Synthesis, Characterization, and Antimicrobial Evaluation of Some Novel Hydrazinecarbothioamides

Efe Doğukan DİNCEL; Ebru Didem KURAN; Harika Öykü DİNÇ; Faika BAŞOĞLU; Emrah GÜLER; Nuray ULUSOY GÜZELDEMİRCİ

doi:10.4274/tjps.galenos.2025.90304

ABSTRACT

Objectives

This study focused on synthesizing and characterizing novel thiosemicarbazide derivatives containing a 1,2,4-triazole moiety and evaluating their antimicrobial activity against several bacterial strains. The research aimed to identify key structural features that enhance antimicrobial efficacy through structure-activity relationship analysis and identify the minimum inhibitory concentration (MIC) of the most potent compounds to assess their potential for further development as antimicrobial agents.

Materials and Methods

Nine novel thiosemicarbazide derivatives containing a 1,2,4-triazole moiety were synthesized by reacting 1,2,4-triazole derivatives with thiosemicarbazide precursors, and the products were characterized using infrared spectroscopy, proton nuclear magnetic resonance (¹H-NMR), carbon-13 nuclear magnetic resonance (¹³C-NMR) spectroscopy, and elemental analysis. The antimicrobial activity of these compounds (5a-i) was tested against Klebsiella pneumoniae (K. pneumoniae),Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa (P. aeruginosa), using microdilution, disk diffusion, and broth microdilution methods. Dimethyl sulfoxide was used as a negative control, and Vancomycin and Meropenem were used as positive controls, with all results converted to µM for consistent analysis.

Results

The synthesized thiosemicarbazide derivatives (5a-i) were confirmed to be structurally correct through Fourier-transform infrared spectroscopy, ¹H-NMR, and ¹³C-NMR spectroscopy. Among the tested compounds, 5e (4-bromophenyl) and 5g (n-propyl) showed significant antimicrobial activity, with 5g exhibiting the strongest effects against S. aureus and P. aeruginosa. Other derivatives, such as 5b (4-NO₂Ph), 5c (4-FPh), and 5d (4-ClPh), showed moderate activity, while no significant activity was observed against K. pneumoniae or E. faecalis.

Conclusion

The study successfully synthesized a series of novel thiosemicarbazide derivatives with a 1,2,4-triazole moiety and evaluated their antimicrobial potential. Compounds 5e and 5g exhibited significant antibacterial activity, particularly against S. aureus and P. aeruginosa, with MIC values in the low micromolar range. These findings suggest that the compounds hold promise as potential antimicrobial agents, and further studies should focus on optimizing their efficacy and exploring their mechanism of action.

Keywords:

Synthesis, hydrazinecarbothioamide, 1,2,4-triazole, antimicrobial activity

INTRODUCTION

The escalating prevalence of antibiotic-resistant bacteria, fueled by the misuse of antibacterial agents, has become a critical global health crisis.^{1, 2} As these resistant strains continue to evolve, an increasing number of infections are no longer responding to conventional treatments. Alarmingly, even last-resort antibiotics are losing their efficacy in some cases.^3-5 This surge in antimicrobial resistance underscores the urgent need for new therapeutic agents, making the development of novel treatments a top priority in medicinal chemistry today.^6-8

Thiosemicarbazides are vital functional groups in medicinal chemistry, known for their role in forming biologically active heterocyclic rings such as 1,3,4-thiadiazoles, 4-thiazolidinones, and 1,2,4-triazole-3-thiones.^9-11 Numerous studies have highlighted the diverse biological activities exhibited by thiosemicarbazides. Triazoles, defined by a five-membered ring containing three nitrogen atoms and two carbon atoms, are particularly noteworthy in this context. Triazole derivatives have garnered significant attention due to their broad spectrum of biological activities.^{9, 12-16}

Several widely used pharmaceuticals incorporate the 1,2,4-triazole scaffold, including the antifungal agents fluconazole, itraconazole, and voriconazole, the antimigraine drug rizatriptan, and the antiviral drug ribavirin (Figure 1). Additionally, the inclusion of fluorine atoms in drug design has become increasingly popular.^17-20 Fluorine’s unique properties, such as its ability to impart diverse physicochemical attributes and its minimal steric impact, make it an attractive choice for enhancing drug interactions with biological systems. Its high electronegativity also significantly alters the physical and chemical properties of molecules, making fluorine incorporation a powerful strategy in medicinal chemistry.^{21, 22}

Motivated by the urgent need to overcome antibacterial resistance, our primary aim was to design agents based on thiosemicarbazide scaffolds bearing a 1,2,4-triazole heterocycle, selected for their well-established medicinal relevance outlined above.

