Mannich Curcuminoids as Potent Anticancer Agents

Article abstract of DOI:10.1002/ardp.201700005

A series of novel curcuminoids were synthesised for the first time via a Mannich-3CR/organocatalysed Claisen–Schmidt condensation sequence. Structure–activity relationship (SAR) studies were performed by applying viability assays and holographic microscopic imaging to these curcumin analogues for anti-proliferative activity against A549 and H1975 lung adenocarcinoma cells. The TNFα-induced NF-κB inhibition and autophagy induction effects correlated strongly with the cytotoxic potential of the analogues. Significant inhibition of tumour growth was observed when the most potent analogue 44 was added in liposomes at one-sixth of the maximally tolerated dose in the A549 xenograft model. The novel spectrum of activity of these Mannich curcuminoids warrants further preclinical investigations.

Full text of DOI:10.1002/ardp.201700005

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Full Paper
Mannich Curcuminoids as Potent Anticancer Agents
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Mario Gyuris , Laszlo Hackler Jr. , Lajos I. Nagy , Robert Alfoldi , Eszter Redei , Annamaria Marton ,
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Tibor Vellai , Nora Farago , Bela Ozsvari , Anasztazia Hetenyi , Gabor K. Toth , Peter Sipos ,
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, and Laszlo G. Puskas
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Ivan Kanizsai
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AVIDIN Ltd., Szeged, Hungary
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Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
Faculty of General Medicine, Department of Medical Chemistry, University of Szeged, Szeged, Hungary
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Department of Genetics, Eotvos Lorand University, Budapest, Hungary
Faculty of Pharmacy, Department of Pharmaceutical Technology, University of Szeged, Szeged, Hungary
A series of novel curcuminoids were synthesised for the first time via a Mannich-3CR/organocatalysed
Claisen–Schmidt condensation sequence. Structure–activity relationship (SAR) studies were performed
by applying viability assays and holographic microscopic imaging to these curcumin analogues for
anti-proliferative activity against A549 and H1975 lung adenocarcinoma cells. The TNFa-induced
NF-kB inhibition and autophagy induction effects correlated strongly with the cytotoxic potential of
the analogues. Significant inhibition of tumour growth was observed when the most potent
analogue 44 was added in liposomes at one-sixth of the maximally tolerated dose in the A549
xenograft model. The novel spectrum of activity of these Mannich curcuminoids warrants further
preclinical investigations.
Keywords: Anticancer / Autophagy / Curcuminoids / Mannich / NF-kB inhibition
Received: January 5, 2017; Revised: April 25, 2017; Accepted: April 25, 2017
DOI 10.1002/ardp.201700005
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
anti-angiogenic and anti-proliferative properties [1–3]. This
broad spectrum of activity has been attributed to its ability to
Introduction
Since cancer arises via multiple pathological or signalling
pathways, certain natural compounds or their derivatives
have the potential to be developed into optimum pharma-
ceuticals for cancer because of their ability to modulate
multiple pathways. Numerous findings have demonstrated
the relevance of herbal medicines for cancer therapy and
prevention, including dietary phytochemicals among
others [1]. Recent attention has focused on curcumin, also
known as diferuloylmethane, a polyphenolic, yellow pigment
found in the rhizome of turmeric (Curcuma longa). Curcumin
has been shown to possess anti-oxidant, anti-inflammatory,
affect multiple targets, including transcription factors,
growth factors, kinases, inflammatory cytokines, adhesion
molecules, apoptosis-related proteins [4] and signalling
pathways involving NF-kB, Akt, MAPK, Wnt, Notch and
p53 [5–7]. One of the main targets of curcumin is the NF-kB
cell signalling pathway [8]. Curcumin directly inhibits IKK and
the 26S proteasome [9, 10] to block NF-kB activation. Several
studies have revealed that curcumin can induce death of cells
that are resistant to apoptosis [11, 12], and treatment with
curcumin has been reported to induce autophagic cell death
in malignant cells [13–26].
Although numerous preclinical and clinical studies have
demonstrated the biological potential of curcumin to treat
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Correspondence: Dr. Ivan Kanizsai, AVIDIN Ltd., Also kikoto sor
11, Szeged H-6726, Hungary.
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Additional correspondence: Dr. Laszlo G. Puskas,
E-mail: i.kanizsai@avidinbiotech.com
Fax: þ3662202108
E-mail: laszlo@avidinbiotech.com
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cancer patients, either alone or in combination with already
existing therapeutic regimens [2], the clinical application of
curcumin has been limited by its low potency and poor
pharmacokinetics [17, 18]. Therefore, in order to improve its
activity and pharmaceutical profile, several structurally
related compounds have recently been synthesised and
evaluated as anticancer agents [19–32].
compound in a human lung cancer/SCID mouse tumour
xenograft model are presented.
Results and discussion
Chemistry
Different synthetic strategies have been reported to
expand the molecular diversity, from side-chain and dike-
tone transformations to alkyl and alkenyl functionalisations
on C-4. Selected reports, including structure–activity rela-
tionship (SAR) studies on C-4-modified curcumin analogues
2–4 have disclosed significantly higher cytotoxic values than
that of curcumin itself (1, Fig. 1) [30–32]. Structural
modification on the central carbon through formation of
a new C–C double bond led to analogue 2, which displays
higher activity against the growth of human lung adenocar-
cinoma cells H1944 and A549, with submicromolar GI50
values [30]. The 4-benzylated derivative 3 exhibits notable
growth suppression of SW480 and other human colon cancer
cells [31]. Furthermore, Michael addition to ethyl propiolate
gave access to derivative 4, which exhibits low micromolar
growth-inhibitory effects against MCF-7 and SKBR3 breast
cancer cell lines [32].
In order to generate more potent curcumin analogues with
favourable medicinal properties, we exploited the potential
of multicomponent reactions. Precursors 5 were synthesized
via a Mannich-type one-pot three-component assembly of
pentane-2,4-dione, an aromatic aldehyde and an alkyl or
alkenyl amide [33]. By a slight modification of the reported
procedure, the multicomponent Mannich transformations
were carried out in the presence of TMSCl, affording
N-(2-acetyl-3-oxo-1-phenylbutyl)amide derivatives 5 in yields
of 22–81% (Scheme 1).
In contrast with literature data, aromatic aldehydes bearing
electron-donating groups did not undergo reaction under the
conditions applied. For further variability, the Mannich
intermediate with R1 ¼ Ph, R2 ¼ ethylene was subjected to
Heck coupling, which resulted in N-(2-acetyl-3-oxo-1-phenyl-
butyl)cinnamamide 6. The curcumin backbone was con-
structed through a morpholinium chloroacetate-catalysed
double aldol (Claisen–Schmidt) condensation (Scheme 1). To
the best of our knowledge, this was the first utilization of
morpholinium chloroacetate as an effective organocatalyst in
synthetic transformations [34]. It should be noted that by the
application of n-butyl amine (Pabon-method) or piperidine in
The present report describes the synthesis of novel
Mannich-type curcumin species as a new class of potential
anticancer agents. These curcumin analogues were screened
for their cytotoxicity in in vitro assays and for their NF-
kB-inhibitory activities. In vivo test results on a selected
Figure 1. Structures of curcumin 1 and compounds 2–4.
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Scheme 1. Reagents and conditions: (a) TMSCl, Et2O/MeCN (1:1), rt, 12 h; 22–81%; (b) B2O3, dry DMF, 75°C, 30 min, R3CHO, B(OBu)3,
75°C, 5 min, morpholinium chloroacetate, 75°C, 4 h, 5% aqueous AcOH solution, 75°C, 1 h; 40–75%; (c) iodobenzene, Pd(OAc)2, PPh3,
TEA, toluene, reflux, 2 h; 71%.
Biological results and SAR
Claisen–Schmidt condensations, no reaction or complex
reaction mixtures were observed.
In vitro cytotoxicity assays were carried out with two different
lung cancer cell lines (A549 and H1975) in order to evaluate
the anticancer activities of the new derivatives 7–52. A total of
95% of all the toxicity measurements exhibited less than 10%
variation. For comparison, concentration–response curves
were obtained to determine GI50 for each compound, using
curcumin (1) and reference compound 2 [35] as positive
controls. The GI50 values obtained after an exposure time of
72 h are reported in Tables 1–5.
The proposed mechanism includes the in situ formation of
an intermediate spirocyclic boroncomplex I and its subsequent
ring opening and closure, involving secondary amine salt-
catalysed multiple aldol condensation via enamine activation,
permitting access to a rosocyanine-type transition state III.
Finally, acid-mediated degradation yielded curcuminoids
7–52 in isolated yields of 40–75%.
However, the application of o-substituted benzaldehydes
led to complex reaction mixtures during the modified Claisen–
Schmidt condensation, presumably due to both steric and
electronic effects.
With this synthetic approach, a 46-membered curcuminoid
library was generated and characterized. Scale-up trials on 44
revealed that the protocol proceeds on a multigram scale
without any alteration in terms of yield, purity or reaction
time (see Experimental section). NMR analysis of 7–52
confirmed the exclusive presence of the 1,3-diketo form in
DMSO-d6; keto-enol tautomerism could not be detected, in
contrast with the reported tautomeric equilibrium of
curcumin (1) [35].
The synthesised analogues were categorised into three
main groups on the basis of their structural similarity and
the modifications of R1, R2 and R3: acetamide analogues
7–36 (group 1 subclasses 1–3), acrylamide derivatives 37–48
(group 2) and cinnamides 49–52 (group 3).
Acetamide derivatives – subclass 1
The levels of in vitro anticancer efficiency of 7–19 were
examined and the results were compared with those of 1 and
2. In this subclass, R1 varied with fixed R2 ¼ Me (-NHAc) and
R3 ¼ 3,4-MeOC6H3 units (Table 1). With two exceptions (18
and 19), the substituted phenyl derivatives 8–17 exerted
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Table 1. AcetamideQ3 derivatives 7–19 (subclass 1) versus A549 and H1975 cancer cell lines.
a)
a)
GI50
IC50
Comp.
A549
3.35
1.98
1.60
H1975
NFkB
7
8
9
1.39
0.77
0.61
4.07
7.90
4.93
10
11
12
1.44
1.78
1.32
0.68
0.73
0.54
6.11
4.55
6.23
13
14
15
1.76
2.28
1.61
0.91 >10
1.10 >10
0.66
7.35
16
17
18
1.65
1.50
>10
0.69
0.61
>10
7.59
10.56
>10
19
3.94
1.68
8.64
a) Concentration in mM; reference GI50 and IC50 values for comparison: curcumin 1 21.37 (A549), 25.80 (H1975), 104.10 (NF-kB) and
compound 2 1.21 (A549), 0.63 (H1975), 9.68 (NF-kB), in mM.
higher in vitro cytotoxic activities on A549 and H1975 human
lung adenocarcinoma cells than those of the unsubstituted
derivative 7 (R1 ¼ Ph) or the parent compound 1. Compounds
9, 12 and 17 displayed levels of efficacy similar to those of 2 on
both cell lines. Introduction of a para-CF3 group (14) resulted
in a lower activity than those of either the regioisomer meta-
CF3 analogue 16 or any mono- or di-halogenated compound.
A para-COOH group (18) completely abolished the cytotoxic
Table 2. Acetamide derivatives 20–27 (subclass 2) versus A549 and H1975 cancer cell lines.
a)
a)
GI50
IC50
Compd. A549
H1975
NFkB
20
21
22
4.49
2.84
3.37
2.10
1.24
1.58
5.76
9.66
8.78
23
24
25
2.15
3.56
>10
1.09
1.63
>10
7.44
8.94
>10
26
27
1.81
4.53
0.88 >10
1.91 7.93
a) Concentration in mM; reference GI50 and IC50 values for comparison: curcumin 1 21.37 (A549), 25.80 (H1975), 104.10 (NF-kB) and
compound 2 1.21 (A549), 0.63 (H1975), 9.68 (NF-kB), in mM.
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Table 3. Acetamide derivatives 28–36 (subclass 3) versus A549 and H1975 cancer cell lines.
a)
a)
GI50
IC50
Compd.
A549
1.22
H1975
1.03
NFkB
28
29
4.01
8.24
3.59 >10
30
31
32
>10
>10
>10
6.81
1.92
3.70 >10
0.92
4.36
33
34
>10
>10
>10
>10
>10
>10
35
36
>10
>10
>10
>10
>10
>10
a) Concentration in mM, reference GI50 and IC50 values for comparison: curcumin 1 21.37 (A549), 25.80 (H1975), 104.10 (NF-kB) and
compound 2 1.21 (A549), 0.63 (H1975), 9.68 (NF-kB), in mM.
Table 4. Acrylamide derivatives 37–48 versus A549 and H1975 cancer cell lines.
a)
a)
GI50
IC50
Compd.
A549
H1975
NFkB
37
38
39
0.80
1.05
1.17
0.36
0.32
0.43
5.16
5.59
8.14
40
41
>10
>10
>10
>10
2.65
1.56
42
43
44
>10
>10
0.56
>10
3.04
0.26
>10
>10
2.80
45
46
>10
>10
>10
>10
3.34
1.94
47
48
0.58
>10
0.29
>10
5.45
>10
a) Concentration in mM; reference GI50 and IC50 values for comparison: curcumin 1 21.37 (A549), 25.80 (H1975), 104.10 (NF-kB) and
compound 2 1.21 (A549), 0.63 (H1975), 9.68 (NF-kB), in mM.
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Table 5. Cinnamide derivatives 49–52 versus A549 and H1975 cancer cell lines.
a)
a)
GI50
IC50
Compd.
A549
H1975
0.32
NFkB
49
50
0.67
>10
4.76
>10
>10
51
52
>10
5.01
0.67
>10
>10
5.76
a) Concentration in mM; reference GI50 and IC50 values for comparison: curcumin 1 21.37 (A549), 25.80 (H1975), 104.10 (NF-kB) and
compound 2 1.21 (A549), 0.63 (H1975), 9.68 (NF-kB), in mM.
activity. The in vitro NF-kB inhibition tests indicated that,
apart from 17, the compounds in this subclass exerted
stronger action than 2. Compounds 7–17 gave rise to a one
order of magnitude higher NF-kB inhibition than that of 1.
lines, with nanomolar GI50 values (Table 4, 37, 44 and 47).
Moreover, derivative 44, in which R1 ¼ Ph and R3 ¼ 3-HOC6H4
exerted excellent NF-kB inhibition also and was therefore
assigned as lead compound for further investigations.
Acetamide derivatives – subclass 2
Cinnamide derivatives
In this subset, R1 and R3 varied, while R2 was fixed (R2 ¼ Me;
-NHAc) (Table 2). Analogues 20–27 demonstrated a lower
level of cytotoxic activity as compared with 8–17, except for
the activities of 23 (R1 ¼ Ph, R3 ¼ 3-HO-4-MeOC6H3) and 26
(R1 ¼ Ph, R3 ¼ 4-HO-3-MeOC6H3). Similarly as in subclass 1, the
introduction of a p-HOOCC6H4 group as R1 (derivative 25) led
to the complete loss of anticancer activity. It should be noted
that the activity was mainly influenced by the nature of R1: the
best results were obtained when it was an unsubstituted
phenyl function (23 and 26) or a hydroxy group. Modification
of R3 to the acylated form of isovanillin diminished the
cytotoxic potential (compound 20).
