Novel indole-based melatonin analogues substituted with triazole, thiadiazole and carbothioamides: studies on their antioxidant, chemopreventive and cytotoxic activities

Abstract

Melatonin (MLT) is a well-known free-radical scavenger, involving in the prevention of cellular damage that can lead to cancer, ageing and a variety of neurodegenerative diseases. Research on MLT-related compounds has been required to optimise the maximum pharmaceutical activity with the lowest side effects. In our ongoing research, we have synthesized new indole-based MLT analogues as potential antioxidant agents by modifying the MLT molecule. In this study, we build on previous findings, through the synthesis, characterization and in vitro antioxidant profiling of a series of new indole-based MLT analogues which possess triazole, thiadiazole and carbothioamides on the third position on the indole ring. In vitro antioxidant activity was investigated by evaluating their reducing effect against oxidation of a redox sensitive fluorescent probe and their radical scavenging activity was assessed via the DPPH assay. In addition, in vitro cytotoxic effects of newly synthesized compounds were investigated in CHO-K1 cells using the MTT assay.

• Antioxidant
• antioxidant activity
• cytotoxicity
• indole
• melatonin

Introduction

Reactive oxygen species (ROS), and reactive nitrogen species (RNS), are products of cellular metabolism, and are well known for having both a harmful and beneficial role1,2. Overproduction of reactive species causes oxidative stress3 which may lead to serious damage to vital cell structures like proteins and DNA4,5. Oxidative stress has been associated with various diseases such as cancer6, heart diseases7, neurological disorders such as Alzheimer’s disease8, Huntington’s disease9,10 and ageing11. Melatonin (MLT) is a hormone primarily secreted by the pineal gland along with many other parts of the human body11–13 and has a significant anti-oxidant property14. MLT (Figure 1) and its metabolites are able to function as endogenous free-radical scavengers and broad-spectrum antioxidants15,16. In addition, MLT and its derivatives have been shown to exhibit the regulation of circadian rhythm and immune functions in many physiological processes and therapeutic functions17,18. In recent years, the many physiological properties of MLT have stimulated much interest in the development of synthetic compounds possessing the indole ring. In addition MLT is a highly lipophilic compound that easily passes through cell membranes to reach intracellular compartments, especially mitochondria which are well-known for having high concentrations of ROS. Also MLT acts in the fixation of oxidant-antioxidant balance in mitochondria19,20. However, MLT has very short half-life in the human body, of approximately 1 h in blood following oral administration. Therefore, the synthesis of MLT analogues with longer half-lives is an interesting topic for researchers20,21.

Figure 1. Modifications on MLT molecule to develop new indole-based analogues.

Recent research has shown that compounds with an indole ring such as indol-3-propionic acid22, indole amine triazole23 and indolehydrazide/hydrazone derivatives24,25 have significant antioxidant effects. The indole ring of these compounds is the reactive centre dealing with oxidants due to its high resonance stability and low activation energy barrier towards free radical reactions24,26. In addition to these compounds, there are some studies that demonstrate the antioxidant effect of 1,2,4-triazoles and 1,3,4-thiadiazoles27–29. The triazole molecule is one of the most significant and well recognized heterocycles which is a common feature of a selection of natural products as well as pharmaceutical agents. Triazoles and their derivatives were investigated in many studies and established to be associated with a variety of biological activities like antibacterial30, antifungals31, antitumor, anti-inflammatory, analgesic32 and antioxidant33. In addition this 1,3,4-thiadiazoles have also been found to be key group of molecules with biological activity particularly antibacterial and antioxidant34. The broad spectrum activities of 1,2,4-triazoles derivatives have attracted the attention of researchers in recent years. Until now, studies showed that modifications of the triazole derivatives have proven highly efficient with better potency and lesser toxicity. Based on the above findings it was planed and synthesized some indole derivatives with 1,2,4-triazole and 1,3,4-thiadiazole with different side chains to improve biological activity of indole derivatives.

In the present study, some modifications were made on the MLT molecule to improve its antioxidant effect. The acetylaminoethyl side chain in the third position of the indole ring was removed and substituted with hydrazinecarbothioamide, triazole and thiadiazole groups. As a part of our ongoing study 31 new compounds were synthesized using four different routes. Synthesis of 1a–h and 3a–h compounds demonstrated high yield with average of 87%. However, synthesis of 4a–g and 2a–h compounds showed low yield, respectively, with 28% and 37%. Biological activity of new synthesis compounds was investigated in vitro. The MTT assay was performed to evaluate their cytotoxic profiles, the DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) assay was carried out for revealing the antioxidant activity of the newly synthesized compounds, and the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity test. All the synthesized compounds except those formerly synthesized (2d35, 2h36, 3c36, 3d36) were characterized on the basis of ¹H and ¹³C NMR, mass spectra and elemental analysis.

Materials and methods

The present study aimed to synthesize, characterize and investigate the potential antioxidant and cytotoxic effects of indole-based MLT analogues containing hydrazinecarbothioamide, triazole and thiadiazoles as side chains attached to the 3-position of the indole ring. Two parts of the MLT molecule were modified to develop new indole-based MLT analogue compounds as shown in Figure 1. These modifications were done mainly on the MLT acylamino group (Figure 1).

These modulations of the lead structures were made at two different points: the methoxy group was replaced with hydrogen at the 5-position of the indole ring as modification 1 and the acetylaminoethyl side chain was replaced by hydrazinecarbothioamide (1a–h), thiadiazole (2a–h) and triazole (3a–h) groups as modification 2. Finally, S-alkylation on the compounds (3a–h) provided analogues (4a–g) (Table 1). These modifications resulted in a new series of compounds having different heterocyclic groups as side chain in the indole nucleus. These modifications helped to investigate the effect of substituents with different electronic and lipophilic properties of heterocyclic groups on the antioxidant activity and biological half-life of new indole derivatives. Synthetic routes to new MLT analogues are outlined in Scheme 1. Reaction of indole 3-methylhydrazine with isothiocyanates in ethanol under reflux gave the corresponding hydrazinecarbothioamides (1a–h) in high yields. Treatment of 1a–h under strongly acidic conditions proceeded with full regiochemical control to gives the corresponding 2-aminothiadiazoles (2a–h), albeit in low to moderate yields. Conversely, treatment of 1a–h under basic conditions (aq. NaOH) with heating produced the 2-thiotriazoles (3a–h) in high yields with full control of cyclization regiochemistry. Triazoles (3a–h) could be further alkylated under basic conditions to produce substituted triazole 4a–g in low to moderate yield.

