Effects of Cu(II) and Zn(II) coordination on the trypanocidal activities of curcuminoid-based ligands

Article abstract of DOI:10.1016/j.ica.2019.119237

Four heteroleptic metal complexes with the general formula [M(L1-2)(phen)Cl] 1a-b, 2a-b, (M = Cu(II) or Zn(II), L = curcuminoid ligand and phen = phenanthroline) have been prepared from the reaction of the ligands in the presence of Et₃N with C

Full text of DOI:10.1016/j.ica.2019.119237

Journal Pre-proofs
Research paper
Effects of Cu(II) and Zn(II) coordination on the trypanocidal activities of cur-
cuminoid-based ligands
Bianca Almeida da Silva, Paulo Pitasse-Santos, Vitor Sueth-Santiago, Antônio
Ricardo Moutinho Monteiro, Roberta Katlen Fusco Marra, Guilherme Pereira
Guedes, Ronny Rocha Ribeiro, Marco Edilson Freire de Lima, Debora
Decoté-Ricardo, Amanda Porto Neves
PII:
S0020-1693(19)31178-8
https://doi.org/10.1016/j.ica.2019.119237
ICA 119237
DOI:
Reference:
Inorganica Chimica Acta
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Accepted Date:
7 August 2019
28 October 2019
28 October 2019
Please cite this article as: B.A. da Silva, P. Pitasse-Santos, V. Sueth-Santiago, A.R.M. Monteiro, R.K.F. Marra, G.P.
Guedes, R.R. Ribeiro, M.E.F. de Lima, D. Decoté-Ricardo, A.P. Neves, Effects of Cu(II) and Zn(II) coordination
on the trypanocidal activities of curcuminoid-based ligands, Inorganica Chimica Acta (2019), doi: https://doi.org/
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Effects of Cu(II) and Zn(II) coordination on the trypanocidal activities of
curcuminoid-based ligands
Bianca Almeida da Silva1, Paulo Pitasse-Santos1, Vitor Sueth-Santiago1,2, Antônio
Ricardo Moutinho Monteiro1, Roberta Katlen Fusco Marra1, Guilherme Pereira Guedes3,
Ronny Rocha Ribeiro4, Marco Edilson Freire de Lima1, Debora Decoté-Ricardo5 and
Amanda Porto Neves1,*
1Instituto de Química, Universidade Federal Rural do Rio de Janeiro, BR-465 Km 7, CEP: 23890-
000, Seropédica, RJ, Brasil. *E-mail: amandanevess@ufrrj.br
2Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro, Rua José Augusto Pereira
dos Santos, CEP 24425-004, São Gonçalo, RJ, Brasil.
3Instituto de Química, Universidade Federal Fluminense, Outeiro de São João Batista, s/n, CEP
24020-150, Niterói, RJ, Brasil.
4Departamento de Química, Universidade Federal do Paraná, Cx. Postal 19081, CEP: 81531980,
Curitiba, PR, Brasil.
5Departamento de Microbiologia e Imunologia Veterinária, Instituto de Veterinária, Universidade
Federal Rural do Rio de Janeiro, BR-465 Km 7, CEP: 23890-000, Seropédica, RJ, Brasil.
1

Abstract
Four heteroleptic metal complexes with the general formula [M(L1-2)(phen)Cl] 1a-b, 2a-
b, (M = Cu(II) or Zn(II), L = curcuminoid ligand and phen = phenanthroline) have been
prepared from the reaction of the ligands in the presence of Et3N with CuCl2.2H2O or
1
ZnCl2. Characterization by IR, H NMR and EPR spectroscopies, and X-ray diffraction
analysis for 1b confirmed the proposed structures for the complexes. The curcuminoid
ligand coordinates in a deprotonated form as an O,O chelate, with phen and chloride
completing the coordination sphere of the metal. The cytotoxic effects of ligands (HL1,
HL2, phen) and complexes (1a-b, 2a-b) on the amastigote form of Trypanosoma cruzi
have been screened. All substances showed significant toxicity. The Zn(II) complexes 1b
(IC50 = 0.47 μM, SI = 4.3) and 2b (IC50 = 0.40 μM, SI = 3.8) were the most potent
compounds being more toxic and selective against the parasite than the ligands and the
copper complexes, thus implying that the coordinating metal is important for the
trypanocidal activity of the curcumin-based compounds.
Keywords: Curcuminoid, Cu(II) and Zn(II) complexes, Trypanosoma cruzi, amastigote,
parasite.
1. Introduction
Chagas disease (CD), also known as American Trypanosomiasis, is a vector-borne
infection transmitted by insects from the triatominae subfamily (Reduviidae) popularly
called kissing bugs [1,2]. The etiological agent of CD, the protozoan Trypanosoma cruzi
(T. cruzi), is endemic to Latin America and according to the World Health Organization
(WHO), approximately 6-7 million people worldwide are infected with Trypanosoma
cruzi [3,4]. Also, 12000 deaths are estimated per year in 21 endemic countries in Latin
America [5]. For almost 50 years, the only drug options available for CD have been
2

