Nine previously reported metabolites, including a chromanol derivative (3), , 3,5-dihydroxyergosta-7,22-dien-6-one (4), byssochlamic acid, hopan-3β,22-diol, chevalone C, sartorypyrone B, helvolic acid, lumichrome and the alkaloid harmane were isolated from the culture of the marine-sponge associated fungus Neosartorya tsunodae KUFC 9213. Paecilin E (1), dankasterone A (2), a chromanol derivative (3), -3,5-dihydroxyergosta-7,22-dien-6-one (4), hopan-3β,22-diol (5), lumichrome (6), and harmane (7) were tested for their antibacterial activity against Gram-positive and Gram-negative reference and multidrug-resistant strains isolated from the environment. While paecilin E (1) was active against S. aureus ATCC 29213 and E. faecalis ATCC 29212, dankastetrone A (2) was only effective against E. faecalis ATCC 29212 and the multidrug-resistant VRE E. faecalis A5/102. Both compounds neither inhibit biofilm mass production in any of the strains at the concentrations tested nor exhibit synergistic association with antibiotics.A previously unreported dihydrochromone dimer, paecilin E ( Neosartorya (Trichocomaceae) have been revealed to be an important source of interesting bioactive metabolites such as polyketides, isocoumarins, ergosterol analogs, meroditerpenes, pyripyropenes, benzoic acid derivatives, prenylated indole derivatives, tryptoquivalines, fiscalins, phenylalanine-derived alkaloids and cyclopeptides +, , which indicated eighteen degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3443 cm−1), conjugated ketone carbonyl (1645 cm−1), ester carbonyl (1790 cm−1), lactone carbonyl (1738 cm−1), and aromatic (1470 cm−1) groups. The 13C NMR spectrum (C 195.3 and 194.9), four ester carbonyl , eight quaternary sp2 , four methine sp2 , two oxyquaternary sp3 (δC 85.4 and 83.9), two oxymethine sp3 (δC 81.9 and 81.7), two methoxyl (δC 53.3 and 53.3), two methine sp3 (δC 32.9 and 32.5), four methylene sp3  and two methyl (δC 14.6 and 14.1) groups. Based on the type and values of their chemical shifts, these carbons were suspected to arise from two structurally similar moieties within compound 1.The molecular formula of spectrum  displayespectrum , can be 1H NMR and COSY spectra /6.61, d (J = 8.6 Hz) and 7.61, d (J = 8.6 Hz)/6.60, d (J = 8.6 Hz), a pair of doublets at δH 4.85, d (J = 7.3 Hz) and δH 4.97, d (J = 6.7 Hz), two pairs of mutually coupled methylene protons at δH 3.58, d (J = 17.4 Hz)/3.05, d (J = 17.4 Hz); δH 3.57, d (J = 17.4 Hz)/3.09, d (J = 17.4 Hz) and δH 2.41, dd /1.75 dd ; δH 2.86, dd /2.33, dd , two methyl singlets at δH 1.06, d (J = 7.1 Hz) and δH 1.17, d (J = 7.1 Hz) and two methoxyl singlets at δH 3.70 and 3.69.The  spectra  exhibiteH-chromen-4-one moiety was substantiated by COSY correlations from H-6  to H-7 , and HMBC correlations (C 107.5) and C-8 (δC 114.6), H-7 to C-5 (δC 160.4) and C-8a (δC 156.1), OH-5  to C-4a, C-5, C-6 (δC 109.2), H-3α  to C-2 (δC 85.4) and C-4 (δC 194.9), H-3β  to C-4 and C-4a. One of the substituents on C-2 was deduced as a methyl formate since both H-3α and the methoxyl singlet (δH 3.70) exhibited HMBC cross peaks to the ester carbonyl at δC 168.9 (C-9). Another substituent was 4-methyldihydrofuran-2-(3H)-one, which linked through C-10, was substantiated by COSY correlations from H-10 /H-11 /H2-12 , and from H-11 to Me-14  as well as by HMBC correlations from H-10 to C-3, C-11 (δC 32.9), C-12 (δC 35.3) and C-13 (δC 174.9), H2-12 to C-10 (δC 81.7), C-13, and Me-14 (δC 14.1) as well as from H-3α to C-10. However, this first monomer constituted only half of the molecular formula, i.e., C16H15O7 and still lacked the substituent on C-8.The existence of a 2,2,8-trisubstituted 5-hydroxy-2,3-dihydro-4elations  from H-6H-chromen-4-one core, but it was 2,2,6-trisubstituted as can be corroborated by COSY correlations from H-7′  to H-8′  as well as by HMBC correlations from H-7′ to C-5′ (δC 158.1), C-8′a (δC 158.2), H-8′ to C-6′ (δC 116.6) and C-4′a (δC 107.0), OH-5′  to C-5′, C-4′a and C-6′, H-3′β  to C-4′ (δC195.3) and C-4′a, and H-3′α  to C-4′ and C-2′(δC 83.9). Similarly, the substituents on C-2′ were methyl formate and 4-methyldihydrofuran-2-(3H)-one, through C-10′, which were based on HMBC correlations from H-3′α to C-9′ (δC 169.3), C-10′ (δC 81.9), H-10′  to C-2′, C-3′, C-11′ (δC 32.5), C-13′ (δC 175.5) and Me-14′ (δC 14.6) as well as the coupling system, as observed in the COSY spectrum, from H-10′, through H-11′  and H2-12′ , and from H-11′ to Me-14′. Like the first monomer, the second monomer also had C16H15O7, and still also lacked the substituent on C-6′. That the two monomers were connected through C-8 and C-6′ was supported by HMBC correlations from H-7 to C-6′ as well as from H-7′ to C-8. The second monomer also consisted of a 5-hydroxy-2,3-dihydro-4H-chromen-4-one with the methyl formate and γ-lactone substituents on C-2 have been previously reported. Guo et al. chromen-3-one.Later on, the same group  describeNMR data . Therefoh et al. ,18. This1H, 13C NMR, COSY, HSQC and HMBC data of 4 revealed that it was -3,5-dihydroxyergosta-7,22-dien-6-one , the hydroxyl group on C-5 was in the α position. However, no optical rotation of this compound was reported. Later on, Ishizuka et al. chromen-3-one (3). Crystals were triclinic, space group P1, cell volume 773.78(18) Å3 and unit cell dimensions a = 9.1295(12) Å, b = 9.2537(14) Å and c = 10.4317(12) Å and angles α = 94.622(11)°, β = 104.310(11)° and γ = 112.486(13)° (uncertainties in parentheses). The refinement converged to R  = 14.12% and wR2  = 29.88%. Diffraction data were collected at 291 K. CCDC 1579876.Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, a clinical isolate S. aureus 40/61/24, MRSA S. aureus 66/1 isolated from public buses [E. faecalis A5/102 and VRE E. faecalis B3/101 isolated from river water [Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and a clinical isolate ESBL E. coli SA/2. Frozen stocks of all strains were grown in Mueller-Hinton agar  at 37 °C. All bacterial strains were sub-cultured in MH agar and incubated overnight at 37 °C before each assay.For reference, a clinical isolate sensitive to the most commonly used antibiotic families, and four multidrug-resistant bacterial strains were used in this study. The Gram-positive bacteria comprised ic buses , and VREer water . The GraThe minimum inhibitory concentration (MIC), which was used for determining the antibacterial activity of each compound, was determined according to the method described previously by May Zin et al. .The effect of the compounds on biofilm formation was assessed using crystal violet staining as previously described by May Zin et al. .Evaluation of the combined effect of the compounds and clinical relevant antimicrobial drugs was performed according to the method previously described by May Zin et al. .Neosartorya fennelliae KUFA 0811, isolated from the marine sponge Clathria reinwardtii, resulted in the isolation of the previously undescribed 6-8 dimer of substituted 3,5-dihydrochromone which we have named paecilin E (1), and the previously reported metabolites including β-sitostenone, ergosta-4,6,8 (14), 22-tetraen-3-one, cyathisterone, byssochlamic acid, dehydromevalonic acid lactone, chevalone B, aszonalenin, dankasterone A (2), helvolic acid, secalonic acid A and fellutanine A. Re-examination of the culture of N. tsunodae KUFC 9213, led to the isolation of the chromanol derivative (3), in addition to sartorypyrone B and helvolic which were previously isolated from this fungus, and other known compounds including byssochlamic acid, hopan-3β,22-diol (5), chevalone C, -3,5-dihydroxyergosta-7,22-dien-6-one (4), the alkaloid harmane (7) and lumichrome (6). The absolute configurations of the stereogenic carbons of the previously undescribed paecilin E (1) and the chromanol derivative (3) were unambiguously established by X-ray analysis. Although -3,5-dihydroxyergosta-7,22-dien-6-one (4) has been reported from several sources, the absolute configuration of its C-5 had never been determined unambiguously by any modern techniques. By comparison of the experimental and calculated ECD spectra, we determined conclusively the absolute configuration of C-5 as 5R. Paecilin E (1), dankasterone A (2), the chromanol derivative (3) and some of the isolated compounds which have not been previously tested for antibacterial activity, i.e., -3,5-dihydroxyergosta-7,22-dien-6-one (4), hopan-3β,22-diol (5), lumichrome (6) and harmane (7) were tested for their antibacterial activity against Gram-positive and Gram-negative bacteria of four reference strains, a clinical isolate sensitive to the most commonly used antibiotic families, and four multidrug-resistant isolates from the environment. Only paecilin E (1) and dankasterone A (2) were able to inhibit growth of Gram-positive bacteria. While paecilin E (1) exhibited an inhibitory effect on both S. aureus ATCC 29213 and E. faecalis ATCC 29212 with MIC values of 32 μg/mL and 16 μg/mL, respectively, dankasterone (2) was only effective against E. faecalis ATCC 29212 and VRE E. faecalis A5/102, with MIC of 32 μg/mL and 64 μg/mL, respectively. Despite a great structural diversity of the secondary metabolites produced by these two marine-derived species of Neosartorya, a majority of them did not possess the antibacterial activity. Nevertheless, it does not mean that they do not have other interesting biological activities. Therefore, more biological assays will be performed in the future.Chemical investigation of the culture of the marine-derived fungus


