Synthesis and Comparison of Antioxidant Properties of Indole-Based Melatonin Analogue Indole Amino Acid Derivatives

Authors


Corresponding author: Sibel Suzen, sibel@pharmacy.ankara.edu.tr

Abstract

Increased levels of reactive oxygen species attributed to oxidative stress have been found to be responsible for the development of some vital diseases such as cardiovascular, neurodegenerative and autoimmune diseases. Recently, it was observed that melatonin is a highly important antioxidant, and melatonin analogues are under investigation to find out improved antioxidant activity. In this study, 14 melatonin -based analogue indole amino acid and N-protected amino acid derivatives were synthesized and elucidated spectrometrically. To investigate the antioxidant activity of the synthesized compounds and to compare with melatonin, butylhydroxytoluene and vitamin E, lipid peroxidation inhibition and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activities were tested. The results indicated that the synthesized new indole amino acid derivatives have similar activities to melatonin in 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity assay but more potent activities in lipid peroxidation inhibition assay.

Reactive oxygen (ROS) and reactive nitrogen species can cause vital damages to all cellular macromolecules, including nucleic acids, proteins, carbohydrates and lipids. Membrane lipids are most important targets of, and lipid peroxidation (LP) may lead to membrane dysfunction and change the cell permeability. Oxidative stress is associated in wide selection of disorders including ischaemia–reperfusion injury, neurodegenerative diseases, diabetes, inflammatory diseases and ageing (1).

It is known that indole derivatives are significant substances for their medicinal and biological features. Some 3-substituted indoles (2), indolyl thiohydantoin derivatROSives (3) and indolyl triazoles (4) have anticancer activity, melatonin has (5) antioxidant activity, indolin-2-one derivatives (6) and indol-5-carbonyl hydrazines (7) show antirheumatoidal activity, and indolyl thiohydantoins have anti-HIV activity (8). Series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulphonamide derivatives (9,10) are known as carbonic anhydrase inhibitors.

Antioxidant effects of the indole ring-containing melatonin (MLT) have been well described and evaluated by Tan et al. (11). It acts as a free radical scavenger and has a broad-spectrum antioxidant (12). Owing to its free radical scavenger and antioxidant properties, MLT-related compounds such as MLT metabolites and synthetic analogues are under investigation to determine which exhibit the highest activity with the lowest side-effects (13–15). Antioxidant activity of synthetic indole derivatives such as indole-3-propionic acid (16), indole amine-triazoles (17) and stobadine (18) was studied extensively. Moreover, our group previously identified the antioxidant activity of MLT analogue indole derivatives such as 2-phenylindole derivatives (19), indole hydrazide and hydrazones (20,21) and indole-amides (22). Recently, we observed the relationship between aldose reductase and superoxide dismutase inhibition capacities of indole-based analogues of MLT derivatives (23).

It is known that many amino acids have potential antioxidant activity. In a study, troloxyl-methionine and Troloxyl-cysteine showed significant antioxidant activity (24,25). Several tryptophan derivatives function as a free radical scavengers and antioxidants. Furthermore, they stimulate a number of antioxidative enzymes and stabilize cell membranes that help to resist free radical damage (26,27). N-(4-pyridoxylmethylene)-l-serine was found as an antioxidant to suppress iron-catalysed ROS generation (28). l-alanine stimulates expression of the antioxidant defence proteins and ferritin in endothelial cells (29). Fullerene-substituted phenylalanine and lysine derivatives were determined to be significantly more potent than Trolox (30). Dehydro amino acids and corresponding peptides can function as radical scavengers (31). In our earlier studies (32,33), we showed that substituted dehydroalanines scavenge ROS. In the first study (32), novel N-acyl dehydroalanine derivatives were studied as antioxidants on rat liver LP and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity. Most of the compounds showed strong inhibitory effect on LP. In the second study (33), comparative effect of N-substituted dehydroamino acids and alpha-tocopherol on rat liver LP activities was investigated. The results indicated that all the synthesized compounds showed very good inhibitory effect on the LP.

