Antibacterial activity of metal complexes based on cinnamaldehyde thiosemicarbazone analogues

Article abstract of DOI:10.1016/j.jinorgbio.2019.110888

The development of microbial antibiotic resistance has become one of the biggest threats to global health and the search for new molecules active against resistant pathogenic strains is a challenge that must be tackled. In many cases nosocomial infections

Full text of DOI:10.1016/j.jinorgbio.2019.110888

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Antibacterial activity of metal complexes based on
cinnamaldehyde thiosemicarbazone analogues
Franco Bisceglie, Cristina Bacci, Alice Vismarra, Elena Barilli,
Marianna Pioli, Nicolò Orsoni, Giorgio Pelosi
PII:
S0162-0134(19)30477-5
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110888
JIB 110888
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
18 July 2019
9 October 2019
10 October 2019
Please cite this article as: F. Bisceglie, C. Bacci, A. Vismarra, et al., Antibacterial activity
of metal complexes based on cinnamaldehyde thiosemicarbazone analogues, Journal of
Inorganic Biochemistry (2019), https://doi.org/10.1016/j.jinorgbio.2019.110888
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Antibacterial activity of metal complexes based on
cinnamaldehyde thiosemicarbazone analogues
Franco Biscegliea,b, Cristina Baccic, Alice Vismarrac, Elena Barillic, Marianna Piolia, Nicolò Orsonia,
Giorgio Pelosia,b*
a Department of Chemistry. University of Parma. Parco Area delle Scienze 17/A, 43124 Parma, Italy
b
C.I.R.C.M.S.B. Consorzio Interuniversitario di Ricerca in Chimica nei Sistemi Biologici, Parma
Local Unit. 43124 Parma, Italy
c Department of Veterinary Sciences, University of Parma, Strada del Taglio 10, 43126 Parma, Italy
* Corresponding author: giorgio.pelosi@unipr.it
Short title: Antibacterial activity of cinnamaldehyde thiosemicarbazone analogues
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Abstract
The development of microbial antibiotic resistance has become one of the biggest threats to global
health and the search for new molecules active against resistant pathogenic strains is a challenge that
must be tackled. In many cases nosocomial infections are caused by bacteria characterized by multi-
drug resistance patterns (MDR) and by their ability to produce biofilms. These properties lead to the
persistence of pathogens in the hospital environment. This paper reports the synthesis and
characterization of three thiosemicarbazone derivatives based on a compound containing the
cinnamaldehyde natural scaffold but possessing different logPow values. These molecules are then
used as ligands to prepare complexes of the Cu(II) and Zn(II) ions. All these compounds, ligands and
complexes, were screened in vitro on stains of Escherichia coli and Klebsiella pneumoniae for their
antibacterial activity. Despite their molecular similarity they revealed variegated behaviors. Only two
of them present interesting antimicrobial properties and have also been studied to verify their
stability in solution. The compound with the lowest partition coefficient is the most promising. The
MBC on K. pneumoniae and E. coli of these substances are very interesting and demonstrate that the
use of metalloantibiotics is a promising device to fight antibiotic resistance.
.
Keywords: thiosemicarbazone, zinc, copper, cinnamaldehyde, Escherichia coli, Klebsiella
pneumoniae
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1.
Introduction
Antibiotic resistance today is one of the biggest threats to global health and food security and
the development of new antibiotics or molecules that present activity against pathogenic strains is
becoming more and more urgent. In many cases nosocomial infections are caused by bacteria
characterized by multi-drug resistance patterns (MDR) and by the ability to produce biofilm, all this
leading to the persistence of pathogens in the hospital environment. The WHO reports that very year
in the European Union alone, about 25000 patients die for resistant bacterial infections acquired in
hospitals. Also in the USA the cost for the management of hospital-acquired infections is extremely
high, reaching US$ 28-34 billion [1] and, therefore, the interest in the development of alternative
therapies and of new molecules effective in combating infections is increasing day by day.
