Journal of Chemistry
Volume 2016 (2016), Article ID 1692540, 7 pages
http://dx.doi.org/10.1155/2016/1692540

Research Article

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

Zafer Asım Kaplancıklı,¹ Mehlika Dilek Altıntop,¹ Belgin Sever,¹ Zerrin Cantürk,² and Ahmet Özdemir¹

¹Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey
²Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey

Received 16 December 2015; Revised 10 January 2016; Accepted 13 January 2016

Academic Editor: Giuseppe Gumina

Copyright © 2016 Zafer Asım Kaplancıklı et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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 against Mycobacterium tuberculosis H37Rv. 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, the most potent anticandidal derivatives were found as compounds 2 and 5. These derivatives and ketoconazole exhibited the same level of antifungal activity against Candida glabrata. According to the Microplate Alamar Blue Assay, the tested compounds showed weak to moderate antitubercular activity.

1. 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 [1–6].

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 [7–12].

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) [8–18].

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 [19–25].

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 [26–28].

Figure 1: Thiacetazone.

On the basis of aforementioned findings, herein, we synthesized a new series of thiosemicarbazone derivatives and investigated their in vitro antimicrobial and antimycobacterial effects.

2. Experimental Section

2.1. Chemistry

All reagents were purchased from commercial suppliers and were used without further purification. Melting points (Mp) were determined on an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. IR spectra were recorded on an IRPrestige-21 Fourier Transform Infrared spectrophotometer (Shimadzu, Tokyo, Japan). ¹H NMR spectra were recorded on a Bruker spectrometer (Bruker, Billerica, MA, USA). Mass spectra were recorded on an Agilent LC-MSD-Trap-SL Mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Elemental analyses (C, H, and N) were performed on a Perkin Elmer EAL 240 elemental analyzer (Perkin-Elmer, Norwalk, CT, USA) and the results were within ±0.4% of the theoretical values. Thin Layer Chromatography (TLC) was performed on TLC Silica gel 60 F₂₅₄ aluminium sheets (Merck, Darmstadt, Germany) to check the purity of the compounds.

2.2. General Procedure for the Synthesis of the Compounds

2.2.1. 4-[4-(Trifluoromethyl)phenyl]thiosemicarbazide

A mixture of 4-(trifluoromethyl)phenyl isothiocyanate (0.1 mol) and hydrazine hydrate (0.2 mol) in ethanol (30 mL) was stirred at room temperature for 4 h and then filtered. The residue was crystallized from ethanol [29].

2.2.2. 4-[4-(Trifluoromethyl)phenyl]-1-(substituted benzylidene)thiosemicarbazides (1–12)

A mixture of 4-[4-(trifluoromethyl)phenyl]thiosemicarbazide (0.01 mol) and aromatic aldehyde (0.01 mol) was refluxed in ethanol for 6 h, filtered, and crystallized from ethanol [29].

2.2.3. 4-[4-(Trifluoromethyl)phenyl]-1-(benzylidene)thiosemicarbazide (1)

IR (cm⁻¹): 3313.71 (N-H stretching), 3140.11 (aromatic C-H stretching), 1614.42, 1539.20, 1504.48 (C=N, C=C stretching and N-H bending), 1327.03, 1278.81, 1099.43 (C-N, C=S stretching and aromatic C-H in plane bending), 846.75, 754.17, 686.66 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 7.43–7.45 (m, 3H, aromatic protons), 7.72 (d,  Hz, 2H, aromatic protons), 7.89–7.91 (m, 4H, aromatic protons), 8.19 (s, 1H, CH=N), 10.29 (s, 1H, N-H), 12.03 (s, 1H, N-H); MS (ESI) (): (M + H)⁺ 324; Anal. Calcd for C₁₅H₁₂F₃N₃S: C, 55.72; H, 3.74; N, 13.00; Found: C, 55.70; H, 3.76; N, 13.00.

2.2.4. 4-[4-(Trifluoromethyl)phenyl]-1-(4-fluorobenzylidene)thiosemicarbazide (2)

IR (cm⁻¹): 3288.63 (N-H stretching), 3153.61 (aromatic C-H stretching), 2983.88 (C-H stretching), 1616.35, 1600.92, 1544.98, 1504.48 (C=N, C=C stretching and N-H bending), 1319.31, 1269.16, 1232.51, 1192.01, 1161.15, 1109.07, 1064.71, 1014.56 (C-N, C=S stretching and aromatic C-H in plane bending), 831.32 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 7.62–7.63 (m, 2H, aromatic protons), 7.72 (d,  Hz, 2H, aromatic protons), 7.89 (d,  Hz, 2H, aromatic protons), 7.97–8.01 (m, 2H, aromatic protons), 8.18 (s, 1H, CH=N), 10.31 (s, 1H, N-H), 12.03 (s, 1H, N-H); MS (ESI) (): (M + H)⁺ 342; Anal. Calcd for C₁₅H₁₁F₄N₃S: C, 52.78; H, 3.25; N, 12.31; Found: C, 52.80; H, 3.24; N, 12.30.

