Synthesis of curcuminoids and evaluation of their cytotoxic and antioxidant properties

Article abstract of DOI:10.3390/molecules22040633

Curcumin (1) and ten derivatives (2-11) were synthesized and evaluated as cytotoxic and antioxidant agents. The results of primary screening by Sulforhodamine B assay against five human cancer cell lines (U-251 MG, glioblastoma; PC-3, human prostatic; HCT-15, human colorectal; K562, human chronic myelogenous leukemia; and SKLU-1, non-small cell lung cancer) allowed us to calculate the half maximal inhibitory concentration (IC₅₀) values for the more active compounds against HCT-15 and K562 cell lines. Compounds 2 and 10 were the most active against both cell lines and were more active than curcumin itself. Thiobarbituric acid reactive substances (TBARS) assay showed that 7 has potent activity; even stronger than curcumin, α-tocopherol, and quercetin.

Full text of DOI:10.3390/molecules22040633

molecules
Article
Synthesis of Curcuminoids and Evaluation of Their
Cytotoxic and Antioxidant Properties
María Concepción Lozada-García 1 , Raúl G. Enríquez , Teresa O. Ramírez-Apán ,
,‡
2
2
2
1
1
Antonio Nieto-Camacho , Juan Francisco Palacios-Espinosa , Zeltzin Custodio-Galván ,
Olivia Soria-Arteche * and Jaime Pérez-Villanueva *
1
, ,†
1, ,†
1
Departamento de Sistemas Biológicos, División de Ciencias Biológicas y de la Salud,
Universidad Autónoma Metropolitana-Xochimilco (UAM-X.), Ciudad de México 04960, Mexico;
mclozada@correo.xoc.uam.mx (M.C.L.-G.); jpalacios@correo.xoc.uam.mx (J.F.P.-E.);
zeltzin_888@hotmail.com (Z.C.-G.)
2
Instituto de Química, Universidad Nacional Autónoma de México (UNAM), Ciudad de México 04510,
Mexico; enriquezhabib@gmail.com (R.G.E.); mtrapan@yahoo.com.mx (T.O.R.-A.);
anieto@unam.mx (A.N.-C.)
*
Correspondence: soriao@correo.xoc.uam.mx (O.S.-A.); jpvillanueva@correo.xoc.uam.mx (J.P.-V.);
Tel.: +52-5483-7259 (O.S.-A. & J.P.-V. )
†
‡
These authors contributed equally to this work.
This paper was written in memory of Dra. María Concepción Lozada-García.
Academic Editor: D. Hadjipavlou-Litina
Received: 23 February 2017; Accepted: 11 April 2017; Published: 14 April 2017
Abstract: Curcumin (1) and ten derivatives (2–11) were synthesized and evaluated as cytotoxic
and antioxidant agents. The results of primary screening by Sulforhodamine B assay against five
human cancer cell lines (U-251 MG, glioblastoma; PC-3, human prostatic; HCT-15, human colorectal;
K562, human chronic myelogenous leukemia; and SKLU-1, non-small cell lung cancer) allowed us
to calculate the half maximal inhibitory concentration (IC ) values for the more active compounds
50
against HCT-15 and K562 cell lines. Compounds
2 and 10 were the most active against both cell lines
and were more active than curcumin itself. Thiobarbituric acid reactive substances (TBARS) assay
showed that 7 has potent activity; even stronger than curcumin, α-tocopherol, and quercetin.
Keywords: antioxidant; azoles; cytotoxic; curcumin
1
. Introduction
Curcumin, a natural yellow pigment present in the rhizomes of several curcuma species, has been
employed in different Asian countries as a dietary condiment, and it has been used for the treatment
of a variety of diseases in traditional Chinese and Indian medicine [ ]. Recent studies have been
published on the broad biological activities of curcumin [ ], which include its anti-inflammatory,
anti-oxidant, antimicrobial, antiviral, anti-cystic fibrosis, and anticancer properties [ ]. Preclinical
studies have suggested curcumin as a drug candidate, and considerable research efforts have been
carried out to understand its mechanisms of action [ ]. Furthermore, curcumin has been considered
an epigenetic agent, which acts by modulating multiple biological processes at low concentration [ ].
This molecule can bind as many as 33 different proteins, including targets such as transcriptional
factors, growth factors, cytokines, and genes regulating cell proliferation and apoptosis [ ]. However,
the clinical potential of curcumin is limited by its extremely low oral bioavailability, due to its poor
aqueous solubility and its chemical instability [ ]. Several strategies have been proposed to overcome
the low bioavailability of curcumin and increase its solubility. These include the use of adjuvants
1
1,2
2,3
4
5
1,5
6
that block metabolic pathways, encapsulation with nanoparticles [7], liposomes [8], cyclodextrins [9],
Molecules 2017, 22, 633; doi:10.3390/molecules22040633
www.mdpi.com/journal/molecules

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bile salt aggregates as carrier [10], etc. An additional strategy has been the synthesis of analogues
or derivatives to increase its bioavailability or improve certain biological activities. In spite of its
drawbacks, curcumin has been considered a promising lead structure [11] to design new derivatives
with enhanced properties, due to its safety [1,3] and pharmacological potential.
In the last decades, a broad number of analogues and derivatives of curcumin have been
synthesized. These new compounds have shown improved effects as free radical scavengers [12],
cytotoxic agents [13,14], anti-inflammatories, and others [15]. Some also have antitumoral properties,
behaving as antitumor agents in multidrug resistant cancer cells [16], NF-kB activators [17],
antioxidants [18], pro-oxidants [19,20], and antivirals [21].
