Activation of NFκB is inhibited by curcumin and related enones

Article abstract of DOI:10.1016/j.bmc.2005.11.035

The transcription factor NFkappaB (NFκB) is up-regulated in many cancer cells where it contributes to development of the pro-survival, anti-apoptotic state. The natural product curcumin is a known inhibitor of activation of NFκB. Enone analogues of curcumin were compared with curcumin for their abilities to inhibit the TNFα-induced activation of NFκB, using the Panomics' NFκB Reporter Stable Cell Line. The enones tested included curcumin analogues that retained the 7-carbon spacer between the aromatic rings, analogues with a 5-carbon spacer, and analogues with a 3-carbon spacer. Inhibitors of NFκB activation were identified in all three series, a number of which were more active than curcumin. Enone analogues in the series with the 5-carbon spacer were especially active, including members that contained heterocyclic rings. 1,5-Bis(3-pyridyl)-1,4-pentadien-3-one was the most active analogue, IC₅₀ = 3.4 ± 0.2 μM. The most active analogues retain the enone functionality, although some analogues devoid of the enone functionality exhibited activity. The activity of the analogues as inhibitors of the activation of NFκB did not correlate with their anti-oxidant activity. The data suggest that the abilities of curcumin and analogues to prevent the stress-induced activation of NFκB result from the inhibition of specific targets rather than from activity as anti-oxidants.

Full text of DOI:10.1016/j.bmc.2005.11.035

Bioorganic & Medicinal Chemistry 14 (2006) 2450–2461
Activation of NFjB is inhibited by curcumin and related enones
Waylon M. Weber,a Lucy A. Hunsaker,b C. Nathaniel Roybal,b
Ekaterina V. Bobrovnikova-Marjon,b Steve F. Abcouwer,b Robert E. Royer,b
Lorraine M. Decka,* and David L. Vander Jagtb,*
aDepartment of Chemistry, University of New Mexico, Albuquerque, NM 87131, USA
bDepartment of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
Received 16 September 2005; revised 11 November 2005; accepted 14 November 2005
Available online 7 December 2005
Abstract—The transcription factor NFkappaB (NFjB) is up-regulated in many cancer cells where it contributes to development of
the pro-survival, anti-apoptotic state. The natural product curcumin is a known inhibitor of activation of NFjB. Enone analogues
of curcumin were compared with curcumin for their abilities to inhibit the TNFa-induced activation of NFjB, using the PanomicsÕ
NFjB Reporter Stable Cell Line. The enones tested included curcumin analogues that retained the 7-carbon spacer between the
aromatic rings, analogues with a 5-carbon spacer, and analogues with a 3-carbon spacer. Inhibitors of NFjB activation were iden-
tified in all three series, a number of which were more active than curcumin. Enone analogues in the series with the 5-carbon spacer
were especially active, including members that contained heterocyclic rings. 1,5-Bis(3-pyridyl)-1,4-pentadien-3-one was the most
active analogue, IC50 = 3.4 0.2 lM. The most active analogues retain the enone functionality, although some analogues devoid
of the enone functionality exhibited activity. The activity of the analogues as inhibitors of the activation of NFjB did not correlate
with their anti-oxidant activity. The data suggest that the abilities of curcumin and analogues to prevent the stress-induced activa-
tion of NFjB result from the inhibition of specific targets rather than from activity as anti-oxidants.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ed IjB. These inhibitory proteins mask a nuclear local-
ization signal. In the classical pathway for activation of
NFjB, the upstream IjB kinase complex (IKK) is first
activated in response to many different signals, resulting
in phosphorylation of IjB, followed by its ubiquitina-
tion and proteosomal degradation. This is followed by
nuclear translocation of NFjB with resulting activation
of a battery of genes, including anti-apoptotic pro-sur-
vival genes. There are also alternative pathways for acti-
vation of NFjB.4,5
The transcription factor NFjB is an established regula-
tor of numerous genes important in the inflammatory
response. More recently however, activation of NFjB
has been shown to have a role in many aspects of onco-
genesis including control of apoptosis as well as regula-
tion of cell cycling and cell migration.1,2 Activated
NFjB has been observed in many cancers and is espe-
cially important in metastasis.3 There are five members
of the NFjB family, distinguished by the presence of
an N-terminal Rel homology domain: Rel (c-Rel), RelA
(p65), RelB, NFjB1 (p50 and its precursor p105), and
NFjB2 (p52 and its precursor p100). NFjB transcrip-
tion factors are homo- or heterodimers of these mem-
bers, with the p65/p50 heterodimer being the most
common. NFjB members are retained in the cytosol
as complexes with a set of inhibitory proteins, designat-
The evidence that links activation of NFjB to oncogen-
esis is compelling. NFjB is activated by a number of
viral transforming proteins,6 and inhibition of NFjB
activation through expression of a dominant negative
IKK can block cell transformation.7 NFjB activation
protects cells from apoptosis induced by cancer che-
mo-therapeutics and oncogenes,8 and activation of
NFjB promotes expression of metastatic factors.2
A number of dietary chemopreventive compounds such
as flavonoids and curcumin block activation of
NFjB.1,9 Curcumin (Fig. 1) is a non-nutritive, non-toxic
compound in turmeric, a spice that has been used for
Keywords: NFjB; Curcumin; Enones; Inhibition.
*
Corresponding authors. Tel.: +1 505 272 5788 (D.L.V.J.); Tel.: +1
505 277 543 (L.M.D.); fax: +1 505 277 5438 (D.L.V.J.); e-mail
addresses: ldeck@unm.edu; dlvanderjagt@ salud.unm.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.035

