Synthesis, crystal structures and antimicrobial activity of dimeric copper(II) complexes with 2-hydroxyphenyl-ethylidene-dithiocarbazates

Article abstract of DOI:10.1016/j.ica.2018.08.032

The current work reports the synthesis and crystals structure of two copper(II) complexes [(CuL¹)₂] (1) and [(CuL²)₂] (2), with S-allyl-2-(2-hydroxyphenyl-ethylidene)dithiocarbazato and S-benzyl-2-(2-hydroxyphen

Full text of DOI:10.1016/j.ica.2018.08.032

Accepted Manuscript
Research paper
Synthesis, crystal structures and antimicrobial activity of dimeric copper(II)
complexes with 2-hydroxyphenyl-ethylidene-dithiocarbazates
Francielle C. Lima, Thayná S. Silva, Carlos H. G. Martins, Claudia C. Gatto
PII:
S0020-1693(18)30832-6
https://doi.org/10.1016/j.ica.2018.08.032
ICA 18432
DOI:
Reference:
Inorganica Chimica Acta
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29 May 2018
17 August 2018
22 August 2018
Please cite this article as: F.C. Lima, T.S. Silva, C. H. G. Martins, C.C. Gatto, Synthesis, crystal structures and
antimicrobial activity of dimeric copper(II) complexes with 2-hydroxyphenyl-ethylidene-dithiocarbazates,
Inorganica Chimica Acta (2018), doi: https://doi.org/10.1016/j.ica.2018.08.032
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Synthesis, crystal structures and antimicrobial activity of
dimeric copper(II) complexes with 2-hydroxyphenyl-
ethylidene-dithiocarbazates
Francielle C. Limaa, Thayná S. Silvab, Carlos H. G. Martinsb, Claudia C. Gattoa*
aLaboratory of Inorganic Synthesis and Crystallography, University of Brasilia
(IQ-UnB). Campus Universitário Darcy Ribeiro, CEP 70904970, Brasília-DF,
Brazil.
bLaboratory of research in Applied Microbiology, University of Franca, CEP
14404-600, Franca-SP, Brazil.
* Corresponding author. Tel.: 55 61 31073872; fax: 55 61 31073900
E-mail address: ccgatto@gmail.com (C. C. Gatto)
1

ABSTRACT. The current work reports the synthesis and crystals structure of two
copper(II) complexes [(CuL1)2] (1) and [(CuL2)2] (2), with S-allyl-2-(2-hydroxyphenyl-
ethylidene)dithiocarbazato
and
S-benzyl-2-(2-hydroxyphenyl-ethylidene)
dithiocarbazato (H2L1 and H2L2, respectively). The single-crystal X-ray analyses reveal
dimeric structures of the complexes with a distorted square planar coordination
geometry. The phenoxo bridging mode observed in these complexes is located between
the two copper atoms with ONS-donor ligands. The crystal packings are stabilized by
hydrogen bonds involving the adjacent molecules. The compounds were also
characterized by physicochemical and spectroscopic methods. The antibacterial and
antifungal properties of the free dithiocarbazates and its Cu(II) complexes have been
evaluated against several multiresistant bacteria and fungi. In all experiments, a superior
antimicrobial activity of the complexes was observed compared to the free ligands.
KEYWORDS. Copper(II) complexes, dithiocarbazate, crystal structure, antimicrobial
activity.
2

1. INTRODUCTION
In recent years, there has been a rapid expansion in the investigation of metal-based
antibacterial drugs, especially regarding the control of microbial proliferation, since it is
considered the main cause of hospital complications, as well as nosocomial infections
[1]. A variety of compounds has attracted great interest in the development of new
compounds with biological activity, which are motivated by structural modifications
caused by the coordination with metallic ions, targeting mechanisms of action that are
more efficient and less toxic [2,3].
Transition metals possess special roles in biology, and copper is a trace metal
essential to animals and plants. Actually, a variety of copper-based complexes
have been investigated, mainly due to the therapeutic potential, which is
increased by coordinating copper(II) complexes with mono- or polidentate Schiff
bases chelates and donor atoms (N, O, S and halides) [4–6]. These derivates from
Cu(II) form stable compounds with a varied coordination number, from four to
six, and the typical d9 electronic configuration promotes d–d transitions. In
general, the activity of a metal complex depends of several factors, as the
oxidation state, geometry, number and types of ligands [7–9].
Dithiocarbazates are Schiff bases with an increasing interest, particularly
because of their properties that can be significantly modified by the introduction
of different organic groups, providing a variety of donor properties relevant to
coordination chemistry. In addition to this fact, the interaction of these donors
with metallic ions, yields complexes with variable geometry and characteristics,
and increases potential biological properties when compared to the free ligands
[4,10,11]. In several cases, the activities of dithiocarbazate derivates are very
3

