Synthesis and evaluation of curcumin-related compounds for anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2012.04.005

Sixty-one curcumin-related compounds were synthesized and evaluated for their anticancer activity toward cultured prostate cancer PC-3 cells, pancreas cancer Panc-1 cells and colon cancer HT-29 cells. Inhibitory effects of these compounds on the growth of PC-3, Panc-1 and HT-29 cells were determined by the MTT assay. Compounds E10, F10, FN1 and FN2 exhibited exceptionally potent inhibitory effects on the growth of cultured PC-3, Panc-1 and HT-29 cells. The IC₅₀ for these compounds was lower than 1 μM in all three cell lines. E10 was 72-, 46- and 117-fold more active than curcumin for inhibiting the growth of PC-3, Panc-1 and HT-29 cells, respectively. F10 was 69-, 34- and 72-fold more active than curcumin for inhibiting the growth of PC-3, Panc-1 and HT-29 cells, respectively. FN1 and FN2 had about the same inhibitory effect as E10 and F10 toward Panc-1 cells but were less active than E10 and F10 toward PC-3 and HT-29 cells. The active compounds were potent stimulators of apoptosis. The present study indicates that E10, F10, FN1 and FN2 may have useful anticancer activity.

Full text of DOI:10.1016/j.ejmech.2012.04.005

European Journal of Medicinal Chemistry 53 (2012) 235e245
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of curcumin-related compounds for anticancer activity
,
,
c
,
a,
Xingchuan Wei a b, Zhi-Yun Du a, Xi Zheng c, Xiao-Xing Cui c, Allan H. Conney a
, Kun Zhang
**
*
a Laboratory of Natural Medicinal Chemistry & Green Chemistry, Guangdong University of Technology, Guangzhou, China
b Department of Chemistry and Chemical Engineering, Guangzhou University, Guangzhou, China
c Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
164 Frelinghuysen Road, Piscataway, NJ 08854, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixty-one curcumin-related compounds were synthesized and evaluated for their anticancer activity
toward cultured prostate cancer PC-3 cells, pancreas cancer Panc-1 cells and colon cancer HT-29 cells.
Inhibitory effects of these compounds on the growth of PC-3, Panc-1 and HT-29 cells were determined
by the MTT assay. Compounds E10, F10, FN1 and FN2 exhibited exceptionally potent inhibitory effects
on the growth of cultured PC-3, Panc-1 and HT-29 cells. The IC50 for these compounds was lower than
Received 18 July 2011
Received in revised form
29 March 2012
Accepted 4 April 2012
Available online 16 April 2012
1 mM in all three cell lines. E10 was 72-, 46- and 117-fold more active than curcumin for inhibiting the
growth of PC-3, Panc-1 and HT-29 cells, respectively. F10 was 69-, 34- and 72-fold more active than
curcumin for inhibiting the growth of PC-3, Panc-1 and HT-29 cells, respectively. FN1 and FN2 had
about the same inhibitory effect as E10 and F10 toward Panc-1 cells but were less active than E10 and
F10 toward PC-3 and HT-29 cells. The active compounds were potent stimulators of apoptosis. The
present study indicates that E10, F10, FN1 and FN2 may have useful anticancer activity.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Anticancer curcumin derivatives
Inhibition of prostate cancer
Inhibition of pancreas cancer
Inhibition of colon cancer
1. Introduction
adequate tissue levels of curcumin necessary for effective cancer
prevention and treatment [20e24]. The weakness of the pharma-
Curcumin, a well-known diarylheptanoid, has been identified as
a major constituent in turmeric. It is used as a spice to give a specific
flavor and yellow color to curry, which is consumed daily by millions
of people. Curcumin has been used in traditional medicine for liver
disease (jaundice), indigestion, urinary tract diseases, rheumatoid
arthritis, and insect bites [1]. Numerous studies demonstrated the
anticancer activity of curcumin in animal models [1e10] as well as
growth inhibition and apoptosis induction in a variety of cancer cell
lines in vitro [11e17]. Because of its anticancer and antiangiogenic
properties, low molecular weight and lack of toxicity, curcumin is
under study as a possible new anticancer drug.
Curcumin has been evaluated in clinical trials for the treatment
of liver disease, rheumatoid arthritis, infectious diseases and
cancers [18]. Despite its efficacy and safety, the clinical usefulness of
curcumin is diminished by its poor oral absorption and extensive
first-pass metabolism, resulting in low oral bioavailability [19].
Several studies suggest that oral consumption may not furnish
cokinetic profile of curcumin in vivo significantly inhibits its clinical
application. Much effort has been devoted to developing useful
curcumin-related compounds to not only circumvent curcumin’s
low bioavailability but also to enhance its selectivity and potency
for addressing the pathological diversity of human cancer [25e35].
Some curcumin-related compounds such as cyclohexanone analogs
were found to have enhanced activity and stability in biological
medium compared to curcumin [34]. Of the many curcumin-related
compounds that were synthesized, a few were found to have-
strong anticancer activity when tested on cultured cancer cells (10-
to 60-fold more potent than curcumin) [25e34]. The 1,5-
diarylpentadienones GO-YO16, GO-YO30 and GO-YO31 were as
much as 60-fold more active than curcumin in certain cancer cell
lines [32].
In our continuing search for potent anticancer agents for the
treatment and prevention of prostate, colon and pancreatic cancer,
several series of curcumin-related compounds were synthesized
and evaluated for their anticancer effects. The main objective of our
study was to systematically test different curcumin-related
compounds for their anticancer activities and to analyze
structureeactivity relationships. Sixty-one curcumin-related
compounds were synthesized and evaluated for their anticancer
effects against three human cancer cell lines. In this paper, we
describe the synthesis of these curcumin-related compounds and
* Corresponding author.
** Corresponding author. Susan Lehman Cullman Laboratory for Cancer Research,
Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The
State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854, USA.
Tel.: þ1 732 445 4940; fax: þ1 732 445 0687.
E-mail addresses: aconney@pharmacy.rutgers.edu (A.H. Conney), kzhang@
gdut.edu.cn (K. Zhang).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.005

236
X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
the in vitro evaluation of their anticancer activities. Fourteen of the
compounds described here were synthesized and characterized
earlier by our laboratory [35]. Since submission of our manuscript,
we became aware of additional papers describing the synthesis of
several curcumin-related compounds described in our paper
[26,36e50]. Some of the curcumin-related compounds studied in
the present manuscript were 34- to 117-fold more active than
curcumin for inhibiting the growth of cultured human prostate,
pancreas and colon cancer cells.
2. Results and discussion
2.1. Chemistry
Fig. 2. Synthesis of groups AN, BN, EN and FN compounds.
A series of curcumin-related compounds were prepared. These
compounds were synthesized by coupling the appropriate
substituted benzaldehyde with cyclohexanone (group A), cyclo-
pentanone (group B), acetone (group C), 4-piperidinone (group D),
tetrahydropyran-4-ones (group E) or tetrahydrothiopyran-4-one
(group F) (Fig. 1). In groups AN, BN, EN and FN, the compounds
were synthesized by coupling the appropriate pyridyl aldehyde
with cyclohexanone (group AN), cyclopentanone (group BN), tet-
rahydropyran-4-one (group EN) and tetrahydrothiopyran-4-one
(group FN) (Fig. 2). In groups AS, BS, ES and FS, the compounds
were synthesized by coupling thiophene aldehyde with cyclohex-
anone (AS), cyclopentanone (BS), tetrahydropyran-4-one (ES) or
tetrahydrothiopyran-4-one (FS) (Fig. 3). The synthesis and charac-
terization of fourteen compounds in the A, B, C and D groups were
described previously by our group [35], and the synthesis of several
additional compounds have been described by others [26,36e50].
E9, E10, E11, F4, F9, F10, F11, EN1, EN3, FN3, AS, BS, ES and FS,
synthesized in the present study, are novel and were not previously
synthesized.
pancreatic cancer cells (Panc-1) and colon cancer cells (HT-29)
were determined by using the MTT assay. For each incubation,
curcumin was evaluated as a positive control. The inhibitory effect
of curcumin did not vary significantly between the different incu-
bations. Data from all curcumin incubations in experiments with
groups A to F compounds were averaged and presented in Table 1.
Average data from curcumin incubations in experiments with
groups AN to FN compounds are presented in Table 2, and average
data from curcumin incubations in experiments with AS, BS, ES and
FS are shown in Table 3. As shown in Table 1, all compounds in
group A (reaction products of different substituted benzaldehydes
and cyclohexanone) had stronger anticancer growth inhibitory
effects on PC-3, Panc-1 and HT-29 cells than curcumin as deter-
mined by the MTT assay. The IC50 for this group of compounds
ranged from 2.6 to 15.9 mM (Table 1). Group B compounds (reaction
products of different substituted benzaldehydes and cyclo-
pentanone) had a weaker growth inhibitory effect on the cells
when compared with group A compounds. Seven compounds in
this group had a similar or weaker effect than curcumin and only 3
compounds had a slightly better effect than curcumin (Table 1). Ten
of the 11 compounds in Group C (reaction products of different
substituted benzaldehydes and acetone) had a stronger anticancer
effect than curcumin and one compound (C3) in this group was
similar to curcumin when tested on PC-3 and Panc-1 cells (Table 1).
As shown in Table 1, compounds in groups D, E and F (reaction
products of different substituted benzaldehydes and piperidone,
pyridine or thiophene furan ketone) all had stronger inhibitory
effects on tumor cell growth than curcumin or group A compounds.
This result indicates that substitution of the cyclohexanone with 4-
piperidinone, tetrahydropyran-4-ones or tetrahydrothiopyran-4-
one strongly increases the effectiveness of these compounds.
2.2. Inhibitory effects of curcumin-related compounds toward
cultured human prostate, pancreas and colon cancer cells
The inhibitory effects of sixty-one curcumin-related compounds
on the growth of cultured human prostate cancer cells (PC-3),
Fig. 1. Synthesis of group A to group F compounds.
Fig. 3. Synthesis of AS, BS, ES and FS compounds.

X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
237
Table 1
Inhibitory effects of groups A to F compounds on the growth of human prostate cancer PC-3, pancreas cancer Panc-1 and colon cancer HT-29 cells.
