New silver (thio)semicarbazide derivatives: Synthesis, structural features, and antimicrobial activity

Article abstract of DOI:10.1039/d0nj02013c

Two novel semicarbazide [HL1, 1, 1-((4-nitrophenyl)(phenyl)methylene)semicarbazide] and thiosemicarbazide [HL2, 2, 1-((4-nitrophenyl)(phenyl)methylene)thiosemicarbazide] ligands were generated from 4-nitrobenzophenone and the corresponding (thio)semicarbazide precursors. Compounds 1 and 2 were fully characterized and their X-ray crystal structures were established. These organic derivatives were further applied as bioactive building blocks for the self-assembly synthesis of silver(i) coordination compounds. The reactions of silver(i) nitrate with HL1/HL2 in the presence of aqueous NH4OH resulted in new silver(i) coordination compounds formulated as [Ag(HL1)(NO3)]·H2O (3) and [Ag6(μ3-L2)6] (4). These complexes were characterized by standard methods (FT-IR, NMR, ESI-MS, and elemental analysis) as well as single-crystal X-ray diffraction. The structure of 3 features a two-coordinate silver(i) center with a distorted linear {AgNO} geometry, wherein an unusual coordination of HL1 through the nitrogen atom of the amine group is realized. The structure of 4 reveals a discrete Ag6 cluster composed of two joined trisilver(i) rings, wherein the three-coordinate Ag(i) centers adopt an {AgNS2} environment. The antimicrobial activity of 1-4 was screened against Gram-positive (S. epidermidis and S. aureus) and Gram-negative (P. aeruginosa and E. coli) bacteria. Compound 4 presented antimicrobial activity against Gram-positive bacteria, including the ability to impair biofilm formation. This study thus contributes to a still limited research area on the development of new silver coordination compounds with potential antibiofilm activity.

Full text of DOI:10.1039/d0nj02013c

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New Silver (Thio)Semicarbazide Derivatives: Synthesis, Structural
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Features, and Antimicrobial Activity†
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Tiago A. Fernandes,a,b Vânia André,a Aliaksandr S. Arol,a,c Ângela França,d Sergei
Mikhalyonok,c Nuno Cercad,* and Alexander M. Kirillova,e,*
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aCentro de Química Estrutural and Departamento de Engenharia Química, Instituto Superior Técnico,
Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; kirillov@tecnico.ulisboa.pt
bDepartamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande,
1749-016 Lisboa, Portugal
cDepartment of Organic Chemistry, Belarusian State Technological University, 13a Sverdlova St., 220006
Minsk, Belarus
dCentre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal;
nunocerca@ceb.uminho.pt
eResearch Institute of Chemistry, Peoples’ Friendship University of Russia (RUDN University), 6 Miklukho-
Maklaya st., Moscow, 117198, Russian Federation
†Electronic supplementary information (ESI) available: data on single-crystal X-ray diffraction (Table S1) and
additional structural representations (Figs. S1, S2); FT-IR (Figs. S3−S6), NMR (Figs. S7−S21) and ESI-MS
(Figs. S22, S23) data.
Abstract
Two novel semicarbazide [HL1, 1, 1-((4-nitrophenyl)(phenyl)methylene)semicarbazide] and
thiosemicarbazide [HL2, 2, 1-((4-nitrophenyl)(phenyl)methylene)thiosemicarbazide]
ligands were generated from 4-nitrobenzophenone and the corresponding
(thio)semicarbazide precursors. Compounds 1 and 2 were fully characterized and their X-
ray crystal structures were established. These organic derivatives were further applied as
bioactive building blocks for the self-assembly synthesis of silver(I) coordination
compounds. The reactions of silver(I) nitrate with HL1/HL2 in the presence of aqueous
NH4OH resulted in new silver(I) coordination compounds formulated as
[Ag(HL1)(NO3)]·H2O (3) and [Ag6(µ3-L2)6] (4). These complexes were characterized by
standard methods (FTIR, NMR, ESI-MS, elemental analysis) as well as single-crystal X-ray
diffraction. The structure of 3 features a two-coordinate silver(I) center with a distorted linear
{AgNO} geometry, wherein an unusual coordination of HL1 through the nitrogen atom of
the amine group is realized. The structure of 4 reveals a discrete Ag6 cluster composed of
two joined trisilver(I) rings, wherein the three-coordinate Ag(I) centers adopt an {AgNS2}
environment. The antimicrobial activity of 1−4 was screened against Gram-positive (S.
epidermidis and S. aureus) and Gram-negative (P. aeruginosa and E. coli) bacteria.
Compound 4 presented an antimicrobial activity against gram-positive bacteria, including
the ability to impair biofilm formation. This study thus contributes to a still limited research
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area on the development of new silver coordination compounds with potential antibiofilm
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activity.
