Agaricus bisporus (AbTYR). We have previously demonstrated that the 4-fluorobenzyl moiety might be effective in interactions with the catalytic site of AbTYR; moreover, the additional chlorine atom exerted beneficial effects in enhancing inhibitory activity. Therefore, we planned the synthesis of new small compounds in which we incorporated the 3-chloro-4-fluorophenyl fragment into distinct chemotypes that revealed the ability to establish profitable contact with the AbTYR catalytic site. Our results confirmed that the presence of this fragment is an important structural feature to improve the AbTYR inhibition in these new chemotypes as well. Furthermore, docking analysis supported the best activity of the selected studied compounds, possessing higher potency when compared with reference compounds.Tyrosinase (EC 1.14.18.1) is implicated in melanin production in various organisms. There is a growing body of evidence suggesting that the overproduction of melanin might be related to several skin pigmentation disorders as well as neurodegenerative processes in Parkinson’s disease. Based on this consideration, the development of tyrosinase inhibitors represents a new challenge to identify new agents in pharmaceutical and cosmetic applications. With the goal of identifying tyrosinase inhibitors from a synthetic source, we employed a cheap and facile preliminary assay using tyrosinase from  Agaricus bisporus (AbTYR) as a surrogate to perform a preliminary screening of potential TYR inhibitors (TYRIs). Despite there being several differences between human and mushroom isoforms, the employment of AbTYR enables a facile screening of new potential TYRIs. Interestingly, some hit compounds identified through AbTYR also demonstrated the ability to inhibit the human tyrosinase (hTYR) and produce antimelanogenic effects in human cell lines, although they proved to be less potent agents sulfanyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-amine, 3a) piperazine (1a)  and 2-(41a) 1a, 2a, 3 piperazine 6 as the central reagent to obtain this series of compounds 2c and 2d, respectively. The parent 4-fluoro-substituted compound 2b was prepared under similar conditions using 1-[(4-fluorophenyl)methyl]piperazine (9) in place of 1-[(3-chloro-4-fluorophenyl)methyl]piperazine (6).The first series of three target compounds ions see . Meanwhi3b–d bearing the 4H-1,2,4-triazol-4-amine core was carried out by the coupling of reactants 10a–b with the suitable benzylbromide 11a–b under basic conditions, as displayed in The synthesis of the compounds 4b–h displaying an alternative fragment to incorporate the pharmacophoric 3-chloro-4-fluorophenyl moiety. To obtain the targeted compounds, the synthetic procedure described in 4b and 4f were synthesized by reacting the suitable thiophenol (12a–b) with diiodomethane and NaOH. Compounds 4c–e and 4g–h were prepared via a simple nucleophilic substitution between the proper thiophenol and the chloro(methylsulfanyl) derivative (14a–c) in a basic environment. The starting materials chloro(methylsulfanyl)methane (14a) and [(chloromethyl)sulfanyl]benzene (14b) were commercially available, whereas 1-[(chloromethyl)sulfanyl]-4-fluorobenzene (14c) was prepared by the reaction of 4-fluorobenzene-1-thiol (13) with paraformaldehyde.Moreover, we prepared compounds All compounds were structurally characterized through spectroscopic measurements, as reported in Mushroom tyrosinase (EC 1.14.18.1) was supplied by Merck (Cat. No. T3824). In vitro assay was performed according to the method of Mirabile et al. . Each sa50 values were determined by interpolation of the dose–response curves.Finally, ICAgaricus bisporus tyrosinase (AbTYR) and tropolone (PDB ID 2Y9X) piperazine-1-carboxylate. Subsequently, a solution of tert-butyl 4-[(3-chloro-4-fluorophenyl)methyl]piperazine-1-carboxylate  in DCM (8 mL) was treated with TFA (15.7 mmol); the resulting mixture was stirred at room temperature for 5 h. After the reaction was completed, it was cooled on ice and diluted with DCM (2 mL) and a solution of K2CO3 . The mixture was extracted with DCM ; the combined organic layers were dried over Na2SO4 and the solvent was removed in vacuo. Finally, the desired intermediate 1-[(3-chloro-4-fluorophenyl)methyl]piperazine (6) was obtained by crystallization with diethyl ether.A mixture of tert-Butyl 4-[(3-chloro-4-fluorophenyl)methyl]piperazine-1-carboxylate (intermediate)1H-NMR : (δ) 1.44 , 2.35 , 3.40 , 3.42 , 7.06 , 7.16 , 7.36 . Anal. Calculated for C16H22ClFN2O2: C 58.44, H 6.74, N 8.52. Found: C 58.39, H 6.71, N 8.56.Yield: 98%. Oily residue. 6)1-[(3-Chloro-4-fluorophenyl)methyl]piperazine : (δ) 2.54 , 3.08 , 3.33 , 3.53 , 7.33 , 7.39 , 7.53 . Anal. Calculated for C11H14ClFN2: C 57.77, H 6.17, N 12.25. Found: C 57.81, H 6.13, N 12.19.Yield: 62%. White powder. M.p. 147–150 °C. 6) (0.66 mmol) in DCM (4 mL), the suitable benzoyl chloride (0.66 mmol) and N,N-diisopropylethylamine  were added. The reaction mixture was stirred at room temperature for 5 h; after the completion of the reaction, the work-up to obtain target compounds 1d and 1e was performed as previously reported for analogues 1a and 1b [Route (a) To a solution of 1-[(3-chloro-4-fluorophenyl)methyl]piperazine uronium hexafluorophosphate (HBTU) (1.05 mmol) in DMF (4 mL) was stirred at room temperature for 1 h. Then, 1-[(3-chloro-4-fluorophenyl)methyl]piperazine (1.05 mmol) and TEA (2.1 mmol) were added and it was stirred for 17 h. The target compound 1f was obtained as previously reported for analog compound 1c thiophen-2-yl(4-(3-chloro-4-fluorobenzyl)piperazin-1-yl)methanone (1f)Benzoethanone  or 2-chloro-1-(4-(4-hydroxyphenyl)piperazin-1-yl)propan-1-one  as powder through treatment with EtOH and Et2O. To a solution of 2-chloro-1-[4-(4-hydroxyphenyl)piperazin-1-yl]ethanone  or 2-chloro-1-(4-(4-hydroxyphenyl)piperazin-1-yl)propan-1-one  in DMF (4 mL), 1-[(3-chloro-4-fluorophenyl)methyl]piperazine (6) (0.82 mmol) or 1-[(4-fluorophenyl)methyl]piperazine  was added. This was followed by the addition of K2CO3 ; then, the reaction mixture was refluxed for 18 h and quenched with a saturated solution of NaHCO3 (5 mL). The aqueous layer was extracted with EtOAc (3 × 10 mL) and the obtained organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated under a reduced pressure. Finally, the desired compounds 2b–d were crystallized with Et2O and EtOH.To a solution of 4-(1-piperazinyl)phenol  in DMF (4 mL), the suitable acyl chloride (1.68 mmol) was slowly added at 0 °C. Then, the reaction mixture was stirred at room temperature for 3 h. After the completion of the reaction, as indicated by TLC, a saturated solution of NaHCO8a)2-Chloro-1-[4-(4-hydroxyphenyl)piperazin-1-yl]ethanone : (δ) 2.92 , 2.98 , 3.58 , 4.41 , 6.66 , 6.81 , 8.89 . Anal. Calculated for C12H15ClN2O2: C 56.59, H 5.94, N 11.00. Found: C 56.65, H 5.83, N 11.07. The structural characterization of the pure intermediate 2-chloro-1-[4-(4-hydroxyphenyl)piperazin-1-yl]ethanone (8a) was in good agreement with previous data reported in the literature -1-fluorobenzene : (δ) 4.24 , 7.09 , 7.29 , 7.46 . 13C NMR : (δ) 42.5 (SCH2), 117.1 , 121.6 , 130.6 , 131.8 , 133.9, 157.8 . Anal. Calculated for C13H8Cl2F2S2: C 46.30, H 2.39. Found: C 46.27, H 2.20.Yield: 93%. Colorless oil. To a mixture of toluene (5 mL) and HCl , paraformaldehyde was added . The resulting mixture was stirred at 50 °C for 10 min and then a solution of 4-fluorobenzene-1-thiol (13)  in toluene (5 mL) was added dropwise at the same temperature. The reaction was stirred at 50 °C for 1 h and then at room temperature for 3 h and then flushed with Ar, and the layers were separated. The water phase was extracted with DCM (3 × 10 mL) and the organic phase was concentrated in vacuo to obtain the desired compound, which was used without further purification.1H NMR : (δ) 4.89 , 7.08 , 7.54 . 13C NMR : (δ) 52.0 (SCH2Cl), 128.1 , 116.4 , 134.3 , 163.0 . Anal. Calculated for C7H6ClFS: C 47.60, H 3.42. Found: C 47.66, H 3.47.Yield: 90%. Yellow oil. 14a–c) and K2CO3 (2.0 mmol) in DMF (5 mL), the proper thiophenol (1.5 mmol) was added. The reaction was stirred at room temperature overnight and then quenched with water and extracted with Et2O (3 × 5 mL). The organic phase was washed with brine and concentrated in vacuo. The desired products 4c–e and 4g–h were obtained after purification through column chromatography on silica gel .To a stirred solution of the suitable chloro(methylsulfanyl) derivative (1.0 mmol) (4c)1-Fluoro-4-{[(phenylsulfanyl)methyl]sulfanyl}benzene : (δ) 4.29 , 7.01 , 7.26 , 7.32 , 7.41 , 7.44 . 13C NMR : (δ) 41.9 , 116.1 , 127.1 (C″-4), 129.0 (C’’-3 and C’’-5), 129.6 , 130.7 (C-2″ and C-6″), 134.1 , 134.2 , 134.8 (C-1″), 162.5 . Anal. Calculated for C13H11FS2: C 62.37, H 4.43. Found: C 62.53, H 4.48.Yield: 91%. Yellow oil. 4d)2-Chloro-1-fluoro-4-{[(phenylsulfanyl)methyl]sulfanyl}benzene : (δ) 4.29 , 7.28 , 7.32 , 7.41 , 7.47 . 13C NMR : (δ) 41.7 (SCH2), 117.0 , 121.4 , 127.4, 129.1, 131.0, 131.1 , 131.7 , 133.7, 134.3, 157.7 . C13H10ClFS2 Anal. Calculated for C13H10ClFS2: C 54.83, H 3.54. Found: C 54.90, H 3.55.Yield: 87%. Colorless oil. 4e)2-Chloro-1-fluoro-4-(([(4-fluorophenyl)sulfanyl]methyl)sulfanyl)benzene : (δ) 4.23 , 7.02 , 7.08 , 7.28 , 7.43 . 13C NMR : (δ) 42.8 (SCH2), 116.2 , 117.0 , 121.4 , 129.1 , 131.0 , 131.5 , 133.6, 134.2 , 157.6 , 162.6 . Anal. Calculated for C13H9ClF2S2: C 51.57, H 3.00. Found: 51.33, H 2.95.Yield: 94%. Colorless oil. 4g)1-Fluoro-4-{[(methylsulfanyl)methyl]sulfanyl}benzene : (δ) 2.22 , 3.93 , 7.02 , 7.44 . 13C NMR : (δ) 15.0 (SCH3), 41.8 (SCH2), 116.0 , 129.8 , 133.9 , 162.4 . Anal. Calculated for C8H9FS2: C 51.04, H 4.82. Found: C 51.34, H 4.75.Yield: 88%. Colorless oil. 4h)1-Fluoro-3-{[(methylsulfanyl)methyl]sulfanyl}benzene : (δ) 2.24 , 4.01 , 6.92 , 7.13 , 7.17 , 7.27 . 13C NMR : (δ) 15.2 (SCH3), 39.8 (SCH2S), 113.7 , 116.7 , 125.4 , 130.1 , 137.6 , 162.7 . Anal. Calculated for C8H9FS2: C 51.04, H 4.82. Found: C 51.11, H 4.78.Yield: 94%. Brown oil. Agaricus bisporus were identified through very simple modifications of 4-fluorophenyl-based compounds that have been previously described. This investigation confirmed that the 3-chloro-4-fluorophenyl moiety was a fragment able to enhance the AbTYR inhibitory activity of distinct chemotypes of molecules targeting AbTYR. Our docking studies suggested that the improvement in activity might be due to additional interactions within the enzyme cavity that are able to reinforce the fluorine bond with crucial residues.In summary, potent inhibitors of TYR from


