untitled European Journal of Chemistry 3 (2) (2012) 214‐219 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2012 EURJCHEM DOI:10.5155/eurjchem.3.2.214‐219.588 European Journal of Chemistry Journal homepage: www.eurjchem.com Novel indole‐2‐carboxylic acid analogues: Synthesis and a new light in to their antioxidant potentials Nagaraja Naika,*, Vishwanath Sharatha and Honnaiah Vijay Kumarb a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, 570006, Karnataka, India b Department of Organic Chemistry, Indian Institute of Science, Bangalore, 560012, India *Corresponding author at: Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, 570006, Karnataka, India. Tel.: +91.948.2959088; fax: +91.948.2959088. E‐mail address: drnaikchem@gmail.com (N. Naik). ARTICLE INFORMATION ABSTRACT Received: 14 January 2012 Received in revised form: 20 February 2012 Accepted: 21 February 2012 Online: 30 June 2012 KEYWORDS Two series of novel indole‐2‐carboxylic acid derivatives is reported. In the first series, N‐ substituted derivatives (3a‐h) were synthesized via acylation of indole‐2‐carboxylic acid followed by aldol condensation reaction. Whereas, in the second series, indole‐2‐ carboxamides (5a‐g) were synthesized through conversion of acid to its acid chloride followed by coupling of substituted anilines. Structures of the newly synthesized compounds were confirmed by elemental analysis and spectral IR, 1H NMR and mass data and were screened for antioxidant activity. Among the first series, compound 3g showed higher antioxidant activity and whereas, in the second series compounds 5b and 5c exhibited potential antioxidant activity. Compounds 3g, 5b and 5c exhibited for its enhanced antioxidant activity. DPPH Antioxidant activity Substituted anilines Indole‐2‐carboxylic acid Indole‐2‐carboxamide Butylated hydroxy anisole 1. Introduction Evidences suggests that free radicals, which are generated in many bioorganic redox processes, may induce oxidative damage in various components of the body (e.g., lipids, proteins and nucleic acids and may also be involved in the processes leading to the formations of mutations [1]. The deleterious effects of an imbalance between reactive oxygen species (ROS) production and the available antioxidant defense capacity, termed oxidative stress, as well as its role in the aggravation of a plethora of pathological conditions, are widely documented in the literature [2]. Efforts to counteract the damage caused by these species are gaining acceptance as a basis for novel therapeutic approaches and the field of preventive medicine is experiencing an upsurge of interest in medically useful antioxidants [3,4]. Antioxidants play a significant role in several important biological processes such as immunity, protection against tissue damage, reproduction and growth or development and can prevent cardiovascular disease, cancer, cataracts and various other ailments associated with ageing [5,6]. Indole and its derivatives are found abundantly in nature and are known to exhibit potent physiological properties [7‐ 10]. Substituted indoles are capable of binding to many receptors with high affinity. Therefore, the synthesis and selective functionalization of indoles have been the focus of active research over the years [11‐17]. Promoted from the above findings and as a continuation of our research interest in synthesis and biological activities of novel derivatives of some heterocyclic compounds [18‐20], the present study aimed to synthesis and to evaluate antioxidant potentials of novel indole‐2‐carboxylic acid analogues. 