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1. Introduction
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One of the major worldwide public health issues identified by the World Health Organization
(WHO’s 2014 report)1 concerns the multiresistant strains of bacteria and fungi that became
resistant to conventional antimicrobials or human’s own defenses. Besides, the formation of
biofilms, multicellular microbial aggregates adhered to each other on a surface, also provides
these microorganisms with an additional resistance to antibiotics and antiseptics.2 Hence,
there is a need for creating new molecules and derived materials that could prevent the
proliferation of bacteria and biofilms.
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Benzophenone and 4-nitrobenzophenone semicarbazides and thiosemicarbazides have been
extensively studied owing to their recognized herbicidal activity and potential inhibiting
action against urease aminohydrolase, a metalloenzyme that is found in bacteria, fungi, and
higher plants.38 On the other hand, it is widely known that silver and its simple salts are
active antimicrobial agents.912 Among all the bioactive metals, silver exhibits the highest
toxicity to bacteria, viruses, and other eukaryotic microorganisms. Simultaneously, silver
shows a relatively low toxicity to mammal cells.9 A large number of bioactive silver
coordination compounds has been reported. In particular, it has been observed that the
derivatives having weak Ag−O and/or Ag−N bonds usually exhibit a significantly higher
bioactivity, lower light stability, and inferior solubility in water than compounds possessing
rather strong Ag−S or Ag−P bonds.10,11 Hence, silver complexes typically feature better
bioactivity in comparison with simple salts or nanoparticles. In many cases, the use of silver
compounds as potential drugs, components of creams, personal care products, and wound
dressings is limited by their light sensitivity and insolubility in aqueous medium.10−12
Considering the above discussion and following our interest in the synthesis and exploration
of novel bioactive coordination compounds,1315 in this study we focused our attention on
two still unexplored semicarbazide and thiosemicarbazide derivatives, and their application
for the synthesis of silver(I) coordination compounds. Hence, herein we report the
preparation, full characterization, structural and topological features, as well as antimicrobial
screening of two closely related (thio)semicarbazide building blocks [HL1, 1, 1-((4-
nitrophenyl)(phenyl)methylene)semicarbazide;
HL2,
2
1-((4-
nitrophenyl)(phenyl)methylene)thiosemicarbazide] as well as their new silver(I) derivatives
[Ag(HL1)(NO3)]·H2O (3) and [{Ag6(µ3-L2)6] (4).
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2. Results and discussion
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2.1 Synthesis and Characterization of 1−4
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Organic compounds 1 and 2. Semicarbazide (1) and thiosemicarbazide (2) were prepared
from 4-nitrobenzophenone via a one-step procedure consisting of a reaction with a
semicarbazide hydrochloride or thiosemicarbazide, respectively. The crude products were
purified by washing with ethanol and were isolated in good yields (1: 72%; 2: 70%). Only
(E)-isomers were crystallized from the reaction mixture as determined by single-crystal X-
ray diffraction. Both compounds were characterized by NMR and FTIR spectroscopy,
electrospray ionization mass spectrometry (ESI-MS) and elemental analysis.
The 1H and 13C{1H} NMR spectra reveal signals typical for 4-nitrobenzophenone moieties.
In 1H NMR, the signals at δH 8.33 and 8.81 ppm indicate the presence of amide protons in 1
and 2, respectively. Two aromatic rings are present in the structures and the protons of the
aromatic ring having the nitro substituent at para position appear at up-field region at δH
8.18 (2H, d, J = 9.0 Hz) and 7.79 ppm (2H, d, J = 9.0 Hz) for 1 and 8.20 (2H, d, J = 8.8 Hz)
and 7.93 ppm (2H, d, J = 8.8 Hz) for 2. This is due an electron withdrawing effect of nitro
group and the decrease of the electron density on the aromatic ring through resonance
withdrawing effect. The remaining aromatic protons can be seen at δH 7.68–7.54 (3H, m)
and 7.31 ppm (2H, d, J = 7.0 Hz) for semicarbazide (1) and at δH 7.70−7.54 (3H, m) and
7.39 ppm (2H, d, J = 7.0 Hz) for thiosemicarbazide (2). The amide signal is observed at δC
~156 ppm for 1, while the thioamide resonance is present at δC ~178 ppm for 2. The
allocation of signals was also supported by the 2D HSQC and HMBC NMR spectra of 1 and
2 (Figs. S7−S14, ESI).
H2N
O
X
N
NH
X
TsCl (X = S)
EtOH
O2N
NH2
+
H2N
N
H
O2N
(HCl for X=O)
X = O (1) or S (2)
X = O or S
Scheme 1 Synthesis of 4-nitrobenzophenone semicarbazide (1, X = O) and thiosemicarbazide (2, X
= S).
