.82 (d,
J = 11.6 Hz, 2H), 2.19 (t, J = 11.5 Hz, 2H), 2.00–2.05 (m,
2H), 1.56 (qd, J = 11.9, 3.3 Hz, 2H); 13C NMR (150 MHz,
CDCl3): d 166.2, 162.9, 161.4, 161.2, 156.5, 149.0, 129.4,
123

Synthesis and structure–activity relationship of N-(piperidin-4-yl)benzamide derivatives as…
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(pyridin-2-
yloxy)benzamide (15a)
J = 5.4, 2.5 Hz, 1H), 7.10 (t, J = 7.7 Hz, 2H), 3.68–3.79
(m, 1H), 3.58 (br s, 2H), 2.83 (d, J = 10.6 Hz, 2H), 2.51 (br
s, 3H), 2.11 (t, J = 11.2 Hz, 2H), 1.78 (d, J = 11.0 Hz,
2H), 1.55 (qd, J = 12.3, 2.6 Hz, 2H); 13C NMR (150 MHz,
DMSO-d6): d 165.5, 165.2, 164.1, 162.7, 161.0, 156.0,
153.1, 151.1, 132.6, 130.4, 130.3, 120.9, 115.1, 113.5,
112.0, 109.8, 56.5, 52.2, 48.8, 47.1, 31.8, 31.2, 26.5, 19.0;
HRMS (ESI): m/z calcd for C26H27F2N4O3 [M ? H?]
481.2051, found 481.2062.
1
White solid; yield: 54%; H NMR (600 MHz, CDCl3): d
8.12 (d, J = 4.8 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.64 (t,
J = 7.4 Hz, 1H), 7.18 (q, J = 7.3 Hz, 1H), 7.10 (d,
J = 8.3 Hz, 2H), 6.96 (t, J = 6.2 Hz, 1H), 6.88 (d,
J = 8.3 Hz, 1H), 6.83 (t, J = 7.6 Hz, 2H), 5.87 (d,
J = 7.7 Hz, 1H), 3.82–3.95 (m, 1H), 3.64 (s, 2H), 2.85 (d,
J = 11.2 Hz, 2H), 2.23 (t, J = 11.1 Hz, 2H), 1.95 (d,
J = 11.4 Hz, 2H), 1.52 (qd, J = 11.0, 3.5 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 166.2, 163.1, 162.9, 161.3,
156.8, 147.7, 139.7, 130.9, 129.4, 128.6, 120.9, 119.1,
112.1, 111.2, 51.5, 48.7, 46.6, 32.1; LCMS (ESI): m/z 425
[M ? H?].
Cell growth inhibition rate (MTT assay)
The compounds were evaluated for their antiproliferative
activity on cancer cell lines like HepG2. All the cell lines
were incubated at 37 °C in 5% CO2. The culture medium
used was the DMEM supplemented with 10% of Fetal
Bovine Serum (FBS).
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(pyridin-3-
yloxy)benzamide (15b)
The cells were seeded on 96-well plate at 5000 cells/
well and incubated overnight. The cells were treated with
compounds at 20 lM concentration, while control cells
were treated with equal DMSO. After 48 h treatment,
75 lL MTT solution (5 mg/mL was added to each well,
and incubated for 4 h, then the culture medium were
removed and dissolved in 100 lL DMSO. After 10 min,
the OD (optical density) was detected at 490 nm on
microplate reader. If the inhibitory rate of tested com-
pounds > 50%, we evaluated their IC50 values.
