A simple and efficient microwave (MW)-assisted method was elaborated for the catalyst-free synthesis of isoindolin-1-one-3-phosphonates by the three-component condensation of 2-formylbenzoic acid, aliphatic primary amines and various dialkyl phosphites. The batch and the continuous flow reactions were optimized in respect of the temperature, the reaction time and the molar ratio of the starting materials. To evaluate the potential of MW irradiation, comparative thermal experiments were also carried out. In order to obtain “real time” information about the condensation, the special Kabachnik–Fields reaction of 2-formylbenzoic acid, butylamine and diethyl phosphite was monitored by in situ FT-IR spectroscopy. The novel title compounds could be prepared in high yields at low temperature under a short reaction time. A suitable method could also be developed for the preparation of the isoindolin-1-one-3-phosphonates at a “few g” scale by using a continuous flow MW reactor. The two series of signals could be separated in the NMR spectra: Isomer A: 31P (CDCl3) δ 35.4; 1H NMR (CDCl3) δ 0.94 3N), 1.23–1.38 2N), 1.43 , 1.54–1.80 , 3.66–3.78 , 4.19–4.39 , 5.05 , 7.12–7.67 , 7.75 ; 13C NMR (CDCl3) δ 13.7 (CH3(CH2)3N), 16.50 , 20.0 (CH2(CH2)2N), 30.02 (CH2CH2N), 41.5 (CH2N), 60.1 , 61.9 , 123.4 , 123.7 , 124.45 , 125.5 , 128.21 , 128.67 , 131.1 , 132.1 , 133.04 , 138.7 , 168.3 ; Isomer B: 31P (CDCl3) δ 39.2; 1H NMR (CDCl3) δ 0.95 3N), 1.23–1.38 2N), 1.45 , 1.54–1.80 , 3.51–3.66 , 3.98–4.19 , 5.05 , 7.12–7.67 , 7.86 ; 13C NMR (CDCl3) δ 13.7 (CH3(CH2)3N), 16.54 , 20.0 (CH2(CH2)2N), 29.98 (CH2CH2N), 41.4 (CH2N), 59.4 , 62.2 , 123.4 , 123.7 , 124.42 , 125.1 , 128.25 , 128.60 , 131.3 , 131.9 , 132.98 , 138.1 , 168.7 .Yield: 78% (0.28 g), colourless oil; [M + H]To 30 mL of ethanol, the starting materials were mixed at room temperature (25 °C) in ten-minute intervals in the following order: 30 mmol (4.5 g) of 2-formylbenzoic acid, 30 mmol (3.9 mL) of diethyl phosphite and 36 mmol (3.6 mL) of butylamine. After the additions, the mixture was heated gradually from 25 °C to 60 °C in an oil bath. Then the temperature was held for an additional 2.5 h.3 volumetric flask, resulting in the aldehyde concentration of 0.84 M. For solution B, amines (50.2 mmol of butylamine (5.0 mL) or 62.7 mmol of butylamine (6.2 mL), 62.7 mmol of cyclohexylamine (7.2 mL) or 62.7 mmol of benzylamine (6.8 mL)) and 41.8 mmol of diethyl phosphite (5.4 mL) or 62.7 mmol of diethyl phosphite (8.1 mL) were dissolved in ethanol in a 50 cm3 volumetric flask, resulting in the amine concentration of 1.01 M or 1.26 M and the dialkyl phosphite concentration of 0.84 M or 1.26 M, respectively. The two solutions (A and B) were pumped separately. The system was flushed with 15 mL of solution A and 15 mL of solution B with a summa flow rate of 10 mL/min (A:B = 1:1) at 25 °C and 17 bar. Next, under the same pressure, the flow rate was set to the desired value (can be seen in H-isoindol-1-yl)phosphonate (7a), diethyl phosphonate (8a) or diethyl phosphonate (9a) were obtained by column chromatography using silica gel as the absorbent and dichloromethane:methanol (97:3) as the eluent. Yields were calculated on the basis of the weights obtained after separation and evaporation taking into consideration the quantity of the limiting starting materials fed in during a given time. After a 3 h flow operation in steady state, 6.9 g of diethyl phosphonate (7a), 4.1 g of diethyl phosphonate (8a) or 5.3 g of diethyl phosphonate (9a) were obtained.Before each experiment, two solutions (A and B) were prepared. For solution A, 41.8 mmol of 2-formylbenzoic acid was dissolved in ethanol in a 50 cmIn conclusion, we have developed a facile, efficient, catalyst-free method for the batch and continuous flow preparation of isoindolin-1-one-3-phosphonates containing alkyl substituents on the nitrogen atom. The novel batch approach enabled the solvent-free synthesis of the target compounds in high yields (70–94%) at low temperature (60 °C) under short reaction times (10–30 min). By the in situ FT-IR study on the condensation of 2-formylbenzoic acid, butylamine and diethyl phosphite, the reaction was followed in “real time” and the time-dependent concentration profiles of the reaction components were determined. The continuous flow MW-assisted method developed for the condensation of 2-formylbenzoic acid, aliphatic primary amines and diethyl phosphite are suitable for the synthesis of the corresponding isoindolin-1-one-3-phosphonates in a “few g” scale. Altogether 16 derivatives were isolated and characterized. Except two, all of them are new compounds.


Further, final compounds (4a–n) were assessed for their anti-inflammatory, analgesic, ulcerogenic, and lipid peroxidation. The compounds 4f, 4g, 4j, 4l, and 4m showed good anti-inflammatory activity, while 4f, 4i, and 4n exhibited very good analgesic activity as compared to the standard drug. The ulcerogenicity of selected compounds was far less than the indomethacin. The ligands had also shown a good docking score  as compared to control indomethacin  against the target protein COX-2. These derivatives have the potential to block this enzyme and can be used as NSAID. The state-of-art DFT theory was used to validate the lipid peroxidation mechanism of the active compounds which was in good agreement with the variations of BDEs and IP of the tested compounds.We have developed a new idea to synthesize a key intermediate molecule by utilizing deep eutectic solvent (DES) and ultrasound in a multistep reaction to ensure process cost-effectiveness. To confirm the stability of reagents with DES, electronic energies were calculated at the B3LYP/6-31+G level of theory. DES stabilized the reagents mainly due to strong intermolecular hydrogen bonding. Key intermediate (3) and final compounds ( Nonsteroidal anti-inflammatory drugs (NSAIDs) are a profound application for the treatment of inflammatory diseases and pain. NSAIDs are the choice of treatment in various inflammation and pain-related problems such as osteoarthritis, rheumatoid arthritis, spondylitis, and gout ,2,3. A mIndole ring and its derivatives have emerged as privileged pharmacophores representing more than thousands of natural isolates with known biological and pharmaceutical activities such as anti-inflammatory, analgesic ,17,18,19The immense application of ultrasound has been highlighted recently in organic and material science ,31. UltrKeeping these things and with extended work ,33,34,353) with substituted aromatic amines and formaldehyde in ethylene glycol as depicted in 1H-NMR, 13C-NMR, mass and elemental analysis. In general, absorption bands due to two -NH groups appeared in the IR spectra at around 3200 cm−1. Other bands due to -C=N and two -C=O functional groups were found at around 1600 and 1700, respectively. In the 1H-NMR spectra, two -NH peaks appeared at around 9 and 10 ppm. The lower value provides information as a singlet due to -NH attached as -CH2NH with indolinone nitrogen as a characteristic peak. Value at δ 5 ppm confirms the presence of -CH2 which is another important peak for identification. Further, characteristics peak of -CH2 of -CH2NH was confirmed by 13C-NMR around δ 69 ppm. 1-(Substitutedphenylaminomethyl)-3-(2-(4-(2-oxochroman-3-yl)thiazol-2-yl)hydrazono)indolin-2-ones were synthesized by treating 3-(2-(4-(2-oxochroman-3-yl)thiazol-2-yl)hydrazono) indolin-2-one 2-(2-Oxoindolin-3-ylidene)hydrazine carbothioamide  cm−1: 3413, 3352, and 3216 (N-H), 1693 (C=O).1H-NMR  ppm: 6.72 , 6.92 , 7.03 , 7.34 , 8.04 , 9.99 , 10.55 ; elemental analysis: Calcd. for (C9H8N4OS), found % : C, 49.07 (49.08); H, 3.65 (3.66); N, 25.43 (25.44).3)3-(2-(4-(2-Oxo-2H-chromen-3-yl)-4,5-dihydrothiazol-2-yl)hydrazono)indolin-2-one cm−1: 1692 and 1703 (C=O), 3315 and 3253 (N-H), 1612 (C=N), 1543 (C=C).1H-NMR  ppm: 7.03 , 7.39 , 8.28 , 10.25 ; elemental analysis: Calcd. for (C20H12N4O3S), found % : C, 61.84 (61.85); H, 3.10 (3.11); N, 14.42 (14.43).4a)3-{[4-(2-Oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1-phenylaminomethyl-1,3-dihydro-indol-2-one  cm−1: 1683 and 1710 (C=O), 3309 and 3251 (N-H), 1613 (C=N), 1546 (C=C); 1H-NMR  ppm: 5.13 , 7.44–8.10 , 9.35 , 10.53 ; 13C-NMR ; 171.0 ,159 and 162 (2CO), 156 , 143.4, 140.9, 139.07, 139.0, 138.6, 131.0, 129.8, 129.5, 127.8, 126.8, 125.5, 124.3, 123.4, 121.2, 117.1, 112.4, (Ar-C), 69.3 ; elemental analysis: Calcd. for (C27H19N5O3S), found % : C, 65.70 (65.71); H, 3.87 (3.88); N, 14.18 (14.19). Mass (m/z): 493 , 200 (C11H6NOS), 175 (C10H7OS), 168 (C12H10N), 159 (C8H7N4), 132 , 106 (C7H8N).4b)1-[(4-Fluoro-phenylamino)-methyl]-3-{[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1,3-dihydro-indol-2-one  cm−1: 1683 and 1704 (C=O), 3312 and 3264 (N-H), 1613 (C=N), 1543 (C=C); 1H-NMR  ppm: 5.10 , 7.42 , 9.15 , 10.55 ; 13C-NMR ; 172.0 , 160 and 161 (2CO), 155 , 145.2, 143.1, 140.2, 139.0, 138.4, 132.5, 130.7, 129.3, 128.9, 127.6, 125.3, 124.4, 122.2, 118.3, 114.3, (Ar-C), 68.9 ; MS (m/z): 511 (M+), 513 (M+ + 2); elemental analysis: Calcd. for (C27H18N5O3SF), found % : C, 63.38 (63.40); H, 3.55 (3.55); N, 13.68 (13.69).4c)1-[(4-Chloro-phenylamino)-methyl]-3-{[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1,3-dihydro-indol-2-one  cm−1: 1689 and 1705 (C=O), 3309 and 3251 (N-H), 1613 (C=N), 1544 (C=C); 1H-NMR  ppm: 5.14 , 7.39 , 9.25 , 10.50 ; 13C-NMR ; 170.0 , 161 and 162 , 157 , 146.1, 143.1, 140.3, 139.1, 138.6, 133.5, 131.7, 129.2, 128.4, 127.7, 126.3, 125.2, 123.1, 117.9, 112.8, (Ar-C), 68.6 ; MS (m/z): 528 (M+), 530 (M+ + 2); elemental analysis: Calcd. for (C27H18N5O3SCl), found % : C, 61.41 (61.42); H, 3.44 (3.44); N, 13.25 (13.26).4d)1-[(4-Bromo-phenylamino)-methyl]-3-{[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1,3-dihydro-indol-2-one  cm−1: 1685 and 1704 (C=O), 3313 and 3251 (N-H), 1611 (C=N), 1547 (C=C); 1H-NMR  ppm: 5.14 , 7.40 , 9.30 , 10.51 ; 13C-NMR ; 171.0 , 162 and 163 , 156 , 145.5, 143.6, 140.3, 139.8, 138.3, 133.6, 131.4, 129.7, 128.1, 127.7, 126.9, 123.6, 118.9, 112.9, (Ar-C), 69.3 ; MS (m/z): 572 (M+), 574 (M+ + 2); elemental analysis: Calcd. for (C27H18N5O3SBr), found % : C, 56.64 (56.65); H, 3.16 (3.17); N, 12.22 (12.23).4e)1-[(2-Nitro-phenylamino)-methyl]-3-{[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1,3-dihydro-indol-2-one  cm−1: 1684 and 1705 (C=O), 3310 and 3255 (N-H), 1613 (C=N), 1543 (C=C); 1H-NMR  ppm: 5.07 , 7.42 , 9.15 , 10.48 ; 13C-NMR ; 170.0 , 163 and 164 (2CO), 157 , 146.4, 143.8, 141.6, 140.4, 139.7, 135.6, 133.4, 130.7, 128.5, 127.5, 126.4, 124.6, 118.6, 112.3, (Ar-C), 70.1 ; MS (m/z): 538 (M+), 540 (M+ + 2); elemental analysis: Calcd. for (C27H18N6O5S), found % : C, 60.21 (60.22); H, 3.36 (3.37); N, 15.60 (15.61).4f)1-[(2-chloro-phenylamino)-methyl]-3-{[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1,3-dihydro-indol-2-one  cm−1: 1689 and 1707 (C=O), 3311 and 3252 (N-H), 1613 (C=N), 1545 (C=C); 1H-NMR  ppm: 5.10 , 7.36 , 9.36 , 10.55 ; 13C-NMR ; 172.0 , 161 and 162 (2CO), 157 , 146.5, 143.9, 140.8, 140.1, 138.7, 135.4, 133.7, 129.7, 128.8, 127.6, 126.3, 124.7, 119.3, 114.1, (Ar-C), 68.3 ; elemental analysis: Calcd. for (C27H18N5O3SCl), found % : C, 61.41 (61.42); H, 3.43 (3.44); N, 13.25 (13.26). Mass (m/z): 527 , 528 (M+ + 1), 202 (C12H9NCl), 175 (C10H7OS), 132 , 111 (C6H4Cl), 59 (C2H3S).4g)1-[-methyl]-3-{[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1,3-dihydro-indol-2-one  cm−1: 1686 and 1704 (C=O), 3292 and 3252 (N-H), 1612 (C=N), 1544 (C=C); 1H-NMR  