Biofilm production is a survival strategy that bacteria use to maintain stable growth in stressful
environmental conditions. A biofilm has been defined as “microbial communities that live together
in a self-produced extracellular polymeric matrix consisting of exopolysaccharides (EPS), proteins
and DNA adherent to abiotic surfaces”[2]. EPS confer structure complexity and a physical barrier
that stops drugs from reaching their bacterial target and limits the action of disinfectants and
detergents [3, 4]. At the same time it allows the exchange of genetic material among
microorganisms, favoring the dissemination of genes and plasmids associated to the antibiotic
resistance phenomenon. Biofilm formation ability by drug-resistance bacteria may result in an
increased tolerance to toxic substances[3], and many biofilm producers show high resistance levels
towards antibiotics[5-7]. In this perspective, studying and developing new molecules effective
against biofilm producing bacteria is an important target in medicinal chemistry.
Transition metal complexes present characteristics that make them good candidates as antimicrobial
agents. It should be noted that the properties required for metal complexes with biological properties
are: an adequately high thermodynamic stability to transport the metal to the active site, a good
hydrolytically stability, a proper molecular weight. In fact low molecular weight compounds with no
charge and very low water solubility have the advantage to be able to cross biological membranes by
passive diffusion[8]. Moreover, the ultimate goal of antimicrobials is the highest efficacy at the
lowest dosage without allowing resistance. A renewed interest in metals as antimicrobial and
biocidal agents is reflected in hopes that they may prevent resistance [9]. Interdisciplinary research is
promoting not only our understanding of metal toxicity but also the design of metal-based
compounds for use as antimicrobial agents and as alternatives to traditional antibiotics [10].
As representative of antimicrobial resistant species, we then selected two bacterial strains commonly
found in the environment, usually commensal of the intestinal and respiratory districts, characterized
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by the ability to form biofilm (Escherichia coli) and by the synthesis of Extended-Spectrum-Beta-
Lactamases enzymes (Klebsiella pneumoniae), that confer them resistance to third generation
cephalosporins. They are Gram-negative bacteria, part of the Enterobacteriaceae family. E. coli was
an intestinal commensal part of the natural microflora of animals and humans, but it can also cause
illnesses as gastroenteritis as well as extra-intestinal infections mainly at the urinary tract, at
respiratory district, at gallbladder. Infections sometimes could evolve to septicaemia and meningitis.
E. coli is a widespread bacteria and its transmission to humans or animals is mainly due to the
ingestion of contaminated water or raw foods (oral-faecal route). K. pneumoniae is a commensal of
intestinal and respiratory tract and can be responsible for pneumonia, infections of the urinary tract,
of wound infections but it can cause also sepsis. Both bacteria are characterised by the presence of a
capsule that they use to resist against phagocytosis.
Following our interest in the compehension of the biological activity of coordination compounds
based on thiosemicarbazones [11-15], recently, antimicrobial activity of Cu(II) and Zn(II) complexes
with aromatic N-substituted thiosemicarbazones have been reported [16]. In a previous study of ours
on the antimycotic activity of a series of thiosemicarbazones, cinnamaldehyde thiosemicarbazone
and its copper complex [15] showed a promising activity with no deleterious effects on human
healthy cells. We have therefore embarked on a new research to verify if they also possess
antibacterial properties. Three analogues with different logPow were synthesised to study the role of
the polarity on their biological effects. Given the preliminary antimycotic activity shown by the
bis(cinnamaldehydethiosemicarbazonato)copper(II) complex, we have also extended the synthesis to
the other copper complexes and to the analogous zinc derivatives. We thus obtained a series of nine
compounds which differ for the polarity of the ligand and the redox nature of the metal centre.
2.
