Synthesis of novel curcuminoids accommodating a central β-enaminone motif and their impact on cell growth and oxidative stress

Article abstract of DOI:10.1016/j.ejmech.2016.07.017

Curcuminoids are high-potential drugs targeting multiple components of vital signaling pathways without being toxic, and are therefore considered to be valuable lead structures in medicinal chemistry. Unfortunately, most curcuminoids poorly reach their site of action because of low bioavailability issues, (partly) associated with the labile β-diketo structure. In that respect, curcumin derivatives bearing a central β-enaminone fragment may have improved solubility and intestinal stability, and therefore may represent a new class of analogs with higher bioactivity. In that mindset, thirteen N-alkyl enaminones were efficiently synthesized via a novel approach, using montmorillonite K10 clay and microwave irradiation. These compounds were then characterized in terms of solubility and chemical anti-oxidant properties, and were applied in screening assays for cell toxicity, growth and oxidative stress using CHO-K1, EA.hy926, HT-29 and Caco-2?cell lines. Compared to native curcumin, many nitrogen derivatives showed a stronger antiproliferative effect, which was highly structure and cell type dependent. In addition, the correlation between cell viability and reactive oxygen species production was limited. Therefore, this set of novel curcumin derivatives may be useful to unravel other mechanisms of oxidative stress-related diseases, and eventually be used as more bioavailable and bioactive alternatives for native curcumin.

Full text of DOI:10.1016/j.ejmech.2016.07.017

Accepted Manuscript
Synthesis of novel curcuminoids accommodating a central β-enaminone motif and
their impact on cell growth and oxidative stress
Rob De Vreese, Charlotte Grootaert, Sander D'hoore, Atiruj Theppawong, Sam Van
Damme, Maarten Van Bogaert, John Van Camp, Matthias D'hooghe
PII:
S0223-5234(16)30565-7
10.1016/j.ejmech.2016.07.017
EJMECH 8735
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 May 2016
Revised Date: 8 July 2016
Accepted Date: 9 July 2016
Please cite this article as: R. De Vreese, C. Grootaert, S. D'hoore, A. Theppawong, S. Van Damme, M.
Van Bogaert, J. Van Camp, M. D'hooghe, Synthesis of novel curcuminoids accommodating a central β-
enaminone motif and their impact on cell growth and oxidative stress, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.017.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and anticancer assessment of novel curcuminoids
accommodating a central β-enaminone motif
Rob De Vreese,a Charlotte Grootaert,b Sander D’hoore,b Atiruj Theppawonga, Sam Van Damme,a
Maarten Van Bogaert,a John Van Campb,* and Matthias D’hooghea,*

ACCEPTED MANUSCRIPT
Synthesis of novel curcuminoids accommodating a central
β-enaminone motif and their impact on cell growth and
oxidative stress
Rob De Vreese,a Charlotte Grootaert,b Sander D’hoore,b Atiruj Theppawonga, Sam Van Damme,a
Maarten Van Bogaert,a John Van Campb,* and Matthias D’hooghea,*
a SynBioC Research Group, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium,
matthias.dhooghe@UGent.be
b Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-
9000 Ghent, Belgium, John.VanCamp@UGent.be
Abstract
Curcuminoids are high-potential drugs targeting multiple components of vital signaling pathways without
being toxic, and are therefore considered to be valuable lead structures in medicinal chemistry.
Unfortunately, most curcuminoids poorly reach their site of action because of low bioavailability issues,
(partly) associated with the labile β-diketo structure. In that respect, curcumin derivatives bearing a
central β-enaminone fragment may have improved solubility and intestinal stability, and therefore may
represent a new class of analogs with higher bioactivity. In that mindset, thirteen N-alkyl enaminones
were efficiently synthesized via a novel approach, using montmorillonite K10 clay and microwave
irradiation. These compounds were then characterized in terms of solubility and chemical anti-oxidant
properties, and were applied in screening assays for cell toxicity, growth and oxidative stress using CHO-
K1, EA.hy926, HT-29 and Caco-2 cell lines. Compared to native curcumin, many nitrogen derivatives
showed a stronger antiproliferative effect, which was highly structure and cell type dependent. In
addition, the correlation between cell viability and reactive oxygen species production was limited.
Therefore, this set of novel curcumin derivatives may be useful to unravel other mechanisms of oxidative
stress-related diseases, and eventually be used as more bioavailable and bioactive alternatives for native
curcumin.
1

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Introduction
The natural extracts of Curcuma Longa, defined as curcuminoids, have attracted a lot of attention in
recent years due to their broad variety of reported biological activities1 and clean safety profile.2
However, despite many efforts and realizations in curcuminoid chemistry and biology, their mode of
action still remains poorly understood.3 This could be related to the fact that curcuminoids are
regarded as pan-assay interference compounds (“PAINS”), and thus often show unspecific activity
across a range of assays.4 Another drawback associated with the curcuminoid scaffold, besides the
mainly unknown mode of action, is its poor bioavailability because of its high intrinsic chemical
reactivity (related to the presence of a β-diketone moiety, two phenolic groups and two α,β-
unsaturated carbonyl entities), water insolubility, pH dependence and metabolic instability.5
Nevertheless, curcuminoids target multiple important biological components of vital signaling
pathways and thus could serve as key building blocks toward the preparation of more drug-like
bioactive compounds.6 In that respect, the replacement of the β-diketone moiety by a β-enaminone
group could possibly improve the bioavailability and/or biological activity of the resulting structures
and could therefore represent a new approach in curcuminoid research.7 β-Enaminones in general
have been reported to display anticonvulsant, antibacterial, anti-inflammatory and antitumor
activities,8 but also comprise excellent starting points for the development of bioactive heterocycles
in medicinal chemistry.9 Curcumin-derived structures with a central β-enaminone motif, however,
have only been studied sparsely,10 and a thorough evaluation of this class of compounds is thus
lacking. This can be attributed to the fact that no convenient synthetic approach toward these β-
enaminone curcumin derivatives is available in the literature. Therefore, the goal of this study was (i)
to successfully convert naturally occurring curcuminoids, i.e. curcumin 1a and
bisdemethoxycurcumin 1b (BDMC), into a set of N-alkyl-substituted β-enaminone derivatives 2
(Figure 1) by means of a general and straightforward synthetic methodology, (ii) to characterize
these compounds in terms of solubility and antioxidant capacity, and (iii) to test the obtained
compounds for their effect on basic cell parameters such as viability, mitochondrial activity, cell
growth and oxidative stress as a preliminary investigation of the biological potential of this new class
of curcumin derivatives.
2

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Figure 1. Article outline – replacing the labile β-diketo structure with a β-enaminone system
Synthesis
Only one synthetic approach toward N-alkyl-substituted enaminone derivatives of curcumin has been
reported so far.10a In that procedure, a Dean-Stark trap with benzene as a solvent and acetic acid as a
proton source has been used, providing the desired compounds 2 (R1 = OMe; R2 = H, CH2Ph, CH(CH3)2,
CH(CH3)Ph, CH2CH3; R3 = H, Figure 1) in rather low yields (25-35%). Applying these reaction conditions
using other aliphatic amines (n-propylamine, n-butylamine) in toluene, however, resulted in a low
conversion of the starting material to the corresponding enaminones after long reaction times (40
hours), pointing to the limitation of this synthetic strategy. To improve these conversions and to
drive the reaction faster to completion, in the present study we enforced microwave irradiation11 and
added montmorillonite K10 clay (MK10 clay) as an additional reagent.12 Microwave irradiation –
considered to be a green approach – often accelerates the reaction speed and gives altered product
distributions compared to conventional heating, and MK10 clay is known to act as an efficient
catalyst for this type of reactions. So far, the combination of MK10 and microwave irradiation has
amply been used in the literature, and almost always resulted in efficient synthesis of the anticipated
products.13 The straightforward synthesis of β-enaminones from methyl acetoacetate, ethyl
acetoacetate and acetylacetone, by using both microwave irradiation and MK10, has also been
reported recently.14 Inspired by these promising results, several attempts were made to produce β-
enaminone analogs of curcumin, employing a microwave irradiation/MK10 strategy. After thorough
evaluation of a variety of reaction condition combinations (by changing the solvent, time,
temperature, microwave conditions, amount of acid, amine and MK10 clay used), full conversion of
the starting material was eventually achieved in rather short reaction times (60-105 minutes, Scheme
1, Table 1). As shown in Table 1, the optimal reaction conditions to obtain curcumin analogs 3a-e
comprise treatment of curcumin with an excess of amine (5 equiv.), 1.2 equiv. of acetic acid and one
mass equiv. of MK10 clay. Stirring of this mixture in chloroform under microwave irradiation at a
temperature of 80°C resulted in the formation of compounds 3a-e in short reaction times (60
minutes) and acceptable yields (40-56%). A double amount of cyclohexylamine (10 equiv.) and acetic
acid (2.4 equiv.) was required to fully convert curcumin to enaminone derivative 3f in a respectable
3

