Synthesis, characterization and antioxidant activities ...

Journal of Saudi Chemical Society (2014) xxx, xxx–xxx

King Saud University

Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Synthesis, characterization and antioxidant activities of Schiff bases are of cholesterol Madasamy Kumar, Tamilenthi Padmini *, Kandasamy Ponnuvel School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu, India Received 6 December 2013; revised 9 March 2014; accepted 21 March 2014

KEYWORDS Antioxidant; Schiff’s base; Cholesterol and radical

Abstract A series of new cholesterol based Schiff base derivatives, namely cholesteryl-n-(4-((E)(40 -cyanobiphenyl-4-ylimino)methyl)phenoxy)alkanoate (3a–j) have been synthesized and characterized by IR, NMR and mass spectral studies. In vitro antioxidant activities of these compounds were evaluated against super oxide anion radical, nitric oxide radical, DPPH radical and hydrogen peroxide and were compared with standard natural antioxidant, ascorbic acid. Our results reveal that these compounds exhibit excellent radical scavenging activities. ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction Currently, numerous investigations are ongoing into the interaction of free radicals with biological systems such as lipids, DNA and protein. The free radicals are responsible for many diseases such as neurodegenerative diseases, atherosclerosis, age-related diseases, rheumatoid arthritis, cancer initiation and tumor [2,18,33]. It is necessary to keep a proper level of natural antioxidants such as vitamin C, vitamin E and glutathione in biological system in order to avoid serious health problems [11,16,14,27]. All of these health problems are caused by the reaction of reactive oxygen species (ROS) and reactive nitrogen species (RNS), commonly known as reactive species (RSs) [5,17]. There are three ways by which RSs are formed: * Corresponding author. Tel.: +91 9095169125. E-mail addresses: [email protected], padimini_tamilenthi@ yahoo.co.in (T. Padmini). Peer review under responsibility of King Saud University.

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(i) metal catalyzed reactions, (ii) presence of pollutants in the atmosphere, (iii) irradiation by UV light, X-ray and gamma rays [8]. Antioxidants are generally hydrogen donors or electron donors to the reactive site in neutralizing free radicals [13,20]. The scavenging activity of different organic compounds can be assessed using DPPH, hydrogen peroxide, superoxide anion radical, and ABTS+. It has been already reported that many organic molecules act as very good antioxidants, thus it is important to understand the mode of action and efficiency of these antioxidants. There are large numbers of natural and synthetic antioxidants which have been explored, and their antioxidant capacity has been assessed by different methods [26]. Realizing the effects of free radicals, it is of paramount significance to develop very efficient antioxidants to prevent damage caused by free radicals. Our group is interested in synthesizing organic new Schiff base molecule to investigate the antioxidants’ property by employing superoxide anion radical, nitric oxide radical, DPPH radical and hydrogen peroxide. Schiff base has a similar structure of trans stilbene and it is derived from the condensation reaction between aldehyde derivatives with aniline. Resveratrol is a well-characterized

1319-6103 ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.jscs.2014.03.006 Please cite this article in press as: M. Kumar et al., Synthesis, characterization and antioxidant activities of Schiff bases are of cholesterol, Journal of Saudi Chemical Society (2014), http://dx.doi.org/10.1016/j.jscs.2014.03.006

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M. Kumar et al.

antioxidant found in grapes. It has been reported that the only structural difference between resveratrol and Schiff base is C‚N and C‚C [34]. The presence of C‚N linkage in Schiff base is very important for biological activity, such as antibacterial, antifungal, anticancer, antioxidant and diuretic activities. These compounds also have applications in food chemistry, dye industry, agro chemical and pharmaceuticals [1,15,35,9,25,30,36]. Some selected examples are given in Fig. 1. [1,21,35]. These Schiff bases can be used for further biological studies such as antimicrobial [28], antifungal [32] and antitumor [23]. Steroids play a very important role in human metabolism by acting as an essential part of the cell membranes (cholesterol) or hormones. Addition or modification in steroidal skeleton would lead to changes in their biological activities [3,24,31]. Selected examples are listed in Fig. 2. Steroids have many biological applications due to their non-toxic activity, bio-availability and less vulnerable to multi-drug resistance (MDR) [4].