Scheme 1. Synthetic route to obtain new indole-based melatonin analogue compounds.

Chemistry – experimental

Uncorrected melting points were determined with a Büchi melting point B-540 apparatus (Buchi Labortechnik, Flawil, Switzerland). The ¹H and ¹³C NMR spectra were measured with a Varian 400 MHz instrument (Palo Alto, CA) using TMS as an internal standard and DMSO-d₆ as solvent. ESI mass spectra were determined on a Waters Micromass ZQ. Elemental analyses were performed using a CHNS-932 instrument (Leco Corporation, St. Joseph, MI). All spectral analysis was performed at Central Laboratory of the Faculty of Pharmacy, Ankara University. Chromatography was carried out using Merck silica gel 60 (230–400 mesh ASTM). The chemical reagents used in synthesis were purchased from Sigma (Germany) and Aldrich (USA).

General procedure for the synthesis of hydrazinecarbothioamide compounds 1a–h

Equal amounts of 2-(1H-indol-3-yl) acetohydrazide (5 mmol) and aryl or alkylisothiocyanates (5 mmol) were dissolved in absolute ethanol (20 ml). The mixture was heated under reflux for 4–5 h at 80–85 °C. After completion of the reaction, the reaction mixture was concentrated on the rotary evaporator under reduced pressure and kept overnight at room temperature. The crystals thus obtained were purified by washing with petroleum ether35.

2-(2-(1H-Indol-3-yl)acetyl)-N-ethylhydrazincarbothioamide (1a). Yield 95%, m.p. 132 °C; ¹H NMR: δ 1.01 (t, 3H, J = 7.6 Hz, CH₃); 3.42 (q2H, CH₂); 3.55 (s, 2H, Ar–CH₂); 6.96 (m, 1H, H-5); 7.05 (m, 1H, H-6); 7.21 (d, 1H, J = 2.4 Hz, H-2); 7.32 (d, 1H, J = 8 Hz, H-7); 7.55 (d, 1H, J = 7.6 Hz, H-4); 7.78, 9.16, 9.84, 10.88 (s, 4H, NH–NH–CS–NH and NH indole); ¹³C NMR: δ 14.87, 31.07, 38.85, 108.44, 110.00, 111.73, 118.76, 119.19, 121.42, 124.38, 127.65, 136.46, 170.84 (C=O); ESI MS m/z 277 (M + H, 100%); Anal. calcd. for C₁₃H₁₆N₄OS: C, 55.59%; H, 5.92%; N, 19.95%. Found: C, 55.21%; H, 6.24%; N, 19.66%.

2-(2-(1H-Indol-3-yl)acetyl)-N-propylhydrazincarbothioamide (1b). Yield 86%, m.p. 143.5 °C; ¹H NMR: δ 0.80 (t, 3H, J = 7.2 Hz, CH₃); 1.46 (m, 2H, CH2); 3.37 (t, 2H, J = 7.6 Hz, NH–CH₂); 3.57 (s, 2H, COCH₂); 6.98 (t, 1H, J = 7.6 Hz, H-5); 7.07 (t, 1H, J = 8.4 Hz, H-6); 7.23 (d, 1H, J = 2.4 Hz, H-2); 7.34 (d, 1H, J = 8 Hz, H-7); 7.57 (d, 1H, J = 7.6 Hz, H-4); 7.75; 9.18; 9.87; 10.90; (s, 4H, NH-NH-CS-NH and NH-indole); ¹³C NMR: δ 11.60, 22.40, 31.10 45.72 (NH–C); 108.46, 111.75, 118.77, 119.19, 121.44, 124.40, 127.65, 136.49, 170.86 (C=O), 175.28 (C=S); ESI MS m/z 291 (M + H, 100%); Anal. calcd. for C₁₄H₁₈N₄OS: C, 57.91%; H, 6.24%; N, 19.29%. Found: C, 58.19%; H, 6.75%; N, 19.14%.

2-(2-(1H-Indol-3-yl)acetyl)-N-benzylhydrazincarbothioamide (1c). Yield 78%, m.p. 142.5 °C; ¹H NMR: δ 3.58 (s, 2H, COCH₂); 4.74 (d, 2H, J = 5.6 Hz, benzyl-CH₂); 6.96 (m, 1H, H-5); 7.06 (m, 1H, H-6); 7.23 (d, 1H, J = 2.4 Hz, H-2); 7.21–7.35 (m, 6H, Ar–H); 7.56 (d, 1H, J = 8 Hz, H-4); 8.44, 9.35, 9.94, 10.88 (s, 4H, NH–NH–CS–NH and NH-indole); ¹³C NMR: δ 30.50; 46.54 (NH-C); 107.84; 111.15; 118.17; 118.68; 120.84; 123.80; 126.50; 126.88, 127.15, 127.94, 135.91, 139.20, 170.36 (C=O) ESI MS m/z 339 (M + H, 100%); Anal. calcd. for C₁₈H₁₈N₄OS: C, 62.55%; H, 5.49; N, 16.21%. Found: C, 63.07%; H, 6.01%; N, 16.68%.

2-(2-(1H-Indol-3-yl)acetyl)-N-phenylhydrazincarbothioamide (1d). Yield 90%, m.p. 178.5 °C; ¹H NMR: δ 3.63 (s, 2H, COCH₂); 6.98 (m, 1H, H-5); 7.07 (m, 1H, H-6); 7.26 (d, 1H, J = 2.4 Hz, H-2); 7.60 (d, 1H, J = 7.6, H-4); 7.14–7.41 (m, 6H, Ar–H); 9.54, 9.64, 10.11, 10.91 (s, 4H, NH–NH–CS–NH and NH indole); ¹³C NMR: δ 31.17, 108.45, 110.00, 111.75, 118.80, 119.27, 121.44, 124.45, 125.44, 126.04, 127.71, 128.55, 136.50, 139.57, 167.71, 171.01 (C=O), 181.33 (S=C); ESI MS m/z 325 (M + H, 100%); Anal. calcd. for C₁₇H₁₆N₄OS: C, 62.94%; H, 4.97%; N, 17.27%. Found: C, 63.05%; H, 5.33%; N, 17.05%.