2. Experimental 2.1. Instrumentation All chemicals used were of laboratory grade (Qualigen, Merck). The melting points were determined by open capillary method on a Campbel electronic apparatus and are uncorrected. The IR spectra of synthesized compounds were recorded on a Shimadzu 8400S FT‐IR in potassium bromide disks. The 1H NMR was recorded in DMSO‐d6 using a NMR Varian‐Mercury 400 MHz spectrometer and chemical shifts are given in units as δ ppm, downfield from tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained on an Electron Impact mass spectrometer using Micromass Q‐Tof‐ 2 mass spectrometers at 70 eV ionizing beam and using a direct insertion probe. The progress of reactions was monitored by thin layer chromatography using chloroform‐methanol and hexane‐ethyl acetate as the solvent systems and spots were visualized after exposure to iodine vapours or under ultraviolet (UV) light. 2.2. Synthesis of 1‐acetyl‐1H‐indole‐2‐carboxylic acid (2) To a well stirred solution of 1H‐indole‐2‐carboxylic acid (1 mM) and triethylamine (1.2 mM) in 15 mL dichloromethane, acetyl chloride (1.3 mM) in 5 mL was added drop by drop for 10 min, then the reaction mixture is stirred at room temperature for about 3 hr. Progress of the reaction was monitored by thin layer chromatography (TLC) using hexane:ethylacetate (6:4) mixture as mobile phase. After the completion of reaction, the reaction mass was quenched in ice cold water and the product was extracted with ethyl acetate. Naik et al. / European Journal of Chemistry 3 (2) (2012) 214‐219 215 Figure 1. Protocol for the synthesis of indole‐2‐carboxylic acid derivatives, (a) N‐substituted indole‐2‐carboxylic acid analogues, (b) indole‐2‐carboxamides. The organic layer was washed with 5% NaHCO3 followed by distilled water. Finally the organic layer was dried over anhydrous Na2SO4. The brown solid product was obtained by desolventation through rotary evaporator. 1‐acetyl‐1H‐indole‐2‐carboxylic acid (2): Brown solid. Yield: 87%. M.p.: 175‐178 oC. FT‐IR (KBr, cm‐1): 1664 (C=O), 3347 (OH), 2853‐2943 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.1 (s, 1H, COOH), 6.92‐7.50 (m, 5H, Ar‐H), 2.36 (s, 3H, COCH3). MS (EI, m/z): 204.10 (M+1)+. Anal. calcd. for C11H9NO3 : C, 65.02; H, 4.46; N, 6.89; O, 23.62%. Found; C, 65.05; H, 4.43; N, 6.85; O, 23.65%. 2.3. Synthesis of 1H‐indole‐2‐carbonyl chloride (4) To a well stirred solution of 1H‐indole‐2‐carboxylic acid (1 mM) in 15 mL dry tetrahydrofuran (THF), thionyl chloride (1.2 mM) in 3mL dry THF was added drop wise at 0 oC, then the reaction mixture is stirred at room temperature for about 3 h. Progress of the reaction was monitored by TLC using hexane:ethylacetate(6:4) mixture as mobile phase. After the completion of reaction, the product was extracted with ethyl acetate. The organic layer was washed with 5% NaHCO3 followed by distilled water. Finally the organic layer was dried over anhydrous Na2SO4. The light brown solid product was obtained by desolventation through rotary evaporator. Further, coupling of substituted aldehydes and substituted anilines to obtain a series analogues of 1H‐indole‐2‐carboxylic acid 3a‐h and 5a‐g in moderate good yield, which were identified by spectroscopic techniques: 1H NMR, FT‐IR and MS‐ EI. The synthetic strategies of the synthesized compounds are depicted in Figure 1 (Table 1). 