A new family of homoleptic copper complexes of curcuminoids: Synthesis, characterization and biological properties

Article abstract of DOI:10.3390/molecules24050910

We report herein the synthesis and crystal structures of five new homoleptic copper complexes of curcuminoids. The scarcity of reports of homoleptic complex structures of curcuminoids is attributed to the lack of crystallinity of such derivatives, and therefore, their characterization by single crystal X-ray diffraction is rare. The ligand design suppressing the phenolic interaction by esterification or etherification has afforded a significant increase in the number of known crystal structures of homoleptic metal complexes of curcuminoids revealing more favorable crystallinity. The crystal structures of the present new copper complexes show four-fold coordination with a square planar geometry. Two polymorphs were found for DiBncOC-Cu when crystallized from DMF. The characterization of these new complexes was carried out using infrared radiation (IR), nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and single crystal X-ray diffraction (SCXRD) and the antioxidant and cytotoxic activity of the obtained complexes was evaluated.

Full text of DOI:10.3390/molecules24050910

molecules
Article
A New Family of Homoleptic Copper Complexes of
Curcuminoids: Synthesis, Characterization and
Biological Properties
William Meza-Morales 1 , Juan C. Machado-Rodriguez 1, Yair Alvarez-Ricardo 1
,
Marco A. Obregón-Mendoza 1, Antonio Nieto-Camacho 1 , Rubén. A. Toscano 1,
Manuel Soriano-García 1, Julia Cassani 2,* and Raúl G. Enríquez 1,
*
1
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria,
Mexico City C.P. 07340, Mexico; willy_meza_morales@hotmail.com (W.M.-M.);
jcmrn.n@gmail.com (J.C.M.-R.); yfar30@hotmail.com (Y.A.-R.);
obregonmendoza@yahoo.com.mx (M.A.O.-M.); camanico2015@yahoo.com (A.N.-C.);
toscano@unam.mx (R.A.T.); soriano@unam.mx (M.S.-G.)
Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Unidad Xochimilco,
Mexico City C.P. 04960, Mexico
2
*
Correspondence: cassani@correo.xoc.uam.mx (J.C.); enriquezhabib@gmail.com (R.G.E.);
Tel.: +52-5554837255 (J.C.); +52-5556224404 (R.G.E.); Fax: +52-5556224404 (R.G.E.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Derek J. McPhee
Received: 20 February 2019; Accepted: 2 March 2019; Published: 5 March 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: We report herein the synthesis and crystal structures of five new homoleptic copper
complexes of curcuminoids. The scarcity of reports of homoleptic complex structures of curcuminoids
is attributed to the lack of crystallinity of such derivatives, and therefore, their characterization by
single crystal X-ray diffraction is rare. The ligand design suppressing the phenolic interaction by
esterification or etherification has afforded a significant increase in the number of known crystal
structures of homoleptic metal complexes of curcuminoids revealing more favorable crystallinity.
The crystal structures of the present new copper complexes show four-fold coordination with a
square planar geometry. Two polymorphs were found for DiBncOC-Cu when crystallized from
DMF. The characterization of these new complexes was carried out using infrared radiation (IR),
nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and single crystal
X-ray diffraction (SCXRD) and the antioxidant and cytotoxic activity of the obtained complexes
was evaluated.
Keywords: crystal structure; homoleptic copper complexes; curcuminoids; antioxidant activity
1. Introduction
Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-diketone) is a metabolite of
the Indian Curcuma species, generated by the rhizome of the perennial herb Curcuma longa, a member
of the gingiberacea family widely cultivated in India and China [
time by Vogel and Pelletier more than two centuries ago, and Milobedzka and Lampe first proposed
its chemical structure in 1910 [ ]. Furthermore, this posed the fundamental interest that curcumin
awoke gradually and expanded to other compounds that retain similar molecular topology and are
denominated curcuminoids. Curcumin and curcuminoids are chelating agents due to the –diketone
functionality, which allow them to form stable complexes with a series of metal ions [ ]. In recent years,
1]. Curcumin was isolated for the first
2,3
β
1
numerous studies have been performed to better understand the medicinal properties of curcumin,
curcuminoids and their metal complexes, with purported antitumor, antimicrobial, anti-inflammatory,
Molecules 2019, 24, 910; doi:10.3390/molecules24050910
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antioxidant, antiviral, anti-Alzheimer and anti-cancer potential [
1,
4–7]. Based on the wide medicinal
applications reported in the literature for copper complexes, we tried to obtain copper complexes
with several curcuminoids as ligands designed to promote homoleptic structures and to investigate
their biological activities. However, there are very few known crystal structures of homoleptic metal
complexes of curcumin and this has been attributed to an inherent low crystallinity [
they are often insoluble in water and in most common organic solvents [ ]. Such property has
precluded the use of single crystal X-ray diffraction as a characterization technique [ 10]. As a
1,4]. In addition,
1
1,4,8–
consequence, the studies of metal complexes of curcumin and related compounds are more focused on
their biological properties [8–21] rather than in detailed structural characterizations [8–10].
In the present work, it was possible to use several curcuminoid as ligands for complexation with
copper (II) i.e., acetylated curcumin (1,7-Bis (3-methoxyl-4-acetoxy) -phenol-1,6-heptadiene-3,5-diketone,
DAC) 1, hydrogenated acetylated curcumin (1,7-Bis (3-methoxyl-4-acetoxy) -phenol-heptane-3,5-diketone,
DACH4)
2, methoxylated curcumin (1,7- Bis (3,4-dimethoxy) -phenol-1,6-heptadiene-3,5-diketone,
DiMeOC)
3, benzylated curcumin (1,7-Bis (3-methoxy-4-benzyl)-phenol-1,6-heptadiene-3,5-diketone)
4
5
, DiBncOC and bisdemethoxy-bisdehydroxy-curcumin (1,7-diphenylhepta-1,6-diene-3,5-dione, PhCurc)
(see Figure 1) which proved to be suitable ligands for the formation of single crystals for X-ray studies.
The characterization of all synthesized homoleptic complexes was carried out using IR, NMR and EPR in
liquid state, magnetic moment, MS as well as the single crystal X-ray diffraction technique. After a full
characterization was carried out, their cytotoxic and antioxidant activity was evaluated.
Figure 1. Ligands used for complexation with copper 1–5.
2. Results and Discussion
2.1. IR Spectra
The IR spectrum of DAC
1
shows two bands, one of high intensity at 1755 cm−1 and another
-diketone, indicating that
of very low intensity at 1795 cm−1 due to the free carbonyl group of the
β
the compound exists mainly in the enolic form. The low-intensity band in the range 1632–1610 is
attributed to the intramolecular hydrogen bridge of the enol. The band at 966 cm−1 that corresponds