ble material, the filtrate collected and the solvent removed under reduced pressure. The resulting mixture was purified by column chromatography (silica) using dichloromethane as eluent. The crude product was recrystallized twice from a mixture of chloroform and ethanol [10]. 2.3. General procedure for the synthesis of cholesteryl aldehyde derivatives (2) A mixture of 4-hydroxy benzaldehyde (3.3 mmol), cholesterol bromoalkyl derivatives 1 (2.9 mmol) and K2CO3 (3 g) in 10 ml of butanone was heated under reflux for 12 h. After removal of the solvent, 25 ml of water was added to the residue and the mixture was extracted with chloroform. The organic layer was washed with water (2 · 15 ml) and dried over Na2SO4. The residue obtained after removing the solvent was subjected to column chromatography over silica gel using petroleum ether/ethyl acetate (19:1) as eluent to get the product [22].

2. Experimental section

2.4. Synthesis of cholesteryl-3-(n-((E)-(40 -cyanobiphenyl-4ylimino)methyl)-phenoxy or 3-hydroxyphenoxy)alkanoates

2.1. General The target compounds (3a–3j) were synthesized by the classical condensation of aldehyde and aniline, with the reaction path shown in Scheme 1. The equal mole ratio of cholesterol aldehyde derivatives 2 and 40 -aminobiphenyl-4-carbonilide were dissolved in ethanol. The reaction mixture was stirred under reflux to give an orange solution. After allowing the solution to stand in cold condition, yellow solid was collected, washed with cold ethanol and recrystallized from absolute ethanol to yield a pure product. All the synthesized compounds were prepared by using the same procedure and shown in Scheme 1. The structural confirmations were done by using 1H NMR, 13 C NMR, EI mass, IR spectra and given as Supplementary information.

The requisite precursors were purchased from Aldrich and Alfa-Aesar and used as received. The solvents were purified and dried as per the literature methods prior to use. The target molecules were purified by recrystallization choosing an appropriate mixture of solvents. The intermediates were purified by column chromatography using silica gel (60–120 mesh) as stationary phase. Thin layer chromatography was performed using aluminum sheets pre-coated with silica gel. Melting points were determined with sigma melting point apparatus. 1H NMR spectra were recorded using Bruker (300 MHz) spectrometer. For the 1H NMR spectra, the chemical shifts are reported in ppm relative to SiMe4 as an internal standard and coupling constants are presented in Hz. Infrared spectra were recorded on JASCO-FT-IR spectrometer 4000– 400 cm1; the spectral positions are given in wave numbers (cm1). Electro spray ionization mass spectrometry (ESI-MS) analysis was performed in the positive ion mode on a liquid chromatography-ion trap mass spectrometer (LCQ Fleet, Thermo Fisher Instruments Limited, US).

2.5. Cholesteryl-4-(4-((E)-(4’-cyanobiphenyl-4-ylimino) methyl) phenoxy) butanoate (3a) Light yellow solid; Mp: 260 C; Y = 93%; IR max (KBr): 2934, 2156, 1720, 1594, 1246, 1164, 824, 726, 546 cm1;1H NMR (300 MHz, CDCl3) d = 8.42 (s, 1H, N‚CH), 7.85 (d, J = 9.0 Hz, 2H, ArH), 7.73–7.67 (m, 4H, ArH), 7.61 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 9.0 Hz, 2H, ArH), 6.96 (d, J = 9.0 Hz, 2H, ArH), 5.4 (d, J = 6.0 Hz, 1H, olefinic H), 4.69–4.53 (m, 1H, CHOCO), 4.07 (t, 2H, J = 6.6 Hz, OCH2), 2.53–2.48(m, 2H, CH2), 2.34–2.30 (m, 2H, CH2), 2.14–0.85 (m, 42H, CH2), 0.67 (s, 3H,CH3); 13C NMR (75 MHz, CDCl3) d = 172.5, 161.8, 160.0, 152.3, 145.1, 139.6, 136.1, 132.6, 130.7, 129.1, 127.9, 127.3, 122.7, 121.7, 118.9, 114.7, 110.6, 74.1, 67.0, 56.7, 56.1, 50.0, 42.3, 39.5,