W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
2451
H
O
O
O
O
H3CO
HO
OCH3
H3CO
OCH3
OH
OH
HO
Curcumin
Figure 1. Curcumin exists in solution as an equilibrium mixture of the symmetrical dienone and the keto–enol tautomer stabilized by intramolecular
H-bonding.
centuries in India and elsewhere as an herbal medicinal
treatment of wounds, jaundice, and rheumatoid arthri-
tis.10 In addition, curcumin inhibits the proliferation of
a variety of tumor cells9 and has anti-metastatic activi-
ty.11 Curcumin also exhibits potent anti-oxidant activity,
which depends upon the presence of phenolic groups in
the aryl rings.12 Previously, we described the anti-oxi-
dant activities of curcumin, as well as enone and dienone
analogues of curcumin.13 In the present study, curcumin
and analogues were compared for their abilities to inhib-
it the TNFa-induced activation of NFjB in cells stably
transfected with an NFjB-dependent reporter construct,
using the commercially available PanomicsÕ Reporter
Stable Cell Line. The role of the enone or dienone
functionality in diaryl systems was examined where the
spacer is 7-carbons (as in curcumin), 5-carbons or 3-car-
bons. The role of the aryl ring substituents was also
examined. The abilities of the analogues to inhibit the
activation of NFjB were compared with their anti-oxi-
dant activities to determine whether both activities de-
rive from general redox chemistry or whether the
inhibition of NFjB is selective and unrelated to anti-oxi-
dant activity, which would point toward inhibition of
specific targets important in the activation of NFjB.
Compound 17 was prepared from compound 15 by reac-
tion with sodium hydroxide and benzyl bromide.13
Three additional series 1 curcumin analogues, 4, 5, and 6,
were prepared as shown in Scheme 1. Compounds 4–6
contain two aryl rings separated by a saturated seven-car-
bon spacer having two carbonyls. Compounds 4, 5, and 6
were prepared by reduction of 3, 17, and 20, respectively,
using a procedure described by Venkateswarlu et al.16 In
the proton and carbon NMRÕs of compounds 4 and 6, a
mixture of the keto- and enol- forms was observed.17
Compounds in series 2, which contain a 5-carbon spacer,
were prepared as shown in Scheme 1. Compounds 23, 29,
34, 36, 38–41, and 55 contain two identical aryl rings sep-
arated by an unsaturated 5-carbon spacer having a single
carbonyl. Compounds 23, 29, 34, 38, 40, and 41 were pre-
pared from acetone and a substituted benzaldehyde in a
base catalyzed aldol reaction as described by Masuda
et al.18 Compounds 39 and 55 were prepared in a similar
fashion by reaction of benzaldehyde or 3,4-dimethoxy-
benzaldehyde with acetone.13 Compound 36 was pre-
pared from compound 55 by reaction with boron
tribromide following a procedure as described by Royer
et al.19
2. Chemistry
Compounds 31 and 44, which are series 2 analogues, were
prepared as shown in Scheme 2. These compounds con-
tain two identical aryl rings separated by a 5-carbon spac-
er containing both a carbonyl and two epoxide rings.
Compounds 31 and 44 were synthesized following the
procedure of Yadav and Kapoor20 by reaction of com-
pound 39 with tert-butyl hydroperoxide and aluminum
oxide–potassium fluoride. A mixture containing the
trans/trans and cis/cis isomers was formed and the trans/
trans isomer, compound 31, was separated from the cis/
cis isomer, compound 44, through recrystallization from
ethanol.20
Curcumin is a relatively simple bisphenolic compound
that can be prepared in a single reaction. The procedures
used to prepare several of the series 1–3 curcumin ana-
logues were described previously.13 The following
schemes summarize the procedures used to prepare
new series 1–3 curcumin analogues. For clarity the num-
bering of the compounds in the schemes correlates to the
compound numbers found in Figure 2.
Compounds in series 1 contain a 7-carbon spacer. Five
series 1 analogues, 2, 3, 15, 17, and 20, were prepared
as shown in Scheme 1. Compounds 2, 3, 15, 17, and 20
contain two aryl rings separated by an unsaturated sev-
en-carbon spacer. Compounds 2, 3, 17, and 20 have
either one or two substituents attached to the central
methylene carbon, whereas compound 15 is devoid of
a central substituent. Compounds 2 and 3 were pre-
pared by reaction of 3-benzyl-2,4-pentanedione or 3-
methyl-2,4-pentanedione with 4-hydroxy-3-methoxy-
benzaldehyde in an aldol-type reaction following the
procedure described by Pabon.14 As expected, in the
proton NMR of compounds 2 and 3 the enol form pre-
dominates, whereas the carbon spectra exhibit both the
keto- and enol-forms. 15 Compounds 15 and 20 were pre-
pared in a similar fashion by reaction of 2,4-pentanedi-
one or 3-methyl-2,4-pentanedione with benzaldehyde.
Compound 63, a series 3 analogue, was prepared as
shown in Scheme 2. This compound contains two identi-
cal aryl rings separated by a 3-atom spacer having both a
carbonyl and a saturated heterocyclic ring. This
compound was synthesized following the procedure of
Selvaraj et al.21 by reaction of compound 39 with
methylamine. Syntheses of all of the other analogues were
described previously.13
3. Results
Inhibition of the TNFa-induced activation of NFjB by
curcumin and analogues was determined with the
PanomicsÕ NFjB Reporter Stable Cell Line 293T/

