SYNTHESIS AND ANTIOXIDANT ACTIVITY OF NOVEL 4,8-DISUBSTITUTED-3,4-DIHYDRO-6-METHYL-IMIDAZO[1,5-b][1,2,4]TRIAZIN-2(8H)-ONE DERIVATIVES.
Atul Baravkar, Sanjay Sawant, Aniruddh Chabukswar, ...
1. *Department of Pharmaceutical Chemistry, Karpagam University, Coimbatore, Tamilnadu, India-641021. 2. Department of Pharmaceutical Chemistry, STES’s SKN College of Pharmacy, University of Pune, Kondhwa, Pune, India-411048. 3. Department of Pharmaceutical Chemistry, Maharashtra Institute of Pharmacy, University of Pune, Kothrud, Pune, India-411038
Keywords: Earlenmeyer-Azlactone synthesis, DPPH, Radical scavenging assay, Ascorbic acid.
Abstract

Purpose: The main objective of the present research study is to synthesize series of novel 4,8-disubstituted disubstituted-3,4-dihydro-6-methyl-imidazo[1,5-b][1,2,4]triazin-2(8H)-one derivatives (5a-5f) and evaluate them for their antioxidant effect. Methods: The said compounds were synthesized in total three steps viz Earlenmeyer-Azlactone synthesis, followed by reaction with substituted and unsubstituted 2, 4-dinitrophenylhydrazine and lastly reaction with chloracetamide. In vitro antioxidant study was performed using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay using various concentrations such as 10, 20, 30, 40 and 50 µg/mL. Synthesis of compounds was confirmed by melting point, HPTLC, FTIR, 1H NMR, 13C NMR and LC-MS. The obtained results (IC50 values) were compared with standard antioxidant agent ascorbic acid. Results: The IC50 values were compared with standard antioxidant ascorbic acid. Compound 5a, 5b and 5c showed very higher significant activity (p<0.01), 5d showed significant activity while compound 5e and 5f showed marginally significant activity. Conclusion: Compounds bearing electron donating group showed higher antioxidant effect as compared to the compounds with electron withdrawing groups.

Article Information

Identifiers and Pagination:
Year:2013
Volume:5
First Page:107
Last Page:117
Publisher Id:JAppPharm (2013 ). 5. 107-117
Article History:
Received:July 10, 2013
Accepted:September 17, 2013
Collection year:2013
First Published:October 1, 2013

INTRODUCTION

Imidazole and triazine heterocycles are important building blocks for development of novel medicinal, pharmaceutical and agricultural agents. Most classes of heterocyclic medicinal compounds contain imidazole ring in their chemical structure such as antioxidant agents [1], anticancer agents [2], antifungal [3], antibacterial [4], antiprotozoal [5]. Coming to the triazine nucleus, it has very importance because of its effectiveness in most of chemical compounds such as drugs, polymers, resins, agricultures and optics [6-10]. Triazine based complexes of copper has superoxide radical scavenging effect in pathological cascade [11]. Various novel fused 1,2,4-triazine aryl analogues antitumor effect [12], ethanol induced antistress effect in mouse brain [13], and adenosine receptor antagonist [14]. The literature survey revealed that when dynamically active 2 hetocycles couples with each other, new molecule development occurs and which can display synergistic effect on biological systems. This encouraged us to synthesize imidazole ring clamped with triazine ring could give entrance to novel antioxidant agents. Therefore an attempt has been made to synthesize newer 4,8-disubstituted-3,4-dihydro-6-methyl-imidazo[1,5-b][1,2,4]triazin-2(8H)-one derivatives as antioxidants.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS), capable of causing damage to DNA, has been associated with carcinogenesis, coronary heart disease, and many other health problems related to advancing age. In low concentrations, synthetic antioxidants are also in use for many industrial processes e.g. inhibition of radical formation for preventing premature polymerization during processing, storage and transportation of unsaturated monomers. They exert their effects by scavenging or preventing the generation of ROS which can protect the formation of free radicals and retard the progress of many chronic diseases including cancer, neurodegenerative, inflammation and cardiovascular diseases [15-17]. Hence the interest for the protective role of antioxidant drugs has been growing over last 14 years. They are considered as potential medicines because of their ability to reduce or stop free radical reactions initiated by ROS/RNS.

