The newly synthesized free ligands and their zinc (II) complexes were characterized using several spectroscopic techniques; their structures were identified by single-crystal X-ray diffraction; and their photophysical properties have been studied in the context of their chemical structure. The 2ZnCl(ImL1)2 and 2ZnCl(ImL2)2 complexes showed good thermal stability at 341 °C and 365 °C, respectively. Photophysical properties, including UV-Vis absorption spectra in ethanol solution and photoluminescence (PL) in both solid state and ethanol solution, were determined. UV-Vis adsorption data indicated that both free ligands had similar UV-Vis absorption properties, while their Zn (II) complexes had distinctive absorption characteristics. The fluorescence spectra show that both ligands and their corresponding Zn (II) complexes emit violet to cyan luminescence in the solid state at room temperature, while in ethanol solution at the same temperature, they exhibit efficient photoluminescence properties in the UV-A emission spectral region. Because of these photophysical properties, the synthesized ligands and their cognate Zn (II) complexes can be used as scaffolds for the potential development of optoelectronic materials.We present here the design, synthesis, and photophysical properties of two novel fluorescent zinc (II) complexes,  N)-containing organic ligands linked by a spacer and/or donors with a certain positional orientation is critical in building and controlling the architecture of complexes with desirable photophysical properties -4-yl)-4-amine ImL1 and 9-(4-(1-octyl-1H-imidazol-4-yl)--4-yl)-9H-carbazole ImL2. The ligands were designed with an imidazole unit as the metal coordinator and two donor moieties, phenylcarbazole and triphenylamine, linked through a benzene moiety. The molecular structures of the newly synthesized free ligands and their corresponding zinc (II) complexes were analyzed and confirmed by proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR), single-crystal X-ray diffraction, and Fourier-transform infrared spectroscopy (FTIR). The photophysical properties were also investigated employing ultraviolet-visible (UV-Vis) spectrophotometry and photoluminescent (PL) analysis.In this framework, we present the synthesis, characterization, and photophysical characteristics of two novel fluorescent zinc (II) complexes, ImL1 and ImL2) and their corresponding zinc (II) complexes, 2ZnCl(ImL1)2 and 2ZnCl(ImL2)2, are shown in ImL1 and ImL2, were synthesized from 4-(4-bromophenyl)-1H-imidazole synthon in a two-step reaction -1-octyl-1H-imidazole intermediate 3 with an isolated yield of 94% -4-yl)-4-amine ImL1 and 9-(4′-(1-octyl-1H-imidazol-4-yl)--4-yl)-9H-carbazole ImL2 were isolated with yields of 74% and 66%, respectively.The molecular structures of the novel ligands (reaction : the fird of 94% . The secer argon . After 42ZnCl(ImL1)2 and 2ZnCl(ImL2)2, were prepared by reacting one equivalent of ZnCl2 with two equivalents of the appropriate ligands ImL1 and ImL2, respectively, and heating under reflux in methanol for 24 h, affording the complexes as solid materials with yields of 56% for 2ZnCl(ImL1)2 and 59% for 2ZnCl(ImL2)2  in comparison to the bond angles of the triphenylamine unit [116.6(3)° for C16-N3-C22 and 118.5 (3)° for C13-N3-C22] 2 crystals for X-ray examination were grown in the same way as Ligand L1 and obtained as pale yellow crystals. The crystallographic data and refinement information for the 2ZnCl(ImL1)2 complex are given in 2ZnCl(ImL1)2 complex are displayed in 2ZnCl(ImL1)2 complex is given in the The 2L12ZnCl complex revealed that the complex crystallized as a monoclinic with space group C2. The 2ZnCl(ImL1)2 complex has a distorted tetrahedral environment in the zinc (II) ion center, with two coordination sites occupied by the bidentate chelation of two ImL1 ligands through the nitrogen atom of each ligand and the remaining two coordination sites occupied by two chlorine atoms. The Zn-N bond lengths between the nitrogen atoms of two ligands and the zinc ion were presented to be at distances of 2.029(2) Å and 2.030(2) Å, which are characteristic of Zn(II) complexes with neutral N donors, while the Zn-Cl length was exhibited to be at 2.2274(8) Å and 2.2453(8) Å, respectively. In addition, the N-Zn-N angles around Zn were found to be around the tetrahedral standard of 104.43(9)°, while the Cl-Zn-Cl angle of 110.74(3)° is also centered on the tetrahedral standard, which confirmed that the Zn center forms a tetrahedral coordination geometry. The N-Zn-Cl angles are shown to be 107,24(7)° for N(1)-Zn(1)-Cl(1), 113.76(7)° for N(4)-Zn(1)-Cl(1)°, 114.91(7)° for N(1)-Zn(1)-Cl(2), and 105.77(7)° for N(4)-Zn(1)-Cl(2), respectively. Furthermore, the N-C-N bond angles for the two imidazole moieties of the complex were at 116.6(3)° for N(1)-C(1)-N(2) and 111.6(2)° for N(4)-C(36)-N(5), while the C-N-C bond angles of the same systems ranged from 1056(3)° to 126.5(3)°. The N-Zn-N and Cl-Zn-Cl bond lengths fall within the range of bond lengths in complexes of tetrahedral coordinated Zn with two N-coordinated ligands and two halide atoms previously reported  using various ratio of ethyl acetate to hexane. FTIR data were acquired using an Attenuated Total Reflectance (ATR) Fourier Transform Infrared spectroscope (Thermo Nicolet 5700 FTIR). NMR data were obtained at 298 K with a 400 MHz Bruker Avance spectrometer equipped with a BBI 5 mm probe. The solvent peaks for proton NMR of 7.26 2.Crystallographic data reported in this article has been deposited with the Cambridge Crystallographic Data Center, under deposition codes 2,270,110 for H-imidazole 1 , 1-bromooctane 2 , NaOH , and benzyltetraethylammonium chloride , was added toluene (30 mL). After 24 h of heating at 110 °C, the mixture was cooled to room temperature. It was then poured into water and extracted with ethyl acetate after cooling. The organic extracts were dried with anhydrous MgSO4 after being rinsed with a brine solution. The crude product was separated by silica gel chromatography (eluting with 1% ethyl acetate in a hexane ratio) to give 4-(4-bromophenyl)-1-octyl-1H-imidazole 3 as a white solid . 1H NMR  δ ppm: 7.62 , 7.45–7.47 , 7.18 , 3.89 , 1.79 , 1.29–1.31 , 0.85  ppm. 13C NMR  δ ppm: 141.0, 137.3, 133.3, 131.5, 126.2, 120.1, 114.8 (ArC), 47.2, 31.6, 30.9, 29.0, 28.9, 26.5, 22.5 (7 × CH2), 14.0 (CH3).In a two-necked round-bottom flask containing a mixture of 4-(4-bromophenyl)-1H-imidazole 3 , 4-(diphenylamino)phenylboronic acid pinacol ester 4 , XPhos Pd G3 , and potassium carbonate  in anhydrous 1,4-dioxane (60 mL) was sonicated under argon for 5 min. To this was added water (1 mL), and the reaction mixture was heated to 100 °C for 20 h. The cooled reaction mixture was diluted with water (250 mL), acidified with HCl (2 M), and extracted with ethyl acetate (4 × 100 mL). The organic layer was filtered through celite, dried over MgSO4, filtered, and concentrated under reduced pressure to afford brown solids. This solid was purified by column chromatography over silica (eluent: 20–100% (1:9 EtOAc: Hexane)), and clean fractions were combined and concentrated under reduced pressure to give 4′-(1-octyl-1H-imidazol-4-yl)-N,N-diphenyl--4-yl)-4-amine ImL1 as a yellow solid . 1H NMR  δ ppm: 7.79 , 7.56 , 7.49–7.50 , 7.20–7.26 , 7.10–7.12 , 6.98 , 3.90 , 1.78 , 1.22–1.30 , 0.84 . 13C NMR  δ ppm: 148.1, 147.3, 143.0, 137.5, 135.8, 132.5, 129.7, 129.3, 127.7, 127.3, 124.7, 124.6, 123.3, 123.2, 122.5, 118.7, 118.6 (ArC), 50.3, 35.3, 31.6, 30.1, 29.0, 26.3, 22.5 (7 × CH2), 14.0(CH3). FTIR νmax/cm−1: 3121, 3032, 2920, 2851, 1705, 1589, 1520, 1481, 1466, 1327, 1285, 1281, 1188, 1103, 1069, 941, 818, 752, 694, 617, 517, 447.A solution of 4-(4-bromophenyl)-1-octyl-1H-imidazole 3 , 9H-Carbazole-9-(4-phenyl) boronic acid pinacol ester 5 , XPhos Pd G3 , and potassium carbonate  in anhydrous 1,4-dioxane (60.00 mL) were sonicated under argon for 5 min. To this was added water (1.00 mL), and the reaction mixture was heated to 100 °C for 20 h. The cooled reaction mixture was diluted with water (250 mL), acidified with HCl (2 M), and extracted with ethyl acetate (4 × 100 mL). The organic layer was filtered through celite, dried over MgSO4, filtered, and concentrated under reduced pressure to afford brown solids. This solid was purified by column chromatography over silica (eluent: 20–100% (1:9 EtOAc: Hexane)), and clean fractions were combined and concentrated under reduced pressure to give-(4’-(1-octyl-1H-imidazol-4-yl)--4-yl)-9H-carbazole ImL2 as a pale yellow solid . 1H NMR  δ ppm: 7.18 & 7.97 , 7.87 91H, d, J = 8.4 Hz, ArH), 7.63–7.65 , 7.52–7.57 , 7.44–7.49 , 7.30–7.49 , 7.19 , 6.61 , 4.16 , 1.88 , 1.29–1.37 , 0.89 . 13C NMR  δ ppm: 141.8, 141.0, 135.8, 135.7, 131.7, 129.1, 128.8, 128.6, 127.2, 125.9, 123.4, 121.1, 120.2, 119.9, 119.6, 110.0, 109.9, 101.4 (ArC), 46.6, 31.8, 30.4, 29.3, 29.1, 27.1, 22.7 (7 × CH2), 14.1 (CH3). FTIR νmax/cm−1: 3109, 3055, 2940, 2855, 1701, 1597, 1520, 1477, 1450, 1362, 1319, 1227, 1188, 1111, 941, 829, 745, 718, 625, 5559, 517, 520.A solution of 4-(4-bromophenyl)-1-octyl-1H-imidazol-4-yl)-N,N-diphenyl--4-yl)-4-Amine ImL1  in methanol (10 mL) was refluxed for 24 h. The reaction mixture was allowed to cool and kept at room temperature for three days. The resulting precipitates were separated by filtration, washed with water (2 × 3 mL), and air dried to obtain the desired novel 2ZnCl(ImL1)2 complex as pale yellow solids. Yield: 375 mg, 55% based on ligand ImL1. 