Ligustrum robustum has been not only used as a heat-clearing and detoxicating functional tea (Ku-Ding-Cha) but also consumed as a hypotensive, anti-diabetic, and weight-reducing folk medicine. From the leaves of L. robustum, ten new monoterpenoid glycosides named ligurobustosides T10 (1a), T11 (1b), T12 (2a), T13 (2b), T14 (3a), T15 (3b), F1 (4b), T16 (5a), T17 (5b), and E1 (6b), together with five known ones , were separated and identified using the spectroscopic method and chemical method in this research. The results of biological tests exhibited that the fatty acid synthase (FAS) inhibitory action of compound 5 (IC50: 4.38 ± 0.11 μM) was as strong as orlistat (IC50: 4.46 ± 0.13 μM), a positive control; the α-glucosidase inhibitory actions of compounds 1–4 and 7–8, and the α-amylase inhibitory actions of compounds 1–8 were medium; the ABTS radical scavenging capacities of compounds 1–3 and 5–8 (IC50: 6.27 ± 0.23 ~ 8.59 ± 0.09 μM) were stronger than l-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM) served as a positive control. This research offered a theoretical foundation for the leaves of L. robustum to prevent diabetes and its complications. N-substituted iminosugar C-glycoside + , confirming that its molecular formula was C31H46O13, which contains nine degrees of unsaturation. The IR spectrum of 1 suggested the presence of hydroxy (3366 cm−1), carbonyl (1693 cm−1), double bond (1632 cm−1), and phenyl . Two stereoisomers, 1a and 1b (3:1), were observed in the NMR spectra of compound 1. The 1H NMR spectrum of 1a , 6.35, 7.64 ], two monosaccharide residues , and a monoterpenoid aglycone . The 13C NMR spectrum of 1a (trans-p-coumaroyl (δC 114.9–169.0), two monosaccharide residues (δC 17.9–102.7), and a monoterpenoid aglycone (δC 16.4–142.4). The above 1H and 13C NMR features of 1a were closely related to those of -1--(3-O-α-l-rhamnopyranosyl)-(6-O-trans-p-coumaroyl)-O-β-d-glucopyranoside (ligurobustoside T8)  in 1b took the place of the trans-p-coumaroyl in 1a. The long-distance correlations between H-1′ of Glc (δH 4.26) and C-1 of aglycone (δC 66.2), between H-1″ of Rha (δH 5.15) and C-3′ of Glc (δC 84.5), and between H-6′ of Glc  and carbonyl of Cou (δC 168.2) were shown in the HMBC spectrum of 1b (1b was identified as (E)-7-hydroxy-3,7-dimethyl-2-octen-1-yl 3-O-(α-l-rhamnopyranosyl)-6-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside. It is a new monoterpenoid glycoside named ligurobustoside T11. In conclusion, compound 1 is made up of ligurobustosides T10 and T11.The NMR characteristics of 1b  were simum of 1b . Thus, c2 was obtained as a white amorphous powder. The molecular formula C31H46O13 containing nine degrees of unsaturation was determined by HRESIMS at m/z 649.2836 [M + Na]+ . The IR spectrum of 2 indicated the existence of hydroxy (3366 cm−1), carbonyl (1693 cm−1), double bond (1632 cm−1), and phenyl . Two stereoisomers, 2a and 2b (3:1), were displayed in the NMR spectra of compound 2. The 1H and 13C NMR characteristics of 2a  and H-10 of aglycone (δH 1.64) was revealed in the NOEDS of 2a  and C-1 of aglycone (δC 66.2), between H-1″ of Rha (δH 5.15) and C-3′ of Glc (δC 84.1), and between H-6′ of Glc  and carbonyl of Cou (δC 168.1) were displayed in the HMBC spectrum of 2a -7-hydroxy-3,7-dimethyl-2-octen-1-yl 3-O-(α-l-rhamnopyranosyl)-6-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside. It is a new monoterpenoid glycoside named ligurobustoside T12.Compound cs of 2a  were verDS of 2a , suggestum of 2a . HMBC anum of 2a  were use2b , 6.31, 7.64 ] in 2b took the place of cis-p-coumaroyl  in 2a. From the HMBC spectrum of 2b (δH 4.32) and C-1 of aglycone (δC 66.2), between H-1″ of Rha (δH 5.17) and C-3′ of Glc (δC 84.0), and between H-6′ of Glc  and carbonyl of Cou (δC 169.3) were observed. Thus, compound 2b was confirmed as (Z)-7-hydroxy-3,7-dimethyl-2-octen-1-yl 3-O-(α-l-rhamnopyranosyl)-6-O-(trans-p-coumaroyl)-O-β-d-glucopyranoside, which is a novel monoterpenoid glycoside named ligurobustoside T13. To sum up, compound 2 is composed of ligurobustosides T12 and T13.The NMR features of 2b  were cloum of 2b , the lon3, a white amorphous powder, was determined to be C31H46O13 by HRESIMS , which contains nine degrees of unsaturation. The hydroxy (3391 cm−1), carbonyl (1698 cm−1), double bond (1632 cm−1), and phenyl  were observed in the IR spectrum of 3. Two stereoisomers, 3a and 3b (3:1), were exhibited in the NMR spectra of compound 3. The 1H and 13C NMR characteristics of 3a  and H-4 of aglycone (δH 2.06) was shown in the NOEDS of 3a  and C-1 of aglycone (δC 66.5), between H-1″ of Rha (δH 5.19) and C-3′ of Glc (δC 81.6), and between H-4′ of Glc (δH 4.92) and carbonyl of Cou (δC 168.3) were revealed in the HMBC spectrum of 3a -7-hydroxy-3,7-dimethyl-2-octen-1-yl 3-O-(α-l-rhamnopyranosyl)-4-O-(trans-p-coumaroyl)-O-β-d-glucopyranoside, which is a new monoterpenoid glycoside named ligurobustoside T14.Compound cs of 3a  were cloDS of 3a , affirmium of 3a . 1H-1H Cnd NOEDS  were app3b , 5.79, 6.95 ] in 3b took the place of the trans-p-coumaroyl  in 3a. The long-distance correlations between H-1′ of Glc (δH 4.33) and C-1 of aglycone (δC 66.5), between H-1″ of Rha (δH 5.16) and C-3′ of Glc (δC 81.9), and between H-4′ of Glc (δH 4.86) and carbonyl of Cou (δC 166.9) were exhibited in the HMBC spectrum of 3b (3b was identified as (E)-7-hydroxy-3,7-dimethyl-2-octen-1-yl 3-O-(α-l-rhamnopyranosyl)-4-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside. It is a new monoterpenoid glycoside named ligurobustoside T15. In sum, compound 3 is made up of ligurobustosides T14 and T15.The NMR features of 3b  were verum of 3b . Therefo4, a white amorphous powder, was analyzed to be C31H44O13 by HRESIMS , which contains ten degrees of unsaturation. The hydroxy (3392 cm−1), carbonyl (1696 cm−1), double bond (1633 cm−1), and phenyl  were observed in the IR spectrum of 4. Two stereoisomers, 4a and 4b (5:3), were shown in the NMR spectra of compound 4. The 1H and 13C NMR data of 4a (E)-6-hydroxy-3,7-dimethyl-2,7-octadien-1-yl 3-O-(α-l-rhamnopyranosyl)-6-O-(trans-p-coumaroyl)-O-β-d-glucopyranoside (ligurobustoside F)  in 4b took the place of the trans-p-coumaroyl  in 4a. The correlation between H-2 of aglycone (δH 5.38) and H-4 of aglycone (δH 2.07) was displayed in the NOEDS of 4b  and C-1 of aglycone (δC 66.4), between H-1″ of Rha (δH 5.15) and C-3′ of Glc (δC 84.0), and between H-6′ of Glc  and carbonyl of Cou (δC 168.2) were exhibited in the HMBC spectrum of 4b -6-hydroxy-3,7-dimethyl-2,7-octadien-1-yl 3-O-(α-l-rhamnopyranosyl)-6-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside. It is a novel monoterpenoid glycoside named ligurobustoside F1. To sum up, compound 4 is made up of ligurobustosides F and F1.Compound ta of 4a  were concs of 4b  were cloDS of 4b , confirmum of 4b . 1H-1H Cnd NOEDS  were use5 was acquired as a white amorphous powder. Its molecular formula C31H46O14, which contains nine degrees of unsaturation, was affirmed by HRESIMS at m/z 665.2790 [M + Na]+ . The presence of hydroxy (3375 cm−1), carbonyl (1693 cm−1), double bond (1632 cm−1), and phenyl  was confirmed with the IR spectrum of 5. Two stereoisomers, 5a and 5b (6:4), were revealed in the NMR spectra of compound 5. The 1H NMR spectrum of 5a , 6.35, 7.64 ], two monosaccharide residues , and a monoterpenoid aglycone . The 13C NMR spectrum of 5a (trans-p-coumaroyl (δC 114.9–169.1), two monosaccharide residues (δC 17.9–102.7), and a monoterpenoid aglycone (δC 16.5–142.5). The above 1H and 13C NMR characteristics of 5a were close to those of (E)-6,7-dihydroxy-3,7-dimethyl-2-octen-1-yl 3-O-(α-l-rhamnopyranosyl)-4-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside (ligurobustoside L)  in 6b took the place of the trans-p-coumaroyl  in 6a. The correlation between H-2 of aglycone (δH 5.35) and H-4 of aglycone (δH 2.03) was shown in the NOEDS of 6b  and C-1 of aglycone (δC 66.2), between H-1″ of Rha (δH 5.15) and C-3′ of Glc (δC 84.0), and between H-6′ of Glc  and carbonyl of Cou (δC 168.1) were revealed in the HMBC spectrum of 6b -3,7-dimethyl-2,6-octadien-1-yl 3-O-(α-l-rhamnopyranosyl)-6-O-(cis-p-coumaroyl)-O-β-d-glucopyranoside. It is a novel monoterpenoid glycoside named ligurobustoside E1. To sum up, compound 6 is made up of ligurobustosides E and E1.Compound ta of 6a  were cones of 6b  were cloDS of 6b , affirmium of 6b . 1H-1H Cnd NOEDS  were use7–8  + .Compound 2: white amorphous powder. UV (MeOH) λmax (log ε): 213 (4.1), 227 (4.2), 316 (4.4) nm; IR (film) νmax: 3366, 2926, 1693, 1632, 1604, 1515, 1447, 1268, 1166, 1038, 912, 834 cm–1; 1H NMR  data, see 13C NMR  data, see m/z 649.2836 [M + Na]+ .Compound 3: white amorphous powder. UV (MeOH) λmax (log ε): 214 (4.1), 228 (4.2), 316 (4.4) nm; IR (film) νmax: 3391, 2935, 1698, 1632, 1605, 1515, 1263, 1169, 1041, 835 cm–1; 1H NMR  data, see 13C NMR  data, see m/z 649.2834 [M + Na]+ .Compound 4: white amorphous powder. UV (MeOH) λmax (log ε): 213 (4.1), 228 (4.2), 317 (4.4) nm; IR (film) νmax: 3392, 2926, 1696, 1633, 1605, 1515, 1263, 1169, 1040, 835 cm–1; 1H NMR  data, see 13C NMR  data, see m/z 647.2684 [M + Na]+ .Compound 5: white amorphous powder. + .Compound 6: yellowish oil. + .Compound 1–6  were hydrolyzed with 1 M H2SO4 aqueous solution at 92–95 °C for 6 h, respectively, and then neutralized with Ba(OH)2 solution. After filtration, the solvent of the hydrolyzed solution was volatilized. The monosaccharides in the concentrated solution were identified by TLC with l-rhamnose and d-glucose references, which were developed with EtOAc-MeOH-HOAc-H2O (8:1:1:0.7) [fR values of l-rhamnose and d-glucose were 0.73 and 0.43, respectively.Compounds 1:1:0.7) . The Rf 5 (20 mg) and cellulase (30 mg) were added to a 12 mL HOAc-NaOAc buffer solution (pH 5.0) and incubated at 37 °C for 6 h. The mixture solution was extracted with EtOAc and isolated on a silica gel column (EtOAc) to give -3,7-dimethyl-oct-2-ene-1,6,7-triol and -3,7-dimethyl-oct-2-ene-1,6,7-triol (6:4), which were affirmed by [αc 0.19, EtOAc) [Compound , EtOAc) .1–8, including FAS inhibitory action, α-glucosidase inhibitory action, α-amylase inhibitory action, DPPH radical scavenging capacity, and ABTS radical scavenging capacity, were measured in this study. The published methods [l-(+)-ascorbic acid served as positive controls, respectively , T11 (1b), T12 (2a), T13 (2b), T14 (3a), T15 (3b), F1 (4b), T16 (5a), T17 (5b), and E1 (6b), together with five known ones , in which nine ingredients belonged to trans-p-hydroxycinnamic acid esters, were separated from the leaves of L. robustum and identified using the spectroscopic method  and chemical method. The results of biological tests exhibited that the FAS inhibitory action of compound 5 (IC50: 4.38 ± 0.11 μM) was as strong as asorlistat (IC50: 4.46 ± 0.13 μM) used as a positive control; the α-glucosidase inhibitory actions of compounds 1–4 and 7–8, and the α-amylase inhibitory actions of compounds 1–8 were medium and weaker than acarbose; the ABTS radical scavenging capacities of compounds 1–3 and 5–8 (IC50: 6.27 ± 0.23 ~ 8.59 ± 0.09 μM) were stronger than l-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM), a positive control. Together with our previous studies [trans-p-hydroxycinnamic acid, were believed to be the main functional constituents of L. robustum to prevent diabetes and its complications. This research provided a theoretical foundation for the leaves of L. robustum to prevent diabetes and its complications. Nevertheless, the activities of the stereoisomer mixture might be different from those of the relevant pure compound, and the activities in vitro might be different from those in vivo. Thus, further research should be carried out to evaluate the activities of pure compounds in vivo in the future.In conclusion, ten new monoterpenoid glycosides named ligurobustosides T


