Their structures and relative configurations were identified through spectral analysis and possible formation mechanisms were also discussed. Among these oligomers, trimers 6 and 7 were obtained for the first time through direct transformation from resveratrol. Results indicated that this reaction is suitable for the preparation of resveratrol oligomers with a complex structure.Biotransformation of  Stilbenes are a class of plant polyphenols that can be divided into two categories, namely, monomeric and oligomeric stilbenes. Resveratrol oligomers possess novel structures and exhibit various biological activities, such as anticarcinogenesis, anti-inflammation, and tyrosinase activity inhibition. These oligomers can be used to treat cancer, AIDS, bacterial infections, and other diseases. Some oligostilbenes exhibit more potent bioactivities than their monomers do ,2,3.ε-viniferin is a promising way to prepare these oligomers. Wilkens et al. −  in its HR-ESI-MS (42H34O10 and indicated that 3 could be a resveratrol trimer. The IR spectrum (max = 3395 and 3187 cm−1) and aromatic rings . Absorption bands were also observed at λmax (log ε) 203 (5.14), 231 (4.66), and 283 (3.92) nm in the UV spectrum a), 7.24 a), 6.54 b), 6.34 b); 6.68 c), and 6.85 c); one set of AB2 system for ring A2 at δH of 6.36  and 6.20 a); two sets of meta-coupled aromatic protons for rings B2 and C2 at δH of 5.98  and 6.19 , and δH 5.17  and 6.24 . The 1H-NMR spectrum also displayed the presence of two mutually coupled benzyl methine protons at δH of 5.33  and 4.83  and a sequence of successively coupled benzyl methine protons at δH of 4.30 , 3.76 , 4.02 , and 2.96 . Moreover, the HSQC spectrum  caused by the anisotropic effect of the aromatic ring suggested that 3 could be a 7c-empimer of davidiol B  and upfield shift of H-8b [2.81 ] in 3 compared with those of davidiol B further substantiated this observation. Therefore, the structure of 3 was determined as shown in The stereochemistry of spectrum b. The in4 was obtained as a light brown amorphous powder. Its corresponding negative HR-ESI-MS  showed a molecular formula of C42H31O9, which implied that 4 could be a resveratrol trimer. The UV (λmax = 203.4 (4.99), 228 (4.69), and 281 (4.22) nm) and IR  spectra  and 3.85 . These signals are characteristic of a 2,3-diaryldihydrobenzofuran moiety, except a cis-1,2-disubstituted olefinic bond at δH 6.21  and 6.05 . Our comparison revealed that the NMR spectroscopic data of 4 were remarkably similar to those of cis-diptoindonesin B reported in the literature +.1  and 2  in acetone (240 mL), 50 mL of water was added under stirring at room temperature. After that, a solution of HRP (10.0 mg) in water (30 mL) was added slowly. The reactant was stirred for 5 min, 30% H2O2 (1.4 mL) was then added. The reactant was stirred for another 7 h at room temperature. Finally, the reaction mixture was suspended in water, and extracted with ethyl acetate. The organic layer was dried on anhydrous Na2SO4 for 24 h, and concentrated in vacuo to yield a residue.To a mixed solution of 3–MeOH  to yield 8  and R-VI-2~R-VI-4. R-VI-3 was then separated by semi-preparative Rp-HPLC  eluted using methanol/water  to afford 9 ; 30 mg R-I  was separated by semi-preparative Rp-HPLC using a mobile phase of methanol/water  to afford 10  and 11 . R-II (30 mg) was further purified by semi-preparative Rp-HPLC eluted with a mobile phase of MeOH–H2O  to afford 5 (1.7 mg). 300 mg R-III  was resolved by Rp-MPLC with a gradient of increasing MeOH (18~30%) in water to give R-III-1~R-III-3. Among them, Fraction R-III-2 (34 mg) were subsequently separated by Rp-HPLC with a mobile phase of methanol/water  to afford 6  and 3 ; R-III-1 and R-III-3 were dealt with the same manner to provide 13 , and 12 , respectively. Furthermore, R-VII (600 mg) was subjected to Rp-MPLC eluting with methanol–water  to yield R-VII-1 and R-VII-2; subsequently, R-VII-1 was purified by semi-preparative Rp-HPLC using a mobile phase of methanol–water  to provide 7 (2 mg); R-VII-2 was dealt with methanol–water  by semi-preparative Rp-HPLC in the same manner to give 4 (6 mg).The residue was subjected to column chromatography over ODS, eluting with a gradient of increasing methanol in water (15~100%) to provide nine fractions (R-I~R-IX) on the basis of HPLC analysis. R-VI (2.5 g) was further fractionated via silica gel column chromatography (CC), eluting with CHClCompound3: Brown amorphous powder. UV (MeOH) λmax (log ε): 203 (5.14), 231.2 (4.66), 283 (3.92) nm; IR (film) νmax: 3395, 3187, 2921, 2850, 1646, 1513, 1469, 1420, 1342, 1245, 1120, 1005, 834, 722, 648 cm−1; 1H-NMR  and 13C-NMR , see m/z: 721.0 [M + Na]+, 736.9 [M + K]+; (−)-ESI m/z: 696.9 [M − H]−, 732.9 [M + Cl]−; HR-ESI-MS m/z: 697.2076, [M − H]− .Compound4: Light brown amorphous powder. UV (MeOH) λmax (log ε): 203.4 (4.99), 228 (4.69), 281 (4.22) nm; IR (film) νmax: 3335, 2924, 2852, 1604, 1516, 1487, 1449, 1349, 1241, 1169, 1004, 836, 694 cm−1; 1H-NMR  and 13C-NMR , see m/z: 703.3 [M + Na]+; (−)-ESI m/z: 678.9 [M − H]−, 715.8 [M + Cl]−; HR-ESI-MS m/z: 679.1973, [M − H]− .Compound5: Brown amorphous powder. UV (MeOH) λmax (log ε): 203.4 (4.73), 229.8 (4.29), 283 (3.70) nm; IR (film) νmax: 3337, 2921, 2852, 1615, 1517, 1488, 1464, 1351, 1235, 1198, 1157, 1105, 1000, 836, 778, 746, 691 cm−1; 1H-NMR  and 13C-NMR , see m/z: 337.1073 [M + H]+ .Davidiol B (6): Brown amorphous powder. 1H-NMR , δ: 7.00 a), 6.87 a), 6.68–6.65 , 6.53 b), 6.45 c), 6.38 , 6.35 c), 6.31 , 6.16 , 6.13 b), 5.99 , 5.91 a), 5.05 , 4.10 , 3.95 , 3.11 , 2.90 , 2.81 . 13C-NMR , δ: 161.74s (C-11b), 159.94s (C-11(13)a), 159.83s (C-13b), 158.71s (C-13c), 157.90s (C-4a), 157.48s (C-4c), 155.84s (C-4b), 154.98s (11c), 150.15s (C-9c), 148.81s (C-9a), 148.01s (C-9b), 136.55s (C-1b), 136.18s (C-1c), 134.35s (C-1a), 129.72d (C-2(6)b), 128.66d (C-2(6)c), 127.57d (C-2(6)a), 122.69s (C-10c), 118.50s (C-10b), 116.41d (C-3(5)c), 116.02d (C-3(5)a), 115.18d (C-3(5)b), 107.41d (C-14c), 106.90d (C-10(14)a, 104.52d (C-14b), 102.11d (C-12c), 101.70d (C-12a), 63.97d (C-8c), 54.57d (C-8b), 54.46d (C-8a), 53.24d (C-7b). (+)-ESI m/z: 721.0 [M + Na]+, 736.9 [M + K]+; (−)-ESI m/z: 696.9 [M − H]−, 732.9 [M + Cl]−.cis-Diptoindonesin B (7): Brown amorphous powder. 1H-NMR , δ: 7.09 , 6.99 b), 6.92 a), 6.75 a), 6.68 b), 6.63 , 6.60 , 6.37 , 6.31 , 6.25 , 6.22 c), 6.19 , 6.12 , 6.10 , 5.85 a), 5.15 , 5.14 , 4.29 , 3.49 . 13C-NMR , δ: 162.61s (C-11b), 160.55s (C-4c), 160.25s (C-13b), 159.86s (C-11(13)a), 159.12s (C-11(13)c), 158.81s (C-4b), 158.41s (C-4a), 147.58s (C-9a), 140.93s (C-9c), 140.29s (C-9b), 133.87s (C-1a), 131.92s (C-1b), 131.73s (C-1c), 131.12s (C-3c), 130.32d (C-6c), 129.53d (C-8c), 128.94d (C-2(6)b), 128.17d (C-2(6)a), 127.23d (C-2c), 122.03s (C-10b), 116.36d (C-3(5)b), 116.28d (C-3(5)a), 109.68d (C-5c), 108.53d (C-10(14)c), 108.13d (C-14b), 107.09d (C-10(14)a), 102.56d (C-12c), 102.18d (C-12a), 96.83d (C-12b), 94.99d (C-7b), 94.55d (C-7a), 56.84d (C-8a), 55.52d (C-8b). (+)-ESI-MS m/z: 681.0 [M + H]+, 703.0 [M + Na]+; (−)-ESI m/z: 678.9 [M − H]−, 714.9 [M + Cl]−.Ampelopsin B (13): Brown amorphous powder. 1H-NMR , δ: 7.02 b), 6.89 a), 6.69 b), 6.60 a), 6.30 , 6.27 , 6.09 , 6.01 , 5.65 , 5.14 , 4.05 , 3.53 , 3.17 . (+)-ESI m/z: 455.0 [M + H]+, 477.0 [M + Na]+; (−)-ESI m/z: 453.0 [M − H]−, 489.0 [M + Cl]−.1 and 2 produced various resveratrol stilbene oligomers, including dimers, trimers, and tetramers. In this reaction mixture, four resveratrol trimers , one new resveratrol derivative (5) with a dihydrofuran skeleton, and six dimers (8–13) were isolated and identified. Among these compounds, 3 and 4 were newly identified in our study. The raceme nature of the dimers was indicated by the zero values of their optical rotations, and this finding suggested that a radical mechanism was involved in HRP-catalyzed biotransformation. Our study favored the enzymatic biotransformation of stilbenes by HRP as a prominent method to produce oligomeric stilbenes for research activity. Considering that these new compounds may occur naturally as minor constituents, we observed that our reference data provided a basis for the detection of the presence of these stilbene oligomers in future investigations. Oligostilbenes were reported to show various activities [The HRP-catalyzed biotransformation of


