Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 3(1), 82-85, January (2013) Res.J.Chem. Sci. International Science Congress Association 82 In-vitro Antioxidant activity of Diethyl malonate adducts of PhenothiazineAkilandapuram Velusamy Saranya, Subban Ravi*, Senniappan Venkatachalapathi Department of Chemistry, Karpagam University, Coimbatore 641021, Tamil Nadu, INDIAAvailable online at: www.isca.in Received 20th October 2012, revised 26th November 2012, accepted 30th December 2012Abstract A series of novel diethyl malonate adducts 2a-g was prepared by the condensation between chalcones 1a-g and diethyl malonate in presence of KOt-Bu. The products were characterized by their spectral data (UV, IR, NMR and elemental analysis). In-vitro antioxidant activity was carried out for 2a-g which showed good anti-oxidant activity with IC50 value between 36.15 to 81.00µg/ml and comparable to the control quercetin with IC50 value 44.53(µg/ml). Compound 2a and 2d showed higher activity with IC50 value of 39.13 and 36.1µg/ml respectively. Keywords: Chalcones, michael addition, diethyl malonate adduct, KO-Bu, antioxidant activity. Introduction Chalcone and its derivatives are medicinally important. Michael addition reaction of appropriate carbanionic reagents to , unsaturated carbonyl compounds such as chalcone is of synthetic interest for C-C bond formation1,2. Addition of 1, 3-dicarbonyl compounds such as malonate esters and acetacetate esters is important for the synthesis of 1, 5-dicarbonyl compounds which are key compounds for the preparation of many biological heterocyclic compounds In recent years, there has been a growing interest pertaining to the synthesis of bioactive compounds in the field of organic chemistry. Molecules that possess sulfur atoms are universal and crucial in living organisms 4. Phenothiazine derivatives constitute an important class of compounds possessing diverse type of biological properties including antiviral, antiparasitic, antiparkinsonian, anticonvulsant, antihistaminic as well as anthelmintic properties. Antioxidant is a term widely used but rarely defined; it is difficult these days to open a popular science magazine or medical journal without seeing an article about the role of free radicals in human diseases. Free radicals and reactive oxygen species in general are no longer seen only a destructive factors but also as messengers involved in intracellular and intercellular signaling. In view of the substantial changes in the understanding of the role of reactive oxygen speciesand antioxidants in living systems, a critical re-evaluation of the methods of determination of the antioxidant activity is necessary. The peroxidase substrate 2, 2'-azino-bis(3-ethyl benzthiazoline-6-sulphonic acid) (ABTS) forming relatively stable radical upon one-electron oxidation has become a popular substrate for estimation of total antioxidant activity. Furthermore synthesis of novel chemical entities, which are still in resemblance with bioactive molecules by virtue of the presence of some critical structural features, is an essential component of the search for new leads in drug designing programs. Hence careful perusal of literature on antioxidant properties and our continued interest