A novel cluster of C5-curcuminoids: design, synthesis, in vitro antiproliferative activity and DNA binding of bis(arylidene)-4-cyclanone derivatives based on 4-hydroxycyclohexanone scaffold

Article abstract of DOI:10.1007/s11164-019-03859-4

A new series (6) of C₅-curcuminoid derivatives (2E,6E-2,6-dibenzylidene-4-hydroxycyclohexanones) is described here with their evaluation for in vitro antiproliferative activities. Evaluation of 31 compounds against human A2780 (ovarian), C33A (cervix) and MDA-MB-231 (breast) cancer cell lines was performed to obtain structure activity relation data. The best performer was (2E,6E)-2,6-bis(3′-nitrobenzylidene)-4-hydroxycyclohexanone (6h) with IC₅₀ values of 0.68?μM (A2780), 0.69?μM (C33A) and 0.92?μM (MDA-MB-231) compared to cisplatin with 1.30?μM, 3.69?μM and 19.13?μM, respectively. According to calculated physicochemical properties some members in series 6, namely (2E,6E)-2,6-bis[(4′-pyridinyl)methylene]-4-hydroxycyclohexanone (6p) [IC₅₀ = 0.76?μM (A2780), 2.69?μM (C33A), 1.28?μM (MDA-MB-231)] seem to have improved bioavailability compared to curcumin. Selected members of series 6 were involved in circular dichroism spectroscopic measurements in order to determine their interaction with natural DNA. Based on these data, we conclude that these derivatives do not bind to DNA in vitro. A proposal is summarized based on mass spectrometric assessment for fingerprint analysis in biological research of such C₅-curcuminoids.

Full text of DOI:10.1007/s11164-019-03859-4

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-019-03859-4
A novel cluster of C5‑curcuminoids: design, synthesis,
in vitro antiproliferative activity and DNA binding
of bis(arylidene)‑4‑cyclanone derivatives based
on 4‑hydroxycyclohexanone scafold
Imre Huber1 · István Zupkó2 · András Gyovai2 · Péter Horváth3 · Eszter Kiss3 ·
Gergely Gulyás‑Fekete4 · János Schmidt4 · Pál Perjési1
Received: 14 February 2019 / Accepted: 15 May 2019
© The Author(s) 2019
Abstract
A new series (6) of C5-curcuminoid derivatives (2E,6E-2,6-dibenzylidene-4-hydrox-
ycyclohexanones) is described here with their evaluation for in vitro antiproliferative
activities. Evaluation of 31 compounds against human A2780 (ovarian), C33A (cer-
vix) and MDA-MB-231 (breast) cancer cell lines was performed to obtain structure
activity relation data. The best performer was (2E,6E)-2,6-bis(3′-nitrobenzylidene)-
4-hydroxycyclohexanone (6h) with IC50 values of 0.68 μM (A2780), 0.69 μM
(C33A) and 0.92 μM (MDA-MB-231) compared to cisplatin with 1.30 μM, 3.69 μM
and 19.13 μM, respectively. According to calculated physicochemical properties
some members in series 6, namely (2E,6E)-2,6-bis[(4′-pyridinyl)methylene]-4-hy-
droxycyclohexanone (6p) [IC50 =0.76 μM (A2780), 2.69 μM (C33A), 1.28 μM
(MDA-MB-231)] seem to have improved bioavailability compared to curcumin.
Selected members of series 6 were involved in circular dichroism spectroscopic
measurements in order to determine their interaction with natural DNA. Based on
these data, we conclude that these derivatives do not bind to DNA in vitro. A pro-
posal is summarized based on mass spectrometric assessment for fngerprint analy-
sis in biological research of such C5-curcuminoids.
Keywords Antiproliferative curcuminoids · Cyclic curcuminoids · C5-
curcuminoids · DNA binding of curcuminoids · MS fngerprint of curcuminoids
* Imre Huber
imre.huber@aok.pte.hu
1
Department of Pharmaceutical Chemistry, University of Pécs, Pecs 7624, Hungary
2
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged 6720,
Hungary
3
Department of Pharmaceutical Chemistry, Semmelweis University, Budapest 1092, Hungary
4
Department of Biochemistry and Medical Chemistry, University of Pécs, Pecs 7624, Hungary
Vol.:(0123456789)
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I. Huber et al.
Introduction
Although there has been great progress in the development of treatment and
prevention, cancer remains a major worldwide health problem. Because of an
increase in worldwide mortality with growing prevalence, according to WHO
reports [1], the successful treatment of cancer is still a challenge this century.
Chemotherapy alongside surgical, radiological and biological treatment of can-
cer played a fundamental role for many decades. Hence, there is a current need
to search for newer and more active anticancer compounds in pharmaceutical
research felds. The research for mimics of natural materials is such a feld in
pharmaceutical science and is considered one of the major sources of lead mol-
ecules and drug candidates [2].
Plants used as spices, favoring and coloring agents in the kitchen (nutraceu-
ticals), or as preventive/curing additives in the Ayurvedic and folkloric medici-
nal practice (pharmaceuticals) are excellent sources for natural compounds. Such
plants are common in the Zingiberaceae (ginger) family. Their powdered rhi-
zome (a modifed subterranean stem), as a yellow powder known as turmeric,
has been used for thousands of years in Asian countries. For example, plants such
as Curcuma longa L. and/or Curcuma domestica L. are also in use to prepare
the well-known Indian spice turmeric currently. The major component of tur-
meric is its secondary metabolite, curcumin (1). Extensive research has shown
that curcumin exhibits many diferent pharmacological efects and has a num-
ber of molecular targets with multiple pathways as an antiproliferative molecule
[3, 4], although curcumin is remarkably non-toxic and has promising anticancer
activities. Clinical studies indicate that its poor bioavailability and pharmacoki-
netic profles due to instability under physiological conditions is a disadvantage
[3]. Curcumin (diferuloylmethane), 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
heptadien-3,5-dion contains a C7 ß-diendione linkage (1,6-heptadien-3,5-dion)
between the two arylidene groups in its structure. It was believed for a long time
that natural curcuminoids (including the metabolites of curcumin) are exclusively
C7-curcuminoids. However, along with curcumin a C5-curcuminoid was also
isolated from both Curcuma longa and Curcuma domestica [5, 6]. This natural
C5-curcuminoid, 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one, as a
truncated analogue of curcumin contains a C5 ß-dienone linker (1,4-pentadiene-
3-one) between the two benzylidene cores in its structure. This compound and
its related derivatives proved to be more potent anticancer molecules compared
to curcumin [7]. There is a great number of cyclic C5-curcuminoid analogues
with excellent antiproliferative potential among the synthetic curcuminoids
with the general structure 2 (Fig. 1). Structural modifcations of curcumin and
C5-curcuminoids focusing on enhancing their bioactivities have been investigated
intensively during the last couple of decades. Numerous studies have been con-
ducted, for example about the (3E,5E)-3,5-dibenzylidene-4-piperidone synthetic
C5-curcuminoid family [8–18]. It is well known about these 4-piperidone deriva-
tives that they exhibit higher cytotoxicity than curcumin towards diferent tumor
cell lines such as breast, prostate, cervix, melanoma [3]. Compound EF24, for
1 3

A novel cluster of C5-curcuminoids: design, synthesis,…
Fig. 1 Curcumin and synthetic cyclic C5-curcuminoids
example (Fig. 1), showed high activity against cisplatin-resistant human ovarian
cancer cells. The inhibitory efect of EF24 on cell-proliferation, as is common
with such compounds, is due to its inductive ability to G2/M arrest and apoptosis
by increasing phosphorylated phosphatase and tensin homolog (PTEN) expres-
sion [19]. In addition to G2/M arrest and apoptosis induction, these 4-piperidone
derivatives show diferential cytotoxicity: they are less toxic to non-cancerous
cells when compared to cancer cells [8, 13–16].
