Full structural characterization of homoleptic complexes of diacetylcurcumin with Mg, Zn, Cu, and Mn: Cisplatin-level Cytotoxicity in Vitro with Minimal Acute Toxicity in Vivo

Article abstract of DOI:10.3390/molecules24081598

At the present time, scientists place a great deal of effort worldwide trying to improve the therapeutic potential of metal complexes of curcumin and curcuminoids. Herein, the synthesis of four homoleptic metal complexes with diacetylcurcumin (DAC), using a ligand designed to prevent the interaction of phenolic groups, rendering metal complexes through the β-diketone functionality, is reported. Due to their physiological relevance, we used bivalent magnesium, zinc, copper, and manganese for complexation with DAC. The resulting products were characterized by ultraviolet-visible (UV-Vis), fluorescence spectroscopy, infrared spectroscopy (IR), liquid and solid-state nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), magnetic moment, mass spectrometry (MS), single crystal, and powder X-ray diffraction (SCXRD and PXRD). Crystallization was achieved in dimethylsulfoxide (DMSO) or N,N-dimethylformamide (DMF) as triclinic systems with space group P-1, showing the metal bound to the β-diketone function, while the ¹H-NMR confirmed the preference of the enolic form of the ligand. Single crystal data demonstrated a 1:2 metal:ligand ratio. The inhibition of lipid peroxidation was evaluated using the thiobarbituric acid reactive substance assay (TBARS). All four metal complexes (Mg, Zn, Cu, and Mn) exhibited good antioxidant effect (IC₅₀ = 2.03 ± 0.27, 1.58 ± 0.07, 1.58 ± 0.15 and 1.24 ± 0.10 μM respectively) compared with butylated hydroxytoluene (BHT) and α-tocopherol. The cytotoxic activity in human cancer cell lines against colon adenocarcinoma (HCT-15), mammary adenocarcinoma (MCF-7), and lung adenocarcinoma (SKLU-1) was found comparable ((DAC)₂Mg), or ca. 2-fold higher ((DAC)₂Zn) than cisplatin. The acute toxicity assays indicate class 5 toxicity, according to the Organization for Economic Co-operation and Development (OECD) guidelines at doses of 3 g/kg for all complexes. No mortality or changes in the behavior of animals in any of the treated groups was observed. A therapeutic potential can be envisaged from the relevant cytotoxic activity upon human cancer cell lines in vitro and the undetected in vivo acute toxicity of these compounds.

Full text of DOI:10.3390/molecules24081598

molecules
Article
Full Structural Characterization of Homoleptic
Complexes of Diacetylcurcumin with Mg, Zn, Cu, and
Mn: Cisplatin-level Cytotoxicity in Vitro with
Minimal Acute Toxicity in Vivo
William Meza-Morales 1 , M. Mirian Estévez-Carmona 2 , Yair Alvarez-Ricardo 1
Marco A. Obregón-Mendoza 1 , Julia Cassani 3, María Teresa Ramírez-Apan 1,
,
Carolina Escobedo-Martínez 4 , Manuel Soriano-García 1, William F. Reynolds 5 and
Raúl G. Enríquez 1,
*
1
Instituto de Qu
í
mica, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria,
CDMX CP 07340, México; willy_meza_morales@hotmail.com (W.M.-M.); yfar30@hotmail.com (Y.A.-R.);
obregonmendoza@yahoo.com.mx (M.A.O.-M.); mtrapan@yahoo.com.mx (M.T.R.-A.);
soriano@unam.mx (M.S.-G.)
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Wilfrido Massieu SN,
CDMX CP 07738, México; mirianestevezc@gmail.com
Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Unidad Xochimilco,
CDMX CP 04960, México; cassani@correo.xoc.uam.mx
Departamento de Farmacia, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Campus
2
3
4
5
Guanajuato, Guanajuato CP 36050, México; karolesma@hotmail.com
Department of Chemistry, University of Toronto, Toronto, ON M5S 1A1, Canada;
wreynold@chem.utoronto.ca
*
Correspondence: enriquezhabib@gmail.com; Tel./Fax: +52-5556224404
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 April 2019; Accepted: 21 April 2019; Published: 23 April 2019
Abstract: At the present time, scientists place a great deal of effort worldwide trying to improve
the therapeutic potential of metal complexes of curcumin and curcuminoids. Herein, the synthesis
of four homoleptic metal complexes with diacetylcurcumin (DAC), using a ligand designed to
prevent the interaction of phenolic groups, rendering metal complexes through the
β-diketone
functionality, is reported. Due to their physiological relevance, we used bivalent magnesium, zinc,
copper, and manganese for complexation with DAC. The resulting products were characterized
by ultraviolet-visible (UV-Vis), fluorescence spectroscopy, infrared spectroscopy (IR), liquid and
solid-state nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), magnetic
moment, mass spectrometry (MS), single crystal, and powder X-ray diffraction (SCXRD and PXRD).
Crystallization was achieved in dimethylsulfoxide (DMSO) or N,N-dimethylformamide (DMF) as
triclinic systems with space group P-1, showing the metal bound to the β-diketone function, while the
1H-NMR confirmed the preference of the enolic form of the ligand. Single crystal data demonstrated
a 1:2 metal:ligand ratio. The inhibition of lipid peroxidation was evaluated using the thiobarbituric
acid reactive substance assay (TBARS). All four metal complexes (Mg, Zn, Cu, and Mn) exhibited
good antioxidant effect (IC50 = 2.03
compared with butylated hydroxytoluene (BHT) and
±
0.27, 1.58
±
0.07, 1.58
±
0.15 and 1.24
±
0.10 µM respectively)
α
-tocopherol. The cytotoxic activity in human
cancer cell lines against colon adenocarcinoma (HCT-15), mammary adenocarcinoma (MCF-7), and
lung adenocarcinoma (SKLU-1) was found comparable ((DAC)2Mg), or ca. 2-fold higher ((DAC)2Zn)
than cisplatin. The acute toxicity assays indicate class 5 toxicity, according to the Organization for
Economic Co-operation and Development (OECD) guidelines at doses of 3 g/kg for all complexes.
No mortality or changes in the behavior of animals in any of the treated groups was observed. A
therapeutic potential can be envisaged from the relevant cytotoxic activity upon human cancer cell
lines in vitro and the undetected in vivo acute toxicity of these compounds.
Molecules 2019, 24, 1598; doi:10.3390/molecules24081598
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1598
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Keywords: homoleptic metal complexes; curcuminoids; crystal structures; antioxidant and
cytotoxic activity
1. Introduction
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenol)-1,6-heptadiene-3,5-diketone) is the key secondary
metabolite present in the yellow rhizome of the perennial herb, Curcuma longa, along with
desmethoxycurcumin, and bis-desmethoxycurcumin. It has been widely studied for its multiple
medicinal properties, e.g., antimicrobial, antioxidant, antibacterial, and anti-inflammatory, including
anticancer activity, Alzheimer and infectious diseases [
in water, and its rapid metabolism and excretion, are related to a poor bioavailability that limits the
scope of its potential benefits in living organisms [ 12]. Curcumin and curcuminoids, both natural
-diketone ligands capable of binding to a variety of metals to produce
1–7]. However, the low solubility of curcumin
1,8–
and synthetic, are examples of
stable complexes [1,2].
β
The use of metals of physiological interest in complexation, such as magnesium, zinc, copper,
and manganese, serves the purpose of exploring the potential benefits of these metal complexes in
medicinal therapies, since the positive biological role of such metals is known to a good extent. For
example, magnesium (Mg(II)) is a cofactor in several enzymatic reactions mainly related to energy
metabolism and the synthesis of nucleic acids [9,13]. Zinc (Zn(II)) is involved in the regulation of
mitochondrial apoptosis in many mammalian cells [14]. Copper (Cu(II)) is an essential micronutrient
related to mitochondrial respiration, as well as the response to cellular stress. It is also involved in
antioxidant and anti-tumoral defense [10,11,15]. Manganese (Mn(II)) is a trace element present in bone,
liver, kidney, and pancreas. Mn(II) helps the body form connective tissue, bones, sex hormones and
assists in blood clotting. It also plays a role in fat and carbohydrate metabolism, calcium absorption,
and blood sugar regulation. This metal is also necessary for normal brain and nerve function and it
is also a component of the antioxidant enzyme superoxide dismutase (SOD), helping to quench free
radicals and reduce their damage [16,17].
The molecule curcumin belongs to a class of naturally occurring
good ligand, like its closely related acetylacetone, and also displays the typical keto-enol tautomerism.
However, unlike acetylacetone, the central -diketone functionality is flanked by two large groups
β-diketones, which can act as
β
–CH=CH-C6H4(OH)(OMe)-3,4 bearing phenolic –OH groups, which are reactive centers that can
interfere in the preparation of metal complexes. Thus, it was necessary to protect the hydroxyl
substituents and use diacetylcurcumin (or 1,7-bis(3-methoxyl-4-acetoxy1)-pheny-1,6-heptadiene-3,5
diketone (DAC)), which proved to be a suitable ligand [
1]. Acetylation turns curcumin into a ligand
with its main binding capacities at the β-diketone function.
Wang et al. (2014) reported, for the first time, the crystal structure of two homoleptic curcumin
metal complexes, using as ligands the O-ethoxy and O-butoxy derivatives, through strong alkaline
conditions in their synthetic procedure [10]. In other work, Aliaga-Alcalde et al. [18,19] reported
the crystal structures of two homoleptic metal complexes with zinc and copper, where the original
aromatic rings of curcumin are replaced by anthracene substituents. The scarcity of homoleptic
crystal structures reported until now, however, it has been attributed to the low crystallinity of the
curcumin-metal association, making the obtention of suitable single crystals difficult [1,10,18–20].
By using diacetylcurcumin, we circumvented this barrier, while preserving the overall structure of
curcumin in its complexes. The copper and manganese complexes of diacetylcurcumin have been
reported previously [8,16], however, the X-ray structures have not been reported. In a recent work
by Meza-Morales et al. (2019), the synthesis of five homoleptic complexes, with copper was reported
using different curcuminoids [21], along with their single crystal structures.
In the present work, the complexation was conducted under mild conditions to form the Mg(II),
Zn(II), Cu(II) and Mn(II) complexes at the β-diketone region, in absence of a strong base. We report the

