Synthesis of some novel heterocylic compounds derived from 2-[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetohydrazide and investigation of their lipase and α-glucosidase inhibition

Abstract

In the present study, 2-[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetohydrazide (1) was used as starting compound for the synthesis of 2-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetyl}-4-thiosemicarbazides (2a–c) and 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-1,3,4-oxadiazole-2-thione (5). The cyclization of compounds 2a–c in the presence of NaOH resulted in the formation of 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thiones (3a–c). Aminomethylation of compounds 3a–c and 5 with formaldehyde and N-methyl/phenylpiperazine furnished Mannich bases (4a–f and 6a–b). The newly synthesized compounds were well-characterized by IR, ¹H NMR, ¹³C NMR, elemental analysis and mass spectral studies. They were also screened for their lipase and α-glucosidase inhibition. Among the tested compound 2c (IC₅₀ = 2.50 ± 0.50 µM) showed the best anti-lipase activity and compounds 2c (IC₅₀ = 3.41 ± 0.16 µM) and 6a (IC₅₀ = 4.36 ± 0.10 µM) showed the best anti-α-glucosidase activity.

• Biheterocyclic compounds
• lipase and α-glucosidase inhibition
• Mannich bases
• 1,3,4-oxadiazoles
• 1,2,4-triazoles
• 1,2,4-triazole-5-thiones

Introduction

Obesity is a major global public health problem, and its prevalence is steadily increasing1. Many serious chronic diseases such as hypertension, high total cholesterol and triglycerides, type II diabetes mellitus, sleep apnea and respiratory problems, cancer, stroke, and coronary heart diseases are attributable to obesity2. Basically, there are two types of anti-obesity agents, classified as inhibitors of lipid (lipase inhibitor) and carbohydrate (α-glucosidase inhibitor) absorption3.

Lipase is the lipid-digesting enzyme that catalyzes the hydrolysis of ester bonds of triacylglycerols (TAGs) to produce free fatty acids, diacylglycerols, monoglycerols and glycerol. The break down and digestion of dietary TAG in the small intestine of mammals is enabled by pancreatic lipase4. The resulting free fatty acids are then absorbed by the body and if not utilized, added to the development of obesity. Therefore, if the breakdown and hydrolysis of TAGs can be stopped or minimized, absorption will be lowered and consequently the prevalence of obesity could be reduced5. Hence, an inhibitor of pancreatic lipases could be a useful agent in the fight against obesity6. Orlistat is the only authorized anti-obesity drug in Europe. However, it has some side effects, including fecal incontinence, flatulence and steatorrhea7.

Carbohydrate digestion is catalyzed by the enzyme α-glucosidase which is an exo-acting enzyme located in the brush-border surface membrane of intestinal cells8. It releases an α-d-glucose from the non-reducing end of the sugar by acting on 1,4-α linkages9. The α-glucosidase inhibitors in the gut work against α-glucosidase in the gut preventing the release of glucose from disaccharides and oligosaccharides, and therefore reducing postprandial glucose levels10. It is considered as a significant strategy in the management of diabetes mellitus if the α-glucosidase inhibitors are used. For example, in the treatment of diabetes mellitus patients, some α-glucosidase inhibitors, such as acarbose and voglibose, are extensively used but often causes severe gastrointestinal side effects such as flatulence and diarrhea11–13.

Heterocyclic compounds containing 1,2,4-triazole skeleton are known to possess a wide range of pharmacological activities such as antimicrobial, antitubercular, anti-inflammatory, antiviral, antioxidant, antidepressant, anticonvulsant, antimalarial, anti-diabetic, anti-obesity, enzyme inhibitory and anticancer activities14–17. Some present day drugs such as fluconazole, itraconazole, terconazole (antifungal agents), anastrozole, letrozole, vorozole (aromatase inhibitors in the treatment of postmenopausal breast cancer), ribavirin (antiviral agent), etoperidone and nefazodone (antidepressant), rizatriptan (antimigraine agent) and rilmazafon (hypnotic, anxiolytic, used in the case of neurotic insomnia) are examples of potent bioactive molecules possessing 1,2,4-triazole moieties16^,18–21. Similarly, heterocyclic compounds bearing 1,3,4-oxadiazole nucleus are also known to possess a wide spectrum of pharmacological properties, including analgesic, anti-inflammatory, antimicrobial, antifungal, anti-convulsant, anti-diabetic, antihelmintic, hypo-glycemic, antiallergic, enzyme inhibitors, antineoplastic, CNS depressant, antitubercular and anticancer22–26. Furthermore, there are a number of drugs containing 1,3,4-oxadiazole ring such as raltegravir (antiretroviral), nesapidil (antiarrhythemic), furamizole (antibacterial), tiodazosin (antihypertensive) and fenadiazole (hypnotic)27. Other important groups of heterocyclic compounds for medicinal chemistry are the thiosemicarbazides and their corresponding cyclized counterpart 1,2,4-triazole-thions which have been extensively reported in the literature for the synthesis of new biologically active molecules28.

Mannich base is a β-amino-carbonyl compound, which is formed in the reaction of an amine (primery or secondary), formaldehyde and a reactive hydrogen atom29. Mannich bases have been reported to be useful agent for potential biological activities such as anticancer, antibacterial, antifungal, analgesic, anti-inflammatory, antimalarial and antitubercular30. Many Mannich bases of 1,2,4-triazoles carrying N-methylpiperazine moiety are reported to possess protozoacidal and bactericidal activities31. The modern drugs, Prazosin, Lidoflazine and Urapidil, carrying piperazine nucleus are used as cardiovascular agents31. Furthermore, Mannich bases containing 1,3,4-oxadiazoles ring are also known to posses a number of biological activities32.

In view of these findings, the aim of this present study is to obtain 1,2,4-triazole and 1,3,4-oxadiazole derivatives containing 3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazole sekeleton incorporating also Mannich base structures. All newly synthesized compounds were screened for the lipase and α-glucosidase inhibition.