This study reports the design, synthesis, structural characterization, and antimicrobial evaluation of nine novel thiosemicarbazides (5a-i), each incorporating a 1,2,4-triazole moiety (Figure 2). The antimicrobial efficacy of the synthesized compounds was tested against a panel of bacterial strains, including Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (BAA-2146), and Escherichia coli (ATCC 25922).

MATERIALS AND METHODS

Materials

Melting points were recorded on a STUART SMP40. Infrared (IR) spectra were recorded on a Shimadzu Fourier-transform infrared (FT-IR) spectrometer using KBr pellets. Affinity-1 FT-IR spectroscopy instrument and an Alpha Bruker FT-IR spectrometer. Proton nuclear magnetic resonance (¹H-NMR) and carbon-13 nuclear magnetic resonance (¹³C-NMR) spectra were measured on a Bruker spectrometer in dimethyl sulfoxide (DMSO)-d₆solution at 500 Megahertz (MHz) and 125 MHz, respectively. Chemical shifts (δ) were reported in ppm, and coupling constants (J) were recorded in hertz (Hz).

Elemental analysis was recorded using Leco CHNS-932. The reactions were monitored using thin-layer chromatography on aluminum plates, which have a silica gel Kieselgel 60 F 254 layer of thickness 0.25 mm (Merck), using ultraviolet light as a visualizing agent. All reagents and solvents were purchased from Merck, Fluka, and Sigma-Aldrich and were used without further purification.

Chemical synthesis

Synthesis of N-(4-fluorophenyl)-2-(furan-2-carbonyl)hydrazine-1-carbothioamide (1)

N-(4-Fluorophenyl)-2-(furan-2-carbonyl)hydrazine-1-carbothioamide (1) was synthesized with a good yield of 86% through the reaction of equimolar amounts (0.005 mol) of 2-furoic acid hydrazide and 4-fluorophenylisothiocyanate. The reaction proceeded smoothly under reflux in boiling ethanol.²³

Synthesis of 4-(4-fluorophenyl)-5-(furan-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2)

A solution of compound 1 (0.005 mol) was prepared in 2N NaOH, and the solution was refluxed for 4-5 hours. Afterward, the solution was neutralized using 12.5% hydrochloric acid (HCl). Lastly, the product was filtered and washed with distilled water (Yield: 93%).²³

Synthesis of ethyl ((4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazole-3-yl)sulfanyl)acetate (3)

A mixture of 0.005 mol of compound 2, K₂CO₃, and 0.0055 mol of ethylbromoacetate (BrCH₂COOEt), in ethanol, was refluxed for 5 hours. After completion, the reaction mixture was cooled and poured onto ice water, yielding ethyl ((4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazole-3-yl)sulfanyl)acetate (3) with a 75% yield.²⁴

Synthesis of 2-[[4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl]sulfanyl]acetohydrazide (4)

A mixture of compound 3 (0.005 mol), ethanol, and NH₂NH₂.H₂O (0.025 mol) was heated under reflux in ethanol for 5-6 hours to yield 2-[[4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl]sulfanyl] acetohydrazide (4), with a 61.1% yield.

The analytical and spectral data were reported in our previous study.²⁵

Synthesis of N-(substituted)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamides (5a-i)

0.005 mol of compound 4 is dissolved in ethanol, and 0.005 mol of appropriate isothiocyanate is added to it. It is boiled in a water bath under reflux for 4 hours. At the end of the time, the precipitated product (5a-i) is filtered, dried, and the yield is calculated.

N-(Phenyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5a)

