Synthesis and evaluation of curcumin-related compounds containing benzyl piperidone for their effects on human cancer cells

Article abstract of DOI:10.1248/cpb.c13-00507

Eleven curcumin-related compounds containing a benzyl piperidone moiety were synthesized and evaluated for their effects on cultured prostate cancer PC-3 cells, pancreas cancer BxPC-3 cells, colon cancer HT-29 cells and lung cancer H1299 cells. Inhibitory effects of these compounds on the growth of PC-3, BxPC-3, HT-29 and H1299 cells were determined by the 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue exclusion assay. Compounds benzyl piperidone 2 (P2), P4, P7, 4-bromo-2-fluoro-benzyl piperidone 2 (PFBr2), PFBr3 and PFBr4 (see syntheses and structures in Figs. 1, 2) exhibited potent inhibitory effects on the growth of cultured PC-3, BxPC-3, HT-29 and H1299 cells. The IC₅₀ for these compounds was lower than 2 μM in all four cell lines. PFBr4 was 41-, 36-, 40- and 46-fold more active than curcumin for inhibiting the growth of PC-3, BxPC-3, HT-29 and H1299 cells, respectively. The benzyl piperidone-containing compounds studied also stimulated apoptosis in PC-3 cells. Mechanistic studies indicate that the effects of both curcumin and PFBr4 on PC-3 cells were associated with a decrease in phospho-Akt and phospho-extracellular signal-regulated kinase (Erk)1/2. The present study indicates that P2, P4, P7, PFBr2, PFBr3 and PFBr4 may have useful effects on human cancer cells.

Full text of DOI:10.1248/cpb.c13-00507

November 2013
Regular Article
Chem. Pharm. Bull. 61(11) 1149–1155 (2013)
1149
Synthesis and Evaluation of Curcumin-Related Compounds Containing
Benzyl Piperidone for Their Effects on Human Cancer Cells
Dai-Ying Zhou,a,b Kun Zhang,a Allan H. Conney,a,c Ning Ding,c Xiao-Xing Cui,c Hui Wang,a
,a,c
,a
Michael Verano,c Su-qing Zhao,a Yan-Xiong Fan,a Xi Zheng,* and Zhi-Yun Du*
a Allan H Conney Laboratory for Research, Institute of Natural Medicinal Chemistry & Green Chemistry, Guangdong
b
University of Technology; Guangzhou 510006, China: Guangdong Food and Drug Vocational College; Guangzhou
c
512520, China: and Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology,
Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey; Piscataway, NJ 08854, U.S.A.
Received June 24, 2013; accepted August 14, 2013; advance publication released online August 27, 2013
Eleven curcumin-related compounds containing a benzyl piperidone moiety were synthesized and
evaluated for their effects on cultured prostate cancer PC-3 cells, pancreas cancer BxPC-3 cells, colon can-
cer HT-29 cells and lung cancer H1299 cells. Inhibitory effects of these compounds on the growth of PC-3,
BxPC-3, HT-29 and H1299 cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) assay and trypan blue exclusion assay. Compounds benzyl piperidone 2 (P2), P4, P7,
4-bromo-2-fluoro-benzyl piperidone 2 (PFBr2), PFBr3 and PFBr4 (see syntheses and structures in Figs. 1,
2) exhibited potent inhibitory effects on the growth of cultured PC-3, BxPC-3, HT-29 and H1299 cells. The
IC50 for these compounds was lower than 2µM in all four cell lines. PFBr4 was 41-, 36-, 40- and 46-fold more
active than curcumin for inhibiting the growth of PC-3, BxPC-3, HT-29 and H1299 cells, respectively. The
benzyl piperidone-containing compounds studied also stimulated apoptosis in PC-3 cells. Mechanistic stud-
ies indicate that the effects of both curcumin and PFBr4 on PC-3 cells were associated with a decrease in
phospho-Akt and phospho-extracellular signal-regulated kinase (Erk)1/2. The present study indicates that
P2, P4, P7, PFBr2, PFBr3 and PFBr4 may have useful effects on human cancer cells.