Molecules 2019, 24, 910
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to the trans -CH=C-double bond is also observed. The IR spectra of DAC-Cu
6
show intense bands
at 1514 cm−1 and ~484 cm−1 due to the interaction of me−ta1l
β-diketone group from M-O vibrations.
−1
The IR spectrum of DACH4
2
shows two bands at 1757 cm (high intensity) and 1797 cm (very low
intensity) showing a small ratio of the free carbonyl group of the
compound exists mainly in its enolic form (see Table 1) [13].
β-diketone, indicating that the
Three bands were observed in the range of 2966-2841 cm−1 of low-intensity due C-H stretch.
The -CH=C- band at 965 cm−1 was not observed. IR spectra of DACH4-Cu (
due to the interaction of the metal with the
-diketone group at 1508 cm−1 and an additional band at
~467 cm−1 due to M-O vibrations. The IR spectrum of DiMeOC
shows the presence of two bands,
one of high intensity at 1620 cm−1 and another with very low intensity at 1663 cm−1 due to the free
7) showed strong bands
β
3
carbonyl group of the β-diketone, revealing that the compound exists in enolic form. The trans -CH=C-
band appears at 964 cm−1. The IR spectra of DiMeOC-Cu
8
shows strong bands due to the interaction
−1
−1
of the metal with the
vibrations. The IR spectrum of DiBncOC
at 1625 cm−1 and the other of very low intensity at 1730 cm−1, due to the free carbonyl group of the
-diketone. Five bands are observed in the range 849–694 cm−1 due C-H bending. The trans -CH=C-
band appears at 970 cm−1. IR spectra of DiBncOC-Cu
shows strong bands due to the interaction
of the metal with the
-diketone group at 1501 cm−1 and an additional band at ~465 cm−1 due to
M-O vibrations. The IR spectrum of PhCurcu shows the presence of two bands, one of high intensity
at 1619 cm−1 and the other of very low intensity at 1670 cm−1, due to the free carbonyl group of the
-diketone. The trans -CH=C- band appears at 968 cm−1. IR spectra of PhCurcu-Cu 10 show strong
bands due to the interaction of the metal with the
-diketone group at 1511 cm−1 and an additional
band at ~420 cm−1 due to M-O vibrations (see Table 1) [13].
β-diketone group at 1506 cm and an additional band at ~463 cm due to M-O
4
shows the presence of two bands, one of high intensity
β
9
β
5
β
β
Table 1. IR spectral data of compounds 1–10.
Keto-enol
-CH=C-
C-H Stretch
C-H Bending
Vibrations
M-O (cm
Compounds
−1
−1
−1
−1
−1
(cm
)
(cm
)
(cm
)
(cm
)
)
DAC
1755 and 1795
966
-
-
-
-
-
-
-
DACH4
1757 and 1797
-
2966–2841
-
-
DiMeOC
1620 and 1663
964
-
-
-
-
-
-
-
-
DiBcOC
1625 and 1730
970
849–694
PhCurcu
1619 and 1670
968
-
-
-
-
-
-
DAC-Cu
-
-
-
-
-
-
-
-
-
-
1514 and 484
1508 and 467
1506 and 463
1501 and 465
1511 and 420
DACH4-Cu
DiMeOC-Cu
DiBcOC-Cu
PhCurcu-Cu
2.2. NMR Spectra
1
The H NMR spectrum of ligand DAC
1 shows one singlet for the OH proton at 16.12 ppm
(strong intramolecular hydrogen bond) and one singlet for the methine proton at ~6.20 ppm
(vinylic proton). Protons to the diketone appear at 6.99 ppm and protons to the diketone at
7.66 ppm, with a trans coupling constant of 15.9 Hz. Methoxyl and acetyl protons appear as singlets at
3.85 ppm and 2.28 ppm, respectively. The 1H NMR spectrum of DACH4 (ligand
) shows a keto-enol
α
β
2
equilibrium with ca. 1:1 ratio. The enol tautomer shows one singlet for the OH proton at 15.53 ppm and
one singlet for the methine proton at 5.78 ppm; both protons are involved in a strong intramolecular
hydrogen bridge. The keto tautomer shows one singlet for the methylene proton at 3.74 ppm. Protons
α
to the diketone group appear at 2.77 and 2.65 ppm while protons appear at 2.85 ppm. Methoxyl and
β