2-(2-(1H-Indol-3-yl)acetyl)-N-(2,4-difluorophenyl)hydrazincarbothioamide (1e). Yield 81%, m.p. 169 °C; ¹H NMR: δ 3.64 (s, 2H, Ar–CH₂); 6.98–7.61 (m, 8H, Ar–H); 9.60 (s, 1H, NH indole); ESI MS m/z 361 (M + H, 100%); Anal. calcd. for C₁₇H₁₄F₂N₄OS: C, 56.66%, H, 3.92%, N, 15.55%. Found: C, 56.68%, H, 4.14%, N, 15.66%.

2-(2-(1H-Indol-3-yl)acetyl)-N-(3-fluorophenyl)hydrazincarbothioamide (1f). Yield 92%, m.p. 183.5 °C; ¹H NMR: δ 3.63 (s, 2H, COCH₂); 6.97 (t, 1H, J = 6.8 Hz, H-5); 7.07 (t, 1H, J = 7.2 Hz, H-6); 7.23–7.61 (m, 7H, Ar-H); 9.60, 9.78, 10.13, 10.90, (s, 4H, NH–NH–CS–1NH and NH indole); ¹³C NMR: δ 31.15, 108.38, 111.76, 118.75, 119.25, 121.41, 124.46, 127.68, 130.10, 136.50, 141.33, 141.44, 160.76, 163.15, 164.568, 165.87, 167.30, 168.76, 170.22 (C=O); 175.49, 181.17 (C=S); ESI MS m/z 343 (M + H, 100%); Anal. calcd. for C₁₇H₁₅FN₄OS: C, 59.63%; H, 4.42%; N, 16.36%. Found: C, 59.91%; H, 4.51%; N, 16.51%.

2-(2-(1H-Indol-3-yl)acetyl)-N-(2,4-dichlorophenyl)hydrazincarbothioamide (1g). Yield 91%, m.p. 172.5 °C; ¹H NMR: δ 3.62 (s, 2H, COCH₂); 6.97 (t, 1H, J = 7.2 Hz, H-5); 7.07 (t, 1H, J = 6.8 Hz, H-6); 7.59 (d, 1H, J = 8 Hz, H-4); 7.25–7.68 (m, 5H, Ar–H); 9.47, 9.87, 10.20, 10.90 (s, 4H, NH–NH–CS–NH and NH indole); ¹³C NMR: δ 31.01, 108.31, 111.71, 118.72, 119.29, 121.41, 124.45, 127.73, 129.23, 132.34, 136.46, 171.07 (C=O); 182.14 (C=S); ESI MS m/z (M⁺, 100%); 395 (M + 2, 85%); 397 (M + 4, 38%); Anal. calcd. for C₁₇H₁₄Cl₂N₄OS: C, 51.92%; H, 3.59%; N, 14.25%. Found: C, 52.30%; H, 3.92%; N, 14.51%.

2-(2-(1H-Indol-3-yl)acetyl)-N-(3-chlorophenyl)hydrazincarbothioamide (1h). Yield 84%, m.p. 168 °C; ¹H NMR: δ 3.64 (s, 2H, COCH₂); 6.98 (t, 1H, J = 6.8 Hz, H-5); 7.07 (t, 1H, J = 7.2 Hz, H-6); 7.21 (d, 1H, J = 7.2 Hz, H-2); 7.60 (d, 1H, J = 1.6 Hz, H-4); 7.26–7.44 (m, 5H, Ar–H); 9.63, 9.81, 10.13, 10.91 (s, 4H, NH–NH–CS–NH and NH indole); ¹³C NMR: δ 25.81, 31.15, 108.35, 111.74, 118.78, 119.24, 121.44, 124.45, 125.18, 127.68, 130.13, 136.49, 141.12, ESI MS m/z 359.5 (M + H, 72%), 361 (M + H + 2, 28%); Anal. calcd. for C₁₇H₁₅N₄SOCl: C, 56.90%; H, 4.21%; H, 15.61%. Found: C, 57.26%; H, 4.41%; N, 15.31%.

General procedure for the synthesis of compounds 2a–h

Conc. sulfuric acid (3 ml) was placed in a conical flask, and 2-(1H-Indol-3-yl-acetyl)-N-(aryl or alkyl)hydrazincarbothioamide (1 mmol) was added in small portions over a period of 2 h under stirring while maintaining the temperature at 0–5 °C. When the reaction was completed the mixture was poured into crushed ice and neutralized with 2 N NH₄OH dropwise until the pH was adjusted to 7. The formed precipitate was filtered, washed with water, dried at room temperature and recrystallized from absolute ethanol35.

5-((1H-Indol-3-yl)methyl)-2-ethylamino-1,3,4-thiadiazole (2a). Yield 42%, m.p. 125 °C; ¹H NMR: δ 1.12 (t, 3H, J = 6.8 Hz, CH₃); 3.21 (q, 2H, CH₂); 4.26 (s, 2H, Ar–CH₂);6.98 (m, 1H, H-5); 7.09 (m, 1H, H-6); 7.30 (d, 1H, J = 2.4, H-2); 7.36 (d, 1H, J = 8 Hz, H-7); 7.46 (d, 1H, J = 8 Hz, H-4); 7.73 (s, 1H, NH); 11.01 (s, 1H, NH indole); ¹³C NMR: δ 14.62, 26.51, 39.31(NH–C); 110.94, 112.02, 118.76, 119.11, 121.73, 124.25, 126.99, 136.75, 159.29, 168.89 (S–C–N); ESI MS m/z 259 (M + H, 100%), 300 (M + H + CH₃CN, 76%); Anal. calcd. for C₁₃H₁₄N₄S: C, 57.43%, H, 5.75%, N, 20.61%. Found: C, 57.55%, H, 5.66%, N, 20.23%.

