The addition of 2-amino-1,3,4-thiadiazole derivatives with parallel iodination of differently protected glycals has been achieved using a double molar excess of molecular iodine under mild conditions. The corresponding thiadiazole derivatives of N-glycosides were obtained in good yields and anomeric selectivity. The usage of iodine as a catalyst makes this method easy, inexpensive, and successfully useable in reactions with sugars. Thiadiazole derivatives were tested in a panel of three tumor cell lines, MCF-7, HCT116, and HeLa. These compounds initiated biological response in investigated tumor models in a different rate. The MCF-7 is resistant to the tested compounds, and the cytometry assay indicated low increase in cell numbers in the sub- G1 phase. The most sensitive are HCT-116 and HeLa cells. The thiadiazole derivatives have a pro-apoptotic effect on HCT-116 cells. In the case of the HeLa cells, an increase in the number of cells in the sub-G1- phase and the induction of apoptosis was observed. Moreover, the amino derivatives of 1,3,4-thiadiazole, which have different biocidal effects, are also investigated, probably due to the toxophore group N=C-S- [1,3,4-Thiadiazole represents an important heterocyclic scaffold due to its pharmacological activity. Their derivatives exhibit various and significant activities such as antibacterial , anti-tup N=C-S- ,9,10. Asp N=C-S- .It is well known that derivatives of 1,3,4-thiadiazoles exhibit anti-tumor activity against human cancer cells (MCF-7 (breast cancer), SMMC-7721 (human hepatocarcinoma), HL-60 (human leukemia), A549 , Hep-G2 (human liver hepatocellular carcinoma), PC-3 (human prostate adenocarcinoma), and HCT-116 ; O-, S-, N-glycosides. The method for entry into 2-deoxy glycosides has been discussed in several reviews [Priebe et al.  reported reviews ,33,34,35Continuing this study, we present the synthesis and biological activity of conjugates possessing different 2-deoxy-2-iodomonosaccharide scaffolds joined with thiadiazole derivatives.2) to an unsaturated sugar (1) in the presence of iodine , 3,4-di-O-benzyl-L-rhamnal (1d), 3,4,6-tri-O-acetyl-D-glucal (1b), and 3,4,6-tri-O-acetyl-D-galactal (1c), followed by a substitution reaction, were isolated by column chromatography. The structures of the obtained compounds were established based on the analysis of their NMR spectra. The signals were identified using literature data [1H NMR spectra, the following were diagnostic: chemical shifts of acetyl groups, hydrogen at the anomeric position, hydrogens at C-6 position for rhamnopyranose derivatives, and hydrogen at C-2. In the 13C NMR spectrum were the C-1, C-2, and S-C=N carbon chemical shifts.The glycosylation of the glycal derivatives was performed in the dark at room temperature. The main products formed by the addition of iodine  to the dure data ,37. AddiTo determine the reaction conditions, we performed preliminary experiments presented in: O-acetyl-L-rhamnal (1a) was treated with 2-amino-5-(4-tert-butylphenyl)-1,3,4-thiadiazole (2a) in different solvents (Entry a–c) and in the absence of iodine, no substrate conversion was observed. In the second set of experiments, the reaction was carried out in the presence of 1 eq. of iodine in the mixture of solvents with different polarities (Entry d–f). Under applied conditions, only traces of the product were detected. The third set of experiments was carried out in THF in the presence of a different concentration of iodine, namely, 0.5, 1.0, and 2.0 eq (Entry g–i). These results indicated that the reaction occurred successfully in the presence of double molar excess of molecular iodine. To determine which of the iodoamination methods would be the most beneficial, an experiment with NIS was also performed (Entry j), this reactant is recommended as conventional in synthesis 2-deoxy-2-halogenosugars starting from glycals [When the 3,4-di- glycals . The pre glycals . UnfortuO-acetyl-L-rhamnal (1a), 3,4,6-tri-O-acetyl-D-glucal (1b), 3,4,6-tri-O-acetyl-D-galactal (1c), and 3,4-di-O-benzyl-L-rhamnal (1d) with 2-amino-5-phenyl-1,3,4-thiadiazole derivatives (2a–2d) containing different substituents in the aromatic ring in anhydrous THF containing molecular iodine. As a result of these experiments, hitherto unknown derivatives 3a–3m were obtained. The results are presented in In a series of experiments, we carried out reactions of 3,4-di-1H and 13C NMR spectra analysis. The main products  obtained from L-rhamnal diacetate (1a) showed large values of the coupling constants , J2,3 (9.6 Hz), and J3,4 (9.6 Hz)), indicating the trans-diaxial disposition between H-1 and H-2, H-2 and H-3, and H-3 and H-4 in the favored 5H4 conformation. Large J1,2 values in compounds 3a–3d signify the L-gluco configuration. The reaction of 3,4-di-O-benzyl-L-rhamnal (1d) with 2-amino-5-phenyl-1,3,4-thiadiazole (2b) led to 1:1 α/β mixture of L-gluco stereoisomers.The anomeric configurations of all products were established based on their d-glucal (1b) with 2-amino-5-phenyl substituted 1,3,4-thiadiazole derivatives (2a and 2d) was highly stereoselective, affording the β-1,2-trans isomer of D-glucose (3e and 3h) in moderate yields (Entry e and h). Under the same conditions, D-galactal (1c) was transformed into an inseparable mixture of 2-deoxy-2-iodo-α/β-galacto N-glycosides  in modest 45%–72% overall yields .Iodine-promoted iodoamination of l yields . These lThe electrophilic addition of halogens to cyclic enol ethers has been investigated earlier by Lemieux and Fraser-Reid , who proTo improve the stereoselectivity of glycosylation reactions involving glycals, directing groups such as Cl, Br, and I are introduced. Most commonly, a directing group is temporarily introduced at C-2, and the acceptor is then incorporated at C-1. An example is NIS- or NBS-mediated glycosylation developed by Thiem et al. These protocols have found widespread use in the construction of complex amine-containing N-glycosides ,43.The addition of halogen to double bonds is expected to occur by a bimolecular process, with the approach of a halogen molecule perpendicularly to the system of the vinyl ether from both site of a molecule plane, and the compound formed may rearrange in the rate-limiting step to intermediate ions  which, according to their relative reactivities, control the stereochemical course of the reaction .4H5, 5H4 conformational equilibrium of glycals. The factor affecting the conformational equilibrium of glycals is the 1,3-diaxial interactions destabilizes the 5H4 form of D-glycals with the C-3 and C-5 substituents in the cis orientation [Unsaturated pyranoid rings are conformationally more flexible than saturated ones. Therefore, even insignificant changes to the spatial disposition of substituents attached to the ring may give rise to changes in the entation .1a exist mainly in the 5H4 conformation  a mixture of 1,2-trans L-gluco and L-manno glycosides was formed, suggesting that the addition of iodine proceeds through the stabilized carbo-oxonium ion  exists mainly in the 5H4 conformation  were determined with MTT assay. After a 72 h incubation with the tested compounds, the cells’ viabilities were measured and presented as Survival Fraction s(SF) in 50 calculation. However, in the case of two compounds (3c and 3j), the SF of the cells decreased significantly at the highest doses . The typical histograms of DNA content in cells are presented in 3a and 3c) influenced the cell cycle distribution and increased the number of cells in the sub-G1 phase, which corresponds to the dead cells .Based on the results, for the MCF-7 cell line , we concIn contrast to the MCF-7, the HCT116 cells respondedSimilarly to the HCT116 cells, the tested compounds have exhibited a pro-apoptotic effect on the HeLa cell line. However, in most of the cases, the effect was much stronger .The efficient formation of glycosidic linkages has played an important role in the development of modern synthetic carbohydrate chemistry. Among the various types of donors that can be employed for the construction of glycosidic bonds, glycosyl iodides should now be viewed as versatile glycosylation reagents ,62.The research found the ineffectiveness of the conventional methods for the preparation of N-glycosides of 2-deoxy monosaccharides in the nucleophilic substitution reaction with the participation of very weakly nucleophilic amine thiadiazole derivatives. The publication presents an efficient method for glycosylation starting from glycals, with the activation leading to the glycosyl donor taking place with 2eq of iodine, preferably in a polar solvent such as THF. It has been shown that by the appropriate optimization of conditions, it is possible to obtain highly stereoselective products of rhamnal and glucal. The advantage of this method is the use of an easily available, cheap, and environmentally friendly reagent, molecular iodine.The biological activity of the tested compounds was determined based on the MTT assay complemented with flow cytometry analysis. The combination of these methods allows for the detection of cytotoxic, cytostatic, and lethal effects of the compounds on the analyzed cell lines.50 parameter nor the selectiveness of the compounds against the tested cell lines could be evaluated for the examined doses. Nevertheless, based on the SF values, we observed that the highest concentrations of the compounds (100 µM) inhibited the cells proliferation. Because of it, we decided to test the effect of the compounds on the cell cycle only for the highest concentrations.The performed MTT assay indicated that the effect of the compounds depended significantly on the tested cell line . Among tAccording to the biological assays, in the case of MCF-7 cells, despite the low cytotoxic effect of the compounds, we did not observe any cytostatic or pro-apoptotic effect. During the cell cycle analysis, we detected a slight increase in the number of cells present in the sub-G1 phase or an increase in cells arrested in the G0/G1 phase. Furthermore, there was no increase in apoptotic nor necrotic cells. Nevertheless, the detected effect was not significant when compared to the untreated control.More promising effects were detected in the case of other cell lines. The cell line, which was the most sensitive in the MTT assay (HCT116) responded with both cytostatic and cytotoxic effects. During the cell cycle analysis, we observed an increase in the number of cells in the sub-G1 fractions, however, the strongest effect was visible in the inhibition of the S phase and the arrest of the cells in the G2/M phase. Additionally, we detected an increase in cells in the early apoptosis, which suggests that the compounds have pro-apoptotic activity against the HCT116 cell line.The most auspicious effects were observed for the HeLa cells. Similarly to the HCT116 cell line, the compounds caused cytotoxic and cytostatic effects on this cell line. Moreover, in the case of the HeLa cells, we detected an increase in the number of cells in the sub-G1 phase. Furthermore, Annexin V staining proved that the cells underwent both apoptotic cell deaths. Based on the carried out experiments we noticed a tissue-dependent effect of the tested compounds and we concluded that they exhibit a cytotoxic effect on the MCF-7, HCT116, and HeLa cell lines, a cytostatic effect on the HCT116 and HeLa cell lines, and a lethal effect on the HeLa cell line.Our results indicate that the tested compounds in their current form cannot be exploited as drugs, however, they stand as promising models for further modification. In order to improve the antitumor activity of the conjugates, structural modifications are necessary. Especially interesting is applying in biological tests deprotected conjugates, it should affect the solubility of these derivatives in the applied medium. Another approach is the synthesis of the derivatives where a sugar moiety is linked to a thiadiazole derivative via an aliphatic, aromatic-aliphatic, or aromatic linker.254 ; detection was carried out with either ultraviolet light (254 nm) or spraying with a solution of phosphomolybdic acid, a basic potassium permanganate solution, or ethanolic solution of concentrated H2SO4, with subsequent heating. Optical rotations were measured with a JASCO P-2000 polarimeter using a sodium lamp (589.3 nm) at room temperature. Melting points were measured on a Stuart SMP3 melting point apparatus. The 1H NMR and 13C NMR spectra were recorded on an Agilent 400 MHz spectrometer and Varian 600 MHz spectrometer in DMSO-6d or CDCl3 using tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported as δ values (ppm). FT-IR spectra were recorded between 4000 and 650 cm−1 using an FT-IR Nicolet 6700 apparatus  with a Smart iTR accessory. HRMS spectra were recorded on a Waters ACQUITY UPLC/Xevo G2QT instrument.General. All reagents were purchased from Merck , Acros Organics , Alpha Aesar , and Sigma-Aldrich  and used without further purification. A column chromatography was per-formed on a silica gel packed column . Solvents for chromatography  were distilled before use. Thin layer chromatography was performed on plates coated with silica gel 60 FGeneral procedure for iodination of glycals:1a–d: 1.0 eq) in THF (2 mL), 2-amino-1,3,4-thiadiazole derivative (2a–d: 2.0 eq) and I2 (2.0 eq) were added. The reaction was stirred at room temperature for 1.5h. The TLC  indicated total consumption of glycal. