Synthesis, molecular modeling, and biological evaluation of 1,2,4-triazole derivatives containing pyridine as potential anti-tumor agents

Abstract

There is an accumulating body of experimental evidences validating focal adhesion kinase (FAK) as a therapeutic target and offering opportunities for anti-tumor drug development. In present study, we sought to synthesize twenty-eight potential FAK inhibitors as anti-tumor agents based on 1,2,4-triazole skeleton. The bioassay assays demonstrated that compounds 3e and 6j showed the most potent activity, 3e inhibited the growth of HCT116 and HepG2 cell lines with IC₅₀ values of 8.17 and 7.04 μM, while compound 6j showed the most potent biological activity against HCT116 cell line (IC₅₀ = 1.99 μM). Besides, compound 6j also exhibited significant FAK inhibitory activity (IC₅₀ = 2.41 μM). The results of flow cytometry and western-blot assay demonstrated that compounds 3e and 6j possessed good anti-proliferative activity. Docking simulations were performed to position compounds 3e and 6j into the active site of FAK to determine the probable binding model.

Introduction

Cancer, the second leading cause of death in the world, is continuing to be a major health problem in developing as well as undeveloped countries (EI-Azab et al., 2010). Cancer chemotherapy targeting tumor progression represents one of the most relevant challenges of chemists and oncologist. In order to gain new insights into the complexity of the disease, robust screening methods for evaluating different natural or synthetic drugs have been carried out from the science community (Stanton et al., 2008). Therefore, there is an increasing need for new therapies, especially those that based on current knowledge of cancer biology as well as that taking advantage of the cancer cells phenotype, described by Hanahan and Weinberg (2000).

The non-receptor protein tyrosine kinase focal adhesion kinase (FAK) was discovered almost 15 years ago (Van Nimwegen and van de Water, 2007). Most studies show an enhanced expression of FAK mRNA and/or protein in a variety of human cancers, including squamous cell carcinoma of the larynx (Aronsohn et al., 2003), invasive colon and breast tumors (Owens et al., 1995), and malignant melanoma (Kahana et al., 2002). Activation of FAK leads to a number of cell biological processes, including cell attachment, migration, invasion, proliferation, and survival (Maroesja and Bob, 2007). The human FAK core promoter contains binding sites for many transcription factors, including but not limited to NF-κB, p53, AP-1, AP-2, PU.1, TCF-1, and EGR-1 (Golubovskaya et al., 2004). Inhibition of FAK by anti-sense techniques or antibody-methods results in the onset of apoptosis and numerous studies have clearly confirmed a protective role for FAK in apoptosis (Sonoda et al., 2000). Given the role of FAK in processes important in tumorigenesis, metastasis, and the link to prominent oncogenes, FAK might be a promising target in the ongoing search for an anti-cancer drug (Maroesja and Bob, 2007).

The chemistry of N-bridged heterocycles derived from 1,2,4-triazole has received considerable attention in recent years due to their usefulness in different areas of biological activities and as industrial intermediates (Bhat et al., 2009). 1,2,4-triazole derivatives are known to exhibit anti-microbial (Ashok and Holla, 2007; Prasad et al., 2009), anti-tubercular (Walczak et al., 2004), anti-cancer (Sztanke and Tuzimski, 2008; Romagnoli and Baraldi, 2010), anti-convulsant (Amir and Shikha, 2004), anti-inflammatory, and analgesic properties (Almasirad et al., 2004). 1,2,4-triazole nucleus has been incorporated into a wide variety of therapeutically interesting drug candidates including H1/H2 histamine receptor blockers, cholinesterase active agents, CNS stimulants, anti-anxiety, and sedatives (Schreier and Helv, 1976), anti-mycotic activity such as fluconazole, itraconazole, and voriconazole (Budavari et al., 1996; Haber, 2001). Some 1,3,4-thiadiazole derivatives have been reported as potential FAK inhibitors (Juan et al., 2011). Pyridine was used to replace benzo[b]dioxin mainly according to the result of CADD (computer assistant drug design) method. The result indicated that a smaller moiety with higher electronic density might improve the bioactivity. Herein, in continuation to extend our research on anti-tumor compounds with FAK structure inhibitory activity, in the present study we sought to synthesis two series of 1,2,4-triazole derivatives containing pyridine as anti-tumor agents. Biological evaluation indicated that some of the synthesized compounds were potent inhibitors of FAK structure.

