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. 2023 Jun 26;8(27):24573–24585. doi: 10.1021/acsomega.3c02797

Synthesis, Antifungal Activities, Molecular Docking and Molecular Dynamic Studies of Novel Quinoxaline-Triazole Compounds

Derya Osmaniye ‡,†,*, Nurnehir Baltacı Bozkurt §, Serkan Levent †,‡, Gamze Benli Yardımcı §, Begüm Nurpelin Sağlık †,‡, Yusuf Ozkay †,‡, Zafer Asım Kaplancıklı †
PMCID: PMC10339406  PMID: 37457491

Abstract

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Uncontrolled use of antifungal drugs affects the development of resistance to existing drugs. Azole antifungals constitute a large part of antifungal therapy. Therefore, there is a need for new azole antifungals. Within the scope of this study, 17 new triazole derivative compounds were synthesized. Structure determinations were clarified by spectroscopic analysis methods (1H-NMR, 13C-NMR, HRMS). In addition, structure matching was completed using two-dimensional NMR techniques, HSQC, HMBC and NOESY. The antifungal effects of the compounds were evaluated on Candida strains by means of in vitro method. Compound 5d showed activity against Candida glabrata with a MIC90 = 2 μg/mL. Compound 5d showed activity against Candida krusei with a MIC90 = 2 μg/mL. This activity value, which is higher than fluconazole, is promising. In addition, the biofilm inhibition percentages of the compounds were calculated. Molecular docking and molecular dynamics simulations performed with compound 5d are in harmony with activity studies.

1. Introduction

Invasive fungal infections (IFIs), especially in the last three decades, pose a serious global threat. Especially immunocompromised patient groups (those living with HIV-AIDS, cancer, organ transplant recipients and autoimmune patients) face this threat.1 It is estimated that the number of invasive fungal infections associated deaths is as high as 1.5 to 2 million worldwide each year.2 As these patients need to use this drug, it highlights the need to synthesize new classes of antimicrobial agents, particularly structurally diverse molecules with unique mechanism of action, high potency, less toxicity, and no or fewer side effects.3 In fact, fungal infections are easier to treat than bacterial and other infections. But unfortunately, resistance to drugs complicates the treatment process.4 Studies have shown that ergosterol biosynthesis is crucial for the survival of fungi, and many antifungal drug targets have been found based on these studies.5

As antifungal agents, azoles act through inhibition of fungal lanosterol 14-α-demethylases (CYP51), an enzyme required to catalyze the oxidative removal of 14-α-demethylases in sterol biosynthesis by fungi.6 Fluconazole is used in the treatment of fungal infections as a drug with high oral bioavailability and therapeutic index. To overcome some of the shortcomings when using these first generation agents, second generation analogues such as voriconazole, ketoconazole, albaconazole and posaconazole have been developed.7

Candida species are commensal organisms that can lead to infection ranging from minor to severe candidemia and invasive candidiasis.8 The most crucial and harmful quality of Candida species is their capacity to create biofilms, which makes finding effective treatments and battling disease more difficult.9 Biofilms are habitats in which microbial organisms are coated in a matrix of extracellular polymeric molecules (EPS). The biofilm manner of life is influenced by the matrix’s composition, characteristics, and dynamics.10 The protective shielding effect of biofilm is countered by the slow diffusion of therapeutic antifungals into its interior, which hinders the eradication of illness. Candida pathogenicity is mostly attributable to the release of biofilm development, and adherence to polymeric medical devices and/or host cells.9

Triazole is a quintuple aromatic ring system containing three nitrogen atoms. Many antifungal drugs are in their structure. It contributes to the activity by the bonds it forms with the HEM in the active site of the 14-α-demethylases enzyme. When the literature is examined, it has been proven by many studies that this ring has antifungal activity.11−19 There are literature studies showing the antifungal activities of not only 1,3,4-triazoles but also 1,2,4-triazoles and 1,2,3 triazoles.20−24

Molecular hybridization is one of the most frequently used methods to develop new compounds. Effective molecules can be reached with the synergistic effect created using two pharmacophore groups or two rings with known activity. In this study, besides the triazole ring, all the compounds also carry the quinoxaline ring system in common. It was thought that the quinoxaline ring would provide stability in the active site of the enzyme as an aromatic bulky group.

2. Results and Discussion

2.1. Chemistry

The synthesis scheme is presented in Scheme 1. A seven-step synthesis procedure was followed to obtain the target compounds. First, the methyl 3,4-diaminobenzoate and hexane-3,4-dione were refluxed for obtained methyl 2,3-diethylquinoxaline-6-carboxylate (compound 1). Quinoxaline ring closure reaction lasted for 5 h. Second, compound 1 was converted to hydrazide derivative (compound 2) by means of reflux system. Third, the thiourea derivative (compound 3) was obtained using ethyl isothiocyanate. Then, triazole ring (compound 4) was closed by using thiourea (compound 3) in basic medium. The one part of target compounds (5a–5l) was obtained by reaction between triazole derivate (compound 4) and appropriate 2-bromoacetophenone. Then, the anilines were acetylated for obtained compounds 6a–6f. The other one part of target compounds (7a–7f) was obtained by reaction between triazole derivate (compound 4) and obtained acetylated anilines (6a–6f).

Scheme 1. Synthesis Pathway for Obtained Compounds (5a–5k and 7a–7f).

Scheme 1

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The structures of the compounds obtained were established by spectroscopic methods, namely 1H-NMR, 13C-NMR, and HRMS (Table 1 and Supplementary Data). Thanks to the 2D NMR analyzes performed on the active compound (5d) (Figure 1), all hydrogen and carbons on the compound were matched. HSQC, HMBC, and NOESY were used as 2D NMR analysis technique.

Table 1. Molecular Structure of Obtained Compounds (5a–5k and 7a–7f).

compounds R1 R2 R3 R4
5a -H -H -CH3  
5b -H -H -OCH3  
5c -H -H -CN  
5d -H -H -NO2  
5e -H -H -F  
5f -H -H -Cl  
5g -H -H -Br  
5h -CH3 -H -CH3  
5i -F -H -F  
5j -Cl -H -Cl  
5k -H -Cl -Cl  
7a       -H
7b       -CH3
7c       -OCH3
7d       -F
7e       -Cl
7f       -CF3
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Figure 1.

Figure 1

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Molecular structure of compound 5d.

Using the HSQC technique, hydrogen NMR and carbon NMR are compared. And in this way, the interactions of hydrogen with the carbon to which it is directly bonded can be detected. Using this technique, it was determined that the carbons at the value of 124.4 ppm were the carbons in the p3 position. The carbon that comes in at 130.4 ppm belongs to the p2 carbons (Figure 2). Using the NOESY technique, where the 130.4 carbon is p2, it was determined by the interaction of the hydrogens of this carbon with the methylene group hydrogens (Figure 3). Again, using the HSQC technique, the value of 128.4 ppm is q5; the value of 129.0 is q8; and the value of 129.7 was determined to be q7. With the HMBC technique, the value of 127.7 is q4a; q8a of 140.3; p4 of 140.5; the value of 141.0 is q6; t5 of the value 150.5; p1 of the value 150.6; It was determined that the t2 value of 154.6 and the values of 158.9 and 159.1 belong to the q2 and q3 positions (Figure 4).

Figure 2.

Figure 2

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HSQC analysis of compound 5d.

Figure 3.

Figure 3

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HMBC analysis of compound 5d.

Figure 4.

Figure 4

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NOESY analysis of compound 5d.

2.2. Anticandidal Activity

2.2.1. Minimum Inhibitory Concentration Values

The MIC values of the compounds against Candida spp. range from 2 to 128 μg/mL, as shown in Table 2. Microbial growth was unaffected by the solvents (DMSO and distilled water) utilized to prepare the chemical stock solutions. Among the compounds used in the study, the strongest MIC effect was seen in the 5d component for four Candida stains. Compound 5d was outperformed fluconazole as a control for the Candida krusei strain in terms of antifungal activity. The obtained results are presented in Table 2.