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography  to give products as a pale yellow oil being a mixture of anomers. To a solution of glycal (5-(4-(tert-buthyl)phenyl)-N--1,3,4-thiadiazol-2-amine (3a): Starting from 3,4-di-O-acetyl-L-rhamnal  and 2-amino-5-(4-tert-buthylphenyl)-1,3,4-thiadiazole , yield 71% (62.9 mg); ratio of anomers (α:β = 1:11); 1H NMR : δ 7.58–7.56 , 7.46–7.43 , 6.04 , 5.90 , 5.44 , 4.87 , 4.69–4.62 , 3.87 , 2.12 , 2.08 , 2.06 , 2.05 , 1.34 3), 1.26 , and 1.19 ; 13C NMR : δ 170.0 (COCH3), 169.7 (COCH3), 167.2 (S-C=N), 154.4 (N-C=N), 127.4 (Ph), 126.2 (Ph), 126.0 (Ph), 81.6 (C-1), 76.3 (C-3), 74.0 (C-5), 72.7 (C-4), 35.1 (C(CH3)3), 31.3 (C(CH3)3), 29.8 (C-2), 21.0 (COCH3), 20.9 (COCH3), and 17.5 (C-6); HRMS m/z calcd for (C22H28IN3O5S + H+): 574.0867; found: 574.0874.5-phenyl-N--1,3,4-thiadiazol-2-amine (3b): Starting from 3,4-di-O-acetyl-L-rhamnal  and 2-amino-5-phenyl-1,3,4-thiadiazole , yield 47% (85.8 mg); ratio of anomers (α:β = 1:1.7); 1H NMR : δ 7.70–7.68 , 7.65–7.63 , 6.28 , 5.91 , 5.44 , 5.32 , 5.10 , 5.04 , 4.88 , 4.69–4.62 , 4.15–4.11 , 3.88 , 2.18 , 2.12 , 2.11 , 2.06 , 1.38 , and 1.28 ; 13C NMR : δ 170.0 (COCH3), 169.8 (COCH3), 169.7 (COCH3), 162.1 (S-C=N), 147.4 (N-C=N), 130.8 (Ph), 130.2 (Ph), 129.1 (Ph), 129.0 (Ph), 128.3 (Ph), 126.4 (Ph), 126.4 (Ph), 76.3 (C-1), 74.0 (C-3), 72.8 (C-2), 72.0 (C-5), 71.3 (C-4), 26.8 (C-2), 21.2 (COCH3), 21.1 (COCH3), 21.0 (COCH3), 20.8 (COCH3), 17.5 (C-6), and 17.1 (C-6); HRMS m/z calcd for (C18H20IN3O5S + H+): 518.0241; found: 518.0251.5-(4-fluorophenyl)-N--1,3,4-thiadiazol-2-amine (3c): Starting from 3,4-di-O-acetyl-L-rhamnal  and 2-amino-5-(4-fluorophenyl)-1,3,4-thiadiazole , yield 55% (68.7 mg); ratio of anomers (α:β = 1:99); α24D = -80 ; 1H NMR : δ 7.65–7.62 , 7.14–7.11 , 5.90 , 5.44 , 4.87 , 4.67–4.60  3.89–3.85 , 2.12 , 2.06 , and 1.28 ; 13C NMR : δ 170.0 (COCH3), 169.7 (COCH3), 166.7 (S-C=N), 164.2 , 154.8 (N-C=N), 132.4 (Ph), 128.4 (Ph), 128.3 (Ph), 116.3 (Ph), 116.2 (Ph), 83.8 (C-1), 76.3 (C-3), 74.0 (C-4), 72.8 (C-5), 27.0 (C-2), 21.0 (COCH3), 20.9 (COCH3), and 17.5 (C-6); HRMS m/z calcd for (C18H19FIN3O5S + H+): 536.0147; found: 536.0153.5-(4-chlorophenyl)-N--1,3,4-thiadiazol-2-amine (3d): Starting from 3,4-di-O-acetyl-L-rhamnal  and 2-amino-5-(4-chlorophenyl)-1,3,4-thiadiazole , yield 49% (58.6 mg); ratio of anomers (α:β = 1:99); α24D = −13.3 ; 1H NMR : δ 7.58–7.56 , 7.42–7.40 , 5.91 , 5.44 , 4.87 , 4.65–4.59  3.89–3.85 , 2.12 , 2.06 , and 1.28 ; 13C NMR : δ 170.0 (COCH3), 169.7 (COCH3), 165.8 (S-C=N), 146.3 (N-C=N), 136.9 (C-Cl), 136.4 (Ph), 129.3 (Ph), 127.6 (Ph), 83.8 (C-1), 76.2 (C-3), 74.0 (C-4), 72.8 (C-5), 42.7 (C-2), 21.0 (COCH3), 20.9 (COCH3), and 17.5 (C-6); HRMS m/z calcd for (C18H19ClIN3O5S + H+): 551.9851; found: 551.9855.5-(4-(tert-buthyl)phenyl)-N--1,3,4-thiadiazol-2-amine (3e): Starting from 3,4,6-tri-O-acetyl-D-glucal  and 2-amino-5-(4-tert-buthylphenyl)-1,3,4-thiadiazole , yield 51% (157.7 mg); ratio of anomers (α:β = 1:99); α24D = 8.6 ; 1H NMR : δ 7.60–7.43 , 6.01 , 5.51 , 5.14 , 4.73–4.68 , 4.35 , 4.15 , 4.10–4.06 , 2.12 , 2.06 , 2.04 , 1.35 3), 1.34 3), and 1.33 3); 13C NMR : δ 170.5 (COCH3), 170.4 (COCH3), 170.1 (COCH3), 169.8 (COCH3), 165.5 (S-C=N), 156.2 (C-C(CH3)3), 154.4 (C-C(CH3)3), 146.2 (S-C=N), 129.3 (Ph), 128.1 (Ph), 127.0 (Ph), 126.8 (Ph), 98.6 (C-1), 80.2 (C-1), 74.2 (C-3), 73.2 (C-3), 70.5 (C-5), 68.2 (C-5), 63.5 (C-4), 63.2 (C-4), 62.2 (C-6), 60.8 (C-6), 36.1 (C-2), 31.5 (C(CH3)3), 29.6 (C(CH3)3), 27.3 (C-2), 21.9 (COCH3), 21.5 (COCH3), 20.9 (COCH3), 20.7 (COCH3), and 20.5 (COCH3); HRMS m/z calcd for (C24H30IN3O7S + H+): 632.09 22; found: 632.0930.5-phenyl-N--1,3,4-thiadiazol-2-amine (3f): Starting from 3,4,6-tri-O-acetyl-D-glucal  and 2-amino-5-phenyl-1,3,4-thiadiazole , yield 68% (107.8 mg); ratio of anomers (α:β = 4.4:1); 1H NMR : δ 7.70–7.68 , 7.47–7.44 , 6.30 , 5.94 , 5.48 , 5.36 , 5.28 , 5.15–5.10 , 4.72–4.67 , 4.37–4.34 , 4.29–4.22 , 4.14 , 4.00 , 2.19 , 2.12 , 2.09 , and 2.07 ; 13C NMR : δ 170.7 (COCH3), 169.5 (COCH3), 169.3 (COCH3), 161.5 (S-C=N), 147.5 (N-C=N), 130.8 (Ph), 129.0 (Ph), 126.2 (Ph), 82.9 (C-1), 72.9 (C-3), 70.8 (C-5), 67.5 (C-4), 61.6 (C-6), 25.7 (C-2), 21.0 (COCH3), 20.8 (COCH3), and 20.7 (COCH3); HRMS m/z calcd for (C20H22IN3O7S + H+): 576.0296; found: 576.0309.5-(4-fluorophenyl)-N--1,3,4-thiadiazol-2-amine (3g): Starting from 3,4,6-tri-O-acetyl-D-glucal  and 2-amino-5-(4-fluorophenyl)-1,3,4-thiadiazole , yield 58% (126.4 mg); ratio of anomers (α:β = 1:1.3); 1H NMR : δ 8.15–8.10 , 7.16–7.12 , 6.27 , 5.99 , 5.50 , 5.33–5.30 , 5.26 , 5.16–5.11 , 4.71–4.66 , 4.44–4.21 , 4.14 , 4.07–4.03 , 2.19 , 2.12 , 2.10 , 2.05 , and 2.04 ; 13C NMR : δ 170.6 (COCH3), 170.0 (COCH3), 169.6 (COCH3), 169.5 (COCH3), 169.3 (COCH3), 166.2 , 163.0 (S-C=N), 145.6 (N-C=N), 132.8 (Ph), 132.7 (Ph), 129.0 (Ph), 128.2 (Ph), 116.3 (Ph), 116.1 (Ph), 115.8 (Ph), 115.5 (Ph), 84.2 (C-1), 83.5 (C-1), 76.1 (C-3), 74.3 (C-3), 73.2 (C-5), 70.6 (C-5), 68.7 (C-4), 67.3 (C-4), 61.8 (C-6), 61.5 (C-6), 25.9 (C-2), 25.4 (C-2), 21.4 (COCH3), 21.0 (COCH3), 20.8 (COCH3), 20.7 (COCH3), and 20.6 (COCH3); HRMS m/z calcd for (C20H21FIN3O7S + H+): 594.0202; found: 509.0209.5-(4-chlorophenyl)-N--1,3,4-thiadiazol-2-amine (3h): Starting from 3,4,6-tri-O-acetyl-D-glucal  and 2-amino-5-(4-chlorophenyl)-1,3,4-thiadiazole , yield 69% (154.6 mg); ratio of anomers (α:β = 1:99); α24D = 177.7 ; 1H NMR : δ 7.59–7.56 , 7.43–7.40 , 5.94 , 5.47 , 5.13 , 4.68–4.62 , 4.35 , 4.14 , 4.01–3.97 , 2.12 , 2.07 , and 2.04 ; 13C NMR : δ 170.6 (COCH3),170.5 (COCH3), 169.8 (COCH3), 169.7 (COCH3), 169.5 (COCH3), 156.6 (S-C=N), 146.5 (N-C=N), 139.3 (C-Cl), 129.8 (Ph), 129.6 (Ph), 129.1 (Ph), 128.3 (Ph), 127.9 (Ph), 98.7 (C-1), 80.9 (C-1), 78.2 (C-3), 73.5 (C-3), 68.2 (C-5), 67.5 (C-5), 64.3 (C-4), 62.5 (C-6), 60.5 (C-6), 29.8 (C-2), 26.7 (C-2), 21.5 (COCH3), 21.3 (COCH3), 20.9 (COCH3), 20.7 (COCH3), and 20.6 (COCH3); HRMS m/z calcd for (C20H21ClIN3O7S + H+): 609.9906; found: 609.9917.5-(4-(tert-buthyl)phenyl)-N--1,3,4-thiadiazol-2-amine (3i): Starting from 3,4,6-tri-O-acetyl-D-galactal  and 2-amino-5-(4-tert-buthylphenyl)-1,3,4-thiadiazole , yield 72% (100.7 mg); ratio of anomers (α:β = 2.3:1); 1H NMR : δ 7.80–7.62 , 7.50–7.46 , 6.72 , 6.55 , 5.80 , 5.55–5.53 , 5.49 , 5.42–5.41 , 5.09 , 4.72–4.71 , 4.54–4.51 , 4.49–4.47 , 4.32–4.30 , 4.27–4.19 , 2.33 , 2.26 , 2.12 , 2.07 , 2.02 , 1.34 3), and 1.33 3); 13C NMR : δ 170.6 (COCH3), 170.3 (COCH3), 170.2 (COCH3), 169.5 (COCH3), 165.7 (S-C=N), 156.4 (C-C(CH3)3), 155.2 (C-C(CH3)3), 145.5 (S-C=N), 129.1 (Ph), 128.3 (Ph), 126.7 (Ph), 126.3 (Ph), 98.9 (C-1), 81.5 (C-1), 73.0 (C-3), 72.9 (C-3), 69.4 (C-5), 66.2 (C-5), 64.0 (C-4), 63.9 (C-4), 62.0 (C-6), 60.3 (C-6), 35.1 (C-2), 31.2 (C(CH3)3), 29.8 (C(CH3)3), 27.1 (C-2), 21.7 (COCH3), 21.1 (COCH3), 20.9 (COCH3), 20.8 (COCH3), and 20.7 (COCH3); HRMS m/z calcd for (C24H30IN3O7S + H+): 632.0922; found: 632.0928.5-phenyl-N--1,3,4-thiadiazol-2-amine (3j): Starting from 3,4,6-tri-O-acetyl-D-galactal  and 2-amino-5-phenyl-1,3,4-thiadiazole , yield 62% (106.5 mg); ratio of anomers (α:β = 3.8:1); 1H NMR : δ 7.65–7.63  7.46–7.42 , 6.23 , 5.94 , 5.76 , 5.45 , 5.34–5.28 , 5.01–4.98 , 4.73–4.68 , 4.52–4.47 , 4.42–4.38 , 4.27–4.11 , 2.24 , 2.20 , 2.09 , 2.08 , and 2.04 ; 13C NMR : δ 169.8 (COCH3), 169.6 (COCH3), 169.5 (COCH3), 162.7 (S-C=N), 147.2 (N-C=N), 131.0 (Ph), 129.5 (Ph), 127.5 (Ph), 83.2 (C-1), 73.7 (C-3), 71.3 (C-5), 68.1 (C-4), 62.6 (C-6), 24.9 (C-2), 21.2 (COCH3), 20.6 (COCH3), and 20.5 (COCH3); HRMS m/z calcd for (C20H22IN3O7S + H+): 576.0296; found: 576.0312.5-(4-fluorophenyl)-N--1,3,4-thiadiazol-2-amine (3k): Starting from 3,4,6-tri-O-acetyl-D-galactal  and 2-amino-5-(4-fluorophenyl)-1,3,4-thiadiazole , yield 45% (68.7 mg); ratio of anomers (α:β = 4:1); 1H NMR : δ 7.67–7.6 , 7.37–7.36 , 6.21 , 5.94 , 5.75 , 5.44 , 5.33–5.28 , 4.98 , 4.81–4.75 , 4.51–4.46 , 4.36 , 4.27–4.12 , 2.24 , 2.24 , 2.20 , 2.09 , and 2.03 ; HRMS m/z calcd for (C20H21FIN3O7S + H+): 594.0202; found: 594.0217.5-(4-chlorophenyl)-N--1,3,4-thiadiazol-2-amine (3l): Starting from 3,4,6-tri-O-acetyl-D-galactal  and 2-amino-5-(4-chlorophenyl)-1,3,4-thiadiazole , yield 58% (90.9 mg); ratio of anomers (α:β = 1.4:1); 1H NMR : δ 7.66–7.63 , 7.45–7.42 , 6.45 , 6.23 , 5.74 , 5.55–5.54 , 5.45 , 5.42–5.41 , 5.00 , 4.73–4.71 , 4.50–4.47 , 4.31 , 4.28–4.19 , 2.26 , 2.12 , 2.08 , and 2.02 ; 13C NMR : δ 170.6 (COCH3),170.4 (COCH3), 170.2 (COCH3), 169.4 (COCH3), 169.3 (COCH3), 165.6 (S-C=N), 145.5 (N-C=N), 138.1 (C-Cl), 129.7 (Ph), 129.5 (Ph), 129.1 (Ph), 128.0 (Ph), 127.6 (Ph), 99.0 (C-1), 80.5 (C-1), 74.1 (C-3), 72.9 (C-3), 69.7 (C-5), 66.2 (C-5), 64.0 (C-4), 62.0 (C-6), 60.2 (C-6), 29.8 (C-2), 25.7 (C-2), 21.2 (COCH3), 21.1 (COCH3), 20.9 (COCH3), 20.8 (COCH3), and 20.7 (COCH3); HRMS m/z calcd for (C20H21ClIN3O7S + H+): 609.9906; found: 609.9917.5-phenyl-N--1,3,4-thiadiazol-2-amine (3m): Starting from 3,4-di-O-benzyl-L-rhamnal  and 2-amino-5-(4-tert-buthylphenyl)-1,3,4-thiadiazole , yield 42% yield (41.5 mg); ratio of anomers (α:β = 1:1); 1H NMR : δ 7.70–7.68 , 7.47–7.42 , 7.37–7.31 , 5.99 , 5.08 , 4.93–4.88 , 4.72–4.66 , 4.62 , 4.21 , 3.90 , 3.82 , 3.72 , 3.62 , 3.33 , 1.37 , and 1.33 ; 13C NMR : δ 160.8 (S-C=N),148.5 (N-C=N), 138.1 (Ph), 138.0 (Ph), 130.7 (Ph), 130.5 (Ph), 129.0 (Ph), 128.7 (Ph), 128.6 (Ph), 128.5 (Ph), 128.3 (Ph), 128.1 (Ph), 126.5 (Ph), 95.5 (C-1), 94.9 (C-1), 86.4 (C-3), 85.1 (C-3), 85.0 (C-4), 75.5 (C-5), 74.3 (-CH2-), 73.9 (-CH2-), 37.6 (C-2), 31.1 (C-2), and 17.9 (C-6); HRMS m/z calcd for (C28H28IN3O3S + H+): 614.0969; found: 614.0969.2 surface bottles , in an incubator  of a solid concentration of 5% carbon dioxide, 37 °C, and constant humidity of 80%. The culture was maintained in vitro by the passaging of cells (every 3 to 4 days). For the experiments, cells were obtained by trypsinization with 0.25% trypsin solution  in phosphate-buffered saline lacking Ca+2 and Mg+2 (PBS) to detach them from the ground. Trypsin was neutralized by adding an equal amount of the culture medium. After centrifugation , the cells were resuspended in a fresh culture medium and counted using an automatic cell counter—Juli Stage .Cytotoxicity of drugs was determined for several cell lines. Experiments were performed on HeLa , HCT 116 , and MCF-7 (human breast adenocarcinoma cell line) according to the standard procedures described previously . The cel4 cells/well/200 μL in completed grown medium. After approx. 