2.2.5. 4-[4-(Trifluoromethyl)phenyl]-1-(4-chlorobenzylidene)thiosemicarbazide (3)

IR (cm⁻¹): 3329.14 (N-H stretching), 3134.33 (aromatic C-H stretching), 2980.02 (C-H stretching), 1616.35, 1593.20, 1541.12, 1489.05 (C=N, C=C stretching and N-H bending), 1321.24, 1274.95, 1109.07, 1085.92, 1062.78, 1012.63 (C-N, C=S stretching and aromatic C-H in plane bending), 823.60 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 7.49–7.51 (m, 2H, aromatic protons), 7.73 (d,  Hz, 2H, aromatic protons), 7.88 (d,  Hz, 2H, aromatic protons), 7.94–7.96 (m, 2H, aromatic protons), 8.16 (s, 1H, CH=N), 10.34 (s, 1H, N-H), 12.07 (s, 1H, N-H); MS (ESI) (): (M + H)⁺ 358; Anal. Calcd for C₁₅H₁₁ClF₃N₃S: C, 50.36; H, 3.10; N, 11.74; Found: C, 50.35; H, 3.10; N, 11.75.

2.2.6. 4-[4-(Trifluoromethyl)phenyl]-1-(4-bromobenzylidene)thiosemicarbazide (4)

IR (cm⁻¹): 3329.14 (N-H stretching), 3142.04 (aromatic C-H stretching), 2981.95 (C-H stretching), 1614.42, 1589.34, 1539.20, 1521.84, 1487.12 (C=N, C=C stretching and N-H bending), 1323.17, 1273.02, 1207.44, 1165.00, 1107.14, 1062.78, 1006.84 (C-N, C=S stretching and aromatic C-H in plane bending), 840.96, 819.75, 808.17 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 7.62–7.64 (m, 2H, aromatic protons), 7.73 (d,  Hz, 2H, aromatic protons), 7.88 (d,  Hz, 4H, aromatic protons), 8.15 (s, 1H, CH=N), 10.34 (s, 1H, N-H), 12.07 (s, 1H, N-H); MS (ESI) (): (M + H)⁺ 403; Anal. Calcd for C₁₅H₁₁BrF₃N₃S: C, 44.79; H, 2.76; N, 10.45; Found: C, 44.78; H, 2.74; N, 10.46.

2.2.7. 4-[4-(Trifluoromethyl)phenyl]-1-(4-nitrobenzylidene)thiosemicarbazide (5)

IR (cm⁻¹): 3307.92 (N-H stretching), 3116.97 (aromatic C-H stretching), 2980.02 (C-H stretching), 1614.42, 1541.12, 1516.05 (C=N, C=C stretching and N-H bending), 1330.88, 1317.38, 1188.15, 1105.21, 1066.64 (C-N, C=S stretching and aromatic C-H in plane bending), 837.11 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 7.75 (d,  Hz, 2H, aromatic protons), 7.87 (d,  Hz, 2H, aromatic protons), 8.18–8.20 (m, 2H, aromatic protons), 8.24–8.27 (m, 3H, CH=N, aromatic protons), 10.48 (s, 1H, N-H), 12.28 (s, 1H, N-H); MS (ESI) (): (M + H)⁺ 369; Anal. Calcd for C₁₅H₁₁F₃N₄O₂S: C, 48.91; H, 3.01; N, 15.21; Found: C, 48.90; H, 3.03; N, 15.20.