Key words cancer cell; benzyl piperidone; curcumin-related compound; apoptosis
Curcumin is a yellow compound isolated from the dried rhi- ing the growth of PC-3, BxPC-3, HT-29 and H1299 cells and
zomes of the plant Curcuma longa, and is used for traditional for stimulating apoptosis in PC-3 cells. We also discuss the
medicine in India and China.1) Numerous studies have shown structure–activity relationship of curcumin-related compounds
that curcumin possesses multifunctional pharmacological with a benzyl piperidone.
properties including anti-oxidant activity,2–4) anti-inflammato-
ry activity5–7) and activity.8–12) Its activity is related in part to
Results and Discussion
its ability to induce apoptosis in cancer cells. Because of its
Chemistry A series of curcumin-related compounds con-
and antiangiogenic properties, low molecular weight and lack taining benzyl piperidone were synthesized by coupling the
of toxicity, curcumin is under study as a possible new drug.13) appropriate substituted benzaldehyde with P or PFBr (Fig. 1).
Curcumin has been evaluated in clinical trials for the The synthesis and characterization of PFBr1, PFBr2, PFBr3
treatment of liver disease, rheumatoid arthritis, infectious and PFBr4 were not previously reported. The synthesis and
diseases and cancer. Despite its safety, the clinical usefulness characterization of P1 to P7 are known, but no report on the
of curcumin is diminished by extensive first-pass metabolism, activity on these compounds was found. Structures of cur-
resulting in low oral bioavailability.14–16) In the last decade cumin and curcumin-related compounds containing a benzyl
curcumin has been much explored and various synthetic piperidone moiety are shown in Fig. 2.
analogues have been prepared and evaluated for various phar-
Inhibitory Effects of Curcumin-Related Compounds
macological activities.17–26) Some analogues have shown good Containing Benzyl Piperidone toward Cultured Human
activities in various models and various cell lines. A recent Prostate, Pancreas, Colon and Lung Cancer Cells The
study demonstrated that curcumin-related compounds with inhibitory effects of eleven curcumin-related compounds con-
a benzyl piperidone had enhanced absorption and biological taining benzyl piperidone on the growth of cultured prostate
activities.27,28) The IC50 of these compounds in different cancer cancer PC-3 cells, pancreas cancer BxPC-3 cells, colon cancer
cell lines ranged from 5.5–8.3µM.
HT-29 cells and lung cancer cells H1299 were determined by
In the present study, eleven curcumin-related compounds using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
using benzyl piperidone (P) or 4-bromo-2-fluoro-benzyl piper- bromide (MTT) assay and the trypan blue exclusion assay.
idone (PFBr) as a linker and various substituents on the aryl For the experiments using the MTT assay, curcumin was
rings (Fig. 1) were synthesized and evaluated for their effects evaluated as a positive control in each incubation. The inhibi-
against four human cancer cell lines. PFBr1, PFBr2, PFBr3 tory effect of curcumin did not vary significantly between the
and PFBr4 are novel compounds with fluorine and bromine. different incubations. Data from all curcumin incubations in
P1 to P7 are known compounds.27–32) The curcumin-related experiments with group P and group PFBr compounds were
compounds containing benzyl piperidone were evaluated in averaged and presented in Table 1.
the present study and they showed a potent effect for inhibit-
All compounds had stronger inhibitory effects than cur-
cumin as determined by the MTT assay. Among the eleven
curcumin-related compounds tested, PFBr4 exhibited excep-
The authors declare no conflict of interest.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: xizheng@rci.rutgers.edu;
zhiyundu@yahoo.com.cn

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Vol. 61, No. 11
Fig. 1. Synthesis of Group P and Group PFBr Compounds
tionally potent inhibitory effects on the growth of cultured cytotoxic effects between our compounds and those reported
PC-3, BxPC-3, HT-29 and H1299 cells. The IC50 for this in the earlier study may be due to different cell lines used in
compound was ≤0.5µM in all four cell lines, indicating that each study. A previous study also indicated that piperidone
PFBr4 was approximately 40-fold more active than curcumin. enhanced the absorption of curcumin.28) Although we did
As shown in Table 1, the IC50 values of all eleven curcumin- not monitor the uptake of the curcumin-related compounds
related compounds ranged from 0.41 to 17.67µM in the four containing benzyl piperidone in the present study, it is pos-
cell lines studied.
sible that enhancement of absorption contribute to increased
Because the achievable concentration of curcumin and cur- activities of these compounds. Further studies are needed to
cumin-related compounds in vivo may be low, we further de- determine the cellular uptake of these compounds.