Results and discussion

Chemistry

In this study, twenty-eight triazole derivatives containing pyridine were synthesized. The synthetic route of compounds 3a–3n and 6a–6n was shown in Scheme 1. It was prepared in six steps. First, a mixture of corresponding isoniazid and phenyl isothiocyanate was refluxed in ethanol for 30 min. The solution was cooled and compound 1 was precipitated. A solution of NaOH (2 N) containing 1 was stirred under refluxing for 30 min, yielding the desired compound 2 according to the reported procedure. Then, fourteen 1,2,4-triazole derivatives (3a–3n) were prepared by refluxing in anhydrous acetonitrile of 2 with different benzyl bromide compounds as shown in Scheme 1 according to the literature. To a solution of compound 2 in acetone, ethyl bromoacetate was added and the mixture was refluxed for 2 h in the presence of K₂CO₃. The solution was cooled and compound 4 appeared. Then, compound 5 was prepared by treatment of 4 with hydrazine hydrate (85 %) in ethanol. Finally, fourteen 1,2,4-triazole derivatives (6a–6n) were collected by stirring in ethanol of 5 with different benzaldehyde compounds as shown in Scheme 1.

Scheme 1

General synthesis of compounds (3a–3n and 6a–6n). Reagents and conditions: a Phenyl isothiocyanate, ethanol, reflux, 30 min; b NaOH (2 N), reflux, 30 min. c Reflux, acetonitrile. d Bromoacetate, K₂CO₃, acetone, reflux, 2 h. e Hydrazine hydrate (85 %), ethanol, rt, 8–12 h; f Ethanol, rt

All of the synthetic compounds gave satisfactory analytical and spectroscopic data, which were in full accordance with their depicted structures. Furthermore, the crystal data, data collection, and refinement parameter for compound are listed in Table 1, and Fig. 1 gives a perspective view of this compound together with the atomic labeling system. The structure was solved by direct methods and refined on F2 by full-matrix least-squares methods using SHELX-97 (Sheldrick, 1997).

Table 1 Crystallographical and experimental data for compound 3g

Fig. 1

Molecule structure of compound 3g

Biological activity

All the synthesized derivatives 3a–3n and 6a–6n were evaluated for their anti-proliferative activity against MCF7 (human breast cancer cells), HCT116 (human colorectal cancer cells), and HepG2 (human hepatoma cells) cell lines. The results were summarized in Table 2. As illustrated in Table 2, compounds 3a–3n and 6a–6n showed all certain activities against the three tumor cells as compared with staurosporine. For the studied compounds, we observed that compounds (3e, 6j) showed potent anti-cancer activities against MCF7, HCT116, and HepG2 cell lines. It was obvious that compound 6j exhibited best activity against HCT116 cells with the IC₅₀ value of 1.99 μM, which was even better than the reference drug staurosporine (IC₅₀ = 13.20 μM).

Table 2 Anti-proliferative activity of the synthesized compounds (3a–3n and 6a–6n)

Structure–activity relationships (SARs) in these 1,2,4-triazole derivatives demonstrated that compounds having halogen atom substituent exhibited high activity. Meanwhile, a comparison of the substitution on benzene ring was demonstrated as follows (3a–3n): when the compounds were bromine-substituted derivatives, the potency order was meta > para > ortho (3c, 3e, 3h), and when the compounds were chlorine-substituted derivatives, the potency order was also meta > para > ortho ( 3b, 3d, 3g ). Interestingly, among the electron-withdrawing substituents compounds, the potency order was almost bromine > chlorine > fluorine > nitro. Another comparison of the substitution on benzene ring was demonstrated as follows (6a–6n): compounds with electron-donating substituents at para-position at A-ring had better inhibitory activity than that with electron-withdrawing substituents at the same position. When the compounds were fluorine-substituted derivatives, the potency order was para > ortho > meta (6g, 6h, 6i), among the electron-withdrawing substituents compounds, the potency order was bromine > chlorine > fluorine > nitro. Compounds with hydroxy at ortho-position had better inhibitory activity (6j, 6k, 6l). Overall, series 6a–6n had better activities than series 3a–3n. That might be caused by the additional hydrazone structure, and it will also be mentioned in the SARs study part.

In addition, we also selected the top 7 compounds which had better anti-proliferative activity to test their FAK inhibitory activity against HepG2 cell line. The results were summarized in Table 3. Most of the tested compounds displayed potent FAK inhibitory. Among them, compound 6j showed the most potent inhibitory with IC₅₀ of 2.41 μM. The results of FAK inhibitory activity of the tested compounds were corresponding to the structure relationships of their anti-tumor activities. This demonstrated that the potent anti-tumor activities of the synthetic compounds were probably correlated to their FAK inhibitory activities.

Table 3 FAK inhibitory activity of selected compounds

Apoptosis assay

Apoptosis is an essential mechanism used to eliminate activated HCT116 cells during the shutdown process of excess immune responses and maintain proper immune homeostasis, while deficient apoptosis of the activated HCT116 cell is associated with a wide variety of immune disorders. We detected the mechanism of compounds 3e and 6j (Fig. 2) inhibition effects by flow cytometry, and found that the compounds could induce the apoptosis of activated HCT116 cells in a dose- dependent manner. The result indicated that compounds 3e and 6j induced apoptosis of anti-tumor stimulated HCT116 cells.

