Design, synthesis, anticancer activity and cytotoxicity of novel 4-piperidone/cyclohexanone derivatives

Article abstract of DOI:10.1007/s11164-016-2583-y

Abstract: Design and synthesis of two series of novel double Schiff-base substituted 4-piperidone/cyclohexanone derivatives, like curcumin analogues, series 1 (1a–d) and 2 (2a–e) were generated and characterized by ¹H NMR, ¹³C NMR, I

Full text of DOI:10.1007/s11164-016-2583-y

Res Chem Intermed
DOI 10.1007/s11164-016-2583-y
Design, synthesis, anticancer activity and cytotoxicity
of novel 4-piperidone/cyclohexanone derivatives
Qin Chen1 Yun Hou1 Gui-Ge Hou1
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Ju-Feng Sun1 Ning Li1 Wei Cong1 Feng Zhao1
Hong Juan Li1 Chun-Hua Wang1
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Received: 8 April 2016 / Accepted: 17 May 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract Design and synthesis of two series of novel double Schiff-base substituted
4-piperidone/cyclohexanone derivatives, like curcumin analogues, series 1 (1a–d) and 2 (2a–
e) were generated and characterized by 1H NMR, 13C NMR, IR, and elemental analysis. The
anticancer activities against human carcinoma cell lines HePG2, HeLa, K562, THP-1, and
their cytotoxicities for LO2 cell lines were subsequently evaluated by the MTT method. The
results show that series 2 (2a–e) have the better anticancer activity than series 1 (1a–d) in spite
of the bigger cytotoxicity of series 2. Structure analysis shows that the N-methyl-4-piperidi-
none group and the introduction of hydroxy is helpful for improving their anticancer activity,
especially 2a, 2d and 2e, and their IC50 values against THP-1 cells can reach 0.69–0.96 lM.
Graphical Abstract
Qin Chen and Yun Hou have contributed equally to this work.
&
&
1
Gui-Ge Hou
guigehou@163.com
Chun-Hua Wang
chunhuawang508@126.com
The Key Laboratory of Prescription Effect and Clinical Evaluation of State Administration of
Traditional Chinese Medicine of China, Binzhou Medical University, Yantai 264003, People’s
Republic of China
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Q. Chen et al.
Keywords 4-Piperidone Á Cyclohexanone Á Schiff-base Á Anticancer activity Á
Cytotoxicity
Introduction
Curcumin (Diferuloylmethane, Fig. 1), a member of the curcuminoid family, is the
yellow-coloured phenolic pigment of the rhizome of Curcuma longa or turmeric
from the Zingiberaceae family. Since antiquity, Curcuma longa or turmeric has been
used to improve colour and flavor of food products [1, 2]. More important,
curcumin, the active principle of turmeric [3, 4], has been reported to possess anti-
inflammatory, anticoagulant, antibacterial, antifungal, anticarcinogenic, antitumor,
anti-HIV, anti-mutagenic, antiproliferative, antioxidant, anti-protozoal, and anti-
venom activities [5–10]. Curcumin and its analogues containing the pharmacophore
of 1,5-diaryl-3-oxo-1,4-pentadienyl, is thought to interact at the primary binding site
and bio-thiols from susceptible neoplasms. The pharmacophore of methoxyphenol
groups align at an auxiliary site and also influence their bio-activities. However,
curcumin and some analogues have many major problems in clinical applications,
such as poor aqueous solubility, relatively low bioavailability, intense staining
colour and so on [11]. In order to improve these defects, many improved curcumin
Fig. 1 Structures of curcumin and some curcumin analogues
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Design, synthesis, anticancer activity and cytotoxicity of…
analogues have been reported, such as demethoxycurcumin, bisdemethoxycur-
cumin, cyclocurcumin, and so on [12–19].