Molecules 2019, 24, 1598
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synthesis (see Scheme 1), structural characterization by ultraviolet-visible (UV-Vis), fluorescence, and
infrared spectroscopy, liquid and solid-state Nuclear Magnetic Resonance, Electronic Paramagnetic
Resonance, magnetic moment and single-crystal X-ray diffraction using diacetylated curcumin as
ligand with the physiologically relevant metals Mg, Zn, Cu and Mn. This approach successfully
afforded the reportedly elusive homoleptic complexes as single crystals. In addition, these metal
complexes of DAC showed enhanced antioxidant and antitumor activity respect to the ligand.
H3CCOO
H3CO
HO
H3CO
H3CCOO
H3CO
OOCCH3
OCH3
H3CCOO
H3CO
Pyridine
Ac2O
O
O
O
O
O
M(OAc)2
O
O
O
O
CH2Cl2
M
OH
MeOH/AcOEt
H3CO
H3CO
HO
Curcumin
H3CO
OCH3
H3CO
M=
Mg,
Zn, Cu
and Mn
H3CCOO
H3CCOO
OOCCH3
H3CCOO
Keto isomer
Enol isomer
Scheme 1. Synthetic route of diacetylcurcumin (DAC) and its DAC-metals complexes.
2. Results and Discussion
2.1. UV-Vis Spectra
The electronic spectra of compounds
temperature. The spectra of compounds
414 and 328 nm. These bands are attributed to
complexes 4, and exhibit three bands at 415, 338, and 439; 414 nm, 338 and 438 nm; 418, 328 and
440 nm, due to *, n– * transitions and charge transfer effects (CT) (see Table 1). The CT bands of
compounds 4, and demonstrate the formation of the complexes. In addition, very high values of
the molar extinction coefficient ( ) were found, indicating a high capacity to interact with light. It was
1–5
were recorded in dimethylsulfoxide (DMSO) at room
1
and
2
exhibit two absorption bands at 408 and 328 nm;
* and n– * transitions. The electronic spectrum of
π
–π
π
3
,
5
π
–π
π
3
,
5
ε
not possible to observe the d-d transitions for the complexes 3, 4, and 5 [9].
Table 1. Transition bands in ultraviolet-visible (UV-Vis) spectra for ligand and complexes.
Ligand &
Complexes
π–π* (nm)
εmax (L/cm mol)
n–π* (nm)
εmax (L/cm mol)
CT (nm)
εmax (L/cm mol)
DAC (1)
408
414
415
414
418
349,187.06
587,658.78
250,821.69
387,091.23
300,551.07
328
328
338
338
328
34,955.35
137,652.06
211,677.96
128,582.44
111,206.39
-
-
-
-
(DAC)2Mg (2)
(DAC)2Zn (3)
(DAC)2Cu (4)
(DAC)2Mn (5)
439
438
440
161,111.68
266,119.18
213,207.90
2.2. Fluorescence Spectra
All compounds emit at the same wavelength ca. 458 nm, when excited between 408–418 nm (see
Table 2). The Mg(II) complex shows an increase in fluorescence intensity, while Zn(II), Cu(II) and Mn(II)
complexes show a decrease in intensity compared to the ligand as shown in Table 2. The increase in
fluorescence intensity for Mg(II) can be attributed to the charge transfer from metal to ligand [22]. On
the contrary, the decrease in intensity for complexes of Zn(II), Cu(II) and Mn(II), can be explained by
the charge transfer from the ligand to the metal. The relative intensities for the complexes go in the
order, Mg(II) > Cu(II) > Mn(II) > Zn(II) [23].

