Synthesis, antitumor activity evaluation of some new N-aroyl-α, β-unsaturated piperidones with fluorescence

Article abstract of DOI:10.3109/14756366.2015.1043296

Novel N-aroyl-α,β-unsaturated piperidones, series 1, series 2 and series 3 (featuring 2-bromo-4,5-dimethoxybenzylidene, 4-dimethylaminobenzylidene and 4-trifluoromethylbenzylidene, respectively), were synthesized as candidate cytotoxins. Most of the compo

Full text of DOI:10.3109/14756366.2015.1043296

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, antitumor activity evaluation of some
new N-aroyl-α,β-unsaturated piperidones with
fluorescence
Jufeng Sun, Suwen Wang, Hongjuan Li, Wenguo Jiang, Guige Hou, Feng Zhao
& Wei Cong
To cite this article: Jufeng Sun, Suwen Wang, Hongjuan Li, Wenguo Jiang, Guige Hou, Feng
Zhao & Wei Cong (2015): Synthesis, antitumor activity evaluation of some new N-aroyl-α,β-
unsaturated piperidones with fluorescence, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2015.1043296
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1043296
!
Synthesis, antitumor activity evaluation of some new
N-aroyl-a,b-unsaturated piperidones with fluorescence
Jufeng Sun1*, Suwen Wang2*, Hongjuan Li1, Wenguo Jiang1, Guige Hou1, Feng Zhao1, and Wei Cong1
2
1School of Pharmacy, Binzhou Medical University, Yantai, Shandong, P.R. China and Yantai Yuhuangding Hospital Emergency,
Yantai, Shandong, P.R. China
Abstract
Keywords
Novel N-aroyl-a,b-unsaturated piperidones, series 1, series 2 and series 3 (featuring 2-bromo-
4,5-dimethoxybenzylidene, 4-dimethylaminobenzylidene and 4-trifluoromethylbenzylidene,
respectively), were synthesized as candidate cytotoxins. Most of the compounds displayed
potent cytotoxicity against the human neoplastic cell lines SK-BR-3, PG-BE1, NCI-H460, MIA
PaCa-2 and SW1990 in vitro, and approximately 64% of the IC50 values were lower than 5 lM.
Among those tested, compound 1b of series 1, 3a, 3d and 3e of series 3 proved to be the most
active. Importantly, 1b displayed marked inhibitory effects on tumor growth in vivo and had no
apparent toxicity to mice; this was evaluated by a nude mouse PG-BE1 xenograft model.
In addition, the fluorescent properties of compounds series 1–3 were investigated. The
interesting fluorescence exhibited by these compounds could be useful for their visualization in
tumor cells, permitting further studies on these a,b-unsaturated piperidones as candidates for
novel fluorescent antitumor agents.
Antitumor agents, cytotoxicity, fluorescence,
a,b-unsaturated piperidones, xenograft
model
History
Received 12 January 2015
Revised 3 April 2015
Accepted 11 April 2015
Published online 15 September 2015
Introduction
anticancer drugs9–11. Moreover, one or more thiol groups are
found in a multitude of cellular constituents, for example,
glutathione, cysteine and thioredoxin. Thus, thiol alkylators
have the potential to covalently bond with multiple biological
targets. The pharmacophore, a 1,5-diaryl-3-oxo-1,4-pentadienyl
group with alicyclic unsaturated enones and cyclic piperidone,
can react at each primary binding site A (Figure 2) and may allow
successive chemical attacks by cellular thiols. The electron-
withdrawing or electron-donating properties of different groups in
the benzene rings could influence the binding effect12,13. In
addition, the nature of the N-substituents at binding site B could
affect the cytotoxic properties of the molecules. The effect could
increase cytotoxic activity by facilitating the approach of the
piperidone to a specific binding site or reduce the cytotoxic
activity by hindering this interaction. Malignant cells are more
susceptible than normal cells to this type of sequential chemical
attack, so these antitumor drugs possess preferential toxicity for
malignant cells over normal tissues14. Thus, this kind of
compounds may have lower toxic side effect which is the main
drawback of the current antitumor drugs to the normal cells.
Moreover, they might activate caspase-3 to induce cell apoptosis
or cause cell autophagy15. These reasons illustrate the importance
of synthesizing and studying new 3,5-bis(benzylidene)-4-piper-
idone derivatives as antitumor drugs, which possess variable
substitution on the aryl groups in the main binding site A and a
range of N-substituents in the additional binding site B.