Intermediate 6 was synthesized via the Heck reaction and was
converted to 49–52 (Table 5). Although the in vitro activity of
49 was much more pronounced than those of 50–52, it was
not significantly different from that of lead compound 44.
Interestingly, 37 (Table 4) was significantly less active than 51.
Replacement of the acrylamide framework by cinnamide
generally leads to a decrease in anticancer activity.
The overall in vitro results allowed the following con-
clusions concerning the SAR: When R1 ¼ Ph, 4-FC6H4 or
3-F3CC6H4, R2 ¼ vinyl and/or R3 ¼ 3-HOC6H4 or 3,4-MeOC6H3,
we observed noteworthy potency in terms of in vitro
cytotoxicity and NF-kB inhibition. Interestingly, introduction
of
a carboxyl functionality at any position completely
Acetamide derivatives – subclass 3
abolished the cytotoxicity. It was not determined that this
lack of activity was due to decreased activity at molecular
targets or due to limited cell permeability of carboxyl group
bearing compounds. Moreover, none of the derivatives with a
3,4-HOC6H3 side-chain, regardless of the nature of R1 and R2,
exhibited activity against lung carcinoma cell growth in the
tested concentration range. The most active novel analogues
displayed 40- to 100-fold better potency than 1 in these assays.
While 1 exhibited GI50 values of 21.4 and 25.8 mM against
A549 and H1975 cancer cell lines, respectively, and the
previously described 2 exhibited improved anti-proliferation
activities (1.2 and 0.56 mM), lead compound 44 exerted even
better anticancer activity (GI50 values of 0.56 and 0.26 mM),
and is probably the most potent synthetic curcumin derivative
reported to date (Fig. 2).
In analogues 28–36, R3 varied, while R1 was Ph or 4-HOOCC6H4
(Table 3). Compounds containing a heteroaromatic R3, e.g. 3-
bromothiophene (29) exhibited relatively low potency, while
furan derivative 36 was inactive in the tested range, similarly
to the compounds containing a 3,5-HOC6H3 (30) or a 2-MeO-4-
vinyl-C6H3 side-chain (34 and 35). Appreciable activity against
the tested lung cancer cells was achieved when R3 was either
3-HOC6H4 or 4-FC6H4 (28 and 32). Conversely, the cytotoxicity
was completely lost when a carboxyl group was present,
regardless of its position (33 and 34).
Acrylamide derivatives
The replacement of acetyl by acryloyl as group R2 resulted in
significantly improved cytotoxic activity against both cell
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Figure 2. Effects on lung cancer cell viability of 1, 2 or 44, applied for 72 h. Cell viability was assessed by the MTS method and
expressed as a fraction of the vehicle control (DMSO). (A) Lung adenocarcinoma, A549. (B) Lung adenocarcinoma, H1975.
Holographic cell analysis
(while cell viability was not affected). 44 proved to be very
potent, inhibiting NF-kB activation at micromolar concentra-
tion (IC50 ¼ 2.8 mM). This value is about 40 times lower than
that of 1 (IC50 ¼ 104 mM).
To visualise the cytotoxic effects of the most potent analogue
44, holographic microscopic analysis was performed. With this
technology, morphological parameters of treated cells such as
area, thickness and volume can be followed in a label-free
way [36, 37]. A549 human lung adenocarcinoma cells were
incubated with 0.5 mM 44 or 25 mM 1 as a control, and
holographic images were taken before and 24 h after
treatment. The morphological changes induced by the
cytotoxic effects after 24 h are illustrated through holo-
graphic images in Fig. 3. A decrease in cell surface and an
increase in average optical thickness were registered after
treatment, as the affected cells started to show the typical
profile of dying cells, changing to a spherical shape and
becoming detached from the surface. Similar shifts in the
parameters of area and thickness were observed after
treatment with 1 or 44, but the effect of 44 was more
pronounced than that of 1, even at a 50 times lower
concentration. The dynamics of the morphology change
indicated that the effect of the treatment was already
manifested after only 8 h of incubation in the case of 44,
although with a slight shift in distribution (data not shown).
The data in Fig. 4, where determined inhibitory IC50 values
were compared with GI50 values obtained in the cell toxicity
assays, suggest an important correlation between the anti-
proliferative activity and the NF-kB-inhibitory activity of the
tested compounds. Active curcumin analogues behaved
consistently in both assays, with 44 being the most potent
in both. Analogues 30, 42 and 45, which did not exert anti-
proliferative activity, were also inactive in NF-kB activation
inhibition. These results strongly support the notion that the
NF-kB pathway is one of the major molecular targets for
curcumin and its analogues.
Unfolded protein response-related gene expression
analysis
Several factors are known to be responsible for the ER stress-
mediated transcriptional activation of HSPA5 (GRP78), includ-
ing XBP-1 and activated ATF-4. ATF-4 is a cAMP response
element-binding transcription factor that activates the
transcription of genes involved in UPR and ER stress
response [40]. Curcumin has been shown to induce apoptotic
cell death initiated by the activation of the UPR signalling
pathway [41]. UPR-related gene expression changes following
treatment with the novel analogues were investigated. A549
cells were treated with the selected compounds at 1 and 5 mM,
and harvested after 6 h of incubation. Table 6 lists the results
of the QRT-PCR measurements. The selected inactive ana-
logue 40 did not alter the expression of XBP-1 or DDIT3,
whereas the active analogues 2, 7, 37, 40 and 41 increased the
expression of both genes significantly, in a dose-dependent
manner. 1 increased the expression of DDIT3 and HSPA5 only
at 25 mM, but no induction was registered for the ATF4 and
XBP1 genes. The expression of HSPA5 also displayed a
significant, dose-dependent increase after treatment.
Inhibition of TNFa induced NF-kB activation by the new
analogues
NF-kB proteins influence the expression of genes involved in a
large number of physiological processes, including immune
response, cell survival, differentiation, and proliferation [38].
One of the main targets of curcumin is the NF-kB cell
signalling pathway [8]. The correlation between the NF-
kB-inhibitory potential of different curcumin analogues and
their cytotoxicity was reported recently [39].
A B16 cell line stably expressing an NF-kB luciferase reporter
construct was used to screen the sythesised analogues for
their NF-kB-inhibitory properties. Preincubation of cells with
increasing concentrations of curcumin and its analogues dose-
dependently inhibited the TNFa-induced NF-kB expression
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Figure 3. (A) Phase-contrast (left) and holo-
graphic image (right) of untreated A549
cells. (B) 25 mM 1-treated cells at 24 h post-
treatment. (C) 0.5 mM 44-treated cells at 24 h
post-treatment. (D) Change in distribution
of phenotypic parameters (cell area vs.
average cell thickness) of 1-treated (left)
and 44-treated (right) cells.
Effects of 44 on the autophagic machinery
It was earlier shown that bis-dehydroxycurcumin not only
experimental setup enabled us to discriminate early auto-
phagic organelles (GFP-positive and mRFP-positive) from
acidified autolysosomes (GFP-negative and mRFP-positive),
due to quenching of the GFP signal exclusively inside acidic
compartments [44]. After a 6 h incubation in the presence of
44 or controls (autophagy activator: rapamycin, autophagy
inhibitor: bafilomycin), images were taken with a fluorescent
confocal microscope in both red and green channels.
Figure 5 shows representative images of 44, rapamycin and
bafilomycin treated cells. 44 treatment, similarly to rapamy-
cin, resulted in marked increase of red puncta that represent
acidic autolysosomes, suggesting an activated autophagic
machinery.
induces ER stress, but, as
a downstream consequence,
together with mitochondrial-dependent apoptosis, induces
the autophagic machinery [42]. Recently, a TFEB activating
novel curcumin analog was shown to promote autophagy [43].
We have investigated the effect of our most potent
compound 44 on autophagy with three methodologies.
First, a cell-based assay was used to investigate the
autophagic induction potential of 44. To show the delivery
of autophagosomes to the lysosomes, an RFP-GFP (red
fluorescent protein–green fluorescent protein)-tagged LC3B
construct expressing HeLa cell line was used [44]. This
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treatment. 44 decreased the amount of LC3B-II while the
cytosolic LC3B-I was still present suggesting an active
autophagic process (Fig. 7B). Bafilomycin treatment inhibited
autophagy evidenced by the accumulation of LC3B-II protein.
A considerable decrease in p62 protein level was detected
only after 24 h in treated cells but not at 6 or 12 h (data not
shown). Here only the 0.5 mM concentration was tested for 44
since cells above this dose were showing massive signs of cell
death preventing protein analysis. At 24 h the used positive
control, rapamycin decreased significantly the level of p62. 44
treatment also resulted in a slight decrease of protein level
(Fig. 7D).
Liposome-encapsulated 44 reduced the size of human
lung tumour xenografts
The effects of the most potent analogue 44 were tested
against subcutaneously implanted human lung cancer (A549)
in the SCID mouse xenograft model. To circumvent bioavail-
ability problems, we used a liposome delivery system for the
encapsulation of 44 and intravenous administration. Liquid
phase 44-containing liposomes were prepared by hydrating
and redispersing a lipid film of CHOL/PC/DSPE-mPEG and 44 in
PBS solution to a final concentration of 1 mg/mL of 44. The
droplet size, SSA (specific surface area) and PDI (polydispersity
index) of the liposomes were measured by laser diffractome-
try (n ¼ 5) by the wet method. The d(0.1), d(0.5) as median and
d(0.9) droplet sizes, SSA and PDI were 75 ꢁ 19 nm,
114 ꢁ 20 nm, 178 ꢁ 24 nm, 54.5 m2/g and 0.901 ꢁ 0.20,
respectively.
Figure 4. Correlation of GI50 values obtained with H1975 cells
and NF-kB induction inhibition IC50 values. GI50 and IC50 values
are plotted for those analogs that were active in both
measurements. A linear regression was fitted on the scatterplot.
Then, autophagy-related genes were selected and their
expression was measured in A549 cells following treatment
with 44 along with rapamycin. Gene expression was measured
at 6, 12 and 24 h after treatment. All tested genes showed
time or dose-dependent activation by 44 (Fig. 6). Expression of
MAP1LC3B a key molecule in autophagy was activated by 44
even after 6 h, while rapamycin activated its expression only
after 12 h. Even the lowest applied concentration of 44
(0.5 mM) activated the expression of BECN1, GABARAP and
SQSTM1.
Administration of 44-containing liposomes, as an intrave-
nous injection, was initiated on day 7 and subsequently
applied three times a week for 4 weeks at one-sixth of the
tolerated dose (3 mg/kg). A significant reduction in tumour
Thereafter, the expression of LC3B and p62 protein was
measured by Western blot analysis in A549 cells following 44
Table 6. Summary of QRT-PCR analyses on UPR-related gene expression.
GI50
Compd.
mM
HSPA5
p-Value
ATF4
p-Value
XBP1
p-Value
DDIT3
p-Value
(mM)
21.37
1.214
3.35
1
2
10
25
1
5
1
5
1
5
1
5
1
5
0.5
1
5
1.18
4.27
4.14
7.01
2.03
6.45
2.30
5.94
2.30
1.75
3.68
9.09
1.26
2.73
6.76
0.465
0.002
0.001
<0.001
0.016
<0.001
0.009
0.001
0.009
0.033
0.002
<0.001
0.138
0.005
0.001
0.85
1.24
3.81
3.89
4.41
3.73
2.79
3.39
1.05
2.14
2.13
2.22
1.43
1.71
2.40
0.713
0.667
0.020
0.019
0.014
0.021
0.045
0.027
0.891
0.099
0.101
0.089
0.254
0.156
0.141
1.02
1.28
2.60
3.27
2.17
4.17
2.16
3.55
1.40
1.57
1.61
4.20
1.13
1.93
3.04
0.911
0.177
0.005
0.002
0.010
0.001
0.011
0.002
0.121
0.056
0.048
0.001
0.471
0.012
0.004
1.24
4.96
19.52
27.79
6.22
89.68
17.11
96.11
0.71
1.60
4.05
9.83
3.32
0.672
0.002
0.001
0.001
0.007
<0.001
0.001
<0.001
0.388
0.255
0.017
0.003
0.014
0.006
>0.000
7
0.8
37
40
41
44
>10
2.65
0.5603
9.62
55.46
Gene expression values are fold changes relative to untreated control cells. Statistically significant relative expression changes
are highlighted in bold. p-Values were calculated with the independent two-sample t-test.
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Figure 5. Representative confocal images of
HeLa cells stably expressing GFP-mRFP-LC3B
after 6 h treatment. Merged images of red
and green fluorescent scans of untreated
cells (A), cells treated with bafilomycin
(100 nM) (B), rapamycin (500 nM) (C) or with
0.5 mM analogue 44 (D). The scale bar on
panel (A) represents 30 mm. (E) Quantifica-
tion of images A–D, error bars represent
standard deviation, ꢀꢀp < 0.01 (Student’s
t-test, control vs. treatment).
size relative to that in the control animals (n ¼ 8) was detected
after day 23 in the treated group (n ¼ 9). The tumour mass
remained significantly lower in the treated animals even
weeks after the final administration (Fig. 8).
Together with the noteworthy in vitro data, this suggests
that 44 may potentially be an effective chemotherapeutic
agent.
Experimental
Conclusion
Chemistry
General procedure for the synthesis of 7–52
The present report has described the synthesis of novel
Mannich-type curcumin derivatives as a new class of potential
anticancer agents. Screening of these analogues for cytotox-
icity, coupled with biochemical and gene expression studies,
revealed that they have significantly better NF-kB-inhibitory
activity than the parent compound curcumin. The favourable
in vitro anticancer results led to in vivo tests with the lead
compound 44. This compound effectively reduced the size of
human lung tumours in SCID mice and displayed little toxicity.
To a stirred solution of the appropriate Mannich precursor 5
(0.79 mmol) in 1.25 mL dry DMF under an argon atmosphere,
B2O3 (55 mg, 1 equiv.) was added. The reaction mixture was
stirred at 75°C for 30 min, followed by the addition of B(OBu)3
(427 mL, 2 equiv.) and the appropriate aldehyde (2 equiv.).
After stirring for 5 min, morpholinium chloroacetate (36 mg,
25 mol%) was added to the solution. The resulting mixture
was stirred for a further 4 h at 75°C and monitored by TLC
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Figure 6. QRT-PCR anylysis of autophagy related genes. A549 cells were treated with rapamycin (500 nM) or with 44 (0.5, 1 or 5 mM).
Expression is plotted on a log2 scale. Error bars represent standard deviation (n ¼ 3), ꢀp < 0.05; ꢀꢀp < 0.01 (Student’s t-test, time
matched control vs. treatment).