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W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
110
100
90
80
70
60
50
40
30
20
10
0
1
2
3
4
5
6
Compound Number (10 µM)
O
OH
O
O
OH
O
O
OH
OH
H3CO
OCH3
H3CO
HO
OCH3
15
1
2
8
9
Curcumin
HO
OH
OH
O
O
OH
OH
H3CO
OCH3
16
Bn
Bn
HO
H3CO
HO
OH
HO
OH
OH
O
O
O
O
OCH3
OH
Bn
Bn
3
4
10
17
H3C CH3
CH3
O
OH
O
OH
OCH3
O
OH
OCH3
H3CO
HO
OCH3
OH
H3CO
OCH3
11
18
CH3
(H3C)2N
N(CH3)2
O
O
O
O
OH
OH
O
O
OH
OH
HO
OH
5
6
Bn
Bn
12
13
19
20
H3CO
OCH3
O
OH
OCH3
OCH3
CH3
CH3
O
OH
O
OH
H3CO
OCH3
OCH3
7
14
H3CO
OCH3
H3CO
Figure 2. Activities of curcumin (1) and analogues in the 7-carbon spacer series as inhibitors of the activation of NFjB by TNFa. Error bars show
SD. The remaining analogues showed little or no activity.
NFjB-luciferase, which contains a luciferase reporter
construct regulated by 6 copies of the NFjB response
element. Initially the three series of analogues were
screened with curcumin or analogues at 10 lM concen-
tration. All compounds that demonstrated significant
activity (26 of the 71 analogues) were then run in tripli-
cate. The results from the analyses of these 26 ana-
logues, compared to curcumin, are summarized in
Figures 2–4. The results with curcumin and analogues in
series 1 with the 7-carbon spacer are shown in Figure 2.

W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
2453
Scheme 1. Reagents: (i) CH3C(O)CH2C(O)CH3 or CH3C(O)CH(CH3)C(O)CH3 or CH3C(O)CH(CH2C6H5)C(O)CH3 (0.5 equiv), B2O3, B(OC4H9)3,
n-BuNH2, EtOAc; (ii) H2, Pd/C, EtOAc; (iii) C6H5CH2Br (2 equiv), NaOH, NH4Cl, CH2Cl2; (iv) CH3C(O)CH3 (0.5 equiv), NaOH, H2O, EtOH;
(v) BBr3, CH2Cl2, N2.
Scheme 2. Reagents: (i) CH3CN, KF–Al2O3, t-BuOOH, ClCH2CH2Cl; (ii) CH3NH2, DMF, H2O.
Curcumin (1) is among the most active members of this
series. Analogues 2 and 3, which contain a single substi-
tuent attached to the central methylene carbon but re-
tain the same aryl ring substituents as curcumin, are
more active than curcumin. Analogue 4, the saturated
form of analogue 3, is no longer an enone and is almost
as active as curcumin; this suggests that the enone or
dienone functionality is not essential for activity. This
is further supported by the activities of analogues 5, 6,
8, and 12 that also are devoid of enone functionality.
An additional conclusion can be drawn regarding the
importance of anti-oxidant activity in the abilities of
these analogues to inhibit the activation of NFjB. Ana-
logues 10 and 12 do not exhibit anti-oxidant activity13
but do inhibit the activation of NFjB.
the analogues that are more active than curcumin retain
the enone functionality.
The results with analogues in series 3 with the 3-carbon
spacer are shown in Figure 4. Analogue 61 was more ac-
tive than curcumin with analogues 62 and 63 exhibiting
activities comparable to those of curcumin. These ana-
logues retain the enone functionality and exhibit anti-
oxidant activity. Analogue 63, which is almost as active
as curcumin, is devoid of the enone functionality.
The analogues that were more active than curcumin
were compared with curcumin in dose–response studies
run in triplicate, from which IC50 values were obtained.
Representative dose–response curves are shown in Fig-
ure 5, and IC50 values are summarized in Table 1. Also
included in Table 1 is reference to whether the active
analogues exhibited anti-oxidant activities. The most ac-
tive analogues exhibit IC50 values in the low micromolar
range. Analogue 24, which contains heterocyclic rings, is
devoid of anti-oxidant activity, but is the most active
against NFjB, with an IC50 of 3.4 0.2 lM. In separate
experiments, the analogues in Table 1 were examined for
cellular toxicity. Cells were treated with analogue,
15 lM, under the same conditions as before but then
The results with analogues in series 2 with the 5-carbon
spacer are shown in Figure 3. Significantly, many of the
analogues are more active than curcumin. This includes
enones that are devoid of anti-oxidant activity13 (21, 24–
26), further supporting the conclusion that there is no
correlation between anti-oxidant activity and inhibition
of the activation of NFjB. A number of analogues in
this series that are devoid of the enone functionality re-
tain measurable activity (31, 42, and 44). Nevertheless,

