Synthesis and cytotoxic potential of heterocyclic cyclohexanone analogues of curcumin

Article abstract of DOI:10.1016/j.bmc.2010.07.063

A series of 18 heterocyclic cyclohexanone analogues of curcumin have been synthesised and screened for their activity in both adherent and non-adherent cancer cell models. Cytotoxicity towards MBA-MB-231 breast cancer cells, as well as ability to inhibit NF-κB transactivation in non-adherent K562 leukemia cells were investigated. Three of these analogues 3,5-bis(pyridine-4-yl)-1- methylpiperidin-4-one B1, 3,5-bis(3,4,5-trimethoxybenzylidene)-1- methylpiperidin-4-one B10, and 8-methyl-2,4-bis((pyridine-4-yl)methylene)-8-aza- bicyclo[3.2.1]octan-3-one C1 showed potent cytotoxicity towards MBA-MB-231, MDA-MB-468, and SkBr3 cell lines with EC₅₀ values below 1 μM and inhibition of NF-κB activation below 7.5 μM. The lead drug candidate, B10, was also able to cause 43% of MDA-MB-231 cells to undergo apoptosis after 18 h. This level of activity warrants further investigation for the treatment of ER-negative breast cancer and/or chronic myelogenous leukemia as prototypical cellular models for solid and liquid tumors.

Full text of DOI:10.1016/j.bmc.2010.07.063

Bioorganic & Medicinal Chemistry 18 (2010) 6701–6707
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cytotoxic potential of heterocyclic cyclohexanone analogues
of curcumin
Babasaheb Yadav a, Sebastien Taurin a, Rhonda J. Rosengren a, Marc Schumacher b, Marc Diederich b,
Tiffany J. Somers-Edgar a, Lesley Larsen c,
*
a Department of Pharmacology & Toxicology, University of Otago, Dunedin, New Zealand
b Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Luxembourg
c The New Zealand Institute for Plant and Food Research Ltd, Dunedin, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 18 heterocyclic cyclohexanone analogues of curcumin have been synthesised and screened for
their activity in both adherent and non-adherent cancer cell models. Cytotoxicity towards MBA-MB-231
breast cancer cells, as well as ability to inhibit NF-jB transactivation in non-adherent K562 leukemia cells
were investigated. Three of these analogues 3,5-bis(pyridine-4-yl)-1-methylpiperidin-4-one B1, 3,5-
bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidin-4-one B10, and 8-methyl-2,4-bis((pyridine-4-
yl)methylene)-8-aza-bicyclo[3.2.1]octan-3-one C1 showed potent cytotoxicity towards MBA-MB-231,
Received 18 June 2010
Revised 21 July 2010
Accepted 27 July 2010
Available online 1 August 2010
Keywords:
MDA-MB-468, and SkBr3 cell lines with EC50 values below 1
lM and inhibition of NF-jB activation below
Curcumin analogues
MDA-MB-231 cells
ER-negative breast cancer
7.5 M. The lead drug candidate, B10, was also able to cause 43% of MDA-MB-231 cells to undergo apop-
l
tosis after 18 h. This level of activity warrants further investigation for the treatment of ER-negative
breast cancer and/or chronic myelogenous leukemia as prototypical cellular models for solid and liquid
NF-jB activation
tumors.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
min itself has limited clinical efficacy due to its low bioavailability
and stability in physiological media,3 it has been the subject of
Breast cancer is the most prevalent form of cancer diagnosed in
women, and its incidence is steadily rising.1 Although there are
effective treatments for estrogen receptor (ER)-positive breast can-
cers unfortunately for ER-negative breast cancers, which account
for approximately 30% of all breast cancer diagnoses, treatments
are limited. Those cancers that overexpress Her-2 can now be trea-
ted with trastuzumab (Herceptin) immunotherapy, however, there
is still a need for more drug treatments for ER-negative breast can-
cers that either overexpress or lack Her-2.
In the search for effective drugs for ER-negative breast cancer
several natural product lead compounds have been identified. Cur-
cumin (diferuloylmethane), the primary bioactive compound iso-
lated from the rhizome of turmeric (Curcuma longa Linn.), is of
particular interest.2 Curcumin has long been known as a chemo-
preventative and chemotherapeutic agent, and in vivo studies have
demonstrated decreased tumorigenesis of many organs, including
the mammary gland.2–8 In vitro studies have also demonstrated
that curcumin has potent cytotoxicity towards many cell lines
including ER-negative human breast cancer cells.9–15 Whilst curcu-
many analogue studies.16–20 From these studies it has been shown
that cyclohexanone analogues of curcumin have enhanced activity
and stability in biological medium compared to curcumin.19 The
cyclohexanone-containing curcumin derivative 2,6-bis((3-meth-
oxy-4-hydroxyphenyl)methylene)-cyclohexanone (BMHPC) A13
was found to be cytotoxic towards ER-negative breast cancer cells
(EC50 of 5.0 l
M),21 although bioavailability and in vivo efficacy
were still problematic. More recently several fluorinated cyclohex-
anone derivatives have been prepared, one of which has shown po-
tent cytotoxicity towards MDA-MB-231 cells (EC50 of 0.8 l
M).16,22
Recently we have reported that the two pyridine analogues, 2,6-
bis(pyridin-4-ylmethylene)-cyclohexanone, A1 and 2,6-bis(pyri-
din-3-ylmethylene)-cyclohexanone, A2 are cytotoxic towards
MDA-MB-231 cells with EC50s of 1.10 and 1.54 l
M, respectively).23
In order to investigate structure–activity relationships of these po-
tent analogues, and in the search for analogues with even greater
activity, we needed to assay a wider range of compounds. We chose
to concentrate on nitrogen heterocycles, since these have the advan-
tage of their ability to exist in both a protonated and neutral form,
allowing both, solubility in aqueous media, as well as the potential
to cross cellular membranes. Our laboratory (Rosengren) uses the
aggressive ER-negative MDA-MB-231 human breast cancer cells,
amongst others, to identify new potent drugs. Our collaborators
(Diederich) use the inhibition of the nuclear transcription factor
* Corresponding author. Address: Plant & Food Research, Department of Chem-
istry, Cnr Union and Cumberland Streets, University of Otago, Dunedin, New
Zealand. Tel.: +64 3 479 7922; fax +64 3 479 7906.
