Design, Synthesis, Biological Evaluation and Molecular Docking Studies of New -Heterocyclic Compounds as Aromatase Inhibitors.

1. Introduction
Breast cancer is the most commonly diagnosed cancer in women in the world [1]. Approximately one in eight women will be diagnosed with invasive breast cancer in their lifetime [2]. According to 2024 data, 310,720 women in the United States were diagnosed with invasive breast cancer, while 42,250 women died from breast cancer. According to studies conducted between 2012 and 2021, the incidence of breast cancer increased by 1% each year [3]. Treatment options for breast cancer include surgical removal of part or all of the cancerous tissue, radiation or hormonal therapy and chemotherapy [4]. As in all types of cancer, the need for highly effective and selective chemotherapeutic agents is increasing day by day in the fight against breast cancer. Approximately 80% of breast cancer cases are estrogen-dependent, meaning that cancerous tissue has the chance to develop in the presence of estrogen [5]. Therefore, molecules that stop estrogen production or block estrogen receptors are needed in the cancer treatment [6].
Aromatase is an enzyme of the cytochrome P450 superfamily that can catalyze the aromatization of androgens to estrogen in humans [7]. Aromatases are commonly found in the ovary, placenta, brain and adipose tissue. The ovaries in premenopausal women and adipose tissue in postmenopausal women are the main sources of aromatase production [8]. Aromatase inhibitors are used in the treatment of breast cancer by suppressing estrogen production [9]. There are three generations of aromatase inhibitors developed to date: the first-generation aromatase inhibitor, aminoglutethimide; second-generation aromatase inhibitors, adrozole and formestane; and third-generation aromatase inhibitors, anastrozole, letrozole and exemestane. The activity, selectivity and reversible binding of molecules against aromatase enzyme increase from first-generation to third-generation inhibitors [10]. According to their chemical structure and mechanism of action, third-generation aromatase inhibitors are divided into two classes: steroidal and non-steroidal [11]. Exemestane is a steroidal and irreversible inhibitor that covalently binds to the enzyme, while anastrozole and letrozole are non-steroidal and reversible inhibitors without covalent interaction [12]. If we divide the chemical structures of the anastrozole and letrozole molecules into two parts, one of these parts carries a 1,2,4-triazole ring in common. The nitrogen atoms in the triazole ring interact with the heme iron of the aromatase enzyme and stop the enzyme from working [13,14]. The other part consists of 2-phenylacetonitrile or benzonitrile moieties containing a cyano group in both inhibitor molecules. Based on this information, potent aromatase inhibitors have been designed as new drug candidates. As in aromatase inhibitors, the target compounds have 2-phenylacetonitrile moieties in their general structure. Half of the target compounds have a 1,2,4-triazole ring in their structure, as in aromatase inhibitors, while the other half of the target compounds have 1,3,4-thiadiazole rings, which are the bioisosters of 1,2,4-triazole structure (Figure 1). Thus, the activities of 1,2,4-triazole ring and 1,3,4-thiadiazole ring can be compared in aromatase enzyme studies.
In accordance with the strategy outlined above, this study aimed to synthesize new 1,2,4-triazole and 1,3,4-thiadiazole structures with high aromatase enzyme inhibitory activity and evaluate their biological activity. First, the cytotoxic activities of the synthesized compounds were examined on MCF-7 and NIH3T3 cell lines. Then, the activity of all compounds against the aromatase enzyme was tested. In silico molecular docking and ADME studies were then conducted to determine the potential of these compounds as drug candidates.

2. Results and Discussion

2.1. Chemistry
The synthesis of the target compounds, 2,5-disubstituted-1,3,4-thiadiazol-5-amine (4a–4j) and 1,2-disubstituted-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl (5a–5j), is completed at four different steps (Scheme 1). Firstly, 4-(cyanomethyl)benzohydrazide (2) is synthesized by the nucleophilic substitution reaction of methyl 4-(cyanomethyl)benzoate (1) with hydrazine monohydrate in ethanol. In the second step, new thiosemicarbazide derivatives (3a–3j) are formed by nucleophilic addition and the reaction of hydrazide compounds (2) with various isothiocyanate derivatives in ethanol. In the third step, the thiosemicarbazide derivatives (3a–3j) are directly stirred with concentrated sulfuric acid and 1,3,4-thiadiazole derivatives (4a–4j) are obtained by ring closure reaction. In the fourth step, the thiosemicarbazide derivatives (3a–3j) are heated in a dilute alkaline medium and 1,2,4-triazole derivatives (5a–5j) are gained as a result of ring closure reaction. The target compounds, 1,3,4-thiadiazole (4a–4j) and 1,2,4-triazole (5a–5j) derivatives, were prepared in high yields (75–85%) and in pure form. Different techniques such as IR, 1H-NMR, 13C-NMR spectroscopies and elemental analysis were used for the structural characterization of the compounds.
The N-H, C=O and C=S bonds in the structure of thiosemicarbazide derivatives (3a–3j) gave specific IR bands in the range of 3130–3342 cm−1, 1662–1678 cm−1 and 1207–1238 cm−1, respectively. The characteristic C=O bands of thiosemicarbazide have been replaced by C=N bands of the 1,3,4-thiadiazole (4a–4j) and 1,2,4-triazole structures (5a–5j) in the range of 1599–1714 cm−1. In addition, N-H bonds of 1,3,4-thiadiazole and 1,2,4-triazole derivatives were detected with distinct bands in the range of 3146–3402 cm−1.
When the 1H-NMR spectra of thiosemicarbazides (3a–3j) and the hydrazide compound (2) are compared, the most important difference is that the peak (NH2) detected with the 2H integral at 4.50 ppm of the hydrazide is not seen in the spectra of thiosemicarbazides. In addition, three NH protons forming the thiosemicarbazide structures were recorded as singlet peaks from 7.74 ppm to 10.70 ppm. These three NH protons were replaced by a single NH proton in the range of 7.83–11.62 ppm in the 1H-NMR spectra of thiadiazole structures (4a–4j). Similarly, NH protons in triazole structures (5a–5j) were found as singlet peaks between 12.42 ppm and 14.22 ppm.
In the 13C-NMR spectra of thiosemicarbazides, thion (C=S) and carbonyl (C=O) carbons were detected in the ranges of 180.64–184.84 ppm and 165.45–166.03 ppm, respectively. The peaks of thiadiazole derivatives (4a–4j) in the range of 155.85–172.32 ppm were attributed to (C=N) carbons forming the heterocyclic structure. Similarly, the carbons forming the triazole ring (5a–5j) showed specific peaks in the range of 150.30–172.74 ppm.
In a previous study, in the IR spectrum of 2,5-disubstituted-1,3,4-thiadiazole structures, N-H and C=N stretching bands were detected in the range of 3134–3481 cm−1 and 1593–1633 cm−1, respectively. The NH proton attached to the 1,3,4-thiadiazole ring was recorded in the range of 10.35–11.28 ppm in the 1H-NMR spectrum. The imine (C=N) carbons of the thiadiazole structure are visualized in the downfield region (156.08–165.97 ppm) in the 13C-NMR spectrum [15].
In another study synthesized based on thiosemicarbazides, the C=S absorption bands of 1,2,4-triazole-3-thione derivatives were shown at 1323–1326 cm−1. The NH protons were observed in the range of 14.15–14.31 ppm in 1H-NMR spectra. The C=S carbon attached to the 1,2,4-triazole ring was reported in the range of 168.97–169.62 ppm in 13C-NMR spectrums [16]. The spectral data in these two recent studies were found to be compatible with the data in this study.