1H NMR  δ ppm: 8.13 , 7.30–7.34 , 7.22–7.24 , 7.02–7.10 , 7.00–7.10 , 6.63 , 1.56 , 1.18–1.25 , 0.80 .13C NMR  δ ppm: 147.5, 147.4, 140.9, 139.8, 139.6, 133.8, 129.4, 129.2, 127.9, 127.2, 125.5, 123.5, 123.2, 115.8 (ArC), 48.2, 31.7, 30.6, 29.1, 28.9, 26.4, 22.6 (7 × CH2), 14.1 (CH3). FTIR νmax/cm−1: 3125, 1531, 1516, 1327, 1288, 1088, 934, 826, 934, 826, 768, 660, 621.A mixture of zinc (II) chloride  and 4-(1-octyl-1H-imidazol-4-yl)--4-yl)-9H-carbazole ImL2  in methanol (10 mL) was refluxed for 24 h. The reaction mixture was allowed to cool and kept at room temperature for three days. The resulting precipitates were separated by filtration, washed with water (2 × 3 mL), and air dried to obtain the desired novel 2ZnCl(ImL2)2 complex as white solids. Yield: 342 mg, 59% based on ligand ImL2. 1H NMR  δ ppm: 8.26 , 7.91 , 7.78 , 7.69–7.77 , 7.45 , 8.26 , 7.30–7.33 , 4.00 , 1.74 , 1.18–1.25 , 0.83 . 13C NMR  δ ppm: 140.1, 140.0, 139.0, 138.2, 136.8, 135.9, 134.0, 128.0, 127.0, 126.8, 126.3, 125.0, 122.8, 1206, 116.3, 109.7 (ArC), 46.4, 31.2, 30.4, 28.6, 28.5, 26.0, 22.1 (7 × CH2), 14.0 (CH3). FTIR νmax/cm−1: 3125, 3044, 2924, 2851, 1593, 1501, 1493, 1450, 1362, 1319, 1227, 1115, 1076, 1003, 818, 745, 721, 637, 563, 536, 478, 420.A mixture of zinc (II) chloride  and 9-(4’-(1-octyl-1H-imidazol-4-yl)-N,N-diphenyl--4-yl)-4-amine ImL1 and 9-(4 -(1-Octyl-1H-imidazol-4-yl)--4-yl)-9H-carbazole ImL2 were synthesized in 74% and 66% yields, respectively. These ligands were later treated with zinc chloride to give their corresponding zinc (II) chloride complexes, 2ZnCl(ImL1)2 and 2ZnCl(ImL2)2, in up to 59% yield. FTIR, 1H, and 13C-NMR confirmed the molecular structures of the free ligands and their corresponding Zn (II) complexes. The crystal structure analysis shows that the ImL1 ligand and the 2ZnCl(ImL1)2 complex crystallized in triclinic with space groups P-1 and C2, respectively, while the ImL2 ligand crystallized in monoclinic with space group P 21/c. In addition, the 2ZnCl(ImL1)2 complex shows a distorted tetrahedron geometry with two coordination sites occupied by the bidentate chelation of two ImL1 ligands through the nitrogen atom of each ligand and the remaining two coordination sites occupied by two chlorine atoms. Both zinc complexes are thermally stable at 341 °C for 2ZnCl(ImL1)2 and 365 °C for 2ZnCl(ImL2)2, corresponding to 2% weight loss. Both the free ligands and their complexes display efficient photoluminescence properties in the violet to cyan spectral range from 410 to 488 nm in the solid state at room temperature, with fluorescence decay lifetimes ranging from 1 to 9 nanoseconds, as well as good photoluminescence properties in the UV-A emission area from 345 to 372 nm in ethanol solutions. Because of these potential photophysical features, additional research is being conducted to assess these products as optoelectronic materials.Two new fluorescent ligands, 4-(1-octyl-1


Fluorescent labels are key tools in a wide range of modern scientific applications, such as fluorescence microscopy, flow cytometry, histochemistry, direct and indirect immunochemistry, and fluorescence in situ hybridization (FISH). Small fluorescent labels have important practical advantages as they allow maximizing the fluorescence signal by binding multiple fluorophores to a single biomolecule. At present, the most widely used fluorescent labels available present small Stokes shifts and are too costly to be used in routine applications. In this work we present four new coumarin derivatives, as promising and inexpensive fluorescent labels for biomolecules, obtained through a cost-effective, efficient, and straightforward synthetic strategy. Density functional theory and time-dependent density functional theory calculations of the electronic ground and lowest-lying singlet excited states were carried out in order to gain insights into the observed photophysical properties. Fluorescent labels, also called fluorescent markers or probes, have become elementary in a broad range of modern scientific practices. The referred fluorophores provide fluorescence when it is absent or inadequate in molecules of interest in many applications, such as fluorescence microscopy, flow cytometry, histochemistry, direct and indirect immunochemistry, and fluorescence in situ hybridization (FISH) ,4,5,6,7.Generally, fluorescent labels are classified into three major groups, i.e., organic dyes, proteins, and nanoparticles, but the vast majority of fluorophores belong to the organic dyes group. Organic dyes have important practical advantages over the other dyes as they allow maximizing the fluorescence signal by binding multiple fluorophores to a single biomolecule, due to their small size. Also, these small fluorescent molecules can be chemically modified through simple and effective organic synthetic methods, which allows the synthesis of labels with a wide range of photophysical properties ,9,10,11.Considering the efficiency and sensitivity of the detection process, the photophysical properties of fluorescent labels  are determinant for the wide range of specific applications available .The combination of fluorescence imaging with fluorophores based on organic molecules has become a powerful tool as it has enabled the understanding of biological events at the molecular level due to its high sensitivity or selectivity, non-invasive character, easy implementation, rapid response rate, real-time detection, spatiotemporal resolution, facile visualization, and in situ detection ,14,15.Due to the abundance of amino groups, or their easy incorporation into biomolecules, the amine-reactive fluorescent labels are most commonly used to prepare stable bioconjugates for a wide range of biological applications. However, for super-resolution multi-color imaging experiments, such as optical microscopy of biomolecules based on stimulated emission depletion (STED) and in Förster-type resonance energy transfer (FRET) applications, the development of new fluorescent labels with large Stokes shifts is essential and indispensable ,19,20,21Currently, the most widely used fluorescent labels available present small Stokes shifts, many of which are under 30 nm and are too costly to be used in routine applications, which is their major handicap. In this context, coumarin derivatives can be a real solution to develop new cost-effective brightness fluorophores, with large Stokes shifts ,22,23.Coumarins, found in many plant species  and ofte9, 14, 15, and 20) with red-shifted fluorescence properties, utilizing the readily available and affordable 7-diethylamino-4-methylcoumarin (1) as our starting material. These newly synthesized compounds hold potential as fluorescent labels for biomolecules. To further enhance our understanding, we have conducted theoretical analyses on the optical, electronic, and geometric characteristics of these fluorescent labels in both their ground and lowest-energy singlet excited states. This analysis was performed using density functional theory (DFT) and time-dependent density functional theory (TD-DFT), complementing the overall investigation.In this study, we aim to establish a cost-effective synthetic approach for producing four novel 4-styrylcoumarin derivatives  as the starting material, are shown in para position could enhance both the π-delocalization and the push–pull nature of the chromophore. These modifications can improve photophysical properties of the coumarin derivatives, such as high-fluorescence quantum yields, as well as higher bathochromic and large Stokes shifts.The synthetic routes followed for the preparation of the fluorescent labels for biomolecules , and 20 , using t3, 11, and 16, were readily synthesized with high yields (85% to 89%). This synthesis involved a thionation reaction on the carbonyl group of 7-diethylamino-4-methylcoumarin (1), followed by the condensation of the resulting thionated coumarin (2) with malononitrile, 4-pyridylacetonitrile hydrochloride, or piperidine, depending on the case.The intermediates mentioned, which had electron-withdrawing groups (EWGs) at position 2, specifically 3, 11, and 16 and the aromatic aldehydes with EDGs at the para position (6-((4-formylphenyl)(methyl)amino)hexanoic acid and 6-(4-formylphenoxy)hexanoic acid) afforded the 4-styrylcoumarins 6, 7, 12, 13, 17, and 18 in good to high yields.The regioselective and highly efficient aldol condensation between intermediates 12 and 13 may have been caused by an acidity decrease in the methyl protons present at position 4 due to the superior electronic delocalization across the molecular structure caused by the pyridine group.The lower yields observed in the pyridinyl derivatives The differences between the electronegativity of the nitrogen or oxygen atom in the electron-donating groups present in the aromatic aldehydes may explain the small difference in reaction yields observed, with a small advantage for the amino aldehydes.6, 7, 12, 13, 17, and 18 produced the N-hydroxysuccinimide (NHS) esters 8, 9, 14, 15, 19, and 20 as promising reactive fluorescent labels for biomolecules, in high yields. The referred fluorescent labels can be obtained through four effective and linear synthetic steps from cheap commercially available precursors (five steps to 14 and 15). All new compounds  were fully characterized by 1D and 2D NMR spectroscopy and HRMS. The spectral data were consistent with the proposed structures.The esterification of coumarin carboxylic acids 16, 3, and 11, respectively. These modifications increased the push–pull character from the electron-donating group NEt2, at position 7, by extending the π-system with the introduction of a new double bond that was conjugated with the aforementioned electron-withdrawing groups (EWGs). The combination between an effective positive charge and the electronic delocalization present in the pyridine moiety of the EWG intermediate 11 allowed the largest bathochromic shift observed. In contrast, the intermediate 16 that only presented a positive charge as EWG had the lowest bathochromic shift value.We conducted a study on the photophysics of the newly synthesized coumarin derivatives, examining their absorption and