Curcumin is a diarylheptanoid, with two o-methoxyphenols attached to a
symmetrical olefinic bonds. Several modifications have been made to the curcumin structure,
including derivatives with 1,2-azole rings instead of the -diketone [ ]. It is worth mentioning
β-diketone by two
β
6
that azole curcumin derivatives have shown to be stable at physiological pH, having good
antiproliferative activity against several cancer cell lines and better free radical scavenging activity
than its predecessor [
curcumin and its derivatives depends upon the presence of phenolic groups [24,25].
Herein we synthesized curcumin and ten of its derivatives, 11, to expand the knowledge about
6,11,14,16,22,23]. Several reports have suggested that the anti-oxidant activity of
1
2–
the biological activity of curcuminoids as cytotoxic and antioxidants agents (Figure 1). All compounds
were evaluated as cytotoxic agents in five human cancer cell lines. In view of the previous assays,
the most active compounds were also tested as antioxidants using the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and thiobarbituric acid reactive substances (TBARS) models.
Figure 1. Chemical structure of curcumin and the analogues synthesized.

Molecules 2017, 22, 633
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2
. Results and Discussion
2
.1. Synthesis of Curcumin Derivatives
Compounds
aromatic aldehyde with 2,4-pentanodione and butylamine, by a slight modification of Pabon synthesis
Scheme 1) [11 26]. First, a complex with boric anhydride and 2,4-pentanodione was formed; then,
a mixture of arylaldehyde and tributylborate was added, followed by addition of n-butylamine under
1–5 were synthesized by aldol condensation of the corresponding substituted
(
,
N atmosphere. Finally, the resulting boron complex was treated in situ with acetic acid to obtain the
2
respective curcumin derivative in moderate yields. The synthesis of azole derivatives of curcumin
is shown in Scheme 1. The isoxazole derivative of curcumin
curcumin with hydroxylamine hydrochloride in acetic acid at 120 C, using a microwave monomodal
reactor under flash heating conditions in sealed vessels [27]. Pyrazole derivatives of curcumin
were prepared in a similar way by heating curcumin with hydrazine hydrate, phenyl hydrazine, and
,4-dinitrophenylhydrazine, respectively. As reported by previous work [28], the synthesis of azoles of
6 was prepared by condensation of
◦
7–9
2
curcumin heated under microwave irradiation decreased the reaction time and in some cases increased
reaction yields.
Scheme 1. Synthesis of curcumin derivatives 1–11.
The azole derivatives 10 and 11 were synthesized as described previously [29], by acetylation
of curcumin with acetic anhydride and pyridine, followed by a catalytic hydrogenation with Pd/C
(5%) and cyclization with hydroxylamine hydrochloride or hydrazine sulfate in EtOH with NaOAc,
respectively. The structure of all synthesized compounds was confirmed using spectroscopic and
spectrometric methods. Additionally, melting points were determined and they were consistent with
the literature reports. The nuclear magnetic resonance and mass spectra of compounds can be found
in Supplementary Materials.
2
.2. Biological Assays
2
.2.1. Cytotoxic Activity against Human Cancer Cell Lines
It is known that curcumin and several curcuminoids possess anti-proliferative activity [14,23,30].
Therefore, these derivatives also have been considered as potential anti-tumor agents [14]. However,
the knowledge of biological properties of curcumin and its derivatives can be expanded through the
exploration of their cytotoxic activity in other cell lines.

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The cytotoxic activity of the synthesized compounds was evaluated employing the
Sulforhodamine B assay against five human cancer cell lines: U-251 MG (glioblastoma), PC-3
(
human prostatic), HCT-15 (human colorectal), K562 (human chronic myelogenous leukemia),
and SKLU-1 (non-small cell lung cancer). Curcumin ( ) and its derivatives 11 were screened
at 50 M concentration [31]. The results of growth inhibition for the five cell lines tested are shown
in Table 1. Since compounds , and exhibit high activity at 50 M (100% of growth inhibition),
they were subsequently tested at lower concentrations of 12.5 and 10 M, as indicated in Table 1.
Compounds , and 10 11 showed strong activity against most of the cancer cell lines tested
% growth inhibition > 50), but particularly on HCT-15 and K562.
1
2–
µ
1
–
2
5–
7
µ
µ
1,
2,
5
–
8
–
(
Table 1. Cytotoxic activity for compounds 1–11 in terms of percentage of growth inhibition against
cancer cell lines.
