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

Accepted Manuscript Title: Thiosemicarbazones and thiadiazines derived from fluorinated benzoylthioureas: synthesis, crystal structure and anti-Trypanosoma cruzi activity Authors: Federico Salsi, Gisele Bulh˜o es Portapilla, Konstantin Schutjajew, Zumira Aparecida Carneiro, Adelheid Hagenbach, S´ergio de Albuquerque, Pedro Iva da Silva Maia, Ulrich Abram PII: DOI: Reference:

S0022-1139(18)30277-X https://doi.org/10.1016/j.jfluchem.2018.08.004 FLUOR 9208

To appear in:

FLUOR

Received date: Revised date: Accepted date:

9-7-2018 3-8-2018 9-8-2018

Please cite this article as: Salsi F, Bulh˜o 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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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

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Ulrich Abrama)*

a)

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Freie Universität Berlin, Institute of Chemistry and Biochemistry, Fabeckstr. 34-36, D-

14195 Berlin, Germany b)

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Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Av.

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do Café - Vila Monte Alegre, Ribeirão Preto - SP, 14040-903, Brazil. c)

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Departamento de Química, Universidade Federal do Triângulo Mineiro, 38025-440,

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Uberaba, MG, Brazil.

Dedicated to Prof. Erhard Kemnitz in recognition of the ACS Award on Creative Work in

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Fluorine Chemistry 2018

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Corresponding Autor: [email protected] (Ulrich Abram)

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Graphical Abstract Fluorinated thiosemicarbazones derived from N-(diethylaminothiocarbonyl)benzimidoyl chlorides show a considerable activity against Trypanosomacruzi, the parasite, which is

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responsible for Chaga’s disease.



Biologically

active

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Highlights

thiosemicarbazones

with

N-(diethylaminothiocarbonyl)-



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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

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

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of the reaction conditions.

Abstract. A series of thiosemicarbazones was obtained by condensation of halogenated Nchlorides

(3b-3h)

with

4,4-dimethyl-3-

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(diethylaminothiocarbonyl)benzimidoyl

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 nonsubstituted 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

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cruzi activity

1. Introduction

Thiosemicarbazones, which are obtained by reactions of 4,4-dialkyl-3-thiosemicarbazide

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with N-(dialkylaminothiocarbonyl)benzimidoyl chlorides (H2L, Scheme 1) have attracted

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attention for their versatile coordination chemistry. They can act as mono- or dianionic

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tridentate ligands and have been shown to stabilize a number of metal ions. Typical

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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.

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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

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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

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available chemotherapy based on a nitrofuran (Nifurtimox©) and a nitroimidazole (Benznidazole©) is unsatisfactory because of their limited efficacy and their toxic side

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effects [16]. Despite recent efforts in the development of new chemotherapeutic agents,

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they are still the only accessible drugs. Thus, more safer and efficacious drugs are urgently

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M

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required [17,18].

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Fig. 1. Gold(III) organometallic complexes with thiosemicarbazide ligands.

The complexation of thiosemicarbazones of the type H2L with the organometallic

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{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

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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

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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

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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].

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In this study, the phenyl group of the thiosemicarbazone 4a has been functionalized with

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halogenides or trifluoromethyl groups, and the anti-parasitic activity of the resulting

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compounds has been investigated. This gives new insights into the chemical properties of

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benzimidoyl chloride-derived thiosemicarbazones and into the influence of fluorination on

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the anti-parasitic properties of such compounds and their metal complexes.

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2. Results and Discussion

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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

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(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

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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)

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in order to obtain 1h at higher concentrations of the reaction mixture failed.

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Scheme 2 Synthesis of the halogenated thiosemicarbazones

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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.

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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

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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,

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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

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addition of a sterically hindered secondary amine and aqueous hydrochloric acid to a

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benzoyl thiocyanate solution [23]. We hypothesized, hence, that the lower pH, which is

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caused by the hydrolysis of the residual benzoyl chloride in acetone, might induce the

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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

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for the cyclisation reaction.

Scheme 3 One-pot synthesis of thiadiazine-2-thiones

The 1H 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. The 1H 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

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C

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spectrum. In the region between 160 and 200 ppm appear the signals related to the

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heterocyclic quaternary carbon atoms.

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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

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planar system. The root-mean-square deviation of the atom coordinates from a fitted plane

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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 S1C9 (1.760 Å) bonds are slightly shortened single bonds, while the C8-S2 bond of 1.660 Å

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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

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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

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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

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fluorinated substances.

Chelation of the molecules with nickel(II) ions lowers the energy barrier for the rotation

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around the (S)C—N bond. Thus, only one triplet and one quartet are observed in the

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aliphatic range of the 1H NMR spectra of the diethylamino groups of the complexes 2b-2h.

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The signals of the NH protons around 9.50 ppm disappear upon coordination, indicating the

upon chlorination in the 1H and

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deprotonation of the ligand. The splittings of the methyl and methylene signals reappear C spectra of compounds 3b-3h. The conversion of the

identified in the

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benzoylthioureas to nickel complexes and then to benzimidoyl chlorides can be easily C NMR spectra through the chemical shifts of the

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C=O signals. They

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are shifted down-field upon complexation from the range of 161-163 ppm to 170-171 ppm,

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while a strong up-field shift to 142-143 ppm is observed, when the C-Cl bonds are formed. The

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F 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

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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-dimethyl3-thiosemicarbazide leads to the formation of 4a [1]. The same reaction can be successfully

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conducted with halogenated starting materials, provided that the conditions are slightly modified. 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.

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In the 1H 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 13

C NMR spectrum is simplified by the

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F-13C coupling which gives well resolved

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the

multiplets. The quaternary carbon atoms can be identified by their relative intensities. The

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C=S peaks are found in the typical region around 180 ppm. The chemical shift of 19F NMR

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signals are not significantly influenced by the substitution with different electron-

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withdrawing moieties, i.e. carbonyl, chloride, thiosemicarbazone, at the phenyl ring (see

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given in the Supporting Information.

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Table 1). More information can be derived from reproductions of the spectra, which are

Table 1 19F NMR data for the fluorinated compounds 1-4. Chemical shifts and multiplicity are reported.

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e -63.2 (s) -62.8 (s) -63.1 (s) -63.0 (s)

f -107.2 (m) -110.2 (m) -107.9 (m) -107.8 (m)

g -111.0 (m) -113.8 (m) -111.9 (m) -111.5 (m)

h -62.8 (s) -63.0 (s) -62.8 (s) -62.8 (s)

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1 2 3 4

d -105.5 (m) -108.7 (m) -105.5 (m) -108.0 (m)

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Fig. 3. Molecular structure of the thiosemicarbazone 4e.

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As a representative compound, the thiosemicarbazone 4e was characterized by X-ray

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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

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switch from the “thiosemicarbazide” tautomeric form, which is observed for 4a [1], to a

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“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

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of the corresponding hydrogen atoms and the fact that they are involved in hydrogen bonds

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(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

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double bond character, which is responsible for the hindered rotation of the two ethyl groups.

Scheme 4. Tautomeric equilibrium

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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

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Benznidazole (BZ) (see Table 1). The ratio between the cytotoxic activity (CC 50) and trypanocidal activity (IC50) is also provided in order to determine the selectivity index for

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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 4a

42.9 ± 2.3

4b

13.6 ± 2.5

4c

16.1 ± 2.9

4e 4f

28.7 ± 5.2

4g 4h

5.4

> 200

> 14.7

> 200

> 12.4 1.6

> 200

> 10.8

> 200

> 7.0

9.0 ± 0.8

> 200

> 22.2

12.5 ± 3.0

165.4 ± 14.0

13.2

> 200

> 53.0

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3.8 ± 0.3

BZ

Mean ± SD of two independent experiments

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a

SI

232.5 ± 6.0

33.7 ± 0.4

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20.6 ± 3.8 18.5 ± 5.3

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4d

CC50 ± SD (µM)

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IC50 ± SD (µM)

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Compound

All tested compounds show a high trypanocidal activity at low concentrations, especially

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compounds 4b, 4g and 4h, which are statistically as active as BZ. Previous

studies

by

Maia

and

coworkers

reported

that

the

non-fluorinated

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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].