emission characteristics, along with fluorescence quantum yields. para position. As expected, the nitrogen group, when compared with the oxygen group, promoted higher bathochromic shifts (approximately more than 40%) due to the superior mesomeric electron-donating character of this substituent.Unsurprisingly, these shifts were further increased to longer wavelengths with the inclusion of a 4-styryl moiety, containing one electron-donating nitrogen or oxygen group at the 8, 9, 14, 15, 19, and 20) were also strongly influenced by the nature of the EWGs at position 2 and the EDGs at position 4 in the coumarin ring  = 24,000 cm−1 M−1 vs. ε (15) = 113,500 cm−1 M−1). Considering only the 4-styryl moiety effect, the presence of the nitrogen group more than doubled the molar absorption coefficients when compared with the counterpart oxygen group.The molar absorption coefficients of the fluorescent labels  process of the emissive excited state, due to the extension of the π-conjugated system in the molecule. Large Stokes shifts minimized the undesirable spectral overlap between absorption and emission, allowing the reduction in the interference and the quenching process, providing an effective detection of the fluorescence emission.14 and 15 could be related to the effective intramolecular charge transfer process due to the increase in the extension of π-conjugated system in these molecules + were 511.2704 and 511.2696. The UV λmax  values were as follows: 500 and 578. The ε (cm−1 M−1) values were as follows: 59,000. ΦF = 0.27.The E)-2,5-dioxopyrrolidin-1-il 6-(4-(2-(2-(dicyanomethylene)-7-(diethylamino)-2H-chromen-4-il)vinyl)phenoxy)hexanoate (8) was performed according to the method described by Eustáquio et al. + were 608.2868 and 608.2863. The UV λmax  values were as follows: 498, 576. The ε (cm−1 M−1) values were as follows: 59,000. ΦF = 0.27.The E)-2-(7-(diethylamino)-4-methyl-2H-chromen-2-ylidene)-2-(pyridin-4-yl)acetonitrile (10) was performed according to the methods described by Gandioso et al. + were 346.1914 and 346.1906. The UV λmax  value was 550. The ε (cm−1 M−1) value was 80,000. ΦF = 0.36.The 11 , 6-(4-formylphenoxy)hexanoic acid 4 , and piperidine  in dry acetonitrile (4 mL) was stirred at 85 °C, for a period of 48 h. The reaction mixture, at room temperature, was then acidified with 6M HCl and was continuously monitored by TLC, using CHCl3/CH3OH/H2O (65:10:1) as the eluent. The reaction mixture was evaporated to dryness, and the residue was purified by flash chromatography, using CHCl3/CH3OH/H2O (65:10:1) as the eluent, to yield 4-(((E)-4-((E)-4-((5-carboxypentyl)oxy)styryl)-7-(diethylamino)-2H-chromen-2-ylidene)(cyano)methyl)-1-methylpyridin-1-ium iodide, as a dark green solid .A mixture of compound 1H NMR 2SO, δ, ppm) values were as follows: 1.16 2), 1.45 , 1.58 , 1.74 , 2.24 , 3.51 2), 4.03 , 4.13 , 6.84 , 6.85 , 6.98 , 6.99 , 7.54 , 7.78 , 7.96 , 8.07 , and 8.47 . The 13C NMR 2SO, δ, ppm) values were as follows: 12.5 , 24.3 (C-32), 25.2 (C-31), 28.4 (C-30), 33.8 (C-33), 44.2 , 45.8 (C-21), 67.6 (C-29), 77.7 (C-16), 96.7 (C-8), 103.3 (C-3), 108.6 (C-4a), 111.6 (C-6), 114.8 , 117.2 (C-9), 118.5 (C-15), 119.9 , 127.0 (C-5), 128.1 (C-23), 130.4 , 139.1 (C-22), 143.3 , 147.7 (C-4), 148.4 (C-14), 151.9 (C-7), 155.1 (C-8a), 160.5 (C-26), 166.0 (C-2), and 174.5 (C-34). The FTMS(+) calc. values for C35H38O4N3 [M-(I-)]+ were 564.28568 and 564.2849. The UV λmax  values were as follows: 593 and 615. The ε (cm−1 M−1) value was 55,000. ΦF = 0.07.The 11 , 6-((4-formylphenyl)(methyl)amino)hexanoic acid 5 , and piperidine  in dry acetonitrile (10 mL) was stirred at 85 °C, for a period of 24 h. The reaction mixture, at room temperature, was then acidified with 6M HCl and was continuously monitored by TLC, using CHCl3/CH3OH/H2O (65:10:1) as the eluent. The reaction mixture was evaporated to dryness, and the residue was purified by flash chromatography, using CHCl3/CH3OH/H2O (65:10:1) and CHCl3/CH3OH/H2O (65:20:2) as eluents, to yield 4-(((E)-4-((E)-4-((5-carboxypentyl)(methyl)amino)styryl)-7-(diethylamino)-2H-chromen-2-ylidene)(cyano)methyl)-1-methylpyridin-1-ium iodide, as a dark blue solid .A mixture of compound 1H NMR 2SO, δ, ppm) values were as follows: 1.14 2), 1.27–1.33 , 1.48–1.56 , 2.17 , 2.99 , 3.40 , 3.45 2), 4.05 , 6.68 , 6.72 , 6.75 , 6.82 , 7.27 , 7.38 , 7.59 , 7.79 , 7.97 , and 8.36 . The 13C NMR 2SO, δ, ppm) values were as follows: 12.5 , 24.8 (C-32), 26.1 (C-31), 26.2 (C-30), 34.6 (C-33), 38.1 (C-35), 44.2 , 45.5 (C-21), 51.3 (C-29), 76.7 (C-16), 96.6 (C-8), 101.6 (C-3), 108.6 (C-4a), 111.4 , 111.5 (C-6), 112.9 (C-9), 118.8 (C-15), 119.1 , 122.6 (C-23), 126.7 (C-5), 130.9 , 140.6 (C-22), 142.8 , 148.0 (C-4), 148.5 (C-14), 150.8 (C-26), 151.7 (C-7), 155.0 (C-8a), 165.3 (C-2), and 175.0 (C-34). The FTMS(+) calc. values for C36H41O3N4 [M-(I)-)]+ were 577.3173 and 577.3161. The UV λmax  value was 634. The ε (cm−1 M−1) value was 113,500. ΦF = 0.05.The 12 , DCC , and DMAP  in dry DMF (2 mL) and dry acetonitrile (8 mL) was stirred at room temperature for 5 min. After this reaction time, N-hydroxysuccinimide  was added, and the reaction was stirred at room temperature for 6 h and 30 min. The reaction was monitored by TLC, using CH2Cl2/CH3OH (9:1) as the eluent. The reaction mixture was evaporated to dryness, and the residue was purified by flash chromatography using CH2Cl2/CH3OH (95:5) and CH2Cl2/CH3OH (9:1) as eluents, to yield 4-(cyano((E)-7-(diethylamino)-4-((E)-4-oxy)-6-oxohexyl)oxy)styryl)-2H-chromen-2-ylidene)methyl)-1-methylpyridin-1-ium iodide, as a dark green solid .A mixture of compound 1H NMR  values were as follows: 1.25 2), 1.54 , 1.83 , 2.67 , 2.86 , 3.59 2), 4.02 , 4.13 , 6.77 , 6.90 , 6.97 , 7.03 , 7.26 , 7.38 , 7.56 , 7.81 , 7.98 , and 8.57 . The 13C NMR  values were as follows: 13.2 , 24.7 (C-32), 25.6 (C-31), 26.0 , 29.0 (C-30), 31.2 (C-33), 46.0 , 46.7 (C-21), 68.1 (C-29), 79.1 (C-16), 98.2 (C-8), 104.2 (C-3), 109.5 (C-4a), 112.5 (C-6), 115.3 , 117.0 (C-9), 119.2 (C-15), 120.6 , 126.6 (C-5), 128.3 (C-23), 130.4 , 139.7 (C-22), 143.1 , 148.5 (C-4), 149.7 (C-14), 152.9 (C-7), 156.1 (C-8a), 161.3 (C-26), 167.0 (C-2), 168.8 (C-34), and 169.6 . The FTMS(+) calc. values for C39H41O6N4 [M + H]+ were 661.3021 and 661.3013. The UV λmax  values were as follows: 589 and 615. The ε (cm−1 M−1) value was 55,000. ΦF = 0.10.The 13 , DCC , and DMAP  in dry DMF (2 mL) and dry acetonitrile (8 mL) was stirred at room temperature for 5 min. After this reaction time, N-hydroxysuccinimide  was added, and the reaction was stirred at room temperature for 23 h. The reaction was monitored by TLC, using CHCl3/CH3OH (9:1) as the eluent. The reaction mixture was evaporated to dryness, and the residue was purified by flash chromatography using CHCl3/CH3OH (9:1) and CHCl3/CH3OH (8:2) as eluents, to yield 4-(cyano((E)-7-(diethylamino)-4-((E)-4-oxy)-6-oxohexyl)(methyl)amino)styryl)-2H-chromen-2-ylidene)methyl)-1-methylpyridin-1-ium iodide, as a dark blue solid .A mixture of compound 1H NMR  values were as follows: 1.25 2), 1.46–1.52 , 1.62–1.72 , 1.77–1.85  2.64 , 2.86 , 3.04 , 3.42 , 3.57 2), 4.11 , 6.66 , 6.77  6.90 , 7.03 , 7.15 , 7.41 , 7.51 , 7.83 , 7.93 , and 8.45 . The 13C NMR values were as follows: 13.0 , 24.6 (C-32), 25.8 , 26.2 (C-31), 26.7 (C-30), 29.8 (C-33), 38.7 (C-35), 45.6 , 46.3 (C-21), 52.3 (C-29), 77.4 (C-16), 97.9 (C-8), 103.0 (C-3), 109.4 (C-4a), 112.0 , 112.2 (C-6), 113.2 (C-9), 119.3 (C-15), 120.0 , 123.2 (C-23), 126.3 (C-5), 130.8 , 141.0 (C-22), 142.4 , 148.9 (C-4), 149.8 (C-14), 151.1 (C-26), 152.5 (C-7), 156.0 (C-8a), 166.3 (C-2), 168.6 (C-34), and 169.4 . The FTMS(+) calc. values for C40H44O5N5 [M-(I-)]+ were 674.3337 and 674.3331. The UV λmax  value was 634. The ε (cm−1 M−1) value was 113,500. ΦF = 0.07.The H-chromen-2-ylidene)-piperidin-1-ium nitrate (16) was performed according to the method described by Eustáquio et al. + were 530.3377 and 530.3369. The UV λmax  value was 489. The ε (cm−1 M−1) value was 64,000. ΦF = 0.54.The E)-1-(7-(diethylamino)-4-oxy)-6-oxohexyl)oxy)styryl)-2H-chromen-2-ylidene)piperidin-1-ium nitrate (19) was performed according to the method described by Eustáquio et al. + were 627.3541 and 627.3531. The UV λmax  value was 487. The ε (cm−1 M−1) value was 640,00. ΦF = 0.56.The To shed light into the observed photophysical properties, density functional theory (DFT) and time-dependent density functional theory (TD-DFT) and calculations were performed by the Gaussian 16 package  with theH-chromen-2-ylidene)malononitrile (3), (E)-4-(cyano(7-(diethylamino)-4-methyl-2H-chromen-2-ylidene)methyl)-1-methylpyridin-1-ium iodide (11), and 1-(7-(diethylamino)-4-methyl-2H-chromen-2-ylidene)piperidin-1-ium nitrate (16) hold significant promise as cost-effective and innovative intermediate compounds in the synthesis of novel fluorescent labels for biomolecules. The utilization of DFT and TDDFT calculations provided valuable insights into the observed photophysical properties, specifically the large Stokes shifts. These shifts were attributed to substantial structural relaxation observed in the excited states of the molecules, offering a rationalization for the phenomenon.In summary, our aim to extend the delocalization the π-electron system led us to create and produce novel derivatives of 4-styrylcoumarin. These derivatives exhibited absorption and emission characteristics at extended wavelengths and featured significant Stokes shifts. All of this was accomplished through a straightforward, efficient, and cost-effective synthetic approach. The results derived from the UV/Vis spectra indicated that the intermediates 2-(7-(diethylamino)-4-methyl-2We are currently working on synthesizing other red-shifted coumarin fluorescent labels for biomolecules, aiming to enhance their characteristics. We anticipate sharing a concise report on the outcomes in the near future.


