Chromenone and quinolinone derivatives as potent antioxidant agents
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The antioxidant activity (AOA) of three different classes of phenolic compounds viz chromen-2-ones, chromen-4-ones, and quinolin-2-ones was systematically studied using DPPH, ABTS, FRAP, and in vitro lipid peroxidation inhibition assays. The effect of incorporation of hydrophobic group on AOA was also studied. In DPPH, ABTS, and FRAP assays, the highest AOA was registered for the dihydroxy chromenones among all the phenolic derivatives. Presence of alkyl group led to reduction in AOA in the above three assays. However, in lipid peroxidation inhibition assay for selected compounds, incorporation of alkyl group led to enhancement in AOA. The AOA of few compounds was observed to be more than three times higher in comparison to standard “Trolox” in lipid peroxidation inhibitory assay.
KeywordsAntioxidant activity Chromen-2-ones Chromen-4-ones Quinolin-2-ones Lipid peroxidation inhibitory activity
In vivo, lipid oxidation may play a role in coronary heart disease, atherosclerosis, cancer, and the aging process. Lipid oxidation occurs when reactive oxygen species (ROS) reacts with lipids in a series of free radical chain reactions that lead to complex chemical changes. Oxidation of lipids in foods causes quality loss and is thus a major concern for such industry (Devasagayam et al., 2004). Antioxidants, the compounds that can delay or inhibit lipid oxidation, when added to foods can minimize rancidity and retard the formation of toxic oxidation products.
The antioxidant property of a compound is attributed to its ability of (a) oxygen radical scavenging, (b) inhibiting cellular microsomal P-450-linked mixed function oxidation (MFO) reaction, and (c) suppressing the formation of ROS. Thus, antioxidants are considered as remedies to overcome the lethal action of oxygen free radicals (Malhotra et al., 2008). A criterion for a good antioxidant is that the compound should contain a highly labile hydrogen atom forming a radical; the radical formed should be stable and non-reactive so that it does not participate in the reactive species (RS) propagation step. Sterically hindered phenolic compounds satisfy most of the above-mentioned requirements. Thus, the anti-oxidant and radical scavenging effects of polyphenols are highly cited and reviewed (Prochazkova et al., 2011; Bubols et al., 2013). Phenolic compounds are also abundant natural products as they occur often in significant concentrations (0.5–1.5 %). In model systems, many natural products show antioxidant activity (AOA), for instance, tocopherols (Seppanen et al., 2010; Halliwell et al., 2005), flavonoids (Terao, 1999; Cao et al., 1997), coumarins (Kumar et al., 2005; Raj et al., 1998), phenolic acids (Kartika et al., 2007; Yoshida et al., 2010), cinnamates (Garrido et al., 2012), etc. The antioxidant potential of dietary phenolic compound from fruits and vegetables has been well explored, and there is always an ever increasing demand to identify more effective antioxidants that are analogs of naturally occurring phenolic compounds. Herein, we have designed and developed a series of phenolic derivatives of chromen-2-ones, chromen-4-ones, and quinolin-2-ones and evaluated their antioxidant potential using DPPH, ABTS, and FRAP assays. The lipid peroxidation inhibitory activity of few select compounds was also studied.
Chromen-2-one (coumarin or benzopyran-2-one) affects the formation and scavenging of ROS and influence free radical-mediated oxidative damage (Raj et al., 1998; Pedersen et al., 2007). The derivatives viz 4-hydroxychromen-2-one, 7-hydroxychromen-2-one, 7,8-dihydroxychromen-2-one, 4-methylesculetin, and 7-hydroxy-4-methylchromen-2-one were found to possess potent radical scavenging ability (Lin et al., 2008). Although the chromenone core does not ensure the radical scanvenging activity by itself, but the phenolic character/hydroxy substitution on these cores is responsible for the radical scavenging activity through the formation of stable radical due to extended resonance stabilization by pyran ring (Raj et al., 1999) and Wenum et al. (2013).
Materials and methods
The hydroxy coumarins 1–8 were synthesized following the procedure reported in the literature (Pechmann and Duisberg, 1883; Kathuria et al., 2009). The pyranocoumarins 9–11 were synthesized according to the earlier published procedure from our group (Kathuria et al., 2011). The hydroxy chromones 12–14 and chromone acrylates 15–20 were synthesized following the earlier published method from our group (Chand et al., 2014). Quinolin-2-ones (22–24) too were synthesized according to the literature procedure (Priya et al., 2010).