2.2.8. 4-[4-(Trifluoromethyl)phenyl]-1-(4-hydroxybenzylidene)thiosemicarbazide (6)

IR (cm⁻¹): 3367.71, 3286.70 (N-H stretching), 3165.19 (aromatic C-H stretching), 1604.77, 1541.12, 1510.26, 1494.83 (C=N, C=C stretching and N-H bending), 1327.03, 1276.88, 1186.22, 1163.08, 1099.43, 1064.71, 1018.41 (C-N, C-O, C=S stretching and aromatic C-H in plane bending), 831.32 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 6.80–6.83 (m, 2H, aromatic protons), 7.70–7.74 (m, 4H, aromatic protons), 7.92 (d,  Hz, 2H, aromatic protons), 8.09 (s, 1H, CH=N), 9.97 (s, 1H, O-H), 10.17 (s, 1H, N-H), 11.85 (s, 1H, N-H); MS (ESI) (): (M + H)⁺ 340; Anal. Calcd for C₁₅H₁₂F₃N₃OS: C, 53.09; H, 3.56; N, 12.38; Found: C, 53.10; H, 3.55; N, 12.37.

2.2.9. 4-[4-(Trifluoromethyl)phenyl]-1-(4-methoxybenzylidene)thiosemicarbazide (7)

IR (cm⁻¹): 3142.04 (aromatic C-H stretching), 2978.09 (C-H stretching), 1606.70, 1541.12, 1490.97 (C=N, C=C stretching and N-H bending), 1246.02, 1207.44, 1165.00, 1107.14, 1060.85, 1028.06, 1016.49 (C-N, C=S stretching and aromatic C-H in plane bending), 829.39, 792.74 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 3.80 (s, 3H, OCH₃), 6.98–7.00 (m, 2H, aromatic protons), 7.71 (d,  Hz, 2H, aromatic protons), 7.83–7.86 (m, 2H, aromatic protons), 7.91 (d,  Hz, 2H, aromatic protons), 8.05 and 8.14 (2s, 1H, CH=N), 10.22 and 10.35 (2s, 1H, N-H), 11.77 and 11.92 (2s, 1H, N-H); MS (ESI) (): (M + H)⁺ 354; Anal. Calcd for C₁₆H₁₄F₃N₃OS: C, 54.38; H, 3.99; N, 11.89; Found: C, 54.37; H, 3.98; N, 11.91.

2.2.10. 4-[4-(Trifluoromethyl)phenyl]-1-(4-methylbenzylidene)thiosemicarbazide (8)

IR (cm⁻¹): 3309.85 (N-H stretching), 3140.11 (aromatic C-H stretching), 2978.09 (C-H stretching), 1616.35, 1541.12, 1492.90 (C=N, C=C stretching and N-H bending), 1325.10, 1278.81, 1109.07, 1062.78 (C-N, C=S stretching and aromatic C-H in plane bending), 806.25 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 2.33 (s, 3H, CH₃), 7.25 (d,  Hz, 2H, aromatic protons), 7.72 (d,  Hz, 2H, aromatic protons), 7.79 (d,  Hz, 2H, aromatic protons), 7.90 (d,  Hz, 2H, aromatic protons), 8.06 and 8.15 (2s, 1H, CH=N), 10.26 and 10.38 (2s, 1H, N-H), 11.81 and 11.96 (2s, 1H, N-H); MS (ESI) (): (M + H)⁺ 338; Anal. Calcd for C₁₆H₁₄F₃N₃S: C, 56.96; H, 4.18; N, 12.46; Found: C, 56.95; H, 4.17; N, 12.45.

2.2.11. 4-[4-(Trifluoromethyl)phenyl]-1-(4-isopropylbenzylidene)thiosemicarbazide (9)

IR (cm⁻¹): 3290.56 (N-H stretching), 3140.11 (aromatic C-H stretching), 2976.16 (C-H stretching), 1616.35, 1541.12, 1492.90 (C=N, C=C stretching and N-H bending), 1323.17, 1276.88, 1107.14, 1064.71 (C-N, C=S stretching and aromatic C-H in plane bending), 839.03, 817.32 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 1.21 (d,  Hz, 6H, 2CH₃), 2.89–2.95 (m, 1H, CH, i-Pr), 7.31 (d,  Hz, 2H, aromatic protons), 7.72 (d, J = 10 Hz, 2H, aromatic protons), 7.81 (d,  Hz, 2H, aromatic protons), 7.90 (d,  Hz, 2H, aromatic protons), 8.07 and 8.15 (2s, 1H, CH=N), 10.25 and 10.39 (2s, 1H, N-H), 11.83 and 11.97 (2s, 1H, N-H); MS (ESI) (): (M + H)⁺ 366; Anal. Calcd for C₁₈H₁₈F₃N₃S: C, 59.16; H, 4.97; N, 11.50; Found: C, 59.17; H, 4.96; N, 11.51.