White powder, mp 178-179 ^oC, yield: 71.42%, Anal. Calcd. for: C₂₁H₁₇FN₆O₂S₂, C, 53.83; H, 3.66; N, 17.94%; Found: C, 53.46; H, 3.785; N, 17.88%. FT-IR υmax (cm^-1): 3300, 3252 (N-H), 3146, 3096, 3046 (ar. C-H), 2930, 2834 (al. C-H), 1706 (C=O). ¹H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.40 (s, 1H, CONH), 9.78 (s, 1H, NH), 9.75 (s, 1H, NH), 7.78 (d, J: 1.64 Hz, 1H, furan C₅-H), 7.67-7.61 (m, 2H, 4FPhC_2,6-H), 7.55 (d, J: 7.73 Hz, 2H, Ph C_2,6-H), 7.49 (t, J: 8.77 Hz, 2H, 4FPhC_3,5-H), 7.35 (t, J: 7.73 Hz, 2H, Ph C_3,5-H), 7.18 (t, J: 7.73 Hz, 1H, Ph C₄-H), 6.54 (dd, J: 3.45; 1.64 Hz, 1H, furan C₄-H), 6.24 (d, J: 3.45 Hz, 1H, furan C₃-H), 3.99 (s, 2H, SCH₂). ¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 181.2 (C=S), 167.2 (C=O), 163.2 (d, J: 148 Hz; 4FPh C₄), 151.7 (triazole C₂), 147.9 (triazole C₅), 145.4 (furan C₅), 141.2 (furan C₂), 139.4, 130.7 (d, J: 9.25 Hz, 4FPh C₂), 130.0 (d, J: 2.69 Hz, 4FPh C₁), 128.4, 126.2, 125.6, 117.5 (d, J: 23.36 Hz; 4FPh C₃), 112.2 (furan C₃), 112.0 (furan C₄), 35.2 (-SCH₂).

N-(4-Nitrophenyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5b)

Light yellow powder, mp 182 ^oC, yield: 64.93%, Anal. Calcd. for: C₂₁H₁₆FN₇O₄S₂ C, 49.12; H, 3.14; N, 19.09%; Found: C, 49.21; H, 3.259; N, 19.04%. FT-IR υmax (cm^-1): 3280, 3210 (N-H), 3147, 3101 (ar. C-H), 2928, 2837 (al. C-H), 1721 (C=O), ¹H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.52 (s, 1H, CONH), 10.22 (s, 1H, NH), 10.09 (s, 1H, NH), 8.23 (d, J: 8.73 Hz, 2H, 4-NO₂Ph C_2,6-H), 8.03 (broad s, 2H, 4-NO₂Ph C_3,5-H), 7.79 (s, 1H, furan C₅-H), 7.68-7.62 (m, 2H, 4FPhC_2,6-H), 7.49 (t, J: 8.54 Hz, 2H, 4FPhC_3,5-H), 6.54 (m, 1H, furan C₄-H), 6.26 (d, J: 3.56 Hz, 1H, furan C₃-H); 3.98 (s, 2H, SCH₂). ¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 180.9 (C=S), 167.5 (C=O), 163.2 (d, J: 248.64 Hz, 4FPh C₄), 151.7 (triazole C₂), 147.9 (triazole C₅), 145.8 (4-NO₂Ph C₄), 145.5 (furan C₅), 144.0 (4-NO₂Ph C₁), 141.2 (furan C₂), 130.7 (d, J: 9.26 Hz, 4FPh C₂), 130.3 (d, J: 2.75 Hz, 4FPh C₁), 125.2 (4-NO₂Ph C₃), 124.1 (4-NO₂Ph C₂), 117.5 (d, J: 23.31 Hz, 4FPh C₃), 112.2 (furan C₃), 112.1 (furan C₄), 35.2 (-SCH₂).

N-(4-Fluorophenyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5c)

White powder, mp 178 ^oC, yield: 77.24%, Anal. Calcd. for: C₂₁H₁₆F₂N₆O₂S₂, C, 51.84; H, 3.31; N, 17.27%; Found: C, 51.48; H, 3.444; N, 17.17%. FT-IR υmax (cm^-1): 3298 (N-H), 3122, 3095 (ar. C-H), 2929, 2833 (al. C-H), 1715 (C=O). ¹H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.33 (s, 1H, CONH), 9.74 (s, 1H, NH), 9.69 (s, 1H, NH), 7.69 (d, J: 1.82 Hz, 1H, furan C₅-H), 7.58-7.52 (m, 2H, 4FPhC_2,6-H), 7.46-7.37 (m, 4H, 4FPhC_2,3,5,6-H), 7.09 (t, J: 8.75 Hz, 2H, 4FPhC_3,5-H), 6.45 (dd, J: 3.46, 1.82 Hz, 1H, furan C₄-H), 6.15 (d, J: 3.46 Hz, 1H, furan C₃-H), 3.89 (s, 2H, SCH₂).¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 181.5 (C=S), 167.2 (C=O), 163.2 (d, J: 248 Hz, 4FPh C₄), 160.0 (d, J: 248 Hz, 4FPh C₄), 151.8 (triazole C₂), 147.9 (triazole C₅), 145.4 (furan C₅), 141.2 (furan C₂), 135.7 (d, J: 2.77 Hz, 4FPh C₁), 130.6 (d, J: 9.27 Hz, 4FPh C₂), 130.0 (d, J: 2.77 Hz, 4FPh C₁), 117.5 (d, J: 23.05 Hz, 4FPh C₃), 115.1 (d, J: 23.05 Hz, 4FPh C₃), 112.2 (furan C₃), 112.0 (furan C₄), 35.2 (-SCH₂).