Melatonin has a role in the regulation of many physiological processes but therapeutic use is limited because of two major problems. The first is very short biological half-life, owing to its fast metabolism to 6-hydroxymelatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine and the second is the non-selectivity of MLT at target sites (34,35). To increase the biological half-life, the acetyl amino ethyl side chain was replaced with bulky amino acid derivatives by making hydrazones. Hydrazone derivatives possess a range of pharmacological activities including antitumoural, anti-microbial, antimalarial, anti-convulsant and anti-inflammatory activity (36). Recently, hydrazones were found as potential antioxidants (37–40). It is possible that the synthesized compounds may undergo some hydrolysis under the in vivo conditions because oxidative stress induces a LP of cellular membranes, resulting in the generation of reactive carbonyl compounds that involves in the ‘carbonyl stress’ (41,42). Hydrazine derivatives also exhibit carbonyl scavenger activity by the reaction of ketones/aldehydes (43).

There has been no research published related to antioxidant properties of indole amino acid derivatives. In this study, 14 MLT-based analogue indole amino acid derivatives and N-protected amino acids (Figure 1) were synthesized. Their antioxidant activity was investigated in vitro by LP inhibition and DPPH radical-scavenging assays. The results were compared with MLT, butylhydroxytoluene (BHT) and vitamin E. All new MLT analogue compounds 4a–i and new amino acid derivatives 2c and 2f were characterized on the basis of 1H- and 13C-NMR, mass and FT-IR spectra.

Figure 1.

 Modifications made on melatonin molecule.

Melatonin molecule was modified in the 5-methoxy and acylamino groups showed in Scheme 1. These chemically significant modulations of the lead structure were made at two different points: the methoxy group at the fifth position of the indole ring and replacement of acetyl with amino acids derivatives. Scientific rationales and perspectives for the synthesis of new MLT analogues can be summarized as combining indole aldehyde and amino acids which have antioxidant properties to create synergistic antioxidant activity, use of bulky amino acid derivatives by making hydrazones with indole aldehyde to create longer biological half-life than MLT, to have carbonyl scavenger activity as well as antioxidant activity from hydrazine in case of the possibility of hydrolysis of the molecules and to see the possibility of antioxidant activity of the molecules without having methoxy on the fifth position of the indole ring.

Figure Scheme 1:.

 Synthetic pathway of indol-aminoacid derivatives.

Methods and Materials

General

Uncorrected melting points were determined using a Büchi SMP-20 apparatus (Büchi Labortechnik, St. Gallen, Zurich, Switzerland). The 1H- and 13C- NMR spectra were measured with a Varian 400 MHz instrument (Agilent Technologies, Santa Clara, CA, USA) using tetramethylsilane (TMS) internal standard and DMSO-d6 as solvent. ESI Mass spectra were determined on a Waters Micromass ZQ. FT-IR spectra were recorded on a Jasco 420 Fourier Transform apparatus (Jasco Corp., Tokyo, Japan). All spectral analysis was performed at the Central Laboratory of the Faculty of Pharmacy, Ankara University. Chromatography was carried out using Merck silica gel 60 (230–400 mesh; Merck KGaA, Darmstadt, Germany). The chemical reagents used in synthesis were purchased from Sigma (Hamburg, Germany) and Aldrich (Burbank, CA, USA).

Experimental

Target compounds were derived from N-protected amino acid esters (4ai) and hydrazine hydrate using simple reaction strategies. The hydrazide derivatives of N-protected amino acids (2ah) were reacted with 1-methyl-1H-indole-3-carboxaldehyde (3) to give the resulting indole amino acid hydrazones (4ai) using a methodology similar to that of Kidwai et al. (44). All new compounds were characterized on the basis of their spectral and analytical data.

General procedure for the synthesis of compounds 2a–h

Compounds 1a–f (1 mmol) were treated with excess hydrazine hydrate in MeOH (10 mL) by refluxing for 3–4 h on the hot water bath. On cooling, the precipitate was collected, washed with cold MeOH and recrystallized from MeOH to give 2a–h with 76–96% yield.