5-((1H-Indol-3-yl)methyl)-2-propylamino-1,3,4-thiadiazole (2b). Yield 44%, m.p. 148 °C; ¹H NMR: δ 0,86 (t, 3H, J = 7.6 Hz, CH₃); 1.52 (m, 2H, CH₂); 3.15 (q, 2H, NH–CH₂); 4.26 (s, 2H, Ar–CH₂); 6.98 (t, 1H, J = 8 Hz, H-5); 7.09 (t, 1H, J = 8 Hz, H-6); 7.31 (d, 1H, J = 2.4 Hz, H-2); 7.36 (d, 1H, J = 8 Hz, H-7); 7.46 (d, 1H, J = 8.4 Hz, H-4); 7,82 (s, 1H, NH); 11.00 (s, 1H, NH-indole); ¹³C NMR: δ 11.78, 22.16, 26.51, 47.05 (NH–C); 110.91, 112.03, 118.77, 119.12, 121.74, 124.29, 127.01, 136.76, 159.22, 169.06 (N–C–S); ESI MS m/z (M + H, 100%), 314 (M + H + CH₃CN, 100%); Anal. calcd. for C₁₄H₁₆N₄S: C, 51.51%; H, 6.79%; N, 17.16%. Found: C, 51.23%; H, 5.57%; N, 16.78%.

5-((1H-Indol-3-yl)methyl)-2-amino-1,3,4-thiadiazole (2c). Yield 39%, m.p. 177 °C; ¹H NMR: δ 4.22 (s, 2H, Ar–CH₂); 6.94 (bs, 2H, NH₂); 6.97 (dt, 1H, J₁=8.4, J₂ =1.2 Hz, H-5); 7.07 (dt, 1H, J₁=8.4, J₂=1.2 Hz H-6); 7.27 (d,1H, J = 2.4 Hz, H-2); 7.35 (d, 1H, J = 8 Hz, H-7); 7.45 (d, 1H, J = 8 Hz, H-4); 10.97 (s, 1H, NH-indole); ¹³C NMR: δ 25.98, 110.98, 111.42, 118.24, 118.48, 121.11, 123.52, 126.47, 136.18, 159.21, 168.41 (N–C–S); ESI MS m/z (M + H, 37%), 272 (M + H + CH₃CN, 100%); Anal. calcd. for C₁₁H₁₀N₄S: C, 55.21%, H, 4.63%, N, 23.41%. Found: C, 54.90%, H, 4.58%, N, 23.35%.

5-((1H-Indol-3-yl)methyl)-2-(2,4-difluorophenyl)amino-1,3,4-thiadiazole (2e). Yield 26%, m.p. 141 °C; ¹H NMR: δ 4.34 (s, 2H, Ar–CH₂); 6.70–8.33 (m,8H, Ar–H); 9.89 (s, 1H, NH); 11.01 (s, 1H, NH indole); ESI MS m/z 343 (M + H, 100%), 384 (M + H + CH₃CN, 44%); Anal. calcd. for C₁₇H₁₂ F₂N₄S: C, 58.11%, H, 3.73%, N, 15.94%. Found: C, 57.98%, H, 3.63%, N, 15.80%.

5-((1H-Indol-3-yl)methyl)-2-(3-fluorophenyl)amino-1,3,4-thiadiazole (2f). Yield 22%, m.p. 183 °C; ¹H NMR: δ 4.36 (s, 2H, Ar-CH₂); 6.73–7.63 (m, 9H, Ar-H); 10.36 (s, 1H, NH); 11.01 (s, 1H, NH indole); ¹³C NMR: δ 26.36; 104.38; 104.64; 108.28; 110.94; 112.06; 113.51; 118.76; 119.18; 121.77; 124.34; 126.97; 131.02; 136.77; 124.75; 161.88; 164.25 (S–C–N); ESI MS m/z 325 (M + H, 100%), 366 (M + H + CH₃CN, 33%); Anal. calcd. for C₁₇H₁₃FN₄S: C, 61.24%, H, 4.23%, N, 16.81%. Found: C, 61.16%, H, 4.07%, N, 16.69%.

5-((1H-Indol-3-yl)methyl)-2-(2,4-dichlorophenyl)amino-1,3,4-thiadiazole (2g). Yield 50%, m.p. 112 °C; ¹H NMR: δ 4.35 (s, 2H, Ar–CH₂); 6.97 (m, 1H, H-5); 7.07 (m, 1H, H-6); 7.39 (d, 1H, J = 2.4 Hz, H-7); 7.48 (d, 1H, J = 7.6 Hz, H-4); 7.21–7.59 (m, 4H, Ar-H); 8.33 (d, 1H, J = 9.2 Hz, NH); 11.02 (s, 1H, NH indole);¹³C NMR: δ 25.78; 110.38; 111.51; 118.22; 118.61; 121.21; 121.89; 122.74; 123.81; 126.08; 126.38; 127.74; 128.76; 136.21; 136.48; 162.66; 164.11 (S–C–N); ESI MS m/z 375 (M⁺, 100%), 377 (M + 2, 68%), 379 (M + 4, 13%), 416 (M⁺+CH₃CN, 77%); Anal. calcd. for C₁₇H₁₂Cl₂N₄S: C, 49.43%, H, 3.95%, N, 13.56%. Found: C, 49.03%, H, 3.54%, N, 14.03%.

General procedure for the synthesis of compounds 3a–h

2-(1H-Indol-3-yl-acetyl)-N-(aryl or alkyl)hydrazincarbothioamide (2 mmol) and 2 N NaOH solution (25 ml) were placed in conical flask. The mixture was heated under reflux for 4–5 h. After completion of the reaction, the reaction mixture was neutralized with 2 N HCl dropwise till pH was adjusted to 7. The mixture was kept aside for a few minutes. The precipitate thus obtained was filtered, washed with water and recrystallized from a mixture of ethanol/water (4:1)35.