Piperidone derivatives are frequently encountered in natural
products and drug candidates because of their interesting
biological activities and are therefore important synthetic targets1.
Many piperidone derivatives are used widely in the synthesis of
new drugs and fine chemicals because of both their pharmaceut-
ical importance and the presence of a keto functionality with
its adjacent active methylene center that can easily accommodate
the introduction of other substituted groups2. To date, some
piperidone derivatives3–7 have been shown to possess antitumor,
antidepression, antimycobacterium, antithrombotic, sedation,
cholesterol-lowering and proteasome-inhibiting activities. In
particular, some a,b-unsaturated piperidones, 3,5-bis(arylidene)-
4-piperidones, have been reported to display cytotoxic activities8.
Therefore, a number of novel N-aroyl-3,5-bis(benzylidene)-4-
piperidones were designed and evaluated for their cytotoxic and
antitumor activities (Figure 1). These compounds feature
2-bromo-4,5-dimethoxy, 4-dimethylamino or 4-trifluoromethyl
functionality in the phenyl ring appendages, as well as various
substituents at the piperidone nitrogen.
An additional consideration inspired the synthesis and evalu-
ation of antitumor effects for these 4-piperidones. Molecules
carrying one or more a,b-unsaturated keto groups show a greater
preference, or sometimes an exclusive affinity, for bio-thiols
over both amino and hydroxy groups. As thiols are not found in
nucleic acids, these benzylidene piperidones may avoid the
genotoxic effects associated with a number of currently available
The noteworthy fluorescent properties of these molecules are
another reason to synthesize and study them. In the case of
cytotoxic molecules, natural fluorescence could permit tracking
of the target organelle in cells. Recent investigations showed
interesting fluorescent properties of a series of 3,5-bis(arylidene)-
4-piperidone derivatives; some of these could be potential two-
photon sensitizers because of the donor-p-acceptor-p-donor
*Jufeng Sun and Suwen Wang contributed equally to this article.
Address for correspondence: Jufeng Sun, School of Pharmacy, Binzhou
Medical University, Yantai, Shandong 264003, P.R. China. Tel: +86
5356913406. Fax: +86 5356913719. E-mail: sjf.xzy@163.com

2
J. Sun et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Figure 1. The structures of 3,5-bis(benzyli-
dene)-4-piperidones synthesized are shown.
using a Bruker Avance-600 MHz spectrometer (Bruker, Billerica,
MA). Chemical shifts are reported in d relative to TMS. All
fluorescence measurements were carried out on a Cary Eclipse
Spectrofluorimeter (Varian, Australia) equipped with a xenon
lamp and quartz carrier at room temperature. Biological evalu-
ation was performed using a Microplate Reader 450 (Bio-Rad,
Hercules, CA) enzyme-labeled instrument.
Synthesis of 1a, 2a, 3a
4-Piperidone hydrochloride monohydrate 2 (0.01 mol) and the
appropriate aryl aldehyde (0.02 mol) were mixed into glacial
acetic acid (50 mL). The mixture was piped into dry hydrogen
chloride gas for 0.5 h, until a clear solution was obtained. After
stirred for about 24 h at room temperature, the precipitate was
collected and washed with 40% sodium hydroxide in water. The
free base was collected and washed with water until pH 7, then
dried under the vacuum at 50–65 ꢁC for 2 h. The compounds were
recrystallized from methanol (1a); methanol–ethanol–chloroform
(2a) and methanol–chloroform (3a). The melting points (ꢁC)
and yields (%) of 1a–3a were as follows: 1a: 246–247, 48%; 2a:
270–272, 72%; 3a: 227–228, 59%. 1H NMR, 13C NMR and
elemental analysis data for compounds 1a, 2a, and 3a are
available in supplementary material.
Figure 2. Binding sites A and B and designation of the torsion angles ꢀ1
and ꢀ2 of the compounds synthesized.
structure of the molecules, and their fluorescent properties may be
affected by the substituents in the benzene ring16–19. As these
types of compounds demonstrate both marked antitumor activities
and fluorescent properties, they have been recognized as
promising antineoplastic drug candidates.