 

MATERIALS AND METHODS

General:

All reagents were used as purchased from E. Merck, Mumbai and used without further purification. Melting points were determined by using a Remi digital melting point determination apparatus and are uncorrected. Purity of compounds were checked by High Performance Thin-layer chromatography (HPTLC) and was performed on CAMAG twin with applicator Linomat-IV and plate specifications are Merck precoated silica gel 60 F254 with 0.2 mm thickness. Spectroscopic data were recorded by using FT-IR (Shimadzu spectrophotometer 8400 using KBr), 1H NMR (Varian Mercury 400, Model- Unity AS400, serial- S0121719, frequency 400 MHz using DMSO as a solvent and tetramethylsilane (TMS) as an internal standard and chemical shifts were expressed as d values in ppm), 13C NMR (INOVA-300 with 75 MHz frequency DMSO as a solvent and tetramethylsilane (TMS) as an internal standard), LC-MS (Benchtop Agilent 1100 series LC–MSD (Agilent Technologies, Waldbronn, Germany), Column: C18, preparation on ODS (octadecylsilica) Hypersil column (Agilent Technologies), Flow-rate was 0.25 mL/min to 0.50 mL/min). Absorbance was recorder using UV Jasco spectrophotometer model V-630. Antioxidant activity was performed by using DPPH radical scavenging assay.

Experimental:

a.Typical procedure for synthesis of compounds (3a-3f) by Erlenmeyer-Azlactone synthesis [18].

Warm a mixture of 29 g (0.25 mol) of N-acetylglycine, 37.5 ml (0.37 mol) of aromatic aldehydes (1a-1g), 15 gm (0.183 mol) of anhydrous sodium acetate and 59 mL (0.62 mol) of acetic anhydride in 500 mL flask equipped with a reflux condenser, on water bath with occasional shaking until solution is complete (10-20 min). Boil the resulting solution for 1 h, cool and leave in a refrigerator overnight. Stir the solid mass of yellow crystals with 60 mL of cold water, transfer to a Buchner funnel and wash well with cold water. Wash with a little ether. Crystallized from carbon tetrachloride and used for next step of synthesis.


Figure 1-Synthetic route.

 

b. Typical procedure for synthesis of compounds (4a-4f)

A solution of 3a-3f (6 mmole) in dry benzene (30 mL) and 2,4-dinitro phenylhydrazine (5 mmole) was heated under reflux for 4 h. Then the mixture was poured upon water. The precipitated solid was filtered off, dried and crystallized from ethanol to get the desired compounds.

c. Typical procedure for synthesis of compounds (5a-5f)

A solution of 4a-4f (8 mmole) and chloroacetamide (8 mmole) was refluxed for 3 h in boiling N, N-dimethylformamide (30 mL). Then the mixture was poured into water. The precipitated solid was filtered off, dried and crystallized from ethanol to get the desired compounds.

Antioxidant activity by DPPH free radical scavenging assay [19]:

Antioxidant activity of all novel synthesized compounds (5a-5f) was done by DPPH free radical scavenging assay as described by Philip Molyneux. Ascorbic acid and the synthesized compounds of different concentrations were prepared in distilled ethanol. 3 mL of each compound solution having different concentrations (10, 20, 30, 40 and 50 µg/mL) were taken in different test tubes of 0.004% DPPH in ethanol was added in required to obtain above mentioned five concentrations and shaken vigorously. The tubes were then incubated in the dark room at RT for 20 min. A DPPH blank was prepared without compound, and ethanol was used for the baseline correction. Decrease in the absorbance at 517 nm was measured using a UV-visible spectrophotometer and the remaining DPPH was calculated. The percent decrease in the absorbance was recorded for each concentration, and percent quenching of DPPH was calculated on the basis of the observed decrease in absorbance of the radical. The radical scavenging activity was expressed as the inhibition percentage and was calculated using the formula:

Radical scavenging activity (%) = [(Ao-A1)/Ao × 100]                     

Where Ao is the absorbance of the control (blank) and A1 is the absorbance of the compound.