The X-ray crystallography of the two complexes evidenced the formation of monomeric complexes with a pentacoordinated geometry around tin via three donor atoms from the ligand, the sulfur of the thiol, the nitrogen of the imine group, and the oxygen of the pyran ring. The geometries of the five-coordinated complexes 3a (Bu2SnL3), 3c (Ph2SnL3), and 3d (Cy2SnL3) acid were intermediate between square pyramidal and trigonal bipyramidal, and complex 1a (Bu2SnL1) adopted a bipyramidal trigonal geometry (BPT). The sulforhodamine B assay assessed the cytotoxicity of organotin(IV) complexes against the MDA-MB-231 and MCF-7 (human mammary adenocarcinoma) cell lines and one normal COS-7 (African green monkey kidney fibroblast). The IC50 values evidenced a significant antiproliferative effect on cancer cells; the complexes were more potent than the positive cisplatin control and the corresponding ligands, dehydroacetic acid thiosemicarbazone (L1), dehydroacetic acid-N(4)-methylthiosemicarbazone (L2), and dehydroacetic acid-N(4)-phenylthiosemicarbazone (L3). The IC50 values also indicated that the organotin(IV) complexes were more cytotoxic against the triple-negative breast cell line MDA-MB-231 than MCF-7, inducing significant morphological alterations. The interactions of organotin(IV) 1c (Ph2SnL1), 1d (Cy2SnL1), and 1e (((CH3)3SiCH2)2SnL1) were evaluated with ss-DNA by fluorescence; intensity changes of the fluorescence were indicative of the displacement of ethidium bromide (EB), confirming the interaction of the organotin(IV) complexes with ss-DNA; the results showed a DNA binding affinity. The thermodynamic parameters obtained through isothermal titration calorimetry showed that the interaction of 1c (Ph2SnL1), with ss-ADN, was exothermic. Molecular docking studies also demonstrated that the organotin(IV) complexes were intercalated in DNA by conventional hydrogen bonds, carbon-hydrogen bonds, and π-alkyl interactions. These complexes furthermore showed a greater affinity towards DNA than cisplatin.Organotin complexes were prepared through a one-pot reaction with three components by reacting thiosemicarbazide or 4-methyl-3-thiosemicarbazide or 4-phenylthiosemicarbazide, dehydroacetic acid (DHA) and dibutyl, diphenyl, dicyclohexyl, and bis[(trimethylsilyl)methyl]tin(IV) oxides; all complexes were characterized by infrared (IR), ultraviolet-visible (UV-vis), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. The  Moreover, the modification of the heteroaromatic ring produces better activity; although some compounds have entered phase I trials, their use has so far been avoided due to their associated gastrointestinal and hematological side effects tin oxide were prepared as described in the literature .−1. Molar conductivity measurements were carried out on a Hanna 6484 instrument using anhydrous methanol as a solvent. The ultraviolet-visible (UV-Vis) absorption spectra were obtained on a Cary 100 series Agilent UV-Vis spectrophotometer, using a methanol solution at a concentration of 2.04510 × 10−5 M. The 1H, 13C, and 119Sn spectra were recorded on Bruker Avance 300 and 400 MHz spectrometers and analyzed using the Mestre Nova software. Two-dimensional (2D) nuclear magnetic resonance (NMR) experiments (COSY (correlated spectroscopy), DEPT-135 (distortionless enhancement by polarization transfer), HSQC (heteronuclear single quantum coherence), and HMBC (heteronuclear multiple bond correlation)) were conducted for a complete assignment of the signals. The DART+  mass spectra were acquired using a JEOL-JMS-X103 spectrometer. The CLogP values were computed via the Molinspiration Cheminformatics free web services at https://www.molinspiration.com in Slovensky Grob, Slovakia.Melting points were measured on a Fisher-Johns MEL-TEMP II apparatus. Infrared (IR) attenuated total reflectance (ATR) spectra were recorded on a Thermo Scientific instrument over a range from 200 to 4000 cmλ(Mo-Kα) = 0.71073 A) at T = 150 K was used to collect diffraction intensity data for the 1a, 3a, 3c, and 3d compounds. The structures were solved by direct methods and refined by full-matrix least-squares on F2. The refinement of the structures was carried out using the SHELXL software. All nonhydrogen atoms were refined anisotropically, and the hydrogen atoms were located at idealized positions. Crystallographic data for structures reported in this manuscript have been deposited with the Cambridge Crystallographic Data Centre under the CCDC numbers: 2241000 (complex 3c), 2241001 (complex 3d), 2241002 (complex 1a), and 2241003 (complex 3a). Copies of these data can be obtained free of charge from https://ccdc.cam.ac.uk/data_request/cif.A Bruker Smart Apex CCD diffractometer with a graphite monochromator , and 1% of nonessential amino acids (Gibco). The cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2. The viability of the cells used in the experiments exceeded 95%, as determined with trypan blue.The compounds were screened in vitro against two cancer cell lines, MCF-7 and MDA-MB-231 (human mammary adenocarcinoma), and the normal cell line Cos-7 (African green monkey kidney fibroblast). The MCF-7 and MDA-MB231 cell lines were cultured in RPMI-1640 medium, and Cos-7 was cultured in DMEM (Dulbecco's Modified Eagle's Medium) and supplemented with 10% of fetal bovine serum, 2 mM L-glutamine, 10000 units/ml penicillin G sodium, 10000 μl containing 5000–10000 cells per well. The cells were then pipetted into 96 microliter plates (Costar) and incubated at 37°C for 24 h in a 5% CO2 atmosphere. Subsequently, 100 μl of a solution of the corresponding organotin complex at different concentrations was added to each well. The cultures were exposed to the compounds for 48 h. After the incubation period, the cells were fixed to the plastic substratum by the addition of 50 μl of cold trichloroacetic acid (50% aqueous).The human tumor cytotoxicity was assessed using the protein-binding dye sulforhodamine B (SRB) in a microculture assay to measure the cell growth as described by . The humμl). The plates were placed on a shaker for 10 min, and the absorption was measured at 515 nm using an ELISA plate reader (Bio-Tex Instruments).The plates were incubated at 4°C for one hour, washed with tap water, and air-dried. The addition of 4% SRB stained trichloroacetic acid-fixed cells. The free SRB was removed by washing with 1% aqueous acetic acid, the plates were air-dried, and the dyed was solubilized by adding ten mM unbuffered Tris base  complexes, MCF-7 and MDA-MB231 cell lines were cultured on a tissue culture flask with or without different treatments for 48 h. After incubation, cells were washed two times with PBS, fixed with methanol 100%, and stained with Wright-Giemsa . The culture flasks were examined under an Olympus IX71 Inverted Fluorescence Phase Contrast Microscope. The images were recorded with a Nikon Coolpix 4300 digital camera using the 20x objectives and analyzed by ImageJ software : 217 (17648) π-π∗ (ligand), 271 (9470) π-π∗ (C=N), 363 (6161) n-π∗ (C=N); IR (ATR) cm−1: 3382, 3177 ν(N-H), 1670 ν(OC=O), 1558 ν(C=N), 1332 ν(C-OPyrone), 1244 ν(C-S), 683 ν(Sn-O), 520 ν(Sn-C), 432 ν(Sn-N), 314 ν(Sn-S); 1H NMR δ: 0.90 3CH3), 1.27–1.36 2CH2CH3), 1.48–1.54 2CH3), 1.59–1.70 , 2.19 , 2.64 , 4.98 , 5.74 ; 13C NMR δ: 178.2 (C-8), 168.6 (C-7), 165.1 (C-4), 163.7 (C-11a), 163.1 (C-10), 105.7 (C-11), 99.1 (C-7a), 22.1 (C-13), 19.9 (C-12), 27.4 (Sn(CH2)2CH2CH3, 3J(13C-119Sn) = 29.47 Hz), 26.5  = 90.68, 86.91 Hz), 24.2 (SnCH2(CH2)2CH3, 1J(13C-119/117Sn) = 556.95, 550.90 Hz), 13.6 (Sn(CH2)3CH3); 119Sn NMR δ: −129.2; 119Sn NMR δ: −173.7; mass spectrometry (MS): (DART+) [m/z] (%):  (100); HR-MS (DART+) [m/z]: 474.0873 ; observed: 474.0884.Compound 1b was obtained from 0.0504 g (0.5537 mmol) of thiosemicarbazide, 0.0931 g (0.5537 mmol) of dehydroacetic acid, and 0.2 g (0.5537 mmol) of dioctyl tin oxide, to produce a yellow oil in a 94% yield (0.3040 g); Rf = 0.61 (70% ethyl acetate/hexane); molar conductance, ΛM : 21.7 ohm−1 cm2 mol−1 (nonelectrolyte); UV-Vis : 217 (20183) π-π∗ (aromatic), 305 (10321), π-π∗ (C=N), 362 (6777) n-π∗ (C=N); IR (ATR) cm−1: 3222, 3179 ν(N-H), 1697 ν(OC=O), 1544 ν(C=N), 1354 ν(C-OPyrone), 1234 ν(C-S), 695 ν(Sn-O), 523 ν(Sn-C), 441 ν(Sn-N), 325 ν(Sn-S); 1H NMR δ: 0.88 7-CH3), 1.21–1.33 4-CH3) 1.47–1.54 6-CH3), 1.61–1.68 4-CH3), 2.18 , 2.64 , 4.97 , 5.73 ; 13C NMR δ: 178.2 (C-8), 168.6 (C-7), 165.1 (C-4), 163.7 (C-11a), 163.1 (C-10), 105.7 (C-11), 99.1 (C-7a), 33.5 (Sn-CH2-CH2-(CH2)5-CH3,2J(13C-119/117Sn) = 84.64, 81.62 Hz), 31.8 (Sn-(CH2)6-CH2-CH3), 29.2 (Sn-(CH2)3-CH2-(CH2)3CH3, 4J(13C-119Sn) = 15.87 Hz), 29.1 (Sn-(CH2)4-CH2-(CH2)2CH3), 25.3 (Sn-(CH2)2-CH2-(CH2)4-CH3, 3J(13C-119Sn) = 29.47 Hz), 24.6 (Sn-CH2-(CH2)6-CH3, 1J(13C-119/117Sn) = 549.4, 525.2 Hz), 22.7 (Sn-(CH2)5-CH2-CH2-CH3), 22.1 (C-13), 19.9 (C-12), 14.1 (Sn-(CH2)7-CH3); 119Sn NMR δ: −129.3; 119Sn NMR  δ: −169.7; MS: (DART+) [m/z] (%):  (100); HR-MS (DART+) m/z: 586.2125 ; observed: 586.2139.Compound 1c was prepared from 0.1164 g (0.6922 mmol) of thiosemicarbazide, 0.0631 g (0.6922 mmol) of dehydroacetic acid, and 0.2 g (0.6922 mmol) of diphenyl tin oxide. A yellow solid was isolated with 77% yield (0.2723 g); m.p. 86–88°C (dec); Rf = 0.63 (70% ethyl acetate/hexane); molar conductance, ΛM : 23.0 ohm−1 cm2 mol−1 (nonelectrolyte); UV-Vis : UV-Vis : 215 (72627) π-π∗ (aromatic), 262 (24119) π-π∗ (C=N), 367 (18410) n-π∗ (C=N); IR (ATR) cm−1: 3312, 3177 ν(N-H), 1694 ν(OC=O), 1595 ν(C=N), 1354 ν(C-OPyrone), 1230 ν(C-S), 693 ν(Sn-O), 527 ν(Sn-C), 439 ν(Sn-N), 326 ν(Sn-S); 1H NMR δ: 2.17 , 2.68 , 5.07 , 5.94 , 7.83–7.85  7.40–7.42 ; 13C NMR δ: 178.4 (C-8), 169.6 (C-7), 163.53 (C-10), 163.47 (C-11a), 163.3 (C-4), 139.9  = 902.31, 870.56 Hz), 135.9  = 58.19 Hz), 130.5  = 16.63 Hz), 128.9  = 84.64, 81.62 Hz), 105.9 (C-11), 99.5 (C-7a), 22.6 (C-13), 19.9 (C-12); 119Sn NMR δ: −239.9; 119Sn NMR δ: −324.2; MS: (DART+) [m/z] (%):  (100); HR-MS (DART+) m/z: 514.0247 ; observed: 514.0246.Compound 1d was obtained from 0.0605 g (0.6644 mmol) of thiosemicarbazide, 0.1117 g (0.6644 mmol) of dehydroacetic acid, and 0.2 g (0.6644 mmol) of dicyclohexyl tin oxide. A yellow solid was isolated with 79% yield (0.2755 g); m.p. 140−142°C (dec); Rf = 0.82 (70% ethyl acetate/hexane); molar conductance, ΛM : 6.3 ohm−1 cm2 mol−1 (nonelectrolyte); UV-Vis : 212 (22955) π-π∗ (ligand), 272 (9324) π-π∗ (C=N), 369 (6234) n-π∗ (C=N); IR (ATR) cm−1: 3384 ν(N-H), 1707 ν(OC=O), 1580 ν(C=N), 1350 ν(C-OPyrone), 1229 ν(C-S), 696 ν(Sn-O), 527 ν(Sn-C), 439 ν(Sn-N), 323 ν(Sn-S); 1H NMR δ: 1.44–2.00 5-), 2.18 5-), 2.27 , 2.70 , 5.04 , 5.85 ; 13C NMR δ: 178.4 (C-8), 168.4 (C-7), 163.8 (C-11a), 163.0 (C-10), 165.1 (C-4), 105.8 (C-11), 99.2 (C-7a), 42.0  = 532.0, 519.9 Hz), 30.44, 30.24 (Sn-CH-CH2-CH2-(CH2)3-, 3J(13C-119Sn) = 16.63 Hz), 28.67, 28.60 (Sn-CH-CH2-(CH2)4-, 2J(13C-119/117Sn) = 92.2, 89.2 Hz), 26.0 (Sn-CH-(CH2)2-CH2-(CH2)2-, 4J(13C-119Sn) = 10.58 Hz); 22.1 (C-13), 19.9 (C-12); 119Sn NMR δ: −172.8; 119Sn NMR δ: −184.6; MS: (DART+) [m/z] (%):  (100); HR-MS (DART+) m/z: 526.1183 ; observed: 526.1192.Compound 1e was prepared from 0.0589 g (0.6470 mmol) of thiosemicarbazide, 0.1487 g (0.6470 mmol) of dehydroacetic acid, and 0.2 g (0.6470 mmol) of bis[(trimethylsilyl)methyl tin] oxide. A yellow solid was isolated with a 69% yield (0.2386 g); m.p. 40−42°C (dec); Rf = 0.74 (70% ethyl acetate/hexane); molar conductance, ΛM : 11.6 ohm−1 cm2 mol−1 (nonelectrolyte); UV-Vis : 210 (52560) π-π∗ (ligand), 265 (24808) π-π∗ (C=N), 369 (20794) n-π∗ (C=N); IR (ATR) cm−1: 3317, 3180 ν(N-H), 1696 ν(OC=O), 1595 ν(C=N), 1320 ν(C-OPyrone), 1246 ν(C-S), 827 ν(Si-CH3), 689 ν(Sn-O), 523 ν(Sn-C), 437 ν(Sn-N), 324 ν(Sn-S); 1H NMR δ: 0.00 3), 0.30, 0.53 , 2.12 , 2.59 , 4.91 , 5.64 ; 13C NMR δ: 176.7 (C-8), 167.9 (C-7), 163.6 (C-10), 162.5 (C-11a), 162.0 (C-4), 104.6 (C-11), 97.7 (C-7a), 21.3 (C-13), 18.7 (C-12), 9.9  = 448.13, 428.48 Hz), 0.00 (Si-(CH3)31J(13C-29Si) = 26.9 Hz); 119Sn NMR δ: −118.0; 119Sn NMR δ: −129.0; MS: (DART+) [m/z] (%):  (63); HR-MS (DART+) m/z: 534.0725 ; observed: 534.0723.Compound 2a was prepared from 0.0845 g of 4-methyl-3-thiosemicarbazide (0.8034 mmol), 0.1351 g of dehydroacetic acid (0.8034 mmol), and 0.2 g of dibutyltin oxide (0.8034 mmol). A yellow solid was isolated with a 62% yield (0.2406 g); m.p. 88–90°C (dec); Rf = 0.35 (70% ethyl acetate/hexane); molar conductance, ΛM : 6.5 ohm–1 cm2·mol−1 (nonelectrolyte); UV-Vis : 211 (39515) π-π∗ (ligand), 267 (22554) π-π∗ (C=N), 367 (16340) n-π∗ (C=N); IR (ATR) cm−1: 3305 ν(N-H), 1684 ν(OC=O), 1552 ν(C=N), 1355 ν(C-OPyrone), 1237 ν(C-S), 582 ν(Sn-O), 523 ν(Sn-C), 446 ν(Sn-N), 352 ν(Sn-S); 1H NMR δ: 0.89 3-CH3), 1.28–1.36 2-CH2-CH3), 1.47–1.52 2-CH3), 1.59–1.67 , 2.18 , 2.67 , 2.96 , 4.89 , 5.73 ; 13C NMR δ: 178.0 (C-8), 167.7 (C-7), 164.4 (C-4), 163.8 (C-11a), 162.8 (C-10), 105.8 (C-11), 99.1 (C-7a), 30.3 (NH-CH3), 27.5 (Sn-(CH2)2-CH2-CH3, 3J(13C-119Sn) = 29.17 Hz), 26.5  = 87.54 Hz), 24.1 (Sn-CH2-(CH2)2-CH3, 1J(13C-119/117Sn) = 564.47, 540.33 Hz), 22.1 (C-13), 19.8 (C-12), 13.6 (Sn-(CH2)3-CH3); 119Sn NMR δ: −135.7; 119Sn NMR δ: −177.1; MS: (DART+) [m/z] (%):  (100); HR-MS (DART+) [m/z]: 488.1030 ; observed: 488.1034.Compound 2b was obtained from 0.0582 g of 4-methyl-3-thiosemicarbazide (0.5537 mmol), 0.0931 g of dehydroacetic acid (0.5537 mmol), and 0.2 g of dioctyl tin oxide (0.5537 mmol). A yellow solid oil was isolated with a 91% yield (0.3025 g); Rf = 0.58 (70% ethyl acetate/hexane); molar conductance, ΛM : 15.8 ohm–1·cm2·mol−1 (nonelectrolyte); UV-Vis : 217 (18153) π-π∗ (ligand), 269 (10879) π-π∗ (C=N), 370 (9588) n-π∗ (C=N); IR (ATR) cm−1: 3345 ν(N-H), 1693 ν(OC=O), 1547 ν(C=N), 1353 ν(C-OPyrone), 1232 ν(C-S), 582 ν(Sn-O), 522 ν(Sn-C), 441 ν(Sn-N), 355 ν(Sn-S); 1H NMR δ: 0.88 7-CH3), 1.26–1.29 4-CH3), 1.47–1.53 6-CH3), 1.61–1.67 2-CH2-(CH2)4CH3), 2.17 , 2.71 , 2.95 , 4.87 , 5.73 ; 13C NMR δ: 178.2 (C-8), 168.7 (C-7), 164.4 (C-4), 163.8 (C-11a), 162.8 (C-10), 106.0 (C-11), 99.5 (C-7a), 33.5 (Sn-(CH2)2-CH2-(CH2)4CH3, 2J(13C-119Sn) = 82.51 Hz), 31.8(Sn-(CH2)6-CH2-CH3), 30.2 (NH-CH3), 29.2 (Sn-(CH2)3-CH2-(CH2)3CH3, 4J(13C-119Sn) = 27.16 Hz), 25.3 (Sn-(CH2)2-CH2-(CH2)4CH3, 3J(13C-119Sn) = 28.17 Hz), 24.5 (Sn-CH2-(CH2)6-CH3, 1J(13C-119/117Sn) = 550.4, 527.3 Hz), 22.7 (Sn-(CH2)5-CH2-CH2-CH3), 22.1 (C-13), 19.8 (C-12), 14.1 (Sn-(CH2)7-CH3); 119Sn NMR δ: −135.8; 119Sn NMR δ: −173.2; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 600.2282 ; observed: 600.2254.Compound 2c was prepared from 0.0728 g of 4-methyl-3-thiosemicarbazide (0.6922 mmol), 0.1164 g of dehydroacetic acid (0.6922 mmol), and 0.2 g of diphenyl tin oxide (0.6922 mmol). A yellow solid was isolated with 81% yield (0.2946 g); m.p. 180–182°C (dec); Rf = 0.33 (70% ethyl acetate/hexane); molar conductance, ΛM : 20.1 ohm–1 cm2·mol−1 (nonelectrolyte); UV-Vis : 212 (41955) π-π∗ (aromatic), 264 (13759) π-π∗ , 370 (9920) n-π∗ (C=N); IR (ATR) cm−1: 3308 ν(N-H), 1681 ν(OC=O), 1547 ν(C=N), 1352 ν(C-OPyrone), 1228 ν(C-S), 581 ν(Sn-O), 528 ν(Sn-C), 448 ν(Sn-N), 364 ν(Sn-S); 1H NMR δ: 2.17 , 2.71 , 2.96 , 5.00 , 5.94 , 7.84–7.86  7.39–7.42 ; 13C NMR δ: 178.2 (C-8), 168.7 (C-7), 163.6 (C-4), 163.2 (C-11a), 162.7 (C-10), 140.2  = 1088.71, 849.23 Hz), 135.9  = 57.35 Hz), 130.4  = 17.10 Hz), 128.9  = 83.51 Hz), 106.0 (C-11), 99.5 (C-7a), 30.1 (NH-CH3), 22.6 (C-13), 19.9 (C-12); 119Sn NMR δ: −246.0; 119Sn NMR δ: −326.8; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 528.0404 ; observed: 528.0386.Compound 2d was obtained from 0.0699 g of 4-methyl-3-thiosemicarbazide (0.6644 mmol), 0.1117 g of dehydroacetic acid (0.6644 mmol), and 0.2 g of dicyclohexyl tin oxide (0.6644 mmol), affording 0.2479 g (71%) of a yellow solid; m.p. 144–146°C (dec); Rf = 0.34 (70% ethyl acetate/hexane); molar conductance, ΛM: 18.7 ohm–1 cm2 mol−1 (nonelectrolyte); UV-Vis : 212 (25395) π-π∗ (ligand), 272 (12311) π-π∗ (C=N), 368 (6596) n-π∗ (C=N); IR (ATR) cm−1: 3344 ν(N-H), 1701 ν(OC=O), 1545 ν(C=N), 1353 ν(C-Opyrone), 1233 ν(C-S), 583 ν(Sn-O), 523 ν(Sn-C), 437 ν(Sn-N), 356 ν(Sn-S); 1H NMR δ: 1.35–1.93 5-) 2.03–2.13 5-), 2.19 , 2.65 , 2.95 , 4.88 , 5.77 ; 13C NMR δ: 178.2 (C-8), 167.5 (C-7), 164.4 (C-4), 163.9 (C-11a), 162.7 (C-10), 105.9 (C-11), 99.2 (C-7a), 41.7 (Sn-CH-(CH2)5-, 1J(13C-119/117Sn) = 535.79, 523.70 Hz), 30.4 (NH-CH3), 30.28, 30.22 (Sn-CH-CH2-CH2-(CH2)3-, 3J(13C-119Sn) = 24.18 Hz), 28.67, 28.61 (Sn-CH-CH2-(CH2)4- 2J(13C-119/117Sn) = 83.88, 80.86 Hz), 26.6 (Sn-CH-(CH2)2-CH2-(CH2)2-, 4J(13C-119/Sn) = 9.82 Hz), 22.1 (C-13), 19.9 (C-12); 119Sn NMR δ: −178.6; 119Sn NMR δ: −189.4; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 540.1343 ; observed: 540.1353.Compound 2e was synthesized from 0.0680 g of 4-methyl-3-thiosemicarbazide (0.6470 mmol), 0.1087 g of dehydroacetic acid (0.6470 mmol), and 0.2 g of bis[(trimethylsilyl)methyl tin] oxide (0.6470 mmol). A yellow solid was isolated with a 60% yield (0.2107 g); m.p. 133–135°C (dec); Rf = 0.34 (70% ethyl acetate/hexane); molar conductance, ΛM : 0.0 ohm–1 cm2 mol−1 (nonelectrolyte); UV-Vis : 210 (41725) π-π∗ (ligand), 266 (22178) π-π∗ (C=N), 379 (15391) n-π∗ (C=N).; IR (ATR) cm−1: 3381 ν(N-H), 1692 ν(OC=O), 1556 ν(C=N), 1355 ν(C-OPyrone), 1250 ν(C-S), 829 ν(Si-CH3), 587 ν(Sn-O), 519 ν(Sn-C), 437 ν(Sn-N), 360 ν(Sn-S); 1H NMR δ: 0.00 3), 0.30, 0.52 , 2.12 , 2.63 , 2.89 , 4.82 , 5.65 ; 13C NMR δ: 176.5 (C-8), 166.9 (C-7), 163.0 (C-4), 162.7 (C-11a), 161.7 (C-10), 104.7 (C-11), 97.8 (C-7a), 28.9 (NH-CH3), 21.4 (C-13), 18.7 (C-12), 9.92  = 450.78, 430.65 Hz), 0.00 (Si-(CH3)3, 1J(13C-29Si) = 26.16 Hz); 119Sn NMR δ: −125.4; 119Sn NMR δ: −133.1; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 505.0881 ; observed: 548.0895.Compound 3a was prepared from 0.1343 g of 4-phenyl-3-thiosemicarbazide (0.8034 mmol), 0.1351 g of dehydroacetic acid (0.8034 mmol), and 0.2 g of dibutyltin oxide (0.8034 mmol). A yellow solid was isolated with 80% yield (0.3504 g); m.p. 118–120°C (dec); Rf = 0.34 (70% ethyl acetate/hexane); molar conductance, ΛM : 13.0 ohm–1 cm2 mol−1 (nonelectrolyte); UV-Vis : 209 (48272) π-π∗ (aromatic), 256 (29619) π-π∗ (aromatic), 274 (26818) π-π∗ (C=N), 376 (23380) n-π∗ (C=N); IR (ATR) cm−1: 3384, 3290 ν(N-H), 1683 ν(OC=O), 1553 ν(C=N), 1353 ν(C-OPyrone), 1238 ν(C-S), 690 ν(Sn-O), 527 ν(Sn-C), 440 ν(Sn-N), 322 ν(Sn-S); 1H NMR δ: 0.89 3-CH3), 1.27–1.37 2-CH2-CH3), 1.53–1.57 2-CH3), 1.62–1.70 , 2.20 , 2.73 , 5.76 , 6.89 , 7.04 , 7.31 , 7.52 ; 13C NMR δ: 178.4 (C-8), 169.6 (C-7), 163.7 (C-119a), 163.3 (C-10), 160.9 (C-4), 139.8 (C-ipso), 128.9 (C-meta), 123.0 (C-para), 119.8 (C-ortho), 105.8 (C-11), 99.2 (C-7a), 22.9 (C-13), 19.9 (C-12), 27.5 (Sn-(CH2)2-CH2-CH3, 3J(13C-119Sn) = 28.17 Hz), 26.5  = 87.54 Hz), 24.1 (Sn-CH2-(CH2)2-CH3, 1J(13C-119/117Sn) = 554.41, 537.31 Hz), 13.6 (Sn-(CH2)3-CH3); 119Sn NMR δ: −141.2; 119Sn NMR δ: −197.5; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 550.1186 ; observed: 550.1194.Compound 3b was obtained from 0.0926 g of 4-phenyl-3-thiosemicarbazide (0.5537 mmol), 0.0931 g of dehydroacetic acid (0.5537 mmol), and 0.2 g of dioctyl tin oxide (0.5537 mmol). A yellow solid was isolated with a 74% yield (0.3074 g); m.p. 73–75°C (dec); Rf = 0.58 (70% ethyl acetate/hexane); molar conductance, ΛM : 5.1 ohm–1 cm2 mol−1 (nonelectrolyte); UV-Vis : 217 (33149) π-π∗ (aromatic), 250 (24877) π-π∗ (aromatic), 283 (18833) π-π∗ (C=N), 379 (22784) n-π∗ (C=N); IR (ATR) cm−1: 3301 ν(N-H), 1687 ν(OC=O), 1551 ν(C=N), 1353 ν(C-OPyrone), 1235 ν(C-S), 690 ν(Sn-O), 523 ν(Sn-C), 442 ν(Sn-N), 324 ν(Sn-S); 1H NMR δ: 0.87 7-CH3), 1.24–1.30 4-CH3), 1.52–1.56 6-CH3), 1.64–1.69 2-CH2(CH2)4-CH3), 2.20 , 2.73 , 5.75 , 6.77 , 7.04 , 7.31 , 7.51 ; 13C NMR δ: 178.4 (C-8), 169.6 (C-7), 163.7 (C-11a), 163.3 (C-10), 160.9 (C-4), 139.7 (C-ipso), 128.9 (C-meta), 123.0 (C-para), 119.7 (C-ortho), 105.8 (C-11), 99.2 (C-7a), 33.5 (Sn-CH2-CH2-(CH2)5-CH3, 2J(13C-119Sn) = 83.51 Hz), 31.8 (Sn-(CH2)6-CH2-CH3), 29.2 (Sn-(CH2)3-CH2-(CH2)3-CH3, 4J(13C-119Sn) = 24.15 Hz), 29.1 (Sn-(CH2)4-CH2-(CH2)2-CH3), 25.3 (Sn-(CH2)2-CH2-(CH2)4-CH3, 3J(13C-119Sn) = 28.17 Hz), 24.6 (Sn-CH2-(CH2)6-CH3, 1J(13C-119/117Sn) = 548.38, 526.24 Hz), 22.7 (Sn-(CH2)5-(CH2)-CH2-CH3), 22.9 (C-13), 19.9 (C-12), 14.1 (Sn-(CH2)7-CH3); 119Sn NMR δ: −141.4; 119Sn NMR δ: −195.3; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 661.5130 ; observed: 590.0559.Compound 3c was synthesized from 0.1157 g of 4-phenyl-3-thiosemicarbazide (0.6922 mmol), 0.1164 g of dehydroacetic acid (0.6922 mmol), and 0.2 g of diphenyl tin oxide (0.6922 mmol). A yellow solid was isolated with a 53% yield (0.2152 g); m.p. 105–107°C (dec); Rf = 0.37 (70% ethyl acetate/hexane); molar conductance ΛM : 38.0 ohm–1 cm2 mol−1 (nonelectrolyte); UV-Vis : 209 (40624) π-π∗ (aromatic), 243 (17797) π-π∗ (aromatic), 280 (13617) π-π∗ (C=N), 382 (10849) n-π∗ (C=N); IR (ATR) cm−1: 3384, 3290 ν(N-H), 1683 ν(OC=O), 1549 ν(C=N), 1352 ν(C-Pyrone), 1227 ν(C-S), 691 ν(Sn-O), 526 ν(Sn-C), 439 ν(Sn-N), 330 ν(Sn-S); 1H NMR δ: 2.19 , 2.77 , 5.96 , 6.94 , 7.04 , 7.31 , 7.51 , 7.85–7.87 ), 7.40–7.42 ); 13C NMR δ: 178.6 (C-8), 170.6 (C-7), 163.8 (C-11a), 163.5 (C-10), 159.2 (C-4), 139.52 (C-ipso), 139.48  = 916.64, 873.38 Hz), 135.9  = 57.35 Hz), 130.6  = 16.09 Hz), 129.0  = 83.51 Hz), 128.9 (C-meta), 123.3 (C-para), 120.0 (C-ortho), 106.0 (C-11), 99.6 (C-7a), 23.4 (C-13), 20.0 (C-12); 119Sn NMR δ: −251.0; 119Sn NMR δ: −347.6; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 590.0560 ; observed: 590.0573.Compound 3d was prepared from 0.1111 g of 4-phenyl-3-thiosemicarbazide (0.6644 mmol), 0.1117 g of dehydroacetic acid (0.6644 mmol), and 0.2 g of dicyclohexyl tin oxide (0.6644 mmol). A yellow solid was isolated with a 62% yield (0.2477 g); m.p. 88–90°C (dec); Rf = 0.34 (70% ethyl acetate/hexane); molar conductance, ΛM: 8.0 ohm–1 cm2 mol−1 (nonelectrolyte); UV-Vis : 208 (41803) π-π∗ (aromatic), 261 (22246) π-π∗ (aromatic), 274 (20921) π-π∗ (C=N), 381 (12140) n-π∗ (C=N); IR (ATR) cm−1: 3302 ν(N-H), 1684 ν(COO), 1549 ν(C=N), 1352 ν(C-OPyrone), 1232 ν(C-S), 634 ν(Sn-O), 523 ν(Sn-C), 440 ν(Sn-N), 325 ν(Sn-S); 1H NMR δ: 1.35–1.94 5-), 2.10–2.17 5-), 2.20 , 2.71 , 5.79 , 6.82 , 7.03 , 7.31 , 7.51 ; 13C NMR δ: 178.6 (C-8), 169.4 (C-7), 163.8 (C-11a), 163.2 (C-10), 160.7 (C-4), 139.9 (C-ipso), 128.9 (C-meta), 122.8 (C-ortho), 119.7 (C-para), 105.8 (C-11), 99.3 (C-7a), 40.0  = 531.27, 507.12 Hz), 30.51, 30.22 (Sn-CH-CH2-CH2-(CH2)3-, 3J(13C-119Sn) = 23.14 Hz), 28.69, 28.63 (Sn-CH-CH2-(CH2)4-, 2J(13C-119Sn) = 82.50 Hz), 26.5 (Sn-CH-(CH2)2-CH2-(CH2)2-), 23.0 (C-13), 19.9 (C-12); 119Sn NMR δ: −184.7; 119Sn NMR δ: −203.0; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 602.1499 ; observed: 602.1510.Compound 3e was prepared from 0.1082 g of 4-phenyl-3-thiosemicarbazide (0.6470 mmol), 0.1087 g of dehydroacetic acid (0.6470 mmol), and 0.2 g (0.6470 mmol) of bis[(trimethylsilyl)methyl tin] oxide. A yellow solid was isolated with 64% yield (0.2514 g); m.p. 58–60°C (dec); Rf = 0.34 (70% ethyl acetate/hexane); molar conductance, ΛM : 43.0 ohm−1 cm2 mol−1 (nonelectrolyte); UV-Vis : 208 (29306) π-π∗ (aromatic), 260 (16746) π-π∗ (aromatic), 279 (14863) π-π∗ (C=N), 382 (11798) n-π∗ (C=N); IR (ATR) cm−1: 3316 ν(N-H), 1684 ν(OC=O), 1557 ν(C=N), 1352 ν(C-Opyrone), 1246 ν(C-S), 830 ν(Si-CH3), 693 ν(Sn-O), 526 ν(Sn-C), 447 ν(Sn-N), 342 ν(Sn-S); 1H NMR  δ: 0.00 3), 0.34, 0.55 , 2.13 , 2.68 , 5.65 , 6.65 , 6.97 , 7.24 , 7.43 ; 13C NMR δ: 176.8 (C-8), 168.8 (C-7), 162.4 (C-11a), 162.2 (C-10), 159.4 (C-4), 138.5 (C-ipso), 127.7 (C-meta), 121.8 (C-para), 118.6 (C-ortho), 104.6 (C-11), 97.8 (C-7a), 22.1 (C-13), 18.7 (C-12), 9.90  = 447.76, 428.64 Hz), 0.00 (Si-(CH3)3, 1J(13C-29Si) = 26.16 Hz); 119Sn NMR δ: −129.5; 119Sn NMR δ: −142.0; MS: (DART+) [m/z] (%):  (100); HR-MS: (DART+) [m/z]: 610.1038 ; observed: 610.1024.Compound −1 cm2·mol−1, indicating the absence of counter ions, where the donor atoms of the ligand were covalently bonded + and [M − 2R1]+ were detected, corresponding to the subsequent loss of the organic groups attached to the tin atom. The peaks at m/z 343, 284, 243, and 240 were observed, indicating the loss of different organic groups. This allowed us to propose the fragmentation pattern as shown in The samples of all complexes were analyzed using direct analysis in real time (DART-MS). The spectra showed a high relative abundance of [M + H]1H NMR spectra of the complexes  were detected in the aliphatic or aromatic region. In addition, two single signals in the aliphatic due to the methyl groups of the DHA moiety were observed in the regions of 2.17–2.20 and 2.64–2.71 ppm for H-12 and H-13, respectively.The  Figures –S50 show13C NMR spectra revealed four signals in the aliphatic region for the organic butyl groups 1a, 