Stable Schiff bases containing a furoxan moiety are synthesized as single regioisomers by the reaction of 3-methyl-2-oxy-furazan-4-carbaldehydewith various amino compounds at room temperature. The structures of synthesized compounds were fully characterized by multinuclear NMR spectroscopy and X-ray crystallography. The effect of synthesized Schiff bases containing a furoxan moiety on biological generation of reactive oxygen species and nitric oxide in plant tissues was investigated for the first time by fluorescence microscopy and the released NO identified as nitrite with Griess reagent. There is a good correlation between the biological generation of NO determined by fluorescence microscopy and with Griess reagent. Some of the synthesized compounds exhibited both nitric oxide and reactive oxygen species generation abilities and represent potential NO donors in plant tissues. S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) and so-called NONOates (spermidine or diethylamine-NONOate) are among the most used nitric oxide donors -for C11H11N4O4S, calcd. 295.0501, found 295.0518.White crystals, yield 86% (5.1 g), mp 163-165°C (from MeOH:H-1): 3199, 2979, 2936, 1608, 1467, 1377, 1330, 1301, 1162, 1098, 1029. 1H NMR ): 2.04 , 2.28 , 2.60 , 7.10 , 7.94 , 12.53 . 13C NMR ): 8.7 (CH3-3), 20.4 (CH3-12), 22.5 (CH3-10/14), 111.1 (C-3), 131.7 (CH-11/13), 132.4 (C-9), 133.3 (CH-6), 139.3 (C-10/14), 142.9 (C-12), 153.3 (C-4). 15N NMR ): 177.5 (NH-8), 338.5 (N-7), 357.6 (N-2), 371.6 (N-5). X-Ray:C13H16N4O4S, (M = 324.36 g/mol): monoclinic, space group P21/n (no. 14), a = 7.8827(6) Å, b = 18.4644(14) Å, c = 10.6000(8) Å, β = 110.634(9), V = 1443.9(2) Å3, Z = 4, T = 200.00(10) K, μ(MoKα) = 0.241 mm-1, Dcalc = 1.469 g/cm3, 5588 reflections measured (4.412 ≤ 2Θ ≤ 50.046), 2552 unique  which were used in all calculations. The final R1 was 0.0556 (I ≥ 2σ(I)) and wR2 was 0.1466 .CCDC – 1556711. HRMS-ESI (m/z): [M-H]- for C13H15N4O4S, calcd.323.0814, found 323.0846.Pale yellow solid, yield 74% (4.8 g), mp 177-179°C (from MeOH). IR : 3422, 3220, 3032, 1638, 1610, 1570, 1490, 1460, 1381, 1331, 1310, 1283, 1186, 1147, 1036. 1H NMR ): 2.40 , 7.38 , 7.85 , 8.49 , 12.33 . 13C NMR ): 9.1 (CH3-3), 21.1 (CH3-13), 111.7 (C-3), 127.8 (CH-11/15), 129.2 (CH-12/14), 129.6 (C-10), 135.8 (CH-6), 142.6 (C-13), 154.1 (C-4), 163.2 (CO-9). 15N NMR ): 174.0 (NH-8). HRMS-ESI (m/z): [M-H]- for C12H11N4O3, calcd. 259.0831, found 259.0851.White solid, yield 71% (3.7 g), mp 212-214°C (from MeOH), IR , IR : 3324, 3128, 2983, 1609, 1518, 1491, 1462, 1383, 1306, 1251, 1169, 1110, 1033. 1H NMR ): 2.44 , 7.24 , 7.40 , 7.59 , 8.24 , 9.87 , 12.32 . 13C NMR ): 9.2 (CH3-3), 111.6 (C-3), 125.1 (CH-12/16), 125.6 (CH-14), 128.3 (CH-13/15), 131.4 (CH-6), 138.7 (C-11), 153.9 (C-4), 176.5 (CS-9). 15N NMR ): 129.1 (NH-10), 177.4 (NH-8), 334.1 (N-7), 357.6 (N-2), 372.3 (N-5). HRMS-ESI (m/z): [M-H]- for C11H10N5O2S, calcd. 276.0555, found 276.0537.Beige solid, yield 69% (3.83 g), mp 184-186°C (from CHCl2O), IR : 3412, 3350, 3127, 2966, 1612, 1540, 1517, 1458, 1379, 1304, 1259, 1205, 1170, 1120, 1024. 1H NMR ): 2.31 , 2.43 , 7.19 , 7.45 , 8.23 , 9.78 , 12.26 . 13C NMR ): 9.2 (CH3-3), 20.5 (CH3-14), 111.6 (C-3), 125.1 (CH-12/16), 128.7 (CH-13/15), 131.2 (CH-6), 134.9 (C-14), 136.1 (C-11), 153.9 (C-4), 176.5 (CS-9). 15N NMR ): 128.8 (NH-10), 177.4 (NH-8), 334.4 (N-7), 357.6 (N-2), 372.6 (N-5). HRMS-ESI (m/z): [M-H]- for C12H12N5O2S, calcd. 290.0712, found 290.0722.Yellow solid, yield 63% (3.67 g), mp 188-190°C  have been seeded in sterilized soil and cultivated for six weeks in a special growth room, at 21-23°C, 70% humidity, light intensity 150 μmol/m2 and a photoperiode of 14/10. Each synthesized compound  dissolved in ethanol was mixed with 0.25 g Tween 20 and demineralized water to prepare 50 mL of each test suspension/solution. The inductor suspensions were kept in spraying glass bottle, in the dark, at room temperature. The Arabidopsis leaves were sprayed with the inductor suspensions (at a rate of 1 mL/plant) andcollected after 24 hours. The leaves were washed with distilled water for histochemical analysis of ROS and NO by fluorescence microscopy, or worked-up according to the reported protocol [In our experiments we used rasystem . Arabidoprotocol , in orde2DCFA) as fluorescent indicator. The collected Arabidopsis leaves were washed with distilled water and incubated with 2.5 μMH2DCFA solution (10 mMin DMSO), for 30 min, in the dark, at room temperature. Then the leave fragments were washed twice with distilled water and the H2DCFA – mediated fluorescence was detected (emission/excitation: 488/525 nm). As negative controls, Arabidopsis leaves untreated with inductor suspensions have been used. Intracellular NO was visualized using 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA)as fluorescent indicator. The collected Arabidopsis leaves were washed with distilled water and incubated with 10 μM DAF-FM diacetate (5mM in DMSO),for 15 min, in the dark, at room temperature. Then the leave fragments were washed twice with phosphate buffer saline (PBS) at pH 7.4 and the fluorescence of the reaction product of DAF-FM DA with NO was captured (emission/excitation: 488/525 nm). As negative controls, Arabidopsis leaves untreated with inductor suspensions have been used.Intracellular ROS was visualized using 2’,7’-dichlorodihydrofluorescein diacetate  formation outside of the plant cells, on tissue level. The 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA) is a cell permeable dye. This dye is converted by cytosolic esterase to DAF-FM, which produces the benzotriazole fluorescent derivative inside the cell. The generation of ROS is a biological effect in plants, mainly due to the released NO, which are redox gasotransmitters. The reactive species formed in plants are NO and, most probably, on physiological conditions, peroxynitrite (due to NO reaction with ROS).The NO specific dye, 4-amino-5-methylamino-2’,7’-difluorofluorescein (DAF-FM), reacts with NN-(1-naphthyl)ethylenediamine dihydrochloride 0.1% (w/v) solution were added to 50 μl Arabidopsis leaves extract supernatant. The leaves, treated with the same amounts of synthesized Schiff bases suspensions, collected after 24 hours, and controls washed with distilled water, were powdered with nitrogen liquid into a mortar. 100 mg of leaves powder was extracted for 30 min in 300 μl of 100 mM phosphate buffer, pH 7.4. The extract was centrifuged for 15 min at 10,000 x g and 4°C. The resulted supernatant was used for indirect NO determination with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride solutions, after incubation for 5-10 min at room temperature protected from light. The color appeared immediately as the Griess reagent is formed. The absorbance was directly measured in a plate reader with a filter between 520 nm and 550 nm. A nitrite standard curve was used to calculate the nitrite concentration in the samples and expressed as μM of nitrite anion. The detection of nitrite concentration in Aradidopsis thaliana leaves was performed according to the reported bioprotocol [50 μl of sulfanilamide 1% (w/v) solution in 5% (v/v) phosphoric acid and 50 μl of protocol .1), already described in literature [1 with phenylsulfonyl hydrazide derivatives 2a,b the corresponding phenylsulfonylhydrazones containing a furoxan moiety 3a,b were obtained . NMR data clearly indicated the presence of only one regioisomer bearing the external oxygen atom on the nitrogen in position 2 of the furoxan ring. The 2- versus5- N-oxidation of the furoxan ring in all compounds  is supported by similar chemical shifts for C3 and C4 in the 13C-NMR spectra. Assigning the site of N-oxidation in various natural or biological active compounds is important both for structural and mechanistic purposes related to metabolisation of these compounds. We have also previously investigated the influence of N-oxidation on 15N- and 13C-NMR spectra for series of octahydroacridines [N-oxidation to C-alpha (C3 in furoxan derivatives) and C-beta (C4 in furoxan derivatives) is consistent with our previous studies [N-oxidation induces a significant shielding of the C-alpha and a slight deshielding of the C-beta in the 13C-NMR spectra.The structures of all Schiff bases containing the furoxan moiety were assigned on the basis of chemical and spectral analysis  and NO generation in plant tissues. Bio-molecules are rapidly damaged by reactive oxygen species produced under a pathogen attack or in abiotic stress conditions. It is known that both ROS and NO together are required to induce the activation of defense-related enzymes in plants [Furoxan derivatives have been of considerable interest to chemists for years but they received relatively little attention from biologists despite their NO-releasing capacities. It is for the first time when Schiff bases containing a furoxan moiety were used as NO donors in plant experiments. We investigated the effect of synthesized compounds on ROS  [Arabidopsis leaves treated with all Schiff bases having a furoxan moiety, at both concentrations, especially at higher concentration of compounds (50 μg/mL). Efficacy of Schiff bases bearing a furoxan moiety  on ROS generation pursues the series: 7a>3b≥7b>5>3a.ROS induction was detected on (H2DCFA) . FluoresArabidopsis leaves infiltrated with suspension of each synthesized compound at the same concentrations (10 μg/mL and 50 μg/mL respectively) in the presence of a specific and sensitive fluorescence indicator, 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA) [3b>7b>5>7a>3a.NO donor properties of the synthesized Schiff bases bearing a furoxan moiety were determined on F-FM DA) –60 and tArabidopsis leaves we used Griess reagent for indirect determination of NO through its oxidized nitrite form [Arabidopsis leaves treated with the same amounts of synthesized Schiff bases bearing a furoxan moiety were incubated at room temperature for 5-10 min. with Griess reagent, protected from light, and the absorbance was immediately measured in a plate reader with a filter between 520 nm and 550 nm. A nitrite standard curve was used to calculate the nitrite concentration in the samples and expressed as μM of nitrite anion Click here for additional data file.S2 File(DOC)Click here for additional data file.