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Figure 7. Western blot analysis of LC3B and
p62 protein expression. (A, B) LC3B expres-
sion. A549 cells were treated with rapamycin
(500 nM), bafilomycin (100 nM), or with 44
(0.5, 1 or 5 mM) and their combinations for
6 h. (C, D) p62 expression. A549 cells were
treated with rapamycin (500 nM), or with 44
(0.5 mM) for 24 h.
(eluent: CHCl3/MeOH mixture). After the mixture had been
cooled to room temperature, 5 mL of 5 w/w% aqueous AcOH
solution was added dropwise in 15 min to quench the
reaction, and the mixture was then stirred for a further 1 h
at 75 °C. In the event of precipitation, the solid formed
was collected by filtration, washed with water (2 ꢂ 25 mL)
and dried in vacuum. Crude products were recrystallised
from either Et2O or Et2O/EtOH mixtures. In the event of
unsuccessful precipitation, the reaction mixture was
extracted with EtOAc (2 ꢂ 20 mL). The combined organic
layers were washed with 40 mL of 5 w/w% aqueous NaHCO3
solution and brine, dried over Na2SO4, evaporated and
purified by column chromatography on silica (eluent:
CHCl3/MeOH mixture).
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
Figure 8. In vivo efficacy study of 44 in the A549 xenograft
model. Error bars represent SEM. ꢀp < 0.05, Student’s t-test.
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Multigram-scale synthesis of lead compound 44 – a
representative example
N-((E)-1-(3-Bromophenyl)-5-(3,4-dimethoxyphenyl)-2-((E)-
3-(3,4-dimethoxyphenyl)acryloyl)-3-oxopent-4-enyl)-
acetamide (10)
Lead compound 44 was synthesised by the general procedure,
starting from 5 g (19.3 mmol) of the corresponding Mannich
precursor 5. A yellow solid precipitated during the acidic
degradation of the boron complex. The solid formed was
collected by vacuum filtration, and washed with water
(2 ꢂ 25 mL). After drying, the crude product was washed with
100 mL Et2O/EtOH mixture (4:1) and left at room temperature
for 3 h. The solid was then filtered, washed with Et2O (50 mL)
and dried in vacuum, yielding 5.2 g (57%) of 44 as a pale-
yellow powder.
C32H32BrNO7, M ¼ 603.1; yield: 48%; m.p.: 215–218°C; 1H NMR
(500.13 MHz, CDCl3): d 1.98 (s, 3H, CH3C(O)), 3.91 (s, 12H,
OCH3), 4.73 (d, 1H, J ¼ 6.5 Hz, C(O)CHC(O)), 5.94 (t, 1H,
J ¼ 8.0 Hz, NHCH), 6.67 (d, 1H, J ¼ 15.4 Hz), 6.74 (d, 1H,
J ¼ 15.7 Hz), 6.84 (t, 2H, J ¼ 8.8 Hz), 7.02 (s, 1H), 7.05 (s, 1H),
7.06–7.12 (m, 2H), 7.12–7.19 (m, 2H), 7.29–7.36 (m, 2H), 7.51 (d,
1H, J ¼ 15.5 Hz), 7.55 (s, 1H) 7.62 (d, 1H, J ¼ 15.9 Hz); 13C NMR
(125.76 MHz, CDCl3): d 22.9, 51.5, 55.5, 66.3, 109.5, 109.6,
110.6, 110.7, 121.1, 121.8, 123.6, 123.7, 125.1, 126.3, 126.4,
129.5, 129.7, 130.3, 142.0, 145.1, 145.2, 148.8, 151.6, 151.7,
169.1, 191.8, 194.4; IR (FTIR, cmꢃ1): 982, 1022, 1142, 1261,
1514, 1591, 1649 and 3433; MS (ES, neg. mode) m/z ¼ 620.1
[MꢃH]ꢃ.
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-dimethoxy-
phenyl)acryloyl)-3-oxo-1-phenyl-pent-4-enyl)acetamide (7)
C32H33NO7, M ¼ 543.2; yield: 43%; m.p.: 191–193°C. 1H NMR
(500.13 MHz, DMSO-d6): d 1.70 (s, 3H, CH3C(O)), 3.78 (s, 12H,
OCH3), 5.03–5.20 (m, 1H, C(O)CHC(O)), 5.66–5.85 (m, 1H,
NHCH), 6.82–7.09 (m, 4H,), 7.00–7.04 (m, 10H), 7.66 (d, 1H,
J ¼ 15.0 Hz), 8.41 (s, 1H, NHC(O)); 13C NMR (125.76 MHz,
DMSO-d6): d 23.6, 52.7, 56.5, 68.1, 111.49, 111.53, 112.5, 112.6,
124.0, 124.3, 124.4, 124.7, 127.6, 127.9, 128.0, 128.4, 129.0,
142.3, 144.85, 144.92, 149.9, 152.3, 152.4, 169.0, 192.8, 193.2;
IR (FTIR, cmꢃ1): 982, 1022, 1142, 1263, 1514, 1591, 1653, 2361
and 3298; MS (ES, neg. mode) m/z ¼ 542.2 [MꢃH]ꢃ.
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-dimethoxy-
phenyl)acryloyl)-1-(2-fluorophenyl)-3-oxopent-4-enyl)-
acetamide (11)
C32H32FNO7, M ¼ 561.2; yield: 41%; m.p.: 209–212°C; 1H NMR
(500.13 MHz, CDCl3): d 1.98 (s, 3H, CH3C(O)), 3.89 (s, 6H, OCH3),
3.92 (s, 6H, OCH3), 4.92 (d, 1H, J ¼ 6.5 Hz, C(O)CHC(O)), 6.18 (t,
1H, J ¼ 7.6 Hz, NHCH), 6.64 (d, 1H, J ¼ 15.7 Hz), 6.75 (d, 1H,
J ¼ 15.8 Hz), 6.82 (d, 1H, J ¼ 8.4 Hz), 6.87 (d, 1H J ¼ 8.4 Hz),
6.97–7.24 (m, 8H), 7.42–7.54 (m, 2H), 7.68 (d, 1H, J ¼ 15.8 Hz);
13C NMR (125.76 MHz, CDCl3) d 22.9, 47.9, 55.5, 64.5, 109.5,
109.6, 110.5, 110.6, 115.0, 115.1, 121.6, 121.8, 123.6, 123.7,
123.9, 126.4, 128.9, 129.0, 129.2, 144.6, 145.2, 148.8, 148.9,
151.5, 151.6, 169.0, 192.3, 194.4; IR (FTIR, cmꢃ1): 984, 1024,
1144, 1267, 1520, 1589, 1647, 1676 and 3296; MS (ES, neg.
mode) m/z ¼ 560.2 [MꢃH]ꢃ.
N-((E)-1-(4-Chlorophenyl)-5-(3,4-dimethoxyphenyl)-2-((E)-
3-(3,4-dimethoxyphenyl)acryloyl)-3-oxopent-4-enyl)-
acetamide (8)
C32H32ClNO7, M ¼ 577.2, yield: 45%; 1H NMR (500.13 MHz,
DMSO-d6): d 1.72 (s, 3H, CH3C(O)), 3.77–3.83 (m, 12H, OCH3),
5.15 (d, 1H, J ¼ 10.7 Hz, C(O)CHC(O)), 5.74 (t, 1H, J ¼ 9.6 Hz,
NHCH), 6.94–7.06 (m, 4H), 7.21–7.46 (m, 9H), 7.71 (d, 1H,
J ¼ 16.2 Hz), 8.46 (d, 1H, J ¼ 8.8 Hz, NHC(O)); 13C NMR
(125.76 MHz, DMSO-d6): d, 23.5, 52.1, 56.5, 67.8, 111.50,
111.52, 112.5, 112.6, 123.8, 124.4, 124.5, 124.8, 127.5, 127.8,
129.0, 130.3, 132.5, 141.3, 145.1, 145.2, 149.8, 149.9, 152.4,
152.5, 169.2, 192.6, 192.9; IR (FTIR, cmꢃ1): 984, 1022, 1142,
1263, 1512, 1594, 1653, 2362 and 3294; MS (ES, neg. mode)
m/z ¼ 576.2 [MꢃH]ꢃ.
N-((E)-1-(3,4-Difluorophenyl)-5-(3,4-dimethoxyphenyl)-2-
((E)-3-(3,4-dimethoxyphenyl)acryloyl)-3-oxopent-4-enyl)-
acetamide (12)
C32H31F2NO7; M ¼ 579.2; yield: 51%; m.p.: 218–220°C; 1H NMR
(500.13 MHz, CDCl3): d 1.97 (s, 3H, CH3C(O)), 4.71 (s, 12H,
OCH3), 4.75 (d, 1H, J ¼ 6.4 Hz, C(O)CHC(O)), 5.91 (t, 1H,
J ¼ 7.8 Hz, NHCH), 6.68 (d, 1H, J ¼ 15.8 Hz), 6.74 (d, 1H,
J ¼ 15.8 Hz), 6.81–6.88 (m, 2H), 7.03 (d, 2H, J ¼ 9.1 Hz),
7.05–7.17 (m, 5H), 7.54 (d, 1H, J ¼ 15.8 Hz), 7.62 (d, 1H,
J ¼ 15.8 Hz); 13C NMR (125.76 MHz, CDCl3): d 22.8, 51.3, 55.5,
66.4, 109.4, 109.7, 110.6, 110.7, 115.6, 115.8, 116.8, 116.9,
121.0, 121.5, 122.6, 123.6, 123.8, 126.3, 145.3, 145.4, 148.9,
151.7, 151.8, 169.2, 191.7, 194.3; IR (FTIR, cmꢃ1): 980, 1022,
1142, 1267, 1516, 1591, 1651 and 3292; MS (ES, neg, mode)
m/z ¼ 578.2 [MꢃH]ꢃ.
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-dimethoxy-
phenyl)acryloyl)-1-(4-fluorophenyl)-3-oxopent-4-enyl)-
acetamide (9)
C32H32FNO7, M ¼ 561.2; yield: 47%; m.p.: 200–202°C; 1H NMR
(500.13 MHz, CDCl3): d 1.95 (s, 3H, CH3C(O)), 3.89 (s, 12H,
OCH3), 4.75 (d, 1H, J ¼ 6.9 Hz, C(O)CHC(O)), 5.97 (t, 1H,
J ¼ 7.8 Hz, NHCH), 6.70 (d, 1H, J ¼ 13.1 Hz), 6.73 (d, 1H,
J ¼ 13.4 Hz), 6.80–6.86 (m, 2H), 6.96 (d, 2H, J ¼ 8.5 Hz), 7.00–
7.04 (m, 2H), 7.05–7.15 (m, 3H), 7.34–7.40 (m, 2H), 7.53 (d, 1H,
J ¼ 15.6 Hz), 7.60 (d, 1H, J ¼ 15.6 Hz); 13C NMR (125.76 MHz,
CDCl3): d 22.9, 51.4, 55.5, 66.8, 109.6, 109.7, 110.6, 114.9,
115.1, 121.2, 121.7, 123.5, 123.7, 126.4, 128.1, 128.2, 135.5,
145.0, 145.1, 148.9, 151.6, 160.6, 162.5, 169.0, 192.0, 194.5; IR
(FTIR, cmꢃ1): 984, 1022, 1142, 1261, 1514, 1591, 1651, 2361 and
3433; MS (ES, neg. mode) m/z ¼ 560.2 [MꢃH]ꢃ.
N-((E)-1-(2,4-Dichlorophenyl)-5-(3,4-dimethoxyphenyl)-2-
((E)-3-(3,4-dimethoxyphenyl)acryloyl)-3-oxopent-4-enyl)-
acetamide (13)
C32H31Cl2NO7; M ¼ 611.2; yield: 55%; m.p.: 224–226°C;
1H NMR (500.13 MHz, CDCl3): d 1.99 (s, 3H, CH3C(O)), 3.87
(s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.92 (s, 6H, OCH3), 4.99 (d, 1H,
J ¼ 4.7 Hz, C(O)CHC(O)), 6.08 (dd, 1H, J ¼ 8.3 and 3.3 Hz,
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NHCH), 6.51 (d, 1H, J ¼ 15.7 Hz), 6.79–6.90 (m, 3H), 6.97 (s, 1H),
7.03–7.11 (m, 2H), 7.18 (t, 2H, J ¼ 8.3 Hz), 7.36 (s, 1H), 7.43 (s,
1H), 7.45 (d, 1H, J ¼ 5.8 Hz), 7.52 (d, 1H, J ¼ 8.3 Hz); 13C NMR
(125.76 MHz, CDCl3): d 22.8, 49.9, 55.5, 62.2, 109.4, 109.8,
110.5, 110.7, 120.8, 122.6, 123.5, 123.7, 126.2, 126.4, 127.0,
129.1, 130.1, 132.3, 133.6, 135.2, 144.8, 145.3, 148.8, 148.9,
151.7, 169.1, 192.0, 194.8; IR (FTIR, cmꢃ1): 982, 1022, 1140,
1265, 1514, 1591, 1649, 1672, 2359 and 3421; MS (ES, neg.
mode) m/z ¼ 610.2 [MꢃH]ꢃ.
145.2, 145.3, 148.8, 151.6, 169.3, 191.8, 194.3; IR (FTIR, cmꢃ1):
982, 1024, 1119, 1163, 1263, 1333, 1514, 1593, 1651, 1670 and
3294; MS (ES, neg. mode) m/z ¼ 610.2 [MꢃH]ꢃ.
N-((E)-1-(4-Butylphenyl)-5-(3,4-dimethoxyphenyl)-2-((E)-3-
(3,4-dimethoxyphenyl)acryloyl)-3-oxopent-4-en-1-yl)-
acetamide (17)
C36H41NO7; M ¼ 599.3; yield: 30%; m.p.: 182–183°C; 1H NMR
(500.13 MHz, DMSO-d6): d 0.80 (t, 3H, J ¼ 7.3 Hz, CH3CH2),
1.16–1.24 (m, 2H, CH2CH2), 1.38–1.47 (m, 2H, CH2CH2), 1.69 (s,
3H, CH3C(O)), 2.45 (t, 2H, J ¼ 7.5 Hz, CH3CH2Ph), 3.76 (s, 3H,
OCH3), 3.77 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.80 (s, 3H, OCH3),
5.08 (d, 1H, J ¼ 10.7 Hz, C(O)CHC(O)), 5.72 (t, 1H, J ¼ 9.9 Hz,
NHCH), 6.89 (d, 1H, J ¼ 15.9 Hz), 6.95 (d, 1H, J ¼ 8.2 Hz),
6.97–7.03 (m, 2H), 7.06 (d, 2H, J ¼ 7.9 Hz), 7.18 (d, 1H,
J ¼ 8.4 Hz), 7.21–7.32 (m, 4H), 7.33 (s, 1H), 7.37 (d, 1H,
J ¼ 15.9 Hz), 7.65 (d, 1H, J ¼ 15.9 Hz), 8.37 (d, 1H, J ¼ 8.9 Hz,
C(O)NH); 13C NMR (125.76 MHz, DMSO-d6): d 14.6, 22.6, 23.6,
33.9, 35.3, 52.4, 56.5, 68.1, 111.47, 111.52, 112.4, 112.6, 124.1,
124.3, 124.4, 124.7, 127.6, 127.9, 128.2, 128.9, 139.5, 142.0,
144.8, 149.8, 149.9, 152.3, 152.4, 169.0, 193.0, 193.3; IR (FTIR,
cmꢃ1): 983, 1022, 1140, 1265, 1514, 1591, 2359, 2933 and 3304;
MS (ES, neg. mode) m/z ¼ 598.2 [MꢃH]ꢃ.