Fig. 2

HCT116 cells isolated from naïve mice were cultured with anti-cancer and various concentrations of 3e and 6j for 24 h. Cells were stained by Annexin VeFITC/PI and apoptosis was analyzed by flow cytometry

Western-blot assay

In an effort to study the preliminary mechanism of the compounds with potent inhibitory activity, the western-blot experiment was performed to explore the effect of compounds 3e and 6j. The western-blot results were summarized in Fig. 3a and b, confirming compounds 3e and 6j’s inhibitory activities. The result indicated that compound 6j showed excellent inhibitory activity.

Fig. 3

a Compounds 3e and 6j were examined by western blotting. Data are representative of three independent experiments. b Compounds 6j were examined by western blotting. Data are representative of three independent experiments

Molecular docking study

To gain better understanding on the potency of the synthesized compounds and guide further SARs studies. The FAK-7PY protein–ligand complex crystal structure (PDB ID: 2ETM) was chosen as the template to compare the docking mode between our compounds and FAK. The molecular docking was performed by embedding potent inhibitors 3e and 6j into binding site of FAK. All docking runs were applied using Discovery Studio3.1 (DS. 3.1). In the binding mode, compound 3e (Fig. 4) is bound to FAK via two π-cation interactions. One π-cation interaction (4.76 Å) is formed between LYS 454 and benzene ring while the other (6.19 Å) is formed between LYS 454 and triazole ring. In addition, compound 6j (Fig. 4) is bound to FAK via one hydrogen bond and two π-cation interactions. Imino group on the acylhydrazone group and oxygen atom of CYS 502 form the hydrogen bond (distance: N–H···O: 2.35 Å, angle: 119.98°). Analogously, one π-cation interaction (4.79 Å) is formed between LYS 454 and benzene ring while the other (6.61 Å) is formed between LYS 454 and triazole ring. These results, along with the data of inhibitory activity assay indicated that compounds 3e and 6j would be a kind of potential inhibitors.

Fig. 4

Molecular docking modeling of compounds 3e and 6j with FAK: for clarity, only interacting residues are displayed. Left 3D model of the interaction between compounds 3e, 6j, and the 2ETM binding site. The H-bond (green lines) is displayed as dotted lines, and the π-cation interactions are shown as orange lines. Right 2D model of the interaction between compound 3e, 6j, and the 2ETM binding site. The H-bond (blue arrows) is displayed as dotted arrows, and the π-cation interactions are shown as orange lines (Color figure online)

As the combinations shown in Fig. 5, Compound 2p (Juan et al., 2011) is nicely bound to FAK with two interactions. The amino hydrogen atom of LYS 454 forms a hydrogen bond with the oxygen atoms of 1,4-dioxane group. The nitrogen atom of amide group forms another hydrogen bond with the oxygen atom of CYS 502. Compound 7YP is also nicely bound to FAK with three interactions. The amino hydrogen atom of LYS 454 forms a hydrogen bond with the nitrogen atom of pyridine group. Also the pyridine ring of compound 7PY forms a π-cation interaction with LYS 454. Besides, the pyrrole ring of compound 7PY forms a π-cation interaction with ARG 550. This insures the binding affinity and results in an increased FAK inhibitory activity.

Fig. 5

2D model of the interaction between compounds 2p, 7PY and the 2ETM binding site. The H-bond is displayed as dotted arrows, and the π-cation interactions are shown as orange lines (Color figure online)

In summary, the chosen 1,2,4-triazole derivatives containing pyridine are nicely combined to the FAK. LYS 454 and CYS 502 play an important part in the combination of the receptor and ligand.

Conclusions

Two series of 1,2,4-triazole derivatives containing pyridine have been synthesized and evaluated for their anti-tumor activities. Compound 3e and 6j demonstrated the most potent inhibitory activity. 3e inhibited the growth of the three cell lines with IC₅₀ values range from 7.04 to 10.04 μM, while 6j inhibited the growth of the three cell lines with IC₅₀ values range from 1.99 to 6.46 μM. Besides 6j also inhibited the activity of FAK with IC₅₀ of 2.41 μM, which was comparable to the positive control staurosporine. In order to gain deeper understanding of the SARs observed at the FAK, molecular docking of the most potent inhibitor 3e and 6j into the binding site of FAK was performed on the binding model based on the FAK complex structure. Analysis of the compound 6j’s binding conformation demonstrated that compound 6j was stabilized by hydrogen bonding interaction with CYS502. Apoptosis assay and western-blot results showed the compound 6j was a potential anti-tumor agent.

Experimental

Cell proliferation assay

The anti-tumor activities of compounds 3a–3n and 6a–6n were determined using a standard (MTT)-based colorimetric assay (Sigma). Seed 10⁴ cells per well into 96-well plates, incubate at 37 °C, 5 % CO₂ for 24 h. Then add 100 μL a series concentration of drug-containing medium into wells to maintain the final concentration of drug as 100, 30, 10, 3 μM. One concentration should be triplicated. And staurosporine was used for the positive control. After 48 h, cell survival was determined by the addition of an MTT solution (25 μL of 5 mg mL⁻¹ MTT in PBS). After 4 h, discard the medium and add 100 μL DMSO; the plates were votexed for 10 min to make complete dissolution. Optical absorbance was measured at 490 nm.