All the compounds have been evaluated for their in vitro antioxidant activities using DPPH free radical scavenging assay as well as FRAP assay. The result showed promising antioxidant activities having IC50 values in the range of 4.74 ± 0.08 to 92.20 ± 1.54 μg/mL taking ascorbic acid (IC50 = 4.57 μg/mL) as standard reference. In this study, compounds 20b and 20t, the most active compound of the series, showed IC50 values of 6.89 ± 0.07 μg/mL and 4.74 ± 0.08 μg/mL, respectively in comparison with ascorbic acid. In addition, the detailed SAR study shows that electron-withdrawing group increases antioxidant activity and vice versa. Furthermore, in the FRAP assay, eight compounds  were found more potent than standard reference BHT (C0.5FRAP = 546.0 ± 13.6 μM). The preliminary cytotoxic study reveals the non-toxic nature of active compounds 20b and 20t in non-cancerous 3T3 fibroblast cell lines in MTT assay up to 250 μg/mL concentration. The results were validated via carrying out in silico molecular docking studies of promising compounds 20a, 20b, and 20t in comparison with standard reference. To the best of our knowledge, this is the first detailed study of C-3 tethered 2-oxo-benzooxazines as potential antioxidant agents.The discovery of C-3 tethered 2-oxo-benzooxazines as potent antioxidants is disclosed. All the analogs  Electrospray ionization mass spectrometry (ESI-MS) and HRMS were recorded on Xevo G2-S Q Tof  Spectrometer. Column chromatography was performed over Merck silica gel (particle size: 60-120 Mesh) procured from Qualigens? (India), flash silica gel (particle size: 230-400 Mesh). All chemicals and reagents were obtained from Sigma Aldrich (USA), Merck (India) or Spectrochem (India) and were used without further purification.All glass apparatus were oven dried prior to use. Melting points were taken in open capillaries on complab melting point apparatus and are presented uncorrected. Ultrasonic irradiation was performed in a Elmasonic S 30 (H) ultrasonic water bath cleaner and the reaction vessel was positioned in the maximum energy area in the cleaner and the removal or addition of water was used to control the temperature of the water bath. Infrared spectra were recorded on a Perkin-Elmer FT-IR Spectrum 2 spectrophotometer 16a-h  were taken in toluene (50 ml) and NaH  was added carefully. After stirring this reaction mixture at 0°C for 30 min dimethyl oxalate  were added and reflux for 6 h. The progresses of the reaction were monitored by TLC using 9:1 Hexane/ethyl acetate as an eluent. After completion of reaction, the reaction mixture was quenched with distilled water and extracted with ethyl acetate (3 × 50 ml); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The organic layer was combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude products were purified by recrystalization using EtOAc/Hexane (v/v = 20:80), which afforded the pure desired diketo-ester 17a-h in 78-92% yields. Compounds 17a-h was used for next step without any further purification.Substituted acetophenone 17a-h  in MeOH:THF: H2O , added LiOH.H2O  into the reaction mixture and stirred it for 4 h at room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction, it was quenched with 3N HCl solution and extracted with ethyl acetate (3 × 30 mL); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The combined organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum to afford the corresponding crude product. These crude products were further purified by recrystalization with EtOAc/Hexane, which afforded diketo-acids 18a-h in excellent yields (up to 97%). Compounds 18a-h were used for next step without any further purification.To a solution of 18a-h  in water (2.0 mL) was added compound 19a-f  and the reaction mixture was irradiated under ultrasonic sonicator at 80°C temperature for about 75-90 min (depending upon the substrate employed). The progress of the reaction was checked by TLC using 9:1 Hexane/ethyl acetate as an eluent. After completion of reaction, the reaction mixture was extracted with ethyl acetate (3 × 50 ml); then with distilled water (2 × 10 mL) followed by brine solution (2 × 20 mL). The organic layers were combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude products were purified either by recrystalization using Hexane/EtOAc (v/v = 90:10) or by flash column chromatography method over silica gel using 7.5:2.5 to 9:1 Hexane/ethyl acetate as an eluent which afforded the pure desired (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b]-oxazin-2-one 20a-ab having good yields (80–98%).To a solution of the compound f  = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 185–186°C; FT-IR  3434, 1754, 1614, 1594, 1270, 1113; 1H NMR  δ 8.00 , 7.55–7.46 , 7.21–7.05 ; 13C NMR  δ 191.6 (C=O), 156.3 (O=C-O), 141.3 (C=CH), 139.1 (Ar-C), 138.3 (Ar-C), 132.8 (NH-C), 128.8 (Ar-C-NH), 127.7 (Ar-C), 126.0 (Ar-C), 124.0 (Ar-C), 123.8 (Ar-C), 117.2 (Ar-C), 116.0 (Ar-C), 94.7 (C=CH); HRMS (ESI) calcd. for C16H11NO3 [M+H]+: 266.0739; found 266.0734.Yellow solid; yield: 51.97 mg (98%), Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. 200–203°C; FT-IR  3435, 1756, 1602, 1112; 1H NMR  δ 7.99 , 7.17 , 7.09–7.05 , 7.02–6.95 , 3.87 ; 13C NMR  δ 190.5 (C=O), 163.5 (Ar-C-OCH3), 156.6 (O=C-O), 141.2 (Ar-C-N), 138.6 (Ar-C-O), 131.2 (C=CH), 130.0 (Ar-C), 125.9 (Ar-C), 124.0 (Ar-C), 123.7 (Ar-C), 117.2 (Ar-C), 115.8 (Ar-C), 114.0 (Ar-C), 94.7 (C=CH), 55.6 (O-CH3); HRMS (ESI) calcd. for C17H13NO4 [M+H]+: 296.0845; found 296.0849.Yellowish solid; yield: 51.2 mg (88%), Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 185–187°C; FT-IR  3434, 1761, 1555, 1622, 1174; 1H NMR  δ 8.00–7.98 , 7.58–7.55 , 7.50-7.47 , 7.13–7.03 ; 13C NMR  δ 191.8 (C=O), 155.8 (O=C-O), 139.8 (Ar-C-N), 138.4 (Ar-C-O), 138.1 (C=CH), 133.0 (Ar-C), 131.1 (Ar-C-Cl), 128.9 (Ar-C), 127.8 (Ar-C), 124.8 (Ar-C), 123.8 (Ar-C), 118.3 (Ar-C), 115.8 (Ar-C), 95.7 (C=CH); HRMS (ESI) calcd. for C16H10ClNO3 [M+2]+: 301.7085; found 301.7089.Yellowish solid; yield: 56.93 mg (95%), Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 155–157°C; FT-IR  3434,1754,1634,1601,1495, 1226,1160; 1H NMR  δ 8.06-8.02 , 7.20-7.13 , 7.08-7.04 ;13C NMR  δ 190.4 (C=O), 167.2 (Ar-C-F), 155.8 (O=C-O), 139.8 (Ar-C-N), 138.6 (Ar-C-O), 131.3 (Ar-C-Cl), 130.5 (C=CH), 130.4 (Ar-C), 124.8 (Ar-C), 123.9 (Ar-C), 118.4 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 115.8 (Ar-C), 95.4 (C=CH); HRMS (ESI) calcd. for C16H9ClFNO3 [M+H]+: 318.0255; found 318.0259.Yellowish solid; yield: 59.08 mg (93%); Rf  = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 182–185°C; FT-IR  3434, 1761, 1631, 1586, 1088; 1H NMR  δ 7.93 , 7.45 , 7.13–7.00 ; 13C NMR  δ 190.4 (C=O), 155.7 (O=C-O), 139.8 (Ar-C-Cl), 139.4 (Ar-C-N), 138.7 (Ar-C-O), 136.3 (C=CH), 131.2 (Ar-C), 129.2 (Ar-C), 129.1 (Ar-C), 124.6 (Ar-C), 124.0 (Ar-C), 118.3 (Ar-C), 115.9 (Ar-C), 95.3 (C=CH); HRMS (ESI) calcd. for C16H9Cl2NO3 [M+2]+: 334.9959; found 334.9956.Yellowish solid; yield: 63.9 mg (96%); Rf  = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 135–137°C; FT-IR  3432, 3075, 2923, 1626, 1583, 1105; 1H NMR  δ 7.54 , 7.47 , 7.34 , 7.17-7.09 , 6.82 ; 13C NMR  δ 192.1 (C=O), 155.4 (O=C-O), 139.9 (Ar-C-Cl), 138.4 (Ar-C-Cl), 137.7 (Ar-C-N), 137.2 (Ar-C-O), 132.7 (C=CH), 131.3 (Ar-C), 130.8 (Ar-C), 130.7 (Ar-C), 127.6 (Ar-C), 124.4 (Ar-C), 118.4 (Ar-C), 116.1 (Ar-C), 99.3 (C=CH); HRMS (ESI) calcd. for C16H8Cl3NO3 [M+2]+: 368.9570; found 368.9577.Yellowish solid; yield: 65.9 mg (89%); Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 175–177°C; FT-IR  3436, 2924, 1755, 1632, 1583, 1007; 1H NMR  δ 7.85 , 7.62 , 7.13 – 7.00 ; 13C NMR  δ 190.5 (C=O), 155.6 (O=C-O), 139.8 (Ar-C-N), 138.8 (Ar-C-O), 136.8 (C=CH), 132.1 (Ar-C-Cl), 131.2 (Ar-C), 129.3 (Ar-C-Br), 128.1 (Ar-C), 124.6 (Ar-C), 124.0 (Ar-C), 118.3 (Ar-C), 115.9 (Ar-C), 95.2 (C=CH); HRMS (ESI) calcd. for C16H9BrClNO3 [M+2]+: 377.9454; found 377.9458.Yellowish solid; yield: 70.7 mg (93%); Rf  = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 160–162°C; FT-IR  3434, 2925, 1624, 1766, 1494, 1178; 1H NMR  δ 7.94–7.92 , 7.30 , 7.14-7.04 , 2.44 ;13C NMR  δ 191.6 (C=O), 156.0 (O=C-O), 144.0 (Ar-C-CH3), 139.8 (Ar-C-N), 138.2 (Ar-C-O), 135.6 (C=CH), 131.2 (Ar-C), 129.7 (Ar-C), 128.0 (Ar-C), 125.0 (Ar-C), 123.7 (Ar-C), 118.3 (Ar-C), 115.8 (Ar-C), 95.9 (C=CH), 21.8 (-CH3); HRMS (ESI) calcd. for C17H12ClNO3 [M+H]+: 314.0506; found 314.0509.Yellowish solid; yield: 56.5 mg (90%); Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9:1) as an eluent; m.p. 178-180°C; FT-IR  3434, 1764, 1628, 1594, 1018; 1H NMR  δ 7.99-7.97 , 7.10-7.06 , 7.02–6.99 , 6.97-6.94 , 3.87 ; 13C NMR  δ 190.5 (C=O), 163.7 (Ar-C-OCH3), 156.1 (O=C-O), 139.6 (Ar-C-N), 137.8 (Ar-C-O), 131.1 (C=CH), 130.9 (Ar-C), 130.1 (Ar-C), 125.0 (Ar-C), 123.4 (Ar-C), 118.2 (Ar-C), 115.6 (Ar-C), 114.1 (Ar-C), 95.7 (C=CH), 55.6 (-OCH3); HRMS (ESI) calcd. for C17H12ClNO4 [M+2]+: 331.7345; found 331.7349.Yellowish solid; yield: 56.6 mg (86%); Rf  = 0.85; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 145–147°C; FT-IR  3433, 2930, 1770, 1624, 1596, 1128; 1H NMR  δ 8.02 , 7.17–7.07 , 6.98–6.90 , 2.36 ; 13C NMR  δ 190.0 (C=O), 166.9 (Ar-C-F), 156.4 (O=C-O), 139.3 (Ar-C-CH3), 136.1 (Ar-C-N), 134.7 (Ar-C-O), 130.3 (C=CH), 130.2 (Ar-C), 124.9 (Ar-C), 123.3 (Ar-C), 116.9 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 115.8 (Ar-C), 94.1 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H12FNO3 [M+H]+: 298.0801; found 298.0807.Yellowish solid; yield: 53.7 mg (90%); Rf  = 0.80; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 142–145°C; FT-IR  3436, 2913, 1755, 1618, 1570, 1083; 1H NMR  δ 7.53 , 7.45 , 7.33–7.30 , 7.10–7.08 , 6.94–6.92 , 6.73 , 2.36 ; 13C NMR  δ 191.6 (C=O), 155.9 (O=C-O), 139.5 (Ar-C-CH3), 139.2 (Ar-C-Cl), 137.5 (Ar-C-Cl), 137.2 (Ar-C-N), 136.2 (Ar-C-O), 132.5 (C=CH), 130.7 (Ar-C), 130.6 (Ar-C), 127.4 (Ar-C), 125.4 (Ar-C), 123.0 (Ar-C), 117.0 (Ar-C), 116.4 (Ar-C), 98.0 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H11Cl2NO3 [M+2]+: 349.0116; found 349.0112.Yellowish solid; yield: 65.4 mg (94%), Rf  = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. 180–182°C; FT-IR  3452, 1755, 1625, 1581; 1H NMR  δ 7.99 , 7.06 , 7.00 , 6.96 , 6.88 – 6.86 , 3.88 , 2.34 ; 13C NMR  δ 190.4 (C=O), 163.4 (Ar-C-OCH3), 156.8 (O=C-O), 139.3 (Ar-C-CH3), 138.7 (Ar-C-N), 136.0 (Ar-C-O), 131.3 (C=CH), 129.9 (Ar-C), 124.4 (Ar-C), 123.6 (Ar-C), 116.8 (Ar-C), 116.0 (Ar-C), 114.0 (Ar-C), 94.5 (C=CH), 55.6 (OCH3), 21.1 (CH3); HRMS (ESI) calcd. for C18H15NO4 [M+H]+: 310.1001; found 310.1009.Yellowish solid; yield: 57.7 mg (93%), R−1) 3417, 1767, 1626, 1583, 1282, 1177; 1H NMR  δ 8.00 , 7.58-7.47 , 7.13 , 7.07 , 6.85 , 2.34 ; 13C NMR  δ 191.7 (C=O), 155.7 (O=C-O), 138.6 (Ar-C-N), 138.1 (Ar-C-O), 136.9 (C=CH), 136.6 (Ar-C-CH3), 132.9 (Ar-C), 128.9 (Ar-C), 128.2 (Ar-C), 127.8 (Ar-C), 124.7 (Ar-C), 115.5 (Ar-C), 110.2 (Ar-C-Br), 95.2 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H12BrNO3 [M+2]+: 359.0001; found 359.0008.Yellowish solid; Yield: 65.7 mg (92%); Rf  = 0.80; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 190–192°C; FT-IR  = 0.80; Purification of crude product was done by recrystalization using EtOAc/Hexane; m.p. 230–232°C; FT-IR  3417, 1771, 1631, 1285, 1159; 1H NMR  δ 8.03-7.99 , 7.17-7.13 , 6.99 , 6.84 , 2.34 ; 13C NMR  δ 190.1 (C=O), 167.0 (Ar-C-F), 164.5 (Ar-C-F), 155.6 (O=C-O), 138.7 (Ar-C-N), 136.9 (Ar-C-O), 136.6 (C=CH), 134.5 (Ar-C-CH3), 134.4 (Ar-C), 130.4 (Ar-C), 130.3 (Ar-C), 128.3 (Ar-C), 124.6 (Ar-C-Br), 116.1 (Ar-C), 115.9 (Ar-C), 115.5 (Ar-C), 110.2 (Ar-C), 94.