5-((1H-Indol-3-yl)methyl)-4-ethyl-4H-1,2,4-triazole-3-thiol (3a). Yield 91%, m.p. 191.5 °C; ¹H NMR: δ 0.92 (t, 3H, J = 7.6 Hz, CH₃); 3.90 (q, 2H, CH₂); 4.20 (s, 2H, Ar–CH₂); 6.98 (dt, 1H, J₁=7.2, J₂=0.8 Hz, H-5); 7.09 (dt, 1H, J₁=7.2, J₂=1.2 Hz H-6); 7.33 (d, 1H, J = 2.4 Hz, H-2); 7.37 (d, 1H, J = 8 Hz, H-7); 7.49 (d, 1H, J = 7.6 Hz, H-4); 11.05 (s, 1H, NH indole); 13.54 (s, 1H, SH); ¹³C NMR: δ 13.31; 22.34; 38.72; 107.70; 112.057; 118.73; 119.16; 121.76; 124.61; 127.14; 136.70; 151.70; 166.67 (N–C–S); ESI MS m/z 259 (M + H, 100%), 300 (M + H + CH₃CN, 8%); Anal. calcd. for C₁₃H₁₄N₄S: C, 57.63%, H, 5.73%, N, 20.68%. Found: C, 57.93%, H, 5.96%, N, 20.51%.

5-((1H-Indol-3-yl)methyl)-4-propyl-4H-1,2,4-triazole-3-thiol (3b). Yield 76%, m.p. 151.5 °C; ¹H NMR: δ 0.73 (t, 3H, J = 7.6 Hz, CH₃); 1.35 (m, 2H, CH₂); 3.78 (t, 2H, J = 7.6 Hz, NH–CH₂); 4.19 (s, 2H, Ar–CH₂); 6.98 (t, 1H, J = 7.2 Hz, H-5); 7.09 (t, 1H, J = 7.2 Hz, H-5); 7.33 (d, 1H, J = 2 Hz, H-2); 7.37 (d, 1H, J = 8 Hz, H-7); 7.47 (d, 1H, J = 7.6 Hz, H-7); 11.047 (s, 1H, NH indole); 13.54 (s, 1H, SH); ¹³C NMR: δ 10.59; 20.51; 21.77; 44.42 (N–C); 107.12; 111.50; 118.14; 118.58; 121.20; 124.00; 126.57; 136.13; 151.27; 166.40 (N–C–S); ESI MS m/z 273 (M + H, 100%); Anal. calcd. for C₁₄H₁₆N₄S: C, 60.73% H, 6.01%, N, 20.23%. Found: C, 60.25%, H, 5.79%, N, 19.77%.

5-((1H-Indol-3-yl)methyl)-4-(2,4-difluorophenyl-4H-1,2,4-triazole-3-thiol (3e). Yield 85%, m.p. 242.5 °C; ¹H NMR: δ 3.96 (q, 2H, Ar–CH₂); 6.70–7.48 (m, 8H, Ar–H); 10.86 (s, 1H, NH indole); 13.85 (s, 1H, SH); ESI MS m/z 343 (M + H, 100%); Anal. calcd. for C₁₇H₁₂ F₂N₄S: C, 59.63%, H, 3.53%, N, 16.36%. Found: C, 59.50%, H, 3.77%, N, 16.21%.

5-((1H-Indol-3-yl)methyl)-4-(3-fluorophenyl)-4H-1,2,4-triazole-3-thiol (3f). Yield 94%, m.p. 134 °C; ¹H NMR: δ 3.96 (s, 2H, Ar–CH₂); 6.67–7.49 (m, 9H, Ar-H); 10.85 (bs, 1H, NH indole); ¹³C NMR: δ 22.76; 107.56; 111.84; 116.08; 116.24; 116.32; 116.45; 118.58; 118.89; 121.51; 124.07; 124.98; 127.04; 130.92; 131.02; 136.28; 136.43; 151.38; 160.98; 163.41; 167.94 (N–C–S); ESI MS m/z 325 (M + H, 100%); Anal. calcd. For C₁₇H₁₃FN₄S: C, 55.54%, H, 4.88%, N, 15.24%. Found: C, 55.03%, H, 3.96%, N, 15.04%.

5-((1H-Indol-3-yl)methyl)-4-(2,4-dichlorophenyl)-4H-1,2,4-triazole-3-thiol (3g). Yield 89%, m.p. 254.5 °C; ¹H NMR: δ 3.91 (q, 2H, Ar–CH₂); 6.92 (t, 1H, J = 8 Hz, H-5); 7.05 (t, 1H, J = 8 Hz, H-6); 7.497 (d, 1H, J = 6.4 Hz, H-7); 7.52 (d, 1H, J = 2.4 Hz, H-4); 6.77–7.78 (m, 4H, Ar-H); 10.91 (s, 1H, NH indole); 13.86 (s, 1H, SH); ¹³C NMR: δ 22.01; 105.87; 111.30; 117.80; 118.39; 120.99; 123.90; 126.52; 128.28; 129.60; 130.13; 132.05; 133.22; 135.23; 135.91; 151.11; 167.735 (N–C–S); ESI MS m/z 375 (M⁺, 100%), 377 (M + 2, 58%), 379 (M + 4, 13%), 426 (M + CH₃CN, 11%); Anal. calcd. for C₁₇H₁₂Cl₂N₄S: C, 53.76%, H, 3.32%, N, 14.75%. Found: C, 53.57%, H, 3.29%, N, 14.69%.

5-((1H-Indol-3-yl)methyl)-4-(3-chlorophenyl)-4H-1,2,4-triazole-3-thiol (3h). Yield 83%, m.p. 140 °C; ¹H NMR: δ 3.96 (s, 2H, Ar–CH₂); 6.91 (t, 1H, J = 7.2 Hz, H-5); 7.028 (t, 1H, J = 7.6 Hz, H-6); 7.19 (d, 1H, J = 7.6 Hz, H-2); 6.71–7.58 (m, 6H, Ar–H); 10.86 (s,1H, NH indole); 13.77 (s, 1H, SH); ¹³C NMR: δ 22.73; 107.03; 111.88; 118.50; 118.97; 121.57; 124.26; 126.99; 127.56; 128.74; 129.78; 131.09; 133.57; 135.56; 136.43; 151.72;168.14 (S–C–N); ESI MS m/z 341.2 (M⁺, 100%); 343.3 (M + 2, 30%), 382 (M + CH₃CN, 20%); Anal. calcd. for C₁₇H₁₃ClN₄S: C, 58.36%, H, 4.03%, N, 16.01%. Found: C, 58.54%, H, 4.01%, N, 15.70%.