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yield (58%). The enaminone analogs 3a-f were purified via column chromatography (SiO2) and finally
1
obtained in high purity (>95%, determined via LC-MS and H NMR). As can be noticed from these
yields, a substantial amount of product was lost during column chromatography because of the
cumbersome separation of the target product from the minor impurities formed during the reaction.
Recrystallization of the products was also evaluated as a purification method, albeit without any
success. For the synthesis of bisdemethoxycurcumin analogs 3g-i, slightly longer reaction times (90-
105 minutes) and another solvent (2-methyltetrahydrofuran) were required to attain full conversion
(Scheme 1, Table 1). The corresponding enaminones 3g-i were isolated in somewhat lower yields (11-
40%), but could be purified more easily through recrystallization from acetone or methanol/water,
providing high purity samples (>95%, determined via LCMS and 1H NMR). Again, a substantial amount
of product was lost during the purification step because of difficult separation of the product from
minor impurities. Although an excess of amine was used in all reactions to effect full conversion, no
formation of di-imines was observed, pointing to the reluctance of the obtained enaminones to
undergo a second imination step. Spectroscopic NMR analysis of compounds 3a-i revealed
characteristic signals for the β-enaminone group, in accordance with previous literature data of
similar structures,10a indicating the dominance of the β-enaminone structure over the β-iminoketone
form. Both the CH proton and the exchangeable NH proton of enaminones 3a-i were clearly present
at δ-values between 5.5-5.6 and 11.5-11.7 ppm, respectively (1H NMR, CDCl3 and D6-DMSO),
confirming the presence of the β-enaminone entity in these molecules. In summary, this new
method was shown to represent a reliable, general approach for the convenient synthesis of N-alkyl-
substituted β-enaminone curcuminoids in acceptable yields and high purities, demonstrated by the
preparation of nine representatives.
Scheme 1. Microwave-assisted synthesis of β-enaminones 3a-i.
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Table 1. Reaction conditions and yields for the synthesis of β-enaminones 3a-i.
Time
60
Equiv.
1.2
Equiv.
Compound
R1
OMe
OMe
OMe
OMe
OMe
OMe
H
R2
Solvent
CHCl3
Yield (%)
40
3a
3b
3c
3d
3e
3f
nPr
5
5
allyl
60
CHCl3
1.2
48
nBu
60
CHCl3
1.2
5
47
iBu
60
CHCl3
1.2
5
56
secBu
cycloHex
iBu
60
CHCl3
1.2
5
48
60
CHCl3
2.4
10
5
58
3g
3h
3i
90
2-methyl-THF
2-methyl-THF
2-methyl-THF
2.4
30
H
secBu
cycloHex
90
2.4
5
40
H
105
2.4
7
11
In a following part, four of the obtained β-enaminones (3d-g) were O-acetylated to assess the effect
of free versus protected phenolic groups on the biological activity of these compounds. To achieve
this, a previously reported procedure for bis-acetylated curcumin synthesis was slightly modified in
terms of the solvent used (the more environmentally friendly solvent 2-methyltetrahydrofuran was
used instead of dichloromethane, Scheme 2).15 Otherwise, no adjustments were made to the
reported procedure and compounds 4a-d were obtained in 42–67% yield upon treatment of
enamines 3d-g with three equiv. of acetic anhydride and three equiv. of pyridine. Bis-acetylated
curcumin 5 was also prepared as a reference compound, to more easily compare the enamine
analogs with their respective oxygenated counterparts. Hence, the previously mentioned modified
procedure was used to synthesize the earlier documented bis-acetylated curcumin 5 in an excellent
yield as well (98%, Scheme 2).15
Scheme 2. Synthesis of acetylated β-enaminones 4a-d and bis-acetylated curcumin 5.
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In conclusion, thirteen β-enaminones (3a-i and 4a-d) were efficiently synthesized, including nine
compounds with free phenolic groups and four O-acetyl-protected structures. Nine of these
molecules were derived from curcumin and four from bisdemethoxycurcumin. This small set of
compounds allows for a preliminary SAR study concerning the influence of free or O-acetylated
phenols, dimethoxy or bisdemethoxy analogs, and the nature of the N-alkyl group on the biological
activity of this class of β-enaminone curcuminoids.
Characterization of solubility and anti-oxidant activity
In a next step, the solubility of the curcumin derivatives in cell culture medium was evaluated to have
an idea of the bioaccessibility of the compounds to the cells, which may then reflect also differences
in bioactivity. To this end, a concentration range of 0, 0.5, 1, 5, 10, 25 and 50 µM was prepared in
serum-free DMEM, transferred to a 96-well plate and incubated for 1 day at 37°C and 10% CO2
conditions. Crystal formation was checked microscopically. Table 2 presents the concentration at
which crystals were detected.
Table 2. Chemical characterization of the curcumin derivatives in terms of solubility in cell culture medium and
antioxidant capacity using the DPPH and FRAP test.
DPPH (% inhibition
FRAP (Trolox
equivalent per µM)
1.00
Compound
R1
R2
R3
Solubility (µM)
per µM)
0.15
1a
1b
OMe
H
n.a.
n.a.
H
H
0.5
25
0.02
0.28
3a
3b
3c
3d
3e
3f
OMe
OMe
OMe
OMe
OMe
OMe
H
nPr
allyl
H
H
H
H
H
H
H
H
H
Ac
50
25
5
0.10
0.10
0.08
0.09
0.13
0.13
0.02
0.03
0.03
0.00
0.87
1.14
1.29
0.89
1.08
0.83
0.19
0.18
0.56
0.04
nBu
iBu
25
50
25
25
25
5
secBu
cycloHex
iBu
3g
3h
3i
H
secBu
cycloHex
iBu
H
4a
OMe
5
4b
4c
4d
5
OMe
OMe
H
secBu
cycloHex
iBu
Ac
Ac
Ac
Ac
5
5
0.00
0.01
0.00
0.00
0.00
0.04
0.01
0.03
25
0.5
OMe
n.a.
6