termined the effects of these compounds at 1µM on the grown
To further determine the mechanisms for growth inhibition
of PC-3 cells by using the trypan blue exclusion assay. As and apoptosis induction in PC-3 cells, we used a nuclear fac-
shown in Table 2, compound-related decreases in the number tor kappa B (NF-κB)-luciferase reporter gene expression assay
of viable cells were observed. Compared to the control group, to determine the effect of curcumin-related compounds (IC50
the number of viable cells decreased by 14.7 to 99.3% when <2µM) on activation of NF-κB. In these experiments, VCaP/N
the cells were treated with curcumin and curcumin-related cells were treated with a low concentration (1µM) of cur-
compounds (Table 2). The effects of curcumin and curcumin- cumin and curcumin-related compounds for 24h. Treatment of
related compounds at 1µM on apoptosis of PC-3 cells were VCaP/N cells with group PFBr4 compounds resulted in strong
determined by morphological assessment of propidium iodide decreases in NF-κB transcriptional activity (Fig. 4). Com-
stained cells. In these experiments, PC-3 cells were treated pounds P2, P4, P7, PFBr2 and PFBr3 had moderate effects for
with curcumin and curcumin-related compounds for 96h. As decreasing NF-κB transcriptional activity while curcumin had
shown in Fig. 3, weak stimulatory effects on the induction of no effect (Fig. 4).
apoptosis were observed in PC-3 cells treated with curcumin
The levels of activated Akt and extracellular signal-regu-
(1 µM), while much more obvious effects were seen in PC-3 lated kinase (Erk)1/2 were evaluated by Western blot analysis
cells treated with PFBr4 (1µM). An earlier study showed that using anti-phosphorylated Akt and anti-phosphorylated Erk1/2
curcumin-related compounds with a benzyl piperidone had en- antibodies (Fig. 5). In these experiments, PC-3 cells were
hanced cytotoxic potential in colon, pancreas, lung and renal treated with curcumin (1, 20µM) or with PFBr4 (0.5, 1µM) for
cancer cells.27) In the present study, we found that compounds 24h and analyzed by Western blotting. The levels of phos-
with similar structure had even stronger cytotoxic effects in phorylated Akt and Erk1/2 in Western blots were analyzed by
prostate, pancreas, colon and lung cancer cells. Differences in optical density measurements and normalized for β-actin. As

November 2013
1151
Fig. 2. Structures of Curcumin and Curcumin-Related Compounds
shown in Fig. 5, treatment of PC-3 cells with PFBr4 resulted activity.33) In the present study, we showed that using benzyl
in a strong decrease in the level of phosphorylated Erk1/2 piperidone as a linker enhanced the cytotoxic potential of the
while curcumin was inactive. Treatment with curcumin and curcumin-related compounds. We also found that PFBr3 and
PFBr4 all caused a decrease in the level of phosphorylated PFBr4 had huge increases in activity as compared to P3 and
Akt. Our results indicate that the effects of both curcumin P4, indicating that the fluorine group in the benzyl piperidone
and PFBr4 on PC-3 cells were associated with a decrease in linker conferred improvement in activity in these compounds.
phospho-Akt and phospho-Erk1/2.