different, although their structure may vary just slightly. In addition, since these
ligands have both hard nitrogen atoms and soft sulfur donor atoms, they are able
to coordinate with a wide range of metal ions, resulting in coordination
compounds with intriguing physicochemical properties and potentiated biological
applications [2,4].
Due to our interest in the study of new compounds with this type of ligand, we
describe the synthesis, crystal structures, spectroscopy and biological activity of a
new dithiocarbazate ligand and two new copper(II) complexes. The complexes
[(CuL1)2] (1) and [(CuL2)2] (2) (where H2L1 and H2L2 are S-allyl-2-(2-
hydroxyphenyl-ethylidene)
dithiocarbazato and S-benzyl-2-(2-hydroxyphenyl-
ethylidene) dithiocarbazato, respectively) were synthesized as Scheme 1,
crystallized and analyzed using a single crystal X-ray diffraction. Moreover, a
detailed assignment of IR, UV-vis of both the dithiocarbazates and their
copper(II) complexes have been performed, as well NMR analysis of the ligands.
The copper(II) complexes were evaluated against antimicrobial activity of
multiresistant clinical isolates and certified microbial strains, and their biological
response were compared with the free ligands.
4

Scheme 1. Synthesis of dithiocarbazates and their copper(II) complexes.
2. EXPERIMENTAL SECTION
2.1. Materials, methods, and instruments. The starting materials were obtained from
a commercial source (Sigma-Aldrich) and used without further purification. The
reagents and solvents were obtained from commercial sources. Elemental analyses were
performed with Perkin Elmer/Series II 2400 analyzer. Fourier-transform infrared spectra
were recorded from KBr pellets (4000-400 cm-1) using an FT-IR Varian 640
spectrometer. UV-Vis spectra were collected on Varian-Cary and the concentration used
1
for all analyses was 10 M and 20 M. H NMR and 13C NMR analyses were
conducted using an NMR YH 600 Oxford. The molar conductance of a 10–3 M solution
of each metal complex in DMSO was measured at room temperature (25 ºC) using an
Ion DDS 12DW microprocessor conductivity meter and a dip-type cell with a platinized
electrode.
5

2.2. Synthesis of the ligand S-allyl-2-(2-hydroxyphenyl-ethylidene) dithiocarbazato
(H2L1). The mixture of 0.15 mL (3 mmol) hydrazine hydrate and 198 mg (3 mmol)
KOH in 30 mL of ethanol was cooled down to 5° C. 0.180 mL (3 mmol) of carbon
disulfide and 0.260 mL (1 mmol) of allyl bromide were added to the above mixture one
by one with continuous stirring. After 1 h, 0.361 mL (3 mmol) of 2-
hydroxyacetophenone was added and the mixture was refluxed and heated for 3 h.
Yellow product was obtained and filtered off from the mother solution. Yield: 546 mg
(70%). Melting Point: 116-118°C. Elemental analysis: calcd for C12H14N2S2O: C, 54.11;
H, 5.28; N, 10.52 and found: C, 53.83; H, 5.14; N, 10.65. Selected IR bands (KBr,
/cm-1): (O–H), 3245; (N–H), 3184; (C=N), 1601; (C–O), 1219; (CSS), 1334;
(C=S), 1101; (N–N), 936. UV-vis (DMF): λmax = 294 and 361 nm; UV-vis (MeOH):
1
3
λmax = 293 and 358 nm. H NMR (DMSO-d6) δ, ppm): 2.47 (s, 3H, CH3), 3.90 (d, J =
2
3
3
7.00 Hz, 2H, S–CH2), 5.34 and 5.17 (d, Jgem = 1.10 Hz, Jcis = 9.90 Hz, Jtrans = 16.87
3
3
3
Hz, 2H, =CH2), 5.88 (m, J = 6.97 Hz, Jcis = 9.90 Hz, Jtrans = 16.87 Hz, H, –CH=),
3
7.64-6.93 (m, J = 7.70 Hz, H, Ar), 11.40 (s, H, Ar–O–H). 13C NMR (DMSO-d6) δ,
ppm): 15.67 (CH3), 40.13 (S–CH2), 117.21 (=CH2), 129.44 (–CH=), 118.83, 119.26,
119.75, 131.94, 132.62 (Ar), 157.44 (CAr–OH), 157.94 (C=N), 195.97 (C=S).
2.3. Synthesis of the S-benzyl-2-(2-hydroxyphenyl-ethylidene)dithiocarbazato
(H2L2). The synthesis procedures for S-benzyl-2-(hydroxyphenyl-ethylidene)
dithiocarbazate (H2L2) were followed according to a related methodology [12]. Melting
Point: 145-147°C. Elemental analysis: calcd for C16H16N2S2O: C, 60.73; H, 5.10; N,
8.85 and found: C, 60.93; H, 5.55; N, 9.12. Selected IR bands (KBr, /cm-1): (O–H),
3418; (N–H), 3179; (C=N), 1601; (C–O), 1228; (CSS), 1384; (C=S), 1057; (N–
N), 948. UV-vis (DMF): λmax = 292 and 376 nm; UV-vis (MeOH): λmax = 290 and 348
nm.
6

