# Synthesis, Characterization, X-Ray Single-Crystal Structure, Potentiometric Measurements, Molecular Modeling, and Bioactivity Screening of Some Thiosemicarbazones

Authors: Abeer A. El-Sisi; Mohamed Ali; Sohair F. Ramdan; Osama AlTaweel; Ahmed I. A. Abd El-Mageed; Ahmed A. El-Sherif
Journal: Journal of Chemistry
 (2022)

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## Abstract

A series of thiosemicarbazone (TSCN) compounds including ((E)-2-((E)-1-(2-(p-tolyl)hydrazono)propan-2-ylidene)hydrazine-1-carbothioamide (TSC1), (E)-N-ethyl-2-((E)-1-(2-(p-tolyl)hydrazono)propan-2-ylidene)hydrazine-1-carbothioamide (TSC2), and (E)-N-phenyl-2-((E)-1-(2-(p-tolyl)hydrazono)propan-2-ylidene)hydrazine-1-carbothioamide) (TSC3) were synthesized and fully characterized by diverse spectroscopies, such as X-ray single-crystal, infrared, mass, proton nuclear magnetic resonance, and ultraviolet-visible. Potentiometric measurements, molecular modeling, and biological and antitumor activity screening were studied. The thermodynamics and protonation constants of TSC1 as a representative of the synthesized TSCs were calculated and discussed. The solution speciation of different species was studied with pH. The molecular parameters of the optimized structures were calculated and discussed. The X-ray single crystals of TSC2 and TSC3 were established. Considering the antimicrobial activities and correlating structure-activity relationship of the synthesized compounds, the TSC1 molecule was considered a promising candidate as an antifungal agent againstCandida albicans. Thus, it would be extremely helpful in the field of medicinal chemistry, particularly as an antimicrobial agent. The results are of significance to the chemistry of antimicrobial agents.


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## Body

## 1. Introduction

Globally, infectious diseases constitute a high burden on public health worldwide. Owing to the high resistance of certain Gram-positive and Gram-negative bacteria to many drugs, a large number of infectious diseases threaten human life worldwide [1]. Globally, approximately 110000 of over 50 million infected people die annually. By the middle of the 21st century, it is expected that the mortality rate caused by Gram-negative bacterial infection alone could increase to 10 million deaths annually [2]. Antibiotics are the main base for microbial (bacterial and fungal) infection therapy. Antibiotic overuse has become the main cause of the appearance and spread of multidrug-resistant strains of many microbes [3]. The emergence and increasing prevalence of antibiotic-resistant bacterial strains urge the discovery of new therapeutic approaches [4]. Additionally, the available drugs are expensive or exhibit many unwanted side effects [5]. Therefore, the development of novel antimicrobial agents is vital, given the fast global spread of resistant clinical isolates. Considering the relationship between bacterial infection and multidrug resistance, this study developed a new effective antimicrobial drug. Nowadays, there is a growing area of interest in the medicinal chemistry of Schiff base compounds, such as hydrazones and thiosemicarbazones (TSCs), owing to their wide range of biological activities [6, 7]. C=N and N–C = S groups are of great interest in chemotherapy and are responsible for the pharmacological activity therein. The most essential step in the metal complex implementation is the synthesis of novel compounds that exhibit unique properties and reactivity. In this regard, TSCs are a topic of interest for researchers of various profiles. TSCN compounds and their complexes have been widely investigated as they exhibit extremely interesting properties for biomedicine and potential medicinal applications [8–12]. These properties comprise antiparasitic [13], antibacterial [14], antitumor [15], antiviral [16], fungicidal [17], antineoplastic [18], and antiamebic [19] activities. TSCs exhibit thione (A) and thiol (B) forms (Scheme 1).Scheme 1
Thione (a) and thiol (b) tautomers of thiosemicarbazones.Hydrazones are a crucial class of compounds with impressive ligation characteristics, owing to the existence of several coordination sites [20]. Previous reports show hydrazine and its derivatives with anti-inflammatory, analgesic [21], antibacterial [22], and antitumor [23] activity.Cd is an extremely toxic metal ion that poses human and animal health hazards. Its toxicity is caused by its easy localization inside the liver. It binds to metallothionein, which eventually forms a complex and is transmitted into the bloodstream to be lodged in the kidney.Cd toxicity is caused by the negative effect on cell enzyme systems that are the consequence of metallic ion substitution (mainly Zn (II), Cu (II), and Ca (II)) into metalloenzymes and their large affinity to thiol group compounds [24]. Zn (II) replacement with a chemically analogous Cd (II) ion usually causes apoprotein catalytic activity to break down [25, 26]. Therefore, it is important to discover novel compounds that can form stable complexes with Cd (II) because they can be used as detoxifiers. TSCN compounds and their complexes are fit for this role because of their pharmacological properties. Recently, experimental studies were supplemented by computational studies [27], owing to their crucial role in recognizing the expected behaviors of the compound during reactions and recognition of valuable information on the compounds under examination, such as the total energy, binding energy, electronic energy, dipole moment, bond length, highest occupied molecular orbital (HOMO), and lowest unoccupied molecular orbital (LUMO) [28–30]. Based on this and in the perpetuation of our studies in the subject area of bioactive compounds, it is of great interest to synthesize and identify novel compounds with TSCN and hydrazone moieties. In addition, we explored the biological activities of the identified compounds.


## 2. Experimental Process

### 2.1. Materials and Reagents

The chemicals used in this study were supplied by Aldrich Chemicals Company and were used without extra purification.


### 2.2. Synthesis

#### 2.2.1. Synthesis of (1-(p-Tolyl)Hydrazono)Propan-2-One (PTHP)

We synthesized PTHP using a reported method [31, 32]. Scheme 2 shows the chemical equations for the preparation.(1)CH3COCH2CO2C2H5+KOH⟶CH3COCH2CO2K+C2H5OH,CH3COCH2CO2K+HCl⟶CH3COCH2CO2H+,CH3COCH2CO2H+p−Me−C6H5N=NCl⟶p−Me−C6H5NH−N=CHCOCH3+CO2+HCl.Scheme 2
Preparation of (1-(p-tolyl)hydrazono)propan-2-one.


#### 2.2.2. Synthesis of TSCN Compounds

Equimolar amounts of PTHP (0.1760 g, 1 mmol) in 30 mL of ethanol with an ethanolic solution (30 mL) of thiosemicarbazide (0.091 g, 1 mmol), N-ethylthiosemicarbazide (0.119 g, 1 mmol), and N-phenylthiosemicarbazide (0.167 g, 1 mmol) were refluxed on a hot plate for 3–5 hrs. The precipitate was separated, filtered off, washed with (C2H5)2O, and desiccated all night using silica gel. The target compounds are shown in Figure 1.Figure 1
Structural formula for the thiosemicarbazone compounds. TSC1, TSC2, and TSC3 withR = H, Et, and ph, respectively.(1) ((E)-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC1). Yield, 77%. Color, brown. Anal. Calc. for C11H15N5S: C, 52.99; H, 6.06; N, 28.09; S, 12.86. Found: C, 52.93; H, 6.01; N, 28.03; S, 12.79%. Infrared (IR) (KBr, cm−1): 3386, 3234 (NH2), 1507, 1251, 1017, 805 (thioamide bands, I–IV, respectively), 3501, 3177 (N2H), 1100 (N–N), 1603 (C = N), 1553 (C = C), 3045 (C–H). MS (m/z): 251 (M++2, 6.03%), 250 (M++1, 18.07%), 249 (M+, 100%), 234 (2.58%), 232 (38.20%), 159 (2.07%), 157 (0.59%), 118 (3.81%). Proton nuclear magnetic resonance (1H NMR) (DMSO): 11.32 (s, 1H, NH); 10.89 (s, 2H, NH); 7.8 (s, 2H, NH2); 7.48 (s, H, CH = N); 6.94–7.01 (m, 4H,–Ar); 2.21 (s, 3H,–CH3); 2.02 (s, 3H,–CH3). 13C-NMR (DMSO): 11.02, 20.09, 112.21, 129.51, 130.80, 136.01, 142.21, 145.62, 178.3.(2) ((E)-N-Ethyl-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC2). Yield, 69%. Color, dark brown. Anal. Calc. for C13H19N5S: C, 55.52; H, 6.17; N, 25.25; S, 11.56. Found: C, 55.48; H, 6.12; N, 25.19; S, 11.51%. IR (KBr, cm−1): 1532, 1251, 1079, 805 (thioamide bands, I–IV, respectively), 3444, 3338, 3230 (3NH), 1079 (N–N), 1615 (C = N), 1535 (C = C), 3019 (C–H). MS (m/z): 279 (M++2, 6.53%), 278 (M++1, 21.11%), 277 (M+, 100%), 262 (0.94%), 173 (2.73%), 118 (3.23%). 1H-NMR: 11.33 (s, 1H, NH); 10.33 (s, 1H, NH); 8.91 (s, 1H, NH); 7.54 (s, H, CH = N); 6.36–7.54 (m, 4H,–Ar); 3.60 (q, 2H,–CH2); 2.4 (t, 2H,–CH3); 2.23 (s, 3H,–CH3); 2.02 (s, 3H,–CH3). 13C-NMR: 11.01, 14.32, 20.09, 39.21, 112.23, 128.07, 129.61, 136.0, 142.23, 148.24, 177.04.(3) (E)-N-Phenyl-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC3). Yield, 67%. Color, brownish yellow. Anal. Calc. for C17H19N5S: C, 62.74; H, 5.89; N, 21.50; S, 9.85. Found: C, 62.75; H, 5.85; N, 21.47; S, 9.81%. IR (KBr, cm−1): 1518, 1250, 1065, 748 (thioamide bands, I–IV, respectively), 3444, 3259, 3020 (3NH), 1065 (N–N), 1603 (C = N), 1551 (C = C), 3020 (C–H). MS (m/z): 326 (M++1, 1.78%), 325 (M+, 7.41%), 118 (5.63%). 1H-NMR: 11.32 (s, 1H, NH); 10.30 (s, H, NH); 7.70 (s, H, NH); 7.61 (s, H, CH = N); 6.4–7.70 (m, 9H,–Ar); 2.21 (s, 3H,–CH3); 2.03 (s, 3H,–CH3). 13C-NMR: 11.33, 20.13, 39.40, 112.24, 115.63, 124.94, 128.28, 129.47, 135.87, 138.92, 142.16, 149.22, 175.9.


### 2.3. Methods and Experimental Measurements

The chemicals used were supplied by Aldrich. A CHNS automatic analyzer (Vario EII-Elementar) was used to conduct elemental microanalysis for C, H, N, and S. A PerkinElmer Fourier transform IR (FTIR) type 1650 spectrophotometer with a potassium bromide disc was used to record the IR spectra. Electronic spectra were recorded on a Shimadzu UV-3101PC spectrophotometer. A Bruker ARX-300 device was applied to record the1H-NMR spectra. Chemical shifts were recorded in ppm comparative to tetramethylsilane (TMS) using deuterated dimethyl sulphoxide (d6-DMSO) as a solvent. Mass spectrometry was conducted using a Shimadzu GCMS-QP1000EX. The X-ray single-crystal diffraction of the TSC2 molecule was performed using a Rigaku VariMax RAPID FR-E diffractometer with monochromatized Mo Kα radiation at a radiation wavelength of λ = 0.71075 Å in ω scan mode. The crystal was cooled with a cold N2 gas flow. We performed diffraction data scaling, cell refinement, indexing, collection, and peak integration using the Rapid Auto software (Rigaku). The molecular structure was analyzed using Mercury 4.1.3 software. However, the X-ray crystallography data of TSC2 and TSC3 were collected by mounting a single sample crystal on glass fiber. The cell parameters and intensity data collection were performed at 298 K using monochromatized Mo Kα radiation at a radiation wavelength of λ = 0.71073 Ǻ. The crystal structure was analyzed using the SIR-92 program [33] and was refined on F2 using the full-matrix least-squares technique with the maXus processor program for the solution and refinement of crystal structures [34]. The Oak Ridge Thermal Ellipsoid Plot (ORTEP) program was used for molecular graphics [35].A Metrohm 848 Titrino supplied with a Dosimat unit (Switzerland-Herisau) was used for potentiometric titrations as previously described in 50% water-DMSO mixture [29, 30, 32].