2.2. General procedure for the synthesis of cholesterylbromo alkyl derivatives (1) A mixture of n-bromoalkanoic acid (0.5 mmol), cholesterol (1.5 mmol), N-N0 -dicyclohexylcarbodiimide (DCC) (2 equiv.) and a catalytic amount of 4-dimethylaminopyridine (DMAP) in dry dichloromethane was stirred overnight at room temperature. The reaction mixture was filtered to remove the insolu-

O

O

R4

O

R1 1

N R2

Figure 1

R2

R3

N

R3

R1

R1

R2

2

N

O N

N 3

Some Schiff base examples.

Please cite this article in press as: M. Kumar et al., Synthesis, characterization and antioxidant activities of Schiff bases are of cholesterol, Journal of Saudi Chemical Society (2014), http://dx.doi.org/10.1016/j.jscs.2014.03.006

Synthesis, characterization and antioxidant activities of Schiff bases are of cholesterol O

3

NH 2 R

CN H H HO

HO

H

5

Ph

H

6

H 2N

n

N 7

Some steroid examples.

Figure 2

O Br

HO

+

n OH

n= 4, 5, 6, 8,11

a

R OH + Br

OHC

()

O

n

O

b

1

OHC NC

NH 2

+

()

O

R

O

n

O

R= H or OH

n= 4, 5, 6, 8,11 2

c NC

N R

3

O

()

O

n

O

3a;R= H, n= 4: 3b; R= OH, n=4: 3c; R= H, n=5: 3d; R= OH, n=5: 3e; R= H, n=6: 3f ; R= OH, n=6: 3g;R= H, n=8: 3h;R= OH, n=8: 3i:R= H, n=10, 3j;R= OH, n=10.

Scheme 1 Reagents and conditions: (a) DCC, DMAP, DCM, (b) anhyd K2CO3, KI, butanone reflux, 12 h and (c) ethanol, AcOH (catalytic), refux, 2 h.

36.6, 36.2, 35.8, 31.9, 31.8, 28.2, 28.0, 23.8, 22.8, 22.6, 19.3, 18.7, 11.8; Ms:m/z 754.417 (M+1). 2.6. Cholesteryl-4-(4-((E)-(4’-cyanobiphenyl-4-ylimino) methyl)-3-hydroxyphenoxy)butanoate (3b) Yellow solid; Mp: 264 C; Y = 94%; IR max (KBr): 3390, 2936, 2226, 1728, 1598, 1258, 1164, 824, 726, 546 cm1; 1H NMR (300 MHz, CDCl3) d = 13.63 (s, 1H, ArOH), 8.59 (s, 1H, N‚CH), 7.74–7.70 (m,4H, ArH), 7.67 (d, J = 6.0 Hz, 2H, ArH), 7.37 (d, J = 6.0 Hz, 2H, ArH), 7.32 (d, J = 6.0 Hz, 2H, ArH), 6.59 (d, J = 6.0 Hz, 1H) 5.46 (d, 1H, olefinic H), 4.69–4.53 (m 1H, CHOCO), 4.03 (t, 2H, J = 6.6 Hz, OCH2), 2.36–2.29 (m, 4H, CH2), 2.38–0.94 (m, 49H, –CH2), 0.76 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d = 172.9, 161.8, 159.5, 148.8, 141.8, 139.6, 138.3, 133.7, 132.6, 128.1, 127.4, 122.6, 121.76, 118.8, 118.1, 112.94, 107.74, 106.3, 101.5, 73.8, 67.8, 56.6, 56.1, 49.9, 42.2, 39.6, 39.4, 36.5, 36.7, 31.8, 27.9, 27.8, 22.7, 22.5, 20.9, 18.6, 68.1, 11.8; Ms: m/z 770.667 (M+1). 2.7. Cholesteryl-5-(4-((E)-(40 -cyanobiphenyl-4-ylimino) methyl)phenoxy) pentanoate (3c) Pale yellow solid; Mp: 264 C; Y = 93%; IR max (KBr): 2937, 2256, 1728, 1593, 1246, 1164, 824, 726, 546 cm1; 1H NMR