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H11BrFNO3 [M+2]+: 376.9906; found 376.9909.Yellowish solid; Yield: 67.6 mg (90 %); Rf  = 0.85; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 224–226°C; FT-IR  3417, 1763, 1626, 1561, 1294, 1127; 1H NMR  δ 7.54 , 7.47 , 7.33 , 7.18 , 6.88 , 6.79 , 2.35 ; 13C NMR  δ 191.9 (C=O), 155.2 (O=C-O), 138.7 (Ar-C-Cl), 137.5 (Ar-C-N), 137.3(Ar-C-O), 137.0 (C=CH), 136.8 (Ar-C-CH3), 132.7 (Ar-C-Cl), 130.8 (Ar-C), 130.7 (Ar-C), 128.8 (Ar-C), 127.5 (Ar-C), 124.3 (Ar-C), 115.7 (Ar-C), 110.3 (Ar-C), 98.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H10BrCl2NO3 [M+2]+: 426.9221; found 426.9227.Yellowish solid; Yield: 79.7 mg (94%); Rf  = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (9.5:0.5) as an eluent; m.p. 259–260°C; FT-IR  3417, 1768, 1629, 1562, 1283, 1138; 1H NMR  δ 7.86 , 7.62 , 7.16 , 7.00 , 6.86 , 2.35 ; 13C NMR  δ 190.4 (C=O), 155.6 (O=C-O), 139.0 (Ar-C-N), 137.0 (Ar-C-O), 136.9 (C=CH), 136.7 (Ar-C-CH3), 132.2 (Ar-C), 129.3 (Ar-C), 128.5 (Ar-C), 128.1 (Ar-C), 124.5 (Ar-C), 115.6 (Ar-C), 110.3 (Ar-C), 94.8 (C=CH), 20.9 (CH3); HRMS (ESI) calcd. for C17H11Br2NO3 [M+2]+: 436.9106; found 436.9100.Yellowish solid; Yield: 77.6 mg (89%); Rf  = 0.90; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8.5:1.5) as an eluent; m.p. 219–220°C; FT-IR  3417, 1764, 1630, 1602, 1281, 1182; 1H NMR  δ 7.86 , 7.26–7.24 , 7.08 , 7.00 , 6.80 , 2.39 , 2.30 ; 13C NMR  δ 191.4 (C=O), 155.8 (O=C-O), 143.9 (Ar-C-CH3), 138.3 (Ar-C-N), 136.8 (Ar-C-O), 136.5 (C=CH), 135.6 (Ar-C-CH3), 129.6 (Ar-C), 128.0 (Ar-C), 127.9 (Ar-C), 124.8 (Ar-C), 115.4 (Ar-C), 110.1 (Ar-C), 95.3 (C=CH), 21.8 (CH3), 20.9 (CH3); HRMS (ESI) calcd. for C18H14BrNO3 [M+2]+: 373.0157; found 373.0152.Yellowish solid; Yield: 70.7 mg (95%); Rf  = 0.75; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 211–213°C; FT-IR  3446, 3072, 1758, 1621, 1515, 1183; 1H NMR  δ 8.32 , 8.13 , 7.12 , 7.01–6.96 , 2.37 ; 13C NMR  δ 188.8 (C=O), 155.9 (O=C-O), 149.9 (Ar-C-NO2), 143.3 (C=CH), 140.5 (Ar-C-N), 139.7 (Ar-C-O), 136.4 (Ar-C), 128.6 (Ar-C), 125.8 (Ar-C), 124.0 (Ar-C), 123.9 (Ar-C), 117.2 (Ar-C), 116.7 (Ar-C), 94.1 (C=CH), 21.1 (CH3); HRMS (ESI) calcd. for C17H12N2O5 [M+H]+: 325.0746; found 325.0748.Yellowish solid; yield: 57.2 mg (82%), Rf  = 0.75; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 207–209°C; FT-IR  3448, 3069, 1758, 1621, 1515; 1453; 1H NMR  δ 8.32 , 8.15 , 7.26 – 7.17 , 7.04 ; 13C NMR  δ 188.9 (C=O), 155.8 (O=C-O), 150.1 (Ar-C-NO2), 143.3 (Ar-C-N), 141.7 (C=CH), 140.5 (Ar-C-O), 128.7 (Ar-C), 126.2 (Ar-C), 125.1 (Ar-C), 124.1 (Ar-C), 123.4 (Ar-C), 117.5 (Ar-C), 116.6 (Ar-C), 94.3 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0596.Yellowish solid; yield: 52.2 mg (84%); Rf  = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 198–200°C; FT-IR  3436, 2928, 1762, 1625, 1581, 1142; 1H NMR  δ 8.03-7.96 , 7.61-7.49 , 7.31 , 7.15 ; 13C NMR  δ 192.1 (C=O), 155.1 (O=C-O), 145.2 (Ar-C-NO2), 144.9 (C=CH), 137.7 (Ar-C-N), 137.6 (Ar-C-O), 133.4 (Ar-C), 128.9 (Ar-C), 127.9 (Ar-C), 124.7 (Ar-C), 118.9 (Ar-C), 118.0 (Ar-C), 111.5 (Ar-C), 96.9 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0593.Yellowish solid; yield: 55.21 mg (89%); Rf  = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (8:2) as an eluent; m.p. > 250°C; FT-IR  3435, 3107, 1759,1622, 1594, 1156; 1H NMR  δ 8.73 , 8.11 , 7.92 , 7.44 – 7.36 , 6.92 ; 13C NMR (100 MHz) δ 188.7 (C=O), 166.6, (Ar-C-F), 156.0 (O=C-O), 145.9 (Ar-C-NO2), 144.6 (C=CH), 139.5 (Ar-C-N), 135.1 (Ar-C-O), 130.9 (Ar-C), 125.8 (Ar-C), 118.9 (Ar-C), 117.7 (Ar-C), 116.6 (Ar-C), 116.4 (Ar-C), 113.0 (Ar-C), 94.5 (C=CH); HRMS (ESI) calcd. for C16H9FN2O5 [M+H]+: 329.0495; found 329.0490.Yellowish solid; yield: 61.71 mg (94%); Rf  = 0.75; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7:3) as an eluent; m.p. 195–197°C; FT-IR  3435, 2926, 1599, 1758, 1633, 1594; 1H NMR  δ 8.02–7.93 , 7.29 , 7.10 , 6.98 , 3.89 ; 13C NMR  δ 190.7 (C=O), 164.0 (Ar-C-OCH3), 155.3 (O=C-O), 145.2 (Ar-C-NO2), 144.9 (Ar-C-N), 137.0 (Ar-C-O), 130.7 (C=CH), 130.3 (Ar-C), 124.9 (Ar-C), 118.6 (Ar-C), 117.8 (Ar-C), 114.2 (Ar-C), 111.2 (Ar-C), 97.0 (C=CH), 55.7 (OCH3); HRMS (ESI) calcd. for C17H12N2O6 [M+H]+: 341.0695; found 341.0692.Yellowish solid; yield: 58.51 mg (86%); Rf  = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 240–242°C; FT-IR  3436, 1763, 1622, 1596, 1268; 1H NMR  δ 8.06 – 8.00 , 7.83–7.81 , 7.62 , 7.54 , 6.99 ; 13C NMR  δ 190.7 (C=O), 156.1 (O=C-O), 142.2 (Ar-C-N), 141.1 (Ar-C-O), 139.2 (Ar-C-NO2), 138.3 (C=CH), 133.6 (Ar-C), 131.3 (Ar-C), 129.6 (Ar-C), 128.0 (Ar-C), 121.4 (Ar-C), 117.4 (Ar-C), 112.6 (Ar-C), 96.0 (C=CH); HRMS (ESI) calcd. for C16H10N2O5 [M+H]+: 311.0590; found 311.0595.Yellowish solid; yield: 55.22 mg (89%); Rf  = 0.70; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 235–237°C; FT-IR  3411, 3090, 1773, 1626, 1516, 1473; 1H NMR  δ 8.18 - 8.08 , 7.88 – 7.86 , 7.42–7.38 , 7.02 ; 13C NMR  188.7 (C=O), 163.7 (Ar-C-F), 155.5 (O=C-O), 141.8 (Ar-C-N), 140.6 (Ar-C-O), 138.7 (Ar-C-NO2), 134.4 (C=CH), 130.7 (Ar-C), 130.6 (Ar-C), 130.5 (Ar-C), 120.9 (Ar-C), 116.9 (Ar-C), 116.2 (Ar-C), 115.9 (Ar-C), 112.0 (Ar-C), 95.3 (C=CH); HRMS (ESI) calcd. for C16H9FN2O5 [M+H]+: 329.0495; found 329.0499.Yellowish solid; yield: 52.7 mg (80%); Rf  = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 205–207°C; FT-IR  3432, 2925, 2860, 1633, 1525, 1776, 1075; 1H NMR  δ 8.08 , 7.89-7.87 , 7.63 , 7.01 ; 13C NMR  δ 188.9 (C=O), 155.4 (O=C-O), 141.9 (Ar-C-N), 140.7 (Ar-C-Cl), 138.9 (Ar-C-O), 136.5 (Ar-C-NO2), 130.7 (C=CH), 129.8 (Ar-C), 129.5 (Ar-C), 129.2 (Ar-C), 120.9 (Ar-C), 117.0 (Ar-C), 112.1 (Ar-C), 95.2 (C=CH); HRMS (ESI) calcd. for C16H9ClN2O5 [M+H]+: 345.0200; found 345.0207.Yellowish solid; yield: 59.7 mg (87%); Rf  = 0.70; Purification of crude product was done by recrystalization using Hexane/ethyl acetate; m.p. 208–210°C; FT-IR  3433, 3088, 1770, 1620, 1522, 1470, 1072; 1H NMR  δ 12.39 , 8.09–8.08 , 7.95–7.93 , 7.78–7.58 , 6.62 ; 13C NMR  δ 190.3 (C=O), 155.3 (O=C-O), 142.2 (Ar-C-N), 140.8 (Ar-C-Cl), 138.5 (Ar-C-O), 137.7 (Ar-C-NO2), 136.2 (C=CH), 131.2 (Ar-C-Cl), 131.0 (Ar-C), 130.5 (Ar-C), 130.1 (Ar-C), 127.9 (Ar-C), 120.8 (Ar-C), 117.3 (Ar-C), 112.1 (Ar-C), 98.8 (C=CH); HRMS (ESI) calcd. for C16H8Cl2N2O5 [M+H]+: 378.9810; found 378.9818.Yellowish solid; yield: 64.6 mg (85%); Rf  = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7.5:2.5) as an eluent; m.p. 230–232°C; FT-IR  3435, 3093, 1769, 1619, 1521; 1H NMR  δ 8.09-7.96 , 7.83-7.44 , 7.01; 13C NMR  δ 188.9 (C=O), 154.7 (O=C-O), 141.8 (Ar-C-N), 140.1 (Ar-C-O), 137.9 (C=CH), 136.6 (Ar-C-NO2), 131.5 (Ar-C), 130.0 (Ar-C), 129.1 (Ar-C-Br), 126.6 (Ar-C), 120.7 (Ar-C), 116.5 (Ar-C), 111.4 (Ar-C), 95.7 (C=CH); HRMS (ESI) calcd. for C16H9BrN2O5 [M+2]+: 389.9695; found 389.9691.Yellowish solid; yield: 67.7 mg (87%); Rf  = 0.70; Purification of crude product was done by flash column chromatography method over silica gel using Hexane/ethyl acetate (7:3) as an eluent; m.p. 218–220°C; FT-IR  3437, 2927, 2854, 1632, 1517; 1H NMR  δ 8.07-8.02 , 7.73 , 7.09 , 7.02 , 3.89 ; 13C NMR  δ 189.1 (C=O), 163.4 (Ar-C-OCH3), 155.8 (O=C-O), 141.7 (Ar-C-N), 140.6 (Ar-C-O), 138.1 (C=CH), 131.1 (Ar-C-NO2), 130.7 (Ar-C), 130.1 (Ar-C), 121.2 (Ar-C), 116.8 (Ar-C), 114.5 (Ar-C), 112.2 (Ar-C), 95.9 (C=CH), 55.8 (OCH3); HRMS (ESI) calcd. for C17H12N2O6 [M+H]+: 341.0695; found 341.0699.Yellowish solid; yield: 55.4 mg (81%); R−1 was mixed with 1.5 mL of a DPPH methanolic solution (20 mg L−1). Pure methanol was taken as control and ascorbic acid (vitamin C), vitamins A and E were used as a reference compounds. The absorbance was measured at 517 nm after 20 min of reaction. The % of DPPH decolouration of the sample was calculated according to the formula  at different concentrations ranging from 10 to 100 μg mL50. The results are expressed as antiradical efficiency (AE), which is 1000-fold inverse of the IC50 value AE=1000/ IC50.The decolouration was plotted against the sample extract concentration and a logarithmic regression curve was established in order to calculate the IC3.6H2O solution, 10.0 mM of ferric-tripyridyltriazine (TPTZ) solution and 300 mM sodium acetate buffer (pH 3.6) in a ratio of 1:1:10 (v/v/v). After that, Sample  was added to 3 ml of freshly prepared FRAP reagent and this reaction mixture was incubated at 37°C temperature for 30 min. and the absorbance was measured at 593 nm. It is also noted that, a freshly prepared solution of FeSO4 was used for calibration of standard curve. The FRAP antioxidant capability were evaluated in terms of C0.5FRAP  . Drawing of structures and simple geometry optimization were performed with Chem Bio-Office suite Ultra v12.0 (2012) . Docking of all compounds was carried out on the human antioxidant enzyme in complex (PDB ID: 3MNG)  were taken from the Protein Data Bank (http://www.rcsb.org/pdb). Program automatically docks ligand into binding pocket of a target protein by using protomol-based algorithm and empirically produced scoring function. The protomol is very important and necessary factor for docking algorithm and works as a computational representation of proposed ligand that interacts into binding site. Surflex-Dock's scoring function have several factors that play an important role in the ligand-receptor interaction, in terms of hydrophobic, polar, repulsive, entropic and solvation, and it is a worldwide well-established and recognized method. The most standard docking protocols have ligand flexibility into the docking process, while counts the protein as