Results and discussion

Chemistry

The reaction sequence employed for synthesis of target compounds is shown in Scheme 1. The starting compound 2-[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetohydrazide (1) was prepared according to our previous report33.

Scheme 1. Reagents and conditions: (i) absolute ethanol, RNCS, reflux; (ii) 2 N NaOH, reflux; (iii) and (v) DMF, HCHO, N-methyl/phenyl piperazine, room temperature; (iv) ethanol, CS₂/KOH, relux.

Reaction of compound 1 with various isothiocyanate resulted in the formation of 2-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetyl}-4alkyl/arylthiosemicarbazides (2a–c)34. The IR spectra of these compounds showed moderately strong bands around 3150–3360, 1688–1689 and 1177–1182 cm⁻¹, characteristic of the NH, C = O and C = S groups, respectively34. The ¹H NMR spectra of the compounds displayed three singlets due to N₁–H, N₂–H and N₃–H groups at δ 8.09–9.78, 9.40–9.89 and 10.26–10.53 ppm35. The ¹³C NMR spectra showed two signals at δ 163.20–166.18 ppm and δ 181.56–182.61 ppm characteristic to C = O and C = S carbons, respectively, which confirm the formation of thiosemicarbazides34^,35. Alkaline cyclization of the compounds (2a–c) using NaOH afforded the corresponding derivatives (3a–c)36. These compounds can exist in two tautomeric forms, 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thioles and 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thiones (3a–c). The spectral analysis (IR, ¹H NMR and ¹³C NMR) shows that these compounds exist in latter tautomeric form. According to IR spectroscopic data of compounds (3a–c), the SH vibration band (2550–2600 cm⁻¹) was absent and C = S stretching bands were observed at 1321–1326 cm⁻¹. Moreover, NH stretching bands of 3a–c were observed at 3169, 3236 and 3240 cm⁻¹, respectively37^,38. In the ¹H NMR spectra of these compounds, NH peaks were observed as a singlet at δ 13.75, 14.02 and 14.07 ppm, respectively38. The C = S group resonated at δ 168.08–169.15 ppm in the ¹³C NMR spectra of compounds (3a–c) and so their thionic form was proved39.

Intramolecular cyclization of the acid hydrazide (1) with carbon disulfide in the presence of potassium hydrtoxide in absolute alcohol resulted in 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-1,3,4-oxadiazole-2(3H)-thione (5)40. The IR spectra of this compound showed NH bands in the region of 3291 cm⁻¹ and C = S absorption band at 1326 cm⁻¹ instead of SH band at around 2550–2600 cm⁻¹ 41^,42. The N–H protons of 1,3,4-oxadiazole ring (5) were seen at δ 13.83 ppm41^,42. The ¹³C NMR data of 5 which showed a peak at δ 160.12 and 178.55 ppm, due to oxadiazole C-5 and oxadiazole C-2 (C = S)41^,42.

Mannich bases 4a–f and 6a–b were synthesized from the compounds 3a–c and 5 by reacting with N-methylpiperazine or N-pheylpiperazine in dimethyl formamide in the presence of formaldehyde33. The IR spectra of these compounds showed moderately strong bands around 1170–1179 and 1325–1339 cm⁻¹, characteristic of the N–CH₂–N and C = S groups, respectively43. In the ¹H NMR spectra, a characteristic signal due to the –N–CH₂–N– protons appeared at δ 5.49–5.74 ppm. The signal due to the N–CH₃ (for 4a–c and 6a) protons appeared at δ 5.50–5.52 ppm43^,44. The signals due to the N–CH₂–N and C = S carbons appeared at δ 68.30–70.06 and 168.95–178.27 ppm in the ¹³C NMR, respectively43^,44.

Biological evaluation

Anti-lipase activity

All compounds were evaluated with regard to pancreatic lipase activity and 2a, 2c and 6a showed anti-lipase activities at various concentrations (Table 1). No significant inhibitory effect was detected for other compounds. Among the tested compounds, 2c showed the best anti-lipase activity. The compound inhibited pancreatic lipase by 100.00 ± 0.49% at concentration of 10 µM (Table 1). Orlistat, known pancreatic lipase inhibitor used as anti-obesity drug, showed inhibitory effect by 99.15 ± 0.35% at concentration of 312 nM (IC₅₀ = 0.85 ± 0.042 nM). Compounds 2a and 2c IC₅₀ values were calculated as 10.25 ± 0.40 and 2.50 ± 0.50 µM, respectively. Compund 2c and Orlistat almost completely inhibited lipase activity, whereas 2a and 6a were less effective. The difference was not statistically significant for compound 2c compared to Orlistat. Synthesized compound 2c has a potential to be an alternative for Orlistat.

Table 1. Inhibitory effects of selected synthesized compounds (at final concentration of 10 µM).

Compound	% Inhibition	IC50 (µM)
2a	96.37 ± 1.03^a	10.25 ± 0.40^b
2c	100.00 ± 0.49^b	2.50 ± 0.50^b
6a	78.34 ± 1.16^a	42.21 ± 4.54^a
Orlistat (312 nM)	99.15 ± 0.35^b	8.5 × 10^−4 ± 4.2 × 10^−5b

Orlistat was used as positive control. All the results are expressed as mean ± standard error of the mean (S.E.M). The significance of differences of synthesized compounds from the control (Orlistat) was determined by ANOVA followed by Dunnett’s test (p < 0.05). Different letters in the same column indicate significant differences in % inhibition or IC50 values as determined by Dunnett’s test (p < 0.05).

α-Glucosidase inhibitory activity

All compounds were evaluated with regard to α-glucosidase activity and 2c, 6a and 6b showed anti-α-glucosidase activity at various concentrations (Figure 1). These compounds exhibited more inhibitory effect than Acarbose, known α-glucosidase inhibitor was used as anti-diabetic drug (Table 2). No significant inhibitory effect was detected for other compounds. Among the tested compounds, 2c and 6a showed the best anti-α-glucosidase activity (p < 0.05). These compounds inhibited α-glucosidase activity by 99.79 ± 1.17% and 99.87 ± 0.38% at a concentration of 100 µM, respectively. Acarbose showed inhibitory effect by 55.4 ± 3.01% at the same concentration (IC₅₀ = 26.12 ± 2.38 µM). Compounds 2c and 6a IC₅₀ values were calculated as 3.41 ± 0.16 and 4.36 ± 0.10 µM, respectively (Table 2). Compunds 2c, 6a and 6b almost completely inhibited α-glucosidase activity. The difference was statistically significant for these compounds compared to Acarbose. Synthesized compounds 2c, 6a and 6b have substantial inhibition potential.