1H‐indole‐2‐carbonyl chloride (4): Light brown solid. Yield: 78%. M.p.: 157‐160 oC. FT‐IR (KBr, cm‐1): 1695 (C=O), 3338 (N‐ H), 2873‐2927 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.91 (s, 1H, N‐H), 6.91‐7.67 (m, 5H, Ar‐H). MS (EI, m/z): 181.08 (M+1)+. Anal. calcd. for C9H6ClNO: C, 60.19; H, 3.37; Cl, 19.74; N, 7.80; O, 8.91%. Found; C, 60.17; H, 3.39; Cl, 19.78; N, 7.84; O, 8.93. 2.4. General procedure for the synthesis of 1‐acetyl‐1H‐ indole‐2‐carboxylic acid analogues (3a‐h) To a solution of 1‐acetyl‐1H‐indole‐2‐carboxylic acid (1 mM) in ethanol (10 mL) substituted benzaldehydes were added in the presence of 10% NaOH at room temperature (Figure 1). Progress of the reaction was monitored by TLC using chloroform:methanol (6:4) mixture as mobile phase. After the completion of reaction, the product was extracted with ethyl acetate. The organic layer was washed with brine solution followed by distilled water. Finally the organic layer was dried over anhydrous Na2SO4. Further the product was obtained by desolventation through rotary evaporator. Table 1. Chemical structures of the synthesized compounds. (Z)‐1‐(3‐phenylacryloyl)‐1H‐indole‐2‐carboxylic acid (3a): Brown semi solid. Yield: 88%. FT‐IR (KBr, cm‐1): 1664 (C=O), 3505 (OH), 2863‐2918 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.3 (s, 1H, COOH), 7.45‐7.50 (m, 5H, Ar‐H), 6.91‐7.10 Entry Entry 3a 5a 3b 5b 3c 5c 3d 5d 3e 5e 3f 5f 3g 5g 3h 216 Naik et al. / European Journal of Chemistry 3 (2) (2012) 214‐219 (m, 5H, Ar‐H of R‐CHO), 6.63 (d, 1H, CH=CH), 6.59 (d, 1H, CH=CH). MS (EI, m/z): 292.10 (M+1)+. Anal. calcd. for C18H13NO3: C, 74.22; H, 4.50; N, 4.81; O, 16.48%. Found; C, 74.25; H, 4.52; N, 4.84; O, 16.45%. (Z)‐1‐(3‐(4‐chlorophenyl)acryloyl)‐1H‐indole‐2‐carboxylic acid (3b): White semi solid. Yield: 81%. FT‐IR (KBr, cm‐1): 1674 (C=O), 3445 (OH), 2853‐2948 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.1 (s, 1H, COOH), 7.21‐8.10 (m, 5H, Ar‐H), 7.22 (d, 4H, Ar‐H of R‐CHO), 6.73 (d, 1H, CH=CH), 6.68 (d, 1H, CH=CH). MS (EI, m/z): 326.12 (M+1)+. Anal. calcd. for C18H12ClNO3: C, 66.37; H, 3.71; Cl, 10.88; N, 4.30; O, 14.73%. Found; C, 66.39; H, 3.71; Cl, 10.85; N, 4.27 O, 14.76%. (Z)‐1‐(3‐(4‐hydroxyphenyl)acryloyl)‐1H‐indole‐2‐carboxylic acid (3c): Light brown solid. Yield: 87%. M.p.: 105‐108 oC. FT‐ IR (KBr, cm‐1): 1624 (C=O), 3338 (OH), 2849‐2923 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.24 (s, 1H, COOH), 7.21‐ 7.86 (m, 5H, Ar‐H), 6.83‐7.10 (d, 4H, Ar‐H of R‐CHO), 7.22 (d, 1H, CH=CH), 6.28 (d, 1H, CH=CH), 5.34 (s, 1H, Phenolic ‐OH). MS (EI, m/z): 308.12 (M+1)+. Anal. calcd. for C18H13NO4: C, 70.35; H, 4.26; N, 4.56; O, 20.83%. Found; C, 70.37; H, 4.24; N, 4.58; O, 20.87%. (Z)‐1‐(3‐(4‐nitrophenyl)acryloyl)‐1H‐indole‐2‐carboxylic acid (3d): Brown solid. Yield: 77%. M.p.: 115‐118 oC. FT‐IR (KBr, cm‐1): 1664 (C=O), 3445 (OH), 2838‐2988 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.1 (s, 1H, COOH), 7.45‐7.50 (m, 5H, Ar‐H), 6.86‐7.48 (d, 4H, Ar‐H of R‐CHO), 6.73 (d, 1H, CH=CH), 6.27 (d, 1H, CH=CH). MS (EI, m/z): 337.20 (M+1)+. Anal. calcd. for C18H12N2O5: C, 64.29; H, 3.60; N, 8.33; O, 23.79%. Found; C, 64.25; H, 3.63; N, 8.35; O, 23.75%. (Z)‐1‐(3‐(4‐methoxyphenyl)acryloyl)‐1H‐indole‐2‐carboxylic acid (3e): Yellow solid. Yield: 85%. M.p.: 124‐127 oC. FT‐IR (KBr, cm‐1): 1683 (C=O), 3447 (OH), 2847‐2943 