in the development of simpler and more convenient synthetic routes for achieving the biologically challenging heterocyclic system5 has induced us to synthesize a class of molecules having diethyl malonate adducts of phenothiazine ring and to evaluate their antioxidant activity. Material and Methods Chemistry: Melting points (uncorrected) were determined using a Guna melting point apparatus. UV spectra were obtained UV 2460 shimadzu spectrophotometer. IR spectra were carried out on a Perkin-Elmer 1650 spectrophotometer. NMR spectra were recorded in CDCl on a Bruker AM 400 MHz spectrometer, using residual CHCl and TMS as an internal standard. Elemental analysis was carried out in a Perkin Elmer 240C model instrument. Column chromatography and TLC were carried out on silica gel 60 -120 mesh and silicagel ‘G’ respectively. All the chemicals are of AR grade. General procedure for the preparartion of compounds1a-g: 2-acetyl phenothiazine (0.01 mol) was dissolved in 25 ml methanol and different benzaldehyde derivatives (0. 01 mol) were added, heated for 6 hrs with constant stirring in a magnetic stirrer and a catalytic amount of NaOH was added in drops by Claisen-Schmidt condensation7-9. The reaction was poured into ice-cold water, neutralized with con.HCl and left over night in a refrigerator. The precipitate was filtered, dried and purity of the compound was checked by TLC using chloroform as the solvent. The compound was purified by column chromatography using silica gel (60-120 mesh). General procedure for the preparartion of diethyl malonate adducts 2a-g: Chalcone 1a-g (0.01 mol) was dissolved in 10 ml methanol and diethyl malonate(0. 01 mol), and a catalytic amount of KOt-Bu was added and refluxed for 4 hrs by Michael addition reaction. The reaction was poured into ice-cold water, neutralized with con.HCl and left over night in a refrigerator. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 3(1), 82-85, January (2013) Res. J. Chem. Sci. International Science Congress Association 83 The precipitate was filtered, dried and purity of the compound was checked by TLC using chloroform as the solvent. The compound was purified by column chromatography using silica gel (60-120 mesh). The analytical data is given in table-1 and spectral data for each compound is given below. Diethyl 2-(1-(4-methoxyphenyl)-3-oxo-3-(10H-phenothiazin-2-yl)propyl)malonate 2a: UV max: 392.00, 280.50, 244.00; IR (KBr) cm-1: 1589 (C = C), 1666 (C =O ketone), 1737 (C = O ester), 3344 (NH); H-NMR (400MHz CDCl) : 3.43, 3.29 (dd, J=7.6, 16Hz, 2H, CH), 4.07-4.04 (td, J=4.4Hz, 1H, CH ), 3.75 (d, J=9.6Hz, 1H, CH), 4.21-4.18 (q, J=7.2Hz, 2H, OCH), 3.98-3.93 ( q, J=7.2Hz, 2H, OCH), 1.00 (t, J=7.2Hz, 3H, CH), 1.02 (t, J=7.2Hz, 3H, CH), 3.73 (1H, s, OCH), 6.76-6.74 (m, 3H, Ar-H), 7.00-6.90 (m, 4H, Ar-H), 7.14-7.12 (m, 2H, Ar-H), 7.33(m, 1H, Ar-H); 13C-NMR (400MHz CDCl) : 13.82, 14.04, 61.37, 61.67, 167.77, 168.48, 55.14, 42.72, 40.44, 196.78, 113.03, 135.97, 114.67, 132.12, 132.12, 118.00, 129.22, 126.36, 142.00, 117.00, 141.00, 126.66, 140.90, 127.76, 113.80, 158.58, 57.81. Diethyl 2-(3-oxo-3-(10H-phenothiazin-2-yl)-1-phenylpropyl) malonate 2b: UV max: 282.00, 248.00, 211.50; IR(KBr) cm-1: 1597 (C = C), 1680 (C =O ketone), 