Table 2. Anticandidal Activity of Synthesized Compounds (5a–5k and 7a–7f) and Standard Drug (Fluconazole) (MIC90: μg/mL).
comp. C. albicans ATCC 10231 C. glabrata ATCC 2951 C. tropicalis ATCC 750 C. krusei ATCC 34135
5a 64 64 64 64
5b 64 64 64 64
5c 128 128 128 128
5d 4 2 4 2
5e 64 128 64 128
5f 64 64 64 64
5g 64 64 128 128
5h 64 64 64 64
5i 64 128 64 128
5j 32 64 64 128
5k 32 64 128 128
7a 64 64 64 64
7b 64 128 64 128
7c 32 64 64 128
7d 32 128 32 64
7e 64 64 64 64
7f 64 128 64 128
fluconazole 0.5 2 4 16
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Compound 5d showed activity against Candida albicans with a value of MIC90 = 4 μg/mL. Fluconazole, the reference drug against the same Candida species, showed activity with a MIC90 = 0.5 μg/mL. Compound 5d showed activity against Candida glabrata with a value of MIC90 = 2 μg/mL. Fluconazole, the reference drug against the same Candida species, showed activity with a value of MIC90 = 2 μg/mL. In other words, it showed the same activity as fluconazole against C. glabrata. Compound 5d showed activity against Candida tropicalis with a value of MIC90 = 4 μg/mL. Fluconazole, the reference drug against the same Candida species, showed activity with MIC90 = 4 μg/mL. Compound 5d showed activity against C. krusei with a value of MIC90 = 2 μg/mL. Fluconazole, the reference drug against the same Candida species, showed activity with a MIC90 = 16 μg/mL. That is, compound 5d showed 8 times more activity compared to the reference drug.

When the structure of the compounds is examined, the compound contains an amide group in the 7a–7f structure. Compounds 5a–5j contain a ketone group. This difference did not make a significant difference in terms of activity. When the structure of the 5d (active compound) is examined, the nitro substituent in the fourth position is remarkable. The inclusion of this substituent in the structure significantly increased the activity. The interaction of this group with the enzyme active site was investigated by in silico methods and presented in Sections 2.3 and 2.4.

2.2.2. Antibiofilm Activity against Candida Species

Structured microbial populations that are connected to a surface and enclosed in an extracellular matrix that they have formed themselves are known as biofilms. A crucial aspect of infection is that Candida spp. can attach to and create biofilms on biological surfaces and implanted medical devices. Compared to planktonic cells, microbial biofilms are considerably more resistant to several antimicrobial treatments because they serve as protective reservoirs for the bacteria.25 In the present study, we evaluated the ability of the synthesized compounds to reduce the formation of fungal mature biofilms.

Candida isolates were exposed to three different dilutions (2 × MIC, MIC, and 1/2 × MIC) of each the compounds to assess its antibiofilm activity using the crystal violet method. The highest biofilm inhibition rates were determined at 2 × MIC values for all compounds. The compound 5d with the lowest MIC value showed the highest biofilm inhibition rate on C. tropicalis at 70%–77%. Biofilm percent inhibitions results are presented in Table 3. In our study, it was found that as the MIC value decreased, the percentage of mature biofilm inhibition also decreased. When the mature biofilm inhibition percentages of the Candida strains of the synthesized compounds were compared, it was determined that they showed the lowest biofilm inhibition effect on C. glabrata. The outcomes demonstrated a dose-dependent relationship between the compounds and antibiofilm activity.

Table 3. Biofilm Percent Inhibitions Results of Synthesized Compounds (5a-5 K and 7a-7f) and Standart Drug (Fluconazole).
  C. albicans ATCC 10231
 C. glabrata ATCC 2951
 C. tropicalis ATCC 750
 C. krusei ATCC 34135
comp. 2 × MIC MIC 1/2 × MIC 2 × MIC MIC 1/2 × MIC 2 × MIC MIC 1/2 × MIC 2 × MIC MIC 1/2 × MIC
5a 66 61 47 30 29 19 71 74 64 79 73 69
5b 58 52 30 41 30 21 72 70 66 75 66 64
5c 59 55 40 33 29 25 70 71 67 77 69 67
5d 61 50 36 33 26 14 72 70 68 77 74 64
5e 63 43 29 33 27 22 77 74 70 74 70 66
5f 56 36 25 29 25 15 73 72 67 77 71 65
5g 61 48 36 36 31 17 72 71 67 72 64 62
5h 61 50 36 33 26 14 72 70 68 77 74 64
5i 63 45 39 38 34 22 73 73 71 71 65 61
5j 61 41 28 37 29 22 70 70 71 77 79 78
5k 57 46 38 40 22 25 72 68 67 75 72 68
7a 50 51 28 27 26 21 70 68 70 73 70 68
7b 49 43 41 25 29 26 69 64 64 70 68 63
7c 43 41 38 44 37 29 71 66 61 77 79 77
7d 46 40 33 37 38 26 71 69 69 69 66 61
7e 50 45 30 43 31 20 71 68 67 65 63 61
7f 59 44 30 45 39 38 69 68 67 73 69 66
fluconazole 67 64 62 84 80 79 77 76 70 78 76 60
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2.3. Molecular Docking

For docking studies, it was carried out with the most active derivative obtained because of in vitro activity. Docking studies were performed both against the 14-α-demethylases (CYP51) (1EA1).26 Docking studies were carried out using compound 5d with anticandidal activity values below MIC90 = 2 μg/ml.

When Figure 5 is examined, two-dimensional interactions of compound 5d in the enzyme active site are seen. Here, it is seen that the NO2 group makes a cation−π bond in addition to the triple salt bridge with HEM460 in the enzyme active site. This interaction clearly revealed the contribution of the NO2 group to the activity. In addition, the phenyl ring also establishes a π–π interaction with HEM460. The triazole ring and the phenyl ring formed a cation−π bond with the amino group of Arg96. Finally, the quinoxaline ring formed a π–π interaction with the phenyl group of Phe78.

Figure 5.

Figure 5

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2D interaction of compound 5d at binding region (PDB ID: 1EA1).

When Figure 6 is examined, the three-dimensional interaction of compound 5d with the enzyme active site will be seen. In addition to the interactions displayed with the two-dimensional pose, aromatic hydrogen bonds are seen in this pose. The carbonyl group of compound 5d formed three aromatic hydrogen bonds with the phenyl groups of Phe255 and Phe83.

Figure 6.

Figure 6

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3D interaction of compound 5d at binding region (PDB ID: 1EA1).

2.4. Molecular Dynamics Simulation

Docking studies do not give a clear answer about the dynamic behavior and stability of proteins and protein-ligand complexes. For this purpose, the MD simulation method is often used. In the present study, to estimate the stability of the docking complex made between the promising molecule (5d) and 14-α-demethylases (CYP51) (1EA1), were taken into consideration for the 100 ns MD simulation study in an explicit hydration environment.

In Figure 7, the results are for the compound 5d-14-α-demethylases enzyme complex. The RMSD and RMSF parameters are used to measure the stability of the model created during the simulation period. Figure 7a presents the RMSD parameter plot. In the RMSD graph we obtained, it ranged from 1.25 to 2.25 Å and was fixed here. Since these values are between 1–3 Å, it would be correct to say that our complex maintains its stability. It is seen that the stability shows a slight fluctuation at 40 ns. However, after a short time after this fluctuation, it becomes stable again. Re-establishment of stability may be due to bonds with Leu100, His101 and Ser252. A similar fluctuation is observed at 682 ns. But this lasts for such a short time that it is negligible. It would be correct to evaluate the reason for the fluctuation here as the breaking of the bonds made with the iron of HEM460. The iron in the middle of HEM460 forms either a salt bridge with a nitro group, a cation−π bond with a triazole, or a cation−π bond with a phenyl ring. None of these bonds are present at 682 ns. However, as of 683 ns, these bonds continue, and stability is provided. The stability established in 880 ns continues until the end of its dynamic studies. This stabilization is thought to be the result of hydrophobic interactions with Phe255 (Figure 7c).

Figure 7.

Figure 7

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MD simulation analysis of compound 5d in complex 14-α-demethylases enzyme (PDB ID: 1EA1). (A) RMSD (Protein RMSD is shown in gray while RMSD of compound 5d are shown in red). (B) Protein RMSF. (C) Amino acid interaction histogram. (D) 2D interaction diagram and (E) protein–ligand contact analysis of MD trajectory.