Figure 1. Dose-dependent inhibitory effect of compounds 2c, 6a and 6b. Acarbose was used as standard inhibitor. Inhibitory effect of the tested compounds and Acarbose was measured at the range of 0.045–100 µM concentrations. Residual activities of the compounds were expressed as the mean ± S.D., measured in triplicate.

Table 2. Inhibition of α-glucosidase by newly synthesized compounds 2–6.

Compound	% Inhibition	IC50 ± SD (µM)
2c	99.79 ± 1.17^a	3.41 ± 0.16^a
6a	99.87 ± 0.38^a	4.36 ± 0.10^a
6b	92.41 ± 1.93^a	17.68 ± 1.01^b
Acarbose	55.4 ± 3.01^b	26.12 ± 2.38^b

All compounds were assayed at concentration of 100 µM. All the results are expressed as mean ± standard error of the mean (S.E.M). The significance of differences of synthesized compounds from the control (Acarbose) was determined by ANOVA followed by Dunnett’s test (p < 0.05). Different letters in the same column indicate significant differences in % inhibition or IC50 values as determined by Dunnett’s test (p < 0.05).

Statistical analysis

Results are expressed as means ± standard error of the mean for each experiment. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s Multiple Comparison Test and p value of less than 0.05 was considered to be statistically significant.

Experimental

Chemistry

Melting points were determined in open capillaries on a Gallenkamp Electrothermal digital melting point apparatus (London, England). IR spectra were recorded in a Perkin-Elmer Frontier FT-IR spectrophotometer (San Jose, CA) using attenuated total reflection (ATR) accessory. ¹H and ¹³C NMR spectra were measured on a BRUKER AVANCE II-400 (400 MHz) (Darmstadt, Germany) using DMSO-d₆ as solvent and TMS as internal standard. The elemental analysis was performed on a Costech Elemental Combustion System CHNS-O elemental analyzer (Valencia, CA). All the compounds gave C, H and N analyses within ± 0.4% of theoretical values. The mass spectra were taken on a Quattro LC–MS (70 eV) instrument (Manchester, UK). The progress of the reaction was monitored by thin layer chromatography (TLC) on silica gel plates (silica gel 60 F 2.54 0.2 mm thickness). Compound 1 was synthesized by the methods reported earlier33.

General procedure for the synthesis of 2-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetyl}-4-alkyl/arylthiosemicarbazide (2a–c)

2-[3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetohydrazide (0.01 mol) was mixed with corresponding isothiocyanate (0.01 mol) in absolute ethanol (50 mL). Then this mixture was refluxed for about 4 h. At the and of the reaction, crude product collapsed. The precipitate formed was filtered off and purified by recrystallization from ethanol.

2-{[3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetyl}-4-metylthiosemicarbazide (2a). Yield (3.98 g, 89.44%); m.p. 184–185 °C;IR (ATR, ν_max, cm⁻¹): 3228, 3150 (3NH), 1688 (C = O), 1182 (C = S); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.90 (d, 3H, CH₃, J = 4.0 Hz), 3.73 (s, 3H, OCH₃), 4.04 (s, 2H, benzyl CH₂), 5.00 (s, 2H, N–CH₂), Ar–H: [6.90 (d, 2H,J = 8.4 Hz), 7.23 (d, 2H, J = 8.4 Hz), 7.50 (d, 2H, J = 8.4 Hz), 7.93 (d, 2H, J = 8.4 Hz)], 8.09 (bs, 1H, NH), 9.40 (s, 1H, NH), 10.26 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.76 (benzyl CH₂), 31.36 (NH–CH₃), 49.91 (N–CH₂), 55.51 (OCH₃), Ar–C: [114.42 (2CH), 127.84 (2CH), 128.36, 129.28 (2CH), 130.32 (2CH), 130.37, 134.11, 158.57], 157.72 (triazole C-3), 159.26 (triazole C-5), 163.20 (C = O), 182.63 (C = S); EI MS m/z (%): 467.35 ([M+Na]⁺, 58), 434.31 (100), 327.51 (60), 229.27 (10), 163.13 (16), 130.28 (18); Anal. Cald for C₂₀H₂₁ClN₆O₂S: C, 53.99; H, 4.76; N, 18.89. Found: C, 54.14; H, 4.70 N, 19.00.

2-{[3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetyl}-4-phenylthiosemicarbazide (2b). Yield (4.70 g, 92.70%); m.p. 193–194 °C; IR (ATR, ν_max, cm⁻¹): 3336, 3252, 3168 (3NH), 1701 (C = O), 1177 (C = S); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 3.72 (s, 3H, OCH₃), 4.12 (s, 2H, benzyl CH₂), 5.08 (s, 2H, N–CH₂), Ar–H: [6.89 (d, 2H, J = 8.4 Hz), 7.19–7.26 (m, 3H), 7.34–7.40 (m, 4H), 7.50 (d, 2H, J = 8.0 Hz), 7.94 (d, 2H, J = 8.4 Hz)], 9.78 (s, 2H, 2NH), 10.51 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.71 (benzyl CH₂), 50.02 (N–CH₂), 55.51 (OCH₃), Ar–C: [114.41 (2CH), 128.56 (CH), 127.84 (2CH), 128.39, 128.67 (2CH), 129.27 (2CH), 130.19, 130.35 (2CH), 130.40 (2CH), 134.08, 139.41, 158.57], 157.74 (triazole C-3), 159.27 (triazole C-5), 166.18 (C = O), 181.56 (C = S); LC–MS/MS m/z (%): 529.36 ([M+Na]⁺, 100), 507.40 ([M]⁺, 62), 436.38 (20), 394.33 (38), 372.43 (32), 358.29 (61), 300.29 (15), 229.46 (18); Anal. Cald. for C₂₅H₂₃ClN₆O₂S: C, 59.22; H, 4.57; N, 16.58. Found: C, 59.45; H, 4.53; N, 16.71.