Molecules 2019, 24, 1598
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Table 2. Fluorescence of diacetylcurcumin (DAC) and metal complexes in dimethylsulfoxide (DMSO).
Excitation
Wavelength (nm)
Fluorescence
Wavelength (nm)
Ligand & Complexes
Intensity (I)
DAC (1)
408
414
415
414
418
457.77
458.64
459.06
458.50
459.08
280,465.66
412,828.49
175,399.40
230,331.13
188,354.49
(DAC)2Mg (2)
(DAC)2Zn (3)
(DAC)2Cu (4)
(DAC)2Mn (5)
2.3. Infrared Spectra
The IR spectrum of DAC shows the presence of two bands, one of high intensity at 1755 cm−1
and the other of very low intensity at 1795 cm−1, due to the free carbonyl group of the
β-diketone
indicating that the compound exists in enolic form. The absorption in the range 1632–1610 cm−1 of low
intensity corresponds to the intramolecular hydrogen bond of the enol. The -CH=C- band at 965.78
cm−1 is also observed. The IR spectra of metal complexes show intense bands for the interaction of
the metal with the
(DAC)2Mg , (DAC)2Zn 3, (DAC)2Cu 4 and (DAC)2Mn 5, respectively. All complexes spectra show
β
-diketonate group at 1447.36, 1509.64, 1514.26 and 1505.53 cm−1 corresponding to
2
additional bands at ~466.01, ~469.59, ~484.78, and ~463.93 cm−1, due to M-O vibrations [12].
2.4. NMR Spectra
The 1H nuclear magnetic resonance (NMR) spectrum of ligand DAC shows one singlet for the
OH proton, at 16 ppm, and one singlet for the methine proton at ~6.20 ppm; both protons are involved
in a strong intramolecular hydrogen bridge (enol tautomer). Protons
α to the diketone function appear
at 6.99 ppm and protons
acetyl protons appear as singlets at 3.85 ppm, and 2.28 ppm, respectively. The H-NMR spectra
of the complexes lack the corresponding signal of the enol proton at 16 ppm, indicating complex
β
at 7.66 ppm, with a trans coupling constant of 15.9 Hz. Methoxyl and
1
formation through the β-diketonate. The chemical shift towards low frequencies by the protons of the
methine, vinyl, and aromatic groups of metal complexes is presumably due to the fact that DMSO
is a highly coordinating molecule that could be forming part of the coordination sphere (being part
of the complex) [
1
], increasing the electron density, and shielding effect at the coordination site. The
1
same behavior has been found in the H-NMR spectra of metal complexes (Cu(II) and Zn(II)) of
curcumin [24]. By contrast, the spectra of the metal complexes of curcumin and curcuminoids in
MeOD-d4 or CDCl3 show the opposite behavior (protons are observed deshielded) [12,25,26]. The
1H-NMR spectra of manganese and copper complexes show overall paramagnetic broadening of
signals. The methine proton in Zn and Mg complexes, receives the highest shielding effect (Table 3)
that can be attributed to an increase in electron density [24]. Methoxyl and acetyl protons are not
affected since they are far away from the ligand-metal site of interaction.
Table 3. 1H-NMR chemical shifts of the methine proton of DAC and their metal complexes.
Ligand & Complexes
δ (ppm) of Methine in DMSO-d6
DAC (1)
6.20
5.71
5.84
(DAC)2Mg (2)
(DAC)2Zn (3)
(DAC)2Cu (4)
(DAC)2Mn (5)
(Not observed)
5.32
2.5. NMR Solid State Spectra
The Cross Polarized Magic Angle Spinning (CP-MAS) 13C-NMR spectrum of ligand DAC shows
signals at 184.61 and 179.35 ppm for the carbonyls of the -diketone system. The diamagnetic metals
complexes (DAC)2Mg and (DAC)2Zn show the overlap of these signals into a singlet (see Suplementary
β

Molecules 2019, 24, 1598
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Information). The CP-MAS spectrum of (DAC)2Zn is shown to illustrate this effect (Figure 1). The
carbonyls overlap into a singlet at 183.44, which can be attributed to the increased symmetry at
the plane of coordination about the metal atom. The CP-MAS spectra of complexes (DAC)2Cu and
(DAC)2Mn prevents such observation, due to the paramagnetic effects.
DAC
184.61
179.35
(DAC)2Zn DAC-Zn
183.44
Figure 1. Comparison between 100 MHz 13C nuclear magnetic resonance (NMR) spectra of DAC and
(DAC)2Zn.
2.6. EPR Spectra
The ligand and their complexes with Mg(II) and Zn(II) were diamagnetic, while the electron
paramagnetic resonance (EPR) spectra of Cu(II) and Mn(II) complexes (
4 and 5) show a typical four
and six lines pattern (see Supplementary Material). The g , g , A and A values were measured from
⊥
⊥
k
k
the EPR spectra of the complexes. The g and g values for DAC-Cu were 2.29, and 2.06, respectively
⊥
k
(see Table 4); indicating that complexes have unpaired electrons in their dx2-y2 molecular orbital. The
value of g is greater than 2.3, suggesting ionic environments in the copper complexes. The A value
k
k
is ca. 160
×
10−4 cm−1 consistent with a typical monomeric distorted square planar structure. The
quotient g /A provides an index of departure from the tetrahedral structure. The quotient value falls
in the range of 105–135 cm−1 for a regular square planar structure but the observed results (141 cm−1
k
k
)
are indicative of strong distortion from planarity. The EPR spectrum of the Mn(II) complex shows one
broad isotropic signal with g value of 2.00 (see Table 5). Magnetic moment values of Cu(II) and Mn(II)
complexes suggest that there are paramagnetic with µeffect values of 1.7 and 4.8 B.M with one and five
unpaired electrons, respectively (see Tables 4 and 5) [9,27–31].