General procedure for the synthesis of compounds 4a–g

5-((1H-Indol-3-yl) methyl)-4-(substituted aryl/alkyl)-4H-1,2,4-triazole-3-thiol (1 mmol) was dissolved in a solution of 2 N KOH (2 ml) and absolute ethanol (20 ml) in a conical flask. R-X (X = Cl) (1 mmol) was added in to the mixture and stirred at room temperature for 2–3 h. After completion of the reaction, the reaction mixture was poured onto crushed ice. The formed precipitate was filtered, washed with water, dried at room temperature and recrystallized from absolute ethanol33.

2-(5-((1H-Indol-3-yl)methyl)-4-ethyl-4H-1,2,4-triazole-3-ylthio)-N-phenylacetamide (4a). Yield 17%, m.p. 120 °C; ¹H NMR: δ 0.96 (t, 3H, J = 6.8 Hz, CH₃); 3.91 (q, 2H, CH₂); 4.09 (s, 2H, Ar–CH₂); 4.25 (s, 2H, S–CH₂–CO); 6.92–7.55 (10H, m, Ar–H); 10.32 (s, 1H, amide-NH); 10.97 (s,1H, NH indole); ¹³C NMR: δ 14.55; 21.43; 28.90; 37.51; 108.30; 111.37; 118.40; 118.45; 119.01; 121.11; 123.42; 123.61; 126.65; 128.69; 136.16; 138.66; 148.32; 154.44 (N–C–S); 165.62 (C=O); ESI MS m/z 392 (M + H, 100%); Anal. calcd. for C₂₁H₂₁N₅OS: C, 56.04%, H, 6.15%, N, 15.56%. Found: C, 55.68%, H, 5.60%, N, 15.22%.

2-(5-((1H-Indol-3-yl)methyl)-4-propyl-4H-1,2,4-triazole-3-ylthio)-N-propylacetamide (4b). Yield 32%, m.p. 79 °C; ¹H NMR: δ 0.71 (t, 3H, CH₃); 0.79 (t, 3H, J = 7.2 Hz, CH₃); 1.34 (m, 2H, CH₂); 1.43 (m, 2H, CH₂); 2.97 (q, 2H, CO–NH–CH₂); 3.78 (t, 2H, J = 7.6, triazole–NH–CH₂); 3.82 (s, 2H, Ar–CH₂); 4.23 (s, 2H, S–CH₂–CO); 6.94 (t,1H, J = 8 Hz, H-5); 7.06 (t,1H, J = 8.4 Hz, H-6); 7.25 (d, 1H, J = 2 Hz, H-2); 7.34 (d, 1H, J = 8.4 Hz, H-7); 7.50 (d, 1H, J = 8 Hz, H-4); 8.16 (bs, 1H, amide–NH); 10.960 (s,1H, NH indole); ¹³C NMR: δ 11.08; 11.72; 22.06; 22.59; 22.99; 37.29; 43.12; 45.26; 79.63; 108.95; 111.90; 118.95; 121.65; 124.07; 127.21; 136.71; 149.14; 155.04; 167.12 (C=O); ESI MS m/z 372.3 (M + H, 100%); Anal. calcd. for C₁₉H₂₅N₅OS: C, 58.18%, H, 7.02%, N, 17.85%. Found: C, 58.77%, H, 6.96%, N, 17.13%.

2-(5-((1H-Indol-3-yl)methyl)-4-benzyl-4H-1,2,4-triazole-3-ylthio)-N-(2-fluorophenyl)acetamide (4c). Yield 30%, m.p. 182.5 °C; ¹H NMR: δ 4.13(s, 2H, indole-CH₂); 4.20 (s, 2H, benzyl-CH₂); 5.18 (s, 2H, amide-CH₂); 6.92–7.88 (m, 14H, Ar-H); 10.16 (s, 1H, amide-NH); 10.91 (s, 1H, NH-indole); ¹³C NMR: δ 22.20; 37.64 (S–C); 47.02 (N–C); 108.48; 111.89; 115.87; 116.07; 118.98; 121.67; 124.09; 124.25; 124.87; 125.86; 126.27; 126.38; 127.02; 127.22; 128.20; 129.08; 135.832; 136.76; 149.86; 152.56; 155.00; 155.46; 166.82 (C=O); ESI MS m/z 472.2 (M + H, 100%); Anal. calcd. for C₂₆H₂₂ FN₅OS: C, 66.22%, H, 4.70%, N, 14.85%. Found: C, 65.73%, H, 5.08% N, 14.36%.

2-(5-((1H-İndol-3-yl)methyl)-4-phenyl-4H-1,2,4-triazole-3-ylthio)-N-propylacetamide (4d). Yield 17%, m.p. 165.5 °C; ¹H NMR: δ 0.89 (t, 3H, J = 7.6 Hz, CH₃); 1.53 (m, 2H, CH₂); 3.20 (q, 2H, CO–NH–CH₂); 3.73 (s, 2H, Ar–CH₂); 4.16 (s, 2H, S–CH₂–CO); 6.61–7.50 (m, 10H, Ar–H); 7.96 (bs, 1H, amide-NH); 8.10 (s, 1H, NH indole); ¹³C NMR: δ 11.39; 22.05; 22.56; 34.56; 41.56; 109.34; 110.00; 111.10; 118.78; 119.71; 122.28; 122.69; 126.63; 127.02; 129.78; 130.19;132.72; 136.02; 152.17; 155.39 (N–C–S); 168.68 (C=O); ESI MS m/z 406 (M + H, 100%); Anal. calcd. for C₂₂H₂₃ N₅OS: C, 64.44%, H, 5.78%, N, 17.08%. Found: C, 64.21%, H, 5.88%, N, 16.85%.