2-{[3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetyl}-4-(4-chlorophenyl)thiosemicarbazide (2c). Yield (5.20 g, 95.94%); m.p. 198–199 °C; IR (ATR, ν_max, cm⁻¹): 3360, 3240, 3157 (3NH), 1689 (C = O), 1178 (C = S); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 3.72 (s, 3H, OCH₃), 4.12 (s, 2H, benzyl CH₂), 5.07 (s, 2H, N–CH₂), Ar–H: [6.84–6.90 (m, 2H), 7.25 (d, 2H, J = 8.4 Hz), 7.40–7.44 (m, 4H), 7.49–7.51 (m, 2H), 7.92–7.94 (m, 2H)], 9.89 (s, 2H, 2NH), 10.53 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.80 (benzyl CH₂), 50.02 (N–CH₂), 55.51 (OCH₃), Ar–C: [114.41 (2CH), 127.83 (2CH), 128.57, 128.60 (2CH), 129.28 (2CH), 130.17, 130.40 (4CH), 134.01, 134.09, 138.42, 158.57], 157.73 (triazole C-3), 159.27 (triazole C-5), 163.24 (C = O), 182.61 (C = S); LC–MS/MS m/z (%): 565.59 ([M+Na]⁺, 48), 541.50 ([M]⁺, 62), 415.67 (22), 359.42 (25), 283.46 (39), 229.33 (100), 214.38 (40), 113.45 (26); Anal. Cald. for C₂₅H₂₂Cl₂N₆O₂S: C, 55.46; H, 4.10; N, 15.52. Found: C, 55.63; H, 4.02; N, 15.64.

General procedure for the synthesis of 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3a–c)

A 10 mL ethanolic solution of corresponding thiosemicarbazide (2a–c) (0.005 mol) was heated in aqueous NaOH solution (2 N, 25 mL) under reflux for 6 h. After completion of reaction, the mixture was cooled and then acidified to pH 3–4 with concentrated HCl. The precipitate formed was filtered, washed with cold water and recrystallized from ethanol–water (3:1) to yield the desired compound. The completion of reaction was monitored by thin-layer chromatography.

5-{[3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-4-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3a). Yield (1.85 g, 86.04%); m.p. 207–208 °C; IR (ATR, ν_max, cm⁻¹): 3169 (NH), 1611, 1578 (C = N), 1326 (C = S); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 3.38 (s, 3H, CH₃), 3.72 (s, 3H, OCH₃), 4.22 (s, 2H, benzyl CH₂), 5.67 (s, 2H, N–CH₂), Ar–H: [6.86 (d, 2H, J = 8.4 Hz), 7.21 (d, 2H, J = 8.8 Hz), 7.50 (d, 2H, J = 8.4 Hz), 7.94 (d, 2H, J = 8.4 Hz)], 13.75 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.59 (benzyl CH₂), 30.64 (NH–CH₃), 43.58 (N–CH₂), 55.52 (OCH₃), Ar–C: [114.37 (2CH), 127.86(2CH), 127.94, 128.07 (2CH), 129.33, 129.91 (2CH), 134.30, 158.59], 148.04 (triazole C-5, second ring), 157.40 (triazole C-3), 159.68 (triazole C-5), 168.08 (triazole C-3, second ring); LC–MS/MS m/z (%): 427.48 ([M+1]⁺, 17), 358.35 (22), 306.48 (15), 219.38 (32), 130.28 (100); Anal. Cald. for C₂₀H₁₉ClN₆OS: C, 56.27; H, 4.49; N, 19.69. Found: C, 56.42; H, 4.55; N, 19.73.

5-{[3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3b). Yield (2.05 g, 80.71%); m.p. 255–256 °C; IR (ATR, ν_max, cm⁻¹): 3236 (NH), 1615, 1578 (C = N), 1321 (C = S); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 3.72 (s, 3H, OCH₃), 3.81 (s, 2H, benzyl CH₂), 5.44 (s, 2H, N–CH₂), Ar–H: [6.84 (d, 2H, J = 8.4 Hz), 7.07 (d, 2H, J = 8.4 Hz), 7.28–7.31 (m, 2H), 7.47–7.49 (m, 5H), 7.84 (d, 2H, J = 8.4 Hz)], 14.02 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.31 (benzyl CH₂), 48.32 (N–CH₂), 55.53 (OCH₃), Ar–C: [114.40 (2CH), 127.46, 127.80 (2CH), 127.92 (2CH), 128.54 (2CH), 129.24 (2CH), 129.87, 130.18 (2CH), 130.42 (CH), 133.35, 134.22, 158.56], 147.43 (triazole C-5, second ring), 156.99 (triazole C-3), 159.49 (triazole C-5), 169.15 (triazole C-3, second ring); LC–MS/MS m/z (%): 489.38 ([M+1]⁺, 18), 478.49 (32), 456.46 (13), 329.32 (20), 157.06 (72), 139.11 (100); Anal. Cald. for C₂₅H₂₁ClN₆OS: C, 61.41; H, 4.33; N, 17.19. Found: C, 61.63; H, 4.41; N, 17.07.