Experimental
Chemistry
2.1
2.1.1 Syntheses and characterizations
1
All reactants used were purchased form Sigma Aldrich. The H-NMR spectra were recorded on a
Bruker Anova spectrometer at 400 MHz. The FT-IR measurements were recorded on a Nicolet 5PC
FTIR analysing products directly on the ATR accessory in the 4000–400 cm−1 range. The relative
intensity of reported FT-IR signals are defined as s = strong, m = medium, and w = weak. Melting
points were determined with a Gallenkamp instrument (Weiss-Gallenkamp). ESI-MS analysis were
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performed using a Waters Acquity Ultraperformance ESI-MS spectrometer with Single Quadrupole
Detector.
2.1.1.1 Preparation of the ligands
The desired thiosemicarbazones were obtained by mixing an equimolar amount of
thiosemicarbazide with the proper aldehyde in absolute ethanol. The mixture was refluxed under
stirring for 8 hours and left overnight at 0°C. The precipitate was filtered out, washed with cold
ethanol and dried under vacuum.
(E)-cinnamaldehydethiosemicarbazone (1)
Thiosemicarbazide (0.34 g, 3.8 mmol), (E)-cinnamaldehyde (0.50 g, 3.8 mmol). White powder.
Yield: 96%. Mp: 139 °C. FT-IR (cm−1): 1533 (s), 1254 (w), 966 (m). 1H-NMR (δ, ppm; DMSO-d6):
6.87 (dd, J = 16.1, J’ = 9.2 Hz, 1H), 7.04 (d, J = 16.1 Hz, 1H), 7.34 (t, J = 8.2 Hz, 1H), 7.39 (t, J =
8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.61 (s, 1H), 7.92 (d, J = 9.2 Hz, 1H), 8.18 (s, 1H), 11.40 (s,
1H). ESI-MS (+) m/z calc. 206.07 found 206.09.
(E)-cinnamaldehyde-4,4-dimethyl-3-thiosemicarbazone (2)
4,4-Dimethyl-3-thiosemicarbazide (0.44 g, 3.8 mmol), (E)-cinnamaldehyde (0.50 g, 3.8 mmol).
Orange powder. Yield: 79%. Mp: 139 °C. FT-IR (cm−1): 1545 (m), 1325 (w), 981 (m). 1H-NMR (δ,
ppm; DMSO-d6): 3.26 (s, 6H), 6.97 (m, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.40 (t, J = 7.2 Hz, 2H), 7. 61
(d, J = 7.2 Hz, 2H), 8.04 (dd, J = 6.1 Hz, J’ = 2.5 Hz, 1H), 10.83 (s, 1H). ESI-MS (+) m/z calc.
234.33 found 204.57.
(E)-1-(4-methoxyphenyl)-1-pentene-3-one-3-thiosemicarbazone (3)
Thiosemicarbazide (0.10 g, 1.1 mmol), 1-(4 methoxyphenyl)-1-pentene-3-one (0.21 g, 1.1 mmol).
Yellow crystals. Yield: 80%. Mp: 187 °C. FT-IR (cm−1): 1577 (m), 1490 (w), 1242 (m). 1H-NMR (δ,
ppm; DMSO-d6): 1.13 (t, J = 7.4 Hz, 3H), 2.56 (q, J = 7.4 Hz, 2H), 3.80 (s, 3H), 6.99 (d, J = 8.8 Hz,
2H), 7.10 (d, J = 16 Hz, 1H), 7.50 (d, J = 16 Hz, 1H), 7.59 (s, 1H), 7,74 (d, J = 8.8 Hz, 2H), 8.13 (s,
1H), 10.93 (s, 1H). ESI-MS (+) m/z calc. 264.36 found 264.09.
2.1.1.2 Preparation of the complexes
Synthetic approach used for the synthesis of copper(II) complexes
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The appropriate ligand was mixed with copper(II) acetate monohydrate in ethanol with a
metal to ligand ratio of 1 : 2. The mixture was left under stirring at room temperature for 2 hours.
Usually, it was observed a change in the solution colour during the reaction. Finally, the solvent was
removed under reduced pressure and the product was washed twice with diethylether, then dried
under vacuum.