E-mail address: lesley.larsen@plantandfood.co.nz (L. Larsen).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.063

6702
B. Yadav et al. / Bioorg. Med. Chem. 18 (2010) 6701–6707
j
B (NF-
ery for ER-negative breast cancer as it is over expressed in these tu-
mors.24 As NF-
B is responsible for increased cell resistance towards
possible cytocidal treatments currently applied in clinics, apoptosis
induction through NF- B inhibition and/or extrinsic and intrinsic
pathway activation is considered as a main aim in cancer research.
NF- B has largely been proven to be implicated in cell death (apop-
j
B) activation. NF-
j
B is an important target in drug discov-
ported compounds are described in Section 4. 13C NMR data are
presented in Table 1.
j
2.2. Cytotoxicity of curcumin analogues towards MDA-MB-231
cells and inhibition of NF-jB in K562 cells
j
j
Cytotoxicity of the analogues towards MDA-MB-231 cells as
well as inhibition of NF- B activity in K562 cells was determined
and the results presented in Table 2. The results demonstrated that
there were 12 analogues with a greater or similar NF- B inhibition
tosis), cell adhesion, cell proliferation, innate- and adaptive-im-
j
mune response and cancer development.25,26
This work describes the preparation and biological assessment
of a series of 18 heterocyclic cyclohexanone curcumin analogues,
in both adherent and non-adherent cancer cell models. Curcumin,
A10, A12, A13 and the two previously studied heterocyclic ana-
logues A1 and A2 were included for comparison purposes. All com-
pounds were initially screened for their cytotoxicity towards ER
j
compared to curcumin and 12 analogues that were more cytotoxic
towards MDA-MB-231 cells than curcumin. Of these, five ana-
logues (A1, B1, B10, B12, and C1) showed potent cytotoxicity
(EC50 values of ꢀ1
lM) and potent NF-jB inhibition (IC50 values
of <7.5 M).
l
negative breast cancer cells as well as their inhibition of NF-
jB
in leukemia cells. By including the inhibition of NF- B we have en-
j
2.3. Structure–activity relationship analysis
sured that only drugs with a dual potency in both adherent and
non-adherent cellular models underwent further testing.
We have previously demonstrated that the two pyridine ana-
logues, 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone, A1 and
2,6-bis(pyridin-3-ylmethylene)-cyclohexanone, A2 were cytotoxic
towards MDA-MB-231 cells.23 In an extension of this work we
wanted to explore the structural requirements for activity, in par-
ticular in relation to other heterocyclic analogues with differing
electronic effects. Previous studies have reported that the six-
membered cyclohexanone ring system is in general superior to
the five-membered cyclopentanone system for inhibitory activity
in a group of seven cancer cell lines.19 Evidence that this was also
the case for MBA-MB-231 cells was found when the five-mem-
bered cyclopentanone derivative D2 was compared with cyclohex-
anone A2 and showed a fivefold decrease in activity. We therefore
decided to restrict our investigation to six-membered ring ketones.
2. Results and discussion
2.1. Chemistry
A series of heterocyclic analogues of curcumin containing a cyc-
lic ketone as a core, with aromatic groups linked to the core via two
methylidene groups (Fig. 1) were prepared. Series A contain a
cyclohexanone core; series B, an N-methylpiperidone core; series
C a tropinone core and series D a cyclopentanone core unit. The
aromatic groups include fluorine substituted pyridines, since there
are examples of fluorine substitution of aromatics giving enhanced
activity.16 We also included the electron rich five-membered aro-
matic units of pyrrole, imidazole and indole, as well as electron
rich trimethoxyphenyl and two dimethoxyphenyl groups because
there has been some evidence that electron rich aromatics can
have enhanced activity.27 Structures are shown in Figure 2. The
synthesis of compounds was carried out via condensation of two
equivalents of the appropriate aldehyde with the core cyclic ketone
In general it was found that the inhibition of NF-jB activation par-
alleled cytotoxicity against MBA-MB-231 cells, so we have exam-
ined structure–activity relationships mainly in terms of the
cytotoxicity against MBA-MB-231 cells. Concentrating initially on
the cyclohexanone core A series we then examined fluorine substi-
tuted pyridine rings. Two examples A3 and A4 were readily pre-
pared from commercially available starting materials. These
derivatives were found to be 2–3 times less active than the parent
pyridine compounds A1 and A2. We next turned our attention to
replacement of the pyridine ring with different heterocyclic rings.