The planar structures of compounds were elucidated via detailed 1D, 2D NMR, and HR-ESI-MS analysis. ECD spectra, optical rotation values calculation, and alkali hydrolysis were applied in the determination of the absolute configuration of the new compounds. In bioassays, 6 and 9 exhibited promising antifungal activities against Penicillium italicum, with an MIC value of 6.25 μM for both compounds. Moreover, 3 displayed moderate AChE inhibitory activity with an IC50 value of 20.6 ± 0.3 μM.Five new polyketides, including two chromones ( Trichoderma species have been widely discovered in marine environments, including soil, decaying wood, and living plants in mangrove forests + 273.0734 . The 1H NMR spectrum showed two aromatic protons at δH 6.59 and 6.50, an oxymethylene at δH 4.57, two methoxy signals at δH 3.91 and 3.90, and a methyl at δH 2.48. The 13C NMR spectrum showed a conjugated carbonyl at δC 178.2, four oxygenated olefinic carbons at δC 166.1, 165.5, 162.2, and 161.1, two quaternary carbons at δC 121.6 and 109.0, two protonated olefinic carbons at δC 97.1 and 93.9, two methoxys at δC 56.5 and 56.4, an oxymethylene at δC 55.5, and a methyl at δC 17.8. Comparing the 1D NMR data of 2 and 3, 2 was highly similar to 3 + 273.0734 . The 1H NMR spectrum acquired in DMSO-d6 showed two exchangeable protons at δH 9.22, four aromatic protons at δH 6.12, 6.10, 6.07, and 6.04, a methylene adjacent to olefinic carbons at δH 3.65, and a methyl δH 2.20. The 13C NMR spectrum showed a conjugated carbonyl at δC 178.7, four oxygenated olefinic carbons at δC 167.6, 165.8, and 158.5 (2C), a quaternary carbon at δC 137.5, five protonated olefinic carbons at δC 113.2, 113.2, 106.9 (2C), and 101.2, a methylene adjacent to olefinic carbons at δC 38.7, and a methyl at δC 19.2. The HMBC correlation signals from H-3 to C-1, C-2, C-4, and C-5, and H-1 and H-5 to C-6 indicated the presence of a 2,4-dihydroxyphenyl moiety. HMBC correlations from H-9 to C-7, C-8, C-10, and C-11; H-11 to C-12; H3-13 to C-11 and C-12 constructed an 8,12-dihydroxyhepta-8,11-dien-10-one moiety. The two moieties connected with C-7 were determined via the analysis of HMBC correlations from H2-7 to C-1, C-5, and C-6. The NOESY correlations of H2-7/H-9 and H-11/H3-13 revealed that these protons were on the same side. Thus, the structure of 4 was established as -7--8,12-dihydroxyhepta-8,11-dien-10-one.Compound 5 was isolated as a yellow oil. Its molecular formula C15H18O6 with seven degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M − H2O + H]+ 277.1070 . The 1H NMR spectrum acquired in DMSO-d6 showed three exchangeable protons at δH 9.22 (2H) and 4.80, four aromatic protons at δH 6.12, 6.10, 6.05, and 6.04, an oxymethine at δH 3.90, two methylenes at δH 3.64 and 2.52, and a methyl at δH 1.06. The 13C NMR spectrum showed a conjugated carbonyl at δC 178.7, four oxygenated olefinic carbons at δC 167.7, 166.9, and 158.5 (2C), a quaternary carbon at δC 137.5, five protonated olefinic carbons at δC 114.0, 113.3, 106.9 (2C), and 101.2, an oxymethine at δC 64.0, two methylenes at δC 42.6 and 38.8, and a methyl at δC 23.2. Compared with 4, compound 5 was identified to possess an iso-propanol group instead of a methyl connecting to C-12, which was verified via the analyses of 1H–1H COSY signals H2-13/H-14/H3-15 and HMBC correlations from 14-OH to C-13, C-14, and C-15; H2-13 to C-11 and C-12. The NOESY correlations H2-7/H-9 and H-11/H2-13 revealed that these protons were on the same side. Therefore, the planar structure of 5 was established, as shown in Compound 5, C-14, experimental and calculated optical rotation values were obtained. The experimental optical rotation value c 0.26, MeOH) was matched with the calculated one for 14S-5, S-5 showed a good agreement with the experimental one, as shown in 5 was determined to be -7--8,12,14-trihydroxynona-8,11-dien-10-one.To identify the configuration of the only chiral center of 6 was isolated as a pale-yellow oil. Its molecular formula C25H38O7 with seven degrees of unsaturation was deduced by the ion peak of HR-ESI-MS m/z [M + Na]+ 473.2509 . Analyses of the 1H and 13C NMR data aided with HSQC revealed the presence of three carbonyls, two quaternary carbons, ten methines, five methylenes, and five methyls (NMR data in 8)  to yield five subfractions (SFrs. 7.1–7.5). SFrs. 7.3 (5.4 g) was applied to silica gel column chromatography (CC)  to give a mixture of 1, 4, and 5 (24 mg). The mixture was isolated using reverse phase C18 silica gel column  to yield 1 (5.1 mg), 4 (2.3 mg), and 5 (4.5 mg). Frs. 3 (13.2 g) was subjected to Sephadex LH-20  to yield three subfractions (SFrs. 3.1–3.3). SFrs. 3.3 (4.6 g) was applied to silica gel CC  to give mixture of 2 and 3 (26 mg). The mixture was isolated utilizing HPLC with the ChiralPak AY-H column, respectively, at tR = 6.0 and 15.5 min  to give 2 (3.2 mg) and 3 (20.6 mg). Frs. 4 (15.5 g) was subjected to Sephadex LH-20  to yield three subfractions (SFrs. 4.1–4.3). SFrs. 4.2 (6.0 g) was applied to silica gel CC  to give mixture of 6−10 (126 mg). The mixture was isolated utilizing HPLC with the ChiralPak AY-H column, respectively, at tR = 15.0, 18.5, 9.0, 12.5, and 4.5 min  to give 6 (10.2 mg), 7 (35.7 mg), 8 (34.3 mg) 9 (12.3 mg), and 10 (6.5 mg).Frs. 7 (10.6 g) was subjected to Sephadex LH-20 + 329.0627 ; 1H and 13C NMR data (5-hydroxy-3-((3’NMR data . SpectraH-chromen-4-one (2): C13H14O5; Light-yellow oil; UV (MeOH) λmax (log ε): 201 (3.81), 230 (3.98), 280 (3.51) nm; HR-ESI-MS: m/z [M + Na]+ 273.0734 ; 1H and 13C NMR data (3-(hydroxymethyl)-5,7-dimethoxy-2-methyl-4NMR data . SpectraZ,11Z)-7--8,12-dihydroxyhepta-8,11-dien-10-one (4): C13H14O5; Yellow oil; UV (MeOH) λmax (log ε): 201 (4.12), 241 (3.74), 276 (3.04) nm; HR-ESI-MS: m/z [M + Na]+ 273.0734 ; 1H and 13C NMR data -7--8,12,14-trihydroxynona-8,11-dien-10-one (5): C15H18O6; Yellow oil; c 0.26, MeOH); UV (MeOH) λmax (log ε): 201 (4.18), 246 (3.76), 277 (3.02) nm; HR-ESI-MS: m/z [M − H2O + H]+ 277.1070 ; 1H and 13C NMR data ((14NMR data . Spectra6): C25H38O7; Pale-yellow oil; c 0.45, MeOH); UV (MeOH) λmax (log ε): 215 (3.82) nm; HR-ESI-MS: m/z [M + Na]+ 473.2509 ; 1H and 13C NMR data  in MeOH (200 μL) was stirred evenly for 5 min, and then 200 μL of aqueous NaOH (1.0 M) was added. The reaction mixture was stirred for 30 min at room temperature. After completion, the reaction mixture was extracted with EA thrice, and the organic layer was concentrated under reduced pressure to afford eujavanicol A (1.9 mg). In order to determine its absolute configuration and exclude potential differences in test conditions with the previous articles, the known compound 8 (5.0 mg) was also hydrolyzed to produce eujavanicol A (3.2 mg) following the same procedure.Compound 1 and 5 for calculation in Initial conformational analysis was carried out using the Merck molecular force field method with the Spartan 14’ software . The conformation with a Boltzmann population larger than 1% was selected for optimization and calculation in MeOH at B3LYP/6-31+G level with the density functional theory (DFT) executed via Gaussian 09 . The ECDThe compounds to be tested were dissolved individually in dimethyl sulfoxide (DMSO), and the antimicrobial activity assays were performed in 96-well plates via a serial dilution test in the range of 0.1–100 μM, according to the methods previously reported ,19. All 1–10 and eujavanicol A were evaluated for AChE inhibitory activity, following the same method previously described [50 was the average value of two independent experiments.Compounds escribed . Donepez1–10 and eujavanicol A on cells were assessed using MTT assay as described previously [The cytotoxicities of compounds eviously . Six cel1–2), two phenyl derivatives (4–5), and a tandyukusin derivative (6), along with five known polyketides (3 and 7–10), were isolated from mangrove endophytic fungus Trichoderma lentiforme ML-P8-2, and it is the first time to report secondary metabolites from this specific species. The planar structures of the isolated compounds were elucidated via detailed 1D, 2D NMR, and HR-ESI-MS analysis. ECD spectra, optical rotation values calculation, and alkali hydrolysis were applied in the determination of the absolute configuration of the new compounds. In bioassays, antimicrobial, AChE inhibitory, and cytotoxic activities tests for compounds 1–10, together with the alkali-hydrolysis treatment product eujavanicol A, were carried out. Compounds 6 and 9 exhibited promising antifungal activities against Penicillium italicum, with MIC, both for 6.25 μM. Moreover, 3 displayed moderate AChE inhibitory activity with an IC50 value of 20.6 ± 0.3 μM.In summary, five new polyketides, including two chromones (


Sophora flavescens led to the isolation of two novel prenylflavonoids with an unusual cyclohexyl substituent instead of the common aromatic ring B, named 4′,4′-dimethoxy-sophvein (17) and sophvein-4′-one (18), and 34 known compounds . The structures of these chemical compounds were determined by spectroscopic techniques, including 1D-, 2D-NMR, and HRESIMS data. Furthermore, evaluations of nitric oxide (NO) production inhibitory activity against lipopolysaccharide (LPS)-treated RAW264.7 cells indicated that some compounds exhibited obvious inhibition effects, with IC50 ranged from 4.6 ± 1.1 to 14.4 ± 0.4 μM. Moreover, additional research demonstrated that some compounds inhibited the growth of HepG2 cells, with an IC50 ranging from 0.46 ± 0.1 to 48.6 ± 0.8 μM. These results suggest that flavonoid derivatives from the roots of S. flavescens can be used as a latent source of antiproliferative or anti-inflammatory agents. The phytochemical investigation of the roots of the traditional Chinese medicinal plant  Sophora flavescens are ordinarily served in the traditional Chinese medicine (TCM), “Ku Shen”, for the curing of skin diseases, cancer, dysentery, hematochezia, jaundice, pruritus vulvae, eczema, and hepatitis + , suggesting the molecular formula C20H24O6 with 9° of unsaturation. The 1H and 13C NMR spectroscopic data of 18 are similar to 17 . Combined with HR-ESI-MS, it was found that the molecular weight of compound 18 is 46 less than that of 17. The above data indicate that the C-4′ of the B-ring of compound 18 has a carbonyl group. This inference was further defined by the HMBC correlation of the δH 2.03 (H-3′b), 2.21 (H-2′), and 2.10 (H-6′) proton signals with C-4′ (δC 210.3). Finally, by comparing the NOE effects in the ROESY spectra of the two compounds, it was found that 18 and 17 have the same configuration. The optical rotation values of the two compounds  for 18 also support this inference. Based on the above data analysis, the structure of 18 was confirmed and named sophvein-4′-one. See 1H and 13C NMR, HMQC, HMBC, 1H-1H COSY, and ROESY of compounds 17 and 18.Compound ar to 17 , with th1–16 and 19–36) were determined based on a comparison with published NMR data in the references to be sophorafiavanone B (1)  ×100%, and SPSS version 21.0  was used for calculating CC50 extract. 