Molecules 2019, 24, 910
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1
acetyl protons appear as singlets at 3.74 ppm and 2.23 ppm, respectively. The H NMR spectrum
of DiMeOC (ligand ) shows one singlet for the OH proton at 16.27 ppm and one singlet for the
methine proton at 5.80 ppm; both protons are involved in a strong intramolecular hydrogen bond
(enol tautomer). Protons to the diketone appear at 6.47 ppm and protons to the diketone at
3
α
β
7.58 ppm, with a trans coupling constant of ca. 15.8 Hz. Methoxyl protons are singlets at 3.91 ppm
and 3.89 ppm. The 1H NMR spectrum of DiBncOC (ligand 4) shows one singlet for the OH proton at
16.30 ppm and one singlet for the methine proton at 6.11 ppm. Unsaturated protons
group appear at 6.84 ppm and the corresponding protons at 7.59 ppm, with trans coupling constant
of 15.78 Hz. Methoxyl protons are singlets at 3.84 ppm. Benzyl protons are singlets at 5.14 ppm.
The 1H NMR spectrum of PhCurcu (ligand
) shows one singlet for the OH proton at 16.11 ppm and
one singlet for the methine proton at 6.21 ppm). Protons
and protons
α to the diketone
β
5
α
to the diketone function appear at 6.96 ppm
1
β
at 7.67 ppm, with a trans coupling constant of 15.97 Hz. The H NMR spectra of
complexes
6–
10 lack signals for the enol proton at ca. 16 ppm, due to paramagnetic effects (see Table 2).
1
Table 2. H NMR Chemical shifts (δ) of compounds 1–10.
α to the
Diketone (δ)
β to the
Diketone (δ)
Compounds
OH (δ)
Methine (δ)
Methoxyl (δ)
Acetyl (δ)
Benzyl (δ)
Aromatic (δ)
DAC
16.12
6.20
6.99
2.77
6.47
6.48
6.96
-
7.66
2.85
7.58
7.59
7.67
-
3.85
2.28
2.23
-
-
7.16–7.52
DACH4
15.53
5.78
3.74
-
5.78–7.00
DiMeOC
16.27
5.80
3.91–3.89
-
6.85–7.05
DiBcOC
16.30
6.11
3.84
-
5.14
7.34–7.59
PhCurcu
16.11
6.21
-
-
-
-
-
-
-
-
7.73
DAC-Cu
-
-
-
-
-
-
-
-
-
-
2.27
4.01
4.01
3.86
-
3.85
-
-
-
-
-
DACH4-Cu
DiMeOC-Cu
DiBcOC-Cu
PhCurcu-Cu
-
-
-
-
-
-
-
-
-
-
6.91
7.66
2.3. EPR Spectra
The EPR spectra of ligands show diamagnetic spectra, while the EPR spectra of copper complexes
of ligands 10 show a typical four lines pattern (see Figure 2). The g , g , A and A values were
obtained directly from the EPR spectra. The g and g values of complexes
6
–
⊥
⊥
k
k
6
-
10 were ca. 2.29 and 2.06,
⊥
k
resulting from unpaired electrons in the dx2−y2 molecular orbital [13]. The values of g greater than 2.3,
suggest a ionic environment for the complexes. The A values ca. 160
with a typical monomeric distorted square planar geometry. The quotient g /A provides an index
of departure from the tetrahedral structure. The quotient values that fall in the range 105–135 cm−1
suggest a regular square planar structure, although the observed values (141–146 cm−1) are indicative
k
×
10−4 cm−1 are consistent
k
k
k
of a strong distortion from planarity (see Table 3) [13,22–24]. The magnetic moment values for Cu(II)
complexes suggest that they are paramagnetic with µeffect values of ca. 2.0 B.M with one unpaired
electron (see Table 3).

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Figure 2. Spectra EPR of 6 (a), 7 (b), 8 (c) 9 (d), and 10 (e).
Table 3. EPR spectra data of copper complexes 6–10.
−4
−1
−1
)
−1
)
(10−4 cm
g
A
(10 cm
)
g /A (cm
µeffec
Complexes
g
A
k
⊥
k
k
k
⊥
DAC-Cu
2.29
2.06
2.06
2.07
2.06
2.07
162.3
161.8
160.8
159.9
157.3
11.3
10.5
9.5
12.4
18.11
141.1
141.5
143.0
143.8
146.3
1.69
2.01
1.99
2.00
2.09
DACH4-Cu 2.29
DiMeOC-Cu 2.30
DiBcOC-Cu 2.30
PhCurcu-Cu 2.30
Parallel g-value = g , perpendicular g-value = g , parallel A-value = A , perpendicular A-value = A .
⊥
⊥
k
k
2.4. Single Crystal X-Ray Diffraction
A rigorous analysis of the crystal structure determinations of the five complexes
6,
7,
8,
9a and
10 and the triclinic polymorph 9b (see Figure 3) reveals a four-fold coordination around copper
atom with a square planar geometry (see supplementary material, Table S1), which is confirmed by
the characteristics of the EPR spectra. However, if we take into consideration the “close contacts”
(26.8 and 33.9% longer than the average Cu-O distances) compound,
(tetragonal pyramid) and 4 + 2 (octahedron), respectively. In all compounds, the transition metal
resides in a special position, except for the triclinic polymorph b where the copper atom resides in a
6 and 7 can be described as 4+1
9

Molecules 2019, 24, 910
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general position. The curcuminoid ligands adopt a fully extended and almost planar conformation
(see supplementary material, Table S2). It is noteworthy that these structural variations place the
methyl substituents on phenols, face to face towards the inner part of the molecule in complexes
b; all pointing outside in complex and two pointing towards the inner and two pointing towards
the outside in complexes and a, revealing high conformational degrees of freedom around the
6 and
9
6
8
9
(1,7-bis-(4-hydroxy-3-methoxyphenol) moiety and giving an approximately overall symmetry C2v for
complexes 6, 7, 9b and 10, while for complexes 8 and 9a the Ci symmetry gives the best description.
(a)
(b)
(c)
Figure 3. Cont.

Molecules 2019, 24, 910
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(d)
(e)
Figure 3. Cont.