ppm: 5.15 , 7.45 , 9.33 , 10.51 ; 13C-NMR ; 169.0 , 159 and 161 (2CO), 155 , 145.7, 142.5, 140.2, 139.1, 137.4, 135.7, 133.5, 129.6, 128.6, 127.3, 124.7, 118.9, 112.3, (Ar-C), 69.7 ; MS (m/z): 583 (M+), 585 (M+ + 2); elemental analysis: Calcd. for (C27H17N7O7S), found % : C, 55.56 (55.57); H, 2.94 (2.94); N, 16.79 (16.80).4h)3-{[4-(2-Oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1--1,3-dihydro-indol-2-one  cm−1: 1685 and 1706 (C=O), 3253 and 3279 (N-H), 1613 (C=N), 1543 (C=C); 1H-NMR  ppm: 5.17 , 7.48 , 9.42 , 10.47 ; 13C-NMR ; 170.0 , 161 and 161 (2CO), 157 , 144.8, 140.2, 137.4, 135.9, 132.4, 129.4, 128.7, 127.5, 124.3, 116.9, 112.3, (Ar-C), 70.2 ; MS (m/z): 484 (M+), 486 (M+ + 2); elemental analysis: Calcd. for (C23H16N8O3S), found % : C, 57.01 (57.02); H, 3.32 (3.33); N, 23.12 (23.13).4i)1-[(3-Chloro-4-fluoro-phenylamino)-methyl]-3-{[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1,3-dihydro-indol-2-one  cm−1: 1684 and 1703 (C=O), 3240 and 3273 (N-H), 1612 (C=N), 1544 (C=C); 1H-NMR  ppm: 5.05 , 7.41 , 9.31 , 10.55 ; 13C-NMR ; 171.0 , 161 and 163 (2CO), 156 , 146.9, 143.4, 140.6, 139.2, 137.6, 135.7, 133.8, 129.3, 128.3, 127.6, 124.2, 117.4, 112.3, (Ar-C), 68.6 ; MS (m/z): 546 (M+), 548 (M+ + 2); elemental analysis: Calcd. for (C27H17N5O3SClF), found % : C, 59.38 (59.40); H, 3.14 (3.14); N, 12.82 (12.83).4j)3-{[4-(2-Oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1-(pyridine-4-ylaminomethyl)-1,3-dihydro-indol-2-one  cm−1: 1687 and 1705 (C=O), 3244 and 3268 (N-H), 1613 (C=N), 1545 (C=C); 1H-NMR  ppm: 5.17 , 7.45 , 9.38 , 10.57 ; 13C-NMR ; 172.0 , 161 and 163 (2CO), 156 , 144.8, 143.5, 140.4, 139.0, 138.3, 132.5, 130.9, 129.3, 128.8, 127.6, 125.1, 124.7, 122.9, 116.9, 112.6, (Ar-C), 68.3 ; MS (m/z): 594 (M+), 596 (M+ + 2); elemental analysis: Calcd. for (C26H18N6O3S), found % : C, 63.14 (63.15); H, 3.66 (3.67); N, 16.98 (16.99).4k)3-{[4-(2-Oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1-(pyridine-3-ylaminomethyl)-1,3-dihydro-indol-2-one  cm−1: 1686 and 1706 (C=O), 3251 and 3277 (N-H), 1612 (C=N), 1543 (C=C); 1H-NMR  ppm: 5.15 , 7.47 , 9.35 , 10.55 ; 13C-NMR ; 172.0 , 161 and 162 (2CO), 156 , 144.9, 143.6, 140.2, 139.0, 138.5, 132.3, 130.7, 129.5, 128.7, 127.3, 125.5, 124.9, 122.8, 116.6, 112.4, (Ar-C), 68.3 ; MS (m/z): 594 (M+), 596 (M+ + 2); elemental analysis: Calcd. for(C26H18N6O3S), found % : C, 63.14 (63.15); H, 3.66 (3.67); N, 16.98 (16.99).4l)1-[(4-Nitro-phenylamino)-methyl]-3-{[4-(2-oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1,3-dihydro-indol-2-one  cm−1: 1684 and 1702 (C=O), 3255 and 3278 (N-H), 1612 (C=N), 1544 (C=C); 1H-NMR  ppm: 5.18 , 7.46 , 9.32 , 10.54 ; 13C-NMR ; 170.0 , 163 and 164 (2CO), 158 , 146.6, 143.7, 141.9, 140.5, 139.7, 135.2, 133.5, 130.1, 128.2, 127.7, 126.1, 124.3, 116.9, 112.3, (Ar-C), 70.2 ; MS (m/z): 538 (M+), 540 (M+ + 2); elemental analysis: Calcd. for (C27H18N6O5S), found % : C, 60.21 (60.22); H, 3.36 (3.37); N, 15.60 (15.61).4m)3-{[4-(2-Oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1-(p-tolylamino-methyl)-1,3-dihydro-indol-2-one  cm−1: 1685 and 1706 (C=O), 3252 and 3273 (N-H), 1608 (C=N), 1544 (C=C); 1H-NMR  ppm: 2.23 , 5.11 , 7.41 , 9.31 , 10.48 ; 13C-NMR ; 172.0 , 160 and 163 (2CO), 156 , 146.8, 144.2, 142.4, 140.6, 139.9, 135.3, 133.8, 130.5, 128.8, 127.1, 126.3, 124.8, 117.4, 114.1, (Ar-C), 70.3 ; MS (m/z): 507 (M+), 509 (M+ + 2); elemental analysis: Calcd. for (C28H21N5O3S), found % : C, 66.25 (66.26); H, 4.16 (4.17); N, 13.79 (13.80).4n)3-{[4-(2-Oxo-2H-chromen-3-yl)-thiazol-2-yl]-hydrazono}-1-(o-tolylamino-methyl)-1,3-dihydro-indol-2-one  cm−1: 1686 and 1703 (C=O), 3251 and 3282 (N-H), 1612 (C=N), 1543 (C=C); 1H-NMR  ppm: 2.21 , 5.13 , 7.43 , 9.34 , 10.53 ; 13C-NMR ; 171.0 , 161 and 162 (2CO), 157 , 145.9, 144.2, 142.6, 140.3, 139.2, 135.6, 133.5, 130.2, 128.9, 127.6, 126.1, 124.9, 118.2, 112.3, (Ar-C), 68.3 ; elemental analysis: Calcd. for (C28H21N5O3S), found % : C, 66.25 (66.26); H, 4.16 (4.17); N, 13.80 (13.80); Mass (m/z): 507 , 387 (C20H11N4O3S), 200 (C11H6NOS), 132 , 120 (C8H10N), 90 (C6H4N), 59 (C2H3S).4a–n) utilizing ultrasound as a source of heating. Some of our earlier work and other related literature also mentioned the significance DES and ultrasound technology as an energy saving process [To develop the efficient method as compared to conventional, we have conducted the synthesis of key intermediate (3) utilizing biocompatible deep eutectic solvent (DES) and ultrasound blend of technique. As a result of combined use of DES and ultrasound, an increase in % yield of key intermediate as high as 95% has been found with the expense of 1 h only. Whereas, a similar type of organic transformation using dioxane and another organic solvent together with conventional heating were reported to have % yield around 44%–68% in 3–4 h ,37,38. F process ,39,40 whThe exact mechanism of formation of the desired intermediate compound is not yet clear. However, it was suggested by some researchers that the urea part of DES  catalyzes the reaction by making hydrogen bond. Thus, urea in a deep eutectic solvent was involved in stabilizing the acetyl moiety of 3-bromoacetylcoumarin via hydrogen bonding, which was further attacked by amide functional group of hydrazine thioamide to form key intermediate, 3-(2-(4-(2-oxochroman-3-yl) thiazol-2-yl) hydrazono) indolin-2-one through cyclization and dehydration process .To emphasize the role of DES in the stability of starting reagents, the electronic energies of the latter were calculated at the B3LYP/6-31+G level of theory in the presence and absence of DES . The preMoreover, ultrasound also played a significant role in the formation of the desired compound. Under the influence of sonic waves inside the reaction vessel, there was the formation of microscopic bubbles, as a result of high temperature and pressure ,29,30,314a–n) was evaluated by the carrageenan-induced paw edema method. An oral dose of 10 mg/kg was used for compounds and compared with the standard. Anti-inflammatory activity was accessed through percentage inhibition after 2 and 4 h (The anti-inflammatory activity of the synthesized compounds ( and 4 h .4f (72.42%), 4g (77.94%), 4j (77.90%), 4k (70.75%), 4l (80.94%), and 4m (78.42%) showed comparable results against the standard drug.Anti-inflammatory activity in terms of percentage inhibition for the test compounds is ranging from 5.57% to 80.94% , whereas4l (Ar = 4-nitrophenyl) exhibited the highest anti-inflammatory activity. Other compounds of the series, namely, 4f (Ar = 2-chlorophenyl), 4g , 4j (Ar = 4-pyridyl), 4k (Ar = 2-pyridyl), and 4m (Ar = 4-methyl phenyl) also displayed significant anti-inflammatory activity. Two compounds, 4a (Ar = phenyl) and 4d (Ar = 4-bromophenyl) displayed negligible anti-inflammatory activity. All other compounds displayed moderate anti-inflammatory activity. Further, the number and position of substituents also count the variation in anti-inflammatory activity. Nitrogen bearing compounds 4g  and 4l (Ar = 4-nitrophenyl) showed highest anti-inflammatory activity. When the chloro substituent present on ortho-position (4f) of the phenyl ring displayed almost double activity as compared to a compound bearing para chloro compound (4c). Similarly, the difference in anti-inflammatory activity was found in compounds 4j and 4k, as well as 4m and 4n due to different arrangements of substituents on the phenyl ring.The structure of 1-(substituted phenyl amino methyl)-3-(2-(4-(2-oxochroman-3-yl) thiazol-2-yl) hydrazono) indolin-2-one derivatives revealed that the compound Compounds under investigation showed analgesic activity ranging from 7.96% to 69.36% with a reference drug of 73.61% .4l) which showed maximum anti-inflammatory activity produces the least analgesic activity, but some selected compounds such as 4f, 4i, and 4n displayed analgesic activity in a similar fashion as the anti-inflammatory activity (4k) which exhibited the least analgesic activity was among the top-ranked anti-inflammatory activity. On the contrary, many compounds exhibiting good analgesic properties were not displayed as good anti-inflammatory activity and vice-versa  that showed negligible anti-inflammatory activity. Compound 4i possessing two electron-withdrawing groups exhibited moderate anti-inflammatory activity but good analgesic activity. Compound (4m) having an electron releasing group (-CH3) at C-4 position exhibited better anti-inflammatory activity but less analgesic activity. On the other hand, a methyl group at C-2 showed better anti-inflammatory and analgesic agents (4n). A steep decrease in analgesic activity was observed when the phenyl ring was replaced by a triazole ring (4h).After a close understanding of anti-inflammatory and analgesic potentials of compounds under the present series, we have made a structure-activity relationship. Compounds possessing a substituted phenyl ring showed better anti-inflammatory and analgesic activity than a compound having an unsubstituted phenyl ring. In most of the cases, the substitution of electron-withdrawing groups at C-2 and C-4 positions of the phenyl ring resulted in potent compounds except compound 4c (Ar = 4-chlorophenyl), 4f (Ar = 2-chlorophenyl), 4i (Ar = 4-fluoro-3-chlorophenyl), and 4n (Ar = 2-methylphenyl) were selected for their ulcerogenic activity. This selection was based on their anti-inflammatory and analgesic activity. Compounds were evaluated at an oral dose of 30 mg/kg relative to 10 mg/kg indomethacin.Four compounds, namely, 4n exhibited the highest severity index of 0.833 but it was only 20% of the severity shown by the standard. Mainly, compounds 4f, 4i, and 4n displayed excellent anti-inflammatory, an analgesic with reduced ulcerogenic potential. Significant reduction in ulcerogenecity is ranging from 0.500 ± 0.129 to 0.833 ± 0.210, whereas standard drug indomethacin showed a high severity index of 4.500 ± 0.316.The ulcerogenic activity of these compounds revealed that all the compounds showed a lesser severity index for ulcerogenicity than indomethacin . Compoun4c, 4f, 4i, 4n). It was measured as the nmol of MDA/100 mg of gastric tissue. We have found the Lipid peroxidation value was maximum 6.71 ± 0.18 for indomethacin, whereas 3.89 ± 0.17, 4.08 ± 0.22, 4.26 ± 0.12, and 4.81 ± 0.13 for compounds 4i, 4c, 4f, and 4n, respectively. It was interesting to mention that all these compounds having electron-withdrawing functionality on the phenyl ring (except 4n) exhibited less ulcerogenecity with reduced lipid peroxidation  ulceration, bleeding, and renal problems are common complications of NSAID’S consumption, which is directly related to lipid peroxidation. It has been evidenced that drugs having less ulcerogenecity showed reduced malondialdehyde (a byproduct of lipid peroxidation) content ,41. We hxidation .As mentioned above, only the synthesized derivatives (In-H) that exhibited maximum anti-inflammatory and analgesic activities derivatives are subjected to the lipid peroxidation (LP) test . The tesThe tested compounds showed similar lipid peroxidation with a small variation between their values. This result is confirmed by the small differences of BDEs of the active 17-NH group and IP energies, where the maximum variations of BDEs and IPs are 0.03 kcal/mol and 0.08 eV, respectively.The 3D structure of target protein COX-2 from two different organisms, i.e., mouse and human were retrieved from the protein data bank having PDB id 3NT1 and 5F91, respectively ,43.The protein structures