Another crucial advantage of these synthetic C5-curcuminoids is that a number of
them are able to revert multi-drug resistance (MDR) [17, 20, 21]. Moreover, there
are reports on the in vivo tolerability and the lack of acute toxicity of these curcumi-
noids on rodents. There are compounds in this family that proved to be safe and did
not cause mortality or convulsion in mice during a 2-week period of oral adminis-
tration in 100–500 mg/kg dosage [22]. A number of C5-curcuminoids possessing
the 1,4-pentadiene-3-one moiety have diferent modes of action, such as inducing
apoptosis, cell cycle arrest, inhibiting the biosynthesis of polypeptides fundamental
to tumor-progression, afecting mitochondrial respiration and stimulation/inhibition
of certain enzymes playing role in tumor-growth [23]. Further, there is continuing
debate on the binding or intercalation of curcumin onto DNA [24, 25] as to whether
binding or intercalation would result in DNA damage in healthy cells as a dramatic
side efect. There are scientifc facts against this “DNA damage” theory in that cur-
cumin binds to the minor groove of DNA, albeit weakly [26]. In another example,
incubation with calf thymus DNA had little efect on either UV/Vis or fuorescence
spectra of curcumin [27].
Based on the above description and the literature available in this feld, we can
say that the number of reports on the structural modifcations on C5-curcuminoids is
very high. So far, however, none of these drug-candidates passed into drug-develop-
ment study or clinical trials.
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I. Huber et al.
In view of the considerations above, we have decided to synthetize new, cyclic
derivatives of natural C5-curcuminoids with the general structure 2 (Fig. 1). In
one of our previous publications, we compared the antiproliferative activity of 25
arylidene cyclanones where the ketone scafold was cyclopentanone, cyclohex-
anone, 1-indanone, 1-tetralone or 4-piperidone (2, X=CH2, NH or N-alkil) [28].
Their cytotoxicity against four human adherent cancer cell lines was evaluated. The
cytotoxicity screen revealed that the diarylidene derivatives in general dominate the
monoarylidene enones and, simultaneously, the nitrogen containing heterocycles
display higher inhibition in cell-proliferation compared with the homocyclic ana-
logs. The best performer was N-(γ-oxobutyl)-(3E,5E)-3,5-bis(4′-chlorobenzylidene)-
4-piperidone (3, Fig. 1) with IC50 values of 0.438–1.048 μM. In the present article
we describe the synthesis and comparative pharmacological evaluation of cyclic
C5-curcuminoid analogues 2 (X=NCH3, CHCH3 or CHOH).
Experimental
Chemistry
Chemicals, solvents and reagents for the study were purchased from Alfa Aesar,
Molar and Merck Ltd (Budapest, Hungary). Melting points were determined on
a Barnstead-Electrothermal 9100 apparatus and are uncorrected. Silica gel 60
(0.2–0.5 mm, MERCK) was used for column chromatography and pre-coated silica
gel 60 (F-254, MERCK) plates for TLC.
1
NMR: H- and 13C-NMR spectra were obtained using a Varian UNITY INOVA
400 WB spectrometer. Chemical shifts are referenced to the residual solvent signal.
Measurements were run at a probe temperature of 298 K in CDCl3 or DMSO-d6
1
solutions. All the H- and 13C-NMR spectra were in accordance with the expected
structures.
MS: The sample solutions were prepared by dissolving 0.2 mg synthesized mol-
ecules in 1000 µL acetonitrile/0.1% TFA (1:1, v/v). The clear sample solutions were
pipetted onto the MALDI target plate (MTP 384 target plate, massive steel, Bruker
Daltonics) After crystallization the mass spectra were acquired with an Autofex
II MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany)
equipped with a 337-nm pulsed nitrogen laser (MNL-205MC; LTB Lasertechnik
Berlin, Berlin, Germany). MALDI measurements were performed in refectron
mode in a detection range of m/z 120 to 1000. Ions were accelerated under delayed
extraction conditions (120 ns) in positive and in negative ion modes with an accel-
eration voltage of 20.00 kV. We used alpha-cyano-4-hydroxycinnamic acid (CHCA)
matrix peaks as external calibration. The instrument was controlled and data was
processed using FlexControl 2.4 and FlexAnalysis 3.4 software packages (Bruker
Daltonics, Bremen, Germany.)
CD: CD and UV measurements were performed on a Jasco J-720 spectropola-
rimeter (Jasco Ltd., Tokyo, Japan) in Jasco cylindrical cuvettes with path length
of 10 mm. One milligram of chicken erythrocyte DNA (Reanal, Budapest, Hun-
gary) was dissolved in 10 mL distilled water (stock solution, 0.1 mg/mL) and
1 3

A novel cluster of C5-curcuminoids: design, synthesis,…
was further diluted to 0.033 mg/mL for the experiments. Tested substances were
dissolved in DMSO (Sigma-Aldrich, Budapest, Hungary) in 5–10 mM concentra-
tion. All experiments were performed at ambient temperature.
General method for the synthesis of compounds 6f, 6g, 6h, 6m, 6o, 6p (Method A:
acid‑catalyzed Claisen‑Schmidt condensation)
A solution of 0.1 mol (1.141 g) 4-hydroxycyclohexanone and 0.2 mol of the
appropriate arylaldehyde in 15 mL of glacial acetic acid was treated with 4 drops
of concentrated sulfuric acid. This mixture was left to stand at room temperature
until the starting materials couldn’t be detected on the TLC plate. The reaction
mixture was then diluted with 100 mL water. After 10 min stirring the yellow
solid was collected on a glass flter, treated with 25 ml of 5% aqueous solution of
potassium hydrogencarbonate and washed with water. The yellow solid was dried
and recrystallized from a suitable solvent.
(2E,6E)‑2,6‑Bis(4′‑nitrobenzylidene)‑4‑hydroxycyclohexanone (6f) Overall yield: 85%.
Mp: 216–218 °C (MeOH/CHCl3); Lit.: 210–213 °C [42]. 1H-NMR (500 MHz, CDCl3)
δ (ppm) 3.06–3.18 (br m, 4H), 5.21 (m, 1H), 7.59 (d, J=9.0 Hz, 4H), 7.92 (s, 2H), 8.29
(d, J=9.0 Hz, 4H). 13C-NMR (125 MHz, CDCl3) δ (ppm) 32.9, 66.9, 123.8, 130.7,
134.1, 137.4, 141.4, 147.6, 187.2. MS: m/z calculated for C20H16N2O6: see Table 1.
(2E,6E)‑2,6‑Bis(2′‑nitrobenzylidene)‑4‑hydroxycyclohexanone (6g) Overall yield: 81%.
1
Mp: 178–179 °C (MeOH/CHCl3). H-NMR (500 MHz, DMSO-D6) δ (ppm) 2.84
(m, 2H), 2.95 (m, 2H), 4.97 (s, 1H), 7.54 (d, J=7.5 Hz, 2H), 7.68 (t, J=7.9 Hz, 2H),
7.83 (t, J=7.5 Hz, 2H), 7.93 (s, 2H), 8.19 (d, J=7.9 Hz, 2H). 13C-NMR (125 MHz,
DMSO-D6) δ (ppm) 31.4, 67.2, 124.8, 129.8, 130.5, 130.9, 133.2, 133.8, 135.5, 147.7,
186.6. MS: m/z calculated for C20H16N2O6: see Table 1.
(2E,6E)‑2,6‑Bis(3′‑nitrobenzylidene)‑41‑hydroxycyclohexanone (6h) Overall yield: 79%.
Mp: 168–169 °C (MeOH/CHCl3). H-NMR (500 MHz, acetone-D6) δ (ppm) 3.29
(m, 2H), 3.35 (m, 2H), 5.27 (m, 1H), 7.79 (t, J=8.0 Hz, 2H), 7.90 (s, 2H), 7.99 (d,
J=8.0 Hz, 2H), 8.27 (d, J=8.0 Hz, 2H), 8.36 (s, 2H). 13C-NMR (125 MHz, acetone-
D6) δ (ppm) 34.2, 69.0, 125.2, 126.2, 126.6, 131.9, 136.4, 137.8, 137.9, 139.0, 188.6.
MS: m/z calculated for C20H16N2O6: see Table 1.