In recent years, some curcumin analogues 3,5-bis(arylidene)-4-piperidone
derivatives were synthesized and evaluated for bioactivity. They incorporate two
a,b-unsaturated ketos and b-aminoketones structures. These possess 1,5-diphenyl-
penta-1,4-dien-3-ones pharmacophore, which could combine with sensitive parts of
the tumor cells and induce apoptosis or autophagy [20]. The nitrogen-containing
heterocyclic dienones, such as 4-piperidone, can display higher inhibitory properties
toward human carcinoma cell lines compared with their homocyclic dienone
analogs [21]. Another advantage is that these analogues have lower toxicity toward
normal cells. In other words, these compounds can be easier to attack malignant
cells rather than normal cells. 3,5-Bis(2-fluorobenzylidene)-4-piperidone (EF24,
Fig. 1) with excellent anti-proliferative activity was first reported by Adams [22].
Another derivative 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-
butenoic acid] (CLEFMA, Fig. 1) with the better anti-proliferative activity in H441
cells than EF24 was reported by Awasthi [23]. Recently, our group has also reported
some novel N-substituted-3,5-bis(arylidene)-4-piperidone derivatives as anticancer
agents with fluorescent properties [24]. The results show an interesting relationship
between cytotoxic activities and fluorescent properties. In addition, some
bispyridyl-substituted piperidone derivatives [25, 26] and their pharmaceutical co-
crystals with coformers were reported recently with interesting luminescent
properties [27] and the better anticancer activities against human neoplastic cell
lines and lower cytotoxicities, which showed their improved anticancer activities
may be dependent on the synergistic effect between pharmaceutical ingredients and
gallic acid [28]. However, the anticancer activities and the water solubility of these
derivatives need to be further improved. The structure-property relationship
between structures and anticancer activities also needs to continue refinement. In
addition, Schiff-base compounds with active –C=N group can be seen as a good
donor site or an active ligand, which exhibits a range of biological activities,
including antifungal, antiradical, antimalarial, antiviral, anticancer, antibacterial,
antiinflammatory, and antipyretic properties [29–32].
In this study, intermediate (2E,6E)-2,6-bis(3-aminobenzylidene)cyclohexanone
(3) and (3E,5E)-3,5-bis(3-aminobenzylidene)-1-methylpiperidin-4-one (4) were
synthesized via the Claisen-Schmidt condensation reaction and the reduction reaction
of stannous chloride. Next, some aromatic aldehydes with phenol substitution groups
were selected to synthesize two series of target compounds 1a–d and 2a–e (Fig. 2).
Their anticancer activities against human HePG2, HeLa, K562, THP-1 cell lines, and
their cytotoxicities for LO2 cell lines were evaluated by the MTT method.
Experimental section
Materials and methods
Cyclohexanone, N-methyl-4-piperidinone, m-nitrobenzaldehyde, tetrabutyl ammo-
nium bromide (TBAB), stannous chloride, and all aromatic aldehydes were
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Fig. 2 Structures of double Schiff-base substituted 4-piperidone/cyclohexanone derivatives
purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China) and were
1
used as obtained without further purification. H NMR data were collected using a
Bruker Avance 400 MHz. 13C NMR data were collected at 100 MHz on a Bruker
Avance 400 MHz spectrometer. Chemical shifts were reported in d relative to TMS.
Elemental analyses were performed on a Perkin–Elmer Model 240c analyzer.
Infrared (IR) spectra were obtained in the 400–4000 cm-1 range using a Perkin-
Elmer Frontier Mid-IR FTIR Spectrometer.
Preparation of 3 and 4
(2E,6E)-2,6-bis(3-aminobenzylidene)cyclohexanone (3)
Cyclohexanone (0.98 g, 0.01 mol) and m-nitrobenzaldehyde (3.32 g, 0.022 mol) was
mixed with 40 mL glacial acetic acid, stirred on a magnetic stirrer at room temperature
for about 30 min, during which time a clear solution was obtained. Dry hydrogen
chloride gas was bubbled into this mixture for about 2 h, at this time a large amount of
precipitation appeared. Then, the hydrogen chloride gas device was removed, and the
mixture continued stirring at room temperature for 24 h (monitored by TLC). The
precipitate was collected and dried under the vacuum to obtain crude intermediates
(2E,6E)-2,6-bis(3-nitrobenzylidene)cyclohexanone, which were untreated and
directly used in the following reaction. The crude intermediates dissolved in a
solution of trichloromethane (10 mL), water (20 mL), and methanol (16 mL) with
TBAB (0.32 g, 0.001 mol). In addition, stannous chloride (6.75 g, 0.03 mol)
dissolved in concentrated hydrochloric acid (12 mL), was slowly added to the
reaction system. The mixture was stirred at 40 °C for 8 h. After cooled to temperature,
the mixture was adjusted to pH 12 by 40 % sodium hydroxide. The precipitate was
collected and dried under vacuum to get yellow powder 3. Yield: 57 %; mp: 70–72 °C.