benznidazole and nifurtimox, which are effective in the acute phase but less efficient in
the chronic phase of the disease. In addition, these drugs are not considered ideal, because
of their severe side effects and the possible development of resistance [5,6]. The search
for novel chemotherapeutics that show toxic effects selective to parasites without
compromising the host organism is the primary paradigm in the search for new
antiparasitic drugs. The approach to antiparasitic chemotherapy can be based both on
biological targets that are specific to the parasite as well as on those shared by the parasites
and their hosts, which have variations that make selectivity possible. In any case, the goal
will always be the development of a new drug exerting greater effects on the parasite and
fewer deleterious effects on the host [7,8].
Diarylheptanoids comprise a class of natural and synthetic compounds of great
interest in medicine, due to their pharmacological profile, such as anticancer,
antimicrobial and antifungal activity [9,10]. The most important compound and one of
the most popular medicinal herb extracts of this class is curcumin [10,11], which has
therapeutic efficacy in skin and autoimmune diseases, as well as proven anti-
inflammatory, antioxidant and antiviral activities [12,13,14]. More recently, its effect
against Aedes aegypti larvae has also been demonstrated [15].
In recent work developed by our group, Sueth-Santiago and co-workers reported
the trypanocidal activity of three natural diarylheptanoids isolated from Curcuma longa
(Zingiberaceae): curcumin, desmethoxycurcumin and bis-desmethoxycurcumin.
Curcumin presented an IC50 value against epimastigotes of T. cruzi (Dm28c strain) of 10
μM and is a potential agent for the treatment of Trypanosoma cruzi infections [16].
Despite the biological relevance exhibited by curcumin, this compound displays some
restrictions on its clinical use, such as low bioavailability and stability, and poor solubility
[17-19]. One of the strategies to overcome these drawbacks has been the coordination of
3

curcumin with metal ions. Such complexes may prevent degradation of curcumin and also
enhance its biological activity [20-25]. For instance, complexes containing curcuminoids
have been reported for a number of applications, including antiarthritis [26], antimicrobial
[27] and, mainly, anticancer [28]. A ruthenium(III) complex containing a curcumin
derivative was reported to be cytotoxic against HeLa cells, with an IC50 = 0.04172 nM,
indicating great anticancer activity [29]. Another important study about oxovanadium(IV)
curcumin complexes containing phen and dppz as auxiliary ligands showed the
remarkable phototoxicity of curcumin (IC50 = 8.2 μM) and its respective complexes (IC50
= 8.1 μM and 3.3 μM) against HeLa cells, in which all were less toxic in the dark (IC50 >
50 μM) [30]. Zinc(II) complexes of curcumin were also shown to induce cell apoptosis
[31] and copper(II) complexes of the type [Cu(curc-deriv)2] were about 3-5 fold more
cytotoxic than the free ligands [32].
Considering that coordination of curcuminoids often results in the improvement
of their biological activities, this work aimed at investigating the trypanocidal activity of
curcumin (HL1) and its carbocyclic analogue (HL2) compared with their corresponding
Cu(II) and Zn(II) complexes of the type [M(L1-2)(phen)Cl] (1a-b, 2a-b) (Figure 1).
Phenanthroline was chosen as the auxiliary ligand based on previous results that showed
greater antibacterial activity of Cu(II) and Zn(II) complexes after coordination with phen
[33,34].
4

Figure 1: Curcuminoid ligands and their Cu(II) and Zn(II) complexes.
The compounds were characterized by spectroscopic techniques, including single
crystal X-ray diffraction for 1b. Finally, their trypanocidal activities were evaluated
towards amastigote forms of T. cruzi, analyzing the effect of complexation on the
activities in vitro.
2. Experimental
2.1. Materials and Methods
All chemicals and solvents were purchased from Sigma-Aldrich, unless otherwise
stated, and were used as received. Curcumin (HL1) was prepared following a procedure
described in the literature [27]. Complex [Zn(L1)(phen)Cl] (1b) has been reported
previously and was synthesized in this work after changing the described methodology
[31]. Melting points were measured in a PFM II capillary. IR spectra were recorded in
1
the ATR mode on a FT-IR Bruker Vertex 70 spectrophotometer. H NMR (500 MHz)
spectra were recorded with a Bruker Ultrashield Plus in DMSO-d6; chemical shifts are
reported in parts per million (ppm) relative to an internal standard of Me4Si. The hydrogen
5