It is known that individual amino acids play a very important role in the stability of the protein-ligand complex during the MD simulation study. The RMSF parameter is important for stability and is used to interpret the conformational changes of individual residues (Figure 7b). α-Helix regions are red in the RMSF plot; β-banded regions are represented by a blue background; the white background represents the loop region. The contribution of contacting residues between each protein chain and ligand is indicated by vertical green lines on the x axis of the plot.27−29

As per RMSF plot, compound 5d contacted 21 amino acids of 14-α-demethylases protein, namely, Gln72 (0.81 Å), Ala75 (0.65 Å), Phe78 (0.64 Å), Met79 (0.77 Å), Phe83 (0.89 Å), Arg96 (2.09 Å), Glu98 (2.58 Å), Met99 (1.73 Å), His101 (1.88 Å), Ser252 (0.63 Å), Phe255 (0.57 Å), Ala256 (0.77 Å), His259 (0.76 Å), Pro320 (0.54 Å), Leu321 (0.81 Å), Ile322 (0.71 Å), Ile323 (0.51 Å), Leu324 (0.49 Å), Cys394 (0.55 Å), Met433 (0.59 Å), Val434 (0.72 Å).

By watching the MD simulation video, aromatic hydrogen bonds were determined for 100 ns seconds. Accordingly, the aromatic hydrogen bonds formed can be listed as follows. Between the carbonyl group of compound 5d and the phenyl group of Phe83; between NO2 group of compound 5d and imidazole group of Hid259; between the phenyl group of compound 5d and the Ala256 carbonyl; between the phenyl group of compound 5d and the Phe255 carbonyl; between the phenyl group of compound 5d and the carbonyl group of Hid259 and between the carbonyl group of compound 5d and the phenyl group of Phe255.

3. Conclusions

Among the antifungal drug groups, azole group antifungals have a very important place. This group of drugs is frequently used in the clinic. However, resistance to the drug, especially in long-term treatments, keeps the need for new azole antifungals constantly. Azole antifungals inhibit 14-α-demethylases enzyme and inhibit ergosterol synthesis necessary for fungal cell wall construction. The azole group heterocyclic ring is essential for its interaction with hemoglobin in the enzyme active site. This is exactly the purpose of the synthesis of new triazole derivatives within the scope of this study. In this study, 17 new triazole derivatives were synthesized. Structure determinations of the compounds were made using 1H-NMR, 13C-NMR and HRMS techniques. In addition, structure determination was validated for compound 5d using the 2D-NMR technique. In addition to the antifungal activities of the compounds, the percent biofilm inhibition values were tested on Candida strains by in vitro methods. As a result of activity studies, compound 5d exhibited a similar antifungal potential with fluconazole. It was even 8 times more active on C. krusei than fluconazole. This promising result has moved the study forward. The obtained in silico studies also confirm the activity studies. Molecular docking and then molecular dynamics studies were carried out as in silico studies. Dynamic studies showed that the interaction with hemoglobin iron, which is necessary for activity, is carried out from three points of our molecule. These structures are nitro substituent, triazole ring and phenyl ring. Especially the nitro group and the triazole ring showed a significant interaction rate. Therefore, the importance of both the triazole ring and the nitro substituent for the activity is clear. The bulky quinoxaline ring showed a continuous and strong interaction with the Tyr76 amino acid for 100 ns. Although this interaction may not be of primary importance for activity, it is thought to play an important role in maintaining the stability of the compound. In future studies, it is planned to develop the compound by keeping these structures constant.

4. Experimental Section

4.1. Chemistry

Purchase processes and analysis methods of all commercial preparations were carried out as previously reported.29

4.1.1. Synthesis of Methyl 2,3-Diethylquinoxaline-6-carboxylate (1)

Methyl 3,4-diaminobenzoate (6 g, 0.036 mol) and hexane-3,4-dione (4.104 g, 0.036 mol) were refluxed in EtOH (30 mL). At the end of the reaction, the precipitated product was filtered, washed with cooled EtOH, dried, and crystallized from ethanol.

4.1.2. Synthesis of 2,3-Diethylquinoxaline-6-carbohydrazide (2)

Methyl 2,3-diethylquinoxaline-6-carboxylate (7.9056 g, 0.032 mol) was dissolved in absolute EtOH. Then the excess of hydrazine hydrate dissolved in EtOH (absolute) at different vessel. The hydrazine hydrate solution was added in reaction medium as portions. After the add process is complete, the reaction mixture was refluxed at 2 h. At the end of the reaction, the precipitated product was filtered, washed with cooled EtOH, dried, and crystallized from ethanol.

4.1.3. Synthesis of 2-(2,3-Diethylquinoxaline-6-carbonyl)-N-ethylhydrazine-1-carbothioamide (3)

2,3-Diethylquinoxaline-6-carbohydrazide (6.6368 g, 0.027 mol) and isothiocyanatoethane (2.3528 g, 0.027 mol) were dissolved in absolute EtOH. Then, the reaction mixture was refluxed at 5 h. At the end of the reaction, the precipitated product was filtered, washed with cooled EtOH, dried, and crystallized from ethanol.

4.1.4. Synthesis of 5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazole-3-thiol (4)

2-(2,3-Diethylquinoxaline-6-carbonyl)-N-ethylhydrazine-1-carbothioamide (7.1591 g, 0.022 mol) was dissolved in absolute EtOH in presence of NaOH. Then, the reaction mixture was refluxed at 5 h. At the end of the reaction, the reaction mixture was poured in iced water. The pH of the reaction content was adjusted to pH = 2. For this adjustment, 20% HCl was used as the acidic agent. Then, the precipitated product was filtered, washed with distilled water, dried, and crystallized from ethanol.

4.1.5. Synthesis of the Target Compounds (5a–5k)

5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazole-3-thiol (0.3 g, 0.001 mol) and appropriate 2-brmoacetophenone (0.002 mol) were stirred in acetone (20 mL) in presence of potassium carbonate for 8 h. At the end of the reaction, the acetone was evaporated. The residue product washed with distilled water, dried, and crystallized from ethanol.

4.1.5.1. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-1-(p-tolyl)ethan-1-one (5a)

Yield: 88%, cream. M.P.: 161.3–162.7 °C. IR (cm –1 band): 2976 (C-H), 1668 (C=O), 817. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 2.40 (3H, s, -CH3), 3.02–3.10 (4H, m, -CH2-), 4.15 (2H, q, J = 7.3 Hz, -CH2-), 4.99 (2H, s, -CH2-), 7.37 (2H, d, J = 7.9 Hz, 1,4-disubstituted benzene), 7.94 (2H, d, J = 8.1 Hz, 1,4-disubstituted benzene), 7.99 (1H, d, J = 1.9 Hz, quinoxalin), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.21 (1H, d, J = 1.8 Hz, quinoxalin). 13C-NMR (75 MHz, DMSO-d6): δ =11.96, 12.01, 15.48, 21.70, 27.86, 40.34, 41.23, 127.81, 128.22, 129.01, 129.04, 129.65, 129.85, 133.25, 140.32, 141.02, 144.81, 150.80, 154.52, 158.95, 159.11, 193.22. HRMS (m/z): [M + H]+ calcd for C25H27N5OS: 446.2009; found: 446.1991.

4.1.5.2. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-1-(4-methoxyphenyl)ethan-1-one (5b)

Yield: 90%, cream. M.P.: 142.9–144.0 °C. IR (cm –1 band): 2972 (C–H), 1660 (C=O), 817. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.39 (6H, m, -CH3), 3.03–3.11 (4H, m, -CH2-), 3.87 (3H, s, -OCH3), 4.16 (2H, q, J = 7.1 Hz, -CH2-), 4.97 (2H, s, -CH2-), 7.09 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.03 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.21 (1H, d, J = 1.8 Hz, quinoxalin). 13C-NMR (75 MHz, DMSO-d6): δ =11.95, 12.01, 27.85, 40.35, 56.14, 114.52, 127.82, 128.20, 128.59, 128.99, 129.65, 131.35, 140.32, 141.02, 150.88, 154.50, 158.95, 159.10, 164.06, 192.03. HRMS (m/z): [M + H]+ calcd for C25H27N5O2S: 462.1958; found: 462.1944.