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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 (CC 50 = 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.

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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

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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

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the non-fluorinated thiosemicarbazone 4a.

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It is not unexpected, that the chemical and physical properties of potential drug molecules

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are drastically influenced by the introduction of fluorine atoms [27]. Especially, the

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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

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degradation pattern of drugs are frequently altered upon fluorination, which may lead to a reduced or increased toxicity.

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In order to determine whether the increased potency and reduced cytotoxicity of the

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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

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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

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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

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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

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chloride. The resulting (thiocarbonyl)benzimidoyl chlorides (3b-3h) react with 4,4dimethyl-3-thiosemicarbazide under formation of the thiosemicarbazones 4b-4h in good

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yields. Compound 4b was characterized by crystal structure analysis, revealing that

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fluorination shifts the tautomeric thiosemicarbazone/thiosemicarbazide equilibrium towards

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the thiosemicarbazone form. Comparison of the anti-parasitic activity of the non-substituted

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thiosemicarbazone 4a with the halide-substituted representatives 4b-4h shows that the introduction of electron-withdrawing substituents like halogens on the aromatic ring

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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 >

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200 µM). The selectivity index (SI = IC50/CC50) of this compound is four times higher than

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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

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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-

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thiadiazine-2-thiones (5). A crystal structure determination of 5c confirms the for-mation of a planar heterocyclic ring.

4. Experimental

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4.1. Biological tests 4.1.1. Mammalian cells, parasites and drugs

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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

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serum (Sigma-Aldrich), 50 U/ml penicillin, 0.05 mg/ml streptomycin. In vitro assays were

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performed against amastigotes forms of Tulahuen strain, which was genetically modified to

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express the β-galactosidase gene from E. coli (lacZ).28 Trypomastigotes were harvested

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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

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in DMSO (final concentration in the assays did not exceed 1.5 %).

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4.1.2. In vitro trypanocidal activity

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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

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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

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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

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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

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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

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Mosmann.29 Briefly, 2.5 mg/mL MTT in PBS was added (50 μL/well) and the plates were

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further incubated for 4 h at 37 °C. The resulting formazan crystals formed were dissolved

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with DMSO (50 μL/well) and the samples were measured in a spectrophotometer at 570 nm

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after 30 min.

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4.1.4. Statistical analysis

Data were expressed as mean ± standard deviation (S.D.) of at least two independent

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experiments performed on two different days. Statistical analysis was performed using one-

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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

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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

measured in dry CDCl3 under argon.

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400 MHz multinuclear spectrometer. The NMR spectra of compounds 4b-4h were

F chemical shifts are given against CFCl3. Positive

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and negative mode ESI mass spectra were measured with an Agilent 6210 ESI-TOF (Agilent Technology) mass spectrometer. Elemental analysis of carbon, hydrogen, nitrogen

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and sulfur were performed using a Heraeus elemental analyser.

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4.2.2. X-Ray crystallography

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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

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the SHELXS 2014 and SHELXL 2014 programs [30,31]. Hydrogen atoms were placed at

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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)

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[32].

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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 orangeyellow oil after the addition of water. The oily material was extracted with 200 ml

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methylene chloride. The extract was dried over MgSO4 and concentrated. The resulting crude residue was dried under vacuum. Recrystallisation and drying under vacuum afforded

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the isolation of the benzoylthioureas 1b-1g as crystalline solids.

N,N-Diethyl-N´-(p-chloro)benzoylthiourea (1b). Recrystallisation from ethanol yielded

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yellowish needles, which were washed with cold EtOH and dried under vacuum. Yield:

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81%. Elemental analysis: calc. for C12H15ClN2OS: C 53.2, H 5.6, N 10.35, S 11.8; found: C

A

53.0, H 5.55, N 10.4, S 11.8. m.p. 151-152° C. 1H-NMR (CDCl3, 400 MHz): 7.78 (m, 2H,

M

Ph), 7.43 (m, 2H, Ph), 4.00 (q, 3JHH = 6.9 Hz, 2H, CH2CH3), 3.59 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 1.35 (t, 3JHH = 7.1 Hz, 3H, CH2CH3), 1.28 (t, 3JHH = 6.9 Hz, 3H, CH2CH3); C{1H}-NMR (CDCl3, 101 MHz): δ 179.21 (s, C=S), 163.03 (s, C=O), 139.41 (s, C-Cl),

ED

13

131.07 (s, quart. C, Ph), 129.42 (s, Ph), 129.22 (s, Ph), 48.07 (s, CH2CH3), 47.75 (s,

PT

CH2CH3), 13.36 (s, CH2CH3), 11.52 (s, CH2CH3). HRMS-ESI (m/z): [M-H]- calc. for

CC E

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.

A

Yield: 75%. Elemental analysis: calc. for C12H15BrN2OS: C 45.72, H 4.80, N 8.89, S 10.17; found: C 45.67, H 4.79, N 8.98, S 9.95. m.p. 158-159° C. 1H-NMR (CDCl3, 400 MHz): δ 8.54 (s, 1H, N-H), 7.69 (m, 2 H, Ph), 7.59 (m, 2H, Ph), 4.00 (q, 3JHH = 6.9 Hz, 2H, CH2CH3), 3.57 (q, 3JHH = 6.9 Hz, 2H, CH2CH3), 1.34 (t, 3JHH = 6.9 Hz, 3H, CH2CH3), 1.27 (t, 3JHH = 6.9 Hz, 3H, CH2CH3);

C{1H}-NMR (CDCl3, 101 MHz): δ 179.19 (s, C=S),

13

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

IP T

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

SC R

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

U

(q, 3JHH = 7.5 Hz, 2H, CH2CH3), 1.23 (t, 3JHH = 7.5 Hz, 3H, CH2CH3), 1.16 (t, 3JHH = 7.5

N

Hz, 3H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –105.49 (m); 13C{1H}-NMR (CDCl3,

A

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, JCF = 9.4 Hz, Ph), 128.83 (s, quat. C, Ph), 115.98 (d, 2JCF = 21.8 Hz, Ph), 48.00 (s,

M

3

CH2CH3), 47.70 (s, CH2CH3), 13.32 (s, CH2CH3), 11.52 (s, CH2CH3). HRMS-ESI (m/z):

ED

[M + Na+] calc. for C12H15FN2OSNa: 277.0787, found: 277.0812. N,N-Diethyl-N´-(p-trifluoromethyl)benzoylthiourea (1e). The crude product was

PT

recrystallized from hexane. Washing with hexane and drying under vacuum yielded 1e as

CC E

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

A

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 9.72. m.p. 92-93° C. 1H-NMR (CDCl3, ppm): δ 8.84 (s, 1H, NH), 7.94 (d, 3JHH = 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 = 6.7 Hz, 2H, CH2CH3), 1.42-1.18 (m, 6H, CH2CH3);

19

F-NMR (CDCl3, 377 MHz): δ –

19

63.15 (s);

13

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, Ph), 123.45 (q, 1JCF = 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.

