Thiosemicarbazones and thiadiazines derived from fluorinated benzoylthioureas: Synthesis, crystal structure and anti-Trypanosoma cruzi activity

Article abstract of DOI:10.1016/j.jfluchem.2018.08.004

A series of thiosemicarbazones was obtained by condensation of halogenated N-(diethylaminothiocarbonyl)benzimidoyl chlorides (3b–3h) with 4,4-dimethyl-3-thiosemicarbazide. The activity of the halogenated compounds against the parasite Trypanosoma cruzi was evaluated and compared to the previously reported activity of the corresponding non-substituted thiosemicarbazone. It was found that the halogen-substitution enhances in most cases the anti-parasitic activity. The meta-fluorinated compound (4g) was identified as the most potent one (IC₅₀= 9.0 μM, CC₅₀ > 200 μM), having a selectivity index (SI = IC₅₀/CC₅₀), which is 4-times higher than that of the non-substituted compound. Slight modification of the reaction conditions employed for the synthesis of some of the benzoylthioureas 3a–3g led to the unexpected formation of novel halogenated 6-amino-1,3,5-thiadiazine-2-thiones.

Full text of DOI:10.1016/j.jfluchem.2018.08.004

Accepted Manuscript
Title: Thiosemicarbazones and thiadiazines derived from
fluorinated benzoylthioureas: synthesis, crystal structure and
anti-Trypanosoma cruzi activity
˜
Authors: Federico Salsi, Gisele Bulho es Portapilla,
Konstantin Schutjajew, Zumira Aparecida Carneiro, Adelheid
´
Hagenbach, Sergio de Albuquerque, Pedro Iva da Silva Maia,
Ulrich Abram
PII:
S0022-1139(18)30277-X
DOI:
Reference:
https://doi.org/10.1016/j.jfluchem.2018.08.004
FLUOR 9208
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Received date:
Revised date:
Accepted date:
9-7-2018
3-8-2018
9-8-2018
˜
Please cite this article as: Salsi F, Bulho es Portapilla G, Schutjajew K, Carneiro ZA,
Hagenbach A, de Albuquerque S, da Silva Maia PI, Abram U, Thiosemicarbazones
and thiadiazines derived from fluorinated benzoylthioureas: synthesis, crystal
structure and anti-Trypanosoma cruzi activity, Journal of Fluorine Chemistry (2018),
https://doi.org/10.1016/j.jfluchem.2018.08.004
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Thiosemicarbazones and thiadiazines derived from fluorinated benzoylthioureas:
synthesis, crystal structure and anti-Trypanosoma cruzi activity
Federico Salsi,a) Gisele Bulhões Portapilla,b) Konstantin Schutjajew,a) Zumira Aparecida
Carneiro,b) Adelheid Hagenbach,a) Sérgio de Albuquerque,b) Pedro Iva da Silva Maia,c) and
Ulrich Abrama)*
a)Freie Universität Berlin, Institute of Chemistry and Biochemistry, Fabeckstr. 34-36, D-
14195 Berlin, Germany
b)Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Av.
do Café - Vila Monte Alegre, Ribeirão Preto - SP, 14040-903, Brazil.
c)Departamento de Química, Universidade Federal do Triângulo Mineiro, 38025-440,
Uberaba, MG, Brazil.
Dedicated to Prof. Erhard Kemnitz in recognition of the ACS Award on Creative Work in
Fluorine Chemistry 2018
Corresponding Autor:ulrich.abram@fu-berlin.de (Ulrich Abram)
1

Graphical Abstract
Fluorinated thiosemicarbazones derived from N-(diethylaminothiocarbonyl)benzimidoyl
chlorides show a considerable activity against Trypanosomacruzi, the parasite, which is
responsible for Chaga’s disease.
Highlights
 Biologically active thiosemicarbazones with N-(diethylaminothiocarbonyl)-
benzamidine fragments have been prepared.
 Fluorination of the compounds enhances their activity against the parasite
Trypanosoma cruzi.
 Fluorinated 6-amino-1,3,5-thiadiazine-2-thiones are formed by slight modifications
of the reaction conditions.
Abstract. A series of thiosemicarbazones was obtained by condensation of halogenated N-
(diethylaminothiocarbonyl)benzimidoyl
chlorides (3b-3h) with
4,4-dimethyl-3-
thiosemicarbazide. The activity of the halogenated compounds against the parasite
Trypanosoma cruzi was evaluated and compared to the previously reported activity of the
corresponding non-substituted thiosemicarbazone. It was found that the halogen-
substitution enhances in most cases the anti-parasitic activity. The meta-fluorinated
compound (4g) was identified as the most potent one (IC50 = 9.0 µM, CC50 > 200 µM),
2

having a selectivity index (SI = IC50/CC50), which is 4-times higher than that of the non-
substituted compound. Slight modification of the reaction conditions employed for the
synthesis of some of the benzoylthioureas 3a-3g led to the unexpected formation of novel
halogenated 6-amino-1,3,5-thiadiazine-2-thiones.
Keywords: Fluorinated ligands, Thiosemicarbazones, Spectroscopy, anti-Trypanosoma
cruzi activity
1. Introduction
Thiosemicarbazones, which are obtained by reactions of 4,4-dialkyl-3-thiosemicarbazide
with N-(dialkylaminothiocarbonyl)benzimidoyl chlorides (H2L, Scheme 1) have attracted
attention for their versatile coordination chemistry. They can act as mono- or dianionic
tridentate ligands and have been shown to stabilize a number of metal ions. Typical
complexes are [MO(L)Cl] (M = Re, Tc) [1,2], [Au(L)Cl] [3], [Pd(HL)Cl] or [Pt(HL)Cl] [4].
Scheme 1. Synthesis of the thiosemicarbazides of the present study.
Moreover, thiosemicarbazones and related compounds are of considerable significance with
respect to their biological and pharmaceutical properties. Many of these substances possess
remarkable activity against a number of diseases, such as cancer [2-6], HIV [7],
tuberculosis [8], or parasitic diseases [9-12]. Modification of the thiosemicarbazone
3

framework and complexation with different transition metal ions have been performed, in
order to tune the biological activity and the stability of these compounds [5,13].
In particular, thiosemicarbazones derived from benzoylthioureas have been reported to have
promising activity against the parasite Trypanosoma cruzi,14 which is responsible for the
American trypanosomiasis or Chagas’ disease, which is one of the major causes of
mortality from cardiovascular diseases in Latin America, and is expanding to other regions
of the world as a result of intensified human migration in the last decades [15]. Currently
available chemotherapy based on a nitrofuran (Nifurtimox©) and a nitroimidazole
(Benznidazole©) is unsatisfactory because of their limited efficacy and their toxic side
effects [16]. Despite recent efforts in the development of new chemotherapeutic agents,
they are still the only accessible drugs. Thus, more safer and efficacious drugs are urgently
required [17,18].
Fig. 1. Gold(III) organometallic complexes with thiosemicarbazide ligands.
The complexation of thiosemicarbazones of the type H2L with the organometallic
{AuIII(damp)}2+ unit (damp- = dimethylaminomethylphenyl) gives stable gold(III)
complexes of the composition [Au(Hdamp)(L)]Cl (Fig. 1). The representative derived from
4,4-dimethyl-3-thiosemicarbazide (4a) possesses a remarkable trypanocidal activity against
the intracellular amastigote form of the parasite and no appreciable toxicity to mice spleen
4

cells [13]. This makes compound 4a and the corresponding metal complex (Fig. 1)
promising candidates for further structure optimization.
The selective fluorination of organic molecules is one of the most effective strategies in the
designing of new drugs, in order to increase pharmaceutical potency, biological half-life
and to improve pharmacokinetic properties. This becomes evident by considering the
continuous increase of the number of fluorinated drugs already approved or drug candidates
currently entering clinical trial. In 2010, it was estimated that about 20% of all administered
drugs contained fluorine atoms. However, the current trend is increasing from 20% to about
30% for all potentially new approved drugs, in the most recent years [19].
In this study, the phenyl group of the thiosemicarbazone 4a has been functionalized with
halogenides or trifluoromethyl groups, and the anti-parasitic activity of the resulting
compounds has been investigated. This gives new insights into the chemical properties of
benzimidoyl chloride-derived thiosemicarbazones and into the influence of fluorination on
the anti-parasitic properties of such compounds and their metal complexes.
2. Results and Discussion
2.1. Synthesis and Structural Characterization
The first step of the syntheses of the thiosemicarbazones 4b-4h is the conversion of the
substituted benzoyl chloride into the corresponding N,N-dialkyl-N’-benzoylthiourea 1b-1h
(Scheme 2). The previously developed methods for the synthesis of N,N-dialkyl-N’-
benzoylthioureas were found to produce the mono- and difluorinated analogues in
low/moderate yields [20,21]. The latter methods involve the dropwise addition of benzoyl
chloride to a hot solution of ammonium or potassium thiocyanate, followed by heating for
2h. In order to improve the yields and extend the synthesis to differently substituted
5