N-(4-Chlorophenyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5d)

White powder, mp 214 ^oC, yield: 82.66%, Anal. Calcd. for: C₂₁H₁₆ClFN₆O₂S₂, C, 50.15; H, 3.21; N, 16.71%; Found: C, 50.04; H, 3.268; N, 16.75%. FT-IR υmax (cm^-1): 3295 (N-H), 3153, 3097 (ar. C-H), 2957 (al. C-H), 1709 (C=O). ¹H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.42 (s, 1H, CONH), 9.89 (s, 1H, NH), 9.81 (s, 1H, NH), 7.78 (d, J: 1.24 Hz, 1H, furan C₅-H), 7.68-7.60 (m, 2H, 4FPhC_2,6-H), 7.59 (d, J: 8.7 Hz, 2H, 4-ClPh C_2,6-H), 7.49 (t, J: 8.60 Hz, 2H, 4FPhC_3,5-H), 7.40 (d, J: 8.7 Hz, 2H, 4-ClPh C_3,5-H), 6.54 (dd, J: 3.56; 1.78 Hz, 1H, furan C₄-H), 6.25 (d, J: 3.56 Hz, furan C₃-H), 3.97 (s, 2H, SCH₂). ¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 181.2 (C=S), 167.3 (C=O), 163.2 (d, J: 248 Hz, 4FPh C₄), 151.8 (triazole C₂), 147.9 (triazole C₅), 145.5 (furan C₅), 141.2 (furan C₂), 138.4 (4-ClPh C₁), 130.7 (d, J: 9.27 Hz, 4FPh C₂), 130.0 (d, J: 2.77 Hz, 4FPh C₁), 129.6 (4-ClPh C₄), 128.3 (4-ClPh C_3,5), 127.8 (4-ClPh C_3,5), 117.5 (d, J: 23.05 Hz, 4FPh C₃), 112.2 (furan C₃), 112.0 (furan C₄), 35.2 (-SCH₂).

N-(4-Bromophenyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5e)

White powder, mp 210 ^oC, yield: 61.66%, Anal. Calcd. for: C₂₁H₁₆BrFN₆O₂S₂.2H₂O C, 43.23; H, 3.46; N, 14.40%; Found: C, 43.71; H, 3.017; N, 14.77%. FT-IR υmax (cm^-1): 3293 (N-H), 3157, 3098 (ar. C-H), 2943 (al. C-H), 1708 (C=O). ¹H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.34 (s, 1H, CONH), 9.82 (s, 1H, NH), 9.71 (s, 1H, NH), 7.70 (d, J: 1.79 Hz, 1H, furan C₅-H), 7.58-7.53 (m, 2H, 4FPhC_2,6-H), 7.47-7.41 (m, 6H, 4FPhC_2,6-H and 4BrPhC_2,3,5,6-H), 6.46 (dd, J: 3.5; 1.79 Hz, 1H, furan C₄-H), 6.17 (d, J: 3.5 Hz, 1H, furan C₃-H), 3.89 (s, 2H, SCH₂).¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 181.2 (C=S), 167.3 (C=O), 163.2 (d, J: 248.64 Hz, 4FPh C₄), 151.8 (triazole C₂), 147.9 (triazole C₅), 145.5 (furan C₅), 141.2 (furan C₂), 138.8, 131.3, 130.6 (d, J: 9.26 Hz, 4FPh C₂), 130.0 (d, J: 2.75 Hz, 4FPh C₁), 117.5 (d, J: 23.31 Hz, 4FPh C₃), 112.2 (furan C₃), 112.0 (furan C₄), 35.2 (-SCH₂).