As substituted groups in the benzene ring might affect both the
cytotoxicity and fluorescent properties of these compounds,
2-bromo-4,5-dimethoxy (having both electron-withdrawing and
Synthesis of 1b, 2b, 3b
electron-donating properties), 4-dimethylamino (electron-donat- Two milliliters of a 25% (w/v) aqueous sodium hydroxide solution
ing) and 4-trifluoromethyl (electron-withdrawing) functionality containing a catalytic amount of tetrabutylammonium bromide
was introduced into these derivatives. In this study, 15 new and a suspension of 1a, 2a or 3a (1 mmol) in 1,2-dichloroethane
N-aroyl-3,5-bis(benzylidene)-4-piperidones are reported. Their (5 mL) were combined and stirred for 15 min at 0–5 ꢁC.
cytotoxic and antitumor activities were evaluated by a CCK-8 4-Nitrobenzoyl chloride (1.5 mmol) in 1,2-dichloroethane
method and xenograft model, and their fluorescent properties (5 mL) was then added to the flask. The resulting mixture was
were investigated.
stirred for approximately 6–24 h at room temperature and detected
by TLC (dichloromethane–methanol ¼ 25:1). The solvent was
removed under vacuum. Then, 10% potassium carbonate was
added to the residue, and this was stirred for 2 h. The precipitate
was collected and dried in vacuo. The compounds were
recrystallized from methanol (1b), dimethylformamide (2b), or
methanol–chloroform (3b). The melting points (ꢁC) and yields
(%) of 1b–2b were as follows: 1b: 236–238, 40%; 2b: 256–259,
73%; 3b: 209–212, 78%. 1H NMR, 13C NMR and elemental
analysis data for compounds 1b, 2b, and 3b are available in
supplementary material.
Experimental
Materials and methods
Samples for ultraviolet (UV) studies were prepared as 10ꢀ4
in a mixture of ethanol and dimethyl sulfoxide (Vethanol
M
–
Vdimethyl
Beckman Du-70 ultraviolet spectrophotometer (Beckman, Reja,
FL). Elemental analyses were performed on a Perkin-Elmer
¼ 4:1), and spectra were obtained with a
sulfoxide
1
Model 240c analyzer (Perkin-Elmer, Wellesley, MA). H NMR
data of 1a, 1e, 2a and 3a were collected using a Bruker Avance-
500 MHz spectrometer (Bruker, Billerica, MA), and others were
collected using a Bruker Avance-400 MHz spectrometer (Bruker,
Billerica, MA). All 13C NMR data were collected at 150 MHz
General synthesis of 1c–1e, 2c–2e, 3c–3e
Two milliliters of a 25% (w/v) aqueous sodium hydroxide solution
was mixed with a suspension of 1a, 2a or 3a (1 mmol) in

DOI: 10.3109/14756366.2015.1043296
Synthesis and evaluation of some new N-aroyl-a,b-unsaturated piperidones
3
1,2-dichloroethane (5 mL). A catalytic amount of tetrabutylam- during the study was permitted by the Animal Ethics Committee
monium bromide was added to the mixture, and this was stirred of the Institute of Medicinal Biotechnology, Chinese Academy of
for 10 min at 0–5 ꢁC. To the mixture was added alternatively Medical Sciences & Peking Union Medical College (permission
4-methylbenzenesulfonyl chloride or 4-acetaminobenzenesulfonyl number: c1-2011-1121).
chloride or 4-nitrobenzenesulfonyl chloride (1.5 mmol) in
PG-BE1 cells growing in exponential phase were implanted
1,2-dichloroethane (5 mL). The resulting solution was stirred for into the 6–8-week-old female athymic nude mice by subcutaneous
approximately 7–24 h at room temperature and detected by TLC injection of 10 ꢂ 106 cells on the right flank. After 3 weeks, the
(chloroform–methanol ¼ 25:1). After the reaction was complete, tumors were dissected aseptically, and pieces of tumor tissue
glacial acetic acid was added to the mixture until achieving pH 3. (2 mm3 in size) were transplanted into athymic mice subcutane-
The precipitate was collected, washed with water and dried in ously. When the size of tumors reached approximately 100 mm3,
vacuo. The compounds were recrystallized from methanol or the mice were divided into groups at random (n ¼ 6 per group)
methanol–chloroform (1c–1e), dimethylformamide or acetone and treated with 5-FU, compound 1b at different doses twice
(2c–2e), or methanol, 95% ethanol or chloroform (3c–3e). The weekly for 3 weeks, respectively. Control group was injected with
melting points (ꢁC) and yields (%) of 1c–1e, 2c–2e, 3c–3e were as saline. The tumor diameter was measured with a caliper, and
follows: 1c: 229–231, 68%; 1d: 255–256, 81%; 1e: 238–239, 49%; tumor volumes were calculated with the following formula:
2c: 239–240, 63%; 2d: 255–256, 47%; 2e: 248–249, 75%; 3c: V ¼ 0.5a ꢂ b2, where a and b are the long and the perpendicular
1
182–183, 79%; 3d: 216–219, 45%; 3e: 235–236, 74%. H NMR, short diameters of the tumor, respectively. Typically, the experi-
13C NMR and elemental analysis data for compounds 1c–1e, 2c– ments were concluded when tumors in the control animals were
2e, and 3c–3e are available in supplementary material.