(2E,6E)‑2,6‑Bis(4′‑hydroxy‑3′‑methoxybenzyliden1e)‑4‑hydroxycyclohexanone
(6m) Overall yield: 86%. Mp: 217–218 °C (MeOH). H-NMR (500 MHz, DMSO-
D6) δ (ppm) 2.89 (m, 2H), 3.06 (m, 2H), 3.81 (s, 6H), 3.98 (s, 1H), 6.86 (m, 2H),
7.03 (m, 2H), 7.11 (s, 2H), 7.60 (s, 2H). 13C-NMR (125 MHz, DMSO-D6) δ (ppm)
36.6, 56.2, 64.2, 115.3, 116.0, 124.5, 127.4, 131.4, 137.8, 147.9, 148.3, 188.3. MS:
m/z calculated for C22H22O6: see Table 1.
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I. Huber et al.
Table 1 MALDI-TOF MS data of the newly synthesized compounds. The presence of [M-H]+ ions
proves the hydride transfer, the [M+H]+ the proton transfer mechanism of fragmentation in positive
mode
Compound
[M-H]+
[M+H]+
[M]+
Calculated
Observed
Calculated
Observed
Calculated
Observed
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
15
18a
18b
18c
18d
19
289.123
357.046
424.967
357.046
357.061
379.093
379.093
379.093
375.207
349.144
409.137
349.144
381.134
317.154
291.113
291.113
341.154
633.228
350.095
418.017
410.116
436.179
365.094
289.127
357.030
424.950
357.059
357.036
379.086
379.865
379.149
375.200
349.146
409.135
349.171
381.138
317.187
291.161
291.089
341.158
633.015
350.160
418.018
410.126
436.149
365.144
291.139
359.061
426.983
359.061
359.061
381.109
381.109
381.109
378.264
351.160
411.181
351.160
383.150
319.170
293.129
293.129
343.170
635.244
352.111
420.033
412.132
438.195
367.110
291.172
359.030
426.953
359.060
359.036
381.025
381.955
381.115
377.256
351.163
411.145
351.176
383.155
319.209
293.179
293.138
343.154
635.201
352.162
420.024
412.127
438.182
367.148
290.131
358.053
425.975
358.053
358.053
380.101
380.101
380.101
376.215
350.152
410.173
350.152
382.142
318.162
292.121
292.121
342.162
634.236
351.103
419.025
411.124
437.187
366.102
290.126
358.030
426.953
358.026
358.034
380.119
380.117
380.155
376.252
350.154
410.145
350.179
382.094
318.196
292.168
292.115
342.169
634.061
351.106
419.020
411.124
437.158
366.146
(2E,6E)‑2,6‑Bis[(3′‑pyridinyl)methylene]‑4‑hydroxycyclohexanone (6o) Overall yield:
77%. Mp: 198–199 °C (MeOH). 1H-NMR (500 MHz, DMSO-D6) δ (ppm) 2.94 (m,
2H), 2.98 (m, 2H), 4.08 (s, 1H), 4.97 (m, 1H), 7.49 (m, 2H), 7.68 (s, 2H), 7.96 (m,
2H), 8.57 (m, 2H), 8.74 (s, 2H). 13C-NMR (125 MHz, DMSO-D6) δ (ppm) 35.5,
62.8, 123.5, 131.1, 133.7, 135.5, 136.8, 149.2, 150.9, 187.8. MS: m/z calculated for
C18H16N2O2: see Table 1.
(2E,6E)‑2,6‑Bis[(4′‑pyridinyl)methylene]‑4‑hydroxycyclohexanone (6p) Overall yield:
82%. Mp: 212–213 °C (MeOH). 1H-NMR (500 MHz, DMSO-D6) δ (ppm) 2.96 (m,
2H), 2.99 (m, 2H), 4.10 (m, 1H), 5.01 (m, 1H), 7.49 (d, J=5.9 Hz, 4H), 7.59 (s, 2H),
8.65 (d, J=5.9 Hz, 4H). 13C-NMR (125 MHz, DMSO-D6) δ (ppm) 35.3, 39.4, 62.5,
124.0, 134.3, 137.2, 142.4, 149.8, 188.1. MS: m/z calculated for C18H16N2O2: see
Table 1.
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A novel cluster of C5-curcuminoids: design, synthesis,…
General method for the synthesis of compounds 6a, 6b, 6c, 6d, 6e, 6i, 6j, 6k, 6l, 6n,
6q (Method B: base‑catalyzed Claisen‑Schmidt condensation)
A solution of 0.1 mol (1.141 g) 4-hydroxycyclohexanone and 0.2 mol of the
appropriate arylaldehyde in 20 mL of methanol was treated with a solution of
0.3 mol (1.683 g) potassium hydroxide in 20 mL of water. This mixture was
stirred at room temperature on a magnetic stirrer until the starting materials
couldn’t be detected on the TLC plate from the mother liquor. The reaction mix-
ture was then diluted with 100 mL water. The precipitated yellow solid was col-
lected on a glass flter and washed with water. The yellow solid was dried, and
recrystallized from a suitable solvent.
(2E,6E)‑2,6‑Bis(benzylidene)‑4‑hydroxycyclohexanone (6a) Overall yield: 88%. Mp:
1
139–140 °C (MeOH). H-NMR (500 MHz, CDCl3) δ (ppm) 1.94 (s, 1H), 2.98
(m, 2H), 3.19 (m, 2H), 4.12 (m, 1H), 7.45 (br m, 10H), 7.88 (s, 2H). 13C-NMR
(125 MHz, CDCl3) δ (ppm) 36.5, 65.6, 128.4, 128.8, 130.3, 132.3, 135.5, 139.3,
188.8. MS: m/z calculated for C20H18O2: see Table 1.
(2E,6E)‑2,6‑Bis(4′‑chlorobenzylidene)‑4‑hydroxycyclohexanone (6b) Overall yield:
1
86%. Mp: 167–168 °C (MeOH). H-NMR (500 MHz, CDCl3) δ (ppm) 2.97 (dd,
J=1.7, 7.8 Hz, 1H), 3.00 (dd, J=1.7, 7.8 Hz, 1H), 3.14 (s, 1H), 3.17 (s, 1H), 4.18
(m, 1H), 7.38 (s, 8H), 7.82 (s, 2H). 13C-NMR (125 MHz, CDCl3) δ (ppm) 36.4,
65.4, 128.8, 131.5, 132.6, 133.9, 135.0, 138.1, 188.3. MS: m/z calculated for
C20H16Cl2O2: see Table 1.
(2E,6E)‑2,6‑Bis(2′,4′‑dichlorobenzylidene)‑4‑hydroxycyclohexanone
(6c) Overall
1
yield: 82%. Mp: 180–182 °C (MeOH/CHCl3). H-NMR (500 MHz, DMSO-D6) δ
(ppm) 2.81 (m, 2H), 2.92 (m, 2H), 4.00 (m, 1H), 4.96 (s, 1H), 7.53 (br m, 4H),
7.71 (s, 2H), 7.76 (s, 2H). 13C-NMR (125 MHz, DMSO-D6) δ (ppm) 35.3, 63.1,
127.2, 129.1, 131.8, 132.3, 132.7, 133.9, 134.8, 136.0, 187.6. MS: m/z calculated for
C20H14Cl4O2: see Table 1.
(2E,6E)‑2,6‑Bis(3′‑chlorobenzylidene)‑4‑hydroxycyclohexanone (6d) Overall yield:
1
85%. Mp: 120–122 °C (EtOH). H-NMR (500 MHz, CDCl3) δ (ppm) 2.99 (d,
J=7.6 Hz, 1H), 3.02 (d, J=7.6 Hz, 1H), 3.14 (s, 1H), 3.17 (s, 1H), 4.19 (m, 1H),
7.32 (br m, 6H), 7.41 (s, 2H), 7.80 (s, 2H). 13C-NMR (125 MHz, CDCl3) δ (ppm)
36.3, 65.3, 128.4, 128.9, 129.7, 129.8, 133.2, 134.4, 137.2, 138.0, 188.2. MS: m/z
calculated for C20H16Cl2O2: see Table 1.