IR (cm-1): 3408(m), 3299(m), 2985(m), 2872(m), 1647(m), 1592(s), 1549(w),
1486(w), 1456(w), 1381(m), 1315(m), 1262(s), 1162(s), 1135(w), 1062(w), 986(m),
1
881(m), 783(m). H NMR (400 MHz, CDCl3): d 7.71 (s, 2H), 7.22 (t, J = 8.0 Hz,
2H), 6.89 (d, J = 8.0 Hz, 2H), 6.78 (s, 2H), 6.69 (d, J = 8.0 Hz, 2H), 3.73 (s, 4H),
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Design, synthesis, anticancer activity and cytotoxicity of…
2.92 (t, J = 8.0 Hz, 4H), 1.85–1.75 (m, 2H). Elemental analysis (%) calcd. for
C20H20N2O (304.38): C 78.92, H 6.67, N 9.20; Found: C 78.87, H 6.70, N 9.23.
(3E,5E)-3,5-bis(3-aminobenzylidene)-1-methylpiperidin-4-one (4)
N-Methyl-4-piperidinone (1.13 g, 0.01 mol) and m-nitrobenzaldehyde (3.32 g,
0.022 mol) was mixed with 40 mL glacial acetic acid. Dry hydrogen chloride gas
was bubbled into this mixture for about 2 h, at this time a large amount of
precipitation appeared. Then, hydrogen chloride gas device was removed, and the
mixture continued stirring at room temperature for 24 h (monitored by TLC). The
precipitate was collected and dried under vacuum to obtain crude intermediates
(3E,5E)-1-methyl-3,5-bis(3-nitrobenzylidene)piperidin-4-one, which were untreated
and directly used in the following reaction. Then, the crude intermediates were
dissolved in a solution of water (20 mL) and methanol (16 mL) with TBAB (0.32 g,
0.001 mol). Stannous chloride (6.75 g, 0.03 mol) was dissolved in concentrated
hydrochloric acid (12 mL), was slowly added to the reaction system. The mixture
was stirred at 40 °C for 8 h. After cooled to temperature, the precipitate was
collected and washed with 5 % sodium hydroxide solution, and dried under vacuum
to get yellow powder 4. Yield: 52 %; mp: 101–103 °C. IR (cm-1): 3355(w),
3347(w), 3222(w), 1670(m), 1600(s), 1583(s), 1452(w), 1316(w), 1259(s), 1171(s),
1
1095(w), 1051(w), 991(w), 915(w), 862(m), 780(s), 688(s). H NMR (400 MHz,
CDCl3) d 7.74 (s, 2H), 7.31–7.17 (m, 2H), 6.81 (d, J = 7.7 Hz, 2H), 6.78–6.66 (m,
4H), 3.76 (d, 8H), 2.47 (s, 3H). 13C NMR (100 MHz, CDCl3) d 187.00, 146.45,
136.76, 136.27, 132.97, 129.38, 120.71, 116.68, 115.85, 57.07, 45.75. Elemental
analysis (%) calcd. for C20H21N3O (319.40): C 75.21, H 6.63, N 13.16; Found: C
75.27, H 6.60, N 13.09.
Preparation of 1a–d and 2a–e
3 (0.30 g, 0.001 mol) or 4 (0.32 g, 0.001 mol) and aromatic aldehyde (0.002 mol)
were dissolved in methanol (5 mL). A drop of formic acid was added into the
mixture. The mixture was stirred for 3–8 h at ambient temperature (monitored by
TLC). The precipitate was collected and recrystallized by methanol to afford 1a–
d and 2a–e.