Molecules 2019, 24, 910
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(f)
Figure 3. Crystal structures for
6,
7,
8,
9a
,
9b and 10 are shown in Figure 3 (a), (b), (c), (d), (e) and (f)
with displacement ellipsoids at 30% probability level.
2.5. Inhibition of Lipoperoxidation (LP) in Rat Brain Homogenate
DAC proved to be the only ligand possessing antioxidant potential comparable to curcumin.
The vinyl groups are essential for the antioxidant activity of the curcumin and curcuminoids, as it
can be appreciated by the significant decrease in TBARS inhibitory percentage when going from
DAC to DACH4 (see Table 4). In this case, when ether groups replace the phenolic groups of
curcumin, a significant decrease in the antioxidant potential is observed. Three copper complexes
showed increased antioxidant activity on the lipoperoxidation in rat brain homogenate model [25
Only copper complex showed a decrease in antioxidant effect respect to its ligand (see Table 4).
The metal complexes and showed high antioxidant activity on the lipoperoxidation of rat brain
homogenate model with values similar α-tocopherol (see Table 5).
6-8
,
26].
9
7
8
·
LP is a process initiated and mediated by reactive oxygen species (ROS), hydroxyl (HO ), peroxyl
(ROO·), alkoxyl (RO·) and hydroperoxyl (HOO·) radicals and it is known that the hydroxyl radical
is an important initiator in lipid peroxidation, while peroxyl and alkoxyl radicals are intermediates
in the propagation phase of lipid peroxidation [27]. The results shown in Table 5 suggest that the
compounds exert moderate to good inhibition of ROS. The data show that Inhibitory Concentration-50
(IC50) of the free ligands DACH4 and DiMeOC are ca. half as large (less active) than those observed
for DACH4-Cu and DiMeOC-Cu (see Table 5), indicating that the copper complexation leads to higher
activity in reducing lipid peroxidation. In general, copper is a good inducer of oxidative stress in
its free form when it has the correct oxidation state. In the complexes studied in this work, copper
has an oxidation state different to +1, and in our experimental conditions (pH 7.4), copper remains
chelated by the ligand. Thus, the IC50 values support the idea that copper remains bound to the
ligands. For complexes, the IC50 value is ca. half of the ligands, indicating that copper is bound to
two ligand molecules. This suggests that copper is not free and does not participate as an inducer of
lipid peroxidation.

Molecules 2019, 24, 910
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Table 4. Lipoperoxidation Screening of ligands and their copper complexes 1–10.
Products
Concentration (uM)
D. O. 540 nm
nmol/mg prot.
Inhibition (%)
Basal
FeSO4
-
-
0.004
0.851
0.000
0.000
0.030
0.012
0.022
0.018
0.313
0.015
0.020
0.024
0.733
0.169
0.321
0.225
0.785
0.612
0.795
0.769
0.712
0.208
0.474
0.217
0.169
11.379
0.116
0.116
0.516
0.275
0.410
0.348
4.257
0.308
0.377
0.427
9.808
2.355
4.360
3.086
10.496
8.217
10.628
10.287
9.530
2.865
6.388
2.990
-
-
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
10
100
98.67
98.67
94.66
97.16
95.75
96.40
55.95
96.81
96.16
95.65
1.47
75.63
55.61
68.57
7.76
27.79
6.60
Curcu
DAC
DAC-Cu
DACH4
DACH4-Cu
DiMeOC
DiMeOC-Cu
DiBncOC
DiBncOC-Cu
PhCurcu
9.59
8.22
72.41
38.48
71.20
PhCurcu-Cu
Table 5. Inhibition of lipid peroxidation IC50 of compounds DAC, DACH, DiMeOC, DAC-Cu,
DACH4-Cu and DiMeOC-Cu.
Concentration
TBARS
(nmol/mg prot.)
Inhibition
(%)
IC50
(µM)
Products
(µM)
Basal
FeSO4 100 µM
-
-
0.24 ± 0.09
-
-
-
-
9.15 ± 0.32
0.1
0.32
1
3.16
10
31.62
100
6.28 ± 0.18
6.04 ± 0.24
4.62 ± 0.57
8.26 ± 1.31
5.21 ± 0.33 *
3.67 ± 0.56 **
2.72 ± 0.33 **
1.84 ± 0.31 **
1.40 ± 0.36 **
21.13 ± 2.56 *
44.84 ± 6.74 **
59.00 ± 3.71 **
72.3 ± 3.87 **
79.09 ± 4.79 **
α-Tocopherol
6.78 ± 2.16
(n = 4)
0.1
0.32
1
3.16
10
9.68 ± 0.43
4.16 ± 1.97
9.29 ± 0.67
8.32 ± 1.49
DAC
7.78 ± 0.53 *
5.22 ± 0.35 **
1.43 ± 0.08 **
23.19 ± 0.92 *
48.46 ± 1.53 **
85.82 ± 0.60 **
3.21 ± 0.16
1.55 ± 0.15
0.1
0.32
1
3.16
10
9.08 ± 0.66
10.34 ± 1.48
8.04 ± 0.70
20.78 ± 2.44
DAC-Cu
6.24 ± 0.71 **
3.08 ± 0.21 **
1.32 ± 0.48 **
38.70 ± 3.82 **
69.57 ± 1.56 **
86.58 ± 5.21 **