FAK inhibitory assay

Seven 1,2,4-triazole derivatives containing pyridine were tested in a search for small molecule inhibitors of FAK. In a typical study, human recombinant full-length FAK was incubated in kinase buffer containing ATP and the substrate for 4 h at room temperature with or without the presence of the triazole derivatives, the final concentration of drug as 100, 30, 10, 3 μM. The remaining ATP in solution was then quantified utilizing the Kinase-Glo-luminescence kit (Promega).

Apoptosis assay

HCT116 cells were treated with various concentrations of compounds 3e and 6j for 24 h and then stained with both Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI). Then samples were analyzed by FACSCalibur flow cytometer (Becton–Dickinson, SanJose, CA).

Western-blot analysis

After incubation, cells were washed with PBS and lysed using lysis buffer (30 mm Tris, pH 7.5, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na₃VO₄, 1 % Nonidet P-40, 10 % glycerol, and phosphatase and protease inhibitors). After centrifugation at 10,000×g for 10 min, the protein content of the supernatant was determined by a BCATM protein assay kit (Pierce, Rockford, IL, USA). The protein lysates were separated by 10 % SDS-PAGE and subsequently electrotransferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5 % non-fat milk for 2 h at room temperature. The blocked membrane was probed with the indicated primary antibodies overnight at 4 °C, and then incubated with a horse radish peroxidase-coupled secondary antibody. Detection was performed using a LumiGLO chemiluminescent substrate system (KPL, Guildford, UK).

Molecular docking modeling

Molecular docking of compounds into the 3D FAK complex structure (PDB code: 2ETM) was carried out using the Discovery Studio (version 3.1) as implemented through the graphical user interface DS-CDocker protocol.

The three-dimensional structures of the aforementioned compounds were constructed using Chem 3D ultra 12.0 software [Chemical Structure Drawing Standard; Cambridge Soft corporation, USA (2010)], then they were energetically minimized using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal structures of FAK complex were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound water and ligands were eliminated from the protein and the polar hydrogen was added. The whole FAK complex was defined as a receptor and the site sphere was selected based on the ligand binding location of 7PY, then the 7PY molecule was removed and 3e and 6j were placed during the molecular docking procedure. Types of interactions of the docked protein with ligand were analyzed after the end of molecular docking.

Chemistry

All the NMR spectra were recorded on a Bruker DPX 300 model Spectrometer in CDCl₃. Chemical shifts (δ) for ¹H NMR spectra were reported in parts per million to residual solvent protons. Melting points were measured on a Boetius micro melting point apparatus. The ESI–MS spectra were recorded on a Mariner System 5304 Mass spectrometer. All chemicals and reagents used in current study were of analytical grade. TLC was run on the silica gel coated aluminum sheets (Silica Gel 60 GF254, E. Merk, Germany) and visualized in UV light (254 nm).

General procedure for synthesis of the target compounds (3a–3n) and (6a–6n)

To a solution of compound 2 (1 mmol) in acetonitrile, the corresponding benzyl bromide compounds (1 mmol) were added and the mixture was stirred under reflux for 4–8 h in the presence of NaOH (2 mmol). Then, the solvent was removed under reduced pressure and a solid obtained. The solid was recrystallized from acetonitrile to afford compounds 3a–3n.

To a stirred solution of compound 5 (1 mmol) and the corresponding benzaldehyde compounds (1 mmol) in ethanol (15 mL), water (1 mL) was added followed by dropwise addition of glacial acetic acid (0.2 mL). The resulting mixture was stirred at room temperature until the target product precipitate from the solvent, which was collected using suction filtration and dried. The solid was recrystallized from acetonitrile to afford compounds 6a–6n.

4-(5-((2-Fluorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)pyridine (3a)

Yellow solid, yield 83 %, mp: 173–174 °C. ¹H NMR (300 MHz, CDCl₃): 4.62 (s, 2H); 7.02–7.12 (m, 2H); 7.24 (d, J = 11.52 Hz, 1H); 7.31 (d, J = 5.28 Hz, 1H); 7.51 (t, J = 7.78 Hz, 1H); 7.61–7.70 (m, 4H); 7.92 (d, J = 6.24 Hz, 2H); 8.64 (d, J = 6.22 Hz, 2H). ESI–MS 363.1 (C₂₀H₁₆FN₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₅FN₄S: C, 66.28; H, 4.17; N, 15.46. Found: C, 66.09; H, 4.16; N, 15.50.