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It was seen that β-diketone 5 and curcumine 1a were highly insoluble (crystal formation at 0.5 µM),
followed by compounds with an acetyl group on the R3 position (crystal formation at 5 µM) with
exception of compound 4d, and compound 3c and 3i. Two compounds 3a and 3e showed a better
solubility then the other compounds and only started to form crystals at a concentration of 50 µM.
To assess the antioxidant properties of the newly synthetized structures, two commonly used tests
were used including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and the ferric reducing ability of
plasma (FRAP) assay. The DPPH assay is based on the neutralization of the stable DPPH radical and
measures the percentage inhibition of the radical activity, whereas the FRAP assay is based on the
reduction of a ferric-tripyridyltriazine complex (Fe3+-TPTZ) to the ferrous form (Fe2+-TPTZ) and is
measured in trolox equivalents. The DPPH results in Table 2 are expressed as percentage inhibition
per µM, based on the slope of the linear curve in the (%) inhibition in function of concentration
graphs. Similarly, the values of the FRAP assay are expressed as trolox equivalents, i.e., the anti-
oxidant potential of the analog compared to that of trolox as a reference molecule, per µM based on
the slope of the linear curve of trolox equivalents in function of concentration of the analog. The
higher the values of the DPPH and FRAP assay, the higher the anti-oxidant capacity of the analog.16
From Table 2 it can be seen that high antioxidant activities were observed by both the FRAP and
DPPH assay for compounds 3a-f (0.08-0.13 % inhibition/µM and 0.83-1.29 trolox equiv./µM,
respectively). Reference compound curcumin 1a showed activity in the same range on both the FRAP
and DPPH assay (0.15 % inhibition/µM and 1 trolox equiv./µM respectively). All compounds exerting
good antioxidant activity (curcumin 1a and enaminones 3a-f) carry a methoxy group in ortho-position
with respect to the phenol group on the aromatic rings. The favorable impact of the ortho-methoxy
group on the antioxidative activity has previously been established for curcumin analogs and
explained by the formation of an intramolecular hydrogen bond between the hydrogen atom of the
phenol and the ortho-methoxy group, leading toward a facilitated removal of the hydrogen atom and
thus an improved antioxidant activity.17 For β-enaminone analogs 3g-i and reference molecule
bisdemethoxycurcumin 1b (not containing an ortho-methoxy group), only small antioxidant effects
were observed in both assays (0.2-0.3% inhibition/µM and 0.18-0.56 trolox equiv./µM), again
pointing to the importance of the ortho-methoxy group for the antioxidant activity of curcumin
analogs. Protection of both phenolic groups through acetylation (in compounds 4a-d and bis-
acetylated curcumin 5) resulted in complete abolition of the antioxidant activity in both assays,
further supporting the established importance of the free phenolic groups for the antioxidant
activity. It should also be noted that the obtained N-alkyl enaminone derivatives of
(bisdemethoxy)curcumin in general display a similar aqueous solubility as compared to the original
substrates.
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Evaluation of cell growth and oxidative stress
It is known that curcuminoids have a beneficial effect on, amongst others, inflammation18 and
chronic diseases such as cancer,19 cardiovascular diseases,20 diabetes,21 metabolic syndrome,22
aging.23 Cell growth, viability and oxidative stress are common cell markers involved in these health
aspects. 24 Therefore, in this study, we have applied the newly synthesized nitrogen analogs 3a-i and
4a-d, as well as the non-nitrogen-containing reference molecules 1a,b and 5 on cell lines and have
performed high throughput assays for protein content as marker for cell growth (SRB), mitochondrial
respiration (MTT) and intracellular ROS production (DCFH). Cell lines included human-derived
intestinal cells such as the HT-29 cell line and the Caco-2 cell line under undifferentiated and
differentiated conditions, to simulate the gut which is considered to be the first barrier exposed to
curcuminoids. In addition, the EA.hy962 cell line, which shows characteristics of endothelial cells, was
used as well because of the influence of angiogenesis in many of the chronic diseases mentioned
above. Finally, the analogs were also applied on the Chinese hamster ovarian cell line CHO-K1,
because this fast-growing and relatively stable cell line is, in contrast to the other cell lines, not from
a cancerous origin. Concentrations of the curcumin analogs applied onto the cells were 0, 1, 5, 10
and 25 µM in cell culture medium.
Table 3 shows the IC50 of the mitochondrial activity (MTT) and protein content of the cells (SRB), two
parameters that indicate cell growth inhibition when they both decrease in parallel. A first
observation is that inhibition of mitochondrial activity and protein content was strongly dependent
on the cell type. Interestingly, none of the enaminones caused cytotoxic effects on differentiated
Caco-2 cells. As differentiated Caco-2 cells show high similarities with in vivo enterocytes lining the
intestinal tract, we may conclude that none of these analogs disturb the intestinal barrier by cell
damage, which is desirable for therapeutic use. In addition, chemical compounds specifically
targeting and killing undifferentiated cells of tumor origin without damaging differentiated cells are
interesting candidates for anti-cancer drugs.25 Secondly, when examining the nitrogen-lacking
reference compounds 1a,b and 5, curcumin decreased the mitochondrial activity of the CHO ovarian
cell line to a large extent, and none of the reference compounds had an effect on the
undifferentiated cell lines from cancerous origin. In contrast, β-enaminone analogs of
bisdemethoxycurcumin 3g-i were cytotoxic for all undifferentiated cell lines at the concentrations
tested. Especially compound 3i, bearing a cyclohexyl-substituted enamine structure, was more
cytotoxic compared to curcumin (3i: IC50 CHO = 9.2 µM, IC50 Caco-2 undiff. = 7.8 µM, IC50 HT-29 = 4.0
µM).
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Table 3. Cell growth inhibition (IC50-values in µM) measured after 72 h of curcumin derivative treatment using
assays for mitochondrial activity (MTT) and protein content (SRB).
IC50 CHO
(µM)
IC50 Caco-2
undiff (µM)
IC50 Caco-2
diff (µM)
IC50 HT-29
(µM)
IC50 EA.hy926
(µM)
MTT SRB
MTT
SRB
>25
>25
22.0
>25
23.8
>25
>25
5.1
MTT
SRB
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
MTT
SRB
>25
>25
>25
24.6
21.2
24.0
22.3
>25
22.5
15.6
5.9
MTT
>25
>25
>25
>25
>25
>25
>25
>25
>25
22.0
3.3
SRB
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
4.2
1a
1b
3a
3b
3c
3d
3e
3f
7.5
>25
>25
>25
>25
>25
>25
>25
>25
>25
23.0
9.4
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
22.7
>25
19.7
24.5
>25
>25
20.1
13.0
4.0
>25 >25
>25 >25
7.4
7.0
>25
20.4
>25 >25
>25 >25
24.7 >25
17.9 23.7
13.1 15.1
3g
3h
3i
18.5
5.8
9.2
11.6
7.8
6.9
4a
4b
4c
4d
5
>25 >25
>25 >25
10.1 >25
>25 >25
>25 >25
>25
>25
9.7
>25
24.0
4.4
>25
>25
>25
13.7
>25
>25
>25
>25
22.6
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
Despite the high potency of these compounds to target undifferentiated cells, their potential use as
anticancer drugs requires further optimization studies, as it is not desirable to destroy other dividing
cells of non-cancerous origin. Therefore, other nitrogen derivatives may be more interesting in this
context. Some of the curcumin nitrogen derivatives 3 and bis-acetylated analogs 4 were more
selective in their cytotoxic activity. Especially compounds 3a, 3d, 3e, 4b and 4d were effective against
cancer-related cell lines without cytotoxicity to the CHO-K1 cell line. In addition, these specific
compounds did not affect the viability of the endothelial-like cell line EA.hy926, which may indicate
that their circulation in the blood stream may not damage the endothelial cells. Interestingly, a
methoxy group on the R1 position in combination with a cyclohexyl group on the R2 position
specifically targeted undifferentiated Caco-2 cells (compounds 3f, and 4c), whereas the viability of
the HT-29 cells was unaffected. A reason why both intestinal cell lines react differently is currently
unclear, although differences in both cell lines in terms of morphology, differentiation behavior and
gene expression have been reported previously.26 Overall, the selectivity of the different curcumin
derivatives towards certain cell types is probably caused by their different uptake mechanisms by the
9