Compound
R1
R2
R3
R4
IC50
(mM)
PC-3
Panc-1
HT-29
Curcumin
A1
A2
A3
A4 (A1)
A6
A7 (A3)
A8 (A5)
A9 (A6)
A10
A11
B1
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
OCH3
H
H
H
H
OH
F
Cl
Br
OH
OCH3
OH
OCH3
OH
OCH3
OCH3
F
Cl
Br
OH
OCH3
OH
OCH3
OH
OCH3
OCH3
F
Cl
Br
OH
OH
OCH3
OH
OCH3
OH
OCH3
OCH3
F
Cl
Br
OH
OH
OCH3
OH
OCH3
OCH3
OH
OCH3
OH
OCH3
OCH3
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
19.37 ꢀ 2.5
11.87 ꢀ 2.2
11.31 ꢀ 1.5
13.55 ꢀ 2.0
13.25 ꢀ 0.5
12.35 ꢀ 1.4
5.89 ꢀ 0.9
5.20 ꢀ 1.0
4.14 ꢀ 0.7
6.17 ꢀ 1.3
8.22 ꢀ 1.5
18.99 ꢀ 3.0
24.98 ꢀ 3.5
>30
22.95 ꢀ 4.3
25.81 ꢀ 4.0
15.54 ꢀ 2.5
9.30 ꢀ 1.5
9.98 ꢀ 1.1
16.42 ꢀ 1.3
18.71 ꢀ 2.9
15.19 ꢀ 0.8
16.67 ꢀ 1.1
23.09 ꢀ 5.4
9.68 ꢀ 1.2
8.99 ꢀ 2.0
11.22 ꢀ 1.8
4.20 ꢀ 0.7
2.98 ꢀ 0.4
3.41 ꢀ 0.4
4.63 ꢀ 1.0
5.93 ꢀ 1.1
2.87 ꢀ 0.4
3.63 ꢀ 0.6
1.95 ꢀ 0.3
5.94 ꢀ 1.3
7.28 ꢀ 1.4
6.12 ꢀ 1.5
0.91 ꢀ 0.1
0.27 ꢀ 0.1
2.74 ꢀ 0.3
8.23 ꢀ 1.5
8.46 ꢀ 1.6
1.13 ꢀ 0.2
0.28 ꢀ 0.1
3.78 ꢀ 0.6
23.68 ꢀ 3.1
10.42 ꢀ 2.0
11.77 ꢀ 1.5
12.24 ꢀ 2.1
13.21 ꢀ 2.1
15.92 ꢀ 1.6
4.43 ꢀ 0.5
4.46 ꢀ 0.2
2.63 ꢀ 0.4
9.66 ꢀ 1.8
11.55 ꢀ 0.8
19.71 ꢀ 2.6
23.17 ꢀ 3.0
25.32 ꢀ 3.4
>30
18.74 ꢀ 2.2
7.56 ꢀ 0.8
7.75 ꢀ 0.8
9.19 ꢀ 0.8
11.70 ꢀ 2.2
13.65 ꢀ 0.8
3.73 ꢀ 0.5
4.00 ꢀ 0.6
2.70 ꢀ 0.2
4.23 ꢀ 0.3
7.14 ꢀ 1.1
14.39 ꢀ 2.0
15.88 ꢀ 2.2
19.88 ꢀ 3.1
21.35 ꢀ 3.9
23.02 ꢀ 4.2
9.70 ꢀ 0.9
13.49 ꢀ 1.8
7.80 ꢀ 1.4
12.72 ꢀ 1.3
19.75 ꢀ 3.8
9.35 ꢀ 0.6
10.86 ꢀ 1.3
14.02 ꢀ 1.5
9.38 ꢀ 0.8
9.02 ꢀ 1.2
11.31 ꢀ 1.2
2.94 ꢀ 0.2
2.17 ꢀ 0.3
2.24 ꢀ 0.1
1.68 ꢀ 0.2
5.09 ꢀ 0.3
3.26 ꢀ 0.5
6.33 ꢀ 1.3
4.13 ꢀ 0.4
6.92 ꢀ 0.8
3.86 ꢀ 0.3
6.05 ꢀ 0.8
1.44 ꢀ 0.2
0.16 ꢀ 0.1
2.45 ꢀ 0.2
4.27 ꢀ 0.5
7.93 ꢀ 0.6
1.73 ꢀ 0.1
0.26 ꢀ 0.1
2.89 ꢀ 0.3
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OH
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
B2
B3
B4 (B1)
B6
B7 (B3)
B8 (B5)
B9 (B6)
B10
B11
C1
C2
C3
C4 (C1)
C5 (C2)
C6
C7 (C3)
C8 (C5)
C9 (C6)
C10
C11
D1
D2
D3
D4 (D1)
E4
E6
E9
E10
E11
F4
F6
F9
>30
15.49 ꢀ 1.9
13.69 ꢀ 0.6
9.63 ꢀ 2.0
18.80 ꢀ 1.0
22.71 ꢀ 1.6
12.53 ꢀ 1.0
14.08 ꢀ 2.8
20.82 ꢀ 2.9
13.59 ꢀ 2.2
13.36 ꢀ 1.8
14.03 ꢀ 1.7
4.62 ꢀ 0.4
3.39 ꢀ 0.5
2.23 ꢀ 0.2
3.38 ꢀ 0.4
9.22 ꢀ 2.3
3.15 ꢀ 0.4
3.73 ꢀ 0.3
2.06 ꢀ 0.3
8.08 ꢀ 1.4
11.05 ꢀ 2.2
7.63 ꢀ 1.8
1.72 ꢀ 0.2
0.52 ꢀ 0.1
2.67 ꢀ 0.4
11.96 ꢀ 1.7
10.91 ꢀ 1.2
2.20 ꢀ 0.5
0.70 ꢀ 0.1
4.71 ꢀ 0.7
H
OCH3
OCH3
H
H
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
H
H
OCH3
OCH3
H
H
H
OCH3
OCH3
H
H
H
H
H
H
OCH3
H
H
H
H
OCH3
OCH3
OCH3
H
H
OCH3
OCH3
OCH3
F10
F11
H
OCH3
Human prostate cancer PC-3, pancreas cancer Panc-1 and colon cancer HT-29 cells were seeded at a density of 0.2 ꢁ 105 cells/ml of medium in 96-well plates (0.2 ml/well) and
incubated for 24 h. The cells were then treated with various concentrations (0.05e30 mM) of the different compounds for 72 h. Effects of the different compounds on the
growth of PC-3, Panc-1 and HT-29 cells were determined by the MTT assay. The labeling of compounds that were synthesized as described in ref 35 are given in parentheses.
Heteroatoms such as nitrogen, oxygen or sulfur in the 4-position of
the central six-carbon ring (groups D, E and F) may be important for
increasing the inhibitory effect of these compounds on tumor cell
growth.
The results for the growth inhibitory effect of compounds in
groups AN, BN, EN and FN on PC-3, Panc-1 and HT-29 cells are
summarized in Table 2. All compounds in these four groups had
a stronger inhibitory effect on tumor cell growth than curcumin,
Among the 61 curcumin-related compounds in our study, IC50
measurements indicated that compounds E10, F10, FN1 and FN2
exhibited the most potent inhibitory effects on the growth of
cultured PC-3, Panc-1 and HT-29 cancer cells (21- to 117-fold more
active than curcumin). The IC50 values for these compounds were
lower than 1 mM in all three cell lines. The higher activity of E10,
F10, FN1 and FN2 were confirmed by using the trypan blue assay for
evaluation of cell growth. E10 was 72-, 46- and 117-fold more active
than curcumin (IC50) for inhibiting the growth of PC-3, Panc-1 and
HT-29 cells, respectively (Table 1), whereas F10 was 69-, 34- and
72-fold more potent than curcumin for inhibiting the growth of PC-
3, Panc-1 and HT-29 cells, respectively (Table 1). FN1 and FN2 had
about the same inhibitory effect as E10 and F10 toward Panc-1 cells
but were less active than E10 and F10 toward PC-3 and HT-29 cells
(Tables 1and 2). The structures of E10, F10, FN1, FN2 and curcumin
are shown in Fig. 4. To the best of our knowledge, these four highly
active compounds are novel and have not previously been
synthesized.
and the IC50 values ranged from 0.51 to 7.88 mM. Compounds in the
AN, EN and FN groups had a stronger inhibitory effect on tumor cell
growth than compounds in the BN group (Table 2). The results for
the growth inhibitory effect of compounds in the AS, BS, ES and FS
groups on PC-3, Panc-1 and HT-29 cells are summarized in Table 3.
AS and BS had a similar or higher IC50 than curcumin. ES and FS had
a stronger inhibitory effect on tumor cell growth than curcumin
(Table 3). Other publications recently pointed out the inhibitory
effect of curcumin and certain curcumin-related compounds on the
growth of colon cancer cell lines [51e53].

238
X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
Table 2
Inhibitory effects of groups AN, BN, EN, FN compounds on the growth of human
prostate cancer PC-3, pancreas cancer Panc-1 and colon cancer HT-29 cells.
Compound
X1
X2
X3
IC50
(mM)
PC-3
Panc-1
HT-29
Curcumin
AN1
AN2
AN3
BN1
BN2
BN3
EN1
EN2
EN3
FN1
FN2
20.16 ꢀ 2.3
1.28 ꢀ 0.2
2.16 ꢀ 0.2
2.12 ꢀ 0.4
2.94 ꢀ 0.3
6.97 ꢀ 0.8
5.58 ꢀ 0.3
1.88 ꢀ 0.3
2.21 ꢀ 0.4
1.83 ꢀ 0.2
0.52 ꢀ 0.1
0.96 ꢀ 0.2
0.70 ꢀ 0.1
22.83 ꢀ 2.7
1.30 ꢀ 0.2
2.50 ꢀ 0.6
2.18 ꢀ 0.5
5.19 ꢀ 0.6
6.22 ꢀ 1.2
7.88 ꢀ 1.1
3.24 ꢀ 0.6
0.98 ꢀ 0.2
2.96 ꢀ 0.3
0.70 ꢀ 0.1
0.51 ꢀ 0.1
1.14 ꢀ 0.2
20.95 ꢀ 2.9
1.43 ꢀ 0.1
1.99 ꢀ 0.1
2.35 ꢀ 0.3
5.36 ꢀ 0.4
5.54 ꢀ 0.3
7.58 ꢀ 0.6
3.28 ꢀ 0.2
1.81 ꢀ 0.1
4.17 ꢀ 0.3
0.76 ꢀ 0.1
0.79 ꢀ 0.1
1.29 ꢀ 0.1
N
C
C
N
C
C
N
C
C
N
C
C
C
N
C
C
N
C
C
N
C
C
N
C
C
C
N
C
C
N
C
C
N
C
C
N
Fig. 4. Structures of curcumin and highly active anticancer analogs of curcumin (from
the present study).