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W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
110
100
90
80
70
60
50
40
30
20
10
0
1
21 24 26 27 28 22 23 25 31 29 30 35 34 36 38 40 32 41
Compound Number (10 µM)
O
O
O
OH
O
H3CO
HO
OCH3 H3CO
OCH3
30
1
41
51
F3
F3
HO
OH
OH
OCH3
OCH3
OCH3
OCH3
OH
O
O
O
O
O
O
O
H3CO
HO
21
22
31
42
43
52
53
trans/trans
O
N
N
O
H3CO
OCH3
OH
F3
32
33
H3CO2C
CO2CH
H3CO
OCH3
HO
F3
OCH3 H3CO
O
3
O
O
O
O
O
O
O
O
O
HO
OH
23
44
54
cis/cis
O
Cl
Cl
OCH3
OCH3
OCH3
O
O
H3CO
H3CO
55
H3CO
OCH3
N
N
24
25
34
35
45
46
OCH3
O
O
CH3
CH3
OCH3
OCH3
OH
OCH3
OCH3
OH
OCH3
OCH3
H3
H3
56
O
O
O
H3CO
OCH3
H3CO
OCH3
HO
HO
OH
OH
26
36
37
47
57
H3CCO2
O2CCH3
Cl
O
O
O
Cl
O
O
O
O
OCH3
O
O
O
OCH3
27
28
29
48
49
58
H3CO
H3CO
OCH3
O
O
F
F
H3CO
HO
OCH3
OH
38
59
60
H3CCO2
O2CCH3
Cl
CF3
CF3
Cl
39
50
H3
H3
O
40
Figure 3. Activities of curcumin (1) and analogues in the 5-carbon spacer series as inhibitors of the activation of NFjB by TNFa.
were examined for cell viability rather than for luciferase
activity. This concentration was the highest concentra-
tion that was generally used in the dose–response stud-
ies. Cell viability was determined colorimetrically with
the CellTiter 96ꢁ AQueous One Solution Cell Prolifera-
tion Assay (Promega). Cell viability was generally great-
er than 90%. Therefore, the values listed in Table 1
represent inhibition of the activation of NFjB and are
not the result of cell death.
A preliminary QSAR analysis of the data was carried out
using the Catalyst program (Accelrys). A wide range of

W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
2455
110
100
90
80
70
60
50
40
30
20
10
0
1
61
62
63
Compound Number (10 µM)
O
O
OH
O
H3CO
HO
OCH3
OH
H3CO
HO
CH3
1
69
70
64
65
Curcumin
COOH
O
O
CH3
O
OCH3
OH
61
NC
H3C
O
O
OH
O
H3CO
HO
OCH3
CH3
62
63
66
71
72
OH
O
O
O
H3CO
HO
CH3
67
68
N
H3CO
O
CH3
HOOC
Figure 4. Activities of curcumin (1) and analogues in the 3-carbon spacer series as inhibitors of the activation of NFjB by TNFa.
structures and activities from the results in Figures 2–4
were used to generate multiple pharmacophores. A single
pharmacophore did not provide a satifactory fit of the
data. Moreover, pharmacaphores that were derived
separately from 5-carbon curcumin analogues or from
3-carbon analogues did not provide satisfactory fits.
However, a single pharmacophore could provide a satis-
factory fit of the data for analogues in the 7-carbon series.
Figure 6 shows a pharmacophore on which curcumin is
superimposed. This pharmacophore provided an excel-
lent fit (correlation 0.9) of analogues on the 7-carbon ser-
ies. The inability of a single pharmacaphore to provide a
satisfactory fit of all of the data or of the data from the 5-
carbon series or 3-carbon series may mean that there are
several different targets of these analogues.
4. Discussion
Curcumin has a broad range of biological activities,
some of which may derive from its anti-oxidant activity
or ability to quench free radical reactions and some that

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W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
120
110
100
90
80
70
60
50
40
30
20
10
0
O
OH
H3CO
HO
OCH3
OH
1
IC50 = 8.2 µM
0
5
10
15
20
25
30
35
40
45
50
55
[Curcumin] (µM)
120
110
100
90
80
70
60
50
40
30
20
10
0
O
OH
OH
27
IC50 = 4.2 µM
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
[Analogue 27] (µM)
Figure 5. Dose–response curves for curcumin (top) and analogue 27 (bottom) are representative of the dose–response curves for the analogues
summarized in Table 1. Curves were generated by Sigma plotÕs regression wizard (curve fitter).
involve inhibition or inactivation of specific targets.
Curcumin can scavenge superoxide radicals, hydrogen
peroxide and nitric oxide, and it has been suggested that
the ability of curcumin to protect against radiation dam-
age, iron-induced hepatic damage, xanthine oxidase
injury, and oxidative stress depends upon the anti-oxi-
dant and free radical-scavenging properties of curcu-
min.22–25 In our previous study of the anti-oxidant
properties of curcumin,13 we determined the abilities
of curcumin and analogues to quench the pre-formed
radical monocation of 2,20-azinobis-(3-ethylbenzothiaz-
oline-6-sulfonic acid), known as the total radical-trap-
ping anti-oxidant parameter (TRAP) assay, and the
abilities of these compounds to reduce the ferric tripyr-
idyltriazine complex, known as the ferric reducing/anti-
oxidant power (FRAP) assay.26 It is noteworthy that
many of the most active analogues in the present study
(21, 23–26) show no activity in the TRAP or FRAP as-
say, which leads us to conclude that there is no correla-
tion between anti-oxidant activity and ability to inhibit
the TNFa-induced activation of NFjB. The lack of cor-
relation between the anti-oxidant activities of curcumin
and analogues, and the abilities of these compounds to
inhibit the TNFa-induced activation of NFjB suggests
that curcumin and analogues inhibit a specific target
(or targets) rather than function through general redox
chemistry.
The anti-inflammatory properties of curcumin and its
ability to inhibit the immune response upon exposure
to a variety of external stimuli may, at least in part, re-
sult from inhibition of the activation of NFjB by these
external signals, since many of the genes that are impli-
cated in the immune/inflammatory response are up-reg-
ulated by NFjB. For example, cucumin inhibits the
LPS-induced production of IL-1b and TNFa27, and