Thiophene A5, N-methylpyrrole A6, N-methylindole A7 and 4-
substituted N-methylimidazole A9 exhibited no activity
following the procedures of Liang et al.28 and Vatsadze et al.29 1
H
NMR spectroscopy showed that the compounds were all isomeri-
cally pure. It is known30 that the Z isomers of @CH are character-
ized by chemical shifts at dꢀ6.8 ppm, whereas the signals for the
E isomers should appear at higher field than 7.2 ppm. For all these
compounds @CH signals in the 7.5–8.5 ppm region indicated the E
configuration. Additionally X-ray crystallography of 8-methyl-2,4-
bis(4-methoxybenzylidene)-8-azabicyclo[3.2.1]octan-3-one,31 3,
5-bis(benzylidene)-1-methyl-4-piperidones, and 3,5-bis(benzyli-
dene)-cyclohexanones29 have all shown that the olefinic double
bonds adopt the E configuration. Hence, the assumption was made
that all of the compounds were E,E isomers. The synthetic yields,
melting points, elemental analyses, 1H NMR, and ESI-MS of unre-
(EC50 >30
cytotoxicity towards MBA-MB-231 cells, but no inhibition of NF-
B activation. We also replaced the pyridyl groups with electron
lM). 2-Substituted N-methylimidazole A8 showed good
j
rich trimethoxyphenyl and dimethoxyphenyl to give analogues
A10 and A12 but neither analogue showed any activity in either
assay.
For the N-methylpiperidone core series B analogues, the hetero-
aromatic analogues generally had similar or slightly increased
activity over cyclohexanone core, series A analogues. Thus 4-pyri-
dyl B1 showed some increased activity over A1, the 3-pyridyl ana-
logue B2 slightly less activity than the corresponding A2, and
thiophene B5 retained the lack of activity of A5. Surprisingly N-
methylimidaz-2-yl analogue B8, showed no activity compared to
A8 which showed good activity. In contrast the tri- and dimethoxy-
phenyl B series derivatives showed good activity compared to the
A series where little activity was observed. In fact the trimethoxy-
phenyl analogue B10 exhibited a level of activity about three times
greater than any other compound examined. The dimethoxyphenyl
analogues were also active, with B11 being less active than B12,
further indicating that the fluorine group conferred no improve-
ment in activity in these compounds.
7
3
8
8
7
X
X
+
Ar-CHO
5
4
2
Ar
Ar
6
1
O
1'
O
series A X = (CH2)n n=1
series B X = NMe
series C X = NMe 7,8 = CH2-CH2
series D X = (CH2)n n=0
Figure 1. Generic structure of curcumin analogues.

B. Yadav et al. / Bioorg. Med. Chem. 18 (2010) 6701–6707
6703
Me
N
Me
N
R
R
R
R
R
R
R
R
O
O
O
O
series A
series B
series C
series D
F
F
N
N
S
N
N
R =
5
4
1
2
3
Me
N
NMe
NMe
NMe
N
N
6
7
9
8
OMe
OMe
OMe
MeO
MeO
F
HO
MeO
MeO
MeO
12
11
13
10
Figure 2. Structures of the heterocyclic curcumin analogues.
Table 1
13C NMR data for heterocyclic curcumin analoguesa
Compound
A3b
A4b
A7
A8
A9
B8
B10
B11b
B12
C1
C2
C10
Position
1
2 and 6
3 and 5
4
188.35
140.79
28.38
188.55
139.4
28.34
22.58
188.09
134.18
28.62
190.23
138.36
28.45
188.71
131.53
29.39
187.39
135.28
57.22
—
186.61
132.40
56.96
—
186.01
133.17
57.06
—
186.83
133.4
57.06
—
187.14
142.33
60.88
—
187.05
140.23
61.00
—
187.28
138.98
61.01
—
22.31
21.65
21.63
22.48
7 and 8
29.99
133.44
36.12
30.11
132.69
36.09
30.38
136.97
35.69
10
127.46
128.32
133.69
130.37
130.35
130.76
46.02
136.71
43.61
129.68
45.47
132.34
45.58
4-NMe
Ar-1
2
3
4
5
6
7
8
9
NMe
OMe
OMe
OMe
131.03
138.81
156.94
—
145.41
124.06
118.67
161.08
—
147.62
121.11
140.85
129.25
—
140.16
—
144.41
—
122.79
119.00
—
128.95
127.59
—
136.56
112.59
122.83
120.64
119.52
109.35
33.29
143.83
—
123.16
117.79
—
130.73
107.93
153.12
139.18
153.12
107.93
114.08
155.76
100.19
151.07
145.01
112.62
125.31
152.89
111.8
114.76
152.89
116.34
141.62
123.97
150.16
—
150.16
123.97
130.87
150.81
—
149.63
123.45
137.06
130.52
107.72
153.10
137.51
153.10
107.72
121.26
31.95
33.25
33.30
56.26
56.26
60.97
56.56
56.21
56.11
55.83
56.12
56.12
60.97
a
Non-systematic numbering has been used to facilitate comparisons between compounds. Numbering is based on numbering shown in Figure 1. Assignments have been
determined by HSQC and HMBC 2D NMR experiments.
b
13C–19F coupling not included.
For the tropinone core, series C analogues which have a more ri-
gid structure, as well as being more sterically hindered, we found
either no change in activity, as in the case of the 4-pyridyl com-
pound C1, or else reduced activity, as seen for compounds C2 or
C10.