2a, and 3a and eight for the octyl groups 1b, 2b, and 3b attached to the metallic center. Signals of the ipso, ortho, meta, and para carbons for phenyl derivatives 1c, 2c, and 3c were identified. However, in the case of the 1d, 2d, and 3d complexes, the cyclohexyl moieties gave rise to eight signals, indicating the inequivalence of substituents due to the high steric demand and conformation of the cyclohexyl rings. The 1e, 2e, and 3e complexes also exhibited nonequivalent protons for the methylene group of the bis(trimethylsilyl)methyl bonded to the tin atom. An AB system was observed alongside a single signal for the methyl groups , 2J (13C-119Sn), 3J (13C-119Sn), and 4J (13C-119Sn) were measured. The Lockhart–Manders and Holeček equations  and N6-Sn2-S3 [75.52(4)°–77.8(1)°] chelate angles which were lower than 90°. The dihedral angles involving these chelate rings ranged from 0.42 (5)° to 45.1(3)°, indicating the nonplanarity of the ligand plane around the tin atom -O(1) at the six-membered ring, the distances ranged from 2.140(4) to 2.276(12) Å; both distances were close to the sum of the covalent radii of the Sn-S (2.44 Å) and Sn-O (2.10 Å) atoms, respectively, but smaller than those of the van der Waals radii between the Sn and S (4.0 Å) and Sn and O (3.75 Å) atoms. These values indicate that the complexes formed strong Sn-S and Sn-O bonds. The Sn (2)-N (6) bond distances ranged from 2.218(2) to 2.193(2), which were slightly larger than the sum of the covalent radii for Sn and N (2.10 Å), suggesting a strong tin-nitrogen interaction comparable to the analogous diorganotin(IV) complexes containing both five- and six-membered chelate rings including a set of ONS donor atoms. }  with a b3a, 3c, and 3d formed 1D polymeric chains in the crystalline lattice (3a had a bifurcated interaction between the hydrogen atom (H14) of the donor amine with two neighboring oxygen atoms (O2 and O9) of the dehydroacetic acid ring and C=O group with bond distances of (O2)⋯H(14) (2.204 Å) and (O9)⋯H(14) (2.611 Å), respectively. On the other hand, only the first interaction with the oxygen atom (O2) of the C=O group was observed with bond distances of 2.081 Å for 3c and 2.308 Å for 3d for compounds 3c and 3d  polymeric chains in compound 3c  between the hydrogen atom H-17 of the phenyl ring with the sulfur atom (S3) of another molecule helped to form a 2D polymeric chain  interaction participated in the formation of a heterosynthon III (eight-membered ring) together with a hydrogen bond of the amine group (N14-H14B O1) and joined by the interaction (O2)⋯Sn (2) with a bond distance of (2.761 Å) forming a 3D polymeric structure . The complexes were solubilized in a DMSO-Tris-HCl buffer (3 : 7 v/v). After 72 h, the absorption pattern bands of the complexes remained unchanged and neither a significant red nor blue shift was observed; additionally, no new bands appeared, indicating that all compounds were stable in the solvent systems used. As representative examples, the spectra of complexes 1a, 1d, 2a, 2d, and 3a, in the DMSO-d6 solution, were acquired from the time of preparation until 72 h later. These complexes displayed good stability, and there were no observed changes in chemical shifts, signal intensity or multiplicity, or the presence of any additional signals  values  complexes, on the MDA-MB231 and the normal cell lines, at a concentration of 20 µM, L1–L2 were found noncytotoxic, whereas L-3 exhibited an antiproliferative effect on both normal and MCF-7 cell lines. However, the organotin complexes were remarkably more cytotoxic. The 1b, 2b, and 3b complexes (Octyl substituents attached to the tin atom) exhibited a less cytotoxic effect against both cancer cell lines. Regarding the N-substitution of the thiosemicarbazone moiety, the N-phenyl derivatives (3a–3e) were found to be more potent than thiosemicarbazone (1a–1e), followed by the N-methyl derivatives (2a–2e), against both MDA-MB-231 and MCF-7 methyl (BTMSM) substituents were the most potent. The steric hindrance provided by the BTMSM substituents increased the cell growth inhibition against the two cancer cell lines. The structure-activity relationship analysis suggests that the order cytotoxicity followed the overall trend BTMSM > Cy > Bu > Ph > Oc  and a nonhuman cell line COS-7 (African green monkey kidney fibroblast). Following an incubation period of 72 h, the ICnd MCF-7 . The ana Ph > Oc . The CLo> Oc -methylthiosemicarbazone and tested on human colorectal cancer cells (HCT 116) with IC50 values ranged from 3.5 to 7.58 µM , which have been shown to have better binding energy. These observations suggest that the synthesized diorganotin(IV) derivatives, when intercalated with DNA fragments, could be used in anticancer therapy, as reported in other studies for other organotins -1,3,5,6,2-oxathiadiazastannonin-8-one system with a radical increases the affinity of the complexes towards the DNA. In this study was found that better binding energies were observed as a result of the presence of the phenyl group, due to the increase in the electronic density on the aromatic ring through a positive mesomeric effect with the amine. This effect increases the electronic density of the aromatic, allowing it to interact favorably with the pyrimidine-type π-electron deficient heterocyclic ring of the DC3 DNA fragment through π-πT-shaped interaction, as seen with 3e. The same effect was observed in interaction 3a with the pyrimidine and imidazole heterocyclic rings of the guanine system in DG4. On the other hand, the substituent on the tin atom affects the affinity; longer alkyl chains result in a weaker interaction due to the different conformations that the alkyl chain can adopt, limiting its ability to intercalate with the DNA in comparison with other radicals, this effect explains the lower affinity of complexes 1b, 2b and 3b with DNA, these findings correlated with biological activity data showing higher IC50 values. However, the presence of the cyclohexyl substituent enhances the interaction with the DNA, by exhibiting additional π-alkyl interactions with the guanine heterocyclic system of DG4 with 1d and DG22 with 3d.In addition, it is important to note that substituting the N4 of thiosemicarbazone of the pyrane[3,4-π-alkyl ones . It should be noted that some of these interactions have been found in other works where tin (IV) complexes exhibited binding affinities with DNA fragments [in vitro studies; these diorganotin(IV) derivatives have better values of IC50 compared to Cisplatin.Of note, some of the DNA fragments were conserved in most of the derivatives , such asragments , 62, 64.https://hdock.phys.hust.edu.cn/). The results are presented in 1a–1e, 2a–2e, and 3a–3e  were significantly higher than the Cisplatin −50.63 kcal/mol. Autodock 4.2 software showed a similar trend with compounds 3d, 3c, 3e, 1d, 2e, and 3a exhibiting the strongest interaction energies, with binding energies of −110.64, −109.83, −109.78, −109.66, −105.82, and −105.74 kcal/mol, respectively. It is worth noting that both programs confirm that organotin(IV) complexes have a higher ability to interact with the DNA compared to Cisplatin and that most of the DNA nucleotides with which tin complexes interact are conserved. This validates the effectiveness of tin complexes in DNA interaction.We also conducted molecular docking studies using HDOCK software  complexes are described in 50 values suggest that the substituents bonded to the tin atom increased the cell growth inhibition, and the CLogP values indicate that the lipophilic-hydrophilic balance facilitated the permeability and absorption through the cell membrane. The selectivity index of the organotin(IV) complexes showed a higher selectivity than Cisplatin on both MCF-7 and MDA-MB-231 cancer cell lines compared to the normal cell line COS-7. Thus, the organotin(IV) complex may have a less toxic effect on normal cells than Cisplatin. The morphological alterations on both cancer cell lines suggested different stages of apoptosis. The fluorescence emission spectra of complexes 1a, 1c, and 1d revealed the interaction of the organotin(IV) complexes with DNA, and the constant Ksv values suggested that the bis-trimethylsilyl methyl substituent on tin had a stronger interaction with the DNA, followed by the phenyl substituent which could be stacked inside the DNA. Thermodynamic parameters obtained by Isothermal Titration Calorimetry showed that the binding reaction 1c with the DNA was exothermic. Molecular docking studies revealed that the organotin(IV) complexes were intercalated in the DNA; in general, most of the compounds showed better binding energy than the Cisplatin, particularly compounds with the phenyl ring on the nitrogen of thiosemicarbazone fraction showed a higher affinity with the dodecamer DNA. Such findings were correlated with the cytotoxicity results. The organotin(IV) complexes exhibited selective cytotoxicity and potency towards breast cancer cell lines, and thus, they could be used in promising applications in the design of anticancer agents.The synthesis of organotin(IV) complexes through a one-pot reaction using a three-component approach provides an efficient strategy to produce monomeric complexes in high yields. All organotin complexes evaluated exhibited higher cytotoxicity than Cisplatin on the two breast cancer cell lines. The organotin complexes were more cytotoxic against the triple-negative breast cancer cell line MDA-MB-23. The phenyl substituent on the nitrogen of the thiosemicarbazone moiety increased the cytotoxicity; the IC


Daucus carota L.) are considered effective medicinal herbs; however, the use of D. carota leaves (DCL) as therapeutic agents has not been explored in depth. Therefore, we aimed to demonstrate the value of DCL, generally treated as waste while developing plants for wide industrial availability. Six flavone glycosides were isolated and identified from DCL, and their constituents were identified and quantitated using an NMR and HPLC/UV method, which was optimized and validated. The structure of chrysoeriol-7-rutinoside from DCL was elucidated for the first time. The method exhibited adequate relative standard deviation (<1.89%) and recovery (94.89–105.97%). The deglycosylation of DCL flavone glycosides by Viscozyme L and Pectinex was assessed. Upon converting the reaction contents to percentages, the luteolin, apigenin, and chrysoeriol groups showed values of 85.8, 33.1, and 88.7%, respectively. The enzyme-treated DCL had a higher inhibitory effect on TNF-α and IL-2 expression than that of the carrot roots or carrot leaves without enzyme treatments. These results highlight the importance of carrot leaves and could be used as baseline standardization data for commercial development.In traditional oriental medicine, carrots ( Daucus carota), an important vegetable consumed by humans worldwide, is a vital source of nutrients and contains large quantities of carotenoids + and 592.9 [M − H]+. In the 13C NMR spectrum, 27 resonances were observed, which were attributed to one CH3, one CH2, sixteen CH, and eight quaternary carbon atoms. Among the carbon resonances, the signal at δC 182.6 (C-4) indicated a ketone, and the chromophore site, C-3, δC 102.9, demonstrated that it was a flavone. It was also revealed at δH 6.56  in the 1H NMR spectrum. In addtion, the aromatic protons in the ABX system were observed at δH 7.37  and 6.90  of the C-ring and at δH 6.70  and 6.48  of the A-ring. Therefore, the aglycone of 1 was determined to be luteolin. Furthermore, two anomeric proton signals at δH 5.01  and 4.69  demonstrated the α-rhamnopyranosyl and β-glucopyranose moiety, respectively. Furthermore, these anomeric protons were verified by proton–carbon correlations