22 was the most toxic composition with a CC50 value of 13.8 ± 0.6 μM.The potential cytotoxicity of compounds on RAW 264.7 macrophages cells was determined before executing further studies. Macrophage cells were treated with compounds, and a mitochondria colorimetric (MTT) assay was used to test cell survival. Cell viability (%) =  × 100%. The determination of IC50 was calculated using probit analysis in SPSS version 21.0. The research results show that some compounds exhibited obvious NO production inhibitory activity, with IC50 ranging from 4.6 ± 1.1 to 14.4 ± 0.4 μM, as shown in 35 displayed the most significant inhibition of NO production, with an IC50 value of 4.6 ± 1.1 μM.NO is one of the immune effectors used by macrophages to defend our bodies against intracellular pathogens. To determine if compounds 1–36 was measured by the MTT assay and compared with a negative control and positive control . Inhibitory rate (%) = [(O.DDrug − O.DBlank)/(O.DControl − O.DBlank)] ×100%. The IC50 values were calculated with probit analysis using software SPSS version 21.0. The results show that eight compounds inhibited the growth of HepG2 cells with an IC50 ranging from 0.46 ± 0.1 to 48.6 ± 0.8 μM (50 > 50 μM). Compound 22 exhibited the best antiproliferative effects on HepG2 cell lines, with an IC50 value of 0.46 ± 0.1 μM. The present work revealed that many secondary metabolites of S. flavescens have antiproliferative and NO production inhibitory activities, indicating its application in traditional Chinese medicine.The inhibitory rate of HepG2 cells growth of compounds ± 0.8 μM , while o1H NMR, 13C NMR, HMBC, HSQC, 1H-1H COSY, and ROESY spectra were measured by a Bruker Advance III HD using tetramethylsilane (TMS) as an internal standard . Chemical shifts are demonstrated in δ (ppm) and relative to the residual solvent signals. Silica gel , polyamide , sephadex LH-20 , and RP-18 reverse-phase silica gel  have been used for column chromatography (CC). The reverse-phase medium-pressure liquid chromatography (RP-MPLC) system comprises a Büchi pump , column and precolumn , and MCI-gel CHP-20P . Thin-layer chromatography (TLC) was performed using precoated silica gel plates , and the spots were visualized by spraying with 10% sulfuric acid ethanolic solution or α-naphthol-sulfuric acid solution and heating at 110 °C for 3–5 min. Murine macrophage (RAW 264.7) cells were acquired from Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). Macrophage cells were placed in a constant temperature incubator and cultured at a temperature of 37 °C and a concentration of 5% CO2. Human hepatoma HepG2 cell lines were also obtained from the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). HepG2 cells were cultured at 37 °C under 5% CO2. Additionally, an inverted phase contrast microscope was used to observe cell morphology.The optical rotation value was recorded with a Jasco DIP-370 polarimeter . The ultraviolet (UV) spectrum was recorded by a UV2700 spectrophotometer . The infrared (IR) spectrum was obtained by an FT-IR spectrophotometer  using KBr pellets. High-resolution electrospray ionization mass spectroscopy (HRESIMS) was obtained with an Agilent 6500 LC/Q-TOF mass spectrometer . The S. flavescens were collected in Honghe, Yunnan Province, People’s Republic of China, in June 2019. The sample was identified by one of the authors (Xuan-Qin Chen). A voucher specimen (number: KUMST20190628) was conserved at the Key Laboratory of Phytochemistry, Kunming University of Science and Technology, China.The roots of S. flavescens (20 Kg) were crushed and extracted with 95% aq. ethanol (24 h × 3 times). The ethanol extracts were percolated and evaporated in vacuo to obtain a residue. This residue was suspended in water and then extracted with ethyl acetate (3 times) and concentrated to produce an ethyl acetate phase (493 g). The ethyl acetate phase was segmented and enriched by macroporous resin to obtain 308 g of total flavonoids. The total flavonoid extract (300 g) was segmented using a normal phase silica gel column chromatography (CC) and stepwise gradient elution with a gradient of petroleum ether-ethyl acetate (4:1–1:1) to obtain 7 subfractions (Fr.1–7).The air-dried roots of 3 (14.0 mg). F1-2 (567.0 mg) was further segmented via Sephadex LH-20 (methanol) to obtain 8 subfractions (F1-2-1~F1-2-8). F1-2-5 (59.0 mg) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-ethyl acetate (9:1–2:1) to obtain compounds 1 (141.4 mg) and 4 (33.2 mg). F1-2-8 (80.0 mg) was segmented by CC (silica gel) and stepwise gradient elution with petroleum ether-ethyl acetate (10:1–5:1) to yield compound 22 (30.0 mg).Fr.1 (3.4 g) was further segmented by a silica gel chromatography column (CC) stepwise gradient elution with petroleum ether-dichloromethane (1:2) to yield two subfractions (F1-1~F1-2). F1-1 (1.9 g) was further segmented via Sephadex LH-20 (methanol) to obtain 8 subfractions (F1-1-1~F1-1-8). F1-1-5 (120.6 mg) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (150:1) to obtain compound 15 (8.3 mg) and 16 (7.5 mg). F2-4-5 (15.0 mg) was further segmented via Sephadex LH-20 (methanol) to obtain compound 11 (6.0 mg). F2-6 (246.0 mg) was further segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (100:1–50:1) to obtain 5 subfractions (F2-6-1~F2-6-5). F2-6-3 (32.5 mg) was further segmented via Sephadex LH-20 (methanol) to yield compound 19 (24.3 mg). F2-6-5 (508.6 mg) was further segmented via Sephadex LH-20 (methanol) to obtain compound 21 (468.8 mg).Fr.2 (1.5 g) was further segmented via Sephadex LH-20 (methanol) to obtain 6 subfractions (F2-1~F2-6). F2-4 (366.0 mg) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (150:1) to obtain 5 subfractions (F2-4-1~F2-4-5). F2-4-4 (28.0 mg) was further segmented via Sephadex LH-20 (methanol) to obtain compounds 10 (5.6 mg). F3-1-4 (1.1 g) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (50:1–10:1) to obtain 6 subfractions (F3-1-4-1~F3-1-4-6). F3-1-4-5 (642.5 mg) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-ethyl acetate (30:1–10:1) to obtain 3 subfractions (F3-1-4-5-1~F3-1-4-5-3). F3-1-5 (2.3 g) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-ethyl acetate (50:1–30:1) to obtain 6 subfractions (F3-1-5-1~F3-1-5-6). F3-1-5-1 (400.4 mg) was further segmented via Sephadex LH-20 (methanol) to obtain 2 subfractions (F3-1-5-1-1~F3-1-5-1-2). F3-1-5-1-1 (119.2 mg) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-acetone (4:1–1:1) to obtain compound 34 (27.8 mg). F3-1-5-1-2 (70.3 mg), followed by semipreparative high-performance liquid chromatography (HPLC) , was used to obtain compound 31 (4.1 mg). F3-2 (25.0 g) was first subjected to reverse-phase RP-18 chromatography with 40–100% methanol-water as the eluent, and the same fractions were combined by thin-layer chromatography to obtain 8 subfractions (F3-2-1~F3-2-8). F3-2-5 (276.5 mg) was further segmented via Sephadex LH-20 (methanol) to yield compound 30 (140.2 mg).Fr.3 (30.0 g) was further segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (50:1–10:1) to obtain 2 subfractions (F3-1~F3-2). F3-1 (4.0 g) was further segmented via Sephadex LH-20 (methanol) to yield 7 subfractions (F3-1-1~F3-1-7). F3-1-3 (501.6 mg) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (50:1–20:1) to obtain 4 subfractions (F3-1-3-1~F3-1-3-4). F3-1-3-2 (61.2 mg) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-ethyl acetate (30:1–10:1) to obtain compound 36 (68.2 mg). F4-3-5 (78.5 mg) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-ethyl acetate (20:1–5:1) to obtain compound 33 (30.1 mg). F4-4 (900.6 mg) was further segmented via Sephadex LH-20 (methanol) to obtain 3 subfractions (F4-4-1~F4-4-3). F4-4-1 (20.3 mg) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-ethyl acetate (3:1) to obtain compound 17 (9.0 mg). F4-4-2 (102.3 mg) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-ethyl acetate (3:1) to obtain compound 18 (12.2 mg). F4-4-3 (500.1 mg) was segmented by CC (silica gel) stepwise gradient elution with chloroform-methanol (80:1–20:1) to obtain 4 subfractions (F4-4-3-1~F4-4-3-4). F4-5 (1.1 g) was further segmented via Sephadex LH-20 (methanol) to obtain 5 subfractions (F4-5-1~F4-5-5). F4-5-5 (350.4 mg) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-ethyl acetate (4:1–1:1) to obtain 4 subfractions (F4-5-5-1~F4-5-5-4). F4-5-5-1 (60.3 mg) was segmented by CC (silica gel) stepwise gradient elution with trichloromethane-methanol (60:1–10:1) to obtain compound 7 (27.5 mg). F4-5-5-3 (200.3 mg) was further segmented via Sephadex LH-20 (methanol) to obtain compound 9 (123.9 mg). F4-6-4 (2.1 g) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-ethyl acetate (4:1–1:1) to obtain 2 subfractions (F4-6-4-1~F4-6-4-2). F4-6-4-1 (1.2 g) was further segmented via Sephadex LH-20 (methanol) to obtain compound 27 (20.7 mg). F4-6-4-2-2 (1.0 g) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-ethyl acetate (3:1–1:1), and then was further segmented via Sephadex LH-20 (methanol) to obtain compounds 5 (60.6 mg) and 35 (40.2 mg).Fr.4 (11.0 g) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (60:1–10:1) to obtain 6 subfractions(F4-1~F4-6). F4-3 (900.7 mg) was further segmented via Sephadex LH-20 (methanol) to obtain 5 subfractions (F4-3-1-F4-3-5). F4-3-4 (163.3 mg) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-ethyl acetate (20:1–5:1) to obtain compound 32 (10.3 mg). F5-2-2 (1.3 g) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-acetone (2:1–1:1) to obtain 5 subfractions (F5-2-2-1~F5-2-2-5). F5-2-2-3 (201.4 mg) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (25:1–10:1) and then was further segmented via Sephadex LH-20 (methanol) to obtain compound 8 (5.0 mg). F5-3 (10.0 g) was segmented by CC (silica gel) stepwise gradient elution with trichloromethane-methanol (80:1–30:1) to obtain 6 subfractions (F5-3-1~F5-3-6). F5-3-3-7 (91.5 mg), followed by semipreparative HPLC , was used to obtain compound 2 (4.0 mg). F5-3-6 (5.0 g) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-acetone (3:1–1:1) to obtain 4 subfractions (F5-3-6-1~F5-3-6-4). F5-3-6-3, (76.1 mg) followed by semipreparative HPLC , was used to obtain compound 26 (4.2 mg). F5-3-6-4 (47.6 mg), followed by semipreparative HPLC , was used to obtain compound 28 (4.2 mg) and 29 (4.0 mg). F5-4 (2.0 g) was further segmented via Sephadex LH-20 (methanol) to obtain 9 subfractions (F5-4-1~F5-4-9). F5-4-1 (614.5 mg) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-acetone (3:1–1:1) to obtain compound 13 (500.1 mg). F5-9 (2.0 g) was further segmented via Sephadex LH-20 (methanol) to obtain 10 subfractions (F5-9-1~F5-9-10). F5-9-1 (1.0 g) was segmented by CC (silica gel) stepwise gradient elution with trichloromethane-methanol (50:1–10:1) to obtain compound 14 (806.2 mg). F5-10 (5.5 g) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-acetone (2:1–1:1) to obtain compound 23 (20.1 mg).Fr.5 (92.0 g) was passed through a normal-phase silica gel column, firstly subjected to reversed-phase RP-18 silica gel eluting with methanol-water (60–100%) to obtain 11 subfractions (F5-1~F5-11). F5-1 (1.1 g) was further segmented via Sephadex LH-20 (methanol) to obtain 4 subfractions (F5-1-1~F5-1-4). F5-2 (2.0 g) was further segmented