(2E,6E)‑2,6‑Bis(2′‑chlorobenzylidene)‑4‑hydroxycyclohexanone (6e) Overall yield:
1
80%. Mp: 152–153 °C (MeOH). H-NMR (500 MHz, CDCl3) δ (ppm) 2.85 (m,
2H), 3.02 (m, 2H), 4.11 (m, 1H), 7.26–7.32 (br m, 6H), 7.44 (m, 2H), 8.02 (s, 2H).
13C-NMR (125 MHz, CDCl3) δ (ppm) 36.5, 65.9, 126.4, 129.8, 129.9, 130.3, 134.0,
134.1, 135.0, 136.7, 188.1. MS: m/z calculated for C20H16Cl2O2: see Table 1.
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I. Huber et al.
(2E,6E)‑2,6‑Bis(4′‑dimethylaminobenzylidene)‑4‑hydroxycyclohexanone (6i) Overall
1
yield: 78%. Mp: 264–265 °C (MeOH/CHCl3). H-NMR (500 MHz, DMSO-D6) δ
(ppm) 2.83 (m, 2H), 2.98 (s, 12H), 3.07 (m, 2H), 3.92 (s, 1H), 4.91 (s, 1H), 6.77 (d,
J=8.3 Hz, 4H), 7.41 (d, J=8.3 Hz, 4H), 7.56 (s, 2H). 13C-NMR (125 MHz, DMSO-
D6) δ (ppm) 36.5, 56.3, 64.0, 111.6, 122.9, 129.0, 132.0, 137.1, 150.3, 187.2. MS:
m/z calculated for C24H28N2O2: see Table 1.
(2E,6E)‑2,6‑Bis(4′‑methoxybenzylidene)‑4‑hydroxycyclohexanone (6j) Overall yield:
1
81%. Mp: 162–163 °C (MeOH/CHCl3). H-NMR (500 MHz, CDCl3) δ (ppm) 2.96
(dd J=1.6, 8.4 Hz, 1H), 2.99 (dd J=1.6, 8.4 Hz, 1H), 3.21 (d, J=3.0 Hz, 1H), 3.24
(d, J=3.0 Hz, 1H), 3.84 (s, 6H), 4.14 (m, 1H), 6.93 (d, J=8.7 Hz, 4H), 7.44 (d,
J=8.7 Hz, 4H), 7.85 (s, 2H). 13C-NMR (125 MHz, CDCl3) δ (ppm) 36.7, 55.3, 66.0,
114.0, 128.3, 130.4, 132.2, 138.9, 160.2, 188.6. MS: m/z calculated for C22H22O4:
see Table 1.
(2E,6E)‑2,6‑Bis(3′,4′‑dimethoxybenzylidene)‑4‑hydroxycyclohexanone (6k) Overall yield:
82%. Mp: 158–159 °C (MeOH). 1H-NMR (500 MHz, CDCl3) δ (ppm)2.99 (dd, J=1.6,
8.2 Hz, 1H), 3.02 (dd, J=1.6, 8.2 Hz, 1H), 3.21 (d, J=2.4 Hz, 1H), 3.24 (d, J=2.4 Hz,
1H), 3.90 (s, 6H), 3.91 (s, 6H), 4.17 (m, 1H), 6.92 (d, J=8.4 Hz, 2H), 6.99 (d, J=1.6 Hz,
2H), 7.09 (dd, J=1.6, 8.4 Hz, 2H), 7.83 (s, 2H). 13C-NMR (125 MHz, CDCl3) δ (ppm)
36.7, 55.9, 56.0, 65.8, 111.0, 113.8, 123.9, 128.5, 130.6, 139.2, 148.7, 149.9, 188.5. MS:
m/z calculated for C24H26O6: see Table 1.
(2E,6E)‑2,6‑Bis(3′‑methoxybenzylidene)‑4‑hydroxycyclohexanone (6l) Overall yield:
1
82%. Mp: 95–96 °C (MeOH). H-NMR (500 MHz, CDCl3) δ (ppm) 3.01 (m, 2H),
3.20 (m, 2H), 3.83 (s, 6H), 4.15 (m, 1H), 6.91 (d, J=8.2 Hz, 2H), 6.97 (s, 2H),
7.04 (d, J=8.2 Hz, 2H), 7.32 (m, 2H), 7.85 (s, 2H). 13C-NMR (125 MHz, CDCl3)
δ (ppm) 36.5, 55.3, 65.7, 114.5, 115.7, 122.7, 129.4, 132.6, 136.9, 139.3, 159.5,
188.7. MS: m/z calculated for C22H22O4: see Table 1.
(2E,6E)‑2,6‑Bis(4′‑methylbenzylide1ne)‑4‑hydroxycyclohexanone (6n) Overall yield:
86%. Mp: 155–156 °C (MeOH). H-NMR (500 MHz, DMSO-D6) δ (ppm) 2.34 (s,
6H), 2.91 (m, 2H), 3.05 (m, 2H), 3.99 (m, 1H), 4.93 (m, 1H), 7.28 (d, J=8.0 Hz,
4H), 7.43 (d, J=8.0 Hz, 4H), 7.63 (s, 2H). 13C-NMR (125 MHz, DMSO-D6) δ
(ppm) 20.9, 35.9, 63.3, 129.1, 130.2, 132.5, 132.9, 138.8, 138.5, 188.1. MS: m/z
calculated for C22H22O2: see Table 1.
(2E,6E)‑2,6‑Bis(cinnamylidene)‑4‑hyd1roxycyclohexanone (6q) Overall yield: 87%.
Mp: 195–196 °C (MeOH/CHCl3). H-NMR (500 MHz, CDCl3) δ (ppm) 2.87 (m,
2H), 3.11 (m, 2H), 4.28 (m, 1H), 7.05 (br m, 4H), 7.31 (m, 2H), 7.36 (m, 4H), 7.50
(m, 4H), 7.56 (m, 2H). 13C-NMR (125 MHz, CDCl3) δ (ppm) 35.2, 65.7, 123.3,
127.3, 128.8, 129.0, 131.6, 136.6, 138.6, 141.7, 187.3. MS: m/z calculated for
C24H22O2: see Table 1.
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A novel cluster of C5-curcuminoids: design, synthesis,…
General method for the synthesis of compounds 18a–d and 19
The appropriate bis(benzylidene)-cyclanone 17a–d or 6a (2 mmol) was dissolved
in a warm (50–60 °C) solution of 35 mL dry toluene and triethylamine (2 mmol,
0.202 g). After removal of heating 2 mmol (0.226 g) chloroacetyl chloride in
10 ml of dry toluene was added dropwise to the above solution in 30 min under
continuous stirring. Magnetic stirring was continued until the disappearance of
the starting materials from the TLC plate. The mixture was evaporated to dry-
ness under reduced pressure. The residue was triturated with 50 mL water and the
precipitated yellow solid fltered of. After washing the solid several times with
water the crystals were dried and recrystallized from a suitable solvent.
1‑(α‑Chloroacetyl)‑(3E,5E)‑3,5‑bis(benzylidene)‑piperidin‑4‑one (18a) Prepared from
(3E,5E)-3,5-bis(benzylidene)-piperidin-4-one (17a) [28]. Overall yield: 83%. Mp:
1
145–146 °C (toluene). H-NMR (500 MHz, CDCl3) δ (ppm) 3.88 (s, 2H), 4.78 (s,
2H), 4.91 (s, 2H), 7.40 (br m, 10H), 7.84 (s, 1H), 7.89 (s, 1H). 13C-NMR (125 MHz,
CDCl3) δ (ppm) 40.6, 43.9, 46.7, 128.7, 128.9, 129.6, 129.7, 130.0, 130.5, 130.9,
134.2, 134.3, 137.8, 138.8, 165.2, 185.9. MS: m/z calculated for C21H18ClNO2: see
Table 1.