1
White solid; yield: 63%; H NMR (600 MHz, CDCl3): d
8.35 (d, J = 2.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.26 (t,
J = 8.4 Hz, 1H), 7.23 (m, J = 7.9, 4.4 Hz, 1H), 7.19 (q,
J = 6.8 Hz, 1H), 6.94 (d, J = 8.4 Hz, 2H), 6.83 (t,
J = 7.6 Hz, 2H), 5.90 (d, J = 7.7 Hz, 1H), 3.83–3.92 (m,
1H), 3.66 (s, 2H), 2.86 (d, J = 11.4 Hz, 2H), 2.23 (t,
J = 11.2 Hz, 2H), 1.95 (d, J = 11.7 Hz, 2H), 1.52 (qd,
J = 10.6, 2.4 Hz, 2H); 13C NMR (150 MHz, CDCl3): d
165.9, 162.9, 162.9, 161.3, 161.2, 159.3, 152.8, 145.3,
142.0, 130.2, 129.4, 129.0, 126.4, 124.3, 118.1, 111.2,
51.5, 48.7, 46.7, 32.1; LCMS (ESI): m/z 425 [M ? H?].
Colony forming assay
The cells were seeded on 6-well plate at 1000 cells/well
and incubated in different concentrations of compound 10b
and 10j for 1 week. Then the cells were washed with PBS,
fixed by 70% ethanol and stained with 0.25% crystal violet.
The number of colonies was analyzed. These experiments
were performed in triplicate.
N-(1-(2,6-Difluorobenzyl)piperidin-4-yl)-4-(pyrimidin-2-
yloxy)benzamide (15c)
1
White solid; yield: 73%; H NMR (600 MHz, CDCl3): d
8.58 (d, J = 4.8 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.27 (t,
J = 7.0 Hz, 2H), 7.26 (d, J = 8.1 Hz, 1H), 7.09 (t,
J = 4.7 Hz, 1H), 6.92 (t, J = 7.5 Hz, 2H), 6.00 (d,
J = 7.5 Hz, 1H), 3.93–4.03 (m, 1H), 3.74 (s, 2H), 2.94 (d,
J = 11.2 Hz, 2H), 2.32 (t, J = 11.1 Hz, 2H), 2.03 (d,
J = 11.6 Hz, 2H), 1.61 (qd, J = 12.1, 2.9 Hz, 2H); 13C
NMR (150 MHz, CDCl3): d 166.0, 165.0, 162.9, 161.3,
159.8, 155.3, 132.0, 129.4, 128.6, 121.8, 116.6, 112.8,
111.2, 51.5, 48.7, 46.7, 32.2; LCMS (ESI): m/z 426
[M ? H?].
Western blotting
Cells were lysed with SDS buffer containing 200 mM Tris
(pH 6.8), 40% glycerol, and 10% mercaptoethanol. Protein
(30 lg) obtained from cell extracts were separated on 10 or
15% SDS/polyacrylamide gel, and then transferred to
Immobilon-P membrane. Membranes were blocked with
10% nonfat milk in Tris-buffered saline containing 0.1%
Tween-20 for 1 h at room temperature and then incubated
overnight at 4 °C with respective primary antibodies dilu-
ted 1:1000–2000 in TTBS containing 5% nonfat milk.
After three times washing with TTBS, membranes were
incubated with a horseradish peroxidase-conjugated anti-
rabbit, anti-mouse serum were used as a secondary anti-
bodies (diluted 1:10,000 in TTBS containing 5% nonfat
milk) for 1 h at room temperature, and the antigen–
4-(4-((1-(2,6-Difluorobenzyl)piperidin-4-
yl)carbamoyl)phenoxy)-N-methylpicolinamide (15d)
White solid; yield: 64%; 1H NMR (600 MHz, DMSO-d6):
d 8.80 (d, J = 4.6 Hz, 1H), 8.55 (d, J = 5.5 Hz, 1H), 8.31
(d, J = 7.7 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.42 (q,
J = 7.5 Hz, 2H), 7.30 (d, J = 8.6 Hz, 2H), 7.21 (dd,
123

Z.-N. Huang et al.
antibody complexes were visualized using the ECL kit. b-
actin was analyzed as a loading control.
hydroxybenzotriazole hydrate). Compounds 4a–j, 9a–r,
and 14a–d were obtained after deprotection (66–90%
yield). Finally, 2,6-difluorobenzyl substituent was intro-
duced to the piperidine ring, yielding compounds 5a–j,
10a–r, and 15a–d (54–91% yield after extraction).