(300 MHz, CDCl3) d = 8.42 (s, 1H, N‚CH), 7.87 (d, J = 9.0 Hz, 2H, ArH), 7.75–7.69 (m, 4H, ArH), 7.61 (d, J = 9.0 Hz, 2H, ArH), 7.29 (d, J = 9.0 Hz, 2H, ArH), 6.99 (d, J = 9.0 Hz, 2H, ArH), 5.38 (d, J = 6.0 Hz, 1H, olefinic H), 4.67–4.57 (m, 1H, CHOCO), 4.06 (t, 2H, J = 6.6 Hz, OCH2), 2.39–2.30 (m, 4H, CH2), 2.03–0.86 (m, 42H, CH2), 0.68 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) d = 172.6, 161.9, 160.0, 152.9, 145.2, 139.6, 136.1, 132.5, 130.6, 129.1, 127.9, 127.3, 122.6, 118.8, 114.8, 110.7, 73.9, 67.7, 56.7, 56.2, 50.1, 42.3, 39.8, 39.5, 38.2, 37.0, 36.6, 36.2, 35.7, 34.2, 31.9, 28.5, 28.2, 27.9, 27.8, 24.2, 23.8, 22.7, 22.5, 21.7, 21.0, 19.3, 18.7, 11.8; Ms: m/z 768.333 (M+1). 2.8. Cholesteryl-5-(4-((E)-(40 -cyanobiphenyl-4-ylimino) methyl)-3-hydroxyphenoxy)pentanoate (3d) Yellow solid; Mp: 267 C; Y = 94%; IR max (KBr): 3391 2936, 2252, 1736, 1556, 1248, 1164, 824, 726, 546 cm1; 1H NMR (300 MHz, CDCl3) d = 13.59 (s, 1H, ArOH), 8.57 (s, 1H, N‚CH), 7.74–7.62 (m, 4H, ArH), 7.62 (d, J = 6.0 Hz, 2H, ArH), 7.37 (d, J = 6.0 Hz, 2H, ArH) 7.34 (d, J = 6.0 Hz, 2H, ArH) 6.46 (d, J = 8.7 Hz, 1H, ArH), 5.36 (d, J = 6.0 Hz, 1H, olefinic H), 4.63–4.57 (m, 1H, CHOCO), 4.15 (t, J = 6.6 Hz, 2H, OCH2), 2.38–2.30 (m, 4H, CH2),