Compound a
U-251 MG d
PC-3 e
HCT-15 f
K562 g
SKLU-1 h
b
1
59.1 ± 4.9
89.7 ± 4.0
41.1 ± 6.2
18.0 ± 3.5
73.7 ± 4.4
50.6 ± 5.4
63.3 ± 5.1
90.6 ± 5.1
15.2 ± 0.7
19.7 ± 4.2
34.0 ± 5.5
22.0 ± 2.4
78.0 ± 2.5
28.2 ± 1.4
26.3 ± 4.6
83.0 ± 6.2
55.8 ± 1.2
64.5 ± 1.7
91.5 ± 4.5
19.9 ± 0.7
63.4 ± 12.1
57.9 ± 3.7
81.2 ± 0.7
93.0 ± 2.3
33.8 ± 8.3
35.3 ± 11.9
94.1 ± 2.8
78.9 ± 7.0
77.0 ± 3.6
95.7 ± 4.2
33.5 ± 8.8
83.7 ± 7.4
69.3 ± 5.0
59.9 ± 5.0
93.0 ± 3.5
43.7 ± 6.2
27.8 ± 2.8
93.7 ± 5.8
84.5 ± 8.6
87.5 ± 10.9
97.3 ± 2.5
39.2 ± 5.7
71.2 ± 6.4
76.8 ± 3.5
72.4 ± 2.8
86.8 ± 2.4
24.2 ± 10.6
18.9 ± 2.1
91.4 ± 1.0
80.1 ± 10.8
88.6 ± 1.2
98.9 ± 1.0
5.2 ± 1.8
2 c
3
4c
5
6
7
c
c
8
9
0
1
1
1
18.9 ± 5.1
51.2 ± 2.5
a
SDE; b Tested at 12.5
Data represents the mean of three or four independent replica measures
±
µ
M due to its
c
d
e
high activity at 50
(
(
µ
M; Tested at 10
µM due to its high activity at 50
µ
M; U-251 MG (glioblastoma); PC-3
human prostatic); f HCT-15 (human colorectal); K562 (human chronic myelogenous leukemia); SKLU-1
g
h
non-small cell lung cancer).
Results from the primary screening allowed us to calculate the inhibitory concentration 50 (IC )
50
values for the more active compounds against K562 and HCT-15 cell lines. Although several studies
have reported cytotoxic activity for curcumin derivatives, including some of the compounds reported
here [14
The calculated IC50 values are shown in Table 2. The results for the HCT-15 cancer cell line show
that all selected compounds ( 10, and 11) were more active than curcumin ( ). The most
active compound was with IC50 = 2.3 M, which is 6-fold more active than curcumin. According to
these results, the methylation of the hydroxyl group at the aromatic ring in compound improved
the cytotoxic effects, however, this was the only successful modification on the aromatic ring that
improved the activity. The introduction of an additional methoxy (compound ) and the presence of
an N,N-dimethylamine group (compound ) decreased the cytotoxic activity in both cases (Table 1).
On the other hand, the substitution of aromatic rings by 3-indolyl moieties (compound ) increased
the cytotoxic effect relative to that of curcumin. To explore the effect of the replacement of -diketone
for a 1,2-azole ring, compounds , and were tested. The results show that substitution of diketone
,23,30], no reports were available for compounds 1–11 against K562 and HCT-15 cell lines.
2
,
5
,
6
,
7
,
8,
1
2
µ
2
3
4
5
β
6,
7
8
for pyrazole, N-phenylpyrazole, and oxazole improves the cytotoxic activity compared with that
of curcumin, but no differences in potency were found among them. Therefore, 1,3-diketone is not
essential for the cytotoxic activity, at least in this compound series. A similar result has been reported
for some curcuminoids tested against other different cancer cell lines [6,23]. Last, compounds 10 and
1
1
are more active than curcumin, however, the oxazole derivative 10 is more active than the pyrazole
analog 11 against HCT-15 cell line (Table 2). It is worth noting that 10 and 11 lack the conjugated
double bounds and the 1,3-diketone, therefore, these characteristics are not essential to obtain active
compounds. For assays on K562 cancer cell line, the activity changes in compounds
show the same tendency as that observed against the HCT-15 cell line. Compound
1
2
,
2
,
7
,
8
,
10, and 11
showed the most
important cytotoxic effect against K562, with IC50 = 2.3
µM, which is 4-fold more active than curcumin.
Also, compounds , and 10 were more active than curcumin. However, some differences were found
7, 8

Molecules 2017, 22, 633
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when compared with the results from the HCT-15 cell line. At this point, inverse structure–activity
relationships (SAR) were found for compounds and ; whereas structural modifications increase
5
6
the activity against HCT-15 relative to curcumin, the same modifications decrease the activity against
K562. Although the compounds tested display cytotoxic activity in the low micromolar range against
K562 and HCT-15, in most cases higher than that of curcumin, they have lower potency than the
Adriamycin standard.
Table 2. IC50 values (
µ
M) for selected compounds (
1
,
2,
5–
8,
10, and 11) against HCT-15 and K562
cell lines.
IC50 µM
Compounds
HCT-15
K562
1
2
5
6
7
8
13.9 ± 0.6
2.3 ± 0.1
4.8 ± 0.4
5.8 ± 0.7
6.2 ± 0.9
5.0 ± 0.4
2.8 ± 0.1
12.7 ± 0.1
9.2 ± 0.4
2.3 ± 0.3
13.3 ± 0.6
22.1 ± 0.9
6.3 ± 0.6
4.5 ± 0.2
3.0 ± 0.4
8.8 ± 0.3
1
0
1
1
Adriamycin
0.0050 ± 0.0009
0.0140 ± 0.0009
Data shown is the mean
inhibitory concentration.
±
standard error from three independent experiments. IC50: half maximal
2
.2.2. Antioxidant Activity in the Thiobarbituric Acid Reactive Substances and 2,2-Diphenyl-1-
picrylhydrazyl Assays
Based on the results of the previous assays, curcumin and the most active compounds as cytotoxic
agents (2 and 10) were selected to test for antioxidant activity. Compounds 6 and 7 were also selected
since no reports on the TBARS assay were available, and they exhibited significant cytotoxic activity,
at least on one of the cell line tested. Two different in vitro assays were used to evaluate the antioxidant
activity of compounds [31]. The TBARS assay measures the amount of malondialdehyde (MDA) as a
product of lipid oxidation and the DPPH measures the efficacy of a hydrogen atom transferred from
the compound to the radical by colorimetric determination. Quercetin and α-tocopherol were used
as reference antioxidants. As shown in Table 3, curcumin possess IC50 values of 3.22 and 23.52
in TBARS and DPPH assays, respectively; which agrees with previous literature reports [32
µM
,
33].