1
1
signals were assigned based on the coupling constant values and 2D H × H – COSY
experiments. 77 K frozen DMSO/H2O solution EPR spectra of the Cu(II) complexes were
obtained employing a Bruker EMX X-band continuous wave spectrometer configured to
300 ms of conversion time, 40.96 ms of time constant, 600 s of sweep time, 1.0 G of
modulation amplitude and 2500 points of spectral resolution. The corresponding spin
Hamiltonian simulations were done using the EasySpin software package [35]. Electronic
spectra were recorded on a UV-1800 Shimadzu spectrophotometer using MeOH, in five
different concentrations in order to calculate the molar absorptivity values (ε). Molar
conductivities were determined from a 1.0 x 10–3 mol L–1 solution of the complexes in
DMSO, using an ion 120 W microprocessor conductivity meter. Elemental analyses of
CHN were performed in a Perkin-Elmer CHN 2400.
2.2. Synthesis
2.2.1. Synthesis of the curcuminoid ligand HL2
2-Acetylcyclopentanone (0.6 mL, 5 mmol), vanillin (1.5 g, 10 mmol) and
tributylborate (5.4 mL, 20 mmol) were added into a suspension of boric anhydride (175
mg, 2.5 mmol) in 15 mL of ethyl acetate, forming a yellow solution. The reaction was
kept under stirring for 5 min at 50 oC. A solution of 2-aminobutane (0.2 mL, 2.5 mmol)
in 2.5 mL of ethyl acetate was added drop wise to the reaction for 15 min until the solution
turned from yellow to orange. The temperature was raised to 80 °C and the reaction was
stirred for 3 h. Afterwards, 15 mL of aqueous hydrochloric acid (1 mol L-1) were added,
and the reaction was stirred for an additional 30 min. The resulting orange precipitate was
filtered under vacuum and washed with hot ethanol.
6

(E)-2-(4-hydroxy-3-methoxybenzylidene)-6-((E)-3-(4-hydroxy-3-
o
methoxyphenyl)acryloyl)cyclopentanone, HL2. Yield: 1.2 g, 62 %; m.p. 222-224 C.
HRMS/ESI(m/z): Calculated for [M+H+]: (394.1416), found: (394.1416). FTIR (ATR,
1
cm-1): 3513 (νOH)ar, 3253 (νOH)enol, 1623 (νC=O). H NMR (500 Mz, DMSO-d6) δ
(ppm): 9.66 (s, 1H); 9.63 (s, 1H), 7.50 (d, 1H, J = 20 Hz); 7.36 (s, 1H); 7.22 (m, 3H) 7.14
(d, 1H, J = 10 Hz); 6.89 (d, 1H, J = 10 Hz); 6.84 (d, 1H, J = 10 Hz); 6.76 (d, 1H, J = 20
Hz); 3.86 (s, 3H); 3.85 (s, 3H); 3.01 (s, 2H); 2.90 (s, 2H). UV-Vis [CH3OH; λ/nm
(deprotonated)]: 263 (260), (345), 450 (450), (472).
2.2.2. Synthesis of Cu(II) and Zn(II) complexes
The curcuminoid ligand (HL1 or HL2) (0.15 mmol) was suspended in CH3CN (5
mL), followed by addition of Et3N (0.18 mmol) and a solution of phen (0.15 mmol) in 2
mL of CH3CN. A solution of the respective metal salt – CuCl2·2H2O or ZnCl2 (0.15
mmol) in 3 mL CH3CN – was added to the resulting suspension. The reaction was allowed
to stir for 5 h at room temperature, and the precipitate was filtrated and washed with
CH3CN, MeOH and diethyl ether.
o
[Cu(phen)(L1)Cl].H2O, 1a. Yield: 80 %, m.p. 215 C (decomposition). Elemental
analysis of CHN: Calculated for C33H27ClN2O6Cu.H2O: C, 59.6; H, 4.4; N, 4.2; Found:
C, 59.6; H, 4.6; N, 4.1. FTIR (ATR, cm-1): 3432 (νOH), 1622 (νC=O), 1589 (νC=N). UV-
Vis [CH3OH; λ/nm(/L mol-1 cm-1)]: 271 (36,230); 294sh; 346; 428 (36,540); 451sh.
o
[Cu(phen)(L2)Cl].H2O, 2a.Yield: 74 %, m.p. 212-213 C (decomposition). Elemental
analysis of CHN: Calculated for C35H29ClN2O6Cu.H2O: C, 60.9; H, 4.5; N, 4.0; Found:
C, 61.1; H, 4.6; N, 3.6. FTIR (ATR, cm-1): 3369 (νOH), 1620 (νC=O), 1587 (νC=N). UV-
Vis [CH3OH; λ/nm (/L mol-1 cm-1)]: 270 (24,798); 294sh; 357; 456 (20,515); 482sh. dd
7