obtained from PDB were modified suitably for the molecular docking studies. The modified protein structures were then validated through the Ramachandran plot. The Ramachandran plot of these two-target proteins is shown in The evaluation of phi/psi angles validates the prepared protein structures as most of the residues are in the most favored region. In the case of 3NT1, 90.8% amino acid residues are in the most favored regions whereas 8.8%, 0.1%, and 0.3% are in additional allowed regions, generously allowed regions, and disallowed regions, respectively. Similarly, in 5F91 90.7%, 9.1%, 0.1%, and 0.1% amino acid residues are in the most favored regions, additionally allowed regions, generously allowed regions, and disallowed regions, respectively. The result obtained allows the use of these structures for further molecular docking studies.The site map application predicts five different drug binding sites in both the target proteins. The site score for the protein 3NT1 was 1.078, 1.053, 1.048, 1.034, and 0.991. Similarly, the site score obtained for the protein 5F19 was 1.082, 1.051, 1.046, 1.034, and 0.990. As a rule of thumb, the binding sites having a score above one are considered as druggable pockets. In the present in silico study, the site with the highest score was selected for the molecular docking studies. The druggable pocket inside the respective target proteins is shown in 4a–4n) was prepared for the docking studies as per the standard guidelines and used in the molecular docking studies.The lowest energy conformation of each test ligand , i.e., the derivatives of 1-(substituted phenyl aminomethyl)-3-(2-(4-(2-oxochroman-3-yl) thiazol-2-yl) hydrazono) indolin-2-ones (4a–n) were docked inside the binding pocket of target proteins COX-2 (PDB ID: 3NT1 and 5F19) using Glide application . To compare the docking efficacy of test ligands, indomethacin was used as a reference/control drug. The molecular docking results were evaluated on the basis of docking score, E-model score, and binding energy as summarized in All the synthesized ligands  against the mouse target protein. A similar pattern of docking score is observed against human target protein where all test ligands (4a–n) have lower docking scores as compared to control . It was believed that a low binding energy dock conformer exhibited maximum stability. The two best compounds on the basis of experimental results is 4n and 4f. 4n and 4f have docking score −7.077 and −6.859 kcal/mol against human target protein, respectively. The same two ligand 4n and 4f have a score −6.693 and −6.071 kcal/mol against mouse target protein, respectively. The docked ligands (4n and 4f) inside the binding pocket of the respective target proteins (3NT1 and 5F19) is shown in The maximum test ligands that are The further efficacy of the docking is interpreted in terms of interaction that exists between the ligand and the surrounding amino acid residues inside the druggable pocket. The overall binding interaction (in terms of bonding) for each ligand is summarized in 4n is more suitable for drug candidates as it possesses a strong affinity towards the target proteins. In 3NT1, it forms two hydrogen bonds with Arg 376, whereas in 5F19 two pi–pi stacking exists with the involvement of Phe 142 and Arg 333. In the case of 4f, there is no hydrogen bonding or pi–pi interaction is observed (whether it is 3NT1 or 5F19). All these interactions are shown in Among the two potent ligands, https://www.mdpi.com/2504-3900/41/1/8). The suitability of test ligands as drug candidates depending on their pharmacokinetic behavior was also assessed using the in silico approach. Many drug candidates fail at a later stage of a clinical trial due to their poor pharmacokinetic performance. In order to avoid such failure and save time, energy, and money, in silico ADME profiling is a good choice . The res4n and 4f have QPPlogCaco value 619.284 and 479.473 that is very good as compared to indomethacin that has a score of 185.783 only. The Madin–Darby canine kidney (MDCK) cell model is used to investigate the apparent MDCK cell permeability [4n and 4f is 586.303 and 809.359 whereas the standard drug has a value of 251.855. The percent human abortion of both the potential ligand is also comparable to the standard and above 80%. The test ligands are also found to be obeying the Lipinski rule of 5.The oral drug absorption is predicted in terms of apparent Caco-2 permeability (QPPlogCaco) that represents the gut-blood barrier. The value above 500 indicates a great absorption while below 25 is considered a poor score . The ligeability . The scop < 0.05.Data used in the experimental pharmacological section was used as the mean ± standard error (SEM). One way analysis of variance (ANOVA) and Dennett’s multiple comparison test techniques was employed to compare between the test, control, and standard group, utilizing statistical software GraphPad Prism version 5.00, California corporation, San Diego, USA. Such results showed significantly different at Melting points were evaluated in open capillary tubes and are uncorrected. 5 PC FTIR spectrometer  spectrophotometer and Jeol-JMS-D-300 mass spectrometer (70 eV)  for IR, NMR, and mass, respectively were used to characterize the compounds. A combination of isatin (0.01 mole) and thiosemicarbazide (0.01 mole) was placed in a 100 mL round bottom flask with 50 mL of methanol as solvent, refluxed for 2 h, and then put onto the ice. The obtained was filtered, dried, and recrystallized using methanol.A mole ratio (1:2) of choline chloride and urea were chosen to prepare DES as per the reported method .In a specially designed sonicating flask, an equimolar (0.01 mole) quantity of 3-bromoacetyl coumarin and 2-(2-oxoindolin-3-ylidene) hydrazinecarbothioamide (2) with 8 g of prepared DES was added. A sonicating probe of 26 kHz frequency at 40% amplitude was submerged into the reaction vessel. The completion of the reaction was monitored by taking TLC at regular intervals. Upon completion, it was poured onto crushed ice. Then, upon completion of the reaction, it was extracted by dichloromethane using a separating funnel. The organic solvent layer was collected and evaporated to get the desired product. DES was isolated and kept for future use.A mixture of 3-(2-(4-(2-oxochroman-3-yl) thiazol-2-yl) hydrazono)indolin-2-one (3) (0.01 mole), substituted aromatic amines (0.01 mole) and formaldehyde (0.02 moles) in 30 ml of ethylene glycol was refluxed from 1 to 3 h. The reaction mixture was transferred onto the crushed ice upon completion, as confirmed by TLC. The solid was decanted, filtered, washed with water, dried, and recrystallized from dioxane to get affored compounds w/v) carrageenan was injected in the plantar region of the hind limb (right) of all the rats in each group including the control group. The paw volume was again measured after the time interval of 2 and 4 h. Using the following formula, inflammation was calculated as percentage inhibition for the test and reference compoundsCompounds produced were assessed for their anti-inflammatory activity using the carrageenan-induced hind paw edema method . The ant × 100 / final foot volume of control)v/v) acetic acid solution in distilled water was injected intraperitoneally in all the mice in each group including the control group. The writhing  was counted after 5 min of acetic acid administration and were counted for a period of 15 min. The percentage of analgesic activity was calculated using the following formula. The analgesic activity of the tested compounds was carried out by acetic acid-induced writhing method as given in the literature  using Sw(Mean wriths of control—mean wriths of std./test) / mean wriths of control) × 100Acute ulcerogenic activity evaluation of the synthesized compounds was carried out according to the method described  using WiThe method adopted for lipid peroxidation is the same as Ohkawaet al.  and the It is well known that almost all phenolic compounds may inhibit the lipid peroxidation process due to their ability to scavenge the chain-carrying lipid peroxyl radicals, LOO•. The lipid, LH, peroxidation process is represented by three main steps initiation, propagation, and terminations. The scavenging of LOO• by the synthesized indolin-2-Ones derivatives (In-H) may refer to hydrogen atoms transferred or an electron transfer from the former to the lipid peroxyl radical. The hydrogen atom transfer is represented by the following reaction: The above lipid peroxidation inhibition is governed by bond dissociation enthalpies (BDE) of i-NH groups of the synthesized indolin-2-ones derivatives (In-H). BDE is calculated using the following equation:Previously, we showed the success of the hybrid functional B3B86 in rationalizing the scavenging of free radicals by synthesized natural polyphenols ,49,50. HAll the in silico experiments of the present study were carried out using the Schrodinger Maestro interface  ,53.https://www.rcsb.org/). There are two different structures of this enzyme available, one from the mouse and another from the human. The two structures obtained have PDB ID 3NT1 (mouse) and 5F91 (human).In the present study, COX-2 is selected as the target protein because the therapeutic response of NSAID’s is generated by blocking/inhibiting this enzyme. The 3D structure of COX-2 was retrieved from Protein Data Bank  using the protein preparation wizard . The protein preparation is a multi-step process that includes the addition of hydrogen atoms, optimization of hydrogen bonds, and elimination of any atomic level clashes. The final step of protein preparation is energy minimization that was performed at the condition 0.3 Å of RMSD and OPLS_2005 force field . The proTo locate the ligand-binding site in the target protein, site map application  was used. The potency of the predicted site is decided on the basis of the site score generated by the tool. The binding site effectiveness is determined by several physical parameters such as size, the degree of enclosure/exposure, hydrophobic/hydrophilic character, opportunities of hydrogen bonding, etc.4a to 4n) that were synthesized chemically in the previous steps were used as ligands. The chemical structure of individual ligand was drawn and prepared using LigPrep . The purpose of ligand preparation is to generate the 3D structure (of each ligand) that has minimum energy conformation.The derivatives of 1-(substituted phenyl aminomethyl) -3-(2-(4-(2-oxochroman-3-yl) thiazol-2-yl)hydrazono)indolin-2-ones  at the respective binding site using the Glide  tool. The extra precision (XP) algorithm was employed for the docking operation and output is obtained in the form of docking score. It determines a possible binding pose between the target and the ligand. It also provides information about the most favorable interactions among them ,56,57.4a to 4n) were assessed for their pharmacokinetic efficacy through the QikProp application . It predicts 51 pharmacokinetic properties but the present study includes a few important parameters that are logP (Octanol/Water), apparent Caco-2 permeability QPP Caco), brain/blood partition coefficient (QPlogBB), apparent MDCK permeability (QppMDCk), (QP%) human oral absorption %, and Lipinski rule of five violations (Rule of 5).The test ligands, i.e., the derivatives of 1-(substituted phenyl aminomethyl)-3- (2-(4-(2-oxochroman- 3-yl)thiazol-2-yl)hydrazono) indolin-2-ones (4a–n) have been evaluated as anti-inflammatory and analgesic activities. Selected compounds were further tested for ulcerogenic and lipid peroxidation potential. Only two compounds claimed to be most potent as anti-inflammatory and analgesic molecule with the highest reduction in GI toxicity. In silico study also supports the utility of these two potent ligands as drug candidates and paves the path for future drug development studies. The active compounds showed similar lipid peroxidation activities, and this was mainly due to their closest BDEs and IP values, i.e., the active compounds have the same potency to inhibit lipid radical by a hydrogen atom transfer from the active site of titled compounds to a lipid radical.In conclusion, an improved synthesis of key intermediate through the combined use of deep eutectic solvent and ultrasound is a rational approach to enhance the yield of desired compounds via an economically viable and environmentally acceptable way. The stability of reagents with DES was verified by calculating electronic energy. Further, all the final compounds (