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Table 5. Cont.
Concentration
TBARS
(nmol/mg prot.)
Inhibition
(%)
IC50
(µM)
Products
(µM)
1.78
3.16
5.62
10
17.78
31.62
56.23
8.32 ± 0.31
7.85 ± 0.41 *
7.25 ± 0.36 **
6.32 ± 0.18 **
4.45 ± 0.22 **
0.36 ± 0.06 **
0.14 ± 0.01 **
7.32 ± 0.30 **
6.67 ± 0.34 **
5.89 ± 0.33 **
3.28 ± 0.37 **
0.28 ± 0.05 **
0.20 ± 0.02 **
0.14 ± 0.007 **
9.05 ± 1.03
14.28 ± 1.59 *
20.86 ± 1.48 **
30.84 ± 0.57 **
51.41 ± 0.92 **
96.02 ± 0.57 **
98.39 ± 0.18 **
DACH4
16.46 ± 0.30
7.93 ± 0.41
1.78
3.16
5.62
10
17.78
31.62
56.23
19.93 ± 2.01 **
27.14 ± 1.30 **
35.74 ± 1.88 **
64.27 ± 2.74 **
96.90 ± 0.63 **
97.73 ± 0.29 **
98.39 ± 0.03 **
DACH4-Cu
17.78
31.62
56.23
100
5.51± 0.13 **
3.40 ± 0.007 **
2.67 ± 0.06 **
2.27 ± 0.09 **
2.08 ± 0.10 **
8.58 ± 0.28
7.42 ± 0.20 **
4.28 ± 0.06 **
3.10 ± 0.12 **
2.89 ± 0.16 **
2.8 0± 0.23 **
38.66 ± 3.26 **
62.16 ± 1.18 **
70.33 ± 0.63 **
74.75 ± 0.92 **
76.91 ± 0.88 **
DiMeOC
23.01 ± 1.37
9.35 ± 0.34
177.83
1
3.16
10
31.62
100
316.23
4.55 ± 4.35
17.51 ± 2.63 **
52.41 ± 1.20 **
65.56 ± 1.49 **
67.90 ± 1.17 **
68.97 ± 1.99 **
DiMeOC-Cu
Data presented as mean ± SEM of three replicates. * p ≤ 0.05 and ** p ≤ 0.05 compared to FeSO4.
2.6. Cytotoxic Activity
DiMeOC is the only ligand possessing higher cytotoxicity [28] than curcumin, a noteworthy
fact since the phenolic groups are blocked. A significant decrease in cytotoxic activity occurs when
going from DAC to DACH4, indicating the importance of conjugated double bonds in the heptanoid
fragment of curcumin or curcuminoids. In general, a significant decrease in the cytotoxic activity of
the copper complexes
6–10 with respect to their free ligands is observed. Interestingly compounds
6
–10 did not show significant cytotoxic effect against the cell lines tested. Although there are several
reports of cytotoxic or antitumor activity of copper complexes with curcumin, this effect appears to be
related to the presence of free phenolic groups (see Table 6) [11,17,29].
Table 6. Cytotoxic Screening of ligands and their copper complexes 1–10.
% of Inhibition
Products (25 µM)
U251
PC-3
K562
HCT-15
MCF-7
SKLU-1
DAC
DAC-Cu
DACH4
DACH4-Cu
DiMeOC
DIMeOC-Cu
DiBncOC
DiBncOC-Cu
PhCurcu
46.2
8.6
77.54
13.19
26.08
31.5
100
100
10.7
4.1
67.8
19.7
39.4
15.4
89.4
75.8
10.9
NC
54.7
4.2
46.88
4.16
31.12
3.9
96.95
74.5
NC
NC
25,0
NC
41.74
NC
7.16
3.0
100
48.4
1.7
NC
48.7
NC
41.74
NC
7.16
11.30
100
48.02
6.4
7.5
22.8
NC
92.6
31.4
3.0
6.6
47.5
3.5
31.2
NC
53.0
NC
PhCurcu-Cu

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3. Materials and Methods
All chemicals were available commercially, and the solvents were purified by conventional
methods prior to use [30]. Curcumin was obtained from natural source by usual extractive procedures
and purified by crystallization.
3.1. Physical Measurements
Melting points were determined on an Electrothermal Engineering IA9100X1 melting point
apparatus and are uncorrected.
3.2. Spectroscopic Determinations
IR absorption spectra were recorded in the range of 4000–230 cm−1 as KBr pellets on a BRUKER
1
Tensor 27 spectrophotometer. H and 13C NMR spectra were recorded in dimethyl sulfoxide (DMSO-d6)
on a Bruker Fourier 300 MHz and Varian Unity Inova 500 MHz spectrometer using TMS as internal
reference. The EPR spectra were recorded in DMF at liquid nitrogen temperature (77 K) on an Electron
Paramagnetic Resonance Spectrometer JEOL, JES-TE300, ITC Cryogenic System, Oxford. Magnetic
moments were determined using a Johnson-Matthey magnetic susceptibility balance type msb model
mk II 13094-3002, with the Gouy method at room temperature. Mass spectra were recorded in a JEOL,
SX 102 A spectrometer on Bruker Microflex equipped with MALDI-Flight time. Single-crystal X-ray
diffractions (SCXRD) were obtained in a Bruker diffractometer, model Smart Apex, equipped with Mo
radiation (λ = 0.71073Å), CCD two-dimensional detector and low-temperature device. Data collection
and data reduction were performed by APEX and SAINT-Plus programs [31]. These structures
were solved by direct methods using SHELX-2013 software and refined by Full-matrix least-squares
procedure on F2 using SHELX-2008 program [32].
3.3. Inhibition of Lipid Peroxidation on Rat Brain
3.3.1. Animals
Adult male Wistar rats (200–250 g) were provided by Instituto de Fisiología Celular,
Universidad Nacional Autónoma de México (UNAM). Procedures and care of animals were conducted
in conformity with the Mexican Official Norm for Animal Care and Handling NOM-062-ZOO-1999.
They were maintained at 23
and water.
±
2 ◦C under a 12/12 h light-dark cycle with ad libitum access to food
3.3.2. Rat Brain Homogenate Preparation
Animal sacrifice was carried out avoiding unnecessary pain. Rats were sacrificed with CO2.
The cerebral tissue (whole brain), was rapidly dissected and homogenized in phosphate-buffered saline
(PBS) solution (0.2 g of KCl, 0.2 g of KH2PO4, 8 g of NaCl, and 2.16 g of NaHPO4.7H2O/L, pH adjusted
to 7.4) as described elsewhere to produce a 1/10 (w/v) homogenate [33,34]. The homogenate was
centrifuged at 800 rcf (relative centrifugal field) for 10 min. The supernatant protein content was
measured using Folin and Ciocalteu’s phenol reagent [35] and adjusted with PBS at 2.666 mg
of protein/mL.
3.3.3. Induction of Lipid Peroxidation and Thiobarbituric Acid Reactive Substances
(TBARS) Quantification
As an index of lipid peroxidation, TBARS levels were measured using rat brain homogenates according
to the method described by Ng and co-workers [36], with some modifications. Supernatant (375
added with 50 L of 20 M EDTA and 25 L of each sample concentration dissolved in DMSO (25
DMSO for the control group) and incubated at 37 ◦C for 30 min. Lipid peroxidation was started adding
50 L of freshly prepared 100 M FeSO4 solution (final concentration 10
M) and incubated at 37 ◦C
µL) was
µ
µ
µ
µL of
µ
µ
µ