benzoyl chlorides, it was necessary to optimize the procedure. The benzoyl chlorides were
dissolved in acetone before the addition and the reactions were carried out at room
temperature. The volume of solvent used in the reaction was found to be crucial for the
success of the reaction. For example, if the synthesis of 1h was carried out with the same
quantity of solvent employed in the syntheses of the other derivatives (1b-1g), only a dark
oil could be isolated and the formation of the desired product was not observed (1H NMR).
Any attempt to modify the reaction conditions (temperature, rate of addition, reaction time)
in order to obtain 1h at higher concentrations of the reaction mixture failed.
Scheme 2 Synthesis of the halogenated thiosemicarbazones
Rasmussen et al. suggest avoiding anhydrous reaction conditions utilizing an excess of
benzoyl chloride in the first step of the reaction [22]. When an excess of 3-fluorobenzoyl
chloride (1.6 equiv.) was treated with ammonium thiocyanate and subsequently with
diethylamine, the reaction mixture turned orange-red. After precipitation with water and
recrystallization from ethanol, orange needles were obtained. Crystal structure analysis
revealed the unexpected formation of the heterocyclic product 5c (Scheme 3). This reaction
6

can be regarded as a one-pot version of the laborious synthesis developed by Weber et al.
[23]. It has been shown that the same reaction can be carried out with different starting
materials, obtaining the corresponding heterocycles (5a, 5b) in moderate yields. The
reaction involves the condensation of one molecule of 3-fluorobenzoyl chloride with two
molecules of thiocyanate. But, surprisingly, when the reagents are introduced with the
stoichiometric ratio benzoyl chloride/thiocyanate 1:2, only the N,N´-diethylbenzoylthiourea
is obtained. Every attempt to change the reaction conditions (temperature, rate of addition,
volume of reaction solvent), in order produce the heterocycle 5c without excess of benzoyl
chloride, failed. It has been reported that the cyclization can be induced through the
addition of a sterically hindered secondary amine and aqueous hydrochloric acid to a
benzoyl thiocyanate solution [23]. We hypothesized, hence, that the lower pH, which is
caused by the hydrolysis of the residual benzoyl chloride in acetone, might induce the
cyclisation. On the contrary, addition of water to the reaction mixture suppressed the
formation of the heterocycle. The presence of an excess of the benzoyl chloride is essential
for the cyclisation reaction.
Scheme 3 One-pot synthesis of thiadiazine-2-thiones
1
The H NMR spectra of the N,N-diethyl-N’-benzoylthiourea derivatives 1b-1h show the
usual nonequivalence of the two ethyl groups. As previously reported [20], the hindered
7

rotation around the (S)C-N bond is due to its partial double bond character. The same
spectral features are observed in the 13C NMR spectra.
1
The H NMR spectra of the heterocycles 5a-5c are similar to those of the corresponding
benzoylthioureas, but the peaks corresponding to the protons in the ortho position with
respect to the heterocycle are shifted down-field. More diagnostic appears to be the 13C
spectrum. In the region between 160 and 200 ppm appear the signals related to the
heterocyclic quaternary carbon atoms.
Fig. 2. Molecular structure of the thiadiazine-2-thione 5c.
Figure 2 depicts the molecular structure of 5c. The heterocyclic ring is a slightly distorted
planar system. The root-mean-square deviation of the atom coordinates from a fitted plane
is 0.0081. The C-N bonds in the ring (C7-N2, C7-N3, C9-N2, C8-N3) can be described as
elongated double bonds with bond lengths of roughly 1.34 Å. The S1-C8 (1.780 Å) and S1-
C9 (1.760 Å) bonds are slightly shortened single bonds, while the C8-S2 bond of 1.660 Å
has clearly double bond character. The C9-N1 bond length (1.318 Å) is also typical for an
elongated double bond, accounting for the partial double bond character of the C-N bond
and, hence, for the splitting of the corresponding NMR signals.
Beyer et al. demonstrated that it is possible to transform benzyolthioureas into benzimidoyl
chlorides by complexation with nickel(II) and treatment of the isolated nickel(II) chelate
8

complexes with thionyl chloride in dry carbon tetrachloride [24]. The toxic carbon
tetrachloride utilized in the chlorination step was substituted by methylene chloride.
Moreover, the higher reactivity of the fluorinated nickel complexes requires milder reaction
conditions than those employed by Beyer. This can be achieved by a very slow addition of
the thionyl chloride to the reaction mixture and its previous dissolution in methylene
chloride. As already observed during the synthesis of the N,N-dialkyl-N’-benzoylthioureas,
high dilution of the reagents is a good approach for controlling the reactivity of the
fluorinated substances.
Chelation of the molecules with nickel(II) ions lowers the energy barrier for the rotation
around the (S)C—N bond. Thus, only one triplet and one quartet are observed in the
aliphatic range of the 1H NMR spectra of the diethylamino groups of the complexes 2b-2h.
The signals of the NH protons around 9.50 ppm disappear upon coordination, indicating the
deprotonation of the ligand. The splittings of the methyl and methylene signals reappear
1
upon chlorination in the H and 13C spectra of compounds 3b-3h. The conversion of the
benzoylthioureas to nickel complexes and then to benzimidoyl chlorides can be easily
13
13
identified in the C NMR spectra through the chemical shifts of the C=O signals. They
are shifted down-field upon complexation from the range of 161-163 ppm to 170-171 ppm,
while a strong up-field shift to 142-143 ppm is observed, when the C-Cl bonds are formed.
The 19F NMR signals are shifted up-field in the nickel complexes. Especially the ones
corresponding to the fluoride groups, since these fluorine atoms are directly bonded to the
aromatic ring.
It is well known that benzimidoyl chlorides undergo nucleophilic substitutions when treated
with nucleophiles such as amines, carbazides, sulfide, alkohols or selenolates [24,25].
Nguyen et al. described that the reaction of the benzimidoyl chloride 3a with 4,4-dimethyl-
3-thiosemicarbazide leads to the formation of 4a [1]. The same reaction can be successfully
9

conducted with halogenated starting materials, provided that the conditions are slightly mo-
dified. While 4a is relatively stable at 20° C under ambient conditions, the purification of
the halogenated substances 4b-4h must be done in anhydrous solvents to avoid degradation.
The dry crystalline products are stable in air at 20° C for a few days and at -20° C for
months.
1
In the H NMR spectrum of 4b-4h, signals are present around 9.5 ppm, which can be
assigned to NH groups, denoting protonation of two nitrogen atoms. The interpretation of
the 13C NMR spectrum is simplified by the 19F-13C coupling which gives well resolved
multiplets. The quaternary carbon atoms can be identified by their relative intensities. The
C=S peaks are found in the typical region around 180 ppm. The chemical shift of 19F NMR
signals are not significantly influenced by the substitution with different electron-
withdrawing moieties, i.e. carbonyl, chloride, thiosemicarbazone, at the phenyl ring (see
Table 1). More information can be derived from reproductions of the spectra, which are
given in the Supporting Information.
Table 1 19F NMR data for the fluorinated compounds 1-4. Chemical shifts and multiplicity are reported.
d
e
f
g
h
1
2
3
4
-105.5 (m)
-108.7 (m)
-105.5 (m)
-108.0 (m)
-63.2 (s)
-62.8 (s)
-63.1 (s)
-63.0 (s)
-107.2 (m)
-110.2 (m)
-107.9 (m)
-107.8 (m)
-111.0 (m)
-113.8 (m)
-111.9 (m)
-111.5 (m)
-62.8 (s)
-63.0 (s)
-62.8 (s)
-62.8 (s)
10