Phyllostachys heterocycla var. pubescens were screened against panel of cancer cell lines and normal one. The cell viability results exhibited that the ethyl acetate extract showed the least vitality percentage of 2.14% of HepG2 cells. Accordingly, it was subjected to chromatographic separation, which resulted in the isolation of a new natural product; 7-hydroxy, 5-methoxy, methyl cinnamate (1), together with four known compounds. The structures of the pure isolated compounds were deduced based on different spectroscopic data. The new compound (1) was screened against the HepG2 and MCF-7 cells and showed IC50 values of 7.43 and 10.65 µM, respectively. It induced apoptotic cell death in HepG2 with total apoptotic cell death of 58.6% (12.44-fold) compared to 4.71% in control by arresting cell cycle progression at the G1 phase. Finally, compound 1 was validated as EGFR tyrosine kinase inhibitor in both enzymatic levels (IC50 = 98.65 nM compared to Erlotinib (IC50 = 78.65 nM). Finally, in silico studies of compound 1 through the molecular docking indicated its high binding affinity towards EGFR protein and the ADME pharmacokinetics indicated it as a drug-like.Different extracts of the Bamboo shoot skin  Phyllostachys edulis, extract proved to induce apoptosis signaling in osteosarcoma cells, associated with AMPK activation −, molecular formula: C11H12O4, NMR data: see 7-Hydroxy, 5-methoxy, methyl cinnamate (2): White amorphous powder; 1H-NMR : δH = 6.79: 6.98 , 5.79 , 5.78 , 3.34 , 5.24 , 3.68 , 5.40 , 4.29 , 4.03 , 3.88 , 3.87 . 13C-NMR : δC = 51.0 (C-1), 85.1 (C-2), 179.9 (C-4), 54.5 (C-5), 87.2 (C-6), 73.8 (C-8), 132.4 (C-1′), 110.6 (C-2′), 149.2 (C-3′), 147.5 (C-4′), 116.2 (C-5′), 119.5 (C-6′), 133.2 (C-1′′), 110.7 (C-2′′), 149.4 (C-3′′), 148.2 (C-4′′), 116.4 (C-5′′), 119.8 (C-6′′), 56.4 (3′-3OCH), 56.5 (3′′-OCH3). 4-Keto-pinoresinol (3): Yellow powder; 1H-NMR : δH 6.29, brs (H-6), 6.64, brs (H -8), 7.39, brs, (H-2′), 7.06, brs, (H-5′), 7.41, brs, (H-6′), 3.88, s, (OCH3), 13.05 (OH). 13C-NMR : δC 148.9 (C-2), 136.0 (C-3), 177.0 (C-4), 162.2 (C-5), 99.5 (C-6), 165.1 (C-7), 94.8 (C-8), 153.9 (C-9), 104.8 (C-10), 123.0 (C-1′), 114.1 (C-2′), 146.3 (C-3′), 149.0 (C-4′), 119.5 (C-5′), 123.8 (C-6′), 56.9 (OCH3). Tamarixetin (4): White amorphous powder; 1H-NMR : δH 7.44, brs (H-2), 6.81, d, J = 10.0 (H-5), 7.46, brd, J = 10.0 (H-6). 13C-NMR : δC 122.0 (C-1), 116.4 (C-2), 144.6 (C-3), 150.0 (C-4), 114.5 (C-5), 122.5 (C-6), 169.9 (CO).3,4-Dihydroxybenzoic acid (5): White amorphous powder; 1H-NMR : δH 6.18, d, J = 15.9 (H-2), 7.41, d, J = 15.9 (H-3), 6.80, brs (H-5), 6.89, d, J = 8.4 (H-8), 7.52, brd, J = 8.4 (H-9), 4.10, s (1-OCH3), 4.20, s, (6-OCH3). 13C-NMR : δC 167.9 (C-1), 114.8 (C-2), 145.0 (C-3), 126.8 (C-4), 109.5 (C-5), 148.1 (C-6), 146.9 (C-7), 114.8 (C-8), 123.1 (C-9), 51.9 (1-OCH3), 55.8 (6-OCH3). Methyl ferulate , the harvested HepG2 cells were subjected to flow cytometric analyses including “FITC/Annexin-V-FITC/PI differential apoptosis/necrosis assessment and DNA content flow cytometry aided cell cycle analysis.”Cell cycle analysis and apoptotic assays were performed to determine in which phase cells would be arrested and to calculate the percentage of apoptotic activity. Full methodologies of flow cytometric assays are provided in the Supplementary File . After t1 against the EGFR [EGFR-TK assay was performed to evaluate the inhibitory potency of novel compound the EGFR . For fulFor the docking studies, the crystal structure of the epidermal growth factor receptor tyrosine kinase (EGFR) was obtained from the Protein data bank (PDB code: 1M17). According to routine work , preparaADME pharmacokinetics parameters of the most active compounds were calculated using a set of software including “MolSoft,” “Molinspiration”, and “SwissADME” websites as previously described by Youssef et al. 2020 .5 cells) are collected by trypsinization and washed twice with ice-cold PBS (pH 7.4). Then, cells are incubated in dark with 0.5 mL of Annexin V-FITC/PI solution for 30 min in dark at room temperature according to manufacturer protocol. After staining, cells are injected via ACEA Novocyte™ flowcytometer  and analyzed for FITC and PI fluorescent signals using FL1 and FL2 signal detector, respectively (λex/em 488/530 nm for FITC and λex/em 535/617 nm for PI). For each sample, 12,000 events are acquired and positive FITC and/or PI cells are quantified by quadrant analysis and calculated using ACEA NovoExpress™ software .Apoptosis and necrosis cell populations are determined using Annexin V-FITC apoptosis detection kit  coupled with 2 fluorescent channels flowcytometry. After treatment with test compounds for 48 h, cells (105 cells) are collected by trypsinization and washed twice with ice-cold PBS (pH 7.4). Cells are re-suspended in two milliliters of 60% ice-cold ethanol and incubated at 4 °C for 1 h for fixation. Fixed cells are washed twice again with PBS (pH 7.4) and re-suspended in 1 mL of PBS containing 50 μg/mL RNAase A and 10 μg/mL propidium iodide (PI). After 20 min of incubation in dark at 37 C, cells are analyzed for DNA contents using flow cytometry analysis using FL2 (λex/em 535/617 nm) signal detector . For each sample, 12,000 events are acquired. Cell cycle distribution is calculated using ACEA NovoExpress™ software .After treatment with test compounds for 48 h, cells . 5ʹ upstream to the EGFR sequence comprised a sequence that encoded (His)6. HepG2 cells were infected for 48h for protein expression. The pellets of HepG2 cells were solubilized in a buffer containing sodium vanadate (100 µM), aprotinin (10 µg/mL), triton (1%), HEPES buffer (50 mM), ammonium molybdate (10 µM), benzamidine HCl (16 µg/mL), NaCl (10 mM), leupeptin (10 µg/mL) and pepstatin (10 µg/mL) at 0 °C for 20 min at pH 7.4, followed by centrifugation for 20 min. To eliminate the nonspecifically bound material, a Ni-NTA superflow packed column was used to pass through and wash the crude extract supernatant first with 10 mM and then with 100 mM imidazole. Histidine-linked proteins were first eluted with 250 and then with 500 mM imidazole subsequent to dialysis against NaCl (50 mM), HEPES (20 mM), glycerol (10%) and 1 µg/mL each of aprotinin, leupeptin and pepstatin for 120 min. The purification was performed either at 4 °C or on ice. To record autophosphorylation level, EGFR kinase assay was carried out on the basis of DELFIA/Time-Resolved Fluorometry. The compound was first dissolved in DMSO absolute, subsequent to dilution to appropriate concentration using HEPES (25 mM) at pH 7.4. Each compound (10 µL) was incubated with recombinant enzyme  for 10 min at 25 °C, subsequent to the addition of 5X buffer  and ATP-MgCl2  and incubation for 1h. The negative and positive controls were included in each plate by the incubation of enzyme either with or without ATP-MgCl2. The liquid was removed after incubation and the plates were washed thrice using wash buffer. Europium-tagged antiphosphotyrosine antibody  was added to each well followed by incubation of 1h and then washing of the plates using buffer. The enhancement solution was added to each well and the signal was recorded at excitation and emission wavelengths of 340 at 615 nm. The autophosphorylation percentage inhibition by compounds was calculated using the following equation:EGFR-TK assay was performed to evaluate the inhibitory potency of novel compound 50 was calculated. Majority of signals detected by antiphosphotyrosine antibody were from EGFR because the enzyme preparation contained low impurities.Using the curves of percentage inhibition of five concentrations of each compound, ICPhyllostachys heterocycla var. pubescens exhibited potent cytotoxic activity and showed vitality percentage of 2.14% of HepG2 cells. This extract afforded five compounds of which one new compound was identified as 7-hydroxy, 5-methoxy, methyl cinnamate. The new compound was screened against the HepG2 and MCF-7 cells and exhibited IC50 values of 7.43 and 10.65 µM, respectively, with non-cytotoxic activity against the THLE2 cells. It induced total apoptotic cell death of 58.6% (12.44-fold) compared to 4.71% in control by arresting cell cycle progression at the G1 phase. Moreover, the EGFR activity was validated by the mechanism of action through both enzymatic and in silico levels. The study demonstrated the edible plant Phyllostachys heterocycla var. pubescens as an excellent promising anticancer agent.The ethyl acetate extract of


The structures of the isolated compounds were elucidated on the basis of NMR , UV, FT-IR and LC-Q-TOF-MS spectrometric data.Chromatographic separation of Treatment with plants is as old as human history. Mankind has benefited from natural herbal resources to protect health and treat against diseases. The isolation and characterization of the active compounds found in plants started to develop from the beginning of the 19th century with developing technology. Numerous natural compounds in the scientific process, compounds such as morphine, penicillin, codeine, atropine, resveratrol, and digoxin, were isolated from natural sources and took place in the treatment processes. The isolation of the compounds from natural sources and the clarification of their structures are extremely important in the scientific process as they have the potential to become a new drug-active substance. Due to the potential of natural compounds to be exemplary molecules in active substance synthesis, studies in this field are considered to be inevitable for serving humanity. Scorzonera L. species are known to be used in complementary medicine and consumed as a vegetable in the world and Turkey [1-4].S. aucherianaknown as “Buz Tekesakalı” in Turkey is caulescent, herbaceous perennial herbs endemic to Turkey [4]. Their rootstock is thick and crowned by decayed leaf remains [2]. Members of Scorzonera includingS. aucherianaare characterized by a rich latex ingredient [2].The genus Scorzonera L. is a member of tribe Cichorieae (Asteraceae), and it is represented by around 180-190 species in the worldwide [1-2]. The major center in terms of diversity is the arid and mountainous Irano-Turanian region [3]. The genus is represented by perennial herbs often having a caudex or tuber under ground [2]. There are 52 species in Turkey and 31 of them are endemic [4]. Some members of this genus are consumed as a vegetable in Turkey [5-6].Scorzonera