In summary, we have found that pyridine heteroaromatic
substituted bismethylene cyclohexanones are the most active of
the heteroaromatic analogues tested to date, and that the five-
membered heteroaromatic in general have no activity in this cell
line. Replacement of the cyclohexanone core with N-methylpiperi-
done can give some improvement in activity for the heteroaro-
matic analogues, although this varied. In contrast the
polymethoxyphenylmethylene N-methylpiperidone derivatives
had good activity compared to the cyclohexanone core derivatives
which had no activity. The use of the more sterically hindered trop-
inone led to equal to or reduced activity in all cases. Additionally,
fluorine substituents on the aromatic groups do not increase the
activity of these analogues. These results suggest that activity in
this cell line is not determined by the electronic effects of the aro-
matic groups. It also appears that a nitrogen heteroatom in either
the aromatic group or in the core cyclic ketone provides analogues
with good activity.

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B. Yadav et al. / Bioorg. Med. Chem. 18 (2010) 6701–6707
Table 2
IC50/cytotoxicity EC50 between both curcumin and A1 cytotoxicity
in the SkBr3 cell line and NF- B inhibitory activity and also be-
tween cytotoxicity of B12 in all cell lines and NF- B inhibition. This
Cytotoxicity and inhibition of NF-jB activation
j
j
Compound
MDA-MB-231
EC50 M)
NF-
IC50
j
(lM)
B activation
(
l
indicates that other factors are involved in the cytotoxicity.
Curcumin
A1
A2
A3
A4
A5
A6
A7
7.6
1.1
1.5
3.3
3.2
>30
>30
>30
1.9
>10
>30
>10
2.6
0.8
2.1
>30
>30
0.3
3.8
1.1
1.1
12.9
>30
8.6
15
5
2.5 0.2
2.5 0.5
2.2 0.3
3.8 0.5
>200
>200
140 20
48
>200
Not tested
>200
10
1.0 0.3
0.8 0.2
>200
2.5. Induction of apoptosis
To determine mechanisms for the cytotoxicity elicited, the two
analogues with the overall greatest potency (B1 and B10) were
examined for their ability to induce apoptosis. The results (Fig. 3)
showed that the two analogues had differing abilities to induce
A8
A9
7
apoptosis in MDA-MB-231 cells. B10 (1
increase in the proportion of apoptotic cells versus control. Treat-
ment of MDA-MB-231 cells with B1 (2 M) induced significant
lM) caused a significant
A10
A12
A13 (BMHPC)
B1
B2
B5
l
3
but much lower apoptosis versus control. Furthermore, B10 had
a higher apoptotic potency compared to A1 and A2, as we have
previously shown that these analogues at concentration of
2–3 lM caused less than 20% of MDA-MB-231 cells to undergo
B8
47
3
apoptosis following 18 h of treatment.23 However, A1 is a stronger
inducer of apoptosis than B1, as A1 exhibited a peak apoptotic
induction of ꢀ40% of cells after 36 h,23 compared to both A2 and
B1 which showed less than 20% of cells undergoing apoptosis at
this time-point. Therefore, B10 displays the greatest apoptotic
induction potential of all the compounds examined.
B10
B11
B12
C1
C2
C10
D2
0.9 0.1
4.0 0.5
6.3 0.3
1.5 0.2
4.0 0.5
>200
34
5
3. Conclusions
2.4. Cytotoxicity of curcumin analogues in MDA-MB-468 and
SKBr3 cells
In this study, a series of 18 heterocyclic cyclohexanone analogues
of curcumin together with two previously studied analogues A1 and
A2 and three non-heterocyclic analogues of curcumin were synthes-
The analogues which showed potent cytotoxicity (EC50 values of
ised and evaluated for inhibitory activity against NF-jB transactiva-
ꢀ1
lM) and potent NF-jB inhibition (IC50 values of <7.5 lM) were
tion in non-adherent K562 leukemia cells as well as cytotoxicity by
using ER-negative breast cancer cell models. A structure–activity
relationship study showed that the more electron poor pyridine het-
eroaromatics were the most active of those tested to date, and that
the electron rich pyrrole based structures in general, had no activity
in this cell line. The N-methylpiperidone analogues did show some
improvement in activity over cyclohexanone analogues, although
this was not consistent. The more sterically hindered tropinone ana-
logues showed reduced activity in all cases, indicating that bulky
groups in this area of the molecule led to decreased activity.
Additionally, the incorporation of a fluorine substituent into the aro-
matic rings did not increase the activity of these compounds. Three
of these analogs 3,5-bis(pyridine-4-yl)-1-methylpiperidin-4-one
B1, 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidin-4-one
B10 and 8-methyl-2,4-bis((pyridine-4-yl)methylene)-8-aza-bicy-
clo[3.2.1]octan-3-one C1 showed potent cytotoxicity towards
MBA-MB-231, MDA-MB-468, and SkBr3 cell lines with EC50 values
further examined for their ability to elicit cytotoxicity in other ER-
negative cell lines. The results are presented in Table 3. A1, B1,
B10, B12, and C1 were examined in cell lines that both contained
(SkBr3) and lacked (MDA-MB-468) the Her-2, isoform of the epi-
dermal growth factor receptor. All five compounds were found to
be more potent than curcumin towards these two breast cancer
cell lines, as all elicited EC50 values that were 2- to 30-fold lower
than that for curcumin. In the SkBr3 cell line A1 showed the great-
est cytotoxicity (EC50 0.2
(0.6 M) whereas in the MDA-MB-468 cell line compound B10
(EC50 0.3 M) showed the greatest cytotoxicity, followed by B1
(0.5 M) and C1 (0.6 M). Within the three cell lines, curcumin
lM), followed by B10 (0.4 lM) and B1
l
l
l
l
and A1 showed a variation in activity between the different cell
lines, with the greatest activity in the SkBr3 cell line, whilst B1,
C1, B10, and B12 showing similar activity in all three cell lines.