### 2.4. Potentiometric Titrations

Using the aforementioned potentiometric technique [36], the formation constant of the complex was estimated. Standard buffer solutions were used to accurately calibrate the glass electrode to the National Bureau of Standards (NBS) [37]. A standard solution of 0.05 mol/dm3 NaOH, free from CO2, was used to titrate the samples in an N2 atmosphere. The sample solution was developed to avoid hydrolysis during titration by mixing equal volumes of DMSO and H2O. The ionic strength was maintained during titration using NaNO3 as a supporting electrolyte.The calculation of the formation constants using the potentiometric method was performed using a concentration of hydrogen ion expressed in molarity. The concentration in the pH meter was expressed in activity coefficient −log aH+ (pH). Thus, Van Uitert and Hass used equation (2) to change the pH meter reading (B) to [H+] as follows [38, 39]:(2)−log10H+=B+log10UH,where log10 UH = solvent composition correction factor and the ionic strength read by B. The pKw values for the titrated samples were estimated as previously described [40]. All precautions and procedures comply with literature requirements [41–43].The protonation constants of TSC1 were estimated potentiometrically by titrating 40 cm3 of a 1.25 × 10−3 mol/dm3 TSC1 solution with a standard sodium hydroxide solution.


### 2.5. Processing of Data

The MINIQUAD-75 computer program was applied to calculate ca. 100–150 readings for each titration [44]. Species distribution diagrams for the studied samples were produced using the SPECIES program [45].


### 2.6. Molecular Modeling Studies

Density functional theory (DFT) calculations were performed using the DMol3 program [46, 47] in the Materials Studio package [48]. The calculations for the DFT semicore pseudopods (DSPP) were created with dual numerical base sets and polarization properties (DNPs) [49]. The revised Perdew–Burke–Ernzerhof (RPBE) model was focused on the generalized gradient approximation (GGA) as the best functional approximation [50, 51].


### 2.7. Biological Activity

#### 2.7.1. Antibacterial and Antifungal Activities

The ability of the synthesized TSCN compounds to suppress bacterial growth was confirmed using the disc diffusion process [52, 53]. Aerobic G+ bacteria including Staphylococcus aureus, Bacillus subtilis, and Streptococcus faecalis and G− aerobic bacteria including Pseudomonas aeruginosa, E. coli, and Neisseria gonorrhoeae were among the bacterial strains used in this study. Additionally, two fungal strains (Aspergillus flavus and Candida albicans) were confirmed. The stock novel compound solutions were prepared in DMSO. First, 100 μL of each of the synthesized TSCN compounds was inserted into discs (0.8 cm) and allowed to dry. The discs were completely saturated with the synthesized compounds and placed in the upper layer of the medium at least 25 mm from the edge. They were gently placed on the same plate surface. The plate was incubated at 37°C for 72 h, and a clear inhibition area was confirmed. Eventually, using the ruler millimeter, we determined the inhibition zone (an area where there is no growth around the discs).


#### 2.7.2.In Vitro Antitumor Activity

The synthesized TSCN compounds were screened for their cytotoxicity against liver cancer (HepG2) and breast cancer (MCF-7) cells using the sulforhodamine B (SRB) assay protocol [54].The potential cytotoxicity of the compounds was tested using the method reported by Skehan and Storeng. The cells were plated in 96-multiwell plates (104 cells/well) for 24 h before treatment with the compounds to allow the attachment of the cell to the wall of the plate. Different concentrations of the compounds under investigation (0, 5, 25, and 50μg/mL) were added to the cell monolayer, and triplicate wells were prepared for each dose. The monolayer cells were incubated with the compounds for 48 h at 37°C in a 5% CO2 atmosphere. Afterward, the cells were fixed, washed, and stained with SRB. The excess stain was washed with acetic acid, and the attached stain was recovered with tris‐ethylenediaminetetraacetic acid (EDTA) buffer. The optical density (OD) of each well was measured spectrophotometrically at 564 nm with an ELISA microplate reader. The mean background absorbance was automatically subtracted, and the mean values of each drug concentration were calculated. The relationship between the drug concentration and surviving fraction was plotted to obtain the survival curve of the breast and liver tumor cell lines for each compound.The percentage of cell survival was calculated as follows. Survival fraction = OD (treated cells)/OD (control cells). The IC50 values are the concentrations of the Schiff base ligand (L) or complexes required to produce 50% inhibition of cell growth. The experiment was performed thrice.


## 2.1. Materials and Reagents

The chemicals used in this study were supplied by Aldrich Chemicals Company and were used without extra purification.


## 2.2. Synthesis

### 2.2.1. Synthesis of (1-(p-Tolyl)Hydrazono)Propan-2-One (PTHP)

We synthesized PTHP using a reported method [31, 32]. Scheme 2 shows the chemical equations for the preparation.(1)CH3COCH2CO2C2H5+KOH⟶CH3COCH2CO2K+C2H5OH,CH3COCH2CO2K+HCl⟶CH3COCH2CO2H+,CH3COCH2CO2H+p−Me−C6H5N=NCl⟶p−Me−C6H5NH−N=CHCOCH3+CO2+HCl.Scheme 2
Preparation of (1-(p-tolyl)hydrazono)propan-2-one.


### 2.2.2. Synthesis of TSCN Compounds

Equimolar amounts of PTHP (0.1760 g, 1 mmol) in 30 mL of ethanol with an ethanolic solution (30 mL) of thiosemicarbazide (0.091 g, 1 mmol), N-ethylthiosemicarbazide (0.119 g, 1 mmol), and N-phenylthiosemicarbazide (0.167 g, 1 mmol) were refluxed on a hot plate for 3–5 hrs. The precipitate was separated, filtered off, washed with (C2H5)2O, and desiccated all night using silica gel. The target compounds are shown in Figure 1.Figure 1
Structural formula for the thiosemicarbazone compounds. TSC1, TSC2, and TSC3 withR = H, Et, and ph, respectively.(1) ((E)-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC1). Yield, 77%. Color, brown. Anal. Calc. for C11H15N5S: C, 52.99; H, 6.06; N, 28.09; S, 12.86. Found: C, 52.93; H, 6.01; N, 28.03; S, 12.79%. Infrared (IR) (KBr, cm−1): 3386, 3234 (NH2), 1507, 1251, 1017, 805 (thioamide bands, I–IV, respectively), 3501, 3177 (N2H), 1100 (N–N), 1603 (C = N), 1553 (C = C), 3045 (C–H). MS (m/z): 251 (M++2, 6.03%), 250 (M++1, 18.07%), 249 (M+, 100%), 234 (2.58%), 232 (38.20%), 159 (2.07%), 157 (0.59%), 118 (3.81%). Proton nuclear magnetic resonance (1H NMR) (DMSO): 11.32 (s, 1H, NH); 10.89 (s, 2H, NH); 7.8 (s, 2H, NH2); 7.48 (s, H, CH = N); 6.94–7.01 (m, 4H,–Ar); 2.21 (s, 3H,–CH3); 2.02 (s, 3H,–CH3). 13C-NMR (DMSO): 11.02, 20.09, 112.21, 129.51, 130.80, 136.01, 142.21, 145.62, 178.3.(2) ((E)-N-Ethyl-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC2). Yield, 69%. Color, dark brown. Anal. Calc. for C13H19N5S: C, 55.52; H, 6.17; N, 25.25; S, 11.56. Found: C, 55.48; H, 6.12; N, 25.19; S, 11.51%. IR (KBr, cm−1): 1532, 1251, 1079, 805 (thioamide bands, I–IV, respectively), 3444, 3338, 3230 (3NH), 1079 (N–N), 1615 (C = N), 1535 (C = C), 3019 (C–H). MS (m/z): 279 (M++2, 6.53%), 278 (M++1, 21.11%), 277 (M+, 100%), 262 (0.94%), 173 (2.73%), 118 (3.23%). 1H-NMR: 11.33 (s, 1H, NH); 10.33 (s, 1H, NH); 8.91 (s, 1H, NH); 7.54 (s, H, CH = N); 6.36–7.54 (m, 4H,–Ar); 3.60 (q, 2H,–CH2); 2.4 (t, 2H,–CH3); 2.23 (s, 3H,–CH3); 2.02 (s, 3H,–CH3). 13C-NMR: 11.01, 14.32, 20.09, 39.21, 112.23, 128.07, 129.61, 136.0, 142.23, 148.24, 177.04.(3) (E)-N-Phenyl-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC3). Yield, 67%. Color, brownish yellow. Anal. Calc. for C17H19N5S: C, 62.74; H, 5.89; N, 21.50; S, 9.85. Found: C, 62.75; H, 5.85; N, 21.47; S, 9.81%. IR (KBr, cm−1): 1518, 1250, 1065, 748 (thioamide bands, I–IV, respectively), 3444, 3259, 3020 (3NH), 1065 (N–N), 1603 (C = N), 1551 (C = C), 3020 (C–H). MS (m/z): 326 (M++1, 1.78%), 325 (M+, 7.41%), 118 (5.63%). 1H-NMR: 11.32 (s, 1H, NH); 10.30 (s, H, NH); 7.70 (s, H, NH); 7.61 (s, H, CH = N); 6.4–7.70 (m, 9H,–Ar); 2.21 (s, 3H,–CH3); 2.03 (s, 3H,–CH3). 13C-NMR: 11.33, 20.13, 39.40, 112.24, 115.63, 124.94, 128.28, 129.47, 135.87, 138.92, 142.16, 149.22, 175.9.


## 2.2.1. Synthesis of (1-(p-Tolyl)Hydrazono)Propan-2-One (PTHP)

We synthesized PTHP using a reported method [31, 32]. Scheme 2 shows the chemical equations for the preparation.(1)CH3COCH2CO2C2H5+KOH⟶CH3COCH2CO2K+C2H5OH,CH3COCH2CO2K+HCl⟶CH3COCH2CO2H+,CH3COCH2CO2H+p−Me−C6H5N=NCl⟶p−Me−C6H5NH−N=CHCOCH3+CO2+HCl.Scheme 2
Preparation of (1-(p-tolyl)hydrazono)propan-2-one.


## 2.2.2. Synthesis of TSCN Compounds

Equimolar amounts of PTHP (0.1760 g, 1 mmol) in 30 mL of ethanol with an ethanolic solution (30 mL) of thiosemicarbazide (0.091 g, 1 mmol), N-ethylthiosemicarbazide (0.119 g, 1 mmol), and N-phenylthiosemicarbazide (0.167 g, 1 mmol) were refluxed on a hot plate for 3–5 hrs. The precipitate was separated, filtered off, washed with (C2H5)2O, and desiccated all night using silica gel. The target compounds are shown in Figure 1.Figure 1
Structural formula for the thiosemicarbazone compounds. TSC1, TSC2, and TSC3 withR = H, Et, and ph, respectively.(1) ((E)-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC1). Yield, 77%. Color, brown. Anal. Calc. for C11H15N5S: C, 52.99; H, 6.06; N, 28.09; S, 12.86. Found: C, 52.93; H, 6.01; N, 28.03; S, 12.79%. Infrared (IR) (KBr, cm−1): 3386, 3234 (NH2), 1507, 1251, 1017, 805 (thioamide bands, I–IV, respectively), 3501, 3177 (N2H), 1100 (N–N), 1603 (C = N), 1553 (C = C), 3045 (C–H). MS (m/z): 251 (M++2, 6.03%), 250 (M++1, 18.07%), 249 (M+, 100%), 234 (2.58%), 232 (38.20%), 159 (2.07%), 157 (0.59%), 118 (3.81%). Proton nuclear magnetic resonance (1H NMR) (DMSO): 11.32 (s, 1H, NH); 10.89 (s, 2H, NH); 7.8 (s, 2H, NH2); 7.48 (s, H, CH = N); 6.94–7.01 (m, 4H,–Ar); 2.21 (s, 3H,–CH3); 2.02 (s, 3H,–CH3). 13C-NMR (DMSO): 11.02, 20.09, 112.21, 129.51, 130.80, 136.01, 142.21, 145.62, 178.3.(2) ((E)-N-Ethyl-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC2). Yield, 69%. Color, dark brown. Anal. Calc. for C13H19N5S: C, 55.52; H, 6.17; N, 25.25; S, 11.56. Found: C, 55.48; H, 6.12; N, 25.19; S, 11.51%. IR (KBr, cm−1): 1532, 1251, 1079, 805 (thioamide bands, I–IV, respectively), 3444, 3338, 3230 (3NH), 1079 (N–N), 1615 (C = N), 1535 (C = C), 3019 (C–H). MS (m/z): 279 (M++2, 6.53%), 278 (M++1, 21.11%), 277 (M+, 100%), 262 (0.94%), 173 (2.73%), 118 (3.23%). 1H-NMR: 11.33 (s, 1H, NH); 10.33 (s, 1H, NH); 8.91 (s, 1H, NH); 7.54 (s, H, CH = N); 6.36–7.54 (m, 4H,–Ar); 3.60 (q, 2H,–CH2); 2.4 (t, 2H,–CH3); 2.23 (s, 3H,–CH3); 2.02 (s, 3H,–CH3). 13C-NMR: 11.01, 14.32, 20.09, 39.21, 112.23, 128.07, 129.61, 136.0, 142.23, 148.24, 177.04.(3) (E)-N-Phenyl-2-((E)-1-(2-(p-Tolyl)Hydrazono)Propan-2-Ylidene)Hydrazine-1-Carbothioamide (TSC3). Yield, 67%. Color, brownish yellow. Anal. Calc. for C17H19N5S: C, 62.74; H, 5.89; N, 21.50; S, 9.85. Found: C, 62.75; H, 5.85; N, 21.47; S, 9.81%. IR (KBr, cm−1): 1518, 1250, 1065, 748 (thioamide bands, I–IV, respectively), 3444, 3259, 3020 (3NH), 1065 (N–N), 1603 (C = N), 1551 (C = C), 3020 (C–H). MS (m/z): 326 (M++1, 1.78%), 325 (M+, 7.41%), 118 (5.63%). 1H-NMR: 11.32 (s, 1H, NH); 10.30 (s, H, NH); 7.70 (s, H, NH); 7.61 (s, H, CH = N); 6.4–7.70 (m, 9H,–Ar); 2.21 (s, 3H,–CH3); 2.03 (s, 3H,–CH3). 13C-NMR: 11.33, 20.13, 39.40, 112.24, 115.63, 124.94, 128.28, 129.47, 135.87, 138.92, 142.16, 149.22, 175.9.