IP T

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

SC R

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:

U

calc. for C12H14F2N2OS: C 52.93, H 5.18, N 10.29, S 11.77; found: C 52.73, H 5.11, N

N

10.42, S 11.72. m.p. 116-117° C. 1H-NMR (CDCl3, 400 MHz): δ 8.67 (s, 1H, NH), 7.36

A

(m, 2H, Ph), 7.00 (tt, 3JHF = 8.4 Hz, 4JHH = 2.2 Hz, 1H, Ph), 4.00 (br. m, 2H, CH2CH3), 3.56

M

(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, JCF = 12.0 Hz, C-F), 135.93 (s, C=O), 111.27 (m, Ph), 109.23 (t, 3JCF = 25.6 Hz, quat. C,

ED

3

Ph), 108.30 (t, 3JCF = 25.3 Hz, Ph), 47.99 (s, CH2CH3), 47.67 (s, CH2CH3), 13.25 (s,

PT

CH2CH3), 11.34 (s, CH2CH3); HRMS-ESI (m/z): [M + Na+] calc. for C12H14F2N2OSNa:

CC E

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.

A

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, Ph), 4.00 (q, 2H, 3JHH = 6.6 Hz, CH2CH3), 3.61 (q, 3JHH = 7.0 Hz, 2H, CH2CH3), 1.35 (t,

20

3

JHH = 6.6 Hz, 3H, CH2CH3), 1.29 (t, 3JHH = 7.0 Hz, 3H, CH2CH3); 19F-NMR (CDCl3, 377

MHz): δ –111.00 (m);

13

C{1H}-NMR (CDCl3, 101 MHz): δ 179.05 (s, C=S), 162.85 (d,

1

JCF = 248.8 Hz, C-F), 162.72 (d, 4JCF = 2.7 Hz, C=O), 134.94 (d, 3JCF = 6.9 Hz, quart. C,

Ph), 130.68 (d, 3JCF = 7.9 Hz, Ph), 123.39 (d, 4JCF = 3.1 Hz, Ph), 120.08 (d, 2JCF = 21.3 Hz, Ph), 115.33 (d, 2JCF = 23.2 Hz, Ph), 48.14 (s, CH2CH3), 48.12 (s, CH2CH3), 13.38 (s,

IP T

CH2CH3), 11.55 (s, CH2CH3). HRMS-ESI (m/z): [M – H]– calc. for C12H14FN2OS: 253,0811, found: 253.0801.

SC R

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-

U

(trifluoromethyl)benzoyl chloride (1 ml, 6.8 mmol, 1.0 equiv) in dry acetone (15 ml) was

N

slowly added dropwise to the first solution under vigorous stirring. This resulted in the

A

formation of a colorless precipitate (NH4Cl). The suspension was stirred at room

M

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

ED

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

PT

ethanol and 3 ml methylene chloride, followed by the addition of 90 ml hexane. The

CC E

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

A

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, N-H), 8.00 (s, 1H, Ph), 7.95 (d, 3JHH = 7.9 Hz, 1H, Ph), 7.72 (d, 3JHH = 7.8 Hz, 1H, Ph),

21

7.51 (t,

3

JHH = 7.8 Hz, 1H, Ph), 3.93 (q,

3

JHH = 7.0 Hz, 2H, CH2CH3), 3.50 (q,

3

JHH = 7.1 Hz, 2H, CH2CH3), 1.27 (t, 3JHH = 7.1 Hz, 3H, CH2CH3), 1.21 (t, 3JHH = 7.1 Hz,

3H, CH2CH3);

19

F-NMR (CDCl3, 377 MHz): δ –62.85 (s);

13

C{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, 2

JCF = 32.5 Hz, C-CF3), 131.12 (s, Ph), 129.40 (s, Ph), 129.29 (q, 3JCF = 3.7 Hz, Ph), 125.13

IP T

(q, 3JCF = 3.8 Hz, Ph), 123.46 (q, 1JCF = 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]–

SC R

C13H14F3N2OS: 303.0779, found: 303.0785.

U

4.2.4. General procedure for the preparation of 2b-2h

N

The nickel complexes were synthetized according to a literature procedure [21].

A

cis-Bis{N,N-diethyl-N´-(p-chloro)benzoylthioureato-2O,S}nickel(II) (2b). Red solid.

M

Yield: 98%. Elemental Analysis: calc. for C24H28Cl2N4NiO2S2: C 48.11, H 4.88, N 9.35, S 10.70; found: C 50.46, H 5.13, N 9.90, S 11.14. m.p. 231-232° C. 1H-NMR (CDCl3, 400

ED

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-

PT

S), 171.46 (s, C-O), 137.76 (s, C-Cl), 135.34 (s, quart. C, Ph), 130.58 (s, Ph), 128.30 (s,

CC E

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

A

9.32; found: C 41.94, H 4.14, N 8.11, S 9.24. m.p. 237-238° C. 1H-NMR (CDCl3, 400 MHz): δ 7.95 (d, 3JHH = 8.2 Hz, 4H, Ph), 7.51 (d, 3JHH = 8.2 Hz, 4H, Ph), 3.77 (m, 8H, CH2CH3), 1.26 (m, 12H, CH2CH3);

13

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 11.34; found: C 50.73, H 4.93, N 9.98, S 11.21. m.p. 186-187° C. 1H NMR (CDCl3, 400

IP T

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)

SC R

δ 172.53 (s, C=S), 171.40 (s, C=O), 165.07 (d, 1JCF = 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

U

(s, CH2CH3), 13.21 (s, CH2CH3), 12.61 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for

N

C24H29F2N4NiO2S2: 565.1053; found: 565.1018.

A

cis-Bis{N,N-diethyl-N´-(p-trifluoromethyl)benzoylthioureato-2O,S}nickel(II) (2e). Red

M

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,

ED

400 MHz): δ 8.20 (d, 3JHH = 8.0 Hz, 4H, Ph), 7.65 (d, 3JHH = 8.0 Hz, 4H, Ph), 3.88-3.77 (8H, m, CH2CH3), 1.36-1.26 (m, 12H, CH2CH3);

19

F-NMR (CDCl3, 377 MHz): δ –62.78

PT

(s); 13C NMR (CDCl3, 101 MHz): δ 172.98 (s, C=S), 171.02 (s, C=O), 140.12 (s, quat. C,

CC E

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:

A

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 10.66; found: C 47.69, H 4.32, N 9.37, S 11.45. m.p. 178-179° C. 1H-NMR (CDCl3, 400 MHz): δ 7.58 (m, 4H, Ph), 6.91 (tt, 3JHF = 8.5 Hz, 4JHH 2.4 Hz, 2H, Ph), 3.86-3.64 (m, 8H,

23

CH2CH3), 1.25-1.14 (m, 12H, CH2CH3);

19

F-NMR (CDCl3, 377 MHz): δ –110.20 (m);

C{1H}-NMR (CDCl3, 101 MHz): δ 173.02 (s, C=S), 169.95 (t, 4JCF = 3.5 Hz, C=O),

13

163.92 (dd, 1JCF = 248.0 Hz, 3JCF = 12.1 Hz, C-F), 140.35 (t, 3JCF = 8.90 Hz, quat. C, Ph), 111.91 (m, Ph), 106.73 (t, 2JCF = 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

IP T

C24H27F4N4NiO2S2: 601.0865, found: 601.0781; [M + Na+] calc. for C24H26F4N4NiO2S2Na: 623.0684, found: 623.0665.

