Synthesis and cytotoxic evaluation of monocarbonyl curcuminoids and their pyrazoline derivatives

Article abstract of DOI:10.1007/s00706-019-02516-1

Abstract: A small set of structurally different monocarbonyl curcuminoids was prepared and screened for cytotoxic activity. In particular, bis-3-methoxy-4-hydroxy- and bis-4-methoxyphenyl-substituted monocarbonyls were synthesized and transformed into the corresponding three-dimensional N-acetylpyrazoline derivatives. In addition, a non-symmetrical indole-based monocarbonyl curcumin was prepared as well. Preliminary cytotoxic evaluation revealed significant effects for 4-hydroxy (pyrazoline) monocarbonyl curcuminoids, whereas the non-phenolic variants displayed rather poor activity. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-019-02516-1

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02516-1
ORIGINAL PAPER
Synthesis and cytotoxic evaluation of monocarbonyl curcuminoids
and their pyrazoline derivatives
Tim Van de Walle1 · Atiruj Theppawong1 · Charlotte Grootaert2 · Steven De Jonghe3 · Leentje Persoons3 ·
Dirk Daelemans3 · Kristof Van Hecke4 · John Van Camp2 · Matthias D’hooghe1
Received: 24 September 2019 / Accepted: 3 November 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
A small set of structurally diferent monocarbonyl curcuminoids was prepared and screened for cytotoxic activity. In particu-
lar, bis-3-methoxy-4-hydroxy- and bis-4-methoxyphenyl-substituted monocarbonyls were synthesized and transformed into
the corresponding three-dimensional N-acetylpyrazoline derivatives. In addition, a non-symmetrical indole-based monocar-
bonyl curcumin was prepared as well. Preliminary cytotoxic evaluation revealed signifcant efects for 4-hydroxy (pyrazoline)
monocarbonyl curcuminoids, whereas the non-phenolic variants displayed rather poor activity.
Graphic abstract
O
MeO
O
N
N
MeO
HO
OMe
OH
HO
OMe
OH
O
cytotoxic
O
synthesis
N
assessment
N
MeO
MeO
OMe
OMe
O
MeO
HO
NH
Keywords Aldol reactions · Antitumor agents · Curcumin · Heterocycles · Real-time proliferation assay
Introduction
Curcumin, a natural compound extracted from the rhizomes
of Curcuma longa, is a popular food additive and a tradi-
tional medicine in South Asia. Curcumin has been explored
by many researchers, focusing on a broad diversity of bio-
logical activities such as antioxidant, anti-infammatory,
antimicrobial, anticancer, and antimalarial properties. Espe-
cially in the feld of oncology, curcumin has attracted par-
ticular attention because of promising prospects for future
applications [1].
* John Van Camp
john.vancamp@UGent.be
* Matthias D’hooghe
Matthias.Dhooghe@UGent.be
1
SynBioC Research Group, Department of Green Chemistry
and Technology, Faculty of Bioscience Engineering, Ghent
University, Coupure Links 653, 9000 Ghent, Belgium
2
nutriFOODchem, Department of Food Technology, Safety
During the last decade, synthetic modifcations of the cur-
cumin molecular framework, mainly aimed at improving its
bioactivities, have been studied intensively [2]. However,
the potential benefcial efects of this natural product on
various diseases, especially on cancer, are overshadowed
by its unstable chemical structure. Due to the β-diketone
moiety, curcumin is rapidly metabolized by aldo–keto
and Health, Faculty of Bioscience Engineering, Ghent
University, Coupure Links 653, 9000 Ghent, Belgium
3
Department of Microbiology, Immunology
and Transplantation, Laboratory of Virology
and Chemotherapy, Rega Institute for Medical Research, KU
Leuven, Herestraat 49, Leuven, Belgium
4
XStruct, Department of Chemistry, Faculty of Sciences,
Ghent University, Krijgslaan 281, S3, 9000 Ghent, Belgium
Vol.:(0123456789)
1 3