W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
2457
Table 1. IC50values for inhibition by curcumin (1) and analogues of the TNFa-induced activation of NFjB
p valuesb
TRAPc
FRAPd
a
Structure
Analogue #
IC50 (lM)
O
OH
H CO
3
OCH
OH
3
1
8.2 0.4
Curcumin
HO
O
OH
H3CO
HO
OCH3
OH
2
3
6.3 0.5
6.7 1.2
3.9 0.3
4.4 0.8
7.0 0.5
3.4 0.2
6.5 0.9
5.3 1.1
4.2 0.3
9.6 0.7
5.0 0.3
8.3 1.5
6.2 0.1
<0.005
NSe
+
+
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
ꢀ
+
Bn
O
OH
H3CO
HO
OCH3
OH
CH3
N
O
21
22
23
24
25
26
27
28
29
31
61
<0.001
<0.002
<0.04
<0.001
<0.05
<0.02
<0.001
NS
ꢀ
+
N
O
H CO
3
OCH
3
HO
OH
O
F3C
CF3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
O
N
N
OCH
O
OCH
3
3
OCH
OCH
3
3
O
H3CO
OCH3
H3CCOO
OOCCH3
OH
O
OH
O
H CO
3
OCH
3
HO
OH
CF3
O
O
CF3
<0.001
NS
O
O
trans/trans
O
OCH
OH
3
<0.001
a IC50 values standard deviation (n = 3).
b t test, p values with curcumin as the reference.
c TRAP, total radical-trapping anti-oxidant parameter assay.
d FRAP, ferric reducing/anti-oxidant power assay.
e NS, not significant.
the IL-1b-induced expression of IL-2,28 as well as the
TNFa-induced expression of ICAM-1, VCAM-1, and
E-Selectin.29 NFjB is implicated in these signaling path-
ways.30,31 However, curcumin has also been shown to be
a direct inhibitor of enzymes that are important in the
inflammatory response, including lipoxygenase and
cyclooxygenase.32
The extensive literature on the anti-cancer properties
of curcumin also includes reports of direct inhibition

2458
W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
Figure 6. Curcumin aligned with the hypothesis (pharmacophor model) generated with the Catalyst program using compounds 1–7 as the training
˚
set. The hypothesis consists of two hydrophobic aromatic regions with centers 11.8 angstroms apart and a hydrogen bond acceptor 6.2 A from the
˚
nearest hydrophobic aromatic region and 7 A from the other. Using this hypothesis, the correlation coefficient for the linear correlation between
predicted activity and actual activity was 0.9.
by curcumin of enzymes that may be important in can-
cer progression, such as inhibition of c-Jun N-terminal
kinase,33 epidermal growth factor receptor34, and
p185neu.35 The pro-apoptotic activity of curcumin in
many types of cancer cells, where the classical hall-
marks of apoptosis are observed including DNA lad-
dering, chromatin condensation, and cleavage of 28S
and 18S ribosomal RNA,36 may be related to the acti-
vation of NFjB since many cancer cells protect against
apoptosis by activating NFjB as a pro-survival strate-
gy. Consequently, much of the interest in the mecha-
nism of curcumin in cancer biology has been directed
at NFjB. Over-expression of p65 renders cells resistant
to the pro-apoptotic effects of curcumin.37 When these
cells are then transiently transfected with a super-re-
pressor form of IjBa, these cells are no longer resis-
tant to curcumin, consistent with an important role
for NFjB in the apoptosis-inducing activity of curcu-
min. Studies designed to identify the specific target(s)
of curcumin in preventing the activation of NFjB
point toward targets that are upstream from IjB.
Singh and Aggarwal reported that curcumin inhibited
TNFa-dependent activation of NFjB at a step before
phosphorylation of IjB but after a point where multi-
ple stimuli converge.38 Brennan and OÕNeill reported
that curcumin inhibited degradation of IjB but also
reacted with p50.39 Several groups reported that curcu-
min inhibited IKK,40,41 or that curcumin targets a ki-
nase that is upstream from IKK.40 The IKK complex
includes IKKa, IKKb, IKKc/NEMO, as well as the
IjB recruiter/regulator ELKS,42 and the chaperone
HSP90 and co-chaperone Cdc37.43 More recently,
c-Src has been reported to be part of the IKK com-
plex.44 In addition, NFjB-inducing kinase (NIK),
which usually is assigned a role in the alternative
activation pathway, has recently been shown to have
a role in the activation of IKK.45 Thus, there are
numerous kinases and other proteins that are associat-
ed with the IKK complex that are potential targets of
the analogues of curcumin.
Recent studies of the biological activities of curcumin and
its analogues have focused on the anti-angiogenic proper-
ties of these compounds.46–50 Analogues of curcumin
retaining the same aryl ring substituents, including the
phenolic groups, were more active than curcumin in their
anti-angiogenic activities. Otheranalogues exhibited anti-
cancer activity against a wide range of cells. An analogue,
designated EF24, induces apoptosis in breast andprostate
cell lines by a redox-dependent process.49 ER24 does not
contain phenolic groups and is structurally related to our
analogues with the 5-carbon spacer. This is consistent
with our conclusion that analogues of curcumin may
exhibit a range of activities, not all of which are related
to the anti-oxidant properties of curcumin.
5. Conclusions
In summary, analogues of curcumin in which the two
aryl rings are separated by 7-carbon, 5-carbon or 3-car-
bon spacers are able to inhibit the TNFa-induced