24 h the culture medium was replaced with a fresh culture medium containing serial dilutions of the tested drugs. After 72 h, a solution of the compound was removed. Next, cell viability was determined by the MTT assay based on mitochondrial dehydrogenase enzyme activity. The basis of the method is the reaction of the reduction of tetrazolium salts to colored formazan carried out by dehydrogenase in living cells. Therefore, the amount of formazan formed is proportional to the number of functional cells. MTT assay was carried out according to the protocol  [2 incubator for 1 to 2 h. After this time, MTT solution was removed, and formazan crystals were dissolved in acidic isopropanol. Next, spectrophotometrically absorbance measuring at 570 nm, using a multi-well plate reader SYNERGY4  was performed. The fraction of viable cells was determined with concern to not treat with any factor control cells, according to the following formula:For the cytotoxicity determination of the tested derivatives, the cells in the exponential growth phase were trypsinized and seeded on 96-well plates  at concentration 5 × 10Germany) ,64. To eSF—survival fraction;A—the absorbance value;K—the absorbance value for control cells.A5 cells/well/2 mL. After 72 h, the growth medium was exchanged for a fresh medium in control groups or medium containing the tested drugs at chosen concentrations. The cells were incubated for 72 h. After this time, the medium was collected and the cells were trypsinized with 1 mL trypsin/EDTA . Trypsin was then neutralized by adding 2 mL of culture medium. The cells were centrifuged at 1300 rpm for 5 min at 4 °C, washed twice with 1 mL PBS, and finally fixed with 1 mL of ice-cold 70% ethanol. The samples were stored at −20 °C until analysis. Directly before the analysis by flow cytometer , cells were centrifuged at 1300 rpm for 3 min, washed with 500 µL PBS, and centrifuged again. Then, cells were resuspended in 50 µL PBS and 50 µL of RNAse solution  at a concentration of 100 µg/mL (in PBS). After 15 min of incubation at 37 °C, cells were stained with 250 µL propidium iodide solution  at a concentration of 100 µg/mL. Fluorescence was measured using a flow cytometer with the PE configuration . The samples were vortexed before analysis [The exponentially growing cells were harvested by trypsinization and seeded on a 6-well plate  in DMEM/F12 medium at concentrations of 1 × 10analysis ,66.For apoptosis assay, the cells after collection were stained directly using FITC conjugated primary Annexin-V antibody  in binding buffer for 20 min in darkness at 37 °C and Annexin with PI solution 100 µg/mL . Fluorescence was measured using a flow cytometer with the PE configuration  for necrotic and late apoptotic cell counting. For apoptotic cells, the FITC channel configuration was used . The samples were vortexed before analysis ,66.p-value less than 0.05 was considered statistically significant.At least three replicates were performed for every kind of experiment. The results were presented as the mean value ± SD. The statistical analysis was based on a t-test, and a


O-methyl ether derivatives were computationally designed aiming to improve the selectivity and potency profiles against monoamine oxidase (MAO) B. The designed compounds were evaluated for their potential to inhibit human MAO-A and -B. Compounds 1c, 2c, 3c, and 4c were the most potent with a Ki of 37 to 68 nM against MAO-B. Compounds 1c–4c displayed more than a thousand-fold selectivity index towards MAO-B compared with MAO-A. Moreover, compounds 1c and 2c showed reversible inhibition of MAO-B. These results provide a basis for further studies on the potential application of these modified flavonoids for the treatment of Parkinson’s Disease and other neurological disorders.A series of dietary flavonoid acacetin 7- Parkinson’s Disease (PD) is one of the most prevalent neurodegenerative disorders , affectiThe etiology of PD remains obscure, but oxidative stress is thought to play a significant role in dopaminergic neurotoxicity. At the cellular level, PD is associated with excessive production of reactive oxygen species (ROS), resulting in metabolic alteration of catecholamine, the mitochondrial electron-transporter chain (METC), or enhancement of the deposition of iron in the substantia nigra pars compacta (SNpc) . RecentlMAO-B is one of the two subtypes of the monoamine oxidases (MAO-A and MAO-B). It is a major monoamine-metabolizing enzyme that is known to oxidize the neurotransmitter dopamine, along with other amines. MAO-B is primarily localized in astrocytes, while MAO-A is largely located in the neurons of the brain . SelectiCalea urticifolia, yielded acacetin, a prominent dietary flavonoid + .IUPAC.:  was treated with 3–4 mol% of iodine crystals with a minimal amount of DMSO at 140 °C for 4–6 h. The resulting white precipitate was extracted with CH2Cl2 and was removed by filtration. The filtrate was transferred into a separatory funnel where the layers were separated as aqueous and organic. The organic layer was washed with sodium thiosulfate (Na2S2O3), dried with sodium sulfate (Na2SO4), and concentrated. The intermediate flavonoid 1b was then purified in a silica column using 100% CH2Cl2, 50:1 CH2Cl2-MeOH, 9:1 CH2Cl2-MeOH, 1:1 CH2Cl2-MeOH, and 100% MeOH . 1H NMR  δH 7.80 , 6.98 , 6.59 , 6.57 , 6.37 , 3.99 , 3.96 , 3.91 , 1.84 , 1.06 . 13C NMR  δC 177.7 (C-4), 163.9 (C-2), 161.6 (C-5), 160.9 (C-4′), 160.7 (C-9), 159.8 (C-7), 127.5 (C-2′ and C-6′), 123.5 (C-1′), 114.8 (C-3′ and C-5′), 109.2 (C-10), 107.6 (C-3), 96.0 (C-8), 92.8 (C-6), 69.7 (C-1″), 56.4 (OCH3), 55.7 (OCH3), 22.5 (C-2″), 10.5 (C-3″). HRESIMS m/z 341.0738 [M+H]+ .IUPAC.: 5,7-Dimethoxy-2-(4-propoxyphenyl)-4H-chromen-4-one. The intermediate 1b  was treated with 1N boron tribromide  in the presence of CH2Cl2 at rt for 1–3 h. The reactant solution was diluted with 10:1 CH2Cl2:MeOH and then washed with brine and water. Na2SO4 was added to the solution and left overnight. The solution was then filtered, dried, and concentrated. The flavonoid 1c was purified in a silica column using 30:1 CH2Cl2-MeOH . 1H NMR  δH 12.83 , 7.82 , 7.00 , 6.56 , 6.47 , 6.35 , 4.00 , 3.88 , 1.86 , 1.08 . 13C NMR  δC 182.4 (C-4), 165.4 (C-7), 164.0 (C-2), 162.2 (C-4′), 162.1 (C-5), 157.6 (C-9), 127.7 (C-2′ and C-6′), 123.5 (C-1′), 115.0 (C-3′ and C-5′), 105.5 (C-10), 104.2 (C-3), 98.0 (C-8), 92.5 (C-6), 69.8 (C-1″), 55.8 (OCH3), 22.5 (C-2″), 10.5 (C-3″). HRESIMS m/z 327.1038 [M+H]+ .IUPAC.: 5-Hydroxy-7-methoxy-2-(4-propoxyphenyl)-4E)-1--3-(4-isopropoxyphenyl)prop-2-en-1-one. The procedure for the preparation of the chalcone 2a is similar to that for the preparation of the chalcone 1a except for the use of 4-isopropoxybenzaldehyde . The chalcone 2a was purified in a silica column using 4:1 Hex-EtOAc, 3:1 Hex-EtOAc, 4:1 EtOAc-Hex, 100% EtOAc, and 100% MeOH . 1H NMR  δH 14.43 , 7.80 , 7.55 , 6.91 , 6.12 , 5.97 , 4.62 , 3.93 , 3.84 , 1.37 . 13C NMR  δC 192.6 (C=O), 168.3 (C-2′), 166.0 (C-4′), 162.4 (C-6′), 159.8 (C-4), 142.6 (C-β), 130.1 (C-2 and C-6), 127.9 (C-1), 124.9 (C-α), 115.9 (C-3 and C-5), 106.4 (C-1′), 93.8 (C-3′), 91.2 (C-5′), 70.0 (C-1″), 55.8 (OCH3), 55.6 (OCH3), 22.0 (C-2″ and C-3″). HRESIMS m/z 343.0876 [M+H]+ .IUPAC.: . 1H NMR  δH 7.78 , 6.95 , 6.56 , 6.53 , 6.34 , 4.62 , 3.93 , 3.89 , 1.36 . 13C NMR  δC 177.6 (C-4), 163.8 (C-2), 160.8 (C-5), 160.7 (C-4′), 160.5 (C-9), 159.8 (C-7), 127.6 (C-2′ and C-6′), 123.3 (C-1′), 115.8 (C-3′ and C-5′), 109.2 (C-3), 109.2 (C-10), 107.5 (C-3), 96.0 (C-8), 92.8 (C-6), 70.1 (C-1″), 56.4 (OCH3), 55.7 (OCH3), 21.9 (C-2″ and C-3″). HRESIMS m/z 341.0796 [M+H]+ .IUPAC.: 2-(4-Isopropoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one. This procedure is similar to that for the preparation of the final flavonoid 1c. The final flavonoid 2c was purified in a silica column using 7:3 Hex-EtOAc and 1:1 Hex-EtOAc . 1H NMR  δH 12.83 , 7.83 , 6.99 , 6.58 , 6.49 , 6.37 , 4.66 , 3.89 , 1.40 . 13C NMR  δC 182.4 (C-4), 165.4 (C-7), 164.1 (C-2), 162.2 (C-4′), 161.1 (C-5), 157.3 (C-9), 128.0 (C-2′ and C-6′), 123.1 (C-1′), 115.9 (C-3′ and C-5′), 105.5 (C-10), 104.2 (C-3), 98.0 (C-8), 92.6 (C-6), 70.2 (C-1″), 55.8 (OCH3), 21.9 (C-2″ and C-3″). HRESIMS m/z 327.1038 [M+H]+ .IUPAC.: 5-Hydroxy-2-(4-isopropoxyphenyl)-7-methoxy-4E)-1--3-(4-isobutoxyphenyl)prop-2-en-1-one. This procedure is similar to that for the preparation of the chalcone 1a except for the use of 4-isobutoxy benzaldehyde . The chalcone 3a was purified in a silica column using CH2Cl2 . 1H NMR  δH 14.45 , 7.82 , 7.57 , 6.94 , 6.13 , 5.99 , 3.94 , 3.86 , 3.79 , 2.13 , 1.06 . 13C NMR  δC 192.6 (C=O), 168.4 (C-2′), 166.0 (C-4′), 162.4 (C-6′), 161.1 (C-4), 142.6 (C-β), 130.1 (C-2 and C-6), 128.0 (C-1), 124.9 (C-α), 114.9 (C-3 and C-5), 106.4 (C-1′), 93.8 (C-3′), 91.2 (C-5′), 74.5 (C-1″), 55.8 (OCH3), 55.6 (OCH3), 28.2 (C-2″), 19.2 (C-3″ and C-4″). HRESIMS m/z 357.0610 [M+H]+ .IUPAC.: . 1H NMR  δH 7.73 , 6.92 , 6.52 , 6.48 , 6.29 , 3.89 , 3.85 , 3.73 , 2.07 , 1.00 . 13C NMR  δC 177.6 (C-4), 163.8 (C-2), 161.7 (C-5), 160.7 (C-4′ and C-9), 159.7 (C-7), 127.4 (C-2′ and C-6′), 123.4 (C-1′), 114.8 (C-3′ and C-5′), 109.0 (C-10), 107.4 (C-3), 96.0 (C-8), 92.8 (C-6), 74.5 (C-1″), 56.3 (OCH3), 55.7 (OCH3), 28.2 (C-2″), 19.2 (C-3″ and C-4″). HRESIMS m/z 355.0958 [M+H]+ .IUPAC.: 2-(4-Isobutoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one. This procedure is similar to that for the preparation of the final flavonoid 1c. The final flavonoid 3c was purified in a silica column using 7:3 EtOAc-Hex and 100% EtOAc . 1H NMR  δH 12.79 , 7.81 , 7.00 , 6.56 , 6.47 , 6.36 , 3.88 , 3.80 , 2.14 , 1.07 . 13C NMR  δC 182.4 (C-4), 165.4 (C-7), 164.1 (C-2), 162.3 (C-4′), 162.1 (C-5), 157.6 (C-9), 127.9 (C-2′ and C-6′), 123.1 (C-1′), 114.9 (C-3′ and C-5′), 105.5 (C-10), 104.1 (C-3), 98.0 (C-8), 92.5 (C-6), 74.6 (C-1″), 55.7 (OCH3), 28.2 (C-2″), 19.2 (C-3″ and C-4″). HRESIMS m/z 341.0737 [M+H]+ .IUPAC.: 5-Hydroxy-2-(4-Isobutoxyphenyl)7-methoxy-4E)-1--3-(4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one. This procedure is similar to that for the preparation of the chalcone 1a except for the use of 4-(prop-2-ynyloxy)benzaldehyde . The chalcone 4a was purified in a silica column using 100% CH2Cl2 . 1H NMR  δH 14.38 , 7.84 , 7.79 , 7.60 , 7.03 , 6.14 , 5.99 , 4.76 , 3.94 , 3.86 , 2.57 . 13C NMR  δC 192.6 (C=O), 168.4 (C-2′), 166.1 (C-4′), 162.5 (C-6′), 159.1 (C-4), 142.1 (C-β), 130.0 (C-2 and C-6), 129.2 (C-1), 125.7 (C-α), 115.3 (C-3 and C-5), 106.4 (C-1′), 93.8 (C-3′), 91.3 (C-5′), 78.1 (C-2″), 75.9 (C-3″), 55.9 , 55.6 (OCH3). HRESIMS m/z 339.0599 [M+H]+ .IUPAC.: . 1H NMR  δH 7.82 , 7.07 , 6.58 , 6.54 , 6.36 , 4.76 , 3.94 , 3.90 , 2.57 . 13C NMR  δC 177.6 (C-4), 163.9 (C-2), 160.9 (C-5), 160.4 (C-4′), 159.8 (C-9), 159.8 (C-7), 127.5 (C-2′ and C-6′), 124.7 (C-1′), 115.2 (C-3′ and C-5′), 109.2 (C-10), 107.9 (C-3), 96.1 (C-8), 92.8 (C-6), 77.9 (C-2″), 76.1 (C-3″), 56.4 (C-1″), 55.9 (OCH3), 55.7 (OCH3). HRESIMS m/z 337.0495 [M+H]+ .IUPAC.: 5,7-Dimethoxy-2-(4-(prop-2-yn-1-yloxy)phenyl)-4H-chromen-4-one. This procedure is similar to that for the preparation of the final flavonoid 1c. The final flavonoid 4c was purified in a silica column with 7:3 Hex-EtOAc, 1:1 Hex-EtOAc, 100% EtOAc, 20:1 EtOAc-MeOH, and 9:1 EtOAc-MeOH . 1H NMR  δH 12.79 , 7.88 , 7.12 , 6.60 , 6.50 , 6.39 , 4.80 , 3.90 , 2.59 . 13C NMR  δC 182.4 (C-4), 165.5 (C-7), 163.8 (C-2), 162.2 (C-4′), 160.4 (C-5), 157.7 (C-9), 128.0 (C-2′ and C-6′), 124.4 (C-1′), 115.4 (C-3′ and C-5′), 105.6 (C-10), 104.6 (C-3), 98.1 (C-8), 92.6 (C-6), 77.7 (C-2″), 76.2 (C-3″), 55.9 (C-1″), 55.8 (OCH3). HRESIMS m/z 323.0767 [M+H]+ .IUPAC.: 5-Hydroxy-7-methoxy-2-(4-(prop-2-yn-1-yloxy)phenyl)-41H NMR  δH 9.82 , 7.76 , 6.94 , 3.84 , 1.27–1.21 , 0.64 – 0.61 , 0.34 – 0.32 . 13C NMR  δC 190.7 (CHO), 164.0 (C-4), 131.9 (C-2 and C-6), 129.8 (C-1), 114.8 (C-3 and C-5), 73.0 (C-1′), 10.0 (C-2′), 3.2 (C-3′ and C-4′).IUPAC.: 4-(Cyclopropylmethoxy)benzaldehyde. (Bromomethyl)cyclopropane  was added to 4-hydroxy benzaldehyde  and was dissolved in 15 mL of acetone and potassium carbonate . The mixture was then refluxed for 36 h, then diluted with EtOAc and washed with water. This compound IV was purified in a silica column using 95:5 Hex-EtOAc . E)-3-(4-(Cyclopropylmethoxy)phenyl)-1-prop-2-en-1-one. This procedure is similar to that for the preparation of the chalcone 1a except for the use of 4-(Cyclopropylmethoxy)benzaldehyde (compound IV) . The chalcone 5a was purified in a silica column with 4:1 CH2Cl2-Hex, 100% CH2Cl2, 20:1 CH2Cl2-MeOH, and 9:1 CH2Cl2-MeOH . 