A mixture of 10 mL hydrobromic acid and acetic acid (7:3) was added to 1 g of 2,3-dimethoxy cinnamic acid. The reaction mixture was refluxed for 12 h and then poured on crushed ice. The resulting precipitate was filtered and washed with water to yield 8-hydroxycoumarin. It was obtained as off white solid in 50 % yield. Mp = 158.9 °C; UV (acetonitrile) λ max: 296 and 326 nm; IR (KBr) ν max: 3,386.1 (OH), 1,706.9 (C=O), 1,576.3 cm−1; 1H NMR (400 MHz, CDCl3): δ 4.67 (1H, brs, OH), 6.39 (1H, d, J = 9.52 Hz, H-3), 6.99–7.02 (1H, m, H-5), 7.14 (2H, m, H-6 and H-7), 7.69 (1H, d, J = 9.52 Hz, H-4); 13C NMR (100.5 MHz, CDCl3): δ 116.31 (C-3), 118.33 (C-7), 118.99 (C-5) 119.14 (C-10), 124.85 (C-6), 141.84 (C-9), 143.57 (C-4), 144.17 (C-8), 160.04 (C-2).; HRMS calcd. for C9H6O3: 162.0317; found: 162.4670.
(E)-Ethyl 3-(7,8-dihydroxy-4-oxo-4H-chromen-3-yl)acrylate (21)
It was synthesized starting from corresponding hydroxyacetophenone by following the method reported earlier from our group (Chand et al., 2014). Mp = 232–234 °C; UV (MeOH) λ max: 270 and 290 nm; IR (KBr) ν max: 3,568.6 (OH), 1,672.0 (OCO), 1,638.0 (C=O), 1,611.2 (C=C) cm−1; 1H NMR (400 MHz, CDCl3): 1.25 (t, 3H, J = 6.6 Hz, H-2”), 4.16 (q, 2H, J = 6.5 Hz, H-1″), 6.97 (d, 1H, J = 8.8 Hz, H-5), 7.18 (d, 1H, J = 16.1 Hz, H-3′), 7.42–7.46 (m, 2H, H-2′ and H-6), 8.70 (s, 1H, H-2); 13C NMR (100.5 MHz, DMSO-d 6 ): 14.24 (C-2″), 59.98 (C-1″), 114.64 (C-6), 115.75 (C-2′), 117.08 (C-5), 117.05 (C-10) 119.69 (C-3), 133.15 (C-8), 136.87 (C-3′), 146.02 (C-9), 150.65 (C-2), 159.41 (C-7), 166.61 (C-1′), 175.06 (C-4).
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), TPTZ (2,4,6,-tripyridyl-s-triazine), and ABTS (2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) were obtained from Sigma-Aldrich Co., USA. DPPH was obtained from Fluka (Buchs, Switzerland). Trolox was used as the reference antioxidant compound in all of the assays.
DPPH radical scavenging assay
The AOA was assessed by modifying the method of Blois (1958) using the DPPH assay. A solution of DPPH in ethanol (2 mL) was added to the solution of substance in ethanol (1 mL) or 1 mL ethanol for the blank. The final radical concentration was 0.09 mmol/L. The absorbance was read at 517 nm after 30 min of incubation. All the analyses were done in three replicates. Percentage inhibition of DPPH activity caused by 1 µM test compound or Trolox was calculated by the formula: % Inhibition = [(Blank OD − sample OD)/Blank OD] × 100. Trolox equivalent antioxidant capacity was calculated by the formula: TEAC = % Inhibition by sample/% Inhibition by Trolox.
ABTS radical scavenging assay
ABTS assay was done following the method of Re et al. (1999) with little modification. The ABTS radical cation was pregenerated by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate (1:1) and incubating for 12–16 h in the dark at room temperature until the reaction gets complete and the absorbance becomes stable. Absorbance of ABTS was equilibrated to 1.5 (±0.02) by diluting with water at room temperature, then 1 mL of this solution was mixed with 1 mL of the test sample, and the absorbance was measured at 734 nm after 6 min. Percentage inhibition of ABTS activity caused by 1 µM test compound or Trolox was calculated by the same formula used for DPPH assay. TEAC too was calculated as per DPPH assay.
The ferric reducing antioxidant power (FRAP) assay was done by the method of Benzie and Strain (1996). The FRAP reagent was freshly prepared by mixing TPTZ (2.5 mL, 10 mM in 40 mM HCl), 25 mL of 300 mM acetate buffer (pH 3.6), and 2.5 mL of FeCl3·H2O (20 mM) and warmed to 37 °C prior use. 150 µL of series of Trolox concentrations (0, 1, 2, 5, 10, 20, 50, 75, and 100 µM) or 10 µM test sample was mixed with 2,850 µL of working FRAP reagent, respectively, and incubated for 30 min in dark. The change in color intensity was read at 593 nm using a spectrophotometer. A plot of Trolox concentration versus absorbance (OD) was used to develop the slope equation. Using this equation, FRAP/10 µM test sample was calculated. TEAC of test compounds was calculated by the formula TEAC = FRAP of sample/FRAP of Trolox.