## 2.3. Methods and Experimental Measurements

The chemicals used were supplied by Aldrich. A CHNS automatic analyzer (Vario EII-Elementar) was used to conduct elemental microanalysis for C, H, N, and S. A PerkinElmer Fourier transform IR (FTIR) type 1650 spectrophotometer with a potassium bromide disc was used to record the IR spectra. Electronic spectra were recorded on a Shimadzu UV-3101PC spectrophotometer. A Bruker ARX-300 device was applied to record the1H-NMR spectra. Chemical shifts were recorded in ppm comparative to tetramethylsilane (TMS) using deuterated dimethyl sulphoxide (d6-DMSO) as a solvent. Mass spectrometry was conducted using a Shimadzu GCMS-QP1000EX. The X-ray single-crystal diffraction of the TSC2 molecule was performed using a Rigaku VariMax RAPID FR-E diffractometer with monochromatized Mo Kα radiation at a radiation wavelength of λ = 0.71075 Å in ω scan mode. The crystal was cooled with a cold N2 gas flow. We performed diffraction data scaling, cell refinement, indexing, collection, and peak integration using the Rapid Auto software (Rigaku). The molecular structure was analyzed using Mercury 4.1.3 software. However, the X-ray crystallography data of TSC2 and TSC3 were collected by mounting a single sample crystal on glass fiber. The cell parameters and intensity data collection were performed at 298 K using monochromatized Mo Kα radiation at a radiation wavelength of λ = 0.71073 Ǻ. The crystal structure was analyzed using the SIR-92 program [33] and was refined on F2 using the full-matrix least-squares technique with the maXus processor program for the solution and refinement of crystal structures [34]. The Oak Ridge Thermal Ellipsoid Plot (ORTEP) program was used for molecular graphics [35].A Metrohm 848 Titrino supplied with a Dosimat unit (Switzerland-Herisau) was used for potentiometric titrations as previously described in 50% water-DMSO mixture [29, 30, 32].


## 2.4. Potentiometric Titrations

Using the aforementioned potentiometric technique [36], the formation constant of the complex was estimated. Standard buffer solutions were used to accurately calibrate the glass electrode to the National Bureau of Standards (NBS) [37]. A standard solution of 0.05 mol/dm3 NaOH, free from CO2, was used to titrate the samples in an N2 atmosphere. The sample solution was developed to avoid hydrolysis during titration by mixing equal volumes of DMSO and H2O. The ionic strength was maintained during titration using NaNO3 as a supporting electrolyte.The calculation of the formation constants using the potentiometric method was performed using a concentration of hydrogen ion expressed in molarity. The concentration in the pH meter was expressed in activity coefficient −log aH+ (pH). Thus, Van Uitert and Hass used equation (2) to change the pH meter reading (B) to [H+] as follows [38, 39]:(2)−log10H+=B+log10UH,where log10 UH = solvent composition correction factor and the ionic strength read by B. The pKw values for the titrated samples were estimated as previously described [40]. All precautions and procedures comply with literature requirements [41–43].The protonation constants of TSC1 were estimated potentiometrically by titrating 40 cm3 of a 1.25 × 10−3 mol/dm3 TSC1 solution with a standard sodium hydroxide solution.


## 2.5. Processing of Data

The MINIQUAD-75 computer program was applied to calculate ca. 100–150 readings for each titration [44]. Species distribution diagrams for the studied samples were produced using the SPECIES program [45].


## 2.6. Molecular Modeling Studies

Density functional theory (DFT) calculations were performed using the DMol3 program [46, 47] in the Materials Studio package [48]. The calculations for the DFT semicore pseudopods (DSPP) were created with dual numerical base sets and polarization properties (DNPs) [49]. The revised Perdew–Burke–Ernzerhof (RPBE) model was focused on the generalized gradient approximation (GGA) as the best functional approximation [50, 51].


## 2.7. Biological Activity

### 2.7.1. Antibacterial and Antifungal Activities

The ability of the synthesized TSCN compounds to suppress bacterial growth was confirmed using the disc diffusion process [52, 53]. Aerobic G+ bacteria including Staphylococcus aureus, Bacillus subtilis, and Streptococcus faecalis and G− aerobic bacteria including Pseudomonas aeruginosa, E. coli, and Neisseria gonorrhoeae were among the bacterial strains used in this study. Additionally, two fungal strains (Aspergillus flavus and Candida albicans) were confirmed. The stock novel compound solutions were prepared in DMSO. First, 100 μL of each of the synthesized TSCN compounds was inserted into discs (0.8 cm) and allowed to dry. The discs were completely saturated with the synthesized compounds and placed in the upper layer of the medium at least 25 mm from the edge. They were gently placed on the same plate surface. The plate was incubated at 37°C for 72 h, and a clear inhibition area was confirmed. Eventually, using the ruler millimeter, we determined the inhibition zone (an area where there is no growth around the discs).


### 2.7.2.In Vitro Antitumor Activity

The synthesized TSCN compounds were screened for their cytotoxicity against liver cancer (HepG2) and breast cancer (MCF-7) cells using the sulforhodamine B (SRB) assay protocol [54].The potential cytotoxicity of the compounds was tested using the method reported by Skehan and Storeng. The cells were plated in 96-multiwell plates (104 cells/well) for 24 h before treatment with the compounds to allow the attachment of the cell to the wall of the plate. Different concentrations of the compounds under investigation (0, 5, 25, and 50μg/mL) were added to the cell monolayer, and triplicate wells were prepared for each dose. The monolayer cells were incubated with the compounds for 48 h at 37°C in a 5% CO2 atmosphere. Afterward, the cells were fixed, washed, and stained with SRB. The excess stain was washed with acetic acid, and the attached stain was recovered with tris‐ethylenediaminetetraacetic acid (EDTA) buffer. The optical density (OD) of each well was measured spectrophotometrically at 564 nm with an ELISA microplate reader. The mean background absorbance was automatically subtracted, and the mean values of each drug concentration were calculated. The relationship between the drug concentration and surviving fraction was plotted to obtain the survival curve of the breast and liver tumor cell lines for each compound.The percentage of cell survival was calculated as follows. Survival fraction = OD (treated cells)/OD (control cells). The IC50 values are the concentrations of the Schiff base ligand (L) or complexes required to produce 50% inhibition of cell growth. The experiment was performed thrice.


## 2.7.1. Antibacterial and Antifungal Activities

The ability of the synthesized TSCN compounds to suppress bacterial growth was confirmed using the disc diffusion process [52, 53]. Aerobic G+ bacteria including Staphylococcus aureus, Bacillus subtilis, and Streptococcus faecalis and G− aerobic bacteria including Pseudomonas aeruginosa, E. coli, and Neisseria gonorrhoeae were among the bacterial strains used in this study. Additionally, two fungal strains (Aspergillus flavus and Candida albicans) were confirmed. The stock novel compound solutions were prepared in DMSO. First, 100 μL of each of the synthesized TSCN compounds was inserted into discs (0.8 cm) and allowed to dry. The discs were completely saturated with the synthesized compounds and placed in the upper layer of the medium at least 25 mm from the edge. They were gently placed on the same plate surface. The plate was incubated at 37°C for 72 h, and a clear inhibition area was confirmed. Eventually, using the ruler millimeter, we determined the inhibition zone (an area where there is no growth around the discs).


## 2.7.2.In Vitro Antitumor Activity

The synthesized TSCN compounds were screened for their cytotoxicity against liver cancer (HepG2) and breast cancer (MCF-7) cells using the sulforhodamine B (SRB) assay protocol [54].The potential cytotoxicity of the compounds was tested using the method reported by Skehan and Storeng. The cells were plated in 96-multiwell plates (104 cells/well) for 24 h before treatment with the compounds to allow the attachment of the cell to the wall of the plate. Different concentrations of the compounds under investigation (0, 5, 25, and 50μg/mL) were added to the cell monolayer, and triplicate wells were prepared for each dose. The monolayer cells were incubated with the compounds for 48 h at 37°C in a 5% CO2 atmosphere. Afterward, the cells were fixed, washed, and stained with SRB. The excess stain was washed with acetic acid, and the attached stain was recovered with tris‐ethylenediaminetetraacetic acid (EDTA) buffer. The optical density (OD) of each well was measured spectrophotometrically at 564 nm with an ELISA microplate reader. The mean background absorbance was automatically subtracted, and the mean values of each drug concentration were calculated. The relationship between the drug concentration and surviving fraction was plotted to obtain the survival curve of the breast and liver tumor cell lines for each compound.The percentage of cell survival was calculated as follows. Survival fraction = OD (treated cells)/OD (control cells). The IC50 values are the concentrations of the Schiff base ligand (L) or complexes required to produce 50% inhibition of cell growth. The experiment was performed thrice.


## 3. Results and Discussion

### 3.1. Characterization of the Synthesized TSCN Compounds

The condensation of PTHP with thiosemicarbazide, N-ethylthiosemicarbazide, and N-phenylthiosemicarbazide readily yielded the corresponding TSC1, TSC2, and TSC3 compounds. The isolated compounds were air-stable and insoluble in H2O but easily soluble in dimethylformamide (DMF) or DMSO. Different analytical tools were used to identify the structure of the prepared compounds. The results from the basic analysis were correlated with the calculated results for the proposed formula. The novel TSCs structures are shown in Figure 1.


#### 3.1.1. IR Spectrum

The preliminary allocations of major IR spectrum bands of the TSCN compounds showed a disappearance ofν (>C = O) and a new band within the range of 1603–1615 cm−1 attributable to ν (C = N) stretching vibration [55], supporting condensation reaction and formation of the TSCN compounds. TSCN compounds exhibit thione ↔ thiol tautomerism [56] because of the existence of an–NH–C = S linkage, but ν (S–H) absorption band within the range of 2500–2600 cm−1 was absent with the appearance of ν (C = S) band around 750 cm−1, indicating the existence of the prepared TSCN compounds in the thione form in the solid state. For the prepared TSCN compounds, vibrational bands with the wave numbers of 3012 cm−1 (νC–H, Ar–H), 1615 cm−1 (νC = N), 1548 cm−1 (νC = C), and 1082 cm−1 (νN–N) were detected.Four vibrational bands with wave numbers in the ranges of 3019–3045 cm−1 (νC–H, Ar–H), 1601–1615 cm−1 (νC = N), 1535–1553 cm−1 (νC = C), and 1065–1100 cm−1 (νN–N) were observed for the TSCN compounds. The νsym and νasym of the TSC1 amino group were observed at 3234 and 3386 cm−1, respectively. In the spectra of the TSCN compounds, the bands observed in the ranges of 1494–1532, 1249–1251, 1017–1088, and 748–817 cm−1 were assigned to the thioamide bands of I, II, III, and IV, respectively [57].