2.4.
Synthesis
of
(µ2-phenoxo)
bis(S-allyl-2-(2-hydroxyphenyl-
ethylidene)dithiocarbazato)-dicopper(II), [(CuL1)2] (1). H2L1 (31.60 mg, 0.1 mmol)
was dissolved in 5 mL of methanol and added to a solution of Cu(CH3COO)2 (18.16
mg, 0.1 mmol) in 5 mL of methanol. The mixture was stirred for 30 min at room
temperature. Dark green crystals, which were appropriate for X-ray diffraction, were
filtered off from the mother solution. Yield: 379 mg (58 %). Melting Point: 187-189°C.
Elemental analysis: calcd for C24H24N4S4O2Cu2: C, 43.95; H, 3.68; N, 8.54 and found:
C, 44.34; H, 3.82; N, 8.94. Selected IR bands (KBr, /cm-1): (C=N), 1593; (C–O),
1159; (CSS), 1300; (N–N), 1003. UV-vis (DMF): λmax = 324 and 405 nm; UV-vis
(MeOH): λmax = 322 and 382 nm. Molar conductivity (10–3 mol.L–1, DMSO): 9.92 Ω–1
cm2 mol–1.
2.5.
Synthesis
of
(µ2-phenoxo)
bis(S-benzyl-2-(2-hydroxyphenyl-
ethylidene)dithiocarbazato)-dicopper(II), [(CuL2)2] (2). H2L2 (24 mg, 0.1 mmol) was
dissolved in 5 mL of methanol and added to a solution of Cu(CH3COO)2 (18.16 mg, 0.1
mmol) in 5 mL of methanol. The mixture was stirred for 30 min at room temperature.
Dark green crystals, which were appropriate for X-ray diffraction, were obtained
directly from the mother liquor upon standing the solution at room temperature for
several days. Yield: 475 mg (63 %). Melting Point: 183-185°C. Elemental analysis:
calcd for C32H28N4S4O2Cu2: C, 50.84; H, 3.73; N, 7.41 and found: C, 50.36; H, 3.46; N,
7.46. Selected IR bands (KBr, /cm-1): (C=N), 1597; (C–O), 1226; (CSS), 1296;
(N–N), 1004. UV-vis (DMF): λmax = 324 and 395 nm; UV-vis (MeOH): λmax = 322
and 380 nm. Molar conductivity (10–3 mol.L–1, DMSO): 28.40 Ω–1 cm2 mol–1.
2.6. Crystal structure determination. The X-ray diffraction data were collected on a
Bruker CCD SMART APEX II single crystal diffractometer with Mo K radiation
7