T. Van de Walle et al.
reductase enzymes in the liver [3]. Evidence suggests that
the β-diketone moiety and its active methylene group in the
structure of curcumin contribute signifcantly to curcumin
instability under physiological conditions and induce rapid
degradation and metabolization [4, 5]. To identify the key
structural motifs responsible for bioactivity and to create
opportunity for designing new analogs with a better activity/
stability profle, monocarbonyl curcumins have been pro-
posed in the literature as simplifed curcumin derivatives
[6–10]. The results of previous research on monocarbonyl
curcuminoids indicate that bioactive representatives are
chemically more stable than curcumin in vitro and provide
interesting anti-infammatory and/or antiproliferative efects
[11]. Moreover, pyrazole-containing analogs and their deriv-
atives are known to act as antitubercular [12] and antitumor
agents [13], rendering the incorporation of this azaheterocy-
clic scafold into the curcumin framework a valuable strat-
egy. Keeping these observations in mind, and elaborating
on our recent contributions to the feld of curcumin research
[14–18], we aimed at synthesizing a few (non-)symmetrical
and cyclized monocarbonyl curcuminoids to preliminarily
assess their potential as cytotoxic agents.
reacted with indole-3-carboxaldehyde in basic conditions
through aldol condensation to deliver (1E,4E)-1-(4-hydroxy-
3-methoxyphenyl)-5-(1H-indol-3-yl)penta-1,4-dien-3-one
(5). The choice for indole-3-carboxaldehyde stems from
our previous exploration of diferent azaheteroaromatic cur-
cumin analogs (indoles, quinolines, isoquinolines, pyridines,
pyrroles, …) and their biological assessment [14–18]. The
low yield of indole 5 relates to a low conversion and a cum-
bersome purifcation via column chromatography to obtain
an analytically pure sample. When the reaction time was
extended (up to 8 days), mainly degradation was observed.
Furthermore, monocarbonyl compounds 2a, 2b were sub-
jected to hydrazine hydrate in acetic acid to efect cyclization
en route to the corresponding N-acetylpyrazoline derivatives
3a, 3b (Scheme 1) [22, 23]. The correct molecular struc-
ture of the latter azaheterocycles 3 was secured by means
of single-crystal X-ray analysis of pyrazoline 3b (Fig. 1).
The added value of transforming curcumins into pyrazolines
relates to the introduction of sp3 carbons in their structure,
conveying three-dimensionality to these scafolds (‘escape
from fatland’ strategy), as it has been established in the
literature that the design of out-of-plane compounds often
results in increased solubility, enhanced target selectivity
and fewer of-target efects [24, 25].
Results and discussion
Previously, non-symmetrical monocarbonyl analogs have
been shown to exhibit good chemical stability in a phosphate
bufer medium [26]. Furthermore, the efects of two repre-
sentative analogs regarding their in vitro anti-infammatory
activity on the apoptosis pathway MAPKs/NF-κB and in
in vivo animal models have been studied [9], demonstrating
their biological relevance.
The synthesis and structures of the target compounds are
shown in Scheme 1. Briefy, saturated NaOH in methanol
was added dropwise to a mixture of either 3-methoxy-
4-hydroxybenzaldehyde (1a, R1=OMe, R2=OH, vanillin)
or 4-methoxybenzaldehyde (1b, R1=H, R2=OMe) and ace-
tone at room temperature to furnish the desired (1E,4E)-1,5-
bisarylpenta-1,4-dien-3-ones 2a, 2b [19, 20]. Moreover, in
the pursuit of a non-symmetrical analog, intermediate (E)-
4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one (4), obtained
by monocondensation of vanillin with acetone [21], was
In a frst round of screening, the obtained (pyrazoline)
monocarbonyl curcumin analogs 2a, 2b, 3a, 3b, and 5 were
evaluated for in vitro cytotoxicity on five different cell
lines (HepG2, HT-29, Caco-2, EAhy.926 and CHO-K1)
by two diferent endpoint-based cytotoxicity assays. The
Scheme 1
1 3

Synthesis and cytotoxic evaluation of monocarbonyl curcuminoids and their pyrazoline…
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay relies on the mitochondrial activity of
living cells to convert the yellow MTT substrate into purple
formazan crystals that can be measured spectrophotometri-
cally. The sulforhodamine B (SRB) assay is based on the
stoichiometric binding of the SRB dye to cellular proteins
under mild acidic conditions and its subsequent extrac-
tion under basic conditions. The amount of dye extracted
is related to the cell mass and thus the number of cells in
a sample. In both assays, curcumin was included as a ref-
erence. The results obtained in this study are reported in
Table 1.