1H NMR  δH 14.42 , 7.84 , 7.55 , 6.93 , 6.11 , 5.97 , 3.86 , 3.92 , 3.84 , 1.31 – 1.27 , 0.67 , 0.38 . 13C NMR  δC 192.6 (C=O), 168.3 (C-2′), 166.0 (C-4′), 162.4 (C-6′), 160.8 (C-4), 142.5 (C-β), 130.1 (C-2 and C-6), 128.2 (C-1), 125.0 (C-α), 114.9 (C-3 and C-5), 106.3 (C-1′), 93.8 (C-3′), 91.2 (C-5′), 72.9 (C-1″), 55.8 (OCH3), 55.5 (OCH3), 10.2 (C-2″), 3.2 (C-3″ and C-4″). HRESIMS m/z 355.0575 [M+H]+ .IUPAC.: . 1H NMR  δH 7.78 , 6.97 , 6.56 , 6.53 , 6.34 , 3.93 , 3.89 , 3.85 , 1.25 , 0.66 , 0.37 . 13C NMR  δC 177.6 (C-4), 163.9 (C-2), 161.5 (C-5), 160.8 (C-4′), 160.7 (C-9), 159.8 (C-7), 127.5 (C-2′ and C-6′), 123.6 (C-1′), 114.9 (C-3′ and C-5′), 109.1 (C-10), 107.5 (C-3), 96.0 (C-8), 92.8 (C-6), 72.9 (C-1″), 56.4 (OCH3), 55.7 (OCH3), 10.1 (C-2″), 3.2 (C-3″ and C-4″). HRESIMS m/z 353.0805 [M+H]+ .IUPAC.: 2-(4-(Cyclopropylmethoxy)phenyl)-5,7-dimethoxy-4E)-3-(4-Bromophenyl)-1-prop-2-en-1-one. A mixture of equimolar amounts of 2′-hydroxy-4′,6′-dimethoxyacetophenone  and 4-bromobenzaldehyde  was taken in a RBF and 10 mL of 50% NaOH in ethanol was added and left for 16 h at rt. The resulting yellow precipitate was then suction filtered and washed with hexanes and CH2Cl2. The brominated chalcone 6a was then purified in a silica column using 10% EtOAc and hexanes . 1H NMR  δH 14.23 , 7.89 , 7.71 , 7.55 , 7.47 , 6.13 , 5.98 , 3.93 , 3.86 . 13C NMR  δC 192.3 (C=O), 168.4 (C-4′), 166.4 (C-6′), 162.5 (C-2′), 140.8 (C-β), 134.5 (C-1), 132.1 (C-3 and C-5), 129.7 (C-2 and C-6), 128.1 (C-α), 124.2 (C-4), 106.3 (C-1′), 93.8 (C-3′), 91.3 (C-5′), 55.9 (OCH3), 55.6 (OCH3). HRESIMS m/z 363.1418 [M+H]+ .IUPAC.:   was treated with 3–4 mol% of iodine crystals with a minimal amount of DMSO at 140 °C for 4–6 h. The resulting white precipitate was extracted with CH2Cl2 and was removed by filtration. The filtrate was transferred into a separatory funnel, where the layers were separated as aqueous and organic. The organic layer was washed with Na2S2O3, dried with Na2SO4, and concentrated. The intermediate flavonoid 6b was purified in a silica column with 1:1 Hex-EtOAc, 3:2 EtOAc-Hex, 4:1 EtOAc-Hex, and 9:1 EtOAc-Hex . 1H NMR  δH 7.72 , 7.62 , 6.64 , 6.55 , 6.37 , 3.95 , 3.91 . 13C NMR  δC 177.3 (C-4), 164.2 (C-2), 160.9 (C-5), 159.8 (C-9), 159.5 (C-7), 132.2 (C-3′ and C-5′), 130.4 (C-1′), 127.3 (C-2′ and C-6′), 125.8 (C-4′), 109.2 (C-10 and C-3), 96.3 (C-8), 92.8 (C-6), 56.4 (OCH3), 55.8 (OCH3). HRESIMS m/z 361.1324 [M+H]+ .IUPAC.: 2-(4-Bromophenyl)-5,7-dimethoxy-4H-chromen-4-one. Sodium tert-butoxide , tris(dibenzylideneacetone)dipalladium , and 1,1′-binaphthalene-2,2′-diylbis(diphenylphosphine)  were dissolved in 5 mL of toluene. To this, the brominated intermediate flavonoid 6b  and propylamine  were added dropwise with stirring at rt, and the mixture was refluxed at 80 °C for 48 h. The nitrogenated intermediate flavonoid was purified in a silica column using 7:3 EtOAc-Hex, 9:1 EtOAc-Hex, 100% EtOAc, and 9:1 EtOAc-MeOH . 1H NMR  δH 7.67 , 6.61 , 6.52 , 6.50 , 6.33 , 3.92 , 3.88 , 3.13 , 1.66 , 1.00 . 13C NMR  δC 177.8 (C-4), 163.6 (C-2), 161.6 (C-5), 160.7 (C-4′), 159.7 (C-9), 150.9 (C-7), 127.5 (C-2′ and C-6′), 119.0 (C-1′), 112.1 (C-3′ and C-5′), 109.2 (C-10), 105.8 (C-3), 95.8 (C-8), 92.8 (C-6), 56.3 (OCH3), 55.7 (OCH3), 45.2 (C-1″), 22.5 (C-2″), 11.5 (C-3″). HRESIMS m/z 340.2991 [M+H]+ .IUPAC.: 5,7-Dimethoxy-2-(4-(propylamino)phenyl)-4H-chromen-4-one. The nitrogenated intermediate flavonoid 6c  was treated with double the moles of 1N boron tribromide  in the presence of CH2Cl2 at rt for 1–3 h. The reactant solution was diluted with 10:1 CH2Cl2:MeOH and then washed with brine and water. Na2SO4 was added to the solution and left overnight. The solution was then filtered into a RBF, dried, and concentrated. The final compound 6d was purified in a silica column using 1:1 Hex-EtOAc . 1H NMR  δH 12.90 , 7.65 , 6.58 , 6.43 , 6.39 , 6.27 , 3.80 , 3.10 , 1.62 , 0.96 . 13C NMR  δC 182.3 (C-4), 165.2 (C-7), 164.4 (C-2), 162.1 (C-5 and C-4′), 157.6 (C-9), 128.0 (C-2′ and C-6′), 127.5 (C-1′), 114.0 (C-3′ and C-5′), 105.4 (C-10), 103.0 (C-3), 97.9 (C-8), 92.5 (C-6), 55.8 (OCH3), 29.7 (C-1″), 22.1 (C-2″), 11.5 (C-3″). HRESIMS m/z 326.2691 [M+H]+ .IUPAC.: 5-Hydroxy-7-methoxy-2-(4-(propylamino)phenyl)-4H-chromen-4-one. This procedure is similar to that for the preparation of the intermediate flavonoid 6c except for the use of isopropylamine . The intermediate flavonoid 7c was purified in a silica column using 9:1 EtOAc-Hex . 1H NMR  7.65 , 6.58 , 6.50 , 6.48 , 6.31 , 3.90 , 3.86 , 3.67 , 1.22 . 13C NMR  δC 177.7 (C-4), 163.6 (C-2), 161.5 (C-5), 160.7 (C-4′), 159.7 (C-9), 150.0 (C-7), 127.5 (C-2′ and C-6′), 118.8 (C-1′), 112.5 (C-3′ and C-5′), 109.1 (C-10), 105.7 (C-3), 95.8 (C-8), 92.8 (C-6), 56.3 (OCH3), 55.6 (OCH3), 43.9 (C-1″), 22.7 (C-2″ and C-3″). HRESIMS m/z 340.3005 [M+H]+ .IUPAC.: 2-(4-(Isopropylamino)phenyl)-5,7-dimethoxy-4H-chromen-4-one. This procedure is similar to that for the preparation of the final compound 6d. The final compound 7d was purified in a silica column with 9:1 Hex-EtOAc . 1H NMR  13.00 , 7.74 , 6.64 , 6.52 , 6.48 , 6.37 , 3.90 , 3.75 , 1.29 . 13C NMR  δC 182.4 (C-4), 165.1 (C-7), 164.9 (C-2), 162.1 (C-4′), 157.6 (C-5), 150.5 (C-9), 128.1 (C-2′ and C-6′), 118.6 (C-1′), 112.6 (C-3′ and C-5′), 105.5 (C-10), 102.4 (C-3), 97.8 (C-8), 92.5 (C-6), 55.7 (OCH3), 44.1 (C-1″), 22.8 (C-2″ and C-3″). HRESIMS m/z 326.2691 [M+H]+ .IUPAC.: 5-Hydroxy-2-(4-(Isopropylamino)phenyl)-7-methoxy-4H-chromen-4-one. This procedure is similar to that for the preparation of the intermediate flavonoid 6c except for the use of (aminomethyl)cyclopropane . The intermediate flavonoid 8c was purified in a silica column using 7:3 EtOAc-Hex, 100% EtOAc, 10:1 EtOAc-MeOH, and 100% MeOH . 1H NMR  7.66 , 6.66 , 6.56 , 6.41 , 6.38 , 3.97 , 3.92 , 3.05 , 1.12 , 0.61 , 0.30 . 13C NMR  δC 177.8 (C-4), 163.7 (C-2), 161.5 (C-5), 160.8 (C-4′), 160.9 (C-9), 159.8 (C-7), 127.5 (C-2′ and C-6′), 119.3 (C-1′), 112.3 (C-3′ and C-5′), 109.3 (C-10), 106.0 (C-3), 96.3 (C-8), 92.8 (C-6), 56.5 (OCH3), 55.7 (OCH3), 48.5 (C-1″), 10.7 (C-2″), 3.6 (C-3″ and C-4″). HRESIMS m/z 352.2586 [M+H]+ .IUPAC.: 2-(4-((Cyclopropylmethyl)amino)phenyl)-5,7-dimethoxy-4H-chromen-4-one. This procedure is similar to that for the preparation of the final compound 6d. The final compound 8d was purified in a sephadex column with 1:1 Hex-EtOAC . 1H NMR  13.00 , 7.75 , 6.67 , 6.53 , 6.48 , 6.37 , 3.90 , 3.07 , 1.14 , 0.63 , 0.31 . 13C NMR  δC 182.4 (C-4), 165.1 (C-7), 164.9 (C-2), 162.1 (C-4′), 157.6 (C-5), 151.3 (C-9), 128.1 (C-2′ and C-6′), 118.9 (C-1′), 112.3 (C-3′ and C-5′), 105.5 (C-10), 102.5 (C-3), 97.8 (C-8), 92.5 (C-6), 55.7 (OCH3), 48.4 (C-1″), 10.7 (C-2″), 3.6 (C-3″ and C-4″). HRESIMS m/z 338.2880 [M+H]+ .2-(4-((Cyclopropylmethyl)amino)phenyl)-5-hydroxy-7-methoxy-4All the designed MAO-B inhibitors were analyzed per the recently published editorial  using thO-methyl ether analogs on human recombinant MAO-A and MAO-B, the kynuramine oxidation deamination assay was performed in 384-well plates as previously reported, with minor modifications [50 values. The kynuramine concentrations for MAO-A and -B were 80 μM and 50 μM, respectively. These concentrations of kynuramine were twice the apparent KM value for substrate binding [O-methyl ether analogs were tested at the concentrations ranging from 0.001 μM to 100 μM for MAO-A and -B inhibition assays. Enzymatic reactions were performed in 50 μL of the assay mixture containing 0.1 M potassium phosphate buffer, pH 7.4. The inhibitors and acacetin 7-O-methyl ether analogs were dissolved in DMSO, diluted in the buffer solution, and pre-incubated at 37 °C for 10 min . Reactions were initiated by the addition of 12.50 μL of MAO-A (to 5 μg/mL) or -B (to 10 μg/mL). The plate was incubated for 20 min at 37 °C, and the enzymatic reaction was terminated by the addition of 18.8 μL of 2N NaOH. Formation of the enzyme product, 4-hydroxyquinoline, was measured fluorometrically using a SpectraMax M5 fluorescence plate reader  with excitation (320 nm) and emission (380 nm) wavelengths and the Soft Max Pro program. The inhibition effects of enzyme activity were calculated as percent of product formation compared to the corresponding control (enzyme–substrate reaction) without inhibitors. Controls included the assays where the enzyme or the substrate was added after terminating the reaction to determine the interference with the fluorescence measurements. The IC50 values for MAO-A and -B inhibition were calculated from the concentration-dependent inhibition curves using XLFit® software.To investigate the inhibitory effect of the acacetin 7-ications . A fixed binding ,18. The ® Excel and the IC50 values were calculated using XLfit software [The enzyme assay was performed at a fixed concentration of the substrate kynuramine (80 μM for MAO-A and 50 μM for MAO-B) and varying concentrations of the inhibitor/test analogs (0.01 μM to 100 μM) for MAO-A and (0.01 μM to 100 μM) for MAO-B. The dose–response curves were generated using Microsoftsoftware .Ki) with MAO-B, the enzyme assays were carried out at different concentrations of kynuramine substrate (1.90 μM to 500 μM) and varying concentrations of the test compound. Acacetin 7-O-methyl ether analogs: 1c, 2c, 3c, and 4c were tested at 0.015–0.500 μM for MAO-B to determine the mK and maxV values of the enzymes in the presence of the inhibitor. The controls without inhibitor were also run simultaneously. Three sets of assays were performed at varying concentrations of the substrate for each experiment: one control without inhibitor and the others at two fixed concentrations of the inhibitor. The data were analyzed by double reciprocal Lineweaver–Burk plots to determine Ki  values and the kinetic data, namely Km, Vmax, and Ki values, were computed by SigmaPlot 12 [For determination of the binding affinity of the inhibitor  enzyme was incubated with each analog, namely acacetin (0.5 μM), 1c (1.5 μM), 2c (1.5 μM), 3c (1.5 μM), 4c (1.5 μM), and deprenyl (0.5 μM), in a total volume of 1 mL of potassium phosphate buffer . After 20 min of incubation at 37 °C, the reaction was stopped by chilling the tubes in the ice bath. All the samples were dialyzed against potassium phosphate buffer  at 4 °C for 14–18 h (with three buffer changes). The control enzyme (without inhibitor) was also run simultaneously using the same procedure and the activity of the enzyme was determined before and after the dialysis.Most of the inhibitors produce inhibition of the target enzyme activity through formation of an enzyme–inhibitor complex. Formation of the enzyme–inhibitor complex may be accelerated in the presence of a high concentration of the inhibitor. The reversibility/irreversibility of binding of acacetin 7-1c (0.100 μM), 2c (0.300 μM), 3c (0.300 μM), 4c (0.400 μM), and deprenyl (0.080 μM) with MAO-B (12.5 μg/mL). The controls, without inhibitors, were also run simultaneously. Activities of the enzyme were determined as described above, and the percentage of the remaining enzyme activity was plotted against the pre-incubation time to determine time-dependent inhibition.To analyze if the nature of MAO-B binding to the inhibitor was time-dependent, the enzyme was pre-incubated for different time periods (0–15 min) with the inhibitor concentrations, which exhibited approximately 70–80% inhibition. The inhibitor concentrations used to test time-dependent inhibition were acacetin (0.080 μM), www.rcsb.org, accessed on 12 January 2017). The protein structural files were prepared using the protein preparation wizard of Schrödinger suite [The crystal structures of MAO-A (PDB ID: 2Z5Y with an