(0.71073 Å). SADABS [13] was used to scale the data and perform the multi-scan
absorption correction. The structures were solved by direct methods using SHELXS-97
[14] and subsequent Fourier-difference map analyses yielded the positions of the non-
hydrogens atoms, the refinement was performed using SHELXL-2018 [15]. Molecular
graphics were generated with POV-Ray [16] and DIAMOND [17] programs. The
crystal data, experimental details and refinement results are summarized in Table 1.
2.7. Antibacterial assay.
2.7.1. Bacterial strains. To evaluate the antibacterial activity of the free ligands
and copper(II) derivatives, multiresistant clinical isolates and bacterial strains
from the American Type Culture Collection (ATCC) and National Collection of
Type Cultures (NCTC) were employed, namely: Staphylococcus epidermidis
(isolated from blood); S. epidermidis (ATCC 14990); Staphylococcus capitis
(ATCC 27840); Enterococcus faecalis (isolated from urine); E. faecalis (NCTC
775); Staphylococcus aureus (isolated from surgical wound), S. aureus (ATCC
29213); Pseudomonas aeruginosa (isolated from urine); Klebsiella pneumoniae
(isolated from urine); Enterococcus faecium (NCTC 7171); and Enterobacter
cloacae (isolated from hospital outbreak). The multiresistant clinical isolates
were kindly supplied by Hospital das Clínicas de Ribeirão Preto (São Paulo State,
Brazil).
2.7.2. Determination of the Minimum Inhibitory Concentration. The Minimum
Inhibitory Concentration (MIC) values corresponds to the minimum concentration of
sample that is able to inhibit the growth of microorganisms, and were determined by the
microdilution broth method in 96-well microplates, in triplicate [18]. The samples were
dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich) at 1.0 µg mL-1, followed by
8

dilution in cation-adjusted Mueller-Hinton broth – (Difco, Kansas City, MO, USA);
until concentrations ranging from 400.0 to 0.195 µg mL-1 were achieved. The final
DMSO content was 5% (v/v). The inoculum was adjusted for each organism, to yield a
cell concentration of 5 × 105 colony forming units (CFU) per mL, according to
guidelines of the Clinical and Laboratory Standards Institute[18]. DMSO 5% (v/v) was
used as negative control; to validate the tests, the positive control tetracycline was tested
against reference strains of S. aureus (ATCC 29213) and Escherichia coli (ATCC
25922) [18] in a range of MIC values between 0.0115 and 5.9 µg mL–1. One inoculated
well was included, to control broth adequacy for bacterium growth. Another well
containing broth free of antimicrobial agent and inoculum was included, to control
medium sterility. The microplates (96 wells) were incubated for 24 h at 37º C. After the
incubation period, 30 µL of resazurin aqueous solution (0.02%) was added to the
microplates [19], for observation of bacterial growth: the blue and red color indicated
absence and presence of bacterial growth, respectively. The microplates were re-
incubated for 30 min and observed and analyzed in a descriptive way [19]. All assays
were carried out in triplicate.
2.7.3. Antifungal activity against Candida spp. The microorganism tested were
Candida albicans (ATCC 28366), C. tropicalis (ATCC 13803) and C. glabrata (ATCC
15126), from American Type Culture Collection (ATCC). The assays were performed
using method of broth microdilution using the standards recommended by Clinical and
Laboratory Standards Institute.[20] The stock solutions were prepared by dissolving the
samples, in DMSO (Sigma-Aldrich) in a concentration of 192,000 µg mL–1. Dilutions
with stock solutions were made using the standard RPMI 1640 medium buffered to pH
7.2 with 0.165 mol L–1 of 3-N-morpholinepropanesulfonic acid (MOPS, Acros
Organics, Geel, Turnhout, Belgium) of 12,000 µg mL–1. The inoculum was prepared
9

using a spectrophotometric method (at 530 nm wavelength), and compared with a 0.5
McFarland scale to obtain the value of 6.0 × 106 CFU mL–1. Then, the dilutions
recommended by CLSI with RPMI until the inoculums reached 1.2 × 103 UFC mL–1.
The MIC values were also evaluated for these microorganisms and all assays were
carried out in triplicate. It was performed using 96-well microplates, where series of
dilutions with final concentrations were made in the range of 1.46 to 3,000.0 µg mL–1.
Each well received 100.0 µL of the inoculum suspension and the final volume in each
well was 200.0 µL. The negative control (DMSO) was tested with concentrations
ranging from 1% to 10% v/v and not influenced in yeast growth. The added inoculum
medium was used as culture growth control. To validate the tests, amphotericin B
(positive control) was tested against reference strains of C. krusei (ATCC 6258) and C.
parapsilosis (ATCC 22019) in a range of MIC values between 0.031 and 16.0 µg mL–1.
If the MIC value is within this range for these yeasts, the methodology and the results
for the other tested yeasts are validated according to the reference protocol M27-
A3.[20] At the end of the procedure, after incubation, 30 μL of a 0.02% aqueous
resazurin (Sigma) solution was added to each well and the microplates were incubated
for further 30 min. For observation and descriptive analysis, the blue and red colors
represent the absence and the presence of microbial growth, respectively [21].
(Table 1)
3. RESULTS AND DISCUSSION
3.1. Synthesis and Crystal Structure Description
Two tridentate ONS - ligands were synthesized by the condensation reaction of 2-
hydroxyacetophenone with S-allyldithiocarbazate and S-phenyldithiocarbazate.
10