Flow cytometric analysis of apoptosis
As described above, cells in the exponential growth phase
were treated with compounds 10b and 10j for 24 h, and
cell death or apoptosis was evaluated. Cell suspensions
(0.3 mL, 1 9 106 cells/mL) were placed in the flow
cytometry apparatus. Before detection, the horizontal
coordinate was set for propidium iodide (PI) and the ver-
tical coordinate was set for SSC (side scatter, side light
scatter). The PI-positive cells were gated to include the
apoptotic and necrotic cells. A map was set to the linear
model, and a horizontal histogram for PI in the third flu-
orescence channel (FL3, 610 nm) was obtained. Finally, a
log ploidy determination of the diploid and tetraploid cells
was performed. A tube of unstained cells was used as a
blank control to adjust the test conditions.
In vitro antitumor activity
Preliminary biological activity screening showed that most
of the compounds in the N-(piperidin-4-yl)benzamide
derivative series have been found to have good biological
potential and inhibitory activity, of which compounds 10b
and 10j are the most prominent.
Activity of synthesized N-(1-(2,6-difluoro)-4-piper-
idinyl) benzamide compounds in inhibiting proliferation of
HepG2 cells in vitro is shown in Tables 1 and 2.
Discussion
Structure–activity relationships
Results
In the N-(1-(2,6-difluoro)-4-piperidinyl)benzamide series,
compared to a single mono- or poly-substituted benzene
ring compounds, diaryl ether derivatives showed higher
cytotoxicity towards human hepatoma HepG2 cells.
Compound 10b was the most potent (IC50 = 0.12 lM).
Comparative analysis of the activity of compounds 5a–
5f (Table 1) and 10a (Table 2) indicated that phenoxy
group (10a) and chlorine atom (5c) in the para-position of
the benzene ring improve activity, whereas compounds
with other substituents (5a–f, except 5c) did not reach 50%
inhibition in the preliminary screening and showed poor
inhibition in HepG2 cells. Furthermore, activity of 10a in
inhibiting HepG2 cell proliferation was significantly higher
than that of 5c, which may indicate that diaryl ether region
A is more likely to be associated with anti-tumor potential
than benzene ring substituents. This analysis suggests that
diaryl ether region A is the main pharmacophore of this
compound series.
Chemistry
Based on our previous work on lead sorafenib N-(1-(2,6-
difluorobenzyl)-4-piperidinyl) benzamide derivatives, we
retained the diaryl ether moiety and optimized the A region
of the substituent to further improve anti-cancer activity.
In order to study structure–activity relationships, three
series of N-(1-(2,6-difluorobenzyl)-4-piperidinyl)benza-
mide derivatives were designed and synthesized
(Schemes 1, 2, 3) (Adeniji et al. 2012; Carato et al. 2007;
Yang et al. 2013).