4.1.5.3. 4-(2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)acetyl)benzonitrile (5c)

Yield: 85%, beige. M.P.: 153.1–154.6 °C. IR (cm –1 band): 2981 (C–H), 2227 (-CN), 1680 (C=O), 829. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.11 (4H, m, -CH2-), 4.16 (2H, q, J = 7.1 Hz, -CH2-), 5.06 (2H, s, -CH2-), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.06 (2H, d, J = 8.6 Hz, 1,4-disubstituted benzene), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.19–8.22 (3H, m, quinoxalin+1,4-disubstituted benzene). 13C-NMR (75 MHz, DMSO-d6): δ =11.97, 12.03, 15.45, 27.86, 40.40, 41.14, 116.04, 118.58, 127.76, 128.26, 129.00, 129.55, 129.67, 133.35, 139.08, 140.33, 141.04, 150.53, 154.61, 158.98, 159.15, 193.37. HRMS (m/z): [M + H]+ calcd for C25H24N6OS: 457.1805; found: 457.1800.

4.1.5.4. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-1-(4-nitrophenyl)ethan-1-one (5d)

Yield: 79%, brown. M.P.: 144.0–145.9 °C. IR (cm –1 band): 2970 (C–H), 1681 (C=O), 1527 (NO2), 1342 (NO2), 854.1H-NMR (300 MHz, DMSO-d6): δ = 1.28 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.10 (4H, m, -CH2-), 4.16 (2H, q, J = 7.5 Hz, -CH2-), 5.09 (2H, s, -CH2-), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.7 Hz, quinoxalin), 8.13 (1H, d, J = 8.7 Hz, quinoxalin), 8.21 (1H, d, J = 1.8 Hz, quinoxalin), 8.29 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 8.38 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene). 13C-NMR (75 MHz, DMSO-d6): δ =11.96, 12.02, 15.46, 27.85, 41.32, 124.39, 127.74, 128.27, 129.00, 129.67, 130.38, 140.32, 140.51, 141.03, 150.52, 150.62, 154.63, 158.98, 159.14, 193.20. HRMS (m/z): [M + H]+ calcd for C24H24N6O3S: 477.1703; found: 477.1691.

4.1.5.5. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-1-(4-fluorophenyl)ethan-1-one (5e)

Yield: 77%, cream. M.P.: 150.4–151.8 °C. IR (cm –1 band): 2978 (C–H), 1670 (C=O), 835. 1H-NMR (300 MHz, DMSO-d6): δ = 1.28 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.11 (4H, m, -CH2-), 4.16 (2H, q, J = 7.1 Hz, -CH2-), 5.02 (2H, s, -CH2-), 7.37–7.44 (2H, m, 1,4-disubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.12–8.17 (3H, m, quinoxalin + 1,4-disubstituted benzene), 8.22 (1H, d, J = 1.8 Hz, quinoxalin). 13C-NMR (75 MHz, DMSO-d6): δ =11.96, 12.02, 15.47, 27.85, 40.37, 41.11, 116.39 (d, J = 21.7 Hz), 127.79, 128.24, 129.00, 129.66, 132.02 (d, J = 9.4 Hz), 132.53, 140.32, 141.03, 150.73, 154.55, 158.97, 159.12, 165.79 (d, J = 250.8 Hz), 192.37. HRMS (m/z): [M + H]+ calcd for C24H24N5OFS: 450.1758; found: 450.1742.

4.1.5.6. 1-(4-Chlorophenyl)-2-((5-(2,3-diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one (5f)

Yield: 80%, cream. M.P.: 184.5–185.9 °C. IR (cm –1 band): 2978 (C–H), 1670 (C=O), 827. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.10 (4H, m, -CH2-), 4.16 (2H, q, J = 7.1 Hz, -CH2-), 5.02 (2H, s, -CH2-), 7.65 (2H, d, J = 8.6 Hz, 1,4-disubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.07 (2H, d, J = 8.6 Hz, 1,4-disubstituted benzene), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.22 (1H, d, J = 1.8 Hz, quinoxalin). 13C-NMR (75 MHz, DMSO-d6): δ =11.97, 12.02, 15.47, 27.87, 40.24, 41.09, 128.22, 128.26, 129.01, 129.44, 129.67, 130.86, 130.87, 140.33, 141.10, 146.69, 150.66, 154.65, 156.11, 159.18, 192.87. HRMS (m/z): [M + H]+ calcd for C24H24N5OSCl: 466.1463; found: 466.1458.

4.1.5.7. 1-(4-Bromophenyl)-2-((5-(2,3-diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one (5g)

Yield: 81%, pink. M.P.: 132.3–134.1 °C. IR (cm –1 band): 2974 (C–H), 1670 (C=O), 823. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.10 (4H, m, -CH2-), 4.16 (2H, q, J = 7.0 Hz, -CH2-), 5.03 (2H, s, -CH2-), 7.79 (2H, d, J = 8.6 Hz, 1,4-disubstituted benzene), 7.98–8.01 (3H, m, quinoxalin + 1,4-disubstituted benzene), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.22 (1H, d, J = 1.8 Hz, quinoxalin). 13C-NMR (75 MHz, DMSO-d6): δ =11.08, 12.01, 15.43, 27.91, 39.09, 40.28, 41.14, 128.43, 128.98, 129.89, 130.91, 131.23, 131.96, 132.40, 133.55, 134.83, 140.28, 141.12, 150.85, 154.53, 159.26, 192.85. HRMS (m/z): [M + H]+ calcd for C24H24N5OSBr: 510.0958; found: 510.0952.

4.1.5.8. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-1-(2,4-dimethylphenyl)ethan-1-one (5h)

Yield: 87%, orange. M.P.: 122.5–123.8 °C. IR (cm –1 band): 2976 (C–H), 1674 (C=O), 839. 1H-NMR (300 MHz, DMSO-d6): δ = 1.26 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 2.33 (3H, s, -CH3), 2.39 (3H, s, -CH3), 3.03–3.10 (4H, m, -CH2-), 4.12 (2H, q, J = 7.0 Hz, -CH2-), 4.89 (2H, s, -CH2-), 7.15–7.18 (2H, m, 1,2,4-trisubstituted benzene), 7.86 (1H, d, J = 7.8 Hz, 1,2,4-trisubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.5 Hz, quinoxalin), 8.14 (1H, d, J = 8.7 Hz, quinoxalin), 8.20 (1H, d, J = 1.9 Hz, quinoxalin). 13C-NMR (75 MHz, DMSO-d6): δ = 11.97, 12.02, 15.44, 21.40, 27.85, 43.05, 126.89, 127.82, 128.21, 129.01, 129.66, 130.23, 132.95, 133.54, 138.67, 140.33, 141.02, 142.71, 150.86, 154.50, 158.97, 159.13, 196.35. HRMS (m/z): [M + H]+ calcd for C26H29N5OS: 460.2166; found: 460.2159.

4.1.5.9. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-1-(2,4-difluorophenyl)ethan-1-one (5i)

Yield: 77%, beige. M.P.: 130.3–131.7 °C. IR (cm –1 band): 2976 (C–H), 1678 (C=O), 821. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.11 (4H, m, -CH2-), 4.15 (2H, q, J = 6.8 Hz, -CH2-), 4.91 (2H, d, J = 2.5 Hz, -CH2-), 7.26–7.32 (1H, m, 1,2,4-trisubstituted benzene), 7.47–7.54 (1H, m, 1,2,4-trisubstituted benzene), 7.96–8.00 (1H, m, quinoxalin), 8.02–8.07 (1H, m, 1,2,4-trisubstituted benzene), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.22 (1H, d, J = 1.7 Hz, quinoxalin). 13C-NMR (75 MHz, DMSO-d6): δ =11.97, 12.03, 15.43, 27.85, 40.35, 44.27 (d, J = 7.8 Hz), 105.82 (t, J = 25.9 Hz), 113.07 (d, J = 22.3 Hz), 122.02 (d, J = 40.1 Hz), 127.77, 128.27, 129.03, 129.66, 133.43, 140.32, 141.03, 150.66, 154.58, 159.05 (d, J = 11.9 Hz), 165.79 (d, J = 251.8 Hz), 165.97 (d, J = 250.3 Hz), 190.30. HRMS (m/z): [M + H]+ calcd for C24H23N5OF2S: 468.1664; found: 468.1651.