Structure–activity relationship analysis for the aromatic rings
Structure–Activity Relationship Earlier studies on showed that reactive groups such as methoxy can enhance
the analysis of the relationship between the structures of activity. P4 and PFBr4 showed some increases in activities
curcumin-related compounds and their ability to inhibit the over the corresponding P1 and PFBr1. P5 had slightly less
growth of cultured cancer cells showed that the linker, the activity than the corresponding P7. For the same linker and
aromatic rings and steric hinderance are all very important for same aromatic rings, such as P3-P4 and PFBr3-PFBr4, steric

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Vol. 61, No. 11
Table 1. Inhibitory Effects of Curcumin-Related Compounds Containing Table 2. Effects of Curcumin-Related Compounds Containing Benzyl
Benzyl Piperidone on the Growth of PC-3, BxPC-3, HT-29 and H1299
Cells
Piperidone on the Growth of PC-3 Cells
No. of viable cells
Treatment
% growth inhibition
IC50 (µM)
(1×10−4
)
Compound
PC-3
BxPC-3
HT-29
H1299
Control
Curcumin
P1
44.2 2.7
37.7 2.4
27.8 1.7
13.2 1.5
28.9 2.1
14.5 1.2
28.7 2.3
26.2 2.0
24.9 1.8
34.3 2.5
10.6 1.2
13.9 1.4
0.3 0.1
—
14.7
37.1
70.1
34.6
67.2
35.1
40.7
43.7
22.4
76.0
68.6
99.3
Curcumin 19.98 2.4
18.25 2.2
6.67 0.9
0.91 0.1
8.93 1.2
1.13 0.2
2.64 0.3
1.97 0.3
1.09 0.2
15.59 2.4
1.70 0.2
1.51 0.2
0.50 0.1
18.74 2.2
7.95 1.1
0.86 0.1
9.16 1.2
1.37 0.3
4.97 0.6
2.79 0.4
1.55 0.2
16.43 2.1
2.38 0.4
1.63 0.3
0.47 0.1
18.93 2.1
5.61 0.7
1.05 0.2
10.95 1.6
1.45 0.2
1.58 0.2
2.01 0.3
1.31 0.2
17.23 2.7
2.16 0.3
2.59 0.4
0.41 0.1
P1
P2
9.13 1.3
1.35 0.2
10.18 2.1
1.75 0.2
5.05 0.7
4.12 0.6
1.82 0.3
17.67 2.6
1.78 0.2
1.75 0.2
0.49 0.1
P2
P3
P3
P4
P4
P5
P5
P6
P6
P7
P7
PFBr1
PFBr2
PFBr3
PFBr4
PFBr1
PFBr2
PFBr3
PFBr4
Human prostate cancer PC-3 cells were seeded at a density of 0.2×105 cells/mL in
35-mm tissue culture dishes and incubated for 24h. The cells were then treated with
curcumin and curcumin-related compounds (all at 1µM) for 96h. The number of vi-
able cells was determined by the trypan blue exclusion assay. Each value represents
the mean S.E. from three separate experiments.
Human prostate cancer PC-3, pancreas cancer BxPC-3, colon cancer HT-29 and
lung cancer cells H1299 were seeded at a density of 0.2×105 cells/mL of medium
in 96-well plates (0.2mL/well) and incubated for 24h. The cells were then treated
with various concentrations (0.1–30µM) of the different compounds for 72h. Effects
of the different compounds on the growth of PC-3, BxPC-3, HT-29 and H1299 cells
were determined by the MTT assay. Each value is the mean S.E. from three separate
experiments.
ArH), 7.32–7.29 (m, 5H, ArH), 6.92 (d, J=8.0Hz, 4H, ArH),
hinderance is very important for activity; P4 had increased 3.87 (s, 4H, –CH2–N–CH2–), 3.84 (s, 6H, –OCH3), 3.75 (s, 2H,
activity as compared to P3, and PFBr3 was slightly less active –CH2–). 13C-NMR (CDCl3, 100MHz) δ (ppm): 54.5, 55.3, 61.5,
than PFBr4.
114.0, 127.3, 128.0, 128.3, 129.0, 131.5, 132.2, 136.0, 137.5,
160.2, 187.6. ESI-MS (m/z): 426 (M+1)+. C28H27NO3: 425.
1-Benzyl-3,5-di(3,4-dimethoxybenzene methylene)piperi-
Experimental
1
General Procedures Melting points were determined din-4-one (P2): Yield 92%. mp 168–170°C. H-NMR (CDCl3,
on a Yanagimoto micro melting apparatus and were uncor- 400MHz) δ (ppm): 7.75 (s, 2H, –CH=), 7.27–7.22 (m, 5H,
rected. The 1H-NMR spectra were measured on a Varian ArH), 6.96 (d, J=8.1Hz, 2H, ArH), 6.86 (s, 2H, ArH), 6.84
Gemini-2000 spectrometer using dimethyl sulfoxide (DMSO) (d, J=8.9Hz, 2H, ArH), 3.87 (s, 12H, –OCH3), 3.85 (s, 4H,
as solvent unless otherwise specified. Chemical shifts for –N–CH2–), 3.73 (s, 2H, –CH2–). 13C-NMR (CDCl3, 100MHz)
1H-NMR (300MHz) and 13C-NMR (75MHz) were expressed δ (ppm): 54.4, 55.8, 55.9, 61.8, 111.0, 113.4, 124.2, 127.4, 128.2,
in ppm units with tetramethylsilane (TMS) as an internal 128.3, 129.0, 131.6, 136.2, 137.3, 148.7, 179.9, 187.3. ESI-MS
standard. Multiplicities were recorded as s (singlet), brs (broad (m/z): 486 (M+1)+. C30H31NO5: 485.
singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).