N-Benzoyl-d-serine hydrazide2c. Yield 92.4%, m.p. 195–196 °C; 1H-NMR: δ 3.73 (2H, t, CH2) 4.29 (2H, s, NH2), 4.52 (1H, m, CH), 5.00 (1H, t, OH), 7.52–7.96 (5H, m, Ar-H), 8.30 (1H, d, NH), 9.24 (1H, s, NH); ESI MS m/z 192 (M- CH2OH, 100%), 224 (M + 1).

N-Benzoyl-l-proline hydrazide2f. Yield 95.1%, m.p. oily compound; 1H-NMR: δ 1.74 (4H, m, (CH2)2), 2.17–3.40 (2H, m, CH2), 3.57 (2H, m, NH2), 4.41 (1H, t, CH), 7.32–7.56 (5H, m, Ar-H), 7.65 (1H, m, NH); ESI MS m/z 202 (M- NH-NH2, 100%), 256 (M+Na), 234 (M + 1); FT-IR (KBr) /cm 1687 (C-N stretch), 3338–3224 (N-H stretch).

General procedure for the synthesis of compounds 4a–i

N-protected amino acid hydrazines 2a–h (1.2 mmol) and 1H-indole-3-carboxaldehyde 3 (1 mmol) in EtOH (10 mL) was refluxed until the starting materials disappeared on TLC plate, on the hot water bath. On cooling, the precipitate was collected, washed with cold EtOH and recrystallized from EtOH to give 4a–i with 22–96% yield.

(E)-N-(2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-2-oxoethyl)benzamide4a. Yield 38.3%, m.p. 239–240 °C; 1H-NMR: δ 3.98 (1H, d, NH), 4.48 (2H, d, CH2), 7.16–8.71 (10H, m, Ar-H), 8.40 (1H, s, azomethine-CH), 11.17 and 11.22 (1H, 2s, NH), 11.57 (1H, brs, indole-NH); 13C-NMR: δ 112.08, 112.61, 121.31, 122.21, 123.32, 124.77, 127.94, 129.03, 131.05, 131.99, 134.87, 137.77, 141.53, 144.57 (azomethine-C), 165.44, 167.29, 170.12; ESI MS m/z 321 (M + 1), 343 (M+Na, 100%). FT-IR (KBr) /cm 1611 (C=N, azomethine stretch), 3365 (NH-CO stretch).

(E)-N-(1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-1-oxopropan-2-yl)benzamide4b. Yield 81.1%, m.p. 139–140 °C; 1H-NMR: δ 1.47 (3H, d, CH3), 3.44 (1H, m, NH), 4.54 (1H, m, CH), 7.16–8.65 (10H, m, Ar-H), 8.40 (1H, s, azomethine-CH), 11.07 and 11.20 (1H, 2s, NH), 11.56 (1H, s, indole-NH); 13C-NMR: δ 17.25, 46.93, 40.07, 56.73, 112.14, 120.99, 121.31, 122.02, 122.59, 123.30, 124.77, 128.17, 131.06, 131.92, 134.85, 137.79, 141.45, 144.73 (azomethine-C), 166.77, 168.93, 173.73; ESI MS m/z 335 (M + 1), 357 (M+Na, 100%); FT-IR (KBr) /cm 1641 (C=N, azomethine stretch), 3249 (NH-CO stretch).

(E)-N-(1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-hydroxy-1-oxopropan-2-yl)benzamide4c. Yield 60.6%, m.p. 123–126 °C; 1H-NMR: δ 3.76 (1H, m, NH), 3.94 (1H, brs, OH), 4.56 (1H, m, CH), 4.99–5.04 (2H, m, CH2) 7.16–8.42 (10H, m, Ar-H), 8.42 (1H, s, azomethine-CH), 11.16 and 11.22 (1H, 2s, NH), 11.57 (1H, brs, indole-NH); 13C-NMR: δ 54.51, 56.19, 56.73, 61.40, 62.26, 112.107, 121.02, 122.26, 123.27, 124.74, 134.71, 137.68, 141.61, 144.90 (azomethine-C), 166.63, 167.02, 170.87; ESI MS m/z 351 (M + 1), 373 (M+Na, 100%); FT-IR (KBr) /cm 1642 (C=N, azomethine stretch), 3260 (NH-CO stretch).

