Halogenated Bis(methoxybenzylidene)-4-piperidone Curcuminoids with Improved Anticancer Activity

Article abstract of DOI:10.1002/cmdc.201800135

A series of readily available curcuminoids with a halogenated bis(4-methoxy/4,5-dimethoxybenzylidene)-4-piperidone structure were prepared and analyzed for their cytotoxic impact on eight human cancer cell lines of five different entities. The known 3,4,5

Full text of DOI:10.1002/cmdc.201800135

Accepted Article
Title: Halogenated bis(methoxy-benzylidene)-4-piperidone
curcuminoids with improved anticancer activity
Authors: Florian Schmitt, Dharmalingam Subramaniam, Shrikant
Anant, Subhash Padhye, Gerrit Begemann, Rainer Schobert,
and Bernhard Biersack
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ChemMedChem
10.1002/cmdc.201800135
FULL PAPER
Halogenated bis(methoxy-benzylidene)-4-piperidone
curcuminoids with improved anticancer activity
[
a]
[b]
[b]
[b]
Florian Schmitt, Dharmalingam Subramaniam, Shrikant Anant, Subhash Padhye, Gerrit
[
c]
[a]
[a]
Begemann, Rainer Schobert, Bernhard Biersack*
[
a]
F. Schmitt, Prof. Dr. R. Schobert, Dr. B. Biersack
Department of Chemistry
University of Bayreuth
Universitätsstraße 30, 95440 Bayreuth (Germany)
E-mail: bernhard.biersack@yahoo.de
Prof. Dr. D. Subramaniam, Prof. Dr. S. Anant, Prof. Dr. S. Padhye
University of Kansas Medical Center
[
[
b]
c]
3901 Rainbow Boulevard, Kansas City 66160 (USA)
Prof. Dr. G. Begemann
Developmental Biology
University of Bayreuth
Universitätsstraße 30, 95440 Bayreuth (Germany)
Abstract: A series of readily available curcuminoids 3a-f with
halogenated bis(4-methoxy/4,5-dimethoxybenzylidene)-4-piperidone
structure was prepared and analyzed for their cytotoxic impact on
eight human cancer cell lines of five different entities. The known
demonstrated by its inhibition or down-regulation of NF-κB,[5]
COX-2,[6,12] STAT-3, Akt,[12] and MMP-2[13]. Apart from EF24, a
multiplicity of mono-carbonyl analogs of curcumin were
synthesized and investigated including bis(arylidene) derivatives
of acetone, cycloalkanones, piperidones, and tetrahydro-4H-
(thio)pyran-4-ones.[14-22] Recently, our group reported on several
mono-carbonyl curcumin analogs with different fluoro and
pentafluorothio substituents on the phenyl rings, which showed
3,4,5-trimethoxybenzylidene curcuminoid 2a and the new bis-(3-
bromophenyl)- and bis-(3,5-dibromophenyl)-derivatives 3c and 3d
proved more strongly antiproliferative than the known curcuminoid
EF24 against six of these cell lines. Compounds 2a and 3c caused a
distinct increase of reactive oxygen species, which eventually elicited
apoptosis in 518A2 melanoma cells. 2a arrested 518A2 melanoma
cells in G1 phase of the cell cycle and had no effect on the expression
of prometastatic matrix metalloproteinases MMP-2 and MMP-9,
whereas 3c led to an accumulation of 518A2 cells in G2/M phase and
to a down-regulation of the MMP-2 expression. In addition, 2a and 3c
treatment resulted in significant inhibitzion of colony formation in HCT-
antiproliferative,
antiangiogenic
and
vascular-disruptive
activity.[23] Since curcuminoids with a piperidin-4-one backbone
displayed the highest cytotoxicities against cancer cells, we now
kept this backbone unaltered and concentrated on the
modification of the phenyl rings starting with the 3,4,5-
trimethoxyphenyl motif of the lead compound 2a (Scheme 1). This
3,4,5-trimethoxyphenyl motif was observed in many biologically
active compounds, such as combretastatin A-4, podophyllotoxin
and colchicine,[24-26] as well as in highly active synthetic
curcuminoids.[27-29] Even though compound 2a has been known
116 cells. Both 2a and 3c showed antiangiogenic activity, e.g. by
inhibiting the formation of subintestinal veins (SIV) in zebrafish
embryos. 3c was also well-tolerated by mice and inhibited the growth
of HCT-116 colon cancer xenografts.
for years, it has up to now only been tested against leukemia,
glioblastoma and laryngeal carcinoma cell lines.[
30-32]
In the
present paper, we investigate its antiproliferative effect on cancer
cells of other entities as well as its impact on other cancer-relevant
parameters such as ROS production, cell cycle progression,
angiogenesis, and the expression of matrix-metalloproteinases
MMP-2 and MMP-9. Since our group had previously disclosed 3-
halo/3,5-dihalo-4,5-dimethoxyphenyl-imidazoles with anticancer
Introduction
EF24 was originally synthesized as a mono-carbonyl derivative of
curcumin, a natural polyphenol extracted from the rhizome of the
plant Curcuma longa (Fig. 1). There is ample literature classifying
curcumin as a multi-potent anticancer agent but also controversial
discussions as to whether its application is meaningful and
effective given its poor water solubility, its rapid metabolic
degradation, and its consequently low bioavailability in plasma
and tissue.[ When compared to curcumin, its mono-carbonyl
analog EF24 showed improved metabolic stability and uptake,
causing a distinctly increased bioavailability.[3] Moreover, the anti-
cancer potential of EF24 exceeded that of curcumin by far. EF24
restricts cell proliferation and induces apoptosis via the intrinsic
pathway and as a consequence of an enhanced reactive oxygen
activities exceeding that of combretastatin A-4 in resistant cancer
[33,34]
cell lines
, we also synthesized a series of halogenated
bis(methoxy-benzylidene)-4-piperidones and evaluated the
influence of halogenation on their anticancer activities in
comparison to 2a.
1,2]
species (ROS) production as it was presented for curcumin
Figure 1. Structure of the mono-carbonyl curcumin analog EF24.
before.[
4-9]
In addition, EF24 was shown to reduce the cell
motility[
10]
and to exert antiangiogenic activity.
[11]
Moreover,
several other cancer-relevant targets are addressed by EF24 as
Results and Discussion
1
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10.1002/cmdc.201800135
FULL PAPER
Curcuminoid 2a and the new derivatives 2b-c and 3a-f were
prepared by condensation of 1-ethyl-4-piperidone (1) with the
soluble in EtOH or DMSO at concentrations suitable for this assay.