1-Benzyl-3,5-di(2,3,4-trimethoxybenzaldehyde methylene)-
Mass spectra were obtained on an LC-MS-2010A spectrometer piperidin-4-one (P3): Yield 89%. mp 166–168°C. 1H-NMR
with electrospray ionization (ESI). Elemental analyses were (CDCl3, 400MHz) δ (ppm): 8.02 (s, 2H, –CH=), 7.22 (d,
performed on an elemental analyser (Vario El). Thin-layer J=8.5Hz, 5H, ArH), 6.87 (d, J=8.7Hz, 2H, ArH), 6.64 (d,
chromatography (TLC) was performed on Merck silica gel J=8.7Hz, 2H, ArH), 3.89 (s, 18H, –OCH3), 3.80 (s, 4H, –
plates (DC-60 F254). Curcumin was isolated from the extract CH2–N), 3.68 (s, 2H, –CH2–). 13C-NMR (CDCl3, 100MHz) δ
of Curcuma longa LINN according to a previous report.34) All (ppm): 54.4, 56.0, 60.8, 60.9, 61.5, 106.8, 122.4, 125.1, 127.1,
reagents (highest grade) were used as received unless other- 128.2, 128.9, 132.0, 132.4, 137.6, 142.3, 153.5, 154.6, 187.5.
wise stated.
ESI-MS (m/z): 546 (M+1)+. C32H35NO7: 545.
Synthesis of Curcumin Analogues A total of 11 cur-
1-Benzyl-3,5-di(3,4,5-trimethoxybenzaldehyde methylene)-
cumin-related compounds containing benzyl piperidone were piperidin-4-one (P4): Yield 78%. mp 140–142°C. 1H-NMR
synthesized as previously described with modification.34,35)
A
(CDCl3, 400MHz) δ (ppm): 7.64 (s, 2H, –CH=), 7.23–7.27
mixture of the appropriate aldehyde (0.01mol) and the ketone (m, 5H, ArH), 6.50 (s, 4H, ArH), 3.86 (s, 18H, –OCH3), 3.84
(0.005mol) was dissolved in glacial acetic acid saturated with (s, 4H, –CH2–N–), 3.80 (s, 2H, –CH2–). 13C-NMR (CDCl3,
anhydrous hydrogen chloride and heated in a water bath at 100 MHz) δ (ppm): 54.2, 56.0, 60.8, 61.8, 103.6, 107.8, 127.5,
25–30°C for 2h. After standing for 2d, the mixture was treat- 128.3, 129.1, 130.6, 132.5, 136.5, 136.8, 139.0, 152.9, 153.1,
ed with cold water and filtered. The solid obtained was then 187.0. ESI-MS (m/z): 546 (M+1)+. C32H35NO7: 545.
washed and dried. The crude product was recrystallized from
1-Benzyl-3,5-bis(4-dihydroxyphenyl methylene)piperidin-4-
1
appropriate solvents (methanol or ethanol). PFBr1 to PFBr4 one (P5): Yield 53%. mp 200–202°C. H-NMR (DMSO-d6,
are novel compounds and were not reported. P1 to P7 were 400MHz) δ (ppm): 9.87 (s, 2H, Ar-OH), 7.81 (s, 2H, –CH=),
known compounds.
7.65 (d, J=8.0Hz, 2H, 2×ArH), 7.37 (d, J=8.0Hz, 2H, ArH),
1-Benzyl-3,5-bis(4-methoxyphenyl methylene)piperidin-4- 7.32–7.37 (m, 5H, ArH), 6.88 (m, 4H, ArH), 4.55 (s, 6H,
one (P1): Yield 90%. mp 148–150°C. 1H-NMR (CDCl3, –CH2–N–). ESI-MS (m/z): 398 (M+1)+. C26H23NO3: 397.