(E)-N-(1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-(methylthio)-1-oxopropan-2-yl)benzamide4d. Yield 33.5%, m.p. 106–108 °C; 1H-NMR: δ 1.04 (2H, m, S-CH2), 2.00 (3H, s, S-CH3), 2.53–2.74 (2H, m, CH2), 4.37 (1H, t, CH), 4.60 (1H, m, NH), 7.16–8.68 (10H, m, Ar-H), 8.42 (1H, s, azomethine-CH), 11.12 and 11.27 (1H, 2s, NH), 11.58 (1H, brs, indole-NH); 13C-NMR: δ 15.36, 30.74, 50.76, 112.06, 121.011, 122.43, 123.35, 124.73, 128.23, 130.98, 134.69, 137.68, 141.77, 144.99 (azomethine-C), 167.36, 172.79; ESI MS m/z 395 (M + 1), 417 (M+Na, 100%); FT-IR (KBr) /cm 1639 (C=N, azomethine stretch), 3258 (NH-CO stretch).

(E)-N-(1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-(indolin-3-yl)-1-oxopropan-2-yl)benzamide4e. Yield 55.6%, m.p. 118 °C (decomp); 1H-NMR: δ 3.29 (2H, m, CH2), 4.36 (1H, m, CH), 4.79 (1H, m, NH), 6.76–8.73 (15H, m, Ar-H), 8.43 (1H, s, azomethine-CH), 10.83 (1H, d, NH), 11.19 and 11.40 (1H, 2s, NH), 11.58 (1H, brs, indole-NH); 13C-NMR: δ 19.22, 56.74, 111.48, 112.22, 118.93, 121.04, 121.55, 122.44, 123.24, 124.05, 124.75, 128.09, 128.87, 131.00, 131.94, 134.84, 136.76, 137.69, 140.70, 142.05, 145.041 (azomethine-C), 66.92, 168.29, 173.30 167.36, 172.79; ESI MS m/z 450 (M + 1), 472 (M+Na, 100%); FT-IR (KBr) /cm 1641 (C=N, azomethine stretch), 3399 (NH-CO stretch).

(E)-N’-((1H-indol-3-yl)methylene)-1-benzoylpyrrolidine-2-carbohydrazide4f. Yield 25.6%, m.p. 105–106 °C; 1H-NMR: δ 1.73–2.12 (6H, m, (CH2)3), 5.45 (1H, m, CH), 7.10–8.24 (10H, m, Ar-H), 8.40 (1H, s, azomethine-CH), 11.09 and 11.21 (1H, 2s, NH), 11.69 (1H, brs, indole-NH); 13C-NMR: δ 112.08, 112.61, 121.31, 122.21, 123.32, 124.77, 127.94, 129.03, 131.05, 131.99, 134.87, 137.77, 141.53, 144.57 (azomethine-C), 165.44, 167.29, 170.12; ESI MS m/z 361 (M + 1), 383 (M+Na, 100%); FT-IR (KBr) /cm 1611 (C=N, azomethine stretch), 3317 (NH-CO stretch).

(E)-tert-butyl 1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-1-oxopropan-2-ylcarbamate4g. Yield 21.7%, m.p. 176–177 °C; 1H-NMR: δ 1.27 (3H, m, CH3), 1.38 (9H, s, (CH3)3), 4.02 (1H, m, NH), 4.90 (1H, m, CH), 6.95–8.90 (5H, m, Ar-H), 8.36 (1H, s, azomethine-CH), 10.96 and 11.07 (1H, 2s, NH), 11.55 (1H, brs, indole-NH); 13C-NMR: δ 17.49, 24.63, 28.93, 47.44, 56.50, 61.82, 709.70, 78.48, 112.08, 120.97, 121.28, 122.57, 123.27, 131.03, 135.99, 137.66, 141.34, 142.83, 145.41 (azomethine-C), 152.96, 155.79, 157.37; ESI MS m/z 331 (M + 1, 100%), 354 (M + 1 + Na); FT-IR (KBr) /cm 1658 (C=N, azomethine stretch), 3396 (NH-CO stretch).