Looking at comparisons between NF-
cytotoxicity for these five compounds, in general these do seem
to be related, with the ratio of the NF- B inhibitory activity IC50
jB inhibitory activity and
j
/
cytotoxicity EC50 being similar for four of the five compounds with
both MDA-MB-231 and MDA-MB-468 cell lines. This implies that
reduction in the level of the cell signalling protein, NF-
jB, is at
least in part related to the cytotoxicity of these analogues. There
are however differences in the ratio of NF-jB inhibitory activity
Table 3
EC50
cells
(lM) values of curcumin analogues in other ER-negative human breast cancer
Compound
MDA-MB-468 EC50
(lM)
SKBr3 EC50 (lM)
Curcumin
A1
B1
B10
B12
C1
9.7
1.9
0.5
0.3
1.1
0.6
2.4
0.2
0.6
0.4
1.1
0.7
Figure 3. Apoptosis following treatment of MDA-MB-231 cells with B1 and B10.
*
Significantly different from control p <0.001.

B. Yadav et al. / Bioorg. Med. Chem. 18 (2010) 6701–6707
6705
below 1
l
M and inhibition of NF-
j
B activation below 7.5
l
M. The
2H), 7.70 (s, 2H), 8.45 (d, J = 5 Hz, 2H), 8.53 (d, J = 1.5 Hz, 2H);
HRMS (+ve ESI) calcd for C18H14F2N2NaO 335.0966 m/z [MNa+],
found 335.0957 m/z.
lead drug candidate, B10 was also able to cause 43% of MDA-MB-
231 cells to undergo apoptosis after 18 h.
However, only B10 was more potent at eliciting apoptosis than
curcumin, BMHPC (A13), and our previously studied cyclohexa-
none curcumin derivatives A1 and A2.32 This level of activity war-
rants further investigation for the treatment of ER-negative breast
cancer and/or chronic myelogenous leukemia as prototypical cellu-
lar models for solid and liquid tumors.
4.1.2.2. (2E,6E)-2,6-Bis((2-fluoropyridine-3-yl)methylene)cyclo-
hexanone (A4). From cyclohexanone and 2-fluoro-3-pyridine-car-
boxaldehyde following general procedure A, yellow solid, 58%
yield; mp 145–146 °C. Anal calcd for C18H14F2N2O 23 H2O: C, 66.66;
H, 4.77; N, 8.64. Found: C, 66.64; H, 4.64; N, 8.70. 1H NMR (CDCl3)
d: 1.82 (quin, J = 6 Hz, 2H), 2.80 (t, J = 6 Hz, 4H), 7.24 (m, 2H), 7.73
(s, 2H), 7.79 (m, 2H), 8.21 (d, J = 5 Hz, 2H); HRMS (+ve ESI) calcd for
4. Experimental section
4.1. Chemical synthesis
C
18H14F2N2NaO 335.0966 m/z [MNa+], found 335.0973 m/z.
4.1.2.3. (2E,6E)-2,6-Bis((1-methyl-1H-imidazol-5-yl)methylene)
cyclohexanone (A7). From cyclohexanone and 1-methyl-5-imidaz-
ole-carboxaldehyde following general procedure B, yellow solid,
68% yield; mp >220 °C. Anal calcd for C16H18N4O: C, 68.06; H,
6.43; N, 19.84. Found: C, 67.95; H, 6.53; N, 20.15. 1H NMR (CDCl3)
d: 1.92 (quin, J = 6 Hz, 2H), 2.83 (t, J = 6 Hz, 4H), 3.73 (s, 6H, NMe),
7.38 (s, 2H), 7.57 (s, 2H), 7.63 (s, 2H); HRMS (+ve ESI) calcd for
Melting points were determined on a Mettler Toledo FP62 melt-
ing block and were uncorrected. High resolution mass spectrome-
try was recorded using a VG70-250S double focusing magnetic
sector mass spectrometer. NMR spectra, at 25 °C, were recorded
at 500 MHz for 1H and 125 MHz for 13C on Varian INOVA-500
spectrometer. Chemical shifts are given in ppm on the d scale ref-
erenced to the solvent peaks CHCl3 at 7.26 and CDCl3 at 77.00.