Molecules 2019, 24, 910
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for 1h. TBARS measurements were obtained as described by Ohkawa and co-workers [37], with some
modifications. 500 L of TBA reagent (0.5% 2-thiobarbituric acid in 0.05 N NaOH and 30% trichloroacetic
µ
acid, in 1:1 ratio) was added to each tube and the final suspension cooled on ice for 10 min, centrifuged at
◦
13,400 rcf for 5 min and heated at 80 C in a water bath for 30 min. After cooling at room temperature, the
absorbance of 200 µL of supernatant was measured at λ = 540 nm in a Microplate Reader Synergy/HT
BIOTEK Instrument, Inc., Winooski, VT, USA. The concentration of TBARS was calculated by interpolation
on a standard curve of tetra-methoxypropane (TMP) as a precursor of MDA [37]. Results are expressed as n
moles of TBARS per mg of protein. The inhibition ratio (IR [%]) was calculated using the formula IR = (C–E)
×
100/C, where C is the control absorbance, and E is the sample absorbance. Butylated hydroxytoluene
(BHT) and –tocopherol were used as positive standards. All data are presented as mean standard
error (SEM). Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test for
comparison against control. Values of p 0.05 (*) and p 0.01 (**) were considered statistically significant.
α
±
≤
≤
3.4. Citotoxic activity in Human Tumor Cells
Citotoxicity of all compounds was tested against six cancer cell lines: U251 (human glioblastoma
cell line), PC-3 (human caucasian prostate adenocarcinoma), K562 (human caucasian chronic
myelogenous leukaemia), HCT-15 (human colon adenocarcinoma), MCF-7 (human mammary
adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). Cell lines were supplied U.S. National
Cancer Institute (NCI). The cell lines were cultured in RPMI-1640 medium supplemented with 10%
fetal bovine serum, 2 mL -glutamine, 10,000 units/mL penicillin G sodium, 10,000 µg/mL streptomycin
sulfate, 25
µ
g/mL amphotericin B (Invitrogen/Gibco
™
, Thermo Fisher Scientific, Waltham, MA, USA),
◦
and 1% non-essential amino acids (Gibco). They were maintained at 37 C in a humidified atmosphere
with 5% CO2. The viability of the cells used in the experiments exceeded 95% as determined with trypan
blue. The human tumor cytotoxicity was determined using the protein-binding dye sulforhodamine B
(SRB) in microculture assay to measure cell growth, as described in the protocols established by the
NCI [38–40].
3.5. Synthesis of Compounds
General synthetic procedure for DAC
1, DACH4 2, DiMeOC 3, DiBncOC 4, DAC-Cu 6, DACH4-Cu
7, DiMeOC-Cu 8 and DiBncOC-Cu 9 is shown in Scheme 1.
Compound 1. 4 g of curcumin in 70 mL of dichloromethane (CH2Cl2) was reacted with 2.6 mL
of pyridine (Py) and 1.6 mL of acetic anhydride (Ac2O) at room temperature for approximately 3 h.
Follow up was done by TLC. Removal of solvent from the reaction was done under reduced pressure,
and the product was extracted three times with ethyl acetate (AcOEt)-water (H2O) in a 3:7 proportion
of until pyridine was eliminated from the organic phase. The product recrystallized in AcOEt (
1
) with
1
70.1% yield. H NMR (600 MHz DMSO-d6):
δ
2.28 (s, 6H), 3.85 (s, 6H), 6.20 (s, 1H), 6.99 (d, 2Hvinyl
,
,
J 15.9 Hz), 7.16 (d, 2Haryl, J 8.1 Hz), 7.33 (dd, 2Haryl, J 8.2; 1.9 Hz), 7.52 (d, 2Haryl, J 2 Hz), 7.66 (d, 2Hvinyl
J 15.9 Hz), 16.1 (br s, 1H,) ppm, 13C NMR (13C {1H} 150 MHz, DMSO-d6):
δ
20.17 (C-H), 55.72 (C-H),
101.62 (C-H), 111.93 (Caryl), 121.27 (Caryl), 123.23 (Caryl), 124.58 (Cvinyl), 133.59 (Caryl), 139.77 (Cvinyl),
140.93 (Caryl), 151.11(Caryl), 168.30 (C=O), 183.10 (C=O) ppm, IR 1755 cm−1, 1596 cm−1, 1506 cm−1
1295 cm−1, 1154 cm−1, 619 cm−1, MS: M+ 453.15; yellow crystals, m.p. 170.5 ◦C.
,

Molecules 2019, 24, 910
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Scheme 1. Synthetic route of curcuminoids and their complexes.
Compound 2. 3.8 g of DAC dissolved in 60 mL of AcOEt was reacted in hydrogen atmosphere
with 380 mg of Pd/C-10%. The reaction mixture was stirred at room temperature until disappearance
of the starting material was complete followed by TLC. The completion of reaction was achieved after
4 h and the reaction was filtered-off through celite; the solvent removed in vacuo. The product was
purified by SiO2 column chromatography eluting with a 7:3 hexane-AcOEt solvent mixture and the
product was dried under high vacuum (2), 80.4% yield. 1H NMR (500 MHz DMSO-d6):
12H), 2.65 (t, 4Haliph, J 7.86 Hz), 2.78 (m, 4Haliph), 2.85 (t, 8Haliph, J 7.02 Hz), 3.92 (d, 14H), 5.78 (s,
1H), 6.76 (dd, 2Haryl, J 8.09; 1.83 Hz), 6.79 (dd, 2Haryl, J 8.09; 1.83 Hz), 6.93 (s, 1Haryl), 6.95 (d, 2Haryl
J 2.44 Hz), 6.96 (m, 3Hvinyl, Hz), 7.00 (d, 2Haryl, J 1.83 Hz), 15.51 (br s, 1H,) ppm. 13C NMR (13C
{1H} 125 MHz, DMSO- d6):
20.17 (C-H), 29.03 (Caliph), 31.02 (Caliph), 39.44 (Caliph), 39.50 (Caliph),
δ 2.23 (s,
,
δ
44.72 (Caliph), 56.19 (C-H), 56.65 (Caliph), 100.11 (C-H), 113.25 (Caryl), 113.32 (Caryl), 120.49 (Caryl),
120.54 (Caryl) 122.90 (Caryl), 122.94 (Caryl), 137.91 (Cvinyl), 138.03 (Caryl), 140.05 (Cvinyl), 140.35 (Caryl),
151.02 (Caryl), 169.07 (C=O), 193.57 (C=O), 204.87 (C=O) ppm, IR 2974 cm−1, 2939 cm−1, 2841 cm−1
1795 cm−1, 1757 cm−1, 1597 cm−1, 1510 cm−1, 1271 cm−1, 1188 cm−1, 599 cm−1, 526 cm−1, 469 cm−1
MS: M+ 455.85; white solid, m.p. 68.3 ◦C.
,
,
Compound 3. 4 g of curcumin in 120 mL of anhydrous acetone was reacted with 0.75 g of
potassium carbonate (K2CO3) and 1.2 mL of dimethyl sulfate (SO2(OCH3)2) and refluxed with stirring
for 48 h until disappearance of the starting material. The solvent was removed under reduced pressure
and the product was extracted with a 3:7 mixture of ethyl acetate AcOEt-water (H2O) and NaOH
10% until SO2(OCH3)2 was removed from the organic phase. The product was purified by SiO2
column chromatography eluting with a 5:4.5:0.5 mixture of hexane-CH2Cl2-MeOH and the product