Each ligand reacted separately with Cu(CH3COO)2 at methanol solution. These
compounds were characterized by elemental analysis and spectroscopic
techniques. These results supported the structures obtained by single crystal X-
ray in which H2L1 and H2L2 behaved as tridentate donor ligands coordinating via
the phenolic oxygen, azomethine nitrogen and thione sulfur atom.
The crystal structure of H2L1 is established accurately by single-crystal X-ray
diffraction (Figure 1), whereas crystal structure of H2L2 ligand has already been
elucidated by Biswal et al. 2015 [12]. These ligands can exhibit thione-thiol
1
tautomerism in the –HN–C=S functional group; however, the H NMR spectra
does not show any peak attributable to the S–H proton and the IR spectra show
the ν(C=S) band around 1100 cm-1, suggesting the presence of the thione form in
solution and in the solid state. The thione form is also predominant in solution in
the presence of metal ions and soft bases, in order to favor the coordination with
the metal ion. The thione tautomer is confirmed both by the presence of the
hydrazinic hydrogen and the characteristic double distance C(9)–S(1) of 1.659(2)
Å [22]. The ligand adopts an E configuration with respect to the C(7)–N(1) and Z
configuration with respect to the N(2)–C(9) bond, and the entire species has
coplanar atoms, with the exception of the atoms of the final portion of the ligand,
due the greater liberty, how found in others similar dithiocarbazates and
thiosemicarbazones [23–27]. The displacement to greater lengths of the bonds
C(9)–N(2) of 1,353(3) Å and C(8)–S(2) of 1,734(2) Å and shorter for N(1)–N(2)
of 1,372 (3) Å, are indicative of the delocalization of electrons through the
dithiocarbazate function by virtue of the coordination with the metal center. The
N–N and C–N bond lengths of the dithiocarbazate moiety are comparable to the
corresponding lengths of other similar compounds [26,28]. The bond length
11

C(1)–O(1) of 1.354(3) Å corresponds to a single bond C–O, evidencing the
protonated form of the hydroxyl as a free ligand [12,24]. Selected bond lengths
and bond angles are given in Table 2.
(Figure 1)
Interestingly, the phenolic oxygen atom can form a bifurcated intramolecular hydrogen
bonds with the azomethine nitrogen atom and the thioether sulfur atom. This interaction
between O(1)–H(1)∙∙∙N(1) [d(O∙∙∙N) = 2.588(3) Å, \(O(1)H(1)∙∙∙N(1)) = 152°] and
O(1)–H(1)∙∙∙S(2) [d(O∙∙∙S) = 3.375(2) Å, \(O(1)H(1)∙∙∙S(2)) = 138°], possesses a
significant role in the structural arrangement of the ligand, stabilizing its structure by
forming five/six membered rings [29,30]. Additionally, an intermolecular hydrogen
bond is observed in the crystal lattices, N(2)–H(2A)∙∙∙S(1)a [d(N∙∙∙S) = 3.580(2)
Å, \(N(2)H(2A)∙∙∙S(1)) = 173° with symmetry operator (a): -x+1, -y+2, -z]
,
different from reported by Zangrando et al. 2015 [10] and Takjoo and Centore
2013 [24] with the formation of a polymer obtained by the repetition of linear
chains of similar ligands.
The crystal structure determination (1) with H2L1 and (2) with H2L2 revealed a
dinuclear centrosymmetric copper(II) complexes. The anisotropic thermal
displacement ellipsoids representations of the complexes are illustrated in Figure
1. The bond lengths and angles (listed in Table 2) are in good agreement with
those reported for other copper(II) dithiocarbazate complexes.
(Figure 2)
In the two complexes, the dithiocarbazates H2L1 and H2L2 acts as doubly
deprotonated ligands, coordinated in the E isomer form, with respect to the N(2)–
12