5-{[3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-4-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3c). Yield (2.35 g, 86.72%); m.p. 247–248 °C; IR (ATR, ν_max, cm⁻¹): 3240 (NH), 1615, 1586 (C = N), 1323 (C = S); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 3.73 (s, 3H, OCH₃), 3.92 (s, 2H, benzyl CH₂), 5.47 (s, 2H, N–CH₂), Ar–H: [6.65 (d, 2H, J = 8.4 Hz), 7.07 (d, 2H, J = 8.6 Hz), 7.33 (d, 2H, J = 8.6 Hz), 7.48–7.55 (m, 4H), 7.82 (d, 2H, J = 8.4 Hz)], 14.07 (s, 1H, NH); ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 29.95 (benzyl CH₂), 43.44 (N–CH₂), 55.08 (OCH₃), Ar–C: [113.95 (2CH), 127.25 (2CH), 127.28, 127.47, 128.84 (2CH), 129.35 (2CH), 129.45 (2CH), 129.88 (2CH), 131.78, 133.77, 134.51, 158.12], 146.92 (triazole C-5, second ring), 156.77 (triazole C-3), 158.92 (triazole C-5), 168.61 (triazole C-3, second ring); LC–MS/MS m/z (%): 523.30 ([M]⁺, 8), 477.49 (5), 389.46 ((12), 375.36 (13), 277.26 (100), 237.18 (86), 159.97 (28), 117.12 (41), 106.04 (48); Anal. Cald. for C₂₅H₂₀Cl₂N₆OS: C, 57.37; H, 3.85; N, 16.06. Found: C, 57.48; H, 3.80; N, 15.99.

General procedure for the synthesis of 4a–f and 6a–b

To the solution of corresponding compound 3a–c/5 (0.001 mol) in DMF (10 mL), formaldehyde (37%, 0.155 mL) and 1-methyl/phenylpiperazine (0.001 mol) were added and the mixture was stirred at room temperature for 16–20 h. Petroleum ether (40–60 °C) was added at the end of the reaction and the mixture was then cooled in refrigerator at 1 °C for 12 h. The resulting solid obtained was filtered, dried and crystallized from benzene:petroleum ether (40–60 °C). The completion of reaction was monitored by thin-layer chromatography.

4-Methyl-2-[(4-methylpiperazin-1-yl)methyl]-5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione (4a). Yield (0.42 g, 77.78%); m.p. 182–183 °C; IR (ATR, ν_max, cm⁻¹): 1610, 1583 (C = N), 1332 (C = S), 1177 (N–CH₂–N); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.16 (s, 3H, N–CH₃), 2.33 (bs, 4H, CH₂–N–CH₂), 2.61 (bs, 4H, CH₂–N–CH₂), 3.45 (s, 3H, N–CH₃), 3.72 (s, 3H, OCH₃), 4.23 (s, 2H, benzyl CH₂), 5.67 (s, 2H, N–CH₂), 5.74 (s, 2H, N–CH₂–N), Ar–H: [6.84 (d, 2H, J = 7.6 Hz), 7.19 (d, 2H, J = 6.8 Hz), 7.51 (d, 2H, J = 7.6 Hz), 7.94 (d, 2H, J = 6.8 Hz)]; ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.54 (benzyl CH₂), 31.73 (N–CH₃), 43.56 (N–CH₂), 46.21 (N–CH₃), 50.02 (2C, CH₂–N–CH₂), 54.93 (2C, CH₂–N–CH₂), 55.49 (OCH₃), 69.98 (N–CH₂–N), Ar–C: [114.37 (2CH), 127.92 (2CH), 128.06, 129.32 (2CH), 129.89, 130.24 (2CH), 134.29, 158.56], 146.16 (triazole C-5, second ring), 157.37 (triazole C-3), 159.61 (triazole C-5), 168.95 (triazole C-3, second ring); LC–MS/MS m/z (%): 539.00 ([M]⁺, 14), 449.24 (12), 327.41 (63), 323.26 (100), 130.14 (14), 113.21 (72); Anal. Cald. for C₂₆H₃₁ClN₈OS: C, 57.93; H, 5.80; N, 20.79. Found: C, 58.21; H, 5.85; N, 20.63.

4-Methyl-2-[(4-phenylpiperazin-1-yl)methyl]-5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione (4b). Yield (0.53 g, 88.33%); m.p. 138–139 °C; IR (ATR, ν_max, cm⁻¹): 1601, 1580 (C = N), 1316 (C = S), 1168 (N–CH₂–N); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.74 (bs, 4H, CH₂–N–CH₂), 3.06 (bs, 4H, CH₂–N–CH₂), 3.49 (s, 3H, N–CH₃), 3.71 (s, 3H, OCH₃), 4.23 (s, 2H, benzyl CH₂), 5.01 (s, 2H, N–CH₂), 5.74 (s, 2H, N–CH₂–N), Ar–H: [6.75–6.88 (m, 4H), 7.18–7.21 (m, 3H), 7.37–7.45 (m, 4H), 7.92 (d, 2H, J = 8.0 Hz)]; ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.64 (benzyl CH₂), 31.74 (N–CH₃), 43.47 (N–CH₂), 48.69 (2C, CH₂–N–CH₂), 50.20 (2C, CH₂–N–CH₂), 55.47 (OCH₃), 68.30 (N–CH₂–N), Ar–C: [114.36 (2CH), 116.02 (2CH), 119.37 (CH), 127.30 (2CH), 127.99, 128.78 (2CH), 129.34 (2CH), 129.89, 130.25 (2CH), 134.28, 151.45, 158.56], 146.80 (triazole C-5, second ring), 157.40 (triazole C-3), 159.68 (triazole C-5), 169.05 (triazole C-3, second ring); LC–MS/MS m/z (%): 603.47 ([M+2]⁺, 18), 601.71 ([M]⁺, 31), 587.55 (100), 585.60 (84), 571.57 (95), 567.60 (15), 547.59 (15); Anal. Cald. for C₃₁H₃₃ClN₈OS: C, 61.94; H, 5.53; N, 18.64. Found: C, 62.17; H, 5.50; N, 18.43.