4-[5-(2-Chloro-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3b)

Yellow solid, yield 87 %, mp: 139–140 °C. ¹H NMR (300 MHz, CDCl₃): 4.63 (s, 2H); 7.12 (d, J = 8.26 Hz, 2H); 7.19–7.23 (m, 2H); 7.28 (t, J = 3.78 Hz, 2H); 7.33–7.37 (m, 1H); 7.49 (t, J = 6.96 Hz, 3H); 7.55–7.59 (m, 1H); 8.53 (d, J = 6.02 Hz, 2H). ESI–MS 379.1 (C₂₀H₁₆ClN₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₅ClN₄S: C, 63.40; H, 3.99; N, 14.79. Found: C, 63.61; H, 4.00; N, 14.75.

4-[5-(2-Bromo-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3c)

Yellow solid, yield 79 %, mp: 145–146 °C. ¹H NMR (300 MHz, CDCl₃): 4.64 (s, 2H); 7.20–7.31 (m, 2H); 7.61–7.70 (m, 5H); 7.80 (s, 2H); 8.18 (d, J = 8.76 Hz, 2H); 8.60 (s, 2H). ESI–MS 423.0 (C₂₀H₁₆BrN₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₅BrN₄S: C, 56.74; H, 3.57; N, 13.23. Found: C, 56.54; H, 3.56; N, 13.20.

4-[5-(3-Chloro-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3d)

Yellow solid, yield 81 %, mp: 149–150 °C. ¹H NMR (300 MHz, CDCl₃): 4.47 (s, 2H); 7.13 (d, J = 6.30 Hz, 2H); 7.18–7.28 (m, 3H); 7.33 (d, J = 6.76 Hz, 3H); 7.49–7.60 (m, 3H); 8.54 (d, J = 6.06 Hz, 2H). ESI–MS 379.1 (C₂₀H₁₆ClN₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₅ClN₄S: C, 63.40; H, 3.99; N, 14.79. Found: C, 63.21; H, 4.00; N, 14.84.

4-[5-(3-Bromo-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3e)

White solid, yield 85 %, mp: 155–156 °C. ¹H NMR (300 MHz, CDCl₃): 4.51 (s, 2H); 7.14–7.26 (m, 3H); 7.37 (dd, J ₁ = 7.68 Hz, J ₂ = 11.70 Hz, 2H); 7.54 (s, 1H); 7.59–7.68 (m, 3H); 7.86 (d, J = 6.76 Hz, 2H); 8.75 (d, J = 6.76 Hz, 2H). ESI–MS 423.0 (C₂₀H₁₆BrN₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₅BrN₄S: C, 56.74; H, 3.57; N, 13.23. Found: C, 56.56; H, 3.58; N, 13.27.

4-(5-((4-Fluorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)pyridine (3f)

Yellow solid, yield 77 %, mp: 142–143 °C. ¹H NMR (300 MHz, CDCl₃): 4.53 (s, 2H); 6.91–7.00 (m, 2H); 7.14 (m, 2H); 7.35–7.40 (m, 4H); 7.50–7.61 (m, 3H); 8.55 (d, J = 6 Hz, 2H). ESI–MS 363.1 (C₂₀H₁₆FN₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₅FN₄S: C, 66.28; H, 4.17; N, 15.46. Found: C, 66.06; H, 4.18; N, 15.51.

4-[5-(4-Chloro-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3g)

White solid, yield 84 %, mp: 143–144 °C. ¹H NMR (300 MHz, CDCl₃): 4.47 (s, 2H); 7.14 (d, J = 7.22 Hz, 2H); 7.26 (d, J = 9.34 Hz, 2H); 7.31 (t, J = 7.01 Hz, 4H); 7.49–7.56 (m, 3H); 8.54 (d, J = 6.04 Hz, 2H). ESI–MS 379.1 (C₂₀H₁₆ClN₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₅ClN₄S: C, 63.40; H, 3.99; N, 14.79. Found: C, 63.24; H, 3.98; N, 14.74.

4-[5-(4-Bromo-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3h)

Yellow solid, yield 78 %, mp: 144–145 °C. ¹H NMR (300 MHz, CDCl₃): 4.46 (s, 2H); 7.14 (d, J = 8.14 Hz, 2H); 7.24 (s, 1H); 7.27 (s, 1H); 7.37 (s, 1H); 7.40 (d, J = 9.50 Hz, 3H); 7.49–7.60 (m, 3H); 8.55 (s, 2H). ESI–MS 423.0 (C₂₀H₁₆BrN₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₅BrN₄S: C, 56.74; H, 3.57; N, 13.23. Found: C, 56.55; H, 3.58; N, 13.28.