Compounds
compounds exhibit their antioxidant activity by a non-radical scavenging mechanism. On the other
hand, compounds and had strong activity in the TBARS assay. Although the antioxidant activity
2 and 10 showed only weak activity in the TBARS assays, which in turn suggests that these
6
7
using the DPPH assay for these compounds was previously reported by Sherin and Rajasekharan,
the same assay was performed in this work to compare experimentally both methods (DPPH and
TBARS) [34]. The values found in the DPPH assay compared to the results in the TBARS assay
show that compounds
6 and 7 exhibit an antioxidant activity mainly by lipid peroxidation inhibition.
A similar conclusion can be achieved if values for DPPH assay reported by Sherin and Rajasekharan
are taken as a reference [34].
Table 3. Antioxidant activity of selected compound.
IC50 (µM) DPPH b
Compound
IC50 (µM) TBARS a
1
2
6
7
3.22 ± 0.32
18.67 ± 2.28
1.31 ± 0.15
0.63 ± 0.02
15.30 ± 0.46
1.49 ± 0.03
6.78 ± 2.16
23.52 ± 1.20
Inactive
25.92 ± 1.73
25.81 ± 2.97
Inactive
1
0
Quercetin
α-Tocopherol
10.89 ± 0.47
31.74 ± 1.04
a
TBARS: thiobarbituric acid reactive substances assay; b DPPH: 2,2-Diphenyl-1-picrylhydrazyl assay.

Molecules 2017, 22, 633
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3
3
3
. Experiment Sections
.1. Chemistry
.1.1. General Considerations
Microwave assisted reactions were carried out in a monomodal reactor equipped with a hydraulic
pressure sensing device and an immersing ruby thermometer for precise control of reaction temperature
◦
(
Anton Parr Monowave 300, Anton Parr, Graz, Austria). Melting points ( C) were determined in
capillary Pyrex using Buchi Melting Point M-565 (Buchi, Flawil, Switzerland). Mass spectra were
obtained in a JEOL JMS-AX505HA mass spectrometer (Jeol, Tokyo, Japan) by electronic impact at
7
0 eV and JEOL SX 102, whereas spectra using electrospray ionization (ESI) were recorded on a
1
Bruker ESI/APCI-TOF (MicroTOF-II-Focus) spectrometer (Bruker, Billerica, MA, USA). H- and
1
3
C-NMR spectra were recorded either on 200, 300, 400, or 600 MHz Varian NMR spectrometer
Palo Alto, CA, USA) or 300 or 500 MHz Bruker NMR spectrometer (Bruker, Billerica, MA, USA),
using tetramethylsilane (TMS) as the internal standard. Chemical shifts ( ) are given in ppm and
(
δ
coupling constants (J) in Hz. TLC (Thin Layer Chromatography) was performed on precoated
Kieselgel 60 F254 plates (Merck, Darmstadt, Germany) and detected with ultraviolet (UV) light.
Flash chromatography was performed with Merck Kieselgel silica gel (60, particle size 40–63 mm
)
(Merck, Kenilworth, NJ, USA). All reagents were purified and the solvents used were of analytical
grade. NMR spectra of compounds
in Supplementary Materials.
1–11 and mass spectra of compounds 1–2 and 4–11 can be found
3
.1.2. General Procedure for the Preparation of 1, 2, 4, and 5
2
,4-Pentanodione (0.204 g, 2.04 mmol) and boric anhydride (0.071 g, 1.02 mmol) were dissolved in
◦
anhydrous ethyl acetate (EtOAc) (4 mL). The solution was stirred for 3 h at 70 C under N atmosphere.
2
Then, the mixture was added to a solution of arylaldehyde (4.9 mmol) and tributyl borate (1.12 g,
4
.9 mmol) in anhydrous EtOAc (10 mL) which was stirred for one hour under N atmosphere; then,
2
n-butylamine (0.245 mmol) dissolved in anhydrous EtOAc (5 mL) was added dropwise. The reaction
◦
mixture was stirred for 10–24 h at 70 C and was quenched by addition of 1 N HCl (20 mL). The reaction
◦
mixture was stirred at 70 C for one hour and the aqueous layer was extracted with EtOAc (4 × 20 mL).
The combined organic layers were washed with brine. The organic phase was dried with Na SO ,
2
4
filtered, and the solvent was removed in vacuo. The residue was purified by column chromatography
SiO , Hexane/EtOAc, 30:70) to give 1, 2, 4, and 5, respectively.
(
2
(
(
7
6
1E,4Z,6E)-5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (
1
). Red light solid
69% yield); m.p.: 180–183 C (lit. [24] 182–184 C); H-NMR (500 MHz, CDCl ) : 16.105 (bs, 1H),
.745 (bs, 2H), 7.574 (d, 2H, J = 15.5 Hz), 7.079 (dd, 2H, J = 8.0, 2 Hz), 7.061 (d, 2H, J = 1.5 Hz),
.916 (d, 2H, J = 8 Hz), 6.473 (d, 2H, J = 15.5 Hz), 5.814 (s, 1H), 3.933 (s, 6H); 13C-NMR (125 MHz,
◦
◦
1
δ
3
CDCl3)
δ
: 183.053, 148.494, 147.32, 145.338, 140.438, 126.955, 122.68, 121.134, 115.205, 109.916, 100.878,
+
5
5.731; EIM m/z (rel. int.): 368 (M , 62%).