1‑(α‑Chloroacetyl)‑(3E,5E)‑3,5‑bis(4′‑chlorobenzylidene)‑piperidin‑4‑one (18b) Pre-
pared from (3E,5E)-3,5-bis(4′-chlorobenzylidene)-piperidin-4-one (17b) [28]. Over-
all yield: 81%. Mp: 169–170 °C (EtOH). 1H-NMR (500 MHz, CDCl3) δ (ppm) 3.91
(s, 2H), 4.77 (s, 2H), 4.88 (s, 2H), 7.28–7.48 (br m, 8H), 7.79 (s, 1H), 7.83 (s, 1H).
13C-NMR (125 MHz, CDCl3) δ (ppm) 40.6, 43.8, 46.9, 129.2, 129.3, 131.2, 131.3,
131.7, 132.7, 132.8, 136.0, 136.1, 136.8, 137.7, 165.2, 185.5. MS: m/z calculated for
C21H16Cl3NO2: see Table 1.
1‑(α‑Chloroacetyl)‑(3E,5E)‑3,5‑bis(4′‑methoxybenzylidene)‑piperidin‑4‑one (18c) Pre-
pared from (3E,5E)-3,5-bis(4′-methoxybenzylidene)-piperidin-4-one (17c) [28].
Overall yield: 80%. Mp: 162–163 °C (MeOH). 1H-NMR (500 MHz, CDCl3) δ (ppm)
3.82 (s, 6H), 3.92 (s, 2H), 4.75 (s, 2H), 4.88 (s, 2H), 6.93 (d, J=7.9 Hz, 2H), 6.96
(d, J=7.9 Hz, 2H), 7.33 (d, J=7.9 Hz, 2H), 7.41 (d, J=7.9 Hz, 2H), 7,77 (s, 1H),
7.80 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm) 40.9, 44.1, 47.0, 55.4, 114.4,
114.5, 127.0, 127.3, 128.9, 129.1, 132.2, 132.7, 137.4, 138.4, 160.9, 165.2, 185.8.
MS: m/z calculated for C23H22ClNO4: see Table 1.
1‑(α‑Chloroacetyl)‑(3E,5E)‑3,5‑bis(4′‑dimethylaminobenzylidene)‑piperidin‑4‑one
(18d) Prepared from (3E,5E)-3,5-bis(4′-dimethylaminobenzylidene)-piperidin-4-
1
one (17d) [28]. Overall yield: 85%. Mp: 223–224 °C (EtOH). H-NMR (500 MHz,
CDCl3) δ (ppm) 3.05 (m, 6H), 3.97 (s, 2H), 4.80 (s, 2H), 4.94 (s, 2H), 6.72 (m,
4H), 7.34 (m, 2H), 7.42 (m, 2H), 7.78 (s, 1H), 7.81 (s, 1H). 13C-NMR (125 MHz,
CDCl3) δ (ppm) 40.0, 41.0, 44.3, 47.1, 111.9, 122.3, 122.8, 126.6, 126.9, 132.4,
133.0, 137.7, 138.8, 151.0, 165.2, 185.7. MS: m/z calculated for C25H28ClN3O2: see
Table 1.
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I. Huber et al.
(2E,6E)‑2,6‑Bis(benzylidene)‑4‑(α‑chloroacetoxy)‑cyclohexanone (19) Prepared from
(2E,6E)-2,6-bis(benzylidene)-cyclohexan-4-one (6a). Overall yield: 85%. Mp: 159–
1
160 °C (MeOH). H-NMR (500 MHz, CDCl3) δ (ppm) 3.22 (m, 4H), 3.97 (s, 2H),
5.31 (m, 1H), 7.35–7.47 (br m, 10 H), 7.94 (s, 2H). 13C-NMR (125 MHz, CDCl3) δ
(ppm) 32.8, 40.8, 69.7, 128.6, 129.1, 130.2, 130.8, 135.2, 140.0, 166.6, 187.8. MS:
m/z calculated for C22H19ClO3: see Table 1.
General method for the synthesis of compounds 15 and 16
Oxaloyl chloride (2.5 mmol) was added to a stirred suspension of 5 mmol of the cor-
responding bis(benzylidene)cyclanone and 50 mL of dry toluene containing 5 mmol
triethylamine at roughly 20 °C. The reaction mixture was stirred and left overnight
at room temperature. Then this the mixture was diluted with 50 mL of water. The
precipitated yellow solid was collected on a glass flter, washed thoroughly with
water, dried and recrystallized from methanol.
1,2‑Bis[(2E,6E)‑2,6‑bis(benzylidene)‑4‑oxycyclohexanone‑4‑yl]ethane‑1,2‑dione
(15) Prepared from compound 6a. Overall yield: 85%. Mp: 247–248 °C (MeOH).
1H-NMR (500 MHz, CDCl3) δ (ppm) 3.17 (m, 4H), 3.30 (m, 4H), 5.24 (s, 2H),
7.30–7.49 (br m, 20H), 7.93 (s, 4H). 13C-NMR (125 MHz, CDCl3) δ (ppm) 32.7,
71.4, 128.6, 129.1, 130.2, 130.5, 135.1, 140.2, 156.6, 187.4. MS: m/z calculated for
C42H34O6: see Table 1.
1,2‑Bis[(3E,5E)‑3,5‑bis(benzylidene)‑4‑piperidon‑1‑yl]ethane‑1,2‑dione (16) Prepared
from (3E,5E)-3,5-bis(benzylidene)-piperidin-4-one (17a) [28]. Overall yield: 88%.
Mp: 243–244 °C; Lit.: 245–246 °C [18].
2,4‑Dimethyl‑1,5‑diphenylpenta‑1,4‑dien‑3‑one (2e) (4) This compound was prepared
according to a known method [45]. Overall yield: 40%. Mp: 125–127 °C; Lit.: 127–
128 °C [45].
1‑Methyl‑(3E,5E)‑3,5‑bis(benzylidene)‑4‑piperidone (5a) and 1‑Methyl‑(3E,5E)‑3,5‑bis(4
′‑chlorobenzylidene)‑4‑piperidone (5b) These compounds were prepared according
to a known method [28]. Mp: 5a – 116–117 °C; Lit.: 116–117 °C [28]. 5b – 183–
184 °C; Lit.: 184–185 °C [28].
(2E,6E)‑2,6‑Bis(benzylidene)‑4‑methylcyclohexanone (7a) and (2E,6E)‑2,6‑Bis(4′‑ch
lorobenzylidene)‑4‑methylcyclohexanone (7b) These compounds were prepared
according to a known methods [46] and [47], respectively. Mp: 7a – 98–99 °C; Lit.:
98–99 °C [46]. 7b – 163–164 °C; Lit.: 156–160 °C [47].
Determination of antiproliferative action in vitro
The antiproliferative properties of the synthesized compounds were determined
against a panel of human adherent cancer cell lines. Briefy, MDA-MB-231 (breast),
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A novel cluster of C5-curcuminoids: design, synthesis,…
C33A (cervical) and A2780 (ovarian) cancer cell lines cell were purchased from
European Collection of Cell Cultures (ECCAC, Salisbury, UK) and maintained
in minimal essential medium (MEM) supplemented with 10% fetal bovine serum
(FBS), 1% non-essential amino acids and an antibiotic–antimycotic mixture (Lonza
Group Ltd., Basel, Switzerland). Near-confuent cancer cells were seeded onto a
96-well microplate (5000/well) and after overnight standing new medium, contain-
ing the tested compounds at 0.3, 3 and 30 µM, was added. After incubation for 72 h
at 37 °C in humidifed air with 5% CO2, the viability of the cells were determined
by the addition of 20 µL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) solution. The mitochondrial reductase of the intact cells
metabolized the MTT and the produced formazan was precipitated as purple crys-
tals during a 4-h contact period. The formazan was dissolved in 100 µL of DMSO
during a 60-min period of shaking at 37 °C assayed at 545 nm, using a microplate
reader [50]. In the case of the most active compounds the assays were repeated with
a set of dilutions (0.03–10 mM), concentration–response curves were ftted to the
generated data and the IC50 values were calculated by means of GraphPad Prism 4.0
(GraphPad Software, San Diego, CA, USA). The experiments were carried out on
two microplates with fve parallel wells. Stock solutions of the tested compounds
(10 mM) were prepared in DMSO. The highest DMSO content of the medium
(0.3%) did not have any substantial efect on the cell proliferation. Cisplatin (Ebewe
Pharma GmbH, Unterach, Austria) was used as a reference agent.