2.2. Biological Activity
The antiproliferative activity of 1,3,4-thiadiazole (4a–4j) and 1,2,4-triazole (5a–5j) derivatives was evaluated against human breast cancer cells (MCF-7) and mouse embryonic fibroblast cells (NIH3T3). The NIH3T3 cells were used to determine the selectivity of all compounds. The compounds with a high selectivity index and cytotoxic effect against MCF-7 were identified as 4a, 4b, 4c, 4e, 4f, and 4g among thiadiazole derivatives and 5a, 5b, 5c, 5d, 5e, 5f, and 5g among triazole derivatives. All compounds were also tested on the aromatase enzyme. Among the compounds, 5b, 5c, 5e, 5f and 5g exhibited significant enzyme inhibitory activity. Doxorubicin and letrozole were used as negative controls in this study (Table 1).

Structure–Activity Relationship Studies
Aromatic (substituted phenyl ring), cycloaliphatic (cyclohexane) and alkyl (methyl, ethyl) groups were preferred as R(Ar) groups in the chemical structures of the compounds. Some interesting structure–activity relationships were obtained based on the cytotoxic and aromatase inhibitory activity results.
✓Based on both cytotoxic and aromatase activity results, compounds bearing a substituted phenyl ring as the R(Ar) group were found to exhibit higher activity (except for the 4-bromophenyl compound). The highest antiproliferative activity among both thiadiazole (4a–4j) and triazole (5a–5j) compounds was observed in derivatives bearing 4-fluorophenyl, 4-chlorophenyl, 4-methylphenyl, 4-methoxyphenyl, and 4-nitrophenyl groups, respectively.

✓1,2,4-Triazole derivatives (5a–5j) generally exhibited higher antiproliferative and anti-aromatase activities than 1,3,4-thiadiazole compounds (4a–4j).

✓Only certain 1,2,4-triazole compounds (5b, 5c, 5e, 5f and 5g) have demonstrated significant inhibitory activity against the aromatase enzyme.

✓No antiproliferative or anti-aromatase activity has been observed in thiadiazole and triazole compounds bearing alkyl and alicyclic hydrocarbon (4h, 4i, 4j, 5h, 5i and 5j).

✓Compounds 5c, 5e and 5f in the series were identified as the most potent aromatase inhibitors with IC50 values lower than 0.1 μM.

✓The anti-aromatase activity was highest with p-Cl substitution as a halogen on the aromatic phenyl ring in the triazole compounds (5a–5j). Anti-aromatase activity also decreased with p-F substitution and disappeared with p-Br substitution. A similar situation was observed in the ranking of the potency of antiproliferative compounds.

2.3. In Silico Studies

2.3.1. Molecular Docking Studies
The pharmacological activity of compounds identified as effective in vitro is further evaluated in silico by analyzing their enzyme–substrate binding profiles through molecular docking studies. To this end, molecular docking simulations were initially conducted on the aromatase enzyme. Upon examination of the aromatase enzyme inhibition assay results, it was observed that several compounds exhibited varying degrees of inhibitory activity. At this stage, the three compounds demonstrating the highest inhibition potential (5c, 5e, and 5f) were selected for detailed molecular docking analysis. The resulting docking poses are presented in Figure 2 and Supplementary Figure S121. Furthermore, the interaction profiles of the selected compounds with the active site of the aromatase enzyme are detailed in Table 2.
As shown in Figure 2, the compounds evaluated in this study exhibited similar interactions. In all three compounds, the triazole ring, which was a common structural motif, formed π–π and cation–π interactions as well as a salt bridge with the HEM molecule. Additionally, a hydrogen bond was established between the nitrogen atom on the triazole ring and the HEM molecule. The sulfur atom attached to the triazole ring also formed another hydrogen bond with the HEM molecule. A π–π interaction was further detected between the HEM molecule and the phenyl ring within the structure. Moreover, this phenyl ring was involved in an additional π–π interaction with the phenyl group of Phe134. Except for compound 5e, π–π interactions were also observed between the 4-Cl-phenyl and 4-OCH3-phenyl rings of compounds 5c and 5f, respectively, and the indole ring of Trp224. In addition to the common interactions described above, compound 5c uniquely formed a halogen bond between the chlorine atom at the 4-position of its phenyl ring and hydroxyl of Ser478. This specific interaction may account for the slightly superior inhibitory potency of 5c compared to the other compounds (IC50 = 0.064 ± 0.003 µM). Collectively, these findings suggest that the studied compounds possess strong inhibitory potential against aromatase enzymes, supporting their further investigation as promising anti-aromatase agents.