mongolicaMaxim. andScorzonera austriacaWilld. are grown in China and are used in the treatment of fever reduction, boils, and nipple inflammation [12-14].Scorzonera divaricataLipsch root and aboveground parts are used in the treatment of ulcers and malignant stomach tumors;Scorzonera pseudodivaricataTurcz is used in febrile conditions caused by pulmonary edema, diuretic, and viral infection [15].Scorzonera creticaWilld., Greek cuisine is used in dishes such as savory [16].S. pseudodivaricataandS. divaricataspecies grow in Mongolia and are used for medical purposes.Scorzonera hispanicais the most commonly grown and cultivated ornamental plant in Europe, as well as being used to treat colds, lung diseases, appetizing, mucolytic, stomatic, diuretic, antipyretic effects, snake bites, and it is also added to coffee [17].Scorzonera humilisL. is used in wound healing and gastrointestinal disorders [18]. Rootstock and fresh shoots of some of the Scorzonera member grown in Turkey are consumed fresh or cooked under the name of ‘manger’. It has been stated that these plants are used for the purpose of removing high blood pressure, kidney diseases, diabetes, moxibustion, infertility, and vascular stiffness .It has been stated that different Scorzonera species are used as pain relievers among the public, and scientific studies in our country have analgesic, antiinflammatory and wound-healing effects [7-11].Phytochemical studies of Scorzonera species gave the guaianolid class sesquiterpene lactones, flavonoids , cafeoyl quinic acids (chlorogenic acid), coumarins, lignans, neolignans, bibenzyl derivates, benzyl phthalides, stilbenoids and triterpenoid class of natural compounds. Studies show that the Scorzonera species is quite rich in terpenic and phenolic compounds .Scorzonera radiatahave reported to have very strong antioxidant activity [21]. Antioxidant (antiproliferative effect on L5178Y mouse lymphoma cells) was investigated in bioactivity directed isolation studies of methanolic extracts prepared from above ground parts ofS. pseudodivaricataandS. divaricataplants. Feruloyl podospermic acid A and B compounds isolated fromS. divaricatahave been investigated; their DPPH radical scavenging effect has been searched and reported to have stronger antioxidant activity standard [15].Bioactivity studies have also been conducted on Scorzonera genus; the dihydrostilben derivative, Skorzodihidrostilben A, B, C, D, and E compounds obtained from the above ground parts ofS. hispanicawas determined [22]. The antinociceptive activities of Scorzonera species have been tested and it has been reported thatScorzonera latifolia(Fisch. & Mey.) DC. is the most effective and those antinociceptive effective taraxasterol acetate and taraxasterol myristate compounds are isolated by bioactivity directed fraction [10]. It was reported that the pilatifillozide compound was isolated from the cytotoxic activated directed study ofS. pseudodivaricataplant [15]. The cytotoxic activity of thyrolobibenzyls obtained fromS. humilis, antibacterial activity against P-388 cells, and antifungal activity againstCandida albicansandTrichophyton mentagrophyteswere investigated, but it was reported that the compounds showed no activity . Flavonoids constitute an important class of natural compounds with polyphenolic structure. Flavonoid compounds are known to have antioxidant activity associated with diseases such as Alzheimer’s, cancer, and atherosclerosis. Phenolic compounds have antiinflammatory, anticarcinogenic, antimutagenic, and antioxidative activities due to their capacity to modulate cellular enzyme functions. Polyphenolic compounds are widely used in complementary therapy in nutraceutical, pharmaceutical, medical, and cosmetic applications [24].The immunomodulatory effect of syringarezinol glycoside I isolated fromS. aucheriana, which we plan to work on. Chemical diversity of Scorzonera species is of great importance in terms of the inclusion of new therapeutic compounds in the scientific world. In addition, the determination of the compounds responsible for the biological activity indicates that the study within this plan will be of special importance.According to the best of our knowledge, there is no any secondary metabolite isolation, structure determination, and biological activity studies withS. aucherianawas collected in July 2018 from Sivas province, Zara district, Yarağıl region in Turkey. The plant was identified by Prof. Kamil Çoşkunçelebi by using Flora of Turkey [2] and taxonomical conspectus of Turkish Scorzonera [4]. Voucher (Makbul 244 & Coşkunçelebi) was deposited in the Herbarium of Biology (KTUB) at Karadeniz Technical University, Turkey. The plant cleaned to remove impurities, dried, and stored in air-tight container before use.-1) spectrometer . The mass spectral analyses were carried out on Agilent 6230A LC-Q-TOF-MS. Shimadzu QP2010 ultra GC-FID/MS was used to identify the FAMEs. Melting points were determined using Thermo-var apparatus fitted with a microscope and uncorrected. 1H and 13C NMR, along with 2D NMR spectra, were obtained on a Bruker 400 MHz NMR spectrometer , using TMS as an internal standard. CD3OD and CDCl3 were used as NMR solvent. ACD NMR program was used for the elucidation of isolated compounds. Chemical shifts were expressed in δ (ppm) and coupling constants (J) were reported in hertz (Hz). CC was carried out on silica gel , RP-18 silica gel, and PTLC . TLCs were carried on silica gel  plates, and the spots were visualized by UV lamp or spraying with 20% H2SO4 and heating.Optical rotations were measured on an Automatic AA-5 series polarimetry. UV spectra were obtained with a Spectrostar nano BMG labtech spectrometer. Infrared spectra were obtained with a PerkinElmer 1600 FT-IR (ATR) (4000-400 cmS. aucheriana(833 g) were macerated three times with methanol (4 L) for 72 h at room temperature on a stirring. Total extracts were evaporated under vacuum at a temperature not exceeding 40 ºC using a chiller (-10 oC) to yield crude extracts (60.6 g). The crude methanol extract (42.96 g) was dissolved in MeOH-H2O (2:8) and then successively fractionated with n-hexane (50 mL × 3), chloroform (50 mL × 3), ethyl acetate (50 mL × 3) and water (80 mL) to yield 12.9 g, 1.8 g, 4.6 g, and 20.9 g, respectively.Dried powdered aerial parts ofn-hexane-AcOEt , and AcOEt-methanol  solvent mixture to give 106 fractions . After the TLC control, some of the fractions were combined and EA-Fr 83-91 (135 mg) was further separated by PTLC  using AcOEt-MeOH-H2O (70:30:4.5) mobile phase to provide A (16 mg), B (15.4 mg), C (20.9 mg), and D (58.5 mg) bands. NMR data of bands A-C seemed to be so complex mixture and not further study was done. Band D was further separated by reverse phase CC  using decreased polarity of H2O:MeOH  mobile phase to give 45 subfraction . After the TLC control, EA-SFr 12 and EA-SFr 18 afforded compounds 1 (6.4 mg) and 2 (6.2 mg), respectively.The crude ethyl acetate fraction (4.6 g) was subjected to CC  using increased polarity ofn-hexane fraction (12.9 g) was subjected to CC  using increased polarity ofn-hexane-chloroform , chloroform- AcOEt , and ethyl acetate-metanol  mobile phase to give 63 fractions (H) (~40 ml each). After the TLC control, some of the fractions were combined and H-Fr 34-42 (610 mg) was purified by repeated CC  with increasing polarity ofn-hexane-CHCl3 , andn-hexane-AcOEt solvent mixture  to give 65 subfractions (SFr) . After the TLC control, H-SFr 7 and H-SFr 13 gave the compounds 6 (12.2 mg) and 4 (25.8 mg), respectively.The cruden-hexane-AcOEt  to give 50 subfractions . After the TLC control, H-SFr 19 and H-SFr 34 afforded compounds 7 (8.1 mg) and 9 (9.8 mg), respectively. After the TLC control, crude n-hexane fractions (H-Fr) 10-14 were combined and purified by CC  with increasing polarity of n-hexane-CHCl3 , and CHCl3-AcOEt  to give 78 subfraction (H-SFr) . After the TLC control, H-SFr 27 afforded compound 3 (30.2 mg). Subfractions (H-SFr1) 35 and 36 combined (100.3 mg) and were further purified by CC  with increasing polarity of petroleum ether-dichloromethane  to give 66 subfraction-2 (H-SFr2) . H-SFr2 16-18 and SFr2 19-21 afforded compounds 8 (4.7 mg) and 5 (4.9 mg), respectively.H-Fr 15-17 (2.24 g) was purified by again CC  increasing polarity ofCompound 1 (Methyl 1-(2-methylcyclopropyl-1-carbonyloxy) chlorogenate)-1): 3368 (-OH), 3050 (=CH), 2949, 2860 (-CH), 1658, 1594 (C=O), 1490, 1450 (C=C) 1268, 1121, 1033 (C-O); C22H26O10, LC-QTOF-MS: m/z (%) [M+Na]+ 473.1412 (08), calc. 473.1417; [M-OCH3]+ 419.1189 (08), calc. 419.1186; [M-CH3COO-CH3-H]+ 375.0661 (30), calc. 375.0663; [M-99+CO2+2Na]+ 353.0841 (100), calc. 353.0838. 1H and 13C NMR data see table 1.Colorless semisolid, m.p. 198-200 °C; [α]D24 +90.9 ; Rf: 0.45 ; UV (Methanol) λmax (nm): 216, 245, 324. FT-IR ] chlorogenic acid)D24 +133 ; Rf: 0.70 ; UV (Methanol) λmax (nm): 218, 243, 327; FT-IR : 3421 (-OH), 3065 (=CH), 2946, 2851 (-CH), 1703, 1657, 1597 (C=O), 1490, 1417 (C=C), 1270, 1033 (C-O); C40H34O15, LC-QTOF-MS: m/z (%) [M-H]+ 753.6709 (10), calc. 753.6704; [M-H2O-H]+ 735.6552 (05), calc. 735.6558; [M-CO2-2H2O+H]+ 675.6651 (100), calc. 675.6669. 1H and 13C NMR data see table 2.Colorless semisolid, m.p. 220-224 °C; [α]Taraxasterol oleate (3)D24 +175.4 ; Rf: 0.68 ; FT-IR : 3055 (=CH), 2935, 2890 (-CH), 1657 (C=O), 1495, 1440 (C=C), 1280 (C-O); C48H82O2, LC-QTOF-MS: m/z (%) [M]+ 691.1509 (6), calc. 691.1512; [M-C4H9-H]+ 633.0808 (12), calc. 633.0818; [M-oleic acid+H]+ 409.7036 (10), calc. 409.7032; [Aglycone+CO2-H]+ 468.7038 (45), calc. 409.7031.Colorless semisolid, m.p. 47-48 °C; [α]Aglycone: 13C NMR  δ (ppm): 38.4 (C-1), 23.7 (C-2) , 80.6 (C-3), 37.8 (C-4), 55.4 (C-5), 18.1 (C-6), 34.0 (C-7), 40.9 (C-8), 50.3 (C-9), 37.0 (C-10), 21.4 (C-11), 26.1 (C-12), 39.1 (C-13), 42.0 (C-14), 26.6 (C-15), 38.3 (C-16), 34.5 (C-17), 48.6 (C-18), 39.3 (C-19), 154.6 (C-20), 25.6 (C-21), 38.8 (C-22), 27.9 (C-23), 16.3 (C-24), 16.6 (C-25), 15.8 (C-26), 14.7 (C-27), 19.5 (C-28), 25.5 (C-29), and 107.2 (C-30); 1H NMR  δ (ppm): 4.42 , 0.78 , 0.77 , 0.81 , 0.81 , 0.83 , 0.86 , 0.89 , 4.44 , 4.40 Glycone:13C NMR  δ (ppm): 173.1 (C=O), 130.1 (=CH), 129.6 (=CH), 34.87 (CH2), 31.95 (CH2), 29.73 CH2), 29.70 (CH2), 29.67 (CH2), 29.61(CH2), 29.49 (CH2), 29.39 (CH2), 29.29 (CH2), 29.20 (CH2), 29.20 (CH2), 27.21(CH2), 25.19 (CH2), 22.72 (CH2), 14.1 (CH3); 1H NMR  δ (ppm): 5.32 , 2.33 , 1.20 .Taraxasterol (4)f : 0.42 ; 13C NMR  δ (ppm): 154.7 (C-20), 107.2 (C-30), 79.0 (C-3), 28.0 (C-23), 15.4 (C-24), 16.3 (C-25), 15.9 (C-26), 15.4 (C-7), 19.5 (C-28), and 25.6 (C-29); 1H NMR  δ (ppm): 4.60 , 4.62 , 3.21 , 0.85 , 0.87 , 1.01 , 0.93 , 0.85, 0.77 , and 1.02 .RTaraxasterol acetate (5)f: 0.39 ; 13C NMR  δ (ppm): 171.0 (OC=O), 154.70 (C-20), 107.1 (C-30), 80.99 (C-3), 27.8 (C-23), 16.5 (C-24), 15.8 (C-25), 16.3 (C-26), 14.6 (C-27), 19.4 (C-28), 25.5 (C-29), and 21.3 (CH3CO); 1H NMR  δ (ppm): 4.62 , 4.60 , 4.45 , 2.05 , 0.85 , 0.87 , 1.01 , 0.93 , 0.85 , 0.77 , and 1.02 .RPtiloepoxide (6)f: 0.32 ; 13C NMR  δ (ppm): 151.3 (C-20), 112.0 (C-30), 78.9 (C-3), 64.0 (C-22), 56.1 (C-21), 28.0 (C-23), 15.4 (C-24), 16.2 (C-25), 15.9 (C-26), 14. 