4-(5-((2,4-Difluorobenzyl)thio)-4-phenyl-4H-1,2,4-triazol-3-yl)pyridine (3i)

Yellow solid, yield 80 %, mp: 172–173 °C. ¹H NMR (300 MHz, CDCl₃): 4.55 (s, 2H); 6.80–6.83 (m, 2H); 7.20–7.23 (m, 2H); 7.58–7.64 (m, 6H); 8.57 (d, J = 6.42 Hz, 2H). ESI–MS 381.1 (C₂₀H₁₅F₂N₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₄F₂N₄S: C, 63.15; H, 3.71; N, 14.73. Found: C, 63.33; H, 3.72; N, 14.78.

4-[5-(2,6-Difluoro-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3j)

Yellow solid, yield 82 %, mp: 142–143 °C. ¹H NMR (300 MHz, CDCl₃): 4.62 (s, 2H); 7.01–7.11 (m, 2H); 7.21–7.29 (m, 2H); 7.51 (t, J = 7.68 Hz, 2H); 7.59–7.68 (m, 2H); 7.87 (s, 2H); 8.62 (d, J = 6.76 Hz, 2H). ESI–MS 381.1 (C₂₀H₁₅F₂N₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₄F₂N₄S: C, 63.15; H, 3.71; N, 14.73. Found: C, 63.37; H, 3.70; N, 14.78.

4-(5-(Benzylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)pyridine (3k)

White solid, yield 86 %, mp: 155–156 °C. ¹H NMR (300 MHz, CDCl₃): 4.53 (s, 2H); 7.12–7.14 (m, 2H); 7.27–7.30 (m, 3H); 7.40–7.42 (m, 4H); 7.49–7.57 (m, 3H); 8.54 (d, J = 6.18 Hz, 2H). ESI–MS 345.1 (C₂₀H₁₇N₄S [M+H]⁺). Anal. Calcd for C₂₀H₁₆N₄S: C, 69.74; H, 4.68; N, 16.27. Found: C, 69.50; H, 4.69; N, 16.33.

4-[5-(2-Nitro-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3l)

Yellow solid, yield 82 %, mp: 141 °C. ¹H NMR (300 MHz, CDCl₃): 4.89 (s, 2H); 7.14 (d, J = 6.78 Hz, 2H); 7.33 (d, J = 4.66 Hz, 2H); 7.43–7.62 (m, 5H); 7.94 (d, J = 7.68 Hz, 1H); 8.08 (d, J = 8.22 Hz, 1H); 8.53 (d, J = 6.24 Hz, 2H). ESI–MS 390.1 (C₂₀H₁₆N₂O₂S [M+H]⁺). Anal. Calcd for C₂₀H₁₅N₂O₂S: C, 61.68; H, 3.88; N, 17.98. Found: C, 61.48, H, 3.89; N, 18.04.

4-[5-(3-Nitro-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3m)

Yellow solid, yield 80 %, mp: 154–155 °C. ¹H NMR (300 MHz, CDCl₃): 4.59 (s, 2H); 7.17 (d, J = 7.50 Hz, 2H); 7.28 (t, J = 5.86 Hz, 2H); 7.46–7.58 (m, 4H); 7.81 (d, J = 7.68, 1H); 8.12 (d, J = 7.32 Hz, 1H); 8.25 (s, 1H); 8.53 (d, J = 5.68 Hz, 2H). ESI–MS 390.1 (C₂₀H₁₆N₅O₂S [M+H]⁺). Anal. Calcd for C₂₀H₁₅N₅O₂S: C, 61.68; H, 3.88; N, 17.98. Found: C, 61.82; H, 3.89; N, 18.04.

4-[5-(4-Nitro-benzylsulfanyl)-4-phenyl-4H-[1,2,4]triazol-3-yl]-pyridine (3n)

Yellow solid, yield 77 %, mp: 90–91 °C. ¹H NMR (300 MHz, CDCl₃): 4.59 (s, 2H); 7.18 (d, J = 6.96 Hz, 2H); 7.38 (d, J = 5.86 Hz, 2H); 7.53–7.63 (m, 5H); 8.16 (d, J = 8.80 Hz, 2H); 8.55 (d, J = 6.04 Hz, 2H). ESI–MS 390.1 (C₂₀H₁₆N₅O₂S [M+H]⁺). Anal. Calcd for C₂₀H₁₅N₅O₂S: C, 61.68; H, 3.88; N, 17.98. Found: C, 61.51; H, 3.89; N, 18.03.

(E)-N′-benzylidene-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6a)

White powder, yield 87 %; mp: 252–253 °C. ¹H NMR (300 MHz, DMSO): 4.52 (s, 2H); 7.30 (s, 2H); 7.45 (s, 1H); 7.50–7.52 (m, 4H); 7.61–7.62 (m, 3H); 7.69–7.73 (m, 2H); 8.01 (s, 1H); 8.57 (s, 2H); 11.73 (s, 1H). ESI–MS: 415.1 (C₂₂H₁₉N₆OS, [M+H]⁺). Anal. Calcd for C₂₂H₁₈N₆OS: C, 63.75; H, 4.38; N, 20.28 %. Found: C, 63.58; H, 4.39; N, 20.22 %.