4 1.97–0.84 (m, 51H, CH2), 0.7 (s, 3H, CH3).13C NMR (75 MHz, CDCl3) d = 172.7, 163.8, 163.6, 161.8, 144.8, 139.6, 136.9, 133.6, 132.6, 128.1, 127.4, 122.6, 121.7, 118.8, 122.9, 110.8, 107.7, 101.6, 73.9, 67.6, 56.6, 56.1, 49.9, 42.2, 39.4, 38.1, 36.6, 35.7, 31.8, 28.1, 27.98, 22.7, 22.5, 21.6, 19.2, 18.6, 11.8; Ms: m/z 784.667 (M+1). 2.9. Cholesteryl-6-(4-((E)-(40 -cyanobiphenyl-4-ylimino) methyl)phenoxy)hexanoate (3e) Pale yellow solid; Mp: 266 C; Y = 93%; IR max (KBr): 2936, 2252, 1736, 1556, 1248, 1164, 824, 726, 546 cm1; 1H NMR (300 MHz, CDCl3) d = 8.42 (s, 1H, N‚CH), 7.84 (d, J = 8.1 Hz, 2H, ArH), 7.77–7.68 (m, 4H, ArH), 7.60 (d, J = 9.0 Hz, 2H), 7.28 (d, J = 9.0 Hz, 1H, ArH), 6.96 (d, J = 9.0 Hz, 2H, ArH), 5.37 (d, J = 6.0 Hz, 1H, olefinic H), 4.64–4.57 (m, 1H, CHOCO), 4.03 (t, 2H, J = 6.6 Hz, OCH2), 2.36–2.31 (m, 4H, CH2), 1.87–0.86 (m, 53H, –CH2), 0.67 (s, 3H, –CH3); 13C NMR (75 MHz, CDCl3) d = 172.9, 161.8, 160.0,152.7, 145.0, 139.6, 136.0, 133.5, 132.6, 128.2, 127.4, 127.3, 122.6, 121.83, 118.9, 113.0, 111.2, 107.8, 101.6, 73.8, 67.9, 56.7, 56.1, 49.9, 42.3, 39.6, 39.4, 36.9,36.5, 35.7, 34.4, 31.9, 28.7, 27.1, 25.5, 24.7, 24.2, 23.7, 22.7, 22.5, 20.9, 19.1, 18.6, 11.8; Ms: m/z 781.667 (M+1). 2.10. Cholesteryl-6-(4-((E)-(40 -cyanobiphenyl-4-ylimino) methyl)-3-hydroxyphenoxy)hexanoate (3f) Yellow solid; Mp: 269 C; Y = 94%; IR max (KBr): 3397, 2936, 2252, 1736, 1556, 1248, 1164, 824, 726, 546 cm1; 1H NMR (300 MHz, CDCl3) d = 13.58 (s, 1H, –OH), 8.58 (s, 1H, N‚CH), 7.75–7.69 (m, 4H, ArH), 7.64 (d, J = 6.0 Hz, 2H, ArH), 7.37 (d, J = 6.0 Hz, 2H, ArH), 7.27 (s, 1H, ArH), 6.46 (d, J = 6.0 Hz, 2H, ArH), 5.37 (d, J = 6.0 Hz, 1H, olefinic H), 4.69–4.53 (m, 1H, CHOCO), 3.98 (t, 2H, J = 6.6 Hz, –OCH2), 2.35–2.31 (m, 4H, CH2), 1.82–0.84 (m, 53H, –CH2), 0.66 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d = 173.0, 161.8, 159.5, 148.9, 141.9, 139.6, 138.5, 133.7, 132.6, 128.2, 127.4, 122.6, 121.7, 118.9,118.1, 112.9, 113.0, 111.2, 107.8,106.6, 101.5, 73.8, 67.96, 56.6, 56.1, 50.0, 42.3, 39.6, 39.4,38.1, 36.6, 36.5,35.8,34.5, 31.9, 28.0, 27.8,25.5,24.2, 23.8, 22.7, 22.5, 21.0,19.2, 18.2, 11.8; Ms: m/z 797.667 (M+1). 2.11. Cholesteryl-8-(4-((E)-(40 -cyanobiphenyl-4-ylimino) methyl)phenoxy)octanoate (3g) Light yellow solid; Mp: 268 C; Y = 93%; IR max (KBr): 2936, 2252, 1736, 1556, 1248, 1164, 824, 726, 546 cm1; 1H NMR (300 MHz, CDCl3) d = 8.41 (s, 1H, N‚CH), 7.82 (d, J = 9.0 Hz, 2H, ArH), 7.75 (m, 4H, ArH), 7.59 (d, J = 9.0 Hz, 2H, ArH), 7.27 (d, J = 9.0 Hz, 2H, ArH), 6.95 (d, J = 9.0 Hz, 2H, ArH), 5.36 (d, J = 6.0 Hz, 1H, olefinic H), 4.69–4.53 (m, 1H, CHOCO), 4.00 (t, 2H, J = 6.6 Hz, ArOCH2), 2.33–2.27 (m, 4H, –CH2), 2.25–0.84 (m, 57H, – CH2), 0.66 (s, 3H, –CH3). 13C NMR (300 MHz, CDCl3) d = 172.3, 161.4, 159.8, 152.8, 152.7, 145.0, 139.5, 135.9, 132.5, 130.6, 129.0, 127.9, 127.2, 122.6, 121.6, 118.9, 114.6, 110.5, 74.2, 67.1, 56.8, 56.2, 50.2, 42.4, 39.6, 36.7, 35.9, 32.0, 31.9, 28.1, 28.1, 23.9, 22.9, 22.7, 19.4, 18.8, 11.9; Ms: m/z 810.667 (M+1).