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-
dimethoxyphenyl)acryloyl)-3-oxo-1-(4-(trifluoromethyl)-
phenyl)pent-4-enyl)acetamide (14)
C33H32F3NO7; M ¼ 611.2; yield: 60%; m.p.: 195–196°C; 1H NMR
(500.13 MHz, CDCl3): d 1.98 (s, 3H, CH3C(O)), 3.89 (s, 3H, OCH3),
3.90 (s, 6H, OCH3), 3.91 (s, 3H, OCH3), 4.77 (d, 1H, J ¼ 6.3 Hz,
C(O)CHC(O)), 6.01 (dd, overlapped peaks, 1H, J ¼ 7.4 Hz,
NHCH), 6.68 (d, 1H, J ¼ 15.7 Hz), 6.75 (d, 1H, J ¼ 15.4 Hz),
6.84 (t, 2H, J ¼ 8.4 Hz), 7.01 (s, 1H), 7.04 (s, 1H), 7.07–7.11 (m,
1H), 7.14 (d, 2H, J ¼ 8.4 Hz), 7.49–7.58 (m, 4H), 7.63 (d, 1H,
J ¼ 15.7 Hz); 13C NMR (125.76 MHz, CDCl3): d 22.8, 51.7, 55.5,
66.3, 109.6, 109.7, 110.6, 110.7, 121.0, 121.5, 123.6, 123.8,
125.1, 125.2, 126.2, 126.3, 126.9, 143.7, 145.3, 145.4, 148.9,
151.7, 151.8, 169.2, 191.8, 194.3; IR (FTIR, cmꢃ1): 982, 1022,
1070, 1122, 1261, 1327, 1514, 1593, 1647, 1678 and 3294; MS
(ES, neg. mode) m/z ¼ 610.2 [MꢃH]ꢃ.
4-((E)-1-Acetamido-5-(3,4-dimethoxyphenyl)-2-((E)-3-(3,4-
dimethoxyphenyl)acryloyl)-3-oxopent-4-en-1-yl)benzoic
acid (18)
N-((E)-1-(4-Bromothiophen-2-yl)-5-(3,4-dimethoxy-
phenyl)-2-((E)-3-(3,4-dimethoxyphenyl)acryloyl)-3-
oxopent-4-enyl)acetamide (15)
C33H33NO9; M ¼ 587.2; yield: 62%; 1H NMR (500.13 MHz,
DMSO-d6): d 1.72 (s, 3H, CH3C(O)), 3.76 (s, 3H, OCH3), 3.77
(s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 5.19 (d, 1H,
J ¼ 10.5 Hz, C(O)CHC(O)), 5.80 (t, 1H, J ¼ 10.2 Hz, NHCH),
6.90–6.99 (m, 3H), 7.00–7.06 (m, 2H), 7.21 (d, 1H, J ¼ 8.4 Hz),
7.24 (s, 1H), 7.32 (d, 1H, J ¼ 8.4 Hz), 7.35 (s, 1H), 7.42 (d, 1H,
J ¼ 15.9 Hz), 7.50 (d, 2H, J ¼ 8.2 Hz), 7.70 (d, 1H, J ¼ 15.9 Hz),
7.85 (d, 2H, J ¼ 8.2 Hz), 8.49 (d, 1H, J ¼ 8.9 Hz, C(O)NH);
13C NMR (125.76 MHz, DMSO-d6): d 22.6, 51.6, 55.5, 66.7,
110.61, 110.64, 111.5, 111.6, 122.9, 123.5, 123.6, 123.8, 126.6,
126.9, 127.7, 129.1, 144.1, 144.2, 146.2, 148.9, 149.0, 151.4,
151.5, 167.0, 168.3, 191.7, 192.0. IR (FTIR, cmꢃ1): 980, 1022,
1140, 1163, 1267, 1515, 1591, 1652, 2361 and 3414; MS (ES,
neg. mode) m/z ¼ 586.2 [MꢃH]ꢃ.
C30H30BrNO7S; M ¼ 627.1; yield: 35%; m.p.: 188–190°C;
1H NMR (500.13 MHz, DMSO-d6): d 1.72 (s, 3H, CH3C(O)),
3.76 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 5.24 (d, 1H, J ¼ 10.8 Hz, C(O)CHC(O)), 5.90 (t, 1H,
J ¼ 9.9 Hz, NHCH), 6.95–7.04 (m, 4H), 7.06 (d, 1H, J ¼ 15.8 Hz),
7.25 (d, 1H, J ¼ 8.2 Hz), 7.28–7.34 (m, 2H), 7.35 (s, 1H), 7.46 (s,
1H), 7.54 (d, 1H, J ¼ 15.8 Hz), 7.72 (d, 1H, J ¼ 15.8 Hz), 8.51 (d,
1H, J ¼ 8.7 Hz, C(O)NH,); 13C NMR (125.76 MHz, CDCl3): d 22.9,
51.7, 55.5, 55.6, 66.3, 109.6, 109.7, 110.6, 110.7, 121.0, 121.5,
123.7, 123.9, 125.2, 126.2, 126.3, 126.9, 143.7, 145.3, 145.4,
148.9, 151.7, 151.8, 169.3, 191.8, 194.3; IR (FTIR, cmꢃ1): 982,
1022, 1140, 1261, 1514, 1589, 1653 and 3427; MS (ES, neg.
mode) m/z ¼ 626.1 [MꢃH]ꢃ.
4-((E)-1-Acetamido-5-(3,4-dimethoxyphenyl)-2-((E)-3-(3,4-
dimethoxy-phenyl)acryloyl)-3-oxopent-4-en-1-yl)-2-
methoxyphenyl acetate (19)
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-dimethoxy-
phenyl)acryloyl)-3-oxo-1-(3-(trifluoromethyl)-phenyl)-
pent-4-enyl)acetamide (16)
1
C35H37NO10; M ¼ 631.2; yield: 49%; m.p.: 180–182°C; H NMR
(500.13 MHz, CDCl3): d 1.94 (s, 3H, CH3C(O)), 2.26 (s, 3H,
CH3C(O)O–Ar), 3.79 (s, 3H, OCH3), 3.89 (bs, overlapped peaks,
12H, OCH3), 4.76 (d, 1H, J ¼ 7.0 Hz, C(O)CHC(O)), 5.99 (t, 1H,
J ¼ 7.7 Hz, NHCH), 6.72 (d, 1H, J ¼ 10.5 Hz), 6.75 (d, 1H,
J ¼ 10.5 Hz), 6.80–6.86 (m, 2H), 6.93 (bs, 2H), 7.04 (bs,
overlapped peaks, 4H), 7.08–7.15 (m, 2H), 7.56 (d, 1H,
J ¼ 15.7 Hz), 7.60 (d, 1H, J ¼ 15.7 Hz); 13C NMR (125.76 MHz,
CDCl3): d 20.2, 22.9, 51.8, 55.5, 66.9, 109.6, 110.6, 111.5, 118.3,
121.4, 121.6, 122.4, 123.5, 123.7, 126.4, 138.7, 144.9, 145.0,
148.8, 151.5, 168.4, 169.0, 192.0, 194.5; IR (FTIR, cmꢃ1): 547,
C33H32F3NO7; M ¼ 611.2; yield: 58%; m.p.: 209–212°C; 1H NMR
(500.13 MHz, CDCl3): d 1.98 (s, 3H, CH3C(O)), 3.89 (s, 12H,
OCH3), 4.77 (d, 1H, J ¼ 6.5 Hz, C(O)CHC(O)), 6.02 (t, 1H,
J ¼ 7.4 Hz, NHCH), 6.68 (d, 1H, J ¼ 16.0 Hz), 6.75 (d, 1H,
J ¼ 16.0 Hz), 6.83 (t, 2H, J ¼ 6.8 Hz), 7.00 (s, 1H), 7.03 (s, 1H),
7.06–7.15 (m, 2H), 7.19 (s, 1H), 7.37–7.44 (m, 2H), 7.44–7.51 (m,
2H), 7.59 (d, 1H, J ¼ 8.6 Hz), 7.67 (s, 1H); 13C NMR (125.76 MHz,
CDCl3): d 22.8, 51.7, 55.5, 66.3, 109.5, 109.6, 110.6, 121.0,
121.6, 122.7, 123.2, 123.6, 123.7, 124.0, 126.3, 128.6, 130.8,
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767, 802, 979, 1024, 1143, 1222, 1514, 1593, 1647 and 2166; MS
OCH3), 3.80 (s, 3H, OCH3), 5.40 (d, 1H, J ¼ 10.8 Hz, C(O)CHC(O)),
5.74 (t, 1H, J ¼ 10.1 Hz, NHCH), 6.72 (d, 1H, J ¼ 15.7 Hz), 6.86 (d,
1H, J ¼ 15.7 Hz), 6.94–6.99 (m, 1H), 7.02–7.08 (m, 2H), 7.02–
7.08 (m, 2H), 7.09–7.18 (m, 2H), 7.25 (t, 2H, J ¼ 7.5 Hz), 7.30 (d,
2H, J ¼ 10.4 Hz), 7.36 (d, 1H, J ¼ 7.5 Hz), 7.55 (d, 1H, J ¼ 15.7
Hz), 8.39 (d, 1H, C(O)NH, J ¼ 9.0 Hz), 9.19 (s, 1H, OH); 9.24 (s,
1H, OH); 13C NMR (125.76 MHz, DMSO-d6): d 22.6, 51.8, 55.6,
67.6, 112.0, 112.1, 114.1, 114.2, 114.3, 122.2, 122.3, 122.6,
126.7, 127.1, 127.5, 128.1, 140.4, 141.3, 144.0, 146.7, 146.0,
150.6, 168.1, 192.0, 192.3; IR (FTIR, cmꢃ1): 982, 1020, 1072,
1130, 1269, 1439, 1512, 1578 and 3325; (ES, neg. mode)
m/z ¼ 514.1 [MꢃH]ꢃ.
(ES, neg. mode) m/z ¼ 630.2 [MꢃH]ꢃ.
4,40-((1E,6E)-4-(Acetamido(phenyl)methyl)-3,5-
dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-4,1-
phenylene)diacetate (20)
C34H33NO9, M ¼ 599.2; yield: 45%; 1H NMR (500.13 MHz,
DMSO-d6): d 1.98 (s, 3H, CH3C(O)), 2.24 (s, 3H, OAc), 2.26 (s,
3H, OAc), 3.78 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 5.21 (d, 1H,
J ¼ 10.7 Hz, C(O)CHC(O)), 5.80 (t, 1H, J ¼ 9.8 Hz, CHNH), 7.03–
7.52 (m, 14H), 7.73 (d, 1H, J ¼ 15.7 Hz), 8.46 (d, 1H, J ¼ 9.1 Hz);
13C NMR (125.76 MHz, DMSO-d6): d 21.2, 23.6, 52.7, 56.9, 68.0,
113.3, 122.6, 122.9, 124.2, 124.3, 126.4, 126.7, 128.1, 128.4,
129.1, 133.8, 134.1, 142.0, 142.2, 142.3, 144.0, 144.1, 152.0,
152.1, 169.1, 169.2, 169.2, 193.1, 193.5; IR (FTIR, cmꢃ1): 594,
702, 985, 1034, 1126, 1200, 1512, 1612, 1767, 2355 and 3319
MS (ES, neg. mode) m/z ¼ 598.2 [MꢃH]ꢃ.
N-((E)-5-(4-Hydroxy-3-methoxyphenyl)-2-((E)-3-(4-
hydroxy-3-methoxyphenyl)acryloyl)-3-oxo-1-(4-
(trifluoromethyl)phenylpent-4-en-1-yl)acetamide (24)
C31H28F3NO7; M ¼ 583.2; yield: 42%; m.p.: 144–147°C; 1H NMR
(500.13 MHz, DMSO-d6): d 1.74 (s, 3H, CH3C(O)), 3.74 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 5.15 (d, 1H, J ¼ 10.9 Hz, C(O)CHC(O)),
5.79 (t, 1H, J ¼ 9.7 Hz, NHCH), 6.77 (d, 1H, J ¼ 8.0 Hz), 6.83 (d,
1H, J ¼ 7.6 Hz), 6.88 (d, 1H, J ¼ 15.2 Hz), 6.96 (d, 1H, J ¼ 17.3
Hz), 7.10 (d, 1H, J ¼ 8.0 Hz), 7.17–7.27 (m, 2H), 7.33 (s, 1H), 7.39
(d, 1H, J ¼ 15.2 Hz), 7.57–7.71 (m, 5H), 8.50 (d, 1H, J ¼ 8.8 Hz,
C(O)NH), 9.74 (bs, 2H, 2xOH); 13C NMR (125.76 MHz, DMSO-
d6): d 22.6, 51.5, 55.6, 55.7, 66.6, 111.6, 115.6, 115.7, 121.9,
122.4, 123.7, 124.0, 124.9, 125.0, 125.3, 125.6, 128.3, 144.6,
144.7, 146.1, 147.8, 147.9, 149.9, 150.0, 168.3, 191.5, 191.8; IR
(FTIR, cmꢃ1): 1070, 1124, 1165, 1279, 1327, 1520, 1574, 1647
and 3460; (ES, neg. mode) m/z ¼ 582.2 [MꢃH]ꢃ.
N-((E)-1-(3-Bromophenyl)-5-(4-hydroxy-3-
methoxyphenyl)-2-((E)-3-(4-hydroxy-3-methoxyphenyl)-
acryloyl)-3-oxopent-4-enyl)acetamide (21)
C30H28BrNO7; M ¼ 593.1; yield: 45%; m.p.: 146–148°C; 1H NMR
(500.13 MHz, CDCl3): d 1.98 (s, 3H, CH3C(O)), 3.90 (bs, 6H,
OCH3), 4.72 (d, 1H, J ¼ 6.3 Hz, C(O)CHC(O)), 5.94 (t, 1H,
J ¼ 7.6 Hz, CHNH), 6.17 (s, overlapped peaks, 2H, 2xOH),
6.65 (d, 1H, J ¼ 15.9 Hz), 6.72 (d, 1H, J ¼ 15.9 Hz), 6.88 (t, 3H,
J ¼ 8.4 Hz), 6.97–7.06 (m, 3H), 7.06–7.18 (m, 2H), 7.31 (d, 1H,
J ¼ 7.9 Hz), 7.50 (d, 1H, J ¼ 15.9 Hz), 7.55 (s, 1H), 7.60 (d, 1H,
J ¼ 15.9 Hz); 13C NMR (125.76 MHz, CDCl3): d 22.9, 51.5, 55.6,
66.3, 109.3, 109.5, 114.4, 114.5, 120.8, 121.4, 122.3, 123.9,
124.2, 125.1, 125.9, 129.5, 129.7, 130.3, 142.0, 145.3, 145.4,
146.4, 148.6, 148.7, 169.3, 191.8, 194.4; IR (FTIR, cmꢃ1): 980,
1032, 1124, 1163, 1273, 1429, 1514, 1578, 1653 and 3394; MS
(ES, neg. mode) m/z ¼ 592.1 [MꢃH]ꢃ.