(E)-N′-(4-methoxybenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6b)

White powder, yield 90 %; mp: 228–229 °C. ¹H NMR (300 MHz, DMSO): 3.79 (s, 3H); 4.50 (s, 2H); 6.97–6.99 (m, 2H); 7.26–7.28 (m, 2H); 7.48 (s, 2H); 7.59–7.64 (m, 5H); 7.94 (s, 1H); 8.54–8.55 (m, 2H); 11.56 (s, 1H). ESI–MS: 445.1 (C₂₃H₂₁N₆O₂S, [M+H]⁺). Anal. Calcd for C₂₃H₂₀N₆O₂S: C, 62.15; H, 4.54; N, 18.91. Found: C, 62.35; H, 4.53; N, 18.85.

(E)-N′-(4-nitrobenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6c)

White powder, yield 91 %; mp: 248–249 °C. ¹H NMR (300 MHz, DMSO): 4.56 (s, 2H); 7.26 (s, 2H); 7.48 (s, 2H); 7.59–7.60 (m, 3H); 7.92–7.97 (m, 2H); 8.11–8.31 (m, 3H); 8.54 (s, 2H); 11.95 (s, 1H). ESI–MS: 460.1 (C₂₂H₁₈N₇O₃S, [M+H]⁺). Anal. Calcd for C₂₂H₁₇N₇O₃S: C, 57.51; H, 3.73; N, 21.34. Found: C, 57.35; H, 3.72; N, 21.41.

(E)-N′-(4-hydroxybenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6d)

White powder, yield 93 %; mp: 284–285 °C. ¹H NMR (300 MHz, DMSO): 4.48 (s, 2H); 6.79 (d, J = 4.95 Hz, 2H); 7.25–7.26 (m, 2H); 7.47–7.51 (m, 4H); 7.59 (s, 3H); 7.89 (s, 1H); 8.53 (s, 2H); 9.88 (s, 1H); 11.45 (s, 1H). ESI–MS: 431.1 (C₂₂H₁₉N₆O₂S, [M+H]⁺). Anal. Calcd for C₂₂H₁₈N₆O₂S: C, 61.38; H, 4.21; N, 19.52. Found: C, 61.58; H, 4.22; N, 19.45.

(E)-N′-(4-bromobenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6e)

White powder, yield 89 %; mp: 232–233 °C. ¹H NMR (300 MHz, DMSO): 4.53 (s, 2H); 7.27–7.29 (m, 2H); 7.50 (s, 2H); 7.62–7.65 (m, 7H); 8.00 (s, 1H); 8.55 (d, J = 3 Hz, 2H); 11.75 (s, 1H). ESI–MS: 493.0 (C₂₂H₁₈BrN₆OS, [M+H]⁺). Anal. Calcd for C₂₂H₁₇BrN₆OS: C, 53.56; H, 3.47; N, 17.03. Found: C, 53.41; H, 3.46; N, 17.07.

(E)-N′-(4-chlorobenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6f)

White powder, yield 92 %; mp: 218–219 °C. ¹H NMR (300 MHz, DMSO): 4.53 (s, 2H); 7.28 (t, J = 5.1 Hz, 2H); 7.50–7.51 (m, 4H); 7.62 (s, 3H); 7.69–7.73 (m, 2H); 8.01 (s, 1H); 8.55 (s, 2H); 11.73 (s, 1H). ESI–MS: 449.1 (C₂₂H₁₈ClN₆OS, [M+H]⁺). Anal. Calcd for C₂₂H₁₇ClN₆OS: C, 58.86; H, 3.82; N, 18.72. Found: C, 58.69; H, 3.83; N, 18.77.

(E)-N′-(4-fluorobenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6g)

White powder, yield 90 %; mp: 227–228 °C. ¹H NMR (300 MHz, DMSO): 4.54 (s, 2H); 7.30 (s, 2H); 7.50–7.51 (m, 4H); 7.61–7.62 (m, 3H); 7.69–7.73 (m, 2H); 8.01 (s, 1H); 8.57 (s, 2H); 11.75 (s, 1H). ESI–MS: 433.1 (C₂₂H₁₈FN₆OS, [M+H]⁺). Anal. Calcd for C₂₂H₁₇FN₆OS: C, 61.10; H, 3.96; N, 19.43. Found: C, 61.29; H, 3.95; N, 19.49.

(E)-N′-(2-fluorobenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6h)

White powder, yield 91 %; mp: 235–236 °C. ¹H NMR (300 MHz, DMSO): 4.41 (s, 2H); 6.95–7.11 (m, 2H); 7.29–7.32 (m, 3H); 7.37–7.39 (m, 2H); 7.51 (s, 2H); 7.60–7.62 (m, 2H); 7.82 (s, 1H); 8.54 (s, 2H); 11.59 (s, 1H). ESI–MS: 433.1 (C₂₂H₁₈FN₆OS, [M+H]⁺). Anal. Calcd for C₂₂H₁₇FN₆OS: C, 61.10; H, 3.96; N, 19.43. Found: C, 61.27; H, 3.95; N, 19.37.