Molecules 2019, 24, 910
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was dried under high vacuum (3), 63.2% yield. The product was recrystallized in MeOH, 65.4% yield.
1H NMR (500 MHz DMSO-d6):
3.89 (s, 6H,) 3.91 (s, 6H), 5.80 (s, 1H), 6.47 (d, 2Hvinyl, J 15.76 Hz),
6.85 (d, 2Haryl, J 8.34 Hz), 7.11 (dd, 2Haryl, J 8.33; 2.00 Hz), 7.05 (d, 2Haryl, J 2.02 Hz), 7.58 (d, 2Hvinyl
J 15.80 Hz), 16.27 (br s, 1H,) ppm. 13C NMR (13C {1H} 125 MHz, DMSO-d6):
60.74 (C-H), 106.16 (C-H),
115.64 (Caryl), 116.84 (Caryl), 127.20 (Caryl), 128.07 (Caryl), 132.73 (Cvinyl), 145.57 (Caryl), 154.19 (Caryl),
δ
,
δ
156.14 (Caryl), 188.37 (C=O) ppm, IR 3005 cm−1, 2926 cm−1, 2831 cm−1, 1624 cm−1, 1583 cm−1
,
1504 cm−1, 1134 cm−1, 802 cm−1, 607 cm−1, 559 cm−1, 544 cm−1, 469 cm−1, MS: M+ 396.77; orange
crystals, m.p. 133.5 ◦C.
Compound 4. 4 g of curcumin in 120 mL of anhydrous acetone was reacted with 0.75 g of K2CO3
and 2.6 mL of benzyl bromide (BnBr) at reflux for approximately 30 h until the disappearance of the
starting material by TLC. The reaction solvent was removed under reduce pressure. The product
was purified by SiO2 column chromatography eluting with a 7:3 hexane-AcOEt solvent mixture.
1
The product was crystallized in AcOEt, 58.7% yield. H NMR (500 MHz DMSO-d6):
δ 3.84 (s, 6H,)
5.14 (s, 4H), 6.11 (s, H), 6.84 (d, 2Hvinyl, J 15.79 Hz), 7.09 (d, 2Haryl, J 8.47 Hz), 7.25 (dd, 2Hvinyl, J 8.37;
2.00 Hz), 7.34 (t, 2Haryl, J 7.22 Hz), 7.38 (d, 2Haryl, J 1.94 Hz), 7.40 (t, 4Haryl, J 7.36 Hz), 7.45 (dd, 4Haryl
J 7.49; 2.19 Hz), 7.59 (d, 2Haryl, J 15.76 Hz), 16.30 (br s, 1H). 13C NMR (13C {1H} 125 MHz, DMSO-d6):
55.65 (C-H), 69.83 (C-H), 101.06 (C-H), 110.80 (Caryl), 113.24 (Caryl), 122.18 (Caryl), 122.70 (Caryl),
,
δ
127.81 (Cvinyl), 128.40 (Caryl), 136.71 (Cvinyl), 140.33 (Caryl), 149.30 (Caryl), 149.91 (Caryl), 183.17 (C=O)
ppm, IR 3061 cm−1, 2922 cm−1, 2856 cm−1, 1726 cm−1, 1628 cm−1, 1585 cm−1, 1510 cm−1, 1126 cm−1
970 cm−1, 739 cm−1, 696 cm−1, 486 cm−1, 459 cm−1, MS: M+ 548.96; yellow crystals, m.p. 159.1 ◦C.
,
Compound 5. PhCurcu was prepared in accordance with a previously reported synthetic
1
method [41], 56.7% yield. H NMR (500 MHz DMSO-d6):
δ 6.21 (s, 1H), 6.96 (d, 2Hvinyl, J 15.97 Hz),
7.45 (m, 6H), 7.67 (d, 2Hvinyl, J 15.92 Hz), 7.73 (dd, 4Haryl, J 7.67; 1.69 Hz), 16.11 (br s, 1H) ppm,
yellow‘crystals, m.p. 140.5 ◦C.
Compound 6. 1 mmol of DAC was dissolved in 30 mL of a 7:3 mixture of ethyl acetate-methanol.
Then, a solution of copper acetate in MeOH and H2O (0.5 mmol) was added dropwise. After 2 h
of stirring at room temperature, a brown powder was formed, which was filtered and crystallized
1
with DMSO, 86.9% yield. H NMR (500 MHz DMSO-d6):
δ 2.27 (s, 6H), 3.85 (s, 6H), 6.26 (br s, 1H),
6.81 (br s, 4H), 7.16 (br s, 1H), 7.34 (br s, 2H), 7.52 (br s, 1H), 7.66 (br s, 1H) ppm, IR 2975 cm−1
2941 cm−1, 1752 cm−1, 1592 cm−1, 1514 cm−1, 1412 cm−, 1299 cm−1, 1156 cm−1, 604 cm−1, 484 cm−1
brown powder, m.p. 242.5 ◦C.
,
,
Compound 7. 1 mmol of DACH4 was dissolved in 25 mL of a 7:3 mixture of ethyl acetate-methanol
and 0.5 mmol of a MeOH/H2O solution of copper acetate was added slowly. After stirring at room
temperature for 2 h, a blue powder was formed, which was filtered and crystallized in DMF/CH3CN,
86% yield. 1H NMR (500 MHz DMSO-d6):
δ 4.01 (br s, 6H), 6.40 (br s, 2H), 7.05 (br s, 2H) ppm,
IR 3020 cm−1, 2960 cm−1, 2926 cm−1, 2870 cm−1, 1759 cm−1, 1734 cm−1, 1574 cm−1, 1508 cm−1
1196 cm−1, 1032 cm−1, 550 cm−1, 515 cm−−1, 469 cm−1, MS: M+ 973.27; blue powder, m.p. 165.8 ◦C.
,
Compound 8. 1 mmol of DiMeOC was dissolved in 30 mL of a 7:3 mixture of ethyl
acetate-methanol and 0.5 mmol of a MeOH/H2O solution of copper acetate was added dropwise.
After stirring for 2 h at room temperature, a dark brown powder was formed, which was filtered
1
and crystallized in DMSO, 93.2% yield. H NMR (500 MHz DMSO-d6):
δ 4.01 (br s, 6), 6.93 (br s, 5H)
ppm, IR 2995 cm−1, 2966 cm−1, 2931 cm−1, 2843 cm−1, 1630 cm−1, 1580 cm−1, 1500 cm−1, 1421 cm−1
1132 cm−1, 1014 cm−1, 968 cm−1, 469 cm−1, MS: M+ 856.258; dark brown powder, m.p. 258.4 ◦C.
,
Compound 9. 1 mmol of DiBncOC was dissolved in a mixture of 50 mL tetrahydrofuran (THF),
later a solution of copper acetate in MeOH and H2O (0.5 mmol) was added slowly. After stirring at
room temperature for 2 h, a brown powder was formed, which was filtered off and recrystallized
1
from DMF, 85.7% yield. H NMR (500 MHz DMSO-d6):
δ 3.86 (br s, 6H), 5.01 (br s, 5H), 6.84 (br s,
6H), 7.35 (br s, 13H) ppm, IR 3030 cm−1, 3001 cm−1, 2936 cm−1, 1622 cm−1, 1502 cm−1, 1132 cm−1
696 cm−1, 498 cm−1, 469 cm−1, MS: M+ 1159.476; brown powder, m.p. 182.5 ◦C.
,