[Zn(phen)(L1)Cl], 1b. Yield: 70 %; m.p. 181 oC. Except for X-Ray diffraction analysis,
this complex has been previously characterized [31].
o
[Zn(phen)(L2)Cl].CH3CN.0.8H2O, 2b. Yield: 73 %; m.p. 215-216 C (decomposition).
Elemental analysis of CHN: Calculated for C35H29ClN2O6Zn.CH3CN.0.8H2O: C, 60.9; H,
4.6; N, 5.8; Found: C, 60.5; H, 4.2; N, 5.6. FTIR (ATR, cm-1): 3307 (νOH), 1623 (νC=O),
1585 (νC=N). 1H NMR (500 Mz, DMSO-d6): δ (ppm): 9.45 (br m, 2H), 8.89 (br m, 2H),
8.29 (s, 2H), 8.07 (br m, 2H), 7.47-6.65 (m, 9H), 3.83 (s, 6H), 2.89 (m, 4H). UV-Vis
[CH3OH; λ/nm (/L mol-1 cm-1)]: 266 (20,100); 291sh; 348; 450 (77,730); 474sh.
2.3. Single-crystal X-ray crystallography
Single crystal X-ray data for 1b were collected on a Bruker D8 Venture diffractometer
using graphite-monochromated MoK radiation (λ = 0.71073 Å) at 150 K. Data
collection, cell refinement and data reduction were performed with Bruker Instrument
Service v4.2.2, APEX2 [36] and SAINT [37], respectively. The absorption correction
using equivalent reflections was performed with the SADABS program [38]. The
structure solutions and full-matrix least-squares refinements based on F2 were performed
with the SHELXS-97 and SHELXL-2014 programs [39]. All atoms except hydrogen
were refined anisotropically. Hydrogen atoms were treated using a constrained
refinement. Structure illustrations were generated using the MERCURY program [40].
The title compound weakly diffracts and in the unit cell large voids filled by disordered
solvent molecules are present. Although the data were collected at low temperature, the
assignment of the intensities related to the solvent molecules failed. Thus, solvent masks
routinely implemented on Olex2 program were applied [41]. The total volume of the
voids and electrons per unit cell are 2638.2 and 651.4 Å3, respectively. The number of
8

electrons per asymmetric unit corresponds to 81.4 (Z=8), which are in good agreement
with one acetonitrile and one chloroform molecule as crystallization solvents. A summary
of the crystal, data collection and refinement is listed in Table 1.
Table 1: Summary of crystal structure, data collection and refinement for 1b
Compound reference
(1b)
Chemical formula
Formula Mass
Crystal system
a/Å
C33H27ClN2O6Zn
648.41
Orthorhombic
22.1942(13)
27.5217(14)
12.6862(8)
7749.0(8)
120(2)
b/Å
c/Å
Unit cell volume/Å3
Temperature/K
Space group
Cmc21
No. of formula units per unit cell, Z
Radiation type
Absorption coefficient, mm-1
No. of reflections measured
No. of independent reflections
Rint
8
MoK
0.74
6809
4830
0.069
0.056
0.125
0.084
0.136
Final R1values (I>2(I))
Final wR(F²) values (I>2(I))
Final R1 values (all data)
Final wR(F²) values (all data)
Goodness of fit on F²
1.04
CCDC Deposition
1945106
2.4. Growth inhibitory activity against T. cruzi amastigotes in vitro
2.4.1. In vitro model for anti-T. cruzi assessment
Monkey kidney fibroblasts (LLC-MK2 cells) were seeded in 96-well transparent
plates at 10,000 cells/well in 100 µL DMEM medium without phenol red (DMEM-WPR)
and with 5% fetal calf serum (FCS). After 12 h incubation for cell adhesion (36 oC, 5%
CO2), 50,000 trypomastigote forms of T. cruzi (Tulahuen C2C4 LacZ strain) were added
per well in 20 µL DMEM-WPR + 5% FCS. After 24 h incubation (36 oC, 5% CO2), the
medium was removed and the wells washed with phosphate buffer saline (PBS, 2 × 150
9