All tested curcuminoids showed dose-dependent inhibition curves
corresponding benzaldehydes under basic conditions (Scheme 1). with IC50 values ranging from triple-digit nanomolar to single-digit
In most cases (2c, 3a-f) treatment with HCl/dioxane and
crystallisation of the curcuminoids as hydrochloride salts was
necessary in order to obtain the pure target compounds as yellow
solids. The logP values of the compounds (free bases) were
compared with that of EF24 and 2a-c were calculated to be more
hydrophilic than EF24 (Tab. S1, cf. Supplementary Data). In
addition, 3a and 3c revealed logP below 5 and, thus, did not
violate Lipinski rule.
micromolar. In the row of curcuminoids substituted with methoxy
groups only, the known bis-3,4,5-trimethoxyphenyl curcuminoid
2a performed best. The effect of the corresponding 2,4-dimethoxy
substituted analog 2b was distinctly weaker followed by the 2,3,4-
trimethoxy substituted compound 2c which was the least active
compound in this series. The introduction of halogen substituents
at the phenyl rings led to distinct changes in the cytotoxicities of
the new compounds 3a, and 3c-f. The bis-3-chloro-4,5-
dimethoxyphenyl substituted 3a and the bis-3-iodo-4,5-
dimethoxyphenyl compound 3e were slightly less active when
compared to 2a whereas in case of 3f a distinct drop in activity
was observed. The introduction of bromine substituents at the
phenyl rings turned out to have the best effect on the cytotoxicities
of the test compounds 3c and 3d. On average, the bis-3,5-
dibromo-4-methoxyphenyl curcuminoid 3d was about as active as
the lead compound 2a whereas the bis-3-bromo-4,5-
dimethoxyphenyl 3c was more active even when compared to 2a.
It has to be emphasized that the antiproliferative activity of the
bromo derivatives 3c and 3d as well as that of 2a exceeded even
that of the well-investigated curcuminoid EF24.[23]
However, there was no specificity for a certain cancer cell line
except for the multidrug resistant MCF-7Topo breast carcinoma,
being on average less sensitive to the test compounds.
Interestingly, 3f is about eight times less efficacious against MCF-
Topo
7
than against the other cell lines. It can be assumed that 3d
is a substrate of the ABC (ATP binding cassette) efflux transporter
BCRP (breast cancer resistance protein) which is overexpressed
in MCF-7Topo cells.
The structure-activity relationships observed in these MTT assays
were confirmed in hexoseaminidase enzyme assays with SW-480
and HCT-116 colon carcinoma cells which took stock of the time-
dependency of the cytotoxicities (Table 2).[36] Compounds 3a-c
as well as 2a strongly inhibited the growth of HCT-116 cells yet
after 24 h, whereas 2b-c and 3d-f showed a delayed and overall
weaker effect. The SW-480 cancer cell line appeared to be slightly
less sensitive to the active test compounds when compared with
the HCT-116 cancer cell line. After 72 h both cell lines had
responded well to compounds 2a and 3a-c which displayed
excellent IC50 values in the range of 0.2-0.3 µM.
Scheme 1. Synthesis of the curcuminoids 2a-c and their halogenated analogs
2 2
3a-f. Reagents and conditions: a) NaOH, MeOH, rt; b) 4 M HCl/dioxane, CH Cl ,
rt for 2c, 3a-f.
Biological evaluation
Antiproliferative activity.
The antiproliferative activity of compounds 2a-c, 3a, and 3c-f was
evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assays against seven human
[35]
cancer cell lines of five different entities (Table 1). 3b was not
Table 1. Inhibitory concentrations IC50 [µM] of 2a-c, 3a, 3c-f and EF24[b] when applied to cells of human 518A2 melanoma, KB-V1Vbl cervix carcinoma, Panc-1
[a]
pancreatic ductular adenocarcinoma, MCF-7Topo breast carcinoma and HT-29, HCT-116 and DLD-1 colon (adeno-)carcinomas
518A2
HT-29
HCT-116
DLD-1
KB-V1Vbl
MCF-7Topo
Panc-1
2
2
2
3
3
3
3
a
b
c
a
c
d
e
0.65 ± 0.03
1.8 ± 0.2
0.89 ± 0.05
1.9 ± 0.2
0.65 ± 0.03
1.3 ± 0.1
1.0 ± 0.1
2.2 ± 0.2
1.8 ± 0.1
1.1 ± 0.1
0.40 ± 0.05
1.2 ± 0.1
0.70 ± 0.03
1.9 ± 0.1
1.3 ± 0.1
0.61 ± 0.06
1.1 ± 0.1
1.8 ± 0.1
1.3 ± 0.1
0.54 ± 0.06
0.43 ± 0.01
1.0 ± 0.1
3.5 ± 0.3
1.1 ± 0.1
1.6 ± 0.2
2.9 ± 0.2
3.7 ± 0.2
3.2 ± 0.2
0.95 ± 0.05
1.3 ± 0.1
3.0 ± 0.4
22 ± 2
0.90 ± 0.08
2.2 ± 0.1
2.3 ± 0.2
1.8 ± 0.1
0.73 ± 0.05
1.7 ± 0.1
1.3 ± 0.1
2.2 ± 0.1
1.5 ± 0.2
5.7 ± 0.3
3.4 ± 0.1
2.7 ± 0.2
0.83 ± 0.08
0.55 ± 0.03
0.49 ± 0.04
3.6 ± 0.2
1.8 ± 0.2
0.89 ± 0.06
0.78 ± 0.02
0.35 ± 0.02
0.73 ± 0.08
1.8 ± 0.1
0.95 ± 0.03
0.93 ± 0.02
0.62 ± 0.05
5.5 ± 0.3
3
f
3.3 ± 0.4
EF24[b]
1.8 ± 0.2
1.6 ± 0.1
1.5 ± 0.2
2.2 ± 0.2
[
a] Values are the means ± SD (standard deviation) of four independent experiments. They were derived from concentration–response curves obtained by measuring
the percentage of vital cells relative to untreated controls after 72 h using MTT-assay. [b] Values of EF24 were taken from ref. [21].
2
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Even at concentrations as low as 1 µM the relative ROS
production in cells treated with 3c exceeded 150% whereas the
treatment with 2a left the cellular ROS levels unaltered. The rapid
increase of ROS is probably causative for the strong
antiproliferative effects of 2a and 3c after 24 h and possibly also
for the arrest of cells in cell cycle progression and for apoptosis.