4.1.5.10. 1-(2,4-Dichlorophenyl)-2-((5-(2,3-diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one (5j)

Yield: 79%, orange. M.P.: 65.9–66.8 °C. IR (cm –1 band): 2976 (C–H), 1685 (C=O), 813. 1H-NMR (300 MHz, DMSO-d6): δ = 1.24 (3H, t, J = 7.3 Hz, -CH3), 1.31–1.37 (6H, m, -CH3), 3.03–3.08 (4H, m, -CH2-), 4.11 (2H, q, J = 7.2 Hz, -CH2-), 4.87 (2H, s, -CH2-), 7.60 (1H, dd, J1 = 3.5 Hz, J2 = 8.4 Hz, 1,2,4-trisubstituted benzene), 7.76 (1H, d, J = 1.9 Hz, 1,2,4-trisubstituted benzene), 7.91 (1H, d, J = 8.4 Hz, 1,2,4-trisubstituted benzene), 7.97 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.12 (1H, d, J = 8.6 Hz, quinoxalin), 8.20 (1H, d, J = 1.8 Hz, quinoxalin). 13C-NMR (75 MHz, DMSO-d6): δ =11.97, 12.01, 15.39, 27.85, 27.87, 40.34, 43.05, 127.71, 128.07, 128.27, 128.99, 129.66, 130.57, 132.01, 132.09, 135.80, 137.28, 140.32, 141.04, 150.44, 154.62, 158.97, 159.14, 194.94. HRMS (m/z): [M + H]+ calcd for C24H23N5OSCl2: 500.1073; found: 500.1057.

4.1.5.11. 1-(3,4-Dichlorophenyl)-2-((5-(2,3-diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one (5k)

Yield: 83%, orange. M.P.: 75.0–76.9 °C. IR (cm –1 band): 2978 (C–H), 1670 (C=O), 835. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.3 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.11 (4H, m, -CH2-), 4.16 (2H, q, J = 7.4 Hz, -CH2-), 5.02 (2H, s, -CH2-), 7.86 (1H, d, J = 8.4 Hz, 1,3,4-trisubstituted benzene), 7.96–8.02 (3H, m, quinoxalin+1,3,4-trisubstituted benzene), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.22 (1H, d, J = 1.9 Hz, quinoxalin), 8.28 (1H, d, J = 1.9 Hz, 1,3,4-trisubstituted benzene). 13C-NMR (75 MHz, DMSO-d6): δ = 11.98, 12.02, 15.47, 27.86, 40.39, 40.99, 127.77, 128.25, 128.29, 128.99, 129.67, 130.89, 130.92, 131.68, 132.38, 136.01, 137.03, 140.33, 141.04, 150.49, 154.61, 159.16, 192.25. HRMS (m/z): [M + H]+ calcd for C24H23N5OSCl2: 500.1073; found: 500.1059.

4.1.6. Synthesis of Acetylated Anilines (6a–6f)

Aniline derivatives (0.009 mol) was dissolved in THF. Triethylamine was used as catalyst. The reaction mixture located in iced bath. The chloroacetyl chloride mixture in THF was added in reaction mixture as dropwise. At the end of the reaction, the THF was evaporated. The residue product washed with distilled water, dried, and crystallized from ethanol.

4.1.7. Synthesis of Target Compounds (7a–7f)

5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazole-3-thiol (0.3 g, 0.001 mol) and appropriate acetylated anilines (6a–6f) (0.002 mol) were stirred in acetone (20 mL) in presence of potassium carbonate for 8 h. At the end of the reaction, the acetone was evaporated. The residue product washed with distilled water, dried, and crystallized from ethanol.

4.1.7.1. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-N-phenylacetamide (7a)

Yield: 81%, white. M.P.: 124.2–125.5 °C. IR (cm –1 band): 3481 (N–H), 2981 (C–H), 1681 (C=O), 758, 694. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.10 (4H, m, -CH2-), 4.16 (2H, q, J = 7.2 Hz, -CH2-), 4.24 (2H, s, -CH2-), 7.04–7.09 (1H, m, monosubstituted benzene), 7.29–7.35 (2H, m, monosubstituted benzene), 7.57–7.60 (2H, m, monosubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.22 (1H, d, J = 1.8 Hz, quinoxalin), 10.38 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =11.97, 12.01, 15.49, 27.85, 27.87, 40.35, 119.56, 124.03, 127.80, 128.28, 129.02, 129.31, 129.65, 139.26, 140.32, 141.03, 150.92, 154.62, 158.97, 159.14, 166.10. HRMS (m/z): [M + H]+ calcd for C24H26N6OS: 447.1962; found: 447.1952.

4.1.7.2. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-N-(p-tolyl)acetamide (7b)

Yield: 86%, cream. M.P.: 92.9–94.1 °C. IR (cm –1 band): 3257 (N-H), 2987 (C–H), 1687 (C=O), 850. 1H-NMR (300 MHz, DMSO-d6): δ = 1.26 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 2.25 (3H, s, -CH3), 3.03–3.10 (4H, m, -CH2-), 4.15 (2H, q, J = 6.9 Hz, -CH2-), 4.22 (2H, s, -CH2-), 7.12 (2H, d, J = 8.3 Hz, 1,4-disubstituted benzene), 7.47 (2H, d, J = 8.5 Hz, 1,4-disubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.14 (1H, d, J = 8.5 Hz, quinoxalin), 8.21 (1H, d, J = 1.9 Hz, quinoxalin), 10.29 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =11.97, 12.02, 15.48, 27.85, 27.88, 37.89, 119.47, 126.67, 126.72, 127.75, 128.28, 129.03, 129.66, 140.31, 141.03, 142.88, 150.83, 154.66, 158.99, 159.16, 166.87. HRMS (m/z): [M + H]+ calcd for C25H28N6OS: 461.2118; found: 461.2105.

4.1.7.3. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-N-(4-methoxyphenyl)acetamide (7c)

Yield: 75%, white. M.P.: 149.3–151.1 °C. IR (cm –1 band): 3496 (N–H), 2976 (C–H), 1678 (C=O), 840. 1H-NMR (300 MHz, DMSO-d6): δ = 1.26 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.10 (4H, m, -CH2-), 3.71 (3H, s, -OCH3), 4.14–4.16 (2H, m, -CH2-), 4.21 (2H, s, -CH2-), 6.89 (2H, d, J = 9.1 Hz, 1,4-disubstituted benzene), 7.49 (2H, d, J = 9.1 Hz, 1,4-disubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.21 (1H, d, J = 1.8 Hz, quinoxalin), 10.24 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =11.96, 12.01, 15.50, 27.87, 37.94, 40.33, 55.63, 114.39, 121.09, 127.80, 128.27, 129.03, 129.65, 132.39, 140.31, 141.03, 150.94, 154.61, 155.83, 158.98, 159.14, 165.55. HRMS (m/z): [M + H]+ calcd for C25H28N6O2S: 477.2067; found: 477.2066.

4.1.7.4. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-N-(4-fluorophenyl)acetamide (7d)

Yield: 74%, white. M.P.: 118.1–119.7 °C. IR (cm –1 band): 3485 (N–H), 2985 (C–H), 1681 (C=O), 902. 1H-NMR (300 MHz, DMSO-d6): δ = 1.26 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.11 (4H, m, -CH2-), 4.16 (2H, q, J = 7.1 Hz, -CH2-), 4.30 (2H, s, -CH2-), 7.15–7.18 (2H, m, 1,4-disubstituted benzene), 7.23–7.28 (1H, m, 1,4-disubstituted benzene), 7.89–7.93 (1H, m, 1,4-disubstituted benzene), 7.99 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.15 (1H, d, J = 8.6 Hz, quinoxalin), 8.22 (1H, d, J = 1.9 Hz, quinoxalin), 10.21 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =11.96, 12.01, 15.50, 27.85, 37.56, 40.22, 115.89, 124.12, 124.94, 125.97, 127.80, 128.28, 129.03, 129.67, 140.32, 141.04, 150.89, 154.63, 158.99, 159.16, 166.79. HRMS (m/z): [M + H]+ calcd for C24H25N6OFS: 465.1867; found: 465.1860.