2.2.12. 4-[4-(Trifluoromethyl)phenyl]-1-(4-dimethylaminobenzylidene)thiosemicarbazide (10)

IR (cm⁻¹): 3275.13 (N-H stretching), 3140.11 (aromatic C-H stretching), 2978.09 (C-H stretching), 1598.99, 1548.84, 1519.91, 1492.90 (C=N, C=C stretching and N-H bending), 1325.10, 1315.45, 1276.88, 1161.15, 1105.21, 1060.85 (C-N, C=S stretching and aromatic C-H in plane bending), 943.19, 840.96, 810.10, 786.96 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 2.97 (s, 6H, N(CH₃)₂), 6.72 (d,  Hz, 2H, aromatic protons), 7.68–7.71 (m, 4H, aromatic protons), 7.94 (d,  Hz, 2H, aromatic protons), 8.07 (s, 1H, CH=N), 10.12 (s, 1H, N-H), 11.79 (s, 1H, N-H); MS (ESI) (): (M + H)⁺ 367; Anal. Calcd for C₁₇H₁₇F₃N₄S: C, 55.73; H, 4.68; N, 15.29. Found: C, 55.72; H, 4.69; N, 15.26.

2.2.13. 4-[4-(Trifluoromethyl)phenyl]-1-(4-trifluoromethylbenzylidene)thiosemicarbazide (11)

IR (cm⁻¹): 3132.40 (aromatic C-H stretching), 2985.81, 2883.58 (C-H stretching), 1598.99, 1541.12, 1492.90 (C=N, C=C stretching and N-H bending), 1394.53, 1319.31, 1064.71 (C-N, C=S stretching and aromatic C-H in plane bending), 825.53 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 7.76 (dd, = 10 Hz, 20 Hz, 4H, aromatic protons), 7.88 (d,  Hz, 2H, aromatic protons), 8.14 (d, = 10 Hz, 2H, aromatic protons), 8.24 (s, 1H, CH=N), 10.42 and 10.62 (2s, 1H, N-H), 12.02 and 12.18 (2s, 1H, N-H); MS (ESI) (): (M + H)⁺ 392; Anal. Calcd for C₁₆H₁₁F₆N₃S: C, 49.11; H, 2.83; N, 10.74; Found: C, 49.10; H, 2.82; N, 10.76.

2.2.14. 4-[4-(Trifluoromethyl)phenyl]-1-(4-cyanobenzylidene)thiosemicarbazide (12)

IR (cm⁻¹): 3309.85 (N-H stretching), 3116.97 (aromatic C-H stretching), 2978.09, 2883.58 (C-H stretching), 2220.07 (C≡N stretching), 1541.12, 1521.84, 1498.69 (C=N, C=C stretching and N-H bending), 1325.10, 1269.16, 1190.08, 1166.93, 1105.21, 1087.85, 1066.64, 1016.49 (C-N, C=S stretching and aromatic C-H in plane bending), 921.97, 831.32 (aromatic C-H out of plane bending); ¹H NMR (500 MHz, DMSO-) δ (ppm): 7.74 (d,  Hz, 2H, aromatic protons), 7.86–7.90 (m, 4H, aromatic protons), 8.12 (d,  Hz, 2H, aromatic protons), 8.20 (s, 1H, CH=N), 10.43 (s, 1H, N-H), 12.22 (s, 1H, N-H); MS (ESI) (): (M + H)⁺ 349; Anal. Calcd for C₁₆H₁₁F₃N₄S: C, 55.17; H, 3.18; N, 16.08; Found: C, 55.15; H, 3.19; N, 16.07.

2.3. Microbiology

2.3.1. 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.

Compounds 1–12 were investigated for their in vitro growth inhibitory activity against pathogenic bacteria and fungi. Microorganisms used in this study were as follows: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Enterococcus faecalis (ATCC 51922), Listeria monocytogenes (ATCC 1911), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35218), Escherichia coli (ATCC 25922), Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), and Candida parapsilosis (ATCC 22019).

2.3.2. Microplate Alamar Blue Assay (MABA) for 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 of the compounds and rifampicin (Sigma, R3501, China) were used: 1.5625–800 μg/mL. All black, clear-bottomed, 96-well plates (Corning 3340, USA) were incubated at 37°C in 5% CO₂ for 7 days. On day 7 of the incubation, freshly prepared 1 : 1 mixture of Alamar Blue reagent (1 : 10 dilution, Invitrogen, 1025, USA) and 10% Tween 80 was added to one well among the positive controls. The plates were further incubated at 37°C for 24 h. If the contents of the well turned pink, the reagent mixture was added to all the wells of the microplate.