(
(
1E,4Z,6E)-5-Hydroxy-1,7-bis(3,4-dimethoxyphenyl)hepta-1,4,6-trien-3-one (
55% yield); m.p.: 129–130 C (lit. [24] 129–131 C); H-NMR (200 MHz, CDCl )
2
).
Dark yellow solid
: 7.609 (d, 2H,
◦
◦
1
δ
3
J = 15.8 Hz), 7.147 (dd, 2H, J = 8.2, 1.6 Hz), 7.078 (d, 2H, J = 2.0 Hz), 6.881 (d, 2H, J = 8.4 Hz),
6
.498 (d, 2H, J = 15.8 Hz); 5.824 (s, 1H), 3.938 (s, 6H), 3.926 (s, 6H); 13C-NMR (50 MHz, CDCl3)
δ
: 183.195, 150.968, 149.147, 140.377, 127.981, 122.625, 121.957, 111.033, 109.622, 101.277, 55.955, 55.864;
EIM (m/z) 396 (38%).
1E,4Z,6E)-5-Hydroxy-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4,6-trien-3-one (
.27 mmol), boric anhydride (0.044 g, 0.63 mmol), trimethoxybenzaldehyde (0.5 g, 2.55 mmol), tributyl
(
3). 2,4-Pentanodione (0.127 g,
1
borate (0.586, 2.55 mmol, 0.68 mL), and n-butylamine (0.0186 g, 0.25 mmol, 0.13 mL) were dissolved in
◦
N,N-dimethylformamide (3 mL) in a 10 mL reaction vial. The reaction mixture was heated to 100 C

Molecules 2017, 22, 633
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under microwave irradiation for 2 min at 400 rpm. After completion of the reaction, the mixture was
◦
poured into ice-water (50 mL) and filtered to give
(
6
3
as an orange solid (40% yield); m.p.: 184–185 C
◦
lit. [35] 189–190 C); δ: H-NMR (500 MHz, CDCl ) δ: 15.907 (bs, 1H), 7.579 (d, 2H, J = 16.0 Hz),
3
.782 (s, 4H), 6.527 (d, 2H, J = 15.5 Hz), 5.863 (s, 1H), 3.907 (s, 12H), 3.893 (s, 6H); C-NMR (125 MHz,
: 183.085, 153.442, 145.432, 140.572, 140.102, 130.485, 123.362, 105.299, 101.508, 60.977, 56.158.
1
13
CDCl3)
δ
(
1E,4Z,6E)-1,7-bis(4-(Dimethylamino)phenyl)-5-hydroxyhepta-1,4,6-trien-3-one ( ). Red solid (50% yield);
: 16.344 (bs, 1H), 7.587 (d, 2H,
J = 16.0 Hz), 7.438 (d, 4H, J = 8.5 Hz), 6.665 (d, 4H, J = 9.0 Hz), 6.407 (d, 2H, J = 16.0 Hz), 5.714 (s, 1H),
4
◦
◦
1
m.p.: 213–215 C (lit. [36] 212–214 C); H-NMR (500 MHz, CDCl )
δ
3
3
1
.009 (s, 12H); 13C-NMR (125 MHz, CDCl3)
δ
: 183.277, 151. 552, 145.766, 140.534, 130.517, 129.774,
+
23.032, 120.671, 119.129, 111.893, 100.831, 40.119; EIM m/z (rel. int.): 362 (M , 84%).
(
2
1E,4Z,6E)-5-Hydroxy-1,7-di(1H-indol-3-yl)hepta-1,4,6-trien-3-one (
04–207 C (lit. [37] 210 C); H-NMR (600 MHz, DMSO-d )
5
).
Red solid (58% yield); m.p.:
δ: 10.731 (s, 2H), 6.914 (d, 2H,
◦
◦
1
6
J = 7.8 Hz), 6.863 (d, 2H, J = 2.4 Hz), 6.794 (d, 2H, J = 15.6 Hz), 6.417 (d, 2H, J = 7.8 Hz), 6.179–6.127
(
m, 4H), 6.156 (d, 2H, J = 15.6 Hz), 5.076 (s, 1H); 13C-NMR (150 MHz, DMSO-d6)
δ: 183.448, 137.682,
1
3
34.603, 132.033, 125.143, 122.877, 121.213, 120.276, 118.431, 112.952, 112.665, 100.262; EIM m/z:
54 (M 8%).