1a: Light yellow powder; Yield: 72 %; mp: 133–135 °C. IR (cm-1): 1667(w),
1619(s), 1596(m), 1568(s), 1282(s), 1169(s), 1149(s), 1031(w), 982(w), 820(m),
1
804(s), 794(m), 747(s), 734(m), 695(m), 684(m), 535(m). H NMR (400 MHz,
CDCl3): d 13.13 (bs, 2H), 8.66 (s, 2H), 7.84 (s, 2H), 7.50–7.20(m, 12H), 7.07 (d,
J = 8.0 Hz, 2H), 6.98 (t, J = 8.0 HZ, 2H), 2.99 (t, J = 4.0 Hz, 4H), 1.90–1.80 (m,
2H). 13C NMR (100 MHz, DMSO): d 188.91, 164.19, 160.24, 148.45, 136.95,
136.59, 135.52, 133.47, 132.65, 129.65, 128.57, 123.01, 121.96, 119.29, 119.20,
116.61, 27.85, 22.33. Elemental analysis (%) calcd. for C34H28N2O3 (512.59): C
79.67, H 5.51, N 5.46; Found: C 79.78, H 5.45, N 5.42.
1b: Light yellow powder; Yield: 72 %; mp: 182–184 °C. IR (cm-1): 1670(m),
1625(w), 1610(w), 1576(m), 1461(m), 1356(m), 1272(m), 1210(s), 1170(s),
1144(s), 1031(w), 992(w), 886(w), 863(m), 784(s), 724(s), 684(s), 540(m),
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Q. Chen et al.
1
504(m). H NMR (400 MHz, DMSO): d 13.65 (bs, 2H), 8.64 (s, 2H), 7.84 (s, 2H),
7.50 (t, J = 8.0 Hz, 2H), 7.45–7.35 (m, 4H), 7.30 (s, 2H), 7.10 (d, J = 8.0 Hz, 2H),
7.02 (d, J = 8.0 Hz, 2H), 6.88 (t, J = 8.0 Hz, 2H), 5.80 (bs, 2H), 2.99 (t,
J = 4.0 Hz, 4H), 1.89–1.80 (m, 2H). 13C NMR (100 MHz, DMSO): d 188.93,
164.60, 149.27, 148.22, 145.61, 136.97, 136.61, 135.32, 129.67, 128.54, 122.93,
121.89, 121.19, 119.36, 119.09, 118.83, 27.86, 22.34. Elemental analysis (%) calcd.
for C34H28N2O5 (544.59): C 74.98, H 5.18, N 5.14; Found: C 75.06, H 5.13, N 5.10.
1c: Light yellow powder; Yield: 74 %; mp: 55–57 °C. IR (cm-1): 3196(br),
1667(m), 1616(s), 1597(s), 1574(s), 1488(w), 1356(m), 1212(s), 1169(s), 1119(m),
978(m), 842(m), 796(s), 689(s), 627(m), 538(m), 511(w). 1H NMR (400 MHz,
DMSO): d 13.35 (bs, 2H), 10.37 (s, 2H), 8.85 (s, 2H), 7.67 (s, 2H), 7.55–7.35 (m,
10H), 6.44 (d, J = 8.0 Hz, 2H), 6.32 (s, 2H), 3.00–2.90 (m, 4H), 1.89–1.80 (m, 2H).
13C NMR (100 MHz, DMSO): d 190.79, 163.29, 163.00, 162.68, 148.39, 136.85,
136.54, 135.42, 134.60, 132.65, 129.60, 122.73, 112.00, 108.59, 107.98, 102.34,
27.86, 22.37. Elemental analysis (%) calcd. for C34H28N2O5 (544.59): C 74.98, H
5.18, N 5.14; Found: C 75.07, H 5.14, N 5.11.