Bis-(E)-cinnamaldehydethiosemicarbazonate of Cu(II) (4)
(E)-Cinnamaldehydethiosemicarbazone (1) (0.10 g, 0.5 mmol), copper(II)acetate (0.05 g, 0.25
mmol). Brown powder. Yield: 60%. FT-IR (cm−1): 1581 (s), 973 (s). ESI-MS (+) m/z calc. 471.09
found 472.16.
Bis-(E)-cinnamaldehyde-4,4-dimethyl-3-thiosemicarbazonate of Cu(II) (5)
(E)-cinnamaldehyde-4,4-dimethyl-3-thiosemicarbazone (2) (0.10 g, 0.4 mmol), copper(II)acetate
(0.04 g, 0.2 mmol). Brown powder. Yield: 73%. FT-IR (cm−1): 1507 (m), 1353 (w), 1137 (m). ESI-
MS (+) m/z calc. 528.20 found 520.20.
(E)-1-(4-methoxyphenyl)-1-pentene-3-one-3-thiosemicarbazonate of Cu(II) (6)
(E)-1-(4-methoxyphenyl)-1-pentene-3-one-3-thiosemicarbazone (3) (0.10 g, 0.4 mmol),
copper(II)acetate (0.04 g, 0.2 mmol). Brown powder. Yield: 87%. FT-IR (cm−1): 1598 (m), 1477 (w).
ESI-MS (+) m/z calc. 587.25 found 588.26.
Synthetic approach used for the synthesis of zinc(II) complexes
The appropriate ligand was mixed with zinc(II)acetate dihydrate in ethanol with a metal to
ligand ratio of 1 : 2. The mixture was stirred at room temperature for 2 hours. The solutions changed
colour gradually and the product precipitated during the reaction. Finally, the solid was filtered out,
washed twice with diethylether, then dried under vacuum.
Bis-(E)-cinnamaldehydethiosemicarbazonate of Zn(II) (7)
(E)-Cinnamaldehydethiosemicarbazone (0.10 g, 0.5 mmol), zinc(II)acetate (0.05 g, 0.25 mmol).
Yellow powder. Yield: 59%. FT-IR (cm−1): 1631 (m), 1460 (s), 1185 (m), 963 (m). 1H-NMR (δ,
ppm; DMSO-d6): 7.07 (d, J = 15.3, 1H), 7.13 (d, J = 15.3 Hz, 1H), 7.44 (m, 7H), 8.27 (d, J = 8.0 Hz,
1H). ESI- ESI-MS (+) m/z calc. 474.92 found 473.15.
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Bis-(E)-cinnamaldehyde-4,4-dimethyl-3-thiosemicarbazonate of Zn(II) (8)
(E)-cinnamaldehyde-4,4-dimethyl-3-thiosemicarbazone (2) (0.10 g, 0.4 mmol), zinc(II)acetate (0.05
g, 0.2 mmol). Yellow powder. Yield: 71%. FT-IR (cm−1): 1579 (m), 1432 (w). 1H-NMR (δ, ppm;
DMSO-d6): 2.52 (s, 12H), 7.03 (dd, J = 15.7 Hz, J’ = 9.7 Hz, 2H), 7.09 (d, J = 17.7 Hz, 2H), 7. 32
(m, 4H), 7.40 (m, 6H), 8.37 (d, J = 9.7 Hz, 2H). ESI-MS (+) m/z calc. 529.03 found 529.22.
(E)-1-(4-methoxyphenyl)-1-pentene-3-one-3-thiosemicarbazonate of Zn(II) (9)
(E)-1-(4-methoxyphenyl)-1-pentene-3-one-3-thiosemicarbazone (3) (0.10 g, 0.4 mmol),
zinc(II)acetate (0.04 g, 0.2 mmol). Dark green powder. Yield: 87%. FT-IR (cm−1): 1598 (m), 1477
(w). 1H-NMR (δ, ppm; DMSO-d6): 0.91 (t, J = 7.5 Hz, 6H), 2.73 (m, 2H), 2.91 (m, 2H), 3.78 (s,
6H), 6.82 (d, J = 16 Hz, 2H), 6.96 (d, J = 8.9, 4H), 7.05 (d, J = 16 Hz, 2H), 7.19 (s, 4H), 7.41 (d, J =