Mesua beccariana has led to the isolation of two new pyranoxanthones, mesuarianone (1) and mesuasinone (2), two anthraquinones, 4-methoxy-1,3,5-trihydroxyanthraquinone (3) and 2,5-dihydroxy-1,3,4-trimethoxyanthraquinone (4), one coumarin, mammea A/AB (5) and three common triterpenes, stigmasterol (6), friedelin (7) and betulinic acid (8). Structural elucidations of these compounds were achieved using 1D and 2D NMR and MS techniques. This is the first report on the phytochemistry of Mesua beccariana.Our recent study on the stem bark extracts of  Mesua is a flowering genus consisting of about 48 species indigenous to south tropical Asia -6”-(2-methyl-2-pentenyl)-6”-methylpyrano-xanthone and named as mesuarianone. The NMR data of this compound are summarized in Hence, the structure of compound 2, mesuasinone (28H30O5 (EIMS m/z 446), as confirmed by HREISIMS 446.2041  had UV and IR spectral data suggestive of a xanthone derivative. The UV spectrum gave maximum absorptions at 208 (3.82), 254 (3.96), 274.0 (3.93), 334 (3.53) nm, while the IR spectrum showed absorptions at 3229 (OH), 2925 (CH3), 2865 (CH2), 1640 (C=O), 1576 and 1496 (C=C aromatic) cm-1.Compound uasinone , which w13C-NMR signals showed the presence of a carbonyl carbon (δ 180.7) along with the presence of twelve quaternary carbons , seven methines , three methylenes  and five methyls . The 13C and DEPT NMR spectra of compound 2 showed some similarity to that of 1, suggesting 2 also has a pyranoxanthone structure. The 1H-NMR spectra also supported the existence of a pyrano ring. Two vinylic protons were observed at δ 6.83  and 5.58  along with two methyl signals at δ 1.45  and 1.66 . The methyl protons H-13 (δ 1.66) demonstrated 3J correlations with δ 27.1 (C-14) and H-14 (δ 1.45) showed 2J correlations with δ 80.7 (C-12) and 3J correlations with δ 126.1 (C-11) in the HMBC experiment. This pyrano ring was connected to the xanthone skeleton at C-3 and C-4 as evidenced by the 3J HMBC correlations between the vinylic proton at δ 6.83 (H-10) and δ 149.2 (C-4a) and δ 158.9 (C-3), while the other vinylic proton at δ 5.58 (H-11) correlates to δ 100.3 (C-4) via another 3J HMBC correlation.Moreover, the 1H NMR spectra also gave a sharp singlet at δ 13.02 indicating a chelated hydroxyl group, while the broad singlet at δ 5.73 was due to the free hydroxyl group. In the HMBC spectra, the chelated hydroxyl proton (δ 13.02) showed cross-peaks with three quaternary carbon signals at δ 103.0 , 111.8  and 160.6 . The deshielded carbon signal at 160.6 (C-1) was found to be attached to the hydroxyl group.The J = 8.3 Hz), 7.77  and 7.29 . The lowest-field aromatic proton (δ 7.77) was assigned to H-8 due to the anisotropic effect of the carbonyl group. This was supported by the 3J and 2J HMBC correlations of H-8 to a carbonyl signal at δ 180.7 (C-9) and two aromatic carbons C-8a (δ 121.2) and C-7 (δ 120.1), while its ortho-coupled proton H-7 correlates to δ 123.9 (C-5) and 121.2 (C-8a) as shown in s) was confirmed to be at C-5 via its HMBC correlations to the carbon signals at δ 120.1  and 144.1 .Three aromatic protons which are arranged in the ortho and meta positions were revealed by the resonances at δ 7.22 , 5.24 , 1.84 , 2.10 , 5.09 , 1.80 , 1.68 , 1.57 . This was further confirmed using the long-range correlation system observed in the HMBC spectra as illustrated in 2J) and 158.9  allowing its placement at C-2.The 2 was elucidated to be 1,6-dihydroxy-2--6”,6”-dimethylpyrano--xanthone and named mesuasinone. The NMR data of this compound are summarized in Hence, the structure of compound 4-Methoxy-1,3,5-trihydroxyanthraquinone (3) , 2,5-dih3 as solvent and tetramethylsilane (TMS) as internal standard. Ultraviolet spectra were recorded in EtOH on a Shimadzu UV-160A, UV-Visible Recording Spectrophotometer.Infrared spectra were measured using the universal attenuated total reflection (UATR) technique on a Perkin-Elmer 100 Series FT-IR spectrometer. EIMS were recorded on a Shimadzu GCMS-QP5050A spectrometer. NMR spectra were obtained using a Unity JEOL 400 MHz FTNMR spectrometer using CDClMesua beccariana was collected from the Sri Aman district in Sarawak, Malaysia. The plant material was identified by Associate Professor Dr Rusea Go, Biology Department, Faculty of Science, Universiti Putra Malaysia.The stem bark of n-hexane, dichloromethane, ethyl acetate and methanol. The extracts were dried under reduced pressure using a rotary evaporator to yield hexane (15.6 g), dichloromethane (21.2 g), ethyl acetate (15.8 g) and methanol (80.5 g) extracts. Each of these extracts was chromatographed over a silica gel column using a stepwise gradient system . The column chromatography of the hexane extract gave mesuarianone , mesuasinone  and friedelin . Meanwhile, the dichloromethane extract gave stigmasterol  and betulinic acid  while the ethyl acetate extract gave two anthraquinones and a coumarin identifed as 4-methoxy-1,3,5-trihydroxyanthraquinone , 2,5-dihydroxy-1,3,4-trimethoxy-anthraquinone  and mammea A/AB . The air-dried and powdered sample was extracted successively with Mesuarianone (1). Yellow solid. m.p. 166-167 °C. 3) +22.5. UV max (cm-1): 3392, 2971, 2923, 1639, 1574, 1475. For 1H- and 13C- NMR spectra, see m/z (rel. int.): 460 [M+] (39), 445 (100), 377 (89), 361 (10), 347 (11), 323 (12), 203 (8), 181 (61), 91 (6), 77 (5), 69 (27), 55 (9). Mesuasinone (2). Yellow solid. m.p. 118-119 °C. UV max (cm-1): 3229, 2925, 2865, 1640, 1576, 1496. For 1H- and 13C-NMR spectra, see m/z (rel. int.): 446 [M+] (14), 431 (4), 391 (8), 363 (100), 307 (21), 154 (12), 69 (10). 4-Methoxy-1,3,5-trihydroxyanthraquinone (3). Orange solid. UV max (cm-1): 3420, 2920, 2860, 1720, 1630, 1470. MS m/z (rel. int.): 286 [M+] (100), 268 (87), 257 (10), 243 (38), 212 (27), 180 (30). The 1H- and 13C-NMR spectral data are consistent with published data [286 [M+] 0, 268 . Orange solid. UV max (cm-1): 3400, 2920, 2840, 1660, 1630,1540. MS m/z (rel. int.): 330 [M+] (100), 315 (60), 312 (5), 297 (20), 287 (22), 272 (24), 227 (20), 58 (23). The 1H- and 13C-NMR spectral data are consistent with published data [Mammea A/AB (5). Colourless solid. UV m/z (rel. int.): 406 [M+] (2), 392 (18), 377 (10), 349 (100), 293 (10). The 1H- and 13C-NMR (CDCl3) spectral data are consistent with the published data  (60), 394 (7), 369 (7), 351 (15), 300 (26), 271 (38), 255 (40), 159 (41), 145 (42), 133 (45), 123 (31), 105 (42), 95 (44), 83 (77), 69 (62), 55 (100). The 1H- and 13C- NMR (CDCl3) spectral data are consistent with literature  (12), 411 (6), 302 (11), 273 (16), 246 (17), 231 (16), 218 (19), 205 (20), 191 (18), 179 (19), 163 (27), 149 (18), 137 (24), 123 (60), 109 (64), 95 (76), 81 (63), 69 (100), 55 (75), 41 (44). The 1H- and 13C-NMR spectral data are consistent with literature  (7), 438 (4), 248 (40), 207 (48), 189 (100), 69 (17). The 1H- and 13C-NMR spectral data are consistent with published data [-292 °C) . IR νmax100), 69 . The 1H-1) and mesuasinone (2), along with two anthraquinones, a coumarin, and three triterpenes were isolated from the stem bark of Mesua beccariana. Future work on the biological activities such as anti-cancer and anti-oxidant properties will be carried out in due course.Two new pyranoxanthones, mesuarianone (