W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
2459
activation of NFjB. However, activities can vary wide-
6.4. 4-Methyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-hep-
tane-3,5- dione (4)
ly. The most active analogues retain the enone function-
ality, although this functionality is not essential for
activity. In addition, analogues with the 5-carbon spacer
are generally the most active. Ring substituents are not
necessary but can affect activity. Moreover, the aryl
rings can be nitrogen heterocycles. The inhibition of
TNFa-induced activation of NFjB by curcumin and
analogues may occur at the level of the IKK complex.
This is currently being investigated.
Prepared according to the method of Venkateswarlu
et al.16 by reaction of compound 3 (0.20 g, 0.5 mmol)
with 10% palladium on activated carbon (25 mg) under
a hydrogen atmosphere at 60 psi to give 0.10 g (48%) of
1
a pale yellow oil: H NMR d 1.23 (d, 3H, J = 7.2 Hz),
1.69 (s, 3H), 2.72 (m, 16H), 3.57 (q, 1H, J = 7.0 Hz),
3.83 (s, 12H), 5.48 (s, 4H), 6.58–6.85 (m, 12H); 13C
NMR: d 12.5, 29.2, 30.6, 43.3, 55.9, 61.4, 110.9,
111.0, 114.2, 114.3, 120.7, 132.4, 132.6, 143.9, 146.3,
206.1; Exact Mass Calcd for C22H26O6: 386.1729, obsd
(M+H) 387.1783.
6. Experimental
6.1. Chemical synthesis
6.5. 4,4-Dibenzyl-1,7-diphenylheptane-3,5-dione (5)
Melting points were determined with a Thomas-Hoover
Uni-Melt apparatus and are uncorrected. NMR spectra
were recorded on a Bruker AC250 NMR spectrometer
in CDCl3 unless otherwise noted. Chemical shifts are in
ppm (d) relative to TMS. High resolution mass spectra
were recorded on a Waters/Micromass LCT-premier.
Analytical data were obtained from Galbraith Laborato-
ries, Knoxville, Tennessee. Compounds 2 and 3 were pre-
pared using a modification of PabonÕs method.14
Compounds 4–6 were prepared following the method of
Venkateswarlu et al.16 Compounds 23, 29, 34, 38, 40,
and 41 were prepared following the method of Masuda
et al.18 Compound 36 was prepared following the proce-
dure of Royer et al.19 Compounds 31 and 44 were pre-
pared following the method of Yadav and Kapoor.20
Compound 63 was prepared following the procedure of
Selvaraj et al.21 Compounds 1, 7–22, 24–28, 30, 32, 33,
35, 37, 39, 42, 43, 45–62, and 64–72 were prepared accord-
ing to procedures described by Weber et al.13
Prepared according to the method of Venkateswarlu
et al.16 by reaction of compound 17 (70 mg, 0.15 mmol)
with 10% palladium on activated carbon (10 mg) under
a hydrogen atmosphere at 60 psi to give 60 mg (86%) of
1
a white solid: mp 100.5–101.5 ꢂC; H NMR d 2.56 (t,
4H, J = 7.4 Hz), 2.74 (t, 4H, J = 6.8 Hz), 3.29 (s, 4H),