in the HMBC spectrum as H-1″/C-8 and H-1‴/C-6″, respectively. In the 1H-1H COSY spectrum, the proton–proton and proton–carbon as H-5″/H-6″ and H-4″/C-6″ correlations in the HMBC, respectively, indicated the arrangement of the α-rhamnopyranosyl and β-glucopyranose moieties as rutinosides. Therefore, compound 1 was determined as luteolin-7-rutinoside (scolymoside) based on the abovementioned experimental results and previously reported literature + and 447.80 [M − H]+, implying the loss of a rhamnose moiety compared with that in compound 1, which has a luteolin aglycone moiety. In the 13C NMR spectrum, 21 resonances were observed. As in the NMR spectrum of 1, the resonances of a glucose moiety at δC 77.7 (C-5″), 76.9 (C-3″), 73.7 (C-2″), and 70.1 (C-4″) and CH2 carbon at 61.2 (C-6″) had one anomeric proton at δC 100.5 (C-1″). This anomeric carbon was also assigned by correlation with a proton in the HMBC spectrum as H-1″ (δH 5.04)/C-8 (δC 100.5). Thus, compound 2 was determined as luteolin-7-O-glucoside (cynaroside) after comparing it with that in previous reports + and 577.1 [M − H]+. The mass value implied the deduction of one hydroxyl group from the structure of compound 1. It was also verified from the resonances of the B-ring aromatic protons that the structure has a para substitution at δH 7.90  and 6.92 . All the other NMR data, besides those for the flavone B-ring, revealed the same structure as of compound 1. Based on the spectroscopic data and literature, compound 3 was determined as apigenin-7-rutinoside + and 607.5 [M − H]+. Compared to the mass data for compound 3, a methoxy group was added, and it was observed in the 1H and 13C NMR spectrum at δH 3.86  and δC 56.6 (C-3′-OCH3). Furthermore, from the anomeric carbon and proton peaks (δC 101.1 (C-1‴), 100.4 (C-1″), δH 5.03 , 4.52 ), and sugar moiety, resonances appeared from ppm: 3.13–3.62 in 1H and 66.6–76.8 in 13C NMR. Thus, the glycoside was determined as rutinoside, the same as that in compounds 1 and 3. The aglycone was determined as chrysoeriol, attributable to one methoxy group attached to C-3′ in the B-ring, assigned based on the HMBC correlation as H-3′-OCH3 (δH 3.86)/C-3′ (δC 148.6). Therefore, compound 4 was identified as chrysoeriol-7-rutinoside + and 592.9 [M − H]+. 1H NMR data in CD3OD (600 MHz): δH 7.37, 6.90 , 6.70 , 6.56 , 6.48 , 5.01 , 4.69 , 4.03–3.31 , and 1.16 . 13C NMR data in CD3OD (600 MHz): δC 182.6 (C-4), 165.6 (C-2), 163.3 (C-8), 161.5 (C-6), 157.5 (C-10), 149.8 (C-4′), 145.6 (C-3′), 122.2 (C-1′), 119.3 (C-6′), 115.6 (C-5′), 113.0 (C-2′), 105.8 (C-5), 102.9 (C-3), 100.8 (C-1‴), 100.3(C-1″), 99.8 (C-7), 94.8 (C-9), 76.5 (C-5″), 75.8(C-3″), 73.4 (C-3‴), 72.7 (C-4‴), 71.1 (C-2‴), 70.8 (C-2″), 70.0 (C-5‴), 68.5 (C-4‴), 66.2 (C-6″), and 16.5 (C-5‴-CH3).ESI-MS ion peaks at O-glucoside (Cynaroside) (2)Luteolin-7-m/z 449.38 [M + H]+ and 447.80 [M − H]+. 1H NMR data in DMSO-d6 (600 MHz): δH 7.40 , 6.88 , 6.75 , 6.69 , 6.40 , 5.04 , and 3.69–3.15 . 13C NMR data in DMSO-d6 (600 MHz): δC 182.4 (C-4), 165.0 (C-2), 163.4 (C-8), 161.6 (C-6), 157.4 (C-10), 150.5 (C-4′), 146.3 (C-3′), 121.9 (C-1′), 119.7 (C-6′), 116.5 (C-5′), 114.1 (C-2′), 105.9 (C-5), 103.7 (C-3), 100.5 (C-1″), 100.1 (C-7), 95.3 (C-9), 77.7 (C-5″), 76.9 (C-3″), 73.7 (C-2″), 70.1 (C-4″), and 61.2 (C-6″).ESI-MS ion peaks at Apigenin-7-rutinoside (3)m/z 579.1 [M + H]+ and 577.1 [M − H]+. 1H NMR data in DMSO-d6 (600 MHz): δH 7.90 , 6.92 , 6.80 , 6.73 , 6.41 , 5.02 , 4.52 , 3.82 , 3.62 , 3.56 , 3.34 , and 1.04 . 13C NMR data in DMSO-d6 (600 MHz): δC 182.5 (C-4), 167.9 (C-2), 163.4 (C-8), 161.8 (C-4′), 161.7 (C-6), 157.4 (C-10), 129.1 (C-2′ and C-6′), 121.6 (C-1′), 116.6 (C-3′ and C-5′), 105.9 (C-5), 103.6 (C-3), 101.0 (C-1‴), 100.5 (C-1″), 100.1 (C-7), 95.3 (C-9), 76.8 (C-5″), 76.2 (C-3″), 73.6 (C-3‴), 72.6 (C-4‴), 71.3 (C-2‴), 70.9 (C-2″), 68.9 (C-5‴), 66.6 (C-4″), and 18.3 (5‴-CH3).ESI-MS ion peaks at Chrysoeriol-7-rutinoside (4)m/z 609.2 [M + H]+ and 607.5 [M − H]+. 1H NMR data in DMSO-d6 (600 MHz): δH 7.54 , 6.93 , 6.92 , 6.76 , 6.42 , 5.03 , 4.52 , 3.86 , 3.81 , 3.62–3.13 , and 1.04 . 13C NMR data in DMSO-d6 (600 MHz): δC 182.5 (C-4), 164.8 (C-2), 163.4 (C-8), 161.7 (C-6), 157.4 (C-10), 151.4 (C-4′), 148.6 (C-3′), 121.9 (C-1′), 121.1 (C-6′), 116.4 (C-5′), 110.8 (C-2′), 105.9 (C-5), 104.0 (C-3), 101.1 (C-1‴), 100.4 (C-1″), 100.0 (C-7), 95.5 (C-9), 76.8 (C-5″), 76.2 (C-3″), 73.6 (C-3‴), 72.6 (C-4‴), 71.3 (C-2‴), 70.1 (C-2″), 68.9 (C-5‴), 66.6 (C-4″), 56.6 (C-3′-OCH3), and 18.3 (5‴-CH3).ESI-MS ion peaks at O-glucoside (5)Apigenin-7-m/z 433.0 [M + H]+ and 430.9 [M − H]+. 1H NMR data in DMSO-d6 (600 MHz): δH 7.91 , 6.90 , 6.81 , 6.79 , 6.41 , 5.03 , 3.68 , and 3.33–3.15 . 13C NMR data in DMSO-d6 (600 MHz): δC 182.5 (C-4), 164.8 (C-2), 163.5 (C-8), 161.9(C-4′), 161.6 (C-6), 157.5 (C-10), 129.1 (C-2′ and C-6′), 121.5 (C-1′), 116.5 (C-3′ and C-5′), 105.9 (C-5), 103.6 (C-3), 100.1 (C-1″), 95.4 (C-7), 77.7 (C-5″), 77.0 (C-3″), 73.7 (C-2″), 70.1 (C-4″), and 61.2 (C-6″).ESI-MS ion peaks at O-glucoside (6)Chrysoeriol-7-m/z 463.0 [M + H]+ and 461.1 [M − H]+. 1H NMR data in DMSO-d6 (600 MHz): δH 7.54, 6.91 , 6.82 , 6.41 , 5.02 , 3.85 , 3.70 , and 3.46–3.16 . 13C NMR data in DMSO-d6 (600 MHz): δC 181.2 (C-4), 163.4 (C-2), 162.1 (C-8), 160.3 (C-6), 156.1 (C-10), 150.1 (C-4′), 147.2 (C-3′), 120.6 (C-1′), 119.7 (C-6′), 115.0 (C-5′), 109.5 (C-2′), 104.6 (C-5), 102.6 (C-3), 99.2 (C-9), 98.7 (C-1″), 94.2 (C-7), 76.5 (C-5″), 75.7 (C-3″), 72.4 (C-2″), 68.9 (C-4″), 59.9 (C-6″), and 55.2 (3′-OCH3).ESI-MS ion peaks at 1), luteolin-7-O-glucoside (2), apigenin-7-rutinoside (3), chrysoeriol-7-rutinoside (4), apigenin-7-O-glucoside (5), and chrysoeriol-7-O-glucoside (6), with >95% purity were isolated and identified in this study. The remaining three aglycones, apigenin (7), luteolin (8), and chrysoeriol (9), with >98% purity, were obtained from Sigma-Aldrich. The mixture of the nine compounds was diluted to 0.5 mg/mL in 70% EtOH and stored at 4 °C as a stock solution. Furthermore, it was consecutively diluted to 18 concentrations (0.00001–0.2 mg/mL) as working solutions before HPLC analysis. Among the nine standards used for HPLC analysis, six glycoside compounds, namely luteolin-7-rutinoside (2O containing 0.1% (v/w) of Viscozyme L or Pectinex, in a water bath set at 50 °C, in five successive treatments: 30 min, 1 h, 3 h, 8 h, and 24 h. EtOH (7 mL extra-pure reagent) was added and the mixture was vortexed for 1 min to deactivate the enzymatic reaction at each time point. All the deactivated samples were sonicated for 5 min and passed through the same disposable syringe filter  before injecting into the HPLC system.Dried DCL and DCR were powdered, and 1 g of each was extracted twice with 10 mL of 70% EtOH for 5 min with sonication to prepare the HPLC samples. The extracted sample solutions were filtered using a disposable syringe filter  from Futecs Co., LTD . For preparing enzyme-treated DCL samples, 1 g of dried powder was extracted with 3 mL of Hr2 (correlation coefficient) values for the calibration curves generated using 10 serial concentrations. The precision of the analysis was examined by intermediate evaluation using measurements of the intra- and inter-day variability. The intra-day variability was determined by analyzing the sample solution during one of the study days (24 h). In contrast, the inter-day variability was assessed over four days by injecting the sample solutions five times daily. Relative standard deviation (RSD) values were estimated for the retention time and peak area in five experiments. RSD was a measure of precision. Recovery tests were performed to evaluate accuracy in the sample solution spiked with each standard compound. Recovery rates were determined by estimating the mean recovery (%) of the standards from the spiked extract solutions vs. the non-spiked extract sample. LOD and LOQ were determined using the signal-to-noise ratio (S/N), in which S/N ratios of 3 for LOD and 10 for LOQ were used.The HPLC analysis was validated in terms of linearity, accuracy, precision, LOD, and LOQ following the guidelines of the International Conference on Harmonization (ICH). The linearity was established by evaluating 2 at 37 °C. PMA and PHA were purchased from Sigma-Aldrich.Human T lymphocyte cells, Jurkat cells, were obtained from American Type Culture Collection (ATCC). The cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin and maintained in a humidified atmosphere containing 5% COGAPDH; 5′-CTCTTCTGCCTGCTGCACTTTG-3′ and 3′-ATGGGCTACAGGCTTGTCACTC-5′ for TNF-α: 5′-AGAACTCAAACCTCTGGAGGAAG-3′ and 3′-GCTGTCTCATCAGCATATTCACAC-5′ for IL-2.Total RNA was extracted using the RNeasy Mini Kit , following the manufacturer’s instructions. cDNA (1 µg) was synthesized from total RNA using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit  and amplified by real-time reverse transcription polymerase chain reaction (RT-PCR) using AmpliTaq Gold DNA polymerase and quantitative real-time PCR. The cDNA was amplified using Premix ExTaq  with SYBR Premix EX Taq (TaKaRa) via the BI PRISM 7500HT Sequence Detection System . The primers were synthesized by Macrogen Inc. . Actin expression was used as the control. The following custom-designed primers were used: 5′-GTCTCCTCTGACTTCAACAGCG-3′ and 3′-ACCACCCTGTTGCTGTAGCCAA-5′ for TNF-α and IL-2 levels in the cell supernatants were measured using a Human TNF-α and IL-2 ELISA kit , according to the manufacturer’s instructions. The OD value was measured at 540 nm using a multi-mode microplate reader .t-test was used to compare the groups and ANOVA with Tukey’s test was used for multiple comparison using the PRISM software . p-values < 0.05 were considered significant.Data are presented as means ± SD. Paired Student’s 1–6, the structure of compound 4, chrysoeriol-7-rutinoside, from the DCL has been elucidated for the first time. Structural elucidations, DCL quality control, and the reproducibility and accuracy of the HPLC/UV analytical method for the six isolated flavone glycosides along with their three aglycones were fully validated for the first time. From the validation study, all standard compounds in the DCL samples were estimated with accuracy and precision, as confirmed by the recoveries of 94.89–105.97% and RSDs < 1.89. The deglycosylation of flavone glycosides from DCL by treatment with Viscozyme L and Pectinex was assessed, and the hydrolyzed compounds were analyzed using the HPLC/UV method. The deglycosylated flavones were gradually produced until 24 h, and their final content was 6.34 ± 0.09 (apigenin), 0.40 ± 0.05 (luteolin), and 1.72 ± 0.03 mg/g (chrysoeriol) after treatment with Viscozyme L and 5.43 ± 0.09 (apigenin), 0.35 ± 0.01 (luteolin), and 1.45 ± 0.09 mg/g (chrysoeriol) after treatment with Pectinex. Upon converting the reaction contents to %, the luteolin, apigenin, and chrysoeriol groups showed values of 85.8, 33.1, and 88.7%, respectively.In this study, six flavone glycosides were isolated from 70% EtOH DCL extract, and their structures were identified via NMR and MS spectroscopy. Among the isolated compounds p < 0.001) and IL-2 (p < 0.0001) expression by enzyme-treated DCL extract was higher than the inhibition by DCR extract and untreated DCL extract. The effects of the extracts of DCR, DCL, and enzyme-treated DCL extract on the expression of TNF-α and IL-2 in human T lymphocyte cells were investigated using PMA/PHA stimulation. These extracts considerably inhibited the expression of TNF-α and IL-2. The inhibition of TNF-α (The new discovery pertaining to the compounds present in DCL may provide valuable data for standardization of raw materials, which is essential for the development of functional foods and drugs and may also provide valuable information for its general use as food.