2.3.2. Molecular Dynamic Studies on Aromatase Enzyme
Molecular dynamics (MD) simulations were carried out for 100 ns using the crystal structures of aromatase (PDB ID: 3EQM) in conjunction with a POPE membrane model. Separate simulations were conducted for the active compound 5c, which was identified as the most potent aromatase enzyme inhibitor. The simulation results revealed that the complexes formed between this ligand and the crystal structures of 3EQM remained structurally stable throughout the entire simulation period. Analyses of root mean square deviation (RMSD) and root mean square fluctuation (RMSF), as well as the interaction profiles of amino acid residues, are presented in Figure 3 and Figure 4, Table 3 and Table 4.
The structural stability of the ligand–enzyme complexes was evaluated using both root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analyses. The overall structural stability of the protein–ligand complex throughout the simulation was first evaluated by calculating the Root Mean Square Deviation (RMSD) of the protein backbone (Cα atoms) and the ligand heavy atoms relative to their initial positions (Figure 3A). The protein Cα RMSD exhibited a gradual increase during the initial 40 ns, after which it achieved a plateau, fluctuating around a stable average of approximately 2.4 Å. This indicates that the protein underwent a period of conformational relaxation before reaching a state of dynamic equilibrium. Similarly, the RMSD of the ligand, when fitted to the protein backbone, followed the protein’s trend, stabilizing at approximately 5.6 Å, which suggests that the ligand remained securely positioned within the binding pocket while accommodating the protein’s conformational adjustments. The ligand’s internal stability was confirmed by fitting its heavy atoms to its initial coordinates, which showed a low RMSD value fluctuating around 1.1 Å after an initial conformational shift at ~20 ns. Collectively, the RMSD data confirm that the protein–ligand complex achieved and maintained a stable equilibrium throughout the latter half of the simulation.
To further investigate the dynamic behavior and flexibility of the protein backbone upon ligand binding, the Root Mean Square Fluctuation (RMSF) of each residue in the 5c-3EQM complex was calculated (Figure 3B). The resulting RMSF profile revealed that residues located within secondary structural elements (α-helices and β-sheets) exhibited minimal fluctuations, typically below 1.5 Å, which is indicative of their structural rigidity. In contrast, the highest fluctuations were observed in the loop regions, consistent with their inherently greater conformational freedom. Notably, the majority of the active site residues that form direct interactions with compound 5c (summarized in Table 3) displayed low RMSF values. This finding strongly suggests that the binding of the ligand confers a significant degree of stabilization to the binding pocket. By restricting the conformational mobility of these key residues, the ligand promotes a highly stable and persistent binding mode throughout the simulation.
A detailed analysis of the protein–ligand interactions was conducted to identify the key residues responsible for anchoring the ligand in the binding pocket. The interactions were monitored throughout the 100 ns trajectory, and their frequencies are summarized in Figure 3C–E.
A detailed analysis of the binding profile was conducted to identify the key interactions stabilizing compound 5c within the aromatase active site. The two-dimensional interaction schematic (Figure 3C) and a comprehensive summary of all observed interactions (Table 4) reveal a persistent network of contacts. The ligand was found to interact consistently with several critical active site residues, including Arg115, Phe116, Trp224, Asp309, Met374, Cys437 and Leu479. Significant contributions to the binding affinity arise from specific hydrogen bonds and non-polar contacts. Critical hydrogen bonds were formed with the side chains of Asp309 (showing ~37% occupancy) and Arg115 (33% occupancy). The complex was further stabilized by several water-mediated contacts, particularly with Asp309. Concurrently, the binding was anchored by hydrophobic interactions with residues such as Phe134 (~40% occupancy) and Ile133 (~23% occupancy). As visualized in the timeline plot, the consistent presence of this specific set of contacts confirms that compound 5c maintains a well-defined and persistent binding mode throughout the simulation.
Figure 3D displays the interaction fractions observed throughout the simulation, classified by interaction type: water-mediated hydrogen bonds (blue), direct hydrogen bonds (green), ionic interactions (pink), and hydrophobic contacts (purple). These visualizations highlight the predominant interactions formed between compound 5c and key residues within the active site of the respective enzyme. Detailed numerical data corresponding to these interactions are provided in Table 4. These interactions further support the structural stability and favorable binding conformations of the aromatase enzyme complex formed by compound 5c.
Figure 3E reveals the number of interactions and the corresponding residue interaction profiles throughout the simulation. Analysis of Figure 3E, corresponding to the 5c-3EQM complex, revealed that the ligand consistently interacted with key residues in the aromatase active site, including Ile133, Trp224, Asp309, and Met374. Persistent interactions with these residues reflect the binding stability of 5c and its potential as a strong aromatase inhibitor.
Finally, to assess the conformational stability of compound 5c upon binding, its intrinsic structural properties were analyzed (Figure 4). The Radius of Gyration (rGyr), a key parameter measuring the ligand’s compactness, was evaluated for the 5c-3EQM complex. The rGyr profile remained highly stable throughout the simulation, fluctuating tightly within a narrow range of 3.7 Å to 3.9 Å (Table 3), with an average value of approximately 3.8 Å. This lack of significant deviation indicates that the ligand maintained a compact conformation, without experiencing any unfolding events or structural instability within the binding site. This conclusion is further corroborated by the stable profiles of other ligand properties, including the Molecular Surface Area (MolSA), Solvent Accessible Surface Area (SASA), and Polar Surface Area (PSA). The consistent fluctuation of these metrics around stable mean values demonstrates that compound 5c adopted a favorable and energetically stable conformation within the binding pocket, free from significant internal strain.
In conclusion, the 100 ns molecular dynamics simulation of the 5c-3EQM complex provides strong evidence that compound 5c adopts and maintains a stable binding conformation within the active site of the aromatase enzyme. This structural stability is comprehensively supported by consistent RMSD, RMSF, and Radius of Gyration (rGyr) values, all of which reached equilibrium and exhibited low fluctuations. The binding is driven by a persistent combination of hydrophobic and π-stacking interactions, primarily with key residues Met374 and Trp224, and is further anchored by key hydrogen bonds with catalytic residues such as Asp309. The minimal fluctuations and the consistent interaction profile confirm that the ligand remains tightly bound, validating the observed binding pose as a highly favorable one. Overall, these findings demonstrate that compound 5c not only exhibits favorable initial binding but also retains structural stability over time, highlighting it as a promising candidate for further investigation in the development of novel, enzyme-targeted therapeutics.

2.3.3. ADME Prediction
In order for active compounds to be developed as new drugs, certain physicochemical, pharmacokinetic, and drug-like properties must be evaluated beforehand [17]. Thus, some problems that may be experienced in the later stages of drug discovery processes will be prevented. In this context, some physicochemical and pharmacokinetic properties of synthesized 1,3,4-thiadiazole (4a–4j) and 1,2,4-triazole compounds (5a–5j) were calculated using SwissAdme online tools. As a result of the physicochemical parameters given in Table 5, it was observed that all compounds were in accordance with drug-likeness properties such as Lipinski, Ghose, Veber, Egan, and Muegge. Of these compounds, only 4g and 5g show one deviation from the Egan rule due to the high TPSA value of the nitro group. On the other hand, log S values, a measure of water solubility properties, indicate that the compounds are moderately soluble or soluble.
The BOILED-Egg model obtained when the gastrointestinal and blood–brain barrier permeation of the compounds were evaluated is shown in Figure 5. Accordingly, it was determined that all of the compounds (except 4g and 5g) had high gastrointestinal permeability because they were found in the white area. The gastrointestinal passage of 4g and 5g carrying bulky nitro groups in the gray area was found to be low. In addition, as seen in Figure 5, none of the compounds entered the yellow area, so there was no blood–brain barrier permeation. The inability of these compounds to cross the blood–brain barrier is a positive feature in terms of central nervous system depression or a low side effect profile [18].

3. Materials and Methods

3.1. Chemistry
All chemicals used in this study were obtained from Sigma-Aldrich. The status of the reactions was monitored by thin-layer chromatography (TLC). For this, silica gel as a stationary phase and a dichloromethane/methanol (1:1, v/v) mixture as mobile a phase were preferred, and all spots were determined under 254 nm UV light. The IR spectra of the compounds were obtained with a Shimadzu FTIR-8400S spectrophotometer. 1H-NMR and 13C-NMR spectra of the compounds were taken with a Bruker Avance III HD 600 instrument against an internal reference standard of TMS in deuterodimethylsulfoxide. Elemental analyses were performed by a Leco CHNS-932 apparatus.

General synthesis method of hydrazide compound (
2
)