C(9), which chelates the copper(II) atom through the phenoxo oxygen O(1), the
imine nitrogen N(1) and thiol sulfur S(2) atoms [23,25,26]. The metal centers
exhibits a square planar geometry and an interesting feature of the compounds is
that the organic ligands act as bridges µ2-phenoxo between two copper atoms in a
fashion that one 5-membered and one 6-membered rings are established. The
oxygen atoms of the phenolate groups form a planar central {Cu2O2} ring. Thus,
the formation of an oxygen bridge between two copper atoms does not result in a
significant lengthening of the corresponding Cu–O bonds that are between
1.908(18) and 2.005(3) Å. The bonding mode results in a Cu–Cu’ distances of
2.926(6) Å for (1) and 2.966(10) Å for (2), which are similar to the values
observed in hydroxo-bridged dimers [31–34].
For both complexes, the coordination environment around the central Cu(II)
atom is best described as tetracoordinated in bonds of atoms O(1), N(1), S(2) and
O(1)’, resulting in the distorted square planar geometry. The bond angles O(1)–
Cu1–N(1), N(1)–Cu1–S(1), S(1)–Cu1–O(1)’, O(1)’–Cu1–O(1), differ from 90°,
as well, the bond angles O(1)–Cu1–S(1), N(1)–Cu1–O(1)’, which differ from
180°, as it is expected for a perfect geometry [23,30,31]. The distorted geometry
can be explained by the rigidity of the ligands and additionally, by the manner in
which the ligands behave around the central atom, with bridged dimers
configuration. The angles between neighboring donor atoms in the chelates rings
are between 88.62(11)° and 93.73(13)° and O(1)–Cu1–O(1)’ of 76.60(9)° for (1)
and 76.67(12)° for (2).
(Table 2)
13

There are slight changes in the bonding lengths for the ligands after their
coordination to the metal center. The bond length C(9)–S(1) increases from
1.659(2) Å for H2L1 to 1.728(3)Å for (1) and 1.661(3) Å for (H2L2) [12] to
1.728(4) Å for (2), evidencing the coordination of the metal on that site [23].
Commonly, the related complexes have their ligands coordinated in their
tautomeric form, a deprotonated thiolate, as already observed for most of the S–
alkyl/aryl dithiocarbazate derivative compounds. In the same way, C(9)–N(2)
suffers a significant decrease from 1.353(3) Å for H2L1 to 1.284(3) Å for the
complex (1) and of 1.352(3) Å for the ligand (H2L2) [12] to 1.284(3) Å for the
complex (2), suggesting a resonance of the electric charge due to the
complexation of the adjacent coordination sites [23]. Considering the structural
similarity of the two complexes, the bond lengths involving the coordination
polyhedron for the copper atoms are in agreement with previous literature studies
and are evidenced by the bond lengths Cu(1)–N(1) of 1.940(2) Å for (1) and
1.935(3) Å for (2), as well as, Cu(1)–S(1) of 2.199(3) Å for (1) and 2.201(13) Å
for (2)[12,23].
3.2. Spectroscopic Analyses. FT-IR spectra of (1) and (2) were compared with the free
dithicarbazates. The bands attributed to of (OH) vibration appear around 3245 cm-1 for
H2L1 and 3418 cm-1 for H2L2, while (NH) are attributed in 3184 cm-1 for H2L1 and
3179 cm-1 for H2L2. These bands disappear with coordination to the copper atoms and
shows the coordination of dithiocarbazate through the phenoxo oxygen and the imine
nitrogen [10,24,35]. Additionally, the presence of a band (C=S) absorption at 1101 cm-
1 and 1057 cm-1 for H2L1 and H2L2, respectively, are not observed in the spectra of the
complexes, indicating an electronic delocalization of the ligands during complexation
[25].
14