2-(5-((1H-Indol-3-yl)methyl)-4-(2,4-difluorophenyl)-4H-1,2,4-triazole-3-ylthio)-N-propylacetamide (4e). Yield 45%, m.p. 140 °C; ¹H NMR: δ 0.81 (t, 3H, J = 7.6 Hz, CH₃); 1.37 (m, 2H, CH₂); 2.99 (q, 2H, CO-NH-CH₂); 3.85 (s, 2H, Ar–CH₂); 4.08 (s, 2H, S–CH₂–CO); 6.69–7.51 (m, 8H, Ar–H); 8.16 (t, 1H, J = 5.2 Hz, amide-NH); 10.81 (s, 1H, NH indole); ESI MS m/z 442 (M + H, 100%); Anal. calcd. for C₂₂H₂₁F₂N₅OS: C, 59.85%, H, 4.79%, N, 15.86%. Found: C, 59.70%, H, 4.97%, N, 15.66%.

2-(5-((1H-Indol-3-yl)methyl)-4-(3-fluorophenyl)-4H-1,2,4-triazole-3-ylthio)-N-propylacetamide (4f). Yield 40%, m.p. 145 °C; ¹H NMR: δ 0.81 (t, 3H, J = 7.6 Hz, CH₃); 1.37 (m, 2H, CH₂); 2.98 (q, 2H, CO-NH-CH₂); 3.84 (s, 2H, Ar–CH₂); 4.11 (s, 2H, S-CH₂-CO); 6.69–7.55 (m, 9H, Ar-H); 8.16 (t, 1H, J = 5.2 Hz, amide-NH); 10.80 (s, 1H, NH indole); ¹³C NMR: δ 11.75; 22.025; 22.61; 36.69; 41.16; 108.36; 111.77; 115.19; 115.43; 117.18; 117.38; 118.63; 118.87; 121.53; 123.84; 124.03; 124.06; 127.02; 131.66; 131.75; 134.87; 134.97; 136.45; 150.10; 155.134; 161.13; 163.58 (N–C–S); 166.85 (C=O); ESI MS m/z 424 (M + H, 100%); Anal. calcd. for C₂₂H₂₂FN₅OS: C, 62.39%, H, 5.24%, N, 16.54%. Found: C, 62.32%, H, 5.28%, N, 16.35%.

2-(5-((1H-Indol-3-yl)methyl)-4-(2,4-dichlorophenyl)-4H-1,2,4-triazole-3-ylthio)-N-phenylacetamide (4g). Yield 15%, m.p. 219 °C; ¹H NMR: δ 4.03 (s, 2H, Ar–CH₂); 4.12(s, 2H, S–CH₂–CO); 6.72–7.80 (m, 13H, Ar–H); 10.27 (s,1H, amide-NH); 10.82 (s, 1H, NH indole); ¹³C NMR: δ 21.47; 37.30; 107.15; 111.19; 117.96; 118.28; 118.99; 120.93; 123.43; 123.47; 126.56; 128.51; 128.69; 129.36; 129.76; 131.13; 132.53; 135.68; 135.90; 138.61; 149.66; 154.80 (N–C–S); 165.26 (C=O); ESI MS m/z 508 (M⁺, 100%), 510 (M + 2, 75%), 512 (M + 4, 16%); Anal. calcd. for C₂₅H₁₉Cl₂N₅OS: C, 59.06%, H, 3.77%, N, 13.77%. Found: C, 59.60%, H, 4.27%. N, 13.47%.

Experimental – biological activity

Cell culture and reagents

CHO-K1 cells were purchased from ATCC and maintained at 37 °C in 5% CO₂ atmosphere. Dulbecco’s modified Eagle’s medium/F12 (Sigma, St. Louis, MO) (1:1) nutrient mixture medium supplemented with 10% foetal bovine serum (FBS), 1% of a 100 U/ml penicillin–streptomycin solution, 2 mM (final concentration) l-glutamine and 1 mM (final concentration) sodium pyruvate was used in all cell incubations. Hyclone characterized FBS is purchased from Thermo Scientific (Waltham, MA). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), DPPH, sodium pyruvate and l-glutamine were obtained from Sigma (St. Louis, MO). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Invitrogen (Waltham, MA). Test materials were dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO never exceeded 0.1% v/v in cell culture medium.

Cytotoxic effects via the MTT assay

CHO-K1 cells were seeded in 96-well plates (5 × 10³ cells/well) and incubated at 37 °C in a humid atmosphere containing 5% CO₂ for 24 h. Cells were treated with the newly synthesized compounds (10 μM) for 24 h. Control (medium only), vehicle control and positive control (15 μM Triton X-100) were included in every experiment. Following the exposure period the medium was removed, cells were washed with phosphate buffered saline (PBS) and then incubated with MTT (1 mg/ml) for 4 h at 37 °C. MTT solution was removed and formazan crystals were dissolved in DMSO. The absorbance was recorded at 550 nm on a microplate reader. The ratio of the absorbance of treated samples to the absorbance of control (taken as 100%) was expressed as % cell viability.

Measurement of intracellular ROS (antioxidant activity on ROS-induced DCFH-DA oxidation)

For estimation of intracellular ROS, DCFH-DA was used as a probe. In cellular systems, the non-fluorescent probe DCFH-DA readily crosses the cell membrane and undergoes hydrolysis by intracellular esterases to non-fluorescent 2′,7′-dichlorofluorescin (DCFH). In the presence of ROS, DCFH is oxidized to highly fluorescent dichlorofluorescein (DCF)37 which can be detected by a fluorescent microplate reader. The DCF fluorescence intensity is believed to be parallel to the amount of ROS formed intracellularly.

Cells were seeded in black 96-well plates at a density of 5 × 10³ cells/well and incubated at 37 °C in a humid atmosphere containing 5% CO₂ for 24 h for cell attachment. The medium was removed and then cells were incubated with DCFH-DA (20 μM) containing medium for 30 min. Cells were washed with PBS to remove excess DCFH-DA. Ten micromolar synthesized compounds and 10 μM cumene hydroperoxide (CMHP) were added into medium. The production of fluorescent DCF was evaluated by monitoring fluorescence intensity at 488 nm excitation, 530 nm emission wavelengths for 60 min.