#### 3.1.2. NMR Spectrum

The1H-NMR spectra of the novel TSC1, TSC2, and TSC3 compounds in DMSO-d6 did not exhibit resonance at approximately 4.0 ppm corresponding to the –SH proton resonance [58], whereas the presence of a peak at 10.77 ppm (signal field of existence of the NH group next to C = S) suggested that they remained in the thione form even in a polar solvent, such as DMSO. The signals for the methine proton of an azomethine group for the TSCN compounds, CH = N, were detected at δ = 7.48–7.61 ppm. In the region of 6.36–7.54 ppm, chemical shifts were allocated for the hydrogen of the aromatic ring. A methyl group was observed as a singlet signal at δ = 2.02–2.20 ppm. The spectrum of TSC1 showed signals at δ = 7.80 ppm, assigned to the NH2 proton.The13C-NMR spectra of the novel TSCN compounds were recorded in DMSO-d6. The peaks of azomethine carbons and C = S of the TSCN compounds were observed as singlet peaks. The signal for the carbon atom of C = S was detected at 178.3, 177.8, and 178.1 in TSC1, TSC2, and TSC3, respectively.


#### 3.1.3. Ultraviolet-Visible (UV-Vis) Spectrum

A strong absorption band detected within the range of 33003–32787 cm−1 was assigned to π⟶π∗ transitions of the (C = N)azomethine group, while the possible assignments for the bands within 26809–27548 cm−1 were attributed to n⟶π∗TSCN compound transitions. Permanent π⟶π∗ transitions occur at a higher energy than n⟶π∗ transitions [59].


#### 3.1.4. Mass Spectra

The proposed formula was further confirmed by mass spectroscopy. The electron impact mass spectrum of TSC1 confirmed the suggested formula by exhibiting a peak at 249 equivalent to C11H15N5S compound moiety, in addition to a series of peaks attributable to different fragments of TSC1. These data suggested that a ketone PTHP group was condensed with the NH2 group of thiosemicarbazide or its derivatives. The mass spectra of TSC1, TSC2, and TSC3 showed peaks at 249, 277, and 325 m/z, confirming the proposed structural formula of the synthesized TSC1, TSC2, and TSC3 compounds, respectively.


#### 3.1.5. Crystallography

The structures of TSC2 and TSC3 were established through X-ray crystallography. The recrystallization of the compounds from hot ethanol, followed by slow evaporation, led to the formation of single crystals. CCDC2026108 and CCDC 2033322 contain the supplementary crystallographic data for this study.The X-ray single-crystal structures of monomeric TSC2 and TSC3 compounds are shown in Figures2(a) and 2(b), respectively. It suggested that TSC2 was crystallized in a monoclinic crystal system with space P21/c, a = 27.985 (12) Å, b = 12.027 (5) Å, c = 9.833 (4) Å, α = 90.0°, β = 93.117 (11)°, γ = 90.0°, V = 3486.57 Å3, and Z = 7, while TSC3 was crystallized also in monoclinic crystal system with space P21/c, a = 11.2343 (6) Å, b = 11.2575 (7) Å, c = 11.8995 (8) Å, α = 90.00°, β = 94.476(6)°, γ = 90.0°, V = 1500.34 (3) Å3, and Z = 4.Figure 2
X-ray structure of (a) TSC2 (b) TSC3 along with the atom numbering scheme.
(a)(b)Note worthily, TSC2 molecules are noncovalently stacked via intermolecular interactions, i.e., van der Waals and H-bonding interactions. This is illustrated in Figure3. In the packing structure of the TSC2 and TSC3 molecules, the unit cell included four and seven molecules stacked to each other per unit cell.Figure 3
Expansion of the intermolecular interactions (i.e., van der Waals interaction) between the TSC2 molecules.


#### 3.1.6. Molecular Modeling and/or Molecular Parameters

Quantum parameters such as EHOMO and ELUMO, in addition to specific parameters such as the ionization potential (IP), absolute softness (σ), absolute hardness (η), electron affinity (EA), separation energy (ΔE), absolute electronegativity (χ), global softness (S), electrophilicity (w), electron-accepting power (w+), electron-donating power (w−), and additional electronic charge (ΔNmax) [60–64], have been computed according to Eqs. 2–11 as shown below [60–65]. The inverse of the global hardness is called softness σ [66].(3)χ=−12ELUMO+EHOMO,(4)IP=−EHOMO,(5)η=12ELUMO−EHOMO,(6)S=12η,(7)ΔNmax=−IEη,(8)σ=1η,(9)EA=−ELUMO,(10)ω=IE22η,(11)ω−=3∗IE+EA216IE−EA,(12)ω−=IE+3∗EA216IE−EA.The geometric optimization of the prepared compounds can be characterized using theoretical calculations; therefore, the optimized structure for the synthesized compounds was obtained by calculating theoretical physical parameters, such as bond lengths and bond angles using DFT calculations.The quantum parameters of the synthesized TSC1, TSC2, and TSC3 compounds were calculated using equations (3)–(12). As shown in Tables 1 and 2, we deduced the following:(a)
HOMO and LUMO are frontier molecular orbitals (FMOs). The HOMO and LUMO energies were negative, indicating that the studied TSC1, TSC2, and TSC3 TSCs were stable molecules [65].(b)
The HOMO and LUMO functioned as electron donor and acceptor, respectively [66, 67].(c)
Hard and soft nucleophiles exhibit low and high HOMO energies, respectively, while hard and soft electrophiles exhibit high and low LUMO energies, respectively.(d)
The HOMO energies of TSC1, TSC2, and TSC3 were closely spaced (EHOMO (TSC1) −8.501 eV; EHOMO (TSC2) −8.561 eV; and EHOMO (TSC3) −8.556 eV).(e)
Soft molecules are characterized by a small energy gap (ELUMO−EHOMO) and higher reactivity than hard molecules because of their easy donation of electrons to an acceptor [68, 69].(f)
Large values of the HOMO–LUMO energy gap (7.410–7.667) indicated good stability and a large chemical hardness for the synthesized TSCN compounds.(g)
Absolute hardness (η) and softness (σ) are essential characteristics in the calculation of molecular stability and reactivity. Hard molecules exhibit a large energy gap with less reactivity (ΔE = EHOMO−ELUMO), whereas soft molecules exhibit a smaller energy space and greater reactivity. This indicated that ΔE was an indicator of stability and can be used to measure the chemical reactivity and kinetic stability of the compound [67, 70].(h)
When the HOMO energy decreased, the ability of the molecule to donate electrons decreased, while a high HOMO energy indicated that the molecule was an efficient electron donor. The LUMO energy indicated the ability of a molecule to receive an electron.(i)
The electrophilicity index (ω) followed this trend: TSC1 (ω = 3.104) > TSC2 (ω = 2.936) > TSC3 (ω = 2.933). Thus, TSC1 exhibited the maximum value of electrophilicity, confirming its high capability to accept electrons.(j)
The HOMO level in the TSCN compounds was commonly localized on the C = N groups, demonstrating that they are the favored sites for Nu attack at the central metal ion.(k)
The negativity of EHOMO and ELUMO indicated the stability of the TSCN compounds [71].Table 1
Some energetic properties of the synthesized thiosemicarbazone compounds.
CompoundTotal energy (kcal/mol)Binding energy (kcal/mol)Electronic energy (kcal/mol)Dipole moment (Debyes)HOMOLUMOTSC1−58321.2−3178.9−368233.16.55−8.501−1.091TSC2−65214.1−3735.7−441527.76.82−8.561−0.915TSC3−79668.2−4295.6−566955.26.94−8.556−0.889Table 2
Calculated quantum chemical parameters of the synthesized thiosemicarbazone compounds.
CompoundΔEIAχηSΔNmaxωω−ω+TSC17.4108.5011.0914.7963.7050.270−2.2943.1045.9651.169TSC27.6468.5610.9154.7383.8230.262−2.2392.9365.7831.045TSC37.6678.5560.8894.7233.8340.261−2.2322.9095.7491.027


### 3.2. Biological Activity

#### 3.2.1. Antibacterial and Antifungal Activities

The antimicrobial activities of the TSCN compounds were screened. The results of the antimicrobial activities of the TSCN compounds against the tested microbes are shown in Table3. TSC3 was inactive against the tested Gram-positive and Gram-negative bacterial species. TSC1 exhibited effective antibacterial activity against the tested bacterial strains. TSC1 exhibited an extremely higher antifungal activity against Candida albicans against Amphotericin, which is a standard antifungal agent. TSC2 exhibited moderate antibacterial activity against Streptococcus faecalis (Gram-positive) and Escherichia coli (Gram-negative). The data showed that the most effective compound was TSC1.Table 3
Antibacterial and antifungal activities of the synthesized thiosemicarbazone compounds.
Concentration (μg/mL)Gram-positiveGram-negativeFungiBacillus subtilisStaphylococcus aureusStreptococcus faecalisE. coliNeisseria gonorrhoeaePseudomonas aeruginosaAspergillus flavusCandida albicansTSC1141317171221030TSC30013110000TSC400000000Ampicillin262127252826——Amphotericin——————1619Ampicillin: standard antibacterial agent, Amphotericin: standard antifungal agent.


#### 3.2.2. Cytotoxic Activities

The cytotoxic activity of the TSCs was investigated against liver cancer cell (HepG2) and breast cancer cell (MCF-7) lines. IC50 is the concentration that can diminish cancer cell growth by 50%. The results of the cytotoxic study indicated that TSC1 exhibited significant activity against the HepG2 and MCF-7 cells with IC50 values of 29.4 and 122 μg/mL, respectively (Table 4), while the IC50 values toward the HepG2 and MCF-7 cells for TSC2 were 46 and 172.9 μg/mL, respectively. Furthermore, the IC50 values toward the HepG2 and MCF-7 cells for TSC3 were 25 and 107 μg/mL, respectively. The antitumor results indicated that the synthesized TSCN compounds were more effective against HepG2 than MCF-7. The antitumor activity of the synthesized TSCN compounds toward HepG2 and MCF-7 cell lines followed this order: TSC3 > TSC1 > TSC2 (Figure 4).Table 4
Antitumor activity of the synthesized thiosemicarbazone compounds.
Concentration (μg/mL)TSC1TSC2TSC3Liver cancer cell line (HepG2)51001001002535.36722.55027.448.719IC5029.44625Breast cancer cell line (MCF-7)583.89577.62579.586.5725070.78365.4IC50122172.9107Figure 4
Cytotoxicity of TSC1, TSC2, and TSC3 on breast and liver cancer cell lines.


#### 3.2.3. Molecular Modeling and Biological Activity

Theoretical calculations were performed to investigate the physicochemical properties that possibly correlated with the antimicrobial action of the investigated TSCs. TSC1, which offered the lowest HOMO energy among the synthesized TSCs, exhibited the highest biological activity among the synthesized compounds. The data showed an inverse relationship between the dipole moment and lipophilicity, indicating that as the dipole moment decreased, the lipophilic nature of the compound increased, which powerfully favored its penetration via a lipid layer of microorganisms [72, 73], thereby violently destroying them. As shown in Table 2, the lipophilicity of TSC1 was higher than those of the other TSCN compounds, which sequentially deactivated the enzymes responsible for the respiration process of the investigated microbes more than the other compounds did, and accordingly increased its cellular uptake by bacterial cells. TSCs exhibit antibacterial activity because of the existence of toxophorically essential imine groups (–C=N) where the action mode of these compounds could include the formation of H-bonds via the azomethine group with an active center of cells that may interfere with ordinary cell processes [74].