Briefly, C-O coupling followed by an ester hydrolysis
reaction was used to obtain intermediates 8a–r and 13a–
d (63–89% yield, Schemes 2 and 3). Compounds 8a–r,
13a–d, and substituted benzoic acids were used as starting
materials for amide formation with 4-amino-1-tert-butyl-
carbonylpiperidine, catalyzed by EDCI (1-(3-dimethy-
laminopropyl)-3-ethylcarbodiimide) and HOBT (1-
Scheme 1 Reagents and conditions: a 4-amino-1-Boc-piperidine, EDCI, HOBt, CH2Cl2, 0 °C 20 min then to rt 12 h; 2 M HCl, EtOAc, 2 h,
73–86% for 2 steps. b 2,6-difluorobenzyl bromide, K2CO3, DMF, rt, 20 h, 67–86%
123

Synthesis and structure–activity relationship of N-(piperidin-4-yl)benzamide derivatives as…
Scheme 2 Reagents and conditions: a K2CO3, DMF, 130 °C, 12 h; NaOH, H2O/EtOH, 80 °C, 1 h, 63–89% for 2 steps. b 4-amino-1-Boc-
piperidine, EDCI, HOBt, CH2Cl2, 0 °C 20 min then to rt 12 h; 2 M HCl, EtOAc, 2 h, 71–90% for 2 steps. c 2,6-difluorobenzyl bromide, K2CO3,
DMF, rt, 20 h, 58–91%
Scheme 3 Reagents and conditions: a CsCO3, DMF, 130 °C, 12 h; NaOH, H2O/EtOH, 80 °C, 1 h, 48–73% for 2 steps. b 4-amino-1-Boc-
piperidine, EDCI, HOBt, CH2Cl2, 0 °C 20 min then to rt 12 h; 2 M HCl, EtOAc, 2 h, 66–83% for 2 steps. c 2,6-difluorobenzyl bromide, K2CO3,
DMF, rt, 20 h, 54–73%
Diaryl ether compound 10a was chosen for further
structure modification studies (10a–r). Most derived com-
pounds showed relatively good anti-proliferative activity
(Table 2). Compounds 10b and 10j outperformed the
unsubstituted diaryl ether structure 10a (IC50 = 0.25 lM).
When comparing compounds containing halogen sub-
stituents, position of the substituent affected inhibition of
HepG2 cell growth (para [ meta). Para-halogen substi-
tuted compounds showed promising anti-tumor activity,
with activity decreasing in order -F [ -Br [ -Cl (IC50
values of compounds 10j, 10l, and 10k were 0.13, 2.29, and
12.08 lM, respectively). Compounds with meta-halogen
substituents were less active without exception, whereas
activity trends of ortho-halogen compounds were irregular,
with only ortho-fluorine compounds showing excellent
activity, even slightly surpassing para-fluorine-substituted
compound 10j.
Para-nitro (10m) or methoxy (10o) substituents
improved activity of the phenoxybenzene compounds.
Introduction of electron-withdrawing groups to the para
123

Z.-N. Huang et al.
Table 1 Inhibition of growth by N-(piperidin-4-yl)benzamide
derivatives in HepG2 cells
Table 2 Inhibition of growth by N-(piperidin-4-yl)benzamide
derivatives in HepG2 cells
Cpd.
R1
R2
HepG2a
Cpd.
R1
R2
R3
R4 HepG2a
IR@20 lM(%)b IC50 (lM)
IR@20 lM(%)b
IC50 (lM)
5a
5b
5c
H
H
H
H
H
H
H
H
Cl
F
H
23.46
14.25
51.74
31.25
31.65
15.90
43.14
46.49
35.11
31.43
40.51
NT
NT
15.45
NT
NT
NT
NT
NT
NT
NT
NT
10a
10b
10c
10d
10e
10f
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
74.25
81.37
30.85
19.46
60.50
44.34
27.75
33.69
69.45
81.32
58.40
69.54
69.40
73.37
69.58
36.89
47.67
0.25c
0.12
NT
F
H
H
Cl
Cl
Br
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
–
H
H
5d
5e
Br
H
H
NT
CH3
OH
OCH3
OCF3
F
H
H
7.26
NT
5f
F
H
5 g
5 h
5i
10g
10h
10i
Cl
Br
CF3
H
H
NT
H
NT
H
2.55
0.13
12.08
2.29c
2.60
0.34c
2.29c
NT
5j
Cl
10j
F
15a
H
10k
10l
H
Cl
Br
NO2
CF3
OCF3
F
H
10m
10n
10o
10p
10q
10r
Sorfenib
H
15b
15c
15d
H
H
H
25.63
42.93
31.85
NT
NT
NT
H
H
F
CF3
F
F
NT
H
Br 34.75
NT
9.61
NT means not tested
aEach compound was tested in triplicate
bIR is the mean inhibitory rate calculated from three independent
experiments measured at 48 h after treatment with the test compound
at the concentration of 20 lM
cHou et al. (2015)
sorfenib
–
9.61
NT means not tested
aEach compound was tested in triplicate
bIR is the mean inhibitory rate calculated from three independent
experiments measured at 48 h after treatment with the test compound
at the concentration of 20 lM
A comparison between the compounds 10a–10r showed
that a mono-substitution effect is generally better for
introducing electron-withdrawing groups such as halogens
on the phenoxybenzene ring; however, double-substitution
will reduce the biological activity of the compound.