Molecules 2019, 24, 910
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Compound 10. 1 mmol of PhCurcu was dissolved in 30 mL of a 7:3 ethyl acetate-methanol mixture
and 0.5 mmol of copper acetate in a MeO/H2O solution was added slowly. After stirring for 2 h of at
room temperature, a brown powder was formed and was filtered and crystallized in DMF, 96.3% yield.
1H NMR (500 MHz DMSO-d6):
δ 6.91 (d, 1Hvinyl, J 15.97 Hz), 7.14 (br s, 4H), 7.45 (d, 4Haryl, J 5.93 Hz),
7.66 (d, 7Hvinyl, J 19.32 Hz), IR 3023 cm−1, 1673 cm−1, 1620 cm−1, 1572 cm−1, 1070 cm−1, 641 cm−1
,
590 cm−1, 510 cm−1, 420 cm−1, MS: M+ 698.99; brown powder, m.p. 275.3 ◦C.
4. Conclusions
The synthesis of 5 (compound
9 has two polymorphs) new homoleptic copper complexes
was achieved with 5 different curcuminoid ligands, and their crystal structures reveal a four-fold
coordination with square planar geometry. The copper ion did not increase the cytotoxic properties
of the complexes with respect to free ligands but instead, high antioxidant activity for compounds
DAC-Cu, DACH4-Cu and DiMeOC-Cu was found. In our results, the presence of free phenolic
groups in curcumin derivatives might not be taken as the sole criterion for antioxidant or cytotoxic
activity. From the pharmacological point of view, dealing with molecular species only composed by the
curcuminoid and the metal atom, thus avoiding the presence of a molecular stabilizer or “spectator”,
might be considered advantageous. Moreover, the synthesis of homoleptic copper complexes of
curcuminoids achieved in the present work demonstrates a feasible approach for the preparation of
new homoleptic complexes of curcuminoids comprising different ligands and metals.
Supplementary Materials: The following are available online. CCDC-1882893, CCDC-1882894, CCDC-1882895,
CCDC-1882896, CCDC-1882897 and CCDC-1882898 contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi, by e-mailing
data_request@ccdc.cam.ac.uk, or by contacting: The Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK, Fax; +44(0)-1223-336033.
Author Contributions: Extensive synthetic laboratory work: W.M.-M. (principal PhD student), J.C.M.-R., Y.A.-R.,
M.A.O.-M.; validation (X-ray extensive studies), M.S.-G., R.A.T.; Biological essays, A.N.-C.; NMR studies and
funding acquisition, J.C.; writing—review and editing, project administration and funding acquisition, R.G.E.
Overlapping tasks from all coauthors is thoroughly acknowledged. CRediT taxonomy.
Funding: We gratefully acknowledge financial support from PAPIIT (DGAPA, UNAM, IN208516) and CONACyT
(CB 252524).
Acknowledgments: Scholarships from CONACYT awarded to WMM (No. 576707), YAR (No. 576706) and
MAOM (No. 603692) are gratefully acknowledged. We are indebted to Dr. Diego Martinez Otero (X-ray), MSc.
Virginia Gómez Vidales (EPR), MSc. María Teresa Ramírez Apan (cytotoxic activity) from Instituto de Química,
UNAM for technical assistance, Claudia Rivera Cerecedo and Hector Malagon Rivero (biological essays) from
Instituto de Fisiología Celular, UNAM. Acknowledgment is made to Dr. Rubén Gaviño (NMR), María del Rocío
Patiño (IR and UV-Vis) and Lucero Rios Ruiz (EM). We gratefully acknowledge assistance from USAI (Facultad de
Química, UNAM).
Conflicts of Interest: The authors declare no competing financial interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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