Molecules 2019, 24, 1598
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Table 4. Electron paramagnetic resonance (EPR) spectral data of complex (DAC)2Cu in DMF.
Complex
g
g
A (10−4 cm−1
)
A
(10−4 cm−1
)
g /A (cm−1
)
µeffect
⊥
⊥
k
k
k
k
(DAC)2Cu (4)
2.29
2.06
162.3
1.13
141.1
1.7
Table 5. EPR spectral data of complex (DAC)2Mn in DMSO.
Complex
g
A (10−4 cm−1
)
g/A (cm−1
)
µeffect
6.2
(DAC)2Mn (5)
2.00
85
235.3
2.7. X-ray Diffraction
Good quality crystals of (DAC)2Mg, (DAC)2Zn, (DAC)2Cu, and (DAC)2Mn were obtained by
controlled nucleation upon standing. The experimental set up was kept undisturbed for crystallization.
Table 6 summarizes the crystal data, collection parameters and refinements for compounds 2, 3, 4,
and 5.
Table 6. Crystal Data, Collection Parameters and Refinements for compounds 2, 3, 4, and 5.
Compound
2
3
4
5
1,506,152
0.10 × 0.04 × 0.040
Yellow/prism
C53H53CuNO17
1039.50
CCDC deposit No.
Crystal size (mm)
Color/shape
Empirical formula
Formula weight
Crystal system
Space group
1,418,638
0.23 × 0.05 × 0.04
Colorless/prism
C54H58 Mg O18S2
1083.43
1,453,160
1,534,704
0.29 × 0.11 × 0.03
Colorless/prism
C54H58MnO18S2
1114.06
0.20 × 0.18 × 0.04
Yellow/prism
C52H52 Zn O17
1046.36
S
Triclinic
P-1
Triclinic
P-1
Triclinic
P-1
Triclinic
P-1
Unit cell dimensions
a, Å
b, Å
c, Å
α, deg
7.4419(5)
12.1480(7)
15.8082(10)
80.701(2)
85.653(2)
81.390(2)
1392.50(15)
1
1.292
0.177
570
2.31 to 25.37
7.4706(2)
11.7637(4)
30.7699(10)
81.6770(10)
86.8260(10)
79.6230(10)
2630.68(14)
2
12.7683(7)
13.2372(7)
15.3255(8)
99.5684(18)
103.0083(19)
94.303(2)
2471.6(2)
2
7.513(5)
12.165(14)
15.688(17)
80.87(10)
85.68(6)
80.40(7)
1394(2)
1
1.327
0.382
583
2.31 to 25.40
β, deg
γ, deg
Volume, Å3
Z
Density (calculated), Mg /m3
Absorption coefficient, mm−1
F (000)
1.321
0.576
1092
1.397
0.517
1086
Θ range for data collection (◦)
Index ranges
2.07 to 25.36
2.41 to 25.37
−8 ≤ η ≤ 8, −14 ≤ κ −8 ≤ η ≤ 8, −14 ≤ κ
−
15 ≤ η ≤ 15,
κ ≤ 15, 18 ≤ λ ≤ 16
40,595
−
15
≤
−8 ≤ η ≤ 9, −14 ≤ κ
≤ 14, −18 ≤ λ ≤ 18
22,968
≤ 14, −19 ≤ λ ≤ 18
≤ 14, −36 ≤ λ ≤ 37
−
Reflections collected
20,701
43,039
Independent reflections
5086 (Rint = 0.1991) 9550 (Rint = 0.0736)
9019 (Rint =
0.16875)
5086 (Rint = 0.1893)
Data/restraints/parameters
Goodness-of-fit on F2
Final R índices
5086/0/346
0.859
9550/90/676
9019/0/659
1.096
5086/20/357
1.054
1.034
R1 = 0.0676, wR2 = R1 = 0.0685, wR2 = R1 = 0.0889, wR2 = R1 = 0.0768, wR2 =
0.1330 0.1517 0.1580 0.1538
R1 = 0.1798, wR2 = R1 = 0.1073, wR2 = R1 = 0.1644, wR2 = R1 = 0.1571, wR2 =
[(I) > 2σ (I)]
R indices (all data)
0.1812
0.300 and −0.430
0.1714
1.354 and −0.495
0.1949
0.539 and −0.606
0.1939
0.337 and −0.373
Largest diff. peak and hole (
ε
/A−3
)
The molecular structures of compounds
ellipsoids at 50% probability level.
2, 3, 4, and 5 are shown in Figures 2–5 with displacement

Molecules 2019, 24, 1598
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Figure 2. Molecular structure of 2. Hydrogen atoms are drawn as circles with an arbitrary radius.
Figure 3. Molecular structure of 3. Hydrogen atoms are drawn as circles with an arbitrary radius.

Molecules 2019, 24, 1598
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Figure 4. Molecular structure of 4. Hydrogen atoms are drawn as circles with an arbitrary radius.
Figure 5. Molecular structure of 5. Hydrogen atoms are drawn as circles with an arbitrary radius.
Selected bond distances and bond angles for compounds 2, 3, 4, and 5 are listed in Table S1.
The values of the M-O bond distances of the metal complexes DAC and the corresponding metal
acetylacetonates are shown in the Table 7, showing great similarity among them [32–38].
The new solid phases of metal complexes were confirmed by powder X-ray diffraction (PXRD),
as can be appreciated from strong differences in the PXRD pattern of magnesium, zinc, copper, and
manganese complexes compared to DAC (see Supplementary Material).

Molecules 2019, 24, 1598
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Table 7. Comparison of bond distances (Å) of metal complexes of diacetylcurcumin and acetylacetonates.
Compounds
Melting Point
Geometry
M-O (Å)
Mg(1)-O(1)
Mg(1)-O(2)
2.022
2.053
(DAC)2Mg (2)
245.6 ◦C
octahedral
Mg(1)-O(1)
Mg(1)-O(2)
2.040
2.027
acac-Mg
265 ◦C
octahedral
Zn(1)-O(1)
Zn(1)-O(2)
Zn(1)-O(9)
Zn(1)-O(10)
2.021
1.987
2.031
1.991
(DAC)2Zn (3)
247.7 ◦C
trigonal bipyramid
Zn(1)-O(11)
Zn(1)-O(12)
Zn(1)-O(21)
Zn(1)-O(22)
2.002
1.971
2.008
2.096
acac-Zn
136.5 ◦C
242.5 ◦C
trigonal bipyramid
square planar
Cu(1)-O(1)
Cu(1)-O(2)
Cu(1)-O(9)
Cu(1)-O(10)
1.906
1.900
1.916
1.913
(DAC)2Cu (4)
Cu(1)-O(1)
Cu(1)-O(2)
1.914
1.912
acac-Cu
(DAC)2Mn (5)
acac-Mn
285.5 ◦C
191 ◦C
249 ◦C
square planar
octahedral
octahedral
Mn(1)-O(1)
Mn(1)-O(2)
2.108
2.132
Mn(1)-O(1)
Mn(1)-O(2)
2.150
2.129
2.8. Inhibition of Lipid Peroxidation (LP) in Rat Brain Homogenate
The four metal complexes of DAC showed high antioxidant activity on the lipid peroxidation in
rat brain homogenate model (see Table 8). The IC50 values of complexes show higher antioxidant
2–5
activity than α−tocopherol and comparable to that of BHT. According to previous works, the antioxidant
effects of curcumin are retained or enhanced in its metal complex form [39,40].
2.9. Cytotoxic Activity
The results indicate that complexes
cytotoxic effects against the cell lines HCT-15, MCF-7, and SKLU-1, the IC50 values of three homoleptic
complexes being similar or lower than cisplatin, and much lower than compound (Table 9). These
2, 3 and 5 (Mg, Zn, and Mn, respectively) had important
1
results agree with several studies of antitumoral activity with heteroleptic metal complexes of curcumin,
where various authors suggest that the metal presence in the complex preserves the therapeutic potential
of curcumin or increases its stability in cellular medium [
application of transition metals in tumor-selective complexes, as drug-delivery agents, has been amply
described previously [ 41]. Interestingly, in this work, compound (copper) did not show a cytotoxic
1,39,41]. Moreover, the importance and
9,
4
effect against the cell lines tested. Although there are several reports of antitumoral activity of copper
complexes with curcumin, the cytotoxic effects seem to be related to the presence of free phenolic
groups [8,15,42].