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cells and/or their influence on different pathways. In a next set of experiments, intracellular ROS was
evaluated in the different cell lines because of its possible role in cytotoxicity-related pathways.
In most of the chronic diseases, such as metabolic syndrome, inflammatory diseases etc., higher ROS
levels are considered to be hazardous and may be counteracted by anti-oxidants. Yet, in cancer
development, high levels of intracellular ROS may have a dual effect. On the one hand, high ROS
levels may cause DNA damage,27 and therefore increase the risk on mutated cells in the first stage of
cancer development. On the other hand, higher ROS levels may also cause cell apoptosis through the
mitochondrial intrinsic pathway, which is the permeabilization of the mitochondrial outer membrane
and the consequent release of pro-apoptotic proteins.28 In general, cancer cells have higher intrinsic
ROS levels compared to normal cells and therefore, pro-oxidant compounds may specifically target
and destroy cancer cells. In general, many polyphenols have both anti-oxidant and pro-oxidant
activities, dependent on structure and intracellular concentration. To investigate whether the
observed cytotoxic effects may be correlated with pathways affected by ROS, we have measured
intracellular ROS using the 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) method.29 We have
applied a mild (1 µM) and high (10 µM) concentration of the curcuminoids, which is in the range of
plasma concentrations reported by Jäger et al.30
A first observation (Table 4) is that the ROS modulating effect of the curcuminoids is strongly
dependent on the cell type. For undifferentiated cells of intestinal cancer origin (Caco-2 and HT-29),
curcuminoids significantly decreased intracellular ROS, especially at a concentration of 10 µM. HT-29
cells were more sensitive than Caco-2 cells, as also at 1 µM, decreases were observed. For CHO-cells,
a mild increase in ROS was observed for some curcuminoids at a concentration of 1 µM. In contrast,
EA.hy926 cells were strongly responsive to 10 µM of most of the curcuminoids, leading towards ROS
increases up to more than 4-fold for 3c and 3h. Interesting exceptions to this effect was compound
4d, which caused a significant decrease of ROS, and 3a and 4b, which showed variable effects.
The comparison of our results with existing literature is not always easy because of differences in
experimental setup including the type of cell lines, the type of analogues and the concentrations and
incubation times with the test compounds. ROS measurements after curcumin incubation has been
described for fibroblast L929,31 HGF32 and CCD-18Co cells,33 human colon carcinoma RKO, SW480,33-34
HT29,35 pancreatic PC-3 and DU145 cells,36 monocyte U937 cells37 and nasopharyngeal CNE1 and
CNE238 cell lines. In all cases, a ROS increase of 2- to even 30-fold was reported depending on cell
line, concentration and incubation time. Our results however showed a limited ROS effect of
curcumin for the tested cell lines. As far as we know, no literature is available on the impact of
curcuminoids on the intracellular ROS production in the Caco-2, CHO-K1 or EA.hy926 cell line. For the
HT-29 cell line, the study of Chen et al. (2012) showed a significant 1.5- to 2.5-fold increase of cellular
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ROS levels after 6 and 12 hours of incubation with 40 µM curcumin. In contrast, our results
demonstrate a ROS-decreasing effect at similar incubation conditions, but at the lower
concentrations of 1 and 10 µM. In addition, we have applied the more frequently used DCFA method
instead of the dihydrorhodamine method, but although small differences exist in the outcome of
these assays,39 we do not expect that this has contributed to the differences in ROS trend.
Furthermore, this decrease could not be correlated to decreased viability. Therefore, we conclude
that the anti-oxidant or pro-oxidant activity of curcumin in the HT-29 cell line is concentration
dependent, and that physiologically relevant concentrations of curcumin result in an anti-oxidant
effect.
So far, the effect of similar nitrogen-containing analogues on ROS production has not been reported,
but the effect of other types of analogues has been published. Gandhy et al. (2012)33 showed that
the concentration of the piperidone analogue (3E,5E)-3,5-bis(2,5-dimethoxybenzylidene)-1-
butoxycarbonylpiperidin-4-one needed for the same ROS stimulating effect on RKO cells was 15 to 30
times lower than curcumin. In a study of Atsumi et al. (2007),32 a one hour incubation with curcumin
resulted in a 4-fold increase of ROS for 10 µM, and a 12-fold increase for 30 µM, whereas no ROS
increase was detected with tetrahydrocurcumin in HSG cells at concentrations of 30 and 100 µM.
This illustrated the importance of the unsaturated bonds in the middle chain of the curcumin
molecule in its ROS-scavenging capacity. Zhang et al. (2015) have demonstrated that a nitrogen-
containing monocarbonyl analogue WZ35 [1-(4-hydroxy-3-methoxyphenyl)-5-(2-nitrophenyl)penta-
1,4-dien-3-one] was able to induce a 2-fold increase in ROS in pancreatic cell lines after 24 hours of
incubation with 10 µM of the compound. These conditions and results are in the range of those
obtained with our experimental setup for the CHO and especially the EA.hy926 cell lines. This study,
and the one from Zou et al. (2015),40 has demonstrated that the anticancer effect of this analogue
WZ35 was mediated through ROS and its subsequent activation of JNK mitochondrial and
endoplasmatic reticulum (ER) stress apoptotic pathways. In our studies, only two compounds, i.e. 3h
and 3i, showed a correlation between ROS induction and decreased cell viability in especially the
EA.hy926 cell line. Therefore, in future research it may be interesting to investigate whether
apoptosis through JNK mitochondrial and ER stress is at the basis of this effect. For the other cell
lines and analogues, other ROS mediated pathways should be explored. For instance, ROS
modulation may also impact NF-κB activity, thereby regulating downstream factors involved in
apoptosis, proliferation, tumor promotion, metastasis, angiogenesis, inflammation and immortality.41
An interesting study in this context is the one from Sandur et al. (2007),42 in which curcumin was
shown to produce ROS while TNF-mediated NF-κB activation was inhibited. The authors suggested a
concentration effect of ROS or the possibility that the apoptotic effects of curcumin are mediated
through ROS while NF-κB activation was supressed in other ways. In future research, pathway
11

ACCEPTED MANUSCRIPT
analysis using molecular techniques may further elucidate the mechanisms by which the nitrogen-
containing curcuminoids may impact cell-related processes in health and disease conditions.
We conclude that in our results, no correlation could be found between chemical anti-oxidant
capacity and anti-oxidant effects in the cells. In addition, no correlation could be found between pro-
oxidant effects and decreased viability, and therefore, none of the curcuminoids seemed to exert
anti-cancer effects through ROS induction. Instead, other mechanisms should be explored in the
future.
Table 4. Intracellular reactive oxygen species (ROS), expressed as percentage compared to the untreated cells
(mean ± standard deviation). *(p<0.05)/**(p<0.01) and §(p<0.05)/§§(p<0.01) indicate significant decreases and
increases, respectively, compared to the untreated cells according to a two-tailed Student t-test with unequal
variances (n≥6).
CHO (%)
Caco-2 undiff (%)
10 µM
HT-29 (%)
EA.hy926 (%)
1 µM 10 µM
1 µM
10 µM
1 µM
1 µM
10 µM
1a 112 (±19.5)
1b 101 (±2.1)
97 (±8.7)
105 (±4.1)
84 (±12.2)**
98 (±19.7)
97 (±21.1)
89 (±13.3)
91 (±8.4)*
91 (±11.5)
91 (±11.7)
88 (±15.8)
100 (±19.0)
102 (±23.2)
77 (±17.6)**
83 (±13.2)**
88 (±11.9)*
76 (±15.6)**
83 (±18.5)*
74 (±15.4)**
66 (±9.5)**
63 (±17.9)**
82 (±20.4)*
102 (±13.8)
82 (±20.4)**
77 (±20.5)**
86 (±18.5)
67 (±9.0)**
75 (±5.3)**
88 (±9.2)*
99 (±5.4)
65 (±10.7)**
69 (±2.7)**
92 (±10.8)
84 (±9.3)**
98 (±6.8)
91 (±8.0)
92 (±6.7)
88 (±2.4)
103 (±16.3)
3a 116 (±12.8)§ 118 (±11.8)
3b 115 (±8.0)§
104 (±7.8)
3c 129 (±20.8)§ 110 (±7.2)
78 (±6.4)** 141 (±3.8)§§
124 (±33.9) 267 (±23.4)§§
110 (±29.9) 439 (±65.0)§§
83 (±22.8) 100 (±16.4)
85 (±18.6) 184 (±55.5)§
99 (±9.3)
3d 104 (±5.1)
118 (±19.0)
112 (±4.1)§
91 (±6.7)**
92 (±8.6)*
90 (±7.0)**
74 (±10.6)**
75 (±8.7)**
76 (±4.5)**
80 (±9.6)**
96 (±7.2)
3e 107 (±13.9)
99 (±6.5)
3f 137 (±36.0)§ 122 (±17.4)
80 (±8.8)**
77 (±3.1)**
76 (±6.4)**
77 (±12.1)**
95 (±4.6)**
92 (±6.2)**
96 (±2.4)*
92 (±6.3)**
79 (±10.0)**
91 (±8.4)
100 (±11.6) 119 (±12.6)§
99 (±4.8)
414 (±46.1)§§
191 (±12.1)§§
3g 109 (±4.9)
3h 104 (±12.7)
3i 111 (±11.2)
4a 111 (±20.5)
4b 88 (±8.2)
97 (±4.2)
111 (±9.0)§
150 (±15.7)§§ 97 (±16.1)
129 (±6.0)§ 299 (±11.4)§§
104 (±10.2) 240 (±55.3)§§
68 (±8.5)** 105 (± 10.1)
101 (±8.3) 113 (±4.6)
60 (±3.2)** 53 (±3.5)**
114 (±18.9) 189 (±57.7)§
111 (±7.6)
92 (± 7.1)
122 (±33.5)
96 (±22.6)
95 (±12.6)
89 (±14.3)
89 (±7.5)**
94 (±6.4)*
99 (±10.2)
76 (±16.8)**
4c 89 (±16.8)
62 (±7.5)**
85 (±15.9)*
90 (±17.7)
4d 152 (±30.4)§ 142 (±28.5)§ 102 (±10.3)
128 (±19.0)§ 112 (±11.7)
93 (±17.9)
5
Conclusion
The efficient synthesis of thirteen β-enaminone analogs of curcumin was established through a new
approach, based on the use of microwave irradiation and montmorillonite K10 clay, thus replacing
the labile β-diketo structure with a β-enaminone fragment. These thirteen structures were subjected
to a preliminary bioactivity screening using different cell lines, and their reactive oxygen species
(ROS)-generating potential and antioxidative activity was evaluated. We conclude that (i) N-analogs
of curcumin strongly increase the cellular responses compared to the non-nitrogen analogs (1a, 1b,
12