atomic resolution of 2.17 Å) and MAO-B (PDB ID: 4A79 with an atomic resolution of 1.89 Å) were downloaded from the protein databank  [O-methyl ether were studied to understand the basis of target selectivity. Molecular constraints were imposed on the system during docking simulations based on our previous investigations [O-methyl ether was mapped with the binding site environment using molecular dynamics (MD) simulations and thermodynamic calculations to better define the most abundant binding mode. The same analysis was carried out for acacetin (Acacetin 7->100 µM) . The binacacetin .O-methyl ether demonstrated a very strong binding interaction with MAO-B and a very weak binding to MAO-A. O-methyl ether with and without constraints to MAO-A and -B. Acacetin 7-O-methyl ether was docked preferentially with a docking score of −8.9 kcal/mol into MAO-B and a docking score of −6.7 kcal/mol into MAO-A, without forcing the constraints. The benzopyranone was located between the aromatic cages (Tyr residues) in both cases, and the terminal phenyl group interacted with the aromatic gating amino acids . Keeping the water molecules close to the gating amino acids clashed with the phenyl group in the case of MAO-A, while it allowed two possible poses for MAO-B.Acacetin 7-O-methyl ether in the case of MAO-B were visualized and are shown in O-methyl ether has benzopyranone facing the cofactor , Gly 101 (11%), and Pro 102 (~5%). More hydrophobic contacts were observed for pose 1, while more hydrogen bonds and water bridges were traced for pose 2 .In conclusion, water molecules are more involved in the binding of pose 2 . SeveralO-methyl ether. To improve ligand binding, the polarity should be decreased at this position. The ligand chemistry of pose 2 did not fit well with the active site hydration map, while pose 1 showed a perfect match , isopropyl-ether (2c), isobutyl-ether (3c), propargylic-ether (4c), and methyl (cyclopropyl)-ether (5c). The role of the bioisosteric change of nitrogen from oxygen was also scrutinized. Thus, propyl amine (6d), isopropyl amine (7d), and methyl (cyclopropyl)-amine (8d) were synthesized.The designed compounds were evaluated for their absorption, distribution, metabolism, and excretion (ADME) properties. They did not show any violation of the rules of five or three; they have a good balance of lipophilicity/hydrophilicity, and are expected to be orally bioavailable . The com1–5) c and (6–b) d were based on reported synthetic procedures [The synthetic routes to access the target compounds (ocedures  Scheme –5 c and 1–4) a were afforded through Schmidt condensation by reacting 2′-hydroxy-4′,6′-dimethoxyacetophenone I and the corresponding 4-benzaldehydes II with 50% sodium hydroxide in ethanol. Treatment of the chalcones (1–4) with iodine crystals in a minimal amount of DMSO underwent cyclization to yield the dimethoxylated flavonoids (1–4) b. To obtain the target flavonoids (1–4) c, selective demethylation of position C-5 of (1–4) b was accomplished using boron tribromide (BBr3) in CH2Cl2  c were prepared by treating compound 6b with sodium tert-butoxide, tris(dibenzylideneacetone) dipalladium, 1,1′-binaphthalene-2,2′-diylbis(diphenylphosphine), and the corresponding amines such as propylamine, isopropylamine, and (aminomethyl)cyclopropane in toluene. Finally, the selective demethylated flavonoids at position 5 (6–8) d were prepared with boron tribromide in dichloromethane  > protected-methylated flavonoids at C-5 (1b–6b) > chalcones (1a–6a) > amine flavonoids at C-4′ . Compounds (1–4) c exhibited more than a thousand-fold SI (1–4) c.The synthesized acacetin 7-er MAO-A . The siger MAO-A . The pre-fold SI , equal t-fold SI , and wer1–4) c were evaluated against MAO-B at varying concentrations of kynuramine, a nonselective substrate, to investigate the nature of inhibition of the enzymes. Based on dose–concentration inhibition, three to five concentrations  were selected for the inhibition kinetics experiment. Modified flavonoids (1–4) c inhibited the enzymatic activity of MAO-B with considerably high affinity Ki at nanomolar units with a range from 43 to 68 nM (Modified flavonoids (to 68 nM .1–4) c with human MAO-B affected mK  as well as maxV (maximum enzyme activity) values, indicating the type of MAO-B inhibition by the analogs: flavonoid 1c , 2c , 3c (mixed/irreversible), and 4c (mixed/irreversible) (Binding of modified flavonoids (ersible) .1–4) c with MAO-B were examined by equilibrium dialysis to measure the dissociation of the enzyme–inhibitor complex (4 (pH 7.4) buffer. The enzyme activities were examined before and after dialysis. Incubation of MAO-B with 1.5 µM of modified flavonoids (1–4) c caused more than 70% inhibition of activity and only the enzyme activities of 1c (30%) and 2c (66%) were recovered after dialysis. Thus, the binding of 1c was partially reversible, while 2c was reversible with MAO-B  c, further computational analysis was conducted. These analogs did not show good docking poses in MAO-A due to the clashes between the R group and the amino acids in the substrate binding site. The size and nature of the R group are essential for selective targeting. In general, these four acacetin 7-O-methyl ether analogs exploited the hydrophobic nature of the amino acids in the binding pocket of MAO-B, particularly Tyr 398 and Tyr 435, near the FAD cofactor by forming π-π stacking with their aromatic functionalities. The non-polar nature of the R group matches the hydrophobic nature of the Leu and Ile amino acids on the other side of the binding site. Considerable hydrophobic contacts were observed between the ligands and Leu 167, Leu 171, Ile 199, Tyr 236, and Phe 343. The ligands placed properly their polar chemistry to form strong hydrogen bonds with the backbones of Leu 171, Ile 199, and the side chain of Gln 206  c with O-alkyl (a short chain with three to four carbon atoms) substituents at position C-4′ yield high selectivity toward MAO-B. The nitrogen amine series did not display any significant selectivity towards MAO-B. This could be attributed to the high electronegativity of oxygen compared with nitrogen. The study of the longer alkyl chain on C-4′ should be explored. It is also worth pursuing the introduction of halogens at position 7, which might help improve the ADMET properties. Introduction of an electronegative atom such as nitrogen at position 8 would be interesting to understand the SAR of ring A in MAO activity. Another chemical modification that would help us to understand the activity of MAO-B versus MAO-A in the flavonoid scaffold that is worth exploring is the substitution of the B ring, introducing O-substituent groups at position C-3′ or C-4′ as on vetulin or diosmetin (see Rasagiline and selegiline are FDA-approved irreversible MAO-B inhibitors that exhibited a selectivity of 103- and 127-fold against MAO-A, while safinamide is a reversible MAO-B inhibitor with a selectivity of 1000–1500-fold towards MAO-B ,9,46. Thetin see , which b


Talaromyces leycettanus. The enzyme is an ando-β-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan . The heteroxylans are hydrolyzed to a mixture of linear as well as branched β-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear β-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the −2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus  They may be decorated with α-D-glucuronic acid, 4-O-methyl-α-D-glucuronic acid and α-L-arabinofuranosyl residues. Moreover, the Xylp residues may be acetylated. The pattern of xylan main chain substitution is species, tissue, and season specific; modulates the physico-chemical properties of the polymer; and renders the hemicellulose water soluble by preventing self-association of the xylan chains X4X4X , X4[A3]X4X4X , X4[A2A3]X4X4X  and a mixture of X4[A3]X4X4X (compound 9) and X4[A2]X4X4X  were purchased from Megazyme International . Xylose was from Serva . MeGlcA3Xyl3 and MeGlcA3Xyl4 were prepared from beechwood GX as described previously -β-D-Xylp-O-Me ), α-L-Araf-1,3-β-D-Xylp-1,4-β-D-Xylp-O-Me ), β-D-Xylp-1,4-β-D-Xylp-O-Me ), β-D-Xylp-1,4-β-D-Xylp-1,4-β-D-Xylp-O-Me ), β-D-Xylp-1,4-β-D-Xylp-1,3-β-D-Xylp-O-Me ), β-D-Xylp-1,4-β-D-Xylp-1,2-β-D-Xylp-O-Me ), β-D-Xylp-1,2-β-D-Xylp-1,4-β-D-Xylp-O-Me ), β-D-Xylp-1,3-β-D-Xylp-O-Me ), β-D-Xylp-1,2-β-D-Xylp-O-Me ), β-D-Xylp-1,4--β-D-Xylp-O-Me ), β-D-Xylp-1,4--β-D-Xylp-O-Me ), β-D-Xylp-1,3-β-D-Xylp-1,4-β-D-Xylp-O-Me ), α-DXylp-1,3-β-D-Xylp-1,4-β-D-Xylp-O-Me ), α-D-Xylp-1,4-β-D-Xylp-1,3-β-D-Xylp-O-Me )) α-D-Xylp-1,4-β-D-Xylp-1,2-β-D-Xylp-O-Me )—were synthesized previously [Beechwood 4- earlier . Isolati earlier . Rhodymeeviously . Methyl TlXyn30A with 1% solution of GX in 40 mM Britton-Robinson (pH 3.5–5.5) or 50 mM sodium phosphate buffers (pH 6.0–8.0). The optimum temperature was determined in the same way in 40 mM Britton-Robinson buffer, pH 3.5, and temperatures ranging from 23 to 90 °C. Temperature stability was tested in 40 mM Britton-Robinson buffer, pH 3.5, at 40–60 °C for up to 5 h. During the incubation, aliquots were taken at different time points and the residual activity was immediately determined as described above .pH optimum was determined at 40 °C using a 15-min incubation of 69 nM w/v) were incubated at 37 °C with TlXyn30A (34 nM) in 50 mM sodium acetate buffer, pH 3.5. At time intervals, 100-μL aliquots were taken for the determination of reducing sugars. One unit of enzyme activity was defined as the enzyme amount liberating in 1 min 1 μmol of reducing sugars expressed as an equivalent of xylose.Specific activity on polysaccharides was determined by the Somogyi–Nelson procedure  as the aTlXyn30A at 40 °C. After 2 h, a fresh enzyme was added to a final concentration of 2.11 μM. During the reaction course, aliquots of 5 μL were spotted on silica gel-coated aluminum sheets . After 24 h, the reaction was terminated by heating at 100 °C for 5 min. Subsequent treatment with β-xylosidase (1 U/mL) was done overnight at 40 °C. Low-molecular-weight substrates, i.e. linear xylooligosaccharides (Xyl2–Xyl6), aldouronic acids MeGlcA3Xyl3 and MeGlcA3Xyl4, arabinoxylooligosaccharides, methyl and 4-nitrophenyl glycosides, were used at 2.5 mM. Their hydrolysis with 6.25 μM TlXyn30A was conducted in 50 mM sodium acetate buffer, pH 3.5, at 35 °C, and 2-μL aliquots were spotted on the TLC plate. The TLC plate was developed either twice in a solvent system of ethyl acetate/acetic acid/2-propanol/formic acid/water 25:10:5:1:15 (v/v)  or once in a solvent system of n-butanol/ethanol/water 10:8:5 (v/v), and the sugars were visualized with the orcinol reagent.For TLC (thin-layer chromatography) analysis, the polysaccharide solutions  were incubated with 0.45 μM + form) and 1 µL was mixed with 1 µL of the matrix  directly on the MS target plate. After air-drying, the samples were analyzed by a UltrafleXtreme MALDI ToF/ToF mass spectrometer  operating in reflectron positive mode.The hydrolysates were decationized by Dowex 50  is smaller by one and two xylosyl residues than the shortest aldouronic acids generated by GH10 and GH11 xylanases, respectively. Unbranched regions of the polysaccharides and larger oligosaccharides as well as linear β-1,4-xylooligosaccharides are degraded mainly to Xyl2. This is important in view of possible biotechnological applications of the enzyme.Although