Two known analogues, pseurotin A (3) and FD-838 (4), as well as four previously reported helvolic acid derivatives, 16-O-propionyl-16-O-deacetylhelvolic acid (5), 6-O-propionyl-6-O-deacetylhelvolic acid (6), helvolic acid (7), and 1,2-dihydrohelvolic acid (8) were also identified. One-dimensional (1D), two-dimensional (2D) NMR, HRMS, and circular dichroism spectral analysis characterized the structures of the isolated compounds.Two new spiro-heterocyclic  Aspergillus fumigatus strains, such as E-β-trans-5,8,11-trihydroxybergamot-9-ene and β-trans-2β,5,15-trihydroxybergamot-10-ene +   and 196.4 (C-17), one amide carbonyl at δC 166.4 (C-6), a mono-substituted benzene ring , two sp2 quaternary carbons at δC 188.5 (C-2) and 109.6 (C-3), four sp3 methylenes , two sp3 oxygenated methines (δC 74.9/δH 4.38 and δC 67.0/δH 4.50), two sp3 oxygenated quaternary carbons at δC 91.2 (C-5) and δC 92.5 (C-8), and three methyls  (1H and 13C NMR data for compound 1 (3) +   and 196.4 (C-17), one amide carbonyl at δC 166.4 (C-6), a mono-substituted benzene ring , two sp2 methines , four sp2 quaternary carbons at δC 172.2 (C-2), 111.6 (C-3), 141.8 (C-10), and 163.4 (C-13), one sp3 methylene (δC 21.0/δH 2.76), one sp3 oxygenated methylene (δC 50.2/δH 4.25), one sp3 oxygenated methine (δC 75.0/δH 4.50), two sp3 oxygenated quaternary carbons (δC 92.1 and δC 92.6), and two methyls (δC 11.8/δH 1.22 and δC 51.7/δH 3.26) (1H and 13C NMR data for compound 2 (4) +; (+)-HRESIMS m/z 440.1684 [M + Na]+ ; 1H and 13C NMR data: See Pale yellow amorphous powder; [α]m/z 428.0 [M + H]+; (+)-HRESIMS m/z 450.1159 [M + Na]+ ; 1H and 13C NMR data: See Pale yellow amorphous powder; [α]S. aureus ATCC 6538, MRSA ATCC 29213, E. coli ATCC 11775, P. aeruginosa ATCC 15692, and C. albicans ATCC 10231 based on a 96 well microplate format in liquid growth. Briefly, the bacteria from glycerol stocks was inoculated on LB agar plate and cultured overnight at 37°C. The glycerol stock of C. albicans was prepared on Sabouraud dextrose agar at 28 °C for 24 h. Afterwards, single colonies were picked and adjusted to approximately 104 CFU/mL with Mueller–Hinton Broth as bacterial suspension and with RPMI 1640 media as fungal suspension. 2 μL of two-fold serial dilution of each compound (in DMSO) were added to each row on 96-well microplate, containing 78 μL of bacterial or fungal suspension in each well. . The 96-well plate was aerobically incubated at 37 °C for 16 h. The 96-well plate of antifungal was aerobically incubated at 35 °C for 24 h. Here, MIC is defined as the minimum concentration of compound at which no bacterial growth is observed.The antimicrobial assays were performed according to the Antimicrobial Susceptibility Testing Standards that were outlined by the Clinical and Laboratory Standards Institute (CLSI) against The anti-BCG assay was carried out by using a constitutive GFP expression strain (pUV3583c-GFP), according to previous published procedure  . The con1 and 2) were isolated and characterized along with the previously reported analogues pseurotin A (3) and FD-838 (4), as well as four known helvolic acid derivatives, namely 16-O-propionyl-16-O-deacetylhelvolic acid (5), 6-O-propionyl-6-O-deacetylhelvolic acid (6), helvolic acid (7), and 1,2-dihydrohelvolic acid (8). All of the structures were confirmed by detailed analysis of the spectroscopic data. Compounds 5–7 showed antibacterial activity against both S. aureus and MRSA. Aanalogue 8 did not exhibit antibacterial activities that indicated that the α,β-unsaturated ketone of 5–7 is the key functional group for antibacterial activity. Structurally, cephalimysins M (1) and N (2) belong to a family of rare natural products with diverse biological activities, which contain an unusual spiro-heterocyclic γ-lactam core. To the best of our knowledge, 28 natural products of this family have been reported, including pseurotin A [O-demethylpseurotin A [2 is the first cephalimysin analogue where the methyl of C-16 was oxidized to hydroxymethyl. The current research diversifies the structures of this class of natural products. As part of our ongoing research program to discover novel secondary metabolites from the marine environment, eight compounds were isolated from the rice solid medium culture of the marine derived fungus CUGBMF170049 isolated from a sediment sample that was collected from the Bohai Sea, China. Two novel compounds (urotin A , 8-O-demurotin A  pseurotiurotin A ,21, pseuurotin A , pseuroturotin A , 14-norpurotin A  synerazourotin A , azaspirurotin A , azaspirurotin A , and FD-urotin A ,28,29. 2


Salvia miltiorrhiza Bunge, as the non-medicinal parts, are always discarded during harvesting, resulting in a huge waste of resources and environmental pressure. Due to the high flavonoid content and their antioxidant activities characteristics, the aerial parts of S. miltiorrhiza can be developed into natural antioxidants and used in foods. A high-speed counter-current chromatography (HSCCC) method, using a two-phase solvent system composed of tert-butyl methyl ether/n-butanol/acetonitrile/water , was the first to successfully isolate five flavonoids from the aerial parts of S. miltiorrhiza in one attempt, and separately categorized as rutin (1), isoquercitrin (2), kaempferol-3-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside (3), kaempferol-3-O-β-d-glucopyranoside (4) and apigenin-7-O-β-d-glucopyranoside (5) after identification. The purities of these plant isolates were 97.3%, 99.5%, 92.8%, 98.1% and 98.7%, respectively. All the flavonoids were identified by HR-ESI-MS, 1D and 2D NMR. Compounds 3 and 5 were firstly isolated from the plant of S. miltiorrhiza. Results from antioxidant assays showed that rutin (1) and isoquercitrin (2) had higher antioxidant capacities compared to L-ascorbic acid as the positive control.The aerial parts of  Salvia miltiorrhiza Bunge (SM), designated as Danshen in China or Tanshen in Japan, are widely distributed in both China and Japan −. 1H-NMR  δ: 12.59 , 7.56 , 7.53 , 6.84 , 6.38 , 6.19 , 5.34 , 4.38 , 0.99 . 13C-NMR  δ: 177.3 (C-4), 164.1 (C-7), 161.2 (C-5), 156.6 (C-9), 156.4 (C-2), 148.4 (C-4′), 144.8 (C-3′), 133.3 (C-3), 121.6 (C-6′), 121.2 (C-1′), 116.3 (C-5′), 115.2 (C-2′), 103.9 (C-10), 101.2 (C-1″), 100.7 (C-1′′′), 98.7 (C-6), 93.6 (C-8), 76.5 (C-3″), 75.9 (C-5″), 74.1 (C-2″), 71.9 (C-4′′′), 70.6 (C-3′′′), 70.4 (C-4″), 70.0 (C-2′′′), 68.2 (C-5′′′), 66.9 (C-6″), 17.7 (C-6′′′). Thus, the structure of 1 was defined as rutin by comparison of its MS, 1H-and 13C-NMR data with literature data −. 1H-NMR  δ: 12.64 , 10.78 , 9.29 , 7.58 , 6.84 , 6.40 , 6.20 , 5.46 . 13C-NMR  δ: 177.4 (C-4), 164.2 (C-7), 161.2 (C-5), 156.3 (C-2), 156.2 (C-9), 148.5 (C-4′), 144.8 (C-3′), 133.2 (C-3), 121.6 (C-6′), 121.2 (C-1′), 116.2 (C-5′), 115.2 (C-2′), 104.0 (C-10), 100.9 (C-1″), 98.7 (C-6), 93.5 (C-8), 77.6 (C-5″), 76.5 (C-3″), 74.1 (C-2″), 69.9 (C-4″), 61.0 (C-6″). Thus, the structure of 2 was defined as isoquercitrin by comparison of its MS, 1H-and 13C-NMR data with literature data −. 1H-NMR  δ: 7.98 , 6.87 , 6.39 , 6.18 , 5.30 , 4.37 , 0.98 . 13C-NMR  δ: 177.2 (C-4), 161.1 (C-5), 160.0 (C-4′), 156.7 (C-9), 156.5 (C-2), 133.1 (C-3), 130.8 , 120.8 (C-1′), 115.1 , 103.7 (C-10), 101.5 (C-1″), 100.8 (C-1′′′), 99.0 (C-6), 93.9 (C-8), 76.4 (C-3″), 75.7 (C-5″), 74.1 (C-2″), 71.8 (C-4′′′), 70.6 (C-3′′′), 70.3 (C-2′′′), 69.9 (C-4″), 68.2 (C-5′′′), 66.8 (C-6″), 17.7 (C-6′′′). Thus, the structure of 3 was defined as kaempferol-3-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside by comparison of its MS, 1H- and 13C-NMR data with literature data −. 1H-NMR  δ: 12.62 , 8.05 , 6.88 , 6.43 , 6.20 , 5.47, 3.75-3.08 . 13C-NMR  δ: 177.5 (C-4), 164.4 (C-7), 161.2 (C-5), 160.0 (C-4′), 156.4 (C-9), 156.2 (C-2), 133.2 (C-3), 130.9 , 124.0 (C-1′), 115.2 , 104.0 (C-10), 101.6 (C-1″), 99.1 (C-6), 98.8 (C-8), 77.5 (C-3″), 76.6 (C-5″), 74.2 (C-2″), 70.2 (C-4″), 61.1 (C-6″). Thus, the structure of 4 was defined as kaempferol-3-O-β-d-glucopyranoside by comparison of its MS, 1H- and 13C-NMR data with literature data −. 1H-NMR δ: 13.00 , 7.95 , 6.95 , 6.87 , 6.84 , 6.44, 5.06 , 3.72~3.19 . 