µL, pre-heated to 36 oC). Fresh medium (120 µL/well) in the presence or absence of the
drugs serially diluted from 100 – 0.41 µM (7 concentrations) was added to the plate prior
to further incubation for 120 h (36 oC, 5% CO2). The drugs were diluted from a 100 µM
DMEM solution containing 0.3% DMSO. After inspection under an inverted microscope
for assurance of cell development and sterility, 50 µL/well of a solution of substrate
CPRG (0.3 mg/mL) and Igepal (8 µL/mL) was added and the absorbance recorded at λ =
570 nm after 1 – 2 h incubation (36 oC, 5% CO2). The data were converted to a percent
cell viability compared to the untreated control and statistically treated using GraphPad
Prism 7.00 software. Benznidazole was used as a reference drug.
2.4.2 In vitro model for LLC-MK2 cytotoxicity assessment
Monkey kidney fibroblasts (LLC-MK2 cells) were seeded in 96-well transparent
plates at 10,000 cells/well in 120 µL DMEM-WPR + 5% FCS. After 24 h incubation (36
oC, 5% CO2), the medium was removed and fresh medium (120 µL/well) in the presence
or absence of the drugs serially diluted from 100 – 0.41 µM (7 concentrations) was added
to the plate, which was then incubated for 120 h (36 oC, 5 % CO2). The drugs were diluted
from a 100 µM DMEM solution containing 0.3 % DMSO. After inspection under an
inverted microscope for assurance of cell development and sterility, 20 µL/well of a
solution of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT
salt, 1.25 mg/mL) and 5-methylphenazinium methyl sulfate (PMS, 0.11 mg/mL) was
o
added, following 2 – 3 h of incubation (36 C, 5% CO2). After medium removal, MTT
formazan crystals were solubilized in 100 µL DMSO and the absorbance recorded at λ =
570 nm. The data were converted to a percent cell viability and compared to the untreated
control and statistically treated using GraphPad Prism 7.00 software. Triton X (0.1% v/v)
was used as positive control.
10

3. Results and Discussion
3.1. Synthesis of the ligand HL2
The curcuminoid ligand HL2 was obtained by reacting vanillin and
acetylcyclopentanone in the presence of tributylborate, boric anhydride and 2-
aminobutane (Scheme 1).
Scheme 1: Synthesis of HL2.
These conditions are classical for the Pabon synthesis, which gives yields higher
than those previously described in literature [42]. Boric anhydride and tributylborate were
used to form a boron-based enolate with the 1,3-dicarbonyl compound, so the
nucleophilic addition to the aldehyde carbonyl is selective for the appropriate carbons,
since the more acidic proton is the one between the carbonyls. After formation of the
complex, the amine reacted with the remaining acidic protons and the condensation with
the aldehyde occurred in the boron-based enolate itself, which was subsequently disrupted
by the addition of the acid solution to generate a yellow precipitate, which was isolated
by filtration. Washing the precipitate with hot ethanol removed both impurities and
byproducts formed. The HL2 carbocyclic derivative was purified by recrystallization
from a hydroalcoholic mixture and suitably characterized by MS, FTIR and NMR, and
subsequently used for the complexation reactions.
11

3.2. Synthesis of the Cu(II) and Zn(II) complexes
The complexes were obtained by mixing a suspension of the curcuminoid ligand
in acetonitrile containing Et3N and the auxiliary ligand (phen), followed by the addition
of the metal salt (CuCl2·2H2O or ZnCl2), also in CH3CN (Scheme 2). Et3N was used with
the aim to deprotonate the curcuminoid. After 5 h, the precipitate was extensively washed
with acetonitrile and methanol to remove unreacted precursors. Complex
[Zn(phen)(L1)Cl] (1b) has been reported previously in the literature [31] and was
1
successfully synthesized in this work. IR (SI, Fig. S4), UV-Vis (SI, Fig. S8), H NMR
(SI, Fig. S10) and melting point (181 °C) analyses agreed well with those described in the
reference [31]. Additionally, single crystals of 1b suitable for X-ray diffraction analysis
were obtained by slow evaporation of a solution of a 1:1 solution of the complex in
CH3CN/CHCl3. This structure has not yet been reported in the literature.
Scheme 2: Synthesis of the complexes [M(L1-2)(phen)Cl] (M = Cu(II) or Zn(II)),
containing curcuminoid and phen ligands. The Zn(II) complex (1b) was previously
reported [31].
12