Disubstituted groups are ortho and meta, or para and meta.
As previously analyzed, the introduction of substituents at
the meta position greatly reduces bioactivity, which may
account for the diminished activity of the disubstituted
compounds. In addition, the introduction of multiple
groups will increase the electron cloud density; increased
position of the phenoxybenzene ring generally improved
activity, with fluorine substitution showing the greatest
effect, probably because fluorine was the most elec-
tronegative and also the substituent with the lowest
molecular weight and electron cloud density. In addition,
introduction of multiple halogen atoms (10p, 10q, and 10r)
on the diaryl ether benzene ring resulted in loss of activity.
123

Synthesis and structure–activity relationship of N-(piperidin-4-yl)benzamide derivatives as…
steric hindrance may also lead to reduced biological
activity.
sharp increased at 0–10 lM (Fig. 2a). Plate clone forma-
tion experiments with compounds 10b and 10j confirmed
MTT results. At a concentration of 1 lM, compound 10b
exhibited [ 50% inhibitory activity after 24 h, whereas at
10 lM, all cells underwent apoptosis within 48 h. Inhibi-
tory activity of different 10j concentrations stabilized with
time, and all cells underwent apoptosis at the concentration
of 10 lM (Fig. 2b). Clonal plate imaging and apoptosis
assays showed a cell inhibition rate of approximately 50%
at 1 lM 10b and 10j and confirmed IC50 values reported
above (Fig. 2c).
Analysis of compounds 15a–15d demonstrated that
replacing the phenoxy substituent with a nitrogen-con-
taining unsaturated six-membered ring such as pyrimidine
or pyridine causes loss of activity, suggesting that the
diaryl ether structure is essential for biological activity.
Proliferation signaling in HepG2 cells
In preliminary MTT assays, compounds 10b and 10j
showed better anti-tumor activity than the parental HJ-47.
Inhibition ratio of HepG2 cells increased continuously with
increased concentration of compounds 10b and 10j, with a
As compounds 10b and 10j displayed optimal activity
against human hepatocarcinoma HepG2 cells in the com-
pound series, they were selected to explore the potential
Fig. 2 Compounds 10j and 10b
inhibit poroliferation of HepG2
cells. a The IC50 curves of
HepG2 cells treatment with
compounds 10j and 10b
respectively. Data are
mean SD (n = 3). b The
growth curves of HepG2 cells
treatment with different dose
compounds 10j and 10b. Data
are mean SD (n = 3).
**P \ 0.01, versus the DMSO
control. (C) Colony forming
assay performed by different
dose compounds 10j and 10b
treatment in HepG2 cells. Data
are mean SD (n = 3).
**P \ 0.01, versus the DMSO
control
123

Z.-N. Huang et al.
involvement of HIF-1 in the mechanism of action of these
derivatives. Human hepatoma HepG2 cell line was treated
with 10 lM 10b or 10j for 24 h and high expression of
HIF-1a protein was observed. Expression levels of HIF-1a
in HepG2 cell line were higher after treatment with 10j,
compared to 10b. These findings are in agreement with the
observed IC50 values and suggest that favorable activity of
compounds 10b and 10j in HepG2 cell line may be due to
their ability to induce apoptosis by increasing HIF-1a
production (Fig. 3a).