+
3
.1.3. Procedure for the Synthesis of Isoxazole Derivative 6
0
4
,4 -((1E,1
'
E)-Isoxazole-3,5-diylbis(ethene-2,1-diyl))bis(2-methoxyphenol) (6). To a solution of curcumin
1
(0.258 g, 0.7 mmol) in 5 mL of glacial acetic acid, hydroxylamine hydrochloride (0.073 g, 1.05 mmol)
◦
was added. The reaction mixture was heated to 70 C with microwave irradiation for 5 min
stirring at 400 rpm. Then, the reaction mixture was dropped into water to precipitate, filtered
in vacuo, and washed with cold water. The solid was purified by recrystallization (MeOH) to
◦
◦
1
give compound 6 (98% yield); m.p. 163–164 C (lit. [28] 162 C); H-NMR (600 MHz, CDCl )
3
δ
: 16.034 (bs, 2H), 7.591 (d, 2H, J = 15.6 Hz), 7.124 (dd, 2H, J = 1.8, 7.8 Hz), 7.052 (d, 2H, J = 1.2 Hz),
6
.936 (d, 2H, J = 8.4 Hz), 6.478 (d, 2H, J = 15.6 Hz), 5.857 (s, 1H), 3.951 (s, 6H); 13C-NMR (125 MHz,
CDCl3)
δ
: 183.479, 148.061, 146.999, 140.748, 127.921, 123.092, 122.008, 115.047, 109.852, 101.386, 56.194.
+
EIM m/z (rel. int.): 365 (M , 100%).
3
.1.4. General Procedure for the Preparation of Pyrazole Analogues of Curcumin (7–9)
To a solution of curcumin (0.105 g, 0.27 mmol) in glacial acid acetic (3 mL), hydrazine hydrate
1
(
0.405 mmol), phenylhydrazine (0.324 mmol), or 2,4-dinitrophenylhydrazine (0.27 mmol) were added.
◦
The reaction mixture was heated to 120 C under microwave irradiation for 10 min and stirred
at 400 rpm. The reaction mixture was dropped into water to precipitate, then filtered in vacuo,
and washed with cold water. The solid was purified by recrystallization.
0
0
4
(
,4 -((1E,1 E)-(1H-Pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (
7
). Light brown solid
EtOH/H O) (71% yield); m.p.: 218–220 C (lit. [38] 218 C); 1H-NMR (500 MHz, CDCl3)
◦
◦
2
δ: 8.067 (bs, 2H), 7.573 (d, 2H, J = 15.5 Hz), 7.387 (s, 1H), 7.075 (d, 2H, J = 7.0 Hz), 7.068 (s, 2H),
.914 (d, 2H, J = 8.5 Hz), 6.477 (d, 2H, J = 15.5 Hz), 5.82 (s, 1H), 3.936 (s, 6H); 13C-NMR (125 MHz,
CDCl3) : 182.979, 148.580, 147.374, 140.385, 122.620, 120.987, 115.867, 115.324, 109.908, 100.797, 55.646.
83.197, 148.798, 147.592, 140.603, 126.99, 122.838, 121.206, 115.452, 110.126, 101.015, 55.864; EIM m/z
6
δ
1
+
(rel. int.): 364 (M , 94%).
0 0
,4 -((1E,1 E)-(1-Phenyl-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (8). Light yellow
4
◦
◦
◦
1
solid (EtOH/H O) (94% yield); m.p.: 104–105 C (lit. [28] 89 C), (lit. [39] 128–129 C); H-NMR
2
(600 MHz, CDCl3)
δ: 7.538 (d, 2H, J = 8.4 Hz), 7.504 (t, 2H, J = 7.8 Hz), 7.412 (t, 1H, J = 7.2 Hz),
7
7
6
.125 (d, 1H, J = 16.8 Hz), 7.086 (d, 1H, J = 1.2 Hz), 7.054 (d, 1H, J = 16.2 Hz), 7.038 (d, 1H, J = 16.2 Hz),
.002 (dd, 1H, J = 8.4, 1.8 Hz), 6.964 (dd, 1H, J = 8.4, 1.8 Hz), 6.897 (t, 3H, J = 8.4 Hz), 6.814 (s, 1H),
.717 (d, 1H, J = 16.2 Hz), 3.923 (s, 3H), 3.893 (s, 3H); 13C-NMR (150 MHz, CDCl ) d: 151.589, 146.971,
3

Molecules 2017, 22, 633
8 of 12
1
1
4
46.462, 145.949, 142.757, 139.798, 132.566, 130.761, 129.996, 129.415, 129.217, 128.013, 125.417, 120.099,
20.785, 118.516, 114.934, 114.719, 113.662, 108.915, 108.081, 100.638, 56.177, 56.061; EIM m/z (rel. int.):
+
40 (M , 100%).
0
0
4
,4 -((1E,1 E)-(1-(2,4-Dinitrophenyl)-1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (
9). Dark
◦
◦
1
orange solid (MeOH/CH Cl ) (60% yield); m.p.: 212–213 C (lit. [39] 203–204 C); H-NMR (600 MHz,
2
2
CDCl3)
δ: 8.827 (d, 1H, J = 2.4 Hz), 8.57 (dd, 1H, J = 8.4, 2.4 Hz), 7.832 (d, 1H, J = 9.0 Hz), 7.449
(bs, 1H), 7.169 (bs, 1H), 7.114 (d, 1H, J = 16.2 Hz), 7.097 (d, 1H, J = 15.6 Hz), 7.065 (d, 1H, J = 1.8 Hz),
6
6
.979 (dd, 1H, J = 8.4, 1.8 Hz), 6.932 (dd, 1H, J = 8.4, 1.8 Hz), 6.899 (d, 1H, J = 15.6 Hz), 6.90–6.87 (m, 4H),
.522 (d, 1H, J = 15.6 Hz), 3.934 (s, 3H), 3.903 (s, 3H); 13C-NMR (150 MHz, CDCl + DMSO-d )
: 154.241,
δ
6
3
1
1
5
47.573, 147.48, 147.385, 146.742, 146.148, 145.303, 143.981, 137.46, 135.344, 132.75, 129.527, 128.823,
27.822, 127.542, 121.253, 120.990, 120.949, 116.773, 115.395, 115.098, 110.337, 109.517, 108.601, 102.244,
+
6.068, 55.945; MS (HR–ESI) for C H N O [M + H] , calcd.: m/z 531.1510, found: m/z 531.1512.