13C-NMR  δ: 182.0 (C-4), 164.4 (C-2), 163.0 (C-7), 161.8 (C-5), 161.1 (C-4′), 157.0 (C-9), 128.6 , 120.7 (C-1′), 116.1 , 105.3 (C-10), 103.0 (C-3), 99.9 (C-6), 99.5 (C-1′′), 94.8 (C-8), 77.2 (C-3′′), 76.5(C-5′′), 73.1 (C-2′′), 69.6 (C-4′′), 60.6 (C-6′′). Thus, the structure of 5 was defined as apigenin-7-O-β-d-glucopyranoside by comparison of its MS, 1H- and 13C-NMR data with literature data [Compound ure data .50 value is a significant standard for antioxidant activity. The smaller the IC50 value, the better the antioxidant activity. As shown in 1) > isoquercitrin (2) > crude flavonoid extract > kaempferol-3-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside (3) > kaempferol-3-O-β-d-glucopyranoside (4) > apigenin-7-O-β-d-glucopyranoside (5), with IC50 values ranging from 16.7 to 177.1 µg/mL.ICtert-butyl methyl ether, n-butyl alcohol and petroleum ether (60–90 °C) used for the preparation of crude extract and counter-current chromatography (CCC) separations were analytical grade . HPLC-grade methanol and acetonitrile were purchased from the Fisher Company . The water used was deionized by an osmosis Milli-Q system . Reverse osmosis Milli-Q water  was used.The ethanol, ethyl acetate, methanol, chloroform, The aerial parts of SM were obtained from a Chinese traditional medicine plantation in Tai′ an  and identified by Dr. Jia Li . A voucher specimen (2018090805) was deposited at Shandong Analysis and Test Center.The HSCCC equipment was TBE-300C  with three multilayer coil separation columns of 300 mL (diameter of the PTFE tube as 2.6 mm) as well as a 20 mL manual sample loop. The HSCCC apparatus was equipped with four other instrument modules, including a TBP-5002 constant-flow pump , a 8823A-UV Monitor at 254 nm , a Model 3057 portable recorder , and a DC-0506 low constant temperature bath  to maintain the temperature at 25 °C. HPLC separation was performed on a 1120 LC system  consisting of a quaternary pump, an online degasser, a diode array detector, an auto plate-sampler and a thermostatically controlled column compartment.n-butanol (3.0 L).The dried aerial parts of SM (1.0 kg) were extracted with 80% ethanol (10.0 L) at room temperature. After being concentrated in vacuo, the ethanol extract (0.15 kg) was suspended in water and extracted with ethyl acetate (3.0 L) and n-butanol soluble fraction (32.3 g) was first separated over a macroporous adsorbent resin column. The column was eluted with water-ethanol , yielding five fractions. Then, the 50% ethanol eluate (17.4 g) was ready to be separated by HSCCC.The K-values). The K-values of the target compounds in the crude flavonoid extract from the aerial parts of SM were determined by HPLC. Five milliliters of each phase of the equilibrated two-phase solvent system were added to approximately 10 mg of the crude flavonoid extract and were shaken vigorously for 1 min. After the phases had fully separated, 1 mL of each layer was removed and dried in a stream of nitrogen. The residues were dissolved in 1 mL of methanol and analyzed by HPLC. The K-values of the target compounds were calculated according to the equation K = AU/AL, where AU and AL were the peak areas of target compound in the upper and lower phases, respectively.The selection of the solvent system is based on the partition coefficient , was placed into a separating funnel. After shaking vigorously, the solution was allowed to stand for several minutes and was separated into two phases for the experiment. The upper phase was the stationary phase, while the lower was the mobile phase. For this HSCCC separation, 200 mg of crude flavonoid extract was dissolved in 20 mL isometric upper and lower phase.For HSCCC separation, a two-phase solvent system, consisting of Firstly, in order to select the optimal flow rate, the retention volume of the upper phase was measured at different flow rates. After the CCC column was filled with the upper phase, the rotation speed was set to 800 rpm, and then the lower phase was used at the flow rate of 2, 4, 6, 8, 10, 12, 14 mL/min successively to achieve equilibrium. The eluent was drained in a measuring cylinder. When the volume of the outflowing upper phase no longer changed, it was regarded that the equilibrium had been achieved.The whole separation procedure had two stages: collection and extrusion. In the first stage, the six-way valve was in the position for collection. This stage encompassed establishment of hydrodynamic equilibrium, sample solution loading, and sample eluent collection. The CCC column was first completely filled with the upper phase at 30 mL/min in head-to-tail elution mode. The lower phase was pumped into the head of the CCC column at 2.0 mL/min, during which the apparatus was rotated at 800 rpm in a clockwise manner. The separation temperature was set at 25 °C. The effluents were continuously monitored at 254 nm by means of a portable recorder. After equilibrium had been reached, the crude flavonoid extract solution was injected using the manual sample loop. After the first target peak was collected, the flow rate was changed to 10 mL/min, while keeping the rotation speed unchanged, and elution was continued. Once the 2nd, 3rd, and 4th target peaks could be collected successively, the flow was stopped, immediately followed by the turning off the rotation speed. The lower phase was used to extrude the solution from the column at a flow rate of 10 mL/min.v/v) with a flowrate of 1.0 mL/min and a wavelength of 254 nm.HPLC analyses of the extract and CCC fractions were performed on Agilent 1120 HPLC equipment with a C18 column . The mobile phase was acetonitrile and 0.1% aqueous solution of formic acid . NMR spectra were performed on a Bruker AV-400 spectrometer  with DMSO-6d as solvent and chemical shifts (δ) being expressed in parts per million (ppm) coupled with the constant (J) in Hz.The separated compounds were identified by ESI-MS, DPPH (12.5 mg) was dissolved in 250 mL ethanol to make a standard solution at a concentration of 0.05 mg/mL. Serial dilution was used to provide standard solutions at concentrations of 0, 5, 10, 15, 20 and 25 μg/mL in ethanol. The absorbance values of the six solutions were measured by ultraviolet spectrophotometry, at 517 nm, to prepare the standard curve.tert-butyl methyl ether/n-butanol/acetonitrile/water , was successfully established to isolate flavonoids from aerial parts of SM. Five flavonoids were obtained in one step, and their purities were 97.3%, 92.8%, 99.5%, 98.7% and 98.1%, respectively. Compounds 3 and 5 were firstly isolated from the plant of S. miltiorrhiza. There was no obvious difference in adding excess water in the two-phase solvent system, which could save a lot of reagents. Antioxidant assays results showed that rutin (1) and isoquercitrin (2) have stronger antioxidant capacities as compared to l-ascorbic acid as the positive control.In conclusion, a new HSCCC method, using a two-phase solvent system composed of


Synthesized compounds  ESI-HRMS m/z: 365.0985 .5′-uridine (2): [α]20D = −82.60° ; 1H-NMR  δ: 4.22–4.27 , 4.52 , 4.68 , 5.56 , 5.83 , 6.32 , 6.37 , 7.64 , 7.70 . 13C-NMR (CD3OD) δ: 64.55 (C-5′), 71.17 (C-3′), 75.26 (C-2′), 82.87 (C-4′), 92.04 (C-1′), 102.82 (C-5), 103.71 (C-3′′), 105.23 (C-1′′), 109.40 (C-5′′), 132.68 (C-6′′), 142.28 (C-6), 152.17 (C-2), 165.20 (C-4′′), 166.11 , 170.87 (C=O). ESI-HRMS m/z: 381.0934 .5′-(5′′-Amino-2′′-hydroxybenzoyl)uridine (3): 1H-NMR  δ: 4.19–4.28 , 4.51 , 4.71 , 5.58 , 5.82 , 6.77 , 6.99 , 7.25 , 7.61 . 13C-NMR (CD3OD) δ: 64.96 (C-5′), 71.159 (C-3′), 75.03 (C-2′), 82.76 (C-4′), 92.27 (C-1′), 102.92 (C-5), 113.16 (C-1′′), 116.29 (C-6′′), 119.04 (C-3′′), 129.29 (C-4′′), 140.34 (C-5′′), 142.44 (C-6), 152.14 (C-2), 155.91 (C-2′′), 169.32 (C-4), 170.84 (C=O). ESI-HRMS m/z: 380.1099 .3,2′,3′-O-tris-(benzyloxycarbonyl)uridine  in DMF (15 mL) 2′-benzyloxy-2-bromoacetophenone  and Cs2CO3  were added. The mixture was stirred at room temperature for 2 h. Then it was diluted with toluene and filtrated to remove solid Cs2CO3. The filtrate was neutralized with Dowex 50WX4 and concentrated. Crude product was purified by column chromatography with a hexane-AcOEt 50:1→5:1 solvent system to yield 14 as light yellow foamy solid . For final deprotection 14 was dissolved in a MeOH/THF 4:1 solvent system (20 mL), 20% Pd(OH)2/C (0.236 g) and cyclohexene (3.0 mL) were added and the mixture was heated under reflux for 50 min. Then the mixture was cooled, the catalyst was removed by filtration and the filtrate was concentrated. Crude product was purified by column chromatography with CHCl3:MeOH 100:1→30:1 solvent system to yield 5′-((2-(2-hydroxyphenyl)-2-oxoethyl)uridine (4) as a cream foamy solid . [α]20D: −3.3° ; 1H-NMR  δ: 3.70 , 3.78 , 4.01 , 4.07 , 4.14 , 4.89–4.93 , 5.20 , 5.42 , 5.62 , 5.84 , 6.95 , 7.00 , 7.50 , 7.78 , 8.06 , 11.20 , 11.30 . 