a rigid structure. Present molecular docking study involves the several steps viz., import of protein structure into Surflex and addition of hydrogen atoms; generation of protomol using a ligand-based strategy. During second step, two parameters first called protomol_bloat, which determines how far the site should extend from a potential ligand; and another called protomol_threshold, which determines deepness of the atomic probes, used to define the protomol penetration into the protein) were specified to form the appropriate binding pocket. Therefore, protomol_bloat and protomol_threshold was set to 0 and 0.50, respectively. In reasonable binding pocket, all the compounds were docked into the binding pocket and 20 possible active docking conformations with different scores were obtained for each compound. During the docking process, all of the other parameters were assigned their default values.To find the possible bioactive conformations of C-3 tethered 2-oxo-benzooxazine derivatives, molecular modeling studies were performed using the Sybyl X 2.0 interfaced for the synthesized compounds, which exhibited promising and lower antioxidant activity 20a-20ab using our reported procedure  solvent. The reaction of nitro/alkyl/halide-substituted 2, 4-dioxo-4-phenylbutanoic acid 18a-h with nitro/alkyl/halide-substituted 2-aminophenol 19a-f in water furnished C-3 tethered 2-oxo-benzooxazines 20a-20ab in 74-98% yields after purification either by flash column chromatography or by recrystallization method  by all the compounds 20a-20ab. The DPPH radical scavenging assay is generally utilized as a quick and reliable parameter to investigate the antioxidant activities of diverse heterocycles  mechanism and the second one is the single electron transfer (SET) mechanism  and/or electron-donating group (EDG) either at ring A or at C. The active group -CO-C=C-NH- enables resonance between the ring B and C, leading to multiple resonance structure, which may be further initiated by the attached substituents of ring A and C and in situ enhances the radical scavenging activity through the removal of hydrogen atom from NH of ring B via HAT mechanism. It has been found that electron withdrawing substituent NO2 at ring A or C increase the antioxidant activity which may be due to resonance based stabilizing effects. Therefore, based on the structures and their antioxidant activities, it was found that the compounds have either no substitution at ring A and C or have EWG/EDG at ring A and C plays a very important role in deciding their DPPH radical scavenging activities. Hence, based on the substituent's (either EWG or EDG) at ring A and C of 2-oxo-benzooxazines 20a-20ab, and their antioxidant activities, their structure-activity relationship can be explained by grouping all compounds into two groups:The generic scaffold of the newly synthesized C-3 tethered 2-oxo-benzo1,4]oxazines, as illustrated in Figure oxazines,No substitution or EDG at ring A or ring C: In the first group of compounds having no substitution at ring A and C i.e., the model compound 20a, exhibited promising antioxidant activity (IC50 = 10.20 ± 0.08 μg/mL) in comparison with standard reference Ascorbic acid (IC50 = 4.57 μg/mL) [entry 1]. Then, by putting EDG (OMe group) at ring C, as in compound 20b, further increases activity (IC50 = 6.89 ± 0.07 μg/mL) [entry 2]. Reversing the order i.e., halogen substitution at ring A and no substitution at ring C do not cause any further increase in antioxidant activity as shown by compound 20c (entry 3). Furthermore, when we incorporated halogen substituents  or CH3 substituent either at A or C as in the case of compounds 20c-l; a decrease in the antioxidant activity was observed due to high electron density in compounds 20d-g, 20j and 20k (entry 4-12). Furthermore, It was observed that two EDG at ring A  exhibited moderate antioxidant activity having IC50 value in the range of 18.86 ± 0.72 to 44.32 ± 0.45 μg/mL. In this series , ring C having Fluorine substituent i.e. compound 20n exhibited good antioxidant activity (IC50 = 16.86 ± 0.72 μg/mL) in comparison with other compounds (20m and 20o-q).2 group at ring C and EDG group i.e., CH3 group at ring A (compound 20r); the antioxidant activity was regained and shows IC50 value of 12.23 ± 0.05 μg/mL nearly equivalent to 20a (entry 18).To our surprise; when we incorporated EWG group i.e., NO20s , which, contrary to our expectations, displayed further decrease in antioxidant activity (IC50 = 21.27 ± 0.28 μg/mL) (entry 19).It is to be noted that EDG at ring A decreases antioxidant activity as shown in entries 3-17; so, we synthesized compound EWG (-NO2Group) at ring A: Since 20r having EWG (NO2) at ring C showed promising antioxidant activity; inspired by this observation, we prepared 20t-v having NO2 group at C-4 position of ring A. 2-oxo-benzooxazine 20t having no substitution at ring C, showed excellent antioxidant activity having IC50 value of 4.74 ± 0.08 μg/mL (entry 20). Since 20b having OMe substituent at ring C, was also found to show excellent antioxidant activity; thus, we synthesized 20u and 20v having Fluoro as well as OMe substituent, respectively at ring C. Unfortunately, antioxidant activity diminishes (entry 21 and 22). In addition, we also prepared 2-oxo-benzooxazines 20w-20ab having EWG group (NO2) at C-5 position of ring A further to investigate SAR study. Compound 20w having no substitution at ring C showed promising antioxidant activity having IC50 value of 12.53 ± 0.09 μg/mL (entry 23). On incorporating Fluoro group at ring C; activity of 20x increases . Furthermore, when we incorporated halogen substituents  at ring C as in the case of compounds 20y-20ab; a decrease in the antioxidant activity was observed (entry 19-23).20a-20ab. Whereas EDG either at ring A or C diminishes antioxidant activity. Our SAR results depict that 20b and 20t, the best compounds of the series, showed antioxidant activity comparable to standard reference Ascorbic acid.Overall, we can interpret that no substitution or EWG at ring A or ring C enhances antioxidant activity of all the synthesized 2-oxo-benzooxazines 3+−TPTZ] and develops a colored ferrous tripyridyltriazine [Fe2+-TPTZ]. The reducing nature of an antioxidant depends on their property to donate a hydrogen atom for the breaking of the free radical chain, which is responsible for oxidative stress etc.The FRAP assay was deliberated using the method as illustrated by Benzie and Strain  were found more potent than BHT (C0.5FRAP = 546.0 ± 13.6 μM).All the synthesized C-3 tethered 2-oxo-benzo1,4]oxazine analogs ,4oxazine20a-20ab) displayed good to moderate activity in comparison with BHT in the range of C0.5FRAP = 328.6 ± 25.8 μM to 916.8 ± 21.4 μM in in-vitro antioxidant FRAP assay except compounds 20e, 20f, 20l, 20q, 20v, and 20ab which showed C0.5FRAP greater than 1000 μM. Compounds having EWG i.e. NO2 substituent at ring C (20r and 20s) showed different potency than standard reference BHT. While 20r having CH3 at C-4 position of ring A displayed potent activity than BHT; 20s was found to be less active. Anomaly was observed in the case of compounds having no substitution at ring C. While 20c and 20m showed greater potency; compounds 20a, 20t and 20w were found less active than BHT.In summary, all the compounds . Since 20b has OMe group at ring C and 20t has NO2 group at ring A; it is worthwhile to compare the docking studies of active compounds with molecules having no substitution either at ring A or C. Therefore, we have also selected compound 20a for our in silico molecular docking simulation studies. For that purpose, Peroxiredoxins (Prdxs), a family of small human antioxidant enzyme, was selected as our target protein. Peroxiredoxins contain essential cysteine residues as catalyst and thioredoxin as an electron donor, which help in scavenging peroxide and are involved in the metabolic cellular response to ROS  .Compounds were docked into the active site of the known the human antioxidant enzyme target peroxiredoxins (Prxs) DTT complex (PDB ID: 3MNG) were taken from the Protein Data Bank  between the ligand and amino acids in the binding site of the protein were used for the ranking of compounds. The mode of interaction of the co-crystallized ligand dithiothreitol (DTT) within the crystal structure of enzyme in complex was used as a reference binding model. The root mean-square deviation (RMSD) of each docking pose was compared to the co-crystallized ligand and used for ranking and for RMSD calculation. The co-crystallized DTT molecule was re-docked onto the same binding site and the most probable binding mode was selected as that with the highest docking total score of 4.8921. An RMSD value 0.6772Å between the predicted and crystal binding mode indicates the high reliability of Surflex-Dock for this protein target.20a, 20b, and 20t against the antioxidant target protein Prxs showed a high binding affinity docking score indicated by a total score of 3.8470  of length 1.8Å to the backbone of hydrophobic aliphatic residue that is, Glycine-46. In the docking pose of the 20a, 20b, and 120t and Prxs complex, the chemical nature of binding site residues within a radius of 3Å with diverse properties was aromatic (hydrophobic), for example, Phe-120, ; hydrophobic, for example, Leu-116, Ile-119, Leu-149, Leu-112(Leucine), Gly-46, Gly-148(Glycine);  residues, for example, Arg-127 (Arginine); nucleophilic , for example, Thr-147 and Thr-44 (Threonine), nucleophilic (polar uncharged), for example Cys-47 (Cysteine); and hydrophobic  residues, for example, Pro-40 and Pro-45 (Proline) as a result, the bound compound showed a strong hydrophobic interaction with Prxs, thus leading to more stability and activity in this compound.On the other hand, docking results for 0 Figure , 3.6567 0 Figure  and 4.270 Figure  forms a The docking results for the ascorbic acid (standard compound) with the antioxidant target protein Prxs showed a low binding affinity docking score, indicated by a low total score of 3.4829 with three H-bond (hydrogen bond) formation of length 2.2, 1.8 and 2.1Å to the Thr-147, Gly-46 and Thr44 Figure . The asc20b and 20t having IC50 value of 6.89 ± 0.07 μg/mL