FN3
Human prostate cancer PC-3, pancreas cancer Panc-1 and colon cancer HT-29 cells
were seeded at a density of 0.2 ꢁ 105 cells/ml of medium in 96-well plates (0.2 ml/
well) and incubated for 24 h. The cells were then treated with various concentra-
cells were all associated with a decrease in phospho-Akt and
phospho-Erk1/2.
tions (0.05e30 mM) of the different compounds for 72 h. Effects of the different
compounds on the growth of PC-3, Panc-1 and HT-29 cells were determined by the
MTT assay.
2.4. Structureeactivity relationship analysis
Based on analysis of the relationship between the structures of
curcumin-related compounds and their ability to inhibit the
2.3. Mechanism studies
Treatment of PC-3 cells with E10, F10, FN1 or FN2 for 96 h had
a dramatic stimulatory effect on apoptosis at concentrations <1 mM
whereas curcumin had little or no effect at these low concentra-
tions (Fig. 5A). E10 was the most potent stimulator of apoptosis
followed by F10 and FN1. Although FN2 was less effective than the
other three compounds, it was also a strong stimulator of apoptosis.
The stimulatory effect of E10, F10, FN1 and FN2 on apoptosis in PC-3
cells paralleled their activities for inhibiting the growth of PC-3
cells (Tables 1and 2).
To further determine the mechanisms for growth inhibition and
apoptosis induction in PC-3 cells, the levels of activated Akt and
Erk1/2 were evaluated by Western blot analysis using anti-
phosphorylated Akt and anti-phosphorylated Erk1/2 antibodies
(Fig. 5B). In these experiments, PC-3 cells were treated with cur-
cumin or with E10, F10, FN1 and FN2 for 24 h and analyzed by
Western blotting. The levels of phosphorylated Akt and Erk1/2 in
Western blots were analyzed by optical density measurements and
normalized for b-actin. As shown in Fig. 5B, treatment of PC-3 cells
with E10 resulted in a strong decrease in the level of phosphory-
lated Akt while the other compounds (F10, FN1 and FN2) were less
active, and curcumin was inactive. Treatment with E10, F10, FN1
and FN2 all caused a decrease in the level of phosphorylated Erk1/2
while curcumin had little or no inhibitory effect (Fig. 5B). Our
results indicate that the effects of E10, F10, FN1 and FN2 on PC-3
Table 3
Inhibitory effects of AS, BS, ES and FS on the growth of human prostate cancer PC-3,
pancreas cancer Panc-1 and colon cancer HT-29 cells.
Compound
IC50
(mM)
Fig. 5. (A) Stimulatory effect of E10, F10, FN1 and FN2 on apoptosis in cultured PC-3
cells. Various concentrations of curcumin, E10, F10, FN1 and FN2 were incubated
with PC-3 cells for 96 h. Apoptosis was determined by morphology as previously
described [56e58] (B) Effect of E10, F10, FN1 and FN2 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 100 mm culture dishes (10 ml/dish) and incubated for 24 h. The cells were then
PC-3
Panc-1
HT-29
Curcumin
19.94 ꢀ 2.2
14.55 ꢀ 2.5
23.01 ꢀ 6.0
6.59 ꢀ 1.4
4.13 ꢀ 1.0
24.12 ꢀ 3.0
17.38 ꢀ 1.8
27.86 ꢀ 2.8
10.82 ꢀ 1.2
7.49 ꢀ 0.6
21.33 ꢀ 2.4
17.34 ꢀ 2.9
19.72 ꢀ 3.5
8.94 ꢀ 1.5
6.75 ꢀ 0.5
AS
BS
ES
FS
treated with curcumin, E10, F10, FN1 or FN2 (all at 0.5 mM) for 24 h. Activated Akt and
Human prostate cancer PC-3, pancreas cancer Panc-1 and colon cancer HT-29 cells
were seeded at a density of 0.2 ꢁ 105 cells/ml of medium in 96-well plates (0.2 ml/
well) and incubated for 24 h. The cells were then treated with various concentra-
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
b
-actin, and the levels of phosphorylated Akt and Erk1/2 in Western
-actin to
tions (0.1e30
mM) of different compounds for 72 h. Effects of the different
blots were analyzed by optical density measurements and normalized for b
compounds on the growth of PC-3, Panc-1 and HT-29 cells were determined by the
MTT assay.
obtain the relative optical densities (RD) for the control (CON), curcumin (CUR), E10,
F10, FN1 and FN2 groups [56,57].

X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
239
growth of cultured cancer cells, the presence of groups on the two
aromatic rings and the linker between the aromatic rings both
played key roles in determining the anticancer activity of the
various analogs.
exceptionally high anticancer activity whereas methoxy groups at
R1, R2 and R3 (compounds E11 and F11) resulted in markedly
reduced activity when compared with E10 and F10. It was observed
that methoxy groups at R2, R3 and R4 in the A, B or C group of
compounds (A10, B10 and C10) had stronger anticancer activity
than curcumin and moving the methoxy groups to R1, R2 and R3
(compounds A11, B11 and C11) decreased activity slightly when
compared with A10, B10 and C10. Compounds with three methoxy
groups on each aromatic ring were also demonstrated to have
strong anticancer activity in previous reports (compounds B10 [28]
and GO-Y016 [32], Fig. 6). The later compound is the same as our
C10 in Table 1 and Fig. 1. In the Ohori et al report, GO-Y016 was 12-
to 60-fold more active than curcumin for inhibiting the growth of
several cancer cell lines [32]. In our study, the same compound
(C10) was only 4- to 11-fold more active than curcumin for inhib-
iting the growth of PC-3, Panc-1 and HT-29 cells (Table 1).
Introduction of halogen at R3 in group A, B or C compounds did
not result in strongly active compounds (compounds A1, A2, A3, B1,
B2, B3, C1, C2 and C3) whereas introduction of halogen at R3 in the
D series (compounds D1, D2 and D3) resulted in compounds that
had substantially more activity than curcumin. Other investigators
have also obtained curcumin-related compounds with high activity
by introduction of fluorine onto the aromatic rings (see compounds
14 and EF24, Fig. 6) [25,33].
An interesting observation was that the growth inhibitory
effects of curcumin became stronger when the normal benzene
rings in curcumin were replaced by distal heterocyclic aromatic
rings (compounds in the AN, BN, EN, FN, ES and FS groups; Table 2).
These results suggest that distal heteroatoms enhance the growth
inhibitory effects of curcumin on cultured prostate, pancreas and
colon cancer cells. A previous report also demonstrated that
curcumin-related compounds with pyridine [30], such as RL91
(Fig. 6, same as our AN3 in Fig. 2 and Table 2), showed strong
inhibitory effects on tumor cell growth. In the earlier report, RL-91
was 7- to 10-fold more active than curcumin for inhibition of the
growth of MDA-MB-231 and SKB-3 cancer cells [30]. In our study,
the same compound was 9- to 10-fold more active than curcumin
for inhibition of the growth of PC-3, Panc-1 and HT-29 cells
(Table 2). Comparing the analogs containing 1-, 2- and 3-pyridine,
we found that the growth inhibitory effects of these compounds
were similar or considerably stronger than curcumin.
Among the different series of curcumin-related compounds,
linear or cyclic linkers between the two aromatic rings of curcumin-
related compounds showed different activity trends. In general, the
activities of compounds with a tetrahydrothiopyran-4-one (group
F) or a tetrahydropyran-4-one (group E) linker exhibited the
strongest activities, and that of compounds with piperidin-4-one
(group D) had moderate activity whereas compounds with cyclo-
hexanone (group A), acetone (group C) or cyclopentanone (group B)
linker were less active. The activities of compounds with a hetero-
atom linker (compounds in groups D, E and F) were better than
those without a heteroatom linker (groups A, B and C), which
suggests that flexibility of curcumin-like compounds may enhance
their antitumor activities by having interactions with the DNA of
cancer cells, whereas compounds with a small and highly rigid
linker would be less active. It is of interest that curcumin-DNA
interaction, a possible explanation for antitumor activity, is asso-
ciated with curcumin flexibility [54,55]. An exception to the
concept of greater flexibility being associated with stronger activity
is with group C compounds that have high flexibility but rather low
anticancer activity. Recent reports in the literature indicate that
some curcumin-related compounds with piperidin-4-one possess
potent anticancer activity, such as compound 14 [25], compound
B10 [28], and compound EF24 [33] (Fig. 6). Our curcumin-related
compounds with a tetrahydropyran-4-one linker (group F) or
a tetrahydrothiopyran-4-one linker (group E) were not previously
reported to have anticancer activity, but E9, E10, F9 and F10 from
groups E and F were strongly active. Comparing compounds with
different heterocyclic ketones as a linker, the inhibitory effect of
certain analogs with a sulfur or oxygen heterocyclic ketone linker
and distal benzene rings (e.g. compounds E10 and F10) on tumor
cell growth were slightly more potent than that of analogs with
a sulfur or oxygen heterocyclic ketone linker and distal nitrogen
heterocyclic rings (e.g. compounds FN1 and FN2) described in this
paper.
Comparing different substituted groups on curcumin-related
compounds with the same linker, we found that introduction of
methoxy groups on the aromatic rings enhanced anticancer
activity. In both the E and F series, compounds with methoxy
groups at R2, R3 and R4 (compounds E10 and F10) had
Taken together, compounds with three methoxy groups at R1,
R2 and R3 on each distal aromatic ring and a heterocyclic ketone
Fig. 6. Structures of selected anticancer of curcumin-related compounds from the literature.