4-Phenyl-2-[(4-methylpiperazin-1-yl)methyl]-5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione (4c). Yield (0.51 g, 85.00%); m.p. 253–254 °C; IR (ATR, ν_max, cm⁻¹): 1610, 1585 (C = N), 1325 (C = S), 1178 (N–CH₂–N); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.16 (s, 3H, N–CH₃), 2.32 (bs, 4H, CH₂–N–CH₂), 2.72 (bs, 4H, CH₂–N–CH₂), 3.72 (s, 3H, OCH₃), 3.84 (s, 2H, benzyl CH₂), 5.03 (s, 2H, N–CH₂), 5.50 (s, 2H, N–CH₂–N), Ar–H: [6.85 (d, 2H, J = 8.4 Hz), 7.07 (d, 2H, J = 8.4 Hz), 7.28–7.32 (m, 2H), 7.47–7.51 (m, 5H), 7.84 (d, 2H, J = 8.4 Hz)]; ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.39 (benzyl CH₂), 43.71 (N–CH₂), 46.18 (N–CH₃), 50.07 (2C, CH₂–N–CH₂), 54.97 (2C, CH₂–N–CH₂), 55.53 (OCH₃), 69.21 (N–CH₂–N), Ar–C: [114.38 (2CH), 127.75, 127.81, 127.92 (2CH), 128.29 (2CH), 129.82 (2CH), 129.91 (2CH), 130.15 (2CH), 130.33 (CH), 133.78, 134.22, 158.56], 146.00 (triazole C-5, second ring), 156.99 (triazole C-3), 159.48 (triazole C-5), 169.96 (triazole C-3, second ring); LC–MS/MS m/z (%): 601.46 ([M]⁺, 100), 589.56 (20), 511.28 (48), 489.45 (22); Anal. Cald. for C₃₁H₃₃ClN₈OS: C, 61.94; H, 5.53; N, 18.64. Found: C, 62.17; H, 5.58; N, 18.43.

4-Phenyl-2-[(4-phenylpiperazin-1-yl)methyl]-5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione (4d). Yield (0.59 g, 89.39%); m.p. 162–163 °C; IR (ATR, ν_max, cm⁻¹): 1600, 1563 (C = N), 1328 (C = S), 1179 (N–CH₂–N);¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.88 (bs, 4H, CH₂–N–CH₂), 3.13 (bs, 4H, CH₂–N–CH₂), 3.70 (s, 3H, OCH₃), 3.80 (s, 2H, benzyl CH₂), 5.15 (s, 2H, N–CH₂), 5.49 (s, 2H, N–CH₂–N), Ar–H: [6.79–6.80 (m, 1H), 6.84 (d, 2H, J = 8.4 Hz), 6.92 (d, 2H, J = 8.4 Hz), 7.06 (d, 2H, J = 8.4 Hz), 7.19–7.23 (m, 2H), 7.28–7.30 (m, 2H), 7.38 (d, 2H, J = 8.4 Hz), 7.48–7.50 (m, 3H), 7.82 (d, 2H, J = 8.4 Hz)]; ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.39 (benzyl CH₂), 43.72 (N–CH₂), 48.68 (2C, CH₂–N–CH₂), 48.76 (2C, CH₂–N–CH₂), 55.61 (OCH₃), 69.12 (N–CH₂–N), Ar–C: [114.39 (2CH), 116.04 (2CH), 119.44 (CH), 127.73, 127.86 (2CH), 128.28 (2CH), 129.22 (2CH), 129.37 (2CH), 129.83, 129.89 (2CH), 130.14 (2CH), 130.38 (CH), 133.78, 134.20, 151.53, 158.66], 146.07 (triazole C-5, second ring), 156.94 (triazole C-3), 159.46 (triazole C-5), 170.03 (triazole C-3, second ring); LC–MS/MS m/z (%): 685.50 ([M+Na]⁺, 47), 663.21 ([M]⁺, 32), 651.29 (27), 585.50 (70), 571.50 (32), 393.47 (28), 323.41 (22), 132.08 (100); Anal. Cald. for C₃₆H₃₅ClN₈OS: C, 65.19; H, 5.32; N, 16.89. Found: C, 65.32; H, 5.29; N, 17.00.

4-(4-Chlorophenyl)-2-[(4-methylpiperazin-1-yl)methyl]-5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione (4e). Yield (0.55 g, 85.94%); m.p. 247–248 °C; IR (ATR, ν_max, cm⁻¹): 1611, 1585 (C = N), 1322 (C = S), 1178 (N–CH₂–N); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.13 (s, 3H, N–CH₃), 2.28 (bs, 4H, CH₂–N–CH₂), 2.70 (bs, 4H, CH₂–N–CH₂), 3.71 (s, 3H, OCH₃), 3.93 (s, 2H, benzyl CH₂), 5.00 (s, 2H, N–CH₂), 5.51 (s, 2H, N–CH₂–N), Ar–H: [6.84 (d, 2H, J = 8.8 Hz), 7.05 (d, 2H, J = 8.8 Hz), 7.29 (d, 2H, J = 8.8 Hz), 7.46 (d, 2H, J = 8.8 Hz), 7.51 (d, 2H, J = 8.8 Hz), 7.80 (d, 2H, J = 8.4 Hz)]; ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.43 (benzyl CH₂), 43.76 (N–CH₂), 46.23 (N–CH₃), 50.06 (2C, CH₂–N–CH₂), 54.98 (2C, CH₂–N–CH₂), 55.50 (OCH₃), 69.26 (N–CH₂–N), Ar–C: [114.36 (2CH), 127.62, 127.89 (2CH), 129.22 (2CH), 129.77, 129.89 (2CH), 130.10 (2CH), 130.28 (2CH), 132.63, 134.20, 135.04, 158.54], 145.87 (triazole C-5, second ring), 156.91 (triazole C-3), 159.29 (triazole C-5), 169.85 (triazole C-3, second ring); LC–MS/MS m/z (%): 637.26 ([M+2]⁺, 72), 635.25 ([M]⁺, 100), 623.54 (12), 587.55 (15); Anal. Cald. for C₃₁H₃₂Cl₂N₈OS: C, 58.58; H, 5.07; N, 17.63. Found: C, 58.77; H, 4.99; N, 17.60.