[
a]
Table 2. Inhibitory concentrations IC50 [µM] of 2a-c and 3a-f when applied to
cells of human HCT-116 and SW-480 colon carcinomas
HCT-116
SW-480
24 h
48 h
72 h
24 h
48 h
72 h
2
2
2
3
3
3
3
3
a
b
c
a
b
c
d
e
0.5
8.0
>10
0.4
0.4
0.4
1.2
1.0
>10
0.3
4.0
5.0
0.3
0.3
0.3
0.5
0.6
3.8
0.3
3.0
1.0
>10
>10
0.5
1.0
0.7
2.0
2.0
>10
0.4
>10
4.0
0.3
0.4
0.3
0.5
0.5
>10
0.3
4.0
1.0
3.0
0.25
0.25
0.2
0.25
0.25
0.25
0.3
0.3
0.6
0.5
3
f
2.0
4.0
[
a] Values are derived from concentration–response curves obtained by
measuring the percentage of vital cells relative to untreated controls after 24 h,
4
±
8 h, and 72 h using hexoseaminidase assay. Values represent the mean (SD
15%) of two independent experiments.
Figure 2. ROS generation in 518A2 melanoma cells induced by test
compounds 2a and 3c. After the pretreatment of the cells with DCFH-DA (20
µM, 30 min), the cells were incubated with the test compounds 2a and 3c (0, 1,
Analogous tests of 2a and 3c against non-malignant FHC colon
epithelial cells revealed a ca. two-fold lower activity in the FHC
cells (IC50 ca. 0.5 µM after 48 h) when compared with their activity
against HCT-116 and SW-480 colon cancer cells (cf.
Supplementary Data).
5, and 10 µM; 1 h). The green fluorescence of DCF as a measure of the
intracellular ROS level was set to 100% for vehicle treated control cells (0 µM).
The ROS generation (%) is depicted as the mean ± SD of five independent
experiments. Significant deviations from control data were determined using a
t-test. *: p < 0.002.
Effect on intracellular ROS levels.
The compound-induced ROS generation has been recognized as
one of the mechanisms underlying the cytotoxicity of compounds
since the ROS overproduction induces ER (endoplasmic
reticulum) stress and mitochondrial apoptosis.[37] Curcuminoids
such as EF24 are known to affect the ROS production and as a
consequence induce apoptosis in cancer cells.[38] Therefore, we
investigated the effect of 2a and 3c on the intracellular ROS levels
in 518A2 melanoma cells using the DCFH-DA (2’,7’-
dichlorohydrofluorescein diacetate) assay. After cellular uptake,
DCFH-DA is deacetylated by cellular esterases to DCFH (2’,7’-
dichlorohydrofluorescein). Then, DCFH is oxidized by ROS to the
Cell cycle analysis.
The effect of the test compounds 2a (800 nM) and 3c (600 nM)
on the cell cycle progression in 518A2 melanoma cells was
assessed after staining the DNA with propidium iodide (PI) by
using flow cytometry (Fig. 3; Table 3). The treatment of the cells
with bis-3,4,5-trimethoxyphenyl derivative 2a caused a distinct
increase of cells in G1 phase, whereas the percentage of cells in
S and G2/M phase was reduced. The change in the proportion of
apoptotic sub-G1 cells was negligible. In contrast to that,
compound 3c caused a significant accumulation of cells in G2/M
phase of the cell cycle. The percentages of cells in G1 and S were
decreased whereas the treatment with 3c gave rise to an increase
of apoptotic sub-G1 events.
fluorescent
DCF
(2’,7’-dichlorofluorescein).
Hence,
its
fluorescence intensity is proportional to the intracellular ROS
levels. Both test compounds, 2a and 3c, caused an increase of
the cellular ROS levels exceeding 200% at 10 µM compared to
that of untreated control cells which was set to 100% (Fig. 2).
Figure 3. Effect of 2a (800 nM), 3c (600 nM) and control (DMSO) on the cell cycle progression of 518A2 melanoma cells after 24 h exposure. The cell cycle profiles
are representative of three independent experiments obtained by flow cytometry after PI staining.
3
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the treated cells were allowed to grow in normal medium for 10
days in order to grow colonies. Both 2a and 3c strongly
suppressed colony formation in colon cancer cells (Fig. 5). These
results suggest that the anticancer effects of 2a and 3c are long-
lasting and irreversible.
Table 3. Effect of 2a (800 nM) and 3c (600 nM) on the cell-cycle progression of
518A2 melanoma cells. Percentages of cells in G1, S and G2/M phase of the
cell cycle as well as the proportion of apoptotic sub-G1 cells as obtained by flow
cytometry after DNA staining with PI are depicted as the means ± SD of three
independent experiments. Control: DMSO.
sub-G1
5.9 ± 1.4
6.2 ± 0.9
11.7 ± 2.4
G1
S
G2/M
control
42.2 ± 1.2
49.4 ± 2.2
31.7 ± 4.2
27.1 ± 0.6
22.5 ± 0.1
22.5 ± 1.8
24.8 ± 1.9
21.9 ± 1.2
34.1 ± 1.9
2
a
c
3
Effects on matrix metalloproteinases.
Matrix metalloproteinases (MMPs) are zinc-dependent
endopeptidases which are expressed and secreted at high
concentrations especially by invasive tumor cells and vascular
endothelial cells.[39,40] They mediate the proteolytic degradation of
the extracellular matrix (ECM) so that endothelial or tumor cells
can egress from or invade into tissues which is a requirement for
tumor angiogenesis or metastasis.[40,41] The essential role of
MMPs in tumor progression is widely recognized so that MMPs
are a promising anticancer target. Since gelatin is a substrate of
MMP-2 and MMP-9, the test compounds’ effects on expression
and secretion of them in 518A2 melanoma cells could simply be
tested by gelatin zymography.[42] Since it is known that
curcuminoids such as EF24[13], Y20[43] and curcumin[44] (cf.
Supplementary Data) inhibit the production of MMP-2, the effect
of 2a and 3c on the intracellular and extracellular levels of MMP-
Figure 5. Compounds 2a and 3c inhibit colony formation. HCT-116 colon
cancer cells were incubated with 0.5 µM 2a or 3c for 48 h and allowed to grow
and form colonies for 10 days. Control probes were treated with DMSO. Results
are representative of three independent experiments.
2
in 518A2 melanoma cells was tested (Fig. 4, cf. Supplementary
Data). Both test compounds 2a and 3c had virtually no effect on
the cellular and extracellular levels of MMP-9. Moreover, 2a did
not alter these levels for MMP-2, either. In contrast, 3c
significantly reduced the levels of MMP-2 in cell medium and
lysate, so that it can be assumed that the treatment with 3c
reduces the expression of MMP-2.
Antiangiogenic effect.