### 3.3. Equilibrium Studies

The protonation constants of the TSC1 ligand were calculated (Table5). These ligands behaved triprotic, as shown in the following equations: (13)L−+H+⇌HL;K1=HLL−H+,(14)HL+H+⇌H2L+;K2=H2L+HLH+,(15)H2L++H+⇌H3L2+;K3=HL2+H+H2L+.Table 5
Protonation constants for TSC1 ligand at different temperatures.
Reactionlog K (±σ)a15°C25°C35°CL + H = HL8.02 (0.09)7.81 (0.05)7.64 (0.03)HL + H = H2L3.31 (0.08)3.22 (0.07)3.14 (0.09)H2L + H = H3L3.17 (0.04)3.08 (0.06)3.01 (0.05)a(σ) is the standard deviation.We concluded that the 1st and 2nd deprotonation constants corresponded to the deprotonation of the two N-imino sites in the TSC ligand, as shown in Scheme3(a). The 3rd deprotonation constant corresponded to the thiolate group site in the TSC ligand, as shown in Scheme 3(b). A similar conclusion was obtained in a previous study [75].Scheme 3
(a) Possible deprotonation pathway of the imino groups and (b) possible deprotonation pathway of the thiolate group.
(a)(b)The log KN-imino values ranging from 2.97 to 3.35 were similar to that observed in a previous study for the imino group (4.40) [76]. The log KSH values ranging from 7.65 to 8.05 were similar to those described in a previous study for the thiolate group (5.5–9.0) [77].A protonation equilibrium study for the TSC ligands in this study cannot be performed in an aqueous solution since it is insoluble in H2O. The DMSO solvent has been extensively used for potentiometric studies of protonation and formation equilibria. The DMSO-water mixture (50%:50%) was best preferred to yield a soluble and stable Schiff base solution [58, 78].Here, three protonation constants were calculated for the TSC1 ligand, and the SPECIES program was used to determine the distribution of the TSC1 ligand species as a function of pH. Figure5(a) shows a classic species distribution diagram; it is possible to highlight, at the mentioned experimental conditions, the pH ranges within which the various species were formed and/or coexisted and their relative formation percentages. Thus, the species distribution graph is a good tool for a thorough observation of the concentration of each species present as a function of pH. It enabled us to obtain the best conditions (pH, concentration, and ligand:metal ratio) for the preparation of a solid complex. At a low pH, TSC1 initially existed in a fully protonated form with a maximum percentage of 100% as H3L below pH < 2. Upon the addition of the base, the pH increased causing the H3L species to lose its first proton from an imino group to form H2L, which is the major species at a pH of 3.3. As the conditions became more alkaline, the second proton released from the second imino group began deprotonation to an HL ligand, thereby achieving the highest percent of 99.1% at a pH of 5.8. A further increase in pH was followed by the liberation of the third H+, forming the fully deprotonated ligand, L, with maximum percent species of 98.4% at a pH of 10.0.Figure 5
(a) Concentration distribution diagram of various species as a function of pH in the TSC1 system (at a concentration of TSC1 = 1.25mM and T = 25°C). (b) Concentration distribution diagram of various species as a function of pH in the Cd-TSC1 system.
(a)(b)


#### 3.3.1. Species Distribution Curves

The calculation of the equilibrium complex concentrations of Cd (II) with TSC1 (Table6) as a function of pH showed metal (II) binding in the biological system. As an illustrative example of metal complexes, Figure 5(b) shows a concentration distribution diagram for the complex, Cd (II)-TSC1. The Cd-TSC1 complex began to form in an acidic pH range reaching a constant concentration of 99.9% at a pH of 5.0, whereas the Cd (TSC1)2 complex species reached a maximum concentration of 45% at a pH of 9.8.Table 6
Stepwise stability for the complexes of TSC1 with Cd (II) metal ion in 50% DMSO-H2O (V/V) solution.
Temp.(°C)logK1 (±σ)alogK2 (±σ)logK1- logK2CdLCdL2Cd (II) complex15°C9.82(0.07)2.80(0.03)7.0225°C9.74(0.03)2.75(0.06)6.9935°C9.57(0.06)2.71(0.09)6.86a (σ) is the standard deviation; definitions of stability constants: K1 = [CdL]/[Cd][L]; K2 = [CdL2]/[CdL][L]; L = TSC1 thiosemicarbazone ligand (charges are omitted for simplicity).


#### 3.3.2. Thermodynamics

The data derived for ΔH°, ΔS°, and ΔG° related to the protonation of TSC1 and Cd (II) complex formation were calculated from the data in Tables7 and 8. For the ligand protonation or complexation, ΔH° was determined from the plot slope (Figures 6(a)–6(b)) through the graphical representation of the Van ’t Hoff equation as follows:(16)−2.303RTlog10K=ΔH°−TΔS°,logK10=−ΔH°2.303R1T+ΔS°R.Table 7
Thermodynamic parameters for the protonation of ligand (TSC1) in 50% DMSO-H2O (V/V) solution.
ParameteraReactionL + H = HLaL + 2H = H2LL + 3H = H3L15oC25°C35°C15°C25°C35°C15°C25°C35°C−ΔG44.2544.5945.0817.4917.5817.8218.2618.3818.47−ΔH32.3315.2912.77ΔS41.1610.3016.27ΔG: Gibbs energy/kJ.mol−1; ΔH: enthalpy/kJ.mol−1; ΔS: entropy/J.mol−1.K−1.Table 8
Thermodynamic parameters for Cd (II)-TSC1 complexes in 50% DMSO-H2O (V/V) solution.
ParameteraReactionCd +L = CdLCd + 2L = CdL215°C25°C35°C15°C25°C35°C−ΔG54.1855.6056.4515.4515.7015.99−ΔH21.157.66ΔS114.8826.99aΔG: Gibbs energy/kJ.mol−1; ΔH: enthalpy/kJ.mol−1; ΔS: entropy/J.mol−1.K−1.Figure 6
(a) Effect of temperature on the protonation constant of TSC1. (b) Effect of temperature on the formation constant of the Cd (II)-TSC1 complexes.
(a)(b)With the well-known relations (6) and (7), from the ΔG and enthalpy change (ΔH) values, we calculated ΔS as follows:(17)ΔG°=−2.303RTlog10K,(18)ΔS°=ΔH°−ΔG°T.The main reasons for the protonation constant determination can be explained as follows:(1)
The ratio and pH of the various substance forms can be determined using their protonation constants.(2)
It is extremely useful in the preparation of newly synthesized compounds. The suggested structure can be reliable where protonation constants are theoretically well-calculated according to the experimental values.(3)
Considering that different types of substances exhibit different UV spectra, quantitative spectrophotometric analyses can be performed by choosing the appropriate pH. To choose the pH, the known protonation constants are required.(4)
The preparation of buffer solutions at different pH levels requires protonation constants [43, 79].(5)
The measurements of the stability constants for the complicated formation reactions of bioactive ion compounds require protonation constants. Additionally, their protonation constants are used to calculate the stability constants of the dynamic formation of bioactive compounds with metal ions [80].(6)
The equilibrium constants of certain compounds must be understood to measure the concentration of each ionized species at a certain pH; this is fundamental to understanding their physiochemical behavior [80].Tables7 and 8 describe the thermodynamic functions measured and can be interpreted as follows.(1)
The corresponding thermodynamic processes for the protonation reactions include the following:(i)
Exothermic processes for neutralization reactions.(ii)
Endothermic processes for ion desolvation.(iii)
Structure alteration and alignment of H-bonds around protonated and free ligands.(2)
When the temperature increased, the value of log10 KH decreased, and its acidity increased as the temperature increased.(3)
The negative ∆H° for the protonation of the TSC1 ligand indicated that this process was exothermic followed by the release of heat.(4)
The positive entropy of the TSC1 protonation reaction indicated increased disorder due to the desolvation processes and breakdown of H-bonds.Table6 lists the stepwise stability constants of the complexes formed at various temperatures. These values decreased and confirmed with an increase in temperature that the complexation phase was preferred at a low temperature.These results provided the following findings:(1)
A negative ∆H° showed that the coordinating process was exothermic, suggesting that the metal-ligand bonds were fairly strong and that the complexity reactions were preferred at low temperatures.(2)
The complexation reactions were spontaneous with a negative ∆G°.(3)
It was widely observed that the ∆G° and ∆H° values for 1 : 1 complexes were more negative than those corresponding to 1 : 2 complexes.(4)
This can be due to the steric hindrances caused by the addition of the 2nd ligand and the principle of charge neutralization.(5)
The electrostatic attraction in the 1 : 1 complex was larger than that in 1 : 2 complex since 1 : 1 complex was formed by a dipositively charged Mn+ and mononegatively charged ligand-anion interaction, while 1 : 2 complex was generated by a monopositively charged 1 : 1 complex and mononegatively charged ligand-anion interaction.(6)
The ∆S° values for the investigated complexes were positive, indicating that the entropy increase resultant from the release of bound solvent molecules during coordination was larger than the decrease resultant from the coordination process due to the ordered arrangement of solvent molecules around the ligand, and Mn + gained a random coordination pattern. This is referred to as an increase in entropy for configuration.


## 3.1. Characterization of the Synthesized TSCN Compounds

The condensation of PTHP with thiosemicarbazide, N-ethylthiosemicarbazide, and N-phenylthiosemicarbazide readily yielded the corresponding TSC1, TSC2, and TSC3 compounds. The isolated compounds were air-stable and insoluble in H2O but easily soluble in dimethylformamide (DMF) or DMSO. Different analytical tools were used to identify the structure of the prepared compounds. The results from the basic analysis were correlated with the calculated results for the proposed formula. The novel TSCs structures are shown in Figure 1.


### 3.1.1. IR Spectrum

The preliminary allocations of major IR spectrum bands of the TSCN compounds showed a disappearance ofν (>C = O) and a new band within the range of 1603–1615 cm−1 attributable to ν (C = N) stretching vibration [55], supporting condensation reaction and formation of the TSCN compounds. TSCN compounds exhibit thione ↔ thiol tautomerism [56] because of the existence of an–NH–C = S linkage, but ν (S–H) absorption band within the range of 2500–2600 cm−1 was absent with the appearance of ν (C = S) band around 750 cm−1, indicating the existence of the prepared TSCN compounds in the thione form in the solid state. For the prepared TSCN compounds, vibrational bands with the wave numbers of 3012 cm−1 (νC–H, Ar–H), 1615 cm−1 (νC = N), 1548 cm−1 (νC = C), and 1082 cm−1 (νN–N) were detected.Four vibrational bands with wave numbers in the ranges of 3019–3045 cm−1 (νC–H, Ar–H), 1601–1615 cm−1 (νC = N), 1535–1553 cm−1 (νC = C), and 1065–1100 cm−1 (νN–N) were observed for the TSCN compounds. The νsym and νasym of the TSC1 amino group were observed at 3234 and 3386 cm−1, respectively. In the spectra of the TSCN compounds, the bands observed in the ranges of 1494–1532, 1249–1251, 1017–1088, and 748–817 cm−1 were assigned to the thioamide bands of I, II, III, and IV, respectively [57].


### 3.1.2. NMR Spectrum

The1H-NMR spectra of the novel TSC1, TSC2, and TSC3 compounds in DMSO-d6 did not exhibit resonance at approximately 4.0 ppm corresponding to the –SH proton resonance [58], whereas the presence of a peak at 10.77 ppm (signal field of existence of the NH group next to C = S) suggested that they remained in the thione form even in a polar solvent, such as DMSO. The signals for the methine proton of an azomethine group for the TSCN compounds, CH = N, were detected at δ = 7.48–7.61 ppm. In the region of 6.36–7.54 ppm, chemical shifts were allocated for the hydrogen of the aromatic ring. A methyl group was observed as a singlet signal at δ = 2.02–2.20 ppm. The spectrum of TSC1 showed signals at δ = 7.80 ppm, assigned to the NH2 proton.The13C-NMR spectra of the novel TSCN compounds were recorded in DMSO-d6. The peaks of azomethine carbons and C = S of the TSCN compounds were observed as singlet peaks. The signal for the carbon atom of C = S was detected at 178.3, 177.8, and 178.1 in TSC1, TSC2, and TSC3, respectively.


### 3.1.3. Ultraviolet-Visible (UV-Vis) Spectrum

A strong absorption band detected within the range of 33003–32787 cm−1 was assigned to π⟶π∗ transitions of the (C = N)azomethine group, while the possible assignments for the bands within 26809–27548 cm−1 were attributed to n⟶π∗TSCN compound transitions. Permanent π⟶π∗ transitions occur at a higher energy than n⟶π∗ transitions [59].


### 3.1.4. Mass Spectra

The proposed formula was further confirmed by mass spectroscopy. The electron impact mass spectrum of TSC1 confirmed the suggested formula by exhibiting a peak at 249 equivalent to C11H15N5S compound moiety, in addition to a series of peaks attributable to different fragments of TSC1. These data suggested that a ketone PTHP group was condensed with the NH2 group of thiosemicarbazide or its derivatives. The mass spectra of TSC1, TSC2, and TSC3 showed peaks at 249, 277, and 325 m/z, confirming the proposed structural formula of the synthesized TSC1, TSC2, and TSC3 compounds, respectively.


### 3.1.5. Crystallography

The structures of TSC2 and TSC3 were established through X-ray crystallography. The recrystallization of the compounds from hot ethanol, followed by slow evaporation, led to the formation of single crystals. CCDC2026108 and CCDC 2033322 contain the supplementary crystallographic data for this study.The X-ray single-crystal structures of monomeric TSC2 and TSC3 compounds are shown in Figures2(a) and 2(b), respectively. It suggested that TSC2 was crystallized in a monoclinic crystal system with space P21/c, a = 27.985 (12) Å, b = 12.027 (5) Å, c = 9.833 (4) Å, α = 90.0°, β = 93.117 (11)°, γ = 90.0°, V = 3486.57 Å3, and Z = 7, while TSC3 was crystallized also in monoclinic crystal system with space P21/c, a = 11.2343 (6) Å, b = 11.2575 (7) Å, c = 11.8995 (8) Å, α = 90.00°, β = 94.476(6)°, γ = 90.0°, V = 1500.34 (3) Å3, and Z = 4.Figure 2
X-ray structure of (a) TSC2 (b) TSC3 along with the atom numbering scheme.
(a)(b)Note worthily, TSC2 molecules are noncovalently stacked via intermolecular interactions, i.e., van der Waals and H-bonding interactions. This is illustrated in Figure3. In the packing structure of the TSC2 and TSC3 molecules, the unit cell included four and seven molecules stacked to each other per unit cell.Figure 3
Expansion of the intermolecular interactions (i.e., van der Waals interaction) between the TSC2 molecules.