Molecules 2019, 24, 1598
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Table 8. IC50 of inhibition of lipid peroxidation of compounds 1, 2, 3, 4, and 5.
Compounds
Concentration
TBARS (nmol/mg
% Inhibition
IC50 (µM)
(µM)
Protein)
Basal
-
-
0.19 ± 0.03
-
-
-
-
Control FeSO4
10.11 ± 0.56
Basal
Control
0.1
0.32
1
3.16
10
31.62
100
0.200 ± 0.011
-
-
6.589 ± 0.213
6.283 ± 0.188
4.62 ± 0.57
8.26 ± 1.31
21.13 ± 2.56 *
44.84 ± 6.74 **
59.00 ± 3.71 **
72.3 ± 3.87 **
79.09 ± 4.79 **
6.048 ± 0.242
α−Tocoferol (n = 4)
6.78 ± 2.16
5.211 ± 0.332 *
3.676 ± 0.569 **
2.725 ± 0.335 **
1.849 ± 0.319 **
1.408 ± 0.364 **
Basal
Control
0.32
0.42
0.56
0.75
1
1.33
1.78
2.37
3.16
0.268 ± 0.053
7.384 ± 0.630
6.690 ± 0.655
6.510 ± 0.551
6.098 ± 0.353
5.559 ± 0.294 *
4.457 ± 0.283 **
3.228 ± 0.572 **
1.315 ± 0.489 **
0.487 ± 0.075 **
0.53 ± 0.044 **
-
-
9.53 ± 2.91
11.62 ± 2.78
16.64 ± 2.86
23.92 ± 2.69 *
37.14 ± 7.44 **
53.59 ± 8.93 **
81.59 ± 6.89 **
93.16 ± 1.16 **
95.08 ± 0.68 **
BHT (n = 5)
1.22 ± 0.44
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 (1)
3.21 ± 0.16
2.03 ± 0.27
1.58 ± 0.07
1.58 ± 0.15
1.24 ± 0.10
7.78 ± 0.53 *
5.22 ± 0.35 **
1.43 ± 0.08 **
23.19 ± 0.92 *
48.46 ± 1.53 **
85.82 ± 0.60 **
0.1
0.33
1
3.28
10.38
9.50 ± 0.63
6.20 ± 1.00
8.48 ± 0.78
16.41 ± 3.31
(DAC)2Mg (2)
(DAC)2Zn (3)
(DAC)2Cu (4)
(DAC)2Mn (5)
6.55 ± 0.67 **
3.56 ± 0.37 **
0.34 ± 0.02 **
35.52 ± 3.78 **
64.52 ± 4.42 **
96.63 ± 0.08 **
0.1
0.32
1
3.22
10.20
9.16 ± 0.90
9.87 ± 3.73
8.02 ± 0.72
20.99 ± 2.56
6.22 ± 0.67 **
2.87 ± 0.14 **
0.35 ± 0.02 **
38.85 ± 3.04 **
71.53 ± 1.67 **
96.53 ± 0.41 **
0.1
0.33
1
3.22
10.20
9.08 ± 0.66
10.34 ± 1.48
8.04 ± 0.70
20.78 ± 2.44
6.24 ± 0.71 **
3.08 ± 0.21 **
1.32 ± 0.48 **
38.70 ± 3.82 **
69.57 ± 1.56 **
86.58 ± 5.21 **
0.1
0.33
1
3.22
10.19
8.79 ± 0.68
7.87 ± 0.69 *
5.71 ± 0.31 **
1.61 ± 0.48 **
0.29 ± 0.01 **
14.18 ± 2.76
23.20 ± 2.76 *
44.10 ± 0.55 **
83.69 ± 5.37 **
97.18 ± 0.14 **
Data are represented as mean ± SEM of thee replicates. * P ≤ 0.05 and ** P ≤ 0.05 compared to FeSO4.

Molecules 2019, 24, 1598
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Table 9. IC50 (µM) for cancer cell lines with compounds 1, 2, 3, 5 and cisplatin.
Compounds
DAC (1)
HCT-15
MCF-7
SKLU-1
14.07 ± 3.3
8.87 ± 1.56 *
4.74 ± 0.31 *
9.23 ± 1.22 *
4.3 ± 0.5 *
22.32 ± 3.0
11.59± 1.97 *
7.21 ± 0.61 *
15.54 ± 1.94 *
10.0 ± 0.9 *
21.79 ± 5.0
5.72 ± 0.31 *
4.92 ± 0.071 *
13.46 ± 1.63 *
9.4 ± 1.0 *
(DAC)2Mg (2)
(DAC)2Zn (3)
(DAC)2Mn (5)
Cisplatin
Data are represented as mean ± standard error on the mean (SEM) of three replicates, * P ≤ 0.05, compared to 1.
2.10. Acute Toxicity Study in Mice
There was no mortality of mice in any of the groups up to a dose of 3 g kg−1 (see Table 10). Only
the oral administration of the (DAC)2Cu and (DAC)2Mn complexes provoked minor bristling of the
fur (see Table 11). The body weight of all mice gradually increased and was not significantly different
to the control groups, which started on the first day of treatment.
Table 10. Body weight (g) and body weight gain (%) of mice given acute oral dose (values are in mean
±SD).
Doses
(mg/kg)
Body Weight at Day
8
% Body Weight Gain
No. of
Animals
Complexes
Sex
1
15
1–8
8–15
(DAC)2Mg
(DAC)2Mg
(DAC)2Zn
(DAC)2Zn
(DAC)2Cu
(DAC)2Cu
(DAC)2Mn
(DAC)2Mn
1000
3000
1000
3000
1000
3000
1000
3000
3
3
3
3
3
3
3
3
Male
Male
Male
Male
Male
Male
Male
Male
25.3
26.5
27.1
28.4
29.3
27.5
25.8
26.1
±
0.3 27.0
0.1 28.6
0.5 28.6
0.4 30.0
0.6 31.4
0.9 29.4
0.2 26.9
1.8 27.6
±
±
±
±
±
±
±
±
0.5 30.0
±
±
±
±
±
±
±
±
0.4
1.5
0.5
0.3
0.3
0.8
0.4
1.3
6.7 ± 0.3
7.5 ± 0.3
5.5 ± 0.7
5.6 ± 0.1
8.5 ± 0.7
6.5 ± 0.6
6.5 ± 0.6
4.2 ± 1.0
10.7
13.3
±
±
0.6
1.5
±
±
±
±
±
±
±
0.2 32.0
0.2 31.3
0.3 33.7
1.1 33.6
1.5 30.5
0.5 29.0
0.8 29.4
9.8 ± 0.4
12.3
±
0.0
8.0 ± 1.1
4.1 ± 0.8
4.1 ± 0.8
5.8 ± 0.5
Table 11. Clinical signs and mortality of mice receiving acute oral dose.
No. of
Animals
Complexes
Doses (mg/kg)
Sex
Clinical Signs
Mortality
(DAC)2Mg
(DAC)2Mg
(DAC)2Zn
(DAC)2Zn
(DAC)2Cu
(DAC)2Cu
(DAC)2Mn
(DAC)2Mn
1000
3000
1000
3000
1000
3000
1000
3000
3
3
3
3
3
3
3
3
male
male
male
male
male
male
male
male
normal
normal
0
0
0
0
0
0
0
0
normal
normal
bristly fur
bristly fur
bristly fur
bristly fur
3. Materials and Methods
All chemicals were available commercially and the solvents were purified with conventional
methods prior to use [43]. Curcumin was obtained from natural source by usual extractive procedures
and purified by crystallization.