SC R

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

U

11.32; found: C 50.83, H 5.03, N 9.82, S 11.44. m.p. 193-194° C. 1H-NMR (CDCl3, 400

N

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),

13

C{1H}-NMR (CDCl3, 101 MHz): δ 172.85 (s, C=S), 171.22 (d,

M

MHz): δ –113.81 (m);

A

7.16 (m, 4H, Ph), 3.78 (m, 8H, CH2CH3), 1.28 (m, 12H, CH2CH3); 19F-NMR (CDCl3, 377

4

JCF = 3.1 Hz, C-O), 162.65 (d, 1JCF = 243.7 Hz, C-F), 139.25 (d, 3JCF = 7.3 Hz, quart. C,

ED

Ph), 129.48 (d, 3JCF = 7.8 Hz, Ph), 124.88 (d, 4JCF = 2.8 Hz, Ph), 118.44 (d, 2JCF = 21.5 Hz, Ph), 115.98 (d, 2JCF = 22.9 Hz, Ph), 46.38 (s, CH2CH3), 45.79 (s, CH2CH3), 13.26 (s,

PT

CH2CH3), 12.60 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C24H29F2N4NiO2S2:

CC E

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).

A

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), 3.913.67 (m, 4H, CH2CH3), 1.39-1.16 (m, 6H, CH2CH3); 62.89 (s);

13

19

F-NMR (CDCl3, 377 MHz): δ –

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 +

IP T

Na+] calc. for C26H28F6N4NiO2S2Na: 687.0809, found: 687.0826.

4.2.5. General procedure for the preparation of 3b-3h

SC R

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

U

a solution of thionylchloride (6.4 mmol, 2 equiv) in dry methylene chloride (6 ml), which

N

resulted in the formation of a green precipitate (NiCl2). The suspension was stirred at room

A

temperature until the red/violet color of the reactant disappeared (1 - 3 hours). The

M

precipitate was filtered off and the filtrate was concentrated by rotary evaporation. N-(Diethylaminothiocarbonyl)-p-chlorobenzimidoyl chloride (3b). The crude product

ED

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

PT

and drying under vacuum, 3b was obtained as yellow solid. Yield: 80%. Elemental

CC E

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, 2H, Ph), 7.43 (d, 3JHH = 8.8 Hz, 2H, Ph), 3.97 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 3.40 (q, JHH = 7.2 Hz, 2H, CH2CH3), 1.34 (t, 3JHH = 7.1 Hz, 3H, CH2CH3), 1.17 (t, 3JHH = 7.2 Hz,

A

3

3H, CH2CH3);

C{1H}-NMR (CDCl3, 101 MHz): δ = 185.05 (s, C=S), 142.25 (s, C-Cl),

13

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

IP T

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

SC R

4.33, N 8.24, S 9.33. m.p. 105-106° C. 1H-NMR (CDCl3, 400 MHz): δ 7.61 (m, 2H, Ph),

7.27 (m, 2H, Ph), 3.65 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 3.07 (q, 3JHH = 7.2 Hz, 2H,

U

CH2CH3), 1.02 (t, 3JHH = 7.1 Hz, 3H, CH2CH3), 0.85 (t, 3JHH = 7.2 Hz, 3H, CH2CH3); C{1H}-NMR (CDCl3, 101 MHz): δ 186.03 (s, C=S), 143.37 (s, C-Cl), 133.06 (s, quart. C,

N

13

A

Ph), 132.00 (s, Ph), 130.75 (s, Ph), 128.16 (s, C-Br), 46.45 (s, CH2CH3), 45.78 (s,

M

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:

ED

370.9387, found: 370.9399.

N-(Diethylaminothiocarbonyl)-p-fluorobenzimidoyl chloride (3d). Recrystallisation

PT

from acetone yielded yellow crystals of 3d, which were dried under vacuum. Yield: 60%.

CC E

Elemental Analysis: calc. for C12H14ClFN2S: C 52.84, H 5.17, N 10.27, S 11.75; found: C 52.61, H 5.18, N 10.18, S 11.79. m.p. 104-105° C. 1H NMR (CDCl3, 400 MHz): δ 8.09 (m, 2H, Ph), 7.14 (m, 2H, Ph), 3.98 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 3.41 (q, 3JHH = 7.1 Hz,

A

2H, CH2CH3), 1.35 (t, 3JHH = 7.1 Hz, 3H, CH2CH3), 1.18 (t, 3JHH = 7.1 Hz, 3H, CH2CH3); 19

F NMR (CDCl3, 377 MHz): δ –105.53 (m);

13

C NMR (CDCl3, 101 MHz): δ 186.15 (s,

C=S), 165.71 (d, 1JCF = 255.5 Hz, C-F)), 143.03 (s, C-Cl), 131.80 (d, 3JCF = 9.5 Hz, Ph), 130.35 (m, C quat. Ph), 115.90 (d, 2JCF = 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.

IP T

Yield: 68%. Elemental Analysis: calc. for C13H14ClF3N2S: C 48.38, H 4.37, N 8.68, S 9.93;

found: C 48.38, H 4.40, N 8.41, S 9.10. m.p. 53-54 ° C. 1H-NMR (CDCl3, 400 MHz): δ

SC R

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

U

(t, 3JHH = 7.1 Hz, 3H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –63.13 (s); 13C{1H}-NMR

N

(CDCl3, 101 MHz): δ 185.75 (s, C=S), 143.00 (s, C-Cl), 137.28 (s, quat. C, Ph), 134.30 (q, JCF = 32.9 Hz, C-CF3), 129.69 (s, Ph), 125.69 (q, 3JCF = 3.8 Hz, Ph), 123.60 (q, 1JCF =

A

2

M

272.7 Hz, CF3), 46.45 (s, CH2CH3), 45.84 (s, CH2CH3), 13.11 (s, CH2CH3), 11.65 (s, CH2CH3).

ED

N-(Diethylaminothiocarbonyl)-m-difluorobenzimidoyl chloride (3f). Recrystallisation from acetone yielded yellow crystals of 3f, which were dried under vacuum. Yield: 70%.

PT

Elemental Analysis: calc. for C12H13ClF2N2S: C 49.57, H 4.51, N 9.63, S 11.03; found: C

CC E

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, Ph), 7.02 (m, 1H, Ph), 3.98 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 3.40 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 1.35 (t, 3JHH = 7.1 Hz, 3H, CH2CH3), 1.19 (t, 3JHH = 7.1 Hz, 3H, CH2CH3); 19F13

C{1H}-NMR (CDCl3, 101 MHz): δ 185.63 (s,

A

NMR (CDCl3, 377 MHz): δ –107.93 (m);

C=S), 162.91 (dd, 1JCF = 250.0 Hz, 3JCF = 12.3 Hz, C-F), 142.04 (t, 4JCF = 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%.

IP T

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,

SC R

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),

3.96 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 3.40 (q, 3JHH = 7.2 Hz, 2H, CH2CH3), 1.34 (t, JHH = 7.1 Hz, 3H, CH2CH3), 1.17 (t, 3JHH = 7.2 Hz, 3H, CH2CH3); 19F-NMR (CDCl3, 377 13

C{1H}-NMR (CDCl3, 101 MHz): δ 184.83 (s, C=S), 161.54 (d,

N

MHz): δ –111.87 (m);

U

3

JCF = 247.6 Hz, C-F), 141.88 (d, 4JCF = 3.4 Hz, C-Cl), 135.16 (d, 3JCF = 7.8 Hz, quart. C,

A

1

M

Ph), 129.22 (d, 3JCF = 8.0 Hz, Ph), 124.08 (d, 4JCF = 2.9 Hz, Ph), 118.89 (d, 2JCF = 21.3 Hz, Ph). 115.12 (d, 2JCF = 24.4 Hz, Ph), 45.35 (s, CH2CH3), 44.71 (s, CH2CH3), 12.04 (s,

ED

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:

PT

295.0471; [M + K+] calc. for C12H14ClFN2SK: 311.0187, found: 311.0212.