8.9 Hz, 4H). ESI-MS (+) m/z calc. 589.08 found 589.30.
2.1.2 Crystallographic data
The crystallographic data of compounds (1) and (2) were collected with a SMART APEX2
diffractometer with Bruker AXS CCD detector using Mo-Kα radiation and a graphite crystal
monochromator [λ(Mo-Kα) 0.71073 Å]. The SAINT [17] software was used for integrating
reflection intensities and scaling, and SADABS [18] for absorption correction. The structure was
solved by direct methods using SHELXS[19] and refined by full-matrix least-squares on all F2 using
SHELXL[20] implemented in the OLEX2 1.2 package [21]. All the non-hydrogen atoms in the
molecules were refined anisotropically. The hydrogen atoms were partly found and partly placed in
the ideal positions using riding models. The structure was solved by direct methods and difference
Fourier synthesis using the SHELX suite of programs as implemented within the OLEX software.
Thermal ellipsoid plots were generated using OLEX.
2.1.2.1 (E)-cinnamaldehyde-4,4-dimethyl-3-thiosemicarbazone (2)
The crystal system is orthorhombic, space group Pbca; cell parameters: a = 10.9446(5), b =
8.2168(4), c =27.9230(13)Å, ,V = 2511.1(2) Å3. The asymmetric unit is formed by a single molecule
of formula C12H15N3S1, Mr = 233.33, Z = 8, Dc = 1.234 g cm-3, μ = 2.35 mm-1, F (000) = 992. A
semi-empirical absorption correction, based on multiple scanned equivalent re-flections, has been
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carried out and gave 0.6069 < T < 0.7454. A total of 39304 reflections were collected up to a θ range
of 32.3° (±16 h,±12 k, ±41 l), 4271 unique reflections (Rint = 0.11). R= 0.0636, wR2 = 0.1594. All
non-hydrogen atoms were refined anisotropically. The hydrogen atoms were placed in ideal positions
and refined using riding models.
2.1.2.2 (E)-1-(4-methoxyphenyl)-1-pentene-3-one-3-thiosemicarbazone (3)
The crystal system is monoclinic, space group Cc; cell parameters: a = 11.364(8), b = 13.751(10) (2),
c =8.695(6)Å, β = 91.824(14)°,V = 1358.2(17)Å3. The asymmetric unit is formed by a single
molecule of formula C13 H17 N3 O1 S1, Mr = 263.37, Z = 4, Dc = 1.288 g cm-3, μ = 2.31 mm-1, F
(000) = 560. A semi-empirical absorption correction, based on multiple scanned equivalent re-
flections, has been carried out and gave 0.6063 < T < 0.7454. A total of 7034 reflections were
collected up to a θ range of 26.69° (±14 h,±17 k, ±10 l), 2827 unique reflections (Rint = 0.07). R=
0.0507, wR2= 0.1135. All non-hydrogen atoms were refined anisotropically. The hydrogen atoms
were placed in ideal positions and refined using riding models.
CCDC1940625-1940626
contain
the
supplementary
crystallographic
data
(http://www.ccdc.cam.ac.uk/data_request/cif; see also ESI).
2.1.3 Stability in solution
Due to their promising antibacterial activity, the stability of complexes 7 and 4 was evaluated in
solution by monitoring their UV-vis absorption spectra with time. It was considered stable a complex
which did not change significantly its profile in a 24 hours period incubated at 37°C. The solvent
used was DMSO, the medium utilised to dissolve the samples for the biological tests. A 100 µM
solution of complex in DMSO was prepared and the corresponding absorption spectra were recorded
to identify the wavelength range to be used for the stability evaluation. For each complex three
different wavelengths were identified: two associated with the absorption maxima and one from the
baseline as a control. Once fixed these wavelengths, the corresponding absorbances were recorded
every 15 minutes for 24 h and the values were plotted versus time to obtain the stability profiles. The
temperature was fixed at 37°C and kept stable during the entire experiment.