Thuja occidentalis (Cupressaceae) showed potent anti-proliferative activity in MT-1 and MT-2 cells. Therefore, we attempted to isolate the active components from this plant. We isolated and identified 32 compounds  from the extracts of the leaves and cones. Their structures were determined by spectroscopic analysis. Several of the isolated compounds inhibited the growth of both cell lines. Lignans showed more potent activity than other classes of compounds. A comparison of the activities of compounds 1–8 revealed that the presence of a trans-lactone (linkage of C-6 to C-7) correlated with increased activity. Diterpenes showed moderate activity, and the presence of a ketone moiety at the C-7 position correlated with increased activity in compounds 12–21. In addition, biflavones showed moderate activity, and the presence of methoxy functions appeared to influence the activity of these compounds. Several lignans were lead compound of anti-cancer reagent (etoposide). In conclusion, not only lignans, but also diterpenes and/or biflavones, may be promising candidates for the treatment of adult T-cell leukemia/lymphoma.During the screening of novel chemotherapeutic candidates from plants against adult T-cell leukemia/lymphoma, we identified that the extracts of  Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature peripheral T-lymphocytes associated with human T-cell lymphotropic virus type I HTLV-1). Conventional chemotherapeutic regimens used to treat other types of malignant lymphoma have been administered to ATL patients, but the therapeutic outcomes of acute- and lymphoma-type ATL remain very poor . Convent, and MT-Thuja occidentalis, a plant belonging to the family Cupressaceae. T. occidentalis is native to North America and widely cultivated as ornamental tree known as white cedar , monoterpenes , and a sesquiterpene alcohol [(+)-occidentalol]  . In tradeumatism . Thujoneeumatism . In in veumatism . The aque stress .T. occidentalis; therefore, we attempted to isolate the active compounds from these extracts. In addition, we attempted to isolate an extract from the cones of T. occidentalis.T. occidentalis was performed with MeOH under reflux, and the extract was initially partitioned between n-hexane and 80% MeOH. The n-hexane layer and 80% MeOH layer were subjected to columns to isolate the compounds. Conversely, extraction of the cones of T. occidentalis was performed with MeOH under reflux and the extracts were subjected to columns to isolate the compounds.Extraction of the leaves of 3–9, 11–18, 20–27, and 29–32 from the leaves; compounds 1–3, 6, 9–10, 19–20, and 27–29 were extracted from the cones  inhibited the growth of both tumor cell lines. But all compounds had weaker antiproliferative activity than positive control except compound 3. The trans-lactone moiety in compounds 1 and 3 increased their anti-proliferative activity compared with compounds 2 and 4, which do not possess a trans-lactone moiety. The presence of a hydroxyl group at position 7α in compounds 1 and 2 appeared to decrease the activity of these compounds compared with compounds 3 and 4. Furthermore, the presence of a ketone moiety at position C-7 in compound 5 was associated with decreased activity in comparison to compound 4, which lacked this moiety. Comparison of compounds 3 and 6 indicated that, the linkage of C-6 to C-7 appeared to enhance the activity  influenced the activities.Compared with compounds 16 appeared to enhance its activity compared with compound 13, which did not have a double bond at this position. Compared with compounds 15 and 16, the presence of a ketone moiety at position C-7 enhanced the activity. Similarly, compared with compounds 18, 19, and 20, the substituents (OH and ketone) influenced their activities  inhibited the proliferation of cell lines. Comparison of compounds 25, 26, 27, 28, and 29, indicated the presence of one or two methoxy groups appeared to influence the activity of the compounds.Several of the isolated flavonoids  and isoginkgetin (26) on apoptosis. The proportion of annexin V-positive cells after the 72 h treatment with deoxypodophyllotoxin (3) and isoginkgetin (26) is shown in 3) and isoginkgetin (26). The concentration of deoxypodophyllotoxin (3) was changed 2 nM to 10 nM increased the early apoptotic cells from 23.20% to 70.32%, the concentration of isoginkgetin (26) was changed 1.4 µM to 35 µM increased the early apoptotic cells from 24.35% to 55.35%. In the case MT-2 had a similar tendency. Therefore, deoxypodophyllotoxin (3) and isoginkgetin (26) significantly induced apoptosis in MT-1 and MT-2 cells.We analyzed the ability of the extracted compounds to induce apoptosis. The following experiments were studied for compound 1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectra were measured in CDCl3 or DMSO-6d using a JNM-ECZ600R spectrometer  at room temperature, and the chemical shifts given as a δ (ppm) scale with tetramethylsilane (TMS) as the internal standard. The FAB-MS was measured using a JEOL JMS-HX110 mass spectrometer and acquired in a glycerol matrix. HPLC was conducted using a Waters machine equipped with a 1525 binary pump and a 2489 UV/Vis detector . Separation was carried out using a Cosmosil 5C18 MS-II column . Apoptosis analyses were carried out using a Muse Cell Analyzer .The The plant materials used in this study were taken from the medicinal plant garden of Fukuoka University, located in Fukuoka, Japan. Voucher specimens were deposited in the Laboratory of Pharmacognosy of Fukuoka University, Fukuoka, Japan (FUN-0270).T. occidentalis (4860 g) was performed with MeOH under reflux. The extract (72.6 g) was initially partitioned using n-hexane and 80% MeOH. The n-hexane phase was subjected to a Diaion HP-20 column using 85% MeOH, 90% MeOH, 95% MeOH, 100% MeOH, and MeOH:acetone (4:1) to obtain fraction (fr.) LH1, LH2, LH3, LH4, and LH5, respectively. The fr. LH1 was subjected to a silica gel column using hexane:acetone (9:1) to obtain fr. LH1-1. The fr. LH1-1 was subjected to a silica gel column using hexane:EtOAc (3:1 and 2:1) to obtain fr. LH1-1-1 and compound 13 (1.9 mg). The fr. LH1-1-1 was subjected to HPLC purification using 55% MeCN to obtain compound 17 (22.0 mg). The fr. LH2 was subjected to a silica gel column using hexane:acetone  to obtain fr. LH2-1, LH2-2, and LH2-3. The fr. LH2-1 was subjected to a silica gel column using hexane to obtain fr. LH2-1-1 and LH2-1-2. The fr. LH2-1-1 was subjected to a silica gel column using hexane:acetone (11:1) to obtain fr. LH2-1-1-1. The fr. LH2-1-1-1 was subjected to HPLC purification using 80% MeCN to obtain compound 21 (13.2 mg) and compound 22 (46.4 mg). The fr. LH2-1-2 was subjected to an ODS column using 60% MeOH to obtain compound 14 (517.0 mg). The fr. LH2-2 was subjected to a silica gel column using hexane:acetone (10:1) to obtain fr. LH2-2-1. The fr. LH2-2-1 was subjected to HPLC purification using 90% MeCN to obtain compound 15 (10.9 mg). The fr. LH2-3 was subjected to a silica gel column using hexane:acetone (4:1 and 3:1) to obtain fr. LH2-3-1 and LH2-3-2, respectively. The fr. LH2-3-1 was subjected to a silica gel column using CHCl3:MeOH (30:1) to obtain fr. LH2-3-1-1. The fr. LH2-3-1-1 was subjected to HPLC purification using 60% MeCN to obtain compound 20 (20.5 mg). The fr. LH2-3-2 was subjected to a silica gel column using CHCl3:MeOH (25:1) to obtain fr. LH2-3-2-1. The fr. LH2-3-2-1 was subjected to HPLC purification using 50% MeCN to obtain compound 16 (18.0 mg). The fr. LH3 was subjected to a silica gel column using hexane:acetone (95:1) to obtain fr. LH3-1. The fr. LH3-1 was subjected to HPLC purification using 80% MeCN to obtain compound 18 (19.8 mg). The fr. LH4 was subjected to a silica gel column using hexane:EtOAC (10:1) and CHCl3:MeOH:H2O (17:3:0.3) to obtain fr. LH4-1 and LH4-2, respectively. The fr. LH4-1 was subjected to a silica gel column using hexane:acetone (50:1) to obtain fr. LH4-1-1. The fr. LH4-1-1 was subjected to HPLC purification using 85% MeCN to obtain compound 11 (13.6 mg). The fr. LH4-2 was subjected to a silica gel column using acetone to obtain compound 32 (18.0 mg). The fr. LH5 was subjected to a silica gel column using hexane:EtOAc (5:1) to obtain fr. LH5-1. The fr. LH5-1 was subjected to a silica gel column using hexane:acetone (10:1) to obtain fr. LH5-1-1. The fr. LH5-1-1 was subjected to HPLC purification using MeOH to obtain compound 31 (26.3 mg).The samples were powdered, and the compounds were extracted in accordance with the procedure described in a previous paper . Extract3:MeOH (100:1) to obtain fr. LM1-1-1-1-1. The fr. LM1-1-1-1-1 was subjected to HPLC purification using 40% MeCN to obtain compound 9 (2.5 mg). The fr. LM1-2 was subjected to a silica gel column using hexane:acetone  to obtain fr. LM1-2-1, LM1-2-2, LM1-2-3, LM1-2-4, and LM1-2-5, respectively. The fr. LM1-2-1 was subjected to a silica gel column using CHCl3:MeOH (84:1) to obtain compound 24 (6.2 mg). The fr. LM1-2-2 was subjected to a silica gel column using hexane:acetone (13:2) to obtain fr. LM1-2-2-1. The fr. LM1-2-2-1 was subjected to an ODS column using 77% MeOH to obtain compound 12 (22.0 mg). The fr. LM1-2-3 was subjected to a silica gel column using CHCl3 and CHCl3:MeOH (100:1) to obtain compound 7 (6.7 mg) and fr. LM1-2-3-1, respectively. The fr. LM1-2-3-1 was subjected to HPLC purification using 50% MeCN to obtain compound 8 (5.7 mg). The fr. LM1-2-4 was subjected to a silica gel column using hexane:acetone (2:1) to obtain fr. LM1-2-4-1. The fr. LM1-2-4-1 was subjected to a silica gel column using CHCl3 to obtain fr. LM1-2-4-1-1. The fr. LM1-2-4-1-1 was subjected to HPLC purification using 40% MeCN to obtain compound 23 (4.1 mg). The fr. LM1-2-5 was subjected to a silica gel column using CHCl3:MeOH:H2O (9:1:0.1) to obtain fr. LM1-2-5-1. The fr. LM1-2-5-1 was subjected to an ODS column using 30% MeCN to obtain compound 25 (9.2 mg). The fr. LM1-3 was subjected to HPLC purification using 50% MeCN to obtain compound 27 (45.7 mg) and compound 26 (6.5 mg). The fr. LM2 was subjected to a silica gel column using hexane:EtOAC (3:2) to obtain fr. LM2-1. The fr. LM2-1 was subjected to a silica gel column using hexane:acetone (3:1 and 5:2) to obtain compound 6 (23.9 mg), compound 3 (730 mg), and fr. LM2-1-1. The fr. LM2-1-1 was subjected to HPLC purification using 35% MeCN to obtain compound 4 (2.7 mg) and compound 5 (4.3 mg). The fr. LM3 was subjected to a silica gel column using hexane:EtOAc (1:1) to obtain fr. LM3-1. The fr. LM3-1 was subjected to a silica gel column using CHCl3:MeOH (80:1) to obtain fr. LM3-1-1. The fr. LM3-1-1 was subjected to HPLC purification using 50% MeCN to obtain compound 29 (2.2 mg). The fr. LM4 was subjected to a silica gel column using CHCl3:MeOH (15:1) to obtain fr. LM4-1. The fr. LM4-1 was subjected to HPLC purification using 45% MeCN to obtain compound 30 (4.6 mg).The 80% MeOH phase was subjected to a Diaion HP-20 column using MeOH, MeOH:acetone (3:2), and acetone to obtain fr. LM1, LM2, LM3, and LM4. The fr. LM1 was subjected to an MCI gel column using 80% MeOH, 90% MeOH, and acetone to obtain fr. LM1-1, LM1-2, and LM1-3, respectively. The fr. LM1-1 was subjected to a silica gel column using hexane:acetone (5:1) to obtain fr. LM1-1-1. The fr. LM1-1-1 was subjected to a silica gel column using hexane:EtOAc (2:1) to obtain fr. LM1-1-1-1. The fr. LM1-1-1-1 was subjected to a silica gel column using CHClT. occidentalis (688 g) was performed with MeOH under reflux. The extract (72.6 g) was subjected to a Diaion HP-20 column using 80% MeOH, 100% MeOH, and acetone to obtain fr. C1, C2, and C3, respectively. The fr. C1 was subjected to a silica gel column using CHCl3:MeOH:H2O (9:1:0.1) to obtain fr. C1-1. The fr. C1-1 was subjected to a silica gel column using hexane:acetone  to obtain fr. C1-1-1, C-1-1-2, and C1-1-3, respectively. The fr. C1-1-1 was subjected to HPLC purification using 50% MeCN to obtain compound 9 (3.2 mg). The fr. C1-1-2 was subjected to a silica gel column using CHCl3:MeOH (80:1) to obtain fr. C1-1-2-1. The fr. C1-1-2-1 was subjected to HPLC purification using 50% MeCN to obtain compound 10 (14.0 mg) and compound 19 (3.0 mg). The fr. C1-1-3 was subjected to a silica gel column using hexane:EtOAc (2:3) to obtain fr. C1-1-3-1. The fr. C1-1-3-1 was subjected to HPLC purification using 30% MeCN to obtain compound 2 (2.8 mg). The fr. C2 was subjected to a silica gel column using CHCl3:MeOH (20:1) to obtain fr. C2-1. The fr. C2-1 was subjected to a silica gel column using hexane:acetone  to obtain fr. C2-1-1, C2-1-2, and C2-1-3, respectively. The fr. C2-1-1 was subjected to a silica gel column using