400MHz) δ (ppm): 7.80 (s, 2H, CH=), 7.34 (d, J=8.0Hz, 4H,
1-Benzyl-3,5-di(3,4-dihydroxyphenyl methylene)piperidin-

November 2013
1153
Fig. 3. Effects of Curcumin-Related Compounds Containing Benzyl Piperidone on Apoptosis of PC-3 Cells
Human prostate cancer PC-3 cells were seeded at a density of 0.2×105 cells/mL in 35-mm tissue culture dishes and incubated for 24h. The cells were then treated with
curcumin and curcumin-related compounds (all at 1µM) for 96h. (A,B) Representative micrographs of propidium iodide-stained controls and PFBr4 treated PC-3 cells. (C)
Percentage of apoptotic cells as determined by morphological assessment in PC-3 cells treated with curcumin and curcumin-related compounds. (D) Caspase-3 activities in
PC-3 cells treated with curcumin and curcumin-related compounds. Each value represents the mean S.E. from three separate experiments.
Fig. 4. Inhibitory Effects of Curcumin-Related Compounds Containing
Benzyl Piperidone on NF-κB Transcriptional Activity in VCaP/N Cells
VCaP/N cells were seeded at a density of 0.2×105 cells/mL of medium in 24-well
plates (1.0mL/well) and incubated for 24h. The cells were then treated with 1µM
of the different compounds for 24h. The NF-κB transcriptional activity was deter-
mined by the luciferase reporter assay. Each value represents the mean S.E. from
three separate experiments.
Fig. 5. Effects of Curcumin and PFBr4 on the Activation of Akt and
Erk1/2 in PC-3 Cells
PC-3 cells were seeded at a density of 1×105 cells/mL of medium in 100mm
culture dishes (10mL/dish) and incubated for 24h. The cells were then treated with
DMSO (final concentration 0.01%) curcumin (CUR; 1, 20µM in DMSO final con-
centration 0.01%), PFBr4 (0.5, 1µM in DMSO final concentration 0.01%) for 24h.
Activated Akt and Erk1/2 were determined by using Western blot analysis with
anti-phosphorylated-Akt or anti-phosphorylated-Erk1/2 antibody. The extent of
protein loading was determined by blotting for β-actin and the levels of phosphory-
lated Akt and Erk1/2 in Western blots were analyzed by optical density measure-
ments and normalized for β-actin to obtain the relative optical densities.

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Vol. 61, No. 11
1
4-one (P6): Yield 51%. mp 232–234°C. H-NMR (DMSO-d6, H1299 cells were seeded at a density of 0.2×105 cells/mL
400MHz) δ (ppm): 7.73 (s, 2H, –CH=), 7.56 (d, J=6.4, 2H, of medium in 96-well plate (0.2mL/well) and incubated for
ArH), 7.40–7.29 (m, 3H, ArH), 6.89 (m, 4H, ArH), 6.80 (d, 24h. The cells were then treated with various concentrations
2H, J=8.4Hz, ArH), 4.52 (s, 6H, –CH2–N). ESI-MS (m/z): 430 (0.2–20µM) of curcumin-related compounds containing benzyl
(M+1)+. C26H23NO5: 429.
piperidone for 72h. After treatment, 3-[4,5-dimethylthiazol-
1-Benzyl-3,5-bis(4-hydroxy-3-methoxybenzoate methylene)- 2-yl]-2,5-diphenyltetrazoliumbromide was added to each well
piperidin-4-one (P7): Yield 66%. mp 210–212°C. 1H-NMR of the plate and incubated for 1h. After careful removal of
(DMSO-d6, 400MHz) δ (ppm): 9.87 (m, 2H, Ar-OH), 7.81 (s, the medium, 0.1mL DMSO was added to each well, and ab-
2H, ArH), 7.58 (s, 2H, ArH), 7.39 (d, J=4.1, 3H, ArH), 7.04 sorbance at 550nm was recorded on a microplate reader. The
(m, 6H, ArH), 4.58 (s, 6H, –CH2–N–), 3.83 (s, 4H, –OCH3). number of viable cells after each treatment was determined
ESI-MS (m/z): 458 (M+1)+. C26H23NO3: 457.
using a hemacytometer under a light microscope (Nikon Op-
1-(4-Bromo-2-fluoro-benzyl)-3,5-bis(4-methoxybenzene tiphot, Japan). Cell viability was determined by the trypan
methylene)piperidin-4-one (PFBr1): Yield 91%. mp 170–172°C. blue exclusion assay, which was done by mixing 80µL of cell
1H-NMR (CDCl3, 400MHz) δ (ppm): 7.80 (s, 2H, –CH=), suspension and 20µL of 0.4% trypan blue stain solution for
7.34 (d, J=8.5Hz, 4H, ArH), 7.22–7.17 (m, 3H, ArH), 6.94 2min. Blue cells were counted as dead cells and the cells that
(d, J=8.6Hz, 4H, ArH), 3.88 (s, 4H, –CH2–N–), 3.86 (s, 6H, did not absorb dye were counted as live cells.