close to an average size of 2000 mm3. The tumor inhibitory rate
A chloroform solution of 1e (7.4 mg, 0.01 mmol) was kept at was calculated as IR% ¼ {1 ꢀ [(tumor volumefinal ꢀ tumor
room temperature. Upon slow evaporation of the solvent about volumeinitial for the treated group)/(tumor volumefinal ꢀ tumor
15 days, bright yellow crystals of 1e were obtained.
volumeinitial for the control group)]} ꢂ 100%.
Biological evaluation
Single-crystal structure determination
Cell cytotoxicity assay by cell counting kit-8
X-Ray intensity data were measured at 298(2) K on a Bruker
SMART APEX CCD-based diffractometer (Mo Ka radiation,
Compounds belonging to series 1–3 were screened against five
tumor cell lines (Peking union medical college), namely SW1990
and MIA PaCa-2 (human pancreatic carcinoma), PG-BE1 and
NCI-H460 (human lung carcinoma) and SK-BR-3 (human breast
cancer) using a cell counting kit-8 (WST-8, Dojindo Laboratories,
Tokyo, Japan) method. Cells were cultured in RPMI-1640
medium (Sigma-Aldrich, St. Louis, MO) supplemented with
10% fetal bovine serum (FBS, HyClone, Logan, UT), 2 mM L-
glutamine and 50 lg/mL gentamycin. Cytotoxicity for each
individual compound was measured after 48 h of cocultivation
with each cell line. Briefly, the different cell lines were seeded
into 96-well plates at a concentration of 1 ꢂ 104 cells/100 mL/well.
The cells were allowed to attach overnight at 37 ꢁC in a
humidified atmosphere containing 5% CO2. The tested com-
pounds were initially dissolved in DMSO (Sigma-Aldrich), and
the working solutions were added to a FBS free culture medium.
The final DMSO concentration in each well was 1%. The
compounds were added to wells with increasing drug concentra-
tions. After a 48-h incubation period, 10 mL of CCK-8 reagent was
added, and the plate was incubated for 4 h at 37 ꢁC. The optical
density (OD) was measured by a multi-well plate reader (TECAN,
˚
k ¼ 0.71073 A) using a SMART and SAINT programs (Bruker,
Billerica, MA). The structures were solved by direct methods and
refined on F2 by full-matrix least-squares methods with
¨
SHELXTL version 6.1 (Gottingen, Germany). Crystal data of
1e: C31H28Br2Cl6N2O9S, M ¼ 977.13, triclinic, space group P-1,
˚
˚
3
yellow block, a ¼ 10.322(9)), b ¼ 11.353(10) A, c ¼ 18.561(16)A,
ꢁ
ꢁ
ꢁ
˚
a ¼ 87.462(13) , b ¼ 86.197(11) , ꢁ ¼ 68.646(10) , V ¼ 2021(3) A ,
Z ¼ 2, Dc ¼ 1.606 g cmꢀ3, l(Mo Ka) ¼ 2.505 mmꢀ1, T ¼ 298(2) K;
7168 unique reflections [Rint ¼ 0.0282]. Final R1 [with
I42ꢂ(I)] ¼ 0.0580, wR2 (all data) ¼ 0.1457.
CCDC 918752 (1e) contains the supplementary crystallo-
graphic data for this article. Copies of the data can be obtained
free of charge on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (Fax: + 44 1223336033; e-mail:
deposit@ccdc.cam.ac.uk).