8 (C-27), 15.1 (C-28), and 27.2 (C-29); 1H NMR  δ (ppm): 5.06 , 4.87 , 3.47 , 3.21 , 2.91 , 0.97 , 0.77 , 0.84 , 1.02 , 0.95 , 0.81 , and 1.05 .RLupeol (7)f: 0.35 ; 13C NMR  δ (ppm): 151.0 (C-20), 109.3 (C-29), 79.0 (C-3), 28.0 (C-23), 15.4 (C-24), 16.1 (C-25), 16.0 (C-26), 14.6 (C-27), 18.0 (C-28), and 19.3 (C-30); 1H NMR  δ (ppm): 4.69 , 4.57 , 3.20 , 1.00 , 0.76 , 0.83 , 1.03 , 0.97 , 0.79 , and 1.68 .RLupeol acetate (8)f: 0.33 ; 13C NMR  δ (ppm): 171.09 (OC=O), 151.0 (C-20), 109.4 (C-29), 80.99 (C-3), 27.6 (C-23), 16.7 (C-24), 16.4 (C-25), 16.2 (C-26), 14.7 (C-27), 18.1 (C-28), 19.6 (C-30), and 21.3 (CH3CO); 1H NMR  δ (ppm): 4.69 , 4.57 , 4.48 , 2.05 , 0.86 , 0.84 , 1.02 , 0.83 , 0.79 , 0.95 , and 1.68 .R-Sitosterol (9)βf: 0.41 ; 13C NMR  δ (ppm): 140.8 (C-5), 121.8 (C-6), 71.8 (C-3), 11.9 (C-18), 19.4 (C-19), 18.8 (C-21), 19.0 (C-26), 19.9 (C-27), and 12.0 (C-29); 1H NMR  δ (ppm): 5.36 , 3.53 , 0.68 , 1.01 , 0.92 , 0.80 , 0.82 , and 0.84 .R22H26O10 from the LC-Q-TOF-MS data . The UV spectrum of 1 showed absorption maxima at 324, 245, and 216 nm, typical of a caffeic acid derivative. The 1H NMR data were very similar to those of the caffeic acid moieties of chlorogenic acid methyl ester, but differed from known compounds with regard to the signals of the quinic acid moiety [25-26]. The 1H NMR spectrum of 1 (Table I) showed two pairs of doublets with coupling constants of 16.0 Hz indicative of trans olefinic protons found in hydroxy cinnamic acid. In the aromatic region, resonances for two ABX systems at δ 7.05 , 6.73 , and 6.92 ppm  were observed, which were assigned to the 1,3,4-trisubstituted phenyl units. From these observations, along with the analysis of the 13C and APT NMR data (Table 1), the presence of one caffeic acid moiety was inferred. The assignments were further supported by analysis of the COSY, HMBC, and HSQC spectrum of 1. The presence of the quinic acid moiety was indicated by 1H NMR resonances of three oxymethine protons at δ 5.39 ppm , 3.70 ppm , and 4.14 , together with two pairs of sp3 methylene protons at δ 2.15/2.05 ppm and 2.20/1.97 ppm for H-2a/2b and H-6a/6b, respectively. The latters are characteristics of a quinic acid unit, with regard to their multiplicity and coupling patterns. The assignments of the protons of the quinic acid nucleus were corroborated by analysis of the COSY and TOCSY spectra of 1. The attachment of caffeoyl moieties to the quinic acid part was deduced from the chemical sifts of H-3. The deshielded resonances of the oxymethine protons in the quinic acid nucleus at δ 5.39 ppm (H-3) implied acylation of the hydroxy group at these positions as reported before for other naturally occurring quinic acid derivatives [25-26]. The 1H NMR spectrum of compound 1 displayed a methoxy at δ 3.33 ppm  which was substitute to the quinic acid C-7 (169.01 ppm) that was shifted upfield. From these observations, the structure of 1 was initially thought to be that of the known compound 3-caffeoylquinic acid methyl ester (chlorogenic acid methyl ester). However, the 1H NMR spectrum of 1 showed slightly but distinctly different peak patterns of the (2-methylcyclopropyl) carbonyl unit at δ 1.78/1.60 , 1.05-0.88 , and 1.02  and carbon peaks at δ 24.21 , 20.98 and 22.18 , and 10.90  which is substituted by ester linkage at C-1 position of quinic acid moiety of compound 1. All of the assigned NMR data of compound 1 is shown in table 1. Therefore compound 1 was elucidated as methyl 1-(2-methylcyclopropyl-1-carbonyloxy) chlorogenate.Compound 1 was obtained as a white solid. The molecular formula was determined as C+, calc. 753.6704). The FT-IR spectrum indicated the presence of an -OH group (3421 cm-1), carbonyl groups (1657 and 1597 cm-1), double bonds (1490 cm-1). The UV spectrum showed absorption maxima at 327, 243, and 218 nm which characteristic to caffeic acid moiety. The presence of a quinic acid moiety was characterized in the 1H NMR spectra of three oxymethine protons at δH 5.40 , 3.71 , and 4.13 , and the two pairs of sp3 methylene protons at δH 2.16/1.97  and 2.12/2.02  for H-2 and H-6 indicating the substitution at C-3 position. The presence of a mono substituted quinic acid moiety was suggested and then confirmed by the 13C NMR spectrum, δ C 76.38 , 37.62 , 71.71 , 73.68 , 71.18 , and 39.29 . The 1H NMR spectrum indicated two protons of trans-configuration for double bonds, δ 7.59  and 6.30 . In the aromatic region, resonances for three ABX systems, δH 7.05 , 6.77 , and 6.95 , were assigned to 1,3,4-trisubstituted phenyl unit. The part of the NMR data of compound 2 were very similar to those of the chlorogenic acid, but differed from known compounds with regard to the carbon signals of the caffeic acid moiety [25-26]. It was illuminated that the groups in the three-cyclic spiro structure were connected to the C-3′ and C-4 ′ positions of the caffeic acid part of compound 2. In the 1H NMR spectrum of compound 2, the acetal proton in ring A was at δ 7.00 ppm , the methylenic protons in ring B were at δ 3.57  and 2.25 , and also the α,β-unsaturated protons in the ring C were at δ 7.11  and 6.25 . The assignments of all moieties of compound 2 were further supported by analysis of the 1H-1H COSY and HMBC correlation . From these observations, along with the analysis of the 13C, APT, the COSY, HMBC, and HSQC NMR data of 2 (Table 2), the presence of two 3,4-bis moiety was inferred. Thus, compound 2 was assigned as 3,4-bis chlorogenic acid, which was a new compound (2) and given the trivial name as scorzonerenone.Compound 2 had a molecular formula of C40H34O15 with 24 unsaturated degrees as determined by NMR and LC-Q-TOF-MS data  and knowns as: taraxasterol (4), taraxasterol acetate (5), ptiloepoxide (6), lupeol (7), lupeol acetate (8), and β-sitosterol (9). The structures of these compounds were elucidated by 1H, 13C, APT, and COSY spectra, and their data were compared with the reported data obtained from the literature. Triterpene compounds containing long chain fatty acids were characterized from natural sources. Of the peaks observed in the proton and carbon NMR spectrum of compound 3, the triterpene ring was detected from the peaks in the proton and carbon NMR spectra (80.6 (C-3), 154.6 (C-20), 107.2 (C-30), and seven methyl peaks at δ 27.9 (C-23), 16.3 (C-24), 16.6 (C-25), 15.8 (C-26), 14.7 (C-27), 19.5 (C-28), 25.5 (C-29) and 4.42 , 0.78 , 0.77 , 0.81 , 0.81 , 0.83 , 0.86 , 0.89 , 4.44 , 4.40 ), where the triterpene ring was taraxasterol. 1H NMR spectra for the aglycone portion of compound 3 revealed peaks at δ 5.32 , 2.33 , 1.20  and the carbonyl peak at δ 171.00 ppm, olefinic carbon peaks at δ 130.1 , 129.6 , and terminal methyl peak at δ 14.1 ppm  in the 13C NMR spectrum suggested that glycone part of compound 3 was oleic acid. Thus, compound 3 was characterized as taraxasterol olaeate (urs-20(30)-en-3-ol, 3β-[(9Z)-9-octadecenoate]) and was not found in the literature. However, the similar compounds (urs-20(30)-en-3-ol, octadecanoate, urs-20(30)-en-3-ol, eicosanoate, urs-20(30)-en-3-ol, 3-hexadecanoate, and urs-20(30)-en-3-ol, 3-dodecanoate) were mentioned in the literature [35-38].In this research, the structure of one new and six known triterpenoids from the aerial parts of1H NMR spectrum of compound 4 revealed a multiple at δ 3.21  for the H-3 and methylene protons at δ 4.60 , and 4.62  ppm as two broad singlets. The same spectrum showed signals for methyls at δ 0.85 , 0.87 , 1.01 , 0.93 , 0.85 , 0.77 , and 1.02 . The 13C NMR spectrum showed 30 signals. The peaks at δ 154.7 (C-20) and 107.2 (C-30) are related to the olefinic carbons. The methine signal at δ 79.0 ppm was for C-3. The peaks at δ 28.0 (C-23), 15.4 (C-24), 16.3 (C-25), 15.9 (C-26), 15.4 (C-27), 19.5 (C-28), and 25.6 (C-29) ppm were related to the methyl carbons. Compound 4 was characterized as taraxasterol [39]. Compound 5 showed similar NMR data as compound 4. However, in the 1H NMR spectra of compound 5, the peaks at δ 4.45 ppm  for H-3 and, the singlet peak at δ 2.05 ppm  for COCH3, and the carbonyl peak at δ 171.00 ppm in the 13C NMR spectrum suggested that compound 5 was taraxasterol acetate [40].The 1H NMR spectrum of compound 6 displayed a dd at δ 3.21  for the H-3. This is deshielded proton as a result of -OH substitution. 1H NMR spectrum of compound 6 displayed methylene protons at δ 5.06  and 4.87  ppm as two singlets and epoxide protons at δ 3.47 , and at δ 2.91 ppm . The same spectrum showed signals for Me-23 at δ 0.97 (s), Me-24 at δ 0.77 (s), Me-25 at δ 0.84 (s), Me-26 at δ 1.02 (s), Me-27 at δ 0.95 (s), Me-28 at δ 0.81 (s), and Me-29 at δ 1.05 . The 13C NMR spectral data showed 30 signals. The peaks at δ 151.3 (C-20) and 112.0 (C-30) ppm are related to the olefinic carbons. The methines signals at δ 78.9 for C-3 and at δ 56.1 (C-21), 64.0 (C-22) ppm related to the epoxide carbons. Compound 6 was identified to be ptiloepoxide [41].The 1H NMR spectrum of compound 7 gave a peak at δ 3.20  for the H-3 and methylene protons at δ 4.69 , and δ 4.57  ppm. The same spectrum showed seven signals for methyl peaks at δ 0.76 , 0.83 , 1.03 , 0.97 , 0.79 , 1.68 , and 1.68  ppm. The 13C NMR spectrum showed 29 signals. The peaks at δ 151.0 (C-19) and 109.3 (C-21) are related to the olefinic carbons. The methine signal at δ 79.0 ppm was for C-3. The peaks at δ 28.0 (C-20), 15.4 (C-24), 16.1 (C-25), 16.0 (C-26), 14.6 (C-27), 18.0 (C-28), and 19.3 (C-29) ppm were related to the methyl carbons. Compound 7 was elucidated as lupeol [42]. Compound 8 revealed the similar NMR data as compound 7. However, in the 1H NMR spectra of compound 8, the peaks at δ 4.48 ppm  for H-3 and, the singlet peak at δ 2.05 ppm  for COCH3, and the carbonyl peak at δ 171.09 ppm in the 13C NMR spectrum supported that compound 8 was lupeol acetate [43].The 1H NMR spectrum of compound 9 showed a multiple peak at δ 3.53  for the H-3 and CH proton at δ 5.36  ppm. The same spectrum showed signals for methyl at δ 0.68 , 1.01 , 0.92 , 0.80 , 0.82 , 0.84 . The 13C NMR spectral data showed 29 signals. The peaks at δ 140.8 (C-5), 121.8 (C-6), are related to the olefinic carbons. The methine signal at δ 71.8 ppm was for C-3. The peaks at δ 11.9 (C-18), 19.4 (C-19), 18.8 (C-21), 19.0 (C-26), 19.9 (C-27), and 12.0 (C-29) ppm were related to the methyl carbons. Compound 9 was identified as β-sitosterol [44].The S. divaricataand exhibited significant cytotoxic activities in vitro against four human cancer cell lines  [45].n-Hexane extract ofS. latifoliaroots has given 3β-hydroxyfern-7-en-6-one-acetate, urs-12-en-11-one-3-acetyl, 3β-hydroxy-fern-8-en-7-one-acetate, olean-12-en-11-one-3-acetyl, and leucodin [8]. D:B-friedoolean-type triterpene has been isolated from the roots ofS. austriacaand in vitro cytotoxicities of triterpenes had tested to cancer cells of human promyelocytic leukemia (HL-60) and human hepatoma (BEL-7404) [46]. Isolation of triterpenes  were reported fromS. mongolica. Lupeol, betulin, betulinic acid, lupeo acetate, and 23Z-3β-acetoxyeupha-7,23-diene-25-ol had demonstrated significant inhibitory effects on A-549 cancer cells at the concentration of 50 mg/L-1 [47]. Two erythrodiol triterpene fatty esters (3β-dodecanoyl erythrodiol and 3β-tetradecanoyl erythrodiol) were characterized fromS. mongolicaand their antitumor effects in vitro has been evaluated with MTT and SRB assays, but showed moderate cytotoxicity on A-549 cell line [48].β-Sitosterol, cholesterol, and other triterpenes have been isolated fromS. mongolica[49]. From the roots ofScorzonera undulatassp.deliciosa(Guss.) Maireβ-amyrin acetate, stigmasterol, andβ-sitosterol were reported [50]. Two new triterpene fatty esters (3β-tetradecanoyloxy-28-hydroxylolean-18-ene and 3β-dodecanoyl-28-hydroxylolean-18-ene) were characterized byS. mongolica[51]. Isolation ofβ-amyrin acetate, methyl oleanate, methyl ursolate, stigmasterol, andβ-sitosterol was given fromS. undulatassp.deliciosaand the methanol extract had examined for in vitro antioxidant properties using DPPH test [52].n-Hexane extract of theS. latifoliahad yieldedα-amyrin, lupeol, lupeol acetate, taraxasteryl acetate, 3β-hydroxy-fern-7-en-6-one acetate, 3-acetyl urs-12-en-11-one, 3β-hydroxy-fern-8-en-7-one acetate, and 3-acetyl olean-12-en-11-one [53].Triterpenes are common natural compounds for the various Scorzonera species, which were mentioned in the liteature [45-53]. Isolation of three tirucallane triterpenes were mentioned from roots ofS. aucherianaafforded nine natural compounds. Two chlorogenic acid derivatives were isolated as new compounds ( 1 and 2). Seven triterpenoids and derivatives were also isolated, and taraxasterol oleate (3) was found to be a new compound along with six known (4-9) ones. All compounds (1-9) were isolated and identified for the first time from this species.Hexane and ethyl acetate fraction of a crude methanol extract of the