Fig. 3. Molecular structure of the thiosemicarbazone 4e.
As a representative compound, the thiosemicarbazone 4e was characterized by X-ray
crystallography. An ellipsoid representation of its molecular structure is given in Fig. 3.
The most notable effect of fluorination on the solid state structure of the molecule is the
switch from the “thiosemicarbazide” tautomeric form, which is observed for 4a [1], to a
“thiosemicarbazone” form, as exemplified in Scheme 4. The protonation of the nitrogen
atoms N2 and N4 is experimentally demonstrated by the detection of the electronic density
of the corresponding hydrogen atoms and the fact that they are involved in hydrogen bonds
(for details, see the Supporting Information). This is also supported by the bond lengths
situation inside the C,N skeleton of 4e with a C2-N3 bond length of 1.289 Å, which can be
regarded as an elongated double bond. The C1-N1 bond is shortened, indicating its partial
double bond character, which is responsible for the hindered rotation of the two ethyl
groups.
Scheme 4. Tautomeric equilibrium
11

2.2. Biological tests
In vitro assays were performed for the evaluation of the biological activity of compounds
4b-4h against mammalian LCC-MK2 cells and amastigotes forms of T. cruzi. Their
trypanocidal activity is given as IC50 values and compared to the reference drug
Benznidazole (BZ) (see Table 1). The ratio between the cytotoxic activity (CC50) and
trypanocidal activity (IC50) is also provided in order to determine the selectivity index for
each thiosemicarbazone (SI = CC50 / IC50).
Table 2 In vitro activity of the thiosemicarbazones against amastigotes forms of T. cruzi Tulahuen lac-Z strain and
cytotoxicity on LLC-MK2 mammalian cells. a
Compound
IC50 ± SD (µM)
42.9 ± 2.3
13.6 ± 2.5
16.1 ± 2.9
20.6 ± 3.8
18.5 ± 5.3
28.7 ± 5.2
9.0 ± 0.8
CC50 ± SD (µM)
232.5 ± 6.0
> 200
SI
5.4
4a
4b
4c
> 14.7
> 12.4
1.6
> 200
33.7 ± 0.4
> 200
4d
4e
> 10.8
> 7.0
> 22.2
13.2
> 200
4f
> 200
4g
4h
BZ
12.5 ± 3.0
3.8 ± 0.3
165.4 ± 14.0
> 200
> 53.0
a Mean ± SD of two independent experiments
All tested compounds show a high trypanocidal activity at low concentrations, especially
compounds 4b, 4g and 4h, which are statistically as active as BZ.
Previous studies by Maia and coworkers reported that the non-fluorinated
thiosemicarbazone 4a possesses moderate trypanocidal activity with IC50 = 42.9 µM and
SI = 5.4 [13]. In the present study, all halogenated thiosemicarbazones, except 4d, show
higher activity and selectivity on amastigote forms than the original molecule 4a. In
particular 4b, 4c, 4g and 4h have SI values higher than 10, as is required for anti-T. cruzi
drug screening [26].
12

The mammalian cells appear to be more sensitive to the para fluorinated derivative 4d,
which is indicated by a strong cytotoxic activity at relatively low concentrations (CC50
=
33.7 µM) in comparison to 4a. However, the introduction of a trifluoromethyl group in the
same position (compound 4e) lowers drastically the cytotoxicity, whereas the trypanocidal
activity remains almost unchanged.
The compound with two fluorine atoms in meta positions (4f) has the lowest inhibitory
capacity in relation to all other tested derivatives. This is particularly interesting in
comparison to compound 4g, which has only one fluorine atom in meta position. This meta
fluorination induces an increase in the selectivity by a factor of five, when compared with
the non-fluorinated thiosemicarbazone 4a.
It is not unexpected, that the chemical and physical properties of potential drug molecules
are drastically influenced by the introduction of fluorine atoms [27]. Especially, the
modulation of the lipophilicity and acid-base properties of molecules exert an important
influence onto the transport across cell membranes. Besides, the metabolic stability and
degradation pattern of drugs are frequently altered upon fluorination, which may lead to a
reduced or increased toxicity.
In order to determine whether the increased potency and reduced cytotoxicity of the
compounds can be ascribed specifically to the introduction of fluorine atoms, also
chlorinated (4b) and brominated (4c) thiosemicarbazones were synthesized and tested. The
obtained molecules have very similar inhibitory effects, due to the similar chemical
properties of chlorine and bromine. But compared with the para-F-substituted compound
4d, a drastic reduction of cytotoxicity is observed. In fact, both 4b and 4c did not show cell
death at the maximum concentration tested (CC50 > 200µM).
Considering the transport of a drug across a cell membrane, the selectivity index represents
an important parameter to predict possible side effects in preliminary studies in vivo. In this
13

sense, 4g was the most promising representative of the tested thiosemicarbazones. It is, in
fact, about 20 times more active agains the amastigote form of the parasite than cytotoxic
for mammalian cells (IC50 = 9 μM and CC50 > 200 μM).
3. Conclusions
The synthesis of a series of halogenated benzoylthioureas (1b-1h) was achieved and they
were chlorinated by complexation with Ni(II) and subsequent reaction with thionyl
chloride. The resulting (thiocarbonyl)benzimidoyl chlorides (3b-3h) react with 4,4-
dimethyl-3-thiosemicarbazide under formation of the thiosemicarbazones 4b-4h in good
yields. Compound 4b was characterized by crystal structure analysis, revealing that
fluorination shifts the tautomeric thiosemicarbazone/thiosemicarbazide equilibrium towards
the thiosemicarbazone form. Comparison of the anti-parasitic activity of the non-substituted
thiosemicarbazone 4a with the halide-substituted representatives 4b-4h shows that the
introduction of electron-withdrawing substituents like halogens on the aromatic ring
enhances the potency of the compounds in almost all cases. The most active substance was
the one bearing a fluorine atom on the meta position of the ring (IC50 = 9.0 µM, CC50 >
200 µM). The selectivity index (SI = IC50/CC50) of this compound is four times higher than
the one of the non-substituted thiosemicarbazone.
The coordination chemistry of these novel compounds with transition metals is currently
under study in our research group with the scope of utilizing the incorporation of metal ions
to a further improvement of the pharmaceutical properties of such molecules.
It was also found that a cyclisation reaction competes with the formation of
benzyolthioureas and leads to 6-amino-1,3,5-thiadiazine-2-thiones. The heterocycles can
selectively be obtained by slightly modified reaction conditions. This alternative reaction
pattern can be exploited for the synthesis of novel fluorine-containing 6-amino-1,3,5-
14

thiadiazine-2-thiones (5). A crystal structure determination of 5c confirms the for-mation of
a planar heterocyclic ring.
4. Experimental
4.1. Biological tests
4.1.1. Mammalian cells, parasites and drugs
Monkey kidney cells LLC-MK2 (ATCC® CCL-7™) were cultivated in RPMI-1640 medium
(Roswell Park Memorial Institute - Sigma-Aldrich), supplemented with 10% fetal bovine
serum (Sigma-Aldrich), 50 U/ml penicillin, 0.05 mg/ml streptomycin. In vitro assays were
performed against amastigotes forms of Tulahuen strain, which was genetically modified to
express the β-galactosidase gene from E. coli (lacZ).28 Trypomastigotes were harvested
from the supernatant of infected LLC-MK2 cultures 5 or 6 days after infection. The
fluorine-substituted thiosemicarbazones and the standard drug Benznidazol were dissolved
in DMSO (final concentration in the assays did not exceed 1.5 %).
4.1.2. In vitro trypanocidal activity
LLC-MK2 were seeded into 96-well plates at a concentration of 5×104 cells/ml and
infected with trypomastigotes at a multiplicity of infection (MOI) of 10. After 48 h of
infection, the plates were washed twice with phosphate-buffered saline (PBS) to remove
extracellular parasites and the fluorine-substituted thiosemicarbazones were added in
different concentrations by serial dilution from 200 µM to 1.56 µM. After an incubation
period of 72 h, viability of amastigotes were assessed from 50 µL of PBS containing 2% of
Triton X-100 and 200 µM Chlorophenol Red-β-D-galactoside (CPRG - Sigma). The plates
were incubated for 4 h at 37°C and the enzyme β-gal activity was set by absorbance values
15