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3.1. Physical Measurements
Melting points were determined on an Electrothermal Engineering IA9100X1 melting point
apparatus and are uncorrected.
3.2. Spectroscopic Determinations
UV-Vis spectra were recorded in a UV-Visible Spectrophotometer Varian Cary 400 (Varian NMR
Instruments Palo Alto, California, CA, USA; www.agilent.com). The fluorescence measurements of the
samples were recorded on the Varian-Cary-Eclipse equipment (Varian NMR Instruments Palo Alto,
California, CA, USA; www.agilent.com). IR absorption spectra were recorded in the range of 4000–
1
230 cm−1 as KBr pellets on a BRUKER Tensor 27 spectrophotometer. H- and 13C-NMR spectra were
recorded in CDCl3 and DMSO-d6 on a JEOL 300 MHz and Unity Varian 500 MHz and an Agilent One
Probe 600 MHz spectrometers using TMS as an internal reference. Solid State CP-MAS NMR spectra
were recorded to monitor complexation and data are included in the Supplementary Material. The
EPR spectra were recorded in DMSO and DMF at liquid nitrogen temperature (77 K) on an Electronic
Paramagnetic Resonance Spectrometer Jeol, JES-TE300, ITC Cryogenic System, Oxford. The magnetic
moment of the compounds was determined using a Johnson-Matthey magnetic susceptibility balance
type msb model mk II 13094-3002 (Thermo Fisher Scientific, Applied Biosystems de M
R.L. M xico, CDMX), with the Gouy method at room temperature. High resolution mass spectra for
complexes of (DAC)2Zn ( ) and (DAC)2Cu ( ) were recorded in a MStation JMS-700 equipment (JEOL
Mexico S.A of C.V., M xico, CDMX) in the fast atom bombardment (FAB+) mode and low resolution
mass spectra for (DAC)2Mg ( ) and (DAC)2Mn ( ) were recorded in a JEOL, SX 102. A spectrometer on
Bruker Microflex equipped with MALDI-Flight time. Elemental analysis were performed in a Thermo
Scientific elemental analyzer (ThermoFisher Scientific, Applied Biosystems de M xico, S. de R.L. de
C.V., M xico, CDMX), model Flash 2000 and a Perkin Elmer elemental analyzer, model 2400CHNSO.
Single-crystal X-ray diffraction (SCXRD) were determined in a Bruker diffractometer (Bruker Mexicana
S.A. de C. V., Mexico, CDMX), model Smart Apex, equipped with Mo radiation ( = 0.71073Å), CCD
éxico, S. de
é
3
4
é
2
5
é
é
λ
two-dimensional detector and a lw temperature device. Data collection and data reduction were
performed by APEX and SAINT-Plus programs (Bruker-Nonius AXS Inc., Madison, WI, USA), [44].
The structures were solved by the direct method with the structure solution program-SHELXS-97 and
Refinement program-SHELXL97 (Dept. of Structural Chemistry, Universität Göttingen, Göttingen,
Germany) and refined by full-matrix least-squares procedure on F2 using SHELX-2008 program [45].
Powder X-ray diffraction (PXRD) were determined in a Bruker diffractometer, model D8 Advance
Davinci, theta-theta configuration Bruker AXS, Lynxeye detector.
3.3. Inhibition of Lipid Peroxidation in Rat Brain
3.3.1. Animals
Adult male Wistar rats (200–250 g) were provided by the Instituto de Fisiolog
ía Celular, Universidad
Nacional Aut noma de M xico (UNAM). Procedures and care of animals were conducted in conformity
ó
é
with the Mexican Official Norm for Animal Care and Handling NOM-062-ZOO-1999. They were
maintained at 23 ± 2 ◦C on a 12/12 h light-dark cycle with free access to food and water.
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 [46 47]. The homogenate was
,
then centrifuged for 10 min at 800 rcf (relative centrifugal field) to yield a pellet that was discarded.
The supernatant protein content was measured using Folin and Ciocalteu’s phenol reagent [48] and
adjusted with PBS at 2.666 mg of protein/mL.