(E)-tert-butyl 1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-hydroxy-1-oxopropan-2-ylcarbamate4h. Yield 91%, m.p. 118–119 °C; 1H-NMR: δ 1.38 (9H, s, (CH3)3), 3.60 (1H, m, NH), 3.78 (1H, brs, OH), 4.05 (1H, m, CH), 4.81–4.99 (2H, m, CH2), 6.58–8.22 (5H, m, Ar-H), 8.39 (1H, s, azomethine-CH), 11.04 and 11.10 (1H, 2s, NH), 11.56 (1H, brs, indole-NH); 13C-NMR: δ 19.21, 28.88, 54.88, 56.64, 62.49, 78.89, 112.23, 112.47, 120.99, 122.14, 122.58, 123.29, 124.71, 130.88, 137.76, 141.53, 144.69 (azomethine-C), 151.88, 166.99, 171.25; ESI MS m/z 347 (M + 1), 369 (M+Na, 100%); FT-IR (KBr) /cm 1668 (C=N, azomethine stretch), 3297 (NH-CO stretch).

(E)-tert-butyl 1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-(1H-imidazol-4-yl)-1-oxopropan-2-ylcarbamate4i. Yield 95.9%, m.p. 169–170 °C; 1H-NMR: δ 1.36 (9H, s, (CH3)3), 2.82 (2H,m, CH2), 3.06 (1H, dd, CH), 4.22 (1H, m, NH), 5.65 (1H, dd, NH), 6.83–8.36 (7H, m, Ar-H), 8.36 (1H, s, azomethine-CH), 10.99 and 11.12 (1H, 2s, NH), 11.54 (1H, brs, indole-NH); 13C-NMR: δ 14.77, 17.02, 21.44, 28.90, 52.00, 54.36, 78.60, 112.25, 121.14, 122.76, 123.24, 124.96, 130.98, 135.38, 137.72, 141.36, 144.61 (azomethine-C), 156.05, 168.04, 171.81; ESI MS m/z 397 (M + 1, 100%), 398 (M+Na); FT-IR (KBr) /cm 1666 (C=N, azomethine stretch), 3247 (NH-CO stretch).

In vitro antioxidant activities

DPPH free radical-scavenging activity

The radical-scavenging assay was determined by the modified method described previously (45). The stock solutions of the synthesized compounds were prepared at 10−2 m in DMSO. A series of stock solution in DMSO were diluted to varying concentrations in 96-well microplates. Then, methanolic DPPH solution (100 μm) was added to each well.

The plate was shaken to ensure thorough mixing before being wrapped with aluminium foil and placed into the dark. After 30 min, the optical density of the solution was read at the wavelength 517 nm. The methanolic solution of DPPH served as a control. Percentage inhibition was calculated using the following formula:

% Inhibition = [(Acontrol − Asample)/Acontrol] × 100

where Acontrol is the absorbance of the control with DMSO and Asample is the absorbance of the sample in the presence of the compounds. Each experiment was performed in triplicate. Melatonin and BHT were used as reference compounds (Table 1).

Table 1.   DPPH radical-scavenging and LP effect of synthesized compoundsa,b
NoDPPH assay (10−3 m) inhibition, %LP (10−3 m) inhibition, %
  1. aThe values represent the average of 2–4 determinations ±SD.

  2. bCompounds were diluted with DMSO (solvent showed no antioxidant activity).