240
X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
linker, such as E10 and F10 as well as compounds with distal
heterocyclic aromatic rings and a heterocyclic ketone linker, such as
FN1 and FN2 (Fig. 4), exhibited more potent anticancer effects than
the other compounds studied.
recrystallized from appropriate solvents (methanol or ethanol).
Fourteen of the compounds studied were synthesized and charac-
terized earlier in our laboratory [35]. The compounds described in
Ref. [35] and their labeling in Ref. [35] are given in parenthesis in
Table 1. Prior synthesis of additional curcumin-related compounds
described in the literature are indicated below by providing
appropriate references.
3. Conclusions
In the present study, several groups of curcumin-related
compounds were synthesized and evaluated for anticancer
activity using cultured human prostate, pancreas and colon cancer
cells. Curcumin-related compounds with a tetrahydropyran-4-one
(groups E and EN) or tetrahydrothiopyran-4-one (groups F and
FN) as the core structure had a more potent effect on cancer cells
than other curcumin-related compounds. Compounds E10, F10,
FN1 and FN2 exhibited particularly potent inhibitory effects on the
growth of cultured prostate cancer PC-3 cells, pancreas cancer
Panc-1 cells and colon cancer HT-29 cells. The IC50 for these
4.2.1. (2E,6E)-2,6-bis (4-fluorobenzylidene)cyclohexanone (A1)
Yield 85%. mp. 155e157 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
7.75(s, 2H, eCH¼), 7.45 (d, J ¼ 8.1 Hz, 4H, arom), 7.10 (d, J ¼ 8.1 Hz,
4H, arom),2.91 (t, J ¼ 5.7 Hz, 4H, eCH2eCeCH2e), 1.82 (q, J ¼ 5.7 Hz,
2H, eCeCH2eCe). 13C NMR (CDCl3, 75 MHz)
d(ppm): 190.0, 164.3,
161.0, 135.9, 132.3, 132.0, 115.7, 28.4, 22.9. ESIeMS (m/z):
311(Mþ1)þ. Anal. Calc. for C20H16F2O: 310, C 77.40, H 5.20. Found: C
77.38, H 5.23 (synthesis also described in Refs. [36,37]).
compounds in all three cell lines was lower than 1
m
M. E10 was 72-,
4.2.2. (2E,6E)-2,6-bis (4-chlorobenzylidene)cyclohexanone (A2)
46- and 117-fold more active than curcumin (comparison of IC50
values) for inhibiting the growth of PC-3 prostate cancer cells, Panc-
1 pancreas cancer cells and HT-29 colon cancer cells, respectively,
whereas F10 was 69-, 34- and 72-fold more potent than curcumin
for inhibiting the growth of cultured PC-3, Panc-1 and HT-29 cells,
respectively. FN1 and FN2 were also highly active inhibitors of the
growth of these cancer cell lines, but overall they were less active
than E10 and F10. Additional research is needed on mechanisms
and the in vivo activity of these compounds on tumor growth in
appropriate animal models. From preliminary studies of
structureeactivity relationships, it was found that (1) tetrahy-
dropyran-4-one or tetrahydrothiopyran-4-one as a core structure,
(2) methoxy groups at R2, R3 and R4 in the aromatic rings and (3)
nitrogen heterocycles in the distal rings may be promising lead
structures for new agents with potent anticancer activity. Four
compounds with particularly potent anticancer activity were
identified, and further research is needed to more fully determine
their anticancer potential.
Yield 83%. mp. 147e149 ꢂC. 1H NMR (CDCl3, 300 MHz)
d
(ppm):7.72(s, 2H, eCH¼),7.42e7.28 (m, 8H, arom), 2.65 (t,
J ¼ 5.7 Hz, 4H, eCH2eCeCH2e), 1.96e1.85 (m, 2H, eCeCH2eCe).
13C NMR (CDCl3, 75 MHz)
d(ppm): 189.8, 136.1, 135.1, 134.3, 133.9,
131.6, 129.0, 28.6, 22.8. ESIeMS (m/z): 344 (Mþ1) þ. Anal. Calc. for
C20H16Cl2O:343; C 69.98, H 4.70. Found: C 69.90, H 4.73 (synthesis
also described in Ref. [38]).
4.2.3. (2E,6E)-2,6-bis (4-bromobenzylidene)cyclohexanone(A3)
Yield 82%. mp. 149e151 ꢂC. 1H NMR (CDCl3, 300 MHz)
d
(ppm):7.70 (s, 2H, eCH¼), 7.53 (d, J ¼ 8.1 Hz, 4H, arom), 7.31 (d,
J ¼ 8.1 Hz, 4H, arom),2.94 (t, J ¼ 5.7 Hz, 4H,eCH2eCeCH2e),
1.88e1.79 (m, 2H, eCeCH2eC). 13C NMR (CDCl3, 75 MHz)
(ppm):
d
189.9, 136.5, 135.9, 134.7, 131.9, 131.7, 123.0, 28.4, 22.8. ESIeMS (m/
z): 433 (Mþ1) þ. Anal. Calc. for C20H16Br2O: 432; C 55.59, H 3.73.
Found: C 55.55, H 3.69 (synthesis also described in Ref. [39]).
4.2.4. (2E,6E)-2,6-bis (4-Hydroxybenzylidene)cyclohexanone (A4)
This compound was characterized earlier as A1 in Ref. [35].
4. Experimental
4.2.5. (2E,6E)-2,6-bis (4-methoxybenzylidene)cyclohexanone(A6)
4.1. General procedures
Yield 90%, mp. 148e149 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 7.54 (s, 2H, eCH¼), 7.48 (d, J ¼ 8.1 Hz, 4H, arom), 6.98 (d,
Melting points were determined on a Yanagimoto micro-
melting apparatus and were uncorrected. The 1H NMR spectra
were measured on a Varian Gemini-2000 spectrometer using
DMSO as solvent unless otherwise specified. Chemical shifts for 1H
NMR (300 MHz) and 13C NMR (75 MHz) were expressed in ppm
units with TMS as an internal standard. Multiplicities were recor-
ded as s (singlet), brs (broad singlet), d (doublet), t (triplet), q
(quartet), m (multiplet). Mass spectra were obtained on an LC-MS-
2010A spectrometer with ESI. Elemental analyses were performed
on an elemental analyser(Vario El). Thin-layer chromatography
(TLC) was performed on Merck silica gel plates (DC-60 F254).
Curcuminoids (1e3) were isolated from the extract of Curcuma
longa Linn according to a previous report [35]. All reagents (highest
grade) were used as received unless otherwise stated.
J ¼ 8.1 Hz, 4H, arom), 3.78 (s, 6H, eOCH3); 2.85 (t, J ¼ 5.7 Hz, 4H,
eCH2eCeCH2e); 1.70 (q, J ¼ 5.7 Hz, 2H, eCeCH2eCe). 13C NMR
(CDCl3, 75 MHz) d(ppm): 189.9, 159.6, 147.1, 141.5, 136.6, 132.2,
114.0, 55.2, 27.9, 23.1, ESIeMS (m/z): 335 (Mþ1) þ. Anal. Calc. for
C22H22O3: 334; C 79.02, H 6.63. Found: C 78.79, H 6.47 (synthesis
also described in Refs. [39, 40]).
4.2.6. (2E,6E)-2,6-bis (4-hydroxy-3-methoxybenzylidene)
cyclohexanone(A7)
This compound was characterized earlier as A3 in Ref. [35].
4.2.7. (2E,6E)-2,6-bis (3,4-dimethoxybenzylidene)cyclohexanone
(A8)
This compound was characterized earlier as A5 in Ref. [35].
4.2. Synthesis of curcumin analogs
4.2.8. (2E,6E)-2,6-bis (4-hydroxy-3,5-dimethoxybenzylidene)
cyclohexanone (A9)
A total of 61 curcumin analogs were synthesized as previously
described with modification [35]. A mixture of the appropriate
aldehyde (0.01 mol) and the ketone (0.005 mol) was dissolved in
glacial acetic acid saturated with anhydrous hydrogen chloride and
heated in a water bath at 25e30 ꢂC for 2 h. After standing for 2d, the
mixture was treated with cold water and filtered. The solid
obtained was then washed and dried. The crude product was
This compound was characterized earlier as A6 in Ref. [35].
4.2.9. (2E,6E)-2,6-bis (3,4,5-trimethoxybenzylidene)cyclohexanone
(A10)
Yield 78%. mp. 196e198 ꢂC. 1H NMR (CDCl3, 300 MHz):
7.72(s, 2H, eCH¼), 6.70(s, 4H, arom), 3.89(s, 18H, eOCH3),
2.96(t, J ¼ 5.7 Hz, 4H, eCH2eCeCH2e), 1.84 (q, J ¼ 5.7 Hz,

X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
241
2H, eCeCH2eCe). 13C NMR (CDCl3, 75 MHz)
138.7, 137.1, 135.8, 131.4, 107.7, 60.9, 56.2, 28.4, 23.0. ESIeMS (m/z):
455(Mþ1) þ. Anal. Calc. for C26H30O7: 454; C 68.71, H 6.65. Found: C
68.78, H 6.63. (synthesis also described in Refs. [37,41]).
d
(ppm): 189.9, 153.0,
4.2.19. (2E,5E)-2,5-bis (3,4,5-trimethoxybenzylidene)
cyclopentanone (B10)
Yield 81%. mp. 202e204 ꢂC 1H NMR (CDCl3, 300 MHz)
d(ppm):
7.52 (s, 2H, eCH¼), 6.85 (s, 4H, arom), 3.91 (s, 18H, eOCH3), 3.16 (s,
4H, eCH2eCH2e). 13C NMR (CDCl3, 75 MHz)
d
(ppm): 195.9, 153.3,
4.2.10. (2E,6E)-2,6-bis (2,3,4 -trimethoxybenzylidene)
cyclohexanone (A11)
139.4, 136.3, 133.5, 131.3, 108.0, 61.0, 56.2, 26.4. ESIeMS (m/z): 441
(Mþ1) þ. Anal. Calc. for C25H28O7: 440; C 68.17, H 6.41. Found: C
68.13, H 6.42 (synthesis also described in Ref. [41]).