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.0 (s, 1H, COOH), 7.45‐7.50 (m, 5H, Ar‐H), 6.91‐7.32 (d, 4H, Ar‐H of R‐CHO), 7.11 (d, 1H, CH=CH), 6.93 (d, 1H, CH=CH), 3.84 (s, 3H, OCH3). MS (EI, m/z): 322.20 (M+1)+. Anal. calcd. for C19H15NO4: C, 71.02; H, 4.71; N, 4.36; O, 19.92%. Found; C, 71.04; H, 4.75; N, 4.33; O, 19.90%. (Z)‐1‐(3‐p‐tolylacryloyl)‐1H‐indole‐2‐carboxylic acid (3f): Brown semi solid. Yield: 85%. FT‐IR (KBr, cm‐1): 1663 (C=O), 3505 (OH), 2853‐2918 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.0 (s, 1H, COOH), 7.45‐7.50 (m, 5H, Ar‐H), 6.83‐7.10 (d, 4H, Ar‐H R‐CHO), 6.79 (d, 1H, CH=CH), 6.63 (d, 1H, CH=CH), 2.33 (s, 3H, CH3). MS (EI, m/z): 306.30 (M+1)+. Anal. calcd. for C19H15NO3: C, 74.74; H, 4.95; N, 4.59; O, 15.72%. Found; C, 74.72; H, 4.96; N, 4.59; O, 15.71%. (Z)‐1‐(3‐(4‐hydroxy‐3‐methoxyphenyl)acryloyl)‐1H‐indole‐2‐ carboxylic acid (3g): Yellow solid. Yield: 88%. M.p.: 162‐165 oC. FT‐IR (KBr, cm‐1): 1675 (C=O), 3388 (OH), 2853‐2918 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.2 (s, 1H, COOH), 7.45‐ 7.68 (m, 5H, Ar‐H), 7.10‐7.22 (d, 3H, Ar‐H of R‐CHO), 7.10 (d, 1H, CH=CH), 6.64 (d, 1H, CH=CH), 5.35 (s, 1H, phenolic OH), 3.83 (s, 3H, ‐OCH3). MS (EI, m/z): 338.20 (M+1)+. Anal. calcd. for C19H15NO5: C, 67.65; H, 4.48; N, 4.15; O, 23.72%. Found; C, 67.63; H, 4.44; N, 4.17; O, 23.76%. (Z)‐1‐(3‐(3,4,5‐trimethoxyphenyl)acryloyl)‐1H‐indole‐2‐ carboxylic acid (3h): White solid. Yield: 82%. M.p.: 126‐129 oC. FT‐IR (KBr, cm‐1): 1653 (C=O), 3433 (OH), 2854‐2938 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.0 (s, 1H, COOH), 6.26‐ 7.11 (m, 5H, Ar‐H), 7.67 (d, 2H, Ar‐H of R‐CHO), 7.42 (d, 1H, CH=CH), 7.46 (d, 1H, CH=CH), 3.83 (m, 9H, OCH3). MS (EI, m/z): 382.23 (M+1)+. Anal. calcd. for C21H19NO6: C, 66.13; H, 5.02; N, 3.67; O, 25.17%. Found; C, 66.15; H, 5.06; N, 3.63; O, 25.14%. 2.5. General procedure for the synthesis of 1H‐indole‐2‐ carbonyl chloride analogues (5a‐g) To a solution of 1H‐indole‐2‐carbonyl chloride (1 mM) in dry THF (10 mL) substituted anilines were added in the presence of TEA (3 mL) under inert (N2) atmosphere. The reaction mixture was refluxed for 4 hr (Figure 1). Progress of the reaction was monitored by TLC using chloroform:methanol (6:4) mixture as mobile phase. After the completion of reaction, the product was extracted with ethyl acetate. The organic layer was washed with 5% NaHCO3 solution followed by distilled water. Finally the organic layer was dried over anhydrous Na2SO4. Further the product was obtained by desolventation through rotary evaporator. N‐phenyl‐1H‐indole‐2‐carboxamide (5a): Yellow solid. Yield: 82%. M.p.: 115‐118 oC. FT‐IR (KBr, cm‐1): 1656 (C=O), 3265 (N‐ H), 2853‐2927 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.9 (s, 1H, NH of indole), 8.35 (s, 1H, NH of amine), 7.1‐7.45 (m, 5H, Ar‐H), 7.5‐7.75 (d, 4H, Ar‐H R‐NH2). MS (EI, m/z): 237.28 (M+1)+. Anal. calcd. for C15H12N2O: C, 76.25; H, 5.12; N, 11.86; O, 6.77%. Found; C, 76.26; H, 5.15; N, 11.88; O, 6.75%. N‐(4‐hydroxyphenyl)‐1H‐indole‐2‐carboxamide (5b): White solid. Yield: 76%. M.p.: 125‐128 oC. FT‐IR (KBr, cm‐1): 1695 (C=O), 3338 (N‐H), 2873‐2956 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.7 (s, 1H, NH of indole), 9.15 (s, 1H, NH of amine), 6.91‐7.54 (m, 5H, Ar‐H), 7.45‐7.70 (d, 4H, Ar‐H R‐NH2), 5.35 (s, 1H, phenolic OH). MS (EI, m/z): 253.21 (M+1)+. Anal. calcd. for C15H12N2O2: C, 71.42; H, 4.79; N, 11.10; O, 12.68%. Found; C, 