1d: Light yellow powder; Yield: 76 %; mp: 210–212 °C. IR (cm-1): 3055(br),
1668(m), 1617(s), 1577(m), 1513(w), 1280(m), 1211(w), 1149(s), 1078(m), 991(m),
1
884(w), 778(m), 680(s), 541(m), 482(w), 438(w). H NMR (400 MHz, DMSO): d
13.51 (s, 2H), 9.77 (s, 2H), 8.83 (s, 2H), 8.52 (s, 2H), 7.68 (s, 2H) 7.55–7.35 (m,
8H), 6.98 (d, J = 8.0 Hz, 2H), 6.45 (d, J = 8.0 Hz, 2H), 2.96 (t, J = 4.0 Hz, 4H),
1.80–1.70 (m, 2H). 13C NMR (100 MHz, DMSO): d 188.95, 163.80, 151.36,
150.48, 148.14, 136.89, 136.58, 135.41, 132.38, 129.62, 127.89, 124.24, 122.71,
121.61, 112.32, 108.36, 107.85, 27.87, 13.49. Elemental analysis (%) calcd. for
C34H28N2O7 (576.59): C 70.82, H 4.89, N 4.86; Found: C 70.75, H 4.92, N 4.90.
2a: Light yellow powder; Yield: 69 %; mp: 114–116 °C. IR (cm-1): 1675(w),
1615(m), 1563(m), 1278(m), 1182(w), 1152(w), 1102(w), 1058(w), 974(w), 922(w),
1
881(w), 800(s), 743(m), 691(m), 539(w), 424(w). H NMR (400 MHz, CDCl3): d
12.95 (s, 2H), 8.50–8.35 (m, 2H), 7.74–7.58 (m, 2H), 7.40–7.00 (m, 12H),
6.95–6.70 (m, 4H), 3.70–3.50 (m, 4H), 2.40–2.20 (m, 3H). 13C NMR (100 MHz,
CDCl3): d 186.62, 163.47, 161.08, 148.94, 136.49, 135.80, 133.73, 133.48, 132.46,
129.63, 128.58, 122.99, 121.58, 119.22, 119.05, 117.29, 56.98, 45.89. Elemental
analysis (%) calcd. for C34H29N3O3 (527.61): C 77.40, H 5.54, N 7.96; Found: C
77.49, H 5.51, N 7.92.
2b: Light yellow powder; Yield: 80 %; mp: 140–142 °C. IR (cm-1): 3253(br),
1672(m), 1614(s), 1573(s), 1460(m), 1363(m), 1273(s), 1209(s), 1183(m), 1028(w),
1
939(w), 850(w), 789(m), 734(s), 684(s), 540(w). H NMR (400 MHz, DMSO): d
13.03 (s, 2H), 9.26 (s, 2H), 8.97 (s, 2H), 7.69 (s, 2H), 7.62–7.50 (m, 4H), 7.50–7.40
(m, 4H), 7.13 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 6.81 (t, J = 8.0 Hz,
2H), 3.82 (s, 4H), 2.42 (s, 3H). 13C NMR (100 MHz, DMSO): d 186.27, 164.75,
149.25, 148.42, 145.63, 135.85, 134.47, 134.04, 129.86, 128.46, 123.32, 122.85,
122.25, 119.38, 119.14, 118.87, 56.17, 45.15. Elemental analysis (%) calcd. for
C34H29N3O5 (559.61): C 72.97, H 5.22, N 7.51; Found: C 72.91, H 5.25, N 7.53.
2c: Light yellow powder; Yield: 72 %; mp: 65–67 °C. IR (cm-1): 3144(br),
1672(w), 1607(s), 1568(s), 1509(w), 1455(w), 1329(w), 1281(w), 1208(s), 1176(m),
1
1127(m), 977(m), 850(m), 788(s), 691(s), 651(w), 538(w). H NMR (400 MHz,
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Design, synthesis, anticancer activity and cytotoxicity of…
DMSO): d 13.38 (s, 2H), 10.50 (s, 2H), 8.84 (s, 2H), 7.66 (s, 2H), 7.53 (t, J = 8 Hz,
2H), 7.46 (d, J = 8 Hz, 4H), 7.38 (t, J = 8 Hz, 4H), 6.43 (d, J = 8 Hz, 2H), 6.31
(s, 2H), 3.78 (s, 4H), 2.40 (s, 3H). 13C NMR (100 MHz, DMSO): d 186.38, 163.44,
162.95, 162.66, 148.70, 135.83, 134.56, 134.46, 134.12, 129.78, 127.77, 123.03,
122.09, 112.06, 107.99, 102.36, 56.26, 45.23. Elemental analysis (%) calcd. for
C34H29N3O5 (559.61): C 72.97, H 5.22, N 7.51; Found: C 72.92, H 5.23, N 7.56.