via Sephadex LH-20 (methanol) to obtain 2 subfractions (F5-2-1~F5-2-2). F5-2-1 (90.8 mg) was repeatedly recrystallized to produce compound 13 (10.0 g) and compound 20 (801.5 mg). F6-5-5 (511.6 mg) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-ethyl acetate (3:1–1:1) to obtain 2 subfractions (F6-5-5-1~F6-5-5-2). F6-5-5-2 (105.3 mg) was further segmented via Sephadex LH-20 (methanol) to obtain compound 24 (89.5 mg).Fr.6 (54.0 g) was firstly subjected to reversed-phase RP-18 silica gel eluting with methanol-water (60–100%) to obtain 7 subfractions (F6-1~F6-7). F6-5 (25 g) was segmented by CC (silica gel) stepwise gradient elution with trichloromethane-methanol (100:1–50:1) to obtain compound 6 (5.2 mg). F7-5-7 (40.9 mg) was segmented by CC (silica gel) stepwise gradient elution with trichloromethane-acetone (15:1–1:1) to obtain compound 25 (30.7 mg).Fr.7 (43.0 g) was segmented by CC (silica gel) stepwise gradient elution with dichloromethane-methanol (15:1–1:1) to obtain 7 subfractions (F7-1~F7-7). F7-5 (4.0 g) was further segmented via Sephadex LH-20 (methanol) to obtain 7 subfractions (F7-5-1~F7-5-7). F7-5-3 (203.5 mg) was further segmented via Sephadex LH-20 (methanol) to obtain 3 subfractions (F7-5-3-1~F9-5-3-3). F7-5-3-3 (30.6 mg) was segmented by CC (silica gel) stepwise gradient elution with petroleum ether-acetone (2:1–1:1) to obtain compound 17): white amorphous powder; c 0.25, MeOH); UV λmax (MeOH) nm (log ε): 214 (4.74), 229 (4.64), 285 (4.58); IR (KBr) νmax: 3456, 2950, 1638, 1400, 1098 cm−1; 1H NMR  and 13C NMR , see m/z 429.1894 [M+Na]+ .4′,4′-Dimethoxy-sophvein (18): white amorphous powder; λmax (MeOH) nm (log ε): 214 (4.48), 231 (4.06), 286 (3.97); IR (KBr) νmax: 3446, 2952, 1635, 1406, 1086 cm−1; 1H NMR  and 13C NMR , see m/z 361.1650 [M+Na]+ .Sophvein-4′-one , 1% HEPES , and 10% fetal bovine serum (FBS), as well as streptomycin (SM)  fluid. In all experiments, cells were left to acclimate for 24 h before any treatments.Murine macrophages (RAW 264.7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with the addition of 10% fetal bovine serum (FBS) and 1% streptomycin -penicillin  at 37 °C in a humidified incubator under 5% CO4 per well) were cultured in 96-well plates for 24 h; then, they were treated with several concentrations of compounds 1–36  for 1 h, and then they were stimulated with LPS (1 μg/mL) for another 24 h. After washing twice with a PBS buffer, 20 μL of the MTT solution was added to each well, and incubation continued for 4 h. Optical density (O.D) was measured at 490 nm by a microplate reader . The relative cell viability was calculated in contrast to the normal control group.To measure cell viability, an MTT assay was performed . Macroph1–36 on treatments on NO production, the accumulation of nitrites (NO2−) in the culture medium was recorded as an indicator of NO production [4 per well) were seeded onto 96-well plates and pretreated by compounds 1–36  1 h prior to treatment by LPS (1 μg/mL). Afterward, costimulation for 24 h at 37 °C was carried out in an incubator under 5% CO2. Then, Griess reagents I and II (100 μL) were mixed with cell culture medium (70 μL). Prior to measuring the optical density, plates were incubated at room temperature for 10 min, and the absorbance at 540 nm was measured using a Thermo Fisher Scientific microplate reader. N(G)-monomethyl-L-arginine, monoacetate salt (L-NMMA), and the medium were used as positive and negative controls, respectively.Activated macrophage cells could express inducible nitric oxide synthase (iNOS), catalyzing the production of NO from L-arginine. To determine the effect of compounds oduction ,43. Macr5 per well) and allowed to adhere for 24 h before drug administration. Then, the cell lines were treated with test compounds 1–36 at five concentrations . At 24 and 48 h of incubation, cells were treated with MTT  and dissolved in the culture medium, for 1 h under at 37 °C under a 5% CO2 humidified atmosphere. The MTT was then removed carefully and resolved with DMSO (150 μL/well). Optical density was recorded using a Thermo Fisher Scientific microplate reader at 490 nm. Cisplatin (CP) and medium were used as positive and negative controls, respectively.Briefly, the cells were incubated in 96-well microplates  software and are presented as the mean ± S.D. values of three different experiments. The results were analyzed via one-way analysis of variance (ANOVA), and statistical significance was defined as S. flavescens led to the isolation of 36 flavonoids (1–36), including 18 dihydroflavonoids (1–18), 7 flavonols (19–25), 4 dihydroflavonols (26–29), 3 isoflavones (30–32), and 4 chalcones (33–36), identified by spectroscopic methods , and chemical and physical methods. Bioactivity assays indicated that nine compounds exhibited the most significant NO inhibitory activity, better than that of the L-NMMA positive control (IC50 = 21.8 ± 0.9 μM). Furthermore, the antiproliferative activities against the HepG2 hepatoma cell lines of all isolates were assessed using the MTT method. The results demonstrated that compound 22 exhibited significant cytotoxic activity, with an IC50 value of 0.46 ± 0.1 μM (positive control cisplatin: IC50 = 24.5 ± 0.8 μM). This study lays the foundation for studying the potential therapeutic applications of flavonoid derivatives from S. flavescens for inflammatory diseases and hepatoma.In summary, the systematic phytochemical study on the roots of


FN-19, a coumarin–thiosemicarbazone analog, exhibited potent anti-tyrosinase activity, with an IC50 value of 42.16 ± 5.16 µM, being more active than ascorbic acid and kojic acid, both reference inhibitors. The kinetic study showed that FN-19 acts as a mixed inhibitor. Still, for this compound, molecular dynamics (MD) simulations were performed to determine the stability of the complex with tyrosinase, generating RMSD, RMSF, and interaction plots. Additionally, docking studies were performed to elucidate the binding pose at the tyrosinase, suggesting that the hydroxyl group of coumarin derivative performs coordinate bonds (bidentate) with the copper(II) ions at distances ranging from 2.09 to 2.61 Å. Then, MM/PBSA calculations revealed that van der Waals interactions are the most relevant intermolecular forces for complex stabilization. Furthermore, it was observed that FN-19 has a binding energy (ΔMME) value similar to tropolone, a tyrosinase inhibitor. Therefore, the data obtained in this study will be useful for designing and developing novel coumarin-based analogs targeting the tyrosinase enzyme.Cancer represents the main cause of morbidity and mortality worldwide, constituting a serious health problem. In this context, melanoma represents the most aggressive and fatal type of skin cancer, with death rates increasing every year. Scientific efforts have been addressed to the development of inhibitors targeting the tyrosinase enzyme as potential anti-melanoma agents due to the importance of this enzyme in melanogenesis biosynthesis. Coumarin-based compounds have shown potential activity as anti-melanoma agents and tyrosinase inhibitors. In this study, coumarin-based derivatives were designed, synthesized, and experimentally evaluated upon tyrosinase. Compound  Cancer represents one of the main causes of morbidity and mortality worldwide, accounting for approximately 10 million deaths only in 2020 . Among mO-dopaquinone via two sequential reactions: the hydroxylation, via monophenolase action, from monophenol L-tyrosine to O-diphenol L-DOPA, followed by its oxidation to O-dopaquinone, via diphenolase action .Yield: 76%, yellow amorphous powder; RDescription of 3. -Acetyl-8-methoxy-2H-chromen-2-one (FN-07)T: 2.84 min; Purity: 95.0%; mp: 169–172 °C; 1H NMR : δ 2.61 ; 3.97 ; 7.35 ; 7.40 ; 7.44 ; 8.50 ; 13C NMR : δ 30.62 (CH3); 56.66 (OCH3); 116.87 (CH); 119.90 (CH); 122.35 (C); 125.65 (CH); 125.93 (C); 145.60 (C); 147.80 (CH); 147.83 (C); 159.33 (OC=O); 195.53 (C=O); ATR-FTIR (υ/cm−1): 1687 v(C=O), 1726 v(C=O), 1566 v(C=Cene), 860 δ(C=Cene); CG RT: 14.6 min; C12H10O4: 218.21 g/mol; MS: 218 m/z [M+].Yield: 80%, white amorphous powder; RDescription of Ethyl 2-oxo-2H-chromene-3-carboxylate (MP-01)1H NMR : δ 1.36 ; 4.36 ; 7.32–7.28 ; 7.63–7.58 ; 8.49 ; 13C NMR  δ: 14.2 (CH3), 61.9 (CH2), 116.6 (C), 117.8 (CH), 118.1 (C), 124.8 (CH), 129.5 (CH), 134.3 (CH), 148.6 (CH), 155.0 (C), 156.8 C=O), 162.9 (C=O). ATR-FTIR (υ/cm−1): 1761 v(OC=O), 1681 v(C=Cene), 795 δ(C=Cene); CG RT: 14.3 min; C20H17NO3: 218.20 g/mol; MS: m/z 219 [M+H]+. All these data are in accordance with Cardoso et al. .Yield: 38%, yellow amorphous powder; RDescription of 3. -Acetyl-7-methyl-2H-chromen-2-one (MP-04)T: 3.16 min; Purity: 99.8%; mp: 157–159 °C; 1H NMR : δ 2.50 ; 2.73 ; 7.17 ; 7.19 ; 7.55 ; 8.51 ; 13C NMR : δ 22.35 (CH3); 30.73 (CH3); 116.20 (C); 117.04 (CH); 123.62 (C); 126.51 (CH); 130.14 (CH); 146.63 (C); 147.69 (CH); 155.80 (C); 159.70 (C=O); 195.73 (C=O); ATR-FTIR (υ/cm−1): 2924 v(C−H), 1732 v(OC=O), 1674 v(C=Cene), 1595 v(C=O), 823 δ(C=Cene); CG RT: 13.4 min; C12H10O3: 202.06 g/mol; MS: m/z 203 [M+H]+.Yield: 88%, pale green amorphous powder; RDescription of 3. -Acetyl-2H-chromen-2-one (MP-05)T: 3.03 min; Purity: 99.8%; mp: 126–127 °C; 1H NMR : δ 2.70 ; 7.32 ; 7.35 ; 7.63–7.62 ; 8.48 ; 13C NMR : δ 30.70 (CH3); 116.92 (CH); 118.52 (C); 124.86 (C); 125.17 (CH); 130.42 (CH); 134.56 (CH); 147.57 (CH); 155.59 (C); 159.41 (C=O); 195.63 (C=O); ATR-FTIR (υ/cm−1): 2924 v(C−H), 1732 v(OC=O), 1676 v(C=Cene), 1610 v(C=O), 823 δ(C=Cene); CG RT: 12.0 min; C11H8O3: 188.0 g/mol; MS: m/z 188 [M+]. All these data are in accordance with He et al. +. All data are in accordance with Helmy et al. .Yield: 60%, red amorphous powder; R(FN-06) or (FN-07) were dissolved in 10.0 mL of ethanol absolute. To this solution, an aqueous-ethanolic (50%) solution of semicarbazide hydrochloride or thiosemicarbazide  was added. Then, after 5 min, 2 drops of glacial acetic acid were added. The reactions were stirred at room temperature for 6–24 hrs. When the consumption of the starting material was verified with TLC, using dichloromethane and methanol as eluent, the reaction was stopped and left in the refrigerator overnight. Finally, the solids formed were filtered and washed with cold water and ethanol to furnish the desired products FN-17, FN-19, FN-25, FN-27, and FN-29.Ketone derivatives Description of (1E)-1-(1-(7-Hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)semicarbazide (FN-17)T: 3.16 min; Purity: 99.9%; mp: 226–229 °C; 1H NMR : δ 2.10 ; 6.50 ; 6.72 ; 6.81 ; 7.58 ; 8.23 ; 9.43 ; 13C NMR : δ 15.89 (CH3); 102.18 (CH); 111.95 (C); 114.01 (CH); 122.20 (C); 130.84 (CH); 142.10 (CH); 143.05 (C); 155.74 (C−O); 157.61 (C=N); 160.17 (C=O); 162.00 (C=O). ATR-FTIR (υ/cm−1): 3149 δ(O−H), 3514 and 3402 v(N−H), 1687 v(C=O), 1622 v(C=Cene), 812 δ(C=Cene); CG RT: 16.1 min; C12H11N3O4: 261.07 g/mol; MS: m/z 218 .Yield: 60%, yellow-green amorphous powder; RDescription of (1E)-1-(1-(7-Hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)thiosemicarbazide (FN-19)T: 2.96 min; Purity: 99.9%; mp: 236–239 °C; 1H NMR : δ 2.22 ; 6.73 ; 6.83 ; 7.58 ; 7.91 ; 8.36 ; 8.38 ; 10.36 ; 13C