M. Kumar et al. 2.12. Cholesteryl-8-(4-((E)-(40 -cyanobiphenyl-4-ylimino) methyl)-3-hydroxyphenoxy)octanoate (3h) Yellow solid; Mp: 273 C; Y = 94%; IR max (KBr): 3396, 2936, 2252, 1736, 1556, 1248, 1164, 824, 726, 546 cm1; 1H NMR (300 MHz, CDCl3) d = 13.62 (s,1H, ArOH) 8.58 (s, 1H, N‚CH), 7.74–7.70 (m, 4H, ArH) 7.63 (d, J = 6.0 Hz, 2H, ArH), 7.H, 7.27 (s, 1H, ArH), 6.41 (d, J = 6.0 Hz, 2H, ArH), 5.38 (d, J = 6.0 Hz, 1H, olefinic H), 4.65–4.59 (m, 1H, CHOCO), 3.90 (t, 2H, J = 6.6 Hz, ArOCH2), 2.32–2.28 (m, 4H, CH2), 2.25–0.84 (m, 57H, –CH2), 0.66 (s, 3H, – CH3). 13C NMR (75 MHz, CDCl3) d = 173.00, 161.87, 159.62, 148.91, 141.90, 139.69, 138.42, 133.76, 132.69, 128.26, 127.47, 122.67, 121.83, 118.93, 113.01, 111.22, 107.82, 101.65, 73.88, 67.96, 56.72, 56.17, 50.06, 42.35, 39.77, 39.56, 36.64, 35.83, 31.90, 28.04, 27.87, 22.86, 22.61, 21.06, 18.76, 11.89; Ms: m/z 826.667 (M+1). 2.13. Cholesteryl-10-(4-((E)-(40 -cyanobiphenyl-4-ylimino) methyl)phenoxy)decanoate (3i) Pale yellow solid; Mp: 271 C; Y = 93%; IR max (KBr): 2936, 2252, 1736, 1556, 1248, 1164, 824, 726, 546 cm1; 1H NMR (300 MHz, CDCl3) d = 8.43 (s, 1H, N‚CH), 7.85 (d, J = 8.1 Hz, 2H, ArH), 7.75–7.70 (m, 4H, ArH), 7.62 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H, ArH), 6.98 (d, J = 8.7 Hz, 2H, ArH), 5.37 (d, J = 6.0 Hz, 1H, olefinic H), 4.61–4.56 (m, 1H, CHOCO), 4.02 (t, 2H, J = 6.6 Hz, ArOCH2), 2.33 (m, 4H, CH2), 2.26–0.86 (m, 63H, -aliphatic H), 0.68 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d = 173.2, 162.1, 160.0, 152.8, 145.0, 139.6, 136.0, 132.5, 130.6, 128.8, 127.9, 127.3, 122.5, 121.6, 118.9, 114.7, 110.5, 73.6, 68.2, 56.6, 56.1, 50.0, 42.2, 39.5,39.4, 38.1, 36.56, 31.8, 29.4, 29.3, 29.2, 29.1, 29.0, 27.9, 24.4, 23.7, 22.7, 22.5,20.9, 19.2, 18.6, 11.7; Ms: m/z 852.227 (M+1). 2.14. Cholesteryl-10-(4-((E)-(40 -cyanobiphenyl-4-ylimino) methyl)-3-hydroxyphenoxy)decanoate (3j) Yellow solid; Mp: 260 C; Y = 94%; IR max (KBr): 3397, 2936, 2252, 1736, 1556, 1248, 1164, 824, 726, 546 cm1; 1H NMR (300 MHz, CDCl3) d = 13.70 (s, 1H, –OH), 8.67 (s, 1H, N‚CH), 7.81–7.72 (m, 6H, ArH), 7.84–7.77 (m, 4H, ArH), 7.72 (d, J = 6.0 Hz, 2H, ArH), 7.42 (d, J = 6.0 Hz, 2H, ArH), 7.39 (s, 1H, ArH) 5.45 (d, J = 6.0 Hz, 1H, olefinic H), 4.74–4.64 (m, 1H, CHOCO), 4.09 (t, 2H, J = 6.6 Hz, OCH2), 2.35–2.33 (m, 4H, CH2), 1.95–0.93 (m, 66H, CH2), 0.75 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) d = 173.0, 161.8, 159.6, 148.9, 141.9, 139.6, 138.4, 133.7, 132.6, 128.2, 127.4, 122.6, 121.8, 118.2, 113.0, 107.8, 106.4, 101.6, 73.8, 67.9, 65.3, 56.7, 56.1, 50.0, 42.3, 39.7, 39.5, 36.6, 35.8, 31.9, 28.0, 23.8, 22.6, 21.06, 18.7, 11.8; Ms: m/z 867.579 (M+1). 2.15. Antioxidant assays The ability of cholesterol derivatives as a free radical scavenger was explored by conducting a series of in vitro antioxidant assays involving DPPH radical, hydroxyl radical, nitric oxide radical, hydrogen peroxide, superoxide anion radical and comparing the results with standard antioxidant vitamin C. The