Yield 75%. mp. 201e204 ꢂC. 1H NMR (CDCl3, 300 MHz): 7.93(s,
2H, eCH¼),7.38 (d, J ¼ 8.1 Hz, 2H,arom),7.27 (d, J ¼ 8.1 Hz, 2H,
arom), 4.12 (s,6H, eOCH3), 4.06 (s,6H, eOCH3), 3.98 (s,6H, eOCH3).
4.2.20. (2E,5E)-2,5-bis (2,3,4 -trimethoxybenzylidene)
cyclopentanone (B11)
2.98(t,
J
¼
5.7 Hz, 4H,eCH2eCeCH2e),1.85 (q,
J
¼
5.7 Hz,
2H, eCeCH2eCe). 13C NMR (CDCl3, 75 MHz)
d
(ppm): 190.1, 154.3,
Yield 66%. mp. 207e209 ꢂC. 1H NMR (CDCl3, 300 MHz)
153.5, 142.2, 135.8, 131.9, 125.2, 123.1, 106.6, 61.5, 60.9, 56.0, 28.8,
23.5. ESIeMS (m/z): 455(Mþ1) þ. Anal. Calc. for C26H30O7: 454; C
68.71, H 6.65. Found: C 68.78, H 6.67 (synthesis also described in
Ref. [26]).
d
(ppm):7.82 (s, 2H, eCH¼), 7.26 (d, J ¼ 8.1 Hz, 2H, arom),7.15 (d,
J ¼ 8.1 Hz, 2H, arom), 4.08 (s,6H, eOCH3), 4.02 (s,6H, eOCH3), 3.96
(s,6H, eOCH3). 3.18 (s, 4H, eCH2eCH2e). 13C NMR (CDCl3, 75 MHz)
d
(ppm): 195.6, 156.1, 154.8, 143.6, 136.6, 132.5, 125.0, 122.8, 106.9,
61.6, 61.1, 56.1, 26.8. ESIeMS(m/z): 441(Mþ1) þ. Anal. Calc. for
C25H28O7: 440; C 68.17, H 6.41. Found: C 68.14, H 6.45 (synthesis
also described in Ref. [43]).
4.2.11. (2E,5E)-2,5-bis (4-fluorobenzylidene)cyclopentanone (B1)
Yield 81%. mp. 212e214 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
7.57(d, J ¼ 8.1 Hz, 4H, arom), 7.56(s, 2H, eCH¼), 7.13 (t, J ¼ 8.1 Hz,
4H, arom), 3.10 (s, 4H, eCH2eCH2e). 13C NMR (CDCl3, 75 MHz)
4.2.21. (1E,4E)-1,5-bis (4- fluorophenyl)-1,4-pentadiene-3-one (C1)
Yield 63%, mp 146e148 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 189.8, 157.7, 143.2, 136.6, 132.6, 132.0, 116.1, 26.4. ESIeMS
(m/z): 297(Mþ1) þ. Anal. Calc. for C19H14F2O: 296; C 77.01, H 4.76.
d
(ppm): 7.71(d, J ¼ 15.9 Hz, 2H, eCH¼), 7.60(d, J ¼ 8.7 Hz, 4H,
Found: C 77.03, H 4.79 (synthesis also described in Ref. [36]).
arom), 7.11(d, J ¼ 8.7 Hz, 4H, arom), 7.00(d, J ¼ 15.9 Hz,
2H, eC ¼ CHe). 13C NMR (CDCl3, 75 MHz)
d(ppm): 188.3, 164.9,
4.2.12. (2E,5E)-2,5-bis (4-chlorobenzylidene)cyclopentanone (B2)
161.6, 141.5, 130.8, 125.4, 115.8. ESIeMS (m/z): 271 (Mþ1) þ. Anal.
Calc. for C17H12F2O: 270; C 75.55, H 4.48. Found: C 75.56, H 4.53
(synthesis also described in Refs. [36, 44]).
Yield 78%. mp. 230e232 ꢂC. 1H NMR (CDCl3, 300 MHz)
d
(ppm):
7.52(d, J ¼ 8.1 Hz, 4H), 7.51(s, eCH¼, arom), 7.41 (d, J ¼ 8.1 Hz, 4H,
arom), 3.10 (s, 4H, eCH2eCH2e). 13C NMR (CDCl3, 75 MHz)
(ppm):
d
189.4, 137.5, 135.5, 134.2, 132.7, 131.9, 129.1, 26.4. ESIeMS (m/
z):329(Mþ1)þ. Anal. Calc. for C19H14Cl2O: 328; C 69.32, H 4.29.
Found: C 69.33, H 4.28 (synthesis also described in Ref. [40]).
4.2.22. (1E,4E)-1,5-bis (4- chlorophenyl)-1,4-pentadiene-3-one
(C2)
Yield 70%, mp. 192e193 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 7.68(d, J ¼ 15.9 Hz, 2H, eCH¼Ce), 7.54(d, J ¼ 8.1 Hz, 4H,
4.2.13. (2E,5E)-2,5-bis (4-bromobenzylidene)cyclopentanone(B3)
arom), 7.38(d, J ¼ 8.1 Hz, 4H, arom), 7.03(d, J ¼ 15.9 Hz,
Yield 82%. mp. 211e213 ꢂC. 1H NMR (CDCl3, 300 MHz)
d
(ppm):
2H,eC ¼ CHe). 13C NMR (CDCl3, 75 MHz)
d(ppm): 188.4, 141.5,
7.57(d, J ¼ 8.1 Hz, 4H, arom), 7.52(s, 2H, eCH¼ ), 7.44(d,J ¼ 8.1 Hz,
135.0, 133.6, 130.2, 129.0, 126.2. ESIeMS (m/z): 303 (Mþ1) þ. Anal.
Calc. for C17H12Cl2O: 302; C 67.35, H 3.99. Found: C 67.36, H 3.93
(synthesis also described in Refs. [38, 45]).
4H, arom), 3.09 (s, 4H, eCH2eCH2e). 13C NMR (CDCl3, 75 MHz)
d
(ppm): 189.8, 137.7, 136.3, 134.6, 132.8, 132.1, 123.9, 26.5. ESIeMS
(m/z): 417 (Mþ1) þ. Anal. Calc. for C19H14Br2O: 416; C 54.58, H
3.37. Found:
Ref. [37]).
C
54.59,
H
3.38 (synthesis also described in
4.2.23. (1E,4E)-1,5-bis (4- bromophenyl)-1,4-pentadiene-3-one
(C3)
Yield 76%, mp. 211e213 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
4.2.14. (2E,5E)-2,5-bis (4-Hydroxybenzylidene)cyclopentanone (B4)
d
(ppm): 7.67(d, J ¼ 15.9 Hz, 2H, eCH¼Ce), 7.55(d, J ¼ 8.1 Hz, 4H,
This compound was characterized earlier as B1 in ref 35.
arom), 7.47(d,
J
¼
8.1 Hz, 4H, arom),7.04(d,J
¼
11.9 Hz,
2H, eCH ¼ Ce). 13C NMR (CDCl3, 75 MHz)
d(ppm): 188.3, 141.6,
4.2.15. (2E,5E)-2,5-bis (4-methoxybenzylidene)cyclopentanone(B6)
135.4, 134.0, 132.0, 130.4, 126.2. ESIeMS (m/z): 391 (Mþ1) þ. Anal.
Calc. for C17H12Br2O: 390; C 52.08, H 3.08. Found: C 52.06, H 3.04
(synthesis also described in Ref. [45]).
Yield: 92%; mp 158e161 ꢂC. 1H NMR (DMSO-d6,
300 MHz)
7.03 (d, J ¼ 8.1 Hz, 4H, arom), 3.79(s, 6H, eOCH3), 3.03 (s,
4H, eCH2eCH2e).13C NMR (CDCl3, 75 MHz)
(ppm): 190.2, 159.8,
d
(ppm): 7.62 (d, J ¼ 8.1 Hz, 4H, arom), 7.35 (s, 2H, eCH¼),
d
4.2.24. (1E,4E)-1,5-bis (4-Hydroxyphenyl)-1,4-pentadiene-3-one (C4)
148.5, 145.6, 138.6, 132.5, 114.5, 55.3, 26.6. ESIeMS (m/
z):321(Mþ1) þ. Anal. Calc. for C21H20O3:320; C 78.73, H 6.29. Found:
C 78.52, H 6.33 (synthesis also described in Ref. [42]).
This compound was characterized earlier as C1 in Ref. [35].
4.2.25. (1E,4E)-1,5-bis (3,4-dihydroxyphenyl) ꢃ1,4-pentadiene-3-
one (C5)
4.2.16. (2E,5E)-2,5-bis (4-hydroxy-3-methoxybenzylidene)
cyclopentanone (B7)
This compound was characterized earlier as C2 in Ref. [35].
This compound was characterized earlier as B3 in Ref. [35].
4.2.26. (1E,4E)-1,5-bis (4-methoxyphenyl)- 1,4-pentadiene-3-one
(C6)
4.2.17. (2E,5E)-2,5-bis (3,4-dimethoxybenzylidene)cyclopentanone
(B8)
Yield 79%. mp. 121e122 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 7.82e7.70(m, 4H, arom), 7.67 (d, J ¼ 15.9 Hz, 2H, eCH¼Ce),
This compound was characterized earlier as B5 in Ref. [35].
7.15 (d, J ¼ 15.9 Hz, 2H, eC ¼ CHe), 7.10e6.98 (m, 4H, arom), 3.78 (s,
6H, eOCH3). 13C NMR (CDCl3, 75 MHz)
d(ppm): 188.1, 161.1, 142.1,
4.2.18. (2E,5E)-2,5-bis (4-hydroxy-3,5-dimethoxybenzylidene)
cyclopentanone (B9)
130.3, 127.3, 123.5, 114.4, 55.3. ESIeMS (m/z): 295 (Mþ1) þ. Anal.
Calc. for C19H18O3:294; C 77.53, H 6.16. Found: C 77.47, H 6.13
(synthesis also described in Ref. [38]).
This compound was characterized earlier as B6 in Ref. [35].