N-(Ethyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5f)

White powder, mp 180 ^oC, yield: 79.36%, Anal. Calcd. for: C₁₇H₁₇FN₆O₂S₂, C, 48.56; H, 4.08; N, 19.99%; Found: C, 48.34; H, 4.218; N, 19.93%. FT-IR υmax (cm^-1): 3311 (N-H), 3050 (ar. C-H), 2972, 2934, 2869 (al. C-H), 1709 (C=O). ¹H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.21 (s, 1H, CONH), 9.33 (s, 1H, NH), 8.24 (s, 1H, NH), 7.79 (d, J: 1.8 Hz, 1H, furan C₅-H), 7.67-7.57 (m, 2H, 4FPhC_2,6-H); 7.49 (t, J: 8.8 Hz, 2H, 4FPhC_3,5-H), 6.55 (dd, J: 3.5; 1.8 Hz, 1H, furan C₄-H), 6.24 (d, J: 3.5 Hz, 1H, furan C₃-H), 3.90 (s, 2H, SCH₂), 3.56 (p, J: 7 Hz, 2H, -CH₂-CH₃), 1.12 (t, J: 7 Hz, 3H, -CH₂-CH₃). ¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 181.5 (C=S), 167.0 (C=O), 163.3 (d, J: 248.5 Hz, 4FPh C₄), 152.1 (triazole C₂), 147.9 (triazole C₅), 145.5 (furan C₅), 141.1 (furan C₂), 130.6 (d, J: 9.16 Hz, 4FPh C₂), 129.9 (d, J: 2.92 Hz, 4FPh C₁), 117.5 (d, J: 23.05 Hz, 4FPh C₃), 112.2 (furan C₃), 112.0 (furan C₄), 39.0 (N-CH₂-CH₃), 34.6 (-SCH₂), 14.9 (N-CH₂-CH₃).

N-(Propyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5g)

White powder, mp 168-169 ^oC, yield: 80.19%, Anal. Calcd. for: C₁₈H₁₉FN₆O₂S₂, C, 49.76; H, 4.41; N, 19.34%; Found: C, 49.39; H, 4.540; N, 19.26%. FT-IR υmax (cm^-1): 3317 (N-H), 3149, 3086 (ar. C-H), 2962, 2930, 2862 (al. C-H), 1704 (C=O). ¹H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.22 (s, 1H, CONH), 9.32 (s, 1H, NH), 8.21 (s, 1H, NH), 7,80 (d, J: 1.7 Hz, 1H, furan C₅-H), 7.65-7.58 (m, 2H, 4FPhC_2,6-H), 7.50 (t, J: 8.69 Hz, 2H, 4FPhC_3,5-H), 6.55 (dd, J: 3.5 ; 1.7 Hz, 1H, furan C₄-H), 6,24 (d, J: 3.5 Hz, 1H, furan C₃-H), 3.90 (s, 2H, SCH₂), 3.46 (m, 2H, N-CH₂-CH₂-CH₃), 1.56 (h, J: 7.4 Hz, 2H, -CH₂-CH₂-CH₃), 0.83 (t, J: 7.4 Hz, 3H, -CH₂-CH₂-CH₃). ¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 181.7 (C=S), 167.0 (C=O), 163.3 (d, J: 148.95 Hz; 4FPh C₄), 152.1 (triazole C₂), 147.9 (triazole C₅), 145.5 (furan C₅), 141.1 (furan C₂), 130.6 (d, J: 9.25 Hz, 4FPh C₂), 129,8 (d, J: 2.69 Hz, 4FPh C₁), 117.5 (d, J: 23.36 Hz; 4FPh C₃), 112.2 (furan C₃), 112.0 (furan C₄), 45.7 (N-CH₂-CH₂-CH₃), 34.5 (-SCH₂), 22.4 (N-CH₂-CH₂-CH₃), 11.5 (N-CH₂-CH₂-CH₃).

N-(Butyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5h)