The zebrafish (Danio rerio) is a commonly used vertebrate model
to study the effect of test substances on angiogenesis since its
circulatory system and its development is quite similar to that of
mammals. The development of the subintestinal veins (SIVs) was
used as a marker for angiogenesis.[45] 48 hours past fertilization
(
hpf) they begin to develop from the common cardinal veins and
[46]
form a network across the yolk of the embryos. At a later stage,
the SIVs provide the blood supply for the digestive system.
Embryos of the line Tg(fli1a:EGFP) crossed into a background of
the unpigmented casper mutant, were used to monitor
fluorescence of the EGFP-positive blood vessels (EGFP,
enhanced green fluorescence protein).[47,48] The area covered by
SIVs of at least 17 identically treated embryos was then quantified
and compared with that of vehicle treated control embryos, which
was set to 100% SIV area (Fig. 6). Both, 2a and 3c showed a
significant reduction of the SIV area to 74% ± 13 and 81% ± 17,
respectively.
Figure 4. Effect of 2a and 3c (0, 0.25, 0.5 and 1 µM) on the cell-associated and
secreted activities of MMP-2 and MMP-9 in 518A2 melanoma cells after 24 h
exposure. Cell lysate and media probes were subjected to gelatin zymography.
Control (DMSO) treated probes are listed as 0 µM. Images are representative
of two independent experiments (negative images of the gelatin gels).
Effects on colony formation of cancer cells.
In order to evaluate the long-term effects of 2a and 3c, HCT-116
colon cancer cells were incubated with 2a or 3c for 48 h. Then,
4
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ChemMedChem
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and P-gp (P-glycoprotein), respectively. The 3-iodo and 3,5-
diiodo substituted curcuminoids 3e and 3f were significantly less
active, possibly because of the large iodo substituents posing a
steric hindrance. The cytotoxicity of 2a and 3c is probably a
consequence of them increasing the intracellular ROS
concentration, which eventually causes apoptosis induction.
Compound 3c led to a rise of the intracellular ROS levels at
distinctly lower concentrations compared to the parent compound
2a. Both curcuminoids, 2a and 3c, showed significant
antiangiogenic activity in vivo in zebrafish embryos, yet only 3c
inhibited the expression of the angiogenesis- and motility-relevant
matrix metalloproteinase MMP-2 in 518A2 melanoma cells. The
combination of its antiangiogenic and anti-migratory effects
highlights the great potential of 3c as a new anticancer drug
candidate, which is underlined by the strong inhibition of
colorectal tumors in mice by non-toxic doses of 3c.
Figure 6. Antiangiogenic effect of test compound 2a and 3c in zebrafish
embryos. Dechorionated 24 hpf old transgenic Tg(fli1a:EGFP) zebrafish with a
casper background were treated with 5 µM of the test compounds or vehicle
(DMSO) for 48 h at 28 °C in embryo medium. A) Images are representative of
at least 17 identically treated embryos (6.3-fold magnification). B) The area
covered by subintestinal veins (SIV) was quantified by using the imageJ
software. Values are the means ± SD of at least 17 independent experiments
with the SIV area of vehicle (DMSO) treated embryos set to 100%. Significant
deviations from the control data were determined using a t-test. *: p < 0.01.
Inhibition of HCT-116 tumor xenograft growth.
To evaluate the effect of 3c on tumor growth in vivo, we next
examined its effects on growth of HCT116 induced xenografts.
Colon cancer xenografts were allowed to develop and grow for
one week before 3c was administered intraperitoneally daily for
three weeks. 3c inhibited the growth of tumor xenografts, (Fig.
7B). The excised tumors from control animals weighed ~1200 mg,
those treated with 3c weighed ~250 mg (Fig. 7C&D). In addition,
tumor volume was significantly decreased (Fig. 7B). There was
no apparent change in liver, spleen, or body weight in the animals
(
data not shown). The treated mice were apparently in relatively
good condition (cf. Supplementary Data). These in vivo results
demonstrate that 3c is a potential therapeutic agent for treating
colon cancer while being relatively non-toxic to the animals.
Conclusions
Figure 7. 3c inhibits HCT116 induced tumor xenograft growth. (A) Experimental plan,
(B) HCT116 cells were injected in to the flanks of nude mice and palpable tumors were
allowed to develop for 7 days. Subsequently, 3c (2 mg/kg bw) was injected daily
intraperitoneally every day for 21 days. Tumor size was measured every week. On day
A
series
of
halogenated
bis(4-methoxy/4,5-
dimethoxybenzylidene)-4-piperidones 3a-f was synthesized as
new derivatives of the known trimethoxy curcuminoid 2a. The
antitumoral properties of compound 2a which was up to now only
tested in leukemia and glioblastoma cells lines, were investigated
in more detail. The effect of halogenation on their anticancer
effects was evaluated for 3a-f. Compound 2a as well as the 3-
bromo compound 3c and the 3,5-dibromo analog 3d, showed
superior antiproliferative activities against seven cancer cell lines
when compared to the extensively investigated curcuminoid EF24.
The new compound 3c in particular exhibited a greater anti-
proliferative effect than 2a on five of the seven cancer cell lines
including the multi-drug resistant cancer cell lines MCF-7Topo and
KB-V1Vbl which overexpress the ABC efflux transporters BCRP
2
2, tumors were excised and subject to further analyses. Tumor volumes of 3c treated
mice were smaller when compared to control (*p<0.05). C and D. 3c treatment
significantly reduced the tumor weight and size when compared to control.
Experimental Section
Chemistry
General: Starting materials and reagents were purchased from Sigma-
Aldrich. The known compounds EF24 and 2a were synthesized according
to literature procedures.[49,50] The following instruments were used: melting
points (uncorrected), Gallenkamp; IR spectra, Perkin-Elmer Spectrum One
5
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FT-IR spectrophotometer with ATR sampling unit; nuclear magnetic
resonance spectra, BRUKER Avance 300 spectrometer; chemical shifts
are given in parts per million (d) downfield from tetramethylsilane as
internal standard; mass spectra, Varian MAT 311A (EI); microanalyses,
Perkin-Elmer 2400 CHN elemental analyzer. All tested compounds were
precipitate was dissolved in CH
added and the reaction mixture was stirred at room temperature for 5 min.
n-Hexane was added and the formed precipitate was collected, washed
2 2
with n-hexane and dried in vacuum. Recrystallization from CH Cl /n-
2
Cl
2
(5 mL), 4 M HCl/dioxane (0.5 mL) was
hexane gave compound 3b as a yellow solid (130 mg, 32%): mp: 226–
1
>
95% pure by elemental analysis.