at 570 nm from a microplate reader (Synergy™ H1). Percentage inhibition of amastigotes
of 50% (IC50) was calculated and untreated cells and Benznidazole were used as assay
controls.
4.1.3. Cytotoxicity on mammalian cells
LLC-MK2 were seeded into 96-well plates at a concentration of 5×104 cells/ml. After 48 h,
the plates were washed twice with PBS and the thiosemicarbazones were added at the same
concentration described above. After 72 h at 37 °C the cytotoxicity effects were set by a 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay as described by
Mosmann.29 Briefly, 2.5 mg/mL MTT in PBS was added (50 μL/well) and the plates were
further incubated for 4 h at 37 °C. The resulting formazan crystals formed were dissolved
with DMSO (50 μL/well) and the samples were measured in a spectrophotometer at 570 nm
after 30 min.
4.1.4. Statistical analysis
Data were expressed as mean ± standard deviation (S.D.) of at least two independent
experiments performed on two different days. Statistical analysis was performed using one-
way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
These analyses were done by GraphPad Prism, V5.0 software (GraphPad Software, San
Diego, CA, USA). p-Values < 0.05 were considered as statistically significant.
4.2. Synthesis and Characterization
4.2.1. General methods and material
Acetone and methylene chloride were dried using an MBRAUN SPS-800 solvent
purification system and stored over 3 Å molecular sieves. THF and diethyl ether were
16

distilled from sodium metal. Triethylamine and CDCl3 were dried over 4 Å and 3 Å
molecular sieves, respectively. All solvents and dry bases were stored and utilized under
argon atmosphere. Thionyl chloride was distilled prior to use. All other chemicals were
purchased and used without further purification. For filtration, 70-230 mesh neutral
aluminium oxide (activity stage I) was employed. NMR spectra were recorded with a JEOL
400 MHz multinuclear spectrometer. The NMR spectra of compounds 4b-4h were
19
measured in dry CDCl3 under argon. F chemical shifts are given against CFCl3. Positive
and negative mode ESI mass spectra were measured with an Agilent 6210 ESI-TOF
(Agilent Technology) mass spectrometer. Elemental analysis of carbon, hydrogen, nitrogen
and sulfur were performed using a Heraeus elemental analyser.
4.2.2. X-Ray crystallography
The intensities for the X–ray determinations were collected on a Bruker D8 Venture
instrument with Mo Kα radiation. Structure solution and refinement were performed with
the SHELXS 2014 and SHELXL 2014 programs [30,31]. Hydrogen atoms were placed at
calculated positions and treated with the ‘riding model’ option of SHELXL. The
representation of molecular structures was done using the program DIAMOND (vers. 4.5.1)
[32].
4.2.3. General procedure for the preparation of 1b-1h
NH4SCN (165.0 mmol, 1.1 equiv) was dissolved in 200 ml of dry acetone under argon. A
solution of the respective benzoyl chloride (150 mmol, 1.0 equiv) in dry acetone (90 ml)
was added dropwise under vigorous stirring, resulting in the formation of a colorless
precipitate (NH4Cl). The suspension was stirred at room temperature for 1 h. Subsequently,
a solution of diethylamine (150 mmol, 1.0 equiv) in acetone (90 ml) was added dropwise.
17

After stirring for two hours, the reaction mixture was concentrated to approximately 200 ml
by rotary evaporation. The products 1b-1f were precipitated by slow addition of 600 ml of
water followed by prolonged stirring. The pale yellow precipitates were isolated by
filtration, thoroughly washed with water and air-dried. 1g was separated as an orange-
yellow oil after the addition of water. The oily material was extracted with 200 ml
methylene chloride. The extract was dried over MgSO4 and concentrated. The resulting
crude residue was dried under vacuum. Recrystallisation and drying under vacuum afforded
the isolation of the benzoylthioureas 1b-1g as crystalline solids.
N,N-Diethyl-N´-(p-chloro)benzoylthiourea (1b). Recrystallisation from ethanol yielded
yellowish needles, which were washed with cold EtOH and dried under vacuum. Yield:
81%. Elemental analysis: calc. for C12H15ClN2OS: C 53.2, H 5.6, N 10.35, S 11.8; found: C
1
53.0, H 5.55, N 10.4, S 11.8. m.p. 151-152° C. H-NMR (CDCl3, 400 MHz): 7.78 (m, 2H,
3
3
Ph), 7.43 (m, 2H, Ph), 4.00 (q, JHH = 6.9 Hz, 2H, CH2CH3), 3.59 (q, JHH = 7.1 Hz, 2H,
3
3
CH2CH3), 1.35 (t, JHH = 7.1 Hz, 3H, CH2CH3), 1.28 (t, JHH = 6.9 Hz, 3H, CH2CH3);
13C{1H}-NMR (CDCl3, 101 MHz): δ 179.21 (s, C=S), 163.03 (s, C=O), 139.41 (s, C-Cl),
131.07 (s, quart. C, Ph), 129.42 (s, Ph), 129.22 (s, Ph), 48.07 (s, CH2CH3), 47.75 (s,
CH2CH3), 13.36 (s, CH2CH3), 11.52 (s, CH2CH3). HRMS-ESI (m/z): [M-H]- calc. for
C12H14ClN2OS, 269.0515, found: 269.0542.
N,N-Diethyl-N´-(p-bromo)benzoylthiourea (1c). Recrystallisation from ethanol yielded
colorless needles of 1c, which were washed with cold EtOH and dried under vacuum.
Yield: 75%. Elemental analysis: calc. for C12H15BrN2OS: C 45.72, H 4.80, N 8.89, S 10.17;
1
found: C 45.67, H 4.79, N 8.98, S 9.95. m.p. 158-159° C. H-NMR (CDCl3, 400 MHz):
3
δ 8.54 (s, 1H, N-H), 7.69 (m, 2 H, Ph), 7.59 (m, 2H, Ph), 4.00 (q, JHH = 6.9 Hz, 2H,
3
CH2CH3), 3.57 (q, 3JHH = 6.9 Hz, 2H, CH2CH3), 1.34 (t, JHH = 6.9 Hz, 3H, CH2CH3), 1.27
3
(t, JHH = 6.9 Hz, 3H, CH2CH3); 13C{1H}-NMR (CDCl3, 101 MHz): δ 179.19 (s, C=S),
18

163.16 (s, C=O), 132.20 (s, Ph), 131.52 (s, quart. C, Ph), 129.54 (s, Ph), 127.95 (s, C-Br),
48.07 (s, CH2CH3), 47.77 (s, CH2CH3), 13.32 (s, CH2CH3), 11.51 (s, CH2CH3). HRMS-ESI
(m/z): [M + Na+] calc. for C12H15BrNaN2OSNa: 336.9986, found: 336.9985.
N,N-Diethyl-N´-(p-fluoro)benzoylthiourea (1d). The crude product was dissolved in
boiling EtOH/acetone 1:1 and stored in the refrigerator. The formed colorless crystals of 1d
were washed with the same cold solvent mixture and dried under vacuum. Yield: 80%.
Elemental analysis: calc. for C12H15FN2OS: C 56.67, H 5.95, N 11.02, S 12.61; found: C
56.52, H 5.88, N 11.12, S 12.31. m.p. 136-137° C. 1H-NMR (CDCl3, 400 MHz): δ 8.61 (br.
s, 1H, NH), 7.73 (m, 2H, Ph), 7.00 (m, 2H, Ph), 3.89 (q, 3JHH = 7.5 Hz, 2H, CH2CH3), 3.45
3
3
3
(q, JHH = 7.5 Hz, 2H, CH2CH3), 1.23 (t, JHH = 7.5 Hz, 3H, CH2CH3), 1.16 (t, JHH = 7.5
19
13
Hz, 3H, CH2CH3); F-NMR (CDCl3, 377 MHz): δ –105.49 (m); C{1H}-NMR (CDCl3,
101 MHz): δ 179.41 (s, C=S), 165.77 (d, 1JCF = 164.2 Hz, C-F), 163.01 (s, C=O), 130.63 (d,
2
3JCF = 9.4 Hz, Ph), 128.83 (s, quat. C, Ph), 115.98 (d, JCF = 21.8 Hz, Ph), 48.00 (s,
CH2CH3), 47.70 (s, CH2CH3), 13.32 (s, CH2CH3), 11.52 (s, CH2CH3). HRMS-ESI (m/z):
[M + Na+] calc. for C12H15FN2OSNa: 277.0787, found: 277.0812.
N,N-Diethyl-N´-(p-trifluoromethyl)benzoylthiourea (1e). The crude product was
recrystallized from hexane. Washing with hexane and drying under vacuum yielded 1e as
colorless crystals. After the addition of water to the reaction mixture, the product may
separate as a slowly crystallizing oil. In this case, it is possible to extract it with methylene
chloride from the aqueous phase as described for derivative 1g. The extract is concentrated
and the product crystallized by addition of hexane. Yield: 80%. Elemental analysis: calc.
for C13H15F3N2OS: C 51.31, H 4.97, N 9.21, S 10.53; found: C 51.19, H 4.96, N 9.29, S
1
3
9.72. m.p. 92-93° C. H-NMR (CDCl3, ppm): δ 8.84 (s, 1H, NH), 7.94 (d, JHH = 8.2 Hz,
2H, Ph), 7.70 (d, 3JHH = 8.2 Hz, 2H, Ph), 4.00 (q, 3JHH = 6.7 Hz, 2H, CH2CH3), 3.57 (q, 3JHH
19
= 6.7 Hz, 2H, CH2CH3), 1.42-1.18 (m, 6H, CH2CH3); F-NMR (CDCl3, 377 MHz): δ –
19