Lipid peroxidation inhibitory activity assay
In vitro lipid peroxidation inhibition was assayed by the method of Bishayee and Balasubramaniyam (1971). 100 μL of rat liver homogenate (25 %, w/v in 20 mM Tris–HCl buffer, pH 7.0) was incubated with Mohr’s salt (0.16 mM), ascorbic acid (0.06 mM), and different concentrations (0, 1, 2, 5, 10, 20, 50, 75, and 100 µM) of Trolox or test compounds in a final volume of 500 μL for 1 h at 37 °C. After incubation, 400 μL of initial reaction mixture was mixed with 200 μL sodium dodecyl sulfate (SDS) (8.1 %), 1.5 mL thiobarbituric acid (0.8 %), 1.5 mL acetic acid (20 %, pH 3.5), and 400 μL distilled water and kept in boiling water bath for 1 h. The reaction mixture was allowed to cool and then added 1 mL distilled water and 4 mL n-butanol:pyridine (15:1) mixture. The contents were shaken vigorously and centrifuged at 4,000 rpm for 10 min. The n-butanol–pyridine layer was removed, and UV absorption was recorded at 532 nm. Percentage inhibition of lipid peroxidation caused by different concentrations of Trolox and test samples was calculated by the same formula used for DPPH assay. The concentration of Trolox or test compound that caused 50 % inhibition was termed as IC50. TEAC was calculated by the formula TEAC = IC50 of Trolox/IC50 of sample.
Results and discussion
The AOA of three different classes of phenolic compounds viz chromen-2-ones (1–11), chromen-4-ones (12–21), and quinolin-2-ones (22–24) was systematically studied.
Synthesis of chromen-2-ones
Synthesis of chromen-4-ones
Synthesis of quinolin-2-ones
Quinolin-2-ones 22–24 were synthesized according to the literature procedure (Priya et al., 2010).
Numerous studies on the AOA of chromen-2-ones and its derivatives have been carried out; however, the effect on AOA by incorporating hydrophobic alkyl group on chromenone/quinolinone skeleton has not been investigated earlier. In this report, we have meticulously compared the antioxidant efficacy of hydroxy derivatives of 3-alkyl-4-methylchromen-2-ones and pyranochromen-2-ones using DPPH, ABTS, and FRAP assays. Also, AOA of chromen-4-ones and quinolin-2-ones was studied. Few select compounds were assayed for their in vitro lipid peroxidation inhibition.
The AOA of compounds was compared with “Trolox” taken as standard and expressed as TEAC. In the present study, three types of chromen-2-one derivatives were screened for AOA, i.e., monohydroxychromen-2-ones (1–5), dihydroxychromen-2-ones (6–8), and pyranochromen-2-ones (9–11) having varied alkyl substituents (ethyl/hexyl/decyl) at C-3 position of coumarin skeleton.
Since the in vitro lipid peroxidation inhibition more closely resembles with the true biological systems, the observance of significant enhancement of AOA of C-3 alkyl substituted chromen-2-ones, pyranochromen-2-ones, chromen-4-ones, and quinolin-2-ones holds promise in the application of these compounds as efficient antioxidants. These results suggest that the incorporation of lipophilic group on a chromenone and quinolinone moiety enhances the interaction of the resulting compound with the lipids which are hydrophobic in nature, and thus an enhancement in AOA was observed. While in DPPH, ABTS, and FRAP assays, simply the radical scavenging effect or ferric reducing activity is studied, and thus hydrophobic alkyl substituents which reduce the aqueous dispersibility also decrease the AOA.
A total of 24 derivatives of chromen-2-ones, chromen-4-ones, and quinolin-2-ones were screened for AOA. It was observed that nature and position of the substituent have a great influence on their AOA. Among the eleven chromen-2-one derivatives and ten chromen-4-one derivatives screened, the dihydroxychromens showed the highest AOA. Pyranochromen-2-ones, on the other hand, exhibited moderate AOA but higher than the monohydroxy counterparts. The presence of an alkyl substituent at C-3 position of chromenone and quinolin-2-one reduces the AOA in DPPH, ABTS, and FRAP assays as compared to corresponding unsubstituted analogs. However, in lipid peroxidation inhibition assay, it was observed that the AOA of chromen-2-ones and quinolin-2-ones having C-3 alkyl substituent is significantly higher in comparison to the corresponding unsubstituted compounds.
We thank University of Delhi, Defence Research Development Organization (DRDO, India), Indo-German Science and Technology Center (IGSTC), and Council of Scientific and Industrial Research (CSIR) for financial support. The award of Junior/Senior Research Fellowships (JRF/SRF) by CSIR (to KC), University Grants Commission (to AKS at Delhi University) and by DRDO (to DAJ) is gratefully acknowledged.
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