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The FT-IR spectrum of 1 shows the ν(NH) vibrations at 3473 and 3351 cm−1. The imine
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DOI: 10.1039/D0NJ02013C
ν(C=N) and aromatic ν(C=C) bands appear at 1598 and 1572 cm , respectively, whereas
the carbonyl ν(C=O) band can be seen at 1708 cm−1. The FT-IR spectrum of 2 reveals similar
features with the ν(NH) vibrations at 3494, 3370, and 3347 cm−1. The imine ν(C=N),
aromatic ν(C=C), and thiocarbonyl ν(C=S) bands appear at 1597, 1591, 1558, and 1272
cm−1, respectively (Figs. S3 and S4, ESI).
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Coordination compounds 3 and 4. A mononuclear silver(I) complex [Ag(HL1)(NO3)]·H2O
(3) and a hexasilver(I) cluster [Ag6(µ3-L2)6] (4) were obtained in the reaction of AgNO3 with
semicarbazide (1, HL1) or thiosemicarbazide (2, HL2) in acetonitrile-acetone (4:1, v/v)
medium and in the presence of aqueous NH4OH. In the used solvent composition, acetone
was required to avoid an instant precipitation of complexes 3 and 4 given their solubility in
acetone. Both silver(I) products were isolated as microcrystalline solids in moderate to good
yields (3: 37%; 4: 77%) upon slow evaporation of solvent in air. A moderate yield of 3 is
explained by its more difficult crystallization that was not prolonged until the end of the
reaction to prevent the contamination of product. The structures of 3 and 4 were confirmed
by standard analytical methods and single-crystal X-ray diffraction (Table S1, ESI).
Compound 3 possesses a two-coordinate silver(I) center with a distorted linear {AgNO}
geometry and an unusual coordination of HL1 through the nitrogen atom of the amine group.
In contrast, the compound 4 reveals a discrete Ag6 cluster composed of two joined trisilver(I)
rings in a chair configuration, wherein the three-coordinate Ag(I) centers adopt an {AgNS2}
environment. Despite probing the reactions of AgNO3 with two closely related
semicarbazide (1, HL1) and thiosemicarbazide (2, HL2) ligands, the resulting coordination
compounds 3 and 4 feature the very distinct structures and different coordination modes of
the ligands. This can be associated with the presence of an S-donor site in the
thiosemicarbazide 2 which, expectedly, shows a stronger coordination affinity to silver(I)
ions in comparison with the O-donor site of 1.
H2N
NH
H2N
O
O
AgNO3
N
N
NH
Ag
NO2
O2N
O2N
O
MeCN, Me2CO (4:1 v/v)
NH4OH
3
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R2
H2N
N
R1
H2N
NH
R
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R2
R1
R2
N
DOI: 10.1039/D0NJ02013C
S
S
N
N
N
N
Ag
Ag
N
Ag
NH2
AgNO3
H2N
H2N
O2N
S
S
NH2
S
S
MeCN, Me2CO (4:1 v/v)
NH4OH
Ag N
N
Ag
N
N
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R2
R1
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S
R2
N
R1
N
R1
NH2
R2
4
R1 = Ph, R2 = 4-NO2-Ph
Scheme 2 Synthesis and structural formulae of silver(I) complexes 3 and 4.
The H and C{1H} NMR spectra of 3 in DMSO reveal signals typical for coordinated
semicarbazide moieties (Fig. 1). In 3, the 1H signals from aromatic rings appear at a lower
field if compared with the free ligand 1, thus suggesting an N-coordination of
thiosemicarbazide as a neutral ligand to Ag. The NH2 protons are observed as a broad signal
in the same region at 6.83 ppm. The amide proton, seen at δH 8.33 ppm for 1, is no longer
observed in 3, probably because of being obscured by protons of atomatic rings at a lower
field (Fig. 1). The 13C NMR spectrum of complex 3 presents the amide carbon atom signal
that is shifted upfield relatively to that in 1, namely from δC ~156 to ~166 ppm (Figs. S8 and
S16, ESI). The signal for a carbon atom between the aromatic rings is slightly shifted from
δC ~147 ppm in 1 to δC ~145 ppm in 3. Aromatic carbon signals appear at a slightly lower
field or do not show any significant shift upon complexation; this behavior is in accord with
related literature for thiosemicarbazide complexes.16 The 2D HSQC and HMBC NMR
spectra of 3 were also collected to support an allocation of signals (Figs. S17 and S18, ESI).