CC E

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

A

solid. Yield: 80%. Elemental Analysis: calc. for C13H14ClF3N2S: C 48.38, H 4.37, N 8.68, S 9.93; found: C 48.33, H 4.40, N 8.51, S 9.83. m.p. 58-59° C. 1H-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

JHH = 7.1 Hz, 2H, CH2CH3), 3.41 (q, 3JHH = 7.2 Hz, 2H, CH2CH3), 1.37 (t, 3JHH = 7.1 Hz,

3H, CH2CH3), 1.20 (t, 3JHH = 7.2 Hz, 3H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –62.83

28

(s);

C{1H}-NMR (CDCl3, 101 MHz): δ 184.79 (s, C=S), 141.91 (s, C-Cl), 134.00 (s,

13

quart. C, Ph), 131.45 (q, 4JCF = 1.1 Hz, Ph), 130.44 (q, 2JCF = 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).

IP T

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

SC R

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

U

argon and treated with dry Et3N (6 mmol, 3 equiv). The respective benzimidoyl chloride

N

3b-3h (2 mmol, 1 equiv) was added. The mixture was stirred until the reaction had

A

completed (4b, 4c, 4g, 4h over night; 4d, 4e, 4f two hours). The colorless precipitate of

M

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

ED

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

PT

recrystallization from a mixture of anhydrous CH2Cl2/hexane. The solid substance is stable

CC E

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)-

A

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. 139141° 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

(CDCl3, 101 MHz): δ 182.33 (s, C=S), 178.91 (s, C=S), 146.87 (s, C=N), 136.40 (s, C-Cl), 130.51 (s, quart. C, Ph), 128.07 (s, Ph), 127.80 (s, Ph), 45.25 (s, CH2CH3), 44.74 (s, NCH3), 43.88 (s, CH2CH3), 11.75 (s, CH2CH3), 11.28 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C15H23ClN5S2,: 372.1083, found: 372.1099; [M + Na+] calc. for C15H22ClN5S2Na: 394.0903, found: 394.0936; [M + K+] calc. for C15H22ClN5S2K:

IP T

410.0642, found: 410.0676.

N-(Diethylcarbamothioyl)-2-(2-(diethylcarbamothioyl)hydrazono)-2-(4-bromophenyl)-

SC R

acetamide (4c). Greenish solid. Yield: 44%. Elemental Analysis: calc. for C15H22BrN5S: C

43.27, H 5.33, N 16.82, S 15.40; found: C 43.18, H 5.33, N 16.87, S 15.12. m.p. 141-142°

U

C. 1H-NMR (CDCl3, 400 MHz): δ 9.59 (br, 2H, N-H), 7.80 (d, 3JHH = 8.4 Hz, 2H, Ph), 7.57

N

(d, 3JHH = 8.4 Hz, 2H, Ph), 3.91 (q, 3JHH = 6.8 Hz, 2H, CH2CH3), 3.53 (q, 3JHH = 6.6 Hz, 2H,

13

C{1H}-NMR (CDCl3, 101 MHz): δ 182.50 (s, C=S), 178.90 (s, C=S),

M

3H, CH2CH3);

A

CH2CH3), 3.28 (s, 6H, N-CH3), 1.26 (t, 3JHH = 6.9 Hz, 3H, CH2CH3), 1.06 (t, 3JHH = 6.8 Hz,

147.17 (s, C=N), 131.04 (s, C-Br), 130.97 (s, Ph), 128.37 (s, Ph), 125.16 (s, quart C, Ph),

ED

45.37 (s, CH2CH3), 44.83 (s, N-CH3), 44.01 (s, CH2CH3), 11.87 (s, CH2CH3), 11.37 (s, CH2CH3).

PT

N-(Diethylcarbamothioyl)-2-(2-(diethylcarbamothioyl)hydrazono)-2-(4-fluorophenyl)-

CC E

acetamide (4d). Colorless crystals. Yield: 30%. Elemental Analysis: calc. for C15H22FN5S: C 50.68, H 6.24, N 19.70, S 18.04; found: C 50.53, H 6.26, N 19.72, S 18.27. m.p. 121123° C. 1H-NMR (CDCl3, 400 MHz): δ 9.73-9.24 (2 br. s, 2H, N-H), 7.90 (m, 2H, Ph), 7.09

A

(m, 2H, Ph), 3.85 (q, 3JHH = 7.0 Hz, 2H, CH2CH3), 3.50 (q, 3JHH = 7.0 Hz, 2H, CH2CH3), 3.24 (s, 4H, N-CH3) 1.21 (t, 3JHH = 7.0 Hz, 3 H, CH2CH3), 1.02 (t, 3JHH = 7.0 Hz, 3H, CH2CH3); 19F-NMR (CDCl3, 377 MHz): δ –108.02 (s); 13C{1H}-NMR (CDCl3, 101 MHz): δ 183.63 (s, C=S), 179.99 (s, C=S), 164.76 (d, 1JCF = 252.6 Hz, C-F), 148.43 (s, C=N), 130.13 (d, 3JCF = 8.9 Hz, Ph), 129.21 (d, 4JCF = 3.4 Hz, quat. C, Ph), 115.88 (d, 2JCF = 22.0

30

Hz, Ph), 46.34 (s, CH2CH3), 45.95 (s, N-CH3), 44.99 (s, CH2CH3), 12.86 (s, CH2CH3), 12.39 (s, CH2CH3). N-(Diethylcarbamothioyl)-2-(2-(diethylcarbamothioyl)hydrazono)-2-(4-(trifluoromethyl)phenyl)acetamide (4e). Colorless solid. Yield: 62%. Elemental Analysis: calc. for C16H22F3N5S2: C 47.39, H 5.47, N 17.27, S 15.81; found: C 47.49, H 5.66, N 17.10, S

IP T

15.52. m.p 137-138° C. 1H-NMR (CDCl3, 400 MHz): δ 9.48 (br. s, 2H, N-H), 8.02 (d, 3

JHH = 8.1 Hz, 2H, Ph), 7.68 (d, 3JHH = 8.1 Hz, 2H, Ph), 3.90 (br. q, 2H, CH2CH3), 3.55 (br.

19

SC R

q, 2H, CH2CH3), 3.25 (s, 6H, N-CH3), 1.24 (br. t, 3H, CH2CH3), 1.08 (br. t, 3H, CH2CH3); F-NMR (CDCl3, 377 MHz): δ –62.96 (s); 13C{1H}-NMR (CDCl3, 101 MHz): δ 182.26 (s,

U

C=S), 178.97 (s, C=S), 146.38 (s, C=N), 135.68 (s, quart. C, Ph), 131.92 (q, 2JCF = 32.5 Hz,

N

C-CF3), 127.24 (s, Ph), 124.68 (q, 3JCF = 3.7 Hz, Ph), 122.73 (q, 1JCF = 272.5 Hz, CF3),

A

45.46 (s, CH2CH3), 44.62 (s, N-CH3), 44.07 (s, CH2CH3), 11.85 (s, CH2CH3), 11.31 (s,

M

CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C16H23F3N5S2: 406.1347, found: 406.1357; [M + Na+] calc. for C16H22F3N5S2Na: 428.1166, found: 428.1194; [M + K+] calc. for

ED

C16H22F3N5S2K: 444.0906, found: 444.0928. N-(Diethylcarbamothioyl)-2-(2-(diethylcarbamothioyl)hydrazono)-2-(3,3-difluorophe-

PT

nyl)acetamide (4f). Yellow solid. Yield: 32%. Elemental Analysis: calc. for C15H21F2N5S2:

CC E

C 48.24, H 5.67, N 18.75, S 17.17; found: C 48.60, H 5.92, N 18.08, S 17.37. m.p. 124125° C. 1H-NMR (CDCl3, 400 MHz): δ 9.44 (br. s, 2H, N-H), 7.39 (br. m, 2H, Ph), 6.87 (br. m, 1H, Ph), 3.85 (br. q, 2H, CH2CH3), 3.50 (br. t, 2H, CH2CH3), 3.20 (s, 6H, N-CH3), 19