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2.1.4 Partition coefficient calculation
Log Pow for the ligands were calculated according to ref [22, 23].
2.2
Biology
2.2.1 Bacterial strains selection
Escherichia coli and Klebsiella pneumoniae were chosen as models to test the molecules. In this case
we selected a common E. coli, non-pathogenic (phylogenetic group A), isolated from poultry meat in
the laboratory of Food Inspection of Parma University characterized by the ability to produce biofilm
and a strain of K. pneumoniae (ATCC 700603) for its ability to produce Extended-Spectrum-Beta-
Lactamases (ESBL) encoded by SHV-18 gene.
Frozen stock bacteria were put into 2 mL of Buffered Peptone Water (BPW), and incubated for 2 h at
37°C. Then, an aliquot was seeded with a sterile calibrated loop on a generic agar plate (Tryptic Soy
Agar, TSA). After an incubation at 37°C for 24 h, bacterial colonies were obtained which were used
for the following experiments.
2.2.2 Study of the antimicrobial effect of the molecules
All the molecules were tested against both E. coli and K. pneumoniae for the definition of the
Minimum Bactericidal Concentration (MBC) as described by the CLSI protocol (2006). MBC is the
lowest concentration of a drug/broth dilution of antimicrobials that results in killing 99.9% of the
bacteria being tested. MBC is determined by the micro-dilution method and it is measured by sub-
culturing the broth dilutions that inhibit growth of a bacterial organism (i.e., those at or above the
MIC- Minimal Inhibitory Concentration) determination onto generic fresh agar plates. The MBC is
the lowest broth dilution of antimicrobial that prevents growth of the organism on the agar plate.
Failure of the organism to grow on the plate implies that only nonviable organisms are present.
In the first step, we diluted the original molecules prepared in DMSO (10 mM)1:100 with sterile
water. Then, in 96-well-plates we made serial two-fold dilutions in water from 50 µM to 24.4 NM
(final concentration) and we added the bacterial solution at the concentration of 0.5 McFarland (~
1.5x108 cells/mL). In this way in each well the final volume is composed by 100 µL of molecules at
different concentrations + 100 µL of bacterial solution. Micro-dilution plates were incubated at 37°C
for 24 h. This part was repeated for each molecules and for both bacterial strains. Positive (bacterial
solution) and negative (water and diluted molecules) controls were added in each plate.
The day after, 50 µL of each overnight cultures were seeded onto Tryptic Soy Agar (TSA) (a
nonselective media that allow the growth of a wide variety of microorganisms) using a L-spatula in
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double and plates were incubated overnight at 37°C. After 24 h plates were read and the MCB was
defined following the scheme reported by CLSI (2006).
The first screening test using all nine molecules onto both bacterial strains was repeated twice. For
the molecules that showed efficacy against both strains, we determined a MBC, a concentration
value confirmed to be effective in reducing 99.9% of bacterial growth in at least three experiments.
The same method was used to evaluate if the single components of the molecules were active
towards the two bacterial strains (metal ions, thiosemicarbazides, and aldehydes). In this way we
may exclude, or not, that the bactericidal action is due to the single parts of each compound, if
eventually it brakes, ant not to the compounds themselves.
3.
Results and Discussion
3.1
Syntheses
(
E)-cinnamaldehydethiosemicarbazone (1) was prepared according to a synthetic procedure
previously described [24] and the other ligands were synthesized in a similar way. The compounds
were obtained in good yields and with a good grade of purity as confirmed by TLC, NMR and X-
Ray data. The metal complexes were obtained as described in the experimental section. A colour
change of the solution containing the ligands and the copper ions was already significative of
complexation. Spectroscopic and spectrometric data of the solid obtained confirmed that the isolated
product was the desired one. Table 1 reports a scheme of the isolated products.