NMR : δ 16.09 (CH3); 101.78 (CH); 111.44 (CH); 113.70 (C); 121.03 (C); 130.62 (CH); 142.66 (CH); 146.62 (C); 155.53 (C=N); 159.55 (COH); 161.92 (C=O); 179.05 (C=S). ATR-FTIR (υ/cm−1): 3340 v(N−H), 3236 δ(O−H), 3311 v(N−H), 1716 v(OC=O), 1683 v(C=Cene), 1217 v(C−N), 813 δ(C=Cene); CG RT: 15.7 min; C13H13N3O3S: 277.30 g/mol; MS: m/z 279 [M+2H]+.Yield: 52%, yellow-orange amorphous powder; RDescription of (1E)-1-(1-(8-Methoxy-2-oxo-2H-chromen-3-yl)ethylidene)semicarbazide (FN-25)T: 2.77 min; Purity: 98.0%; mp: 240–242 °C; 1H NMR : δ 2.12 ; 3.91 ; 6.52 ; 7.30 ; 8.30 ; 9.52 ; 13C NMR : δ 15.20 (CH3); 56.07 (OCH3); 114.25 (CH); 119.56 (C); 120.06 (CH); 124.52 (CH); 126.42 (C); 141.05 (CH); 141.92 (C); 142.51 (C); 146.20 (C=N); 156.93 (C−O); 158.90 (C=O). ATR-FTIR (υ/cm−1): 3384 v(N−H), 3188 v(N−H), 1716 v(OC=O), 1653 v(C=Cene), 1681 v(C=O), 864 δ(C=Cene); CG RT: 17.8 min; C13H13N3O4: 337.11 g/mol; MS: m/z 260 .Yield: 46%, yellow amorphous powder; RDescription of (1E)-1-(1-(8-Methoxy-2-oxo-2H-chromen-3-yl)ethylidene)thiosemicarbazide (FN-27)T: 3.42 min; Purity: 97.5%; mp: 220–222 °C; 1H NMR : δ 2.24 ; 3.91 ; 7.31 ; 7.95 ; 8.39 ; 8.44 ; 10.42 ; 13C NMR : δ 15.89 (CH3); 56.10 (OCH3); 114.56 (CH); 119.44 (C); 120.18 (CH); 124.60 (CH); 125.83 (C); 142.09 (CH); 142.66 (C); 145.82 (C); 146.23 (C=N); 158.73 (C=O); 179.25 (C=S). ATR-FTIR (υ/cm−1): 3456 and 3344 v(N−H), 3207 v(N−H), 1710 v(OC=O), 1612 v(C=Cene), 1240 and 1097 v(C−O), 1220 v(C−N), 835 δ(C=Cene); CG RT: 18.3 min; C13H13N3O3S: 291.07 g/mol; MS: m/z 291 [M+].Yield: 46%, yellow amorphous powder; RDescription of (13E)-N’-(1-(8-Methoxy-2-oxo-2H-chromen-3-yl)ethylidene)isonicotinohydrazide (FN-29)T: 2.78 min; Purity: 98.9%; mp: 202–206 °C; 1H NMR : δ 2.3 ; 3.9 ; 7.3–7.2 ; 7.7 ; 8.2 ; 8.7 ; 11.0 . ATR-FTIR (υ/cm−1): 3188 v(N−H), 1703 v(OC=O), 1606 v(C=Cene), 1678 v(C=O), 1278 v(C−O), 835 δ(C=Cene); CG RT: 18.3 min; C18H15N3O4: 337.33 g/mol; MS: m/z 232 .Yield: 64%, orange amorphous powder; R50 values were calculated from the equation generated with an exponential fit of the experimental data. The effectiveness of inhibition was expressed for the investigated compounds as the percentage of concentration necessary to achieve 50% inhibition (IC50), calculated using the following Equation (1):0 = absorbance of L-DOPA + tyrosinase at t = 0 min, B30 = absorbance of L-DOPA + tyrosinase at t = 30 min, A0 = absorbance of L-DOPA + tyrosinase + inhibitor at t = 0 min, and A30 = absorbance of L-DOPA + tyrosinase + inhibitor at t = 30 min.The mushroom tyrosinase (Sigma-Aldrich) inhibition was performed following previously reported methods ,87. All FN-06 and FN-27 compounds, concentrations between 0.15 and 0.3 mM were used. Maximal initial velocity was determined from the initial linear portion of absorbance up to ten minutes after the addition of the enzyme. The inhibition type of the enzyme, Michaelis constant (Km), and maximal velocity (Vmax) were determined using Lineweaver–Burk plots of the inverse of velocities (1/V) versus the inverse of substrate concentration 1/[L-DOPA] mM−1.A series of experiments was performed to determine the inhibition kinetics of the tested compounds following the already reported method ,89. The ® notebook, , model 5500U, Intel® CoreTM 5th generation, i-7 processer, CPU 2.4 GHz, 16 GB RAM, and running at Windows® 8.1 . The in-house chemolibrary of coumarins was drawn, converted into three-dimensional structures, and energetically minimized by applying the Parameterized Model 3 (PM3) semi-empirical method, using ArgusLab® v. 4.0.1  [Agaricus bisporus fungus with tropolone inhibitor (PDB: 2Y9X) was obtained at the Research Collaboratory for Structural Bioinformatics Protein Data Bank , website . GOLD® v. 5.8.1 software  [® software, v. 0.99 [FN19–tyrosinase complex were assumed as start points for our MD simulations to determine their stabilities during the simulated time. Lastly, all these procedures performed in this study are in accordance with recently published works by our research team involving in silico approaches [® desktop , with a Xeon® Core 2nd generation processer, CPU 3.2 GHz, 12 GB RAM, Video card Asus TUF NVIDIA® GeForce RTX 2060—6GB GDDR6 192 bits Dual, 1920 NVIDIA CUDA® cores, and running a Linux® platform. All MD simulations were performed using the Desmond module in Maestro® v. 2022.1 . Initially, the molecular system was preprocessed by adding missing hydrogens and assuming a pH of 7.4 (PROPKA). The FN19–tyrosinase complex was placed into a 10 Å3 triclinic box, containing water molecules and Na+ and Cl- ions at the physiological concentration (0.15 M). In total, fourteen sodium counterions (Na+) were added into the system to obtain electroneutrality. The FN19–tyrosinase complex, including water molecules and ions, was minimized using the OPLS2005 force field. The Simple Point Charge (SPC) model was used to describe the water solvation model. The complete system described as a total of 37,034 atoms was subsequently energetically minimized to relax potential steric clashes in the structure, and then the constant Number of particles, Pressure, and Temperature (NPT) equilibration was assumed at 300 K and 1 bar pressure. Posteriorly, MD simulations of 100 ns were performed to obtain data on the stability of the resulting complexes, using a recording interval (trajectory) of 100 ps, and approximately 1000 frames. ®UCSF Chimera software was used to perform a cluster analysis for the FN19–tyrosinase complex to obtain the most stable representative structure for further investigations, considering each 5 ns interval of trajectory snapshots comprised within 40–85 ns, which was found to be the most stable period of simulation. Furthermore, Root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and interactions plots were generated using Maestro® v. 2022.1. This protocol is in agreement with other previously published works from our research team [FN19–tyrosinase complex was determined with Ramachandran plots using the web software SAVES  by applying the PROCHECK tool [All molecular docking simulations were performed using a DellWA, USA) . The 3D-dge, UK)  was useddge, UK) . All ill v. 0.99 . Moreoveproaches ,99,100. rch team ,100,101.ECK tool .®GROMACS v. 2018.3 software [.xtc and .tpr files, which are required for this type of calculation. This protocol is in agreement with other previous studies performed by our research team [FN-19 in the presence of explicit water, using the MM/PBSA method to estimate their accurate binding energies. To perform MM/PBSA calculations, the g_mmpbsa package was used via GROMACS, which includes several python scripts to determine the energetic contributions of each amino acid residue in a protein–ligand interaction . Thus, their binding energy can be decomposed on a per residue basis, in which the energy components MM,E in the bonded and unbonded forms (Equation (2)) [To perform MM/PBSA calculations, a 100 ns MD dynamics simulation was performed using software  to generrch team ,81. The ion (2)) ,105,106.FN-19, a coumarin–thiosemicarbazone derivative, was the most potent inhibitor. This compound showed better inhibitory activity (IC50: 42.16 ± 5.16 µM) than ascorbic acid and kojic acid, which are used as reference compounds, having IC50 values of 386.50 ± 11.95 and 72.27 ± 3.14 µM, respectively. However, it was also observed that compounds FN-06, FN-10, and FN-27 could be considered promising inhibitors as well. These three coumarin analogs are better tyrosinase inhibitors than ascorbic acid, whereas only FN-10 showed equivalent activity to kojic acid. Kinetic studies indicated that FN-19 performs tyrosinase inhibition by a mixed mode, displaying maxV and mK values of 0.23 and 0.05, respectively. Similarly, FN-06, FN-10, and FN-27 are mixed inhibitors of the tyrosinase enzyme, according to the Lineweaver–Burk plots. Still, it was verified that FN-19 can significantly reduce the signal for L-DOPA oxidation by tyrosinase in UV-vis studies. In addition, the in silico studies involving docking and MD simulations were carried out for this compound. Thus, it was verified that the FN-19-tyrosinase complex has good stability throughout the simulated time (100 ns), showing that the ionic ligand–target interactions are the most frequent ones. Furthermore, the binding mode studies  suggested a possible bidentate interaction for FN-19, being responsible for its great biological activity, remaining complexed with the copper(II) ions during the entire simulation. Moreover, hydrophobic interactions involving Ans260, Phe264, Gly281, Ser282, and Val283 residues were observed, while a H-bond is performed with Glu256 residue. Finally, the MM/PBSA calculations demonstrated that both FN-19 and tropolone, a tyrosinase inhibitor, have similar binding energy values of −76.7 ± 0.87 and −78.0 ± 2.64 kcal/mol, respectively. This work developed an in silico and experimental model capable of contributing to a greater elucidation of the interactions between the target and ligand, allowing to understand how FN-19 interacts at the binding site. Concerning the promising results presented herein, FN-19 is a promising prototype for additional modification aiming to improve its activity.In this study, a series of coumarin-based compounds were designed, synthesized, and evaluated as inhibitors of the tyrosinase enzyme. Among the compounds tested,


Seseli mairei H. Wolff roots were determined by GC-MS. A total of 37 components were identified, (E)-beta-caryophyllene (10.49%), β-geranylgeranyl (6.64%), (E)-2-decenal (6.17%) and germacrene-D (4.28%). The essential oil of Seseli mairei H. Wolff had nematicidal toxicity against Bursaphelenchus xylophilus with a LC50 value of 53.45 μg/mL. The subsequent bioassay-guided investigation led to the isolation of three active constituents: falcarinol, (E)-2-decenal, and octanoic acid. The falcarinol demonstrated the strongest toxicity against B. Xylophilus (LC50 = 8.52 μg/mL). The octanoic acid and (E)-2-decenal also exhibited moderate toxicity against B. xylophilus . The LC50 of falcarinol for the toxicity of B. xylophilus was 7.7 and 21 times than that of octanoic acid and (E)-2-decenal, respectively. Our findings demonstrate that the essential oil from Seseli mairei H. Wolff roots and their isolates may be developed as a promising natural nematicide.The essential oil (EO) was extracted from aerial parts with insecticidal and fungicidal activity. Herein, the hydro-distilled essential oils of  Meloidogyne spp., Bursaphelenchus spp., Heterodera and Globodera spp.) may cause severe damage to plants and crops. It is estimated that the annual loss in global crop production due to plant-parasitic nematodes is around 8.8–14.6% of crop yields  and ACF300 [300 MHz (1H)] instruments . Tetramethylsilane was used as an internal standard.The crude EO of obtained . The str8–C24) were employed as reference points for calculating retention indices. Component relative percentage was determined according to GC peak areas without using the correction factors.GC-MS analysis was conducted using an Agilent system containing an Agilent Chem Station, a model 5973N mass selective detector, and a model 6890N gas chromatograph . The GC column was HP-5ms Capillary GC column . The oven temperature was initiated at 60 °C for 60 s, increased at 10 °C/min to 180 °C for 60 s, and further increased at 20 °C/min to 280 °C for 15 min. The injector temperature was kept at 270 °C. The sample  was diluted with acetone (1:100), and then injected at a ratio of 1:10. Helium was employed as the carrier gas, and the flow rate was 1.0 mL/min. Spectral scanning was conducted within the range of 20–550 m/z at 2 scans/s. The EO components were identified by comparing their mass spectra and retention index according to previous literature ,33 and p50 values and their 95% confidence intervals, Probit analysis was conducted on the obtained results using the PriProbit Program version 1.6.3  [Abbott’s formula was used to correct the mortality data. To calculate LC, Japan) .. Colorless oil, C8H16O2, 1H-NMR  δ (ppm): 0.88 , 1.21–1.38 , 1.59–1.67 , 2.32–2.37 . 