  3. NE, no effect; DPPH, 2,2-diphenyl-1-picrylhydrazyl; LP, lipid peroxidation.

1b10 ± 1.2NE
1c13 ± 1.0NE
1d2 ± 1.2NE
1e12 ± 1.6NE
1f13 ± 1.4NE
4a9.0 ± 0.833 ± 2.2
4b17 ± 0.425 ± 1.7
4c17 ± 0.817 ± 2.2
4d17 ± 0.830 ± 2.2
4e26 ± 0.842 ± 3.5
4f45 ± 2.134 ± 2.0
4g8.0 ± 1.242 ± 2.1
4h8 ± 1.026 ± 3.4
4i12 ± 0.625 ± 1.1
BHT 10−3 m77 ± 1.1
BHT 10−4 m82 ± 0.8
Melatonin19 ± 2.821 ± 2.1
Vitamin E75 ± 2.2

Assay of lipid peroxidation

The effect of the synthesized indol-amino acids on rat liver homogenate induced with FeCl3-ascorbic acid, and LP was determined by the method of modified Mihara et al. (46). Wistar rats (200–225 g) were fed with standard laboratory rat chow and tap water. The animals were starved for 24 h prior to sacrifice and then killed by decapitation under anaesthesia. The study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals. The livers were removed immediately and washed in ice-cold distilled water and homogenized straight away with teflon homogenizer in ice chilled. Lipid peroxidation was measured spectrophotometrically by estimation of thiobarbituric acid reactive substances (TBARS). Amounts of TBARS were expressed in terms of mmol malondialdehyde/g tissue. A typical optimized assay mixture contained 0.5 mL of liver homogenate, 0.1 mL of Tris–HCl buffer (pH 7.2), 0.05 mL of 0.1 mm ascorbic acid, 0.05 mL of 4 mm FeCl3 and 0.05 mL of various concentration of synthesized compounds, BHT/α-tocopherol or MLT, were incubated for 1 h at 37 °C. After incubation, 3.0 mL of H3PO4 and 1 mL of 0.6% TBA were added and shaken vigorously. The mixture was boiled for 30 min. After cooling, n-butanol was added, and the mixture was shaken vigorously. The n-butanol phase was separated by centrifugation at 1006 g for 10 min. The absorbance of the supernatant was read at 532 nm against a blank, which contained all reagents except liver homogenate. Melatonin and vitamin E were used as positive control. Lipid peroxidation inhibitory activity (%) is expressed as follows:

Lipid peroxidation inhibitory activity (%): (Acontrol − Asample)/(Acontrol − Ablank) × 100

where Acontrol is the absorbance of the control, Asample is the absorbance of the sample and Ablank is the absorbance of the blank, to which the sample and the free radical-generating system (Fe+2/ascorbate) were not added (Table 1).

Statistical analysis

All data are the average of duplicate analyses. The data were recorded as mean ± standard deviation and analysed by spss (version 11.0 for Windows 98; SPSS Inc., Chicago, IL, USA). One-way analysis of variance was performed by anova procedures. Significant differences between means were determined by Duncan’s multiple range tests. Values of p, 0.05 were regarded as statistically significant.

Results

N-protected amino acids and MLT-based analogue indole amino acids derivatives were synthesized and tested for their antioxidant activities using DPPH radical-scavenging and LP inhibitory activity tests. All the results were compared with standard antioxidants BHT, vitamin E and MLT. The results are shown in Table 1.

In DPPH radical-scavenging test, compound 4f showed better activity (approximately two times higher) than MLT at 10−3 m concentration (45%). Compound 4e also showed higher activity than MLT at 10−3 m concentration (26%). Compounds 4b–d possessed similar scavenging activity against the DPPH radical like MLT. Percentages of inhibition of rest of the tested compounds except 1d and 4h were found similar to MLT. None of the compounds were found more potent than BHT.

In LP inhibition test, except non-active compounds 1b–f and compound 4c, all the tested compounds showed very strong antioxidant activity compared with MLT. The most potent compounds were 4e and 4g at 10−3 m concentration with same inhibition value (42%). The second potent compound was 4f (34%). This was followed by compounds 4a, 4d, 4h, 4b and 4i with 33%, 30%, 26%, 25% and 25%, respectively. The results of two assays were found similar to compare the most potent activities. None of the compounds were found more potent than vitamin E.

Discussion

The present work was designed to synthesize, characterize and investigate the potential antioxidant effects of indole-based MLT analogue hydrazide/hydrazone derivatives.