These observations point toward the crucial importance of
the phenolic hydroxyl group for cytotoxic activity. Finally,
the indole-based monocarbonyl curcuminoid 5 is endowed
with promising cytotoxicity, except on the liver cancer cell
line HepG2.
To complement these endpoint viability assays, the
most promising compounds from this initial screen-
ing (compounds 2a, 3a, 5, and curcumin as a reference
compound) were also evaluated in a real-time IncuCyte
proliferation assay against an array of solid and hemato-
logical cancers including LN-229 (glioblastoma), Capan-1
(pancreatic adenocarcinoma), Hap-1 (chronic myeloid
leukemia), HCT-116 (colorectal carcinoma), NCI-H460
(lung carcinoma), DND-41 (acute lymphoblastic leuke-
mia), HL-60 (acute myeloid leukemia), K-562 (chronic
myeloid leukemia) and Z-138 (non-Hodgkin lymphoma)
cell lines (Table 2). Whereas curcumin displayed potent
cytotoxic activity against a selection of cancer cell lines
(Capan-1, Hap-1, HCT-116, Z-138), its cytotoxicity is
much less pronounced in other cells (e.g., LN-229, NCI-
H460 and K-562). On the other hand, the monocarbonyl
These data demonstrate that the vanillin-derived mono-
carbonyl curcumin derivative 2a exhibits an improved
cytotoxic efect on all cell lines, when compared to cur-
cumin. On the other hand, the p-methoxy analog 2b shows
a similar or slightly decreased antiproliferative activity
than curcumin. Among the N-acetylpyrazoline congeners
3, the vanillin derivative 3a displayed a similar cyto-
toxicity profle as curcumin, whereas the non-phenolic
p-methoxy analog 3b is a rather weak cytotoxic agent.
Fig. 1 X-ray molecular structure
of compound 3b, showing
O
thermal displacement ellipsoids
at the 30% probability level.
N
N
The solvent water molecule is
MeO
omitted for clarity
OMe
3b
Table 1 IC50 values of cell growth inhibition of monocarbonyl curcuminoids, measured after 72 h treatment as tested by mitochondrial activity
(MTT) and protein content (SRB) assays, n≥3 (mean SD)
Compound HepG2
MTT/µM
HT-29
Caco-2
EA.hy926
MTT/µM
CHO-K1
MTT/µM
SRB/µM
5.5 0.0
MTT/µM
SRB/µM
<0.1
MTT/µM
SRB/µM
<0.1
SRB/µM
<10
SRB/µM
2a
7.0 0.0
<0.1
<0.1
<10
2.9 0.5
3.1 0.3
>75
2b
71.0 0.2 71.7 0.1 20.6 0.4 22.4 0.4 40.1 0.7 34.5 1.3 43.0 1.2 48.7 2.1 >75
3a
24.0 0.3 23.2 1.2 46.7 1.2 34.6 1.6 3.1 0.0
≥75 73.9 0.9 67.3 1.5 69.4 0.7 >75
66.6 0.7 51.1 2.1 16.2 0.2 14.6 0.7 8.0 0.9
5.9 0.3
>75
9.0 0.1
10.5 0.5 26.2 1.5 11.3 0.1 20.7 0.2
>75 >75 66.8 0.6 58.3 0.4
11.0 0.4 21.7 0.0 16.9 0.4 17.6 1.1
3b
5
Curcumin
25.3 0.9 22.1 2.6 43.4 4.6 52.3 3.8 47.6 1.5 39.1 0.1 46.8 0.7 58.4 4.2 44.9 0.6 38.8 0.3
Table 2 Cytotoxic activity of monocarbonyl curcuminoids in various cancer cell lines, as measured by real-time imaging, n = 2 (mean SD)
Compound
IC50/µM
LN-229
Capan-1
Hap-1
HCT-116
NCI-H460
DND-41
HL-60
K-562
2.5 0.2
3.3 0.5
4.5 2.2
21.8 6.0
Z-138
2a
1.8 0.2
2.0 0.1
2.0 0.0
1.3 0.7
1.9 0.2
1.8 0.3
2.0 0.5
1.5 1.0
1.4 0.7
1.5 0.9
2.4 0.1
1.1 0.4
1.3 0.7
1.8 0.5
3.6 0.2
1.6 1.1
2.1 0.1
2.5 0.2
2.0 0.0
1.4 0.8
3.8 0.1
5.7 4.9
2.0 0.5
2.5 0.3
2.9 0.2
7.4 6.9
1.1 0.5
2.3 0.4
2.5 0.0
1.4 0.8
3a
5
Curcumin
41.4 14.7
33.4 32.5
1 3