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X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
4.2.27. (1E,4E)-1,5-bis (4-hydroxy-3-methoxphenyl) penta-1,4-
dien-3-one (C7)
(d, J ¼ 8.1 Hz, 4H, arom), 6.85 (d, J ¼ 8.1 Hz, 4H, arom), 4.85
(s, 4H, eCH2eOeCH2e). 13C NMR (CDCl3, 75 MHz)
(ppm):
d
This compound was characterized earlier as C3 in Ref. [35].
184.3, 159.0, 134.9, 132.8, 130.5, 125.3, 116.2, 50.3. ESIeMS (m/
z): 307(M ꢃ 1) þ. Anal. Calc. for C19H16O4: 308; C 74.01, H
4.2.28. (1E,4E)-1,5-bis (3,4-dimethoxyphenyl) penta-1,4-dien-3-
one (C8)
5.23. Found:
Ref. [49]).
C 74.00, H 5.24 (synthesis also described in
This compound was characterized earlier as C5 in Ref. [35].
4.2.37. (3E,5E)-3,5-Bis(4-methoxybenzylidene)-tetrahydropyran-4-
one (E6)
4.2.29. (1E,4E)-1,5-bis (4-hydroxy-3,5-dimethoxyphenyl) penta-
1,4-dien-3-one (C9)
Yield: 78%; mp. 177e180 ꢂC; 1H NMR (DMSO-d6, 300 MHz)
This compound was characterized earlier as C6 in Ref. [35].
d
(ppm): 7.60(s, 2H, eCH¼); 7.40 (d, J ¼ 8.7 Hz, 4H, arom); 7.02
(d, J ¼ 8.7 Hz, 4H, arom), 4.88(s, 4H, eCH2-OeCH2e); 3.81 (s,
6H, eOCH3). 13C NMR (CDCl3, 75 MHz)
(ppm): 185.2, 159.7,
4.2.30. (1E,4E)-1,5-bis (3,4,5-trimethoxyphenyl) penta-1,4-dien-3-
one (C10)
d
135.0, 132.5, 130.9, 126.8, 114.7, 68.5, 55.0. ESIeMS (m/z):337
(Mþ1) þ. Anal. Calc. for C21H20O4 :336; C 74.98, H 5.99. Found: C
74.92, H 6.01 (synthesis also described in Ref. [48]).
Yield 66%,mp. 118e120 ꢂC. 1H NMR(CDCl3, 300 MHz)
d:7.65 (d,
J ¼ 15.9 Hz, 2H,eCH¼Ce), 6.98 (d, J ¼ 15.9 Hz, 2H,eC ¼ CHe), 6.84
(s, 4H, arom), 3.89 (s, 18H, eOCH3). 13C NMR (CDCl3, 75 MHz)
d
(ppm): 188.7, 153.6, 143.4, 140.1, 130.8, 125.7, 106.6, 60.6, 56.6.
4.2.38. (3E,5E)-3,5-Bis(4-hydroxy-3,5-dimethoxybenzylidene)-
tetrahydropyran-4-one (E9)
ESIeMS (m/z): 415(Mþ1) þ. Anal. Calc. for C23H26O7: 414; C 66.65, H
6.32. Found: C 66.67, H 6.36 (synthesis also described in Ref. [41]).
Yield 63%. mp. 226e228 ꢂC 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 9.03 (brs, 2H, eOH), 7.58 (s, 2H, eCH¼), 6.70 (s, 4H, arom),
4.2.31. (1E,4E)-1,5-bis (2,3,4 -trimethoxyphenyl) penta-1,4-dien-3-
one (C11)
4.95 (s, 4H, eCH2eOeCH2e), 3.81 (s, 12H, eOCH3). 13C NMR (CDCl3,
75 MHz) d(ppm): 184.3, 147.8, 137.7, 135.7, 131.0, 124.6, 108.6, 67.7,
Yield 56%. mp.169-171 ꢂC. 1H NMR (CDCl3, 300 MHz)
d
ppm: 7.92
56.0. ESIeMS (m/z): 427 (M ꢃ 1) þ. Anal. Calc. for C23H24O8: 428; C
(d, J ¼ 15.9 Hz, 2H,eCH¼Ce), 7.34 (d, J ¼ 8.1 Hz, 2H,arom), 7.08 (d,
J ¼ 15.9 Hz,2H,eCH¼Ce), 6.72 (d, J ¼ 8.1 Hz, 2H, arom), 3.95
(s,6H, eOCH3), 3.91 (s,6H, eOCH3), 3.89 (s,6H, eOCH3). 13C NMR
64.48, H 5.65. Found: C 64.43, H 5.68.
4.2.39. (3E,5E)-3,5-Bis(3,4,5-trimethoxybenzylidene)
tetrahydropyran-4-one (E10)
(CDCl3, 75 MHz) d(ppm): 184.7, 152.8, 138.7, 135.3, 132.8, 129.7,
108.2, 67.6, 60.1, 56.0. ESIeMS (m/z): 415(Mþ1) þ. Anal. Calc. for
C23H26O7: 414; C 66.65, H 6.32. Found: C 66.66, H 6.35 (synthesis
also described in Ref. [46]).
Yield 61%. mp. 248e250 ꢂC. 1H NMR (CDCl3, 300 MHz)
d
ppm:7.76(s, 2H, eCH¼), 6.55 (s, 4H, arom), 4.97 (s, 4H, eCH2-
OeCH2e) 3.90 (s, 18H, eOCH3). 13C NMR (CDCl3, 75 MHz)
d(ppm):
184.7, 152.0, 139.5, 137.7, 133.8, 130.2, 108.5, 67.8, 61.7, 56.2.
ESIeMS(m/z): 457(Mþ1) þ. Anal. Calc. for C25H28O8: 456; C 65.78, H
6.18. Found: C 65.83, H 6.13.
4.2.32. (3E,5E)-3,5-bis (4-fluorobenzylidene)-4-piperidone (D1)
Yield: 78%, mp. 208-210 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 9.61 (br, 2H, eNþ-H), 7.85(s, 2H, eCH¼),7.63e7.50 (m, 4H,
arom), 7.40e7.25 (m, 4H, arom), 4.45 (s, 4H, eCH2e). 13C NMR
4.2.40. (3E,5E)-3,5-Bis(2,3,4 -trimethoxybenzylidene)
tetrahydropyran-4-one (E11)
(CDCl3, 75 MHz) d(ppm): 182.0, 163.2, 138.2, 132.6, 130.5, 128.4,
115.8, 47.4. ESIeMS (m/z): 312 (Mþ1-HCl)þ. Anal. Calc. for
C19H16ClF2NO: 347; C 65.62, H4.64. Found: C 65.56, H 4.61
(synthesis also described in Ref. [47]).
Yield 53%. mp. 162e164 ꢂC.) 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 8.00(s, 2H, eCH¼), 6.81 (d, J ¼ 8.7 Hz, 2H, arom),6.69(d,
J ¼ 8.7 Hz, 2H, arom), 4.82 (s, 4H, eCH2-OeCH2e) 3.95e3.87 (m,
18H, eOCH3). 13C NMR (CDCl3, 75 MHz)
(ppm): 184.5, 155.1, 152.9,
d
4.2.33. (3E,5E)-3,5-Bis(4-chlorobenzylidene) -4-piperidone (D2)
141.7, 132.2, 129.9, 125.6, 120.7, 107.8, 67.9, 61.4, 60.5, 55.9.
ESIeMS(m/z): 457(Mþ1) þ. Anal. Calc. for C25H28O8: 456; C 65.78, H
6.18. Found: C 65.75, H 6.14.
Yield: 80%, mp. 240-243 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 9.80 (br, 2H, eNþ-H), 7.84(s, 2H, eCH¼), 7.60e7.42 (m,
8H, arom), 4.45 (s, 4H, eCH2e). 13C NMR (CDCl3, 75 MHz)
d(ppm):
182.2, 172.0, 137.9, 134.8, 132.3, 128.9, 116.0, 43.7. ESIeMS (m/z):
344 (Mþ1-HCl)þ .Anal. Calc. for C19H16Cl3NO:379; C 59.94, H 4.24.
Found: C 59.90, H4.16 (synthesis also described in Ref. [48]).
4.2.41. (3E,5E)-3,5-Bis(4-hydroxybenzylidene)-
tetrahydrothiopyran-4-one (F4)
Yield 65%. mp. 242e245 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 9.95 (brs, 2H, eOH), 7.51 (s, 2H, eCH¼), 7.38 (d, J ¼ 8.1 Hz,
4H, arom), 6.83 (d, 8.1 Hz, 4H, arom), 3.92 (s, 4H,
eCH2eSeCH2e). 13C NMR (CDCl3, 75 MHz)
4.2.34. (3E,5E)-3,5-Bis(4-bromobenzylidene)-4-piperidone (D3)
J
¼
Yield: 83%, mp. 258e259 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
d
(ppm): 187.6, 158.þ6,
d
(ppm): 9.65 (br, 2H, eNþ-H), 7.82(s, 2H, eCH¼), 7.72 (d, J ¼ 8.4 Hz,
135.5, 132.4, 131.3, 125.6, 115.6, 29.5. ESIeMS (m/z): 323(M ꢃ 1)
.
4H, arom), 7.47 (d, J ¼ 8.4 Hz, 4H, arom), 4.44 (s, 4H, eCH2e). 13
C
Anal. Calc. for C19H16O3S: 324; C 70.35, H 4.97. Found: C 70.31, H
4.99.
NMR (CDCl3, 75 MHz) d(ppm): 182.2, 172.0, 138.0, 132.4, 128.4,
123.8, 116.1, 43.7. ESIeMS (m/z): 434 (Mþ1-HCl)þ. Anal. Calc. for
C19H16Br2ClNO:469; C 48.60, H 3.43. Found: C 48.58, H3.35
(synthesis also described in Ref. [48]).