 

RESULTS

Novel compound of each series were identified by determining their melting points, % yield. They were also confirmed spectrophotometrically using HPTLC, IR, 1H NMR, 13C NMR, LC-MS etc.

4-(2-hydroxybenzylidene)-2-methyloxazol-5(4H)-one (3a)

Mol. Form. C11H9NO3; Ar = (-C6H5-o-OH); mp 305-307 ºC; yield: 89%; IR (KBr, ?max, cm -1): 855 (C-H bend), 1294 (C-O str), 1355 (C-N str), 1520 (C=C str), 1605 (C=N str), 1671 (C=O str), 2970 (CH3 str), 3041 (C-H str), 3324 (O-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz): 1.42 (s, 1H, CH3), 6.68 (s, 1H, CH), 6.71 (d, 1H, ArH), 6.77 (t, 1H, ArH), 6.97 (t, 1H, ArH), 7.13 (d, 1H, ArH), 11.32 (s, 1H, OH).

4-(3-methoxybenzylidene)-2-methyloxazol-5(4H)-one (3b)

Mol. Form. C12H11NO3; Ar = (-C6H5-m-OCH3); mp 195-197 ºC; yield: 88%; IR (KBr, ?max, cm -1): 881 (C-H bend), 1170 (C-O str), 1300 (C-N str), 1509 (C=C str), 1680 (C=N str), 1772 (C=O str), 2902 (CH3 str), 3068 (C-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz): 2.34 (s, 1H, CH3), 3.73 (s, 1H, OCH3), 6.75 (d, 1H, ArH), 6.81 (s, 1H, ArH), 6.86 (d, 1H, ArH), 7.10 (t, 1H, ArH), 7.64 (s, 1H, CH).

5-(2-hydroxybenzylidene)-2-methyl-3-(phenylamino)-3,5-dihydro-4H-imidazol-4-one (4a)

Mol. Form. C17H15N3O2; Ar = (-C6H5-o-OH); mp 90-92 ºC, yield: 72%; HPTLC: Rf 0.62, Chloroform: methanol: water (6:2:2); IR (KBr, ?max, cm -1): 884 (C-H bend), 1133 (C-O str), 1231 (C-N str), 1617 (C=C str), 1637 (C=N str), 1747 (C=O str), 2971 (CH3 str), 3128 (C-H str), 3431 (N-H str), 3674 (O-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.13 (s, 3H, CH3), 6.21 (s, 1H, NH), 6.32 (s, 1H, CH), 6.64 (d, 3H, ArH), 6.74 (t, 2H, ArH), 6.96 (t, 1H, ArH), 7.17 (d, 1H, ArH), 7.19 (t, 2H, ArH), 11.71 (s, 1H, OH); 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 21.31, 108.55, 113.31, 115.86, 116.12, 119.21, 121.23, 127.81, 129.65, 130.61, 144.72, 151.81, 158.91, 166.13; LC-MS (m/z): 294.37 [M++1].

5-(3-methoxybenzylidene)-2-methyl-3-(phenylamino)-3,5-dihydro-4H-imidazol-4-one (4b)

Mol. Form. C18H17N3O2; Ar = (-C6H5-m-OCH3); mp 176-178 ºC, yield: 83%; HPTLC: Rf 0.61, Chloroform: methanol: water (8:1:1); IR (KBr, ?max, cm -1): 851 (C-H bend), 1334 (C-N str), 1519 (C=C str), 1634 (C=N str), 1723 (C=O str), 3032 (CH3 str), 3134 (C-H str), 3485 (N-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.34 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 6.65 (d, 1H, ArH), 6.66 (t, 1H, ArH), 6.71 (d, 2H, ArH), 6.81 (s, 1H, ArH), 6.86 (d, 1H, ArH), 7.10 (t, 1H, ArH), 7.19 (t, 2H, ArH), 7.56 (s, 1H, CH), 7.94 (s,  1H, NH); 13C NMR (d, ppm, DMSO-d6, 75 MHz): 20.87, 55.09, 108.04, 112.67, 113.02, 113.85, 118.45, 119.54, 129.63, 129.73, 130.40, 137.54, 144.76, 161.89, 166.43; LC-MS (m/z): 308.92 [M++1].