Cyclohexanone, N-methylpiperidone, tropinone, pyridine-3-carbox
aldehyde, pyridine-4-carboxaldehyde, 2-fluoropyridine-3-carbox-
aldehyde, 2-fluoropyridine-4-carboxaldehyde, thiophen-2-carbox
aldehyde, 1-methylpyrrole-2-carboxaldehyde, 1-methylindole-3-
carboxaldehyde, 1-methylimidazole-2-carboxaldehyde, 1-methyl-
imidazole-5-carboxaldehyde, 3,4,5-trimethoxybenzaldehyde, 2-flu
oro-4,5-dimethoxybenzaldehyde, and 2,5-dimethoxybenzaldehyde
were all purchased from the Aldrich Chemical Company. 2,6-
bis(pyridin-4-ylmethylene)-cyclohexanone (A1), 2,6-bis(pyridin
-3-ylmethylene)-cyclohexanone (A2), 2,6-bis(thiophen-2-ylmeth-
ylene)-cyclohexanone (A5) and 2,6-bis((3,4,5-trimethoxyphenyl)
methylene)-cyclohexanone (A10), were prepared following the
method of Bhagat et al.33 2,6-Bis((1-methyl-1H-pyrrol-2-yl)methy-
lene)-cyclohexanone (A6) was prepared as described by Liang et
al.19 2,6-Bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexa-
none (A13) was prepared as described previously.34 1-Methyl-
3,5-bis[(E)-(4-pyridyl)methylidene]-4-piperidone (B1), 1-methyl
-3,5-bis[(E)-(3-pyridyl)methylidene]-4-piperidone (B2), 1-methyl-
3,5-bis[(E)-(2-thienyl)methylidene]-4-piperidone (B5) and 2,5-
bis(3-pyridylmethylene)-cyclopentanone (D2) were prepared
according to the method of Vatsadze et al.29
C
16H19N4O: 283.1553 m/z [MH+], found: 283.1560 m/z.
4.1.2.4. (2E,6E)-2,6-Bis((1-methyl-1H-imidazol-2-yl)methylene)
cyclohexanone (A8). From cyclohexanone and 1-methyl-2-imid-
azole-carboxaldehyde following general procedure B, yellow solid,
68% yield; mp 217–219 °C. Anal calcd for C16H18N4O: C, 68.06; H,
6.43; N, 19.84. Found: C, 67.81; H, 6.52; N, 20.04. 1H NMR (CDCl3)
d: 1.86 (quin, J = 6 Hz, 2H), 3.35 (t, J = 6 Hz, 4H), 3.78 (s, 6H, NMe),
6.97 (s, 2H), 7.26 (s, 2H), 7.54 (s, 2H); HRMS (+ve ESI) calcd for
C
16H19N4O: 283.1553 m/z [MH+], found: 283.1560 m/z
4.1.2.5. (2E,6E)-2,6-Bis((1-methyl-1H-indol-3-yl)methylene)
cyclohexanone (A9). From cyclohexanone and indole-3-carboxal-
dehyde following general procedure B, orange yellow solid, 23%
yield; mp >220 °C. Anal calcd for C26H24N2O.H2O: C, 78.24; H,
6.58; N, 7.03. Found: C, 78.24; H, 6.32; N, 6.95. 1H NMR (CDCl3)
d: 1.96 (quin, J = 6 Hz, 2H), 2.87 (br t, J = 6 Hz, 4H), 3.85 (s, 6H,
NMe), 7.25 (m, 2H), 7.31 (m, 2H), 7.34 (m, 2H), 7.35 (s, 2H) 7.94
(d, J = 7.5 Hz, 2H), 8.24 (s, 2H);HRMS (+ve ESI) calcd for
C
26H25N2O 381.1961 m/z [MH+], found 381.1978 m/z.
4.1.2.6. (3E,5E)-1-Methyl-3,5-bis((1-methyl-1H-imidazol-2-yl)
methylene)piperidin-4-one (B8). From 1-methylpiperidone and
1-methyl-2-imidazole-carboxaldehyde following general proce-
dure B, orange yellow solid, 63% yield; mp 196–198 °C. Anal calcd
for C16H19N5O: C, 64.63; H, 6.44; N, 23.55. Found: C, 64.58; H, 6.50;
N, 23.69. 1H NMR (CDCl3) d: 2.58 (s, 3H, NMe), 3.80 (s, 6H,
2 ꢁ NMe), 4.15 (s, 4H), 6.99 (s, 2H), 7.28 (s, 2H), 7.51 (s, 2H);. HRMS
4.1.1. General procedure A29
To a mixture of the appropriate ketone (0.75 mmol) and alde-
hyde (1.50 mmol) in ethanol (5 ml) was added sodium hydroxide
(1 M in water, 1 ml) and the mixture was stirred for 18 h at room
temperature. The resulting precipitate was removed by filtration,
washed with cold ethanol, and purified by recrystallisation from
ethanol.
(+ve ESI) calcd for
C
16H20N5O: 298.1662 m/z [MH+], found
298.1662 m/z.
4.1.2. General procedure B28
4.1.2.7. (3E,5E )-3,5-Bis(3,4,5-trimethoxybenzylidene)-1-meth-
ylpiperidin-4-one (B10)35. From 1-methylpiperidone and 3,4,5-
trimethoxybenzaldehyde following general procedure A, orange
yellow solid 35% yield; mp 151–152 °C. Anal calcd for
To a mixture of the appropriate ketone (0.75 mmol) and alde-
hyde (1.50 mmol) in methanol (10 ml) was added sodium methox-
ide (5 M in methanol, 0.15 ml) and the mixture was stirred for 18 h
at room temperature. The resulting precipitate was removed by fil-
tration then washed with cold methanol and purified by recrystal-
lisation from ethanol.
C
26H31NO7 1 H2O: C, 65.67; H, 6.71; N, 2.95. Found: C, 65.70; H,
3
6.61; N, 3.00. 1H NMR (CDCl3) d: 2.48 (s, 3H, NMe), 3.80 (s, 4H),
3.89 (s, 12H, 4 ꢁ OMe), 3.90 (s, 6H, 2 ꢁ OMe), 6.63 (s, 4H), 7.75
(s, 2H); HRMS (+ve ESI) calcd for C26H32NO7 470.2173 m/z [MH+],
found 470.2161 m/z.