### 3.1.6. Molecular Modeling and/or Molecular Parameters

Quantum parameters such as EHOMO and ELUMO, in addition to specific parameters such as the ionization potential (IP), absolute softness (σ), absolute hardness (η), electron affinity (EA), separation energy (ΔE), absolute electronegativity (χ), global softness (S), electrophilicity (w), electron-accepting power (w+), electron-donating power (w−), and additional electronic charge (ΔNmax) [60–64], have been computed according to Eqs. 2–11 as shown below [60–65]. The inverse of the global hardness is called softness σ [66].(3)χ=−12ELUMO+EHOMO,(4)IP=−EHOMO,(5)η=12ELUMO−EHOMO,(6)S=12η,(7)ΔNmax=−IEη,(8)σ=1η,(9)EA=−ELUMO,(10)ω=IE22η,(11)ω−=3∗IE+EA216IE−EA,(12)ω−=IE+3∗EA216IE−EA.The geometric optimization of the prepared compounds can be characterized using theoretical calculations; therefore, the optimized structure for the synthesized compounds was obtained by calculating theoretical physical parameters, such as bond lengths and bond angles using DFT calculations.The quantum parameters of the synthesized TSC1, TSC2, and TSC3 compounds were calculated using equations (3)–(12). As shown in Tables 1 and 2, we deduced the following:(a)
HOMO and LUMO are frontier molecular orbitals (FMOs). The HOMO and LUMO energies were negative, indicating that the studied TSC1, TSC2, and TSC3 TSCs were stable molecules [65].(b)
The HOMO and LUMO functioned as electron donor and acceptor, respectively [66, 67].(c)
Hard and soft nucleophiles exhibit low and high HOMO energies, respectively, while hard and soft electrophiles exhibit high and low LUMO energies, respectively.(d)
The HOMO energies of TSC1, TSC2, and TSC3 were closely spaced (EHOMO (TSC1) −8.501 eV; EHOMO (TSC2) −8.561 eV; and EHOMO (TSC3) −8.556 eV).(e)
Soft molecules are characterized by a small energy gap (ELUMO−EHOMO) and higher reactivity than hard molecules because of their easy donation of electrons to an acceptor [68, 69].(f)
Large values of the HOMO–LUMO energy gap (7.410–7.667) indicated good stability and a large chemical hardness for the synthesized TSCN compounds.(g)
Absolute hardness (η) and softness (σ) are essential characteristics in the calculation of molecular stability and reactivity. Hard molecules exhibit a large energy gap with less reactivity (ΔE = EHOMO−ELUMO), whereas soft molecules exhibit a smaller energy space and greater reactivity. This indicated that ΔE was an indicator of stability and can be used to measure the chemical reactivity and kinetic stability of the compound [67, 70].(h)
When the HOMO energy decreased, the ability of the molecule to donate electrons decreased, while a high HOMO energy indicated that the molecule was an efficient electron donor. The LUMO energy indicated the ability of a molecule to receive an electron.(i)
The electrophilicity index (ω) followed this trend: TSC1 (ω = 3.104) > TSC2 (ω = 2.936) > TSC3 (ω = 2.933). Thus, TSC1 exhibited the maximum value of electrophilicity, confirming its high capability to accept electrons.(j)
The HOMO level in the TSCN compounds was commonly localized on the C = N groups, demonstrating that they are the favored sites for Nu attack at the central metal ion.(k)
The negativity of EHOMO and ELUMO indicated the stability of the TSCN compounds [71].Table 1
Some energetic properties of the synthesized thiosemicarbazone compounds.
CompoundTotal energy (kcal/mol)Binding energy (kcal/mol)Electronic energy (kcal/mol)Dipole moment (Debyes)HOMOLUMOTSC1−58321.2−3178.9−368233.16.55−8.501−1.091TSC2−65214.1−3735.7−441527.76.82−8.561−0.915TSC3−79668.2−4295.6−566955.26.94−8.556−0.889Table 2
Calculated quantum chemical parameters of the synthesized thiosemicarbazone compounds.
CompoundΔEIAχηSΔNmaxωω−ω+TSC17.4108.5011.0914.7963.7050.270−2.2943.1045.9651.169TSC27.6468.5610.9154.7383.8230.262−2.2392.9365.7831.045TSC37.6678.5560.8894.7233.8340.261−2.2322.9095.7491.027


## 3.1.1. IR Spectrum

The preliminary allocations of major IR spectrum bands of the TSCN compounds showed a disappearance ofν (>C = O) and a new band within the range of 1603–1615 cm−1 attributable to ν (C = N) stretching vibration [55], supporting condensation reaction and formation of the TSCN compounds. TSCN compounds exhibit thione ↔ thiol tautomerism [56] because of the existence of an–NH–C = S linkage, but ν (S–H) absorption band within the range of 2500–2600 cm−1 was absent with the appearance of ν (C = S) band around 750 cm−1, indicating the existence of the prepared TSCN compounds in the thione form in the solid state. For the prepared TSCN compounds, vibrational bands with the wave numbers of 3012 cm−1 (νC–H, Ar–H), 1615 cm−1 (νC = N), 1548 cm−1 (νC = C), and 1082 cm−1 (νN–N) were detected.Four vibrational bands with wave numbers in the ranges of 3019–3045 cm−1 (νC–H, Ar–H), 1601–1615 cm−1 (νC = N), 1535–1553 cm−1 (νC = C), and 1065–1100 cm−1 (νN–N) were observed for the TSCN compounds. The νsym and νasym of the TSC1 amino group were observed at 3234 and 3386 cm−1, respectively. In the spectra of the TSCN compounds, the bands observed in the ranges of 1494–1532, 1249–1251, 1017–1088, and 748–817 cm−1 were assigned to the thioamide bands of I, II, III, and IV, respectively [57].


## 3.1.2. NMR Spectrum

The1H-NMR spectra of the novel TSC1, TSC2, and TSC3 compounds in DMSO-d6 did not exhibit resonance at approximately 4.0 ppm corresponding to the –SH proton resonance [58], whereas the presence of a peak at 10.77 ppm (signal field of existence of the NH group next to C = S) suggested that they remained in the thione form even in a polar solvent, such as DMSO. The signals for the methine proton of an azomethine group for the TSCN compounds, CH = N, were detected at δ = 7.48–7.61 ppm. In the region of 6.36–7.54 ppm, chemical shifts were allocated for the hydrogen of the aromatic ring. A methyl group was observed as a singlet signal at δ = 2.02–2.20 ppm. The spectrum of TSC1 showed signals at δ = 7.80 ppm, assigned to the NH2 proton.The13C-NMR spectra of the novel TSCN compounds were recorded in DMSO-d6. The peaks of azomethine carbons and C = S of the TSCN compounds were observed as singlet peaks. The signal for the carbon atom of C = S was detected at 178.3, 177.8, and 178.1 in TSC1, TSC2, and TSC3, respectively.


## 3.1.3. Ultraviolet-Visible (UV-Vis) Spectrum

A strong absorption band detected within the range of 33003–32787 cm−1 was assigned to π⟶π∗ transitions of the (C = N)azomethine group, while the possible assignments for the bands within 26809–27548 cm−1 were attributed to n⟶π∗TSCN compound transitions. Permanent π⟶π∗ transitions occur at a higher energy than n⟶π∗ transitions [59].


## 3.1.4. Mass Spectra

The proposed formula was further confirmed by mass spectroscopy. The electron impact mass spectrum of TSC1 confirmed the suggested formula by exhibiting a peak at 249 equivalent to C11H15N5S compound moiety, in addition to a series of peaks attributable to different fragments of TSC1. These data suggested that a ketone PTHP group was condensed with the NH2 group of thiosemicarbazide or its derivatives. The mass spectra of TSC1, TSC2, and TSC3 showed peaks at 249, 277, and 325 m/z, confirming the proposed structural formula of the synthesized TSC1, TSC2, and TSC3 compounds, respectively.


## 3.1.5. Crystallography

The structures of TSC2 and TSC3 were established through X-ray crystallography. The recrystallization of the compounds from hot ethanol, followed by slow evaporation, led to the formation of single crystals. CCDC2026108 and CCDC 2033322 contain the supplementary crystallographic data for this study.The X-ray single-crystal structures of monomeric TSC2 and TSC3 compounds are shown in Figures2(a) and 2(b), respectively. It suggested that TSC2 was crystallized in a monoclinic crystal system with space P21/c, a = 27.985 (12) Å, b = 12.027 (5) Å, c = 9.833 (4) Å, α = 90.0°, β = 93.117 (11)°, γ = 90.0°, V = 3486.57 Å3, and Z = 7, while TSC3 was crystallized also in monoclinic crystal system with space P21/c, a = 11.2343 (6) Å, b = 11.2575 (7) Å, c = 11.8995 (8) Å, α = 90.00°, β = 94.476(6)°, γ = 90.0°, V = 1500.34 (3) Å3, and Z = 4.Figure 2
X-ray structure of (a) TSC2 (b) TSC3 along with the atom numbering scheme.
(a)(b)Note worthily, TSC2 molecules are noncovalently stacked via intermolecular interactions, i.e., van der Waals and H-bonding interactions. This is illustrated in Figure3. In the packing structure of the TSC2 and TSC3 molecules, the unit cell included four and seven molecules stacked to each other per unit cell.Figure 3
Expansion of the intermolecular interactions (i.e., van der Waals interaction) between the TSC2 molecules.


## 3.1.6. Molecular Modeling and/or Molecular Parameters

Quantum parameters such as EHOMO and ELUMO, in addition to specific parameters such as the ionization potential (IP), absolute softness (σ), absolute hardness (η), electron affinity (EA), separation energy (ΔE), absolute electronegativity (χ), global softness (S), electrophilicity (w), electron-accepting power (w+), electron-donating power (w−), and additional electronic charge (ΔNmax) [60–64], have been computed according to Eqs. 2–11 as shown below [60–65]. The inverse of the global hardness is called softness σ [66].(3)χ=−12ELUMO+EHOMO,(4)IP=−EHOMO,(5)η=12ELUMO−EHOMO,(6)S=12η,(7)ΔNmax=−IEη,(8)σ=1η,(9)EA=−ELUMO,(10)ω=IE22η,(11)ω−=3∗IE+EA216IE−EA,(12)ω−=IE+3∗EA216IE−EA.The geometric optimization of the prepared compounds can be characterized using theoretical calculations; therefore, the optimized structure for the synthesized compounds was obtained by calculating theoretical physical parameters, such as bond lengths and bond angles using DFT calculations.The quantum parameters of the synthesized TSC1, TSC2, and TSC3 compounds were calculated using equations (3)–(12). As shown in Tables 1 and 2, we deduced the following:(a)
HOMO and LUMO are frontier molecular orbitals (FMOs). The HOMO and LUMO energies were negative, indicating that the studied TSC1, TSC2, and TSC3 TSCs were stable molecules [65].(b)
The HOMO and LUMO functioned as electron donor and acceptor, respectively [66, 67].(c)
Hard and soft nucleophiles exhibit low and high HOMO energies, respectively, while hard and soft electrophiles exhibit high and low LUMO energies, respectively.(d)
The HOMO energies of TSC1, TSC2, and TSC3 were closely spaced (EHOMO (TSC1) −8.501 eV; EHOMO (TSC2) −8.561 eV; and EHOMO (TSC3) −8.556 eV).(e)
Soft molecules are characterized by a small energy gap (ELUMO−EHOMO) and higher reactivity than hard molecules because of their easy donation of electrons to an acceptor [68, 69].(f)
Large values of the HOMO–LUMO energy gap (7.410–7.667) indicated good stability and a large chemical hardness for the synthesized TSCN compounds.(g)
Absolute hardness (η) and softness (σ) are essential characteristics in the calculation of molecular stability and reactivity. Hard molecules exhibit a large energy gap with less reactivity (ΔE = EHOMO−ELUMO), whereas soft molecules exhibit a smaller energy space and greater reactivity. This indicated that ΔE was an indicator of stability and can be used to measure the chemical reactivity and kinetic stability of the compound [67, 70].(h)
When the HOMO energy decreased, the ability of the molecule to donate electrons decreased, while a high HOMO energy indicated that the molecule was an efficient electron donor. The LUMO energy indicated the ability of a molecule to receive an electron.(i)
The electrophilicity index (ω) followed this trend: TSC1 (ω = 3.104) > TSC2 (ω = 2.936) > TSC3 (ω = 2.933). Thus, TSC1 exhibited the maximum value of electrophilicity, confirming its high capability to accept electrons.(j)
The HOMO level in the TSCN compounds was commonly localized on the C = N groups, demonstrating that they are the favored sites for Nu attack at the central metal ion.(k)
The negativity of EHOMO and ELUMO indicated the stability of the TSCN compounds [71].Table 1
Some energetic properties of the synthesized thiosemicarbazone compounds.
CompoundTotal energy (kcal/mol)Binding energy (kcal/mol)Electronic energy (kcal/mol)Dipole moment (Debyes)HOMOLUMOTSC1−58321.2−3178.9−368233.16.55−8.501−1.091TSC2−65214.1−3735.7−441527.76.82−8.561−0.915TSC3−79668.2−4295.6−566955.26.94−8.556−0.889Table 2
Calculated quantum chemical parameters of the synthesized thiosemicarbazone compounds.
CompoundΔEIAχηSΔNmaxωω−ω+TSC17.4108.5011.0914.7963.7050.270−2.2943.1045.9651.169TSC27.6468.5610.9154.7383.8230.262−2.2392.9365.7831.045TSC37.6678.5560.8894.7233.8340.261−2.2322.9095.7491.027