(E)-N′-(3-fluorobenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6i)

White powder, yield 89 %; mp: 227–228 °C. ¹H NMR (300 MHz, DMSO): 4.55 (s, 2H); 7.30 (s, 2H); 7.50–7.52 (m, 4H); 7.60–7.62 (m, 3H); 7.70–7.73 (m, 2H); 8.01 (s, 1H); 8.55 (s, 2H); 11.71 (s, 1H). ESI–MS: 433.1 (C₂₂H₁₈FN₆OS, [M+H]⁺). Anal. Calcd for C₂₂H₁₇FN₆OS: C, 61.10; H, 3.96; N, 19.43. Found: C, 61.28; H, 3.95; N, 19.47.

(E)-N′-(2-hydroxybenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6j)

White powder, yield 88 %; mp: 242–243 °C. ¹H NMR (300 MHz, DMSO): 4.52 (s, 2H); 6.85–6.92 (m, 2H); 7.23–7.30 (m, 3H); 7.50–7.66 (m, 6H); 8.42 (s, 1H); 8.50 (s, 2H); 10.95 (s, 1H); 11.99 (s, 1H). ESI–MS: 331.1 (C₂₂H₁₉N₆O₂S, [M+H]⁺). Anal. Calcd for C₂₂H₁₈N₆O₂S: C, 61.38; H, 4.21; N, 19.52. Found: C, 61.57; H, 4.20; N, 19.46.

(E)-N′-(5-bromo-2-hydroxybenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6j)

White powder, yield 90 %; mp: 290–291 °C. ¹H NMR (300 MHz, DMSO): 4.50 (s, 2H); 6.86–6.91 (m, 2H); 7.25–7.30 (m, 2H); 7.53–7.67 (m, 6H); 8.46 (s, 1H); 8.51 (s, 2H); 10.97 (s, 1H); 11.97 (s, 1H). ESI–MS: 509.0 (C₂₂H₁₈BrN₆O₂S, [M+H]⁺). Anal. Calcd for C₂₂H₁₇BrN₆O₂S: C, 51.87; H, 3.36; N, 16.50. Found: C, 51.68; H, 3.37; N, 16.55.

(E)-N′-(3,5-dibromo-2-hydroxybenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6k)

White powder, yield 88 %; mp: 270–271 °C. ¹H NMR (300 MHz, DMSO): 4.53 (s, 2H); 6.83–6.87 (m, 2H); 7.26–7.30 (m, 2H); 7.55–7.65 (m, 5H); 8.43 (s, 1H); 8.52 (s, 2H); 10.99 (s, 1H); 12.01 (s, 1H). ESI–MS: 586.9 (C₂₂H₁₇Br₂N₆O₂S, [M+H]⁺). Anal. Calcd for C₂₂H₁₆Br₂N₆O₂S: C, 44.92; H, 2.74; N, 14.29. Found: C, 44.78; H, 2.73; N, 14.34.

(E)-N′-(3,4-dihydroxybenzylidene)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetohydrazide (6m)

White powder, yield 92 %; mp: 255–256 °C. ¹H NMR (300 MHz, DMSO): 3.41–3.45 (m, 2H); 4.54 (s, 2H); 6.77 (d, J = 4.83 Hz, 1H); 6.89 (t, J = 5.88 Hz, 1H); 7.16 (d, J = 10.44 Hz, 1H); 7.49–7.50 (m, 2H); 7.53–7.54 (m, 2H); 7.62–7.64 (m, 3H); 7.84 (s, 1H); 8.67–8.69 (m, 2H); 11.47 (s, 1H). ESI–MS: 447.1 (C₂₂H₁₉N₆O₃S, [M+H]⁺). Anal. Calcd for C₂₂H₁₈N₆O₃S: C, 59.18; H, 4.06; N, 18.82. Found: C, 59.01; H, 4.05; N, 18.77.

(E)-2-((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)-N′-((E)-3-phenylallylidene)acetohydrazide (6n)

White powder, yield 92 %; mp: 235–236 °C. ¹H NMR (300 MHz, DMSO): 4.45 (s, 2H); 6.93–7.10 (m, 2H); 7.29–7.33 (m, 3H); 7.36–7.39 (m, 2H); 7.51 (s, 2H); 7.61–7.62 (m, 5H); 7.83 (d, J = 5.88 Hz, 1H); 8.54 (s, 2H); 11.59 (s, 1H). ESI–MS: 441.1 (C₂₄H₂₁N₆OS, [M+H]⁺). Anal. Calcd for C₂₄H₂₀N₆OS: C, 65.44; H, 4.58; N, 19.08. Found: C, 65.59; H, 4.57; N, 19.14.