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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 [49], with some modifications. Supernatant
(375
in DMSO (50
was started adding 50
and incubated at 37 ◦C for 1 h. The TBARS content was determined, as described by Ohkawa and
co-workers [50], with some modifications. A volume of 500 L of TBA reagent (1% 2-thiobarbituric
µ
L) was added with 50
L of DMSO for control group) and incubated at 37 ◦C for 30 min. Lipid peroxidation
L of freshly prepared 100 M FeSO4 solution (final concentration 10 M),
µL of 20 µM EDTA and 50 µL of each sample concentration dissolved
µ
µ
µ
µ
µ
acid in 0.05 N NaOH and 30% trichloroacetic acid, in 1:1 proportion) was added to each tube and
the final suspension was 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 UltraMicroplate (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 [51]. Results are expressed as nmoles 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 were represented as mean
×
α
±
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.
3.4. Citotoxic Activity in Human Tumor Cells
Cytotoxicity of all compounds was tested against three cancer cell lines: HCT-15 (human
colon adenocarcinoma), MCF-7 (human mammary adenocarcinoma) and SKLU-1 (human lung
adenocarcinoma). Cell lines were supplied by the U.S. National Cancer Institute (NCI). The cell lines
were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 2 mM l-glutamine,
10,000 units/mL penicillin G sodium, 10,000
µg/mL streptomycin sulfate, 25
µg/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 [52–54]. The
results were expressed as inhibitory concentration 50 (IC50) values. They were calculated according
to the protocol of Monks [52], where a dose–response curve was plotted for each compound and the
concentration (IC50), resulting in an inhibition of 50% estimated through non-linear regression analysis.
3.5. Acute Toxicity Study In Mice
The experiments were performed using adult male CD1 mice (body-weight range, 25–30 g). Food
was withheld for 12 h prior to experiment with animals having free access to drinking water. The
concentration of the tested compounds (DAC, (DAC)2Mg, (DAC)2Zn, (DAC)2Cu and (DAC)2Mn) was
adjusted for oral administration of 1000 and 3000 mg/kg (three animals per dose) according to the
Lorke method [55]. The mice were observed daily for a period of 14 days for mortality, toxic effects,
and/or changes in weight and behavioral patterns.
All experiments were performed in accordance with ethical standards for experimental
pain research in animals [56] and the Mexican Official Standard for animal care and handling
(NOM-062-ZOO-1999). The experimental protocol (CIBIUG-P15-2018) was approved and overseen
by the Institutional Ethics Committee for Care and Use of Laboratory Animals of the Universidad
de Guanajuato, and the CD1 mice were obtained from the Natural and Exact Science Vivarium at
Universidad de Guanajuato, under controlled temperature (23
±
2 ◦C) and humidity (55
±
10%), with a

Molecules 2019, 24, 1598
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12-h light/darkness cycle, and were familiarized with the environment one week before the experiments.
The animals were allowed food and water ad libitum. Immediately after the experiments, all animals
were sacrificed in a CO2 chamber.
3.6. Synthesis of Compounds
General synthesis procedure of DAC
1, DAC-Magnesium
2, DAC-Zinc
3, DAC-Copper 4 and
DAC-Manganese 5 are shown in Scheme 1.
1
DAC (
(300 MHz CDCl3):
CarylH), 7.61 (d, 2H, J 16.2 CvinylH), 15.85 (br s, 1H,), 13C-NMR (13C {1H} 75 MHz, CDCl3):
1
). DAC was obtained in accordance to a synthetic method reported [57]. 70.1% yield. H-NMR
δ
2.32 (s, 6H), 3.87 (s, 6H), 5.85 (s, 1H), 6.56 (d, 2H, J 16.2 CvinylH), 7.11 (m, 6H,
20.74
δ
(C-H), 56.01 (C-H), 101.88 (C-H), 111.56 (CarylH), 121.16 (CarylH), 123.39 (CarylH), 124.36 (CvinylH),
134.06 (CarylH), 140.04 (CvinylH), 141.41 (Caryl), 151.51(Caryl), 168.88 (C=O), 183.19 (C=O), 1H-NMR
(600 MHz DMSO-d6):
J 8.1 CarylH), 7.33 (dd, 2H, J 8.2; 1.9 CarylH), 7.52 (d, 2H, J 2 CarylH), 7.66 (d, 2H, J 15.9 CvinylH), 16.1
(br s, 1H,), 13C-NMR (13C {1H} 150 MHz, DMSO-d6):
20.17 (C-H), 55.72 (C-H), 101.62 (C-H), 111.93
δ 2.28 (s, 6H), 3.85 (s, 6H), 6.20 (s, 1H), 6.99 (d, 2H, J 15.9 CvinylH), 7.16 (d, 2H,
δ
(CarylH), 121.27 (CarylH), 123.23 (CarylH), 124.58 (CvinylH), 133.59 (CarylH), 139.77 (CvinylH), 140.93
(Caryl), 151.11(Caryl), 168.30 (C=O), 183.10 (C=O), IR (1755.84 cm−1, 1596.49 cm−1, 1506.17 cm−1, 1295.51
cm−1, 1154.04 cm−1, 619.80 cm−1), HRMS: M+ 453.1541 (calculated exact mass 453.1549), yellow crystals
m.p.: 170.5 ◦C. Elemental Analysis: Calculated for C25H24O8: C 66.36, H 5.35; experimental: C 66.27;
H 5.33.
DAC-Magnesium (2). 1 mmol of DAC was dissolved in a mixture of 25 mL ethyl acetate-methanol
(7:3), and later a solution of magnesium acetate in methanol (0.5 mmol) was added slowly. After 2 h of
stirring at room temperature, a yellow powder was formed in the flask (m.p. 245.6 ◦C), which was
1
filtered and crystallized with DMSO (m.p. 239.0 ◦C), 83.1% yield. H-NMR (600 MHz DMSO-d6):
δ
2.26 (s, 6H), 3.83 (s, 6H), 5.71 (s, 1H), 6.85 (d, 2H, J 15.6 CvinylH), 7.09 (d, 2H, J 8.1 CarylH), 7.20 (dd,
2H, J 8.1; 1.8 CarylH), 7.41 (d, 2H, J 1.9 CarylH), 7.41 (d, 2H, J 15.6 CvinylH), 13C-NMR (13C {1H} 150
MHz, DMSO-d6):
δ 20.32 (C-H), 55.63 (C-H), 103.33 (C-H), 111.81 (CarylH), 120.31 (CarylH), 123.01
(CarylH), 131.08 (CvinylH), 134.58 (CarylH), 135.18 (CvinylH), 139.67 (Caryl), 150.91 (Caryl), 168.04 (C=O),
180.96 (C=O)), IR (3263.94 cm−1, 1754.19 cm−1, 1595.53 cm−1, 1509.41 cm−1, 1457.34 cm−1, 1204.07 cm−1
,
1119.45 cm−1, 630.36 cm−1) LRMS: M+ 927.347. (calculated exact mass 927.2709). Elemental Analysis:
Calculated for C50H46MgO16·2H2O: C 62.35, H 5.23; experimental: C 62.54; H 5.42.
DAC-Zinc (3). 1 mmol of DAC was dissolved in a mixture of 25 mL ethyl acetate-methanol (7:3),
and later, a solution of zinc acetate in methanol (0.5 mmol) was added slowly. After 2 h of stirring
at room temperature, a yellow powder was formed in the flask (m.p. 247.7 ◦C), which was filtered
1
and crystallized with DMSO (m.p of crystal: 258.9 ◦C), 95.6% yield. H-NMR (600 MHz DMSO-d6):
δ
2.26 (s, 6H), 3.83 (s, 6H), 5.84 (s, 1H), 6.91 (d, 2H, J 15.7 CvinylH), 7.11 (d, 2H, J 8.1 CarylH), 7.26 (dd,
2H, J 8.3; 1.8 CarylH), 7.47 (d, 2H, J 1.9 CarylH), 7.53 (d, 2H, J 15.6 CvinylH), 13C-NMR (13C {1H} 150
MHz, DMSO-d6):
δ 20.24 (C-H), 55.74 (C-H), 102.92 (C-H), 111.40 (CarylH), 120.58 (CarylH), 123.11
(CarylH), 129.89 (CvinylH), 134.20 (CarylH), 137.49 (CvinylH), 140.07 (Caryl), 150.90 (Caryl), 168.83 (C=O),
183.89 (C=O)), IR (3325.95 cm−1, 1757.91 cm−1, 1587.25 cm−1, 1508.59 cm−1, 1444.73 cm−1, 1203.53 cm−1
,
1119.43 cm−1, 631.93 cm−1), HRMS: M+ 967.2168 (calculated exact mass 967.2156). Elemental Analysis:
Calculated for C50H46O16Zn·H2O: C 60.89, H 4.91; experimental: C 60.23; H 5.09.
DAC-Copper (4). 1 mmol of DAC was dissolved in a mixture of 25 mL ethyl acetate-methanol (7:3),
and later, a solution of copper acetate in methanol and water (0.5 mmol) was added slowly. After
2 h of stirring at room temperature, a brown powder was formed in the flask (m.p. 242.5 ◦C), which
1
was filtered and crystallized with DMF (m.p 240 ◦C.), 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), 7.03 (br s, 1H), 7.16 (br s, 1H), 7.34 (br s,
2H), 7.52 (br s, 1H), 7.66 (br s, 1H), 13C-NMR (13C {1H} 125 MHz, DMSO-d6):
δ
20.37 (C-H), 55.89