1
13
1
Figure 1. H NMR spectra (5−9 ppm) of compounds 1 and 3 with proton assignment (DMSO-d6,
400 MHz).
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In contrast, the 1H and 13C{1H} NMR spectra of 4 are poorly resolved at room temperature
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and feature very broad signals, what can be caused by chemical shift anisotropy due the
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presence of six identical thiosemicarbazide ligands.17 However, through rapid molecular
reorientation at higher temperature, isotropic chemical shifts can become equal due to
averaging of the chemical shift anisotropy to zero at 70 °C. As a result, more resolved spectra
can be obtained and the average values of chemical shifts can be established (Fig. 2). Even
at 70 °C, the averaging of the chemical shift anisotropy of six ligands is still noticeable and
the presence of equivalent aromatic atoms is evident within the same range of chemical
shifts.
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1
Figure 2. H NMR spectra (6.5−9 ppm) of compound 4 over a range of temperatures (DMSO-d6,
400 MHz, 30−70 ºC).
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Figure 3. H NMR spectra (5−9 ppm) of compounds 2 and 4 with proton assignment (DMSO-d6,
400 MHz, acquired at room temperature for 2 and at 70 ºC for 4). In 4, Ph and 4-NO2-Ph
functionalities of µ3-L2 were omitted for clarity.
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As observed for compound 3, the 1H NMR spectrum of 4 in DMSO reveals all proton signals
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from aromatic rings at a lower field if compared with the free ligand 2 (Fig. 3). The amide
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proton is no longer observed, as confirmed by structural analysis (Fig. 4d), while the NH2
amine protons are probably obscured by protons from aromatic rings at a lower field (Fig.
3). The 13C NMR spectrum of complex 4 shows the signal for thioamide carbon atom (C=S,
δC ~178 ppm) in the same region as in 2 (Figs. S12 and S20, ESI). The signal for a carbon
atom between the aromatic rings (C=N) as well as those within the rings appear in the same
region as in 2. No other information on the carbon atoms in the aromatic rings was possible
to collect due to the anisotropic effect of the six equivalent ligands.
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The ESI-MS(+) spectrum 3 shows the hydrated molecular ion [Ag(HL1)NO3 + H2O + H]+
(m/z = 473) that is in accord with the crystal structure of this compound. A (1+)-charged
molecular ion of the Ag6 cluster 4 (m/z = 2443) could not be detected since its m/z is above
the device's standard operating window. Nevertheless, the mass spectrometry pattern of 4
reveals a characteristic (2+)-charged molecular fragment [Ag6(L2)6 + H]2+ (m/z = 1221),
along with some adducts such as [Ag5(L2)4]+ (m/z = 1735) and [Ag2(L2)3]+ (m/z = 1114); all
these feature an expected isotopic distribution pattern (Figs. S22 and S23, ESI).
Hence, the above-discussed NMR and ESI-MS data of 3 and 4 support their structural
integrity in solution. In fact, as can be seen from Fig. 2, compound 4 is stable in solution for
hours even at 70 °C, allowing an accumulation of good quality 13C NMR data. Besides, both
compounds can be dissolved and then recrystallized from acetone and/or acetonitrile upon
slow evaporation, producing parent crystalline samples in 1-2 weeks.
The amide functionalities of semicarbazide or thiosemicarbazide moieties are manifested in
the FT-IR spectra of both complexes as the characteristic ν(C=N) bands with maxima at
1595 and 1596 cm–1, respectively. For 3, the carbonyl ν(C=O) band is observed at 1705 cm−1
and has a lower intensity due to the proximity of a silver center. The thiocarbonyl ν(C=S)
band in 4 appears at a higher wavelength value (1300 cm−1) if compared to 2, what can also
be explained by a close presence of a silver center (Figs. S5, S6, ESI).
2.2 X-ray Crystal Structures
The molecular structures of 1 and 2 were also confirmed by single crystal X-ray diffraction
analysis (Figs. 4a,b). Both structures are extended by strong intermolecular N−H…O
hydrogen bonds (Fig. S1, ESI) to give either H-bonded dimers (in 1) or 1D chains (in 2). In
the latter case, such chains can be classified within the 2C1 topological type.
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The crystal structure of [Ag(HL1)(NO3)]·H2O (3) was also confirmed by single crystal X-
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ray diffraction analysis. It is composed of a monosilver(I) molecular unit and a water
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molecule of crystallization (Fig. 4c). The Ag1 center is two-coordinate and adopts a distorted
linear {AgNO} geometry with a representative O−Ag−N angle of 174.40(13)°. The
coordination sphere of Ag1 atom is filled by an O atom of the terminal nitrate ligand and the
N atom of the HL1 block (Fig. 4c). The respective Ag−O [2.0998(4) Å] and Ag−N [2.118(2)
Å] distances are within the expected values for such silver(I) coordination compounds. It
should be mentioned that the observed coordination of HL1 to Ag(I) through the N atom of
the amine group in 3 is unusual for semicarbazide ligands. For example, Han et al.18 observed
the chelation of semicarbazide to ytterbium and erbium through oxygen and the amine
nitrogen atoms. Interestingly, the adjacent [Ag(HL1)(NO3)]·units in 3 are H-bonded into 1D
ladder motifs, which additionally participate in hydrogen bonding interactions with
crystallization water molecules, thus giving rise to the generation of a 2D H-bonded layer
network (Fig. S2a, ESI). From a topological perspective (Fig. S5b), such a network can be
defined as a uninodal 4-connected net with the sql [Shubnikov tetragonal plane net] topology
and point symbol of (44.62).