71.40; H, 4.77; N, 11.13; O, 12.65%. N‐(2‐hydroxyphenyl)‐1H‐indole‐2‐carboxamide (5c): White solid. Yield: 85%. M.p.: 176‐179 oC. FT‐IR (KBr, cm‐1): 1695 (C=O), 3348 (N‐H), 2863‐2956 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.9 (s, 1H, NH of indole), 9.13 (s, 1H, NH of amine), 6.81‐7.59 (m, 5H, Ar‐H), 7.44 ‐7.99 (d, 4H, Ar‐H R‐NH2), 5.3 (s, 1H, phenolic OH). MS (EI, m/z): 253.18 (M+1)+. Anal. calcd. for C15H12N2O2: C, 71.42; H, 4.79; N, 11.10; O, 12.68%. Found; C, 71.41; H, 4.77; N, 11.14; O, 12.66%. N‐(4‐methoxyphenyl)‐1H‐indole‐2‐carboxamide (5d): Bright yellow solid. Yield: 82%. M.p.: 111‐114 oC. FT‐IR (KBr, cm‐1): 1689 (C=O), 3317 (N‐H), 2873‐2933 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.62 (s, 1H, NH of indole), 9.41 (s, 1H, NH of amine), 7.11‐7.57 (m, 5H, Ar‐H), 7.51‐7.88 (d, 4H, Ar‐H R‐NH2), 3.83 (s, 3H, OCH3). MS (EI, m/z): 267.32 (M+1)+. Anal. calcd. for C16H14N2O2: C, 72.16; H, 5.30; N, 10.52; O, 12.02%. Found; C, 72.17; H, 5.27; N, 10.55; O, 12.06%. N‐(2‐methoxyphenyl)‐1H‐indole‐2‐carboxamide (5e): White solid. Yield: 72%. M.p.: 185‐188 oC. FT‐IR (KBr, cm‐1): 1695 (C=O, 3378 (N‐H), 2823‐2986 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.6 (s, 1H, NH of indole), 9.43 (s, 1H, NH of amine), 6.86‐7.86 (m, 5H, Ar‐H), 7.51‐7.90 (d, 4H, Ar‐H R‐NH2), 3.83 (s, 3H, OCH3). MS (EI, m/z): 267.30 (M+1)+. Anal. calcd. for C16H14N2O2: C, 72.16; H, 5.30; N, 10.52; O, 12.02%. Found; C, 72.18; H, 5.29; N, 10.55; O, 12.05%. N‐(4‐bromophenyl)‐1H‐indole‐2‐carboxamide (5f): White semi solid. Yield: 77%. FT‐IR (KBr, cm‐1): 1775 (C=O), 3407 (N‐ H), 2833‐2936 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.91 (s, 1H, NH of indole), 9.5 (s, 1H, NH of amine), 7.1‐7.67 (m, 5H, Ar‐H), 7.61‐8.11 (d, 4H, Ar‐H R‐NH2), MS (EI, m/z): 316.36 (M+1)+. Anal. calcd. for C15H11BrN2O: C, 57.16; H, 3.52; Br, 25.35; N, 8.89; O, 5.08%. Found; C, 57.16; H, 3.54; Br, 25.37; N, 8.85; O, 5.05%. N‐(4‐nitrophenyl)‐1H‐indole‐2‐carboxamide (5g): White solid. Yield: 70%. M.p.: 106‐109 oC. FT‐IR (KBr, cm‐1): 1677 (C=O), 3438 (N‐H), 2873‐2956 (Ar‐H). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 11.76 (s, 1H, NH of indole), 9.1 (s, 1H, NH of amine), 7.33 ‐7.71 (m, 5H, Ar‐H), 7.64‐7.97 (d, 4H, Ar‐H R‐NH2). MS (EI, m/z): 282.10 (M+1)+. Anal. calcd. for C15H11N3O3: C, 64.05; H, 3.94; N, 14.94; O, 17.07%. Found; C, 64.09; H, 3.92; N, 14.97; O, 17.03%. 2.6. Antioxidant activity studies The novel synthesized molecules were further subjected for the antioxidant evaluation by various in vitro assays like 2,2‐ diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging, 2,2‐azino bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS•+) radical cation decolorization assays, Ferric ion (Fe3+) reducing antioxidant power assay (FRAP) and Cupric ion (Cu2+) reducing ability (CUPRAC method). Naik et al. / European Journal of Chemistry 3 (2) (2012) 214‐219 217 2.6.1. Free radical scavenging activity The newly synthesized compounds were screened for free radical scavenging activity by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) method [21]. Compounds of different concentrations were prepared in distilled ethanol, 1 mL of each compound solutions (3a‐h) and (5a‐g) having different concentrations (10, 25, 50, 100, 200 and 500 µM) were taken in different test tubes, 4 mL of 0.1 mM ethanol solution of DPPH was added and shaken vigorously. The test tubes were then incubated in the dark room