3.3. General characterization
The IR spectra of HL1-2 (SI, Fig. S1-S2) showed a sharp band around 3500 cm-1
associated with the OH from the aromatic rings [27]. In addition, a broad band was
observed at about 3200 cm-1, due to the OH from the enolic form of the ligands [27]. For
1a-b, 2a-b (SI, Figs. S3-S6), the OH frequencies were detected in the range of 3300-3200
cm-1. All complexes exhibited absorption in the region of 1620 cm-1 associated with
stretching vibrations of νC=O. These signals were not significantly shifted compared to
the ligands [27] and could be explained by the intermediate character between a double
and a single C–O bond, both in the enolic form of the ligand and in the complex. 1a-b,
2a-b also displayed a new absorption around 1585 cm-1 attributed to νC=N [31] indicating
coordination with phen. Finally, a sharp signal within 1500-1510 cm-1 was associated to
the C–C–C chelated ring [31] and appeared in the spectra of all compounds.
The 1H NMR spectra of the free ligands (HL1 and HL2) displayed singlets at δ
9.71 (for HL1) and δ 9.66 and 9.63 (for HL2) attributed to phenolic OH, which are absent
in the spectra of the corresponding complexes [Zn(L1-2)(phen)Cl] 1b [31] and 2b (SI,
Fig. S9-10). Both ligands are in the enolic form in solution, confirmed by the lack of the
signal corresponding to C–H10 in the spectra (SI, Fig. S9-10). The aromatic C–H and
Csp2–H peaks were found in the expected region (~ δ 7.5-6.5) for the ligands, with small
shifts upon coordination. Finally, the presence of coordinated phen was confirmed by the
characteristic signals around δ 9.50, 8.90, 8.30 and 8.10 for 1b and 2b.
The electronic spectra of the complexes, recorded in MeOH (SI, Fig. S7-S8),
showed typical π-π* transitions of the extended conjugation system from the curcuminoid
ligand [27], between 426 and 453 nm with a shoulder around 448-478 nm. The intense
absorption observed at 270 with a shoulder at 290 nm was associated with the ligands
(curcuminoid and phen) [43].
13

To further investigate the complexes in solution, conductivity data were
determined in DMSO. The results are compiled in Table 2.
Table 2: Conductivity data (S cm–1) of complexes 1a–b, 2a–b in DMSO at 1.0 x
10–3 mol L–1.
Time
Compound
0 h
11
12.
12.
12
24 h
12
16
11
15
1a
1b
2a
2b
The conductivity values indicate that the complexes behave as non-electrolytes
[44] and confirm their solution stability within 24 h.
The copper complexes were studied by electron paramagnetic resonance (EPR)
and the spectra from the frozen DMSO/H2O 1:1 solution of both 1a and 2a along with
simulations are presented in Figure 2.
Figure 2: 77 K frozen DMSO/H2O 1:1 solution spectra of complexes 1a (a) and 2a (b).
The black line is the experimental spectrum (Exp), the red line the simulated spectrum
(Sim).
14

Employing the approach devised by Peisach and Blumberg [45] it is demonstrated
that the spectra from both complexes comprise one major component derived from a
2
2
distinct static Jahn-Teller elongated configuration, with the orbital 3푑푥 −푦 being the
lowest in energy. This static configuration renders both g and A tensors axial, which is
confirmed by the spin Hamiltonian parameters obtained from the simulations, presented
in Table 3.
Table 3: EPR spectra simulation parameters for 1a and 2a.
Complex
g//
2.3142
2.3145
A// [MHz]
480.32
481.03
g
2.0712
2.0684
A [MHz]
< 50
1a
2a
< 50
The concerned conformation emerges when the static Jahn-Teller axis is set
perpendicular to the plane formed by the two ligands, which corresponds to an equatorial
coordination ring comprising two oxygen atoms from the curcuminoid moiety and two
nitrogen atoms from the phen moiety (2N2O). This attribution is validated by comparing
the parameters of the spin Hamiltonian simulations to the values on the g//A// diagrams on
[45].
The results of the EPR studies agreed to the proposed crystallographic structure
for the analyzed complexes.
3.4. Crystal structure of 1b
Single crystals were obtained by slow evaporation of a CH3CH/CHCl3 1:1 solution
containing 1b. The compound crystallized in the chiral orthorhombic Cmc21 space group,
displaying two crystallographic independent molecules in the asymmetric unit, as shown
15

separately in Figure. 3. For the sake of comparison, from now on the molecules will be
labelled as A and B. Selected bond lengths and angles are listed in Table 4.
Figure 3: Crystal structures of the independent molecules in the asymmetric unit of
compound 1b (molecule A: left, molecule B: right).
16