13C-NMR (DMSO) δ: 70.85 (C-3′), 70.86 (C-5′), 73.86 (C-2′), 75.60 (C-6′), 83.61 (C-4′), 88.21 (C-1′), 102.27 (C-5), 117.92 (C-3′′), 119.71 (C-5′′), 120.72 (C-1′′), 130.10 (C-6′′), 135.96 (C-4′′), 141.55 (C-6), 151.25 (C-2), 159.85 (C-2′′), 163.56 (C-4), 199.62 (C=O). ESI-HRMS m/z: 401.0848 .To a solution of N2)6-dansyl as glycosyl acceptor [2, 2.0 mg/mL BSA, 200 µM β-GlcNAc-O-(CH2)6-dansyl, 40 µM UDP-Gal and potential inhibitors 1–4 at a range of concentrations from 0 mM (control) to 2.0 mM. The enzymatic reactions were started by the addition of 0.2 mU β4GalT I and incubated at 30 °C for 14 min. Inactivation was quickly done by immersion of the reaction solutions for 2 min in a boiling water bath. The solutions were diluted with water (200 µL) and centrifuged for 10 min, and the supernatant was injected into RP-HPLC system. The percentage of inhibition was evaluated from the fluorescence intensity of the peaks referring to product 6-dansyl). Detailed results of the enzymatic assays are given in the β4GalT I activity was assayed using UDP-Gal as glycosyl donor and β-GlcNAc-O-. As a positive and negative controls the cultures without addition of tested compounds and medium without tested microorganism were used, respectively. The plates were incubated at 35 °C for 24–48 h. The tests were read visually  and/or by measure the absorbance of the samples at 530 nm (Candida). The tests were repeated three times for each strain.Examined compounds 1–4, in silico target screening using PharmMapper Server a reverse pharmacophore mapping approach was performed using an in-house pharmacophore database (PharmTargetDB) [1–4 were found using AM1 quantum semiempirical method. The default mapping parameters were used; maximum generated conformation: 300.To identify potential target candidates of compound argetDB) . Uploade1–4 were designed as donor substrate analogues of GTs. The β-ketoenol motif was expected to imitate the diphosphate bridge in UDP at the active site of GTs by chelating the divalent metal ion. No activity against β4GalT suggests that the β-ketoenol motif connected with uridine is not sufficient to ensure binding at the active site. Anyway the lack of activity against β4GalT opens the possibility of selectivity when other GTs would be tested.Synthesized new derivatives of uridine


Gerbera anandria (Compositae) was extracted with 75% ethanol and the residue was fractionated using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by column chromatography employing solvents of different polarity. Column chromatography of the light petroleum fraction resulted in the isolation of methyl hexadecanoate, while the chloroform fraction afforded xanthotoxin, 2-hydroxy-6-methylbenzoic acid, 7-hydroxy-1(3H)-isobenzofuranone, a mixture of β-sitosterol and stigmasterol, and 8-methoxysmyrindiol and the ethyl acetate fraction gave gerberinside, apigenin-7-O-β-d-glucopyranoside and quercetin. A new coumarin, 8-methoxysmyrindiol, was found. The chemical structures of the isolated compounds were established by MS and NMR . Free radical scavenging and cytotoxic activities of crude extracts and 8-methoxysmyrindiol were further investigated. The ethyl acetate phase exerted the strongest DPPH free radical scavenging activity in comparison to the other fractions. The coumarin 8-methoxysmyrindiol demonstrated cytotoxicity against multiple human cancer cell lines, with the highest potency in HepG2 cells. Gerbera anandria is a plant from the Compositae family, a perennial herb of the Gerbera genus, a well-known traditional Chinese medicinal herb and widely grown in China. It is used in Chinese folk medicine to treat coughs, sore throats, as an effective anti-inflammatory, for detoxification, and expelling wind, and is mainly used for rheumatic pains, arthralgia, bruises, sprains, and dysentery. A review of the literature indicates that only a few studies have been conducted on Gerbera anandria. Coumarins have been isolated and detected, such as 4-hydroxy-5-methylcoumarin − ; 1H-NMR , δ (ppm): 0.88 , 1.26 , 1.62 , 2.30 , 3.66 . 13C-NMR (CDCl3), δ (ppm): 174.30 (C-1), 51.42 (C-1'), 34.09 (C-2'), 31.92 (C-14), 29.69 , 29.59 (C-12), 29.45 (C-13), 29.36 (C-6), 29.25 (C-5), 29.14 (C-4), 24.94 (C-3), 22.68 (C-15), 14.11 (C-16). Compound 1 was identified as methyl hexadecanoate from these spectral data and physical properties +, 239.4 [M+Na]+, 455.4 [2M+Na]+. 1H-NMR , δ (ppm): 7.76  , 7.69 , 7.35 , 6.82 , 6.37 , 4.30 . 13C-NMR (CDCl3), δ (ppm): 160.43 (C-2), 147.70 (C-7), 146.63 (C-2'), 144.30 (C-4), 143.02 (C-9), 132.82 (C-8), 126.12 (C-6), 116.50 (C-10), 114.77 (C-3), 112.89 (C-5), 106.72 (C-3'), 61.33 (OCH3). Compound 2 was identified as xanthotoxin from these spectral data and physical properties −. 1H-NMR , δ (ppm): 11.06  ,7.34 , 6.87 , 6.77 , 2.63 . 13C-NMR (CDCl3), δ (ppm): 174.66 (COOH), 163.35 (C-2), 142.44 (C-6), 134.58 (C-4), 123.50 (C-5), 115.89 (C-3), 114.67 (C-1), 23.51 (CH3). Compound 3 was identified as 2-hydroxy-6-methylbenzoic acid from these spectral data and physical properties −. 1H-NMR , δ (ppm): 7.76 , 7.57 , 6.98 , 6.94 , 5.33 , 13C-NMR (CDCl3), δ (ppm): 172.59 (C-1), 156.63 (C-7a), 146.72 (C-7), 136.97 (C-5), 115.32 (C-4), 113.37 (C-6), 110.93 (C-3a), 70.62 (C-3). The structure of was supported by the HMBC correlations of 7-OH(δ7.76) with C-6,C-7; H-5,(δ7.57 ) with C-3a,C-7; H-4,(δ6.98) with C-3, C-7a, C-6; H-6 (δ6.94 ) with C-7, C-4, C-7a, C-1; H2-3 (δ5.33) with C-3a, C-1, C-5, C-7a, C-4, C-6, C-7. Compound 4 was identified as 7-hydroxy-1(3H)-isobenzofuranone from these spectral data and physical properties −, 383.0 [M+Cl]  −, 400.0 [M+HCOOH]−. 1H-NMR , δ (ppm): 7.47 , 7.19  , 7.14 , 5.98 , 5.23 , 3.91 , 3.72 , 3.60 , 3.55 , 3.50 , 3.43 , 2.77 , 13C-NMR (CDCl3), δ (ppm): 168.99 (C-4), 164.84 (C-2), 156.12 (C-9), 138.99 (C-5), 133.14 (C-7), 129.15 (C-6), 116.05 (C-8),115.41 (C-10), 101.29 (C-1'), 94.11 (C-3),78.66 (C-3'), 78.31 (C-5'), 74.59 (C-2') , 70.88 (C-4'), 62.21 (C-6') , 23.81 (5-CH3). The structure of was supported by the HMBC correlations of H-1'(δ5.23) with C-4, C-2',C-5 and H-6'(δ3.72) with C-4', C-5'. Compound 7 was identified as gerberinside [erinside .Quercetin (8). C15H10O7, yellow needle-shaped crystals, with the same Rf values of quercetin using three different TLC developing systems.Apigenin-7-O-β-D-glucopyranoside (9). C21H20O10, yellow needle-shaped crystals 1H-NMR , δ (ppm): 12.98 , 10.41 , 7.95 , 6.94 , 6.88 ,6.81 , 6.46 , 5.08 . 13C-NMR (DMSO-d6), δ (ppm): 182.15 (C-4), 164.38 (C-2), 163.08 (C-7), 161.51 (C-4'), 161.25 (C-5), 157.08 (C-9), 128.89 (C-6'), 128.62 (C-2'), 121.15 (C-1'), 116.19 (C-3'), 116.19 (C-5'), 105.46 (C-10), 103.19 (C-3), 99.64 (C-6), 99.64 (C-1''), 92.9 (C-8), 77.30 (C-3''), 76.57 (C-5''), 73.21 (C-6''), 69.64 (C-4''), 60.50 (C-7''). Compound 9 was identified as apigenin-7-O-β-d-glucopyranoside from these spectral data and physical properties [−1 2,2-diphenyl-1-picrylhydrazyl (DPPH). The mixtures were vigorously shaken and allowed to stand in the dark for 15 min at room temperature. Absorbance was measured at 515 nm against methanol as blank. The inhibition rate of each extract against DPPH radical was calculated according to the following formula:Stock solutions of the crude methanol extract and fractions  were prepared by dissolving 10 mg in 1 mL methanol. Serial dilution was done from each stock solutions and 1mL from each dilution was tested individually by adding to 2 mL of 0.05 mg·mLiA: The absorbance of adding the sample solutions; 0A: The absorbance of adding 1mL methanol instead of the sample solutions; jA: The absorbance of adding 2 mL methanol instead of the DPPH solutions.Y as ordinate and the mass concentration m as abscissa. The free radical scavenging activity, expressed as IC50 (μg·mL−1), was compared with standard antioxidants such as ascorbic acid.The curve was drawn with the clearance 2 at 37 °C.HeLa, HepG2, HT-1080, HCT116, A375-S2, MCF-7, A549, HL60 cells were purchased from American Type Culture Collection  and maintained in RPMI-1640 (2% glutamine) supplemented with 10% foetal bovine serum (FBS), Cells were grown in a humidified atmosphere of 5% CO4/mL ) were seeded in a 96-well plate  after trypsinization [−1 MTT. During incubation for further 4 h, MTT is reduced by mitochondrial dehydrogenases of viable cells to a purple formazan product. The medium was discarded and 150 μL DMSO was added. The plates were shaken for 10 min at room temperature, and the spectrophotometric absorbance was monitored at 492 nm using Tecan-ELISA plate reader.Sensitivity of the cells to drugs was determined in triplicate using the MTT cell viability assay ,20. Exponization , 100 μL 5/mL)were seeded in a 96-well plate, 50 μL Each hole (Corning). After incubation for 24 h the cells