Synthesis and Antimicrobial Evaluation of the Potassium Salts of Benzhydrazine Dithiocarbamates

The lack of personal and social hygiene has led to the spread of infectious diseases in society (1). In addition, drug-resistant bacterial and fungal pathogenic strains have increasingly spread because of the inappropriate and unnecessary use of antibiotics and antifungal agents (2). As a result, the discovery, design, and synthesis of new antimicrobial compounds have become an inevitable necessity in the field of hygiene and healthcare (3). Dithiocarbamates are biologically active organic compounds containing the -N(C=S)Smoiety, which are usually prepared in the presence of a base from the reaction of amines or their equivalent derivatives with carbon disulfide (4). According to (5), numerous available functional groups in dithiocarbamates have given them diverse therapeutic properties (Figure 1). Thiram is a potent scabicidal drug, which is especially prescribed for the treatment of skin diseases. It is also used as a fungicide in agriculture (6). In addition, dithiocarb as a chelating agent helps to remove toxic metals from body tissues (7). Further, (8) confirmed the inhibitory effects of brassinin derivatives on cancer cell lines, especially human clones (Caco-2). Furthermore, the blocking properties of valine dithiocarbamate zinc(II) complex against breast cancer cells (MCF-7) were more pronounced compared to cisplatin (9). Moreover, the excellent inhibitory effects against two β-carbonic anhydrase enzymes belonging to pathogenic Mycobacterium tuberculosis were observed with N-monoand N-,N-disubstituted dithiocarbamates (10). The varied biological properties of dithiocarbamates encouraged us to synthesize several derivatives of this family via the reaction of different benzhydrazines and carbon disulfide, and their antimicrobial properties were assessed against a wide range of bacterial and fungal pathogens.

F254. Additionally, the Kruss type KSP1N melting point meter (Germany) and UV-2100 RAY Leigh UV-Vis spectrophotometer (China) were used to determine uncorrected melting points and the absorption spectra, respectively. The Fourier-transform infrared (FT-IR) spectra of all synthesized dithiocarbamates were recorded on a Bruker Tensor-27 FT-IR spectrometer (Germany) available in the instrumental analysis laboratory of University of Zabol. Their 1 H-and 13 C-NMR (400 & 100 MHz) spectra were collected via a Bruker FT-NMR Ultra Shield-400 spectrometer (Germany).
General Process for the Preparation of Dithiocarbamates 3a-f A mixture containing 10 mmol of hydrazines 1a-f, 10 mmol of potassium hydroxide (0.56 g), and 25 mL of diethyl ether in a 50-mL round-bottom flask was stirred in an ice-bath. Next, 10 mmol of carbon disulfide (0.76 g) was added dropwise to it for 1 hour under these conditions. The reaction continued for another 3 hours at room temperature. Eventually, the solids were collected, washed with cold ethanol (5 mL) and diethyl ether (5 mL), respectively, and then dried over P 2 O 5 in the vacuum desiccator to give potassium salts of dithiocarbamate 3a-f.  13

Minimum Inhibitory Concentration Experiment
To this end, 20 μL of each dithiocarbamate was added to twelve wells in a row of a 96-well microliter plate at concentrations of 20480, 10240, 5120, 2560, 1280, 640, 320, 160, 80, 40, 20, and 10 μg.mL -1 in distilled water, respectively. Then, 80 μL of RPMI 1640 or MHB, as well as 100 μL of microbial suspensions were added to all wells. Next, the plates were incubated at 37 and 35°C for 20 and 48 hours for bacteria and fungi on an orbital shaker (100 rpm), respectively. Eventually, the minimum inhibitory concentration (MIC) value was the lowest concentration of salts which stopped microbial growth (11).

MBC and Minimum Fungicidal Concentration Experiments
The samples of all non-turbid wells in the MIC experiment were re-cultured in RPMI 1640 or MHA agar media plates. Then, the plates were incubated for 24 hours at appropriate temperatures except that 45%-55% relative humidity was supplied for fungi. Finally, the Minimum Fungicidal Concentration (MFC) or MBC value was determined as the lowest concentration of compounds that killed microorganisms (11).

Results and Discussion
Potassium dithiocarbamates 3a-f were generated via the reaction of benzhydrazines 1a-f and carbon disulfide (2). In addition, potassium hydroxide and diethyl ether were used as base and solvent, respectively (Scheme 1). The synthetic data are reported in Table 1. The chemical structure of synthesized dithiocarbamates was characterized by spectral data (Figure 2).
In vitro blocking potential of all synthesized dithiocarbamates was assessed on some important pathogenic bacteria and fungi including four gram-positive and 6 Gram-negative bacterial strains, 2 molds and one yeast. Further, ampicillin and clotrimazole were used as control drugs. As shown in Table 2, a wide variety of activities was obtained with dithiocarbamates that had substituted or unsubstituted phenyl. All dithiocarbamates except for salts 3c and 3d inhibited the tested bacteria and no blocking effects on Escherichia coli and Klebsiella pneumoniae strains were observed with derivatives 3c and 3d. On the other hand, good inhibitory effects against gram-negative Shigella flexneri and Listeria monocytogenes, as well as gram-positive Bacillus cereus and Staphylococcus aureus strains were observed with the potassium salts of morpholine dithiocarbamate, along with its Cu(II) and Ni(II) complexes. More precisely, complexation decreased antibacterial activities (15). Furthermore, the disk diffusion method was applied to determine the blocking properties of potassium 3-dithiocarboxy-3-aza-5-aminopentanoate on nine hospital bacteria (16). Pyrrolidine dithiocarbamates are potent chelating agents and ionophores that easily enter into the cell (17). Moreover, the antibacterial effects of their Zn(II) complexes are usually higher compared to Cu(II) equivalents.
The in vitro blocking effects of synthetic salts were investigated on three pathogenic fungi (Table 3). Based on the results, all synthesized dithiocarbamates affected Candida albicans and Fusarium oxysporum strains. No inhibitory activity against Aspergillus fumigatus was observed with dithiocarbamates 3b and 3d. Additionally, the antifungal activity of some synthesized Pt(II), Pd(II), and Ni(II) dithiocarbamate complexes was studied against Penicillium citrinum, Aspergillus flavus, Aspergillus niger, and Aspergillus parasiticus. They were more efficient at blocking fungi than nystatin (18). Some nontoxic dirutheniumpentadithiocarbamate complexes were synthesized to treat invasive fungal infections. In other words, their MIC values were very similar to those of fluconazole (19). It was proposed that dithiocarbamates act as nonspecific and multisite antifungal agents (20). They further inactivate cellular thiol enzymes and disrupt the energy supply process of fungal cells.

Conclusions
In general, dithiocarbamates are multi-functionalized compounds with a variety of biological properties and thus their synthesis is important. This study evaluated the antibacterial and antifungal effects of 6 synthesized potassium dithiocarbamates (three new salts) on pathogenic bacteria and fungi. These dithiocarbamates showed relatively acceptable antibacterial effects against Gram-negative S. enterica and S. dysenteriae, as well as Gram-positive S. epidermidis and L. monocytogenes. In addition, good to excellent antifungal activities were observed with dithiocarbamates. More precisely, they are potent chelating ligands and complexation may improve    their antimicrobial properties. Eventually, these watersoluble salts can be applied as pesticides and plant growthpromoting agents although their toxic effects must be studied in future studies.