## 3.2. Biological Activity

### 3.2.1. Antibacterial and Antifungal Activities

The antimicrobial activities of the TSCN compounds were screened. The results of the antimicrobial activities of the TSCN compounds against the tested microbes are shown in Table3. TSC3 was inactive against the tested Gram-positive and Gram-negative bacterial species. TSC1 exhibited effective antibacterial activity against the tested bacterial strains. TSC1 exhibited an extremely higher antifungal activity against Candida albicans against Amphotericin, which is a standard antifungal agent. TSC2 exhibited moderate antibacterial activity against Streptococcus faecalis (Gram-positive) and Escherichia coli (Gram-negative). The data showed that the most effective compound was TSC1.Table 3
Antibacterial and antifungal activities of the synthesized thiosemicarbazone compounds.
Concentration (μg/mL)Gram-positiveGram-negativeFungiBacillus subtilisStaphylococcus aureusStreptococcus faecalisE. coliNeisseria gonorrhoeaePseudomonas aeruginosaAspergillus flavusCandida albicansTSC1141317171221030TSC30013110000TSC400000000Ampicillin262127252826——Amphotericin——————1619Ampicillin: standard antibacterial agent, Amphotericin: standard antifungal agent.


### 3.2.2. Cytotoxic Activities

The cytotoxic activity of the TSCs was investigated against liver cancer cell (HepG2) and breast cancer cell (MCF-7) lines. IC50 is the concentration that can diminish cancer cell growth by 50%. The results of the cytotoxic study indicated that TSC1 exhibited significant activity against the HepG2 and MCF-7 cells with IC50 values of 29.4 and 122 μg/mL, respectively (Table 4), while the IC50 values toward the HepG2 and MCF-7 cells for TSC2 were 46 and 172.9 μg/mL, respectively. Furthermore, the IC50 values toward the HepG2 and MCF-7 cells for TSC3 were 25 and 107 μg/mL, respectively. The antitumor results indicated that the synthesized TSCN compounds were more effective against HepG2 than MCF-7. The antitumor activity of the synthesized TSCN compounds toward HepG2 and MCF-7 cell lines followed this order: TSC3 > TSC1 > TSC2 (Figure 4).Table 4
Antitumor activity of the synthesized thiosemicarbazone compounds.
Concentration (μg/mL)TSC1TSC2TSC3Liver cancer cell line (HepG2)51001001002535.36722.55027.448.719IC5029.44625Breast cancer cell line (MCF-7)583.89577.62579.586.5725070.78365.4IC50122172.9107Figure 4
Cytotoxicity of TSC1, TSC2, and TSC3 on breast and liver cancer cell lines.


### 3.2.3. Molecular Modeling and Biological Activity

Theoretical calculations were performed to investigate the physicochemical properties that possibly correlated with the antimicrobial action of the investigated TSCs. TSC1, which offered the lowest HOMO energy among the synthesized TSCs, exhibited the highest biological activity among the synthesized compounds. The data showed an inverse relationship between the dipole moment and lipophilicity, indicating that as the dipole moment decreased, the lipophilic nature of the compound increased, which powerfully favored its penetration via a lipid layer of microorganisms [72, 73], thereby violently destroying them. As shown in Table 2, the lipophilicity of TSC1 was higher than those of the other TSCN compounds, which sequentially deactivated the enzymes responsible for the respiration process of the investigated microbes more than the other compounds did, and accordingly increased its cellular uptake by bacterial cells. TSCs exhibit antibacterial activity because of the existence of toxophorically essential imine groups (–C=N) where the action mode of these compounds could include the formation of H-bonds via the azomethine group with an active center of cells that may interfere with ordinary cell processes [74].


## 3.2.1. Antibacterial and Antifungal Activities

The antimicrobial activities of the TSCN compounds were screened. The results of the antimicrobial activities of the TSCN compounds against the tested microbes are shown in Table3. TSC3 was inactive against the tested Gram-positive and Gram-negative bacterial species. TSC1 exhibited effective antibacterial activity against the tested bacterial strains. TSC1 exhibited an extremely higher antifungal activity against Candida albicans against Amphotericin, which is a standard antifungal agent. TSC2 exhibited moderate antibacterial activity against Streptococcus faecalis (Gram-positive) and Escherichia coli (Gram-negative). The data showed that the most effective compound was TSC1.Table 3
Antibacterial and antifungal activities of the synthesized thiosemicarbazone compounds.
Concentration (μg/mL)Gram-positiveGram-negativeFungiBacillus subtilisStaphylococcus aureusStreptococcus faecalisE. coliNeisseria gonorrhoeaePseudomonas aeruginosaAspergillus flavusCandida albicansTSC1141317171221030TSC30013110000TSC400000000Ampicillin262127252826——Amphotericin——————1619Ampicillin: standard antibacterial agent, Amphotericin: standard antifungal agent.


## 3.2.2. Cytotoxic Activities

The cytotoxic activity of the TSCs was investigated against liver cancer cell (HepG2) and breast cancer cell (MCF-7) lines. IC50 is the concentration that can diminish cancer cell growth by 50%. The results of the cytotoxic study indicated that TSC1 exhibited significant activity against the HepG2 and MCF-7 cells with IC50 values of 29.4 and 122 μg/mL, respectively (Table 4), while the IC50 values toward the HepG2 and MCF-7 cells for TSC2 were 46 and 172.9 μg/mL, respectively. Furthermore, the IC50 values toward the HepG2 and MCF-7 cells for TSC3 were 25 and 107 μg/mL, respectively. The antitumor results indicated that the synthesized TSCN compounds were more effective against HepG2 than MCF-7. The antitumor activity of the synthesized TSCN compounds toward HepG2 and MCF-7 cell lines followed this order: TSC3 > TSC1 > TSC2 (Figure 4).Table 4
Antitumor activity of the synthesized thiosemicarbazone compounds.
Concentration (μg/mL)TSC1TSC2TSC3Liver cancer cell line (HepG2)51001001002535.36722.55027.448.719IC5029.44625Breast cancer cell line (MCF-7)583.89577.62579.586.5725070.78365.4IC50122172.9107Figure 4
Cytotoxicity of TSC1, TSC2, and TSC3 on breast and liver cancer cell lines.


## 3.2.3. Molecular Modeling and Biological Activity

Theoretical calculations were performed to investigate the physicochemical properties that possibly correlated with the antimicrobial action of the investigated TSCs. TSC1, which offered the lowest HOMO energy among the synthesized TSCs, exhibited the highest biological activity among the synthesized compounds. The data showed an inverse relationship between the dipole moment and lipophilicity, indicating that as the dipole moment decreased, the lipophilic nature of the compound increased, which powerfully favored its penetration via a lipid layer of microorganisms [72, 73], thereby violently destroying them. As shown in Table 2, the lipophilicity of TSC1 was higher than those of the other TSCN compounds, which sequentially deactivated the enzymes responsible for the respiration process of the investigated microbes more than the other compounds did, and accordingly increased its cellular uptake by bacterial cells. TSCs exhibit antibacterial activity because of the existence of toxophorically essential imine groups (–C=N) where the action mode of these compounds could include the formation of H-bonds via the azomethine group with an active center of cells that may interfere with ordinary cell processes [74].


## 3.3. Equilibrium Studies

The protonation constants of the TSC1 ligand were calculated (Table5). These ligands behaved triprotic, as shown in the following equations: (13)L−+H+⇌HL;K1=HLL−H+,(14)HL+H+⇌H2L+;K2=H2L+HLH+,(15)H2L++H+⇌H3L2+;K3=HL2+H+H2L+.Table 5
Protonation constants for TSC1 ligand at different temperatures.
Reactionlog K (±σ)a15°C25°C35°CL + H = HL8.02 (0.09)7.81 (0.05)7.64 (0.03)HL + H = H2L3.31 (0.08)3.22 (0.07)3.14 (0.09)H2L + H = H3L3.17 (0.04)3.08 (0.06)3.01 (0.05)a(σ) is the standard deviation.We concluded that the 1st and 2nd deprotonation constants corresponded to the deprotonation of the two N-imino sites in the TSC ligand, as shown in Scheme3(a). The 3rd deprotonation constant corresponded to the thiolate group site in the TSC ligand, as shown in Scheme 3(b). A similar conclusion was obtained in a previous study [75].Scheme 3
(a) Possible deprotonation pathway of the imino groups and (b) possible deprotonation pathway of the thiolate group.
(a)(b)The log KN-imino values ranging from 2.97 to 3.35 were similar to that observed in a previous study for the imino group (4.40) [76]. The log KSH values ranging from 7.65 to 8.05 were similar to those described in a previous study for the thiolate group (5.5–9.0) [77].A protonation equilibrium study for the TSC ligands in this study cannot be performed in an aqueous solution since it is insoluble in H2O. The DMSO solvent has been extensively used for potentiometric studies of protonation and formation equilibria. The DMSO-water mixture (50%:50%) was best preferred to yield a soluble and stable Schiff base solution [58, 78].Here, three protonation constants were calculated for the TSC1 ligand, and the SPECIES program was used to determine the distribution of the TSC1 ligand species as a function of pH. Figure5(a) shows a classic species distribution diagram; it is possible to highlight, at the mentioned experimental conditions, the pH ranges within which the various species were formed and/or coexisted and their relative formation percentages. Thus, the species distribution graph is a good tool for a thorough observation of the concentration of each species present as a function of pH. It enabled us to obtain the best conditions (pH, concentration, and ligand:metal ratio) for the preparation of a solid complex. At a low pH, TSC1 initially existed in a fully protonated form with a maximum percentage of 100% as H3L below pH < 2. Upon the addition of the base, the pH increased causing the H3L species to lose its first proton from an imino group to form H2L, which is the major species at a pH of 3.3. As the conditions became more alkaline, the second proton released from the second imino group began deprotonation to an HL ligand, thereby achieving the highest percent of 99.1% at a pH of 5.8. A further increase in pH was followed by the liberation of the third H+, forming the fully deprotonated ligand, L, with maximum percent species of 98.4% at a pH of 10.0.Figure 5
(a) Concentration distribution diagram of various species as a function of pH in the TSC1 system (at a concentration of TSC1 = 1.25mM and T = 25°C). (b) Concentration distribution diagram of various species as a function of pH in the Cd-TSC1 system.
(a)(b)


### 3.3.1. Species Distribution Curves

The calculation of the equilibrium complex concentrations of Cd (II) with TSC1 (Table6) as a function of pH showed metal (II) binding in the biological system. As an illustrative example of metal complexes, Figure 5(b) shows a concentration distribution diagram for the complex, Cd (II)-TSC1. The Cd-TSC1 complex began to form in an acidic pH range reaching a constant concentration of 99.9% at a pH of 5.0, whereas the Cd (TSC1)2 complex species reached a maximum concentration of 45% at a pH of 9.8.Table 6
Stepwise stability for the complexes of TSC1 with Cd (II) metal ion in 50% DMSO-H2O (V/V) solution.
Temp.(°C)logK1 (±σ)alogK2 (±σ)logK1- logK2CdLCdL2Cd (II) complex15°C9.82(0.07)2.80(0.03)7.0225°C9.74(0.03)2.75(0.06)6.9935°C9.57(0.06)2.71(0.09)6.86a (σ) is the standard deviation; definitions of stability constants: K1 = [CdL]/[Cd][L]; K2 = [CdL2]/[CdL][L]; L = TSC1 thiosemicarbazone ligand (charges are omitted for simplicity).