Aspergillus versicolor 170217 resulted in the isolation of six new (1–6) and 45 known (7–51) compounds. The structures of the new compounds were established on the basis of exhaustive analysis of their spectroscopic data and theoretical–statistical approaches including GIAO-NMR, TDDFT-ECD/ORD calculations, DP4+ probability analysis, and biogenetic consideration. Citriquinolinones A (1) and B (2) feature a unique isoquinolinone-embedded citrinin scaffold, representing the first exemplars of a citrinin–isoquinolinone hybrid. Dicitrinones K–L (3–4) are two new dimeric citrinin analogues with a rare CH-CH3 bridge. Biologically, frangula-emodin (32) and diorcinol (17) displayed remarkable anti-food allergic activity with IC50 values of 7.9 ± 3.0 μM and 13.4 ± 1.2 μM, respectively, while diorcinol (17) and penicitrinol A (20) exhibited weak inhibitory activity against Vibrio parahemolyticus, with MIC values ranging from 128 to 256 μM.A systematic chemical investigation of the deep-sea-derived fungus  Aspergillus has been the most studied, as it is ubiquitous among almost all ecosystems and it is rich in bioactive secondary metabolites, with multifarious and intricate structures −, requiring 12 degrees of unsaturation (DOU). The 1H and 13C NMR spectroscopic data , 1.19 , 2.05 , 2.67 , 2.70 , 1.60 ; δC 21.2 , 19.8 , 12.3 , 20.0 , 16.7 , 29.3 ]; one methylene ; three methines ; fourteen non-hydrogenated carbons, including twelve olefinic , one 3sp [δC 74.7 ]; and one ketone at δC 196.2 . Diagnostic HMBC correlations observed from H3-11 to C-2/3/4, H3-12 to C-4/5/6, H2-1″ to C-6/7/8, and from H-2/3 to C-9, together with the COSY cross-peaks H-2/H3-10/H-3/H3-11, supported a 2,3,5-trimethyl-6,8-dihydrobenzofuran fragment, A − ionic peak at m/z 426.1996 in its negative HRESIMS spectrum, suggestive of a homologue of 1. Comparison of NMR data of 2 , as well as the shielding of C-2 (ΔδC -5.5). HMBC correlations from H-1″ to C-6/7/8, H-9 to C-5/C-7, together with signals observed from H3-11 to C-4 and COSY correlations of H3-10/H-2/H-3/-H3-11, inferred the penta-substituted benzene fragment A. Considering the molecular formula, the planar structure of 2 was assigned as shown in Compound ata of 2 , Table 12 contains a citrinin moiety similar to that of 1, namely a phenol A unit; we therefore examined the biogenetic relationship between these two compounds. Capon et al. documented a plausible mechanism for the transformation from citrinin to 2,3,5-trimethyl-6,8-dihydrobenzofuran and phenol A, in which the stereo configurations of C-2/3 were retained in the whole process +, requiring eight DOU. The 1H, 13C NMR, and DEPT spectroscopic data  and C-2″  examples, existed in pairs, suggesting that 3 was likely a dimeric compound. The comparison of the 1D and 2D NMR data of 3 with those of 16 showed that they closely resembled each other, with the primary difference being the replacement of a methylene in 2 with a methine and methyl in 3. Further confirmation was obtained using the COSY correlations of H3-10 /H-2 /H-3 /H3-11 , and HMBC cross-peaks from H3-12  to C-4 (δC 143.2 s) C-5 (δC 117.6 s) and C-6 (δC 154.6 s), H3-11 to C-4 (δC 143.2 s), H-9  to C-3 (δC 42.9 s), C-5, and C-7 (δC 116.7 s). The COSY correlations of H-1″ /H3-2″ , along with the diagnostic HMBC correlations from H-2″ to C-7, H-1″ to C-6, C-7 and C-8 (δC 152.9 s), proved that the linkage between the two monomers were C-1″-2″, thereby assigning the planar structure of 3, as shown in Compound pic data  revealed1–2, based on a biogenetic consideration with citrinin (30) and phenol A (14). Finally, by comparison of its calculated and experimental ECD spectra MS signal m/z 395.1841 [M−H]−. The 1H, 13C NMR and DEPT spectroscopic data revealed 24 carbons, including seven methyl singlets, five methines, and 12 protonated carbons. Similar to those of 3, the carbon signals of 4 also existed in pairs, with the exception of C-1″ (δC 25.6 d) and C-2″ (δC 23.0 q). A detailed comparison of the 1D and 2D NMR data for 4 and 3 revealed that they were closely related, with the major difference being the replacement of an aromatic methine in 3 with a non-hydrogenated carbon  in 4. The diagnostic HMBC correlations from H-2  to C-9 revealed the presence of a dihydrofuran ring. Further connections were confirmed by the COSY correlations of H3-10 /H-2 /H-3 /H3-11, and H-2″ /H-1″ , as well as the HMBC correlations observed from H3-12  to C-4 (δC 130.2 s), C-5 (δC 113.0 s) and C-6 (δC 146.3 s), from H3-11 to C-4, from H-3  to C-4, C-9 (δC 141.9 s), C-5, and from H-1″ to C-7 (δC 116.4 s), C-8 (δC 136.5 s), C-6 (δC 146.3 s)  . The rel′R,3/3′S , and was5 was isolated as a yellow amorphous solid. Its molecular formula was established as C13H12O6, according to the pseudo molecular ion at m/z 263.0547 [M−H]− in its HRESI(-)MS spectrum, requiring eight DOU. The 1H-NMR and 13C NMRDEPT spectroscopic data exhibited 13 carbons  to C-3 (δC 110.5 s), C-2 (δC 156.4 s) and C-4 (δC 140.7 s), H3-2′  to C-1′ (δC 172.5 s), H2-11  to C-2, C-3 and C-1′, H-7  to C-6 (δC 167.5 s), C-8 (δC 165.3 s), C-5 (δC 102.6 s) and C-9 (δC 99.7 s), H-5  to C-6, C-3, C-7 (δC 101.8 d) and C-9, along with the consideration of the rest of the DOU, the structure of 5 was assigned, as shown in 5 was identified as  methyl acetate.Compound  carbons , includi6 was obtained as a yellow oil. Its molecular formula was established as C11H21NO3 on the basis of the pseudo molecular ion at m/z 238.1435 [M + Na]+ in its HRESI(+)MS spectrum, requiring two degrees of unsaturation. The 13C NMR spectrum, in association with the DEPT spectrum, indicated 11 carbon signals ascribed to four methyls , two 3sp methylenes , two 3sp methines , one 3sp quaternary carbon , and one carbonyl . The 2D NMR spectra revealed COSY correlations of H2-2 /H2-3 /H2-4 , H3-1′ /H-2′ /H-3′ /H-4′ . These, along with the HMBC correlation from H2-3 and H3-6  to C-5, allowed for the assignment of the planar structure, as shown in 3-1′/H-3′ and H3-4′/H-2′ established a trans-relationship between the two methyls. Additionally, a DFT calculation of the OR value of the two possible enantiomers was performed, in which the predicted OR of the 2’S3’S isomer showed the same sign as the experimental result -2′,3′,5′-trimethyl-1,3-dioxolan-2-yl)propyl)acetamide.Compound l result . Therefo17) and frangula-emodin (32) showed potent degranulation-inhibitory activity, with IC50 values of 13.4 and 7.9 μM, respectively, while diorcinol (17) and penicitrinol A (20) exhibited mild inhibition against Vibrio parahemolyticus, with MIC values of 128 μM and 256 μM, respectively (methicillin-resistant Staphylococcus aureus).All isolates were tested for anti-food allergic activity under the concentration of 50 μM and for antibacterial activity up to 256 µM. As a result, diorcinol . Column chromatography was performed on silica gel, Sephadex LH-20, and ODS. Analytical-grade solvents, purchased from Sinopharm Chemical Reagent Co., Ltd. or Xilong Scientific Co., Ltd., were used for solvent extractions Chromatography solvents were HPLC grade, supplied by Xilong Scientific Co., Ltd. or Sigma-Aldrich. Deuterated solvents were purchased from Cambridge Isotopes.NMR spectra were recorded on a Bruker 400 MHz spectrometer. The HRESIMS spectra were recorded on a Waters Xevo G2 Q-TOF mass spectrometer . Optical rotations were obtained with an Anton Par polarimeter (MCP100). ECD spectra were measured on a Chirascan spectrometer. The semi-preparative HPLC was conducted on an Agilent instrument (1260) equipped with a 1260 Diode Array Detector (DAD) and column COSMOSIL 5 CMesoplodon densirostris stranded in Ningde of the East China Sea. It was identified to be an Aspergillus versicolor (GenBank accession number SUB13826338), as the 18S rRNA gene sequence alignment demonstrated that it was 100% identical to the Aspergillus versicolor TF34 (GenBank accession number MN515366.1). For scale-up fermentation, the A. versicolor 170217 was grown under static conditions at 25 °C in 85 × 1 L Erlenmeyer flasks, each containing 200 g oatmeal agar, including 100 g of oatmeal, 15% sea salt, and 120 mL of distilled H2O. After 30 days, the fermentation product was extracted, in triplicate, using 95% ethanol. Then, the organic solvent was combined and concentrated to a small volume. The latter was then extracted, in triplicate, using ethyl acetate. Finally, the solvent was removed under vacuum to provide the crude extract (153 g).The fungus strain 170217 was isolated from the intestinal contents of a whale via medium pressure liquid chromatography  on silica gel, with a gradient of CH2Cl2-MeOH (100%→85%). Fraction Fr.2 (17 g) was separated into eight subfractions (Fr.2-1–Fr.2-8) by column chromatography (CC) over ODS . Subfraction Fr.2-1 was purified by CC over Sephadex LH-20 (MeOH) and HPLC  to yield 5 , 36 , and 43 . Subfraction Fr.2-2 was separated by CC over Sephadex LH-20 (MeOH) and CC on silica gel, followed by HPLC  to afford 27 . Compound 35  was isolated from Fr.2-3 by CC over Sephadex LH-20 (MeOH) and HPLC . Subfraction Fr.2-4 was separated by CC over Sephadex LH-20 (MeOH) to obtain 30  and 20 . Subfraction Fr.2-5 was separated by CC over Sephadex LH-20 (MeOH) to yield 31 , and 32  was isolated from Fr.2-6 by CC over Sephadex LH-20 (MeOH). Compound 4  was isolated from Fr.2-7 by CC over Sephadex LH-20 (MeOH) and HPLC . Subfraction Fr.2-8 was separated by CC over Sephadex LH-20 (MeOH) and HPLC  to obtain 17 , 44 , 45 , and 46 .The crude extract was separated into four fractions (Fr.1−Fr.4) 2O, 10%→100%). Subfraction Fr.3-3 was purified by CC over Sephadex LH-20 (MeOH) and silica gel to yield 6 , 13 , 26 , 10 , and 48 . Subfraction Fr.3-7 was purified by CC over Sephadex LH-20 (MeOH) and HPLC  to yield compound 1 . Subfraction Fr.3-5 was purified by CC over Sephadex LH-20 (MeOH) and silica gel to yield 23  and 39 . Compound 21  was obtained from Fr.3-6 by CC over Sephadex LH-20 (MeOH), 18  and 19  were obtained from Fr.3-11 by CC over Sephadex LH-20 (MeOH), and HPLC. Subfraction Fr.3-9 was purified by CC over Sephadex LH-20 (MeOH) and silica gel to yield 33 , 37 , 40 , 41 , and 42 . Subfraction Fr.3-4 was purified by CC over Sephadex LH-20 (MeOH), silica gel, and HPLC to yield 24 , 29 , and 47 .Fraction Fr.3 (42 g) was separated into 21 subfractions by CC over ODS . Subfraction Fr.4-1 was purified by CC over Sephadex LH-20 (MeOH) and silica gel to yield 8 , 9 , 11 , 12 , and 49 . Subfraction Fr.4-2 was purified by CC over Sephadex LH-20 (MeOH) and HPLC to yield 28  and 7 . Compound 15  was isolated from Fr.4-3 by CC over Sephadex LH-20 (MeOH). Compounds 14 , 50 , and 51  were isolated from Fr.4-4 by CC over Sephadex LH-20 (MeOH) and silica gel. Compound 25  was isolated from Fr.4-5 by CC over Sephadex LH-20 (MeOH). Subfraction Fr.4-6 was repeatedly separated by CC over Sephadex LH-20 (MeOH), silica gel to provide 2 . Subfraction Fr.4-7 was subjected toCC over Sephadex LH-20 (MeOH). Final purification by HPLC afforded 16  and 3 .Fraction Fr.4 (11.8 g) was separated into seven subfractions (Fr.4-1-Fr.4-7) by CC over ODS : Yellow powder; − .Citriquinolione A (2): Yellow amorphous solid; − .Citriquinolione B (3): Yellow oil; + .Dicitrinone K (4): Yellow amorphous solid; − .Dicitrinone L (H-isochromen-3-yl) methyl acetate (5): Yellow amorphous solid; 1H and 13C NMR data, see m/z 263.0547 [M−H]− .