Synthesis and In Vitro Evaluation of New Thiosemicarbazone Derivatives as Potential Antimicrobial Agents

In an effort to develop potent antimicrobial agents, new thiosemicarbazone derivatives were synthesized via the reaction of 4-[4(trifluoromethyl)phenyl]thiosemicarbazide with aromatic aldehydes.The compounds were evaluated for their inhibitory effects on pathogenic bacteria and yeasts using the CLSI broth microdilution method. Microplate Alamar Blue Assay was also carried out to determine the antimycobacterial activities of the compounds againstMycobacterium tuberculosisH37Rv. Among these derivatives, compounds 5 and 11 were more effective against Enterococcus faecalis (ATCC 29212) than chloramphenicol, whereas compounds 1, 2, and 12 and chloramphenicol showed the same level of antibacterial activity against E. faecalis. Moreover, compound 2 and chloramphenicol exhibited the same level of antibacterial activity against Staphylococcus aureus. On the other hand, themost potent anticandidal derivativeswere found as compounds2 and 5.These derivatives and ketoconazole exhibited the same level of antifungal activity againstCandida glabrata. According to theMicroplate Alamar Blue Assay, the tested compounds showed weak tomoderate antitubercular activity.


Introduction
Throughout history, it has been a major worldwide problem to treat microbial diseases caused by bacteria and fungi due to impetuous development of resistance to antibacterial and antifungal drugs.Antimicrobial resistance is observed in pathogenic bacteria before exposure to antimicrobial agent or related to uncontrolled infection after a patient receives that antimicrobial.Bacteria could be resistant inherently staying in variable temperatures and in highly acidic or alkaline conditions or could develop resistance by de novo mutation or through the transmission of resistance genes from other organisms.Gram-positive bacterial infections particularly caused by Staphylococcus aureus and Enterococcus spp.represent a major health problem hampering the therapeutic efficacy of antibacterial drugs due to their frequent multidrug resistance [1][2][3][4][5][6].
In the past two decades, the incidence of fungal infections has gone up all over the world.Fungal infections classified as allergic reactions to fungal proteins and toxic reactions to fungi toxins and mycoses pose a constant and serious threat to human life.Infections caused by Candida species represent the main reason of opportunistic fungal infections worldwide.Candidemia and other forms of invasive candidiasis are important causes of the increasing rate of morbidity and mortality.Candida albicans is the most common etiological agent of candidiasis and Candida glabrata is a growing concern in clinical settings because it causes mucosal and systemic bloodstream infections [7][8][9][10][11][12].
The World Health Organization (WHO) evaluates tuberculosis as one of the deadliest global diseases associated with the occurrence of multiple drug resistant strains of Mycobacterium tuberculosis discovered by Robert Koch in 1882.Mycobacterium tuberculosis H37Rv, the pathogenic agent of tuberculosis, is recognized to reproduce in host cells by pulling through host cell defenses supplied by macrophages and dendritic cells.In 2013, approximately 9 million people were diagnosed with active tuberculosis and 1.5 million deaths attributed to this disease.Diagnosis and treatment are crucial for tuberculosis, particularly in high-risk populations.Early diagnosis of tuberculosis with radiography and computed tomography methods is essential for effective treatment and leads to a reduced onward transmission of tuberculosis.Despite the discovery of tuberculin test and Bacillus-Calmette Guérin (BCG) vaccine and some main drugs called streptomycin, isoniazid, and rifampicin for the treatment of tuberculosis, it has remained one of the most dangerous diseases especially associated with mycobacterial resistance in chemotherapy and the presence of coinfectious diseases such as acquired immune deficiency syndrome (AIDS) [8][9][10][11][12][13][14][15][16][17][18].
Thiosemicarbazones have been investigated for medicinal studies for a long while due to their wide range of biological activities including antineoplastic, antimycobacterial, antibacterial, antifungal, antiviral, and antimalarial effects and versatility as nitrogen and sulfur donors allowing them to bring on a great variety of coordination modes.Thiosemicarbazone is also known as an iron-chelating group, bonding the sulfur and azomethine nitrogen atoms.The complexes of nitrogen and sulfur atoms with metal ions may be considered potential biological agents.In recent years, many thiosemicarbazone derivatives have been synthesized and evaluated for their antibacterial activity.The current use of these agents in bacterial infections has led to the development of novel antibacterial drugs [19][20][21][22][23][24][25].
Thiacetazone, p-acetamidobenzaldehyde thiosemicarbazone, is one of the oldest and cheapest agents used as a second line drug for tuberculosis treatment (Figure 1).This agent is estimated to show antimycobacterial activity inhibiting mycolic acid biosynthesis.Thiacetazone shows only bacteriostatic activity and develops resistance easily during tuberculosis therapy.It is partly cross-resistant to ethionamide and it is forbidden to be used in patients with human immunodeficiency virus (HIV) owing to high frequency of Stevens-Johnson syndrome [26][27][28].