F-NMR (CDCl3, 377 MHz): δ –

A

1.20 (br. t, 3H, CH2CH3), 1.05 (br. t, 3H, CH2CH3);

107.81 (br); 13C{1H}-NMR (CDCl3, 101 MHz): δ 182.37 (s, C=S), 178.96 (s, C=S), 161.88 (dd, 1JCF = 250.0 Hz, 3JCF = 12.5 Hz, C-F), 145.67 (s, C=N), 135.55 (s, quart. C, Ph), 109.96 (dd, 2JCF = 21.7 Hz, 4JCF = 7.3 Hz, Ph), 105.58 (t, 2JCF = 25.8 Hz, Ph), 45.46 (s, CH2CH3), 44.07 (s, CH2CH3), 39.89 (s, N-CH3), 11.81 (s, CH2CH3), 11.26 (s, CH2CH3). HRMS-ESI

31

(m/z): [M + H+] calc. for C15H22F2N5S2: 374.1285, found: 374.1293; [M + Na+] calc. for C15H21F2N5S2Na: 396.1104, found: 396.1127; [M + K+] calc. for C15H21F2N5S2K: 412.0844, found: 412.0874. N-(Diethylcarbamothioyl)-2-(2-(diethylcarbamothioyl)hydrazono)-2-(3-fluorophenyl)acetamide (4g). Yellow solid. Yield: 44%. Elemental Analysis: calc. for C15H22FN5S2: C

IP T

50.68, H 6.24, N 19.70, S 18.04; found: C 50.53, H 6.26; N 19.66, S 17.90. m.p. 125-126° C. 1H-NMR (CDCl3, 400 MHz): δ 9.68 (br. s, 2H, N-H), 7.72 (m, 1H, Ph), 7.64 (m, 1H, 3

JHH = 8.1 Hz,

4

JHF = 5.7 Hz, 1 H, Ph), 7.19 (m, 1H, Ph), 3.92 (q,

SC R

Ph), 7.43 (td, 3

JHH = 7.0 Hz, 2H, CH2CH3), 3.55 (q, 3JHH = 6.9 Hz, 2H, CH2CH3), 3.30 (s, 6H, N-CH3), 19

F-NMR

U

1.26 (t, 3H, 3JHH = 7.0 Hz, CH2CH3), 1.09 (t, 3JHH = 6.9 Hz, 3H, CH2CH3);

1

JCF = 247.4 Hz, C-F), 146.82 (s, C=N), 134.36 (d,

A

178.88 (s, C=S), 161.79 (d,

N

(CDCl3, 377 MHz): δ –111.52 (br); 13C{1H}-NMR (CDCl3, 101 MHz): δ 182.53 (s, C=S),

JCF = 7.6 Hz, quart. C, Ph), 129.51 (d, 3JCF = 8.1 Hz, Ph), 122.13 (d, 4JCF = 3.1 Hz, Ph),

M

3

117.58 (d, 2JCF = 24.2 Hz, Ph), 114.34 (d, 2JCF = 26.6 Hz, Ph), 45.46 (s, CH2CH3), 44.88 (s,

ED

N-CH3), 44.13 (s, CH2CH3), 11.94 (s, CH2CH3), 11.44 (s, CH2CH3). HRMS-ESI (m/z): [M – H]– calc. for C15H21FN5S2: 354.1222, found: 354.1225.

PT

N-(Diethylcarbamothioyl)-2-(2-(diethylcarbamothioyl)hydrazono)-2-(3-(trifluorome-

CC E

thyl)phenyl)acetamide (4h). Colorless solid. Yield: 61%. Elemental Analysis: calc. for C16H22F3N5S2: C 47.39, H 5.47, N 17.27, S 15.81; found: C 46.85, H 5.45, N 17.07, S 15.06. m.p. 128-130° C. 1H-NMR (CDCl3, 400 MHz): δ 9.62 (br. s, 2 H, N-H), 8.18 (s, 1 H,

A

Ph), 8.10 (m, 1H, Ph), 7.73 (m, 1H, Ph), 7.58 (m, 1H, Ph), 3.91 (br. q, 2H, CH2CH3), 3.56 (br. q, 2H, CH2CH3), 3.25 (s, 6H, N-CH3), 1.26 (br. t, 3H, CH2CH3), 1.08 (br. t, 3H, CH2CH3).; 19F-NMR (CDCl3, 377 MHz): δ –62.77 (s); 13C{1H}-NMR (CDCl3, 101 MHz): δ 182.68 (s, C=S), 179.05 (s, C=S), 146.69 (s, C=N), 133.15 (s, quart. C, Ph), 129.85 (q, 2

JCF = 32.9 Hz, C-CF3), 129.56 (s, Ph), 128.37 (s, Ph), 126.92 (s, Ph), 124.20 (s, Ph),

32

122.72 (q, 1JCF = 272.6 Hz, CF3), 45.47 (s, CH2CH3), 44.97 (s, N-CH3), 44.13 (s, CH2CH3), 11.84 (s, CH2CH3), 11.32 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C16H23F3N5S2: 406.1347, found: 406.1367; [M + Na+] calc. for C16H22F3N5S2Na: 428.1166, found: 428.1199; [M + K+] calc. for C16H22F3N5S2K: 444.0906, found: 444.0937.

IP T

4.2.7. General procedure for the preparation of 5b-5c

NH4SCN (5.26 mmol, 1.0 equiv) was dissolved in 10 ml of dry acetone under argon. A

SC R

solution of the respective benzoyl chloride (8.32 mmol, 1.6 equiv) in dry acetone (5 ml)

was added dropwise to the first solution under vigorous stirring, which resulted in the

U

formation of a colorless precipitate (NH4Cl). The suspension was stirred at room

N

temperature for 1 h. Subsequently, a solution of diethylamine (4.78 mmol, 0.9 equiv) in

A

acetone (5 ml) was added dropwise. After stirring for two hours, the reaction mixture was

M

halved in volume by rotary evaporation. The products 5a-5c were precipitated by slow addition of water (30 ml) followed by prolonged stirring. The orange precipitate was

ED

isolated by filtration, thoroughly washed with water and air-dried. 6-(Diethylamino)-2H-1,3,5-thiadiazine-2-thione (5a). After purification by silica gel

PT

column chromatography (ethyl acetate/hexane 1:5), compound 5a was isolated as an

CC E

orange-red solid. Yield: 20%. Elemental Analysis: calc. for C13H15N3S2: C 56.29, H 5.45, N 15.15, S 23.11; found: C 55.21, H 5.60, N 14.28, S 22.36. m.p. 119-120° C. 1H-NMR (CDCl3, 400 MHz): δ 8.53 (m, 2H, Ph), 7.57 (m, 1H, Ph), 7.47 (m, 2H, Ph), 3.96 (q, 3JHH =

A

7.2 Hz, 2H, CH2CH3), 3.56 (q, 3JHH = 7.2 Hz, 2H, CH2CH3), 1.37 (t, 3JHH = 7.2 Hz, 3H, CH2CH3), 1.33 (t, 3JHH = 7.2 Hz, 3H, CH2CH3);

13

C{1H}-NMR (CDCl3, 101 MHz):

δ 196.64 (s, S-C=S), 170.68 (s, N-C=N), 164.40 (s, N-C-S), 136.44 (s, quart. C, Ph), 133.34 (s, Ph), 130.55 (s, Ph), 128.43 (s, Ph), 45.29 (s, CH2CH3), 43.78 (s, CH2CH3), 13.10

33

(s, CH2CH3), 12.29 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C13H16N3S2: 278.0786, found: 278.0761. 4-(4-Chlorophenyl)-6-(diethylamino)-2H-1,3,5-thiadiazine-2-thione

(5b).