27
22
4
8
3
.1.5. Synthesis of Compounds 10 and 11
The synthesis of compounds 10 and 11 were carried employing the methodology previously
described by Lozada et al. [29].
(
(
Isoxazole-3,5-diylbis(ethane-2,1-diyl))bis(2-methoxy-4,1-phenylene) diacetate
(
10).
White solid
◦
◦
1
CH Cl /CH OH) (80% yield); m.p. 80–82 C (lit. [29] 80–82 C); H-NMR (300 MHz, CDCl ):
2
2
3
3
2
6
5
.3 (s, 6H), 2.94 (s, 4H), 2.99 (2t, J = 4.5, 4H), 3.79 (s, 3H), 3.80 (s, 3H), 5.73 (s, 1H), 6.73–6.79 (m, 2H),
.81 (d, J = 1.8, 2H), 6.94 (d, J = 7.8, 2H); 13C-NMR (75 MHz, CDCl ): 20.62, 27.83, 34.26, 28.44, 33.47,
3
5.78, 101.14, 112.46, 112.55, 120.30, 120.39, 122.60, 122.66, 138.07, 138.19, 139.02, 139.64, 150.89, 163.04,
+
+
+
169.10, 169.13, 171.91. MS m/z (rel. int. %): 453 M (7), 411 [M
−
42] (100), 381 [M 72] (8), 369
−
+
[M − 84] (97), 137 (96), 43 (8).
(
(
1H-Pyrazole-3,5-diyl)bis(ethane-2,1-diyl))bis(2-methoxy-4,1-phenylene) diacetate (11).
White solid
◦
◦
1
CH Cl /CH OH) (80% yield); m.p. 77–79 C (lit. [29] 77–79 C); m.p.; H-NMR (300 MHz, CDCl ):
2
2
3
3
2
.30 (s, 6H), 2.92 (s, 8H), 3.77 (s, 6H), 5.86 (s, 1H), 6.73 (dd, 2H, J = 8.1, 2.1), 6.76 (d, J = 1.8, 2H), 6.92
13
(d, J = 8.1, 2H); C-NMR (75 MHz, CDCl ): 20.63, 28.65, 35.48, 55.76, 102.74, 112.58, 120.37, 122.53,
3
+
+
140.03, 137.98, 148.07, 150.79, 169.22; MS m/z (rel. int. %): 452 M (10), 410 [M
−
42] (100), 368
+
+
[M − 84] (56), 274 [M − 178] (21), 232 (43), 137 (84), 43 (6).
3
.2. Biological Assays
3
.2.1. Cytotoxic Activity
The cytotoxicity of
1–11 on tumor cells was determined using the protein-binding dye
sulforhodamine B (SRB) assay in micro culture to determine cell growth [31]. The National Cancer
Institute (NCI), USA, supplied the cell lines employed in these experiments (PC-3, K562, HCT-15,
and SKLU-1). The cell lines were cultured in RPMI-1640 culture medium (Roswell Park Memorial
Institute medium) with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10,000 units/mL penicillin
G, 10,000
µ
g/mL streptomycin sulfate, and 25
µ
g/mL amphotericin B (GIBCO). They were kept
◦
at 37 C in an atmosphere of 5% CO with 95% humidity. With the exception of K562 cell line,
2
the rest of the adherent cell lines were removed from the tissue culture flask by adding 1 mL of
0.05% trypsin-ethylenediaminetetraacetic acid (GIBCO laboratories) and diluted with fresh media.
The viability of the cells used in the experiments exceeded 95% as determined with trypan blue.
4
4
For the assays, 100
µL/well of 5
×
10 cell/mL (K562), 7.5
×
10 cell/ mL (PC-3, SKLU-1),
4
and 10 × 10 cell/mL (HCT-15) of cell suspension were seeded in 96-well micro-titer plates and
incubated to allow for cell attachment. Test samples were dissolved in DMSO to provide stock
solutions (20 mM). After 24 h of incubation, 100 µL of each tested compound (1–11) and the reference
substance (Adriamycin) was added to each well. After 48 h, adherent cell culture was fixed in situ by
◦
adding 50
µ
L of cold 50% (w/v) trichloroacetic acid (TCA) and incubated for 60 min at 4 C. For K562,

Molecules 2017, 22, 633
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the suspension cell lines were fixed to the bottom of a microtiter well by gently adding 50
µL of 80% cold
TCA. The supernatant was discarded and the plates were washed three times with water and air-dried.
Cultures fixed with TCA were stained for 30 min with 100 L of 0.4% SRB solution. Protein-bounded
µ
dye was extracted with 10 mM of unbuffered tris base (tris(hydroximethyl)aminomethane) and the
optical density was measured on an Ultra Microplate Reader (Synergy/HT, BIO-TEK Instrument, Inc.,
Winooski, VT, USA) with a tested wavelength of 515 nm. Results were expressed as half maximal
inhibitory concentration (IC ) values. They were calculated according to the protocol of Monks [40],
50
where a dose-response curve was plotted for each compound and the concentration (IC ) that resulted
50
in an inhibition of 50% was estimated through non-linear regression analysis.