were incubated with 50 μL fresh medium containing various concentrations of compounds at 37 °C for 48 h. The medium was removed and cells were incubated with 10 μL fresh medium containing 5 mg·mL−1 MTT. During incubation for further 4 h, MTT is reduced by mitochondrial dehydrogenases of viable cells to a purple formazan product. The medium was discarded and formazan crystal dissolved in 100 μL triple solution . After incubation for further 12 h at 37 °C, the spectrophotometric absorbance was monitored at 492 nm using Tecan-ELISA plate reader.Exponentially growing cell lines  was calculated according to the following formula:0A: The absorbance of Negative control; iA: The absorbance of adding the sample solutions.Gerbera anandria. The chemical structures of the isolated compounds were established by MS and NMR . Free radical scavenging and cytotoxic activities of crude extracts and 8-methoxysmyrindiol were further investigated. The ethyl acetate phase exerted the strongest DPPH free radical scavenging activity in comparison to other fractions, this may be due to the fact that the majority of the flavonoids are normally present in the ethyl acetate phase. The coumarin 8-methoxysmyrindiol demonstrated cytotoxicity against multiple human cancer cell lines, with the highest potency in HepG2 cells.A new coumarin 8-methoxysmyrindiol and eight other compounds were isolated from the 75% aqueous ethanol extract of the whole plants of


The structures of the new compounds were elucidated primarily by 1D and 2D NMR spectroscopy, as well as by the HR-ESI-MS spectrometry. All compounds were isolated from the Linaceae family for the first time. The in vitro cytotoxic activity of compounds 1, 3–5, 8–18 was evaluated against the Hela, HepG2 and K562 cell lines. Among them, compounds 3, 9, 11, 12, 13, 14, 15, 17, 18 showed moderate inhibitory activities.Seven new diterpenes, named ovoideal A ( Tirpitzia ovoidea Chun et How ex Sha (Linaceae family) is distributed in Chongzuo, Jingxi, Longzhou, Mashan in Guangxi of China, while Vietnam has some distribution too + 315.1985 .ent-14α-Hydroxy-7α,8α-epoxypimara-9(11),15-diene-3,12-dione : Colorless crystals (MeOH); UV, λmax (MeOH) 238 nm; 1H-NMR and 13C-NMR data, see max 3367, 2971, 2936, 1705, 1667, 1605, 1558, 1455, 1387, 1268, 1079, 1048, 921, 880, 827 cm−1; HRESIMS negative mode m/z [M−H]− 329.1755 .ent-12α-Hydroxypimara-8(14),15-diene-3-one : Light yellow gum (MeOH); UV, λmax (MeOH) 190 nm; IR, νmax 3461, 3080, 2941, 2869, 2107, 1704, 1633, 1535, 1456, 1432, 1385, 1367, 1313, 1196, 1143, 1106, 1054, 997, 973, 917, 891, 846, 731 cm−1; 1H-NMR and 13C-NMR data, see m/z [M−H]− 301.2190 .8β-Hydroxycleistanth-9(11),13,15-triene-3,12-dione : Colorless crystals (MeOH); [α]23D −10.0 ; UV, λmax (MeOH) 200 nm; IR, νmax 3441, 2961, 2922, 2866, 1706, 1649, 1619, 1459, 1432, 1376, 1190, 1040, 1012, 957, 934, 888 cm−1; 1H-NMR and 13C-NMR data, see m/z [M+H]+ 315.1959 .3β,8β-Dihydroxycleistanth-9(11),13,15-triene-12-one : Colorless gel (MeOH); UV, λmax (MeOH) 207 nm; IR, νmax 3429, 2943, 2868, 1650, 1616, 1456, 1362, 1191, 1095, 1043, 1003, 929, 888 cm−1; 1H-NMR and 13C-NMR data, see m/z [M+H]+ 317.2115 .7β-Hydroxycleistanth-8,11,13,15-tetraene-3-one : Colorless crystals (MeOH); UV, λmax (MeOH) 220 nm; IR, νmax 3369, 2969, 2929, 1703, 1671, 1605, 1553, 1455, 1383, 1263, 1076, 1047, 920, 880, 825 cm−1; 1H-NMR and 13C-NMR data, see m/z [M−H]− 297.1862 .3α-Hydroxy-12-methyl-podocarpa-8,11,13-triene-7-one : Light yellow crystals (MeOH); UV, λmax (MeOH) 260 nm; IR, νmax 3423, 2963, 2874, 1672, 1604, 1559, 1456, 1411, 1388, 1295, 1267, 1210, 1186, 1137, 1051, 992, 943, 881, 825, 690, 642, 590, 545, 507 cm−1; 1H-NMR and 13C-NMR data, see m/z [M+H]+ 273.1855 .2 atmosphere in an incubator for 24 h. The compounds were dissolved with limited DMSO and diluted to five different concentrations  with RPMI 1640 before adding to the corresponding well. After 48 h cultivation, MTT was added to each well for 4 h cultivation. Finally, the supernatant was discarded and limited DMSO was added to the well to dissolve the blue-violet crystal, then the optical density (OD) values were read on the microplate reader at 492 nm. All tests and analyses were carried out in triplicate. DMSO, 5-FU and taxol were applied as the blank control and positive control, respectively.The cytotoxic effects of compounds were estimated against the Hela, HepG2 and K562 cancer cell lines by using the MTT assay method. The cell suspensions were distributed into 96-well cell culture plates and cultured at 37 °C under a 5% COent-14α-hydroxypimara-7,9(11),15-triene-3,12-dione, named ovoideal A (1), ent-14α-hydroxy-7α,8α-epoxypimara-9(11),15-diene-3,12-dione, named ovoideal B (2), ent-12α-hydroxypimara-8(14),15-diene-3-one, named ovoideal C (3), 8β-hydroxycleistanth-9(11),13,15-triene-3,12-dione, named ovoideal D (4), 3β,8β-dihydroxycleistanth-9(11),13,15-triene-12-one, named ovoideal E (5), 7β-hydroxycleistanth-8,11,13,15-tetraene-3-one, named ovoideal F (6) and 3α-hydroxy-12-methylpodocarpa-8,11,13-triene-7-one, named ovoideal G (7), were isolated from the petroleum ether soluble fraction of ethanol extract of the aerial parts of Tirpitzia ovoidea along with the eleven known diterpenes 8–18. The cytotoxity of these compounds was tested against the Hela, HepG2 and K562 cancer cell lines using the MTT assay method, which indicated some compounds showed moderate inhibitory activities.Seven new diterpenes:


Uncaria macrophylla afforded a new ursolic triterpene, 3β,6β,19α-trihydroxy-urs-12-en-28-oic acid-24-carboxylic acid methyl ester (1), named uncariursanic acid, and three known ursolic triterpenes including 3β,6β,19α-trihydroxy-23-oxo-urs-12-en-28-oic acid (2), 3β,6β,19α-trihydroxy-urs-12-en-28-oic acid (3) and ursolic acid (4). Their structures were elucidated by extensive spectral methods, including 1D and 2D NMR and HR-ESI-MS. The cytotoxicities of the four compounds were evaluated against two cancer cell lines (MCF-7 and HepG2) by the MTT method, and only compound 4 exhibited potent activity.Our ongoing investigations on the stem bark of  Uncaria genus have been broadly used as drugs for curing wounds, ulcers, fevers, headaches, hypertension, and gastrointestinal illness +  corresponding to the molecular formula of C31H48O7Na. [α]25D +4.86°.Compound 1  was isol1H-NMR spectral data showed peaks at δ 1.07 , 1.20 , 1.32 , 1.33 , 1.48  and δ 0.93  due to five methyl groups on quaternary carbons and one methyl group on a tertiary carbon. In the 13C APT spectrum, signals of the methyl groups at δ 18.65, 27.27, 25.03, 17.63, 12.88 and 16.77 were clearly observed. An signal oleﬁnic proton at δ 5.32 in the 1H-NMR spectrum along with signals at δ 129.72 and 139.51 in the 13C-NMR spectrum suggested 1 was a characteristic ursolic pentacyclic triterpene posessing a Δ12,l3 moiety  + 555. For 1H-NMR and 13C-NMR (CD3OD) spectra, see 3β,6β,19α-Trihydroxy-23-oxo-urs-12-en-28-oic acid (2). White solid. IR νmax (cm−1): 3419, 2930, 1709, 1689. ESI-MS m/z: [M+Na]+ 525. The 1H-NMR and 13C-NMR (CD3OD) spectral data were consistent with the published data  +511. The 1H-NMR and 13C-NMR (CD3OD) spectral data were consistent with the published data + 479. The 1H-NMR and 13C-NMR (pyridine-d5) spectral data were consistent with the published data [hed data .cis-platin as positive control. Cells were plated in the appropriate media on 96-well plates in a 100 μL total volume at a density of 6 × 104 cells/mL. The final concentrations of each compound were 0.625, 1.25, 2.5, 5.0, 10 μg/mL. The plates were incubated at 37 °C in 5% CO2 for 72 h. Cell viability was determined based on the mitochondrial conversion of MTT to formazan.Antitumor activity was assayed on HepG2 liver tumor cells and MCF-7 breast tumor cells using 1), along with three known triterpenes were isolated from the stem bark of U. macrophylla. The isolation of the new compound was a new addition to the molecular diversity of U. macrophylla. Ursolic acid (4) exhibited antitumor activity.A new triterpene, 3β,6β,19α-trihydroxy-urs-12-en-28-oic acid-24-carboxylic acid methyl ester (