Results and discussion
Synthesis and structures
Fifteen new compounds 1a–1e (series 1), 2a–2e (series 2) and
3a–3e (series 3) were prepared by the synthetic route shown in
Scheme 1. Dry hydrogen chloride was used to catalyze the
reaction of various aryl aldehydes with 4-piperidone via a
Claisen–Schmidt condensation, leading to the preparation of 1a,
2a and 3a. Treatment of these compounds with each of
4-nitrobenzoyl chloride, 4-methylbenzenesulfonyl chloride,
4-acetamidobenzenesulfonyl chloride or 4-nitrobenzenesulfonyl
chloride produced the corresponding N-acylated or N-sulfonylated
derivatives 1b–1e, 2b–2e and 3b–3e. The yields of the com-
pounds were between 40% and 81%. Their structures were fully
characterized by 1H NMR, 13C NMR and elemental analysis.
¨
Mannedorf, Switzerland) at 450 nm. The results are expressed as a
decrease in the cell viability (%) in comparison to untreated
controls. The concentration of each compound was examined in
triplicate, and the IC50 values are expressed graphically. The
concentrations of the compounds used were 200, 100, 10, 1, 0.1
and 0.01 mg/mL. 5-Fluorouracil (5-FU, Sigma-Aldrich) was used
as a positive control. The concentrations of 5-FU used were 250,
25, 2.5, 0.25 and 0.025 mg/mL.
Tumor xenograft model
The human lung carcinoma PG-BE1 xenograft model was set up. 1H NMR spectroscopy indicated that molecules in series 1–3 were
The BALB/c female athymic nude mice (BALB/c, nu/nu) were stereoisomerically pure. The NMR signals of CH2 groups in the
purchased from the Institute for Experimental Animals, Chinese central piperidone ring were split due to the influence of
Academy of Medical Sciences & Peking Union Medical College. noncoplanarity between two aryl rings of the parent NH-3,5-
The study protocols were according to the regulations of Good bis(benzylidene)-4-piperidones and the adjacent olefinic linkages
Laboratory Practice for non-clinical laboratory studies of drugs for some compounds, most notably for 1b, 2b, 3b and 3e. Some
issued by the National Scientific and Technologic Committee of carbon signals in the 13C NMR spectra for the two aryl rings
People’s Republic of China. The treatment and use of animals (attached to the olefinic bonds) were similarly influenced

4
J. Sun et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Scheme 1 Synthetic chemical pathway of
compounds in series 1–3. The reagents used
in the syntheses were as follows: (I) 2-bromo-
4,5-dimethoxy-benzaldehyde/CH3COOH/dry
HCl; (II) 4-dimethylamino-benzaldehyde/
CH3COOH/dry HCl; (III) 4-trifluoromethyl-
benzaldehyde/CH3COOH/dry HCl; (IV)
4-nitrobenzoyl chloride/NaOH/ClCH2CH2Cl/
K2CO3/tetrabutylammonium bromide or
4-methylbenzene sulfonyl chloride/
4-acetamidobenzenesulfonyl chloride/
4-nitrobenzenesulfonyl/NaOH/ClCH2CH2Cl/
tetrabutylammonium bromide/CH3COOH.
Table 1. Cytotoxicity of 1a–1e, 2a–2e and 3a–3e against human SK-BR-3, PG-BE1, NCI-H460, MIA PaCa-2 and SW1990
cell lines.
IC50 (mM)
Compound
SK-BR-3
PG-BE1
NCI-H460
MIA PaCa-2
SW1990
1a
1b
1c
1d
1e
2a
2b
2c
4.38 0.48
3.19 0.53
10.8 1.9
2.86 0.35
23.1 1.1
64.0 10.5
4100
1.31 0.04
0.382 0.098
3.16 0.73
1.77 0.28
2.27 0.54
11.3 2.77
4100
0.960 0.108
0.402 0.056
3.44 0.67
1.39 0.51
1.41 0.30
11.8 3.6
4100
0.377 0.027
0.167 0.017
1.24 0.35
0.29 0.06
1.33 0.07
8.23 0.33
4100
1.91 0.37
0.978 0.496
3.66 0.28
1.72 0.37
4.74 0.76
36.7 5.0
4100
13.6 1.5
23.4 2.4
9.66 3.79
8.82 1.48
12.6 3.4
2d
2e
4100
4100
4100
4100
4100
4100
4100
4100
4100
4100
3a
3b
3c
3d
3e
0.843 0.092
3.70 0.14
4.40 0.15
1.12 0.24
0.417 0.080
1297 330
0.661 0.046
4.08 0.32
1.75 0.44
0.820 0.085
0.565 0.042
94.3 23.9
1.04 0.21
3.38 0.11
0.544 0.243
0.321 0.081
0.126 0.031
35.8 11.6
0.417 0.094
0.230 0.016
0.593 0.132
0.353 0.016
0.107 0.006
1.94 0.24
1.51 0.48
3.69 0.21
0.755 0.553
0.365 0.109
474 96
5-FU
102
9
and split, for example in compound 2b8,13,20–22. An X-ray change in the appearance of the compounds. Series 3 compounds
crystallographic structure of 1e showed that it adopted an E,E are mainly pale yellow crystals, series 1 compounds are generally
configuration.