at room temperature for 20 min. A DPPH blank was prepared without the compound and ethanol was used for the baseline correction. Changes (decrease) in the absorbance at 517 nm were measured using a UV‐visible spectrometer (Shimadzu 160 A). The radical scavenging activities were expressed as the inhibition percentage and were calculated using the formula (Equation 1): Radical scavenging activity (%) = [(Ac‐As)/Ac)x100] (1) where Ac is absorbance of the control (without compound) and As is absorbance of the compounds 3a‐h and 5a‐g. The radical scavenging activity of BHA and ascorbic acid was also measured and compared with that of the different synthesized compounds. 2.6.2. ABTS•+ radical scavenging activity The synthesized indole‐2‐carboxylic acid analogues were subjected to 2,2‐azino bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS•+) radical scavenging activity [22]. The ABTS•+ cation was produced by the reaction between 7 mM ABTS in H2O and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 hr. Before the usage, the ABTS•+ solution was diluted to get an absorbance of 0.700±0.025 at 734 nm with phosphate buffer (0.1 M, pH = 7.4). Then, 1 mL of ABTS•+ solution was added to the compounds 3a‐h and 5a‐g solution in ethanol at different concentrations (1.5 mL, 10, 25, 50, 100, 200, 500 µM/mL). After 30 min, the percentage inhibition at 734 nm was calculated for each concentration relative to a blank absorbance (ethanol). The scavenging capability of ABTS•+ radical was calculated using the equation 2. ABTS•+ scavenging effect (%) = [(Ac‐As) / Ac] x 100 (2) where, Acontrol is the initial concentration of the ABTS•+ and Asample is the absorbance of the remaining concentration of ABTS•+ in the presence of the compounds 3a‐h and 5a‐g. 2.6.3. Ferric ion (Fe3+) reducing antioxidant power assay (FRAP) All the novel indole‐2‐carboxylic acid analogues were screened for ferric reducing antioxidant power [23]. The compounds 3a‐h and 5a‐g having concentration (10 μM/mL) were mixed with phosphate buffer (2.5 mL, 0.2 M, pH = 6.6) and potassium ferric cyanide (2.5 mL, 1%). The mixture was incubated at 50 oC for 20 min. Later, the reaction mixture was acidified with trichloroacetic acid (2.5 mL, 10%). After FeCl3 (0.5 mL, 0.1%) was added to this solution, the absorbance was measured at 700 nm. The increased absorbance of the reaction mixture indicates an increased reducing power. 2.6.4. Cupric ion (Cu2+) reducing ability (CUPRAC method) All the synthesized compounds were performed cupric ion reducing ability assay [24]. Briefly, a mixture of CuCl2 (1 mL, 0.01 M) solution, ethanolic neocuproine (Nc) (1 mL, 7.5 x 10‐3 M) solution and ammonium acetate (1 mL, 1.0 M) in a test tube were added to a solution of compounds 3a‐h and 5a‐g (1 mL, 10 µM) along with 0.1 mL distilled water. The mixture was incubated for 30 min. Then the absorbance was measured at 450 nm against reagent blank. 2.6.5. Statistical analysis Tests were carried out in triplicate for 3‐5 separate experiments. The amount of compound needed to inhibit DPPH free radicals and ABTS radicals concentration by 50%, (IC50) was graphically estimated using a linear regression algorithm. 