BACKGROUND

1.1. Thiosemicarbazones

Thiosemicarbazones (TSs) are an extremely versatile class of compounds. They possess a variety of interesting physico-chemical properties: peculiar of most of these sulphur-containing organic molecules, are the extensive electronic delocalization and the presence of a thione-thiol tautomerism (Figure 13).

Figure 13: Thione-thiol tautomerism.

The idea was to conjugate the already known antimicrobial properties of some natural aldehydes and ketones (the active component of spices, used for centuries to preserve food) and those of metal ions (for example some metal salts, in particular copper, have been extensively used in agriculture to protect plants from molds). Therefore, TSs are usually obtained from the condensation between the hydrazine group of a thiosemicarbazide with the carbonyl group of an aldehyde or ketone in protic solvents, like ethanol or methanol. The thiosemicarbazide easily reacts with the carbonyl group of the natural aldehydes, possessing the right donor atoms to chelate metal ions and thus allowing the incorporation in a single molecule of three potentially active components.

One of the major assets of these compounds is the ease with which the TS structure can be modified, simply by using differentially substituted aldehydes (or ketones) or different aldehydes. The synthesis is usually fast, and the product is obtained usually in 12-24 hours. The most common nomenclature adopted to describe a TS structure follows a scheme in which every nitrogen is numbered as reported in Figure 14.

Figure 14: Thiosemicarbazone general structure.

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Another crucial characteristic of TSs is the fact that they effectively act as bidentate ligands, using the sulphur and the iminic nitrogen, hence forming a 5-term coordination 2 ring. When the N² is not alkylated, TSs can also act as an anionic ligand through the deprotonation of the hydrazinic nitrogen (N²). The TSs show high affinity to many different metals thanks to the presence of mixed hard-soft N-S donor atoms. Moreover, thanks to the N² deprotonation, Thiosemicarbazone’s can also balance the positive charges of the metal. This electrostatic effect makes TSs very effective in chelating not only transition elements, but also alkaline and alkaline earth metals. Sulphur creates effectively metal-to- ligand charge-transfer (MLCT), an additional electrostatic stabilizing force for the final complex. The formation of a metal complex between a TS and a transition metal is usually indicated by a color transition which can be explained by the well-known crystal field theory.

1.2. Applications of TSs

TSs (and their metal complexes) have attracted scientists for many years, as they have been successfully adapted for different applications in many fields. For example in Figure 15, the acetonethiosemicarbazone is commonly used in the plastic industry as “stopper” in the polyvinyl chloride polymerization, while the Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) is a patented drug used in the treatment of cancer which has successfully reached the phase II of clinical trials (Ocean et al.2011) .

Figure 15: Example of TSs

Thiosemicarbazones find also vast applications in analytical chemistry as sensing agents to detect metals in solution, acting as spectrophotometric or electrochemical indicators (Raman et al. 2009).

In particular, the most recent application explored in this field is the use of TS’s as sensing agents to detect metals in food (Janardhan Reddy et al. 2007).

Triapine®, that as stated before showed promising anticancer ability, it is not the only TS with interesting biological properties: since their discovery in the 1950s, TSs have proven to be a very

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interesting bioactive agents, possessing antibacterial and antiviral activity (Vahma et al. 1967, Padmanabhan et al. 2017), antifungal and anti-aflatoxigenic properties (Gingras et al. 1965, Rogolino et al. 2017;Degola et al. 2015) and antitumoral capacity (Pelosi et al. 2010).

1.3. Thiosemicarbazones as antifungal agents

To our knowledge, in 1960 Benns et al. published the first example of TS designed and tested as antifungal agents:they evaluated the antifungal activity of a panel of 40 TSs and copper complexes on A. niger and Chaetomium globosum cultures; the compounds were compared with two commercial fungicides: the 2,2'-dihydroxy-5,5'-dichlorodiphenylmethane and the copper/8-hydroxyquinolinolate mixture. The results showed that several TSs resulted significantly more effective than their corresponding copper complexes, indicating that the antifungal potential was not only due to the metal ion but also to the TS structure. Starting from this discovery, the exploration of TSs capacities for antifungal purposes has grown rapidly, and many other interesting TSs were characterized. Their mechanism of action is still debated: some results ascribed the antifungal activity to the ability of TSs to modify the redox equilibrium in target cells, acting both as anti-oxidant or ROS stimulating agents. Other studies revealed that TSs seem to induce changes in the biosynthesis of cell membrane sterols: in particular, it was reported that specific TSs can influence the regulation and biosynthesis of ergosterol, an essential vitamin for fungal cells strictly connected with the metabolism of lipids and membrane structure (De Araújo Neto et al. 2017).

As previously indicated, TSs are also interesting because of their effectiveness in chelating metal ions; the synthesis of TS metal complexes can thus be used to improve the TS biological effects through the modification of the original scaffold chemical and physical properties, such as water solubility, membrane permeability, bioavailability and cell uptake. In this context, and with regard to our research interest, the addition of a metallic nucleus to the molecule has been explored for the improvement of TS antifungal and anti-aflatoxigenic effect.

34 1.4. Metal ions in agriculture

In agriculture, metals are usually applied as fertilizers but also as fungicides and antibacterial agents.

Since some metals are essential micro-nutrients, their biological effects are strictly connected with their amount, and the level of each metal must be balanced to grant its homeostasis.

Copper compounds are the most employed, especially copper(II)sulphate. It has been known since the XVIII century that the use of a mixture of copper(II)sulphate and lime in water – called “Bordeaux mixture” - has a strong fungistatic effect. If sprayed on crops, it inhibits mould growth and makes seeds unattractive for birds. The fungicidal action of copper is often explained in terms of capacity to interfere with the redox processes which regulate respiration in cells. In fact, due to the easy redox interconversion between Cu(I)/Cu(II), it raises the number of reactive oxygen species (ROS) in cells inducing high levels of oxidative stress.

Zinc is widely used as fertilizer combined with other macronutrients (like potash, phosphate and nitrogen) and supports root growth increasing leaf size and resilience during stressful growing conditions. However, zinc is not only applied as fertilizer. Zinc dimethyldithiocarbamate is a broad-spectrum fungicide applied on the plant surface where it forms a barrier which inhibits the fungal growth on plant.

From the fungus perspective, the role of copper and zinc in the aflatoxin biosynthesis is still poorly investigated, but there are evidences that they play a significant role (Cuero et al. 2005). In addition, the production of mycotoxins is well known to be strictly connected with the redox equilibrium in fungal cells. Even though the molecular details of this correlation are still unclear, evidence is coming out that the production of ROS (from both fungus and host) during the mould/plant interaction are able to modulate the biosynthesis of aflatoxins (Rogolino et al. 2017).

35 2. Newly synthesized compounds

With regard to the above mentioned evidences, the Inorganic Chemistry unit synthesized newly TSs derived from specific natural aldehydes and ketones, chosen for their previously assessed biological properties. In the Table 3 were reported the complete list of the natural starting materials.

Table 3: Natural ketones and aldehydes used in the Aflatox Project.

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In order to better describe the results obtained, all compounds have been grouped in families on the basis of their structure, including natural starting compounds, ligands, and structural

modifications.

The families are listed below:

1. Cuminaldehyde derivatives