6.88–7.20 (m, 20H); 13C NMR: d 29.6, 37.3, 42.5, 71.1,
126.1, 126.8, 128.4, 129.6, 136.0, 140.6, 207.8. Anal.
Calcd for C33H32O2: C, 86.05; H, 7.00. Found: C,
86.28; H, 7.11.
6.6. 4-Methyl-1,7-diphenylheptane-3,5-dione (6)
Prepared according to the method of Venkateswarlu
et al.16 by reaction of compound 20 (0.96 g, 3.3 mmol)
with 10% palladium on activated carbon (25 mg) under
a hydrogen atmosphere at 60 psi to give 0.71 g (73%) of
1
a clear oil: H NMR d 1.20 (d, 3H, J = 7.2 Hz), 1.66 (s,
3H), 2.61–2.85 (m, 16H), 3.55 (q, 1H, J = 7.0 Hz), 7.08–
7.25 (m, 20H); 13C NMR: d 12.3, 29.3, 31.0, 37.6, 42.8,
60.9, 104.2, 125.9, 128.1, 128.2, 140.4, 140.8, 174.6,
191.5, 205.6; Exact Mass Calcd for C20H22O2:
294.1620, obsd (M+H) 295.1693.
6.2. 4-Benzyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
heptadiene-3,5-dione (2)
Yield: 61%. Orange-yellow solid: mp 144–146 ꢂC [lit.15
1
139–141 ꢂC]; H NMR d (DMSO) 3.81 (s, 6H), 4.11 (s,
2H), 6.78 (d, 2H, J = 8.1 Hz), 7.10–7.30 (m, 11H), 7.58
(d, 2H, J = 15.1 Hz); 13C NMR: d 30.2, 33.7, 55.6, 55.7,
63.0, 109.9, 111.3, 115.5, 117.9, 122.4, 123.2, 123.7,
125.5, 125.7, 126.0, 126.5, 127.7, 128.1, 128.3, 128.7,
139.1, 141.7, 142.3, 144.1, 147.8, 149.2, 149.7, 183.0,
194.0; Exact Mass Calcd for C28H26O6: 458.1729, obsd
(M+H) 459.1798.
6.7. 1,7-Diphenyl-1,6-heptadiene-3,5-dione (15)
Yield: 38%. Yellow solid: mp 140–142 ꢂC [lit.13,15 139–
140 ꢂC].
6.8. 4,4-Dibenzyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (17)
Yield: 75%. White solid: mp 182–183 ꢂC [lit.13,15 181 ꢂC].
6.9. 4-Methyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (20)
6.3. 4-Methyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
heptadiene-3,5-dione (3)
Prepared according to the method of Pabon14 by reaction
of 3-methyl-2,4-pentanedione (1.14 g, 10 mmol) and 4-
hydroxy-3-methoxybenzaldehyde (3.04 g, 20 mmol) to
give 1.08 g (28%) of an orange solid: mp 180–183 ꢂC; 1H
NMR (DMSO) d 2.20 (s, 3H), 3.84 (s, 6H), 6.81 (d, 2H,
J = 8.14 Hz), 7.17 (d, 2H, J = 15.7 Hz), 7.19 (d, 2H,
J = 7.2 Hz), 7.57 (d, 2H, J = 15.3 Hz), 9.62 (s, 2H); 13C
NMR: d 11.5, 13.1, 55.2, 55.6, 55.8, 105.6, 111.4, 111.6,
115.5, 117.7, 122.1, 123.2, 123.5, 125.6, 126.6, 141.4,
143.7, 147.8, 149.1, 149.6, 182.1, 196.0. Anal. Calcd for
C22H22O6: C, 69.10; H, 5.80. Found: C, 69.19; H, 5.89.
Yield: 59%. Orange solid: mp 154–157 ꢂC [lit.13 154–
157 ꢂC].
6.10. 1,5-Bis-(3-trifluoromethylphenyl)-1,4-pentadien-3-
one (23)
Prepared according to the method of Masuda et al.18 by
reaction of a,a,a-trifluoro-m-tolualdehyde (0.5 ml,
3.8 mmol) and acetone (0.14 ml, 1.9 mmol) to give
0.25 g (36%) of yellow crystals: mp 116–117 ꢂC; 1H
NMR: d 7.12 (d, 2H, J = 15.9 Hz), 7.72 (m, 10H); 13C