1730 (C = O ester), 3350 (NH); H-NMR (400MHz CDCl) : 3.45, 3.29 (dd, J=7.6, 16Hz, 2H, CH), 4.14-4.06 (td, J=4.4Hz, 1H, CH ), 3.74 (d, J=9.6Hz, 1H, CH), 3.98 (q, J=7.2Hz, 2H, OCH), 4.15 ( q, J=7.2Hz, 2H, OCH), 1.04 (t, J=7.2Hz, 3H, CH), 1.22 (t, J=7.2Hz, 3H, CH), 6.74-6.70 (m, 3H, Ar-H), 7.00-6.84 (m, 5H, Ar-H), 7.12-7.10 (m, 2H, Ar-H), 7.28(m, 1H, Ar-H); 13C-NMR (400MHz CDCl) : 13.89, 14.10,61.37, 61.43, 169.00, 168.00, 57.00, 25.40, 45.67, 199.00, 136.00, 115.89, 118.90, 132.00, 122.0, 118.40, 132.10, 117.76, 127.5, 119.7, 142.50, 143.34, 149.65, 126.23, 128.50, 126.12. Diethyl 2-(1-(4-chlorophenyl)-3-oxo-3-(10H-phenothiazin-2-yl)propyl)malonate 2c: UV max: 282.00, 245.00, 207.50; IR (KBr) cm-1: 1595 (C = C), 1660 (C =O ketone), 1741 (C = O ester), 3332 (NH); 1H-NMR (400MHz CDCl) : 3.45, 3.27 (dd, J=7.6, 16Hz, 2H, CH), 4.13-4.07 (td, J=4.4Hz, 1H, CH ), 3.75 (d, J=9.6Hz, 1H, CH), 3.98 (q, J=7.2Hz, 2H, OCH), 4.15 ( q, J=7.2Hz, 2H, OCH), 1.04 (t, J=7.2Hz, 3H, CH), 1.24 (t, J=7.2Hz, 3H,CH), 6.52-6.50 (dd, 1H, Ar-H), 7.33-7.30 (dd, 1H, Ar-H), 6.83-6.79 (td, 1H, Ar-H), 7.00-6.90 (m, 5H, Ar-H), 7.21-7.16 (m, 4H, Ar-H); 13C-NMR (400MHz CDCl) : 13.81, 14.03, 61.55, 61.81, 167.55, 168.19, 57.37, 42.27, 40.36, 196.23, 112.88, 135.77, 114.65, 132.98, 132.98, 122.91, 125.63, 122.54, 141.68, 116.92, 140.67, 126.69, 140.90, 127.81, 128.59, 129.64. Diethyl 2-(3-oxo-3-(10H-phenothiazin-2-yl)-1-p-tolylpropyl) malonate 2d: UV max: 278.50, 243.50, 218.50; IR(KBr) cm-1: 1597 (C = C), 1668 (C =O ketone), 1738 (C = O ester), 3356 (NH); 1H-NMR (400MHz CDCl) :3.43, 3.27 (dd, J=7.6, 16Hz, 2H, CH), 4.09 (td, J=4.4Hz, 1H, CH ), 3.75 (d, J=9.6Hz, 1H, CH), 3.98-3.93 (q, J=7.2Hz, 2H, OCH), 4.21-4.17 ( q, J=7.2Hz, 2H, OCH), 1.00 (t, J=7.2Hz, 3H, CH), 1.24 (t, J=7.2Hz, 3H,CH), 2.23 (s, 3H, CH), 6.52-6.50 (dd, 1H, Ar-H), 6.82-6.78 (td, 1H, Ar-H), 7.10 (d, 2H, Ar-H), 7.33-7.31 (dd, 1H, Ar-H), 7.02-6.90 (m, 6H, Ar-H); 13C-NMR (400MHz CDCl) : 13.77, 14.03, 61.35 , 61.64, 167.76, 168.48, 57.70, 42.58, 40.72 , 196.68, 113.03, 135.99, 114.65, 129.10, 129.10, 122.81, 125.27, 122.61, 141.60, 116.96, 140.78 , 126.66, 140.90, 126.35, 128.00 , 136.71, 21.00. Diethyl 2-(1-(3-nitrophenyl)-3-oxo-3-(10H-phenothiazin-2-yl)propyl)malonate 2e: UV max: 280.00, 245.00, 206.00; IR(KBr) cm-1: 1591 (C = C), 1660 (C =O ketone), 1737 (C = O ester), 3340 (NH); H-NMR (400MHz CDCl) : 3.53, 3.39 (dd, J=7.6, 16Hz, 2H, CH), 4.17 (td, J=4.4Hz, 1H, CH ), 3.83 (d, J=9.6Hz, 1H, CH), 4.00 (q, J=7.2Hz, 2H, OCH), 4.23 ( q, J=7.2Hz, 2H, OCH), 1.07 (t, J=7.2Hz, 3H, CH), 1.26 (t, J=7.2Hz, 3H,CH),6.52-6.49 (dd, 1H, Ar-H), 6.82-6.78 (td, 1H, Ar-H), 6.92-6.90 (dd, 1H, Ar-H), 7.00-6.94 (m, 3H, Ar-H), 7.32-7.30 (dd, 1H, Ar-H), 7.44 (t, 1H, Ar-H), 7.66 (d, 1H, Ar-H), 8.06-8.03 (dd, 1H, Ar-H), 8.15-8.14 (t, 1H, Ar-H); 13C-NMR (400MHz CDCl) : 13.83, 14.02, 61.73, 62.01, 167.35, 167.88, 56.91, 41.83, 40.26, 195.73, 112.77, 135.52, 114.65, 129.32, 129.32, 122.96, 125.99, 122.45, 141.74, 116.85, 140.58, 126.48, 148.21, 122.83, 135.16, 142.81, 127.85, 116.85. Diethyl 2-(1-(4-bromophenyl)-3-oxo-3-(10H-phenothiazin-2-yl)propyl)malonate 2f: UV max: 281.50, 244.50, 206.50; IR(KBr) cm-1: 1598 (C = C), 1660 (C =O ketone), 1743 (C = O ester), 3334 (NH); H-NMR (400MHz CDCl) : 3.44, 3.29 (dd, J=7.6, 16Hz, 2H, CH), 4.09 (td, J=4.4Hz, 1H, CH ), 3.73 (d, J=9.6Hz, 1H, CH), 4.05 (q, J=7.2Hz, 2H, OCH), 4.21 ( q, J=7.2Hz, 2H, OCH), 1.04 (t, J=7.2Hz, 3H, CH), 1.24 (t, J=7.2Hz, 3H,CH), 7.32 (dd, 1H, Ar-H), 6.52 (d, iH, Ar-H), 6.80 (t, 1H, Ar-H), 6.99-6.90 (m, 3H, Ar-H), 7.13-7.10 (m, 2H, Ar-H), 7.36-7.33 (m, 2H, Ar-H); 13C-NMR (400MHz CDCl) : 13.81, 14.03, 61.56, 61.81, 167.54, 168.18, 57.31, 42.20, 40.44, 196.20, 112.91, 135.81, 114.65, 132.24, 131.55, 122.54, 125.67, 122.93, 141.69, 116.96, 140.68, 126.45, 140.90, 129.79, 131.55, 121.13. Diethyl 2-(1-(4-formylphenyl)-3-oxo-3-(10H-phenothiazin-2-yl)propyl)malonate 2g: UV max: 280.00, 244.00, 211.50; IR (KBr) cm-1: 1598 (C = C), 1689 (C =O ketone), 1756 (C = O ester), 3358 (NH); H-NMR (400MHz CDCl) : 3.50, 3.33 (dd, J=7.6, 16Hz, 2H, CH), 4.18 (td, J=4.4Hz, 1H, CH ), 3.80 (d, J=9.6Hz, 1H, CH), 3.97 (q, J=7.2Hz, 2H, OCH), 4.21 ( q, J=7.2Hz, 2H, OCH), 1.00 (t, J=7.2Hz, 3H, CH), 1.26 (t, J=7.2Hz, 3H,CH), 9.91 (s, 1H, CHO), 6.51 (d, 1H, Ar-H), 6.79 (td, 1H, Ar-H), 6.97-6.77 (m, 4H, Ar-H), 7.29 (m, 1H, Ar-H), 7.43 (d, 2H, Ar-H), 7.75 (d, 2H, Ar-H); 13C-NMR (400MHz CDCl) : 13.78, 14.01, 61.50, 61.61, 167.41, 168.02, 57.04, 42.02, 40.94, 196.04, 112.81, 135.64, 114.67, 129.83, 129.83, 122.44, 125.78, 122.87, 141.77, 116.81, 140.66, 126.65, 166.64, 126.39, 129.04, 135.08, 191.75. Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 3(1), 82-85, January (2013) Res. J. Chem. Sci. International Science Congress Association 84 In-vitroABTSradical scavenging assay: ABTS radical scavenging activity was determined for compounds (2a-g) accordingly10. ABTS+ radical was freshly prepared by adding 5 ml of 4.9 mM ammonium persulfate solution to 5 ml of 14 mM ABTS solution and kept for 16 h in dark. This solution was diluted with ethanol (99.5%) to yield an absorbance of 0.70±0.02 at 734 nm and the same was used for the assay. To 950 l of ABTS radical solution, added 50 l of prepared solutions (25-500 g/ml) and the reaction mixture was vortexed for 10 sec. After 6 minutes the absorbance was recorded at 734 nm and compared with the control ABTS solution. Quercetin was used as a control. The experiment was carried in triplicates. Percentage inhibition was calculated from the formula Percentage inhibition= [1 (absorbance of test/absorbance of control)] Results and Discussion Spectral values of diethyl malonate adduct 2a-g: A series of seven chalcone diethyl malonate derivatives 2a-g was prepared by Michael addition reaction of diethyl malonate to chalcones 1a-g as shown in scheme-1. Diethyl malonate adducts were obtained in good yields of 68-83%. The analytical data were summarized in Table-1. The structures of all seven adducts of diethyl malonate 2a-g synthesized were characterized by UV, IR, H, 13C-NMR, elemental analysis. The addition of diethyl malonate to chalcones 1a-g leads to the generation of a chiral center in the structure of adducts 2a-g and confirmed by its H-NMR spectral data. In H-NMR spectrum the protons present in the CH group appeared as a doublet of doublets at 3.43 (1H, J = 7.6Hz) and 3.29 (1H, J = 16Hz) with different coupling constant values. For this two signals at 1.00 and 1.24 were attributed to the methyl groups. The two OCH groups