Methyl 4-(cyanomethyl)benzoate (1 mmol, 0.35 g) is dissolved in 10 mL of ethanol. 3 mmol of Hydrazine monohydrate (0.3 mL) is added to this mixture. The mixture is refluxed for 6 h, and the reaction is terminated by TLC [19].
4-(Cyanomethyl)benzohydrazide (2) CAS number: 1388215-23-3) was previously synthesized in our study [20].
General synthesis method of thiosemicarbazide derivatives (3a–3j)
4-(Cyanomethyl)benzohydrazide (2 mmol, 0.35 g) is dissolved in 15 mL of ethanol by heating. Isothiocyanate derivatives in equal molar ratios are added dropwise. The mixture is refluxed for 8–10 h. The excess solvent is evaporated under vacuum to give the solid thiosemicarbazides [21].
2-[4-(Cyanomethyl)benzoyl]-N-phenylhydrazine-1-carbothioamide (3a)
Yield: 93%; Color: white powder; Melting Point: 172.0–172.7 °C; FT-IR (cm−1): 3319 and 3277 (N-H), 3047 (=C-H), 2993 and 2953 (C-H), 2247 (C≡N), 1668 (C=O), 1220 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.59 (s, 1H, Ar-NH-CS), 9.83 and 9.74 (2s, 2H, CO-NHNH-CS), 7.99 (d, J = 8.0 Hz, 2H, Ar-H), 7.49 (d, J = 8.1 Hz, 3H, Ar-H), 7.45 (s, 1H, Ar-H), 7.34 (t, J = 7.8 Hz, 2H, Ar-H), 7.23–7.11 (m, 1H, Ar-H), 4.15 (s, 2H, Ar-CH2). 13C NMR (DMSO-d6, 75 MHz, δ ppm): 180.64 (C=S), 165.49 (C=O), 139.19, 135.17, 131.86, 128.52, 127.94, 118.90 (C≡N), 22.33 (Ar-CH2). Anal. calcd for C16H14N4OS: C 61.92, H 4.55, N 18.05, S 10.33%. Found: C 61.60, H 4.52, N 18.10, S 10.37%.
2-[4-(Cyanomethyl)benzoyl]-N-(4-fluorophenyl)hydrazine-1-carbothioamide (3b)
Yield: 79%; Color: white powder; Melting Point: 179.6–180.5 °C; FT-IR (cm−1): 3313 and 3174 (N-H), 3045 (=C-H), 2993 (C-H), 2251 (C≡N), 1664 (C=O), 1207 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.60 (s, 1H, Ar-NH-CS), 9.82 (s, 2H, CO-NHNH-CS), 8.00 (d, J = 6.0 Hz, 2H, Ar-H), 7.54–7.37 (m, 4H, Ar-H), 7.17 (t, J = 9.0 Hz, 2H, Ar-H), 4.15 (s, 2H, Ar-CH2). 13C-APT NMR (DMSO-d6, 150 MHz, δ ppm): 183.27 (C=S), 166.02 (C=O), 159.96 (d, J = 241.5 Hz), 136.03 (d, J = 3.0 Hz), 135.69, 132.35, 129.02, 128.44 (d, J = 12.0 Hz), 128.08, 119.36 (C≡N), 115.12 (d, J = 21.0 Hz), 22.85 (Ar-CH2). Anal. calcd for C16H13FN4OS: C 58.53, H 3.99, N 17.06, S 9.76%. Found: C 58.99, H 3.95, N 17.71, S 9.46%.
2-[4-(Cyanomethyl)benzoyl]-N-(4-chlorophenyl)hydrazine-1-carbothioamide (3c)
Yield: 80%; Color: yellow powder; Melting Point: 201.4–202.0 °C; FT-IR (cm−1): 3321 and 3213 (N-H), 3016 (=C-H), 2914 (C-H), 2249 (C≡N), 1668 (C=O), 1217 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.62 (s, 1H, Ar-NH-CS), 9.87 (s, 2H, CO-NHNH-CS), 7.99 (d, J = 6.0 Hz, 2H, Ar-H), 7.50 (d, J = 6.0 Hz, 4H, Ar-H), 7.40 (d, J = 9.0 Hz, 2H, Ar-H), 4.15 (s, 2H, Ar-CH2). 13C-APT NMR (DMSO-d6, 150 MHz, δ ppm): 181.62 (C=S), 166.03 (C=O), 138.71, 135.73, 132.31, 129.02, 128.47, 128.08, 119.35 (C≡N), 22.87 (Ar-CH2). Anal. calcd for C16H13ClN4OS: C 55.73, H 3.80, N 16.25, S 9.30%. Found: C 54.69, H 3.74, N 16.37, S 9.45%.
2-[4-(Cyanomethyl)benzoyl]-N-(4-bromophenyl)hydrazine-1-carbothioamide (3d)
Yield: 86%; Color: yellow powder; Melting Point: 201.0–201.7 °C; FT-IR (cm−1): 3325 and 3207 (N-H), 3010 (=C-H), 2958 and 2906 (C-H), 2252 (C≡N), 1668 (C=O), 1217 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.61 (s, 1H, Ar-NH-CS), 9.86 (s, 2H, CO-NHNH-CS), 7.99 (d, J = 8.2 Hz, 2H, Ar-H), 7.50 (q, J = 8.0, 7.1 Hz, 6H, Ar-H), 4.15 (s, 2H, Ar-CH2). 13C NMR (DMSO-d6, 75 MHz, δ ppm): 181.01 (C=S), 165.49 (C=O), 138.63, 135.24, 131.77, 130.80, 128.51, 127.97, 118.89 (C≡N), 22.35 (Ar-CH2). Anal. calcd for C16H13BrN4OS: C 49.37, H 3.37, N 14.39, S 8.24%. Found: C 49.05, H 3.43, N 14.45, S 8.17%.
2-[4-(Cyanomethyl)benzoyl]-N-(p-tolyl)hydrazine-1-carbothioamide (3e)
Yield: 85%; Color: white powder; Melting Point: 144.6–145.9 °C; FT-IR (cm−1): 3308 and 3211 (N-H), 3005 (=C-H), 2918 (C-H), 2256 (C≡N), 1664 (C=O), 1219 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.56 (s, 1H, Ar-NH-CS), 9.76 and 9.66 (2s, 2H, CO-NHNH-CS), 7.98 (d, J = 7.9 Hz, 2H, Ar-H), 7.49 (d, J = 7.9 Hz, 2H, Ar-H), 7.31 (d, J = 8.0 Hz, 2H, Ar-H), 7.14 (d, J = 8.0 Hz, 2H, Ar-H), 4.15 (s, 2H, Ar-CH2), 2.29 (s, 3H, CH3). 13C NMR (DMSO-d6, 75 MHz, δ ppm): 180.91 (C=S), 165.49 (C=O), 136.60, 135.15, 134.19, 131.89, 128.50, 127.92, 118.90 (C≡N), 22.33 (Ar-CH2), 20.53 (Ar-CH3). Anal. calcd for C17H16N4OS: C 62.94, H 4.97, N 17.27, S 9.88%. Found: C 63.67, H 5.00, N 17.15, S 9.95%.
2-[4-(Cyanomethyl)benzoyl]-N-(4-methoxyphenyl)hydrazine-1-carbothioamide (3f)
Yield: 86%; Color: white powder; Melting Point: 151.3–152.5 °C; FT-IR (cm−1): 3298 and 3198 (N-H), 3010 (=C-H), 2995 and 2835 (C-H), 2249 (C≡N), 1670 (C=O), 1238 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.55 (s, 1H, Ar-NH-CS), 9.72 and 9.62 (2s, 2H, CO-NHNH-CS), 7.98 (d, J = 9.0 Hz, 2H, Ar-H), 7.48 (d, J = 9.0 Hz, 2H, Ar-H), 7.28 (d, J = 9.0 Hz, 2H, Ar-H), 6.90 (d, J = 9.0 Hz, 2H, Ar-H), 4.15 (s, 2H, Ar-CH2), 3.75 (s, 3H, OCH3). 13C NMR (DMSO-d6, 75 MHz, δ ppm): 180.95 (C=S), 165.52 (C=O), 156.74, 135.13, 131.95, 128.52, 127.91, 118.90 (C≡N), 113.20, 55.17 (Ar-OCH3), 22.33 (Ar-CH2). Anal. calcd for C17H16N4O2S: C 59.98, H 4.74, N 16.46, S 9.42%. Found: C 59.34, H 4.78, N 16.51, S 9.47%.
2-[4-(Cyanomethyl)benzoyl]-N-(4-nitrophenyl)hydrazine-1-carbothioamide (3g)
Yield: 92%; Color: orange powder; Melting Point: 184.2–185.5 °C; FT-IR (cm−1): 3311 and 3184 (N-H), 3099 (=C-H), 2960 and 2850 (C-H), 2251 (C≡N), 1662 (C=O), 1219 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.70 (s, 1H, Ar-NH-CS), 10.15 (s, 2H, CO-NHNH-CS), 8.20 (d, J = 9.0 Hz, 2H, Ar-H), 7.95 (d, J = 8.6 Hz, 4H), 7.50 (d, J = 9.0 Hz, 2H, Ar-H), 4.16 (s, 2H, Ar-CH2). 13C NMR (DMSO-d6, 75 MHz, δ ppm): 180.74 (C=S), 165.57 (C=O), 145.46, 144.16, 135.36, 132.32, 128.47, 128.05, 123.56, 118.88 (C≡N), 22.34 (Ar-CH2). Anal. calcd for C16H13N5O3S: C 54.08, H 3.69, N 19.71, S 9.02%. Found: C 53.75, H 3.71, N 19.78, S 9.07%.
2-[4-(Cyanomethyl)benzoyl]-N-methylhydrazine-1-carbothioamide (3h)