1
2
4
5
7
8
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3
6
9
Table 1. Scheme of the isolated ligands (1-3), copper(II) (4-6) and zinc(II) (7-9) metal complexes.
3.2
Stability in solution
The spectroscopic results are reported in Figure 1. In both complexes the stability profiles did
not show significant changes during the 24 hours of the experiment. In detail, 7 showed a slight
decrease of absorbance at 400 nm during the first 2 hours, however this effect did not last and the
stability of the values from 2 to 24 hours ensured the stability of the complex.
Figure 1. UV-visible spectra of compound 7 (box A) and 4 (box C) under study together with the
analysis of absorbance variation for selected wavelength vs time (box B and D for compound 7 and 4
respectively)
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Complex 4 underwent a lowering of the absorbance intensity at 390 nm, passing from 0.63 to 0.50.
This effect, having a constant slope, can be assigned to small aggregations of the compound or to a
progressive precipitation from the solution.
In both cases the absorbance of the baseline of the spectra remained stable on zero.
3.3
Crystal structures
Among the compounds presented in this paper, two of them and namely 2 and 3, were obtained as
single crystals of size suitable for an X-ray study and were structurally characterized for the first
time.
3.3.1. X-ray structure of compound 2
The molecular structure of compound 2, as determined by single-crystal X-ray crystallography, is
reported in Fig. 2.
Figure 2. ORTEP representation of the asymmetric unit of compound 2.
The molecule is overall planar, as expected for systems containing an extended system of conjugated
double bonds. Since thiosemicarbazones are subject to a thione-thiol tautomerism phenomenon, this
conjugation stabilises the thiol form and enhances the predisposition of these ligands to
deprotonation, and consequently their acidity (scheme 1).
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Scheme 1. Thiosemicarbazone thione-thiol tautomerism.
In the literature, the structures of very similar molecules are reported, which differ for the substituent
on the terminal nitrogen, and namely the non substituted, the methyl, and the phenyl derivatives [24-
26]. While all three structures known are superimposable, 2 presents a substantial difference in the
fact that the terminal nitrogen and the sulfur are swapped. The three structures reported in the
literature are in fact characterized by an intramolecular hydrogen bonding between the terminal
amino group and the imino nitrogen (Scheme 2A), which is not possible in 2 since both terminal
hydrogens are replaced by methyl groups (Scheme 2B).
X= H, R
B
A
Scheme 2. Effect of the terminal amino group substitution on the thiosemicarbazone configuration.
In addition, the presence of two methyls on the thiosemicarbazone terminal nitrogen, probably due to
sterical hindrance, contributes to stabilize the molecule in a cis configuration of the NN-CS bond
system (Scheme 2B). This pattern is consistently observed in all thiosemicarbazones found in the
Cambridge Structural Database [27] with a doubly methylated terminal nitrogen.
As regards the geometrical parameters, in compound 2 the C-S distances of 1.68(2) is very close to
the average value of non-deprotonated thiosemicarbazones found in the CSD [27]which is C=S =
1.69(2) Å. The N(hydrazine)-C(thione) bond distance of 1.336(2)Å is instead markedly shorter than
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the average of 1.40(1)Å of the free thiosemicarbazones of the same set, suggesting a stronger double
bond-like character.
The packing is characterized by a network of hydrogen bonds between the sulphur atom and the
hydrazinic NH. This bond allows the formation of zigzag strands which in turn interact with each
other through rather loose edge-to-face π-π interactions (Figure 1S).
3.3.2 X-ray structure of compound 3
The molecular structure of compound 3, as determined by single-crystal X-ray crystallography, is
reported in Figure 3.
Figure 3. ORTEP representation of the asymmetric unit of compound 3.