-2′,3′,5′-Trimethyl-1,3-dioxolan-2-yl)propyl)acetamide (6): yellow oil; + .Conformational analysis was first performed via random searching in the stochastic algorithm using the MMFF94 force field, with an energy cutoff of 7.0 kcal/mol and an RMSD threshold of 0.5 Å. The predominant conformers were relocated and confirmed at the B3LYP/6-31G(d) level. The theoretical ECD spectra were calculated using the time-dependent density functional theory (TD-DFT) in methanol. The ECD spectrum was obtained by averaging each conformer using the Boltzmann distribution theory. NMR calculation and conformational optimization were performed under the HF/6-31G(d) level; then NMR was calculated at the mPW1PW91/6-311G  level. For ORD calculation, the conformations of the compounds were optimized at the B3LYP/6-31G(d) level to obtain the energy-minimized conformers. Then, the optimized conformers were subjected to the calculations of specific rotation value using the B3LYPspAug-cc-pVDZ level (λ = 589.3 nm). The calculated specific rotations were later obtained according to the Boltzmann weighting of each conformer.The in vitro anti-food allergic assay was conducted following our previous protocol ,52. BrieVibrio parahemolyticus, were used as the test targets, following the methods included in the literature [5 CFU/mL and added into the 96-well plates, and equal volumes of the compound solutions were added into each well. After culturing for 24 h at 37 °C, the plates were observed by the naked eye. Each experiment was repeated three times. An equal amount of DMSO was used as a negative control.The antibacterial assay (MICs) was evaluated by a broth microdilution in 96-well plates, and two bacterial strains, MRSA and terature . The tesA. versicolor 170217 led to the isolation of six new (1–6) and 45 known (7–51) compounds, enriching the diversity of secondary metabolites from the deep-sea-derived Aspergillus. The structures, including absolute configurations of new compounds, were elucidated by the analysis of comprehensive spectroscopic data, quantum chemical calculations, and biogenetic considerations. Biologically, compounds 32 and 17 showed remarkable anti-food allergic activity, with IC50 values of 7.9 ± 3.0 μM and 13.4 ± 1.2 μM, respectively, while displaying no cytotoxicity. Moreover, diorcinol (17) and penicitrinol A (20) exhibited mild inhibition against Vibrio parahemolyticus, with MIC values of 128 μM and 256 μM, respectively.In the present study, the chemical investigation of the deep-sea-derived fungus


Implications on the stereochemical course of the terpene cyclisation by 2-methylisoborneol synthase are discussed.Two aspects of the biosynthesis of the non-canonical terpene synthase for 2-methylisoborneol have been studied. Several 2-methylisoborneol synthases have a proline-rich N-terminal domain of unknown function. The results presented here demonstrate that this domain leads to a reduced enzyme activity, in addition to its ability to increase long-term solubility of the protein. Furthermore, the substrate scope of the 2-methylisoborneol synthase was investigated through enzyme incubations with several substrate analogs, giving access to two C Th. Th1 hashosphate , followe (2MIBS) –24. Inte-Me-IPP) , followeelicolor   to more than 550 amino acids . The long versions of 2MIBSs exhibit a proline-rich N-terminal domain of unknown function that appears disordered in the crystal structure – 327.0768; found, 327.0762.Z)-2-methylbut-2-en-1-ol [DA-3  that was obtained as a white powder. 1H NMR  δ 5.44 , 4.40 , 1.69 , 1.58  ppm; 13C NMR  δ 132.34 , 124.64 (CH), 64.18 , 20.52 , 12.62  ppm; 31P NMR  δ −7.0 , −10.2  ppm.The same procedure was used to convert -3-methylpent-2-en-1-ol [DA-4  that was obtained as a white powder. 1H NMR  δ 5.36 , 4.38 , 1.98 , 1.63 , 0.92  ppm; 13C NMR  δ 145.09 (Cq), 118.13 , 62.46 , 31.60 (CH2),15.58 (CH3), 11.62 (CH3) ppm; 31P NMR  δ −6.6 , −10.2  ppm.The same procedure was used to convert -3-methylpent-2-en-1-ol [DA-5  that was obtained as a white powder. 1H NMR  δ 5.38 , 4.41 , 2.09 , 1.72 , 0.94  ppm; 13C NMR  δ 145.52 (Cq), 119.35 , 62.05 , 24.53 (CH2), 22.12 (CH2), 12.44 (CH3) ppm; 31P NMR  δ −6.4 , −10.3  ppm.The same procedure was used to convert (-en-1-ol  (215 mg,File 1Biosynthesis and enzymatic preparation of the non-natural analogs, analytical data and spectra.


GZWMJZ-606 in Houttuynia cordata Thunb. Furanpydone A and B had unusual 5-(7-oxabicyclo[2.2.1]heptane)-4-hydroxy-2-pyridone skeleton. Their structures including absolute configurations were determined on the basis of spectroscopic analysis, as well as the X-ray diffraction experiment. Compound 1 showed inhibitory activity against ten cancer cell lines  with IC50 values from 4.35 to 9.72 µM. Compounds 1, 3 and 4 showed moderate inhibitory effects against four Gram-positive strains  and one Gram-negative strain  with MIC values from 1.56 to 25 µM. However, compounds 1–4 showed no obvious inhibitory activity against two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two pathogenic fungi  at 50 µM. These results show that compounds 1–4 are expected to be developed as lead compounds for antibacterial or anti-tumor drugs.Two new 4-hydroxy-2-pyridone alkaloids furanpydone A and B ( Septoria pistaciarum heptane)-4-hydroxy-2-pyridone skeleton. Compared with the previously reported 1,2-epoxyhexane + . Its IR (KBr) spectrum exhibited absorptions at 3434 cm−1 (hydroxy), 1649 cm−1 (carbonyl), and 1605/1552/1446 cm−1 (aromatic heterocycle). Compound 1 had the same molecular formula with N-hydroxyapiosporamide (3) and showed a high degree of similarity in UV absorption. The NMR spectra displayed two methyls, five sp3-methylenes, eight sp3-methines, three sp2-methines, one sp3-quaternary carbon, five sp2-quaternary carbons (including two carbonyls)  and H3-12 (δH 0.82), a single special hydrogen signal at H-16 (δH 7.93), two olefinic protons at H-6 (δH 5.41) and H-7 (δH 5.60). The above evidence suggested that compound 1 has a similar skeleton with compound 3. The 1H-1H COSY correlations (2-1 (δH 0.88 and 1.95) to H-10 (δH 1.58), H-3 (δH 1.50) to H3-11, and H-8 (δH 2.85) to H-12 proved the existence of a decalin moiety. The relative configurations of this part were confirmed by the NOESY correlations (3-11/H3-12 and H-5 (δH 1.83) to H-3/H-9 (δH 4.45), and indicated that compound 1 has the same decalin moiety as compound 3. The 1H-1H COSY correlations from H-20 (δH 3.87) to H2-24 (δH 1.63 and 2.25) and the HMBC correlations (δH 4.00) and H2-23 (δH 1.70 and 2.25) to C-19 (δC 89.4) confirmed the presence of an oxygenated cyclohexane moiety in compound 1. However, there was a large chemical shift difference between these two compounds at C-19/20/21/22 . Nevertheless, there is still one degree of unsaturation in the structure 1, implying an oxygen bridge in this cyclohexane moiety, but the HMBC correlations cannot be used to confirm it. The key HMBC correlations from H-9 to C-13 (δC 211.4), H-16 to C-15 (δC 159.9)/C-18 (δC 173.2)/C-19 indicated that the decalin and hexane moieties substituted at C-13 and C-17 of the 4-hydroxy-2-pyridinone part. The crystal of compound 1 was fortunately acquired in methanol/water  solution. The results of the X-ray  , which welations  from H2-elations  from H-1elations  from H-2he X-ray  analysis2 was obtained as a yellow powder. The molecular formula was deduced as C24H31O6N based on the HRESIMS peak at m/z 452.20319 , which has ten degrees of unsaturation as furanpydone A (1), but one less oxygen atom than it. According to IR (KBr) spectrum data, they seem to have similar functional groups at 3445 cm−1 (hydroxy), 1652 cm−1 (carbonyl), and 1604/1557/1456 cm−1 (aromatic heterocycle). According to 1D NMR and HSQC data, compound 2 displayed two methyl , five sp3-methylene , eight sp3-methines , three sp2-methines , one sp3-quaternary carbon (δC 88.0), and five sp2-quaternary carbons , which were extremely similar to compound 1 (1H-1H COSY correlations (2-1 (δH 0.81 and 1.83) to H-10 (δH 1.45), H-3 (δH 1.46) to H3-11 (δH 0.87), H-8 (δH 2.74) to H3-12 (δH 0.74), H-20 (δH 3.58) to H2-24 (δH 1.47 and 2.04), the key HMBC correlations (δH 4.33) to C-13 (δC 209.6), H-16 (δH 7.33) to C-15 (δC 161.8)/18 (δC 175.7)/19 (δC 88.0), H-21 (δH 3.78) to C-19 further confirmed that compound 2 and 1 have the same skeleton structure. Analysis of the NMR spectral data revealed that the chemical shift of C-15 (δC 161.8) moved to a lower field, which was similar to compound 4, the key 1H-1H COSY correlation between H-16 and H-NH (δH 11.38) confirmed the absence of an N-hydroxy group in 2. The key NOESY correlations from H-10 to H3-11/H3-12, H-5 (δH 1.75) to H-3 (δH 1.47)/H-9 (δH 4.33), as well as the same chemical shift for C-19/20/21/22/23/24 with furanpydone A (1) suggested that these two compounds had the same relative configuration. The similarity of electronic circular dichroism (ECD) curve of compound 2 (213 (−5.83), 228 (−11.74), 265 (+5.40), 310 (+7.09), 341 (−1.63) to 1 (217 (−2.73), 242 (−3.51), 270 (+2.00), 316 (+2.75), 343 (−0.27))  indicated the same absolute configuration for 2 and 1. This novel 4-hydroxy-2-pyridone was named furanpydone B.Compound mpound 1  suggesteelations  from H2-elations  from H-9(−0.27))  along wi1–4. Didymellamide B was the key intermediate in the biosynthesis of these compounds +  Å, b = 14.5093 (6) Å, c = 21.7608 (11) Å, α = 90°, β = 90°, γ = 90°, V = 2416.60 (18) Å3, T = 150 K, space group P21 21 21, Z = 4, μ (Cu Kα) = 0.807 mm−1, 8074 reflections measured, 4534 independent reflections (intR = 0.019). The final 1R values were 0.0724 (I > 2σ (I)). The final wR (F2) values were 0.1921 (I > 2σ (I)). The final 1R values were 0.0767 . The final wR(F2) values were 0.1991 . The goodness of fit on F2 was 1.022. Flack parameter = −0.15 (11). CCDC: 2218951.X-ray crystallographic analyses of 2: yellow powder; ECD  λmax (Δε) 213 (−5.83), 228 (−11.74), 265 (+5.40), 310 (+7.09), 341 (−1.63) +  determined by ESIMS. heptane part. Compound 1 exhibited broad-spectrum cytotoxicity against 10 cancer cell lines with the IC50 values of 4.35–9.72 µM, and showed selective activities against S. aureus and MRSA S. aureus with MIC values of 12.5 µM. The discovery of novel 4-hydroxy-2-pyridinone alkaloids can provide a material basis for the discovery of potential drug molecules.Two new 4-hydroxy-2-pyridone alkaloids were isolated from an endophytic fungus