4-(4-Chlorophenyl)-2-[(4-phenylpiperazin-1-yl)methyl]-5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione (4f). Yield (0.65 g, 92.86%); m.p. 162–163 °C; IR (ATR, ν_max, cm⁻¹): 1601, 1584 (C = N), 1326 (C = S), 1170 (N–CH₂–N); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.86 (bs, 4H, CH₂–N–CH₂), 3.10 (bs, 4H, CH₂–N–CH₂), 3.71 (s, 3H, OCH₃), 3.92 (s, 2H, benzyl CH₂), 5.11 (s, 2H, N–CH₂), 5.51 (s, 2H, N–CH₂–N), Ar–H: [6.75–6.78 (m, 1H), 6.83 (d, 2H, J = 8.4 Hz), 6.89 (d, 2H, J = 8.0 Hz), 7.05 (d, 2H, J = 8.8 Hz), 7.16–7.20 (m, 2H), 7.31 (d, 2H, J = 8.8 Hz) 7.39 (d, 2H, J = 8.4 Hz), 7.52 (d, 2H, J = 8.4 Hz), 7.79 (d, 2H, J = 8.8 Hz)]; ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.43 (benzyl CH₂), 43.77 (N–CH₂), 48.74 (2C, CH₂–N–CH₂), 50.29 (2C, CH₂–N–CH₂), 55.48 (OCH₃), 69.16 (N–CH₂–N), Ar–C: [114.34 (2CH), 116.04 (2CH), 119.44 (CH), 127.60, 127.83 (2CH), 129.22, 129.35 (2CH), 129.75, 129.92 (2CH), 130.10 (2CH), 130.28 (2CH), 132.63, 133.78, 134.18, 135.08, 151.50, 158.53], 145.94 (triazole C-5, second ring), 156.91 (triazole C-3), 159.31 (triazole C-5), 169.94 (triazole C-3, second ring); LC–MS/MS m/z (%): 699.18 ([M+2]⁺, 64), 697.36 ([M]⁺, 32), 685.25 (100), 677.59 (15), 635.47 (12), 591.46 (25), 585.40 (26); Anal. Cald. for C₃₆H₃₄Cl₂N₈OS: C, 61.98; H, 4.91; N, 16.06. Found: C, 62.17; H, 5.00; N, 15.92.

5-{[(3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl)]methyl]}-3-[(4-methylpiperazin-1-yl)methyl]-1,3,4-oxadiazole-2(3H)-thione (6a). Yield (0.35 g, 66.04%); m.p. 87–88 °C; IR (ATR, ν_max, cm⁻¹): 1611, 1586 (C = N), 1339 (C = S), 1177 (N–CH₂–N); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.18 (s, 3H, N–CH₃), 2.70 (bs, 4H, CH₂–N–CH₂), 2.86 (bs, 4H, CH₂–N–CH₂), 3.70 (s, 3H, OCH₃), 4.19 (s, 2H, benzyl CH₂), 5.33 (s, 2H, N–CH₂), 5.49 (s, 2H, N–CH₂–N), Ar–H: [6.87 (d, 2H, J = 8.8 Hz), 7.23 (d, 2H, J = 8.8 Hz), 7.45 (d, 2H, J = 8.8 Hz), 7.92 (d, 2H, J = 8.8 Hz)]; ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.57 (benzyl CH₂), 43.63 (N–CH₂), 46.21 (N–CH₃), 49.91 (2C, CH₂–N–CH₂), 51.47 (2C, CH₂–N–CH₂), 55.47 (OCH₃), 69.78 (N–CH₂–N), Ar–C: [114.32 (2CH), 127.93 (2CH), 128.43, 128.54, 129.23 (2CH), 130.37 (2CH), 134.20, 158.57], 157.13 (triazole C-3), 157.80 (oxadiazole C-5), 159.57 (triazole C-5), 171.15 (oxadiazole C-2); LC–MS/MS m/z (%): 526.49 ([M]⁺, 8), 524.48 (10), 496.57 (13), 460.22 (38), 458.34 (100), 450.33 (41), 438.51 (18); Anal. Cald. for C₂₅H₂₈ClN₇O₂S: C, 57.08; H, 5.36; N, 18.64. Found: C, 57.21; H, 5.27; N, 18.52.

5-{[(3-(4-Chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl)]methyl]}-3-[(4-phenylpiperazin-1-yl)methyl]-1,3,4-oxadiazole-2(3H)-thione (6b). Yield (0.48 g, 81.36%); m.p. 118–119 °C; IR (ATR, ν_max, cm⁻¹): 1610, 1599 (C = N), 1326 (C = S), 1175 (N–CH₂–N); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.77 (bs, 4H, CH₂–N–CH₂), 3.07 (bs, 4H, CH₂–N–CH₂), 3.70 (s, 3H, OCH₃), 4.23 (s, 2H, benzyl CH₂), 4.92 (s, 2H, N–CH₂), 5.74 (s, 2H, N–CH₂–N), Ar–H: [6.74–6.83 (m, 3H), 6.85–6.90 (m, 2H), 7.16–7.22 (m, 4H), 7.48 (d, 2H, J = 8.0 Hz), 7.94 (d, 2H, J = 8.4 Hz)];¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.42 (benzyl CH₂), 43.58 (N–CH₂), 48.66 (2C, CH₂–N–CH₂), 49.91 (2C, CH₂–N–CH₂), 55.45 (OCH₃), 70.06 (N–CH₂–N), Ar–C: [114.29 (2CH), 116.09 (2CH), 119.49 (CH), 127.81 (2CH), 127.95, 129.35 (2CH), 129.73, 130.23 (2CH), 130.44 (2CH), 134.40, 151.42, 158.56], 157.64 (triazole C-3), 159.35 (oxadiazole C-5), 159.81 (triazole C-5), 178.27 (oxadiazole C-2); LC–MS/MS m/z (%): 588.37 ([M]⁺, 11), 578.62 (21), 558.42 (15), 548.41 (43), 546.46 (100); Anal. Calcd. for C₃₀H₃₀ClN₇O₂S: C, 61.27; H, 5.14; N, 16.67. Found: C, 61.41; H, 5.10; N, 16.53.