hexane:EtOAc (7:2) to obtain fr. C2-1-1-1. The fr. C2-1-1-1 was subjected to HPLC purification using 60% MeCN to obtain compound 20 (9.2 mg). The fr. C2-1-2 was subjected to a silica gel column using CHCl3:MeOH (40:1) to obtain fr. C2-1-2-1. The fr. C2-1-2-1 was subjected to HPLC purification using 55% MeCN to obtain compound 1 (10.5 mg). The fr. C2-1-3 was subjected to a silica gel column using hexane:EtOAc (2:1) to obtain fr. C2-1-3-1. The fr. C2-1-3-1 was subjected to HPLC purification using 40% MeCN to obtain compound 3 (63.0 mg) and compound 6 (16.2 mg). The fr. C3 was subjected to a silica gel column using hexane:EtOAc (1:1) to obtain fr. C3-1. The fr. C3-1 was subjected to a silica gel column using CHCl3:MeOH (25:1) to obtain fr. C3-1-1 and C3-1-2. The fr. C3-1-1 was subjected to HPLC purification using 60% MeCN to obtain compound 29 (3.1 mg) and compound 28 (2.0 mg). The fr. C3-1-2 was subjected to HPLC purification using 45% MeCN to obtain compound 27 (5.3 mg).Extraction of the cones of 1–32 were identified as podophyllotoxin (1) [2) [3) [4) [5) [6) [7) [8) [9) [10) [11) [12) [epi-pimara-15-en-18-oic acid (13) [14) [15) [epi-pimara-8,15-dien-19-oic acid (16) [epi-pimara-15-ene-18-oic acid methyl ester (17) [epi-dehydroabietinol (18) [19) [20) [21) [22) [E-communic acid (23) [24) [25) [26) [27) [28) [29) [30) [31) [32) [Compounds oxin (1) , picropo (1) [2) , deoxypo [2) [3) , deoxypi [3) [4) , picropo [4) [5) , (−)-yat [5) [6) , podorhi [6) [7) , podorhi [7) [8) , oplopan [8) [9) , oplodio[9) [10) , phytol 10) [11) , 7α-hydr11) [12) , 7-oxo-ecid (13) ,28, isop13) [14) , macroph14) [15) , 7-oxo-enol 18) , 8,11,1319) [20) , ferrugi20) [21) , (+)-tot21) [22) , E-commu , 8,11,123) [24) , amentof24) [25) , isogink25) [26) , ginkget26) [27) , 7,4′,4′27) [28) , 7,4′,7″28) [29) , hinokif29) [30) , β-sitos30) [31) , and β-s31) [32) , respectL-glutamine and sodium bicarbonate containing 15% fetal bovine serum  and 1% kanamycin. Cells were cultured at 37 °C in humidified 5% CO2/95% air.Two HTLV-1-infected T-cell lines, MT-1 and MT-2, were kindly provided by Dr Isao Miyoshi of Kochi University in Nankoku, Japan. Culture conditions were as previously described . The cel2. After cultivation, 10 μL of 3--dimethyl-2-thiazoyl-2,5-diphenyl-2H-tetrazolium bromide (MTT reagent) solution was added to the microtiter wells. After incubation for 4 h at 37 °C, 100 μL of isopropanol was added to solubilize the MTT-formazan product. The absorbance at 450 nm was measured with a microplate reader.Viability was determined using the MTT assay. The MT-1 and MT-2 cells were maintained in RPMI-1640 medium containing fetal bovine serum (15%). A 50-μL aliquot of the cell suspension (5000 cells per well) and 50 μL of the test sample solution or suspension were plated in flat-bottomed microtiter wells  and incubated for 72 h at 37 °C in a humidified atmosphere containing 5% CO3 and 26 for 72 h were collected by centrifugation (310× g at 4 °C for 10 min), suspended in 100 μL of RPMI 1640 medium, and incubated with 100 μL of Annexin V reagent at room temperature for 20 min. Cells were measured by a Muse Cell Analyzer .Detection of apoptosis was performed using The Muse Annexin V & Dead Cell Assay Kit  according to the manufacturer’s protocol. MT-1 and MT-2 cells incubated in the presence or absence of compound T. occidentalis showed potent inhibitory effects against MT-1 and MT-2 cell lines. We isolated 32 compounds  from the extract and examined their structure–activity relationships. Almost all of the isolated lignans inhibited the growth of both tumor cell lines. Several diterpene compounds had moderate activity, of which some have been previously reported to exhibit cytotoxicity in cancer cells. Moreover, several flavonoids showed moderate activity; some of these biflavones have been reported to show anti-cancer properties in some cancers and may induce cell cycle arrest, and inhibitory activities against amyloid-β peptide 42 cytotoxicity in PC-12 cells [3) and isoginkgetin (26) enhanced apoptosis. Therefore, not only lignans but also diterpenes and/or biflavones may be promising candidates for the treatment of ATL.In conclusion, as part of our investigations of Cupressaceae plants, extracts of 12 cells . We cont


Citrus reticulata Blanco), a by-product of the juice-making industrial process known to contain large amounts of polymethoxyflavones, was exploited for its content in high added value natural coumarins. The study herein afforded a method referring to the evaporation of CPEO volatile fraction under mild conditions (reduced pressure and temperature below 35 °C) as azeotrope with isopropanol. This allowed the isolation of high added value coumarins from the non-volatile fragment using preparative High Performance Liquid Chromatography (HPLC). Pilot-scale application of this procedure afforded for each kg of CPEO processed the following natural bioactive coumarins in chemically pure forms: heraclenol (38–55 mg), 8-gerayloxypsoralen (35–51 mg), auraptene (22–33 mg), and bergamottin (14–19 mg). The structures of coumarins were verified by Nuclear Magnetic Resonance (NMR) spectroscopy and HPLC co-injection with authentic standards. Thus, the low market value mandarin CPEO with current value of 17 to 22 EUR/kg can be valorized through the production of four highly bioactive natural compounds worth 3479 to 5057 EUR/kg, indicating the great potentials of this methodology in the terms of the circular economy.Cold pressed essential oil (CPEO) of mandarin ( Coumarins belong to plant-derived secondary metabolites that structurally are classified into benzopyrone derivatives. They are known as molecules exhibiting a broad spectrum of bioactivities, including anticancer , anti-HIDipteryx (Coumarouna) odorata, Fabaceae/Leguminosae family. It was named from the French term coumarou, which is used for naming the plant. The first synthesis of coumarin was accomplished in 1868, initiating a campaign for its utilization by pharmaceutical industries as a precursor for the synthesis of various synthetic anticoagulants [The molecule of natural coumarin  was firstly isolated in 1820 from the Tonka bean seeds of agulants . Ever siagulants : simple Psoralea corylifolia as described in their sacred book Atharva Veda and by ancient Egyptians from Ammi majus according to their writings [Furanocoumarins comprise the larger family of coumarin derivatives, which are present in higher plants. Their structural skeleton is characterized by a furan ring, attached on carbons C-6 and C-7 (linear type) or C-7 and C-8 (angular type) of the coumarin backbone . The firwritings . Furanocwritings ,32. Undewritings . Therefowritings ,35. Finawritings ,5, anticwritings ,36, antiwritings ,10 activwritings ,38.Citrus crops are included among the richest sources of natural compounds, including coumarins and furanocoumarins [Citruses juice-making process, since it is widely used as a flavoring and fragrance agent by food, beverage, cosmetic, and pharmaceutical industries due to its characteristic aroma profile [Citruses CPEOs, while the non-volatile residue, consisting of sterols, fatty acids, waxes, carotenoids, coumarins, psoralens, and flavonoids [Citruses CPEOs as the source of bioactive psoralens, coumarins, and polymethoxyflavones (PMFs). The respective data revealed the presence of these compounds in the essential oils of oranges, bergamot, lemons, and grapefruits. On the contrary, the literature reports for mandarin (Citrus reticulata) CPEO, a well-known agricultural commodity, reveal only the presence of PMFs. The latter belong to an important class of bioactive molecules that are isolated in large amounts from the peel of various citrus fruits , making economically unprofitable their recovery from the essential oil of mandarin.oumarins . Their goumarins . This enoumarins . The remoumarins  is indicoumarins . To date profile ,45. Its  profile , dependiavonoids , remainsavonoids  collecteThe aim of this study is to exploit, for the first time, the mandarin CPEO as a source for the efficient retrieval of bioactive coumarins and furanocoumarins. It must be noted that the presence of these bioactive molecules in mandarin fruits is well known but there are no reports concerning their presence in the essential oil of mandarin ,49,50. Td-limonene [Coumarins and furanocoumarins (1), 8-geranyloxypsoralen which is also known also as xanthotoxol geranyl ether (2), auraptene (3) and bergamottin (4); Polymethoxyflavones , 5,6,7,4′-tetramethoxyflavone (6), 3,5,6,7,8,3′,4′-heptamethoxyflavone (7), and tangeretin (8).The volatile fraction of mandarin CPEO, consisting mainly of limonene , was remoumarins , eluted s , elutedThe efficacy of the method developed was also tested and verified in pilot scale experiments using a 25 L capacity rotary evaporator for the separation of the non-volatiles fraction of 4 kg of mandarin CPEO and the calculation of the retrieval yields for each compound.1H–NMR spectra revealed the existence of two methyl groups attached on carbon 6′ as singlets at 1.29 and 1.35 ppm, the protons on carbons 4′ and 5′  and the furanocoumarin skeletal protons that resonate in the area 6.00–8.50 ppm. These assignments were based on the respective correlated spectroscopy (COSY) spectrum which verified the connections between vicinal carbons as signals between protons H-3 and H-4, H-2′ and H-3′, H-4′ and H-5. Finally, the existence of carbons 3, 4, 5, 2′, 3′, 4′, 5′, 7′, and 8′ was determined with the heteronuclear multiple-quantum coherence (HMQC) spectrum, while the remaining carbons were located with the aid of heteronuclear multiple bond correlation (HMBC) spectrum.The chemical structures of the retrieved molecules were elucidated by NMR spectroscopy and verified through HPLC co-injection with solutions containing the standard authentic compound. Specifically, for heraclenol, the 1H–NMR spectra of 8-geranyloxypsoralene, the two methyl groups attached on carbon 10′ resonate as singlets at 1.57 and 1.63 ppm, while the methyl group on carbon 6′ appears as a singlet at 1,66 ppm. The double bond protons (H-5′ and H-9′), along with methylene protons on C-4′ that are attached to the oxygen atom were detected at chemical shifts between 5–5.60 ppm, while the protons of furanocoumarin skeleton appear in the area 6–8.20 ppm. On the other hand, the COSY spectrum revealed the connections between protons H-4′ and H-5′, H-7′ and H-8′, H-8′ and H-9′, all indicative of their vicinity and the signals between H-3 and H-4, H-2′ and H-3′ that are characteristic of the furanocoumarin carbon skeleton. The HMQC spectra contributed to the determination of carbons bearing protons and indicated that H-4′, H-5′, H-9′, H-7′, and H-8′ although displaying similar chemical shift values in 1H–NMR, were attached to different carbons. The remaining carbon atoms of the molecule were determined with the HMBC spectrum.In the 1H–NMR spectra as singlets at 1.62, 1.66, and 1.77 ppm respectively, while the methylene protons attached to carbons 4′, 5′ resonate as multiplets at 2.10 and 2.13 ppm. The double bond protons on C-2′ and C-6′ are recorded as triplets at 5.48 and 5.11 ppm, while the methylene protons H-1′ linked to carbon attached to oxygen appear as a broad doublet at 4.66 ppm. Finally, coumarin protons resonate in the area between 6.20–7.80 ppm. The COSY spectrum verified the vicinal interactions between H-1′, H-2′, and H-4′, H-6′ as well as those on coumarin rings as H (6.90 ppm), H (7.51 ppm), and H (6.21 ppm), H (7.80 ppm). The HMQC spectrum was used for the determination of carbons bonded directly with protons in order to reveal the methylene protons and HMBC was used for the designation of the remaining carbons of the molecule.The protons of the three methyl groups of auraptene, attached to carbons 8′, 9′, and 10′ appear in 1H–NMR spectrum revealed three methyl groups, two attached on carbons 10′ (1.60 and 1.66 ppm) and 6′ (1.68 ppm) and methylene protons attached to carbons 7′ and 8′ appear as multiplets at 2.10 and 2.08 ppm. The double bond protons H-5′ and H-9′ attached to double bonds and resonated as triplets at 5.55 and 5.07, respectively, while H-4′ protons on a carbon vicinal to oxygen atom appeared as a doublet at 5.00 ppm. All furanocoumarin protons were detected at 6.20–8.20 ppm. The COSY spectrum is indicative of vicinal interactions between H-5′, H-9′, and H-8′, H-5′, along with those of furanocoumarin carbon skeleton as H (6.25 ppm), H (8.18 ppm), and H (7.13 ppm), H (7.75 ppm). The HMQC spectrum contributed to the determination of carbons bonded directly with protons and especially the designation of methylene protons, while HMBC was used for the designation of the remaining carbons of the molecule.For the bergamottin molecule, the Heraclenol (1):1H–NMR  δ: 8.27 , 7.77 , 7.26 , 7.14 , 6.30 , 4.66 , 3.30 , 1.35 , 1.29 .13C–NMR  δ: 161.6 (C-2), 158.6 (C-7), 153.3 (C-9), 149.4 (C-8), 146.4 (C-3′), 139.9 (C-4), 115.0 (C-6), 113.5 (C-3), 108.1 (C-10), 105.5 (C-2′), 94.9 (C-5), 73.4 (C-4′), 61.6 (C-5′), 58.5 (C-6′), 24.2 (C-7′), 19.0 (C-8′).8-Geranyloxypsoralen (2): 1H–NMR  δ: 7.95 , 7.84 , 7.55 , 6.96 , 6.35 , 5.55 , 5.00 , 5.00 , 2.04 , 2.04 , 1.66 , 1.63 , 1.57 . 