ACCEPTED MANUSCRIPT
5), (ii) all compounds have no toxic effect on differentiated intestinal cells, which is desirable, (iii) no
correlation between chemical anti-oxidant capacity and intracellular ROS could be found, which also
shows the limitations of the chemical anti-oxidant assays to predict biological effect, and (iv) viability
and ROS results are strongly cell type dependent, thereby indicating the involvement of other targets
than ROS-dependent pathways. Because of their strong cytotoxic effects, compounds 3c, 3g, 3h and
3i are considered to be suitable candidates for further elaboration in terms of lead scaffold
optimization studies. In addition, we also recommend to investigate other oxidative stress-related
diseases than cancer. Table 5 gives an overview of the properties of the tested substrates.
Table 5. Overview of the properties of the tested substrates. For chemical characteristics, - and + indicate
ranking according to the characteristic. For cell effects, 0 = no effect, - = decrease and + = increase compared to
untreated cells.
Intestinal
(cancer)
Ovarian
(non-cancer)
Endothelial (cancer)
R1
R2
n.a.
n.a.
nPr
R3 Solub. Anti-ox. Viability ROS Viability ROS
Viability
ROS
0
1a OMe
H
H
-
++
+++
++
+
++
-
0, 0
0, 0
-, -
--,--
--,--
-,-
0
0
0
0
-
0
+
+
+
+
+
+
++
0
+
+
+
0
+
+
+
0
0
0
0
0
0
0
0
0
0
---
0
0
0
0
0
1b
H
0
3a OMe
3b OMe
3c OMe
3d OMe
3e OMe
H
+
--, +
++
+++
0
allyl
nBu
iBu
H
++
++
+
0, -
-,-
H
-, -
-, 0
-, -
H
++
+++
++
++
++
-
0, -
0
0
0
-
secBu
H
++
+
0, -
--, 0
--,--
--,--
0, --
--,--
--, -
0, -
-, 0
-, 0
0, -
+
3f OMe cycloHex
H
---, 0
-,-
+
3g
3h
3i
H
H
H
iBu
secBu
cycloHex
iBu
H
-
+
H
-
--,--
--,---
0, 0
-, 0
--
--
0
0
0
0
0
+++
++
++
-, 0
0
H
+
4a OMe
4b OMe
Ac
Ac
Ac
Ac
Ac
+
--
--
--
--
--
secBu
+
4c OMe cycloHex
+
---, 0
0, --
0, 0
4d
5
H
iBu
++
-
--
OMe
n.a.
+
In summary, this study serves as a valuable starting point for the design of new curcuminoids bearing
a β-enaminone motif and their deployment as promising drugs for oxidative stress-related diseases.
Future research should focus on elaborate bioactivity and bioavailability optimization through
chemical modification of the lead structures identified in this work.
13

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Experimental section
Chemistry
General methods
1H NMR spectra were recorded at 300 MHz (Jeol Eclipse+) or 400 MHz (Bruker Advance III Nanobay)
with CDCl3 or D6-DMSO as solvent. 13C NMR spectra were recorded at 75 MHz (Jeol Eclipse+) or 100.6
MHz (Bruker Anvance III Nanobay) with CDCl3 or D6-DMSO as solvent. Mass spectra were obtained
with a mass spectrometer Agilent 1100, 70 eV. IR spectra were measured with a Spectrum One FT-IR
spectrophotometer. High resolution electron spray (ES) mass spectra were obtained with an Agilent
Technologies 6210 series time-of-flight instrument. Melting points of crystalline compounds were
measured with a Kofler Bench, type WME Heizbank of Wagner & Munz. Microwave reactions were
carried out with a CEM Discover microwave at fixed temperature. The purity of all tested compounds
was assessed by 1H NMR analysis and/or HPLC analysis, confirming a purity of ≥95%.
Representative procedure for the synthesis of β-enaminones 3a-i
Curcumin 1a (5 mmol, 1.84 g) was dissolved in chloroform (15 mL) and mixed with five equiv. of
isobutylamine (25 mmol, 2.48 mL) and 1.2 equiv. of glacial acetic acid (6 mmol, 0.34 mL).
Montmorillonite K10 clay (MK10, 1.83 g) was added and the resulting mixture was stirred for 60
minutes at 80°C under microwave irradiation. After reaction, the mixture was filtered over celite and
the filter cake was thoroughly rinsed with chloroform (200 mL). The filtrate was extracted with
sodium bicarbonate (50 mL) and brine (50 mL), and dried over magnesium sulfate. Evaporation of
chloroform and purification by means of column chromatography on silica gel (EtOAc/PE 3/2)
resulted in 1.19 g (1E,4Z,6E)-1,7-bis-(4-hydroxy-3-methoxyphenyl)-5-isobutylamino-1,4,6-heptatrien-
3-one 3d (2.81 mmol, 56%).
(1E,4Z,6E)-1,7-Bis-(4-hydroxy-3-methoxyphenyl)-5-propylamino-1,4,6-heptatrien-3-one 3a
1H NMR (300 MHz, CDCl3): δ 1.04 (3H, t, J = 7.2 Hz), 1.71 (2H, sextet, J = 7.2 Hz), 3.37-3.39 (2H, m),
3.92 and 3.95 (2 × 3H, 2 × s), 5.52 (1H, s), 6.63 and 6.67 (2 × 1H, 2 × d, J = 15.4 Hz), 6.91-7.09 (6H, m),
7.21 and 7.46 (2 × 1H, 2 × d, J = 15.4 Hz), 11.54 (1H, bs). 13C NMR (75 MHz, CDCl3): δ 11.6, 23.6, 45.5,
56.0, 56.1, 93.4, 109.4, 114.7, 115.0, 118.1, 121.8, 122.3, 126.9, 128.2, 128.8, 137.1, 137.6, 146.8,
146.9, 147.0, 147.4, 162.9, 185.5. IR (ATR, cm-1): νNH/OH = 3000; νC=O = 1567. MS (70 eV): m/z (%) 410
([M+1]+, 100). HRMS (ESI): m/z for C24H28NO5 [M+H]+ calcd 410.1962, found 410.1916. Orange
crystals. Column chromatography Rf (SiO2) = 0.18, EtOAc/PE (3/2), yield 40%. Mp = 160˚C.
(1E,4Z,6E)-5-Allylamino-1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one 3b
1H NMR (300 MHz, CDCl3): δ 3.93 and 3.95 (2 × 3H, 2 × s), 4.04 (2H, t, J = 5.3 Hz), 5.22-5.37 (2H, m),
5.57 (1H, s), 5.80 and 5.85 (2 × 1H, 2 × bs), 5.90-5.99 (1H, m), 6.65 (2H, d, J =15.9 Hz), 6.89-6.97 (3H,
m), 7.04-7.11 (3H, m), 7.20 and 7.47 (2 × 1H, 2 × d, J = 15.9 Hz), 11.48 (1H, bs). 13C NMR (75 MHz,
CDCl3): δ 45.9, 56.0, 56.1, 93.8, 109.3, 109.4, 114.7, 114.9, 116.7, 118.1, 121.8, 122.4, 126.8, 128.2,
14