228°C; H NMR (300 MHz, CDCl
3
): δ=1.22 (t, J=7.1 Hz, 3H), 3.12 (q, J=7.1
Hz, 2H), 3.92 (s, 6H), 3.93 (s, 6H), 4.4-4.7 (m, 4H), 7.3-7.4 (m, 4H), 7.93
ppm (s, 2H); 13C NMR (75.5 MHz, CDCl
): δ=9.4, 48.2, 49.8, 60.7, 124.8,
30.1, 130.3, 140.8, 153.8, 180.4 ppm; IR(ATR): ν=2938, 2871, 2281,
3
Synthesis
dimethoxybenzylidene)-4-piperidone (2b). 1-Ethyl-4-piperidone (95 mg,
.75 mmol) was dissolved in MeOH (5 mL) and 2,4-
dimethoxybenzaldehyde (249 mg, 1.5 mmol) was added. NaOH (40 mg, 1
mmol) and H O (1 mL) were added and the reaction mixture was stirred at
of compounds
2b-c,
3a-f:
(E)-1-Ethyl-3,5-bis(2,4-
1
1
1
7
4
683, 1617, 1594, 1542, 1474, 1424, 1407, 1358, 1269, 1254, 1219, 1191,
147, 1121, 1085, 1042, 1005, 978, 956, 938, 925, 906, 873, 864, 806,
0
-
1
+
80, 766, 745, 729 cm ; MS (EI, 70 eV) m/z (%): 503 (50) [M ], 501 (100),
2
99 (80), 488 (20), 486 (45), 484 (33).
room temperature for 16 h. The formed precipitate was collected, washed
with MeOH and dried in vacuum which gave compound 2b as a yellow
solid (120 mg, 37%): mp: 127–128°C; 1H NMR (300 MHz, [D
]DMSO):
δ=0.93 (t, J=7.1 Hz, 3H,), 2.4-2.5 (m, 2H), 3.65 (s, 4H), 3.82 (s, 6H), 3.85
(E)-1-Ethyl-3,5-bis(3-bromo-4,5-dimethoxybenzylidene)-4-piperidone
HCl (3c). 1-Ethyl-4-piperidone (95 mg, 0.75 mmol) was dissolved in MeOH
x
6
(
(
1
2
1
7
s, 6H), 6.6-6.7 (m, 4H), 7.24 (d, J=8.3 Hz, 2H), 7.80 ppm (s, 2H); 13C NMR
75.5 MHz, [D ]DMSO): δ=12.2, 50.5, 54.1, 55.4, 55.7, 98.4, 105.3, 116.1,
29.8, 131.2, 131.4, 159.6, 161.8, 186.4 ppm; IR(ATR): ν 2971, 2938,
837, 2747, 1664, 1597, 1568, 1498, 1454, 1437, 1417, 1308, 1279, 1252,
(5 mL) and 3-bromo-4,5-dimethoxybenzaldehyde (368 mg, 1.5 mmol) was
added. NaOH (40 mg, 1 mmol) and H O (1 mL) were added and the
2
reaction mixture was stirred at room temperature for 3 h. The formed
precipitate was collected, washed with MeOH and dried in vacuum. The
2 2
precipitate was dissolved in CH Cl (5 mL), 4 M HCl/dioxane (0.5 mL) was
6
=
-
1
207, 1160, 1123, 1094, 1027, 990, 938, 918, 889, 822, 772 cm ; MS (EI,
+
0 eV) m/z (%): 423 (92) [M ], 395 (77), 393 (100), 218 (52), 161 (54).
added and the reaction mixture was stirred at room temperature for 5 min.
n-Hexane was added and the formed precipitate was collected, washed
2 2
with n-hexane and dried in vacuum. Recrystallization from CH Cl /n-
hexane gave compound 3c as a yellow solid (150 mg, 32%); mp: 197–
(
1
E)-1-Ethyl-3,5-bis(2,3,4-trimethoxybenzylidene)-4-piperidone x HCl (2c).
-Ethyl-4-piperidone (95 mg, 0.75 mmol) was dissolved in MeOH (5 mL)
and 2,3,4-trimethoxybenzaldehyde (294 mg, 1.5 mmol) was added. NaOH
40 mg, 1 mmol) and H O (3 mL) were added and the reaction mixture was
1
1
6
7
1
98°C; H NMR (300 MHz, [D
6
]DMSO): δ=1.24 (t, J=7.2 Hz, 3H), 3.81 (s,
H), 3.91 (s, 6H), 3.3-3.5 (m, 2H), 4.63 (s, 4H), 7.27 (s, 2H), 7.40 (s, 2H),
.85 ppm (s, 2H); 13C NMR (75.5 MHz, [D
]DMSO): δ=9.0, 50.4, 56.4, 60.3,
15.2, 117.1, 126.2, 130.8, 138.5, 146.1, 153.3, 181.4 ppm; IR(ATR):
(
2
stirred at room temperature for 3 h. The formed precipitate was collected,
washed with MeOH and dried in vacuum. The precipitate was dissolved in
CH Cl (5 mL), 4 M HCl/dioxane (0.5 mL) was added and the reaction
2 2
mixture was stirred at room temperature for 5 min. n-Hexane was added
and the formed precipitate was collected, washed with n-hexane and dried
in vacuum. Recrystallization from CH
6
ν=2937, 2831, 2362, 1679, 1610, 1588, 1552, 1487, 1464, 1428, 1412,
-
1
1
314, 1286, 1252, 1201, 1143, 1044, 997, 937, 854, 819, 768, 729 cm ;
+
MS (EI, 70 eV) m/z (%): 583 (52) [M ], 581 (100), 579 (50), 568 (22), 566
(43), 564 (23).