In the spectra of the free dithiocarbazates one peak was observed at 1601 cm-1, which
can be attributed to (C=N) and the metal complexes (1) and (2) exhibited a (C=N)
absorption at 1593 cm-1 and 1597 cm-1, respectively [29,35,36]. The band shif attributed
to (C=N) was lower for the complexes when compared to the free ligand, indicating
the electronic delocalization along the structure as soon as N(2) was coordinated to the
copper centers. In the IR spectra of the complexes (1) and (2), a band (C–N) were
observed at 1468 cm-1 and 1568 cm-1, respectively [25]. In the complexes, the bands
observed at 1003 cm-1 for (1) and 1004 cm-1 for (2) are assigned to a (N–N). Therefore,
the IR data for the complexes indicate a coordination of the dithiocarbazate ligands
through the ONS-donor atoms, in accordance with the crystal structures determination.
The UV-Vis experiment was made at room temperature, using two different solvents
(MeOH and DMF), for organic ligands and copper complexes (Figure 3). The results
are summarized in Table 3. Regardless of the solvent, all compounds spectra presented
–* and n–* bands correspondent to the transitions of the dithiocarbazate moiety, as
well as the –* transition, characteristic of the azomethine chromophore, for which the
metallic complexes presents a hypochromic displacement attributed to the coordination
of the copper atom to azomethine nitrogen [10,24,25]. The transitions in the median
region of the spectra (300-400 nm) correspond to the ligand–metal charge transitions of
the complexes as well as the transitions in the ultraviolet region are between the orbitals
of the metal. The presence of a discrete signal at 265 nm H2L1 and 264 nm H2L2 refers
to the –* transition of the aromatic ring, while the strong absorption bands in the
range of 358–376 nm are presumably attributed from the occurrence of –* and n–*
transitions of the characteristic moiety of the dithiocarbazate ligand itself, the band
being present in the 290–293 nm range attributed to the –* transition of the
15

chromophore azomethine of the obtained Schiff base [10,26]. As already predicted, the
mentioned transitions in the complexes presented lower wavelengths, partially due to
the strong nitrogen absorption of the azomethine fragment, being evidenced at 287–289
nm and 322–324 nm. Additionally, the bands in the lower energy region, 380–405 nm,
for both complexes, are attributed to the charge transfer S  M (II) (LMCT), affording
one more evidence of coordination by this sites to the metallic center [10]. The band at
583 nm for (1) and 580 nm for (2) are related to the d–d transition of the metal ions,
2
2
being the predicted A1  B1 spectroscopic term agreeing with a square planar
geometry [10,24]. In addition, the UV-vis of (1) and (2) was evaluated at different times
(see Supporting Information, Figure S1). Over time, the characteristic absorption of
each complex displayed no significant change in the absorption bands. The results of
UV–vis spectra analysis reveal that (1) and (2) are stable in solution in a 72 h period,
indicating no dissociation in the room temperature.
(Figure 3)
(Table 3)
1
The H NMR spectra of the ligand in DMSO–d6 displays the presence of an
unshielded signal at 11.40 ppm, ascribed to the –NH resonance of the thioamide
function of the ligand. The methylene protons (–CH2) of the dithiocarbazate moiety
appears as a doublet (d) (3J = 7) with an integrated value of 2 at 3.90 ppm. The protons
–CH2 and –CH of the allyl group present multiple signals at 5.34–5.37 (dd) (2Jgem = 1.10
3
3
Hz and Jtrans = 16.87 Hz), 5.17–5.19 (dd) (2Jgem = 1.10 Hz and Jcis = 9.90 Hz) and
3
3
5.88–5.95 ppm (m) (3J = 6.97 Hz, Jcis = 9.90 Hz and Jtrans = 16.87 Hz), respectively
[23]. A single signal for methyl appears in the most shielded region at 2.47 ppm [26].
The aromatic protons appear as doublets and double doublets in the range of 6.93-7.64
16

ppm (3J = 7.70) [37]. Due to the higher acidity and, consequently, hydroxyl proton
lability, it was not possible to measure its resonance signal.
The 13C NMR spectra shows twelve signals for the ligand, the main ones being
characterized in the most unshielded region of the spectra, where it is possible to assign
the signals of the carbon bonded to sulfur of the thione (C=S) function in 195.47 ppm,
the carbon of the azomethine (C=N) in 157.98 ppm, C–O at 157.44 ppm. The moiety of
allyl substituent, methylene, –CH= e =CH2 are characterized in 40.13, 117.21 and
129.44 ppm, which are in agreement with the literature [24,26].
The molar conductances of 10−3 M solutions of the compounds in DMSO are in the
range 9.92–28.40 Ω–1 cm2 mol–1 therefore the complexes behave as non-electrolytes in
DMSO, since the measured conductivities are below the limit proposed by Geary [38]
of 35 Ω–1 cm2 mol–1 for non-electrolytic species. It can be concluded, therefore, that the
ligands are tightly bound to the metal center.
3.3. Biological activity. According to our interest, the biological activity of the
synthesized compounds was tested against Gram-negative bacteria, Gram-positive and
fungi. The results for the tests were expressed by the minimal inhibitory concentration
(MIC) according to Tables 4 and 5, through which it evaluates the lowest concentration
(μg.mL-1) capable of inhibiting microbial growth.
With the exception of the strains P. aeruginosa, E. cloacae, E. faecalis and S.
aureus that did not present any satisfactory response, the inhibitory activity of
the complexes was superior compared to the ligands. The complexes manifested
considerable antimicrobial activity to S. aureus (MIC = 1.56 μg.mL-1 of (1) and
-1
-1
3.12 μg.mL of (2)), S. capitis (MIC = 3.12 μg.mL-1 of (1) and 6.25 μg.mL of
(2)) and S. epidermidis (MIC = 3.12 μg.mL-1 of (1) and 6.25 μg.mL-1 of (2)). The
17