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128.7, 134.3, 137.5, 137.7, 146.8, 146.9, 147.0, 147.4, 163.0, 186.0. IR (ATR, cm-1): νNH/OH = 3006; νC=O
= 1562. MS (70 eV): m/z (%) 408 ([M+1]+, 100). HRMS (ESI): m/z for C24H26NO5 [M+H]+ calcd
408.1806, found 408.1816. Orange crystals. Column chromatography Rf (SiO2) = 0.15, EtOAc/PE (7/6),
yield 48%. Mp = 160˚C.
(1E,4Z,6E)-5-Butylamino-1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one 3c
1H NMR (300 MHz, CDCl3): δ 0.96 (3H, t, J = 7.2 Hz), 1.47 (2H, sextet, J = 7.2 Hz), 1.67 (2H, quin, J = 7.2
Hz), 3.41 (2H, q, J = 6.6 Hz), 3.92 and 3.95 (2 × 3H, 2 × s), 5.52 (1H, s), 6.01 (2H, bs), 6.63 and 6.68 (2 ×
1H, 2 × d, J = 15.4 Hz), 6.89-7.09 (6H, m), 7.21 and 7.46 (2 × 1H, 2 × d, J = 15.4 Hz), 11.54 (1H, bs). 13
C
NMR (75 MHz, CDCl3): δ 13.9, 20.1, 32.3, 43.4, 56.0, 56.1, 93.3, 109.4, 114.7, 115.0, 118.1, 121.8,
122.3, 126.9, 128.2, 128.8, 137.1, 137.6, 146.8, 146.9, 147.0, 147.4, 162.9, 185.4. IR (ATR, cm-1):
νNH/OH = 3004; νC=O = 1567. MS (70 eV): m/z (%) 424 ([M+1]+, 100). HRMS (ESI): m/z for C25H30NO5
[M+H]+ calcd 424.2119, found 424.2129. Orange crystals. Column chromatography Rf (SiO2) = 0.14,
EtOAc/PE (3/2), yield 47%. Mp = 169˚C.
(1E,4Z,6E)-1,7-Bis-(4-hydroxy-3-methoxyphenyl)-5-isobutylamino-1,4,6-heptatrien-3-one 3d
1H NMR (400 MHz, CDCl3): δ 1.04 (6H, d, J = 6.6 Hz), 1.94 (1H, nonet, J = 6.6 Hz), 3.24 (2H, t, J = 6.6
Hz), 3.92 and 3.96 (2 × 3H, 2 × s), 5.52 (1H, s), 6.64 (1H, d, J = 15.7 Hz), 6.67 (1H, d, J = 15.9 Hz), 6.90
(1H, d, J = 8.2 Hz), 6.95 (1H, d, J = 8.2 Hz), 6.98-6.99 (1H, m), 7.05-7.10 (3H, m), 7.21 (1H, d, J = 15.9
Hz), 7.47 (1H, d, J = 15.7 Hz), 11.64-11.67 (1H, m). 13C NMR (100 MHz, CDCl3): δ 20.4, 29.4, 51.5, 56.0,
56.1, 93.5, 109.40, 109.42, 114.7, 115.0, 118.4, 121.8, 122.3, 126.9, 128.3, 128.8, 137.1, 137.6, 146.8,
146.9, 147.0, 147.4, 163.1, 185.5. IR (cm-1): νC=O = 1567. MS (70 eV): m/z (%) 424 ([M+1]+, 100). HRMS
(ESI): m/z for C25H30NO5 [M+H]+ calcd 424.2119, found 424.2119. Orange crystals. Column
chromatography Rf (SiO2) = 0.18, EtOAc/PE (3/2), yield 56%. Mp = 84°C.
(1E,4Z,6E)-1,7-Bis-(4-hydroxy-3-methoxyphenyl)-5-sec-butylamino-1,4,6-heptatrien-3-one 3e
1H NMR (400 MHz, CDCl3): δ 1.00 (3H, t, J = 7.4 Hz), 1.29 (3H, d, J = 6.5 Hz), 1.61-1.69 (2H, m), 3.67-
3.74 (1H, m), 3.93 and 3.96 (2 × 1H, 2 × s), 5.48 (1H, s), 5.77 and 5.84 (2 × 1H, 2 × bs), 6.63 (1H, d, J =
15.7 Hz), 6.69 (1H, d, J = 15.9 Hz), 6.90 (1H, d, J = 8.1 Hz), 6.95 (1H, d, J = 8.1 Hz), 6.97-6.98 (1H, m),
7.05-7.10 (3H, m), 7.21 (1H, d, J = 15.9 Hz), 7.47 (1H, d, J = 15.7 Hz), 11.56 (1H, d, J = 8.8 Hz). 13C NMR
(100 MHz, CDCl3): δ 10.6, 21.8, 30.8, 50.9, 56.0, 56.1, 93.4, 109.3, 109.4, 114.7, 115.0, 118.5, 121.7,
122.4, 127.0, 128.4, 128.9, 137.1, 137.4, 146.8, 146.89, 146.92, 147.3, 162.4, 185.4. IR (cm-1): νC=O
=
1564. MS (70 eV): m/z (%) 424 ([M+1]+, 100). HRMS (ESI): m/z for C25H30NO5 [M+H]+ calcd 424.2119,
found 424.2123. Orange crystals. Column chromatography Rf (SiO2) = 0.16, EtOAc/PE (3/2), yield 48%.
Mp = 88°C.
(1E,4Z,6E)-5-Cyclohexylamino-1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,4,6-heptatrien-3-one 3f
1H NMR (400 MHz, CDCl3): δ 1.27-1.52 (5H, m), 1.57-1.62 (1H, m), 1.78-1.83 (2H, m), 1.93-1.96 (2H,
m), 3.55-3.64 (1H, m), 3.92 and 3.96 (2 × 3H, 2 × s), 5.48 (1H, s), 6.63 (1H, d, J = 15.7 Hz), 6.69 (1H, d, J
15

ACCEPTED MANUSCRIPT
= 15.9 Hz), 6.90 (1H, d, J = 8.1 Hz), 6.95 (1H, d, J = 8.1 Hz), 6.97-6.98 (1H, m), 7.05-7.09 (3H, m), 7.21
(1H, d, J = 15.9 Hz), 7.47 (1H, d, J = 15.7 Hz), 11.67 (1H, d, J = 8.3 Hz). 13C NMR (100 MHz, CDCl3): δ
24.4, 25.5, 34.0, 52.0, 56.0, 56.2, 93.5, 109.4, 109.6, 114.7, 115.0, 118.4, 121.6, 122.3, 127.0, 128.4,
128.9, 137.0, 137.4, 146.8, 146.9, 147.0, 147.3, 161.8, 185.3. IR (cm-1): νNH/OH = 3008; νC=O = 1565. MS
(70 eV): m/z (%) 450 ([M+1]+, 100). HRMS (ESI): m/z for C27H32NO5 [M+H]+ calcd 450.2275, found
450.2275. Orange-yellow crystals. Column chromatography Rf (SiO2) = 0.16, EtOAc/PE (4/3), yield
58%. Mp = 99°C.
(1E,4Z,6E)-1,7-Bis-(4-hydroxyphenyl)-5-isobutylamino-1,4,6-heptatrien-3-one 3g
1H NMR (400 MHz, D6-DMSO): δ 0.96 (6H, d, J = 6.5 Hz), 1.82 (1H, nonet, J = 6.5 Hz), 3.28 (2H, t, J =
6.5 Hz), 5.61 (1H, s), 6.67 (1H, d, J = 15.7 Hz), 6.77 and 6.80 (2 × 2H, 2 × d, J = 8.6 Hz), 6.88 (1H, d, J =
16.0 Hz), 7.27 (1H, d, J = 15.7 Hz), 7.32 (1H, d, J = 16.0 Hz), 7.44 and 7.55 (2 × 2H, 2 × d, J = 8.6 Hz),
9.79 (2H, bs), 11.67-11.70 (1H, m). 13C NMR (100 MHz, D6-DMSO): δ 19.8, 28.8, 49.9, 92.1, 115.7,
116.7, 126.4, 126.6, 126.7, 129.2, 129.4, 135.9, 137.2, 158.5, 158.9, 162.4, 184.1. IR (cm-1): νC=O
=
1565. MS (70 eV): m/z (%) 364 ([M+1]+, 100). HRMS (ESI): m/z for C23H26NO3 [M+H]+ calcd 364.1907,
found 364.1922. Orange-yellow crystals. Recrystallization from methanol/water, yield 30%. Mp =
140°C.
(1E,4Z,6E)-1,7-Bis-(4-hydroxyphenyl)-5-sec-butylamino-1,4,6-heptatrien-3-one 3h
1H NMR (400 MHz, D6-DMSO): δ 0.91 (3H, t, J = 7.4 Hz), 1.18 (3H, d, J = 6.4 Hz), 1.46-1.59 (2H, m),
3.85-3.95 (1H, m), 5.57 (1H, s), 6.66 (1H, d, J = 15.7 Hz), 6.77 and 6.80 (2 × 2H, 2 × d, J = 8.5 Hz), 6.90
(1H, d, J = 16.0 Hz), 7.25 (1H, d, J = 15.7 Hz), 7.32 (1H, d, J = 16.0 Hz), 7.43 and 7.55 (2 × 2H, 2 × d, J =
8.5 Hz), 9.78 (2H, bs), 11.57 (1H, d, J = 8.9 Hz). 13C NMR (100 MHz, D6-DMSO): δ 9.9, 21.5, 30.1, 49.2,
92.0, 115.7, 116.9, 126.5, 126.6, 126.7, 129.2, 129.5, 135.8, 137.1, 158.5, 159.0, 161.7, 184.1. IR (cm-
1): νNH/OH = 3021; νC=O = 1561. MS (70 eV): m/z (%) 364 ([M+1]+, 100). HRMS (ESI): m/z for C23H26NO3
[M+H]+ calcd 364.1907, found 364.1903. Orange-yellow crystals. Recrystallization from aceton, yield
40%. Mp = 133°C.
(1E,4Z,6E)-5-Cyclohexylamino-1,7-bis-(4-hydroxyphenyl)-1,4,6-heptatrien-3-one 3i
1H NMR (400 MHz, D6-DMSO): δ 1.29-1.42 (5H, m), 1.52-1.55 (1H, m), 1.66-1.70 (2H, m), 1.84-1.87
(2H, m), 3.76-3.84 (1H, m), 5.57 (1H, s), 6.66 (1H, d, J = 15.7 Hz), 6.77 and 6.81 (2 × 2H, 2 × d, J = 8.6
Hz), 6.90 (1H, d, J = 15.9 Hz), 7.26 (1H, d, J = 15.7 Hz), 7.32 (1H, d, J = 15.9 Hz), 7.43 and 7.55 (2 × 2H,
2 × d, J = 8.6 Hz), 9.80 (2H, bs), 11.70 (1H, d, J = 8.6 Hz). 13C NMR (100 MHz, D6-DMSO): δ 23.6, 25.0,
33.5, 50.2, 92.1, 115.7, 116.7, 126.5, 126.6, 126.8, 129.2, 129.6, 135.9, 137.2, 158.5, 159.0, 161.2,
184.1. IR (cm-1): νNH/OH = 3244; νC=O = 1567. MS (70 eV): m/z (%) 390 ([M+1]+, 100). HRMS (ESI): m/z
for C25H28NO3 [M+H]+ calcd 390.2064, found 390.2066. Orange-yellow crystals. Recrystallization from
aceton, yield 11%. Mp = 254°C.
16