Physiscochemical calculations
For the prediction of some physicochemical parameters of our compounds we have
used the Calculator Plugins of ChemAxon. The ClogP values were calculated with
the Klopman method and database of the software. The ClogP, pKa and solubility
values are results at physiological pH=7.4. See the corresponding homepage for
detailed conditions of the calculations [43]. To explain the relationship between bio-
data and calculated physicochemical parameters the Lipinski approaches have been
used [44].
Results and discussion
Design and synthesis
Our purpose is to give further insight into the infuence of structural modifca-
tions at position 4 of the cyclic ketone scafold on the biological efect. We proved
among other things [28] that this position, being an auxiliary binding ability (sec-
ondary pharmacophore) to the in vivo biological site of action, has great infu-
ence on the cytotoxicity of these compounds. In other words, the binding strength
of the C5-curcuminoids to the biological site of action is weak when merely the
pentadienone moiety reacts. The substituent at position 4 on the cyclanone ring is
able to enhance the interaction between the C5-curcuminoids and their biological
1 3

I. Huber et al.
place of action in the cancer cell. Therefore, we set out to synthesize compounds
4–7 for comparative structure–activity relationship (Fig. 2) investigation.
Syntheses of compounds in series 5–7 and 15–19 are outlined in Schemes 1,
2, 3 and 4. Briefy, compound 4 and the bis(arylidene)-cyclanones 5–7 were syn-
thesized via acid- or base-catalyzed condensation of diethylketone, 4-hydroxy-
cyclohexanone, 4-methylcyclohexanone or 4-piperidone hydrochloride with aryl
aldehydes according to the Claisen-Schmidt condensation protocol [28].
Another objective was the synthesis of a new branch of cyclic C5-curcuminoid
with a hydroxyl functionality at position 4 (6) and derivatives of series 6 with
diferent aryl substituents (Schemes 1,2, 4.) to receive data on the impact of the
corresponding structural changes on cytotoxicity.
The optimal para substituents (H, Cl, OCH3, NO2, OH, dimethylamino) on the
arylidene moiety was selected according to a previous disclosure on this substitu-
ent efect [29]. “Optimal” means that the chosen substituents on the arylidene
group have diferent electronic, hydrophobic and steric properties ftting well to
Craig plot for para substituents assuring the divergence of the σp and π values
[30, 31]. Special aromatic aldehydes like pyridincarbaldehyde and cinnamalde-
hyde were also used (Scheme 2).
The synthesis of the dimeric compound 15 was performed by acylation of the
monomer 6a with oxaloyl chloride in the presence of triethylamine analogously
to compound 16, which was prepared by others earlier [18] (Scheme 3).
Chloroacetyl derivatives 18 and 19 were obtained from their corresponding
dibenzylidene precursors 6a and 17a–d with chloroacetyl chloride in the presence
of trimethylamine (Scheme 4).
Fig. 2 Acyclic (4) and cyclic (5–7) C5-curcuminoids
1 3

A novel cluster of C5-curcuminoids: design, synthesis,…
Scheme 1 The synthesis of series 6 from 4-hydroxycyclohexanone and aromatic aldehydes. Compound
12 was prepared according to a known method [38]
Scheme 2 The synthesis of derivatives 6o-q
Structural characterization of the title compounds
1
Structures of the compounds synthesized were established by means of H, 13C
NMR and MS measurements. The NMR spectra of compounds in series 6 were
similar and simple in appearance. The E,E-confguration of the newly synthesized
compounds was determined by 1H NMR [32]. The E,E-confguration is due to the
1 3

I. Huber et al.
Scheme 3 The synthesis of the diester derivative 15
Scheme 4 The synthesis of chloroacetyl derivatives 18 and 19
anisotropic efect of the carbonyl group on the vinyl protons, which is not possi-
ble in the Z,Z-confgurational forms.
Our research group is using, among others, matrix-assisted laser desorption ioni-
zation (MALDI) technique for single-stage MS analysis. Because of its large mass
range, the device is a time-of-fight spectrometer (MALDI-TOF). This technique
1 3

A novel cluster of C5-curcuminoids: design, synthesis,…
was used also in our previous study on cyclic C5-curcuminoids [28]. We have inves-
tigated a number of analytes in the series of 3,5-dibenzylidene-4-piperidones and
2,6-dibenzylidene-1-cyclanones. All compounds showed characteristic molecular
ion peaks (Table 1). In general we can state that the diagnostic product ions were not
only fragments, but also dimers or sometimes trimers of the molecular ions in their
MS spectra. The dimerization is a common photochemical [2+2] cycloaddition.
The photodimerisation, as a type of autodimerization, is well-known in this family
of compounds [33–36]. The reaction proceeds via a stepwise sequence. The product
is a dispiro cyclobutane derivative in general, as is compound 20 in our case. We
have selected compound 6a to demonstrate what happens due to our general opinion
(Scheme 5) regardless of the type of the central cyclanone core in the structure.
Mention must be made about the fact that it was not necessary to use a matrix.
These curcuminoids are able to ionize themselves. They can behave as their own
matrix in the presence of the laser shots.
There is a recent report on the unexpected formation of [M-H]+ ion dur-
ing MALDI MS analysis of such cyclic C5-curcuminoids [37]. This ion can form
under chemical experimental conditions [33–36] in the photochemical reaction too,
together with the [M+H]− ion in a hydride transfer reaction. Therefore, it is not a
surprise that the dimerization (2+2 cycloaddition) takes place. A very similar reac-
tion is observable if we look at the proton transfer reaction. These two processes,
hydride and/or proton transfer makes the cycloaddition possible. The molecular ion
of dimeric compound 20 (or the dimeric fragment after loss of Cl or NO2 in the case
of derivatives 18 or 6f) is there in the MALDI-TOF MS spectra of 6a. Similarly, all
the product ions in Scheme 5 are there in the corresponding negative or positive ion
mode records. The analytically useful diagnostic product ions are of importance for
example when we analyze samples from complex biological matrices for identity or
quantity of these C5-curcuminoids. That is why we propose to consider the dimeric
ion or its fragment too for the mass spectrometric fngerprint analysis of these
C5-curcuminoids in a MALDI-TOF technique for the future biological research.
Antiproliferative efects in vitro
The primary objective of the present work stems from our interest to study
structure–activity relationships concerning these antiproliferative synthetic
C5-curcuminoids. The biodata presented in Fig. 3 reveals that the majority of the
newly described C5-curcuminiod derivatives exert pronounced antiproliferative
activities as evaluated against the following three human adherent cancer cell lines:
A2780 (ovarian), C33A (cervix) and MDA-MB-231 (breast). Data in Fig. 3 indicate
the percentage of cell-growth-inhibition of the diferent cell lines in three diferent
concentrations (0.3 μM, 3 μM and 30 μM) of the corresponding compound.
Comparisons were made between the cytotoxic properties of compounds 4–7
(Fig. 2). Firstly, we can see a clear sequence among the structures in Fig. 2 regard-
ing their activity. The heterocyclic derivatives 5a and 5b dominates the others in
the case of all three cell lines. The second position is shared by the two homocyclic
derivatives 6 and 7 favoring the hydroxyl-substituted compounds 6a and 6b in the
1 3

I. Huber et al.
Scheme 5 The formation of dimer 20 from 6a under MALDI-TOF conditions (see experimental) in MS
analysis. The related product ions of 6a observed: 289.127/[M-H]+; 291.172/[M+H]+; 290.126/[M]
and the dimer 577.124/[M-3H]+; 580.119 [M]. In negative mode: 289.062/[M-H]−; 291.176/[M+H]−;
290.120/[M]
1 3

A novel cluster of C5-curcuminoids: design, synthesis,…
case of the C33A (cervix) and A2780 (ovarian) cancer cells. The inhibition activ-
ity of the acyclic compound 4 in these two concentrations is negligible. Hence, the
structural modifcations in the ketone core of compounds 4–7 presented in Fig. 2
provided the following sequence in cell-growth-inhibition: N-methylpiperidones
(5a, b)>4-hydroxycyclohexanones (6a, b)>4-methylcyclohexanones (7a, b)>acy-
clic ketone (4).