and 4.74 ± 0.08 μg/mL, showed the binding affinity docking score of 3.6567 and 4.2709, respectively  i.e., 3.8470 . Whereas in FRAP assay, eight compounds  were found more potent than BHT (C0.5FRAP = 546.0 ± 13.6 μM). The active compounds were also found non-toxic in 3T3 fibroblast cell lines in MTT assay. Our in silico molecular docking results reveal that 20b and 20t showed excellent docking total scores against human antioxidant enzyme target as compared to ascorbic acid. Thus, the in silico docking simulation studies effectively validated the in vitro experimental results.In summary, we disclose C-3 tethered 2-oxo-benzo oxazine analogs in vitro activity evaluation of synthesized compounds. DKY and Saloni performed docking studies. SM and MHK provided molecular modeling facility. SC and PKJ drafted the manuscript. All authors read and approved the final manuscript.SC was responsible for the study of concept and design of the project. VS and PKJ were responsible for the performing synthetic reactions, acquisition and analysis of data. MS, MM, and AKS were responsible for the pharmacological The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The new structures were elucidated through extensive spectroscopic analyses and by comparison with previously reported data. Some of the isolates were evaluated for their cytotoxic activities against MCF-7 cell line by an MTT assay, and compounds 15, 16, 19, and 23–25 exhibited inhibitory activities against MCF-7 with IC50 values ranging from 7.1 to 31.3 μM.Six new triterpenoid saponins, aesculusosides A–F (The online version of this article (10.1007/s13659-017-0148-4) contains supplementary material, which is available to authorized users. Hippocastanaceae is known to be a rich source of escins, a group of structurally diverse natural products characterized by a pentacyclic triterpenoid framework combined with a oligoglycoside chain . Modern Aesculus chinensis Bge. (Hippocastanaceae), abundant in the northwestern China, is a medicinal plant and its dried ripe seeds have been used as a stomachic and analgesic in the treatment of ditension and pain in the chest and abdomen [β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid.Compound skeleton . In addiluside-B , except -3), b C-/C-16, c 2, obtained as an amorphous powder, was assigned to have a molecular formula of C52H82O24 by negative-ion HR-ESI–MS (m/z 1089.5107 ([M-H]−), calc. 1089.5123). The 1H and 13C NMR data of 2 /C-1″″ (δC 173.5). The downfield shift of H-22 further supported the above conclusion. Therefore, 2 was established as 22,28-O-diacetyl-protoaescigenin 3-O-[β-d-glucopyranosyl-(1→2)][β-d-glucopyranosyl-(1→4)]-β-d-glucuronopyranosyl acid.Compound 3 was determined to have the same molecular formula of C50H80O23 as 1 by its negative-ion HR-ESI–MS (m/z 1047.5005 ([M−H]−), calc. 1047.5018). The characteristic NMR data suggested that 3 had the similar structure as 1. The significant differences in 1H and 13C-NMR spectra from those of 1 and 3 were chemical shifts of C-21(δC 82.8) and C-22 (δC 73.9) with corresponding protons at δH 5.50 and 3.97. The downfield shifts of C-21 implied that the acetyl group at the C-22 in 1 was transferred to C-21 in 3, which was further confirmed by the key HMBC cross-peak of H-21(δH 5.50)/C-1″″ (δC 174.0). In addition, the alkaline hydrolysis of 3 yielded aesculuside-B [3 was determined as 21-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid.Compound luside-B . Hence, 4 showed a [M−H]− ion at m/z 1117.5425 , corresponding to a molecular formula of C54H86O24. The similar NMR data between 4 and 2 indicated that they are very close in the structure. Furthermore, the 1H-NMR spectrum of 4 displayed one methine proton (δH 2.62) and two doublet methyl protons (δH 1.18 and 1.19), with the corresponding carbon resonances at δC 35.7, 19.5 and 19.9 in its 13C-NMR spectrum. An isopropyl group was easily assembled by 1H-1H COSY correlations of H-2″″ (δH 2.62) with H-3″″ (δH 1.18) and H-4″″ (δH 1.19). The aforementioned data, together with the key HMBC cross-peaks of H-22 (δH 5.23)/C-1″″ (δC 179.3), H-2″″ (δH 2.62)/C-1″″ (δC 179.3), H-3″″ (δH 1.18)/C-1″″ (δC 179.3) and H-4″″ (δH 1.19)/C-1″″ (δC 179.3), suggested that the isopropyl group in 4 was installed at the C-1″″. Based on the above evidence, compound 4 was characterized as 22-O-isobutyryl-28-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid.The HR-ESI–MS data of 5 was isolated as an amorphous powder. Its molecular formula was determined by negative HR-ESI–MS [m/z 1075.5406 ] as C52H84O23, requiring 11 degrees of unsaturation. Careful comparison of the 1H and 13C-NMR spectra of 5 with those of 4 indicated a high degree of similarity. The differences between 5 and 4 were the absence of an acetic group at the C-28 and that the isopropyl group was transferred to C-21 other than C-22 in compound 4, as confirmed by the combinational analysis of 2D NMR data ][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid.Compound 6 was obtained as an amorphous powder and gave a molecular formula of C52H82O23, as determined by HR-ESI–MS at m/z 1073.5162 . The 1H-NMR spectrum of 6 , 0.86 , 0.93 , 0.97 , 1.00 , 1.09 , 1.44 , 2.06 , 2.09 ] and one olefinic proton (δH 5.31).The above data, together with its 13C-NMR data ][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid.Compound O-tigloylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Escin Ivg] (7) [O-diacetylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Escin Iv] (8) [O-angeloylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Escin Ivh] (9) [O-diacetylprotoaescigenin 3-O-[β-d-glucopyranosyl (1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Aesculiside A] (10) [O-tigloyl-28-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Escin IVc] (11) [O-angeloyl-28-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl (1→2)] [β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Escin IVd] (12) [O-tigloylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Aesculioside A] (13) [O-angeloylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Aesculioside B] (14) [O-isobutyryl-22-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Escin V] (15) [O-tigloyl-22-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl (1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid (Escin Ia) (16) [O-isobutyryl-28-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl(1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Isoescin V] (17) [O-angeloyl-22-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl (1→2)] [β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid (Escin Ib) (18) [O-isovaleryl-22-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl (1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Esvin VI] (19) [O-tigloyl-28-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl (1→2)] [β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid (isoescin Ia) (20) [O-angeloyl-28-O-acetylprotoaescigenin 3-O-[β-d-glucopyranosyl (1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid (isoescin Ib) (21) [O-tigloyl-22-O-acetylprotoaescigenin 3-O-[β-d-galactopyranosyl(1→2)] [β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid (22) [O-tigloyl-22-O-acetylbarringtogenol C 3-O-[β-d-glucopyranosyl (1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Escin IIIa] (23) [O-tigloyl-28-O-acetylbarringtogenol C 3-O-[β-d-glucopyranosyl (1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Isoescin IIIa] (24) [O-angeloyl-28-O-acetylbarringtogenol C 3-O-[β-d-glucopyranosyl (1→2)][β-d-glucopyranosyl(1→4)]-β-d-glucopyranosiduronic acid [Isoescin IIIb] (25) [The 19 known compounds were identified as 22-Ivg] (7) , 21,22-O Iv] (8) , 22-O-anVc] 11) , 22-O-anVd] (12) , 21-O-tioside A]  13], 21, 21O-tig V] (15) , 21-O-tiIa) (16) , 21-O-is V] (17) , 21-O-anscin Ib)  [O-isovascin Ia)  20], 21, 21O-tig , 22-O-a7-9, 12-17, 19, and 22–25 were evaluated for their in vitro cytotoxicities against human cancer cell line (MCF-7) using the MTT method as reported previously D25 − 2.5 ; IR (KBr) 3423, 2951, 1721, 1380, 1261, 1031 cm−1; 1H and 13C NMR data: see Tables m/z 1047.5035 ([M−H]−), calc. 1047.5018).Amorphous powder; D25 − 3.5 ; IR (KBr) 3421, 2945, 1725, 1384, 1272, 1077 cm−1; 1H and 13C NMR data: see Tables m/z 1089.5107 ([M−H]−), calc. 1089.5123).Amorphous powder; D25 − 34.5 ; IR (KBr) 3422, 2928, 1720, 1383, 1273, 1076 cm−1; 1H and 13C NMR data: see Tables m/z 1047.5005 ([M−H]−), calc. 1047.5018).Amorphous powder; D25 − 32.1 ; IR (KBr) 3421, 2946, 1723, 1383, 1277, 1074 cm−1; 1H and 13C NMR data: see Tables m/z 1117.5425 ([M−H]−), .Amorphous powder; [α]D25 − 2.5 ;) IR (KBr) 3423, 2930, 1721, 1375, 1277, 1075 cm−1; 1H and 13C NMR data: see Tables m/z 1075.5406 ([M−H]−), .Amorphous powder; D25 − 2.8 ; IR (KBr) 3423, 2929, 1721, 1384, 1273, 1075 cm−1; 1H and 13C NMR data: see Tables −), .Amorphous powder; [ 2.8 c 0., MeOH; I2. Briefly, cells were seeded into each well of a 96-well cell culture plate. After 12 h of incubation at 37 °C, the test compound (40 μM) was added. After incubation for 48 h, cells were subjected to the MTT assay. Compounds with a growth inhibition rate of 50% were further evaluated with cis-platin  as positive control.Compounds were tested in vitro for their cytotoxicities against proliferation of MCF-7 (breast cancer) using the MTT method . The hum1 (4 mg) in H2O (1 mL) was treated with 20% aqueous H2SO4 (1 mL), and the mixture was heated under reflux for 2 h. It was then neutralized by saturated NaHCO3 and extracted three times with EtOAc. Glucose and glucuronic acid were obtained from the H2O layer, and identified by comparison with authentic samples and by PC (Paper Chromatography) behavior, solvent: CHCl3-MeOH-H2O, 10:6:1.A solution of compound 1 (5 mg) was added to a MeOH solution (5 ml) of NaOMe (1 mg). The mixture was stirred at room temperature for 4 h and then neutralized with 20% aqueous HCl. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (CH2Cl2/MeOH 2:1–1:1) to furnish aesculuside-B , which was identical with authentic sample by TLC, 1H- and 13C-NMR spectra comparisons [Compound parisons .Supplementary material 1 (DOCX 2192 kb)Below is the link to the electronic supplementary material.