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The structure of 4 (Fig. 4d) features a discrete [Ag6(µ3-L2)6] cluster that is assembled from
two joined trisilver(I) rings in a chair conformation.19 The structure is composed of six Ag
centers and is driven by the six µ3-L2 ligands. All the silver atoms are 3-coordinate and adopt
an {AgNS2} environment with the Ag−N and Ag−S distances in the 2.239(13)−2.292(16)
and 2.435(5)−2.572(5) Å ranges, respectively. Each silver center is simultaneously bound
by three different µ3-L2 moieties. There are also some weak argentophilic interactions
[Ag…Ag 2.798(3)−2.845(2) Å] that provide an additional interlinkage between the two
trisilver(I) rings.
(a)
(b)
(c)
(d)
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Figure 4. X-ray crystal structures of 1 (a), 2 (b), 3 (c), and 4 (d). Color codes: C (gray), H (white),
N (blue), O (red), S (yellow), Ag (cyan), In 4, Ph and 4-NO2-Ph functionalities of µ3-L2 were omitted
for clarity.
2.3 Antimicrobial Activity
The antimicrobial efficiency of the compounds was first assessed by determining the
minimum inhibitory concentration and minimum lethal concentration against four major
nosocomial pathogens.2023 Both 1 and 3 & 2 and 4 pairs of compounds have a low solubility
in DMSO, with maximum concentrations of 0.125 and 0.250 g/mL respectively. At these
concentrations, only silver cluster 4 revealed antimicrobial activity, as shown in Table 1.
However, this antimicrobial activity was species dependent and only Gram-positive bacteria
were affected. S. epidermidis seems to be more susceptible than S. aureus, since half of the
concentration was needed to inhibit bacterial growth. Interestingly, the antimicrobial activity
of 4 was bactericidal (MLC), but it required twice the concentration needed to inhibit (MIC)
the bacterial growth (Table 1).
The observed antimicrobial activity of 4 can be compared with other Ag-containing
compounds, namely the N-substituted imidazolidine-2-thione silver complexes that feature
some structural similarities with 4 and present the MIC values from 0.005 to 1.0 mg/mL
against S. aureus and S. epidermidis,24 and the silver camphorimine and camphor
sulphonylimine complexes that show the MIC values in the 0.047−0.257 mg/mL range
against S. aureus.25 Besides, several silver(I) coordination polymers were reported in the last
decade with excellent activity against E. coli and S. aureus, which is superior to that of 4.15,26
This can be associated with a higher stability of silver-organic networks in comparison with
discrete complexes as well as the presence of distinct types of ligands, resulting in different
coordination geometries around silver(I) centers.
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Table 1. Minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) for
View Article Online
1−4 (mg/mL) against several bacterial pathogens.
DOI: 10.1039/D0NJ02013C
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MIC
MLC
n/aa
n/a
MIC
>0.125
>0.125
>0.125
>0.125
MLC MIC
MLC MIC
MLC
0.250
0.125
n/a
>0.125
n/a
n/a
n/a
n/a
>0.250
>0.250
>0.250
>0.250
n/aa
n/a
n/a
n/a
0.125
S. aureus
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>0.125
>0.125
>0.125
0.063
S. epidermidis
P. aeruginosa
E. coli
n/a
>0.250
>0.250
n/a
n/a
aNon applicable, no growth of inhibitory activity was observed.
Taken into consideration the obtained MIC and MLC results, another experiment was
performed. As shown before with antibiotics, sub-inhibitory concentrations might impair
biofilm formation.27 As such, both S. epidermidis and S. aureus were induced to form
biofilms in the presence or absence of half the respective MIC concentration of silver
complex 4. Interestingly, even at a half the concentration required to inhibit the growth, S.
aureus biofilm formation was strongly impaired, with a 50% reduction in the total biomass,
as shown in Figure 5. Curiously, S. epidermidis biofilm formation was not affected.