13
63.15 (s); C{1H}-NMR (CDCl3, 101 MHz): δ 179.02 (s, C=S), 162.88 (s, C=O), 135.91
(s, quat. C, Ph), 134.28 (q, 2JCF = 32.9 Hz, C-CF3), 128.54 (s, Ph), 125.76 (q, 3JCF = 3.3 Hz,
1
Ph), 123.45 (q, JCF = 273.0 Hz, CF3), 48.13 (s, CH2CH3), 47.79 (s, CH2CH3), 13.37 (s,
CH2CH3), 11.50 (s, CH2CH3). HRMS-ESI (m/z): [M + Na+] calc. for C13H15F3N2OSNa:
327.0755, found: 327.0794.
N,N-Diethyl-N´-(m-difluoro)benzoylthiourea (1f). The crude product was dissolved in
boiling hexane/ethanol 2:1. The solid that formed was washed with the same mixture and
then with hexane, until the orange oil had been completely removed. After drying under
vacuum, 1f was obtained as colorless crystalline material. Yield: 65%. Elemental analysis:
calc. for C12H14F2N2OS: C 52.93, H 5.18, N 10.29, S 11.77; found: C 52.73, H 5.11, N
1
10.42, S 11.72. m.p. 116-117° C. H-NMR (CDCl3, 400 MHz): δ 8.67 (s, 1H, NH), 7.36
(m, 2H, Ph), 7.00 (tt, 3JHF = 8.4 Hz, 4JHH = 2.2 Hz, 1H, Ph), 4.00 (br. m, 2H, CH2CH3), 3.56
(br. m, 2H, CH2CH3), 1.48-1.33 (m, 6H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –107.17
(br. m); 13C{1H}-NMR (CDCl3, 101 MHz): δ 178.71 (s, C=S), 162.91 (dd, 1JCF = 251.4 Hz,
3
3JCF = 12.0 Hz, C-F), 135.93 (s, C=O), 111.27 (m, Ph), 109.23 (t, JCF = 25.6 Hz, quat. C,
3
Ph), 108.30 (t, JCF = 25.3 Hz, Ph), 47.99 (s, CH2CH3), 47.67 (s, CH2CH3), 13.25 (s,
CH2CH3), 11.34 (s, CH2CH3); HRMS-ESI (m/z): [M + Na+] calc. for C12H14F2N2OSNa:
295.0693, found: 295.0721.
N,N-Diethyl-N´-(m-fluoro)benzoylthiourea (1g). The crude product was dissolved in a
minimum quantity of methylene chloride and treated with a 20-times volume of hexane.
After standing for 2 h, colorless crystals of 1g were filtered off, washed with hexane and
dried under vacuum. Yield: 80%. Elemental analysis: calc. for C12H15FN2OS: C 50.90, H
5.16, N 9.89, S 11.32; found: C 50.83, H 5.03, N 9.82, S 11.44. m.p. 120-121° C. 1H-NMR
(CDCl3, 400 MHz): δ 7.56 (m, 1H, Ph), 7.49 (m, 1H, Ph), 7.37 (m, 1H, Ph), 7.21 (m, 1H,
3
3
Ph), 4.00 (q, 2H, JHH = 6.6 Hz, CH2CH3), 3.61 (q, JHH = 7.0 Hz, 2H, CH2CH3), 1.35 (t,
20

3
19
3JHH = 6.6 Hz, 3H, CH2CH3), 1.29 (t, JHH = 7.0 Hz, 3H, CH2CH3); F-NMR (CDCl3, 377
MHz): δ –111.00 (m); 13C{1H}-NMR (CDCl3, 101 MHz): δ 179.05 (s, C=S), 162.85 (d,
4
3
1JCF = 248.8 Hz, C-F), 162.72 (d, JCF = 2.7 Hz, C=O), 134.94 (d, JCF = 6.9 Hz, quart. C,
3
4
2
Ph), 130.68 (d, JCF = 7.9 Hz, Ph), 123.39 (d, JCF = 3.1 Hz, Ph), 120.08 (d, JCF = 21.3 Hz,
2
Ph), 115.33 (d, JCF = 23.2 Hz, Ph), 48.14 (s, CH2CH3), 48.12 (s, CH2CH3), 13.38 (s,
CH2CH3), 11.55 (s, CH2CH3). HRMS-ESI (m/z): [M – H]– calc. for C12H14FN2OS:
253,0811, found: 253.0801.
N,N-Diethyl-N´-(m-trifluoromethyl)benzoylthiourea (1h). NH4SCN (569 mg, 7.5 mmol,
1.1 equiv) was dissolved in 20 ml of dry acetone under argon atmosphere. A solution of m-
(trifluoromethyl)benzoyl chloride (1 ml, 6.8 mmol, 1.0 equiv) in dry acetone (15 ml) was
slowly added dropwise to the first solution under vigorous stirring. This resulted in the
formation of a colorless precipitate (NH4Cl). The suspension was stirred at room
temperature for 1 h and, subsequently, a solution of diethylamine (500 mg, 1.0 equiv) in
acetone (25 ml) was added dropwise. After stirring for two hours, the reaction mixture was
diluted with 150 ml water and extracted with 60 ml methylene chloride. The extract was
dried over MgSO4 and concentrated. The oily residue was dissolved in a mixture of 10 ml
ethanol and 3 ml methylene chloride, followed by the addition of 90 ml hexane. The
solution was allowed to stand at –20° C for 48 h. The resulting crystals were filtered off,
washed with hexane and dissolved in 30 ml of boiling hexane by addition of a small
quantity of ethanol. After cooling, some crystals obtained in the first crystallization were
added to the solution, which was allowed to stand at –20°C for 24 h. The resulting crystals
of 1h were filtered off, washed with hexane and dried under vacuum. Yield: 68%.
Elemental analysis: calc. for C13H15F3N2OS: C 51.31, H 4.97, N 9.21, S 10.53; found: C
51.26, H 5.05, N 9.08, S 10.36. m.p. 92-93° C. 1H-NMR (CDCl3, 400 MHz): δ 9.01 (s, 1H,
3
3
N-H), 8.00 (s, 1H, Ph), 7.95 (d, JHH = 7.9 Hz, 1H, Ph), 7.72 (d, JHH = 7.8 Hz, 1H, Ph),
21