O-β-d-glucopyranoside) is known for strong chemoprotectant activities. Acylation of flavonoid glucosides with carboxylic acids containing an aromatic ring brings entirely new properties to these compounds. Here, we describe the chemical and enzymatic synthesis of a series of IQ derivatives at the C-6″. IQ benzoate, phenylacetate, phenylpropanoate and cinnamate were prepared from respective vinyl esters using Novozym 435 (Lipase B from Candida antarctica immobilized on acrylic resin). The enzymatic procedure gave no products with “hydroxyaromatic” acids, their vinyl esters nor with their benzyl-protected forms. A chemical protection/deprotection method using Steglich reaction yielded IQ 4-hydroxybenzoate, vanillate and gallate. In case of p-coumaric, caffeic, and ferulic acid, the deprotection lead to the saturation of the double bonds at the phenylpropanoic moiety and yielded 4-hydroxy-, 3,4-dihydroxy- and 3-methoxy-4-hydroxy-phenylpropanoates. Reducing capacity of the cinnamate, gallate and 4-hydroxyphenylpropanoate towards Folin-Ciocalteau reagent was significantly lower than that of IQ, while other derivatives displayed slightly better or comparable capacity. Compared to isoquercitrin, most derivatives were less active in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, but they showed significantly better 2,2′-azinobis- scavenging activity and were substantially more active in the inhibition of tert-butylhydroperoxide induced lipid peroxidation of rat liver microsomes. The most active compounds were the hydroxyphenylpropanoates.Isoquercitrin, (IQ, quercetin-3- For 1H and 13C NMR data see m/z: [M − H]− calcd for C29H25O13 581.1; found 581.1 -6--5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3-phenylpropanoate : Yellow solid . For 1H and 13C NMR data see m/z: [M − H]− calcd for C30H27O13 595.1; found 595.1 -6--5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl cinnamate : Yellow solid . For 1H and 13C NMR data see m/z: [M − H]− calcd for C30H25O13 593.1; found: 593.1 -6-(((tert-Butyldimethylsilyl)oxy)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-2--5,7-dihydroxy-4H-chromen-4-one (32): To a stirred solution of 1  and imidazole  in dry DMF (20 mL) at 0 °C and under Ar, a solution of TBDMSCl  in DMF (20 mL) was added dropwise. After stirring at RT for 17 h the reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (3 × 300 mL). Combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography using gradient (EtOAc/MeOH from 50:1 to 25:1) to produce 32 as yellow oil . For 1H and 13C NMR data see m/z: [M − H]− calcd for C27H33O12Si 577.2; found 577.2.5,7-bis(Benzyloxy)-2-phenyl)-3--3,4,5-tris(benzyloxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-4-one (33): To an argon-purged solution of 32  and NaH  in dry DMF (80 mL) at 0 °C, a solution of BnBr  was added dropwise. The solution was stirred at RT for 24 h and then extracted with EtOAc (3 × 300 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (toluene/EtOAc = 20:1) to produce title compound as a brownish oil . For 1H and 13C NMR data see m/z: [M + H]+ calcd for C76H77O12Si 1209.5; found 1209.5.5,7-bis(Benzyloxy)-2-phenyl)-3--3,4,5-tris(benzyloxy)-6-(hydroxyme-thyl)tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-4-one (34): To an argon-purged flask containing a solution of 33  in anhydrous THF (30 mL) at 0 °C a solution of tetrabutylammonium fluoride in THF  was added dropwise. Reaction mixture was stirred under an Ar atmosphere at 0 °C for 30 min and then at RT for next 17 h. The reaction mixture was diluted with H2O (100 mL) and then extracted with EtOAc (3 × 300 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (toluene/EtOAc = 10:1) to yield yellow oil . For 1H and 13C NMR data see m/z: [M + H]+ calcd for C70H63O12 1095.4; found 1095.4.2Cl2 (10 mL) was added to a flask containing 34 , benzyl-protected acids 26–31 , DCC  and DMAP  and the reaction mixture was stirred at RT for 48 h. Then solvent was evaporated in vacuo and the residue was purified by column chromatography using toluene/EtOAc (99:1) and products 35–40 were isolated.Anhydrous CH-3,4,5-tris(Benzyloxy)-6--2-phenyl)-4-oxo-4H-chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 4-(benzyloxy)benzoate (35): Yellow solid . For 1H and 13C NMR data see m/z: [M + H]+ calcd for C84H73O14 1305.5; found 1305.7.-3,4,5-tris(Benzyloxy)-6--2-phenyl)-4-oxo-4H-chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 4-(benzyloxy)-3-methoxybenzoate (36): Yellow solid . For 1H and 13C NMR data see m/z: [M + H]+ calcd for C85H75O15 1335.5; found 1335.8.-3,4,5-tris(Benzyloxy)-6--2-phenyl)-4-oxo-4H-chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3,4,5-tris(benzyloxy)benzoate (37): Yellow solid . For 1H and 13C NMR data see m/z: [M + H]+ calcd for C98H85O16 1517.6; found 1517.9.(E)--3,4,5-tris(Benzyloxy)-6--2-phenyl)-4-oxo-4H-chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3-(4-(benzyloxy)phenyl)acrylate (38): Yellow solid . For 1H and 13C NMR data see m/z: [M + Na]+ calcd for C86H74O14Na 1353.5; found 1353.7.(E)--3,4,5-tris(Benzyloxy)-6--2-phenyl)-4-oxo-4H-chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3-phenyl)acrylate (39): Yellow solid . For 1H and 13C NMR data see m/z: [M + H]+ calcd for C93H81O15 1437.6; found 1437.7.(E)--3,4,5-tris(Benzyloxy)-6--2-phenyl)-4-oxo-4H-chromen-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3-(4-(benzyloxy)-3-methoxyphenyl)acrylate (40): Yellow solid . For 1H and 13C NMR data see m/z: [M + H]+ calcd for C87H77O15 1362.5; found 1361.7.35–40 (200 mg) was performed with Pd/C-H2  in EtOH/THF 1:1 (40 mL) and was stirred for 12 h. Then reaction mixtures were filtrated and evaporated in vacuo to yield 6–11.Hydrogenolysis of -6--5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4-hydroxybenzoate : Yellow solid . For 1H and 13C NMR data see m/z: [M − H]− calcd for C28H23O14 583.1; found 583.1 -6--5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4-hydroxy-3-methoxybenzoate : Yellow solid . For 1H and 13C NMR data see m/z: [M − H]− calcd for C29H25O15 613.1; found 613.1 -6--5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3,4,5-trihydroxybenzoate : Yellow solid . For 1H and 13C NMR data see m/z: [M − H]− calcd for C28H23O16 615.1; found 615.1 -6--5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3-(4-hydroxyphenyl)propanoate : Yellow solid . For 1H and 13C NMR data see m/z: [M − H]− calcd for C30H27O14 611.1; found 611.1 -6--5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3-propanoate : Yellow solid . For 1H and 13C NMR data see m/z: [M − H]− calcd for C30H27O15 627.1; found 627.2 -6--5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 3-(4-hydroxy-3-methoxyphenyl)propanoate : Yellow solid . For 1H and 13C NMR data see m/z: [M − H]− calcd for C31H29O15 641.2; found 641.0 . Differences were considered statistically significant when 2), phenylacetate (3), phenylpropanoate (4), cinnamate (5), 4-OH benzoate (6), vanillate (7), gallate (8), 4-OHPh propanoate (9), 3,4-diOHPh propanoate (10), and 4-OH-3-OMePh propanoate (11). The FCR reducing and DPPH scavenging capacity of most derivatives was lower or similar to IQ. In contrast, they mostly showed significantly better ABTS scavenging activity—the compounds 2, 7, 9, 10 and 11 displaying the strongest activity. Almost all derivatives were much more efficient inhibitors of microsomal lipid peroxidation. Compounds 8 and 3 (IC50 ≈ 7 µM) were the most active, but strong activity was noted also for phenylpropanoates 9–11 (IC50 ≈ 9.4 µM), which proved to be the most active compounds from the whole series.The chemoenzymatic and chemical approach have been used for the esterification at C-6″ of IQ with acyls containing an aromatic ring. First, the lipase catalyzed preparation of the derivatives was accomplished with Novozym 435 and the vinyl esters of benzoic, phenyl acetic, phenylpropanoic and cinnamic acids, but “hydroxyaromatic” acids or their vinyl esters were not substrates for the lipase. The reaction of protected “hydroxyaromatic” acids with selectively protected IQ lead to the fully protected C-6″ esterified products. The deprotection of the benzyl groups lead to the parallel saturation of the double bond yielding respective hydroxyphenylpropanoic derivatives. Several deprotection methods were attempted to overcome this problem but they proved unsuccessful. We have therefore obtained a panel of following IQ derivatives: IQ benzoate (