In this molecule, we find the common intramolecular hydrogen bond between the terminal amino
group and the iminic nitrogen that characterizes the thiosemicarbazones with the non substituted
terminal nitrogen and conferring the E configuration on to the NNCS atom system. The planarity, as
can be inferred by the angle of 23.69° between the average plane of the aromatic ring and the plane
of the thiourea fragment, is much less extended than in the previous molecule. Nevertheless, an
extended conjugated double bond system is still present as can be seen from the bond lengths which
fall in the range 1.305(6) to 1.516(8). The distortion from the plane is probably due to constraints
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imposed by the packing in which the sulfur forms hydrogen bonds with the terminal NH group of an
adjacent molecule and a looser one with the hydrazinic NH from another adjacent molecule.
3.4. Partition coefficient study
In order to verify if the difference in the biological activity among the three ligands could be related
to an enhanced ability to diffuse in an apolar phase, the logP were calculated. The logP values found
were 2.74 for (E)-cinnamaldehydethiosemicarbazone (1), 3.16 for (E)-cinnamaldehyde-4,4-dimethyl-
3-thiosemicarbazone
(2),
and
3.11
for
(E)-1-(4-methoxyphenyl)-1-pentene-3-one-3-
thiosemicarbazone (3). Due to the difference between the logP of (1) compared to (2) and (3), it
seems that a low octanol/water partition coefficient ratio can concur in the bactericidal activity of
thiosemicarbazone metal complexes.
3.5
Biological tests
Among the nine molecules synthesized, only 4 and 7 showed an inhibitory or bactericidal activity
against the strains under study. Compound 4 showed in particular its best activity against E. coli with
a 8 µM MBC, while compound 7 showed its best activity against K. pneumoniae with a 14 µM MBC
(Table 2).
Molecule Bacterial strain Concentration for MBC
8 µM
E. coli
4
7
30 µM
25 µM
14 µM
K. pneumoniae
E. coli
K. pneumoniae
Table 2. Minimum Bactericidal Concentration (MBC) of compounds 4 and 7 against E. coli and K.
pneumoniae bacterial strains.
In order to understand the causes of the effectiveness of compounds 4 and 7, the constituents of the
thiosemicarbazones, aldehyde and thiosemicarbazide, were also tested but neither showed any
bactericidal action. The results are really interesting and promising, in particular if compared with
the activity shown by analogous compounds recently studied [16].
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4. Conclusions
Thiosemicarbazones bearing the same aromatic scaffold with a different degree of polarity were
synthesized and characterized together with their Cu(II) and Zn(II) metal complexes. From the
biological studies, it results that among the compounds under scrutiny, the one with the lowest
partition coefficient is the most promising. The investigations on the antibacterial activity show that
the metal complexes and the ligands own an antibiotic activity stronger than that of the constituents.
Moreover, the metal thiosemicarbazonates are stable in solution at least for 24 hours and this
suggests that the observed biological activity can be ascribed to the non-dissociated metal
complexes. The MBC found against Klebsiella pneumoniae and Escherichia coli are very interesting
since they fall in the range 8 – 30 µM and therefore encourage an in-depth study to verify if they are
compatible for use on human epithelial tissues.
Acknowledgements
This work has been carried out in the frame of the activities of the ‘COMP-HUB’ initiative, funded
by the ‘Department of Excellence’ Project of the Italian Ministry for Education, University and
Research (MIUR). The authors wish also to thank for the facilities the Centro Interdipartimentale
Misure (CIM) of the University of Parma.
Funding
This research was funded by the University of Parma (FIL).
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Conflicts of interest
There are no conflicts to declare.
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Graphical abstract
Cinnamaldehyde thiosemicarbazone derivatives with different degree of octanol/water ratio were
synthesized and characterized together with their Cu(II) and Zn(II) complexes. The compound with
the lower partition coefficient is the most promising. The minimum bactericidal concentration found
against Klebsiella pneumoniae and Escherichia coli fall in the range 8 – 30 µM.
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Highlights
 Thiosemicarbazone metal complexes are good candidate as metalloantibiotics
 LogPow is a discriminating parameter for a good antimicrobial activity
 Cu(II) and Zn(II) cinnamaldehyde thiosemicarbazonate show a good stability in solution
21