Yield: 92%; Color: lilac powder; Melting Point: 156.3–157.0 °C; FT-IR (cm−1): 3342 and 3230 (N-H), 3025 (=C-H), 2978 and 2937 (C-H), 2245 (C≡N), 1662 (C=O), 1219 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.38 (s, 1H, CO-NHNH-CS), 9.34 (s, 1H, CO-NHNH-CS), 8.06 (q, J = 4.3 Hz, 1H, CS-NH-CH3), 7.93 (d, J = 9.0 Hz, 2H, Ar-H), 7.48 (d, J = 9.0 Hz, 2H, Ar-H), 4.15 (s, 2H, Ar-CH2), 2.88 (d, J = 4.4 Hz, 3H, NH-CH3). 13C NMR (DMSO-d6, 75 MHz, δ ppm): 182.25 (C=S), 165.52 (C=O), 135.14, 131.81, 128.43, 127.93, 118.90 (C≡N), 30.91 (NH-CH3), 22.32 (Ar-CH2). Anal. calcd for C11H12N4OS: C 53.21, H 4.87, N 22.56, S 12.91%. Found: C 53.88, H 4.84, N 22.65, S 12.85%.
2-[4-(Cyanomethyl)benzoyl]-N-ethylhydrazine-1-carbothioamide (3i)
Yield: 95%; Color: white powder; Melting Point: 210.0–210.7 °C; FT-IR (cm−1): 3311 and 3254 (N-H), 3093 (=C-H), 2980 and 2937 (C-H), 2243 (C≡N), 1662 (C=O), 1213 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.35 (s, 1H, CO-NHNH-CS), 9.27 (s, 1H, CO-NHNH-CS), 8.11 (t, J = 5.7 Hz, 1H, CS-NH-CH2CH3), 7.94 (d, J = 9.0 Hz, 2H, Ar-H), 7.47 (d, J = 9.0 Hz, 2H, Ar-H), 4.14 (s, 2H, Ar-CH2), 3.55–3.40 (m, 2H, NH-CH2CH3), 1.07 (t, J = 7.1 Hz, 3H, NH-CH2CH3). 13C NMR (DMSO-d6, 75 MHz, δ ppm): 181.84 (C=S), 165.45 (C=O), 135.14, 131.83, 128.45, 127.93, 118.89 (C≡N), 38.47 (NH-CH2CH3), 22.32 (Ar-CH2), 14.46 (NH-CH2CH3). Anal. calcd for C12H14N4OS: C 54.94, H 5.38, N 21.36, S 12.22%. Found: C 55.53, H 5.30, N 21.50, S 12.10%.
2-[4-(Cyanomethyl)benzoyl]-N-cyclohexylhydrazine-1-carbothioamide (3j)
Yield: 98%; Color: lilac powder; Melting Point: 207.1–207.9 °C; FT-IR (cm−1): 3211 and 3130 (N-H), 2926, 2852 (=C-H and C-H), 2256 (C≡N), 1678 (C=O), 1230 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.31 (s, 1H, CO-NHNH-CS), 9.22 (s, 1H, CO-NHNH-CS), 7.94 (d, J = 9.0 Hz, 2H, Ar-H), 7.74 (d, J = 8.3 Hz, 1H, CS-NH-cyclohexyl), 7.48 (d, J = 9.0 Hz, 2H, Ar-H), 4.14 (s, 2H, Ar-CH2), 1.80 (d, J = 8.9 Hz, 2H, cyclohexyl protons), 1.69 (dd, J = 9.5, 4.2 Hz, 2H, cyclohexyl protons), 1.58 (d, J = 12.2 Hz, 1H, cyclohexyl protons), 1.38–1.15 (m, 5H, cyclohexyl protons), 1.14–1.01 (m, 1H, cyclohexyl protons). 13C-APT NMR (DMSO-d6, 150 MHz, δ ppm): 184.84 (C=S), 165.85 (C=O), 135.62, 132.41, 128.92, 128.46, 119.35 (C≡N), 53.48, 32.31, 25.65, 25.39 (cyclohexyl carbons), 22.83 (Ar-CH2). Anal. calcd for C16H20N4OS: C 60.73, H 6.37, N 17.71, S 10.13%. Found: C 60.19, H 6.29, N 17.79, S 10.01%.
General synthesis method of 2,5-disubstituted-1,3,4-thiadiazol-5-amine (4a–4j)
Weigh 1 mmol of thiosemicarbazide derivatives and put into a beaker. Concentrated sulfuric acid (1–1.5 mL) is added dropwise until the substance is dissolved. The mixture is stirred at room temperature for 4 h. The mixture is poured into ice-water and neutralized with a dilute base solution to obtain thiadiazole derivatives. The solid material is crystallized from ethanol [22].
2-{4-[5-(Phenylamino)-1,3,4-thiadiazol-2-yl]phenyl}acetonitrile (4a)
Yield: 80%; Color: orange powder; Melting Point: 270.0–270.5 °C; FT-IR (cm−1): 3333 and 3184 (N-H), 3053 (=C-H), 2947 and 2890 (C-H), 2366 (C≡N), 1658 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.57 (s, 1H, NH), 7.92–7.76 (m, 2H, Ar-H), 7.70–7.59 (m, 2H, Ar-H), 7.45–7.32 (m, 3H, Ar-H), 7.15–6.91 (m, 2H, Ar-H), 4.38 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.26 (C=N), 164.43, 158.23 (C=N), 142.45, 141.03, 139.34, 130.43, 129.61, 128.92, 127.08, 122.54, 118.01 (C≡N), 116.89, 42.44 (Ar-CH2). Anal. calcd for C16H12N4S: C 65.73, H 4.14, N 19.16, S 10.97%. Found: C 65.10, H 4.19, N 19.29, S 10.87%.
2-(4-{5-[(4-Fluorophenyl)amino]-1,3,4-thiadiazol-2-yl}phenyl)acetonitrile (4b)
Yield: 75%; Color: white powder; Melting Point: 192.7–194.0 °C; FT-IR (cm−1): 3352 and 3146 (N-H), 3039 (=C-H), 2899 and 2891 (C-H), 2258 (C≡N), 1651 and 1606 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.55 (s, 1H, NH), 7.92–7.86 (m, 1H, Ar-H), 7.82–7.75 (m, 1H, Ar-H), 7.75–7.65 (m, 1H, Ar-H), 7.50–7.47 (m, 2H, Ar-H), 7.43–7.37 (m, 1H, Ar-H), 7.28–7.16 (m, 2H, Ar-H), 4.14 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 75 MHz, δ ppm): 171.72 (C=N), 163.97, 157.51 (C=N), 138.85, 137.01 (d, J = 2.25 Hz), 129.92, 129.59, 128.93, 128.37, 127.24, 126.58, 119.12 (C≡N), 115.65 (d, J = 21.75 Hz), 41.94 (CH2CN). Anal. calcd for C16H11FN4S: C 61.92, H 3.57, N 18.05, S 10.33%. Found: C 62.55, H 3.53, N 18.09, S 10.25%.
2-(4-{5-[(4-Chlorophenyl)amino]-1,3,4-thiadiazol-2-yl}phenyl)acetonitrile (4c)
Yield: 80%; Color: white powder; Melting Point: 216.9–217.5 °C; FT-IR (cm−1): 3236 and 3180 (N-H), 3030 (=C-H), 2995 and 2937 (C-H), 2251 (C≡N), 1666 and 1599 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.69 (s, 1H, NH), 7.85 (d, J = 8.1 Hz, 2H, Ar-H), 7.75–7.67 (m, 2H, Ar-H), 7.46–7.34 (m, 3H, Ar-H), 6.96 (s, 1H, Ar-H), 4.14 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.22 (C=N), 164.05, 158.45 (C=N), 139.91, 139.46, 133.98, 130.44, 130.02, 129.45, 128.81, 127.81, 127.14, 125.90, 119.55 (C≡N), 42.60 (CH2CN). Anal. calcd for C16H11ClN4S: C 58.81, H 3.39, N 17.14, S 9.81%. Found: C 58.67, H 3.40, N 17.17, S 9.76%.
2-(4-{5-[(4-Bromophenyl)amino]-1,3,4-thiadiazol-2-yl}phenyl)acetonitrile (4d)
Yield: 80%; Color: yellow powder; Melting Point: 211.6–212.7 °C; FT-IR (cm−1): 3236 and 3176 (N-H), 3030 (=C-H), 2945 and 2850 (C-H), 2366 (C≡N), 1662 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.67 (s, 1H, NH), 7.89–7.77 (m, 2H, Ar-H), 7.70–7.62 (m, 2H, Ar-H), 7.57–7.50 (m, 2H, Ar-H), 7.43–7.36 (m, 2H, Ar-H), 4.40 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 171.72 (C=N), 163.48, 157.99 (C=N), 139.79, 138.97, 131.79, 129.94, 128.27, 126.64, 119.39 (C≡N), 113.24, 41.94 (Ar-CH2). Anal. calcd for C16H11BrN4S: C 51.76, H 2.99, N 15.09, S 8.64%. Found: C 51.42, H 3.01, N 15.01, S 8.74%.
2-(4-{5-[(4-Methylphenyl)amino]-1,3,4-thiadiazol-2-yl}phenyl)acetonitrile (4e)