H-pyrazole-4-carbaldehyde has been synthesized using a microwave-assisted reaction.The new compound 5-butylamino-3-methyl-1-(pyridin-2-yl)-1 14H18N4O, synthesized from an unconventional microwave-assisted method using caesium carbonate as catalyst, has an approximately planar conformation with the pyridyl and pyrazole rings inclined by a dihedral angle of 7.94 (3)°, allowing the formation of an intramolecular N—H⋯N hydrogen bond. The supramolecular assembly has a three-dimensional arrangement controlled mainly by weak C—H⋯O and C—H⋯π interactions.The title compound, C This structural feature is also observed in its analog 5-cyclohexylamino-3-methyl-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde, which even shows a smaller dihedral angle between the pyridyl and pyrazole rings , see Fig. 2B⋯Cg1ii  and C53—H53A⋯Cg2iii  interactions, which help to define a three-dimensional array.In the crystal structure, C15—H15⋯O41et al., 2016H-pyrazole nucleus with the possibility of any group bonded to C3, C4 or C5 gave 12 hits of which 10 correspond to organometallic compounds, one to 2-phenyl)pyrazol-1-yl)pyridine and the last to 2,6-bis(pyrazolyl)pyridine. Any other search considering the presence of the butylamino or carbaldehyde groups gave no hits. However, two related compounds 5-cyclohexylamino-3-methyl-1-(pyridin-2-yl)-1H-pyrazole-4-carbaldehyde and (Z)-4-[(cyclohexylamino)methylidene]-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one have been published recently  in Fig. 3H-pyrazole-4-carbaldehyde [(III) in Fig. 3M + H]+ calculated for C14H19N4O+ 259.1553, found 259.1546. Yield 0.109 g, 94%; m.p. 348–350 K; IR νmax (KBr): 3448, 3211, 3096, 2924, 2858, 1643, 1596, 1563, 1436, 1002 cm−1; 1H NMR (CDCl3): 0.95 , 1.44 , 1.68 , 2.44 , 3.60 , 7.10 , 7.78 , 7.93 , 8.28 , 9.82 ; 13C NMR (CDCl3): 13.7 (CH3), 14.5 (CH3), 19.9 (CH2), 32.0 (CH2), 46.4 (CH2), 106.6 (C), 114.0 (CH), 119.8 (CH), 138.8 (CH), 145.8 (CH), 152.8 (C), 153.0 (C), 154.3 (C), 182.0 (CH); MS (EI) m/z 258 , 215 (67), 187 (59), 134 (32), 93 (47), 78 (76), 51 (24), 32 (100); HRMS m/z (ESI) calculated for [C14H18N4O+H]+: 259.1553; found 259.1546 [(M + H)+].All reactive and solvents, including caesium carbonate , were purchased from commercial sources and used as received. A mixture of 5-chloro-3-methyl-1-(pyridin-2-yl)-1Ueq value of the parent atom. H atoms belonging to NH groups were located in difference density maps and were freely refined.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016017187/bg2597sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016017187/bg2597Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016017187/bg2597Isup3.cmlSupporting information file. DOI: 1511522CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Transcription factor NF-κB and relevant cytokines IL-6 and IL-8 play a pivotal role in the pathogenesis of inflammation. Sinapic acid is a natural product and was demonstrated to possess anti-inflammatory activity. In this paper, we synthesized a series of sinapic acid derivatives and evaluated their anti-inflammatory effects. The result suggested that all of the targets compounds 7a-j inhibit NF-κB activation and decrease IL-6 and IL-8 expression in BEAS-2B cells. By our biological assays, we found that all of the prepared compounds displayed stronger anti-inflammatory activities than their precursor sinapic acid. Especially, compounds 7g and 7i, with electron-drawing groups (nitro and fluoro moieties) in the benzimidazole ring, exhibited remarkable anti-inflammation activity, which was even stronger than the reference drug dexamethasone.  Inflammation, characterized by redness, swelling, heat and pain, is close to many diseases such as cancer , arthritBrassica alba (L.) Boiss and Brassica juncea (L.) coss. Pharmacological investigations have revealed sinapic acid can attenuate the carbon tetrachloride-induced acute hepatic injury  was obtained from PeproTech . Dexamethasone was purchased from Sigma Chemical Co. . The medium and reagents for cells culture were supplied by Life Technologies/Gibco, Thermo Fisher Scientific Inc. . The luciferase reporter assay system was purchased from Promega Co. . The lipofectamine 2000 transfection reagent was obtained from Invitrogen life technologies, Thermo Fisher Scientific Inc. . Enzyme-linked immunosorbent assay kits (ELISA) were from Thermo Scientific Pierce Protein Biology Products, Thermo Fisher Scientific Inc. . The other solvents employed in this study were analytical purity grade.-coated silica gel plates. 1H NMR spectra were recorded on a Bruker AV 300 spectrometer using TMS as an internal standard. The chemical shifts were reported in parts per million (ppm), the signals were described as singlet (s), doublet (d), triplet (t), as well as multiplet (m). The high-resolution mass spectra (HRMS) were recorded on an IonSpec FT-ICR mass spectrometer with ESI resource. All other chemicals and solvents were commercially available, and were used without further purification.TLC analysis was done using preCell culture2 atmosphere at 37 °C.BEAS-2B cells, derived from human bronchial epithelial cells, were obtained from American type culture collection  and cultured using DMEM/F12 medium with 10% fetal bovine serum in 96-well plates and placed in a humid, 5% COTransfectionBEAS-2B cells were co-transfected with NF-κB luciferase reporter plasmid pGL4.32 and Renilla luciferase reporter vector plasmid pRL-TK at 100 and 9.6 ng per well respectively. Transfection was performed for 24 h using lipofectamine 2000 according to the manufacturer’s instructions. The medium was replaced with fresh serum-free medium. The Cells cells were pretreated with dexamethasone (0.01 mM) or target compounds (0.1 mM) before the addition of TNF-α (10 ng/mL).Luciferase reporter assay for inhibition of NF-κBAfter stimulation, BEAS-2B cells were washed, lysed, and assayed by luciferase activity using luciferase reporter assay system according to the manufacturer’s instructions. Relative luciferase activity was obtained by normalizing the firefly luciferase activity against that of the internal control (Renilla luciferase).Measurements of IL-6 and IL-8Commercial ELISA kits were employed to measure the release of IL-6 and IL-8 in the supernatants of BEAS-2B cells after the administration of dexamethasone (0.01 mM) or target compounds (0.1 mM) and stimulation of TNF-α. The absorbance of each sample was determined at 450 nm by a Bio-Rad model 680 microplate reader. Levels of IL-6 and IL-8 were measured from standard curves and expressed as picograms per milliliter.Statistical analysist-test for single comparisons. And, and the level p < 0.05 was considered as statistical significance.All values are expressed as the means ± standard deviation. SPSS software was employed for the statistical analysis. Significant differences of experimental data from different groups were compared by one way analysis of variance (one-way ANOVA) for multiple comparisons and Student’s Synthesis of the intermediates and target compoundsSynthesis of (E)-3- acrylic acid (3)To pyridine (6.4 mL) was added commercial syringaldehyde , malonic acid , and piperidine . After 22 h of stirring at room temperature, the reaction was quenched by addition of 7.5 mL of concentrated aqueous HCl solution. After addition of 94 mL water, the formed precipitate was filtered and dried to get compound 3 . Synthesis of intermediates (5)Benzimidazole 4a  and potassium carbonate  were mixture together in acetonitrile (20 mL) for 0.5 h. Then, 2-chloroacetonitrile  was added dropwise, and the mixture was stirred at 70 °C. After the reaction was completed, the mixture was cooled to room temperature, then the solvent was evaporated under vacuum, and water (30 mL) was added. The resulting mixture was extracted with dichlormethane (3 × 20 mL), then the combined organic phase was dried over anhydrous sodium sulfate and subsequently the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography  to give the desired compound 5a .2-(6-bromo-1H-benzo[d]imidazol-1-yl) acetonitrile (5b)1H NMR : δ 8.05 , 7.89 , 7.43 , 7.32 , 5.23 ; HRMS (ESI) calcd. For C9H7N3Br [M+H]+, 237.0760; found, 237.0762. Compound 5b was prepared according to the procedure depicted for compound 5a, starting from compound 4b , potassium carbonate  and 2-chloroacetonitrile . The product 5b (1.51 g) was obtained as white solid. Yield: 64%; 2-(4-chloro-2-methyl-1H-benzo[d]imidazol-1-yl) acetonitrile (5c)1H NMR : δ 7.47 , 7.41 , 5.25 , 2.37 ; HRMS (ESI) calcd. For C10H9N3Cl [M+H]+, 206.0485; found, 206.0479. Compound 5c was prepared according to the procedure depicted for compound 5a, starting from compound 4c , potassium carbonate  and 2-chloroacetonitrile . The product 5b (1.16 g) was obtained as white solid. Yield: 57%; 2-(2-bromo-1H-benzo[d]imidazol-1-yl) acetonitrile (5d)1H NMR : δ 7.87 , 7.46 , 7.42 , 5.26 ; HRMS (ESI) calcd. For C9H7N3Br [M+H]+, 237.0760; found, 237.0756.Compound 5d was prepared according to the procedure depicted for compound 5a, starting from compound 4d , potassium carbonate , and 2-chloroacetonitrile . The product 5d (1.13 g) was obtained as white solid. Yield: 48%; 2-(2-(3-hydroxypropyl)-5-methyl-1H-benzo[d]imidazol-1-yl) acetonitrile (5f)1H NMR : δ 7.89 , 7.33 , 5.25 , 3.77 , 2.62 , 2.35 , 2.30 ; HRMS (ESI) calcd. For C14H18N3O [M+H]+, 244.3180; found, 244.3185.Compound 5f was prepared according to the procedure depicted for compound 5a, starting from compound 4f , potassium carbonate  and 2-chloroacetonitrile . The product 5d (0.99 g) was obtained as white solid. Yield: 41.0%; 2-(5-nitro-1H-benzo[d]imidazol-1-yl) acetonitrile (5g)1H NMR : δ 8.32 , 8.13 , 7.64 , 7.39 , 5.24 ; HRMS (ESI) calcd. For C9H7N4O2 [M+H]+, 203.0569; found, 203.0572. Compound 5g was prepared according to the procedure depicted for compound 5a, starting from compound 4g , potassium carbonate  and 2-chloroacetonitrile . The product 5g (1.01 g) was obtained as white solid. Yield: 50.0%; 2-(4-fluoro-1H-benzo[d]imidazol-1-yl) acetonitrile (5i)1H NMR : δ 8.29 , 8.06 , 7.61 , 7.40 , 5.22 ; HRMS (ESI) calcd. For C9H7N3F [M+H]+, 176.0624; found, 176.0629.Compound 5i was prepared according to the procedure depicted for compound 5a, starting from compound 4i , potassium carbonate  and 2-chloroacetonitrile . The product 5g (1.20 g) was obtained as white solid. Yield: 68.6%; 2-(6-chloro-4-methyl-1H-benzo[d]imidazol-1-yl) acetonitrile (5j) 1H NMR : δ 8.22 , 7.40 , 7.22 , 5.22 , 2.02 ; HRMS (ESI) calcd. For C10H9N3Cl [M+H]+, 206.0485; found, 206.0486.Compound 5j was prepared according to the procedure depicted for compound 5a, starting from compound 4j , potassium carbonate  and 2-chloroacetonitrile . The product 5j (1.60 g) was obtained as white solid. Yield: 80.0%; Synthesis of intermediates (6)4 , dropwise at 0 °C, and the resulting reaction mixture was brought to room temperature overnight. Methanol (2 mL) was slowly added to quench the reaction at 0 °C, followed by 1 N NaOH (3 mL) at room temperature. The product was extracted with ethyl ether (30 mL × 3). Organics were washed with water, brine and dried over Na2SO4 and concentrated. The crude mass was subjected to silica gel chromatography, eluting with 0−5% methanol in dichloromethane to provide 6a .To a solution of 5a  in THF was added LiAlH2-(6-bromo-1H-benzo[d]imidazol-1-yl) ethan-1-amine (6b)4 . The product 6b (1.1 g) was obtained as white solid. Yield: 95.2%. 