–OCH3), 3.76 (s, 2H, –CH2–). 13C-NMR (CDCl3, 100MHz) δ
Assessment of Apoptotic Cells by Morphology and Ac-
(ppm): 53.5, 54.4, 55.5, 111.1, 118.8, 119.0, 121.3, 123.5, 127.4, tivation of Caspase-3 Apoptotic cells were determined by
127.8, 131.1, 132.3, 136.3, 160.3, 162.2, 187.2. ESI-MS (m/z): morphological assessment in cells stained with propidium
523 (M+1)+. C28H25BrFNO3: 522.
iodide. Cytospin slides were prepared after each experiment
1-(4-Bromo-2-fluoro-benzyl)-3,5-bis(3,4-methoxyben- and cells were fixed with acetone–methanol (1:1) for 10min
zenemethylene)piperidin-4-one (PFBr2): Yield 93%. mp at room temperature, followed by 10min with propidium io-
1
211–213°C. H-NMR (CDCl3, 400MHz) δ (ppm): 7.74 (s, 2H, dide staining (1µg/mL in phosphate buffered saline (PBS))
–CH=), 7.17 (s, 3H, ArH), 6.92 (d, J=16.0Hz, 6H, ArH), 3.91 and analyzed using a fluorescence microscope (Nikon Eclipse
(s, 4H, –CH2–N), 3.87 (s, 12H, –OCH3), 3.76 (s, 2H, –CH2–). TE200, Japan). Apoptotic cells were identified by classical
13C-NMR (CDCl3, 100MHz) δ (ppm): 53.6, 54.3, 55.8, 55.9, morphological features including nuclear condensation, cell
111.0, 115.5, 118.7, 121.4, 123.0, 124.1, 127.4, 128.1, 131.2, shrinkage, and formation of apoptotic bodies.36)
132.5, 136.5, 148.8, 150.0, 162.2, 186.8. ESI-MS (m/z): 583
Caspase-3 activation was measured using an EnzoLyte
AMC Caspase-3 Assay Fluorimetric kit (AnaSpec, Fremont,
(M+1)+. C30H29BrFNO5: 582.
1-(4-Bromo-2-fluoro-benzyl)-3,5-bis(2,3,4-methoxyben- CA, U.S.A.) according to the manufacturer’s instructions.37)
zene methylene)piperidin-4-one (PFBr3): Yield 82%. mp A total of 0.1×105 cells were plated in triplicate in a flat-
151–153°C. 1H-NMR (CDCl3, 400MHz) δ (ppm): 8.01 (s, bottomed 96-well plate. Cells were treated with different
2H, –CH=), 7.16–7.12 (m, 3H, ArH), 6.87 (d, J=8.5Hz, 2H, curcumin-related compounds containing benzyl piperidone
ArH), 6.66 (d, J=8.6Hz, 2H, ArH), 3.90 (s, 18H, –OCH3), for 72h. Following treatment, caspase-3 substrate was added
3.79 (s, 4H, –CH2–N–CH2–), 3.69 (s, 2H, –CH2–). 13C-NMR to each well. Plates were incubated at room temperature for
(CDCl3, 100MHz) δ (ppm): 52.9, 54.4, 56.0, 60.9, 61.5, 106.8, 30min. Fluorescence intensity was measured in a Tecan Inifi-
118.6, 118.9, 122.2, 123.6, 123.7, 125.1, 127.2, 132.1, 132.2, nite M200 plate reader (Tecan US Inc., Durham, NC, U.S.A.).
142.3, 153.5, 154.8, 159.7, 187.0. ESI-MS (m/z): 643 (M+1)+.
C32H33BrFNO7: 642.