T. Van de Walle et al.
curcuminoids 2a, 3a, and 5 are endowed with potent cyto-
toxic activity against all tumor cell lines tested with IC50
values in the 1–5 µM range.
against a broad panel of cancer cell lines using a real-time
assay system. This revealed excellent broad spectrum cyto-
toxic activity for the 4-hydroxy-pyrazoline-monocarbonyl
curcuminoid (compound 3a). Given the fact that this com-
pound lacks apoptogenic activity in normal, healthy cells, it
can be considered as a promising lead structure for further
elaboration of antitumoral curcumin analogs.
To assess the selectivity of these monocarbonyl deriva-
tives (compounds 2a, 3a, and 5) versus normal, non-cancer
cells, their toxicity toward peripheral blood mononuclear
cells (PBMCs), isolated from two healthy donors, was evalu-
ated, by measuring in real-time the apoptosis induced by the
compounds. Addition of the IncuCyte Caspase-3/7 reagent
to apoptotic cells, leads to its cleavage by the activated cas-
pase-3/7, resulting in the release of a DNA dye and allowing
the green fuorescent staining of nuclear DNA at two difer-
ent time points (24 and 72 h). Staurosporine, a well-known
inducer of apoptosis, was included as a positive control, at
a concentration of 1 µM and the fuorescence (and hence,
the number of apoptotic cells) measured under these condi-
tions was set at 100%. The IC50 data as shown in Table 3 are
relative to this value. Compounds 2a and 5 are potent apop-
togenic agents, displaying IC50 values in the 1–4 µM range,
whereas curcumin itself is not a potent inducer of apopto-
sis. Compound 3a is a promising compound, because of its
excellent antiproliferative activity against a broad range of
cancer cell lines, whereas it lacks apoptogenic activity in
healthy cells.
Experimental
All reagents were purchased from commercial suppliers
without further purifcation. 1H NMR spectra were recorded
at 400 MHz (Bruker Avance III Nanobay) with CDCl3,
DMSO-d6, or MeOD-d4 as solvent. 13C NMR spectra were
recorded at 100.6 MHz (Bruker Avance III Nanobay) with
CDCl3, DMSO-d6, or MeOD-d4 as solvent. Low resolution
mass spectra (LR-MS) were recorded by injection on an
Agilent 1100 Series LC/MSD type SL mass spectrometer
with electrospray ionization (ESI 70 eV) and using a mass-
selective detector (quadrupole). When crude reaction mix-
tures were analyzed, the mass spectrometer was preceded
by a HPLC reversed-phase column (Ascentis® Express C18,
HPLC column 3 cm×4.6 mm, 2.7 µm) with a diode array
UV–Vis detector. The purity of all tested compounds was
assessed by 1H NMR analysis and/or HPLC analysis, con-
frming a purity of≥95%.
Conclusion
Curcumins are promising antitumoral agents, but the meta-
bolic instability of the β-dicarbonyl entity hampers their
further development. Therefore, in this manuscript, the
potential of monocarbonyl curcuminoids to act as surrogates
for curcumins was explored by the synthesis of a small set
of representative examples. To that end, 3-methoxy-4-hy-
droxy- and 4-methoxyphenyl-substituted monocarbonyls
2a, 2b were constructed, as well as the corresponding out-
of-plane N-acetylpyrazoline derivatives 3a, 3b. In addition,
an indole-containing monocarbonyl curcumin analog 5 was
synthesized as well. All compounds were initially screened
for cytotoxic activity using two diferent endpoint cell viabil-
ity assays. The most promising congeners were further tested
Synthesis of symmetrical and non‑symmetrical
monocarbonyl curcuminoids 2, 4, and 5
To a solution of diferent benzaldehydes (5.6 mmol) in ace-
tone (2.8 mmol), saturated NaOH in methanol was added
dropwise, and the mixture was stirred at room tempera-
ture for 3–6 h. The resulting solution was then quenched
with 10 cm3 water, while the residual acetone (if any) was
removed under reduced pressure followed by extraction with
EtOAc. The combined organic layers were washed with
30 cm3 brine and dried over anhydrous MgSO4, fltered,
and concentrated under reduced pressure. The residue was
further purifed by either normal-phase (SiO2) or reversed-
phase (C18) column chromatography to obtain compounds
2a, 2b.