4.1.2.1. (2E,6E)-2,6-Bis((2-fluoropyridine-4-yl)methylene)cyclo-
hexanone (A3). From cyclohexanone and 3-fluoro-4-pyridine-car-
boxaldehyde following general procedure A, yellow solid, 12%
yield; mp 152–153 °C. Anal calcd for C18H14F2N2O 14 H2O: C, 68.24;
H, 4.61; N, 8.84. Found: C, 68.28; H, 4.59; N, 8.72. 1H NMR (CDCl3)
d: 1.82 (quin, J = 6 Hz, 2H), 2.80 (t, J = 6 Hz, 4H), 7.27 (t, J = 6 Hz,
4.1.2.8. (3E,5E)-3,5-Bis(2-fluoro-4,5-dimethoxybenzylidene)-1-
methylpiperidin-4-one (B11). From 1-methylpiperidone and 2-
fluoro-4,5-dimethoxybenzaldehyde following general procedure
A, orange yellow solid, 41% yield; mp 189–191 °C. Anal calcd for
C24H25F2NO5: C, 64.71; H, 5.66; N, 3.14. Found: C, 64.77; H, 5.85;

6706
B. Yadav et al. / Bioorg. Med. Chem. 18 (2010) 6701–6707
N, 2.97. 1H NMR (CDCl3) d: 2.42 (s, 3H, NMe), 3.65 (s, 4H), 3.87 (s,
6H, 2 ꢁ OMe), 3.90 (s, 6H, 2 ꢁ OMe), 6.68 (d, J = 12 Hz, 2H), 6.76 (d,
J = 7 Hz, 2H), 7.82 (s, 2H); HRMS (+ve ESI) calcd for C24H26F2NO5
446.1774 m/z [MH+], found 446.1782 m/z.
4.2.2. Cytotoxicity assays
Human breast cancer cells were seeded in 12-well plates
(7 ꢁ 104 cells/well) in 1 ml DMEM/HamF12 supplemented with
5% FBS, 100 U/ml penicillin, 100 lg/ml streptomycin, 25 ng/ml
amphotericin B and 2.2 g/l NaHCO3, and incubated for 24 h at
37 °C. For dose–response assays, cells were treated with a range
of concentrations of curcumin, or analogues for 5 days. Vehicle
control cells were treated with DMSO (0.1%). Cell number in each
well was determined using the sulforhodamine B (SRB) assay.36
The concentration of each compound required to decrease the cell
number by 50% of vehicle control (EC50) was determined by non-
linear regression using Prism software.
4.1.2.9. (3E,5E)-3,5-Bis(2,5-dimethoxybenzylidene)-1-methylpi-
peridin-4-one (B12). From 1-methylpiperidone and 2,5-dime-
thoxybenzaldehyde following general procedure A, orange yellow
solid, 66% yield; mp 133–134 °C. Anal calcd for C24H27NO5: C,
70.40; H, 6.65; N, 3.42. Found: C, 70.38; H, 6.81; N, 3.29. 1H NMR
(CDCl3) d: 2.38 (s, 3H, NMe), 3.66 (s, 4H), 3.78 (s, 6H, 2 ꢁ OMe),
3.81 (s, 6H, 2 ꢁ OMe), 6.76 (d, J = 3 Hz, 2H), 6.84 (d, J = 8 Hz, 2H),
6.88 (dd, J = 3, 8 Hz, 2H), 8.00 (s, 2H); HRMS (+ve ESI) calcd for
4.2.3. Apoptosis analysis by flow cytometry
C
24H28NO5 410.1962 m/z [MH+], found 410.1954 m/z.
Cells were seeded in six-well culture plates (2 ꢁ 105 cells per
well) in 2 ml of DMEM/HamF12 supplemented with 5% fetal
4.1.2.10. (2E,4E)-8-Methyl-2,4-bis((pyridine-4-yl)methylene)-8-
aza-bicyclo[3.2.1]octan-3-one (C1). From tropinone and pyridine-
4-carboxaldehyde following general procedure A, yellow solid, 92%
yield; mp 175–176 °C. Anal calcd for C20H19N3O: C, 75.69; H, 6.03;
N, 13.24. Found: C, 75.44; H, 6.10; N, 13.54. 1H NMR (CDCl3) d: 2.00
(d, J = 7 Hz, 2H), 2.31 (s, 3H, NMe), 2.61 (t, J = 7 Hz, 2H), 4.31 (d,
J = 7 Hz, 2H), 7.23 (d, J = 6 Hz, 4H), 7.68 (s, 2H), 8.68 (d, J = 6 Hz,
4H); HRMS (+ve ESI) calcd for C20H20N3O 318.1601 m/z [MH+],
found 318.1602 m/z.
bovine serum, 100 U/ml penicillin, 100 lg/ml streptomycin,
25 ng/ml amphotericin B and 2.2 g/l NaHCO3. Cells were treated as
above for 12–36 h. Apoptosis was assessed using Annexin-V-FLU-
OS/propidium iodide staining, as described.8 Samples were analyzed
using a FACScalibur flow cytometer (Becton Dickinson) and the pro-
portion of apoptotic cells was determined using CellQuest Pro soft-
ware. MDA-MB-231 cells were treated with B1 (2
lM) or B10
(1 M) for 12, 18, 24 or 36 h. Vehicle control cells were treated with
l
0.1% DMSO. Values are expressed as the number of apoptotic cells as
a % of the total number of cells SEM from three independent exper-
iments conducted in triplicate. Data were analyzed using a two-way
ANOVA coupled with a Bonferroni post-hoc test.