4-((E)-1-Acetamido-5-(4-hydroxy-3-methoxyphenyl)-2-
((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)-3-oxopent-4-
en-1-yl)benzoic acid (25)
C31H29NO9, M ¼ 559.2; yield: 48%; m.p.: 199–201°C; 1H NMR
(500.13 MHz, DMSO-d6): d 1.72 (s, 3H, CH3C(O)), 3.77 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 5.14 (d, 1H, J ¼ 10.6 Hz, C(O)CHC(O)),
5.78 (t, 1H, J ¼ 10.0 Hz, NHCH), 6.78 (d, 1H, J ¼ 8.2 Hz), 6.81–
6.89 (m, 2H), 6.97 (d, 1H, J ¼ 16.0 Hz), 7.09 (dd, overlapped
peaks, 1H, J ¼ 8.2 Hz), 7.18–7.24 (m, 2H), 7.33 (bs, 1H), 7.38 (d,
1H, J ¼ 15.7 Hz), 7.50 (d, 2H, J ¼ 8.2 Hz), 7.65 (d, 1H, J ¼ 16.0
Hz), 7.84 (d, 2H, J ¼ 8.2 Hz), 8.50 (d, 1H, J ¼ 9.0 Hz, C(O)NH),
9.80 (bs, 2H, 2xOH); 13C NMR (125.76 MHz, DMSO-d6): d 22.6,
51.6, 55.6, 55.7, 66.7, 111.5, 111.6, 115.6, 115.7, 122.0, 122.5,
123.7, 124.0, 125.3, 125.7, 127.6, 129.1, 144.5, 144.6, 146.1,
147.9, 148.0, 150.0, 150.1, 167.1, 168.3, 191.6, 191.9; IR (FTIR,
cmꢃ1): 982, 1032, 1124, 1167, 1273, 1429, 1514, 1579, 1647,
2357 and 3396; (ES, neg. mode) m/z ¼ 558.2 [MꢃH]ꢃ.
N-((E)-1-(4-Chlorophenyl)-5-(4-hydroxy-3-methoxy-
phenyl)-2-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)-3-
oxopent-4-en-1-yl)acetamide (22)
C30H28ClNO7, M ¼ 549.2; yield: 47%. 1H NMR (500.13 MHz,
DMSO-d6): d 1.70 (s, 3H, CH3C(O)), 3.77 (s, 3H, OCH3), 3.81 (s,
3H, OCH3), 5.07 (d, 1H, J ¼ 10.6 Hz, C(O)CHC(O)), 5.71 (t, 1H,
J ¼ 9.9 Hz, NHCH), 6.76 (d, 1H, J ¼ 8.2 Hz), 6.81 (d, 1H,
J ¼ 8.0 Hz), 6.86 (d, 1H, J ¼ 15.7 Hz), 6.94 (d, 1H, J ¼ 15.7 Hz),
7.09 (d, 1H, J ¼ 8.0 Hz), 7.19 (d, 1H, J ¼ 8.0 Hz), 7.22 (bs, 1H),
7.29–7.43 (m, 6H), 7.64 (d, 1H, J ¼ 15.7 Hz), 8.42 (d, 1H, C(O)NH,
J ¼ 8.9 Hz), 9.73 (2xs, overlapped peaks, 2H, 2xOH); 13C NMR
(125.76 MHz, DMSO-d6): d 21.7, 51.3, 55.7, 67.0, 111.6, 115.6,
115.7, 122.0, 122.5, 123.7, 124.1, 125.4, 125.7, 128.1, 129.4,
131.6, 140.5, 144.6, 144.7, 147.9, 148.0, 150.0, 150.1, 168.3,
191.7, 192.0; MS (ES, neg. mode) m/z ¼ 548.1 [MꢃH]ꢃ.
N-((E)-5-(4-Hydroxy-3-methoxyphenyl)-2-((E)-3-(4-
hydroxy-3-methoxyphenyl)acryloyl)-3-oxo-1-phenylpent-
4-en-1-yl)acetamide (26)
N-((E)-5-(3-Hydroxy-4-methoxyphenyl)-2-((E)-3-(3-
hydroxy-4-methoxyphenyl)acryloyl)-3-oxo-1-phenyl-pent-
4-en-1-yl)acetamide (23)
C30H29NO7; M ¼ 515.2; yield: 40%; m.p.: 145–147°C; 1H NMR
(500.13 MHz, CDCl3): d 1.97 (s, 3H, CH3C(O)), 3.89 (bs, 6H,
OCH3), 4.78 (d, 1H, J ¼ 6.5 Hz, C(O)CHC(O)), 6.02 (t, 1H,
J ¼ 8.2 Hz, CHNH), 6.22 (s, 1H, OH), 6.30 (s, 1H, OH), 6.70 (t,
2H, J ¼ 16.9 Hz), 6.87 (t, 2H, J ¼ 8.9 Hz), 6.97–7.04 (m, 3H), 7.06
C30H29NO7; M ¼ 515.2; yield: 45%; m.p.: 182–185°C; 1H NMR
(500.13 MHz, DMSO-d6): d 1.65 (s, 3H, CH3C(O)), 3.78 (s, 3H,
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(d, 1H, J ¼ 8.4 Hz), 7.10 (d, 1H, J ¼ 9.1 Hz), 7.18–7.24 (m, 1H),
7.29 (t, 2H, J ¼ 7.3 Hz), 7.35–7.41 (m, 1H), 7.50 (d, 1H,
J ¼ 15.7 Hz), 7.59 (d, 1H, J ¼ 15.7 Hz); 13C NMR (125.76 MHz,
CDCl3): d 22.9, 51.9, 55.5, 66.6, 109.3, 109.5, 114.4, 114.5,
121.0, 121.5, 123.9, 124.2, 126.0, 126.3, 127.2, 128.2, 139.6,
145.0, 145.1, 146.5, 148.6, 169.2, 192.2, 194.7; IR (FTIR, cmꢃ1):
979, 1032, 1122, 1163, 1269, 1429, 1514, 1579, 1655, 2362 and
3390; (ES, neg. mode) m/z ¼ 514.2 [MꢃH]ꢃ.
1070, 1290, 1392, 1502, 1593, 1980, 2177 and 3277; MS (ES,
neg. mode) m/z ¼ 589.9 [MꢃH]ꢃ.
N-((E)-5-(3,5-Dihydroxyphenyl)-2-((E)-3-(3,5-
dihydroxyphenyl)acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)-
acetamide (30)
C28H25NO7; M ¼ 487.16; yield: 35%; m.p.: 145–148°C; 1H NMR
(500.13 MHz, DMSO-d6): d 1.71 (s, 3H, C(O)CH3), 5.09 (d, 1H,
J ¼ 11.3 Hz, C(O)CHC(O)), 5.75 (t, 1H, J ¼ 9.1 Hz, NHCH), 6.31 (d,
2H, J ¼ 16.9 Hz), 6.45 (s, 2H), 6.55 (s, 2H), 6.74 (d, 1H,
J ¼ 15.9 Hz), 6.91 (d, 1H, J ¼ 15.9 Hz), 7.18 (t, 1H, J ¼ 7.5 Hz),
7.24–7.30 (m, 3H), 7.37 (d, 2H, J ¼ 7.9 Hz), 7.47 (d, 1H,
J ¼ 15.8 Hz), 8.40 (d, 1H, J ¼ 8.9 Hz), 9.45 (2H, 2 ꢂ OH) and
9.49 (2H 2 ꢂ OH); 13C NMR (125.76 MHz, DMSO-d6): d 22.9,
51.8, 64.9, 105.4, 105.5, 106.6, 124.7, 124.8, 127.2, 127.5, 128.1,
135.5, 135.8, 141.0, 144.1, 144.2, 158.7, 158.8, 168.1, 192.3 and
192.6; IR (FTIR, cmꢃ1): 516, 603, 669, 839, 960, 1147, 1300,
1598, 1975, 2164 and 3209; MS (ES, neg. mode) m/z ¼ 486.2
[MꢃH]ꢃ.
N-((E)-1-(3,4-Difluorophenyl)-5-(4-hydroxy-3-
methoxyphenyl)-2-((E)-3-(4-hydroxy-3-methoxyphenyl)-
acryloyl)-3-oxopent-4-en-1-yl)acetamide (27)
C30H27F2NO7; M ¼ 551.2; yield: 40%; m.p.: 163–166°C; 1H NMR
(500.13 MHz, DMSO-d6): d 1.73 (s, 3H, CH3C(O)), 3.78 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 5.09 (d, 1H, J ¼ 10.4 Hz, C(O)CHC(O)),
5.71 (t, 1H, J ¼ 9.5 Hz, NHCH), 6.78 (d, 1H, J ¼ 7.6 Hz), 6.83 (d,
1H, J ¼ 8.0 Hz), 6.90 (dd, overlapped peaks, 2H, J ¼ 16.5 Hz),
7.11 (d, 1H, J ¼ 7.6 Hz), 7.17–7.27 (m, 3H), 7.32 (bs, 2H), 7.40 (d,
1H, J ¼ 16.1 Hz), 7.67 (d, 1H, J ¼ 15.8 Hz), 8.41 (d, 1H, C(O)NH,
J ¼ 8.2 Hz), 9.71 (bs, 2H, 2xOH); 13C NMR (125.76 MHz,
DMSO-d6): d 22.6, 51.0, 55.6, 55.7, 66.7, 111.6, 115.6, 115.7,
116.3, 116.4, 116.9, 117.1, 121.9, 122.5, 123.7, 124.0, 125.3,
125.6, 139.2, 144.6, 144.8, 147.9, 148.0, 149.9, 150.1, 168.3,
191.6, 191.8; IR (FTIR, cmꢃ1): 987, 1124, 1211, 1263, 1419, 1516,
1587, 1622, 2366 and 3442; (ES, neg. mode) m/z ¼ 550.2
[MꢃH]ꢃ.
N-((E)-5-(4-Hydroxyphenyl)-2-((E)-3-(4-hydroxyphenyl)-
acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)acetamide (31)
C28H25NO5; M ¼ 455.17; yield: 47%; m.p.: 179–182°C. 1H NMR
(500.13 MHz, DMSO-d6): d 1.70 (s, 3H, C(O)CH3), 5.01 (d, 1H,
J ¼ 10.2 Hz, C(O)CHC(O)), 5.76 (t, 1H, J ¼ 9.3 Hz, NHCH), 6.74–
6.80 (m, 3H), 6.83 (d, 2H, J ¼ 8.6 Hz), 6.91 (d, 1H, J ¼ 15.7 Hz),
7.16 (t, 1H, J ¼ 7.3 Hz), 7.27 (t, 2H, J ¼ 7.6 Hz), 7.33–7.40 (m,
3H), 7.50 (d, 2H, J ¼ 8.6 Hz), 7.58 (d, 2H, J ¼ 8.6 Hz), 7.62 (s, 1H),
8.39 (d, 1H, J ¼ 9.1 Hz), 10.11 (s, 2H, 2 ꢂ OH); 13C NMR
(125.76 MHz, DMSO-d6): d 22.6, 51.8, 67.6, 115.8, 115.9,
121.9, 124.9, 125.2, 127.0, 127.5, 128.0, 130.8, 130.9, 141.3,
143.8, 143.9, 160.3, 160.4, 168.1, 191.9 and 192.2; IR (FTIR,
cmꢃ1): 978, 1092, 1171, 1275, 1439, 1513, 1578, 1601, 1641,
2355 and 3408; MS (ES, neg. mode) m/z ¼ 454.2 [MꢃH]ꢃ.
N-((E)-5-(3-Hydroxyphenyl)-2-((E)-3-(3-hydroxyphenyl)-
acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)acetamide (28)
C28H25NO5; 455.2; yield: 51%; m.p.: 200–202°C; 1H NMR
(500.13 MHz, DMSO-d6): d 1.70 (s, 3H, C(O)CH3), 5.14 (d, 1H,
J ¼ 10.7 Hz, C(O)CHC(O)), 5.77 (t, 1H, J ¼ 10.1 Hz, NHCH), 6.77–
6.93 (m, 3H), 7.00 (d, 1H, J ¼ 9.7 Hz), 7.05 (d, 1H, J ¼ 10.6 Hz),
7.08 (bs, 1H), 7.13–7.21 (m, 4H), 7.22–7.30 (m, 3H), 7.32 (s, 1H),
7.37 (t, 2H, J ¼ 5.3 and 7.6 Hz), 7.60 (d, 1H, J ¼ 15.9 Hz), 8.42 (d,
1H, J ¼ 9.4 Hz), 9.61 (s, 1H, OH), 9.66 (s, 1H, OH); 13C NMR
(125.76 MHz, DMSO-d6): d 22.7, 51.9, 67.4, 114.8, 114.9, 118.2,
118.3, 119.9, 125.1, 125.2, 127.2, 127.5, 128.2, 130.0, 130.1,
135.2, 135.5, 141.1, 143.9, 157.7, 157.8, 168.2, 192.4 and 192.7;
IR (FTIR, cmꢃ1): 980, 1072, 1173, 1234, 1267, 1452, 1545, 1645
and 3369; MS (ES, neg. mode) m/z ¼ 454.2 [MꢃH]ꢃ.
N-((E)-5-(4-Fluorophenyl)-2-((E)-3-(4-fluorophenyl)-
acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)acetamide (32)
C28H23F2NO3; M ¼ 459.16; yield: 56%; m.p.: 209–212°C.
1H NMR (500.13 MHz, DMSO-d6): d 1.70 (s, 3H, C(O)CH3),
5.14 (d, 1H, J ¼ 10.8 Hz, C(O)CHC(O)), 5.78 (t, 1H, J ¼ 9.5 Hz,
NHCH), 6.98 (d, 1H, J ¼ 16.2 Hz), 7.10 (d, 1H, J ¼ 15.8 Hz), 7.17
(t, 1H, J ¼ 7.1 Hz), 7.21–7.34 (m, 6H), 7.39 (d, 2H, J ¼ 8.1 Hz),
7.44 (d, 1H, J ¼ 16.2 Hz), 7.69 (s, 1H), 7.71–7.76 (m, 2H), 7.82 (t,
2H, J ¼ 8.1 Hz), 8.42 (d, 1H, J ¼ 8.6 Hz); 13C NMR (125.76 MHz,
DMSO-d6): d 22.6, 51.8, 67.3, 115.9 (d), 116.1 (d), 125.1, 125.3,
127.2, 127.4 (2 ꢂ CHAr), 128.1 (2 ꢂ CHAr), 130.6 (m), 130.9,
131.0 (d), 131.1 (d), 141.0, 142.4 (2 ꢂ CHAr), 162.5, 164.5, 168.1,
192.2 and 192.5; IR (FTIR, cmꢃ1): 980, 1092, 1161, 1238, 1511,
1597, 1658, 1678 and 3313; MS (ES, neg. mode) m/z ¼ 458.2
[MꢃH]ꢃ.