White powder, mp 197 ^oC, yield: 74.46%, Anal. Calcd. for: C₁₉H₂₁FN₆O₂S₂, C, 50.88; H, 4.72; N, 18.74%; Found: C, 50.71; H, 4.942; N, 18.70%. FT-IR υmax (cm^-1): 3340 (N-H), 3163, 3095 (ar. C-H), 2955, 2933, 2872 (al. C-H), 1712 (C=O). 1H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.22 (s, 1H, NH); 9.33 (s, 1H, NH); 8.18 (s, 1H, NH); 7.79 (d, J: 1.15 Hz, 1H, furan C₅-H); 7.66-7.59 (m, 2H, 4FPhC_2,6-H); 7.49 (t, J: 8.7 Hz, 2H, 4FPhC_3,5-H); 6.54 (dd, J: 3.5; 1.8 Hz, 1H, furan C₄-H); 6.25 (d, J: 3.5 Hz, 1H, furan C₃-H); 3.91 (s, 2H, SCH₂); 3.51 (q, J: 7.4 Hz, 2H, N-CH₂-CH₂-CH₂-CH₃); 1.54 (p, J: 7.4 Hz, 2H, N-CH₂-CH₂-CH₂-CH₃); 1.27 (h, J: 7.4 Hz, 2H, N-CH₂-CH₂-CH₂-CH₃); 0.87 (t, J: 7.4 Hz, 3H, N-CH₂-CH₂-CH₂-CH₃). ¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 181.8 (C=S); 167.0 (C=O); 163.3 (d, J: 248.62 Hz, 4FPh C₄); 152.1 (triazole C₂); 147.9 (triazole C₅); 145.5 (furan C₅); 141.1 (furan C₂); 130.6 (d, J: 9.19 Hz, 4FPh C₂); 129.8 (d, J: 2.93 Hz, 4FPh C₁); 117.5 (d, J: 23.28 Hz; 4FPh C₃); 112.2 (furan C₃); 112.0 (furan C₄); 43.8 (N-CH₂-CH₂-CH₂-CH₃), 34.5 (-SCH₂), 31.3 (N-CH₂-CH₂-CH₂-CH₃), 19.8 (N-CH₂-CH₂-CH₂-CH₃), 14.2 (N-CH₂-CH₂-CH₂-CH₃).

N-(Allyl)-2-{[(4-(4-fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetyl}hydrazine-1-carbothioamide (5i)

White powder, mp 151-152 ^oC, yield: 69.76%, Anal. Calcd. for: C₁₈H₁₇FN₆O₂S₂, C, 49.99; H, 3.96; N, 19.43%; Found: C, 49.50; H, 4.063; N, 19.31%. FT-IR υmax (cm^-1): 3316 (N-H), 3156, 3083, 3012 (ar. C-H), 2921, 2855 (al. C-H), 1705 (C=O). ¹H-NMR (500 MHz) (DMSO-d₆) δ (ppm): 10.28 (s, 1H, CONH), 9.44 (s, 1H, NH), 8.37 (s, 1H, NH), 7.79 (d, J: 1.80 Hz, 1H, furan C₅-H), 7.65-7.58 (m, 2H, 4FPhC_2,6-H), 7.48 (t, J: 8.7 Hz, 2H, 4FPhC_3,5-H), 6.54 (dd, J: 3.53; 1.80 Hz, 1H, furan C₄-H), 6.24 (d, J: 3.53 Hz, 1H, furan C₃-H), 5.85 (ddt, J: 17.20; 10.30; 5.3 Hz, 1H, -NCH₂CH=CH₂), 5.13 (dd, J: 17.20; 1.70 Hz, 1H, -NCH₂CH=CH₂), 5.02 (dd, J: 10.30; 1.7 Hz, 1H, -NCH₂CH=CH₂), 4.17 (s, 2H,-NCH₂CH=CH₂), 3.95 (s, 2H, SCH₂). ¹³C-NMR (APT) (125 MHz) (DMSO-d₆) δ (ppm): 182.1 (C=S), 167.1 (C=O), 163.3 (d, J: 148 Hz; 4FPh C₄), 152.1 (triazole C₂), 147.9 (triazole C₅), 145.5 (furan C₅), 141.1 (furan C₂), 135.2 (-NCH₂CH=CH₂), 130.6 (d, J: 9.25 Hz, 4FPh C₂), 129,8 (d, J: 2.69 Hz, 4FPh C₁), 117.5 (d, J: 23.36 Hz; 4FPh C₃), 115.7 (-NCH₂CH=CH₂), 112.2 (furan C₃), 112.0 (furan C₄), 46.3 (-NCH₂CH=CH₂), 34.6 (-SCH₂).

Antibacterial activity studies

The antimicrobial activity of a series of thiosemicarbazides bearing 1,2,4-triazole (5a-i) was investigated against K. pneumoniae (BAA-2146), E. coli (ATCC 25922), S. aureus (ATCC 25923), E. faecalis (ATCC 29212), and P. aeruginosa (ATCC 27853). Both disk diffusion and broth microdilution methods were employed to assess activity, ensuring consistency across experiments.²⁶^,²⁷

For the broth microdilution method, a stock solution of each compound was prepared at a concentration of 100 micromolar (μM), followed by serial dilutions in sterile 96-well microplates. Muller, Muller-Hinton broth was added to each well, and bacterial suspensions were adjusted to a 0.5 McFarland standard. The plates were incubated at 35-37 °C for 18-24 hours under appropriate conditions, and the minimum inhibitory concentrations (MICs) were determined manually.