Microcera larvarum led to the isolation of a γ-lactone with a furopyran-5-one bicyclic ring system (1) and three known compounds, -4-hydroxymellein (2), -4-hydroxymellein (3) and 7-hydroxy-3-(1-hydroxyethyl)isobenzofuran-1(3H)-one (4). Structure elucidation was conducted by NMR spectroscopic methods. Absolute configuration of 1  was established using the chiral derivatizing agent MPA and was fully supported by calculated specific rotation and ECD spectra. The spectroscopic data observed for 1 were identical to those previously reported for theissenolactone A (7), necessitating a correction of the latter (from C-5/C-8 trans ring fusion to cis). Compounds 1–4 were evaluated for antimicrobial activity against a panel of pathogens.Antimicrobial bioassay-guided fractionation of  Penicillium. Aotearoa New Zealand has a treasure trove of unique fungi that have not been exhaustively searched for new antibiotics. Thus, we have recently begun investigating fungal isolates from the International Collection of Microorganisms from Plants (ICMP), which contains cultures derived from plants and soil from Aotearoa New Zealand and the South Pacific, in pursuit of novel antimicrobials. Bioassay-guided investigation of the ascomycete fungus Microcera larvarum led to the isolation of a γ-lactone with a furopyran-5-one bicyclic ring system (1) and three other known natural products including two hydroxymelleins (2 and 3) and an isobenzofuranone (4)  .b]pyran-5-one bicyclic ring system as 1 identified three isomeric compounds, 5–7 , reported by Liang et al. from the fermentation broth of Theissenia cinerea 89,091,602 + which was consistent with the molecular formula C11H16O4  and 5.42 ), four oxymethines , 4.04 , 3.45  and 3.34 ), a methine ), a diastereotopic methylene  and 1.73 ) and two methyl groups  and 1.33 ). The 13C-NMR data (C 176.0 (C-6)), two olefinic (δC 127.8 (C-10) and 133.0 (C-11)), four oxymethine (δC 66.6 (C-3), 77.7 (C-8), 80.5 (C-7), 81.5 (C-2)), one methine (δC 39.0 (C-5)), one methylene (δC 28.3 (C-4)) and two methyl carbons (δC 17.6 (C-9) and 18.2 (C-12)). The COSY spectrum established the connectivity order as H3-12 to H-11 to H-10 to H-2 to H-3 to H2-4 to H-5 to H-8 to H-7 to H3-9  showed that the data were identical D28 = +47.7  D22 = +23.7 , but was of the same sign. Taken together, these results indicate 1 and 7 are the same compound and thus the correct structure of theissenolactone A is 1.Comparison of the in CD3OD  with thedentical . This wa singlet . This wa1, the chiral derivatizing agent α-methoxyphenylacetic acid (MPA) was used to prepare diastereomeric esters at C-3. Treatment of 1 with (S)-MPA and (R)-MPA, respectively, in the presence of EDC.HCl and DMAP afforded derivatives 1a and 1b. Comparison of the 1H-NMR spectra of the two compounds -δ(S) as positive, establishing S configuration at H-3 pyran-5-one (1) (3.09 mg) after further purification by silica gel column chromatography, eluting with gradient n-hexane/EtOAc. Purification of F4 by Diol-bonded silica gel column chromatography, eluting with gradient n-hexane/EtOAc, afforded  4-hydroxymellein (3) (4.0 mg).Forty PDA plates were inoculated with ICMP 5444 and incubated at room temperature for 4 weeks. Fully grown fungal plates were freeze-dried  and extracted with MeOH (2 × 500 mL) for 4 h followed CHD22 = +23.7 ; UV (MeOH) λmax [log ε] 318.5 (2.30), 224.0 (2.93), 204.5 (3.38); ECD (MeOH) λmax (Δε) 216 (+1.94); m.p. 129–131 °C; Rf 0.70 (EtOAc); IR (ATR) vmax 2959, 2949, 2917, 1649, 1457, 1377, 666 cm−1; Refer to 1H and 13C-NMR data; (+)-HRESIMS m/z 235.0940 [M + Na]+ .White solid; [α]D19 = +38.2  [lit [α]D20 = +32  + .White solid; [α]1, MeOH) ]; m.p. 1D19 = +21.2  [lit [α]D = +37.4  + .White solid; [α]3, MeOH) ]; 1H-NMRD21 = −14.7  [lit [α]D20 = −50.0  + .White solid; [α], CHCl3) ]; m.p. 1, CHCl3) ,9]; 1H-N1  in anhydrous CH2Cl2 (0.5 mL) under nitrogen atmosphere was added (S)-(+)-α-methoxyphenylacetic acid , EDC.HCl  and DMAP  successively and the reaction was stirred overnight. Water was added and the crude product was extracted with diethyl ether (2 × 5 mL). The combined organic extracts were washed with 10% HCl (5 mL), water (5 mL), sat aq. NaHCO3 (5 mL) and water (5 mL) successively and dried over anhydrous MgSO4. Solvent was then removed under pressure to afford (S)-MPA derivative (1a) of 1 as a colorless oil . 1H-NMR  δ 7.39–7.32 , 5.37 , 5.11 , 4.72 , 4.69 , 4.55 , 3.92 , 3.51 , 3.40 , 2.85 , 2.54 , 1.90 , 1.40 , 1.29 ; (+)-HRESIMS m/z 383.1503 [M + Na]+ .To 1  in anhydrous CH2Cl2 (0.5 mL) under nitrogen atmosphere was added (R)-(–)-α-methoxyphenylacetic acid , EDC.HCl  and DMAP  successively and the reaction was stirred overnight. Water was added and the crude product was extracted with diethyl ether (2 × 5 mL). The combined organic extracts were washed with 10% HCl (5 mL), water (5 mL), sat aq. NaHCO3 (5 mL) and water (5 mL) successively and dried over anhydrous MgSO4. Solvent was then removed under pressure to afford (R)-MPA derivative (1b) of 1 as a colorless oil . 1H-NMR  δ 7.41–7.33 , 5.66 , 5.29 , 4.71 , 4.70 , 4.57 , 3.98 , 3.67 , 3.40 , 2.74 , 2.38 , 1.84 , 1.54 , 1.29 ; (+)-HRESIMS m/z 383.1468 [M + Na]+ .To 1 was performed using the MMFF94 molecular mechanics force field via the Spartan ‘08 program. Seven conformers were identified in a 15 kJ/mol window, which, upon initial geometry optimization at the DFT B3LYP/6-31G(d) level of theory and removal of duplicates, afforded four stable conformers (1a–d) (1a–d at the sodium D line (589.3 nm) using the B3LYP-D3(BJ)/6-311+G optimised geometries, with IEFPCM (MeOH), at two levels of theory: B3LYP/6-311+G and CAM-B3LYP/aug-cc-PVTZ. The Boltzmann-population weighted specific rotation values, +49.5 and +45.8, respectively (1 (+47.7) pyran-5-one bicyclic ring system (1), two hydroxymelleins (2 and 3) and an isobenzofuranone (4) from the fungal isolate Microcera larvarum. Upon structure elucidation by NMR, the structure of theissenolactone A (7) was reassigned as 1. The absolute configuration of 1 was then determined using the chiral derivatizing agent MPA and further supported by a combination of experimental and TDDFT calculated ECD spectroscopy.Screening of the ICMP collection in pursuit of novel antimicrobials led to the isolation of a γ-lactone with a furo[3,4-


Penicillium brevicompactum, recovered from a snakelocks sea anemone (Anemonia sulcata), was investigated through the parallel application of LC-ESI-HRMS, GC-MS, and NMR. Our strategy allowed the identification of mycophenolic acid, brevianamide A, and several compounds belonging to the thiosilvatins. Among the latter, five products are reported for the first time in this species. The main product of this series, cis-bis(methylthio)silvatin, was also tested for antiproliferative activity on both cancer and non-tumoral colon cell lines.Marine-derived fungi are an important source of many valuable compounds with original structures and diverse physico-chemical properties. In this work, the metabolomic profile of a strain of  Penicillium is particularly widespread in association with any kind of marine organism, and represents one of the most prolific taxa with reference to the production of bioactive compounds D25 -47.5 ; HR ESI MS (+) spectrum m/z: 839.2969 +, 431.1433 +, 361.1579 +. GC-MS data: RI 3229; m/z: 361 [M-CH3S]+ (26), 293 [M-C6H11S]+ (23), 245 [M-C5H8NO2S-H2O]+ (46), 233 [M-C12H16O]+ (60), 217 [M-C13H21O]+ (21), 186 [M-C13H19OS]+ (40), 107 [M-C13H21N2O2S2]+ (100).Saroclazine A/B (2). HR ESI MS (+) spectrum m/z: 395.1623 +. GC-MS data: RI 2892, 340 [M-C4H7]+ (3), 293 [M-C6H14O]+(76), 245 [M-C4H8N2OS-H2O]+ (28), 235 [M-C11H14N2O]+ (35), 218 [M-C11H14O-CH3]+ (33), 187 [M-C12H20N2O2S2]+ (100).Fusaperazine E/F (3). HR ESI MS (+) spectrum m/z: 383.1822 +, 361.1574 +. GC-MS data: RI 2083, 361 [M]+ (100), 312 [M-CH4S]+ (47), 234 [M-C7H13NO]+ (52), 187 [M-C8H16NOS]+ (76).6-Oxo-methylthiosilvatin (4). HR ESI MS (+) spectrum m/z: 775.2817 +, 399.1354 +, 377.1528 +, 361.1575 [M-CH3calcd. 361.1222]+. GC-MS data: RI 3203, m/z: 360 [M-CH4]+ (4), 292 [M-C5H8O]+ (23), 245 [M-C4H4NO3-H2O]+ (100), 217 [M-C5H5NO3S]+ (44), 146 [M-C8H12N2O3S-CH3]+ (36).Bilain B (5). HR ESI MS (+) spectrum m/z: 443.3332 +. GC-MS data (silylil derivative): RI 3047, 280 [M-C13H31SSi2]+ (50), 218 [M-C15H18N2O2S2-H2O]+ (25).Deprenyl-bis(methylthio)silvatin (6). HR ESI MS (+) spectrum m/z: 363.3094 +, 341.2656 +. GC-MS data (silylil derivative): RI 2014, 412 [M]+ (25), 369 [M-C2H4O]+ (25), 321 [M-C3H10S-H2O]+ (47), 294 [M-C4H9NOS]+ (12).Fusaperazine A (7). HR ESI MS (+) spectrum m/z: 335.2791 +, 313.2351 +. GC-MS data (silylil derivative): RI 1876, 384 [M]+ (5), 369 [M-CH3]+ (100), 353 [M-2(CH3)]+ (5), 295 [M-C3H8Si-H2O]+ (12), 237 [M-C4H7NO2S-CH3]+ (10).Mycophenolic acid (8). HR ESI MS (+) spectrum m/z: 663.24092 +, 343.1153 +, 321.1331 +, 303.1228 +. GC-MS data: RI 2871, m/z: 320 [M]+ (19), 302 [M-H2O]+ (15), 247 [M-C3H6O2]+ (57), 207 [M-C6H10O2]+ (100).Brevianamide A/B (9). HR ESI MS (+) spectrum m/z: 753.3359 +, 731.3549 +, 388.1620 +, 366.1810 +. GC-MS data (silylil derivative): RI 3227, 361 [M-C6H5]+ (45), 245 [M-C9H10N2O2-CH3-CH3]+ (48), 233 [M-C11H15NOS]+ (73), 217 [M-C12H17N2O2]+ (18), 186 [M-C12H20N2O2Si]+ (50).−1 penicillin, and 100 μg mL−1 streptomycin; 1% non-essential amino acids; 2 mM l-glutamine; and 1 M HEPES, in conformity with the manufacturer’s protocols. The immortalized HCEC, derived from human colon biopsies and used as a comparison with tumor cells, was cultured in DMEM supplemented with 10% FBS, 100 U mL−1 penicillin, 100 μg mL−1 streptomycin, 200 mM l-glutamine, 100 mM Na-pyruvate, and 1 M HEPES.For in vitro experiments, two human colon adenocarcinoma cell lines  and a non-tumoral colonic epithelial cell  were used. Caco-2 and HCT116 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U mL4 cells per well and allowed to adhere for 24 h. After this period, cells were incubated with medium containing 10% FBS in the presence or absence of increasing concentrations of cis-bis(methylthio)silvatin  or DMSO 20% used as a positive control, for 24 h. Subsequently, the treatment medium was replaced with fresh medium containing MTT . After solubilization in DMSO, the mitochondrial reduction of MTT to formazan was quantitated at 490 nm . All results are expressed as the percentage of cell viability.Cell viability was evaluated by measuring the mitochondrial reductase activity (MTT assay). Cells were seeded in the presence of 10% FBS in 96-well plates at a density of 1050 values were calculated by non-linear regression analysis using the equation for a sigmoid concentration–response curve (GraphPad Prism). P-value < 0.05 was considered to be significant.Data are expressed as the mean ± SEM of three experiments. Statistical analysis was performed with the software GraphPad Prism (ver. 7.00) using one-way ANOVA, followed by a Tukey multiple comparisons test . ECP. brevicompactum to produce several compounds belonging to the thiosilvatin series [P. brevicompactum, were detected as secondary metabolites of strain AN4.Our metabolomic analysis, carried out by combining several instrumental techniques and structural elucidation approaches, disclosed the ability by strain AN4 of n series , in evid