Table 3 Induction of apoptosis in PBMCs by monocarbonyl curcumi-
For the preparation of analog 4, vanillin (5.6 mmol) was
used in a similar procedure as for compounds 2, followed by
the addition of an excess of acetone (3.0 cm3) to inhibit the
formation of compound 2a and to furnish the expected inter-
mediate 4 for further use. Compound 4 (1.5 mmol) was frst
dissolved in methanol, followed by the addition of indole-
3-carboxaldehyde (1.7 mmol) to the solution. Sequentially,
saturated NaOH in methanol was added dropwise, and the
mixture was stirred at room temperature. After 1 day, the
reaction was neutralized and concentrated under reduced
noids
Compound
IC50/µM
Donor 1
24 h
Donor 2
24 h
72 h
72 h
2a
1.4
>100
2.6
1.7
48.4
1.8
1.6
>100
4.0
1.5
31.3
2.4
3a
5
Curcumin
>100
>100
>100
>100
1 3

Synthesis and cytotoxic evaluation of monocarbonyl curcuminoids and their pyrazoline…
pressure, followed by extraction with EtOAc. The combined
organic layers were washed with 30 cm3 brine and dried over
anhydrous MgSO4, fltered, and concentrated under reduced
pressure. Finally, the crude mixture was further purifed by
reversed-phase (C18) column chromatography in order to
obtain indole derivative 5.
16–24 h. The reaction progress was monitored by either
TLC or LC–MS. After completion of the reaction, the reac-
tion mixture was neutralized by pouring it into an ice-cold
NaHCO3 solution. The resulting solid was suction fltered,
washed with water, dried over vacuum, and subjected to
column chromatography, as previously described in the lit-
erature [22, 23].
(1E,4E)‑1,5‑Bis(4‑hydroxy‑3‑methoxyphenyl)penta‑1,4‑
dien‑3‑one (2a) [20] Yellow-orange solid; yield: 137 mg
(15%); 1H NMR (400 MHz, CDCl3): δ=7.67 (d, J=15.8 Hz,
2H), 7.17 (dd, J=8.2, 1.6 Hz, 2H), 7.10 (d, J=1.8 Hz, 2H),
6.94 (d, J=8.2 Hz, 2H), 6.93 (d, J=15.8 Hz, 2H), 3.94 (s,
6H) ppm; 13C NMR (100 MHz, CDCl3): δ=188.8, 148.3,
146.9, 143.3, 127.5, 123.4, 123.3, 114.9, 109.8, 56.0 ppm;
MS (70 eV): m/z (%)=327 (M+, 100).
(E)‑1‑[5‑(4‑Hydroxy‑3‑methoxyphenyl)‑3‑(4‑hydroxy‑3‑
methoxystyryl)‑4,5‑dihydro‑1H‑pyrazol‑1‑yl]‑ethan‑1‑one
(3a, C21H22N2O5) Pale-yellow solid; yield: 306 mg (40%);
1H NMR (400 MHz, DMSO-d6): δ = 9.36 (br s, 1H), 8.90
(br s, 1H), 7.25 (d, J = 1.8 Hz, 1H), 7.06 (d, J = 16.3 Hz,
1H), 6.99 (dd, J = 8.2, 1.8 Hz, 1H), 6.89 (d, J = 16.3 Hz,
1H), 6.76 (d, J=8.2 Hz, 1H), 6.73 (d, J=2.0 Hz, 1H), 6.70
(d, J=8.1 Hz, 1H), 6.50 (dd, J=8.1, 2.0 Hz, 1H), 5.38 (dd,
J=11.5, 3.9 Hz, 1H), 3.82 (s, 3H), 3.74 (s, 3H), 3.58 (dd,
J=17.5, 11.5 Hz, 1H), 2.95 (dd, J=17.5, 3.9 Hz, 1H), 2.23
(s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6): δ = 167.3,
156.3, 148.4, 148.0, 146.1, 138.6, 133.9, 127.9, 122.0,
117.9, 117.7, 116.0, 115.9, 110.5, 110.3, 59.2, 56.08, 56.06,
41.5, 22.3 ppm; MS (70 eV): m/z (%)=383 (M+, 100).