3
3
7.51 (t, JHH = 7.8 Hz, 1H, Ph), 3.93 (q, JHH = 7.0 Hz, 2H, CH2CH3), 3.50 (q,
3
3
3JHH = 7.1 Hz, 2H, CH2CH3), 1.27 (t, JHH = 7.1 Hz, 3H, CH2CH3), 1.21 (t, JHH = 7.1 Hz,
3H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –62.85 (s); 13C{1H}-NMR (CDCl3, 101
MHz): δ 179.06 (s, C=S), 162.75 (s, C=O), 133.28 (s, quart. C, Ph), 132.27 (q,
2JCF = 32.5 Hz, C-CF3), 131.12 (s, Ph), 129.40 (s, Ph), 129.29 (q, 3JCF = 3.7 Hz, Ph), 125.13
3
1
(q, JCF = 3.8 Hz, Ph), 123.46 (q, JCF = 272.8 Hz, CF3), 47.96 (s, CH2CH3), 47.69 (s,
CH2CH3), 13.32 (s, CH2CH3), 11.38 (s,CH2CH3). HRMS-ESI (m/z): calc. for [M – H]–
C13H14F3N2OS: 303.0779, found: 303.0785.
4.2.4. General procedure for the preparation of 2b-2h
The nickel complexes were synthetized according to a literature procedure [21].
cis-Bis{N,N-diethyl-N´-(p-chloro)benzoylthioureato-2O,S}nickel(II) (2b). Red solid.
Yield: 98%. Elemental Analysis: calc. for C24H28Cl2N4NiO2S2: C 48.11, H 4.88, N 9.35, S
1
10.70; found: C 50.46, H 5.13, N 9.90, S 11.14. m.p. 231-232° C. H-NMR (CDCl3, 400
MHz): δ 8.01 (d, 3JHH = 8.6 Hz, 4H, Ph), 7.34 (d, 3JHH = 8.6 Hz, 4H, Ph), 3.82-3.70 (m, 8H,
CH2CH3), 1.31-1.20 (m, 12H, CH2CH3); 13C{1H}-NMR (CDCl3, 101 MHz): δ 172.67 (s, C-
S), 171.46 (s, C-O), 137.76 (s, C-Cl), 135.34 (s, quart. C, Ph), 130.58 (s, Ph), 128.30 (s,
Ph), 46.34 (s, CH2CH3), 45.73 (s, CH2CH3), 13.25 (s, CH2CH3), 12.63 (s, CH2CH3).
cis-Bis{N,N-diethyl-N´-(p-bromo)benzoylthioureato-2O,S}nickel(II) (2c). Red solid.
Yield: 98%. Elemental analysis: calc. for C24H28Br2N4NiO2S2: C 41.89, H 4.25, N 8.14, S
1
9.32; found: C 41.94, H 4.14, N 8.11, S 9.24. m.p. 237-238° C. H-NMR (CDCl3, 400
3
3
MHz): δ 7.95 (d, JHH = 8.2 Hz, 4H, Ph), 7.51 (d, JHH = 8.2 Hz, 4H, Ph), 3.77 (m, 8H,
13
CH2CH3), 1.26 (m, 12H, CH2CH3); C{1H}-NMR (CDCl3, 101 MHz): δ 172.70 (s, C-S),
171.57 (s, C-O), 135.80 (s, quart. C, Ph), 131.28 (s, Ph), 130.79 (s, Ph), 126.43 (s, C-Br),
22

46.36 (s, CH2CH3), 45.75 (s, CH2CH3), 13.25 (s, CH2CH3), 12.62 (s, CH2CH3). HRMS-ESI
(m/z): [M + H+] calc. for C24H29Br2N4NiO2S2: 684.9452, found: 684.9427.
cis-Bis{N,N-diethyl-N´-(p-fluoro)benzoylthioureato-2O,S}nickel(II) (2d). Red solid.
Yield: 95%. Elemental Analysis: calc. for C24H28F2N4NiO2S2: C 50.99, H 4.99, N 9.91, S
1
11.34; found: C 50.73, H 4.93, N 9.98, S 11.21. m.p. 186-187° C. H NMR (CDCl3, 400
MHz) δ 8.09 (m, 4H, Ph), 7.04 (m, 4H, Ph), 3.83-3.63 (m, 8H, CH2CH3), 1.33-1.11 (m,
12H, CH2CH3); 19F-NMR (CDCl3, 377 MHz) δ –108.74 (m); 13C NMR (CDCl3, 101 MHz)
1
δ 172.53 (s, C=S), 171.40 (s, C=O), 165.07 (d, JCF = 251.7 Hz, C-F), 133.00 (s, quat. C,
Ph), 131.47 (d, 3JCF = 8.9 Hz, Ph), 114.92 (d, 2JCF = 21.0 Hz, Ph), 46.26 (s, CH2CH3), 45.64
(s, CH2CH3), 13.21 (s, CH2CH3), 12.61 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for
C24H29F2N4NiO2S2: 565.1053; found: 565.1018.
cis-Bis{N,N-diethyl-N´-(p-trifluoromethyl)benzoylthioureato-2O,S}nickel(II) (2e). Red
solid. Yield: 91%. Elemental Analysis: calc. for C26H28F6N4NiO2S2: C 46.94, H 4.24, N,
8.42, S 9.64; found: C 46.77, H 4.26, N 8.35, S 10.28. m.p. 218-219° C. 1H-NMR (CDCl3,
3
3
400 MHz): δ 8.20 (d, JHH = 8.0 Hz, 4H, Ph), 7.65 (d, JHH = 8.0 Hz, 4H, Ph), 3.88-3.77
19
(8H, m, CH2CH3), 1.36-1.26 (m, 12H, CH2CH3); F-NMR (CDCl3, 377 MHz): δ –62.78
13
(s); C NMR (CDCl3, 101 MHz): δ 172.98 (s, C=S), 171.02 (s, C=O), 140.12 (s, quat. C,
Ph), 132.87 (q, 2JCF = 32.4 Hz, C-CF3), 129.43 (s, Ph), 125.30 (q, 3JCF = 4.2 Hz, Ph), 124.04
(q, 1JCF = 272.7 Hz, CF3), 46.48 (s, CH2CH3), 45.90 (s, CH2CH3), 13.23 (s, CH2CH3), 12.54
(s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C26H29F6N4NiO2S2: 665.0990, found:
665.0969; [M + Na+] calc. for C26H28F6N4NiO2S2Na: 687.0809, found: 687.0787.
cis-Bis{N,N-diethyl-N´-(m-difluoro)benzoylthioureato-2O,S}nickel(II) (2f). Red solid.
Yield: 93%. Elemental Analysis: calc. for C24H26F4N4NiO2S2: C 47.94, H 4.36, N 9.32, S
1
10.66; found: C 47.69, H 4.32, N 9.37, S 11.45. m.p. 178-179° C. H-NMR (CDCl3, 400
3
4
MHz): δ 7.58 (m, 4H, Ph), 6.91 (tt, JHF = 8.5 Hz, JHH 2.4 Hz, 2H, Ph), 3.86-3.64 (m, 8H,
23

CH2CH3), 1.25-1.14 (m, 12H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –110.20 (m);
4
13C{1H}-NMR (CDCl3, 101 MHz): δ 173.02 (s, C=S), 169.95 (t, JCF = 3.5 Hz, C=O),
1
3
3
163.92 (dd, JCF = 248.0 Hz, JCF = 12.1 Hz, C-F), 140.35 (t, JCF = 8.90 Hz, quat. C, Ph),
2
111.91 (m, Ph), 106.73 (t, JCF = 25.7 Hz, Ph), 46.50 (s, CH2CH3), 45.93 (s, CH2CH3),
13.20 (s, CH2CH3), 12.48 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for
C24H27F4N4NiO2S2: 601.0865, found: 601.0781; [M + Na+] calc. for C24H26F4N4NiO2S2Na:
623.0684, found: 623.0665.
cis-Bis{N,N-diethyl-N´-(m-fluoro)benzoylthioureato-2O,S}nickel(II) (2g). Red solid.
Yield: 91%. Elemental Analysis: calc. for C24H28F2N4NiO2S2: C 50.90, H 5.16, N 9.89, S
1
11.32; found: C 50.83, H 5.03, N 9.82, S 11.44. m.p. 193-194° C. H-NMR (CDCl3, 400
MHz): δ 7.90 (d, 3JHF = 7.6 Hz, 2H, Ph), 7.78 (d, 3JHH = 8.7 Hz, 2H, Ph), 7.35 (m, 4H, Ph),
7.16 (m, 4H, Ph), 3.78 (m, 8H, CH2CH3), 1.28 (m, 12H, CH2CH3); 19F-NMR (CDCl3, 377
MHz): δ –113.81 (m); 13C{1H}-NMR (CDCl3, 101 MHz): δ 172.85 (s, C=S), 171.22 (d,
3
4JCF = 3.1 Hz, C-O), 162.65 (d, 1JCF = 243.7 Hz, C-F), 139.25 (d, JCF = 7.3 Hz, quart. C,
3
4
2
Ph), 129.48 (d, JCF = 7.8 Hz, Ph), 124.88 (d, JCF = 2.8 Hz, Ph), 118.44 (d, JCF = 21.5 Hz,
2
Ph), 115.98 (d, JCF = 22.9 Hz, Ph), 46.38 (s, CH2CH3), 45.79 (s, CH2CH3), 13.26 (s,
CH2CH3), 12.60 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C24H29F2N4NiO2S2:
565.1053, found: 565.1058; [M + Na+] calc. for C24H28F2N4NiO2S2Na: 587.0873, found:
587.0859.
cis-Bis{N,N-diethyl-N´-(m-trifluoromethyl)benzoylthioureato-2O,S}nickel(II)
(2h).
Red solid. Yield: 92%. Elemental Analysis: calc. for C26H28F6N4NiO2S2: C 46.94, H 4.24,
N 8.42, S 9.64; found: C 46.92, H 4.17, N 8.34, S 9.62. m.p. 207-208° C. 1H-NMR (CDCl3,
400 MHz): δ 8.42 (m, 2H, Ph), 8.31 (m, 2H, Ph), 7.72 (m, 2H, Ph), 7.52 (m, 2H, Ph), 3.91-
3.67 (m, 4H, CH2CH3), 1.39-1.16 (m, 6H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –
13
62.89 (s); C{1H}-NMR (CDCl3, 101 MHz): δ 184.74 (s, C=S), 141.86 (s, C-Cl), 133.95
24