yellow or bright yellow crystals and series 2 compounds are a red
The UV spectra of the molecules were inspected at a concen- or red-orange powder. The melting points for compounds in these
tration of 10ꢀ4 mol Lꢀ1 in a mixture of ethanol and dimethyl series decrease according to the following: 24143 when
sulfoxide. From the spectra, two kmax values could be seen for comparing three compounds having identical substituents
almost all the derivatives except 3a and 3e. The main kmax values attached to the piperidyl nitrogen atom. This is related to
ranged from 374 to 383 nm for series 1, 461–472 nm for series 2 increases in electron-donating effects of aryl substituents in the
and 303–311 nm for series 3. The kmax values increased from parent benzene rings.
series 2 to series 1 to series 3 due to the extension of the
p-conjugation system in the molecules. This was caused mainly Antitumor activity
by the change of substituents in the benzene rings of the parent
NH-3,5-bis(4-dimethylaminobenzylidene)-4-piperidone for series
Cell cytotoxicity
2, NH-3,5-bis(2-bromo-4,5-dimethoxybenzylidene)-4-piperidone The cytotoxic activities of the synthetic compounds were
for series 1 and NH-3,5-bis(4-trifluoromethylbenzylidene)-4- evaluated against five human carcinoma cell lines: SW1990,
piperidone for series 3. The electron-withdrawing effects of the MIA PaCa-2, PG-BE1, NCI-H460 and SK-BR-3. The results are
substituents strengthened gradually from series 2, to series 1, to shown in Table 1, which summarizes the corresponding IC50
series 3; thus, the p-conjugation system lengthened in the order of values (the IC50 represents the drug concentration resulting in
series 3, series 1 and series 2. These differing properties caused a 50% growth inhibition).

DOI: 10.3109/14756366.2015.1043296
Synthesis and evaluation of some new N-aroyl-a,b-unsaturated piperidones
5
Figure 3. The inhibitory effect of compounds 1a–1e and 3a–3e against five human carcinoma cell lines.
Figure 4. The ORTEP figure of 1e
(displacement ellipsoids with 30% probabil-
ity, omitting two molecules of CHCl3).
As shown in Table 1, the biological evaluation data showed compounds 2a–2e, which have strong electron-donating dimethy-
that 96% of the IC50 values of the compounds in series 1 and 3 lamino groups in the para-position of the parent benzene rings,
against SW1990, NCI-H460, PG-BE1, MIA PaCa-2 and SK-BR-3 were the least active among the three series. Comparing
cells were lower than 5 mM, while 44% of these values were compounds 3a–3e, with only electron-withdrawing trifluoro-
submicromolar. These data revealed that almost all the com- methyl groups in the parent benzene rings, and compounds
pounds in series 1 and 3 were potent inhibitors of the growth of 1a–1e, bearing both electron-withdrawing bromine groups and
various malignant cells; the especially high potencies of 1b, 3a, electron-donating methoxy groups in the parent benzene rings,
3d and 3e should be noted. In the case of series 2, only 2a and 2c more than 80% of the IC50 values for 3a–3e were higher than or
showed moderate potency, while the IC50 values of 2b, 2d and 2e similar to those of the corresponding compounds of 1a–1e.
were all greater than 100 mM. As displayed in Figure 3, among Compound 3b was the only compound in series 3 that exhibited
compounds 1a–1e and 3a–3e, the SW1990, MIA PaCa-2, lower IC50 values against all five human carcinoma cell lines than
NCI-H460 and SK-BR-3 cell lines were the most sensitive compound 1b in series 1. In addition, the nature of N-substituted
toward compound 3e, while the PG-BE1 cell line was the most groups may also be one of the factors that can affect the cytotoxic
sensitive to compound 1b.