4.1.7.5. N-(4-Chlorophenyl)-2-((5-(2,3-diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)acetamide (7e)

Yield: 81%, white. M.P.: 177.4–179.1 °C. IR (cm –1 band): 3506 (N–H), 2978 (C–H), 1680 (C=O), 827. 1H-NMR (300 MHz, DMSO-d6): δ = 1.26 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.10 (4H, m, -CH2-), 4.15 (2H, q, J = 7.3 Hz, -CH2-), 4.24 (2H, s, -CH2-), 7.38 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.62 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.14 (1H, d, J = 8.6 Hz, quinoxalin), 8.21 (1H, d, J = 1.9 Hz, quinoxalin), 10.53 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =11.97, 12.02, 15.48, 27.85, 37.89, 40.36, 121.09, 127.55, 127.77, 128.29, 129.03, 129.25, 129.66, 138.24, 140.31, 141.03, 150.86, 154.64, 158.98, 159.15, 166.31. HRMS (m/z): [M + H]+ calcd for C24H25N6OSCl: 481.1572; found: 481.1565.

4.1.7.6. 2-((5-(2,3-Diethylquinoxalin-6-yl)-4-ethyl-4H-1,2,4-triazol-3-yl)thio)-N-(4-(trifluoromethyl)phenyl)acetamide (7f)

Yield: 85%, white. M.P.: 175.5–176.7 °C. IR (cm –1 band): 3500 (N–H), 2978 (C–H), 1681 (C=O), 823. 1H-NMR (300 MHz, DMSO-d6): δ = 1.27 (3H, t, J = 7.2 Hz, -CH3), 1.33–1.38 (6H, m, -CH3), 3.03–3.10 (4H, m, -CH2-), 4.15 (2H, q, J = 7.1 Hz, -CH2-), 4.29 (2H, s, -CH2-), 7.69 (2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.80 (2H, d, J = 8.4 Hz, 1,4-disubstituted benzene), 7.98 (1H, dd, J1 = 1.9 Hz, J2 = 8.6 Hz, quinoxalin), 8.13 (1H, d, J = 8.7 Hz, quinoxalin), 8.21 (1H, d, J = 1.6 Hz, quinoxalin), 10.76 (1H, s, -NH). 13C-NMR (75 MHz, DMSO-d6): δ =11.97, 12.02, 15.49, 20.89, 27.87, 37.99, 40.49, 119.54, 127.79, 128.26, 128.30, 129.03, 129.62, 129.68, 132.96, 136.77, 140.31, 141.03, 150.93, 154.61, 158.98, 159.15, 165.83. HRMS (m/z): [M + H]+ calcd for C25H25N6OF3S: 515.1835; found: 515.1815.

4.2. Activity Studies

4.2.1. Strains and Chemicals

Candida strains used in this study are reference strains, respectively C. albicans ATCC 10231, C. glabrata MYA 2951, C. tropicalis ATCC 750, C. krusei ATCC 34135. All strains were stored as 15% (v/v) glycerol stocks at −80 °C. The frozen stocks were subcultured on Sabouraud dextrose agar (SDA, Oxoid, Basingstoke, UK) and incubated at 37 °C for 24 h. Fresh cultures were made in RPMI-1640 (HiMedia, India) and incubated at 37 C for 24 h. Drug solutions were prepared by dissolving fluconazole (Abcr, Germany) in sterile distilled water with a final concentration of 128–0.125 μg/mL and by dissolving the synthesized compounds in dimethyl sulfoxide (DMSO, Sigma, USA) with a final concentration of 1024–1 μg/mL.

4.2.2. Determination of the Minimum Inhibitory Concentration

The antifungal activity of the synthesized compounds was examined using 96-well microtiter plates and the broth microdilution method from Clinical and Laboratory Standards Institute (CLSI) document M27-A3. Briefly, the inoculum size was adjusted to 0.5–2.5 × 103Candida cells/mL using RPMI1640 media (pH: 7.0) supplemented with 2% glucose. The microplates were incubated at 37 °C for 24–48 h. The first well with total growth inhibition was referred to as the minimum inhibitory concentration (MIC).8

4.2.3. Effects on Mature Biofilm Formation

Antibiofilm activity of the synthesized compounds were tested. To form mature biofilm formation, sterile 96-well flat-bottomed microplate was seeded with standard inoculum (0.5 McFarland) of Candida reference strains (100 μL each well), followed by incubation for 24 h at 37 °C. After the incubation, unattached cells were removed by washing the microplates three times in phosphate-buffered saline (PBS). Based on their capacity to produce biofilms, synthesized compounds were tested at sub-inhibitory concentrations of 1/2 × MIC, MIC, and 2 × MIC. The compounds were added to each well and incubated for 24 h at 37 °C. Incubated overnight, the microplates were washed three times with PBS and 100 μL of 0.1% crystal violet was added to each well. The microplates were washed three times with PBS after 5 min at room temperature. Then, 150 μL of 0.04 N HCl-isopropanol and 50 μL of 0.25% sodium dodecyl sulfate (SDS) were added to each well. A microplate reader was used to read the absorbance values at 590 nm. Untreated biofilm served as the growth control. The values of the experimental group and growth control were used to determine the percentage of inhibition. Biofilm inhibition rate = (OD control – OD sample/OD control) × 100.30

4.3. Molecular Docking

X-ray crystal structures of the human 14-α-demethylases (PDB ID: 1EA1)26 were retrieved from the Protein Data Bank server (www.pdb.org, accessed 01 May 2021). Docking procedures were performed using standard procedure and same program interfaces as previously reported by our team.29,31−33

4.4. Molecular Dynamics Simulation

Molecular dynamics (MD) simulations, which are considered an important computational tool to evaluate the time-dependent stability of a ligand at an active site for a drug-receptor complex, were performed for compound 5d within the scope of this study.34 Dynamic procedures were performed using same program interfaces as previously reported by our team.35−43

Acknowledgments

As the authors of this study, we thank Anadolu University Faculty of Pharmacy Central Research Laboratory (MERLAB), for their support and contributions.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c02797.

  • Figure S1. Compound 5a IR spectrum. Figure S2. Compound 5a1H-NMR spectrum. Figure S3. Compound 5a13C-NMR spectrum. Figure S4. Compound 5a HRMS report. Figure S5. Compound 5b IR spectrum. Figure S6. Compound 5b1H-NMR spectrum. Figure S7. Compound 5b13C-NMR spectrum. Figure S8. Compound 5b HRMS report. Figure S9. Compound 5c IR spectrum. Figure S10. Compound 5c1H-NMR spectrum. Figure S11. Compound 5c13C-NMR spectrum. Figure S12. Compound 5c HRMS report. Figure S13. Compound 5d IR spectrum. Figure S14. Compound 5d1H-NMR spectrum. Figure S15. Compound 5d13C-NMR spectrum. Figure S16. Compound 5d HRMS report. Figure S17. Compound 5e IR spectrum. Figure S18. Compound 5e1H-NMR spectrum. Figure S19. Compound 5e13C-NMR spectrum. Figure S20. Compound 5e HRMS report. Figure S21. Compound 5f IR spectrum. Figure S22. Compound 5f1H-NMR spectrum. Figure S23. Compound 5f13C-NMR spectrum. Figure S24. Compound 5f HRMS report. Figure S25. Compound 5 g IR spectrum. Figure S26. Compound 5 g1H-NMR spectrum. Figure S27. Compound 5 g13C-NMR spectrum. Figure S28. Compound 5 g HRMS report. Figure S29. Compound 5 h IR spectrum. Figure S30. Compound 5 h1H-NMR spectrum. Figure S31. Compound 5 h13C-NMR spectrum. Figure S32. Compound 5 h HRMS report. Figure S33. Compound 5i IR spectrum. Figure S34. Compound 5i1H-NMR spectrum. Figure S35. Compound 5i13C-NMR spectrum. Figure S36. Compound 5i HRMS report. Figure S37. Compound 5j IR spectrum. Figure S38. Compound 5j1H-NMR spectrum. Figure S39. Compound 5j13C-NMR spectrum. Figure S40. Compound 5j HRMS report. Figure S41. Compound 5 k IR spectrum. Figure S42. Compound 5 k1H-NMR spectrum. Figure S43. Compound 5 k13C-NMR spectrum. Figure S44. Compound 5 k HRMS report. Figure S45. Compound 7a IR spectrum. Figure S46. Compound 7a1H-NMR spectrum. Figure S47. Compound 7a13C-NMR spectrum. Figure S48. Compound 7a HRMS report. Figure S49. Compound 7b IR spectrum. Figure S50. Compound 7b1H-NMR spectrum. Figure S51. Compound 7b13C-NMR spectrum. Figure S52. Compound 7b HRMS report. Figure S53. Compound 7c IR spectrum. Figure S54. Compound 7c1H-NMR spectrum. Figure S55. Compound 7c13C-NMR spectrum. Figure S56. Compound 7c HRMS report. Figure S57. Compound 7d IR spectrum. Figure S58. Compound 7d1H-NMR spectrum. Figure S59. Compound 7d13C-NMR spectrum. Figure S60. Compound 7d HRMS report. Figure S61. Compound 7e IR spectrum. Figure S62. Compound 7e1H-NMR spectrum. Figure S63. Compound 7e13C-NMR spectrum. Figure S64. Compound 7e HRMS report. Figure S65. Compound 7f IR spectrum. Figure S66. Compound 7f1H-NMR spectrum. Figure S67. Compound 7f13C-NMR spectrum. Figure S68. Compound 7f HRMS report.