Yield: 75%; Color: yellow powder; Melting Point: 212.2–213.2 °C; FT-IR (cm−1): 3342 and 3174 (N-H), 3030 (=C-H), 2916 and 2858 (C-H), 2254 (C≡N), 1633 and 1610 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.44 (s, 1H, NH), 7.91–7.75 (m, 2H, Ar-H), 7.59–7.51 (m, 2H, Ar-H), 7.40 (d, J = 8.3 Hz, 2H, Ar-H), 7.23–7.14 (m, 2H, Ar-H), 4.24 (s, 2H, CH2CN), 2.27 (s, 3H, CH3). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.26 (C=N), 164.66, 157.86 (C=N), 139.25, 138.65, 133.75, 131.57, 130.41, 130.01, 129.42, 128.98, 127.69, 127.03, 118.18 (C≡N), 42.45 (CH2CN), 20.84 (Ar-CH3). Anal. calcd for C17H14N4S: C 66.64, H 4.61, N 18.29, S 10.46%. Found: C 66.97, H 4.65, N 18.37, S 10.41%.
2-(4-{5-[(4-Methoxyphenyl)amino]-1,3,4-thiadiazol-2-yl}phenyl)acetonitrile (4f)
Yield: 77%; Color: lilac powder; Melting Point: 198.6–199.9 °C; FT-IR (cm−1): 3335 and 3173 (N-H), 3053 (=C-H), 2953 and 2835 (C-H), 2283 (C≡N), 1656 and 1599 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 10.34 (s, 1H, NH), 7.90–7.73 (m, 2H, Ar-H), 7.62–7.52 (m, 2H, Ar-H), 7.51–7.35 (m, 2H, Ar-H), 6.99–6.93 (m, 2H, Ar-H), 4.13 (s, 2H, CH2CN), 3.75 (s, 3H, OCH3). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.30 (C=N), 165.14, 156.72 (C=N), 155.02, 139.15, 134.46, 133.66, 130.75, 129.41, 129.05, 127.63, 127.03, 119.79 (C≡N), 114.85, 55.70 (Ar-OCH3), 42.44 (CH2CN). Anal. calcd for C17H14N4OS: C 63.34, H 4.38, N 17.38, S 9.94%. Found: C 63.78, H 4.42, N 17.30, S 9.99%.
2-(4-{5-[(4-Nitrophenyl)amino]-1,3,4-thiadiazol-2-yl}phenyl)acetonitrile (4g)
Yield: 85%; Color: yellow powder; Melting Point: 225.0–226.2 °C; FT-IR (cm−1): 3335 and 3186 (N-H), 3053 (=C-H), 2951 and 2835 (C-H), 2258 (C≡N), 1658 and 1608 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 11.62 and 11.28 (2s, 1H, NH), 8.32–8.22 (m, 2H, Ar-H), 7.88–7.81 (m, 3H, Ar-H), 7.47–7.36 (m, 2H, Ar-H), 6.95 (d, J = 8.9 Hz, 1H, Ar-H), 4.40 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.21 (C=N), 163.27, 160.14 (C=N), 146.68, 141.35, 139.84, 130.86, 130.51, 129.68, 128.50, 127.38, 125.98, 117.49 (C≡N), 42.58 (CH2CN). Anal. calcd for C16H11N5O2S: C 56.97, H 3.29, N 20.76, S 9.50%. Found: C 56.67, H 3.25, N 20.71, S 9.54%.
2-{4-[5-(Methylamino)-1,3,4-thiadiazol-2-yl]phenyl}acetonitrile (4h)
Yield: 85%; Color: white powder; Melting Point: 204.4–205.6 °C; FT-IR (cm−1): 3336 and 3161 (N-H), 3028 (=C-H), 2978 and 2879 (C-H), 2330 (C≡N), 1633 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 7.90–7.83 (m, 1H, NH), 7.77–7.64 (m, 2H, Ar-H), 7.53–7.32 (m, 2H, Ar-H), 3.47 (s, 2H, CH2CN), 2.93 (d, J = 4.4 Hz, 3H, CH3). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.32 (C=N), 169.62, 156.27 (C=N), 138.65, 130.31, 129.52, 126.67, 42.41 (CH2CN), 31.79 (CH3). Anal. calcd for C11H10N4S: C 57.37, H 4.38, N 24.33, S 13.92%. Found: C 57.88, H 4.33, N 24.43, S 13.83%.
2-{4-[5-(Ethylamino)-1,3,4-thiadiazol-2-yl]phenyl}acetonitrile (4i)
Yield: 82%; Color: white powder; Melting Point: 226.6–227.1 °C; FT-IR (cm−1): 3342 and 3163 (N-H), 3041 (=C-H), 2999 and 2866 (C-H), 2339 (C≡N), 1633 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 7.94–7.86 (m, 1H, NH), 7.73–7.64 (m, 2H, Ar-H), 7.56–7.32 (m, 2H, Ar-H), 3.47 (s, 2H, CH2CN), 3.43 (s, 2H, CH2CH3), 1.20 (t, J = 7.2 Hz, 3H, CH2CH3). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.29 (C=N), 168.70, 156.10 (C=N), 138.63, 130.29, 129.74, 129.54, 126.62, 42.59 (Ar-CH2), 39.64 (CH2CH3), 14.74 (CH2CH3). Anal. calcd for C12H12N4S: C 58.99, H 4.95, N 22.93, S 13.12%. Found: C 59.19, H 4.97, N 23.04, S 13.01%.
2-{4-[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]phenyl}acetonitrile (4j)
Yield: 82%; Color: white powder; Melting Point: 242.5–243.3 °C; FT-IR (cm−1): 3288 and 3161 (N-H), 3018 (=C-H), 2939 and 2856 (C-H), 2341 (C≡N), 1678 and 1633 (C=N). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 7.88 (d, J = 7.2 Hz, 1H, NH), 7.72–7.63 (m, 2H, Ar-H), 7.39–7.30 (m, 2H, Ar-H), 3.65–3.50 (s, 2H, CH2CN), 1.99 (m, 2H, cyclohexyl protons), 1.79–1.48 (m, 3H, cyclohexyl protons), 1.40–1.13 (m, 6H, cyclohexyl protons). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.30 (C=N), 167.91, 155.85 (C=N), 138.56, 130.62, 130.28, 129.57, 126.63, 119.44 (C≡N), 54.25, 42.42 (Ar-CH2), 32.58, 25.71, 24.77, 22.68. Anal. calcd for C16H18N4S: C 64.40, H 6.08, N 18.78, S 10.74%. Found: C 64.82, H 6.00, N 18.99, S 10.67%.
General synthesis method of 1,2-disubstituted-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl (5a–5j)
Weigh 1 mmol of thiosemicarbazide derivatives and put into a reaction flask. 2–3 mL of NaOH solution (2N) is added on it until the substance is dissolved. The mixture is refluxed at 100 °C for 6–10 h. Ice pieces are poured onto the reaction mixture and neutralization is carried out with 1N HCl to obtain the triazole derivatives in solid form. The solid residue is crystallized from ethanol [22].
2-[4-(4-Phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl]acetonitrile (5a)
Yield: 75%; Color: gray powder; Melting Point: 257.0–258.3 °C; FT-IR (cm−1): 3309 (N-H), 3037 (=C-H), 2978 and 2914 (C-H), 2339 (C≡N), 1707 (C=N), 1236 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 14.12 (s, 1H, NH), 7.54–7.47 (m, 3H, Ar-H), 7.39–7.34 (m, 2H, Ar-H), 7.28–7.19 (m, 4H, Ar-H), 3.57 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.18 (C=N), 168.54, 150.43 (C=N), 137.46, 134.55, 129.62, 129.41, 129.33, 128.73, 128.02, 124.07, 40.20. Anal. calcd for C16H12N4S: C 65.73, H 4.14, N 19.16, S 10.97%. Found: C 64.89, H 4.09, N 19.30, S 10.83%.
2-{4-[4-(4-Fluorophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]phenyl}acetonitrile (5b)
Yield: 80%; Color: white powder; Melting Point: 244.1–245.0 °C; FT-IR (cm−1): 3379 and 3296 (N-H), 3039 (=C-H), 2931 and 2885 (C-H), 2254 (C≡N), 1672 (C=N), 1225 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 14.03 (s, 1H, NH), 7.49–7.41 (m, 2H, Ar-H), 7.40–7.31 (m, 2H, Ar-H), 7.26 (s, 4H, Ar-H), 3.58 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.67 (C=N), 169.16, 162.52 (d, J = 244.5 Hz), 150.98 (C=N), 138.06, 131.62 (d, J = 9.0 Hz), 131.37 (d, J = 3.0 Hz), 130.17, 128.62, 124.51, 116.78 (d, J = 22.5 Hz), 40.76. Anal. calcd for C16H11FN4S: C 61.92, H 3.57, N 18.05, S 10.33%. Found: C 62.60, H 3.50, N 18.19, S 10.45%.