13C-NMR  δ 14.05 (C-1), 22.58 (C-2), 24.90 (C-6), 29.05* (C-4), 29.08* (C-5), 31.64 (C-3), 34.38 (C-7), 176.95 (C-8).Octanoic acid 1,. ColorleE)-2-Decenal . Colorless oil, C10H16O, 1H-NMR  δ (ppm): 0.89 , 1.20–1.38 , 1.51 , 2.34 , 6.12 , 6.86 , 9.15 . 13C-NMR  δ 14.1 (C-10), 22.60 (C-9), 27.90 (C-8), 29.00* (C-7), 29.10* (C-6), 31.70 (C-5), 32.70 (C-4), 194.10 (C-1), 159.00 (C-2), 133.00 (C-3).. Yellow oil, 1H-NMR  δ (ppm): 0.90 , 1.29–1.31 , 1.36–1.40 , 2.04 , 2.06 , 3.06 , 4.94 , 5.24- 5.28 , 5.40 , 5.47 , 5.50–5.53 , 5.96 . 13C-NMR  δ (ppm): 136.10 (C-2), 133.10 (C-10), 121.90 (C-9), 117.10 (C-1), 80.30 (C-7), 74.20 (C-4), 71.30 (C-5), 64.00 (C-6), 63.60 (C-3), 31.80 (C-15), 29.20 , 27.20 (C-11), 22.60 (C-16), 17.70 (C-8), 14.10 (C-17).Falcarinol 3,. Yellow


Sargassum macrocarpum, and the molecular mechanisms underlying ACE inhibition. Four fractions of S. macrocarpum were prepared using hexane, chloroform, ethyl acetate, and water as solvents and analyzed for their potential ACE-inhibitory effects. The chloroform fraction showed the strongest ACE-inhibitory effect, with an IC50 value of 0.18 mg/mL. Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, were isolated from the chloroform fraction. Meroterpenoids isolated from S. macrocarpum had IC50 values of 0.44, 0.37, and 0.14 mM. The molecular docking study revealed that the ACE-inhibitory effect of the isolated meroterpenoids was mainly attributed to Zn-ion, hydrogen bonds, pi-anion, and pi–alkyl interactions between the meroterpenoids and ACE. These results suggest that S. macrocarpum could be a potential raw material for manufacturing antihypertensive nutraceutical ingredients.Angiotensin I-converting enzyme (ACE) is an important blood pressure regulator. In this study, we aimed to investigate the ACE-inhibitory effects of meroterpenoids isolated from the brown alga,  Hypertension is a chronic health problem related to cardiovascular diseases, including myocardial infarction, arteriosclerosis, and stroke, and is affected by various factors, such as salt intake, smoking, stress, and obesity . Among tSargassum species contain a variety of active compounds such as sterols, sulfated polysaccharides, polyphenols, meroterpenoids, and terpenoids [Natural resources as alternative treatments for hypertension are gaining attention, and studies on herb-derived compounds are ongoing ,13. Marirpenoids , which prpenoids , anticanrpenoids , antimicrpenoids , advancerpenoids , and neurpenoids  propertirpenoids ,26. DespSargassum macrocarpum based on the analytical data results and evaluate their ACE-inhibitory effects and the molecular mechanism underlying ACE inhibition.This study aimed to isolate meroterpenoids from the brown alga, S. macrocarpum were tested for their potential ACE-inhibitory effects. The 80% aqueous methanol extract of S. macrocarpum inhibited ACE activity in a dose-dependent manner by 6.1, 15.7, 40.7, 62.7, and 74.5% at concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mg/mL, respectively : δH 6.46 , 6.30 , 6.22 , 5.96 , 5.55 , 5.12 , 5.07 , 2.58 , 2.24 , 2.11 , 2.11 , 2.05 , 1.65 , 1.64 , 1.56 , 1.55 , 1.33  : δC 173.4 (C-15′), 148.7 (C-6), 145.8 (C-7′), 144.9 (C-8a), 134.5 (C-4′), 132.4 (C-12′), 130.8 (C-3), 130.7 (C-8′), 126.5 (C-8), 125.0 (C-3′), 123.6 (C-11′), 123.1 (C-4), 121.5 (C-4a), 117.2 (C-7), 110.5 (C-5), 77.9 (C-2), 40.9 (C-1′), 39.2 (C-5′), 34.7 (C-9′), 28.3 (C-6′), 28.0 (C-10′), 26.0 (C-17′), 25.8 (C-13′), 22.8 (C-2′), 17.9 (C-14′), 15.9 (C-18′), 15.7 (C-16′)   ; 13C NMR (C-16′) ; HRESIMS23.2535) .1H NMR : δH 6.29 , 6.21 , 5.97 , 5.52 , 5.11 , 5.06 , 2.58 , 2.23 , 2.12 , 2.11 , 2.10 , 2.05 , 1.65 , 1.65 , 1.56 , 1.55 , 1.33  : δC 172.6 (C-15′), 147.0 (C-6), 145.7 (C-7′), 144.9 (C-8a), 134.5 (C-4′), 132.4 (C-12′), 130.6 (C-8′), 130.0 (C-3), 128.3 (C-8), 125.2 (C-3′), 123.7 (C-11′), 123.6 (C-7), 123.0 (C-4), 118.9 (C-4a), 110.0 (C-5), 77.8 (C-2), 40.9 (C-1′), 39.2 (C-5′), 34.7 (C-9′), 28.3 (C-6′), 28.0 (C-10′), 26.0 (C-17′), 25.8 (C-13′), 22.8 (C-2′), 17.9 (C-14′), 15.9 (C-16′), 12.3 (C-19′), 11.8 (C-18′)  (7-methyl sargachromenol (2): brown oils; , H-17′) ; 13C NMR (C-18′) ; HRESIMS37.2691) .1H NMR : δH 6.53 , 6.44 , 5.98 , 5.13 , 5.11 , 5.07 , 3.11 , 2.58 , 2.24 , 2.10 , 2.07 , 2.05 , 2.04 , 1.65 , 1.60 , 1.58 , 1.56  : δC 188.3 (C-3), 188.2 (C-6), 172.7 (C-18′), 148.7 (C-2), 146.1 (C-4), 145.6 (C-10′), 140.0 (C-3′), 134.8 (C-7′), 133.3 (C-5), 132.49 (C-1), 132.44 (C-15′), 130.7 (C-11′), 124.6 (C-6′), 123.6 (C-14′), 118.1 (C-2′), 39.7 (C-4′), 39.2 (C-8′), 34.7 (C-12′), 28.4 (C-9′), 28.0 (C-13′), 27.7 (C-1′), 26.5 (C-5′), 25.9 (C-17′), 17.9 (C-16′), 16.3 (C-21′), 16.2 (C-19′), 16.1 (C-20′)  (Sargaquinoic acid (3): brown oils; , H-16′) ; 13C NMR (C-20′) ; HRESIMS23.2535) .50 value was observed for sargaquinoic acid at a concentration of 0.14 mM, and IC50 value of sargaquinoic acid was 3-fold lower than that of sargachromenol, which was the weakest inhibitor among the three meroterpenoids. It has previously been found that plant phenolic compounds such as quercetin, kaempferol, rutin, apigenin, epicatechin, phloretin, and resveratrol exhibit ACE-inhibitory effects and have IC50 values of 0.415, 0.512, 0.472, 0.667, 1.381, 1.110, and 0.970 mM, respectively [50 value = 2.57 mM), triphlorethol-A , eckol , dieckol , and eckstolonol  [50 value = 0.1 mM) from Terminalla chebula tree [50 value = 0.076 mM) from rice bran [50 value = 0.0086 mM) from bitter melon seed [50 value = 0.064 mM) from Pea [S. macrocarpum exhibited a significantly weaker effect than that of lisinopril , enalapril , and captopril , currently the most widely used synthetic ACE-inhibitor [R- and S-isomers, and structures of meroterpenoids are described in R-isomer interacted with GLU376 , ALA354 , VAL379 , VAL380 , VAL518 , PHE527 , HIS353 , HIS383 , HIS513 , PHE457 , and TYR523  , VAL380 , VAL518 , HIS353 , HIS383 (carbon hydrogen bond interaction), HIS513 , HIS387 , and TRP279  , GLU376 , VAL380 , LEU161 , ALA354 , HIS353 , PHE512 , and TRP279  , LEU161 , HIS353 , HIS387 , HIS410 , HIS513 , PHE512 , TRP279 , ALA354 , GLU411 (carbon hydrogen bond interaction), CYS352 , and CYS370   and S2 (HIS353 and HIS513) active site pockets and that sargachromenol S-isomer forms with S1 (ALA354) and S2  active site pockets. In the case of 7-methyl sargachromenol, R-isomer forms with S1 (TYR523) and S2 (HIS353) active site pockets of ACE, and S-isomer forms with S1  and S2  active site pockets of ACE. In addition, sargaquinoic acid forms an active site with the S1 , S’1 (GLU162), and S2 (HIS353) active site pockets of ACE.Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, with different chemical structures and molecular weights were isolated. The ACE-inhibitory effects of the three meroterpenoids are shown in ectively . Additioula tree , Tyr-Serice bran , Val-Serlon seed , and Lysfrom Pea . A previnhibitor . Diet thnhibitor ,38. Addinhibitor . S1 (antnhibitor . Lisinopnhibitor . The mosraction) A. Sargacraction) B. 7-methraction) C. 7-methraction) D. In theraction) E. The moectively . Among td HIS387 . The mol50 values 0.44, 0.37, and 0.14 mM, respectively. Among the three meropterpenoids, sargachromenol and 7-methyl-sargachromenol do exist as R- and S-isomers, and molecular docking studies of the respective other isomers were performed. These other isomers showed the similar docking poses and binding energies with those of isomers. Molecular docking studies revealed that these meroterpenoids in the ACE active site contributed to the stabilization of the docking complex, and it was confirmed that sargaquioic acid contributed the most to stabilization. These findings provide a partial molecular explanation for the ACE-inhibitory properties of meroterpenoids isolated from S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods.In conclusion, among the various organic solvent fractions, the chloroform fraction exhibited the highest ACE inhibitory effect. Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, having ACE-inhibitory effects were isolated, with ICS. macrocarpum was collected from Jeju Island, South Korea. The alga was washed three times with tap water to remove salt and epiphytes attached to the surface, followed by careful rinsing with fresh water. The algae were stored in a medical refrigerator at −80 °C. Frozen algae were lyophilized and homogenized using a grinder prior to extraction. The ACE assay kit (ACE kit-WST) was obtained from Dojindo Laboratories .S. macrocarpum powder was extracted with 80% aqueous methanol at room temperature, and the liquid layer was obtained via filtration. The filtrate was evaporated to obtain the methanol extract, which was suspended in distilled water and partitioned with hexane, chloroform, ethyl acetate, and butanol. The chloroform fraction exhibited a stronger ACE-inhibitory effect than the other fractions. Thus, the compounds were extracted from the chloroform fraction using a silica gel column and medium-pressure liquid chromatography . Finally, the compounds were purified using preparative high-performance liquid chromatography , and the structures of the compounds were identified by comparing the nuclear magnetic resonance  spectral data and high-resolution electrospray ionization mass spectrometry  analysis results with those in the existing literature.The dried 50 value was defined as the concentration of extract and fractions (mg/mL), and compounds (µM) required for 50% reduction of the ACE activity.The ACE-inhibitory activities of the extract, fractions, and compounds were determined using an ACE kit-WST (Dojindo Laboratories). The assay was performed according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader . ICMolecular docking was performed using CDOCKER and Calculate Binding Energies tools in Discovery Studio 2022  to assess the binding positions of meroterpenoids within the active site of ACE. A docking mechanism based on CHARMm  was usedp < 0.05 was considered statistically significant.Data are presented as mean ± standard error. Statistical comparisons of the mean values were performed using analysis of variance, followed by Duncan’s multiple-range test using the SPSS software (Version 21). A