NF-κB-Dependent Reporter Gene Expression Assay An
NF-κB luciferase construct (#CLS-013L, SABiosciences, CA,
1-(4-Bromo-2-fluoro-benzyl)-3,5-bis(3,4,5-methoxyben- U.S.A.) was stably transfected into VCaP/N cells and a single
zene methylene)piperidin-4-one (PFBr4): Yield 83%. mp stable clone, VCaP/N was obtained and used in the present
169–172°C. 1H-NMR (CDCl3, 400MHz) δ (ppm): 7.70 (s, study. In brief, VCaP/N cells were treated with different cur-
2H, –CH=), 7.18 (s, 3H, ArH), 6.59 (s, 4H, ArH), 3.88 (s, cumin analogues for 24h, and the NF-κB-luciferase activities
4H, –CH2–N–CH2–), 3.87 (s, 18H, –OCH3), 3.78 (s, 2H, – were measured using the luciferase assay kits from Promega
CH2–). 13C-NMR (CDCl3, 100MHz) δ (ppm): 53.8, 54.2, 56.1, (Madison, WI, U.S.A.) as described previously.
60.9, 107.8, 118.8, 119.0, 122.5, 122.6, 127.5, 130.5, 132.2,
136.6, 139.1, 153.1, 159.8, 186.7. ESI-MS (m/z): 643 (M+1)+. PFBr4 for 24h, PC-3 cells were washed with ice-cold PBS
C32H33BrFNO7: 642.
and lysed with 800µL of lysis buffer (10mM Tris–HCl, pH
Western Blot Analysis After treatment with curcumin,
Cell Culture and Reagents PC-3, BxPC-3, HT-29 and 8.0, 10mM ethylenediamine tetraacetic acid (EDTA), 150mM
H1299 cells were obtained from the American Type Culture sodium chloride, 1% NP-40, 0.5% sodium dodecyl sulfate
Collection (ATCC, Rockville, MD, U.S.A.). RPMI-1640 tis- (SDS), in deionized water). The homogenates were centrifuged
sue culture medium, penicillin–streptomycin, L-glutamine and at 12000×g for 15min at 4°C. The protein concentration of
fetal bovine serum (FBS) were from Gibco (Grand Island, whole cell lysates was determined with a Bio-Rad protein
NY, U.S.A.). The cells were maintained in RPMI-1640 culture assay kit (Bio-Rad, Hercules, CA, U.S.A.). Equal amounts
medium, which were supplemented with 10% FBS, penicil- (50 µg) of protein were then resolved on a 10% Criterion
lin (100units/mL)–streptomycin (100µg/mL) and L-glutamine Precast Gel (Bio-Rad, Hercules) and transferred to a poly-
(300 µg/mL). Cultured cells were grown at 37°C in a humidi- vinylidene difluoride (PVDF) membrane using a semi-dry
fied atmosphere of 5% CO2 and were passaged twice a week. transfer system. The membrane was then probed with with
Curcumin-related compounds were dissolved in DMSO and anti-phosphorylated Erk1/2 (#4376, Cell Signaling Technology,
the final concentration of DMSO in all experiments was 0.1%. U.S.A.) primary antibody. After hybridization with primary
MTT, Trypan Blue Assays PC-3, BxPC-3, HT-29 and antibody the membrane was washed with Tris-buffered saline

November 2013
1155
17) Adams B. K., Ferstl E. M., Davis M. C., Herold M., Kurtkaya S.,
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three times, then incubated with horseradish peroxidase-
conjugated secondary antibody (Santa Cruz Biotechnology,
Santa Cruz, CA, U.S.A.) and washed with Tris-buffered saline
three times. Final detection was performed with enhanced
chemiluminescent reagents. The extent of protein loading
was determined by blotting for β-actin. The membrane was
incubated in stripping buffer (100mM β-mercaptoethanol, 2%
SDS, and 62.5mM Tris–HCl at pH 6.7) at 50°C for 30min
with occasional agitation before incubating in blocking buffer
and re-probing using anti-β-actin (Santa Cruz Biotechnology).
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Acknowledgments The present study was supported by
the 2011 Guangdong Province Leadership Grant, China Na-
tional Science Foundation Grants 81272452 and 21272043,
and by department funds from the Department of Chemical
Biology in the Ernest Mario School of Pharmacy at Rutgers
University.
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