3. Results and discussion 3.1. Chemistry To keep the electron‐withdrawing and electron‐donating character of the substituents in the molecule, we decided to study several indole‐2‐carboxylic acid analogues bearing electron rich and deficient substituents. In the first series, the key intermediate (2) was obtained by N‐acylation using acetyl chloride in the presence of triethylamine as base. Further, aldol condensation of the key intermediate (2) with various substituted aldehydes in the presence of sodium hydroxide as base, afforded the corresponding N‐substituted indole‐2‐ carboxylic acid analogues (3a‐h) (Figure 1 (a)). Further, in the second series indole‐2‐carboxamides (5a‐g) was obtained by conversion of acid to acid chlorides in the presence of thionyl chloride followed by coupling of substituted anilines through base condensation reaction in good yield (Figure 1 (b)). The newly synthesized compounds were purified by column chromatography using silica gel 60‐120 mesh and chloroform:methanol (60:40) as eluent. The synthesized compounds were characterized by various physico‐chemical and spectroscopic techniques like IR, 1H NMR, mass and elemental analysis. The absence of a broad absorption band at 3300 cm‐1 corresponding to ‐NH absorption and a sharp band at 3347 cm‐1 corresponding to acid group in IR spectrum of key intermediate (2) and the absence of signal at 11.91 ppm in 1H NMR confirms the acylation to ‐NH proton. Similarly, in the IR spectra of key intermediate (4) exhibited the absence of a broad absorption band at 3300 cm‐1 which corresponds to acid group and the presence of a sharp absorption band at 3338 cm‐1 for the NH absorptions confirms the acid to acid chloride reaction successfully. The IR spectra of all the substituted aldehyde analogues (3a‐h) showed the absence of sharp N‐H band ie., indole (N‐H) band at around 3300‐3500 cm‐1 and also reveals the presence of aromatic peaks (Ar‐H) at the respective region 2853‐2943 cm‐1. Similarly, the IR spectra of all the substituted aniline analogues (5a‐g) showed a sharp N‐H band ie., indole (N‐H) band at around 3304‐3438 cm‐1 and also reveals the presence of aromatic peaks (Ar‐H) at the respective region 2823‐2986 cm‐1. 1H NMR spectra of all conjugated analogues (3a‐h) showed the absence of N‐H protons as singlet at 11.91 ppm. The signal due to acid ‐OH in all the analogues appeared as singlet at about 11.0‐11.4 ppm. In addition to acid ‐OH, the phenolic ‐OH resonated at 5.34 ppm in compound 3c and ‐OCH3 protons present in the compound 5e resonated as singlet at 3.83 ppm, other aromatic protons were observed at expected regions 6.26‐8.10 ppm for (3a‐h) and 6.81‐8.11 ppm for (5a‐g). 3.2. Antioxidant activities 3.2.1. DPPH radical scavenging activity The DPPH radical scavenging activity assay is a simple method for measuring the antioxidant ability to trap free radicals. The scavenging effects of compounds (3a‐h) and (5a‐ g) and two controls, BHA and ascorbic acid are evaluated (Table 2). Compound 3g containing hydroxy and methoxy group and 5b and 5c containing a hydroxy moiety on phenyl 218 Naik et al. / European Journal of Chemistry 3 (2) (2012) 214‐219 ring showed the highest activity (IC50 = 23 μM, 35 μM and 45 μM) among the synthesized analogues. Compound 5e possessing methoxy group and 3c having hydroxy group on phenyl moiety displayed promising activity (IC50 = 50 μM and 65 μM). Table 2. IC50 (Concentration required for 50% inhibition) values of DPPH• and ABTS•+ radical scavenging activities of the compounds (3a‐h) and (5a‐g) and the standard antioxidant compounds such as BHA and Ascorbic acid. Tested compounds DPPH• Scavenging activity (IC50)* ABTS•+ BHA 12 15 Ascorbic acid 10 12 3a 190 140 3b 290 265 3c 65 52 3d 400 200 3e 74 86 3f 145 123 3g 23 20 3h 200 315 5a 190 175 5b 35 28 5c 45 40 5d 85 68 5e 50 62 5f 285 375 5g 195 164 * The values are expressed as μM concentration. Lower IC50 values indicate higher radical scavenging activity. 