C. elegans, the reuptake of serotonin (5-HT) is facilitated by mod-5, which encodes a 5-HT transporter that is orthologous to a human 5-HT transporter (SLC6A4). mod-5 has been shown to effect both feeding and locomotion in C. elegans . We obtained and analyzed EPG data using a microfluidic device (NemaMetrix) for mod-5 null mutant strains, mod-5(n3314)  and mod-5(n822),  and N2 control worms  in M9 saline buffer (2-minute recording duration). Mutations to the C. elegansserotonin reuptake transporter, mod-5, lead to an accumulation of serotonin at the synaptic cleft, which results in a significant increase in baseline pharyngeal pumping frequency in three out of four experiments =1.41 ± 0.44 and 2.26 ± 0.13 Hz; B, N2=0.98± 0.42 and 0.44 ± 0.08; mod-5(n822)=1.64 ± 0.33 and 3.76 ± 0.19 Hz; **p<0.005, ***p<0.0001, 2-tailed students t-test).In mod-5(n822) I; MT9772: mod-5(n3314) IControl Strain: N2Strains: MT8944:


The reaction provides succinimide-substituted dispiropyrrolidine derivatives with high regio- and diastereoselectivities under mild reaction conditions. The stereochemistry of these N-heterocycles has been confirmed by four X-ray diffraction studies. Several synthetized compounds show higher inhibition on acetylcholinesterase (AChE) than butyrylcholinesterase (BChE). Of the 17 synthesized compounds tested, five exhibit good AChE inhibition with IC50 of 11.42 to 22.21 µM. A molecular docking study has also been undertaken for compound 4n possessing the most potent AChE inhibitory activity, disclosing its binding to the peripheral anionic site of AChE enzymes. A novel  Spiroheterocyclic scaffolds have evoked immense research interest in the area of synthetic organic chemistry and medicinal chemistry ,2 since α-aminoacids and 1,2-diketones, is the most widely reported one -cycloaddition reaction of (E)-3-arylidene-1-phenyl-succinimides 1, α-aminoacids 2 and the cyclic 1,2-diketones isatin 3a–b or acenaphtenequinone 3c. Furthermore, some selected heterocyclic compounds were screened in vitro to evaluate their cholinesterase inhibitory propensity. Herein, and in continuation of our interest in azomethine ylide [3+2]-cycloaddition reaction and in the synthesis of novel spiroheterocycles ,51,52,53three-component 1,3-dipolar cycloaddition reaction of (E)-3-benzylidene-1-phenyl-succinimide 1a to azomethine ylide generated in situ from 2-phenylglycine 2 and the cyclic 1,2-diketone isatin 3a was chosen as a testing system to optimize the synthetic conditions  along with fairly high regiostereoselectivity , 2-phenylglycine 2 , and isatin 3a  in MeOH/H2O  under reflux temperature for 6 h. Unfortunately, dispiropyrrolidine 22 ratio , entry 4ectivity , entry 7α-arylidene-succinimides 1 bearing different substituents at the p-position of the aryl group or with heterocyclic group, as well as with various 1,2-diketones 3 , electron-rich  group or electron-withdrawing substituent (4-Cl or 3-Cl) at the phenyl ring reacted well yielding the dispiropyrrolidine derivatives 4a–4e in good to excellent yields 2.8622(15) Å) with a N1-H1····O1’ angle of 169.3(18)°. As illustrated in 4m  3.064(3), dN-H····O 2.230(2) Å) compared to 4b. Additionally, the O3 atom interacts weakly in an intramolecular manner with the H13 atom attached to C13 giving rise to a five-membered cycle (dC13-H····O13··2.391 Å)  .cis-relationship between the carbonyl of the oxindole or acenaphthenequinone ring and the proton attached at C-10b0, and that (ii) the two carbonyl carbons of 1,2-diketone 3a–c moieties and the enone part are in trans-relationship. Thus, the cycloadducts are formed through an exo-approach between the -dipole and enones 1 as outlined in exo-diastereoselectivity affording in each case only one diastereomer. Elucidation of the four structures reveals (i) a 3d are generated in situ by decarboxylative condensation of cyclic 1,2-diketones 3 with aminoacids 2 or 6. The -dipole 3d then undergoes a 1,3-dipolar cycloaddition reaction with the dipolarophile 1 in a regio- and stereoselective manner (path A). The formation of the exo-regioisomer 5 via path A is more favorable because of the presence of a secondary orbital interaction (SOI) -N-phenylsuccinimide-4,5-diphenyl-pyrrolidine-spirooxindole-spiro+ 500.1974, found 500.1975.″]-N-phenylsuccinimide-3,5-diphenyl-pyrrolidine-spirooxindole-spiro+ 500.1974, found 500.1973.″]-N-phenylsuccinimide-4-(p-methylphenyl)-5-phenyl-pyrrolidine-spirooxindole-spiro+ 514.2131, found 514.2131.″]-N-phenylsuccinimide-3-(p-methylphenyl)-5-phenyl-pyrrolidine-spirooxindole-spiro 514.2131, found 514.2129.″]-N-phenylsuccinimide-4-(p-methoxylphenyl)-5-phenyl-pyrrolidine-spirooxindole-spiro+ 530.2080, found 530.2079.″]-N-phenylsuccinimide-3-(p-methoxylphenyl)-5-phenyl-pyrrolidine-spirooxindole-spiro+ 530.2080, found 530.2080.″]-N-phenylsuccinimide-4-(p-chlorophenyl)-5-phenyl-pyrrolidine-spirooxindole-spiro 534.1584, found 534.1587.″]-N-phenylsuccinimide-4-(m-chlorophenyl)-5-phenyl-pyrrolidine-spirooxindole-spiro+ 534.1584, found 534.1583.″]-N-phenylsuccinimide-4-(2-pyridyl)-5-phenyl-pyrrolidine-spirooxindole-spiro-N-phenylsuccinimide-4-(2-furfuryl)-5-phenyl-pyrrolidine-spirooxindole-spiro-N-phenylsuccinimide-4,5-diphenyl-pyrrolidine -spiro-4’-chlorooxindole-spiro-N-phenylsuccinimide-3,5-diphenyl-pyrrolidine-spiro-4’-chlorooxindole-spiro-N-phenylsuccinimide-4-(p-methylphenyl)-5-phenyl-pyrrolidine-spiro-4’-chlorooxindole-spiro-N-phenylsuccinimide-3-(p-methylphenyl)-5-phenyl-pyrrolidine-spiro-4’-chlorooxindole-spiro-N-phenylsuccinimide-4-(p-methoxyphenyl)-5-phenyl-pyrrolidine-spiro-4’-chlorooxindole-spiro-N-phenylsuccinimide-4-(p-chlorophenyl)-5-phenyl-pyrrolidine-spiro-4’-chlorooxindole-spiro-N-phenylsuccinimide-4-(m-chlorophenyl)-5-phenyl-pyrrolidine-spiro-4’-chlorooxindole-spiro-N-phenylsuccinimide-4,5-diphenyl-pyrrolidine-spiroacenaphthene-1’-one-spiro+ 535.2022, found 535.2016.″]-N-phenylsuccinimide-4,5-diphenyl-pyrrolidine-spiroacenaphthene-1’-one-spiro+ 535.2022, found 535.2020.″]-N-phenylsuccinimide-4-(p-methylphenyl)-5-phenyl-pyrrolidine-spiroacenaphthene-1’-one-spiro+ 549.2178, found 549.2183.″]-N-phenylsuccinimide-4-(p-methoxyphenyl)-5-phenyl-pyrrolidine-spiroacenaphthene-1’-one-spiro+ 565.2127, found 565.2127.″]-N-phenylsuccinimide-4-(p-chlorophenyl)-5-phenyl-pyrrolidine-spiroacenaphthene-1’-one-spiro-N-phenylsuccinimide-4-(m-chlorophenyl)-5-phenyl-pyrrolidine-spiroacenaphthene-1’-one-spiro-N-phenylsuccinimide-4-(2-pyridyl)-5-phenyl-pyrrolidine-spiroacenaphthene-1’-one-spiro-N-phenylsuccinimide-4-phenyl-N-methylpyrrolidine-spirooxindole-spiro-N-phenylsuccinimide-4-(p-methylphenyl)-N-methylpyrrolidine-spirooxindole-spiro-N-phenylsuccinimide-4-(p-methoxyphenyl)-N-methylpyrrolidine-spirooxindole-spiro-N-phenylsuccinimide-4-(p-chlorophenyl)-N-methylpyrrolidine-spirooxindole-spiro-N-phenylsuccinimide-4-phenyl-N-methylpyrrolidine-spiro acenaphthene-1’-one-spiro-N-phenylsuccinimide-4-(p-methylphenyl)-N-methylpyrrolidine-spiroacenaphthene-1’-one-spiro-N-phenylsuccinimide-4-(p-methoxyphenyl)-N-methylpyrrolidine-spiroacenaphthene-1’-one-spiro+ 503.1971, found 503.1975.″]-N-phenylsuccinimide-4-(p-chlorophenyl)-N-methylpyrrolidine-spiroacenaphthene-1’-one-spiro[3.3 (7h). White solid ; mp 125–126 °C; 1H-NMR: δ 2.22 , 2.4 , 2.59 , 3.73 , 4.11 , 4.61 , 6.68–6.72 , 7.28–7.37 , 7.42, 7.53 , 7.61–7.82 , 7.93–7.99 , 8.20 ; 13C-NMR: δ 35, 36.2, 49.7, 59.4, 61.8, 80.9, 121.2, 124, 126.1, 126.3, 128.5, 128.6, 128.9, 129.4, 130.7, 131.2, 131.3, 131.5, 132.7, 133.8, 134.5, 136.3, 143.1, 173.2, 178.7, 207.5; Anal Calcd for C31H23ClN2O3: C, 73.44; H, 4.57; N, 5.53%; found: C, 73.39; H, 4.62; N, 5.47%.4b, 4m, 5m, and 7f was selected and mounted on an Xcalibur, Sapphire3 diffractometer . The crystals were kept at 150(2) K during data collection. Using Olex2 [4b, 4m, 5m, and 7f have been deposited at CCDC with deposition numbers 1958732, 1958733, 1958730, and 1958731. These data may be obtained free of charge from CCDC through www.ccdc.cam.ac.uk/data_request/cif. The main crystallographic data together with refinement details are summarized in A suitable crystal of ng Olex2 , the strng Olex2 , structung Olex2 , refinemS-butyrylthiocholine chloride, and 5, 50-dithiobis(2-nitrobenzoicacid)  were purchased from Sigma–Aldrich .Acetylthiocholine iodide (ATCl), acetylcholinesterase , butyrylcholinesterase , 50) for the compounds showing more than 50% inhibition at 5µg/mL concentration.Cholinesterase enzyme inhibitory potential of the test samples was determined following the method of Khaw et al. (2014) with slight modifications on the vehicle used. Briefly, test samples and galantamine were prepared in DMSO at the initial concentration of 0.5 mg/mL. The final concentration of DMSO in reaction mixture was 1%. At this concentration, DMSO has no inhibitory effect on both AChE and BChE enzymes. For AChE inhibitory assay, 140 µL of 0.1 M sodium phosphate buffer (pH 8) was added to a 96 wells microplate followed by 20 µL of test samples and 20 µL of 0.09 units/mL AChE enzyme. Then, 10 µL of 10 mM DTNB was added into each well followed by 10 µL of 14 mM of acetylthiocholine iodide. The absorbance of the colored end product was measured using Tecan Infinite 200 Pro Microplate Spectrophotometer at 412 nm for 30 min. Each test was conducted in triplicate. For BChE inhibitory assay, the same procedures were applied as AChE except for the use of enzyme and substrate, which were BChE from equine serum and S-butyrylthiocholine chloride. Absorbencies of the test samples were corrected by subtracting the absorbance of their respective blank . A set of five concentrations was used to estimate the 50% inhibitory concentration  along with AutoDockTools (ADT) [4m was built using Hyperchem 8 and energy minimization was performed with convergence criterion of 0.05 kcal/molA. Crystal structure of AChE from Torpedo californica in complex with galanthamine was obtained from Protein Data Bank with PDB ID: 1W6R [Molecular docking for ls (ADT) . 4m was ID: 1W6R . The pro(E)-3-benzylidene-1-phenyl-succinimides, 1,2-cyclic diketones, and diverse α-aminoacids. This protocol enables the convenient synthesis of a novel class of dispiropyrrolidines fused succinimide in good to high yields with high diastereoselectivity. Noteworthy, some of the synthetized compounds showed good AChE inhibition. Compound 4n bearing a methyl substituent at the para-position of the phenyl ring exhibited the most potent AChE inhibition. The molecular docking simulation of this compound disclosed its interesting binding interaction to the active site channel of AChE enzymes. To conclude, we have developed an efficient and practical strategy for the multicomponent 1,3-dipolar cycloaddition of