(1E,4E)‑1,5‑Bis(4‑methoxyphenyl)penta‑1,4‑dien‑3‑one
1
(2b) [19] Pale-orange solid; yield: 585 mg (71%); H
NMR (400 MHz, CDCl3): δ = 7.68 (d, J = 15.8 Hz, 2H),
7.54 (d, J=8.2 Hz, 4H), 6.94 (d, J=15.8 Hz, 2H), 6.91 (d,
J = 8.2 Hz, 4H), 3.82 (s, 6H) ppm; 13C NMR (100 MHz,
CDCl3): δ=188.8, 161.6, 142.7, 130.1, 127.6, 123.5, 114.4,
55.4 ppm; MS (70 eV): m/z (%)=295 (M+, 100).
(E)‑1‑[5‑(4‑Methoxyphenyl)‑3‑(4‑methoxystyryl)‑4,5‑dihy‑
dro‑1H‑pyrazol‑1‑yl]ethan‑1‑one (3b, C21H22N2O3) Pale-
(E)‑4‑(4‑Hydroxy‑3‑methoxyphenyl)but‑3‑en‑2‑one (4) [21]
Pale-yellow solid; yield: 861 mg (80%); 1H NMR (400 MHz,
CDCl3): δ = 7.45 (d, J = 16.2 Hz, 1H), 7.09 (dd, J = 8.1,
1.9 Hz, 1H), 7.06 (d, J= 1.9 Hz, 1H), 6.93 (d, J= 8.1 Hz,
1H), 6.59 (d, J=16.2 Hz, 1H), 5.92 (br s, 1H), 3.94 (s, 3H),
2.37 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3): δ=198.4,
148.2, 146.9, 143.7, 126.9, 125.0, 123.5, 114.8, 109.3, 56.0,
27.3 ppm; MS (70 eV): m/z (%)=193 (M+, 100).
1
white solid; yield: 694 mg (99%); H NMR (400 MHz,
DMSO-d6): δ=7.57 (d, J=8.7 Hz, 2H), 7.08 (d, J=8.6 Hz,
2H), 7.06 (d, J =15.8 Hz, 1H), 6.96 (d, J= 15.8 Hz, 1H),
6.95 (d, J=8.7 Hz, 2H), 6.88 (d, J=8.6 Hz, 2H), 5.43 (dd,
J=11.6, 4.0 Hz, 1H), 3.78 (s, 3H), 3.73 (s, 3H), 3.62 (dd,
J=17.6, 11.6 Hz, 1H), 2.96 (dd, J=17.6, 4.0 Hz, 1H), 2.22
(s, 3H) ppm; 13C NMR (100 MHz, DMSO-d6): δ = 172.1,
165.2, 163.6, 160.9, 142.7, 139.7, 134.0, 133.7, 131.9,
123.3, 119.5, 119.2, 63.7, 60.4, 60.3, 46.2, 27.0 ppm; MS
(70 eV): m/z (%)=351 (M+, 100).
(1E,4E)‑1‑(4‑Hydroxy‑3‑methoxyphenyl)‑5‑(1H‑indol‑3‑yl)‑
penta‑1,4‑dien‑3‑one (5, C20H17NO3) Dark-red solid; yield:
1
54 mg (3%); H NMR (400 MHz, CDCl3): δ = 8.68 (1H,
br s), 8.03 (d, J = 15.6 Hz, 1H), 8.04–8.02 (m, 1H), 7.70
(d, J =15.8 Hz, 1H), 7.60 (d, J =2.7 Hz, 1H), 7.46–7.44
(m, 1H), 7.32–7.30 (m, 2H), 7.20 (dd, J=8.3, 1.7 Hz, 1H),
7.15 (d, J=15.8 Hz, 1H), 7.14 (d, J=1.7 Hz, 1H), 6.965 (d,
J=15.6 Hz, 1H), 6.956 (d, J=8.3 Hz, 1H), 5.92 (br s, 1H),
3.97 (s, 3H) ppm; 13C NMR (100 MHz, CDCl3): δ=189.1,
148.0, 146.8, 142.5, 137.2, 137.0, 129.9, 127.7, 125.4,
124.1, 123.5, 123.3, 121.69, 121.67, 120.7, 114.8, 114.4,
111.9, 109.7, 56.0 ppm; MS (70 eV): m/z (%) = 320 (M+,
100). For a related indole curcuminoid compound, see the
literature [27].