present in the structures 2a-g appeared as a pair of quartets at 4.00 and 4.23. All the aromatic protons appeared between 6.51 to 8.06. In 13C-NMR spectrum, keto carbonyl group appeared at 196.00 and two ester carbonyl groups appeared at 167.00 and 168.00. All the aromatic or C = C unsaturated carbon appeared between 100.00 to 160.00. IR spectra showed a sharp strong band at 1730cm-1 (C=O ester), at 1680cm-1 C=O keto), at 1598cm-1 for C = C andat 3358cm-1 N-H group. SH O + SH 1a-g2a-g R CH(COOEt)CHClKOt-Bu4 hrs O EtOOC COOEt Entry R2a 4-OCH2b 4-H2c 4-Cl2d 4-CH32e 3-NO2f 4-Br2g 4-CHOScheme-1 Synthesis of diethyl malonate adducts 2a-g Table-1 Analytical data for compounds 2a-g Compound Molecular formula Yield (%) Mp \rC Elemental analysis Calculated Found C H N C H N 2a 29 H 29 O 6 NS 72 132 67.06 5.58 2.69 67.14 5.52 2.60 2b 28 H 27 O 5 NS 78 143 68.72 5.51 2.86 68.79 5.45 290 2c 28 H 26 O 5 NSCl 83 137 64.20 4.96 2.67 64.23 4.85 2.73 2d 29 H 29 O 5 NS 79 128 68.73 5.72 2.76 68.77 5.82 2.68 2e 28 H 26 O 7 N 2 S 68 166 62.54 4.83 5.20 62.66 4.75 5.15 2f 28 H 26 O 5 NSBr 75 149 58.82 4.54 2.45 58.94 4.47 2.49 2g 29 H 27 O 6 NS 80 150 66.88 5.18 2.68 66.97 5.12 2.74 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 3(1), 82-85, January (2013) Res. J. Chem. Sci. International Science Congress Association 85 Anti-Oxidant Activity (ABTS method): In vitro anti-oxidant activity was evaluated by ABTS radical scavenging assay for 2a-g and IC50 value was determined as shown in table-2. Quercetin was used as a control. Synthesized compounds 2a-g showed a good anti-oxidant activity with IC50 value between 36.15 to 81.00µg/ml when compared to the control quercetin with IC50 value 44.53µg/ml. From the table-2, Compound 2b does not have any substituents and the IC50 value is 40.00µg/ml. When electron donating groups is introduced in fourth position in 2a and 2d it increases the electron density and have marginally high anti-oxidant property with IC50 value of 39.13 and 36.15µg/ml respectively. The compound with electron donating group contributes more to the anti-oxidant activity; with respect to electron withdrawing groups when they are in para position the effect is more. This may be due to the resonance effect. When electron withdrawing group is in the m-positon the effect is less and showed a better activity. The presence of NH and CO group in diethyl malonate adducts of phenothiazine nucleus helps in antioxidant activity. Table-2 In-vitro antioxidant assay of compounds 9a-g S.No Compound IC 50 value in (µg/ml) 1 2a 39.13 2 2b 40.00 3 2c 61.02 4 2d 36.15 5 2e 41.23 6 2f 81.00 7 2g 43.00 8 Quercetin 44.53 ConclusionIn conclusion, a series of diethyl malonate adducts 2a-g were synthesized by Michael addition reaction. In-vitro antioxidant activity was evaluated for all synthesized compounds which showed a good activity with an IC50 value between 36.15 to 81.00µg/ml, when compared to the control quercetin with IC50value of 44.53µg/ml. AcknowledgementsWe are thankful to the Management, Vice-chacellor, Registrar and HOD, Dept. of chemistry, Karpagam University, Coimbatore, Tamil Nadu, India for providing the facilities to carry out the research work. 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