### 3.3.2. Thermodynamics

The data derived for ΔH°, ΔS°, and ΔG° related to the protonation of TSC1 and Cd (II) complex formation were calculated from the data in Tables7 and 8. For the ligand protonation or complexation, ΔH° was determined from the plot slope (Figures 6(a)–6(b)) through the graphical representation of the Van ’t Hoff equation as follows:(16)−2.303RTlog10K=ΔH°−TΔS°,logK10=−ΔH°2.303R1T+ΔS°R.Table 7
Thermodynamic parameters for the protonation of ligand (TSC1) in 50% DMSO-H2O (V/V) solution.
ParameteraReactionL + H = HLaL + 2H = H2LL + 3H = H3L15oC25°C35°C15°C25°C35°C15°C25°C35°C−ΔG44.2544.5945.0817.4917.5817.8218.2618.3818.47−ΔH32.3315.2912.77ΔS41.1610.3016.27ΔG: Gibbs energy/kJ.mol−1; ΔH: enthalpy/kJ.mol−1; ΔS: entropy/J.mol−1.K−1.Table 8
Thermodynamic parameters for Cd (II)-TSC1 complexes in 50% DMSO-H2O (V/V) solution.
ParameteraReactionCd +L = CdLCd + 2L = CdL215°C25°C35°C15°C25°C35°C−ΔG54.1855.6056.4515.4515.7015.99−ΔH21.157.66ΔS114.8826.99aΔG: Gibbs energy/kJ.mol−1; ΔH: enthalpy/kJ.mol−1; ΔS: entropy/J.mol−1.K−1.Figure 6
(a) Effect of temperature on the protonation constant of TSC1. (b) Effect of temperature on the formation constant of the Cd (II)-TSC1 complexes.
(a)(b)With the well-known relations (6) and (7), from the ΔG and enthalpy change (ΔH) values, we calculated ΔS as follows:(17)ΔG°=−2.303RTlog10K,(18)ΔS°=ΔH°−ΔG°T.The main reasons for the protonation constant determination can be explained as follows:(1)
The ratio and pH of the various substance forms can be determined using their protonation constants.(2)
It is extremely useful in the preparation of newly synthesized compounds. The suggested structure can be reliable where protonation constants are theoretically well-calculated according to the experimental values.(3)
Considering that different types of substances exhibit different UV spectra, quantitative spectrophotometric analyses can be performed by choosing the appropriate pH. To choose the pH, the known protonation constants are required.(4)
The preparation of buffer solutions at different pH levels requires protonation constants [43, 79].(5)
The measurements of the stability constants for the complicated formation reactions of bioactive ion compounds require protonation constants. Additionally, their protonation constants are used to calculate the stability constants of the dynamic formation of bioactive compounds with metal ions [80].(6)
The equilibrium constants of certain compounds must be understood to measure the concentration of each ionized species at a certain pH; this is fundamental to understanding their physiochemical behavior [80].Tables7 and 8 describe the thermodynamic functions measured and can be interpreted as follows.(1)
The corresponding thermodynamic processes for the protonation reactions include the following:(i)
Exothermic processes for neutralization reactions.(ii)
Endothermic processes for ion desolvation.(iii)
Structure alteration and alignment of H-bonds around protonated and free ligands.(2)
When the temperature increased, the value of log10 KH decreased, and its acidity increased as the temperature increased.(3)
The negative ∆H° for the protonation of the TSC1 ligand indicated that this process was exothermic followed by the release of heat.(4)
The positive entropy of the TSC1 protonation reaction indicated increased disorder due to the desolvation processes and breakdown of H-bonds.Table6 lists the stepwise stability constants of the complexes formed at various temperatures. These values decreased and confirmed with an increase in temperature that the complexation phase was preferred at a low temperature.These results provided the following findings:(1)
A negative ∆H° showed that the coordinating process was exothermic, suggesting that the metal-ligand bonds were fairly strong and that the complexity reactions were preferred at low temperatures.(2)
The complexation reactions were spontaneous with a negative ∆G°.(3)
It was widely observed that the ∆G° and ∆H° values for 1 : 1 complexes were more negative than those corresponding to 1 : 2 complexes.(4)
This can be due to the steric hindrances caused by the addition of the 2nd ligand and the principle of charge neutralization.(5)
The electrostatic attraction in the 1 : 1 complex was larger than that in 1 : 2 complex since 1 : 1 complex was formed by a dipositively charged Mn+ and mononegatively charged ligand-anion interaction, while 1 : 2 complex was generated by a monopositively charged 1 : 1 complex and mononegatively charged ligand-anion interaction.(6)
The ∆S° values for the investigated complexes were positive, indicating that the entropy increase resultant from the release of bound solvent molecules during coordination was larger than the decrease resultant from the coordination process due to the ordered arrangement of solvent molecules around the ligand, and Mn + gained a random coordination pattern. This is referred to as an increase in entropy for configuration.


## 3.3.1. Species Distribution Curves

The calculation of the equilibrium complex concentrations of Cd (II) with TSC1 (Table6) as a function of pH showed metal (II) binding in the biological system. As an illustrative example of metal complexes, Figure 5(b) shows a concentration distribution diagram for the complex, Cd (II)-TSC1. The Cd-TSC1 complex began to form in an acidic pH range reaching a constant concentration of 99.9% at a pH of 5.0, whereas the Cd (TSC1)2 complex species reached a maximum concentration of 45% at a pH of 9.8.Table 6
Stepwise stability for the complexes of TSC1 with Cd (II) metal ion in 50% DMSO-H2O (V/V) solution.
Temp.(°C)logK1 (±σ)alogK2 (±σ)logK1- logK2CdLCdL2Cd (II) complex15°C9.82(0.07)2.80(0.03)7.0225°C9.74(0.03)2.75(0.06)6.9935°C9.57(0.06)2.71(0.09)6.86a (σ) is the standard deviation; definitions of stability constants: K1 = [CdL]/[Cd][L]; K2 = [CdL2]/[CdL][L]; L = TSC1 thiosemicarbazone ligand (charges are omitted for simplicity).


## 3.3.2. Thermodynamics

The data derived for ΔH°, ΔS°, and ΔG° related to the protonation of TSC1 and Cd (II) complex formation were calculated from the data in Tables7 and 8. For the ligand protonation or complexation, ΔH° was determined from the plot slope (Figures 6(a)–6(b)) through the graphical representation of the Van ’t Hoff equation as follows:(16)−2.303RTlog10K=ΔH°−TΔS°,logK10=−ΔH°2.303R1T+ΔS°R.Table 7
Thermodynamic parameters for the protonation of ligand (TSC1) in 50% DMSO-H2O (V/V) solution.
ParameteraReactionL + H = HLaL + 2H = H2LL + 3H = H3L15oC25°C35°C15°C25°C35°C15°C25°C35°C−ΔG44.2544.5945.0817.4917.5817.8218.2618.3818.47−ΔH32.3315.2912.77ΔS41.1610.3016.27ΔG: Gibbs energy/kJ.mol−1; ΔH: enthalpy/kJ.mol−1; ΔS: entropy/J.mol−1.K−1.Table 8
Thermodynamic parameters for Cd (II)-TSC1 complexes in 50% DMSO-H2O (V/V) solution.
ParameteraReactionCd +L = CdLCd + 2L = CdL215°C25°C35°C15°C25°C35°C−ΔG54.1855.6056.4515.4515.7015.99−ΔH21.157.66ΔS114.8826.99aΔG: Gibbs energy/kJ.mol−1; ΔH: enthalpy/kJ.mol−1; ΔS: entropy/J.mol−1.K−1.Figure 6
(a) Effect of temperature on the protonation constant of TSC1. (b) Effect of temperature on the formation constant of the Cd (II)-TSC1 complexes.
(a)(b)With the well-known relations (6) and (7), from the ΔG and enthalpy change (ΔH) values, we calculated ΔS as follows:(17)ΔG°=−2.303RTlog10K,(18)ΔS°=ΔH°−ΔG°T.The main reasons for the protonation constant determination can be explained as follows:(1)
The ratio and pH of the various substance forms can be determined using their protonation constants.(2)
It is extremely useful in the preparation of newly synthesized compounds. The suggested structure can be reliable where protonation constants are theoretically well-calculated according to the experimental values.(3)
Considering that different types of substances exhibit different UV spectra, quantitative spectrophotometric analyses can be performed by choosing the appropriate pH. To choose the pH, the known protonation constants are required.(4)
The preparation of buffer solutions at different pH levels requires protonation constants [43, 79].(5)
The measurements of the stability constants for the complicated formation reactions of bioactive ion compounds require protonation constants. Additionally, their protonation constants are used to calculate the stability constants of the dynamic formation of bioactive compounds with metal ions [80].(6)
The equilibrium constants of certain compounds must be understood to measure the concentration of each ionized species at a certain pH; this is fundamental to understanding their physiochemical behavior [80].Tables7 and 8 describe the thermodynamic functions measured and can be interpreted as follows.(1)
The corresponding thermodynamic processes for the protonation reactions include the following:(i)
Exothermic processes for neutralization reactions.(ii)
Endothermic processes for ion desolvation.(iii)
Structure alteration and alignment of H-bonds around protonated and free ligands.(2)
When the temperature increased, the value of log10 KH decreased, and its acidity increased as the temperature increased.(3)
The negative ∆H° for the protonation of the TSC1 ligand indicated that this process was exothermic followed by the release of heat.(4)
The positive entropy of the TSC1 protonation reaction indicated increased disorder due to the desolvation processes and breakdown of H-bonds.Table6 lists the stepwise stability constants of the complexes formed at various temperatures. These values decreased and confirmed with an increase in temperature that the complexation phase was preferred at a low temperature.These results provided the following findings:(1)
A negative ∆H° showed that the coordinating process was exothermic, suggesting that the metal-ligand bonds were fairly strong and that the complexity reactions were preferred at low temperatures.(2)
The complexation reactions were spontaneous with a negative ∆G°.(3)
It was widely observed that the ∆G° and ∆H° values for 1 : 1 complexes were more negative than those corresponding to 1 : 2 complexes.(4)
This can be due to the steric hindrances caused by the addition of the 2nd ligand and the principle of charge neutralization.(5)
The electrostatic attraction in the 1 : 1 complex was larger than that in 1 : 2 complex since 1 : 1 complex was formed by a dipositively charged Mn+ and mononegatively charged ligand-anion interaction, while 1 : 2 complex was generated by a monopositively charged 1 : 1 complex and mononegatively charged ligand-anion interaction.(6)
The ∆S° values for the investigated complexes were positive, indicating that the entropy increase resultant from the release of bound solvent molecules during coordination was larger than the decrease resultant from the coordination process due to the ordered arrangement of solvent molecules around the ligand, and Mn + gained a random coordination pattern. This is referred to as an increase in entropy for configuration.


## 4. Conclusions

PTHP was condensed with thiosemicarbazide, N-ethylthiosemicarbazide, and N-phenylthiosemicarbazide in the molar ratio (1 : 1) affording the novel TSC1, TSC2, and TSC3 TSCN compounds. The novel TSCN series were characterized by different analyses. The IR spectra indicated that the TSCN compounds existed in the thione form in the solid state. TSC1 showed equipotent or even more antifungal activity when matched with the reference standardamphotericin reference drug against Candida albicans. Thus, TSC1 was considered a potential compound for extra variation as a clinically valuable antifungal agent against Candida albicans. The structure-activity relationship (SAR) investigation confirmed that there was an inverse relationship between the dipole moment and antimicrobial activity, which could help in the design of powerful antibacterial substances. The potentiometric studies showed that TSC1 formed complexes (1 : 1 or 1 : 2) with the Cd (II) ion. The logK1 and –ΔH1 for the Cd (II)-TSC1 TSCN complexes were to an extent larger than log K2 and –ΔH2, indicating a change in the ligand dentate character from the tridentate (SNN donors) in 1 : 1 chelates to the bidentate (SN-donors) in 1 : 2; M:L chelates in addition to the steric hindrance generated by the entry of the 2nd molecule. The coordination of Cd (II) to TSC1 was spontaneous, exothermic, and entropically favorable. The molecular properties of the synthesized TSCN compounds were investigated by DFT calculations. These compounds may be considered a good remedy for Cd2+ ion toxicity because they formed a highly stable complex with it. The variation in the substitution of the N-thiosemicarbazide moiety was considered vital for the functionalization of the synthesized compounds, owing to its significance in the inhibition process.



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*Source: 1241470-2022-07-09.xml*