The authors declare no competing financial interest.

Supplementary Material

ao3c02797_si_001.pdf (6.9MB, pdf)

References

  1. Ni T.; Chi X.; Xie F.; Li L.; Wu H.; Hao Y.; Wang X.; Zhang D.; Jiang Y. Design, synthesis, and evaluation of novel tetrazoles featuring isoxazole moiety as highly selective antifungal agents. Eur. J. Med. Chem. 2023, 246, 115007 10.1016/j.ejmech.2022.115007. [DOI] [PubMed] [Google Scholar]
  2. Xie F.; Hao Y.; Bao J.; Liu J.; Liu Y.; Wang R.; Chi X.; Chai X.; Wang T.; Yu S.; Jin Y.; Yan L.; Zhang D.; Ni T. Design, synthesis, and in vitro evaluation of novel antifungal triazoles containing substituted 1, 2, 3-triazole-methoxyl side chains. Bioorg. Chem. 2022, 129, 106216 10.1016/j.bioorg.2022.106216. [DOI] [PubMed] [Google Scholar]
  3. Chahal M.; Kaushik C. P.; Luxmi R.; Kumar D.; Kumar A. Synthesis, antimicrobial, and antioxidant activities of disubstituted 1, 2, 3-triazoles with amide-hydroxyl functionality. Med. Chem. Res. 2023, 32, 85–98. 10.1007/s00044-022-02993-w. [DOI] [Google Scholar]
  4. Faazil S.; Malik M. S.; Ahmed S. A.; Alsantali R. I.; Yedla P.; Alsharif M. A.; Shaikh I. N.; Kamal A. Novel linezolid-based oxazolidinones as potent anticandidiasis and antitubercular agents. Bioorg. Chem. 2022, 126, 105869 10.1016/j.bioorg.2022.105869. [DOI] [PubMed] [Google Scholar]
  5. Guo M.-b.; Yan Z.-z.; Wang X.; Xu H.; Guo C.; Hou Z.; Gong P. Design, synthesis and antifungal activities of novel triazole derivatives with selenium-containing hydrophobic side chains. Bioorg. Med. Chem. Lett. 2022, 78, 129044 10.1016/j.bmcl.2022.129044. [DOI] [PubMed] [Google Scholar]
  6. Ni T.; Ding Z.; Xie F.; Hao Y.; Bao J.; Zhang J.; Yu S.; Jiang Y.; Zhang D. Design, synthesis, and in vitro and in vivo antifungal activity of novel triazoles containing phenylethynyl pyrazole side chains. Molecules 2022, 27, 3370. 10.3390/molecules27113370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Yin W.; Liu L.; Jiang H.; Wu T.; Cui H.; Zhang Y.; Gao Z.; Sun Y.; Qin Q.; Zhao L.; Su X.; Zhao D.; Cheng M. Design, synthesis, and evaluation of novel 3-thiophene derivatives as potent fungistatic and fungicidal reagents based on a conformational restriction strategy. Eur. J. Med. Chem. 2022, 233, 114195 10.1016/j.ejmech.2022.114195. [DOI] [PubMed] [Google Scholar]
  8. Aydin M.; Ozturk A.; Duran T.; Ozmen U. O.; Sumlu E.; Ayan E. B.; Korucu E. N. In vitro antifungal and antibiofilm activities of novel sulfonyl hydrazone derivatives against Candida spp. J. Med. Mycol. 2023, 33, 101327 10.1016/j.mycmed.2022.101327. [DOI] [PubMed] [Google Scholar]
  9. Sadanandan B.; Ashrit P.; Nataraj L. K.; Shetty K.; Jogalekar A. P.; Vaniyamparambath V.; Hemanth B. High throughput comparative assessment of biofilm formation of Candida glabrata on polystyrene material. Korean J. Chem. Eng. 2022, 39, 1277–1286. 10.1007/s11814-021-1054-3. [DOI] [Google Scholar]
  10. Flemming H. C.; Van Hullebusch E. D.; Neu T. R.; Nielsen P. H.; Seviour T.; Stoodley P.; Wingender J.; Wuertz S. The biofilm matrix: Multitasking in a shared space. Nat. Rev. Microbiol. 2023, 70–86. 10.1038/s41579-022-00791-0. [DOI] [PubMed] [Google Scholar]
  11. El-Hazek R. M.; Elkenawy N. M.; Zaher N. H.; El-Gazzar M. G. Green synthesis of novel antifungal 1, 2, 4-triazoles effective against γ-irradiated Candida parapsilosis. Arch. Pharm. 2022, 355, 2100287 10.1002/ardp.202100287. [DOI] [PubMed] [Google Scholar]
  12. Huang M.; Duan W. G.; Lin G. S.; Li B. Y. Synthesis, antifungal activity, 3D-QSAR, and molecular docking study of novel menthol-derived 1, 2, 4-triazole-thioether compounds. Molecules 2021, 26, 6948. 10.3390/molecules26226948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Zhu T.; Chen X.; Li C.; Tu J.; Liu N.; Xu D.; Sheng C. Lanosterol 14α-demethylase (CYP51)/histone deacetylase (HDAC) dual inhibitors for treatment of Candida tropicalis and Cryptococcus neoformans infections. Eur. J. Med. Chem. 2021, 221, 113524 10.1016/j.ejmech.2021.113524. [DOI] [PubMed] [Google Scholar]
  14. Amin N. H.; El-Saadi M. T.; Ibrahim A. A.; Abdel-Rahman H. M. Design, synthesis and mechanistic study of new 1, 2, 4-triazole derivatives as antimicrobial agents. Bioorg. Chem. 2021, 111, 104841 10.1016/j.bioorg.2021.104841. [DOI] [PubMed] [Google Scholar]
  15. Bitla S.; Gayatri A. A.; Puchakayala M. R.; Bhukya V. K.; Vannada J.; Dhanavath R.; Kuthati B.; Kothula D.; Sagurthi S. R.; Atcha K. R. Design and synthesis, biological evaluation of bis-(1, 2, 3-and 1, 2, 4)-triazole derivatives as potential antimicrobial and antifungal agents. Bioorg. Med. Chem. Lett. 2021, 41, 128004 10.1016/j.bmcl.2021.128004. [DOI] [PubMed] [Google Scholar]
  16. Nesaragi A. R.; Kamble R. R.; Bayannavar P. K.; Shaikh S. K. J.; Hoolageri S. R.; Kodasi B.; Joshi S. D.; Kumbar V. M. Microwave assisted regioselective synthesis of quinoline appended triazoles as potent anti-tubercular and antifungal agents via copper (I) catalyzed cycloaddition. Bioorg. Med. Chem. Lett. 2021, 41, 127984 10.1016/j.bmcl.2021.127984. [DOI] [PubMed] [Google Scholar]
  17. Qi L.; Li M. C.; Bai J. C.; Ren Y. H.; Ma H. X. In vitro antifungal activities, molecular docking, and DFT studies of 4-amine-3-hydrazino-5-mercapto-1, 2, 4-triazole derivatives. Bioorg. Med. Chem. Lett. 2021, 40, 127902 10.1016/j.bmcl.2021.127902. [DOI] [PubMed] [Google Scholar]
  18. Al-Wabli R. I.; Alsulami M. A.; Bukhari S. I.; Moubayed N. M.; Al-Mutairi M. S.; Attia M. I. Design, synthesis, and antimicrobial activity of certain new indole-1, 2, 4 triazole conjugates. Molecules 2021, 26, 2292. 10.3390/molecules26082292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gondru R.; Kanugala S.; Raj S.; Kumar C. G.; Pasupuleti M.; Banothu J.; Bavantula R. 1, 2, 3-triazole-thiazole hybrids: Synthesis, in vitro antimicrobial activity and antibiofilm studies. Bioorg. Med. Chem. Lett. 2021, 33, 127746 10.1016/j.bmcl.2020.127746. [DOI] [PubMed] [Google Scholar]
  20. Hassan M. Z.; Alsayari A.; Asiri Y. I.; Bin Muhsinah A. 1, 2, 4-Triazole-3-Thiones: Greener, One-Pot, Ionic Liquid Mediated Synthesis and Antifungal Activity. Polycyclic Aromat. Compd. 2023, 43, 167–175. 10.1080/10406638.2021.2009887. [DOI] [Google Scholar]