 

1-(2,4-dinitrophenylamino)-4-(2-hydroxybenzylidene)-2-methyl-1H-imidazol-5(4H)-one (4c)

Mol. Form. C17H13N5O6; Ar = (-C6H5-o-OH); mp 190-193 ºC, yield: 67%; HPTLC: Rf 0.58, Toluene: ethyl acetate (8:2); IR (KBr, ?max, cm -1): 849 (C-H bend), 1261 (C-O str), 1324 (C-N str), 1672 (C=C str), 1672 (C=N str), 1763 (C=O str), 2966 (CH3 str), 3041 (C-H str), 3476 (N-H str), 3623 (OH str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.54 (s, 3H, CH3), 6.43 (s, 1H, CH), 6.64 (s, 2H, NH), 6.69 (d, 1H, ArH), 6.74 (t, 1H, ArH), 6.94 (t, 1H, ArH), 7.18 (d, 1H, ArH), 7.21 (d, 1H, ArH), 8.51 (d, 1H, ArH), 9.03 (s, 1H, ArH), 11.72 (s, 1H, ArH); 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 20.93, 40.87, 108.98, 113.93, 114.65, 119.45, 124.65, 127.35, 129.46, 130.76, 144.24, 149.67, 151.87, 166.13; LC-MS (m/z): 383.31 [M++1].

1-(2,4-dinitrophenylamino)-4-(3-methoxybenzylidene)-2-methyl-1H-imidazol-5(4H)-one (4d)

Mol. Form. C18H15N5O6; Ar = (-C6H5-m-OCH3); mp 90-91ºC, yield: 57%; HPTLC: Rf 0.73, Toluene: ethyl acetate (7:3); IR (KBr, ?max, cm -1): 842 (C-H bend), 1167 (C-O str), 1323 (C-N str), 1534 (C=C str), 1624 (C=N str), 1742 (C=O str), 2965 (CH3 str), 3097 (C-H str), 3454 (N-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.35 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 6.65 (d, 1H, ArH), 6.81 (s, 1H, ArH), 6.86 (d, 1H, ArH), 7.11 (t, 1H, ArH), 7.19 (d, 1H, ArH), 7.45 (s, 1H, CH), 8.50 (d, 1H, ArH), 9.05 (s, 1H, ArH), 9.12 (s, 1H, NH); 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 20.87, 55.09, 108.04, 112.67, 113.02, 113.85, 118.45, 119.54, 129.63, 129.73, 130.40, 137.54, 144.76, 161.89, 166.43; LC-MS (m/z): 397.34 [M++1].

4-(2-hydroxybenzylidene)-1-(4-fluorophenylamino)-2-methyl-1H-imidazol-5(4H)-one (4e)

Mol. Form. C17H14FN3O2; Ar = (C6H5-o-OH); mp 105-106ºC, yield: 71%; HPTLC: Rf 0.61, Toluene: ethyl acetate: formic acid (8:1:1); IR (KBr, ?max, cm -1): 726 (C-H bend), 1080 (C-F str), 1183 (C-O str), 1364 (C-N str), 1483 (C=C str), 1683 (C=N str), 1764 (C=O str), 2965 (CH3 str), 3069 (C-H str), 3644 (N-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.61 (s, 3H, CH3), 6.33 (s, 1H, CH), 6.61 (d, 2H, ArH), 6.64 (d, 1H, ArH), 6.74 (t, 1H, ArH), 6.79 (s, 1H, NH), 6.83 (d, 2H, ArH), 6.94 (t, 1H, ArH), 7.18 (d, 1H, ArH), 11.32 (s, 1H, OH); 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 20.71, 108.65, 114.81, 115.51, 116.11, 116.89, 121.31, 127.76, 129.76, 130.76, 144.78, 146.56, 153.31, 158.98, 166.34.