(s, quart. C, Ph), 131.40 (q, 4JCF = 1.1 Hz, Ph), 130.39 (q, 2JCF = 33.2 Hz, C-CF3), 128.42 (s,
Ph), 128.30 (q, 3JCF = 3.6 Hz, Ph), 125.02 (q, 3JCF = 3.9 Hz, Ph), 122.52 (q, 1JCF = 272.6 Hz,
CF3), 45.39 (s, CH2CH3), 44.79 (s, CH2CH3), 12.07 (s, CH2CH3), 10.58 (s, CH2CH3).
HRMS-ESI (m/z): [M + H+] calc. for C26H29F6N4NiO2S2: 665.0990, found: 665.1009; [M +
Na+] calc. for C26H28F6N4NiO2S2Na: 687.0809, found: 687.0826.
4.2.5. General procedure for the preparation of 3b-3h
The respective nickel complex (3b-3h) (3.2 mmol, 1 equiv) was dissolved under Ar and
magnetical stirring in dry methylene chloride (20 ml). To this mixture was added dropwise
a solution of thionylchloride (6.4 mmol, 2 equiv) in dry methylene chloride (6 ml), which
resulted in the formation of a green precipitate (NiCl2). The suspension was stirred at room
temperature until the red/violet color of the reactant disappeared (1 - 3 hours). The
precipitate was filtered off and the filtrate was concentrated by rotary evaporation.
N-(Diethylaminothiocarbonyl)-p-chlorobenzimidoyl chloride (3b). The crude product
was dissolved in ethyl acetate and filtrated though aluminum oxide. Elution was carried out
first with ethyl acetate and then with methylene chloride. After concentration of the filtrate
and drying under vacuum, 3b was obtained as yellow solid. Yield: 80%. Elemental
Analysis: calc. for C12H14Cl2N2S: C 49.84, H 4.88, N 9.69, S 11.09; found: C 49.85, H
4.98, N 9.72, S 10.21. m.p. 97-99°C. 1H-NMR (CDCl3, 400 MHz): δ 8.00 (d, 3JHH = 8.8 Hz,
3
3
2H, Ph), 7.43 (d, JHH = 8.8 Hz, 2H, Ph), 3.97 (q, JHH = 7.1 Hz, 2H, CH2CH3), 3.40 (q,
3
3
3JHH = 7.2 Hz, 2H, CH2CH3), 1.34 (t, JHH = 7.1 Hz, 3H, CH2CH3), 1.17 (t, JHH = 7.2 Hz,
13
3H, CH2CH3); C{1H}-NMR (CDCl3, 101 MHz): δ = 185.05 (s, C=S), 142.25 (s, C-Cl),
138.53 (s, CPh-Cl), 131.57 (s, quart. C, Ph), 129.65 (s, Ph), 128.04 (s, Ph), 45.47 (s,
CH2CH3), 44.79 (s, CH2CH3), 12.14 (s, CH2CH3), 11.70 (s, CH2CH3). HRMS-ESI (m/z):
[M + H+] calc. for C12H15Cl2N2S: 289.0333, found: 289.0343; [M + Na+] calc. for
25

C12H14Cl2N2SNa: 311.0152, found: 311.0183; [M + K+] calc. for C12H14Cl2N2SK:
326.9892, found: 326.9905.
N-(Diethylaminothiocarbonyl)-p-bromobenzimidoyl chloride (3c). The crude product
was dissolved in ethyl acetate and filtrated though aluminum oxide. Elution was carried out
first with ethyl acetate and then with methylene chloride. After concentration of the filtrate
and drying under vacuum, 3c was obtained as yellow solid. Yield: 73%. Elemental
Analysis: calc. for C12H14BrClN2S: C 43.20, H 4.23, N 8.40, S 9.61; found: C 43.11, H
1
4.33, N 8.24, S 9.33. m.p. 105-106° C. H-NMR (CDCl3, 400 MHz): δ 7.61 (m, 2H, Ph),
3
3
7.27 (m, 2H, Ph), 3.65 (q, JHH = 7.1 Hz, 2H, CH2CH3), 3.07 (q, JHH = 7.2 Hz, 2H,
3
3
CH2CH3), 1.02 (t, JHH = 7.1 Hz, 3H, CH2CH3), 0.85 (t, JHH = 7.2 Hz, 3H, CH2CH3);
13C{1H}-NMR (CDCl3, 101 MHz): δ 186.03 (s, C=S), 143.37 (s, C-Cl), 133.06 (s, quart. C,
Ph), 132.00 (s, Ph), 130.75 (s, Ph), 128.16 (s, C-Br), 46.45 (s, CH2CH3), 45.78 (s,
CH2CH3), 13.13 (s, CH2CH3), 11.69 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for
C12H15BrClN2S: 332.9828, found: 332.9838; [M + K+] calc. for C12H14BrClN2SK:
370.9387, found: 370.9399.
N-(Diethylaminothiocarbonyl)-p-fluorobenzimidoyl chloride (3d). Recrystallisation
from acetone yielded yellow crystals of 3d, which were dried under vacuum. Yield: 60%.
Elemental Analysis: calc. for C12H14ClFN2S: C 52.84, H 5.17, N 10.27, S 11.75; found: C
1
52.61, H 5.18, N 10.18, S 11.79. m.p. 104-105° C. H NMR (CDCl3, 400 MHz): δ 8.09 (m,
3
3
2H, Ph), 7.14 (m, 2H, Ph), 3.98 (q, JHH = 7.1 Hz, 2H, CH2CH3), 3.41 (q, JHH = 7.1 Hz,
3
3
2H, CH2CH3), 1.35 (t, JHH = 7.1 Hz, 3H, CH2CH3), 1.18 (t, JHH = 7.1 Hz, 3H, CH2CH3);
13
19F NMR (CDCl3, 377 MHz): δ –105.53 (m); C NMR (CDCl3, 101 MHz): δ 186.15 (s,
1
3
C=S), 165.71 (d, JCF = 255.5 Hz, C-F)), 143.03 (s, C-Cl), 131.80 (d, JCF = 9.5 Hz, Ph),
2
130.35 (m, C quat. Ph), 115.90 (d, JCF = 21.9 Hz, Ph), 46.45 (s, CH2CH3), 45.75 (s,
26