N-((E)-5-(4-Bromothiophen-2-yl)-2-((E)-3-(4-
bromothiophen-2-yl)acryloyl)-3-oxo-1-phenylpent-4-en-1-
yl)acetamide (29)
C24H19Br2NO3S2; M ¼ 590.9; yield: 39%; m.p.: 148–150°C;
1H NMR (500.13 MHz, DMSO-d6): d 1.71 (s, 3H, C(O)CH3),
5.07 (d, 1H, C(O)CHC(O), J ¼ 10.7 Hz), 5.69 (t, 1H, NHCH,
J ¼ 10.3 Hz), 6.75 (d, 1H, J ¼ 15.7 Hz), 6.89 (d, 1H, J ¼ 15.8 Hz),
7.16–7.22 (m, 1H), 7.27 (t, 2H, J ¼ 7.5 Hz), 7.36 (d, 2H,
J ¼ 7.5 Hz), 7.52 (d, 1H, J ¼ 15.4 Hz), 7.59 (s, 1H), 7.64 (s, 1H),
7.80 (d, 1H, J ¼ 5.8 Hz), 7.84 (s, 1H), 7.88 (s, 1H), 8.41 (d, 1H,
J ¼ 8.8 Hz). 13C NMR (125.76 MHz, DMSO-d6): d 23.4, 48.1, 56.5,
67.5, 108.5, 111.5, 112.5, 112.6, 123.5, 123.9, 124.3, 124.6,
124.9, 127.6, 127.8, 128.0, 145.4, 145.5, 147.6, 149.86, 149.89,
152.4, 152.5, 169.4, 192.3; IR (FTIR, cmꢃ1): 578, 696, 825, 954,
4,40-((E)-5-Acetamido-4-((E)-3-(4-carboxyphenyl)acryloyl)-
3-oxopent-1-ene-1,5-diyl)dibenzoic acid (33)
C31H25NO9; M ¼ 555.18; yield: 49%; m.p.: 220°C (decomposed).
1H NMR (500.13 MHz, DMSO-d6): d 1.71 (s, 3H), 5.29 (d, 1H,
J ¼ 10.7 Hz), 5.80 (t, 1H, J ¼ 9.7 Hz), 7.14 (d, 1H, J ¼ 15.7 Hz),
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7.24 (d, 1H, J ¼ 16.8 Hz), 7.45–7.55 (m, 3H), 7.60 (d, 1H,
J ¼ 8.1 Hz), 7.72–7.80 (m, 3H), 7.82–7.88 (m, 3H), 7.91 (d, 2H,
J ¼ 7.7 Hz), 7.98 (d, 2H, J ¼ 8.2 Hz), 8.52 (d, 1H, J ¼ 9.0 Hz);
13C NMR (125.76 MHz, DMSO-d6): d 22.6, 51.7, 66.8, 127.1,
127.6, 127.7, 128.8, 128.9, 129.3, 129.7, 129.8, 132.4, 137.9,
138.3, 142.4, 142.5, 145.8, 166.7, 166.8, 167.0, 168.4, 192.3,
192.6; IR (FTIR, cmꢃ1): 541, 781, 1234, 1610, 1695 and 2164; MS
(ES, neg. mode) m/z ¼ 554.2 [MꢃH]ꢃ.
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-
dimethoxyphenyl)acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)-
acrylamide (37)
C33H33NO7; M ¼ 555.2; yield: 57%; m.p.: 186–189°C; 1H NMR
(500.13 MHz, CDCl3): d 3.88 (bs, 12H, 4xOCH3), 4.86 (d, 1H,
J ¼ 6.3 Hz, C(O)CHC(O)), 7.06–7.16 (m, 2H) 6.21 (d, 1H,
J ¼ 17.1 Hz), 6.74 (t, 2H, J ¼ 15.8 Hz), 6.79–6.87 (m, 2H)
6.99–7.04 (m, 2H), 7.07 (d, 1H, J ¼ 8.2 Hz), 7.10 (d, 1H,
J ¼ 8.2 Hz), 7.16–7.23 (m, 1H), 7.28 (t, 2H, J ¼ 7.6 Hz), 7.36 (d,
1H, J ¼ 9.0 Hz), 7.41 (d, 2H, J ¼ 7.5 Hz), 7.52 (d, 1H, J ¼ 15.8 Hz),
7.61 (d, 1H, J ¼ 15.8 Hz); 13C NMR (125.76 MHz, CDCl3): d 52.1,
55.5, 66.6, 109.6, 109.7, 110.5, 110.6, 121.3, 121.9, 123.5,
123.7, 126.40, 126.48, 126.52, 127.2, 128.2, 130.3, 139.5,
144.9, 145.0, 148.8, 151.54, 151.56, 164.5, 192.1, 194.7; IR
(FTIR, cmꢃ1): 545, 700, 759, 979, 1022, 1141, 1258, 1512, 1593,
1652, 1967, 2054 and 2166; MS (ES, neg. mode) m/z ¼ 554.2
[MꢃH]ꢃ.
4-((4E,6E)-1-Acetamido-7-(4-hydroxy-3-methoxyphenyl)-
2-((2E,4E)-5-(4-hydroxy-3-methoxyphenyl)penta-2,4-
dienoyl)-3-oxohepta-4,6-dienyl)benzoic acid (34)
C35H33NO9; M ¼ 611.17; yield: 39%; m.p.: 137–139°C. 1H NMR
(500.13 MHz, DMSO-d6): d 1.72 (s, 3H), 3.78 (s, 3H), 3.81 (s, 3H),
4.91 (d, 1H, J ¼ 10.6 Hz), 5.91 (t, 1H, J ¼ 9.7 Hz), 6.31 (d, 1H,
J ¼ 15.8 Hz), 6.44 (d, 1H, J ¼ 14.8 Hz), 6.70–7.26 (m, 11H), 7.41–
7.51 (m, 3H), 7.82–7.88 (m, 2H), 8.45 (d, 2H, J ¼ 9.5 Hz), 9.51 (bs,
2H); 13C NMR (125.76 MHz, DMSO-d6): d 23.5, 52.6, 56.5, 56.5,
68.1, 111.2, 116.5, 123.1, 123.2, 124.6, 124.8, 127.0, 127.1,
128.36, 128.43, 128.6, 130.1, 130.5, 144.6, 144.7, 146.2, 146.3,
147.0, 148.8, 148.8, 149.5, 167.9, 169.1, 192.5, 192.7; IR (FTIR,
cmꢃ1): 995, 1032, 1124, 1284, 1514, 1572, 1653, 2357 and 3367;
MS (ES, neg. mode) m/z ¼ 610.2 [MꢃH]ꢃ.
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-
dimethoxyphenyl)acryloyl)-1-(4-fluorophenyl)-3-oxopent-
4-en-1-yl)acrylamide (38)
C33H32FNO7; M ¼ 573.2; yield: 55%; 1H NMR (500.13 MHz,
DMSO-d6): d 3.76 (s, 6H, 2xOCH3), 3.79 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 5.20 (d, 1H, J ¼ 10.9 Hz, C(O)CHC(O)), 5.52 (d, 1H,
–
N-((4E,6E)-7-(4-Hydroxy-3-methoxyphenyl)-2-((2E,4E)-5-
(4-hydroxy-3-methoxyphenyl)penta-2,4-dienoyl)-3-oxo-1-
phenylhepta-4,6-dienyl)acetamide (35)
J ¼ 10.2 Hz, CHH CH), 5.82 (t, 1H, J ¼ 9.3 Hz, CHNH), 5.99 (d,
–
–
–
–
1H, J ¼ 16.8 Hz, CHH CH), 6.13 (dd, 1H, J ¼ 10.2 Hz, CHH CH),
–
6.90–6.98 (m, 2H), 6.98–7.05 (m, 2H), 7.11 (bs, 3H), 7.17–7.27
(m, 2H), 7.27–7.37 (m, 2H), 7.38–7.49 (m, 2H), 7.69 (d, 1H,
J ¼ 16.1 Hz), 8.66 (d, 1H, J ¼ 8.6 Hz, C(O)NH); 13C NMR
(125.76 MHz, DMSO-d6): d 51.3, 55.6, 67.0, 110.6, 111.6,
111.7, 114.8, 115.0, 123.0, 123.4, 123.7, 123.9, 125.7, 126.7,
126.9, 129.5, 129.6, 131.5, 137.4, 144.3, 144.4, 148.9, 149.0,
151.5, 151.6, 163.7, 191.7, 192.0; IR (FTIR, cmꢃ1): 980, 1022,
1142, 1263, 1512, 1589, 1649, 1674 and 3415; MS (ES, neg.
mode) m/z ¼ 572.2 [MꢃH]ꢃ.
C34H33NO7; M ¼ 567.12; yield: 41%; m.p.: 125°C (decomposed).
1H NMR (500.13 MHz, DMSO-d6): d 1.71 (s, 3H), 3.78 (s, 3H),
3.81 (s, 3H), 4.83 (d, 1H, J ¼ 11.1 Hz), 5.82 (t, 1H, J ¼ 9.8 Hz), 6.29
(d, 1H, J ¼ 15.5 Hz), 6.45 (d, 1H, J ¼ 15.2 Hz), 6.72–6.80 (m, 3H),
6.84–7.39 (m, 14H), 8.37 (d, 1H, J ¼ 7.3 Hz), 9.50 (s, 2H);
13C NMR (125.76 MHz, DMSO-d6): d 23.5, 52.7, 56.5, 56.5, 68.7,
111.2, 116.5, 123.1, 123.1, 124.7, 124.8, 126.9, 127.3, 128.0,
128.4, 128.5, 129.0, 142.1, 144.4, 144.5, 146.0, 146.0, 148.8,
148.8, 149.5, 168.9, 192.7, 193.0; IR (FTIR, cmꢃ1): 1001, 1030,
1120, 1161, 1282, 1514, 1566, 1655, 2362 and 3313; MS (ES,
neg. mode) m/z ¼ 566.1 [MꢃH]ꢃ.
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-
dimethoxyphenyl)acryloyl)-3-oxo-1-(3-(trifluoromethyl)-
phenyl)pent-4-en-1-yl)acrylamide (39)
N-((E)-5-(5-(Hydroxymethyl)furan-2-yl)-2-((E)-3-(5-
(hydroxymethyl)furan-2-yl)acryloyl)-3-oxo-1-phenylpent-
4-en-1-yl)acetamide (36)
C34H32F3NO7; M ¼ 623.2; yield: 54%; m.p.: 184–186°C;
1H NMR (500.13 MHz, DMSO-d6): d 3.75 (s, 3H, OCH3),
3.76 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 5.28
(d, 1H, J ¼ 10.8 Hz, C(O)CHC(O)), 5.54 (d, 1H, J ¼ 10.2 Hz,
C26H25NO7; M ¼ 463.16; yield: 38%; m.p.: 129–132°C; 1H NMR
(500.13 MHz, DMSO-d6): d 1.70 (s, 3H, C(O)CH3), 4.45 (d, 4H,
J ¼ 16.8 Hz), 4.96 (d, 1H, J ¼ 10.7 Hz, C(O)CHC(O)), 5.41 (bs, 2H,
2 ꢂ OH), 5.78 (t, 1H, J ¼ 10.0 Hz, NHCH), 6.45 (d, 1H, J ¼ 2.2 Hz),
6.49 (d, 1H, J ¼ 2.2 Hz), 6.58 (d, 1H, J ¼ 15.8 Hz), 6.74 (d, 1H,
J ¼ 15.7 Hz), 6.92 (d, 1H, J ¼ 2.2 Hz), 6.99 (d, 1H, J ¼ 2.2 Hz),
7.15–7.30 (m, 4H), 7.35 (d, 2H, J ¼ 7.2 Hz), 7.45 (d, 1H,
J ¼ 15.7 Hz), 8.39 ((d, 1H, J ¼ 9.1 Hz); 13C NMR (125.76 MHz,
CDCl3): d 22.9, 51.9, 55.7, 55.8, 68.2, 110.2, 110.3, 119.3, 119.5,
120.5, 120.8, 127.1, 127.4 (2 ꢂ CHAr), 128.1 (2 ꢂ CHAr), 129.9,
130.1, 140.1, 149.5, 149.8, 159.4, 159.6, 168.1, 191.4,191.9; IR
(FTIR, cmꢃ1): 541, 698, 964, 1016, 1599, 2019, 2166 and 3298;
MS (ES, neg. mode) m/z ¼ 462.2 [MꢃH]ꢃ.
–
CHH CH), 5.88 (dd, overlapped peaks, 1H, J ¼ 9.5 Hz,
–
CHNH), 6.01 (d, 1H, J ¼ 17.2 Hz, CHHCH), 6.16 (dd, 1H,
J ¼ 10.2 Hz, CHHCH), 6.92–7.09 (m, 5H), 7.20 (d, 1H, J ¼ 8.3
Hz), 7.24 (s, 1H), 7.31 (d, 1H, J ¼ 8.3 Hz), 7.35 (s, 1H), 7.43 (d,
1H, J ¼ 15.7 Hz), 7.51–7.57 (m, 2H), 7.69 (s, 1H), 7.78 (s, 1H),
8.74 (d, 1H, C(O)NH, J ¼ 8.5 Hz; 13C NMR (125.76 MHz,
DMSO-d6): d 51.7, 55.6, 66.6, 110.6, 110.7, 111.6, 111.7,
122.8, 123.5, 123.8, 124.0, 124.1, 126.0, 126.6, 126.9, 129.3,
131.3, 131.9, 142.5, 144.4, 144.6, 148.9, 149.0, 151.6, 151.7,
163.9, 191.6, 191.8; IR (FTIR, cmꢃ1): 982, 1024, 1122, 1140,
1163, 1265, 1333, 1514, 1593, 1655, 1670 and 3288; MS
(ES, neg. mode) m/z ¼ 622.2 [MꢃH]ꢃ.