Table 4. Selected bond lengths (Å) and angles (º) for compound 1b (i = -x, y, z)
Atom labels
1b
Zn1—Cl1
Zn1—O1
Zn1—N1
Zn2—Cl2
Zn2—O4
Zn2—N2
O1—C2
C1—C2
O4—C13
C12—C13
2.280(3)
2.010(5)
2.157(5)
2.408(5)
1.992(5)
2.280(3)
1.307(9)
1.354(9)
1.200(8)
1.462(9)
N1—Zn1—Cl1
O1—Zn1—Cl1
N1—Zn1—O1
O1—Zn1—O1i
O1i—Zn1—N1i
N1—Zn1—N1i
Cl1—Zn1—O1i
Cl1—Zn1—N1i
N1—Zn1—O1i
103.5(2)
106.41(2)
88.4(2)
91.2(3)
88.4(2)
76.5(3)
106.4(2)
103.5(2)
148.9(3)
N2—Zn2—O4
N2—Zn2—Cl2
O4—Zn2—Cl2
N2—Zn2—N2i
N2—Zn2—O4i
O4—Zn2—N2i
O4—Zn2—O4i
Cl2—Zn2—N2i
Cl2—Zn2—O4i
157.8(3)
98.9(2)
102.1(2)
80.1(3)
89.7(2)
89.7(2)
92.8(3)
98.9(2)
102.1(2)
In both molecules the metal ion is pentacoordinated to one deprotonated curcumin
and one phenanthroline molecules in the basal plane, while the axial position is filled by
one chlorido ligand. The coordination geometry of the zinc(II) ions is closer to a trigonal
bipyramid, confirmed by the Addison’s trigonality parameter [46], , for both molecules:
17

0.92 and 0.76 for A and B, respectively. Although they presented a similar coordination
environment, the geometry around the zinc ion in molecule B is slightly more distorted
from ideality than in A. The bond lengths Zn–Cl and Zn–Ocurcumin and Zn–Nphen are typical
for other similar compounds reported elsewhere [47]. It is important to highlight that the
curcumin moiety is planar, suggesting its coordination is in the diketonate form due to
electronic delocalization. However, differences could also be observed in the curcumin
coordination when A and B are compared. In molecule B, the metal ion is contained in
the curcumin plane, while in A, the zinc(II) ion is displaced by ca. 2 Å from it, as a
consequence of the more pronounced trigonal bipyramidal geometry.
The crystal packing is stabilized by a network of hydrogen bonds involving the
curcumin hydroxyl group and the chlorido ligands from A and B, leading to a
supramolecular chain running along the [1 0 0] direction, as seen in Figure 4a. The
distances between the acceptor and donor atoms are 3.096(11) for Cl1…O2ii, and
3.138(10) for Cl2iii…O5 (ii = -1/2+x, 3/2-y, -1/2+z and iii = -1/2-x, 3/2-y, -1/2+z).
Moreover, in the bc plane, the layers of molecules A (pink) and B (blue) are positioned
in an alternating way (Figure 4b).
18

Figure 4: Details of the crystal packing of compound 1b: (a) intermolecular
interactions involving chlorido and curcumin hydroxyl groups (above). Symmetry
operations to generate equivalent atoms: i = -x, y, z; ii = -1/2+x, 3/2-y, -1/2+z; iii = -1/2-
x, 3/2-y, -1/2+z. (b) Layers of molecules A and B in the crystallographic bc plane
(bottom). Hydrogen atoms were omitted for the sake of clarity.
3.5. In vitro activity assessment against amastigotes of T. cruzi and cell line LLC-MK2
The trypanocidal activity of the compounds has been evaluated in vitro using the
Tulahuen-C2C4 LacZ strain in the amastigote form internalized in the mammalian cell
line LLC-MK2 (rhesus monkey kidney fibroblasts). The inhibitory concentrations of cell
proliferation at 50% (IC50) are presented in Table 5, expressed as the average of three
independent experiments and the standard deviation (σ).
Table 5: IC50 values against T. cruzi and LLC-MK2 cells.
T. cruzi
IC50 ± σ (μM)
9.35 ± 1.31
1.99 ± 0.61
2.30 ± 0.10
0.47 ± 0.12
1.65 ± 0.20
0.40 ± 0.12
0.94 ± 0.20
LLC-MK2
IC50 ± σ (μM)
12.54 ± 1.78
6.15 ± 0.35
2.74 ± 0.29
2.02 ± 0.51
2.73 ± 0.13
1.52 ± 0.28
2.92 ± 0.63
Compound
SIa
1.3ns
3.1**
1.2ns
4.3**
1.7*
3.8**
3.1*
HL1
HL2
1a
1b
2a
2b
Phen
19