This study offers a new approach to structure-antioxidant activity relationship of five substituted indole rings.

It is likely that the synthesized indole derivatives may perhaps experience some hydrolysis under the in vivo conditions. On the other hand, in our previous studies in vitro electrochemical data showed that oxidation starts on the nitrogen atom in the indole ring, which leads finally to the hydroxylation of the benzene ring (47–50). Hydrazones can be chemically hydrolysed to relevant hydrazines and carbonyl compounds under acidic conditions. However, the biological system metabolizes hydrazones via the more complex NAD+-dependent oxidation reaction (51). This is a remarkable difference between the chemical and biological systems for degrading hydrazones. It is clear that synthesized compounds have free radical-scavenging activity, and they might have carbonyl scavenging activity if they metabolize into hydrazine and aldehyde. This gives synthesized compounds advantage over MLT.

In general, all the synthesized indole derivatives 4a–i were found to have reasonable antioxidant activity according to the results of LP inhibition assay against MLT. Interestingly, no significant antioxidant activity was observed in compounds 1b–f. These compounds are the N-protected amino acid esters, and they have no indole ring in their structure. Structure activity investigation of the rest of the active compounds showed that presence of indole ring in the molecule increases the antioxidant activity (except 4c). This finding is in the same direction of the literature. It is noteworthy that the most potent antioxidant and free radical scavenger amino acids are attached to a known molecule such as trolox (24), fullerene (30) or Pyridoxal (28).

Compounds 4a–i (except 4c) are the most promising compounds that should be kept in mind for designing new MLT-based indole derivatives for our ongoing study. These results suggest a new approach for the in vitro antioxidant activity properties and structure activity relationships of 3-substituted indole rings. Lack of a methoxy group in the fifth position did not affect the antioxidant capacity of the new indole derivatives. In fact, the in vitro assays showed that a lack of a methoxy group in the indole ring and an amino acid side chain resulted in much more active compounds than MLT itself. This may be due to increased stability of the indole ring and delocalization of the electrons to help to scavenge free radicals by forming stable indolyl cation radicals. The real function of methoxy group in fifth position of the indole ring of MLT is still not clear. Despite the removal of the methoxy group, the antioxidant capacity of the molecule may be enhanced (15); interestingly, all the synthesized indole derivatives (H in the fifth position of the indole ring) showed more potent antioxidant activity in LP inhibition assay.

In our previous studies, lack of methoxy group and introduction of Cl (52) or Br (33) in the fifth position did not make a dramatic change on the antioxidant capacity of the new indole derivatives infect the in vitro assays showed that many of the compounds were much more active than MLT itself. Literature data also suggest that melatonin shows antioxidant activity without methoxy group. Regarding the structure-antioxidant activity relationship, the 5-methoxy group of melatonin do not seem to significantly affect capacity of radical trapping. Related indoles being very similar to that obtained for melatonin (53).

Conclusion

Several experimental reports show that high concentrations of melatonin are needed to effectively scavenge free radicals, suggesting that these are unlikely mechanisms of action of physiological levels of melatonin in vivo (54,55). More research is needed to develop MLT analogues to find out improved antioxidant activity.

The results indicated significant strong antioxidant activity for most of the compounds, when compared to melatonin. Lack of a methoxy group in the fifth position did not affect the antioxidant capacity of the new indole derivatives. Structural investigation of the active compounds showed that having tryptophan (4e) and proline (4g) as side chains increase the antioxidant activity in two antioxidant activity tests. This result is very similar to our earlier findings (13,14) and the data in literature (11) and confirmed that for antioxidant activity, not only the indole-type aromatic ring is important, but so is the side chain especially at the third position to have better antioxidant activity than MLT.

The hydroxyl radical is considered to be the most reactive, harmful of all free radicals (56). The high LP inhibition results of the new indole amino acid derivatives in particular, comprises of the reactivity towards OH radical. Our results have shown that the combination of indole and amino acid groups is, as we expected, decisive for melatonin analogue compound’s antioxidant activity.

Ancillary