2
Cl
2
/n-hexane gave compound 2c as
1
a yellow solid (150 mg, 39%): mp: 152–154°C; H NMR (300 MHz, CDCl
3
):
δ=1.12 (t, J=7.1 Hz, 3H), 2.9-3.0 (m, 2H), 3.85 (s, 6H), 3.88 (s, 6H), 3.89
s, 6H), 4.3-4.5 (m, 4H), 6.69 (d, J=8.7 Hz, 2H), 6.83 (d, J=8.7 Hz, 2H),
(E)-1-Ethyl-3,5-bis(3,5-dibromo-4-methoxybenzylidene)-4-piperidone
HCl (3d). 1-Ethyl-4-piperidone (95 mg, 0.75 mmol) was dissolved in MeOH
(5 mL) and 3,5-dibromo-4-methoxybenzaldehyde (441 mg, 1.5 mmol) was
2
added. NaOH (40 mg, 1 mmol) and H O (1 mL) were added and the
reaction mixture was stirred at room temperature for 3 h. The formed
precipitate was collected, washed with MeOH and dried in vacuum. The
2 2
precipitate was dissolved in CH Cl (5 mL), 4 M HCl/dioxane (0.5 mL) was
x
(
8
4
1
1
9
.27 (s, 2H), 13.0-13.1 ppm (m, 1H); 13C NMR (75.5 MHz, CDCl
3
): δ=9.8,
6.2, 50.3, 56.1, 60.9, 61.5, 107.3, 119.9, 122.5, 125.4, 140.3, 142.3,
53.4, 156.4, 181.6 ppm; IR(ATR): ν=3396, 2941, 2846, 2445, 1664, 1588,
493, 1459, 1446, 1415, 1303, 1281, 1230, 1200, 1091, 1051, 1015, 958,
-
1
38, 928, 883, 820, 797, 731, 693, 667 cm ; MS (EI), 70 eV) m/z (%): 483
+
(88) [M ], 452 (100).
added and the reaction mixture was stirred at room temperature for 5 min.
n-Hexane was added and the formed precipitate was collected, washed
2 2
with n-hexane and dried in vacuum. Recrystallization from CH Cl /n-
(
E)-1-Ethyl-3,5-bis(3-chloro-4,5-dimethoxybenzylidene)-4-piperidone
x
hexane gave compound 3d as a yellow solid (200 mgl, 37%): mp: 198–
HCl (3a). 1-Ethyl-4-piperidone (95 mg, 0.75 mmol) was dissolved in MeOH
5 mL) and 3-chloro-4,5-dimethoxybenzaldehyde (301 mg, 1.5 mmol) was
added. NaOH (40 mg, 1 mmol) and H O (1 mL) were added and the
reaction mixture was stirred at room temperature for 1 h. The formed
precipitate was collected, washed with MeOH and dried in vacuum. The
precipitate was dissolved in CH Cl (5 mL), 4M HCl/dioxane (0.5 mL) was
2 2
added and the reaction mixture was stirred at room temperature for 5 min.
n-Hexane was added and the formed precipitate was collected, washed
1
1
99°C; H NMR (300 MHz, CDCl
3
): δ=1.26 (t, J=7.2 Hz, 3H), 3.0-3.1 (m,
(
2
H), 3.92 (s, 6H), 4.3-4.7 (m, 4H), 7.48 (s, 4H), 7.96 ppm (s, 2H); 13C NMR
): δ=9.9, 47.5, 49.7, 60.9, 119.2, 124.8, 131.3, 134.0,
2
(75.5 MHz, CDCl
3
1
1
8
41.2, 156.1, 180.6 ppm; IR(ATR): ν=3455, 2928, 2864, 2383, 1679, 1611,
529, 1469, 1420, 1394, 1252, 1189, 1120, 1067, 1035, 990, 945, 925,
-
1
90, 872, 808, 785, 769, 740, 724 cm ; MS (EI, 70 eV) m/z (%): 681 (64)
+
[M ], 679 (100), 677 (72), 667 (22), 665 (31), 663 (22).
2 2
with n-hexane and dried in vacuum. Recrystallization from CH Cl /n-
hexane gave compound 3a as a yellow solid (120 mg, 31%): mp: 124–
(E)-1-Ethyl-3,5-bis(3-iodo-4,5-dimethoxybenzylidene)-4-piperidone x HCl
(3e). 1-Ethyl-4-piperidone (64 mg, 0.51 mmol) was dissolved in MeOH (5
mL) and 3-iodo-4,5-dimethoxybenzaldehyde (297 mg, 1.02 mmol) was
added. NaOH (40 mg, 1 mmol) and H O (1 mL) were added and the
2
reaction mixture was stirred at room temperature for 3 h. The formed
precipitate was collected, washed with MeOH and dried in vacuum. The
1
126°C; H NMR (300 MHz, [D
6
]DMSO): δ=1.25 (t, J=7.2 Hz, 3H), 3.3-3.5
(
(
1
m, 2H), 3.83 (s, 6H), 3.91 (s, 6H), 4.64 (s, 4H), 7.2-7.3 (m, 4H), 7.84 ppm
s, 2H); 13C NMR (75.5 MHz, [D
]DMSO): δ=9.0, 50.2, 56.5, 60.4, 114.6,
23.4, 127.1, 127.3, 130.1, 138.6, 146.0, 153.5, 181.5 ppm; IR(ATR):
ν=2940, 2836, 2476, 1677, 1611, 1560, 1490, 1463, 1429, 1413, 1315,
6
-
1
1
289, 1253, 1204, 1145, 1049, 995, 942, 852, 810, 770, 750, 730 cm ;
2 2
precipitate was dissolved in CH Cl (5 mL), 4 M HCl/dioxane (0.5 mL) was
added and the reaction mixture was stirred at room temperature for 5 min.
+
MS (EI, 70 eV) m/z (%): 493 (63) [M ], 491 (100), 478 (32), 476 (52).
n-Hexane was added and the formed precipitate was collected, washed
2 2
with n-hexane and dried in vacuum. Recrystallization from CH Cl /n-
hexane gave compound 3e as a yellow solid (120 mg, 33%): mp: 206–
(
E)-1-Ethyl-3,5-bis(3,5-dichloro-4-methoxybenzylidene)-4-piperidone
HCl (3b). 1-Ethyl-4-piperidone (95 mg, 0.75 mmol) was dissolved in MeOH
5 mL) and 3,5-dichloro-4-methoxybenzaldehyde (308 mg, 1.5 mmol) was
added. NaOH (40 mg, 1 mmol) and H O (1 mL) were added and the
x
1
2
2
2
1
08°C; H NMR (300 MHz, CDCl
3
): δ=1.25 (t, J=7.2 Hz, 3H), 3.0-3.1 (m,
(
H), 3.89 (s, 12H), 4.4-4.6 (m, 4H), 6.82 (s, 2H), 7.31 (s, 2H), 8.03 ppm (s,
H); 13C NMR (75.5 MHz, CDCl
): δ=10.0, 47.3, 49.9, 56.3, 60.7, 93.1,
14.4, 123.4, 130.7, 132.4, 143.0, 151.1, 152.6, 181.0 ppm; IR(ATR):
2
3
reaction mixture was stirred at room temperature for 1 h. The formed
precipitate was collected, washed with MeOH and dried in vacuum. The
6
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201800135
FULL PAPER
ν=3395, 2935, 2863, 2831, 1356, 1678, 1608, 1583, 1545, 1478, 1464,
serum-free DMEM was added. The cells were treated with various
concentrations of 2a and 3c (0, 1, 5, and 10 µM) for 1 h at 37 °C. Then,
the cells were washed twice with 100 µL PBS and the plate immediately
placed in a microplate reader (TECAN). The DCF fluorescence (λex = 485
nm; λem = 535 nm) as a measure of intracellular ROS levels was
determined and that of untreated control cells was set to 100%. Values
exceeding 100% indicate increased ROS concentrations in the cells. The
ROS generation (%) was depicted as the mean ± SD of five independent
experiments.