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(C-H), 101.59 (C-H), 112.29 (CarylH), 121.51 (CarylH), 123.21 (CarylH), 124.72 (CvinylH), 133.57 (CarylH),
139.99 (CvinylH), 140.87 (Caryl), 151.13 (Caryl), 168.48 (C=O), 183.31 (C=O), IR (2975.04 cm−1, 2941.34
cm−1, 1752.55 cm−1, 1592.19 cm−1, 1514.26 cm−1, 1412.95 cm−1, 1299.11 cm−1, 1156.63 cm−1, 604.42
cm−1), HRMS: M+ 966.2164 (calculated exact mass 966.2160). Elemental Analysis: Calculated for
C50H46CuO16·H2O: C 61.00, H 4.91; experimental: C 60.96; H 5.04.
DAC-Manganese (5). 1 mmol of DAC was dissolved in a mixture of 25 mL ethyl acetate-methanol
(7:3), and later, a solution of manganese acetate in methanol (0.5 mmol) was added slowly. After 2 h
of stirring at room temperature, a brown powder was formed in the flask (m.p.191.5 ◦C), which was
1
filtered and crystallized from DMSO (m.p. 201.3◦), 85.9% yield. H-NMR (500 MHz DMSO-d6):
δ 2.27
(br, 6H), 3.76 (br, 6H), 5.32 (br s, 1H), 7.98 (br s, 2H), IR (3264.94, 1752.62, 1629.42, 1592.61, 1505.53,
1203.53, 1155.12, 602.92, 463.93 cm−1), LRMS: M+ 958.167. (calculated exact mass 958.2239). Elemental
Analysis: Calculated for C50H46MnO16·H2O: C 61.54, H 4.96; experimental: C 61.42; H 4.79.
4. Conclusions
Four homoleptic complexes of Mg(II), Zn(II), Cu(II), and Mn(II), with DAC, were synthesized and
characterized by physicochemical means as amorphous powders and single crystals. All complexes
crystallized in the P-1 space group show that metals are linked to the β-diketone functionality of
DAC in its enolic form. While the ligand design preserving the curcumin motif had the choices
of etherification or esterification, it was acetylation that proved to be synthetically advantageous.
The NMR spectra in solid state (CP-MAS) of non-paramagnetic amorphous complexes, allowed the
assessment of complexation through the maximum change in the chemical shifts of carbonyls at
the β-diketone function once the 1:2 metal:ligand ratio is reached. It was found also that the use of
DMSO and DMF as solvents for crystallization was important for the formation of single crystals. The
approach reported here provides an accesible route for the synthesis of homoleptic metal complexes
of DAC overcoming the fact that curcumin and its derivatives have been described as elusive in
the formation of single crystals. From a pharmacological point of view, it can be considered more
convenient to handle a homoleptic complex composed exclusively of two curcuminoid moieties rather
than a heteroleptic one (stabilizer, curcuminoid and metal ion).
The four homoleptic complexes studied inhibited lipid peroxidation in the rat brain tissue model
with higher activity than
α-tocopherol and comparable to BHT. It is noteworthy that all copper
complexes synthesized in our previous report [21] as well as the copper complex in the present one,
exhibited considerable antioxidant effect but negligible cytotoxicity. Of special significance is the
remarkable cytotoxic effect found for (DAC)2Mg and (DAC)2Zn against HCT-15, MCF-7 and SKLU-1
human cancer cell lines, which surprisingly equated or surpassed that of cisplatin. Moreover, the
preliminary in vivo experiments showed that at a dose of 3 g/kg the complexes fall within category
5 (2000–5000 mg/kg, i.e., the less toxic category for chemical compounds, according to the OECD
protocols). Considering the powerful antioxidant effects and the significant cytotoxicity produced by
the metal complexes of DAC reported here upon three human cancer cell lines, together with their
minimal in vivo acute toxicity found, further research can be expected to provide a deeper insight into
this findings.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Extensive synthetic laboratory work, W.M.-M. (principal PhD student), M.M.E.-C., Y.A.-R.,
and M.A.O.-M.; validation (X-ray extensive studies), M.S.-G.; biological essays, M.T.R.-A. and C.E.-M.; NMR
studies and funding acquisition, J.C.; writing, review and editing, project administration, and funding acquisition,
R.G.E., W.F.R., and NMR in liquids. Overlapping tasks from all co-authors is thoroughly acknowledged.
Funding: We gratefully acknowledge financial support from PAPIIT (DGAPA, UNAM, IN208516) and CONACyT
(CB 252524).
Acknowledgments: The scholarships from CONACYT to WMM (No. 576707), YAR (No. 576706) and MAOM (No.
603692) are gratefully acknowledged. We are indebted to Simón Hernández-Ortega from Instituto de Química,
UNAM (X-ray), Juan Carlos Poveda (NMR) Universidad Industrial de Santander (UIS), and Shane Pawsey (NMR)

Molecules 2019, 24, 1598
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from Bruker Biospin Corporation, Billerica, Massachusetts, USA for technical support. Acknowledgements are
also awarded to: Rosa Isela Del Villar (CP-MAS) and Victor Hugo Lemus Neri (EA) from Facultad de Qu mica
(USAI) and Virginia G mez Vidales (EPR), Mar a de la Paz Orta (EA), Mar a del Roc o Patiño (IR), Isabel Ch vez
í
ó
í
í
í
á
(NMR) and Luis Velazco (EM) from Instituto de Química, UNAM.
Conflicts of Interest: The authors declare no conflicts of interest.
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