4.2.42. (3E,5E)-3,5-Bis(4-methoxybenzylidene)-
tetrahydrothiopyran-4-one (F6)
Yield: 73%; mp. 179e181 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
4.2.35. (3E,5E)-3,5-Bis(4-hydroxybenzylidene)-4-piperidone (D4)
d
(ppm): 7.54(s, 2H, eCH¼), 7.49 (d, J ¼ 8.1 Hz, 4H, arom), 7.00
(d, J ¼ 8.1 Hz, 4H, arom), 3.96(s, 4H, eCH2eSeCH2e), 3.80 (s,
6H, eOCH3). 13C NMR (CDCl3, 75 MHz)
(ppm): 187.5, 160.3,
This compound was characterized earlier as D1 in Ref. [35].
d
4.2.36. (3E,5E)-3,5-Bis(4-hydroxybenzylidene)-tetrahydropyran-4-
one (E4)
136.5, 132.8, 131.2, 125.6, 114.3, 67.4, 29.8. ESIeMS (m/
z):353 (Mþ1) þ. Anal. Calc. for C21H20O3S : 352; C 71.56, H
Yield 75%. mp. 271e273 ꢂC 1H NMR (DMSO-d6, 300 MHz)
5.72. Found:
Ref. [48]).
C 71.52, H 5.80 (synthesis also described in
d
(ppm): 10.03 (brs, 2H, eOH), 7.55 (s, 2H, eCH¼), 7.28

X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
243
4.2.43. (3E,5E)-3,5-Bis(4-hydroxy-3,5-dimethoxybenzylidene)-
4.2.50. (2E,5E)-2,5-Bis(pyridin-3-methylene)cyclopentanone (BN2)
Yield 80%. mp. 233e235 ꢂC. 1H NMR (CDCl3, 300 MHz)
(ppm):
tetrahydrothiopyran-4-one (F9)
d
Yield 71%. mp. 149e152 ꢂC. 1H NMR (DMSO-d6, 300 MHz)
8.90e8.80 (m, 2H, arom), 8.65e8.55 (m, 2H, arom), 7.92e8.82 (m,
2H, arom),7.57(s, 2H, eCH¼), 7.42e7.32(m, 2H, arom), 3.16 (s,
d
(ppm): 8.93 (brs, 2H, eOH), 7.53 (s, 2H, eCH¼), 6.80 (s, 4H, arom),
4.03 (s, 4H, eCH2-SeCH2e), 3.80 (s, 12H, eOCH3). 13C NMR (CDCl3,
4H, eCH2-CH2-). 13C NMR (CDCl3, 75 MHz)
d(ppm): 195.3, 151.7,
75 MHz)
d
(ppm): 187.7, 147.8, 137.2, 136.2, 131.8, 124.9, 108.4, 56.0,
150.0, 138.8, 137.1, 131.5, 130.6, 123.7, 26.4. ESIeMS (m/z): 263
(Mþ1) þ. Anal. Calc. for C17H14N2O : 262; C 77.84, H 5.38. Found: C
77.83, H 5.39. (synthesis also described in Ref. [50]).
29.4. ESIeMS (m/z): 443(M ꢃ 1) þ. Anal. Calc. for C23H24O7S:444; C
62.15, H 5.44. Found: C 62.13, H 5.46.
4.2.44. (3E,5E)-3,5-Bis(3,4,5-trimethoxybenzylidene)
-tetrahydrothiopyran-4-one (F10)
4.2.51. (2E,5E)-2,5-Bis(pyridin-4-methylene)cyclopentanone (BN3)
Yield 70%. mp. 243e246 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
Yield 65%. mp. 220e222 ꢂC. 1H NMR (CDCl3, 300 MHz)
8.70 (d, J ¼ 6.3 Hz, 4H, arom), 7.49(s, 2H, eCH¼), 7.41(d, J ¼ 6.3 Hz,
d
(ppm):7.71 (s, 2H, eCH¼), 6.63(s, 4H, arom), 3.96 (s, 4H, eCH2-
4H, arom), 3.17 (s, 4H, eCH2-CH2-).13C NMR (CDCl3, 75 MHz)
SeCH2e),3.89 (s, 18H, eOCH3). 13C NMR (CDCl3, 75 MHz)
d(ppm):
d(ppm): 195.4, 150.4, 142.4, 140.8, 131.6, 124.1, 26.4. ESIeMS (m/z):
187.9, 152.8, 138.7, 135.6, 133.6, 130.0, 107.9, 60.1, 56.0, 29.3.
ESIeMS(m/z): 473(Mþ1) þ. Anal. Calc. for C25H28O7S: 472; C 63.54,
H 5.97. Found: C 63.50, H 5.92.
263 (Mþ1) þ. Anal. Calc. for C17H14N2O: 262; C 77.84, H 5.38. Found:
C 77.82, H 5.37 (synthesis also described in Ref. [50]).
4.2.52. (3E,5E)-3,5-Bis(pyridin-2-methylene)-tetrahydropyran-4-
one (EN1)
4.2.45. (3E,5E)-3,5-Bis(2,3,4 -trimethoxybenzylidene)
-tetrahydrothiopyran-4-one (F11)
Yield 74%. mp200-202 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
Yield 61%. mp.173e175 ꢂC. 1H NMR (CDCl3, 300 MHz)
d
(ppm):
8.73e8.63(m, 2H, arom), 7.75e7.65(m, 2H, arom), 7.63(s,
2H, eCH ¼ ), 7.49e7.41(m, 2H, arom), 7.23e7.15(m, 2H, arom),
7.84(s, 2H, eCH¼), 6.98 (d, J ¼ 8.7 Hz, 2H, arom), 6.69 (d,
J ¼ 8.7 Hz,2H, arom), 3.89 (s, 18H, -OCH3), 3.83 (s, 4H, eCH2-
5.31(s, 4H, eH2CeOeCH2e). 13C NMR (CDCl3, 75 MHz)
d(ppm):
SeCH2e). 13C NMR (CDCl3, 75 MHz)
d
(ppm): 187.8, 155.0, 152.6,
184.5, 152.8, 149.5, 137.6, 136.5, 131.7, 128.1, 123.3, 68.5. ESIeMS (m/
z): 279 (Mþ1) þ. Anal. Calc. for C17H14N2O2:278; C 73.37, H 5.07.
Found: C 73.39, H 5.08.
141.5, 132.6, 130.6, 126.2, 120.8, 107.6, 61.3, 60.5, 55.9, 29.3.
ESIeMS(m/z): 473(Mþ1) þ. Anal. Calc. for C25H28O7S: 472; C 63.54,
H 5.97. Found: C 63.51, H 5.96.
4.2.53. (3E,5E)-3,5-Bis(pyridin-3-methylene)-tetrahydropyran-4-
one (EN2)
4.2.46. (2E,6E)-2,6-Bis(pyridin-2-methylene)cyclohexanone (AN1)
Yield 85%. mp. 124e127 ꢂC. 1H NMR (CDCl3, 300 MHz)
Yield 71% .mp. 169e171 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
d
(ppm): 8.73e8.62 (m, 2H, arom), 7.75e7.60 (m, 4H, arom), 7.43(s,
8.65e8.55(m, 2H, arom), 8.59 (s, 2H, arom), 7.80(s, 2H, eCH ¼ ),
2H, eCH¼), 7.20e7.08 (m, 2H, arom), 3.30 (t,
J
¼
6.3 Hz,
7.67e7.57(m, 2H, arom), 7.40e7.30(m, 2H, arom), 4.94 (s,
4H, eCH2eCeCH2e), 1.85(q, J ¼ 6.3 Hz, 2H, eCeCH2eCe). 13C NMR
4H, eH2CeOeCH2e). 13C NMR (CDCl3, 75 MHz)
d(ppm): 184.7,
149.5, 148.8, 136.1, 133.9, 132.0, 130.1, 122.8, 68.5. ESIeMS (m/z):
279(Mþ1) þ; Anal. Calc. for C17H14N2O2:278; C 73.37, H 5.07. Found:
C 73.31, H 5.02 (synthesis also described in Ref. [48]).
(CDCl3, 75 MHz)
d(ppm): 191.4, 155.4, 149.5, 140.1, 136.2, 134.0,
127.1, 122.5, 28.5, 22.2. ESIeMS (m/z): 277 (Mþ1) þ. Anal. Calc. for
C18H16N2O:276; C 78.24, H 5.84. Found: C 78.28, H 5.83 (synthesis
also described in Ref. [50]).
4.2.54. (3E,5E)-3,5-Bis(pyridin-4-methylene)-tetrahydropyran-4-
one (EN3)
4.2.47. (2E,6E)-2,6-Bis(pyridin-3-methylene)cyclohexanone(AN2)
Yield 81%. Mp.140e143 ꢂC 1H NMR (DMSO-d6, 300 MHz)
Yield: 55%. mp. 182e183 ꢂC. 1H NMR (CDCl3, 300 MHz)
d
(ppm):
8.75e8.65 (m, 4H, arom), 7.72(s, 2H, eCH¼), 7.17(s, 4H, arom),
4.91(s, 4H, eH2CeOeCH2e). 13C NMR (CDCl3, 75 MHz)
(ppm):
d(ppm): 8.75e8.65 (m,2H, arom), 8.60e8.47(m, 2H, arom),
7.80e7.68(m, 4H, arom), 7.34 (s, 2H, eCH¼), 2.95 (t, J ¼ 6.3 Hz, 4H,
d
eCH2eCeCH2e), 1.85(q, J ¼ 6.3 Hz, 2H, eCeCH2eCe). 13C NMR
184.7, 150.1, 141.0, 136.9, 132.6, 124.3, 67.5. ESIeMS (m/z):
279(Mþ1) þ, Anal. Calc. for C17H14N2O2 : 278; C 73.37, H 5.07.
Found: C 73.38, H 5.09.
(CDCl3, 75 MHz) d(ppm): 189.1, 151.1, 149.3, 137.7, 137.0, 133.2, 131.6,
123.3, 28.3, 22.7. ESIeMS (m/z): 277 (Mþ1) þ. Anal. Calc. for
C18H16N2O:276; C 78.24, H 5.84. Found: C 78.23, H 5.81 (synthesis
also described in Ref. [50]).