In the disk diffusion assay, blank antibiotic disks were impregnated with the compounds, and the zone diameters of inhibition were measured after incubation. The tests were standardized with DMSO as a negative control and specific antibiotics as positive controls: Vancomycin (20 µM) for S. aureus and E. faecalis, and Meropenem (78 µM) for P. aeruginosa. All experiments were conducted following European Committee on Antimicrobial Susceptibility Testing guidelines, and bacterial strains were maintained at -80 °C until use.

Statistical analysis

The antimicrobial assay results were expressed in µM units to enable reliable comparison among the synthesized derivatives and reference compounds. The obtained data were descriptively evaluated, with MIC values serving as the primary measure of antimicrobial potency. This ensured a consistent and reproducible framework for interpreting the biological activity across all tested compounds. No further statistical testing was applied.

RESULTS

Chemistry

All designed novel thiosemicarbazide compounds (5a-i) were obtained according to the methods depicted in the experimental section. The chemical pathway shown in Scheme 1 outlines a synthesis process that involves five steps. Furan-2-carbohydrazide and 4-fluorophenylisothiocyanate were boiled under reflux in ethanol to yield N-(4-Fluorophenyl)-2-(furan-2-carbonyl)hydrazine-1-carbothioamide (1). Solution of (1) in NaOH (2N) was heated under reflux for 4 h, then it was neutralized with 12.5% HCl to yield 4-(4-Fluorophenyl)-5-(furan-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2). A mixture of (2), K₂CO₃ and BrCH₂COOEt was refluxed in acetone to obtain Ethyl ((4-Fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazole-3-yl)sulfanyl)acetate (3). Compound 3 and NH₂NH₂.H₂O were heated under reflux in ethanol to yield 2-[[4-(4-Fluorophenyl)-5-(furan-2-yl)-4H-1,2,4-triazol-3-yl]sulfanyl]acetohydrazide (4). A solution of 4 and the appropriate isothiocyanate in absolute ethanol was heated under reflux to yield 5a-i. The designed compounds (5a-i) were synthesized with good yields (61.10-82.66%).

Antimicrobial activity

According to the in vitro assay results, against P. aeruginosa compound 5g showed the lowest MIC (<0.78 μM), at least 100-fold more potent than Meropenem (MIC = 78 μM), while 5i (MIC = 1.56 μM) was ~50-fold more potent. For S. aureus, 5e (MIC = 12.5 μM) outperformed Vancomycin (MIC = 20 μM), and 5b and 5h (MIC = 25 μM) were comparable to Vancomycin. No compound was active (MIC >100 μM) against E. coli, K. pneumoniae, or E. faecalis, as can be seen in Table 1.

DISCUSSION

IR spectra provide a straightforward diagnostic of the hydrazide-to-thiosemicarbazide transformation: in compound 4, the terminal -NH₂ group exhibits the characteristic pair of N-H stretching bands, whereas in 5a-i these bands disappear and are replaced by a single N-H stretch consistent with conversion of -NH₂ to a secondary -NH- within the thiosemicarbazide framework.²⁸

In the ¹H-NMR spectra, the terminal NH₂ protons of compound 4, initially observed at 4.32 ppm, disappeared in the thiosemicarbazide derivatives (5a-i). However, the NH₂ protons of the newly formed thiosemicarbazide group in 5a-i appeared in the range of 10.22-8.18 ppm, confirming their formation.²⁹ Additionally, the aryl protons of the isothiocyanate groups in 5a-i further supported the confirmation of the structure.

In the ¹³C (APT) NMR spectra, the carbonyl carbons (-C=O) of compounds 4 and 5a-i were detected between 166.4 and 167.0 ppm. Carbons associated with the -C=S group in 5a-i, indicative of the thiosemicarbazide functionality, appeared at 182.1-180.9 ppm.³⁰ The aryl carbons of the isothiocyanate groups also reinforced the structural integrity of the compounds.