Synthesis of 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-1,3,4-oxadiazole-2(3H)-thione (5)

To solution of the compound 1 (0.01 mol) in ethanol (25 mL) at 0 °C, CS₂ (6 mL, 0.01 mol) and a solution of KOH (0.56 g, 0.001 mol) in 50 mL H₂O and 50 mL ethanol were added, and the reaction mixture was refluxed for about 6 h until the evolution of H₂S gas ceased. Excess solvents were evaporated under reduced pressure to dryness, a solid was obtained. This was dissolved in 200 mL H₂O and acidified with conc. HCl to pH ∼5. The precipitate was filtered off, washed with H₂O and recrystallized from ethanol to afford the desired compound. Yield (3.12 g, 75.36%); m.p. 180–181 °C; IR (ν_max, cm⁻¹): 3294 (NH), 1610, 1586 (C = N), 1323 (C = S); ¹H NMR (DMSO-d₆, 400 MHz) δ (ppm): 3.71 (s, 3H, OCH₃), 4.23 (s, 2H, benzyl CH₂), 5.72 (s, 2H, N–CH₂), Ar–H: [6.84 (d, 2H, J = 8.2 Hz), 7.19 (d, 2H, J = 8.6 Hz), 7.50 (d, 2H, J = 8.2 Hz), 7.95 (d, 2H, J = 8.6 Hz)], 13.83 (bs, 1H, NH); ¹³C NMR (DMSO-d₆, 100 MHz) δ (ppm): 30.64 (benzyl CH₂), 43.62 (N–CH₂), 55.71 (OCH₃), Ar–C: [114.62 (2CH), 128.09, 128.21 (2CH), 129.63 (2CH), 129.95, 130.45 (2CH), 134.64, 158.80], 157.92 (triazole C-3), 159.00 (triazole C-5), 160.12 (oxadiazole C-5), 178.55 (oxadiazole C-2); LC–MS/MS m/z (%): 436.31 ([M+Na]⁺, 100), 412.40 ([M−1]⁺, 81), 327.31 (36), 174.39 (31), 130.35 (79); Anal. Cald. for C₁₉H₁₆ClN₅O₂S: C, 55.14; H, 3.90; N, 16.92. Found: C, 55.34; H, 4.07; N, 16.87.

Biological assays

Anti-lipase activity assay

The inhibitory effects of those compounds were evaluated against Porcine Pancreatic Lipase (Applichem, Germany) (15 ng/mL). Lipase activity assay were done according to Kurihara et al45. The lipase activity was measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate. Briefly, compounds were mixed with Porcine Pancreatic Lipase (PPL) 1:3 (v/v) and incubated for 30 min. The microtiter plates containing, 50 µL 0.1 mM 4-MU oleate, 25 µL diluted compound-lipase solution, 25 µL dH₂O and assay buffer (13 mM Tris–HCl, 150 mM NaCl and 1.3 mM CaCl₂, pH 8.0) were incubated at 37 °C for 20 min. After incubation, in order to stop the reaction, 0.1 mL 0.1 M pH 4.2 citrate buffer was added reaction mixture. The amount of 4-methylumbelliferone released by the lipase was measured by using a spectroflourometer (SpectraMax M5, Molecular Devices) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The inhibitory activity of those compounds and Orlistat (Xenical, Hoffman, La Roche, Segrate, Italy), an inhibitor control of pancreatic lipase, was measured at various concentration. Residual activities were calculated by comparing to control without inhibitor. The assays were done in triplicate. The IC₅₀ value was determined as the concentration of compound that give 50% inhibition of maximal activity.

α-Glucosidase ınhibition assay

α-Glucosidase inhibition assay was performed spectrophotometrically46. α-Glucosidase from Saccharomyces cerevisiae (Sigma-Aldrich, St. Louis, MO) was dissolved in phosphate buffer (pH 6.8, 50 mM). Test compounds were dissolved in DMSO. In 96-well microtiter plates, 20 μL of test sample, 20 μL of enzyme (20 mU/mL) and 135 μL of buffer were added and incubated for 15 min at 37 °C. After incubation, 25 μL of p-nitrophenyl-α-d-glucopyranoside (2 mM, Sigma Aldrich) was added and change in absorbance was monitored for 30 min at 400 nm. Test compound was replaced by DMSO (7.5% final) as control. Acarbose (Sigma-Aldrich) was used as a standard inhibitor46.

Conclusion

In this current study, new 2-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetyl}-4-alkyl/arylthiosemicarbazides, 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thiones, 5-{[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]methyl}-1,3,4-oxadiazole-2(3H)-thione were synthesized starting from 2-[3-(4-chlorophenyl)-5-(4-methoxybenzyl)-4H-1,2,4-triazol-4-yl]acetohydrazide. In addition to these, new N-Mannich bases of 1,2,4-triazole-3-thiones and 1,3,4-oxadiazole-2(3H)-thione were synthesized.

The newly synthesized compounds were tested in vitro for lipase and α-glucosidase inhibitory activity. Among the tested compounds 2a (IC₅₀ = 10.25 ± 0.40 µM), 2c (IC₅₀ = 2.50 ± 0.50 µM)and 6a (IC₅₀ = 42.21 ± 4.54 µM) showed anti-lipase activities. In addition, compounds 2c (IC₅₀ = 3.41 ± 0.16 µM), 6a (IC₅₀ = 4.36 ± 0.10 µM) and 6b (IC₅₀ = 17.68 ± 1.10 µM) showed the high anti-α-glucosidase activity. Compounds 2c and 6a showed the best anti-α-glucosidase activity. These compounds inhibited α-glucosidase activity by 99.79 ± 1.17% and 99.87 ± 0.38% at a concentration of 100 µM, respectively. Based on the obtained results, compounds 2c and 6a can be considered as lead in the search of alternative drugs to Orlistat and Acarbose.

Declaration of interest

The support provided by Karadeniz Technical University, BAP, Turkey (Project No. 10020) is gratefully acknowledged.