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4-((E)-1-Acrylamido-5-(3,4-dimethoxyphenyl)-2-((E)-3-(3,4-
dimethoxyphenyl)acryloyl)-3-oxopent-4-en-1-yl)benzoic
acid (40)
N-((E)-5-(3,5-Dihydroxyphenyl)-2-((E)-3-(3,5-
dihydroxyphenyl)acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)-
acrylamide (43)
C34H33NO9; M ¼ 599.2; yield: 63%; m.p.: 218–220°C; 1H NMR
(500.13 MHz, DMSO-d6): d 3.76 (s, 3H, OCH3), 3.78 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 5.27 (d, 1H,
J ¼ 10.6 Hz, C(O)CHC(O)), 5.54 (dd, overlapped peaks, 1H,
C29H25NO7; M ¼ 499.16; yield: 31%; m.p.: 149°C (decom-
posed); 1H NMR (500.13 MHz, DMSO-d6): d 5.19 (d, 1H,
J ¼ 10.1 Hz, C(O)CHC(O)), 5.53 (d, 1H, J ¼ 9.0 Hz), 5.81 (t, 1H,
J ¼ 10.1 Hz, NHCH), 6.00 (d, 1H, J ¼ 15.8 Hz), 6.14 (dd, 1H,
J ¼ 10.1 and 9.0 Hz), 6.20–6.61 (m, 5H), 6.77 (d, 1H, J ¼ 15.8 Hz),
6.91 (d, 1H, J ¼ 16.3 Hz), 7.10–7.54 (m, 6H), 8.62 (d, 1H,
J ¼ 7.9 Hz), 9.34–9.55 (4H, 4 ꢂ OH); 13C NMR (125.76 MHz,
DMSO-d6): d 51.9, 67.1, 105.5, 106.7 (2 ꢂ CHAr), 124.7, 124.8,
125.8, 127.3, 127.5 (2 ꢂ CHAr), 128.3 (2 ꢂ CHAr), 131.4, 135.5,
135.8, 140.8, 144.4, 144.5, 158.6, 158.7, 163.6, 192.2 and 192.4;
IR (FTIR, cmꢃ1): 970, 1151, 1275, 1511, 1591, 1653, 2357 and
3375; MS (ES, neg. mode) m/z ¼ 498.2 [MꢃH]ꢃ.
–
J ¼ 10.2 Hz, CHH CH), 5.88 (t, 1H, J ¼ 9.5 Hz, CHNH), 6.01 (dd,
–
–
overlapped peaks, 1H, J ¼ 17.0 Hz, CHH CH), 6.16 (dd, 1H,
–
–
J ¼ 6.8 and 17.0 Hz, CHH CH), 6.93–6.99 (m, 2H), 6.99–7.06 (m,
–
2H), 7.21 (d, 1H, J ¼ 8.3 Hz), 7.24 (s, 1H), 7.31 (d, 1H, J ¼ 8.3 Hz),
7.35 (s, 1H), 7.43 (d, 1H, J ¼ 15.9 Hz), 7.53 (d, 2H, J ¼ 8.3 Hz),
7.71 (d, 1H, J ¼ 15.9 Hz), 7.86 (d, 1H, J ¼ 7.8 Hz), 8.71 (d, 1H,
J ¼ 8.9 Hz, C(O)NH), 12.8 (bs, 1H, C(O)OH); 13C NMR
(125.76 MHz, DMSO-d6): d 51.7, 55.5, 66.5, 110.6, 111.5,
111.6, 122.9, 123.4, 123.6, 123.9, 125.8, 126.6, 126.9, 127.7,
129.2, 129.6, 131.3, 144.2, 144.4, 145.9, 148.9, 149.0, 151.5,
151.6, 163.7, 166.9, 191.5, 191.8; IR (FTIR, cmꢃ1): 555, 800,
1024, 1131, 1261, 1512, 1593, 1716 and 2160; MS (ES, neg.
mode) m/z ¼ 598.2 [MꢃH]ꢃ.
N-((E)-5-(3-Hydroxyphenyl)-2-((E)-3-(3-hydroxyphenyl)-
acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)-acrylamide (44)
C29H25NO5; M ¼ 467.17; yield: 50%; m.p.: 194–196°C; 1H NMR
(500.13 MHz, DMSO-d6): d 5.22 (d, 1H, J ¼ 10.6 Hz, C(O)CHC
(O)), 5.52 (d, 1H, J ¼ 11.1 Hz), 5.82 (t, 1H, J ¼ 10.3 Hz, NHCH),
5.99 (d, 1H, J ¼ 16.6 Hz), 6.13 (dd, 1H, J ¼ 10.3 Hz), 6.83 (dd, 2H,
J ¼ 7.5 and 8.0 Hz), 6.91 (d, 1H, J ¼ 16.3 Hz), 6.96–7.44 (m, 13H),
7.60 (d, 1H, J ¼ 16.3 Hz), 8.64 (d, 1H, J ¼ 9.1 Hz), 9.61 (s, 1H,
OH), 9.65 (s, 1H, OH); 13C NMR (125.76 MHz, DMSO-d6): d 51.9,
67.1, 114.8, 114.9, 118.3 (2 ꢂ CHAr), 119.9 (2 ꢂ CHAr), 125.1
(2 ꢂ CHAr), 125.8, 127.3, 127.6 (2 ꢂ CHAr), 128.3 (2 ꢂ CHAr),
130.0, 130.1, 131.5, 135.2, 135.5, 140.9, 144.0, 144.1, 157.7,
157.8, 163.7, 192.3 and 192.5; IR (FTIR, cmꢃ1): 978, 1093, 1157,
1263, 1579, 1610, 1684, 2359 and 3290; MS (ES, neg. mode) m/
z ¼ 466.2 [MꢃH]ꢃ.
N-((E)-5-(4-Hydroxy-3-methoxyphenyl)-2-((E)-3-(4-
hydroxy-3-methoxyphenyl)acryloyl)-3-oxo-1-phenylpent-
4-en-1-yl)acrylamide (41)
C34H33NO9; M ¼ 599.22; yield: 63%; m.p.: 218–220°C; 1H NMR
(500.13 MHz, DMSO-d6): d 3.76 (s, 3H, OCH3), 3.78 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 5.27 (d, 1H,
J ¼ 10.6 Hz, C(O)CHC(O)), 5.54 (dd, overlapped peaks, 1H,
–
J ¼ 10.2 Hz, CHH CH), 5.88 (t, 1H, J ¼ 9.5 Hz, CHNH), 6.01 (dd,
–
–
overlapped peaks, 1H, J ¼ 17.0 Hz, CHH CH), 6.16 (dd, 1H,
–
–
J ¼ 6.8 and 17.0 Hz, CHH CH), 6.93–6.99 (m, 2H), 6.99–7.06 (m,
–
2H), 7.21 (d, 1H, J ¼ 8.3 Hz), 7.24 (s, 1H), 7.31 (d, 1H, J ¼ 8.3 Hz),
7.35 (s, 1H), 7.43 (d, 1H, J ¼ 15.9 Hz), 7.53 (d, 2H, J ¼ 8.3 Hz),
7.71 (d, 1H, J ¼ 15.9 Hz), 7.86 (d, 1H, J ¼ 7.8 Hz), 8.71 (d, 1H,
J ¼ 8.9 Hz, C(O)NH), 12.8 (bs, 1H, C(O)OH); 13C NMR
(125.76 MHz, DMSO-d6): d 51.7, 55.5, 66.5, 110.6, 111.5,
111.6, 122.9, 123.4, 123.6, 123.9, 125.8, 126.6, 126.9, 127.7,
129.2, 129.6, 131.3, 144.2, 144.4, 145.9, 148.9, 149.0, 151.5,
151.6, 163.7, 166.9, 191.5, 191.8; IR (FTIR, cmꢃ1): 978, 1028,
1122, 1165, 1207, 1269, 1514, 1589, 1662, 2359 and 3404; MS
(ES, neg. mode) m/z ¼ 598.2 [MꢃH]ꢃ.
4-((E)-1-Acrylamido-5-(3,5-dihydroxyphenyl)-2-((E)-3-(3,5-
dihydroxyphenyl)acryloyl)-3-oxopent-4-en-1-yl)benzoic
acid (45)
C30H25NO9, M ¼ 543.15; yield: 34%; m.p.: 122°C (decom-
posed); 1H NMR (500.13 MHz, DMSO-d6): d 5.27 (d, 1H, C(O)
CHC(O), J ¼ 11.0 Hz), 5.54 (dd, 1H, J ¼ 10.8 and 1.7 Hz), 5.85 (t,
1H, J ¼ 9.5 Hz, NHCH), 6.00 (d, 1H, J ¼ 17.1 Hz), 6.15 (dd, 1H,
J ¼ 10.2 and 10.5 Hz), 6.20–6.27 (m, 1H), 6.30 (bs, 1H), 6.33 (bs,
1H), 6.37 (t, 1H, J ¼ 8.1 Hz), 6.41–6.45 (m, 1H), 6.47 (d, 2H,
J ¼ 2.2 Hz), 6.55 (d, 2H, J ¼ 2.2 Hz), 6.79 (d, 1H, J ¼ 15.9 Hz), 6.91
(d, 1H, J ¼ 15.9 Hz), 7.28 (d, 1H, J ¼ 15.9 Hz), 7.35–7.45 (m, 1H),
7.47–7.54 (m, 3H), 7.85 (d, 2H, J ¼ 8.5 Hz), 8.69 (d, 1H,
J ¼ 8.9 Hz), 9.53 (bs, 1H, COOH); 13C NMR (125.76 MHz,
DMSO-d6): d 52.0, 67.5, 105.5, 105.6, 106.6, 106.8 (2 ꢂ CHAr),
124.7, 124.8, 127.3, 127.6 (2 ꢂ CHAr), 128.2 (2 ꢂ CHAr), 135.6,
135.9, 141.1, 144.3, 144.4, 158.6, 158.7, 158.8, 168.2, 192.4 and
192.8; IR (FTIR, cmꢃ1): 514, 1151, 1269, 1587, 1652, 1975, 2158
and 3238; MS (ES, neg. mode) m/z ¼ 542.2 [MꢃH]ꢃ.
4,40-((1E,6E)-4-(Acrylamido(phenyl)methyl)-3,5-
dioxohepta-1,6-diene-1,7-diyl)dibenzoic acid (42)
C31H25NO7; M ¼ 523.16; yield: 42%; m.p.: 179–182°C;1H NMR
(500.13 MHz, DMSO-d6): d 5.32 (d, 1H, J ¼ 11.3 Hz), 5.50 (d, 1H,
J ¼ 11.2 Hz), 5.85 (t, 1H, J ¼ 9.9 Hz), 5.99 (d, 1H, J ¼ 16.5 Hz),
6.14 (dd, 1H, J ¼ 9.9 and 16.5 Hz), 7.10–7.24 (m, 3H), 7.25–7.33
(m, 3H), 7.42 (d, 2H, J ¼ 7.24 Hz), 7.50 (d, 1H, J ¼ 15.8 Hz), 7.72–
7.80 (m, 2H), 7.84 (d, 2H, J ¼ 7.9 Hz), 7.92 (d, 2H, J ¼ 8.1 Hz),
7.98 (d, 2H, J ¼ 8.2 Hz), 8.69 (d, 1H, J ¼ 8.9 Hz); 13C NMR
(125.76 MHz, DMSO-d6): d 52.0, 67.1, 125.9, 127.2, 127.4,
127.5, 127.6, 128.3, 128.8, 128.9, 129.7, 129.8, 131.4, 132.4,
138.0, 138.2, 140.7, 142.3, 142.4, 163.7, 166.7, 166.8, 192.3,
192.6; IR (FTIR, cmꢃ1): 985, 1084, 1173, 1288, 1410, 1533, 1610,
1659, 1693 and 3063; MS (ES, neg. mode) m/z ¼ 522.2 [MꢃH]ꢃ.
N-((E)-3-Oxo-5-(4-oxo-4H-chromen-3-yl)-2-((E)-3-(4-oxo-
4H-chromen-3-yl)acryloyl)-1-phenylpent-4-en-1-yl)-
acrylamide (46)
C35H25NO7; M ¼ 571.16; yield: 45%; m.p.: 185–187°C;1H NMR
(500.13 MHz, DMSO-d6): d 5.05 (d, 1H, J ¼ 11.2 Hz), 5.52 (d, 1H,
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J ¼ 10.2 Hz), 5.81 (t, 1H, J ¼ 10.2 Hz), 5.99 (d, 1H, J ¼ 17.1 Hz),
6.12 (dd, 1H, J ¼ 10.0 and 16.9 Hz), 7.17 (t, 1H, J ¼ 7.4 Hz), 7.24–
7.30 (m, 2H), 7.39 (d, 2H, J ¼ 7.4 Hz), 7.48 (d, 1H, J ¼ 15.6 Hz),
7.53 (dd, 3H, J ¼ 13.5 and 6.9 Hz), 7.60 (d, 1H, J ¼ 15.8 Hz), 7.66
(d, 1H, J ¼ 8.4 Hz), 7.69 (d, 1H, J ¼ 8.4 Hz), 7.76–7.85 (m, 3H), 8.1
(d, 1H, J ¼ 8.4 Hz), 8.12 (d, 1H, J ¼ 7.4 Hz), 8.67 (d, 1H,
J ¼ 8.9 Hz), 8.83 (s, 1H), 8.90 (s, 1H); 13C NMR (125.76 MHz,
DMSO-d6): d 52.0, 68.6, 117.8, 118.1, 118.5, 123.4, 123.5, 125.4,
125.5, 125.7, 126.2, 126.8, 127.3, 127.6, 128.2, 131.5, 134.7,
136.1, 136.3, 140.8 155.0, 163.5, 175.1, 175.2, 192.3, 192.6; IR
(FTIR, cmꢃ1): 978, 1261, 1292, 1354, 1406, 1464, 1614, 1655,
2341 and 3350; MS (ES, neg. mode) m/z ¼ 570.2 [MꢃH]ꢃ.
N-((4E,6E)-7-(4-Hydroxy-3-methoxyphenyl)-2-((2E,4E)-5-
(4-hydroxy-3-methoxyphenyl)penta-2,4-dienoyl)-3-oxo-1-
phenylhepta-4,6-dienyl)cinnamamide (50)
C41H37NO7; M ¼ 655.26; yield: 36%; m.p.: 128–131°C; 1H NMR
(500.13 MHz, DMSO-d6): d 3.79 (s, 3H), 3.80 (s, 3H), 4,97 (d, 1H,
J ¼ 10.9 Hz), 5.83 (t, 1H, J ¼ 9.9 Hz), 6.34 (d, 2H, J ¼ 14.9 Hz),
6.47 (d, 2H, J ¼ 15.2 Hz), 6.54 (d, 2H, J ¼ 15.9 Hz), 6.74–6.79 (m,
3H), 6.87–7.54 (m, 17H), 8.64 (d, 1H, J ¼ 8.9 Hz), 9.50 (s, 1H),
9.51 (s, 1H); 13C NMR (125.76 MHz, DMSO-d6): d 52.9, 56.47,
56.49, 68.4, 111.2, 116.5, 122.8, 123.10, 123.14, 124.7, 124.8,
127.0, 127.3, 128.1, 128.4, 129.1, 129.7, 130.4, 135.6, 140.1,
141.9, 144.5, 144.6, 146.1, 146.2, 148.8, 149.5, 149.5, 164.8,
192.7, 193.0; IR (FTIR, cmꢃ1): 997, 1034, 1165, 1282, 1514, 1564,
1660, 2355 and 3327; MS (ES, neg. mode) m/z ¼ 654.2 [MꢃH]ꢃ.