4.2.55. (3E,5E)-3,5-Bis(pyridin-2-methylene)-tetrahydrothiopyran-
4-one (FN1)
4.2.48. (2E,6E)-2, 6-bis (pyridin-4-methylene)cyclohexanone (AN3)
Yield 82%. mp. 153e156 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
Yield 69%. mp. 148e149 ꢂC 1H NMR (DMSO-d6, 300 MHz)
8.94e8.83 (m,2H arom), 8.70e8.58 (m, 2H arom), 7.96e7.87 (m, 2H,
arom), 7.62 (s, 2H, eCH¼),7.48e7.39(m, 2H, arom), 3.80 (s,
d
(ppm): 8.72e8.60 (m, 4H, arom), 7.66(s, 2H, eCH¼), 7.32e7.18(m,
4H, arom), 2.93(t, J ¼ 6.3 Hz, 4H, eCH2eCeCH2e), 1.84(q, J ¼ 6.3 Hz,
4H, eH2CeSeCH2e). 13C NMR (CDCl3, 75 MHz)
d(ppm): 189.4, 154.1,
2H, eCeCH2eCe), 13C NMR (CDCl3, 75 MHz)
d
(ppm): 189.2, 149.þ9,
149.5, 137.5, 137.0, 131.9, 128.0, 123.4, 29.1. ESIeMS (m/z): 295
(Mþ1) þ.Anal. Calc. for C17H14N2OS: 294; C 69.36, H 4.79. Found: C
69.38, H4.74 (synthesis also described in Ref. [50]).
143.1, 139.2, 134.3, 124.1, 28.2, 22.4. ESIeMS (m/z): 277 (Mþ1)
.
Anal. Calc. for C18H16N2O:276; C 78.24, H 5.84. Found: C 78.26, H
5.87 (synthesis also described in Ref. [50]).
4.2.56. (3E,5E)-3,5-Bis(pyridin-3-methylene)-tetrahydrothiopyran-
4-one (FN2)
4.2.49. (2E,5E)-2,5-Bis(pyridin-2-methylene)cyclopentanone(BN1)
Yield 83%. mp. 195e196 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
Yield 77%. mp. 174e177 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
8.77e8.68 (m. 2H, arom); 7.75e7.65 (m, 2H, arom); 7.58e7.46 (m,
8.72e8.60 (m, 2H, arom), 8.63e8.52 (m, 2H, arom), 7.80 (s,
2H, eCH¼),7.68e7.57 (m, 2H, arom), 7.43e7.32(m, 2H, arom), 3.91
4H, arom), 7.20(s, 2H, eCH¼), 3.37 (s, 4H, eCH2eCH2e). 13C NMR
(CDCl3, 75 MHz)
d
(ppm): 197.8,155.1,150.0,141.9,136.4,131.5,127.2,
(s, 4H, eH2CeSeCH2e). 13C NMR (CDCl3, 75 MHz)
d(ppm): 189.5,
122.8, 27.2. ESIeMS (m/z):263(Mþ1) þ. Anal. Calc. for C17H14N2O:
262; C 77.84, H 5.38. Found: C 77.83, H 5.39 (synthesis also
described in Ref. [50]).
151.6, 150.1, 137.3, 136.5, 135.3, 132.2, 128.8, 29.5. ESIeMS (m/z): 295
(Mþ1) þ. Anal. Calc. for C17H14N2OS: 294; C 69.36, H 4.79. Found: C
69.38, H4.74 (synthesis also described in Ref. [48]).

244
X. Wei et al. / European Journal of Medicinal Chemistry 53 (2012) 235e245
4.2.57. (3E,5E)-3,5-Bis(pyridin-4-methylene)-tetrahydrothiopyran-
4-one (FN3)
dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (5 mg/ml
in PBS) was added to each well of the plate and incubated for 2 h.
Then the plate was centrifuged at 1000 rpm for 5 min at 4 ꢂC. After
careful removal of the medium, 0.1 ml DMSO was added to each
well. The absorbance was recorded on a microplate reader at
540 nm. The effect of different curcumin-related compounds on
growth was assessed as percent cell growth as compared to the
DMSO-treated cells. The concentration of DMSO as a solvent for the
different curcumin-related compounds was 0.1% in the culture
medium used and was without any effect on cell growth. Apoptosis
was determined by a morphological assay as described earlier
[56e58].
Yield 57%, mp. 140e142 ꢂC. 1H NMR (CDCl3, 300 MHz)
d(ppm):
9.02e8.90(m, 4H, arom), 7.70(s, 2H, eCH ¼ ), 7.50e7.38(m, 4H,
arom), 3.85 (s, 4H, eH2CeSeCH2e). 13C NMR (CDCl3, 75 MHz)
d
(ppm): 193.3, 149.5, 138.9, 136.4, 132.7, 124.0, 27.5. ESIeMS (m/z):
295 (Mþ1)þ .Anal. Calc. for C17H14N2OS: 294; C 69.36, H 4.79.
Found: C 69.35, H4.77.
4.2.58. (2E,6E)-2,6-Bis(thiophen-3-methylene)cyclohexanone (AS)
Yield: 87%. mp 139e141 ꢂC. 1H NMR (CDCl3, 300 MHz)
d (ppm):
7.93e7.84(m, 2H, arom), 7.72e7.58 (m, 4H, eCH¼, arom),
7.42e7.32(m, 4H, arom), 2.87(t, J ¼ 6.7 Hz, 4H, eCH2eCH2eCH2e),
1.78(q, J ¼ 6.7 Hz,2H, eCeCH2eC-). 13C NMR (CDCl3, 75 MHz)
4.5. Western blotting
d
(ppm): 190.0, 137.7, 134.6, 130.5, 129.5, 128.2, 125.7, 28.4, 22.2.
ESIeMS (m/z): 286 (M þ).Anal. Calc. for C16H14OS2:286; C 67.10, H
After treatment with curcumin, E10, F10, FN1 and FN2 for 24 h,
4.93. Found: C 67.13, H 4.91.
PC-3 cells were washed with ice-cold PBS and lysed with 800
lysis buffer (10 mM TriseHCl, pH 7.4, 50 mM sodium chloride,
30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100
mL of
4.2.59. (2E,5E)-2,5-Bis(thiophen-3-methylene)cyclopentanone (BS)
m
M
Yield: 73%. mp. 210e212 ꢂC. 1H NMR (CDCl3, 300 MHz)
sodium orthovandate, 2 mM iodoacetic acid, 5 mM ZnCl2, 1 mM
phenylmethylsulfonyl fluoride and 0.5% Triton-X 100). The
homogenates were centrifuged at 12,000ꢁg for 15 min at 4 ꢂC. The
protein concentration of whole cell lysates was determined with
a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Equal amounts
d
(ppm): 7.65e7.55(m, 4H, eCH¼, arom), 7.36(s, 4H, arom), 3.06(s,
4H, eCH2eCH2e). 13C NMR (CDCl3, 75 MHz)
d(ppm): 196.3, 138.2,
136.3, 129.1, 128.8, 127.0, 126.3, 26.1. ESIeMS (m/z): 272(M þ). Anal.
Calc. for C15H12OS2 : 272; C 66.14, H 4.44. Found: C 66.18, H 4.46.
(20 mg) of protein were then resolved on a 10% Criterion Precast Gel
4.2.60. (3E,5E)-3,5-Bis(thiophen-3-methylene)-tetrahydropyran-4-
one (ES)
(Bio-Rad, Hercules, CA) and transferred to a PVDF membrane using
a semi-dry transfer system. The membrane was then probed with
either anti-phosphorylated Erk1/2 (#4376, Cell Signaling Tech-
nology, Beverly, MA) or anti-phosphorylated Akt (#4501, Cell
Signaling Technology) primary antibody. After hybridization with
primary antibody the membrane was washed with Tris-buffered
saline three times, then incubated with horseradish peroxidase-
conjugated secondary antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) 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
Yield: 76%. mp. 189e192 ꢂC. 1H NMR (CDCl3, 300 MHz)
d (ppm):
7.89e7.80(m, 2H, arom), 7.72e7.58(m, 2H, arom), 7.63(s,
2H, eCH¼), 7.31(s, 2H, arom), 4.89(s, 4H, eCH2eOeCH2e). 13C NMR
(CDCl3, 75 MHz) d(ppm): 184.8, 135.9, 131.8, 130.5, 129.5, 128.1,
127.3, 67.7. ESIeMS (m/z): 288(M þ).Anal. Calc. for C15H12O2S2:288;
C 62.47, H 4.19. Found: C 62.43, H 4.23.
4.2.61. (3E,5E)-3,5-Bis(thiophen-3-methylene)-
tetrahydrothiopyran-4-one (FS)
Yield: 78%. mp. 161-163 ꢂC. 1H NMR (CDCl3, 300 MHz)
d (ppm):
for
(100 mM
6.7) at 50 ꢂC for 30 min with occasional agitation before incubating
b
-actin. The membrane was incubated in stripping buffer
7.98e7.88(m, 2H, arom), 7.70e7.60 (m, 2H, arom), 7.58(s,
b
-mercaptoethanol, 2% SDS, and 62.5 mM TriseHCl at pH
2H, eCH¼), 7.39(s, 2H, arom), 3.99(s, 4H, eCH2-SeCH2e). 13C NMR
(CDCl3, 75 MHz) d(ppm): 187.8, 136.2, 132.3, 129.7, 129.4, 129.1,
in blocking buffer and re-probing using anti-
Biotechnology, Santa Cruz, CA).
b-actin (Santa Cruz
127.0, 29.3. ESIeMS (m/z): 304(M þ).Anal. Calc. for C15H12OS3: 304;
C 59.18, H 3.97. Found: C59.15, H 3.94.
Acknowledgment
4.3. Cell culture and reagents
This work was financially supported by grants from the National
Natural Science Foundation of PR China, the Guangdong Provincial
Foundation for the third batch of 211 key projects, and by depart-
mental funds from the Department of Chemical Biology in the
Ernest Mario School of Pharmacy at Rutgers University. The authors
thank Ms. Annette Dionisio for her excellent help in the preparation
of this manuscript.
PC-3, Panc-1 and HT-29 cells were obtained from the American
Type Culture Collection (ATCC, Rockville, MD, USA). RPMI-1640 and
DMEM tissue culture medium, penicillin-streptomycin, L-glutamine
and fetal bovine serum (FBS) were from Gibco (Grand Island, NY).
PC-3 cells were maintained in RPMI-1640 culture medium, and
Panc-1 and HT-29 cells were maintained in DMEM medium. Both
RPMI-1640 and DMEM media were supplemented with 10% FBS,
penicillin (100 units/ml)-streptomycin (100
mg/ml) and L-glutamine
(300
m
g/ml). Cultured cells were grown at 37 ꢂC in a humidified
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