potencies. As presented in Table 1, the potencies of N-aroyl
To verify the discrepancy between potencies, some investiga- analogs 1b, 1d, 3b, 3d and 3e against SW1990 were higher than
tions were conducted. As mentioned previously, biological those of the parent NH-4-piperidones 1a and 3a. This difference
potencies may be influenced by the nature of the aryl substituents demonstrated that about half of the N-aroyl analogs had potencies
in rings A and B (Figure 2). It can be seen in Table 1 that that were intensified in series 1 and 3. In particular, the IC50

6
J. Sun et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Figure 5. (A) The tumor growth inhibition of compound 1b against nude mice with PG-BE1 xenograft (in the light of tumor volume). (B) The effect of
compound 1b on the body weight of nude mice with PG-BE1 xenograft.
Figure 6. (A) The fluorescence spectra of 1a–1e at room temperature. (B) The fluorescence spectra of 2a–2e at room temperature.
(C) The fluorescence spectra of 3a–3e at room temperature.
values of compounds 1b and 3e against all five carcinoma cell nitrobenzensulfonyl)-3,5-bis(2-bromo-4,5-dimethoxybenzyli-
lines were higher than those of 1a and 3a separately.
dene)-4-piperidone (1e) was investigated. The ORTEP diagram of
Moreover the noncoplanarity between the two aryl rings of the 1e is presented in Figure 4. The X-ray crystallographic structure
parent NH-3,5-bis(benzylidene)-4-piperidones and the adjacent revealed that compound 1e crystallizes in the triclinic space group
olefinic linkages might allow a favorable topography for the P-1 with two CHCl3 molecules. As shown in Figure 4, a single-
alignment of these compounds with their targets at cellular crystal structure of 1e shows the E stereochemistry of the olefinic
binding sites (Figure 2), thereby increasing the cytotoxicity of double bonds (E,E isomer), which is confirmed by the C12–C15–
the molecules22. To confirm this, the X-ray crystallographic C16–C17 torsion angle value (ꢀ1 ¼ 28.915(7)ꢁ) and C10–C9–C7–
structure
of
the
representative
compound
N-(4- C8 torsion angle value (ꢀ2 ¼ ꢀ30.15(8)ꢁ). It is noteworthy that, in

DOI: 10.3109/14756366.2015.1043296
Synthesis and evaluation of some new N-aroyl-a,b-unsaturated piperidones
7
this structure, the 4-nitrophenylsulfonyl group is almost parallel to activities. It can be seen from Table 1 and Figure 6(A)–(C) that
the C14–C10–C11–C12–C13 plane and the two aryl groups in compounds 2a–2e, with stronger fluorescence intensity, demon-
3,5-bis(benzylidene)-4-piperidones. The corresponding dihedral strated lower cytotoxic activities, while compounds 1a–1e and
angles are ca. 25.9(2)ꢁ, ca. 6.52(2)ꢁ and ca. 9.35(2)ꢁ. In addition, 3a–3e, with weaker fluorescent intensity, demonstrated higher
the N1–S1–C24 angle is only 108.0(2)ꢁ, which demonstrates that cytotoxic activities. Further investigation into the structure–
the olefinic double bonds are close to the 4-nitrophenylsulfonyl cytotoxicity–fluorescence relationships of the compounds is
group. The corresponding C12ꢃ ꢃ ꢃC24, C15ꢃ ꢃ ꢃC29, C10ꢃ ꢃ ꢃC24 warranted.
and C9ꢃ ꢃ ꢃC25 distances are 3.372(2), 3.612(2), 3.541(3) and
˚
3.777(3) A. Such short distances may result in inter-conjugated-
Conclusions
system interactions between the 4-nitrophenylsulfonyl groups and
3,5-bis(benzylidene)-4-piperidones, further influencing the fluor-
escent properties of the molecules.