antifungal tests followed the same tendency in which the complexes presented
-1
higher activity when compared to the ligands, C. glabrata (MIC = 375 μg.mL
of (1) and 375 μg.mL-1 of (2)), C. tropicalis (MIC = 375 μg.mL-1 of (1) and 750
μg.mL-1 of (2)) and C. albicans (MIC = 1500 μg.mL-1 of (1) and 750 μg.mL-1 of
(2)).
Moreover, the results evidently show that the antimicrobial activity of the
metal ion became more pronounced when coordinated to the dithiocarbazate
derivatives. The complexation of dithiocarbazates with metal ions reduces its
polarity by partially sharing the positive charge with the donor atoms and π-
electron delocalization, and this can result in an improvement of the lipophilic
characteristics of the metallic atom, improving the activity of the compounds,
since it allows, in part, the blocking of cellular enzymatic activity [37,39].
Additionally, the compounds showed greater activity against Gram-positive
bacteria according the tendency of most reports [2,5,40]. In general, compounds
(1) and (2) presented superior activity for all the evaluated assays, a result that
can be attributed mainly to the presence of the metal coordination, which
contributes to the antibiotic activity.
(Table 4)
(Table 5)
4. CONCLUSIONS
In this paper, we have described the synthesis and crystal structure of a new ligand and
two stable copper(II) complexes of dithiocarbazate derivatives. Both X-ray crystal
analysis and the spectral characterization of the H2L1 ligand ratify its structure in a
18

thione tautomer with bifurcated intramolecular hydrogen bonds. The dithiocarbazates
are doubly-deprotonated and tridentate coordinated to copper(II) atoms in the formation
of the dimeric complexes. The biological analyses show satisfactory results of the
copper complexes, with positive response of microbiological assay and greater activity
against Gram-positive bacteria. The successful synthesis and characterization and the
promising results against the antibacterial agents of copper(II) complexes with
dithiocarbazates constitute a starting point to the development of new compounds and
their applications as promising antimicrobial agents.
ASSOCIATED CONTENT
Supporting Information
Crystallographic data for the structures in this work have been deposited to the
Cambridge Crystallographic Data Centre, CCDC 1822218-1822220. Copies of the
available material can be obtained free of charge by application to the Director, CCDC,
12 Union Road, Cambridge CH2 1EZ, UK (Fax: +44 1223 336033; E-mail:
deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk). Supplementary tables, figures,
NMR and IR spectra are as detailed in the text (PDF). Crystallographic data are in CIF
files.
AUTHOR INFORMATION
Corresponding Authors
*E-mail for Claudia C. Gatto: ccgatto@gmail.com
Author Contributions
19

The manuscript was written with contributions from all authors. All authors have
approved the final version of the manuscript.
Funding Sources
This work has been partially supported by FAPDF, FINEP/CTINFRA, CNPq, CAPES,
FINATEC and DPG-UnB. The authors wish to thank FAPESP (process number
2015/23850-5).
ACKNOWLEDGMENTS
All funding agencies are acknowledged for partial financial support.
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Figure Captions
Figure 1. Molecular structures of H2L1 with crystallographic labelling (30% probability
displacement ellipsoids). Intramolecular hydrogen bonds are shown as dashed lines.
Figure 2. Molecular structures of (1) and (2) with crystallographic labelling (30%
probability displacement ellipsoids).
Figure 3. UV-vis absorption spectra of the ligands and copper(II) derivatives in MeOH
and DMF (concentration 20 M).
27

Figure 1. Molecular structures of H2L1 with crystallographic labelling (30 %
probability displacement ellipsoids). Intramolecular hydrogen bonds are shown as
dashed lines.
28

(1)
(2)
Figure 2. Molecular structures of (1) and (2) with crystallographic labelling (30%
probability displacement ellipsoids).
29