4-(3-methoxybenzylidene)-1-(4-fluorophenylamino)-2-methyl-1H-imidazol-5(4H)-one (4f)

Mol. Form. C18H16FN3O2; Ar = (-C6H5-m-OCH3); mp 139-141ºC, yield: 67%; HPTLC: Rf 0.47, Toluene: ethyl acetate: formic acid (7:2:1); IR (KBr, ?max, cm -1): 768 (C-H bend), 1057 (C-F str), 1186 (C-O str), 1374 (C-N str), 1468 (C=C str), 1663 (C=N str), 1775 (C=O str), 2971 (CH3 str), 3044 (C-H str), 3468 (N-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.35 (s, 3H, CH3), 3.71 (s, 3H, OCH3), 6.63 (d, 2H, ArH), 6.66 (d, 1H, ArH), 6.81 (s, 1H, ArH), 6.87 (d, 1H, ArH), 6.89 (d, 2H, ArH), 7.11 (t, 1H, ArH), 7.58 (s, 1H, CH), 7.94 (s, 1H, NH) ; 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 21.09, 55.90, 108.86, 110.65, 112.87, 114.87, 116.65, 118.86, 129.54, 130.06, 136.14, 144.65, 146.43, 153.65, 161.76, 166.18

8-(2-hydroxybenzylidene)-3,4-dihydro-6-methyl-4-phenylimidazo[1,5-b][1,2,4]triazin-2(8H)-one (5a)

Mol. Form. C19H16N4O2; Ar = (-C6H5-o-OH); m.p: 82-83 ºC; yield: 79%; HPTLC: Rf 0.63, Chloroform: methanol: water (7:1:2); IR (KBr, ?max, cm -1):  885 (C-H bend), 1110 (C-O str), 1223 (C-N str), 1617 (C=C str), 1631 (C=N str), 1764 (C=O str), 2928 (=CH2 str, sym), 2851 (=CH2 str, asym), 3074 (CH3 str), 3164 (C-H str), 3613 (O-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz): 2.82 (s, 3H, CH3), 4.23( s, 2H, CH2), 6.64 (d, 3H, ArH), 6.78 (t, 1H, ArH), 6.89 (t, 1H, CH & 2H, ArH), 6.93 (t, 1H, ArH), 7.18 (d,1H, ArH), 7.22 (d, 1H, ArH), 11.72 (s, 1H, ArH); 13C NMR (d, ppm, DMSO-d6, 75 MHz): 21.38, 62.55, 102.66, 113.21, 116.12, 115.81, 119.23, 121.33, 127.11, 127.98, 129.21, 129.99, 144.72, 151.44, 158.31, 164.56, 200.21; LC-MS (m/z): 333.16 [M++1].

8-(3-methoxybenzylidene)-3,4-dihydro-6-methyl-4-phenylimidazo[1,5-b][1,2,4]triazin-2(8H)-one (5b)

Mol. Form. C20H18N4O2; Ar = (-C6H5-m-OCH3); mp 120-122 ºC; yield: 90%; HPTLC: Rf 0.61, Chloroform: methanol: water (8:1:1); IR (KBr, ?max, cm -1): 876 (C-H bend), 1365 (C-N str), 1523 (C=C str), 1633 (C=N str), 1745 (C=O str), 2872 (=CH2 str, sym), 2902 (=CH2 str, asym), 3054 (CH3 str), 3121 (C-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.34 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 4.17 (s, 2H, CH2), 6.62 (s, 1H, CH), 6.66 (d, 1H, ArH), 6.67 (d, 2H, ArH), 6.72 (t, 1H, ArH), 6.80 (s, 1H, ArH), 6.85 (d,1H, ArH), 7.11 (t, 1H, ArH), 7.19 (t, 1H, ArH); 13C NMR (d, ppm, DMSO-d6, 75 MHz): 21.30, 56.65, 62.40, 102.62, 113.65, 119.65, 123.26, 126.86, 127.70, 128.26, 128.62, 129.43, 136.20, 138.26, 144.70, 151.42, 164.86, 200.56; LC-MS (m/z): 347.64 [M++1].

