Thiosemicarbazones as antifungal agents

Mycotoxin contamination in food represents a public health problem. Mycotoxins are secondary metabolites produced by fungi under specific conditions, such as high humidity, poor agricultural practices, or damaged and contaminated crops.

Among mycotoxins, aflatoxins are the most dangerous and are produced by fungal species from the genus Aspergillus, notably A. flavus, A. parasiticus and A. nomius, which develop naturally in food products. These fungi usually infect cereal crops and can lead to serious risks to human and animal health by causing hepatotoxicity, teratogenicity, and immunotoxicity (Kumar et al., 2017). There are more than 20 types of aflatoxin molecules, although the most prominent are aflatoxins B1, B2, G1, G2, M1, and M2. Aflatoxins are classified by the International Agency for Research on Cancer (IARC, 2012) as group 1 carcinogens. The most severe human health impact of aflatoxins is hepatocellular carcinoma, which is recognized as the 9th and 7th leading type of cancer in women and men, respectively (Ismail et al., 2018).

Aflatoxins exhibit great resistance to conventional treatments usually applied to food or feed processing, including pasteurization, sterilization and other thermal applications.

Different approaches have been proposed to remove or degrade the aflatoxins in foods

117 and the most prominent can be categorized into physical, chemical, and biological methods.

In this context, the “Aflatox project” represents a new approach to develop new typologies of inhibitors of Aspergillus proliferation and particularly of aflatoxins production, harmless to the environment and to human health.

The first step of the project was the synthesis of a wide range of bioactive molecules, obtained starting from compoundsof natural origin, which then were used as ligands for coordination with metal ions. We functionalized these compounds in order to modulate their main properties, such as solubility, complexing ability, and lipophilicity. Endogenous metal ions, mainly copper and zinc, were used to obtain the relative metal complexes (Zani et al., 2015). We set up a method to determine their effects on fungal germination and growth and aflatoxin biosynthesis. The new compounds were tested to detect mycelia growth inhibition and mycotoxin production against different strains of A. flavus (Zani et al., 2015).

Once the chemical-physical properties and the molecular structures of the new complexes have been elucidated and their antifungal and antimycotoxigenic activity determined, we developed an approach to assess toxicity and genotoxicity of the new compounds, before their diffusion in the environment. This approach was exploited for the design of new potential antifungal molecules without toxic side effects on humans.

To assess the potential toxic and genotoxic risks of the new molecules for the environment, a battery of tests with different genetic end-points was carried out: the Ames test to detect point mutations on bacteria and the micronucleus Allium cepa test to detect chromosomal damage (Zani et al., 2015).

The last step of the project was the toxicity and genotoxicityevaluation in human cells to determine the risk for human health. The biological activity of the new active molecules was assessedon normal cells deriving from human districts related to possible xenobiotic ways of interaction with the human body, to investigate the risks linked to specific exposition (Zani et al., 2015).

Starting from molecules of natural origin, we synthesized both semicarbazones (SCs) and thiosemicarbazones. SCs differ from TSCs only by replacing the sulfur atom with an oxygen atom. We observed that semicarbazones showed less activity in terms of inhibition of fungal growth and mycotoxin production than the corresponding TSCs. We can assume that

118 the sulfur atom could be essential for the biological activity of the compounds. We introduced the thio- group yielding the thiosemicarbazones to obtain more potent compounds with antifungal and antiaflatoxigenic ability.

In order to protect human health, one of the main goals of the project was to identify molecules without toxic effects against human cells. With regards to the cytotoxic potential, in this study semicarbazones and thiosemicarbazones did not present differences and generally they showed a very low antiproliferative activity.

To improve the capability of the free ligands to inhibit toxin production, copper and zinc complexes were then synthesized. Metal ions play a role in the aflatoxin biosynthesis.

Unfortunately, the metal complexes displayed also a strong cytotoxic activity against human cell lines: in particular, the copper complexes were more cytotoxic than the zinc complexes.

Interestingly, different human cells showed different sensitivities against the complexes.

The cytotoxicity of the compounds could depend on their ability to cross the phospholipid bilayer. The most of TSCsare molecules with a polar head, the thiosemicarbazide part, and an aromatic hydrophobic part. The metal coordination allows the ligand to hide the thiosemicarbazone polar part around the metal and the metal complex thus exposes the hydrophobic moiety to the solvent endowing it the features necessary to cross the cell membrane (Pelosi et al., 2010).

We do not yet know the mechanism involved in cellular uptake. Our molecules have molecular weight ranging from 150.0 g/mol to 1000.0 g/mol: therefore, the diffusion could be the main mechanism involved in cellular uptake. In addition, we can assume that cells utilize membrane transporters to facilitate the entry or export of molecules that are insufficiently permeable. The expression of these transporters may depend on cell type and consequently cellular sensitivity could depend on these mechanisms of active transport.

We did not exclude that the complex formation is a strategy by which a charged ion and a polar ligand molecule form a structure that exposes the hydrophobic part to the exterior and allows the two to enter the cell. This could explain the reason why complexes result to be more active than the parent ligands. Once inside, the metal ion and the thiosemicarbazone could act separately, the first altering the metal homeostasis within the cell and the other by interacting with enzymes or interfering with cellular pathways (Pelosi

119 et al., 2010). Indeed, several studies reported that TSCs could affect different cellular targets.

Alkaline Comet Assay was performed in order to identify the genotoxic load of molecules that showed any toxic activity. In general, our data indicate that both ligands and metal complexes could induce DNA damage at subtoxic concentration.

After the toxicological studies, the most promising compounds were F62 and F66, a cinnamaldehyde and an anthraquinone derivative, respectively.

All the results were registered in a database correlating chemical structures and biological/toxicological activities. Overall, we designed and synthesized a panel of 180 thiosemicarbazones: 76 ligands, 75 complexes and 29 starting materials. Among the ligands, we tested 5 semicarbazones and 71 thiosemicarbazones. The 75 metal complexes were synthesized using copper and zinc as main metal ions. The other complexes were obtained after complexation with nickel, manganese, iron and magnesium. The starting materials include 9 aldehydes, 6 ketones and 7 raw chemical materials.

The database reports the chemical and physical features including:

▪ Structure: chemical formula and molecular weight;

▪ Type: parent or derivative or raw material; ligand or metal complexes;

▪ Group: natural starting aldehyde;

▪ Chemical properties: LogP, H-bond acceptor and H-bond donator.

We also reported the ability to inhibit A. flavus growth and mycotoxin production and data related to cytotoxicity, genotoxicity and mutagenicity of the new molecules.

The preliminary analysis on the structure activity correlation did not identify single structural motifs responsible for the toxic activities of the compounds. Further analyses will be necessary to better understand these relationships.

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