Expression of apoptotic factors p21 and cleaved caspase-3
also increased with increased drug concentration, with
compound 10j displaying more prominent effects.
Taken together, these data suggest that compounds 10b
and 10j promote tumor cell apoptosis by upregulating the
expression of HIF-1a, p21, and cleaved caspase-3 and
inhibit proliferation of hepatic cancer cells.
In conclusion, a series of N-(1-(2,6-difluorobenzyl)4-
piperidinyl)benzamides was designed and synthesized
based on our previous work. Synthesized compounds
inhibited the proliferation of HepG2 cells, especially
compounds 10b and 10j with IC50 values of 0.12 and
0.13 lM, respectively, which are five times lower than the
IC50 value of the commercial drug sorafenib. Compounds
10b and 10j were found to inhibit tumor cell proliferation
by upregulating the expression of HIF-1a, p21, and cleaved
caspase-3. We speculate that compounds 10b and 10j exert
antitumor effects by activating HIF-1 and promoting
Increased HIF-1a expression was reported to promote
tumor cell apoptosis under normoxic culture conditions
(Rankin and Giaccia 2008). Western blotting suggested
that HIF-1a protein expression in HepG2 cells (Fig. 3a)
rapidly increased over time after treatment with 10b or 10j,
reaching a peak after 24 h. Changes in HIF-1a expression
were dose-dependent (Fig. 3b), with expression increasing
with increased concentration of compounds 10b and 10j.
Fig. 3 Compounds 10j and 10b
promote apoptosis of hepatoma
carcinoma cell by inducing the
expression of HIF-1a and
downstream gene p21. a The
expression of HIF-1a was
evaluated in a time-dependent
manner in HepG2 cells after
incubation with 10 lM
compounds 10j and 10b. b The
expression of HIF-1a and
downstream gene p21 were
evaluated in a dose-dependent
manner in HepG2 cells after
incubation with compounds 10j
and 10b for 24 h. **P \ 0.01,
versus the DMSO control.
c Apoptosis were induced by
compounds 10j and 10b. After
48 h compounds 10j, 10b or
DMSO treatments, positive
apoptotic cells were determined
by flow cytometry. Percentage
of apoptotic cells data are
Mean SD (n = 3).
**P \ 0.01, versus the DMSO
control
123

Synthesis and structure–activity relationship of N-(piperidin-4-yl)benzamide derivatives as…
N-(piperidine-4-yl)benzamide derivatives as potential cell cycle
apoptosis and cell cycle arrest. Therefore, amide com-
pounds 10b and 10j may potentially introduce new anti-
cancer drugs targeting HIF-1 and cyclins.
inhibitors in HepG2 cells. Chem Biol Drug Des 86:223–231
Lina J, Brown EA (2006) Bafilomycin induces the p21-mediated
growth inhibition of cancer cells under hypoxic conditions by
expressing hypoxia-inducible factor-1alpha. Mol Pharmacol
70:1856–1865
Majmundar AJ, Wong WJ, Simon MC (2010) Hypoxia-inducible
factors and the response to hypoxic stress. Mol Cell 40:294–295
Rankin EB, Giaccia AJ (2008) The role of hypoxia-inducible factors
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Acknowledgements This work was supported by the National Nat-
ural Science Foundation of China (NSFC-81472230), the National
Training Program of Innovation and Entrepreneurship for Under-
graduates (2016x0644), and the Natural Science Foundation of Fujian
Province of China (2015Y0081, 2015J01350, 2015J01545).
Sendoel A, Kohler I, Fellmann C, Lowe SW, Hengartner MO (2010)
HIF-1 antagonizes p53-mediated apoptosis through a secreted
neuronal tyrosinase. Nature 465:577–581
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