8-(2-hydroxybenzylidene)-3,4-dihydro-6-methyl-4-(2,4-dinitrophenyl)imidazo[1,5-b][1,2,4]triazin-2(8H)-one (5c)

Mol. Form. C19H14N6O6; Ar = (-C6H5-o-OH); mp 82-83 ºC, yield: 89%; HPTLC: Rf 0.77, Toluene: ethyl acetate (9:1); IR (KBr, ?max, cm -1): 861 (C-H bend), 1274 (C-O str), 1372 (C-N str), 1561 (C=C str), 1692 (C=N str), 1762 (C=O str), 2837 (=CH2 str, sym), 2915 (=CH2 str, asym), 2972 (CH3 str), 3061 (C-H str), 3634 (OH str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.34 (s, 3H, CH3), 4.18 (s, 2H, CH2), 6.67 (d, 1H, ArH), 6.77 (t, 1H, ArH), 6.81 (s, 1H, CH), 6.92 (t, 1H, , ArH), 7.17 (d, 1H, ArH), 7.21 (d, 1H, ArH), 8.51 (d, 1H, ArH), 9.04 (s, 1H, ArH), 11.79 (s, 1H, OH); 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 20.65, 61.44, 102.75, 116.98, 117.54, 119.21, 121.24, 127.11, 127.89, 129.88, 132.65, 139.98, 143.23, 144.28, 158.87, 166.43, 164.56, 200.45; LC-MS (m/z): 422.35 [M++1].


Figure 2. % DPPH radical scavenging effect.

 


Figure 3. IC50 value of compound 5a.

 


Figure 4. IC50 value of ascorbic acid.

8-(3-methoxybenzylidene)-3,4-dihydro-6-methyl-4-(2,4-dinitrophenyl)imidazo[1,5-b][1,2,4]triazin-2(8H)-one (5d)

Mol. Form. C20H16N6O6; Ar = (-C6H5-o-OCH3); mp 105-108 ºC, yield: 77%; HPTLC: Rf 0.73, Toluene: ethyl acetate (7:3); IR (KBr, ?max, cm -1): 843 (C-H bend), 1133 (C-O str), 1375 (C-N str), 1512 (C=C str), 1645 (C=N str), 1734 (C=O str), 2848 (=CH2 str, sym), 2925 (=CH2 str, asym), 2971 (CH3 str), 3069 (C-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.35 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 4.17 (s, 2H, CH2), 6.60 (s, 1H, CH), 6.66 (s, 1H, ArH), 6.81 (d, 1H, ArH), 6.86 (d, 1H, ArH), 7.11 (t, 1H, ArH), 7.19 (d, 1H, ArH), 8.51 (d,1H, ArH), 9.05 (s, 1H, ArH); 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 21.98, 55.90, 61.86, 102.56, 110.85, 113.56, 115.76, 118.76, 119.54, 127.65, 128.05, 131.56, 134.87, 138.87, 141.65, 143.54, 144.12, 161.45, 165.25, 200.65; LC-MS (m/z): 436.37 [M++1].

8-(2-hydroxybenzylidene)-4-(4-fluorophenyl)-3,4-dihydro-6-methylimidazo[1,5-b][1,2,4]triazin-2(8H)-one (5e)

Mol. Form. C19H15FN4O2; Ar = (-C6H5-o-OH); mp 97-99ºC, yield: 71%; HPTLC: Rf 0.62, Toluene: ethyl acetate: formic acid (6:2:2); IR (KBr, ?max, cm -1): 784 (C-H bend), 1050 (C-F str), 1178 (C-O str), 1376 (C-N str), 1465 (C=C str), 1653 (C=N str), 1798 (C=O str), 2851 (CH2 str, sym), 2921 (CH2 str, asym), 2970 (CH3 str), 3041 (C-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.69 (s, 3H, CH3), 4.20 (s, 2 H, CH2), 6.61 (t, 2H, ArH), 6.64 (d, 1H, ArH), 6.74 (t, 1H, ArH), 6.79 (s, 1H, CH), 6.83 (t, 2H, ArH), 6.94 (t, 1H, ArH), 7.18 (d, 1H, ArH), 11.58 (s, 1H, OH) ; 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 21.31, 62.44, 102.66, 114.98, 115.08, 116.54, 116.99, 121.34, 127.12, 127.98, 129.65, 144.72, 147.98, 153.25, 158.34, 164.24, 200.13

 


Figure 5. UV absorption plots for compound 5a at conc of 10 and 20 µg/mL.