N-((E)-1-(4-Fluorophenyl)-5-(3-hydroxyphenyl)-2-((E)-3-(3-
hydroxyphenyl)acryloyl)-3-oxopent-4-en-1-yl)acrylamide
(47)
N-((E)-5-(3,5-Dihydroxyphenyl)-2-((E)-3-(3,5-
C29H24FNO7; M ¼ 485.16; yield: 53%; m.p.: 200–201°C;1H NMR
(500.13 MHz, DMSO-d6): d 5.22 (d, 1H, J ¼ 10.9 Hz), 5.52 (d,
1H, J ¼ 11.6 Hz), 5.81 (t, 1H, J ¼ 9.7 Hz), 5.99 (d, 1H, J ¼ 17.4
Hz), 6.13 (dd, 1H, J ¼ 9.7 and 16.8 Hz), 6.84 (t, 2H, J ¼ 10.3 Hz),
6.92 (d, 1H, J ¼ 15.5 Hz), 7.00 (s, 2H), 7.02–7.26 (m, 7H), 7.37
(d, 1H, J ¼ 16.1 Hz), 7.40–7.47 (m, 2H), 7.62 (d, 1H, J ¼ 16.1 Hz),
8.65 (d, 1H, J ¼ 9.0 Hz), 9.62 (s, 1H), 9.66 (s, 1H); 13C NMR
(125.7 MHz, DMSO-d6): d 51.3, 67.1, 114.9, 115.0, 115.1,
118.2, 118.3, 119.9, 120.0, 125.0, 125.1, 125.9, 129.5, 129.6,
130.0, 130.1, 131.4, 135.1, 135.4, 137.1, 144.1, 144.2, 157.7,
157.8, 163.7, 192.1, 192.3; IR (FTIR, cmꢃ1): 982, 1093, 1163,
1225, 1511, 1581, 1601, 1659, 1678 and 3286; MS (ES, neg.
mode) m/z ¼ 484.2 [MꢃH]ꢃ.
dihydroxyphenyl)acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)-
cinnamamide (51)
C35H29NO7; M ¼ 575.19; yield: 52%; m.p.: 206–209°C; 1H NMR
(500.13 MHz, DMSO-d6): d 5.23 (d, 1H, J ¼ 10.8 Hz), 5.87 (t,1H,
J ¼ 9.7 Hz), 6.31 (d, 2H, J ¼ 8.2 Hz), 6.47 (s, 2H), 6.54 (s, 2H), 6.81
(d, 1H, J ¼ 16.1 Hz), 6.93 (d, 1H, J ¼ 16.1 Hz), 7.18 (t, 1H,
J ¼ 7.1 Hz), 7.24–7.38 (m, 8H), 7.42 (d, 2H, J ¼ 6.4 Hz), 7.45–7.52
(m, 3H), 8.66 (d, 1H, J ¼ 8.9 Hz), 9.46 (s, 2H), 9.48 (s, 2H));
13C NMR (125.76 MHz, DMSO-d6): d 52.0, 67.1, 105.4, 105.5,
106.7, 121.9, 124.8, 124.9, 127.3, 127.5, 127.6, 128.2, 128.9,
129.5, 134.7, 135.5, 135.8, 139.3, 140.9, 144.4, 144.5, 158.7,
158.8, 164.0, 192.3, 192.6; IR (FTIR, cmꢃ1): 974, 1088, 1159,
1271, 1350, 1452, 1506, 1601, 1606, 2353 and 3263; MS (ES,
neg. mode) m/z ¼ 574.2 [MꢃH]ꢃ.
N-((4E,6E)-7-(4-Methoxyphenyl)-2-((2E,4E)-5-(4-
methoxyphenyl)penta-2,4-dienoyl)-3-oxo-1-phenylhepta-
4,6-dienyl)acrylamide (48)
N-((E)-5-(3,4-Dimethoxyphenyl)-2-((E)-3-(3,4-
dimethoxyphenyl)acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)-
cinnamamide (52)
C35H33NO5; M ¼ 547.24; yield: 31%; m.p.: 172–175°C; 1H NMR
(500.13 MHz, DMSO-d6): d 4.97 (d, 1H, J ¼ 8.2 Hz), 5.52 (d, 1H,
J ¼ 6.7 Hz), 5.76 (t, 1H, J ¼ 9.6 Hz), 5.94–6.17 (m, 3H), 6.29–6.52
(m, 3H), 6.74–7.66 (m, 23H), 8.60 (s, 1H); 13C NMR (125.76 MHz,
DMSO-d6): d 52.8, 56.2, 68.3, 115.3, 125.3, 125.5, 126.5, 127.6,
127.8, 128.1, 128.4, 129.0, 129.37, 129.43, 129.8, 130.0, 132.4,
141.8, 143.6, 143.7, 145.9, 146.0, 161.3, 164.4, 192.6, 192.9; IR
(FTIR, cmꢃ1): 999, 1028, 1174, 1254, 1510, 1578, 1660, 2359 and
3338; MS (ES, neg. mode) m/z ¼ 546.2 [MꢃH]ꢃ.
C39H37NO7; M ¼ 631.26; yield: 51%; m.p.: 212–215°C; 1H NMR
(500.13 MHz, DMSO-d6): d 3.73–3.82 (m, 12H), 5.24 (d, 1H,
J ¼ 10.5 Hz), 5.90 (t, 1H, J ¼ 9.5 Hz), 6.56 (d, 1H, J ¼ 15.2 Hz),
6.90–7.08 (m, 4H), 7.14–7.39 (m, 11H), 7.39–7.52 (m, 5H), 7.68
(d, 1H, J ¼ 16.2 Hz), 8.67 (d, 1H, J ¼ 9.5 Hz); 13C
NMR
(125.76 MHz, DMSO-d6): d 52.0, 55.6, 66.9, 110.6, 111.6, 111.7,
122.0, 123.1, 123.5, 123.6, 123.9, 126.7, 127.0, 127.2, 127.5,
127.6, 128.2, 128.9, 129.5, 134.7, 139.2, 141.2, 144.1, 144.2,
148.9, 149.0, 151.5, 151.6, 163.2, 191.9, 192.3; IR (FTIR, cmꢃ1):
984, 1020, 1138, 1263, 1516, 1591, 1659, 1676, 2337 and 3415;
MS (ES, neg. mode) m/z ¼ 630.2 [MꢃH]ꢃ.
N-((E)-5-(3-Hydroxyphenyl)-2-((E)-3-(3-hydroxyphenyl)-
acryloyl)-3-oxo-1-phenylpent-4-en-1-yl)cinnamamide (49)
C35H29NO5; M ¼ 543.20; yield: 47%; m.p.: 187–190°C; 1H NMR
(500.13 MHz, DMSO-d6): d 5.27 (d, 1H, J ¼ 10.2 Hz, C(O)CHC
(O)), 5.90 (t, 1H, J ¼ 9.4 Hz), 6.56 (d, 1H, J ¼ 15.5 Hz), 6.77–7.50
(m, 22H), 7.61 (d, 1H, J ¼ 15.5 Hz), 8.68 (d, 1H, J ¼ 9.0 Hz), 9.62
(s, 1H, OH), 9.68 (s, 1H, OH); 13C NMR (125.76 MHz, DMSO-d6):
d 52.0, 67.1, 114.8, 114.9, 118.2, 118.3, 119.9, 121.9, 125.1,
125.2, 127.3, 127.5, 127.6, 128.2, 128.8, 129.5, 130.0, 130.1,
134.7, 135.2, 135.4, 139.3, 140.9, 144.0, 144.1, 157.7, 157.8,
164.0, 192.3, 192.6; IR (FTIR, cmꢃ1): 545, 696, 972, 1258, 1512,
1573, 1977, 2164 and 3280; MS (ES, neg. mode) m/z ¼ 542.2
[MꢃH]ꢃ.
Biological evaluation
Cell toxicity measurements with novel curcumin analogues
A549 and H1975 lung adenocarcinoma cell lines were
maintained in DMEM supplemented with 10% FBS. Cell
toxicity measurements were carried out in 96-well microtitre
plates with 6000 cells seeded in each well. Test measurements
were carried out in duplicate, and control measurements in
quadruplicate. After an overnight incubation, cells were
treated with an increasing concentration of the analogue. At
72 h post-treatment, the cell viability was measured by means
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of the homogeneous, colourimetric MTS assay (Promega). Cell
viability of treated cells was calculated relative to that of
DMSO vehicle controls. GI50 values were calculated using
GraphPad Prism 5 software, non-linear regression fitting with
variable slope was applied.
family A (Hsp70) member 5, HSPA5_F: 50-agcagggccttcaccaat-
30; HSPA5_R: 50-cgtcgcctactcggcttat-30; DNA damage inducible
transcript 3, DDIT3_F: 50-cagagctggaacctgaggag-30; DDIT3_R:
50-tggatcagtctggaaaagca-30; activating transcription factor 4,
ATF4_F: 50-tctccagcgacaaggctaa-30; ATF4_R: 50-caatctgtcccg-
gagaagg-30; X-box binding protein 1, XBP1_F: 50-ggagttaa-
gacagcgcttgg-30; ATF4_F: 50-tctccagcgacaaggctaa-30; ATF4_R:
50-caatctgtcccggagaagg-30; XBP1_R: 50-cactggcctcacttcattcc-30;
actin beta, ACTB_F: 50-attggcaatgagcggttc-30; ACTB_R: 50-
cgtggatgccacaggact-30) and the SybrGreen protocol [45].
Curves were analysed by using dynamic tube and slope
correction methods, data from cycles close to the baseline
being ignored. Relative expression ratios were normalized to
beta-actin. Statistical analysis: Student’s t-test was performed
in Excel.
Holographic cell analysis
Digital holographic images were taken with the HoloMonitor
M3 instrument (Phase Holographic Imaging AB, Phiab,
Sweden). Cells of lung cancer line A549 were treated with
either 25 mM 1 or 0.5 mM 44 in T25 flasks and incubated for
24 h. Holographic images of the same culture area were
captured before and after treatment. Time-matched un-
treated cells were imaged as controls. Cell morphological
changes were analysed with Holostudio 2.4 software (Phase
Holographic Imaging AB, Phiab, Sweden). Each point in the
scatterplot represents data on a single cell. Parameters
reflecting changes in cell morphology (e.g. area, thickness,
roughness, eccentricity, irregularity and volume) were
recorded [37].
Autophagy-related gene expression analysis
Total RNA was purified from 2 ꢂ 105 A549 cells. Three
biological replicates were applied for all conditions. Treat-
ments included: 500 nM rapamycin, 0.5, 1 and 5 mM 44 and
untreated controls. Samples were collected after 6, 12 and
24 h after treatments (6 h after treatment) with the AccuZolTM
RNA purification kit (Bioneer, Daeleon, Korea) according to
the manufacturer’s protocol. All treatments and controls
involved three biological replicates. Total RNA (1.5 mg) was
converted into cDNA with the High-Capacity cDNA Archive Kit
(Applied Biosystems, Foster City, CA, USA) in a total volume of
20 mL; and without purification, the mixture was diluted
fivefold, and 2 mL was subjected to QRT-PCR analysis. QRT-PCR
was performed on the Light Cycler Nano Instrument (Roche)
with gene-specific primers (autophagy related 5, ATG5_F: 50-
tggatttcgttatatcccctttag-30; ATG5_R: 50-cctagtgtgtgcaactg
tcca-30; beclin 1, BECN1_F: 50-ggatggtgtctctcgcagat-30;
BECN1_R: 50-ttggcactttctgtggacat-30; GABA type A receptor-
associated protein, GABARAP_F: 50-ggtgccttctgatctcacagt-30;
GABARAP_R: 50-tgggtggaatgacattgttg-30; microtubule associ-
ated protein 1 light chain 3 beta, MAP1LC3B_F: 50-cgcaccttc-
gaacaaagag-30; MAP1LC3B_R: 50-ctcacccttgtatcgttctattatca-30;
sequestosome 1, SQSTM1_F: 50-agctgccttgtacccacatc-30;
SQSTM1_R: 50-cagagaagcccatggacag-30) and the SybrGreen
protocol [45]. Relative expression ratios were normalised to
beta-actin. Statistical analysis: Student’s t-test was performed
in Excel.
NF-kB activation assay
The B16 cell line was maintained in RPMI medium (Lonza)
supplemented with 10% FCS (Lonza). The NF-kB reporter cell
lines were created by transfection with the pNF-kB-Luc/neo
reporter construct with the Lipofectamine 2000 reagent
(Invitrogen). Stable cell lines were selected by G418 (Sigma)
treatment. One-day-old cultures of B16/NF-kB-Luc cells,
grown on luminoplates (Corning-Costar) were used. Cells
(5 ꢂ 104 cells/well) were induced with 100 units/mL TNF-alpha.
Each measurement was carried out in duplicate. After 6 h of
incubation with inducers, the medium was removed, and cells
were washed and lysed for 10 min at room temperature in Cell
Culture Lysis Reagent (20 mL/well; Promega). Substrate was
then added (20 mL/well; Promega), and luciferase activity was
measured in a Luminoskan Ascent (Thermo Electron Corpora-
tion) scanning luminometer. Cell viability was routinely
determined through use of the Trypan blue exclusion test
during the assays to make sure that assays were always carried
out on viable cells. A total of 80% of all measurements
displayed a variation of less than 20%. IC50 values were
calculated using GraphPad Prism 5 software, non-linear
regression fitting with variable slope was applied.
UPR-related gene expression analysis
Western blot analysis
Total RNA was purified from 4 ꢂ 105 1-treated, analogue-
treated or control (0.2% DMSO) cells (6 h after treatment)
with the AccuZolTM RNA purification kit (Bioneer, Daeleon,
Korea) according to the manufacturer’s protocol. All treat-
ments and controls involved three biological replicates. Total
RNA (2 mg) was converted into cDNA with the High-Capacity
cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) in
a total volume of 20 mL; and without purification, the mixture
was diluted fivefold, and 2 mL was subjected to QRT-PCR
analysis. QRT-PCR was performed on the Exicycler instrument
(Bioneer) with gene-specific primers (heat shock protein
Western blot samples were obtained by scraping 1 ꢂ 106 A549
cells from 6-well plates in 200 mL of hot Laemmli buffer. A
total of 20 mL samples were run on a 20% SDS-PAGE and
blotted onto Immobilon
P PVDF membrane (Millipore,
0.45 mm pore size). After blocking with 0.5% non-fat dry
milk powder in PBS containing 0.05% Tween 20, membranes
were labeled 1:175 with MAP1LC3B antibody (ABfinityTM
rabbit monoclonal, ThermoFisher Scientific, #700712). Pro-
teins were visualised using the ECL system (Luminata Forte
Western HRP substrate, Millipore, #WBLUF0100). Quantifica-
tion was carried out using ImageJ software by referring the
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relative density of LC3B bands to the untreated sample
relative density. Data are shown adjusted relative density of
these ratios. For loading control Ponceau staining was
applied [46].
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The authors have declared no conflicts of interest.
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