X‑ray crystallography analysis of 3b
For the structure of 3b, X-ray intensity data were collected
at 100 K, on a Rigaku Oxford Difraction Supernova Dual
Source (Cu at zero) difractometer equipped with an Atlas
CCD detector using ω scans and CuKα (λ=1.54184 Å) radi-
ation. The images were interpreted and integrated with the
program CrysAlisPro [28]. Using Olex2 [29], the structure
was solved by direct methods using the ShelXS structure
solution program and refned by full-matrix least-squares
on F2 using the program package [30, 31]. Non-hydrogen
atoms were anisotropically refned and the hydrogen atoms
in the riding mode and isotropic temperature factors fxed
at 1.2 times U(eq) of the parent atoms (1.5 times for methyl
groups). Hydrogen atoms on the water solvent molecule
Synthesis of pyrazoline analogs 3
A mixture of (1E,4E)-1,5-bisarylpenta-1,4-dien-3-one 2
(2 mmol) and hydrazine hydrate (85%, 10 mmol) in 5 cm3
glacial acetic acid was heated at reflux temperature for
1 3

T. Van de Walle et al.
were located from a diference Fourier electron density map
and restrained refned (O–H distance of 0.84 Å).
used to screen for cytotoxicity and cell density. The cells
were seeded at a concentration of 2×104 cells per well, incu-
bated for 24 h and treated with or without (control) com-
pounds. Three days after this treatment, the cells were fxed
by the addition of 50 mm3 of 50% TCA in Milli-Q water for
1 h in the cold room, 4 °C. The plate was washed at least
three times with tap water and dried, after which the cells
were stained with an SRB solution (0.4% sulforhodamine
B in 1% glacial acetic acid) at 4 °C. After 30 min, the plate
was rinsed for fve times with 1% glacial acetic acid and
dried. Sequentially, Tris bufer in a concentration of 10 mM
was used to re-dissolve the stain. Finally, the absorbance
was measured using the microplate spectrophotometer at a
wavelength of 490 nm. Each condition was performed in
triplicate.
Crystal data for compound 3b. C21H24N2O4, M = 368.42,
monoclinic, space group P21/c (No. 14), a = 5.7163(2)
Å, b = 12.5766(5) Å, c = 25.7120(10) Å, β = 95.089(4)˚,
V=1841.19(12) Å3, Z=4, T=100 K, ρcalc =1.329 g cm−3,
μ(Cu-Kα) = 0.753 mm−1, F(000) = 784, 13698 refections
measured, 3734 unique (Rint =0.0676) which were used in
all calculations. The fnal R1 was 0.0742 (I>2σ (I)) and wR2
was 0.2133 (all data). The asymmetric unit shows chirality
at C3 (S). But obviously, because of the centro-symmetric
space group, also the inverse (R) confguration is equally
present in the crystal structure. Hydrogen bonds are formed
between the C=O group and a solvent water molecule.
CCDC 1899076 contains the supplementary crystal-
lographic data for this paper and can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html
(or from the Cambridge Crystallographic Data Centre, 12,
Union Road, Cambridge CB2 1EZ, UK; fax: +44-1223-
336033; or deposit@ccdc.cam.ac.uk).