In summary, we synthesized 15 new a,b-unsaturated piperidones
(N-aroyl-3,5-bis(benzylidene)-4-piperidones), 1a–1e (series 1),
2a–2e (series 2) and 3a–3e (series 3), and investigated their
cytotoxic and fluorescent properties. In addition, we assessed the
antitumor activity in vivo of compound 1b (having better potency)
using a nude mouse PG-BE1 xenograft model. Among the
derivatives assayed, the majority of compounds 1a–1e (possessing
strong electron-withdrawing trifluoromethyl groups in the aryl
rings of A and B) and 3a–3e (possessing both electron-
withdrawing bromine and electron-donating methoxy groups in
aryl rings A and B) displayed significant cytotoxic properties, but
their fluorescent activities were a little weaker. Alternatively,
molecules 2a–2e (bearing strong electron-donating dimethyla-
mino groups in the aryl rings A and B) illustrated poor cytotoxic
properties, but their fluorescent activities were stronger than those
of compounds 1a–1e and 3a–3e. Nevertheless, all the compounds
that were investigated had interesting fluorescent properties
overall. Compounds 1b, 3a, 3d and 3e displayed remarkable
cytotoxic activities towards five human carcinoma cell lines.
Importantly, the results of a PG-BE1 xenograft model showed that
compound 1b possessed marked inhibitory effects on tumor
growth and had no obvious toxicity toward the nude mice.
Compounds 1b, 3a, 3d and 3e represent possible lead molecules
for synthesizing new derivatives with the aim to increase their
cytotoxic potencies, while producing low-toxicity antitumor drug
candidates that undergo fluorescence.
Antitumor activity in vivo
The antitumor activity in vivo is the most important factor to
evaluate for new antitumor agents. The sensitive PG-BE1
xenograft model was used to investigate the antitumor activity
in vivo of compound 1b, which had higher potency. The diameters
of tumors were measured two times per week after treatment of
1b in athymic nude mice with PG-BE1 xenografts (Figure 5A).
With doses of 60, 30 and 15 mg/kg, the corresponding tumor
inhibitory rates were 58.8%, 38.3% and 31.0%, respectively,
which demonstrated that compound 1b could inhibit the growth of
the tumor. Moreover, toxicity is one of the metrics to judge
antitumor agents, and the body weight changes of nude mice post-
therapy can be influenced by the toxicity of the drugs indirectly.
From Figure 5(B), it was shown that the body weight of mice did
not decreased obviously compared with the control group,
indicating that compound 1b has no apparent toxicity to nude
mice bearing PG-BE1 xenografts.
Fluorescent properties
Lengthening the conjugated spacer by inserting additional
substituted groups into 3,5-bis(arylidene)-4-piperidone may
result in increasing the electron-transporting effects and fluores-
cent properties of the molecules23,24. Taking this into account, the
N-aroyl-3,5-bis(arylidene)-4-piperidone derivatives, discussed
above, were designed and synthesized. Their fluorescent proper-
ties were then explored (Figure 6A–C).
Declaration of interest
Financial support of this work was from National Natural Science
Foundation of China (No. 21402010), the Foundation of Shandong
Province (Nos. ZR2014BL008, ZR2010HL065). The authors report no
declarations of interest.
The compounds were excited at 435 nm for series 1, 365 nm for
series 2 and 373 nm for series 3 to obtain the fluorescence spectra.
The solutions (10ꢀ4 mol Lꢀ1) were prepared in a mixture of
ethanol and dimethyl sulfoxide (Vethanol–Vdimethyl sulfoxide ¼ 4:1).
The fluorescence emission was measured at room temperature
from 460 to 650 nm for series 1, 480–700 nm for series 2 and 390–
590 nm for series 3. Figure 6(A)–(C) displays contrasts in the
emission spectra from series 1 to series 3, respectively.
Compounds of series 1 exhibit an emission maximum at
approximately 521 nm, compounds of series 2 at approximately
572 nm and compounds of series 3 at approximately 424 nm.
The results of Figure 6(A)–(C) revealed that compounds 2a–
2e, with strong electron-donating dimethylamino groups, pos-
sessed the strongest fluorescence. The fluorescence intensities of
compounds 1a–1e and 3a–3e were weaker due to the influence of
the heavy atom effect from bromine and the electron-withdrawing
effects of trifluoromethyl groups, respectively. In addition, the
emission intensity of the parent NH-3,5-bis(benzylidene)-4-piper-
idones (1a, 2a, 3a) is stronger than that of N-(4-nitrobenzoyl)
derivatives (1b, 2b, 3b) and N-(4-nitrobenzenesulfonyl)
derivatives (1e, 2e, 3e), but weaker than that of N-(4-acetamino-
benzenesulfonyl) derivatives (1d, 2d, 3d) and N-(4-methylbenze-
nesulfonyl) derivatives (1c, 2c, 3c).
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Supplementary material available online
Supplementary information