3
.2.2. Antioxidant Activity
Scavenging Activity on Free Radical DPPH
Free radical scavenging activity was measured using an adapted method of Mellors and
Tappel [41]. The test was carried out on 96-well microplates. A 50 L aliquot of the solution of
µ
the test compound was mixed with 150
µ
L of an ethanolic solution of DPPH (final concentration
◦
1
00
µM). This mixture was incubated at 37 C for 30 min, and the absorbance was then measured at
5
15 nm using an Ultra Microplate Reader (Synergy/HT, BIO-TEK Instrument, Inc., Winooski, VT, USA).
The percentage of inhibition of each compound was determined by comparison with a 100
ethanolic blank solution.
µM DPPH
Lipid Peroxidation Inhibition (TBARS)
The compounds
homogenate [31].
1–11 were evaluated employing the lipid peroxidation test (TBARS) in rat brain
Animals: Adult male Wistar rats (200–250 g) were provided by Metropolitan Autonomous
◦
University-Xochimilco (UAM-X) animal facility. They were maintained at 25 C on 12 light/12 h dark
cycle with free access to food and water. The procedures with animals and their care were conducted in
conformity with Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999; project
ID: 124 “Evaluation of the antioxidant and antiinflammatory activities of curcumin and indazole
derivatives”; date of approval: 28 May 2014; approved by the Intern Committee for the Use and Care
of Laboratory Animals from UAM-X).
The rat brain homogenate: The animals were killed with CO and the whole brain was rapidly
2
extracted, dissected, and homogenized into phosphate-buffer solution (PBS to 9.5 mM, pH = 7.4) to
produce 10% (w/v) rat brain homogenate following Rossato’s et al. methodology [42].
The homogenate was centrifuged 10 min at 805 rpm and the resulting pellet was discarded.
The protein content in the supernatant was determined according to the Lowry method [43]. Samples
were adjusted to 2.66 mg/mL with PBS.
Lipid peroxidation: A mixture containing 375
incubated 30 min at 37 C in the presence of each compound-test. 50
µ
L of the supernatant and 50
L of 100
M and then each mixture
µ
L of 10
µM EDTA was
◦
µ
µM FeSO solution
4
was added to the mixture in order to obtain a final concentration of 10
µ
◦
was incubated at 37 C for 60 min. TBARS was determined with slight modifications to Ohkawa
´
s
methodology [44]. Then, 500 µL of thiobarbituric acid (TBA) solution (1% TBA in 0.05 N NaOH and
3
0% trichloroacetic acid in 1:1 proportion) was added to the mixture. After cooling on ice for 10 min,
◦
it was centrifuged at 12,879 rpm for 5 min and then incubated at 90 C for 30 min. At room temperature,
the absorbance of 200 µL of supernatant was measured at 540 nm in a Bio-Tek Microplate Reader
Elx808. 1,1,3,3-tetramethoxypropane (TMP) was used as an external standard and the level of lipid
peroxidation was expressed as nmol of malondialdehyde (MDA) per mg of protein.
Inhibition of the lipid test peroxidation was measured as percentage against a blank that did
not contain any test compound. Quercetin and
The inhibition ratio (IR) was calculated using the following formula IR (%) = [(C
α
-tocopherol were used as positive standards.
E)/C] 100
−
×

Molecules 2017, 22, 633
10 of 12
where C represents the control of absorbance and E is the absorbance of the test sample. All data were
represented as mean
followed by Dunnett
±
standard error of the mean (SEM). Data were analyzed by one-way ANOVA
´
s test for comparison against control. Values of p 0.05 ( ) and p 0.01 (**)
≤
*
≤
were considered statistically significant. Half maximal inhibitory concentration (IC ) values were
50
estimated by means of a linear regression.
4
. Conclusions
A series of 11 compounds were synthesized in this work. Five compounds,
curcumin and four derivatives were obtained in moderate yields employing an aldol condensation
reaction. Additionally, six heterocyclic curcumin derivatives, 11, were obtained in moderate to
excellent yields. The cytotoxicity assays revealed that several compounds were more active than
curcumin against HCT-15 and K562 cell lines. Mainly, and 10 were the most active compounds
1–5, including
6–
2
against both cell lines. The results showed that most of the compounds have similar SAR against both
cell lines. The SAR analyses suggest that a 3,4-dimethoxyphenyl substituent improves the cytotoxic
activity. A change from a
in most cases. Finally, the results of TBARS showed that
particularly compound is the best, even better than references curcumin,
β
-diketone group for a pyrazole or oxazole ring results in a gain in potency
and have strong antioxidant activity;
-tocopherol, and quercetin.
6
7
7
α
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This work was supported by project 34610788/911010 from Secretaría de Educación Pública
(
SEP). RGE acknowledges financial support from PAPIIT (DGAPA, UNAM) projects # IN115713 and IN208516.
We are grateful to Marcela Hurtado y de la Peña, Ernesto Sánchez Mendoza and Mónica A. Rincón for the
analytical support.
Author Contributions: María Concepción Lozada-García, Olivia Soria-Arteche, and Jaime Pérez-Villanueva
conceived and designed the experiments and wrote the paper; Zeltzin Custodio-Galván performed the
experiments; Raúl G. Enríquez and Juan Francisco Palacios-Espinosa performed the characterization of compounds
and analyzed the data; Teresa O. Ramírez Apán performed the cytotoxic activity assays; Antonio Nieto-Camacho
performed the antioxidant activity assays. All the authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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