> 100
> 100
1.45 ± 0.20
> 100
> 100
> 100
-
-
CuCl2·2H2O
ZnCl2
Benznidazole
> 70
SI = selectivity index = (IC50T. cruzi)/(IC50LLC-MK2); a P value from Welch's t test: ns = P >
0.05 (not significant), * = P < 0.05, ** P < 0.01
The ligands and complexes generally displayed high toxicity against both host cell
LLC-MK2 and T. cruzi. This result was expected. Curcuminoid compounds have had
their anti-proliferative profiles previously described against tumor cell lines [48-50] as
well as trypanosomatids [16,51,52]. Our findings also demonstrated that metal
complexation improved the biological activity of both curcuminoid ligands, as initially
intended.
Comparatively, the zinc complexes 1b (IC50 = 0.47 μM, SI = 4.3) and 2b (IC50 =
0.47 μM, SI = 4.3) were more toxic towards the parasite and more selective than the
ligands (HL1, HL2, phen) as well as the copper complexes (1a, 2a), implying that the
complexing metal is important to the mode of action of these compounds.
The varying selectivity profiles of the compounds are depicted in Figure 5. The
graphs show the average cell viability vs concentration, with error bars representing the
standard deviation. One can observe a higher superposition for the graphs representing
the compounds with the lowest SI (HL1, 1a, 2a). Zinc complexes (1b, 2b) on the other
hand, besides showing a higher selectivity for the parasite, were able to eradicate T. cruzi
in concentrations as low as 1.3 µM, while preserving around 50-60% of host cells. Even
though the Zn(II) complexes have been more potent than the benznidazole, they were not
as selective as the reference drug.
20

Figure 5: Comparative in vitro cytotoxicity against T. cruzi and LLC-MK2 cells.
21

4. Conclusions
A series of Cu(II) and Zn(II) complexes containing curcuminoids and phen
ligands were obtained and characterized. The crystal structure determined for
[Zn(L1)(phen)Cl] (1b) confirmed the structure previously proposed in the literature. The
curcuminoid (L) coordinates in the anionic form through the -diketonate group in a 1:1
ligand:metal proportion and phen is bound through the nitrogen atoms with a chlorido
ligand completing the coordination sphere of the metal. NMR and EPR spectroscopy
evidenced that the other complexes exhibited the same structure of 1b. Anti-parasitic
investigation against T. Cruzi revealed that the Zn(II) complexes were the most potent
compounds against the parasite, and also exhibited the best selectivities - except when
compared to benznidazole. Finally, complexation of the curcuminoid ligands lead to an
enhancement of trypanocidal activities, besides the presence of phenanthroline that also
contributed to the great toxicity exhibited by the complexes.
Acknowledgements
The authors thank LDRX-UFF (www.ldrx.uff.br/) for the X-ray diffraction analysis and
PPGQ-UFRRJ (http://cursos.ufrrj.br/posgraduacao/ppgq/) for the facilities. This study
was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - Brasil (CAPES) - Finance Code 001, CNPq (BR) and FAPERJ (BR) (grant:
E26/200.991/2014).
22

Appendix A. Supplementary data
Supporting information to this article contains additional data for the spectroscopic
characterization of the compounds. X-ray crystallographic data in cif format available at
CCDC 1945106 can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif.
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27

Highlights
● Four Cu(II) and Zn(II) complexes with curcuminoid-based ligands (L1-2) have been
synthesized
● Complexes are neutral with general formula [M(L1-2)(phen)Cl], M = Cu(II) or Zn(II)
● Ligands and complexes have shown significant toxicity against amastigote form of T.
cruzi
● The Zn(II) compounds were the most potent and selective towards the parasite
28

Graphical Abstract
A series of curcuminoid metal complexes of the type [M(L1-2)(phen)Cl] 1a-b, 2a-b,
where M= Cu(II) or Zn(II) were investigated against amastigote form of T. Cruzi and the
Zn(II) complexes exhibited better activities and selectivity indexes (IC50 1b = 0.47 μM,
SI = 4.3 and IC50 2b = 0.40 μM, SI = 3.8) compared to the free ligands and Cu(II)
complexes. As well as the curcuminoid ligand, the presence of phenantroline contributed
to the trypanocidal activity of the complexes.
29