After

purification by silica gel column chromatography (ethyl acetate/hexane 1:1), compound 5b was isolated as an orange-red solid. Yield: 27%. Elemental Analysis: calc. for

IP T

C13H14ClN3S2: C 50.07, H 4.53, N 13.47, S 20.56; found: C 49.76, H 5.09, N 12.69, 20.44. m.p. 145-146° C. 1H-NMR (CDCl3, 400 MHz): δ 8.41 (m, 2H, Ph), 7.40 (m, 2H, Ph), 3.92

SC R

(q, 3JHH = 7.0 Hz, 2H, CH2CH3), 3.52 (q, 3JHH = 7.0 Hz, 2H, CH2CH3), 1.32 (t, 3JHH = 7.0

Hz, 3H, CH2CH3), 1.28 (t, 3JHH = 7.0 Hz, 3H, CH2CH3); 13C{1H}-NMR (CDCl3, 101 MHz):

U

δ 195.63 (s, S-C=S), 169.72 (s, N-C=N), 162.41 (s, N-C-S), 138.69 (s, C-Cl), 133.99 (s,

N

quart. C, Ph), 130.82 (s, Ph), 127.75 (s, Ph), 44.36 (s, CH2CH3), 42.88 (s, CH2CH3), 12.11

A

(s, CH2CH3), 11.30 (s, CH2CH3). HRMS-ESI (m/z): [M + H+] calc. for C13H15ClN3S2,:

M

312.0396, found: 312.0396; [M + Na+] calc. for C13H14ClN3S2Na: 334.0215, found: 334.0213; [M + K+] calc. for C13H14ClN3S2K: 349.9955, found: 349.9951; [2M + Na+] calc.

ED

for C26H28Cl2N6S4Na: 645.0533, found: 645.0526. [2M + K+] calc. for C26H28Cl2N6S4K: 661.0272, found: 661.0258.

PT

4-(3-Fluorophenyl)-6-(diethylamino)-2H-1,3,5-thiadiazine-2-thione (5c). The crude

CC E

product was recrystallized from ethanol and quantitatively precipitated by the addition of diethyl ether. Orange-red needles of 5c were filtered off, washed with diethyl ether and airdried. Yield: 37%. Elemental Analysis: calc. for C13H14FN3S2: C 50.07, H 4.53, N 13.47, S

A

20.56; found: C 52.90, H 4.80, N 14.20, S 21.70. m.p. 127-128° C. 1H-NMR (CDCl3, 400 MHz): δ 8.33 (m, 1H, Ph), 8.17 (m, 1H, Ph), 7.44 (td, 3JHH = 8.0 Hz, 4JHF = 5.7 Hz, 1H, Ph), 7.26 (m, 1H, Ph), 3.96 (q, 3JHH = 7.1 Hz, 2H, CH2CH3), 3.57 (q, 2H, 3JHH = 7.3 Hz, CH2CH3), 1.40-1.31 (m, 6H, CH2CH3);

19

F-NMR (CDCl3, 377 MHz): δ –112.87 (m);

C{1H}-NMR (CDCl3, 101 MHz): δ 196.58 (s, S-C=S), 170.47 (s, N-C-S), 162.92 (d, 4JCF

13

34

= 3.3 Hz, N-C=N), 162.52 (d, 1JCF = 245.6 Hz, C-F), 138.84 (d, 3JCF = 7.7 Hz, quart. C, Ph), 129.72 (d, 3JCF = 7.8 Hz, Ph), 125.89 (d, 4JCF = 2.9 Hz, Ph), 119.83 (d, 2JCF = 21.4 Hz, Ph), 116.59 (d, 2JCF = 23.3 Hz, Ph), 45.31 (s, CH2CH3), 43.84 (s, CH2CH3), 12.91 (s, CH2CH3),

IP T

12.04 (s, CH2CH3).

SC R

5. Acknowledgements

This work was generously supported by the Graduate School (GK 1582) ‘Fluorine as a key

N

U

element’ of the Deutsche Forschungsgemeinschaft.

A

6. Appendix. Supplementary data

M

NMR spectra of the isolated compounds, Ellipsoid representations of the molecular structures of the crystallographically studied compounds, Tables containing selected bond

ED

lengths and angles. CCDC 1841309 and CCDC 1841310 contain the supplementary

PT

crystallographic data for 5b and 4e. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic

CC E

Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-

A

mail: [email protected].

35

7. References [1] H. H. Nguyen, P. I. S. Maia, V. M. Deflon, U. Abram, Oxotechnetium(V) Complexes with a Novel Class of Tridentate Thiosemicarbazide Ligands, Inorg. Chem. 2009, 48, 25-27. [2] P. I. S. Maia, H. H. Nguyen, A. Hagenbach, S. Bergemann, R. Gust, V. M. Deflon, U. Abram, Rhenium mixed-ligand complexes with S,N,S-tridentate thiosemi–

IP T

carbazone/thiosemicarbazide ligands, Dalton Trans. 2013, 42, 5111-5121.

[3] P. I. S. Maia, H. H. Nguyen, D. Ponader, A. Hagenbach, S. Bergemann, R. Gust, V.

carbazide Ligands, Inorg. Chem. 2012, 51, 1604-1613.

SC R

M. Deflon, U. Abram, Neutral Gold Complexes with Tridentate SNS Thiosemi-

[4] P. I. da Silva Maia, PhD Thesis, University of Sao Paulo, 2011, Sao Paulo, Brazil. [5] H. H. Nguyen, J. J. Jegathesh, P. I. S. Maia, V. M. Deflon, R. Gust, S. Bergemann,

U

U. Abram, Rhenium and Technetium Complexes with Tridentate N-[(N′′,N′′-

N

Dialkylamino)(thiocarbonyl)]-N′-substituted Benzamidine Ligands, Inorg. Chem. 2009, 48, 9356-9364.

A

[6] K. S. Ferraz, J. G. da Silva, F. M. Costa, B. M. Mendes, B. L. Rodrigues, R. G. dos

M

Santos, H. Beraldo, N(4)-Tolyl-2-acetylpyridine thiosemicarbazones and their platinum(II,IV) and gold(III) complexes: cytotoxicity against human glioma cells

ED

and studies on the mode of action, Biometals, 2013, 26, 677-691. [7] P. N. Fonteh, F. K. Keter, D. Meyer, New bis(thiosemicarbazonate) gold(III) complexes inhibit HIV replication at cytostatic concentrations: Potential for

PT

incorporation into virostatic cocktails, J. Inorg. Biochem. 2011, 105, 1173-1180. [8] S. D. Khanye, B. Wan, S. G. Franzblau, J. Gut, P. J. Rosenthal, G. S. Smith, K.

CC E

Chibale, Synthesis and in vitro antimalarial and antitubercular activity of gold(III) complexes containing thiosemicarbazone ligands, J. Organomet. Chem., 2011, 696, 3392-3396.

A

[9] N. Fujii, J. P. Mallari, E. J. Hansell, Z. Mackay, P. Doyle, Y. M. Zhou, J. Gut, P. J. Rosenthal, J. H. McKerrow, R. Kiplin Guy, Discovery of potent thiosemicarbazone inhibitors of rhodesain and cruzain, Bioorg. Med. Chem. Lett. 2005, 15, 121-123.

[10] S. D. Khanye, G. S. Smith, C. Lategan,, P. J. Smith, J. Gut, P. J. Rosenthal, , K. Chibale, Synthesis and in vitro evaluation of gold(I) thiosemicarbazone complexes for antimalarial activity, J. Inorg. Biochem. 2010, 104, 1079-1083.