  21. Sadeghian S.; Emami L.; Mojaddami A.; khabnadideh S.; Faghih Z.; Zomorodian K.; Rashidi M.; Rezaei Z. 1, 2, 4-Triazole derivatives as novel and potent antifungal agents: Design, synthesis and biological evaluation. J. Mol. Struct. 2023, 1271, 134039 10.1016/j.molstruc.2022.134039. [DOI] [Google Scholar]
  22. Ahuja R.; Sidhu A.; Bala A.; Arora D.; Sharma P. Structure based approach for twin-enzyme targeted benzimidazolyl-1, 2, 4-triazole molecular hybrids as antifungal agents. Arabian J. Chem. 2020, 13, 5832–5848. 10.1016/j.arabjc.2020.04.020. [DOI] [Google Scholar]
  23. Suárez-García J.; Cano-Herrera M. A.; María-Gaviria M.; Osorio-Echeverri V. M.; Mendieta-Zerón H.; Arias-Olivares D.; Benavides-Melo J.; García-Sánchez L. C.; García-Ortíz J.; Becerra-Buitrago A.; Valero-Rojas J.; Rodríguez-González M. A.; García-Eleno M. A.; Cuevas-Yañez E. Synthesis, characterization, in-vitro biological evaluation and theoretical studies of 1, 2, 3-triazoles derived from triclosan as difenoconazole analogues. J. Mol. Struct. 2023, 1280, 135053 10.1016/j.molstruc.2023.135053. [DOI] [Google Scholar]
  24. Wani M. Y.; Alghamidi M. S. S.; Srivastava V.; Ahmad A.; Aqlan F. M.; Al-Bogami A. S. Click synthesis of pyrrolidine-based 1, 2, 3-triazole derivatives as antifungal agents causing cell cycle arrest and apoptosis in Candida auris. Bioorg. Chem. 2023, 136, 106562 10.1016/j.bioorg.2023.106562. [DOI] [PubMed] [Google Scholar]
  25. Peixoto L. R.; Rosalen P. L.; Ferreira G. L. S.; Freires I. A.; de Carvalho F. G.; Castellano L. R.; de Castro R. D. Antifungal activity, mode of action and anti-biofilm effects of Laurus nobilis Linnaeus essential oil against Candida spp. Arch. Oral Biol. 2017, 73, 179–185. 10.1016/j.archoralbio.2016.10.013. [DOI] [PubMed] [Google Scholar]
  26. Podust L. M.; Poulos T. L.; Waterman M. R. Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proc. Natl. Acad. Sci. 2001, 98, 3068–3073. 10.1073/pnas.061562898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zrieq R.; Ahmad I.; Snoussi M.; Noumi E.; Iriti M.; Algahtani F. D.; Patel H.; Saeed M.; Tasleem M.; Sulaiman S.; Aouadi K.; Kadri A. Tomatidine and patchouli alcohol as inhibitors of SARS-CoV-2 enzymes (3CLpro, PLpro and NSP15) by molecular docking and molecular dynamics simulations. Int. J. Mol. Sci. 2021, 22, 10693. 10.3390/ijms221910693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ahmad I.; Kumar D.; Patel H. Computational investigation of phytochemicals from Withania somnifera (Indian ginseng/ashwagandha) as plausible inhibitors of GluN2B-containing NMDA receptors. J. Biomol. Struct. Dyn. 2022, 40, 7991–8003. 10.1080/07391102.2021.1905553. [DOI] [PubMed] [Google Scholar]
  29. Osmaniye D.; Karaca Ş.; Kurban B.; Baysal M.; Ahmad I.; Patel H.; Özkay Y.; Kaplancıklı Z. A. Design, synthesis, molecular docking and molecular dynamics studies of novel triazolothiadiazine derivatives containing furan or thiophene rings as anticancer agents. Bioorg. Chem. 2022, 122, 105709 10.1016/j.bioorg.2022.105709. [DOI] [PubMed] [Google Scholar]
  30. Ozturk A.; Abdulmajed O.; Aydin M. Investigation of antifungal, antibiofilm and anti-filamentation activities of biocides against Candida isolates. Ann. Med. Res. 2020, 27, 2041–2046. 10.5455/annalsmedres.2020.05.478. [DOI] [Google Scholar]
  31. Maestro, 10.6; Schrödinger, LLC: New York, NY, USA, 2020. [Google Scholar]
  32. Schrödinger LigPrep, version 3.8; Schrödinger, LLC: New York, NY, USA, 2020. [Google Scholar]
  33. Schrödinger Glide, version 7.1; Schrödinger, LLC: New York, NY, USA, 2020. [Google Scholar]
  34. Liu X.; Shi D.; Zhou S.; Liu H.; Liu H.; Yao X. Molecular dynamics simulations and novel drug discovery. Expert Opin. Drug Discovery 2018, 13, 23–37. 10.1080/17460441.2018.1403419. [DOI] [PubMed] [Google Scholar]
  35. M.D.I. Tools Schrödinger, LLC: New York, NY, 2020, Schrödinger Release 2018–3: Prime, (2018). [Google Scholar]
  36. Sureshkumar B.; Mary Y. S.; Resmi K.; Suma S.; Armaković S.; Armaković S. J.; Van Alsenoy C.; Narayana B.; Sobhana D. Spectroscopic characterization of hydroxyquinoline derivatives with bromine and iodine atoms and theoretical investigation by DFT calculations, MD simulations and molecular docking studies. J. Mol. Struct. 2018, 1167, 95–106. 10.1016/j.molstruc.2018.04.077. [DOI] [Google Scholar]
  37. Schrödinger Release 1: Desmond molecular dynamics system, version 3.7; DE Shaw Research, New York, NY, Maestro-Desmond Interoperability Tools, version, 3, 2014. [Google Scholar]
  38. Humphreys D. D.; Friesner R. A.; Berne B. J. A multiple-time-step molecular dynamics algorithm for macromolecules. J. Phys. Chem. 1994, 98, 6885–6892. 10.1021/j100078a035. [DOI] [Google Scholar]
  39. Hoover W. G. Canonical dynamics: Equilibrium phase-space distributions. Phys. Rev. A 1985, 31, 1695. 10.1103/PhysRevA.31.1695. [DOI] [PubMed] [Google Scholar]
  40. Martyna G. J.; Tobias D. J.; Klein M. L. Constant pressure molecular dynamics algorithms. J. Chem. Phys. 1994, 101, 4177–4189. 10.1063/1.467468. [DOI] [Google Scholar]
  41. Essmann U.; Perera L.; Berkowitz M. L.; Darden T.; Lee H.; Pedersen L. G. A smooth particle mesh Ewald method. J. Chem. Phys. 1995, 103, 8577–8593. 10.1063/1.470117. [DOI] [Google Scholar]
  42. Silveira F. F.; de Souza J. O.; Hoelz L. V.; Campos V. R.; Jabor V. A.; Aguiar A. C.; Nonato M. C.; Albuquerque M. G.; Guido R. V.; Boechat N.; Pinheiro L. C. S. Comparative study between the anti-P. falciparum activity of triazolopyrimidine, pyrazolopyrimidine and quinoline derivatives and the identification of new PfDHODH inhibitors. Eur. J. Med. Chem. 2021, 209, 112941 10.1016/j.ejmech.2020.112941. [DOI] [PubMed] [Google Scholar]
  43. Osmaniye D.; Evren A. E.; Karaca Ş.; Özkay Y.; Kaplancıklı Z. A. Novel thiadiazol derivatives; design, synthesis, biological activity, molecular docking and molecular dynamics. J. Mol. Struct. 2023, 1272, 134171 10.1016/j.molstruc.2022.134171. [DOI] [Google Scholar]

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