2-{4-[4-(4-Chlorophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]phenyl}acetonitrile (5c)
Yield: 85%; Color: white powder; Melting Point: 255.5–256.0 °C; FT-IR (cm−1): 3358 and 3277 (N-H), 3032 (=C-H), 2925 and 2877 (C-H), 2278 (C≡N), 1672 (C=N), 1230 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 13.92 (s, 1H, NH), 7.63–7.53 (m, 2H, Ar-H), 7.48–7.38 (m, 2H, Ar-H), 7.26 (s, 4H, Ar-H), 3.58 (s, 2H, CH2CN). 13C-APT NMR (DMSO-d6, 150 MHz, δ ppm): 172.62 (C=N), 169.03, 150.87 (C=N), 138.10, 134.51, 133.97, 131.20, 130.20, 129.89, 128.66, 124.44, 40.76. Anal. calcd for C16H11ClN4S: C 58.81, H 3.39, N 17.14, S 9.81%. Found: C 57.91, H 3.45, N 17.20, S 9.87%.
2-{4-[4-(4-Bromophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]phenyl}acetonitrile (5d)
Yield: 80%; Color: white powder; Melting Point: 238.8–239.8 °C; FT-IR (cm−1): 3228 (N-H), 3097 (=C-H), 2974 and 2897 (C-H), 2272 (C≡N), 1703 (C=N), 1259 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 14.18 and 12.52 (2s, 1H, NH), 7.77–7.64 (m, 2H, Ar-H), 7.43–7.30 (m, 2H, Ar-H), 7.27 (s, 4H, Ar-H), 3.59 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.12 (C=N), 168.39, 150.30 (C=N), 137.58, 133.87, 132.34, 130.97, 129.72, 128.16, 123.91, 122.63, 40.23. Anal. calcd for C16H11BrN4S: C 51.76, H 2.99, N 15.09, S 8.64%. Found: C 51.08, H 3.05, N 14.98, S 8.73%.
2-{4-[4-(4-Methylphenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]phenyl}acetonitrile (5e)
Yield: 82%; Color: yellow powder; Melting Point: 304.6–305.4 °C; FT-IR (cm−1): 3302 (N-H), 3037 (=C-H), 2941 and 2877 (C-H), 2254 (C≡N), 1672 (C=N), 1232 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 14.09 (s, 1H, NH), 7.33–7.27 (m, 3H, Ar-H), 7.27–7.20 (m, 5H, Ar-H), 3.57 (s, 2H, CH2CN), 2.36 (s, 3H, CH3). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.67 (C=N), 169.17, 150.97 (C=N), 139.47, 137.94, 137.82, 132.48, 130.23, 128.92, 128.52, 124.66, 118.58, 40.72, 21.26. Anal. calcd for C17H14N4S: C 66.64, H 4.61, N 18.29, S 10.46%. Found: C 66.16, H 4.63, N 18.22, S 10.41%.
2-{4-[4-(4-Methoxyphenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]phenyl}acetonitrile (5f)
Yield: 80%; Color: purple powder; Melting Point: 257.8–259.0 °C; FT-IR (cm−1): 3402 (N-H), 3076 (=C-H), 2910 and 2850 (C-H), 2312 (C≡N), 1697 (C=N), 1255 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 14.07 and 12.42 (2s, 1H, NH), 7.35–7.19 (m, 6H, Ar-H), 7.10–6.98 (m, 2H, Ar-H), 3.80 (s, 3H, OCH3), 3.58 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.67 (C=N), 169.34, 160.02, 151.08 (C=N), 137.88, 130.41, 130.13, 128.51, 127.58, 124.72, 114.96, 55.87, 40.69. Anal. calcd for C17H14N4OS: C 63.34, H 4.38, N 17.38, S 9.94%. Found: C 63.88, H 4.43, N 17.30, S 9.99%.
2-{4-[4-(4-Nitrophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]phenyl}acetonitrile (5g)
Yield: 85%; Color: brown powder; Melting Point: 357.8–358.9 °C; FT-IR (cm−1): 3279 (N-H), 3053 (=C-H), 2893 and 2821 (C-H), 2312 (C≡N), 1681 (C=N), 1282 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 14.22 and 12.90 (2s, 1H, NH), 8.05 (s, 4H, Ar-H), 7.94–7.87 (m, 2H, Ar-H), 7.43–7.35 (m, 2H, Ar-H), 3.69 (s, 2H, CH2CN). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.16 (C=N), 167.17, 166.64, 159.02 (C=N), 144.77, 140.11, 134.40, 129.62, 129.42, 129.22, 129.09, 125.48, 40.48. Anal. calcd for C16H11N5O2S: C 56.97, H 3.29, N 20.76, S 9.50%. Found: C 56.30, H 3.33, N 20.66, S 9.40%.
2-[4-(4-Methyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl]acetonitrile (5h)
Yield: 85%; Color: white powder; Melting Point: 224.5–225.3 °C; FT-IR (cm−1): 3294 (N-H), 3051 (=C-H), 2895 and 2870 (C-H), 2351 (C≡N), 1712 (C=N), 1213 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 13.93 and 12.53 (2s, 1H, NH), 7.75–7.63 (m, 2H, Ar-H), 7.51–7.41 (m, 2H, Ar-H), 3.69 (s, 2H, CH2CN), 3.54 (s, 3H, NHCH3). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.29 (C=N), 167.41, 151.31 (C=N), 137.86, 130.02, 128.35, 124.36, 40.37, 31.60. Anal. calcd for C11H10N4S: C 57.37, H 4.38, N 24.33, S 13.92%. Found: C 56.76, H 4.45, N 24.20, S 14.05%.
2-[4-(4-Ethyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl]acetonitrile (5i)
Yield: 77%; Color: white powder; Melting Point: 199.5–200.5 °C; FT-IR (cm−1): 3225 (N-H), 3047 (=C-H), 2995 and 2858 (C-H), 2351 (C≡N), 1662 (C=N), 1255 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 13.92 and 12.49 (2s, 1H, NH), 7.68–7.58 (m, 2H, Ar-H), 7.51–7.43 (m, 2H, Ar-H), 4.04 (q, J = 7.1 Hz, 2H, NHCH2CH3), 3.70 (s, 2H, CH2CN), 1.16 (t, J = 7.1 Hz, 3H, NHCH2CH3). 13C NMR (DMSO-d6, 150 MHz, δ ppm): 172.28 (C=N), 166.73, 150.99 (C=N), 137.90, 130.16, 128.40, 124.40, 40.28, 38.29, 13.41. Anal. calcd for C12H12N4S: C 58.99, H 4.95, N 22.93, S 13.12%. Found: C 59.87, H 4.87, N 22.79, S 13.01%.
2-[4-(4-Cyclohexyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenyl]acetonitrile (5j)
Yield: 80%; Color: white powder; Melting Point: 273.1–274.3 °C; FT-IR (cm−1): 3200 (N-H), 3053 (=C-H), 2929 and 2854 (C-H), 2312 (C≡N), 1714 (C=N), 1294 (C=S). 1H NMR (DMSO-d6, 300 MHz, δ ppm): 13.87 and 12.50 (2s, 1H, NH), 7.47 (q, J = 8.3 Hz, 4H, Ar-H), 4.25 (t, J = 12.3 Hz, 1H, cyclohexyl proton), 3.71 (s, 2H, CH2CN), 2.20 (s, 2H, cyclohexyl protons), 1.72 (d, J = 11.9 Hz, 4H, cyclohexyl protons), 1.54 (d, J = 12.6 Hz, 1H, cyclohexyl proton), 1.28–1.07 (m, 2H, cyclohexyl protons), 0.94 (q, J = 12.9 Hz, 1H, cyclohexyl proton). 13C-APT NMR (DMSO-d6, 150 MHz, δ ppm): 172.74 (C=N), 166.65, 151.97 (C=N), 138.31, 130.29, 130.08, 125.54, 57.46, 40.84, 29.77, 25.87, 25.08. Anal. calcd for C16H18N4S: C 64.40, H 6.08, N 18.78, S 10.74%. Found: C 64.89, H 6.12, N 18.65, S 10.80%.