2d: Light yellow powder; Yield: 78 %; mp: 152–154 °C. IR (cm-1): IR:
3173(br), 1674(w), 1615(s), 1568(s), 1485(s), 1433(w), 1373(w), 1349(w), 1292(s),
1217(s), 1148(s), 874(m), 824(m), 787(s), 727(w), 681(s), 642(w), 615(w), 530(m).
1H NMR (400 MHz, DMSO): d 12.10 (s, 2H), 8.89 (s, 2H), 7.68 (s, 2H), 7.55 (t,
J = 8.0 Hz, 2H), 7.50 (s, 2H), 7.43 (t, J = 7.6 Hz, 4H), 7.07 (d, J = 4.0 Hz, 2H),
6.90 (dd, J = 7.6 Hz, 2H), 6.82 (d, J = 7.8 Hz, 2H), 3.79 (s, 4H), 2.41 (s, 3H). 13
C
NMR (100 MHz, DMSO): d 186.46, 163.90, 153.05, 149.67, 149.03, 135.85,
134.35, 134.23, 129.79, 128.30, 123.19, 122.35, 121.35, 119.30, 117.23, 116.85,
56.28, 45.25. Elemental analysis (%) calcd. for C34H29N3O5 (559.61): C 72.97, H
5.22, N 7.51; Found: C 72.90, H 5.25, N 7.54.
2e: Light yellow powder; Yield: 72 %; mp: 160–162 °C. IR (cm-1): 3061(br),
1672(m), 1613(m), 1578(m), 1516(w), 1452(w), 1276(w), 1207(m), 1169(m),
1082(m), 1057(w), 979(m), 935(w), 875(w), 771(w), 681(w), 606(w), 592(w),
573(m), 564(m), 553(m), 538(w), 532(w). 1H NMR (400 MHz, DMSO): d 13.47 (s,
2H), 9.79 (s, 2H), 8.83 (s, 2H), 8.54 (s, 2H), 7.68 (s, 2H), 7.54 (t, J = 8.0 Hz, 2H),
7.48 (s, 2H), 7.45–7.35 (m, 4H), 6.98 (d, J = 7.8 Hz, 2H), 6.46 (d, J = 7.8 Hz, 2H),
3.82 (s, 4H), 2.43 (s, 3H). 13C NMR (100 MHz, DMSO): d 193.04, 163.97, 151.30,
150.54, 148.33, 135.81, 132.39, 129.83, 127.83, 124.26, 123.04, 121.98, 112.33,
107.89, 56.17, 45.14. Elemental analysis (%) calcd. for C34H29N3O7 (591.61): C
69.03, H 4.94, N 7.10; Found: C 69.10, H 4.90, N 7.05.
Anticancer testing with MTT method
Compounds 1a–d and 2a–e were screened against human neoplastic cell lines such
as human cervical carcinoma cells (HeLa), human liver hepatocellular carcinoma
cell line (HePG2), human chronic myelogenous leukemia cell line (K562), human
acute mononuclear granulocyte leukemia (THP-1), and human normal heptical cell
line (LO2) (Table 1) using modified the MTT assay (3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2-H-tetrazolium bromide, MTT, Dojindo Laboratories, Tokyo, Japan).
The cells were seeded in a 96-well plate in 200 lL medium per well at a density of
19104 cells/well for 24 h. The cells were treated with serial concentrations of
compound and incubated for 24 h. Cells with only culture media were used as
control. After the media was removed, 20 lL of MTT (5 mg/mL) was added, then
the plates were incubated for 4 h at 37 °C in a cell culture incubator. The MTT
containing media were removed and then 150 lL of DMSO was added to dissolve
the dark-blue formazan crystals. The optical density (OD) was measured by a multi-
¨
well plate reader (TECAN, Mannedorf, Switzerland) at 570 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 in Table 1. The concentrations of the compounds were 10, 8, 5,
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Design, synthesis, anticancer activity and cytotoxicity of…
3, 2, 1, 0.5, 0.1, 0.05 and 0.01 lg/mL. Doxorubicin (DOX) was used as a positive
control. The concentrations of DOX used were 1.5, 1.2, 1.0, 0.8, 0.5, 0.3, 0.1, 0.05,
and 0.01 lg/mL.