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Representative procedure for the synthesis of O-acetylated compounds 4a-d
(1E,4Z,6E)-1,7-Bis-(4-hydroxy-3-methoxyphenyl)-5-isobutylamino-1,4,6-heptatrien-3-one 3d (3 mmol,
1.27 g), acetic anhydride (9 mmol, 0.85 mL) and pyridine (9 mmol, 0.73 mL) were dissolved in 2-
methyltetrahydrofuran and refluxed for two hours. When the reaction was finished, the liquids were
evaporated and the resulting solid was recrystallized from methanol, which lead to the formation of
1.02 g (1E,4Z,6E)-1,7-bis-(4-acetoxy-3-methoxyphenyl)-5-isobutylamino-1,4,6-heptatrien-3-one 4a
(2.01 mmol, 67%).
(1E,4Z,6E)-1,7-Bis-(4-acetoxy-3-methoxyphenyl)-5-isobutylamino-1,4,6-heptatrien-3-one 4a
1H NMR (400 MHz, CDCl3): δ 1.04 (6H, d, J = 6.6 Hz), 1.94 (1H, nonet, J = 6.6 Hz), 2.32 and 2.34 (2 ×
3H, 2 × s), 3.23 (2H, t, J = 6.6 Hz), 3.87 and 3.89 (2 × 3H, 2 × s), 5.52 (1H, s), 6.71 (1H, d, J = 15.7 Hz),
6.77 (1H, d, J = 15.9 Hz), 7.02 (1H, d, J = 8.6 Hz), 7.05-7.06 (1H, m), 7.07 (1H, d, J = 8.6 Hz), 7.11-7.15
(3H, m), 7.23 (1H, d, J = 15.9 Hz), 7.50 (1H, d, J = 15.7 Hz), 11.62-11.65 (1H, m). 13C NMR (100 MHz,
CDCl3): δ 20.3, 20.8, 29.5, 51.6, 56.0, 56.1, 94.1, 111.1. 111.2. 120.1, 120.9, 121.2, 123.1, 123.4,
129.2, 134.7, 135.2, 136.5, 137.0, 140.4, 140.8, 151.3, 151.5, 162.9, 169.1, 185.3. IR (cm-1): νNH/OH
=
3358; νC=O = 1758, 1579. MS (70 eV): m/z (%) 508 ([M+1]+, 100). HRMS (ESI): m/z for C29H34NO7
[M+H]+ calcd 508.2330, found: 508.2326. Orange-yellow crystals. Recrystallization from methanol,
yield 67%. Mp = 90°C.
(1E,4Z,6E)-1,7-Bis-(4-acetoxy-3-methoxyphenyl)-5-sec-butylamino-1,4,6-heptatrien-3-one 4b
1H NMR (400 MHz, CDCl3): δ 0.99 (3H, t, J = 7.2 Hz), 1.29 (3H, d, J = 6.5 Hz), 1.65 (2H, pentet, J = 7.2
Hz), 2.32 and 2.34 (2 × 3H, 2 × s), 3.64-3.75 (1H, m), 3.87 and 3.89 (2 × 3H, 2 × s), 5.48 (1H, s), 6.70
(1H, d, J = 15.7 Hz), 6.80 (1H, d, J = 16.0 Hz), 7.02 (1H, d, J = 8.6 Hz), 7.05-7.06 (1H, m), 7.07 (1H, d, J =
8.6 Hz), 7.11-7.14 (3H, m), 7.24 (1H, d, J = 16.0 Hz), 7.49 (1H, d, J = 15.7 Hz), 11.55 (1H, d, J = 8.9 Hz).
13C NMR (100 MHz, CDCl3): δ 10.5, 20.8, 21.8, 30.8, 51.2, 56.0, 56.1, 93.9, 111.1, 111.3, 120.1, 120.9,
121.3, 123.1, 123.4, 129.2, 134.7, 135.3, 136.5, 136.9, 140.4, 140.8, 151.3, 151.5, 162.2, 169.1, 169.2,
185.2. IR (cm-1): νNH = 3017; νC=O = 1760, 1573. MS (70 eV): m/z (%) 508 ([M+1]+, 100). HRMS (ESI):
m/z for C29H34NO7 [M+H]+ calcd 508.2330, found 508.2325. Orange-yellow crystals. Recrystallization
from methanol, yield 44%. Mp = 84°C.
(1E,4Z,6E)-1,7-Bis-(4-acetoxy-3-methoxyphenyl)-5-cyclohexylamino-1,4,6-heptatrien-3-one 4c
1H NMR (400 MHz, CDCl3): δ 1.30-1.51 (5H, m), 1.57-1.61 (1H, m), 1.79-1.82 (2H, m), 1.93-1.96 (2H,
m), 2.32 and 2.34 (2 × 3H, 2 × s), 3.54-3.62 (1H, m), 3.87 and 3.89 (2 × 3H, 2 × s), 5.48 (1H, s), 6.70
(1H, d, J = 15.7 Hz), 6.79 (1H, d, J = 16.0 Hz), 7.02 (1H, d, J = 8.4 Hz), 7.05-7.06 (1H, m), 7.07 (1H, d, J =
8.4 Hz), 7.11-7.14 (3H, m), 7.24 (1H, d, J = 16.0 Hz), 7.49 (1H, d, J = 15.7 Hz), 11.67 (1H, d, J = 8.3 Hz).
13C NMR (100 MHz, CDCl3): δ 20.8, 24.4, 25.5, 34.0, 52.2, 56.0, 56.1, 94.0, 111.2, 111.4, 120.1, 120.9,
121.3, 123.1, 123.5, 129.3, 134.8, 135.3, 136.5, 136.8, 140.5, 140.9, 151.3, 151.6, 161.6, 169.1, 185.2.
IR (cm-1): νC=O = 1761, 1572. MS (70 eV): m/z (%) 534 ([M+1]+, 100). HRMS (ESI): m/z for C31H36NO7
17