Although limited examples of silver derivatives were reported to show antibiofilm activity,
the comparison of the obtained results is not straightforward due to the use of different strains
and/or species of biofilm forming bacteria. An interesting example concerns an ethanediyl
bis(isonicotinate) silver coordination polymer that exhibited a strong bactericidal activity on
biofilms and prevented murine S. epidermidis infection.28 The contact-killing effect on
amoxicillin-resistant E. coli biofilms and methicillin-resistant S. aureus biofilms was also
described for some materials containing an Ag(I) coordination polymer based on 1,3-
bisimidazolyl benzene blocks.29
Figure 5. Inhibition of biofilm formation by ½ MIC concentration of 4 against S. epidermidis and S. aureus.
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3. Experimental section
View Article Online
DOI: 10.1039/D0NJ02013C
3.1 Materials and Methods
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Synthesis of silver(I) complexes was performed under dinitrogen using Schlenk techniques.
AgNO3 was purchased from Sigma-Aldrich. The solvents were purchased from Lab-Scan,
further purified by conventional techniques and distilled before use. FT-IR spectra were
obtained on a JASCO FT-IR 4100 Type A or Shimadzu IRAffinity-1S apparatus equipment
using KBr pellets (abbreviations: vs − very strong, s − strong, m – medium, w – weak, br –
broad, sh − shoulder). NMR spectra (1H, 13C, DEPT, HSQC, and HMBC) were obtained on
Bruker Avance II+ Spectrometers (300 or 400 MHz) using DMSO-d6 ( = 2.50 ppm for 1H,
 = 39.52 ppm for 13C) as a reference. EA (elemental analyses) were performed on a Perkin
Elmer PE 2400 Series II analyzer (Laboratory of Analyses, IST). ESI-MS(±) were measured
using a LCQ Fleet mass spectrometer with an ESI source (Thermo Scientific). Details on
single-crystal X-ray diffraction are described in Electronic Supplementary Information
(ESI).
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3.2 Synthetic Procedures and Analytical Data
(E)-1-((4-nitrophenyl)(phenyl)methylene)semicarbazide (HL1, 1).
A
mixture of
semicarbazide hydrochloride (0.223 g, 2 mmol), 4-nitrobenzophenone (0.454 g, 2 mmol) in
7 mL ethanol was refluxed under stirring. The progress of the reaction was monitored by
TLC. The reaction was complete in 72 h and the reaction mixture was concentrated under a
reduced pressure. The obtained solid was collected by filtration and washed several times
with ethanol to give 1 as colorless microcrystals, yield 72%. 1H NMR (400 МHz, DMSO-
d6): δ, ppm: 8.33 (1H, s, NH), 8.18 (2H, d, J = 9.0 Hz), 7.79 (2H, d, J = 9.0 Hz), 7.68–7.54
(3H, m), 7.31 (2H, dd, J = 7.0 Hz), 6.83 (2H, s, NH). 13C NMR (100 MHz, DMSO-d6): δ,
ppm: 155.49, 147.08, 144.25, 143.50, 133.56, 131.12, 129.88, 128.49, 127.66, 123.49.
Elemental Analysis Found, %: C, 59.14; H, 4.16; N, 19.20; Calculated for [C14H12N4O3], %:
C, 59.15; H, 4.25; N, 19.17. FT-IR (KBr): 3473 (vs), 3351 (s), 3275 (s br), 3213 (s br), 1708
(vs), 1598 (m), 1572 (s), 1561 (s), 1507 (vs), 1455 (s), 1438 (s), 1391 (m), 1345 (vs), 1191
(s), 1107 (s), 867 (m), 849 (m), 783 (w), 759 (m), 710 (m), 699 (m), 689 (w), 583 (w).
ESI−MS(±) (Me2CO), selected fragments: MS(+), m/z: 589 (70%) [(HL1)2 + H]+, 343 (30%)
[HL1 + (CH3)2CO + H]+, 285 (100%) [HL1 + H]+; MS(−), m/z: 283 (100%) [L1]−.
(E)-1-((4-nitrophenyl)(phenyl)methylene)thiosemicarbazide (HL2, 2). A mixture of
thiosemicarbazide (0.182 g, 2 mmol), 4-nitrobenzophenone 0.454 g, 2 mmol) in 7 mL
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ethanol was refluxed under stirring and in the presence of a few crystals (catalytic amount)
View Article Online
DOI: 10.1039/D0NJ02013C
of p-toluenesulfonyl chloride (TsCl). The progress of the reaction was monitored by TLC.