Results and discussion
Structural analysis
As described in the experimental section, the intermediate (2E,6E)-2,6-bis(3-
nitrobenzylidene)cyclohexanone and (3E,5E)-1-methyl-3,5-bis(3-nitrobenzyli-
dene)piperidin-4-one were obtained via a Claisen-Schmidt condensation reaction
from m-nitrobenzaldehyde with cyclohexanone or N-methyl-4-piperidinone. They
were untreated and directly used to synthesize intermediate 3 and 4 by the reduction
reaction of stannous chloride (Scheme 1). Then, some aromatic aldehydes with
phenol substitution groups were introduced into a -NH2 fragment of 3 or 4 to
generate a serie of target compounds 1a–d and 2a–e (Fig. 2) through a Schiff-base
condensation reaction (Scheme 1), from which target compounds can be obtained
1
from precipitate in high yield. Their structures were confirmed by H NMR, 13C
NMR, IR and elemental analysis.
According to the 1H NMR spectra, the chemical shift 3.73 ppm in 3 and
3.76 ppm in 4 are attributed to the amino groups, respectively. After Schiff-base
condensation reaction, two of them disappear, while single bands in the range of
8.50–8.97 ppm appear and are attributed to the protons of –CH=N– groups. In target
compounds, the chemical shift of ortho-hydroxyl proton is in the range of
12.10–13.65 ppm. The single band of –C=CH– in the a,b-unsaturated ketone is
about 8.97–8.50 ppm, which proves the existence of 1,5-diphenylpenta-1,4-dien-3-
ones pharmacophore. In addition, the target compounds of 1a–d have the chemical
shift of methylene of that around ca. 2.96 and ca. 1.85 ppm, while compounds 2a–
e show the chemical shifts of N-methyl and methylene around d ca. 3.78 and ca.
2.40 ppm. From the FTIR spectra, the characteristic band at around
1667–1675 cm-1 are attributed to –C=O group in cyclohexanone or piperidone.
O
O
CHO
O
R
R
iii
i, ii
N
N
R
NO2
OH
R1
HO
R1
NH2
NH2
R3
R3
R2
R2
3 or 4
1a-d, 2a-e
Scheme 1 Synthesis of 1a–d and 2a–e. The reaction conditions as follows: i dry HCl, HAc, ii SnCl2/
HCl, CH3OH/H2O, iii aromatic aldehyde, formic acid, CH3OH
123

Q. Chen et al.
The strong bands at around 1619–1607 cm-1 are attributed to –C=C bonds of a,b-
unsaturated ketone and –C=N bonds of Schiff-base groups. Additionally, 13C NMR
and element analysis further confirm the correctness of their structures.
The solubility of the synthesized compounds 1a–d and 2a–e was evaluated by
dispersing the samples in different solvents at room temperature. They can be
soluble in many organic solvents, such as methanol, alcohol, chloroform, DMSO,
and DMF. In water, their solubility is very small. Only 1b, 2d, and 2e can reach
0.6–0.78 mg/mL (Table 1). Fortunately, all of them exhibit slightly improved
solubility in a 20 mM PBS buffer system (pH 7.4), and 1b, 2c, 2d, and 2e can reach
2.12, 1.23, 2.20, and 1.66 mg/mL (Table 1), respectively. Compared with 1a, the
solubility of 1b in the PBS buffer system is obviously improved because of the
joining of the adjacent hydroxyl group in 1b. Compared with 2a, the solubilities of
2b–e are also obviously improved in water and the PBS buffer system, which may
be caused by additional phenol substitution groups of 2b–e.
Anticancer activity and cytotoxicity
The anticancer activities against human carcinoma cell lines HePG2, HeLa, K562,
THP-1, and their cytotoxicities toward LO2 cell lines were evaluated by the MTT
method. Doxorubicin (DOX) was selected as positive control. The results of the
biological activity evaluation can be found in Table 1. The results suggest that about
80 % of the IC50 values of the compounds against HeLa, HePG2, K562, and THP-1
are lower than 5 lM.