1
425, 1409, 1309, 1282, 1249, 1200, 1142, 1041, 997, 939, 521, 799, 433,
-
1
+
728, 699, 661 cm ; MS (EI, 70 eV) m/z (%): 675 (100) [M ], 660 (35).
(
(
E)-1-Ethyl-3,5-bis(3,5-diiodo-4-methoxybenzylidene)-4-piperidone x HCl
3f). 1-Ethyl-4-piperidone (66 mg, 0.52 mmol) was dissolved in MeOH (5
mL) and 3,5-diiodo-4-methoxybenzaldehyde (400 mg, 1.03 mmol) was
added. NaOH (40 mg, 1 mmol) and H O (1 mL) were added and the
2
reaction mixture was stirred at room temperature for 3 h. The formed
precipitate was collected, washed with MeOH and dried in vacuum. The
precipitate was dissolved in CH
added and the reaction mixture was stirred at room temperature for 5 min.
n-Hexane was added and the formed precipitate was collected, washed
2 2
with n-hexane and dried in vacuum. Recrystallization from CH Cl /n-
2
Cl
2
(5 mL), 4 M HCl/dioxane (0.5 mL) was
Cell cycle analysis: 518A2 melanoma cells (5 × 104 cells/mL, 3 mL/well)
were seeded in 6-well plates and incubated for 24 h. Then, the cells were
treated with 2a (800 nM), 3c (600 nM), or vehicle (DMSO) for 24 h. After
harvesting the cells by trypsination, the cells were fixed with 70% EtOH in
water for at least 24 h at 4 °C. Then, cells were pelleted (300 × g, 5 min,
4 °C), washed with PBS, and stained with PI staining solution (50 µg/mL
propidium iodide, 0.1% sodium citrate, 50 µg/mL RNase A in PBS) for 30
min at 37 °C. The fluorescence intensity of 10,000 single cells was
recorded with a Beckmann Coulter Cytomics FC 500 flow cytometer at λem
= 630 nm, and λex = 488 nm. The percentages of cells in G1, S, and G2/M
phase as well as the proportion of apoptotic cells in sub-G1 were analyzed
by using the CXP analysis software (Beckmann Coulter).
hexane gave compound 3f as a yellow solid (140 mg, 31%): mp: 193–
1
1
94°C; H NMR (300 MHz, [D
6
]DMSO): δ=1.21 (t, J=7.2 Hz, 3H), 3.2-3.4
(
m, 2H), 3.80 (s, 6H), 4.5-4.6 (m, 4H), 7.76 (s, 2H), 7.99 (s, 4H), 11.5-11.6
ppm (m, 1H); 13C NMR (75.5 MHz, [D
]DMSO): δ=9.1, 49.9, 60.4, 66.3,
2.1, 127.8, 133.5, 136.8, 141.1, 159.6, 181.4 ppm; IR(ATR): ν=2935,
323, 1683, 1622, 1593, 1518, 1459, 1412, 1378, 1252, 1187, 1145, 1119,
6
9
2
1
-1
058, 1035, 987, 968, 940, 887, 869, 805, 782, 765, 725, 705, 655 cm ;
+
MS (EI, 70 eV) m/z (%): 867 (100) [M ], 852 (20), 142 (69).
5
Biological evaluation
Gelatin zymography: 518A2 melanoma cells (1 × 10 cells/mL, 3 mL/well)
were grown in 6-well plates for 24 h at 37 °C. Then, the medium was
aspirated, exchanged for 1.5 mL of cell medium supplemented with 0.1%
BSA (bovine serum albumin) and 200 KIU/mL aprotinin and treated with
2a and 3c (0, 0.25, 0.5, and 1.0 µM) for 24 h. After collection of the cell
medium containing the secreted proteins, the cells were washed with PBS,
and collected by scraping them off in 500 µL lysis buffer (0.1 M Tris-HCl,
Cell culture conditions: 518A2 (Department of Radiotherapy, Medical
University of Vienna, Austria) melanoma, Panc-1 (ACC-783) pancreatic
ductular adenocarcinoma, KB-V1Vbl (ACC-149) cervix carcinoma, MCF-
7Topo (ACC-115) breast carcinoma, HT-29 (ACC-299), HCT-116 (ACC-
581), DLD-1 (ACC-278), SW-480 (CCL-228) colon carcinoma cells were
0.2% triton X-100, 200 KIU/mL aprotinin, pH 7.4). For complete lysis cells
cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented
were first vortexed and then incubated for 20 min on ice. After
centrifugation (25,000 × g, 20 min, 4 °C) the supernatant of medium and
cell lysate probes were collected. Equal amount of total protein was
subjected to 10% SDS-PAGE co-polymerized with 0.1 mg/mL gelatin.
Then, the gels were washed with washing buffer I (50 mM Tris-HCl, 2%
triton X-100, pH 7.4) and washing buffer II (50 mM Tris-HCl, pH 7.4) to
exchange SDS for triton X-100 so that the partial refolding of proteins was
achieved. After incubating the gel for 20 h at 37 °C in MMP assay buffer
with 10% fetal bovine serum, and 1% antibiotic-antimycotic at 37 °C, 5%
, and 95% humidity. To keep MCF-7Topo and KB-V1Vbl cells resistant,
CO
2
the maximum-tolerated dose of topotecan or vinblastine were respectively
added to the cell culture medium 24 h after every passage. Non-malignant
colon epithelial cells (FHC, CRL-1831) were grown in Ham’s F12 medium
(45%), DMEM (45%), 25 mM HEPES, 10 ng/mL cholera toxin, 0.005
mg/mL insulin, 0.005 mg/mL transferrin, 100 ng/mL hydrocortisone, 10%
fetal bovine serum (Sigma Aldrich, St. Louis, MO) and 1% antibiotic-
antimycotic solution (Corning, Manassas, VA) at 37 °C in a humidified
2
(50 mM Tris-HCl, 1% triton X-100, 5 mM CaCl , pH 7.4), the gels were
stained with Coomassie blue and destained until the relevant bands
became visible.
2
atmosphere of 5% CO . Only mycoplasma-free cell cultures were used.