Synthesis, characterization and antioxidant activities of Schiff bases are of cholesterol DPPH radical scavenging activity of the compounds was measured according to the method of Blios [7,19]. The assay of nitric oxide (NO) scavenging activity was carried out based on the method reported by Green [12]. The ability of the compounds to scavenge hydrogen peroxide was determined using the method available in the literature [29]. The sueroxide anion radical (O 2 ) scavenging assay was based on the capacity of the complexes to inhibit formazan formation by scavenging the superoxide radicals generated in the riboflavin-light-NBT system [6].

3. Results and discussion 3.1. Synthesis The target cholesterol derivatives (3a–j) were synthesized as depicted in Scheme 1. Initially the cholesterol aldehyde derivatives were prepared by Williamson ether synthesis. The target compound can be prepared by acid catalyzed condensation of cholesterol aldehyde derivatives and 40 -aminobiphenyl-4-carbonitrile in ethanol, then it was refluxed for 3 h. The product was formed by cooling and it was purified by recrystallization with ethanol. The target compounds (3a–j) were synthesized by the classical condensation of aldehyde and aniline as depicted in Scheme 1. The equal mole ratio of cholesterol aldehyde derivatives and 40 -aminobiphenyl-4-carbonilide was dissolved in ethanol. The reaction mixture was stirred under reflux to give an orange color solution. After allowing the solution to stand in cold condition, yellow solid was collected, washed with cold ethanol and recrystallized from ethanol. All the synthesized compounds were prepared by the same procedure. The structures were confirmed by using 1H NMR, 13C NMR, EI mass, IR spectra and given as Supplementary information. 3.2. Spectrophotometric determination of DPPH radical scavenging activity Most of the investigations are taken as DPPH assay to evaluate the antioxidant activity of their targets due to its simple procedure and reliability. The results of the DPPH radical scavenging activity of the compounds are shown in Table 1. Lower IC50 value reflects better DPPH radical-scavenging activity. From the table, it was clear that almost all the compounds showed radical scavenging activities in DPPH assay. It was important to note that compounds 3b, 3d and 3j have shown better antioxidant activity than vitamin C with IC50 values 12.53, 13.75 and 13.90 lM respectively. The DPPH activities of the tested compounds were found to be in the decreasing order of 3b, 3d, 3j, 3f, 3h, 3e, 3a, 3g, 3i and 3c. The presence of Schiff base and –OH groups also has influence on the DPPH radical scavenging activity and ethylene spacer does not have any effect on antioxidant property. 3.3. Superoxide anion radical scavenging assay The results of O 2 assay were expressed as IC50 values as shown in Table 1 revealed that compounds 3b, 3g, 3a and 3d showed better superoxide anion radical scavenging activity than natural antioxidant vitamin C with IC50 of 29.08, 27.82,