2460
W. M. Weber et al. / Bioorg. Med. Chem. 14 (2006) 2450–2461
NMR: d 123.7, 124.7, 126.7, 126.8, 129.5, 131.5, 131.6,
135.4, 141.8, 187.8. Anal. Calcd for C19H12OF6: C,
61.63; H, 3.27. Found: C, 61.82; H, 3.28.
6.18. 1,5-Bis-(4-trifluoromethylphenyl)-1,4-pentadien-3-one
(41)
Yield: 84%. Yellow solid: mp 152–154 ꢂC [lit.56 156–
157 ꢂC]; 1H NMR: d 7.12 (d, 2H, J = 15.9 Hz), 7.69
(m, 10H); 13C NMR: d 121.6, 125.9, 127.2, 128.5,
132.1, 138.0, 141.8, 187.9.
6.11. 1,5-Bis-(2-trifluoromethylphenyl)-1,4-pentadien-3-one
(29)
Yield: 88%. Yellow solid: mp 131–133 ꢂC [lit.51 131 ꢂC].
6.19. 1,5-Bis(3,4-dimethoxyphenyl)-1,4-pentadien-3-one (55)
6.12. trans, trans-1,2,4,5-Diepoxy-1,5-diphenylpentan-3-
one (31) and cis,cis-1,2,4,5-diepoxy-1,5-diphenylpentan-3-
one (44)
Yield: 75%. Yellow solid: mp 72–75 ꢂC [lit.13,53 68–70 ꢂC].
6.20. 1-Methyl-2,6-diphenyl-4-piperidone (63)
Prepared according to the method Yadav and Kapoor20
by reaction of compound 39 (0.47 g, 2.0 mmol) with
tert-butyl hydroperoxide (1.90 g, 21 mmol) and alumi-
num oxide–potassium fluoride (0.48 g) to give a mixture
of trans/trans and cis/cis isomers. Recrystallization from
ethanol gave 0.21 g (40%) of the trans/trans isomer as
white crystals: mp 117–119 ꢂC (lit.52 118–118.5 ꢂC).
Evaporation of the filtrate gave 0.25 g (47%) of the cis/
Yield: 42%. White solid: mp 147–149 ꢂC [lit.21 147–
149 ꢂC].
6.21. Cell assay
An NFjB reporter stable cell line from human 293T
embryonic kidney cells (293T/NFjB-luc) (Panomics,
Inc., Redwood City, CA) was grown in a humidified
atmosphere at 37 ꢂC in 5% CO2/95% air. The cells
were maintained in DulbeccoÕs modified EagleÕs medi-
um (DMEM—high glucose containing 4 mM gluta-
mine) supplemented with 10% fetal bovine serum
(FBS), 1 mM sodium pyruvate, 100 U/ml penicillin,
100 lg/ml streptomycin, and 100 lg/ml hygromycin
(Gibco/Invitrogen, Carlsbad, CA) to maintain cell
selection.
1
cis isomer as an orange-yellow oil: H NMR: d 3.71
(d, 2H, J = 1.6 Hz), 4.12 (d, 2H, J = 1.6 Hz), 7.28–7.35
(m, 10H); 13C NMR: d 58.8, 60.3, 125.8, 128.6, 129.1,
134.4,199.0.
6.13. 1,5-Bis-(3-fluorophenyl)-1,4-pentadien-3-one (34)
Prepared according to the method of Masuda et al.18
by reaction of 3-fluorobenzaldehyde (0.5 ml, 4.8 mmol)
and acetone (0.17 ml, 2.4 mmol) to give 0.26 g (41%) of
1
yellow crystals: mp 96–97 ꢂC; H NMR: d 7.03 (d, 2H,
One day prior to treatment, the 293T/NFjB-luc cells
were plated into 24-well cell culture plates (Costar, Cam-
bridge, MA) at approximately 70% confluency in the
above media without hygromycin. The following day
cells were fed fresh media 1 h prior to treatment. Media
with or without recombinant tumor necrosis factor al-
pha (TNFa) (R&D Biosciences/Clontech, Palo Alto,
CA) were then applied to the cells at 20 ng/ml followed
by immediate treatments with curcumin or analogue.
The cells were placed again in a humidified atmosphere
at 37 ꢂC in 5% CO2/95% air for 7 h. Plate wells were
gently washed with phosphate-buffered saline, pH 7.4,
and lysed with 1· passive lysis buffer (Promega, Madi-
son, WI). The subsequent lysates were analyzed with
the Luciferase Assay System (Promega) utilizing a TD-
20/20 luminometer (Turner Designs, Sunnyvale, CA).
The firefly luciferase relative light units were normalized
to protein (mg/ml) with BCATM Protein Assay Kit (Pierce,
Rockford, IL) and standardized to percent of control
(TNFa control). For assays of cell viability, cells were
treated similarly as above and with 15 lManalogue. After
washing, cells were treated with 100 ll media and 20 ll
CellTiter 96ꢁ AQueous One Solution reagent for 1 h and
then read at 490 nm with a Spectromax plate reader.
J = 16.1 Hz), 7.09 (m, 2H), 7.35 (m, 6H), 7.67 (d, 2H),
J = 15.9 Hz); 13C NMR: d 114.4, 117.5, 124.4, 126.3,
130.4, 136.9, 142.0, 162.9, 188.1. Anal. Calcd for
C17H12OF2: C, 75.55; H, 4.48. Found: C, 75.26; H,
4.65.
6.14. 1,5-Bis-(3,4-dihydroxyphenyl)-1,4-pentadien-3-one (36)
Yield: 76%. Orange solid: mp >250 ꢂC [lit.53 221–
223 ꢂC]; 1H NMR:
d
(DMSO) 6.78 (d, 2H,
J = 8.1 Hz), 6.99 (d, 2H, J = 15.9 Hz), 7.06 (d, 2H,
J = 7.9 Hz), 7.14 (s, 2H), 7.55 (d, 2H, J = 15.7 Hz),
9.15 (s, 2H), 9.63 (s, 2H); 13C NMR: d 114.9, 115.6,
121.5, 122.5, 126.2, 142.5, 145.4, 148.2, 187.6.
6.15. 1,5-Bis-(2-fluorophenyl)-1,4-pentadien-3-one (38)
Yield: 42%. Yellow crystals: mp 68–71 ꢂC [lit.49 68–70 ꢂC].
6.16. 1,5-Diphenyl-1,4-pentadien-3-one (39)
Yield: 86%. Yellow crystals: mp 110–112 ꢂC [lit.13,54
112–114 ꢂC].
6.17. 1,5-Bis-(4-fluorophenyl)-1,4-pentadien-3-one (40)
Acknowledgment
Yield: 87%. Yellow solid: mp 150–152 ꢂC [lit.55 152–
154 ꢂC]; 13C NMR: d 116.1, 1125.1, 130.2, 130.9,
142.0, 164.0, 188.3.
This work was supported by NIH Grant EY13695 from
the National Eye Institute and Grant BC043125 from
the U.S. Army/DOD Breast Cancer Program.

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