1H NMR : δ 8.02 , 7.86 , 7.41 , 7.31 , 4.40 , 3.08 , 1.10 ; HRMS (ESI) calcd. For C9H11N3Br [M+H]+, 241.1120; found, 241.1123. Compound 6b was prepared according to the procedure depicted for compound 6a, starting from compound 5b  and LiAlH2-(4-chloro-2-methyl-1H-benzo[d]imidazol-1-yl) ethan-1-amine (6c)4 . The product 6c (0.94 g) was obtained as desired compound. Yield: 90.0%. 1H NMR : δ 7.46 , 7.40 , 4.43 , 3.06 , 2.37 , 1.14 ; HRMS (ESI) calcd. For C10H13N3Cl [M+H]+, 210.6850; found, 210.6854.Compound 6c was prepared according to the procedure depicted for compound 6a, starting from compound 5c  and LiAlH2-(2-bromo-1H-benzo[d]imidazol-1-yl) ethan-1-amine (6d)4 . The product 6d (1.2 g) was obtained as desired compound. Yield: 93.0%. 1H NMR : δ 7.89 , 7.45 , 7.41 , 4.45 , 3.08 , 1.14 ; HRMS (ESI) calcd. For C9H11N3Br [M+H]+, 241.1120; found, 241.1118.Compound 6d was prepared according to the procedure depicted for compound 6a, starting from compound 5d  and LiAlH2- ethan-1-amine (6e)4 . The product 6e (0.83 g) was obtained as desired compound. Yield: 87.0%. Compound 6e was prepared according to the procedure depicted for compound 6a, starting from compound 5e  and LiAlH3-(1-(2-aminoethyl)-5, 6-dimethyl-1H-benzo[d]imidazol-2-yl) propan-1-ol (6f)4 . The product 6f (0.96 g) was obtained as desired compound. Yield: 77.1%. 1H NMR : δ 7.88 , 7.36 , 4.44 , 3.76 , 3.06 , 2.62 , 2.31 , 2.28 , 1.14 ; HRMS (ESI) calcd. For C14H22N3O [M+H]+, 248.3500; found, 248.3503. Compound 6f was prepared according to the procedure depicted for compound 6a, starting from compound 5f  and LiAlH2-(5-nitro-1H-benzo[d]imidazol-1-yl) ethan-1-amine (6g)4 . The product 6g (1.32 g) was obtained as desired compound. Yield: 64.0%. 1H NMR : δ 8.33 , 8.09 , 7.67 , 7.40 , 4.43 , 3.10 , 1.15 ; HRMS (ESI) calcd. For C9H11N4O2 [M+H]+, 207.0882; found, 207.0884.Compound 6g was prepared according to the procedure depicted for compound 6a, starting from compound 5g  and LiAlH2- ethan-1-amine (6h)4 . The product 6f (1.20 g) was obtained as desired compound. Yield: 86%. Compound 6g was prepared according to the procedure depicted for compound 6a, starting from compound 5g  and LiAlH2-(4-fluoro-1H-benzo[d]imidazol-1-yl) ethan-1-amine (6i)4 . The product 6i (1.13 g) was obtained as desired compound. Yield: 62.8%. 1H NMR : 1H NMR : δ 8.30 , 8.09 , 7.59 , 7.39 , 4.41 , 3.11 , 1.12 ; HRMS (ESI) calcd. For C9H11N3F [M+H]+, 180.0937; found, 180.0942.Compound 6i was prepared according to the procedure depicted for compound 6a, starting from compound 5i  and LiAlH2-(6-chloro-4-methyl-1H-benzo[d]imidazol-1-yl) ethan-1-amine (6j)4 . The product 6i (0.85 g) was obtained as desired compound. Yield: 40.7%. 1H NMR : δ 8.21 , 7.43 , 7.21 , 4.42 , 3.08 , 2.03 , 1.13 ; HRMS (ESI) calcd. For C10H13N3Cl [M+H]+, 210.0798; found, 210.0794.Compound 6j was prepared according to the procedure depicted for compound 6a, starting from compound 5j  and LiAlHSynthesis of the target compounds 7a-jN, N-dimethylformamide was added. Then, compound 6a  in dichloromethane was added. The resulting reaction mixture was stirred at room temperature for 8 h. The reaction was quenched with water (10 mL), and the product was extracted with ethyl acetate (20 mL × 3). Organics were washed with water (20 mL), brine solution (20 mL) and dried over Na2SO4. The crude product was purified by silica gel chromatography, eluting with 0−35% ethyl acetate in hexane to provide the target compound 7a . 1H NMR : δ 7.63 , 7.28 , 6.89 , 6.63 , 6.41 , 6.25 , 4.43 , 3.79 , 3.77 , 3.70 , 2.6 ; HRMS (ESI) calcd. For C20H22N3O4S [M+H]+, 400.4714; found, 400.4715.To a solution of 3  in dichloromethane (15 mL) were added oxalic dichloride  dropwise, the mixture was stirred at ice bath. One drop of (E)-N-(2-(7-bromo-1H-benzo[d]imidazol-1-yl) ethyl)-3- acrylamide (7b) 1H NMR : δ 8.01 , 7.83 , 7.38 , 7.32 , 6.89 , 6.63 , 6.41 , 6.25 , 4.42 , 3.77 , 3.74 , 3.68 ; HRMS (ESI) calcd. For C20H21N3O4Br [M+H]+, 447.3024; found, 447.3021. Compound 7b was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6b . The product 7b (1.30 g) was obtained as desired compound. Yield: 73%. (E)-N-(2-(4-chloro-2-methyl-1H-benzo[d]imidazol-1-yl)ethyl)-3- acrylamide (7c)1H NMR : δ 7.45 , 7.38 , 6.89 , 6.63 , 6.41 , 6.25 , 4.45 , 3.77 , 3.74 , 3.63 , 2.37 ; HRMS (ESI) calcd. For C21H23N3O4Cl [M+H]+] +, 417.8780; found, 417.8782. Compound 7c was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6c . The product 7c (1.30 g) was obtained as desired compound. Yield: 78%. (E)-N-(2-(2-bromo-1H-benzo[d]imidazol-1-yl) ethyl)-3- acrylamide (7d)1H NMR : δ 7.88 , 7.43 , 7.37 , 6.88 , 6.62 , 6.40 , 6.27 , 4.45 , 3.76 , 3.72 , 3.63 , 2.37 ; HRMS (ESI) calcd. For C20H21N3O4Br [M+H]+, 447.3024; found, 447.3021. Compound 7d was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6d . The product 7d (1.23 g) was obtained as desired compound. Yield: 69%. (E)-N-ethyl)-3- acrylamide (7e) 1H NMR : δ 7.72 , 7.36 , 7.16 , 6.88 , 6.62 , 6.40 , 6.27 , 4.45 , 3.76 , 3.72 , 3.63 , 2.18 , 2.15 ; HRMS (ESI) calcd. For C22H26N3O4 [M+H]+] +, 396.4595; found, 396.4597. Compound 7e was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6e . The product 7e (1.40 g) was obtained as desired compound. Yield: 89%. (E)-3--N-(2-(2-(3-hydroxypropyl)-5, 6-dimethyl-1H-benzo[d]imidazol-1-yl) ethyl) acrylamide (7f)1H NMR : δ 7.88 , 7.36 , 6.87 , 6.63 , 6.42 , 6.28 , 4.45 , 3.79 , 3.76 , 3.72 , 3.63 , 2.62 , 2.31 , 2.28 ; HRMS (ESI) calcd. For C25H32N3O5 [M+H]+] +, 454.5387; found, 454.5386. Compound 7f was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6f . The product 7f (0.69 g) was obtained as desired compound. Yield: 38%. (E)-3--N-(2-(5-nitro-1H benzo[d]imidazol-1-yl) ethyl) acrylamide (7g)1H NMR : δ 8.32 , 8.10 , 7.70 , 7.38 , 6.89 , 6.63 , 6.41 , 6.25 , 4.45 , 3.77 , 3.74 , 3.63 ; HRMS (ESI) calcd. For C20H21N4O6 [M+H]+, 413.1461; found, 413.1464.Compound 7g was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6h . The product 7h (0.7 g) was obtained as desired compound. Yield: 42.4%. (E)-N- ethyl)-3- acrylamide (7h)1H NMR : δ 7.88 , 7.36 , 7.05 , 6.87 , 6.63 , 6.42 , 6.28 , 4.45 , 4.25 , 3.79 , 3.76 , 3.72 , 2.28 ; HRMS (ESI) calcd. For C29H32N3O4 [M+H]+, 486.5821; found, 486.5823. Compound 7h was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6h . The product 7h (1.63 g) was obtained as desired compound. Yield: 84%. (E)-N-(2-(5-fluoro-1H-benzo[d]imidazol-1-yl) ethyl)-3- acrylamide (7i)1H NMR : δ 8.28 , 8.07 , 7. 69 , 7.39 , 6.88 , 6.63 , 6.41 , 6.23 , 4.44 , 3.79 , 3.71 , 3.62 ; HRMS (ESI) calcd. For C20H21FN3O4 [M+H]+, 386.1516; found, 386.1519. Compound 7g was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6g . The product 7g (0.78 g) was obtained as desired compound. Yield: 50.6%. (E)-N-(2-(5-chloro-7-methyl-1H-benzo[d]imidazol-1-yl) ethyl)-3- acrylamide (7j) 1H NMR : δ 8.23 , 7.41 , 7.22 , 6.88 , 6.62 , 6.41 , 6.22 , 4.44 , 3.79 , 3.71 , 3.62 , 2.03 ; HRMS (ESI) calcd. For C21H23ClN3O4 [M+H]+, 416.1377; found, 416.1376.Compound 7j was prepared according to the procedure depicted for compound 7a, starting from compound 3  and compound 6j . The product 7j (1.0 g) was obtained as desired compound. Yield: 60.2%. Chemistry1H NMR and HRMS spectra.The synthetic route of the target sinapic acid derivatives was outlined in Inhibitory effects on NF-κBTo investigate the anti-inflammatory activity of the target compounds, the luciferase reporter assay system has been employed to evaluate the inhibitory effects on NF-κB. Comparing with the model group, all of the prepared compounds gave stronger inhibitory effects. Compounds 7a, 7e, 7f, 7h, and 7j, with electron-donating groups in benzimidazole group, displayed relatively weaker activities than the clinic drug dexamethasone . To our Levels of IL-6 and IL-8To further confirm the inhibitory effects of the target compounds on NF-κB, the levels of IL-6 and IL-8 in the supernatants of BEAS-2B cells treated with TNF-α were measured by ELISA kits. As shown in 1H NMR and HRMS spectra. The in-vitro anti-inflammatory activity was evaluated by the luciferase reporter assay system. The results suggest that the compounds 7g and 7i with electron-drawing groups showed the best inhibitory effects on the NF-κB in BEAS-2B cells stimulated by TNF-α. The ELISA assay revealed that compounds 7g and 7i could decrease the expression of IL-6 and IL-8 significantly, and their inhibitory effects were much better than their precursor sinapic acid. In conclusion, a novel series of benzimidazole sinapic acid hybrids were synthesized via an easy, convenient, and efficient synthetic route starting from commercially available syringaldehyde and benzimidazoles in good yields, and all the new compounds were characterized by