8-(3-methoxybenzylidene)-4-(4-fluorophenyl)-3,4-dihydro-6-methylimidazo[1,5-b][1,2,4]triazin-2(8H)-one (5f)

Mol. Form. C20H17FN4O2; Ar = (-C6H5-m-OCH3); mp 109-111ºC, yield: 65%; HPTLC: Rf 0.47, Toluene: ethyl acetate: formic acid (7:2:1); IR (KBr, ?max, cm -1): 771 (C-H bend), 1049 (C-F str), 1178 (C-O str), 1354 (C-N str), 1459 (C=C str), 1661 (C=N str), 1770 (C=O str), 2871 (CH2 str, sym), 2915 (CH2 str, asym), 2955 (CH3 str), 3039 (C-H str); 1H NMR (d, ppm, DMSO-d6, 400 MHz) : 2.33 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 4.18 (s, 2H, CH2), 6.61 (s, 1H, CH), 6.62 (d, 2H, ArH), 6.64 (d, 1H, ArH), 6.78 (s, 1H, ArH), 6.85 (d, 1H, ArH), 6.89 (d, 2H, ArH), 7.13 (t, 1H, ArH) ; 13C NMR (d, ppm, DMSO-d6, 75 MHz) : 21.56, 55.96, 62.56, 103.35, 111.05, 113.45, 114.27, 116.12, 118.74, 127.76, 130.27, 136.26, 144.65, 147.98, 153.98, 160.62, 164.26, 200.12

Antioxidant activity of the synthesized compounds is depicted in tabular and graphical form as follows.

 

Table 1. % DPPH radical scavenging effect.

Conc. (µg/mL)

% DPPH radical scavenged

Ascorbic acid

5a

5b

5c

5d

5e

5f

10

76.52

80.27

72.61

68.81

64.62

63.30

58.94

20

78.45

97.41

73.30

69.11

67.04

63.42

59.38

30

79.76

97.76

73.61

70.02

67.45

63.54

59.71

40

81.15

98.4

76.99

70.15

69.83

72.30

59.98

50

90.88

98.43

79.47

70.26

77.76

76.95

61.01

 

STATISTICAL ANALYSIS

Experimental results were expressed as mean ± SD of 3 parallel readings. Results were analyzed using ANOVA followed by Student’s‘t’ test analysis.

DISCUSSION

All synthesized compounds (5a-5d) were evaluated using physical data such as melting point and Rf values as well as spectroscopic methods IR, 1H NMR, 13C NMR, LC-MS etc and showed best correlation with the same. All these novel compounds were screened for in vitro antioxidant activity by DPPH radical scavenging effect at various concentrations of 10, 20, 30, 40 and 50 µg/mL and IC50 values had been determined for each compound and compared with standard antioxidant. Compound 5a is more active than that of ascorbic acid, compound 5b showed comparable activity as that of ascorbic acid while other compounds such as 5c, 5d, 5e and 5f are less active.

CONCLUSION

IC50 value of compound 5a was found to be smaller than that of ascorbic acid which shows that compound 5a is strong DPPH radical scavenger than ascorbic acid, compound 5b is comparable DPPH radical scavenger while all other are less DPPH radical scavenger than ascorbic acid. Stronger DPPH scavenging effect of compound 5a and 5b is owing to presence of electron donating groups viz hydroxyl and methoxy in their structure, which increases stability of phenoxy radical. In comparison, compound 5a is more active that compound 5b as former contain hydroxyl group at ortho position of benzylidene moiety while later contain methoxy group at meta position of benzylidene moiety and o-hydroxyl group shows resonating effect while m-methoxy group shows +I effect. Former is stronger than later, hence compound 5a is more active than compound 5b and ascorbic acid. Weaker DPPH scavenging effect of remaining compounds is owing to presence of electron withdrawing groups such as nitro and fluoro.

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Editor in Chief
Prof. Dr. Cornelia M. Keck (Philipps-Universität Marburg)
Marburg, Germany

Bibliography

Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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Abbreviation: J App Pharm
doi: http://dx.doi.org/10.21065/19204159
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