5

Table 1 Antioxidant activity of cholesterol derivatives (3a–j) and vitamin C against various radicals. Compound

Vitamin C 3a 3b 3c 3d 3e 3f 3g 3h 3i 3j

IC50 (lM) DPPH

SO

NO

H2O2

32.50 15.48 12.53 16.76 13.75 15.18 14.00 15.51 14.83 15.71 13.90

52.00 29.08 27.82 31.06 30.12 36.07 32.88 28.72 32.91 33.42 35.82

55.00 32.33 29.61 30.00 31.25 32.25 28.65 27.32 25.62 31.48 29.34

43.00 26.69 25.25 30.17 28.00 25.28 24.20 26.40 25.88 29.46 26.76

28.72 and 27.91 lM respectively. Surprisingly IC50 values of all synthesized compounds were lower than IC50 of vitamin C. The super oxide anion radical scavenging activities were found to be in the order of 3b, 3g, 3a, 3d, 3c, 3f, 3h, 3i, 3j and 3e. 3.4. Nitric oxide radical scavenging assay In general scavenging studies, this radical is very essential. Reactive species are responsible for altering the structural and functional behavior of many cellular compounds. The results of the NO assay expressed as IC50 values as shown in Table 1 revealed that compounds 3h, 3g, 3f, 3j and 3b have shown better nitric oxide radical scavenging activity than natural antioxidant vitamin C with IC50 of 25.62, 27.32, 28.65, 29.34 and 29.61 lM respectively. Surprisingly IC50 values of all synthesized compounds were lower than IC50 of vitamin C. The nitric oxide radical scavenging activities of the tested compounds were found to be in the decreasing order of 3h, 3g, 3f, 3j, 3b, 3c, 3d, 3i, 3e, 3a and vitamin C. 3.5. H2O2 scavenging assay Hydrogen peroxide is not very reactive itself, but it can cross the cell membrane and produce the hydroxide radical, which is highly reactive. The IC50 values of hydrogen peroxide assay of 3f, 3b, 3e, 3h and 3g have revealed better scavenging potential than natural antioxidant vitamin C with IC50 of 24.20, 25.25, 25.28, 25.88 and 26.40 lM respectively. The hydrogen peroxide radical scavenging activities of the tested compounds found to be in the decreasing order of 3f, 3b, 3e, 3h, 3g, 3a, 3j, 3d, 3i, 3c, and vitamin C. 4. Conclusions A series of cholesteryl-11-(4-((E)-(40 -cyanobiphenyl-4-ylimino)methyl)phenoxy)alkanoate have been synthesized. The structures were confirmed by 1H NMR, 13C NMR, FT-IR and mass spectral studies. These compounds exhibited excellent radical scavenging activities against super oxide anion radical, nitric oxide radical, DPPH radical and hydrogen peroxide. Of all these targeted compounds 3b, 3d and 3j showed better antioxidant activity than vitamin C in DPPH


6 assay; compounds 3b, 3g, 3a and 3d showed better superoxide anion radical scavenging activity than natural antioxidant vitamin C; compounds 3h, 3g, 3f, 3i, 3j and 3b showed better nitric oxide radical scavenging activity than natural antioxidant vitamin C: compounds 3f, 3b, 3e, 3f, 3h and 3g have revealed that they have better hydrogen peroxide scavenging potential than natural antioxidant vitamin C. Based on the result, it is clear that these compounds can be used as good antioxidants in the field of medicinal and food industry. Acknowledgments The authors gratefully acknowledge DST for financial assistance and also thank DST-IRPHA for providing NMR spectroscopy. PT thanks UGC for the award of Raman Post Doctoral fellowship.

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