On the other hand, there is also a sequence in the structure–activity relation of
the diferent aromatic substituents in series 6. There is a hydroxyl substituent in all
the structures at position 4 of the newly synthesized 2,6-dibenzylidene-4-hydroxy-
cyclohexanone 6a-m derivatives. The most active compounds are the nitro-substi-
tuted 6f (para), 6g (ortho) and 6h (meta) derivatives with nearly 100% inhibition
in almost all cases even at 3 μM. The meta-nitro 6h counterpart shows the high-
est score, the other two isomers 6f and 6g are slightly weaker on C33A (cervix)
cells. The second in this sequence is the pyridylidene substituent in derivatives 6o
and 6p with better inhibition of the 4′-pyridyl derivative 6p on all three cell lines.
The third position is occupied by the chloro substituent in compounds 6b (para), 6c
(ortho,para–dichloro), 6d (meta) and 6e (ortho). The chloro-substituted subgroup
is pharmacologically dominated by the meta-chloro 6d isomer with close to 100%
inhibition on all cell lines. Interestingly, two of the most active cytotoxic derivative
6d and 6h has an electron-withdrawing meta-substituent in its structure. Their calcu-
lated IC50 values are as follows (see also Table 2): 6d: 2.46 (A2780), 1.38 (C33A),
1.71 (MDA-MB-231); 6h: 0.68 (A2780), 0.69 (C33A), 0.92 (MDA-MB-231).
Unexpectedly, the replacement of the previously mentioned substituents to meth-
oxy group(s) in case of compounds 6j-m led to a lower inhibition percentage. These
derivatives showed substancial inhibition only in the 30 μM concentration in almost
all cases. Finally, the replacement of the para substituent on the aromatic rings by
dimethylamino (6i), methyl (6n) or the exchange of the benzylidene groups to a
longer aromatic obtained from cinnamaldehyde (6q), respectively, resulted in virtu-
ally inactive compounds. With the increase of the number of double bonds and the
length of the spacer (from C5 up to C9) between the two aromatic rings in the struc-
ture of 6q the antiproliferative activity dropped to negligible.
These observations reveal that the electronic, hydrophobic and steric properties
of the aryl substituents infuence the magnitude of the inhibition ability of the com-
pounds. The relative potencies of the most active compounds in series 6 with respect
of the substituents on the aryl rings is meta-NO2 (6h)>4′-pyridyl (6p)>meta-Cl
(6d)>ortho-NO2 (6g)>para-NO2 (6f)>3′-pyridyl (6o)>hydrogen (6a). Some
selected IC50 calculated values (see also Table 2) - 6d: 2.46 (A2780), 1.38 (C33A),
1.71 (MDA-MB-231); Average: 1.85. 6p: 0.76 (A2780), 2.69 (C33A), 1.28 (MDA-
MB-231); Average: 1.58. 6h: 0.68 (A2780), 0.69 (C33A), 0.92 (MDA-MB-231);
Average: 0.76.
Only one of the members in series 6 was described earlier. Namely, compound
6f has been identifed as a potent nonselective isopeptidase inhibitor intermediate
in vivo against glioblastoma U87MG cells [42].
Derivatives of compound 6a were also prepared in order to change the structure
of the secondary pharmacophore [28] at position 4 of the molecules with what the
molecule binds to its biological place of action. Two structural modifcations were
1 3

I. Huber et al.
Fig. 3 Evaluation of compounds 4–7, 15, 16, 18 and 19 against human A2780 (ovarian), C33A (cervix)
and MDA-MB-231 (breast) cancer cell lines. The biodata reported here are percentage of cell-growth-
inhibition in diferent concentrations. Cisplatin was used as a reference drug: IC50 =1.30 (A2780); 3.69
(C33A); 19.13 (MDA-MB-231). Some selected IC50 calculated values (see also Table 2)=6d: 2.46
(A2780), 1.38 (C33A), 1.71 (MDA-MB-231); 6p: 0.76 (A2780), 2.69 (C33A), 1.28 (MDA-MB-231);
6h: 0.68 (A2780), 0.69 (C33A), 0.92 (MDA-MB-231); 16 [18]: 0.22 (A2780), 0.43 (C33A), 0.37 (MDA-
MB-231); 18c: 1.34 (A2780), 1.51 (C33A), 1.00 (MDA-MB-231). There was only negligible inhibition
noted in all cases of compounds in 0.3 μM concentration except for compounds 18 and 19. Inhibition
data of curcumin: A2780, 85% in 30 μM [39]; C33A, 22% in 30 μM [40]; MDA-MB-231, 50% in 30 μM
[41]
▸
accomplished on 6a: one of these acylation reactions provided the dimeric form
15 (Scheme 3) while the other one the chloroacetyl derivative 19 (Scheme 4). It
was possible to increase the efcacy of 6a with the introduction of the chloroacetyl
moiety into the structure and inhibition ability grew dramatically from the 30 μM
concentration of 6a down to the 0.3 μM of 19. The diference in inhibition abil-
ity of compounds 5 and 6 (Fig. 2) could have been equalized due to this transfor-
mation from 6a to 19 comparing biodata of chloroacetyl derivatives 18a–d and 19.
The ester type compound 19, according to biodata in Fig. 3, reached the relative
inhibition potential of the corresponding heterocyclic amide analogues 18a–d on all
the three cell cultures. The diference in their inhibition activity can be seen in the
0.3 μM concentration range, only (Fig. 3). The other attempt to modify the second-
ary binding part of 6a, however (Scheme 4), did not bring a positive change in anti-
proliferative action. The diester 15 proved to be less active compared to the analo-
gously synthesized [18] diamide 16.
An evaluation of the biodata was undertaken to see if there was any selectivity
of the compounds on the diferent cell cultures. The results obtained from the IC50
calculations of representative compounds (see the footnotes in Fig. 3) in this inves-
tigation shows there are diferences in inhibition ability on the three cell lines. For
example, compound 6h inhibits the cell-proliferation very actively in both A2780
and C33A, but it is less active in the case of MDA-MB-231 cells. Similarly, the most
refractory cells against compound 6d were in the A2780 culture, and compound 6b
showed the highest inhibition against the A2780 cell line. A similar tendency can be
see among the derivatives with lower performances in the in vitro anticancer screen-
ing process. All in all, we can state there is a selectivity demonstrated by our com-
pounds on the diferent tumor cell lines.
The reference compound of this study was cisplatin. The best performer com-
pounds among the newly synthesized derivatives are 6d, 6p, 6h and 18c. Here we
provide their calculated IC50 values (see also Table 2): cisplatin=1.30 (A2780);
3.69 (C33A); 19.13 (MDA-MB-231); 6d=2.46 (A2780), 1.38 (C33A), 1.71 (MDA-
MB-231); 6p=0.76 (A2780), 2.69 (C33A), 1.28 (MDA-MB-231); 6h=0.68
(A2780), 0.69 (C33A), 0.92 (MDA-MB-231); 18c=1.34 (A2780), 1.51 (C33A),
1.00 (MDA-MB-231). In the case of the MDA-MB-231 cancer cell line, all the new
compounds exceeded the antiproliferative efect of cisplatin, just as in the case of
the C33A tumor cells. The ovarian A2780 cell line showed a similar efect in case of
compound 18c, lower activity with compound 6d while increased cell growth inhi-
bition appeared in the case of 6p and 6h as compared to the reference compound.