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The reaction was complete in 72 h and the reaction mixture was concentrated under a
reduced pressure. The obtained solid was collected by filtration and washed several times
with ethanol to give 2 as orange microcrystals, yield 70%. 1H NMR (400 МHz, DMSO-d6)
δ, ppm: 8.81 (1H, s, NH), 8.64 (1H, s, NH), 8.59 (1H, s, NH), 8.20 (2H, d, J = 8.8 Hz), 7.93
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(2H, d, J = 8.8 Hz), 7.70–7.54 (3H, m), 7.39 (2H, d, J = 6.5 Hz). C NMR (100 MHz,
DMSO-d6): δ, ppm: 178.21, 147.65, 146.64, 142.55, 130.35, 130.05, 128.51, 128.41, 124.64,
123.49. Elemental Analysis Found, %: C, 56.24; H, 4.10; N, 18.64; S, 10.65; Calculated for
[C14H12N4SO2], %: C, 55.99; H, 4.03; N, 18.65; S, 10.68. FT-IR (KBr): 3494 (vs), 3370 (s),
3347 (s), 3105 (w), 3058 (w), 1652 (s), 1595 (s), 1588(s), 1558 (m), 1515 (vs), 1503 (vs),
1467 (s), 1440 (s), 1405 (m), 1340 (vs), 1272 (vs), 1182 (m), 1097 (s), 1079 (s), 943 (w),
929 (w), 865 (w), 853 (m), 843 (m), 706 (m), 699 (m), 529 (w). ESI−MS(±) (Me2CO),
selected fragments: MS(+), m/z: 301 (30%) [HL2 + H]+; MS(−), m/z: 299 (100%) [L2]−.
[Ag(HL1)(NO3)]·H2O (3): AgNO3 (75 mg, 0.44 mmol) was dissolved in a mixture of
acetonitrile-acetone (40 mL:10 mL). Then, solid HL1 (150 mg, 0.5 mmol) and 2.0 mL of
ammonium hydroxide solution (33%) were introduced. The obtained mixture was stirred for
18 h at 50 ºC, producing a dark orange crystalline solid. It was filtered off, washed with n-
hexane (5 mL), and dried in air to give 3 as an orange crystalline solid; yield 37%. A longer
1
reaction time may result in a better product yield but lower overall purity. H NMR (400
МHz, DMSO-d6): δ, ppm: 8.08 (2H, d, J = 9.0 Hz), 7.58 (1H, d, J = 8.9 Hz), 7.54-7.50 (3H,
m), 7.40-7.38 (1H, m), 7.17-7.15 (2H, m). 13C NMR (100 MHz, DMSO-d6): δ, ppm: 165.56
146.65, 144.82, 137.71, 133.90, 129.51, 128.74, 128.09, 125.35, 123.42 ppm. Elemental
Analysis Found, %: C, 41.11; H, 3.96; N, 17.94; Calculated for [C14H12AgN5O63CH3CN],
%: C, 41.61; H, 3.67; N, 18.68. FT-IR (KBr): 3549 (m), 3519 (s), 3400 (m), 3313 (m), 3199
(m br), 1705 (m), 1658 (s), 1607 (vs), 1597 (s), 1591 (s), 1544 (s), 1507 (s), 1498 (vs), 1483
(vs), 1440 (m), 1413 (m), 1397 (s), 1332 (vs), 1294 (s), 1246 (w), 1076 (vs), 855 (m), 782
(w), 760 (m), 698 (m). ESI−MS(±) (Me2CO), selected fragments: MS(+), m/z: 473 (20%)
[Ag(HL1)NO3 + H2O + H]+.
[Ag6(µ3-L2)6] (4) This compound was obtained following a procedure described for 3 but
using HL2 (300 mg, 1.0 mmol) instead of HL1. Orange crystalline solid, yield 77%. 1H NMR
at 70ºC (400 МHz, DMSO-d6): δ, ppm: 8.18 (2H, d, J = 8.5 Hz), 7.77 (1H, d, J = 8.7 Hz),
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7.54-7.50 (3H, m), 7.40-7.38 (2H, m), 7.27-7.25 (2H, m). C NMR at 70ºC (100 MHz,
DMSO-d6): δ, ppm: 178.41, 147.55, 146.49, 142.29, 130.02, 129.66, 129.53, 129.14, 128.03,
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127.43, 127.20, 126.58, 124.20, 123.20, 123.00. 13C NMR at 70ºC (100 MHz, Acetone-d6):
View Article Online
DOI: 10.1039/D0NJ02013C
δ, ppm: 176.61, 149.23, 142.12, 140.36, 132.10, 129.66, 129.15, 128.91, 124.55, 124.07.
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Elemental Analysis Found, %: C, 39.69; H, 2.69; N, 13.26; S, 7.50; Calculated for
[{C14H11AgN4O2S}6], %: C, 41.29; H, 2.72; N, 13.76; S, 7.87. FT-IR (KBr): 3466 (s br),
3349 (s br), 1596 (m), 1559 (vs), 1512 (vs), 1456 (vs), 1441 (vs), 1402 (w), 1343 (vs), 1300
(vs), 1177 (w), 1107 (m), 1044 (m), 1024 (m) 860 (s), 851 (s), 770 (w), 756 (w), 731 (w),
698 (s). ESI−MS(±) (Me2CO), selected fragments: MS(+), m/z: 1221 (5%) [Ag6(L2)6 + H]2+,
1735 (55%) [Ag5(L2)4]+, 1114 (10%) [Ag2(L2)3]+.