MTT assay: The assay was performed as described previously.[35] Briefly,
cells (5 × 10 cells/mL, 100 µL/well) were grown in 96-well plates for 24 h.
4
Colony formation assay: Briefly, six-well dishes were seeded with 500
HCT-116 cells per well and treated with 0.5 µM of 2a or 3c in 10% fetal
bovine serum containing DMEM for 48 h. The compound-containing
medium was removed and the cells were incubated for additional 10 days
in complete medium to allow colonies to form. The colonies were fixed in
formalin followed by staining with crystal violet. Experiments were done in
triplicate.
Then, they were treated with various concentrations of the test compounds,
or vehicle (DMSO, or EtOH) for 72 h at 37 °C. After the addition of 12.5 µL
of a 0.5% MTT solution in PBS the cells were incubated for 2 h at 37 °C
so that the water-soluble MTT could be converted to formazan crystals.
Then, the plates were centrifuged (300 × g, 5 min, 4 °C), the medium
withdrawn, and the formazan dissolved in 25 µL of DMSO containing 10%
SDS and 0.6% acetic acid for at least 2 h at 37 °C. The absorbance of
formazan (λ = 570 nm), and background (λ = 630 nm) was measured with
a microplate reader (Tecan). The IC50 values were derived from dose-
inhibition curves as the means ± SD of four independent experiments with
respect to vehicle treated control cells set to 100%.
Zebrafish angiogenesis assay: The assay was performed as previously
described.[51] Transgenic animals of the strain Tg(fli1a:EGFP) in t casper-
mutant background were raised under standard conditions at 27-
28 °C.[47,48] 24-26 hpf the embryos were manually dechorionated,
transferred into 6-well plates [5 embryos/well in 5 mL E3 medium (5 mM
2 4
NaCl, 0.17 mM KCl, 0.33 mM CaCl , 0.33 mM MgSO , 0.01% methylene
Hexoseaminidase enzyme assay: To assess proliferation, cells were
seeded onto 96-well plates at a density of 5,000 cells per well and allowed
to adhere and grow overnight in 10% heat-inactivated FBS containing
DMEM. The cells were then treated with increasing doses of the test
compounds in 10% FBS containing DMEM. Analysis of cell proliferation
was performed by enzymatic assay as described previously.[36]
blue, pH 7.2)] and treated with 2a (5 µM), 3c (5 µM), or vehicle (DMSO)
for 48 h. The development of the SIV was documented by fluorescence
microscopy (λem = 509 nm, λex = 488 nm; Leica MZ10F with a ZEISS
AxioCam Mrc and the Mrc-ZEN pro 2012 software). The SIV length of at
least 17 identically treated embryos was quantified with the ImageJ
software and depicted as the mean ± SD with vehicle treated controls set
to 100%. Significant deviations from the control data were determined
using a t-test.
5
DFCH-DA assay: 518A2 melanoma cells (10 cells/mL, 50 µL/well) were
grown in a black half-area 96-well plate for 24 h. Then, the medium was
exchanged for serum-free DMEM supplemented with 20 µM DCFH-DA
Induction of HCT-116 xenografts in mice: Five-week-old male athymic
nude mice purchased from Charles River Laboratory were utilized for in
(2’,7’-dichlorohydrofluorescein diacetate, Sigma Aldrich) and incubated for
30 min at 37 °C. After washing the cells twice with 100 µL PBS, 100 µL of
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201800135
FULL PAPER
vivo experiments. They were maintained with water and standard mouse
chow ad libidum and used in protocols approved by the University of
Kansas Animal Studies Committee. Animals were injected with 1x106
HCT116 cells in the left and right flank and allowed to form xenograft. One
week following implantation, and after observing the presence of a
palpable tumor, 3c (2 mg/kg body weight mixed with DMSO and 0.5%
hydroxypropyl methyl cellulose with 0.1% Tween 80) was administered
intraperitoneally daily for 21 d. Tumors were measured weekly. At the end
of treatment the animals were euthanized, and the tumors were removed
and weighed.
[14] B. K. Adams, E. M. Ferstl, M. C. Davis, M. Herold, S. Kurtkaya, R. F.
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P. Snyder, D. C. Liotta, M. Shoji, Bioorg. Med. Chem. 2004, 12, 3871–
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R. Ee, Eur. J. Med. Chem. 2014, 71, 67–80.
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Molina, N. Ni, B. Wang, D. S. Rawat, Bioorg. Med. Chem. Lett. 2013, 23,
All values are expressed as the mean ± SEM. Data was analyzed using
an unpaired 2-tailed t test. A P value of less than 0.05 was considered
statistically significant.
1
12–116.
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[
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Acknowledgements
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Malhotra, D. S. Rawat, Med. Chem. Commun. 2014, 5, 576–586.
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This work was financially supported by grant 2014-2668-SI-01/1
from Bayern Innovativ, Gesellschaft für Innovation und
Wissenschaft mbH (B. Biersack) and by National Institute of
Health Grant R01CA190291 and Thomas O’Sullivan Foundation
23] F. Schmitt, M. Gold, G. Begemann, I. C. Andronache, B. Biersack, R.
Schobert, Bioorg. Med. Chem. 2017, 25, 4894–4903.
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Conflict of Interest
27] B. Yadav, S. Taurin, R. J. Rosengren, M. Schumacher, M. Diederich, T.
J. Somers-Edgar, L. Larsen, Bioorg. Med. Chem. 2010, 18, 6701–6707.
The authors declare no conflict of interest.
[28] S. Das, U. Das, D. Michel, D. K. J. Gorecki, J. R. Dimmock, Eur. J. Med.
Chem. 2013, 64, 321–328.
[
29] F. A. M. Al-Omary, G. S. Hassan, S. M. El-Messery, H. I. El-Subbagh,
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Keywords: piperidone
•
halogenation
•
curcuminoids
•
angiogenesis • anticancer agents
[
30] D. M. Lonard, L. Wang, B. W. O’Mailley, J. Xu, Y. Song, X. Li, T. G.
Palzkill (Baylor College of Medicine, Houston, TX, USA), Int. PCT Pub.
No. WO 2016/109470 A1, 2016.
References:
[
[
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Entry for the Table of Contents
The curcumin halo effect: The new 3-bromo-4,5-dimethoxyphenyl curcuminoid 3c showed higher antiproliferative activity than the
known fluoro-curcuminoid EF24. 3c revealed great potential as promising anticancer drug candidate due to its superior antiproliferative,
ROS-inducing, and anti-invasive activity as well as its strong tumor growth inhibitory activity in HCT-116 mouse xenografts.
1
0
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