For series 1, their IC50 values against Hepg2 and Hela cells are larger, which can
exceed 5.0 lM, except 1a and 1b against Hepg2. But for K562, THP-1, the
anticancer activity of series 1 is significantly better and their IC50 values are lower
than 5.0 lM, especially for 1a, its IC50 value is 1.47 lM. More importantly,
cytotoxicity of series 1 is very small. Their IC50 values toward the LO2 cell line are
in the range of 6.94–9.39 lM, which has cytotoxicity lower than that of DOX
(6.93 lM), except 1b.
For series 2, 87.5 % of the compounds are more active than the analogs in series
1 against HeLa, HePG2, K562, and THP-1 (Table 1). For example, for HeLa,
HePG2, and THP-1, all of the compounds in series 2 have the lower IC50 values
than that of series 1 with the same aryl substitution pattern, respectively. Only for
K562, the anticancer activities of 2b and 2c weaken compared with 1b and 1c,
respectively. In addition, 85 % of the compounds in series 2 show lower IC50 values
(\3 lM) against experimental human carcinoma cell lines. kWhat is more, target
compounds 2a, 2c–e against THP-1 cells are the best compared with other
experimental human carcinoma cell lines. Especially, the IC50 values of compounds
2a, 2d, and 2e against THP-1 cells can reach 0.69–0.96 lM. Compounds 2b and 2c
against THP-1 cells have small IC50 values (1.41 and 1.04 lM) compared with 1b
and 1c. For Hepg2, compound 2e displays the best anticancer activity with the IC50
value 0.92 lM, while 2b and 2d also have the lower IC50 value (1.32 lM for 2b and
1.04 lM for 2d). For Hela and K562, the IC50 values can be below 2.0 lM for 2d,
2e toward Hela and 2e toward K562, respectively. According to these analyses,
compounds 2d and 2e display the best anticancer activities against HeLa, HePG2,
123

Design, synthesis, anticancer activity and cytotoxicity of…
K562, and THP-1. For the LO2 cell line, their cytotoxicity of series 2 is higher than
that of DOX and series 1 with the same aryl substitution pattern, respectively. Their
IC50 values are in the range of 2.32–4.81 lM. This shows that the compounds in
series 2 have great cytotoxicity.
In general, series 2 have better anticancer activity than series 1 against
experimental human carcinoma cell lines. However, the cytotoxicity of series 2 is
slightly greater than that of series 1 and DOX. Structure analysis shows that series 1
are 2,6-bis(3-benzylidene)cyclohexanone derivatives, while series 2 are 3,5-bis(3-
benzylidene)-1-methylpiperidin-4-one derivatives; their anticancer activity and
cytotoxicity are very different in spite of the same phenol substitution groups. The
results show the N-methyl-4-piperidinone group is more beneficial for anticancer
activity than the cyclohexanone group [21]. In addition, the introduction of second
or third hydroxy through Schiff-base condensation reaction is helpful for improving
their anticancer activity for 4-piperidinone derivatives, especially 2d and 2e.
Conclusions
In this study, some aromatic aldehydes with phenol substitution groups were
introduced into the -NH2 fragment of 3 or 4 to generate two series of novel double
Schiff-base substituted 4-piperidone/cyclohexanone derivatives (1a–d, 2a–e). Their
1
structures were characterized by H NMR, 13C NMR, IR, and elemental analysis.
The anticancer activities against human carcinoma cell lines HePG2, HeLa, K562,
THP-1, and their cytotoxicities for LO2 cell lines were subsequently evaluated by
the MTT method. The results show that 2a–e have better anticancer activity than
1a–d against experimental cell lines. But the cytotoxicities of 2a–e are slightly
greater than that of 1a–d and DOX. Structure analysis shows that N-methyl-4-
piperidinone group and the introduction of hydroxy is helpful for improving their
anticancer activity, especially 2d and 2e.
Acknowledgments We are grateful for financial support from the National Natural Science Foundation
of China (No. 21402010), the Foundation of Shandong province (Nos. ZR2013BM022, ZR2014HP006,
ZR2014BL008, 2014CGZH1316, 2015GGX102013), and the Foundation of Shandong Provincial Key
Laboratory of Clean Production of Fine Chemicals (No. ZDSYS-KF201505).
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