3.2.2. ABTS radical cation scavenging activity The ABTS assay is a widely used method for measuring the antioxidant ability to trap free radicals [22]. The ABTS radical cation scavenging capacity of the synthesized compounds (3a‐ h) and (5a‐g) are screened Table 2. Scaffold 3g containing hydroxy and methoxy group and 5b and 5c containing a hydroxy moiety on phenyl ring showed good ABTS scavenging capacity (IC50 = 20 μM, 28 μM and 40 μM). Whereas, compounds 3c, 5e and 5d displayed moderate activity (IC50 = 52 μM, 62 μM and 68 μM). 3.2.3. Ferric reducing antioxidant power (FRAP) Ferric reducing power was determined using the iron(III) to iron(II) reduction assay. Since the antioxidant activity of a substance is usually correlated directly to its reducing capacity, the FRAP assay provides a reliable method to study the antioxidant activity of various compounds [23]. Ferric reducing ability of compounds (3a‐h) and (5a‐g) are studied Table 3. Compounds 3g, 5b and 5c showed the best reducing power (absorbance value = 0.4144, 0.3989 and 0.3791) among the synthesized analogues but slightly less compared to that of the standards BHA and ascorbic acid. This may be due to the presence of electron donating capacity of hydroxy and methoxy groups on phenyl ring. While the reducing power of the other synthesized compounds 3c, 5d and 3e showed moderate activity and compounds 3b, 5g, 3d and 5f containing electron withdrawing groups like ‐Cl, ‐NO2 and Br showed least activity. The reducing power of all compounds and standards showed an increase by rising concentrations. 3.3.4. Cupric ion (Cu2+) reducing ability (CUPRAC method) The CUPRAC method is based on Cu(II)‐Cu(I) reduction by antioxidants in the presence of neocuproine. The copper reducing ability of newly synthesized compounds are examined Table 3. Among the synthesized analogues, compound 3g, containing hydroxy and methoxy group and 5b and 5c containing a hydroxy moiety on phenyl ring showed marked cupric ion reducing ability (absorbance value= 0.2980, 0.2132 and 0.2104). Whereas, compounds 3c and 5d showed average activity. Table 3. Comparison of ferric ions (Fe3+) reducing ability by Fe3+‐Fe2+ transformation methods and Cu2+‐Cu+ reducing ability of the compounds 3a‐ h and 5a‐g and the standard antioxidant compounds such as BHA and Ascorbic acid at the concentration of 10 µM. Tested compounds Fe3+‐Fe2+ reducing ability* Cu2+‐Cu+ reducing ability* BHA 0.5462 0.3534 Ascorbic acid 0.6362 0.4391 3a 0.1577 0.0562 3b 0.0971 0.0486 3c 0.3556 0.2022 3d 0.1137 0.0862 3e 0.3410 0.1598 3f 0.1520 0.1462 3g 0.4144 0.2980 3h 0.2362 0.1639 5a 0.2163 0.1430 5b 0.3989 0.2132 5c 0.3791 0.2104 5d 0.3218 0.1980 5e 0.3164 0.1532 5f 0.1116 0.1429 5g 0.1034 0.1110 * The values are expressed as absorbance. High absorbance indicates high reducing power. 4. Conclusion In the present investigation, two series of novel indole‐2‐ carboxylic acid analogues (3a‐h) and (5a‐g) have been synthesized by a simple and convenient method. All the synthesized analogues were screened for their antioxidant activity by various in vitro assays. 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