4.1.2.11. (2E,4E)-8-Methyl-2,4-bis((pyridine-3-yl)methylene)-8-
aza-bicyclo[3.2.1]octan-3-one (C2). From tropinone and pyridine-
3-carboxaldehyde following general procedure A, pale yellow solid,
85% yield; mp 181–182 °C. Anal calcd for C20H19N3O: C, 75.69; H,
6.03; N, 13.24. Found: C, 75.63; H, 6.18; N, 13.45. 1H NMR (CDCl3)
d: 2.02 (d, J = 7 Hz, 2H), 2.31 (s, 3H, NMe), 2.63 (t, J = 7 Hz, 2H), 4.33
(d, J = 7 Hz, 2H), 7.38 (dd, J = 5, 7 Hz, 2H), 7.69 (dm, J = 8 Hz 2H),
7.77 (s, 2H), 8.61 (dd, J = 1.5, 5 Hz, 2H), 8.67 (d, J = 1.5 Hz, 2H);
HRMS (+ve ESI) calcd for C20H20N3O m/z 318.11601 m/z [MH+],
found 318.1605 m/z.
4.2.4. Measurement of NF-
j
B-inhibitory activity
B was determined using a
The inhibitory activity against NF-
j
Dual-Glo™ Luciferase assay system from Promega. Electroporation
of K562 cells was realised as described previously.37 Equal
amounts (each 5
l
g per pulse (2.5 ꢁ 106 cells)) of a NF-
jB plasmid
jB containing five repeats of a consensus NF-
(Stratagene, p5 ꢁ NF-
jB site) and of a Renilla plasmid (phRG-TK from Promega) were
added to each pulse. After transient transfection cells were trans-
ferred to culture medium. After 24 h, transfected K562 cells were
re-solubilized at a concentration of 1 ꢁ 106 cells/ml in RPMI
1640-medium containing 0.1% FCS and 1% antibiotic–antimycotic.
After 30 min, the cells were incubated with different concentra-
4.1.2.12. (2E,4E )-2,4-Bis(3,4,5-trimethoxybenzylidene)-8-meth
yl-8-aza-bicyclo[3.2.1]octan-3-one (C10). From tropinone and
3,4,5-trimethoxybenzaldehyde following general procedure A, pale
yellow solid, 55% yield; mp 185–186 °C. Anal calcd for C28H33NO7:
C, 67.86; H, 6.71; N, 2.83. Found: C, 67.75; H, 6.91; N, 2.72. 1H NMR
(CDCl3) d: 2.07 (d, J = 7 Hz, 2H), 2.35 (s, 3H), 2.62 (m, 2H), 3.90 (s,
6H), 3.90 (s, 12H), 4.47 (d, J = 7 Hz, 2H), 6.65 (s, 4H), 7.78 (s, 2H);
HRMS (+ve ESI) calcd for C28H34NO7 496.2330 m/z [MH+], found
496.2291 m/z.
tions of curcumin analogues for 2 h. TNF-
a (20 ng/ml) was then
added for 6 h. Expression of NF- B was assessed according to the
j
manufacturer’s protocol by luminescence measurement using a
Berthold Orion Luminometer (integration time 10 s).
4.3. Statistical analysis
4.2. Cell line and reagents
All data presented were analyzed using a two-way ANOVA cou-
pled with a Bonferroni post-hoc test, where p <0.05 denotes a sta-
tistically significant difference.
MDA-MB-231, SkBr3 and MDA-MB-468 cells were purchased
from ATCC (Manassas, VA). Curcumin, 99% purity, was purchased
from Cayman Chemical (Ann Arbor, MI). Fetal bovine serum and
trypsin were purchased from Life Technologies (Auckland, New
Acknowledgments
This work was financed by the Breast Cancer Research Trust,
The New Zealand Institute of Plant and Food Research and Télévie,
the ‘‘Fondation de Recherche Cancer et Sang” and ‘‘Recherches Sci-
entifiques Luxembourg” asbl. MS, was supported by a Télévie
grants (Fonds National de la Recherche Scientifique, Belgium).
The authors thank «Een Häerz fir Kriibskrank Kanner» association
and the Action Lions ‘‘Vaincre le Cancer” for generous support.
Zealand). Tumor necrosis factor
a (TNFa) was purchased from Sig-
ma. K562 (human chronic myeloid leukemia) cells were obtained
from Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH.
4.2.1. Cell maintenance
Breast cancer cells were maintained in Modified Eagle’s Med-
ium-a modification media (MEM) (pH 7.4) supplemented with
10% FBS, 1% antibiotic/antimycotic solution and 0.2% NaHCO3. Leu-
kemia cells were cultured in RPMI 1640 medium supplemented
with 10% fetal bovine serum, penicillin (100 U/ml) and streptomy-
Supplementary data
Supplementary data (1H and 13C NMR spectra for all previously
unreported compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2010.07.063.
cin (100
lg/ml). All cells were cultured in 5% CO2/95% humidified
air at 37 °C.

B. Yadav et al. / Bioorg. Med. Chem. 18 (2010) 6701–6707
6707
19. Liang, G.; Shao, L.; Wang, Y.; Zhao, C.; Chu, Y.; Xiao, J.; Zhao, Y.; Li, X.; Yang, S.
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