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[M+H]+ calcd 534.2486, found 534.2489. Orange crystals. Recrystallization from methanol, yield 45%.
Mp = 88°C.
(1E,4Z,6E)-1,7-Bis-(4-acetoxyphenyl)-5-isobutylamino-1,4,6-heptatrien-3-one 4d
1H NMR (400 MHz, CDCl3): δ 1.03 (6H, d, J = 6.6 Hz), 1.94 (1H, nonet, J = 6.6 Hz), 2.31 and 2.32 (2 ×
3H, 2 × s), 3.23 (2H, t, J = 6.6 Hz), 5.51 (1H, s), 6.72 (1H, d, J = 15.4 Hz), 6.79 (1H, d, J = 16.1 Hz), 7.09
and 7.14 (2 × 2H, 2 × d, J = 8.6 Hz), 7.24 (1H, d, J = 15.4 Hz), 7.52 (1H, d, J = 16.1 Hz), 7.52 and 7.55 (2
× 2H, 2 × d, J = 8.6 Hz), 11.63-11.66 (1H, m). 13C NMR (100 MHz, CDCl3): δ 20.4, 21.3, 29.5, 51.6, 94.0,
121.1, 122.0, 122.3, 128.6, 128.9, 129.2, 133.5, 134.0, 136.1, 136.5, 151.2, 151.6, 162.9, 169.4, 169.5,
185.4. IR (cm-1): νNH = 3044; νC=O = 1752, 1571. MS (70 eV): m/z (%) 448 ([M+1]+, 100). HRMS (ESI):
m/z for C27H30NO5 [M+H]+ calcd 448.2119, found 448.2117. Orange crystals. Recrystallization from
methanol, yield 42%. Mp = 149°C.
Biology
Reagents and cell lines
Dimethylsulfoxide, triethylamine, trichloroacetic acid, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), trolox,
sulforhodamine
B
(SRB),
MEM
non-essential
amino
acid
solution
(NEAA),
tris(hydroxymethyl)aminomethane, 2,2-diphenyl-1-picrylhydrazyl (DPPH), penicillin /streptomycin
(P/S), formic acid and 2,7-dichlorofluorescein diacetate were purchased from Sigma-Aldrich. Both
thiazolyl blue tetrazolium bromide and trypan blue were obtained from Amresco. Dulbecco's
phosphate-buffered saline (PBSD-, no calcium and no magnesium), Dulbecco's modified eagle
medium (DMEM, high glucose + glutamax and RPMI 1640) and trypsin/EDTA solution were obtained
from Life Technologies (Merelbeke, Belgium) and fetal bovine serum (FBS) from Greiner Bio-one
(Vilvoorde, Belgium).
The cell lines, Caco-2 (intestinal), HT-29 (intestinal), CHO (ovarian), and EA.hy926 (endothelial) cells
were originated from ATCC. These cells were cultivated in the same growth medium containing
DMEM + glutamax, 1% NEAA, 1% P/S and 10% FBS in order to avoid interferences of specific medium
compounds with the results. Upon treatment, no FBS was added to the medium. Curcumin and its
compounds were dissolved in DMSO to a concentration of 100 mM. When the cells were exposed,
solutions of the compounds were prepared from this stock solution in medium immediately before
treatment. The final concentration of DMSO in this growth medium was less than 0.1% to avoid
cytotoxic effects caused by DMSO.
MTT assay
An aliquot of cells were counted in a Bürker counting chamber after trypan blue staining. They were
seeded at a concentration of 20000 cells in a 96 well plate and after one day the cells were treated
without (control group) and with curcumin or its analogs 3a-i, 4a-d and 5 at a concentration of 1, 5,
18

ACCEPTED MANUSCRIPT
10 and 25 µM. After three days of treatment, 100 µL of culture medium was removed and 20 µL of
MTT solution (5 mg MTT /ml PBS) was added. After two hours of incubation in the dark at 37°C and
10% CO2, the medium was removed and the 96 well plate was dried. Solubilisation of the formazan
crystals was achieved by adding 200 µL DMSO to each well. Finally, the absorbance was recorded at
570 nm using a Bio-Rad Benchmark Plus microplate spectrophotometer. Each condition was
performed in triplicate.
SRB assay43
Cells were seeded at a concentration of 20000 per well and treated with or without (control)
compound at 1, 5, 10 and 25 µM after one day. Three days after this treatment the cells were fixed
by addition of 50 µL of 50% trichloroacetic acid in milliQ-water for one hour at 4°C. The plate was
carefully rinsed for 5 times with tap water and dried, after which the cells were stained with an SRB
solution (0.4% sulforhodamine B in 1% glacial acetic acid). After 30 minutes the plate was rinsed for
five times with 1% glacial acetic acid and dried. Tris buffer in a concentration of 10 mM was used to
dissolve the stain. Finally the absorbance was measured using the Bio-Rad Benchmark Plus
microplate spectrophotometer at a wavelength of 490 nm. Each condition was performed in
triplicate.
Reactive oxygen species (ROS) assay
Cells were seeded in a black 96 well plate with transparent bottom at a concentration of 20000 cells
per well. At confluency, the cells were treated overnight with or without (control) the compounds.
After one day, the cells were washed and incubated at 37°C and 10% CO2 with 2’,7’-dichloro-dihydro-
fluorescein diacetate (DCFH-DA, 20 µM) for 30 minutes. After one hour, the cells were washed again
and culture medium without phenol red was added. The plate was incubated for one hour, and again
the medium was replaced by serum-free medium. Fluorescence of the wells was immediately
measured with a Gemini XPS Microplate Reader with an excitation of 485 nm and an emission of 535
nm.
DPPH radical scavenging assay
A solution of 40 µg/ml DPPH in methanol was prepared. This solution was mixed in a 9:1 ratio with
the solution of the compound in DMSO having a concentration ranging between 1 and 500 µM. This
mixture was incubated at 30 °C in the dark for 30 minutes. Subsequently, this mixture was added to a
96 well plate and measured in the microplate spectrophotometer at a wavelength of 517 nm. Blanks
without derivative and blanks without DPPH were run simultaneously. The last blank was necessary
to check for absorption of the derivatives themselves. Percentage inhibition was calculated using the
formula:
ꢃꢄꢅꢆꢇꢈꢉꢊꢆꢋꢌꢍꢎꢏꢐ − ꢃꢄꢅꢆꢇꢈꢉꢊꢆꢋꢑꢒꢓꢔꢒꢕꢏꢖ
ꢁ ꢂ
ꢀ % =
∗ 100
ꢃꢄꢅꢆꢇꢈꢉꢊꢆꢋꢌꢍꢎꢏꢐ
19

ACCEPTED MANUSCRIPT
Ferric reducing ability of plasma (FRAP) assay
Acetate buffer of 300 mM was prepared by adding 3.1 g of sodium acetate trihydrate to 16 mL of
acetic acid, which was diluted to 1000 mL with milliQ-water. The TPTZ (2,4,6-tripyridyl-s-triazine)
solution of 10 mM was prepared by adding 0.156 g of TPTZ to 50 mL of ethanol. Lastly, a 20 mM
solution of iron(III) chloride hexahydrate was prepared by mixing 0.54 g of FeCl3.6H2O with 2 mL 37%
HCl and 98 mL milliQ-water. The TPTZ and iron solutions were freshly prepared on the day of the
assay. These three mixtures were added in a 10:1:1 ratio to obtain the FRAP reagent. Finally, 100 µL
of the samples were mixed with 900 µL of the FRAP reagent and after four minutes the absorbance
was measured at 593 nm in a Cary 50 bio UV-visible spectrophotometer. Trolox (a water soluble
analog of vitamin E) was used as a standard and the FRAP value was calculated as trolox equiv.
(µmol/L) via a linear regression of the trolox standard curve. This method is similar to the protocol
used by previous workers.16b, 44
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Highlights
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A new and convenient synthetic access to N-alkyl β-enaminone curcuminoids
Strong increase of the cellular responses compared to the native molecules
No correlation between chemical anti-oxidant capacity and intracellular ROS
Viability and ROS results are strongly cell type dependent
Valuable new building blocks for oxidative stress-related diseases research