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A novel cluster of C5-curcuminoids: design, synthesis,…
MDA-MB-231
(Cell growth inhibiꢀon (%) SEM)
120
100
80
60
40
20
0
-20
0.3 µM
3 µM
30 µM
A2780
(Cell growth inhibiꢀon (%) SEM)
120
100
80
60
40
20
0
-20
0.3 µM
3 µM
30 µM
C33A
(Cell growth inhibiꢀon (%) SEM)
120
100
80
60
40
20
0
-20
0.3 µM
3 µM
30 µM
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I. Huber et al.
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A novel cluster of C5-curcuminoids: design, synthesis,…
Inhibition percentage data of curcumin are known from the literature: A2780,
85% in 30 μM [39]; C33A, 22% in 30 μM [40]; MDA-MB-231, 50% in 30 μM
[41]. We can state, that the majority of the new compounds in series 6, 18 and
derivative 15 and 19 are exceeding these values of curcumin. The compounds
with lower antiproliferative activity are 6i, 6j and 6q in A2780 culture, 6i and 6q
in C33A while 6i and 6q in MDA-MB-231.
DNA binding
CD is a reliable tool for the detection of DNA binding of ligands. The appearance
of the induced circular dichroism signal (ICD) is defnitive proof of the interac-
tion. In addition, shifts in the DNA bands are also indicative of the binding [48,
49].
There is little known about the DNA binding of cyclic C5-curcuminoids. About
curcumin, however, we know more in this respect. It is known from in vitro experi-
ments, for example, that curcumin appears in the nucleus of cultured glioma cells
after incubation. It was revealed that nuclear homing is not a result of curcumin’s
DNA binding [27]. The temporal relationship of curcumin’s apoptotic induction
efect and its nuclear homing is under investigation to acquire details about the
mechanism of action. This fact among others prompted us to initiate measurements
to see possible interactions between C5-curcuminoids and DNA. For this reason, we
conducted circular dichroism spectroscopic investigations on the newly synthesized
cyclic C5-curcuminoid derivatives in series 6 and 18 using natural DNA.
Curcumin, as well as 28 C5-curcuminoid derivatives were tested on chicken
erythrocyte DNS. In the case of curcumin, a large ICD band appeared in the ligand’s
absorption region, showing strong interaction between the molecule and the polynu-
cleotide (Fig. 4). It also caused some minor shift in the DNA band, meaning the dou-
ble helical structure is slightly distorted (Fig. 4). Out of the tested C5-curcuminoid
derivatives (6, 18), those containing either aliphatic or aromatic nitrogen showed
signs of interaction—a weak ICD sign appeared in the recorded spectra for example
in the case of 6i (Fig. 4). DNA bands did not change signifcantly in the same time,
indicating that the polynucleotide remains in its native B-form. The binding is most
probably the result of the ionic bondage of the nitrogen atoms in the ligand structure
with the phosphate groups of DNA. In the case of other derivatives neither an ICD
signal, nor shift in the DNA bands were detected, marking that no interaction occurs
between the molecules (e.g. compounds 6h and 18c in Fig. 4).
Although it would be consistent with an ability of these curcuminoids to adopt
a close-to-planar molecular shape, none of them showed stronger interaction with
the DNA used in this study. It would be conceivable that the very active inhibi-
tor 6h or 18c show diverse interaction comparing to the practically inefective 6i.
However, compounds in series 6 and 18 showed similar properties under CD con-
ditions, that makes possible to generalize: based on these data we conclude that
these derivatives do not bind to DNA in vitro. Hence, their antiproliferative efect
is not due to their interaction with DNA.
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I. Huber et al.
Physicochemical calculations on derivatives of series 6
For an evaluation of some physicochemical properties compared to curcumin the
synthesized compounds were computed. ChemAxon’s Marvin Suite was used for all
of the calculations [43].
The most promising compound from physicochemical point of view (Table 2) is
the 4′-pyridylidene derivative 6p. This compound is one of those, which revealed
the highest cytotoxic activity (6d, 6p, 6h and 18c) against the cancer cell lines, with
a relatively low partition coefcient (ClogP), with the possibility for ionization and
with the highest water-solubility. Its ionization is due to the presence of the two pyr-
idine nitrogen atoms which can be protonated under physiological conditions. The
protonation can increase the solubility in water and decrease lypophilicity resulting
in a possible better penetration through membranes into biological tissues, such as
tumors. These data about this drug-candidate 6p are suggesting its possible higher
bioavailability compared to curcumin consistent with Lipinski rules [44]. Similar
physicochemical properties could be determined on the 3′-pyridylidene counterpart
6o too.
In general, the molecular weight of the newly synthesized derivatives are in the
range of that of curcumin except the highly chlorinated 6c, 18b, the methoxy or
dimethylamino substituted 6k, 18c, 18d and the dimer 15. The calculated partition
coefcient (ClogP) values are usually under 5 (in the majority of cases between 3
and 4) or nearly 5 (6q, 18b, 19), except chlorinated compounds 6b-e and the para-
tolyl substituted 6n. The moderately active dimeric derivative 15 exhibited an
extremely high ClogP value of 9.505.
Those compounds with a slightly basic nitrogen (6i, 6o, 6p and 18d) or with a
phenolic OH group (6m) on the arylidene substituents are slightly less lypophilic
than curcumin. The reason for this diminished lypophilicity can be protonation or
deprotonation. The calculated water-solubility value, however, is increased only in
the case of pyridylidene derivatives 6o and 6p. All of the compounds in Table 2
are able to form 3–7 H-bonds giving the chance to develop more or less weak inter-
actions with macromolecules on the place of biological action. There is no visible
physicochemical reason on the practical inefectiveness of compounds 6i and 6q.
Further investigations about the relationship of biological properties with physico-
chemical behaviors will be undertaken in the near future in our laboratories.
Conclusions
A newly designed branch of bis(arylidene)-4-cyclanones is described here in this
article. The structure of this new group is based on 4-hydroxycyclohexanone, which
was converted to the title-compounds, 2,6-bis(arylidene)-4-hydroxycyclohex-
anone (6) species under the conditions of the Claisen-Schmidt condensation reac-
tion. Series 6 and its derivatives 15, 18, 19 were evaluated against three malignant
experimental cancer cell lines in vitro: A2780 (ovarian), C33A (cervix) and MDA-
MB-231 (breast). Antiproliferative biodata were obtained beside cisplatin as a ref-
erence drug in the form of tumor-growth-inhibition percentage in three diferent
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A novel cluster of C5-curcuminoids: design, synthesis,…
Fig. 4 CD and UV titration of 0.033 mg/ml chicken erythrocyte DNA (black curve) with 5 mM cur-
cumin or curcuminoid stock solution (fnal concentrations are: 16.66 µg/mL, 25 µg/mL and 33.33 µg/mL,
respectively)
concentrations. Structure–activity analysis revealed, that the best performer in this
study is (E,E)-2,6-bis(3′-nitrobenzylidene)-4-hydroxycyclohexanone (6h) with IC50
values of 0.68, 0.69 and 0.92 μM, respectively, compared to cisplatin with 1.30, 3.69
and 19.13 μM. Calculations were performed on computed compounds in order to
obtain a relationship/correlation between biodata and physicochemical parameters.
The best performer in this respect was 6p and 6o, in which case it was possible to
predict improved bioavailability compared to curcumin. Acylation of the functional
group at position 4 with chloroacetyl chloride was successful from pharmacological
point of view. It led us to very efective acyl derivatives. It was possible for instance
to increase the efcacy of 6a with the introduction of the chloroacetyl moiety into
the structure of compound 19: inhibition ability grew dramatically from the 30 μM
concentration down to the 0.3 μM. We have selected some of these derivatives
for CD spectroscopic investigation in order to see whether they show any interac-
tion to natural DNA. Based on our CD spectroscopic data, we conclude that these
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I. Huber et al.
derivatives do not bind to DNA in vitro. Their antiproliferative efect is not due to
their interaction with DNA.
Acknowledgements Open access funding provided by University of Pécs (PTE). This work was sup-
ported by the University of Pécs, Faculty of Medicine Research Fund PTE ÁOK-KA-34039-12/10-11.
Compliance with ethical standards
Confict of interest The authors declare that they have no confict of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Interna-
tional License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if changes were made.
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