DPPH free radical scavenging activity

The free radical scavenging activities of newly synthesized compounds were tested by their ability to bleach the stable radical DPPH38. DPPH presents a maximum of absorbance at 515 nm; when DPPH reacts with an antioxidant compound, which can donate hydrogen, this absorbance diminishes and can be measured on a visible spectrophotometer.

Twenty microlitres of the samples (100 μM) was added to 180 μl of DPPH solution (150 μM) in methanol–water (80:20, v/v) in 96-well plates, incubated for 30 min at room temperature then DPPH reduction was estimated at 517 nm. Percentage inhibition by the sample treatment was determined by comparison with a DMSO-treated control group. All experiments were carried out in triplicate. MLT and BHT were used as reference compounds. The radical scavenging activities were expressed as the inhibition percentage and were calculated using the formula: Radical scavenging activity (%) = [(A₀−A₁/A₀) × 100], where A₀ is the absorbance of the control (blank, without compound) and A₁ is the absorbance of the compound.

Results and discussion

Cytotoxic effects of the synthesized compounds

The cytotoxic effect of the synthesized compounds in CHO-K1 cells was evaluated by the MTT assay (Figure 2). CHO-K1 cells are used because many studies as well as our previous study suggested them as a relevant model for in vitro antioxidant screening studies39. Furthermore, CHO cells are advantageous since they have active CYP enzymes that make them a good in vitro model for detecting the toxicity of chemicals as well as some of their metabolites without adding S9 fraction.

Figure 2. Effect of samples (10 μM) on CHO-K1 cell viability evaluated by MTT assay. Bars represent “medium ± standard deviation” values from five individual experiments. *p < 0.05, **p < 0.005, ***p < 0.0005, groups compared to vehicle control group. Triton X-100 used as a positive control group.

Antioxidant effects of the synthesized compounds

The protective effect of newly synthesized MLT analogues against CMHP induced DCFH-DA oxidation was determined in CHO-K1 cells that were preloaded with the fluorescent probe. Oxidation of the probe that locates in the cytosol was screened at various time intervals up to 60 min (Figure 3). Several compounds were found to have antioxidant activity, whereas 1b and 2e were found to be the most potent two compounds among all. Therefore, their reducing activity against DCFH oxidation was further investigated at their various concentrations (Figure 4)

Figure 3. DCFH oxidation in CHO cells after 60 min incubation with compounds (10 μM) in the presence of CMHP. Bars represent “medium ± standard deviation” values obtained from five individual experiments. Statistical comparisons of samples with CMHP were compared to CMHP alone control group. *p < 0.05, **p < 0.005 (AA, ascorbic acid).

Figure 4. DCFH oxidation in CHO cells after 60 min incubation with MLT, compounds 1b and 2e in the presence of CMHP. Bars represent “medium ± standard deviation” values from five individual experiments. The values above the bars represent percentage of the absorbance values comparing with CMHP group used as control. All the statistical analysis of samples with CMHP was performed by comparing to the CMHP alone group. *p < 0.05, **p < 0.005, ***p < 0.0005 (MLT, melatonin).

.

DPPH free radical scavenging activity

Free radical scavenging activity of the newly synthesized compounds was further investigated in a cell free in vitro model where an active radical, DPPH was used. The radical scavenging activity of a compound can be detected via decrease in the absorbance of DPPH. As can be seen in Figure 5 compounds which named as 1a–h and 3a–h are more potent radical scavengers then 2 and 4 coded compounds.

Figure 5. Radical scavenging activity of compounds (100 μM) determined by DPPH assay. Bars represent “medium ± standard deviation” values from four different experiments. BHT and MLT were used as a reference (BHT, butylated hydroxytoluene; MLT, melatonin).

Conclusion

In this study, 31 new indole-based MLT analogues were synthesized and their antioxidant and cytotoxic effects were tested by three different assays. The MTT assay was used for determining the cytotoxic profiles in CHO-K1 cells, and the DCFH-DA and DPPH assays were preformed to reveal the antioxidant activity. According to the results of the MTT assay 1a, 1d, 2b, 2h and 4c (10 μM) showed significant proliferative effect on CHO cells. However, other compounds have demonstrated cytotoxic effects at their 10 μM concentrations. Compounds with cytotoxic effect can be tested for a possible anticancer activity in further studies due to the similarity of chemical structures to sunitinib which is a receptor tyrosine kinase inhibitor40 and was approved by the FDA for the treatment of renal cell carcinoma. According to the DCFH-DA assay in the presence of CMHP with p < 0.05 degree of significance, 1b and 2e were observed with the highest antioxidant effect, respectively, with values of 74% and 81%. This was followed by 1c, 3 g, 4d, 1d, respectively, with the rate of 83%, 88% and 89%. The concentration dependence effect of the most active compounds 1b and 2e was investigated against MLT at the same concentration. Depending on the dose of 1b and 2e when the concentration is increasing the antioxidant effect is also increasing and this effect is more potent than MLT at concentrations of 1 and 10 μM.

The DPPH assay is a cell-free in vitro screening test for the radical scavenging activity. Among newly synthesized compounds generally hydrazinecarbothioamide (1a–h) and 1,2,4-triazole-3-thiol (3a–h) derivatives were found to have high scavenging activity whereas (4a–g) derivatives were found to have the least scavenging activity. Compound 1b which was found to have the highest antioxidant effect in the cell based in vitro DCFH assay was found to have high scavenging activity in DPPH assay too. This finding indicates that the antioxidant effect of 1b is probably is a result of its radical scavenging activity. However, compound 2e which was also found to have antioxidant activity according to the DCFH assay, and was found to have very low scavenging activity in the DPPH assay which suggests an alternative mechanism for its antioxidant activity. However, almost all hydrazincarbothioamide and 1,2,4-triazole-3-thiol derivatives were found to have radical scavenging activity in the DPPH assay where no antioxidant activity was observed in the cell based DCFH assay. This result could be the result of limited availability of the compounds in cell cytosol because of the possible membrane passage. This speculation needs to be further verified experimentally.

Declaration of interest

The authors report no conflicts of interest. The chemical synthesis and characterization section of this work were supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) Research and Development Grant 112S599. The cell culture facility was established by the support of a TUBITAK 108S202 Grant.