3. Results and Discussion

The synthesis of new thiosemicarbazone derivatives (1–12) was performed as described in Scheme 1. 4-[4-(Trifluoromethyl)phenyl]thiosemicarbazide was synthesized via the reaction of 4-(trifluoromethyl)phenyl isothiocyanate with hydrazine hydrate. The treatment of 4-[4-(trifluoromethyl)phenyl]thiosemicarbazide with aromatic aldehydes afforded the thiosemicarbazone derivatives (1–12). The yields and melting points (Mp) of the compounds were given in Table 1.

Table 1: The yields and melting points (Mp) of the thiosemicarbazone derivatives (1–12).

Compound
R
Yield (%)
Mp (°C)

1
H
80
198

2
F
82
169

3
Cl
87
211

4
Br
90
224

5
NO₂
94
233

6
OH
72
204

7
OCH₃
78
200

8
CH₃
79
203

9
CH(CH₃)₂
72
180

10
N(CH₃)₂
83
214

11
CF₃
80
205

12
CN
96
232

Scheme 1: The synthetic route for the preparation of the thiosemicarbazone derivatives (1–12). Reagents and conditions: (i) NH₂NH₂·H₂O, ethanol, rt, 4 h; (ii) ArCHO, ethanol, reflux, 6 h.

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 ¹H 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 ¹H 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 μg/mL. In general, the tested compounds exhibited more potent inhibitory activity towards Gram-positive bacteria compared to Gram-negative bacteria (Table 2).

Table 2: Antibacterial activity of the thiosemicarbazone derivatives (MIC values in µg/mL).

Compound
A
B
C
D
E
F
G
H
I

1
400
200
400
400
400
400
400
400
200

2
100
200
1600
1600
800
1600
1600
400
400

3
400
400
400
800
400
400
400
400
400

4
400
400
400
400
400
400
800
400
400

5
400
100
400
400
400
400
400
400
400

6
400
400
400
400
400
400
400
400
400

7
400
400
400
800
400
400
800
400
200

8
400
400
400
800
400
400
800
400
200

9
400
400
400
400
400
400
800
400
200

10
400
400
400
400
400
400
400
400
200

11
400
100
400
800
400
400
400
400
200

12
400
200
400
400
400
400
400
400
200

Chloramphenicol
100
200
200
100
200
200
100
25
Nt

Rifampicin
Nt
Nt
Nt
Nt
Nt
Nt
Nt
Nt
25

A: S. aureus (ATCC 25923), B: E. faecalis (ATCC 29212), C: E. faecalis (ATCC 51922), D: L. monocytogenes (ATCC 1911), E: K. pneumoniae (ATCC 700603), F: P. aeruginosa (ATCC 27853), G: E. coli (ATCC 35218), H: E. coli (ATCC 25922), and I: M. tuberculosis H37Rv.

Nt: not tested.

Among these derivatives, fluorosubstituted compound 2 exhibited the highest antibacterial activity against S. aureus with a MIC value of 100 μg/mL when compared with chloramphenicol (MIC = 100 μg/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 μg/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 μg/mL, whereas other derivatives showed antimycobacterial activity with a MIC value of 400 μg/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 μg/mL when compared with ketoconazole (MIC = 200 μg/mL). It can be concluded that fluorine and nitro substituents increase antifungal activity against C. glabrata.

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

Compound
A
B
C
D

1
400
400
200
200

2
400
200
200
200

3
400
400
400
200

4
400
400
200
200

5
400
200
200
200

6
400
400
200
200

7
400
400
200
200

8
400
400
200
200

9
400
400
200
200

10
400
400
200
200

11
400
400
400
200

12
400
400
400
200

Ketoconazole
200
200
3.125
50

A: C. albicans (ATCC 90028), B: C. glabrata (ATCC 90030), C: C. krusei (ATCC 6258), and D: C. parapsilosis (ATCC 22019).

4. 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 μg/mL when compared with chloramphenicol (MIC = 100 μg/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 μg/mL when compared with chloramphenicol (MIC = 200 μg/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 μg/mL. This outcome pointed out the importance of fluorine and nitro substituents for antifungal activity against C. glabrata.

Conflict of Interests

The authors have declared no conflict of interests.

Acknowledgment

This study was supported by Anadolu University Scientific Research Projects Commission under Grants nos. 1404S137 and 1409S386.

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