3.2. Biological Activity

3.2.1. Cytotoxicity Assay
To evaluate the cytotoxic effects on NIH3T3 and MCF-7 cell lines, the MTT assay was employed following established protocols [23,24]. This method quantifies cell viability based on the enzymatic conversion of MTT into formazan crystals, a process indicative of metabolic activity in living cells [25].

3.2.2. Aromatase Inhibition Assay
The in vitro aromatase inhibitory potential of the synthesized compounds was evaluated using a fluorometric screening kit (BioVision, CYP19A), following the methodology established in our previous studies [26,27,28]. Test compounds were prepared in 2% DMSO across a broad concentration range (10−3–10−9 M), with Letrozole serving as the reference inhibitor. Each analysis, including blanks and controls, was performed in quadruplicate, and data were expressed as mean ± standard deviation (SD). For the determination of IC50 values, dose–response curves were generated by plotting inhibition percentages against logarithmic concentrations using GraphPad Prism software (v. 5.0).

3.3. In Silico Studies

3.3.1. Molecular Docking Studies
To elucidate the binding orientations of compounds 5c, 5e, and 5f within the aromatase enzyme’s active site, in silico molecular docking simulations were conducted. The human aromatase X-ray crystal structure (PDB ID: 3EQM) [29] was obtained from the Protein Data Bank (http://www.pdb.org), chosen specifically for its high-resolution data, human origin, and established reliability in the literature. Protein refinement was executed via the Protein Preparation Wizard within the Schrödinger Suite 2020, utilizing the Maestro [30] interface. Concurrently, the LigPrep module [31] was employed for ligand preparation, ensuring accurate atom typing and protonation states. Following the addition of hydrogen atoms and the assignment of bond orders, the Glide module [32] was used for grid generation. Finally, docking simulations were completed employing the Standard Precision (SP) mode.

3.3.2. Molecular Dynamics Simulation
To evaluate the structural stability and temporal behavior of compound 5c within the aromatase active site, molecular dynamics (MD) simulations were conducted [33]. These computational analyses were executed through the same software interfaces as described in our previous methodologies [28,34], ensuring a consistent approach to modeling the ligand–receptor complex.

3.3.3. ADME Studies
The physicochemical, pharmacokinetic, and drug-likeness properties of the compounds, as well as the BOILED-Egg model, were calculated using the SwissAdme program (https://www.swissadme.ch/). Table 5 and Figure 5 were obtained using the SwissAdme program.

4. Conclusions
The aromatase enzyme is one of the most important targets in the treatment of breast cancer. This is because the aromatase enzyme is one of the enzymes involved in estrogen biosynthesis, which leads to the growth of tumor tissue. Resistance to existing aromatase inhibitors limits their clinical use, increasing the need for new and effective aromatase inhibitors. In this study, some new thiadiazole (4a–4j) and triazole structures (5a–5j) were designed and synthesized based on the structures of anastrazole and letrozole. Their antiproliferative activities against MCF7 and NIH3T3 cells were investigated. The aromatase enzyme inhibitory activity of the compounds was tested. Among the triazole compounds, those bearing fluorine, chlorine, methyl, methoxy, and nitro substituents on the phenyl ring (5b, 5c, 5e, 5f and 5g) were found to exhibit the most promising antiproliferative and anti-aromatase activities within the series. In silico studies showed that compound 5c formed stable interactions with the active site of the aromatase enzyme. Therefore, the chemical structure of compound 5c was considered to be a guide for further research in the design of new anti-aromatase enzyme inhibitors for breast cancer treatment.