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3.3 Antimicrobial Activity Studies
Four biofilm-associated bacterial species were tested in this study: Gram positive
Staphylococcus aureus strain ATCC25923 and Staphylococus epidermidis strain RP62A and
Gram negative P. aeruginosa strain PA14 and Escherichia coli strain ATCC25922.
Antimicrobial activity was first assessed by determining the minimum inhibitory
concentration (MIC), according to NCCLS standards30 and also the minimum bactericidal
concentration (MLC), as described before.31 Briefly, all bacteria were grown overnight in
commercially available growth media TSB (Merck) at 37ºC and with agitation at 120 rpm.
Then, each culture density was adjusted by spectrophotometry to and OD640nm of 0.1.
Afterwards, a 100 fold dilution of each culture was incubated in TSB for 24 hours, at 37 ºC,
in 96 well tissue culture plates (Orange Scientific), containing 2 fold dilutions of each
compound used dissolved in DMSO. In some cases, it was not possible to prepare and test
more concentrated solutions due to a limited solubility of the samples. A positive control
included the growth media supplemented only with DMSO at the same concentration used
to dissolve the compounds. MIC value was defined as the lowest concentration that inhibited
bacterial growth. MLC were obtained following MIC determination by inoculating 10 μl of
the content of each well on TSA plates and incubated for 48 h at 37 ºC. Each experiment
was performed 3 independent times. Afterwards, the biofilm formation inhibition activity
was determined for those compounds presenting antimicrobial activity, as described
previously, with minor adaptations.32 Briefly, bacterial cultures were incubated for 24 h in
either TSB supplemented in 0.4% glucose (TSBg), or in TSBg supplemented with ½ of the
respective MIC concentration of each compound, for each bacteria. The 0.4% glucose
addition was performed to induce biofilm formation.33 Biofilms were quantified using and
ELISA plate reader, at 595 nm, after staining with 0.1% crystal violet, as described before.34
For each species, 4 independent experiments were performed with at least 6 technical
replicates per experiment.
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4. Conclusions
View Article Online
DOI: 10.1039/D0NJ02013C
In this study, we synthesized and characterized two novel semicarbazide and
thiosemicarbazide ligands. Their crystal structures were established and H-bonding features
were highlighted. These organic derivatives were further applied as bioactive building
blocks for the self-assembly generation of silver(I) complexes. Two distinct Ag(I) products
were obtained, namely a mononuclear complex [Ag(HL1)(NO3)] (3) and a hexasilver(I)
cluster [Ag6(µ3-L2)6] (4), and fully characterized. Compound 3 features a two-coordinate
silver(I) center with a distorted linear {AgNO} geometry. Interestingly, there is an unusual
coordination of the semicarbazide HL1 ligand in 3 through the nitrogen atom of the amine
group. The structure of 4 discloses a discrete Ag6 cluster assembled from two trisilver(I)
rings adopting a chair conformation, wherein the three-coordinate Ag(I) centers possess an
{AgNS2} environment. Both organic ligands and silver complexes were screened against S.
epidermidis, S. aureus, P. aeruginosa and E. coli and compound 4 was found to be active
against the gram-positive bacteria, including the ability to impair biofilm formation. The
observed antimicrobial activity of 4 is comparable to that of other silver(I) complexes.
Besides, this compound extends a still limited number of silver(I) derivatives that are able
to impair the formation of biofilms. Future work on widening the family of such silver
compounds and particularly multimetallic clusters as well as on establishing their
antimicrobial and antibiofilm performance is currently in progress.
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Acknowledgements
This work was supported by the Fundação para a Ciência e Tecnologia (FCT) and Portugal 2020
(projects PTDC/QUI-QIN/29697/2017, LISBOA-01-0145-FEDER-029697, UIDB/00100/2020,
UIDP/00100/2020, SFRH/BSAB/150368/2019 and REM2013). The publication was also prepared
with the support of the RUDN University Program 5-100. TAF acknowledges the FCT for BPD grant
SFRH/BPD/119980/2016. We thank Dr. M. C. Oliveira and Ms. A. Dias for ESI-MS experimental
assistance.
Conflict of interests
The authors declare no conflict of interests.
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Graphical Abstract
New silver(I) (thio)semicarbazide coordination compounds were prepared, fully
characterized, and tested as antimicrobial and antibiofilm agents.
17