CH2CH3), 13.10 (s, CH2CH3), 11.68 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for
C12H15ClFN2S: 273.0629, found: 273.0626.
N-(Diethylaminothiocarbonyl)-p-(trifluoromethyl)benzimidoyl chloride (3e). The crude
yellow product was dissolved in ethyl acetate and filtered through aluminum oxide. After
concentration of the solution and drying under vacuum, 3e was obtained as a yellow solid.
Yield: 68%. Elemental Analysis: calc. for C13H14ClF3N2S: C 48.38, H 4.37, N 8.68, S 9.93;
1
found: C 48.38, H 4.40, N 8.41, S 9.10. m.p. 53-54 ° C. H-NMR (CDCl3, 400 MHz): δ
8.19 (d, 3JHH = 8.6 Hz, 2H, Ph), 7.72 (d, 3JHH = 8.6 Hz, 2H, Ph), 3.99 (q, 3JHH = 7.1 Hz, 2H,
CH2CH3), 3.41 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 1.35 (t, 3JHH = 7.1 Hz, 3H, CH2CH3), 1.18
(t, 3JHH = 7.1 Hz, 3H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –63.13 (s); 13C{1H}-NMR
(CDCl3, 101 MHz): δ 185.75 (s, C=S), 143.00 (s, C-Cl), 137.28 (s, quat. C, Ph), 134.30 (q,
3
1
2JCF = 32.9 Hz, C-CF3), 129.69 (s, Ph), 125.69 (q, JCF = 3.8 Hz, Ph), 123.60 (q, JCF
=
272.7 Hz, CF3), 46.45 (s, CH2CH3), 45.84 (s, CH2CH3), 13.11 (s, CH2CH3), 11.65 (s,
CH2CH3).
N-(Diethylaminothiocarbonyl)-m-difluorobenzimidoyl chloride (3f). Recrystallisation
from acetone yielded yellow crystals of 3f, which were dried under vacuum. Yield: 70%.
Elemental Analysis: calc. for C12H13ClF2N2S: C 49.57, H 4.51, N 9.63, S 11.03; found: C
49.43, H 4.51, N 9.69, S 10.97. m.p. 88-90° C. 1H-NMR (CDCl3, 400 MHz): δ 7.62 (m, 2H,
3
3
Ph), 7.02 (m, 1H, Ph), 3.98 (q, JHH = 7.1 Hz, 2H, CH2CH3), 3.40 (q, JHH = 7.1 Hz, 2H,
3
CH2CH3), 1.35 (t, 3JHH = 7.1 Hz, 3H, CH2CH3), 1.19 (t, JHH = 7.1 Hz, 3H, CH2CH3); 19F-
13
NMR (CDCl3, 377 MHz): δ –107.93 (m); C{1H}-NMR (CDCl3, 101 MHz): δ 185.63 (s,
1
3
4
C=S), 162.91 (dd, JCF = 250.0 Hz, JCF = 12.3 Hz, C-F), 142.04 (t, JCF = 4.0 Hz, C-Cl),
137.41 (t, 3JCF = 9.7 Hz, quat. C, Ph), 112.54 (dd, 2JCF = 20.1 Hz, 4JCF = 8.2 Hz, Ph), 108.35
(t, 3JCF = 25.3 Hz, Ph), 46.54 (s, CH2CH3), 45.95 (s, CH2CH3), 13.24 (s, CH2CH3), 11.75 (s,
CH2CH3); HRMS-ESI (m/z): [M + H+] calc. for C12H14ClF2N2S: 291.0534, found:
27

291.0535; [M + Na+] calc. for C12H13ClF2N2SNa: 313.0354, found: 313.0330; [M + K+]
calc. for C12H13ClF2N2SK: 329.0093, found: 329.0083.
N-(Diethylaminothiocarbonyl)-m-fluorobenzimidoyl chloride (3g). The crude product
was dissolved in ethyl acetate and filtrated though aluminium oxide. After concentration of
the filtrate and drying under vacuum, 3g was obtained as a yellow solid. Yield: 80%.
Elemental Analysis: calc. for C12H14ClFN2S: C 52.84, H 5.17, N 10.27, S 11.75; found: C
52.78, H 5.22, N 10.12, S 11.65. m.p. 107-108° C. 1H-NMR (CDCl3, 400 MHz): δ 7.86 (m,
1H, Ph), 7.75 (m, 1H, Ph), 7.43 (td, 3JHH = 8.1 Hz, 4JHF = 5.7 Hz, 1H, Ph), 7.25 (m, 1H, Ph),
3
3
3.96 (q, JHH = 7.1 Hz, 2H, CH2CH3), 3.40 (q, JHH = 7.2 Hz, 2H, CH2CH3), 1.34 (t,
3
19
3JHH = 7.1 Hz, 3H, CH2CH3), 1.17 (t, JHH = 7.2 Hz, 3H, CH2CH3); F-NMR (CDCl3, 377
MHz): δ –111.87 (m); 13C{1H}-NMR (CDCl3, 101 MHz): δ 184.83 (s, C=S), 161.54 (d,
4
3
1JCF = 247.6 Hz, C-F), 141.88 (d, JCF = 3.4 Hz, C-Cl), 135.16 (d, JCF = 7.8 Hz, quart. C,
3
4
2
Ph), 129.22 (d, JCF = 8.0 Hz, Ph), 124.08 (d, JCF = 2.9 Hz, Ph), 118.89 (d, JCF = 21.3 Hz,
2
Ph). 115.12 (d, JCF = 24.4 Hz, Ph), 45.35 (s, CH2CH3), 44.71 (s, CH2CH3), 12.04 (s,
CH2CH3), 10.59 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C12H14ClFN2S:
273.0629, found: 273.0650; [M + Na+] calc. for C12H14ClFN2SNa: 295.0448, found:
295.0471; [M + K+] calc. for C12H14ClFN2SK: 311.0187, found: 311.0212.
N-(Diethylaminothiocarbonyl)-m-(trifluoromethyl)benzimidoyl chloride (3h). The
crude product was dissolved in methylene chloride and filtrated though aluminium oxide.
After concentration of the filtrate and drying under vacuum, 3h was obtained as a yellow
solid. Yield: 80%. Elemental Analysis: calc. for C13H14ClF3N2S: C 48.38, H 4.37, N 8.68,
1
S 9.93; found: C 48.33, H 4.40, N 8.51, S 9.83. m.p. 58-59° C. H-NMR (CDCl3, 400
MHz): δ 8.34 (m, 1H, Ph), 8.27 (m, 1H, Ph), 7.82 (m, 1H, Ph), 7.62 (m, 1H, Ph), 3.99 (q,
3
3
3JHH = 7.1 Hz, 2H, CH2CH3), 3.41 (q, JHH = 7.2 Hz, 2H, CH2CH3), 1.37 (t, JHH = 7.1 Hz,
3H, CH2CH3), 1.20 (t, 3JHH = 7.2 Hz, 3H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –62.83
28

(s); 13C{1H}-NMR (CDCl3, 101 MHz): δ 184.79 (s, C=S), 141.91 (s, C-Cl), 134.00 (s,
4
2
quart. C, Ph), 131.45 (q, JCF = 1.1 Hz, Ph), 130.44 (q, JCF = 33.2 Hz, C-CF3), 128.47 (s,
Ph), 128.35 (q, 3JCF = 3.6 Hz, Ph), 125.07 (q, 3JCF = 3.9 Hz, Ph), 122.57 (q, 1JCF = 272.6 Hz,
CF3), 45.44 (s, CH2CH3), 44.84 (s, CH2CH3), 12.12 (s, CH2CH3), 10.63 (s, CH2CH3).
HRMS-ESI (m/z): [M + H+] calcd for C13H15ClF3N2S,: 323.0597, found: 323.0602.
4.2.6. General procedure for the preparation of 4b-4h
For the synthesis of the thiosemicarbazones, a modified literature procedure was used.3 4,4-
Dimethyl-3-thiosemicarbazide (2 mmol, 1 equiv) was dissolved in dry THF (10ml) under
argon and treated with dry Et3N (6 mmol, 3 equiv). The respective benzimidoyl chloride
3b-3h (2 mmol, 1 equiv) was added. The mixture was stirred until the reaction had
completed (4b, 4c, 4g, 4h over night; 4d, 4e, 4f two hours). The colorless precipitate of
NEt3·HCl was filtered off under argon using a syringe filter and the solvent was removed
under reduced pressure. The residue was dissolved in dry diethyl ether (10 mL) and stored
at −20° C over night. The deposited solid was isolated by decantation, washed with dry
diethyl ether and dried under vacuum. The product can be further purified through
recrystallization from a mixture of anhydrous CH2Cl2/hexane. The solid substance is stable
in air for days at 25° C and can be stored for months at -20 ° C without significant
decomposition.
N-(Diethylcarbamothioyl)-2-(2-(diethylcarbamothioyl)hydrazono)-2-(4-chlorophenyl)-
acetamide (4b). Greenish solid. Yield: 60%. Elemental Analysis: calc. for C15H22ClN5S2:
C 48.44, H 5.96, N 18.83, S 17.24; found: C 48.44, H 5.80, N 18.78, S 15.85. m.p. 139-
141° C. 1H-NMR (CDCl3, 400 MHz): δ 9.41 (br. m, 2 H, N-H), 7.79 (d, 3JHH = 8.6 Hz, 2H,
Ph), 7.33 (d, 3JHH = 8.6 Hz, 2H, Ph), 3.83 (br. m, 2H, CH2CH3), 3.47 (br. m, 2H, CH2CH3),
3.19 (s, 6H, N-CH3), 1.17 (br. m, 3H, CH2CH3), 1.00 (br. m, 3H, CH2CH3); 13C{1H}-NMR
29