In Vitro Evaluation of Antimicrobial Activity.
The microbiological assay was carried out according to the CLSI reference M7-A7 broth microdilution method as described previously [30].Chloramphenicol and ketoconazole were used as reference agents.
The structures of new compounds were confirmed by spectroscopic data and elemental analysis.In the IR spectra of compounds 1-12, the N-H stretching bands were observed in the region 3367-3275 cm −1 .The aromatic C-H stretching vibrations gave rise to bands at 3165-3116 cm −1 .C=N, C=C stretching and N-H bending bands were observed in the region 1616-1487 cm −1 .In the IR spectra of compound 12, the stretching band for C≡N group occurred at 2220.07 cm −1 .
In the 1 H NMR spectra of compounds 1-12, the signal due to the CH=N proton was observed in the region 8.0-8.3 ppm.The N-H protons appeared in the region 10-13 ppm.
In the 1 H NMR spectra of some compounds, N-H and CH=N protons gave rise to two singlet peaks in accordance with the presence of the  and  isomers [31].Other aromatic and aliphatic protons were observed at expected regions.The mass spectral data of the synthesized compounds were found in full agreement with the proposed structures.The compounds were tested in vitro against a number of Gram-positive and Gram-negative bacteria and yeasts using broth microdilution method.Based on this assay, the minimum inhibitory concentrations (MICs) of the compounds were determined.The observed MIC values were in the range of 100-1600 g/mL.In general, the tested compounds exhibited more potent inhibitory activity towards Gram-positive bacteria compared to Gram-negative bacteria (Table 2).
Among these derivatives, fluorosubstituted compound 2 exhibited the highest antibacterial activity against S. aureus with a MIC value of 100 g/mL when compared with chloramphenicol (MIC = 100 g/mL).This outcome pointed out the importance of fluorine substituent for antibacterial activity against S. aureus.
Compounds 1, 2, and 12 and chloramphenicol showed the same level of antibacterial activity against E. faecalis (ATCC 29212) with a MIC value of 200 g/mL, whilst compounds 5 and 11 were more effective against E. faecalis than chloramphenicol.Generally electron withdrawing groups such as nitro and trifluoromethyl enhanced antibacterial activity, whereas electron donating substituents decreased antibacterial activity against E. faecalis.
In addition, in vitro antimycobacterial effects of the compounds were investigated against the drug resistant M. tuberculosis H37Rv strain using Microplate Alamar Blue Assay (MABA).Compounds 1, 7, 8, 9, 10, 11, and 12 exhibited antimycobacterial activity with a MIC value of 200 g/mL, whereas other derivatives showed antimycobacterial activity with a MIC value of 400 g/mL.As shown in Table 3, compounds 2 and 5 were the most potent antifungal derivatives against C. glabrata with a MIC value of 200 g/mL when compared with ketoconazole (MIC = 200 g/mL).It can be concluded that fluorine and nitro substituents increase antifungal activity against C. glabrata.

Conclusion
In the present paper, new thiosemicarbazone derivatives were synthesized and evaluated for their inhibitory effects on pathogenic bacteria including M. tuberculosis H37Rv.The antifungal effects of the compounds on Candida species were also investigated.
In general, the tested compounds exhibited more potent inhibitory effects on Gram-positive bacteria compared to Gram-negative bacteria.Among these derivatives, compound 2 was the most potent antibacterial derivative against S. aureus with a MIC value of 100 g/mL when compared with chloramphenicol (MIC = 100 g/mL).This outcome indicated that fluorine substituent increased antibacterial activity against S. aureus.
Compounds 5 and 11 exhibited the highest antibacterial activity against E. faecalis (ATCC 29212) with a MIC value of 100 g/mL when compared with chloramphenicol (MIC = 200 g/mL).This result demonstrated that nitro and trifluoromethyl groups increased antibacterial activity against E. faecalis.
According to MABA, the tested compounds exhibited weak to moderate antimycobacterial activity against M. tuberculosis H37Rv.
Compounds 2 and 5 and ketoconazole showed the same level of antifungal activity against C. glabrata with a MIC value of 200 g/mL.This outcome pointed out the importance of fluorine and nitro substituents for antifungal activity against C. glabrata.
Antitubercular Activity.Mycobacterium tuberculosis H37Rv (ATCC 27294) was provided from American Type Culture Collection (ATCC) cell bank.The cells were grown in ATCC® Medium 1395: Middlebrook 7H9 broth with ADC enrichment at 37 ∘ C for 30 days.The turbidity of the cultures was adjusted to McFarland standard number 1.The following concentrations

Table 3 :
Anticandidal activity of the thiosemicarbazone derivatives (MIC values in g/mL).