IncuCyte proliferation assay
All tumor cell lines were acquired from the American Type
Culture Collection (ATCC, Manassas, VA, USA), except
for the DND-41 cell line, which was purchased from the
Deutsche Sammlung von Mikroorganismen und Zellkul-
turen (DSMZ Leibniz-Institut, Germany), and the Hap-1
cell line which was ordered from Horizon Discovery (Hori-
zon Discovery Group, UK). All cell lines were cultured
as recommended by the suppliers. Media were purchased
from Gibco Life Technologies, USA, and supplemented
with 10% fetal bovine serum (HyClone, GE Healthcare
Life Sciences, USA). Adherent cell lines HCT-116, NCI-
H460, LN-229, Hap-1 and Capan-1 cells were seeded at a
density between 500 and 1500 cells per well, in 384-well,
black-walled, clear-bottomed tissue culture plates (Greiner).
After overnight incubation, cells were treated with the test
compounds at seven diferent concentrations ranging from
100 to 6.4×10−3 µM. Suspension cell lines HL-60, K-562,
Z-138, and DND-41 were seeded at densities ranging from
2500 to 5500 cells per well in 384-well, black-walled, clear-
bottomed tissue culture plates containing the test compounds
at the same seven concentration points. The plates were
incubated and monitored at 37 °C for 72 h in an IncuCyte®
(Essen BioScience Inc., Ann Arbor, MI, USA) for real-time
imaging. Images were taken every 3 h, with one feld imaged
per well under 10× magnifcation.
Mitochondria activity (MTT) experiments
Throughout the experiment, standard procedures were used
to maintain all cell lines at 37 °C with 95% humidity and
10% CO2. The MTT assay was performed to determine the
number of viable cells in this assay. Briefy, 2× 104 cells
suspended in 200 mm3 DMEM medium were frst inoculated
into each well of 96-well microplates and incubated for 24 h.
Then, the medium in each well was removed and an equal
volume of serum-free medium (200 mm3/well) containing
either test compounds or reference control (curcumin) at
various concentrations was added for 72 h. Each compound
was performed in triplicate. Afterwards, the cell viability
was determined by removing 100 mm3 of medium and add-
ing 20 mm3 of MTT solution (5 mg/mm3 in PBS) followed
by 2 h of incubation. Finally, the DMEM medium with MTT
solution was then removed and replaced with DMSO to dis-
solve the formazan crystal. The 96-well microplate was
then measured at 570 nm using a Spectramax (Molecular
Devices) microplate spectrophotometer. For the data analy-
sis, the percentage of surviving cells after a 72 h exposure to
various concentrations of each test compound was calculated
to obtain the IC50 value of each compound.
Apoptosis induction assay in PBMC
Protein content (SRB) analysis
Bufy coat preparations from healthy donors were obtained
from the Blood Transfusion Center in Leuven, Belgium.
PBMCs were isolated by density gradient centrifugation
over Lymphoprep (d = 1.077 g/cm3) (Nycomed, Oslo, Nor-
way) and cultured in cell culture medium (DMEM/F12,
Gibco Life Technologies, USA) containing 8% FBS.
The SRB assay is based on the measurement of cellular pro-
teins. Sulforhodamine B binds electrostatically with basic
amino acid residues if the cells are fxed with trichloroacetic
acid (TCA) and can be solubilized by weak bases. Because
of this quantitative staining capacity of SRB, the assay is
1 3

Synthesis and cytotoxic evaluation of monocarbonyl curcuminoids and their pyrazoline…
12. Chovatia P, Akabari JD, Kachhadia PK, Saicic RN, Zalavadia PD,
PBMCs were seeded at 28,000 cells per well in 384-well,
black-walled, clear-bottomed tissue culture plates containing
the test compounds at seven diferent concentrations ranging
from 100 to 6.4 ×10−3 µM. IncuCyte® Caspase 3/7 Green
Reagent was added as recommended by the supplier, and the
plates were incubated and monitored at 37 °C for 3 days in
an IncuCyte® (Essen BioScience Inc., Ann Arbor, MI, USA)
for real-time imaging. Images were taken every 3 h, with one
feld imaged per well under 10× magnifcation.
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Acknowledgements The authors are indebted to Ghent University and
to the Lotus + Erasmus Mundus Programme of the European Union
for fnancial support. KVH thanks the Hercules Foundation (project
AUGE/11/029 “3D-SPACE: 3D Structural Platform Aiming for Chemi-
cal Excellence”) and the Special Research Fund (BOF)–UGent (project
01N03217) for funding.
18. Theppawong A, Van de Walle T, Van Hecke K, Grootaert C, Van
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