36

[11] R. Arancibia, A. H: Klahn, M. Lapier, J. D. Maya, A. Ibanez, T. Garland, S. Carrere-Kremer, L. Kremer, C. Biot, Synthesis, characterization and in vitro antiTrypanosoma cruzi and anti-Mycobacterium tuberculosis evaluations of cyrhetrenyl and ferrocenyl thiosemicarbazones, J. Organomet. Chem. 2014, 755, 1-6. [12] A. R. Rettondin, Z. A. Carneiro, A. C. R. Goncalves, V. F. Ferreira, A. N. Lima, R. J. Oliveira, S. de Albuquerque, V. M. Deflon, P. I. S. Maia, Gold(III) complexes

IP T

with ONS-Tridentate thiosemicarbazones: Toward selective trypanocidal drugs, Eur. J. Med. Chem. 2016, 120, 217-226.

[13] P. I. S. Maia, Z. A. Carneiro, C. D Lopes, C. G. Oliveira, J. S. Silva, S. de

gold(iii) complexes

SC R

Albuquerque, A. Hagenbach, R. Gust, V. M. Deflon, U. Abram, Organometallic with hybrid SNS-donating thiosemicarbazone

ligands:

cytotoxicity and anti-Trypanosoma cruzi activity, Dalton Trans. 2017, 46, 2559-

U

2571.

[14] C. D. Lopes, A. P. S. Gaspari, R.J. Oliveira, U. Abram, J. P. A. Almeida, P. I. S.

N

Maia, S. de Albuquerque, Z. A. Carneiro, In Vivo Trypanocidal Activity of the

A

Organometallic gold(III) complex [Au(Hdamp)(L14)]+ (L = SNS-donating thiosemi-

M

carbazone) Antimicrobial Agents and Chemotherapy, submitted. [15] E. Chatelain, Chagas disease drug discovery: toward a new era, J. Biomol. Screen. 2015, 20, 22-35.

ED

[16] J. Bermudez, C. Davies, A. Simonazzi, J. P. Real, S. Palma, Current drug therapy and pharmaceutical challenges for Chagas disease, Acta Tropica 2016, 156, 1-16.

PT

[17] J. A. Urbina, R. Docampo, Specific chemotherapy of Chagas disease: controversies and advances, Trends in Parasitology 2003, 19, 495-501.

CC E

[18] R. L. Tarleton, Chagas Disease: A Solvable Problem, Ignored, Trends in Molecular Medicine 2016, 22, 835-838.

[19] Y. Zhou, J. Wang, Z. Gu, S. Wang, W. Zhu, J. L. Acena, V. A. Soloshonok, K. Izawa, H. Liu, Next Generation of Fluorine-Containing Pharmaceuticals,

A

Compounds Currently in Phase II–III Clinical Trials of Major Pharmaceutical Companies: New Structural Trends and Therapeutic Areas, Chem. Rev. 2016, 116, 422-518.

[20] (a) L. Hennig, K. Ayala-Leon, J. Angulo-Cornejo, R. Richter, L. Beyer, Fluorine hydrogen short contacts and hydrogen bonds in substituted benzamides, J. Fluor. Chem. 2009, 130, 453-460; (b) H. Liu, W. Yang, W. Zhou, Y. Xu, J. Xie, M. Li,

37

Crystal structures and antimicrobial activities of copper(II) complexes of fluorinecontaining thioureido ligands, Inorg. Chim. Acta, 2013, 405, 387-394; (c) I. B. Douglass, F. B. Dains, The Preparation and Hydrolysis of Mono- and Disubstituted Benzoylthioureas, J. Am. Chem. Soc., 1934, 56, 719-721; (d) M. K. Rauf, S. Yaseen, A. Badshah, S. Zaib, R. Arshad, Imtiaz-ud-Din, M. N. Tahir, J. Iqbal, Synthesis, characterization and urease inhibition, in vitro anticancer and

IP T

antileishmanial studies of Ni(II) complexes with N,N,N′‑trisubstituted thioureas, J. Biol. Inorg. Chem. 2015, 20, 541-554; (e) W. Yang; H. Liu; M. Li; F. Wang; W.

Zhou; J. Fan, Synthesis, structures and antibacterial activities of benzoylthiourea

SC R

derivatives and their complexes with cobalt, J. Inorg. Biochem. 2012, 116, 97-105.

[21] E. Rodriguez-Fernandez, E. Garcia, M. R. Hermosa, A. Jimenez-Sanchez, M. M. Sanchez, E. Monte, J. Criado, Chloride and ethyl ester morpholine thiourea

U

derivatives and their Ni(II) complexes. Crystal and molecular structures of the thiourea derivative L-leucine methyl ester and its complexes with Cu(II) and Pt(II).

N

Growth of the pathogenic fungus Botrytis cinereal. J. Inorg. Biochem. 1999, 75,

A

181-188.

M

[22] C. R. Rasmussen, F. J. Jr. Villani, L. E. Weaner, B. E. Reynolds, A. R. Hood, L. R. Hecker, S. O. Nortey, A. Hanslin, M. J. Costanzo, Improved Procedures for the Preparation of Cycloalkyl-, Arylalkyl-, and Arylthioureas, Synthesis, 1988, 6, 456-

ED

459.

[23] G. Weber, J. Hartung, L. Beyer, Zur Reaktion von N‐(N′,N′‐Dialkyl(aryl)amino‐

PT

thiocarbonyl)benzimidoylchloriden mit Kaliumthiocyanat, J. Prakt. Chem. 1988, 330, 241-247.

CC E

[24] L. Beyer, J. Hartung, R. Widera, Reaktionen an nickel(II)koordinierten NAcylthioharnstoffen

mit

Säurechloriden:

ein

einfacher

Zugang

für

neue

Thioharnstoffderivate, Tetrahedron, 1984, 40, 405-412.

[25] a) G. Weber, J. Hartung, L. Beyer, Umsetzungen von N‐(Morpholino‐

A

thocarbonyl)‐benzimidchlorid mit Dinucleophilen, Z. Chem., 1986, 26, 70-71; b) R. Köhler, L. Beyer, A. Hantschmann, E. Hoyer, N‐(Dialkylamino‐thiocarbonyl)benzimidothio‐ und ‐selenoester, Z. Chem., 1990, 30, 102-103.

[26] E. Chatelain and N. Konar, Translational challenges of animal models in Chagas disease drug development: a review, Drug Design Development and Therapy, 2015, 9, 4807-4823.

38

[27] J. Wang, M. Sanchez-Rosello, J. L. Acena, C. del Pozo, A. E. Sorochinsky, S. Fustero, V. A. Soloshonok, H. Liu, Fluorine in Pharmaceutical Industry: FluorineContaining Drugs Introduced to the Market in the Last Decade (2001–2011), Chem. Rev. 2014, 114, 2432-2506. [28] F. S. Buckner, C. L. Verlinde, A. C. La Flamme, W. C. Van Voorhis, Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites

IP T

expressing beta-galactosidase, Antimicrob. Agents Chemother. 1996, 40, 25922597.

[29] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:

SC R

application to proliferation and cytotoxicity assays, J. Immunol. Methods, 1983, 65, 55-63.

[30] G. M. Sheldrick, A short history of SHELX, Acta Crystallogr. Sect. A, 2008, 64,

U

112-122.

[31] G. M Sheldrick, Crystal structure refinement with SHELXL, Acta Crystallogr.

N

Sect. C, 2015, 71, 3-8.

A

[32] K. Brandenburg, Diamond- Crystal and Molecular Structure Visualization, Crystal

A

CC E

PT

ED

M

impact GbR, vers. 4.5.1, 2018, Bonn (Germany).

39