These spectroscopic results (¹H NMR, ¹³C NMR, and FT-IR), along with elemental analysis, unequivocally confirmed the successful transformation of compound four into the thiosemicarbazide derivatives 5a-i. No significant side reactions were observed, and the target compounds were obtained in moderate to good yields, with all data aligning with the assigned structures and literature.^{23, 24}

Based on our MIC data, we can describe the structure-activity relationship (SAR) in more detail - see Table 1 and Figure 3. Against P. aeruginosa, the three-carbon n-propyl group on the thiosemicarbazide nitrogen gave the best result (compound 5g, MIC <0.78 μM), which is more than 100-fold better than Meropenem (MIC 78 μM). When n-propyl was changed to allyl, the activity dropped about two-fold (compound 5i, 1.56 μM). Shorter (ethyl) or longer (n-butyl) chains were inactive (MIC >100 μM), and replacing the aliphatic chain with an aryl (phenyl) group also removed the activity. These trends suggest that P. aeruginosa needs a simple, three-carbon saturated chain at this position, probably because it gives the right size and shape for the target and also helps the compound pass the outer membrane; adding an aromatic ring may be too bulky or too rigid, and changing to an allyl group may change the geometry or polarity in a way that weakens binding.

Against S. aureus, activity increased when the para substituent on the phenyl ring was less electronegative: p-Br (5e, 12.5 μM) was stronger than p-NO₂ (5b, 25 μM), while p-F and p-Cl were inactive (MIC >100 μM). One simple explanation is that bromine is less electronegative and more polarizable, so it may improve hydrophobic contacts in the binding site, while very electron-withdrawing or very small substituents reduce those contacts. In the alkyl series for S. aureus, only n-butyl (5h, 25 μM) was active; ethyl, n-propyl, and allyl analogs were inactive, which may indicate that a slightly longer hydrophobic tail helps interaction with the Gram-positive cell envelope or with the target pocket.

None of the compounds were active against E. coli, K. pneumoniae, or E. faecalis (MIC >100 μM). The reason is not clear at this stage; common possibilities include poor permeability, strong efflux, or a different/absent target in these organisms. In future work we plan to check simple permeability and efflux effects and do basic target-engagement tests to understand these findings and to guide the design of more active analogs (for example, small changes around n-propyl for P. aeruginosa and around p-Br for S. aureus).

Upon evaluating all data thoroughly, to clarify the SAR key observations and insights are given as follows, see in Table 1 and Figure 3.

1. The three-carbon alkyl chain (n-propyl, compound 3g) is optimal for activity against Pseudomonas aeruginosa. Shorter (ethyl, compound 3f) or longer (n-butyl, compound 3h) chains are inactive

2. Unsaturation and aryl substitution reduce activity against P. aeruginosa.

• n-propyl > allyl (≈2× loss in potency when changing to allyl)

• n-propyl > allyl > phenyl (aryl) (phenyl = inactive)

3. Less-electronegative para substituents improve activity against Staphylococcus aureus.

• p-Br > p-NO₂, while p-F and p-Cl are inactive

• Among alkyls, only n-butyl shows activity

CONCLUSION

There is still a significant danger arising from various bacteria today. Because many bacterial types, such as S. aureus and P. aeruginosa, are quickly resistant to the current antibiotics, the design and evaluation of novel potent antimicrobial molecules are vital. In this study, nine novel thiosemicarbazide-bearing 1,2,4-triazole compounds were synthesized using simple and practical methods, and their structure was characterized using methods such as FT-IR, ¹H-NMR, ¹³C-NMR, elemental analysis, and mass spectroscopy. All synthesized compounds were investigated for their antibacterial activities against diverse bacteria including E. coli, K. pneumoniae, P. aeruginosa, S. aureus, and E. faecalis. Especially the compounds carrying 4-bromophenyl substitution at the N position of the thiosemicarbazide nucleus ring showed high antibacterial activities against S. aureus with 12.5 µM, whereas the n-propyl substituted derivative displayed the most activity against P. aeruginosa with 0.78 µM. Consequently, we believe that the biological assay data and SAR evaluation obtained from this study may assist in the future discovery of new and more potent antimicrobial compounds.

Ethics

Ethics Committee Approval: Not required.

Informed Consent: Not required.

Authorship Contributions

Concept: E.D.D., E.D.K., H.Ö.D., F.B., E.G., N.U.G., Design: E.D.D., E.D.K., H.Ö.D., F.B., E.G., N.U.G., Data Collection or Processing: E.D.D., E.D.K., H.Ö.D., F.B., E.G., N.U.G., Analysis or Interpretation: E.D.D., E.D.K., H.Ö.D., F.B., E.G., N.U.G., Literature Search: E.D.D., E.D.K., H.Ö.D., F.B., E.G., N.U.G., Writing: E.D.D., E.D.K., H.Ö.D., F.B., E.G., N.U.G.

Conflict of Interest: The authors declare no conflicts of interest.

Financial Disclosure: This work was supported by Bezmialem Vakıf University Scientific Research Project Number: 20211216.

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