13C–NMR  δ: 161.0 (C-2), 149.1 (C-7), 147.7 (C-2′), 145.0 (C-4), 144.4 (C-9), 143.0 (C-6′), 132.3 (C-10′), 131.4 (C-8), 126.6 (C-6), 124.4 (C-9′), 120.2 (C-5′), 117.4 (C-10), 114.9 (C-3), 114.7 (C-5), 107.5 (C-3′), 70.4 (C-4′), 34.8 (C-7′), 26.9 (C-8′), 25.4 (C-12′), 17.4 (C-11′), 16.4 (C-13′).Auraptene (3): 1H–NMR  δ: 7.80 , 7.51 , 6.90 , 6.89 , 6.21 , 5.48 , 5.11 , 4.66 , 2.13 , 2.10 , 1.77 , 1.66 , 1.62 . 13C–NMR  δ: 163.1 (C-7), 161.6 (C-2), 156.9 (C-9), 144.2 (C-4), 143.0 (C-3′), 132.7 (C-7′), 129.5 (C-5), 124.2 (C-6′), 119.4 (C-2′), 113.8 (C-10), 113.1 (C-6), 112.9 (C-3), 101.9 (C-8), 65.8 (C-1′), 39.6 (C-4′), 26.4 (C-5′), 25.2 (C-9′), 17.2 (C-8′), 16.3 (C-10′).Bergamottin (4): 1H–NMR  δ: 8.18 , 7.75 , 7.20 , 7.13 , 6.25 , 5.55 , 5.07 , 5.00 , 2.10 , 2.08 , 1.68 , 1.66 , 1.60 . 13C–NMR  δ: 161.4 (C-2), 158.6 (C-7), 153.7 (C-9), 150.1 (C-5), 146.2 (C-3′),143.9 (C-6′), 140.7 (C-4), 133.2 (C-10′), 125.0 (C-9′), 120.4 (C-5′), 115.5 (C-6), 113.8 (C-3), 106.1 (C-2′), 108.6 (C-10), 94.7 (C-8), 70.8 (C-4′), 40.3 (C-7′), 27.2 (C-8′), 25.8 (C-11′), 17.9 (C-12′), 17.0 (C-13′).Accordingly, the respective NMR data for the isolated of PMFs shown in Nobiletin (5): 1H–NMR  δ: 7.62 , 7.49 , 7.09 , 6,62 , 4.05 , 4.02 , 3.93 , 3.91 , 3.91 , 3.85 . 13C–NMR  δ: 177.0 (C-4), 161.6 (C-2), 152.9 (C-6), 152.2 (C-7), 150.2 (C-3′), 148.7 (C-8), 147.5 (C-9), 144.6 (C-4′), 138.9 (C-5), 124.7 (C-1′), 119.5 (C-6′), 115.4 (C-10), 111.8 (C-5′), 109.1 (C-2′), 106.4 (C-3), 61.6 (5-OMe), 61.6 (8-OMe), 61.4 (6-OMe), 61.2 (7-OMe), 55.6 (3′-OMe), 55.6 (4′-OMe).5, 6, 7, 4′-Tetramethoxyflavone (6): 1H–NMR  δ: 7.95 , 7.95 , 7.08 , 7.08 , 7.03 , 6,54 , 3.97 , 3.89  3.88 , 3.85 . 13C NMR  δ: 176.9 (C-4), 162.8 (C-4′), 161.7 (C-2), 158.6 (C-7), 155.2 (C-9), 152.8 (C-5), 144.4 (C-5′), 144.4 (C-3′), 140.9 (C-6),127.7 (C-6′), 127.7 (C-2′), 124.4 (C-1′), 113.1 (C-10), 106.4 (C-3), 96.9 (C-8), 61.5 (5-OMe), 61.5 (6-OMe), 56.1 (7-OMe), 55.2 (4′-OMe).3, 5, 6, 7, 8, 3′,4′-Heptamethoxyflavone (7): 1H–NMR  δ: 7.95  7.53 , 6.89 , 3.84  3.65 , 3.76 , 3.69 , 3.67 , 3.67 , 3.63 . 13C–NMR  δ: 172.3 (C-4), 151.5 (C-2), 151.4 (C-7), 151.3 (C-3′), 149.9 (C-4′), 147.9 (C-9), 147.8 (C-3), 143.8 (C-6), 140.6 (C-8), 137.9 (C-5), 123.5 (C-1′), 121.7 (C-6′), 114.4 (C-10), 111.5 (C-5′), 111.1 (C-2′), 61.5 (5-OMe), 61.2 (6-OMe), 61.1 (7-OMe), 59.0 (8-OMe), 55.3 (3′-OMe), 53.5 (4′-OMe).Tangeretin (8): 1H–NMR  δ: 7.95 , 7.95 , 7.13 , 7.09 , 6,56 , 4.08 , 4.01 , 3.91 , 3.89 , 3.85 . 13C–NMR  δ: 177.1 (C-4), 163.0 (C-4′), 161.8 (C-2), 152.3 (C-7), 148.7 (C-8), 147.7 (C-9), 144.8 (C-6), 139.2 (C-5), 128.3 (C-2′), 128.3 (C-6′), 124.5 (C-1′), 115.9 (C-3′), 115.9 (C-5′), 115.6 (C-10), 107.2 (C-3), 62.3 (5-OMe), 62.2 (8-OMe), 61.9 (6-OMe), 61.6 (7-OMe), 55.9 (4′-OMe).citrus fruits, a byproduct of the juice-making process, has been known for a longtime [Citrus reticulata) remains somehow less studied. For this byproduct, only the presence of PMFs has been reported until today [5), 5, 6, 7, 4′-tetramethoxyflavone (6), 3, 5, 6, 7, 8, 3′, 4′-heptamethoxyflavone (7) and tangeretin (8)—the following natural coumarins: heraclenol (1), 8-geranyloxypsoralen (2), auraptene (3), and bergamottin (4). The utilization of preparative HPLC allowed their retrieval as pure compounds (purity >95% for each isolated molecule) in the form of amorphous colorless solids. Their retrieval yields from 1 kg of mandarin CPEO were depended on the fruit variety and the industrial procedure and determined as: heraclenol (1), 38–55 mg; 8-geranyloxypsoralen (2), 35–51 mg; auraptene (3), 22–33 mg; bergamottin (4), 14–19 mg, nobiletin (5), 4–11 mg; 5,6,7,4′-tetramethoxyflavone (6), 8,5–18 mg; 3,5,6,7,8,3′4′-heptamethoxyflavone (7), 51–103 mg; tangeretin (8), 39–80 mg. It must be noted that the retrieval yields of PMFs are comparable to those of the literature, while for natural coumarins this study constitutes the first literature report concerning their presence in mandarin CPEO [The retrieval of natural products from agro-industrial byproducts constitutes one of the cornerstones of the circular economy. The application of different isolation techniques allows the retrieval of a large variety of bioactive molecules from complex mixtures. Although the extraction and isolation of coumarins from CPEOs of longtime ,47,48, tAngelica spp., Citrus spp., and Heracleum spp. [With respect to the economic aspects of this study, because PMFs isolation has already been studied in detail previously , our inteum spp. , is knoweum spp. ; it alsoeum spp.  and exhieum spp. . Recentleum spp. , highligeum spp. , which iStaphylococcus epidermidis. This molecule also possesses intense antifungal properties against Candida kruzei and Candida kefyr [Bursaphelenchus xylophilus [The molecule 8-geranyloxypsoralen, also known as xanthotoxol geranyl ether, is a linear furanocoumarin displaying potent antimicrobial activity against da kefyr  and nemalophilus . Additiolophilus . In 2012lophilus .Porphyromonas gingivalis adherence to oral epithelial cells [Auraptene possesses intense activities such as antidiabetic , antiproal cells . In addial cells .Salmonella typhimurium microsome test [Citrus aurantifolia hexane extract pointed out bergamottin as one of the most active constituents [Bergamottin exhibits various bioactivities, such as intense anticancer activity that is attributed to its anti-proliferation, anti-invasion, and anti-migration properties ,69,70,71ome test . Finallytituents .www.ChemFaces.com, accessed on 30 March 2021) for 1 mg of each natural compound are: EUR 54 for heraclenol, EUR 25 for 8-geranyloxypsoralen, EUR 20 for auraptene, and EUR 8 for bergamottin. Thus, in respect the obtained yields for each natural compound, it is estimated that the economic outcome of their retrieval from 1 kg of mandarin CPEO ranges from EUR 2052 to 2970 for heraclenol, EUR 875 to 1275 for 8-geranyloxypsoralen, EUR 440 to 660 for auraptene, and EUR 112 to 152 for bergamottin. Consequently, the overall financial result of these four compounds retrieval from each kg of mandarin CPEO ranges between EUR 3479 to 5057, revealing the economic importance and viability of the method, considering that the current market value of 1 kg of mandarin CPEO is between EUR 17 to 22 per 1 kg [The market values of the retrieved natural compounds are considerably high, reflecting their bioactivities and respective demand by pharmaceutical industries. The lowest market prices identified on the internet . The CPEOs of mandarin used in this study were derived from the industrial juice-making process of Analytical grade isopropanol and methanol, which were used as solvents and HPLC grade water, methanol, and formic acid, used for the HPLC analyses, were obtained from Fisher  Chemicals. Deuterated acetonitrile used for NMR spectroscopy was provided by Sigma-Aldrich . All chemical standards used for the verification of natural products structures were purchased from Sigma-Aldrich .The non-volatile fraction of mandarin CPEO was separated through the exclusive evaporation of the volatile fraction. This was achieved under mild conditions (low temperature and reduced pressure), developed in terms of this endeavor. More specifically, 100 g of CPEO was placed into a Büchi Rotary Evaporator . and 100 mL of isopropanol was added. The alcohol forms an azeotropic mixture with D-limonene , the pre2O with 0.1% formic acid (solvent B) in accordance with the following gradient program: 0.0–5.0 min, A 60%, 5.0–25.0 min A 60%→80%, 25.0–30.0 min A 80%, 30.0–45.0 min A 80→90%, 45.0–50.0 min A 90%, 50.0–65.0 min A 90→100%, 65.0–75.0 min A 100%, 75.1–80.0 min A 60%. The outcome detection was achieved with a diode array detector of the same company  set at 280 nm. The retrieved natural coumarin and furanocoumarins were collected in respect to the peaks determined for the following retention times: heraclenol (1), 40.7 min; nobiletin (5), 50.2 min; 5, 6, 7, 4′-tetramethoxyflavone (6), 50.7 min; 3, 5, 6, 7, 8, 3′, 4′-heptamethoxyflavone (7), 51.9 min; tangeretin (8), 56.3 min; 8-geranyloxypsoralene (2), 68.1 min; auraptene (3), 72.9 min; bergamottin (4), 76.5 min. Then, the solvent of each fraction was evaporated under reduced pressure to afford the corresponding natural product as a colorless amorphous solid which was then stored in the freezer.The non-volatile fraction was dissolved in methanol at a concentration of approximately 50 mg/mL and the mixture was introduced via manual injection into an HPLC system , with an injection volume of 200 μL and flow rate of 1.2 mL/min. A reverse-phase column Kromasil C18, 5 μm, 250 × 10 mm  was used, with a respective guard column of the same material and company, all operated at room temperature. The mobile phase consisted of MeOH (solvent A) and H2O with 0.1% formic acid (solvent B), in accordance with the following gradient program: 0.0−17.5 min A 80→100%, 17.6−20.0 min A 80%. The injected volume was 20 μL, with a flow rate of 1.0 mL/min and a column temperature of 30 °C. The detector was set to monitor at two wavelengths, 280 and 254 nm.The above HPLC system was also used, in analytical mode, for the identification of natural compounds and the verification of their purity. Specifically, the respective samples obtained from preparative HPLC were injected into a Nucleosil 100-5 C18, 5 μm, 4.6 × 250 mm column obtained from Macherey-Nagel  and a guard column of the same material and company. The mobile phase consisted of methanol (solvent A) and HJ in Hz) were recorded on a Bruker Avance III 600 (600 MHz) NMR spectrometer  using tetramethylsilane  as the internal reference δ = 0.00). The determination of chemical structures of the retrieved molecules was performed using data obtained from the following experiments: 1H–NMR, 13C–NMR, and the two-dimensional NMR experiments COSY (correlation spectroscopy), HMQC (heteronuclear multiple quantum coherence), and HMBC (heteronuclear multiple bond coherence).Nuclear magnetic resonance (NMR) spectra (δ in ppm, The exploitation of mandarin CPEO, a scarcely investigated industrial byproduct, indicated its potential as a rich source of bioactive natural coumarins and furanocoumarins. The method developed herein refers to the evaporation, under mild conditions, of the volatile fraction and subsequent retrieval, using preparative HPLC, of the following natural coumarins and furanocoumarins in pure form: heraclenol, 8-geranyloxypsoralen, auraptene, and bergamottin. These molecules are known to possess significant bioactivities, displaying high demand by pharmaceutical companies. Thus, 1 kg of mandarin CPEO with a current market value ranging between EUR 17 to 22 is capable of producing high added value natural compounds worth EUR 3479 to 5057, indicating the great potential of this methodology for valorization in terms of the circular economy.