Genipa americana is a medicinal plant popularly known as “jenipapo”, which occurs in Brazil and belongs to the Rubiaceae family. It is a species widely distributed in the tropical Central and South America, especially in the Cerrado biome. Their leaves and fruits are used as food and popularly in folk medicine to treat anemias, as an antidiarrheal, and anti-syphilitic. Iridoids are the main secondary metabolites described from G. americana, but few studies have been conducted with their leaves. In this study, the aim was to chemical approach for identify the main compounds present at the extract of G. americana leaves. The powdered leaves were extracted by maceration with EtOH: water , following liquid-liquid partition with petroleum ether, chloroform, ethyl acetate and n-butanol. A total of 13 compounds were identified. In addition three flavonoids were isolated from the ethyl acetate fraction: quercetin-3-O-robinoside (GAF 1), kaempferol-3-O-robinoside (GAF 2) and isorhamnetin-3-O-robinoside (GAF 3) and, from n-butanol fraction more two flavonoids were isolated, kaempferol-3-O-robinoside-7-O-rhamnoside (robinin) (GAF 4) and isorhamnetin-3-O-robinoside-7-rhamnoside (GAF 5). Chemical structures of these five flavonoids were elucidated using spectroscopic methods . These flavonoids glycosides were described for the first time in G. americana. Genipa americana L. belongs to Rubiaceae family, which contains approximately 140 genera and 2120 species occurring and in various places around the world, mainly in tropical and subtropical regions pyran-4-carbaldehyde, and 7-(hydroxymethyl)-1-methoxy-1H,4aH,5H,7aH-cyclopenta[c]pyran-4-carbaldehyde −. 1H-NMR  δ: 8.05 , 6.86 , 6.41 , 6.19 , 5.31 , 4.40 , 3.09 , 1.06 . 13C-NMR  δ: 177.39 (C-4), 164.58 (C-7), 161.18 (C-5), 159.97 (C-3′), 156.54 (C-2), 156.47 (C-9), 149.41 (C-4′), 133.29 (C-3), 130.96 (C-6′), 121.2 (C-2′), 120.85 (C-1′), 115.07 (C-5′), 103.79 (C-10), 100.06 (C-1′′′), 102.07 (C-1′′), 98.85 (C-6), 93.79 (C-8), 73.52 (C-5′′), 72.97 (C-3′′), 71.90 (C-4′′′), 71.10 (C-2′′), 70.61 (C-2′′′), 70.42 (C-3′′′), 68.29 (C-5′′′), 68.00 (C-4′′), 65.30 (C-6′′), 17.93 (C-6′′′).binoside ,41  leaves; Atropa beladona (Solanaceae) leaves −. 1H-NMR  δ: 8.00 , 7.51 , 6.90 , 6.42 , 6.19 , 5.45 , 4.42 , 3.85 , 3.08 , 1.05 . 13C-NMR  δ: 177.29 (C-4), 164.65 (C-7), 161.19 (C-5), 156.46 (C-9), 156.33 (C-2), 149.45 (C-4′), 146.99 (C-3′), 133.08 (C-3), 121.96 (C-6′), 121.05 (C-1′), 115.16 (C-5′), 113.44 (C-2′), 103.84 (C-10), 101.85 (C-1′′), 100.06 (C-1′′′), 98.92 (C-6), 93.82 (C-8), 73.56 (C-5′′), 72.94 (C-3′′), 71.14 (C-2′′), 70.6 (C-4′′′), 70.4 (C-2′′′), 68.3 (C-5′′′), 67.98 (C-4′′), 65.18 (C-6′′), 55.94 (C-3′-O-CH3), 17.90 (C-6′′′). binoside   −. 1H-NMR  δ: 8.10 , 6.88 , 6.81 , 6.46 , 5.56 , 5.36 , 4.40 , 3.09 , 1.12 , 1.05 . 13C-NMR  δ: 177.66 (C-4), 161.64 (C-5), 160.19 (C-4′), 157.08 (C-2), 156.04 (C-9), 133.57 (C-3), 131.11 (C-6′), 120.71 (C-1′), 115.14 (C-5′), 105.60 (C-10), 103.29 (C-1′′), 100.03 (C-1′′′), 99.73 (C-6), 98.39 (C-1′′′′), 94.88 (C-8), 73.61 (C-5′′), 72.96 (C-3′′), 71.92 (C-4′′′), 71.62 (C-4′′′), 71.10 , 70.60 (C-3′′′), 70.43 (C-2′′′), 70.27 (C-3′′′′), 69.85 (C-5′′′′), 68.28 (C-5′′′), 68.00 (C-4′′), 17.93 (C-6′′′).robinin)   leaves −. 1H-NMR  δ: 8.02 , 7.60–7.57 , 6.93–6.90 , 6.46 , 5.57 , 5.48 , 3.87 , 3.12–3.05 , 1.13 , 1.06 . 13C-NMR  δ: 178.01 (C-4), 162.08 (C-7), 161.34 (C-5), 157.35 (C-9), 156.46 (C-2), 150.12 (C-4′), 147.50 (C-3′), 133.84 (C-3), 122.69 (C-6′), 121.33 (C-1′), 115.64 (C-5′), 113.87 (C-2′), 106.08 (C-10), 102.16 (C-1′′), 98.81 (C-6), 95.18 (C-8), 73.38 (C-5′′), 72.34 (C-4′′′), 71.58 (C-4′′′′), 71.05 (C-2′′), 70.73 (C-3′′′′), 70.30 (C-3′′′), 56.40 (C-3′-O-CH3), 18.38 (C-6′′′′), 18.34 (C-6′′′).amnoside   leaves [Blackstonia perfoliata (L.) (Gentianaceae) aerial parts [The compounds ) leaves ; and Blaal parts .1H- and 13C-NMR and to differentiate the types of sugar present, being possible to identify the final portion as galactose. In addition, it can be confirmed that the sugars are present in positions-3 (GAF 1–5) and-7 (GAF 4–5).Only after the isolation was it possible to analyze the compounds by GAF 1 exhibited in vitro inhibitory activities against leukemia K562 cells in different extents [C-rings of the flavonoid compounds like quercetin aglycone, play an essential role in their cytotoxic activities, especially the inhibition of protein kinase antiproliferation activity [Compound  extents , and cytactivity .Kaempferol-3-O-robinoside (GAF 2) significantly inhibited the human lymphocyte proliferation in vitro, to a greater extent (IC50 ≅ 25 μg mL−1) and were twice more active than crude extract of Alternanthera brasiliana [GAF 2 showed radical scavenging activities when evaluated using the DPPH method. The IC50 values of the DPPH radical were 286.7 mΜ [asiliana . The com286.7 mΜ .O-robinoside (GAF 3) showed a protective effect against lipid peroxidation induced by H2O2 and antigenotoxic potential on human chronic myelogenous leukemia cell line K562 [The isorhamnetin 3-ine K562 .GAF 4). Similar compound have been previously isolated from leaves of Atropa belladonna [GAF 4) displayed a marked activity, inhibiting edema (38.8%) at a concentration of 0.0027 mmol/kg of body weight, four hours after injection of carrageenin [GAF 4 was also able to inhibit lymphocyte proliferation to a greater extent (IC50 ≅ 25 μg mL−1) and were twice more active than crude extract of Alternanthera brasiliana [Flavonoids are considered as a class of natural products of high pharmacological potency but, unfortunately, many of them have a low solubility in water. We have also isolated one flavonol glycosides very soluble in water: robinin  and a voucher specimen was deposited at the Herbarium of the Federal University of Rio Grande do Norte (UFRN), Brazil, with an identification number 12251.The leaves of The collection of the plant material was conducted under authorization of Brazilian Authorization and Biodiversity Information System (SISBIO) (process number 35017) and National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SISGEN) (process number A618873).G. americana was evaporated at 40 °C in a circulating air oven and powdered leaves (600 g) were extracted by maceration with ethanol:water  for five days . Then, the organic solvent was evaporated under reduced pressure in rotary evaporator (temperature below 45 °C) and water residue was freeze-dried, obtaining the hydroethanolic extract (HE).The leaves of n-butanol (3 × 200 mL). The fractions were evaporated under reduced pressure (temperature below 45 °C) and respectively afforded the PE (0.70 g), CHCl3 (2.9589 g), EtOAc (3.27 g), and n-BuOH (14.51 g) fractions of the leaves of G. americana.The HE was submitted to a liquid-liquid extraction with organic solvents in order of increasing polarity: petroleum ether (PE) (3 × 200 mL), was sequentially partitioned with chloroform (3 × 200 mL), ethyl acetate (3 × 200 mL) and v/v/v/v) as mobile phase. The TLC was analyzed under 254 and 365 nm ultraviolet (UV) light and then sprayed with vanillin sulfuric acid (4%) or natural product reagent (0.5%)—NP reagent.In the phytochemical screening the HE and fractions were analyzed by TLC using aluminum sheets, coated with silica gel F254 as absorbent and chromatographed with ethyl acetate:formic acid:water: methanol , equipped with a degasser (DGU-20A3), auto-sampler (SIl-20AC XR), column oven (CTO-20AC), and diode-array detectors (SPD-M20A), with Software LC Solution  controlled system. A C18 column Shim-pack XR-ODS 30 × 2 mm, 2.2 µm ; a temperature of 25 ± 2 °C was used for the analysis and separation of the compounds and was achieved using a solvent system mixture of acetonitrile: acidified water (with 0.3% formic acid) as the mobile phase with a flow rate of 0.3 mL/min, and a detector was set at 254 nm.The samples were analyzed by ultra-high performance liquid chromatography coupled with a diode array detector (UHPLC/HPLC-DAD), model UFLC , containing a quaternary pump system (LC-20A18 column (Kromasil—250 mm × 4.6 mm × 5 μm) and the following gradient elution: solvent A: water and formic acid ; solvent B: acetonitrile; injection volume of 20 μL, and flow rate of 0.6 mL/min.The hydroethanolic extracts and EtOAc and BuOH fractions were analyzed by HPLC-IT-MS/MS. UFLC  containing two LC20AD solvent pumps, a SIL20AHT auto sampler, a SPD-M20A detector and a CBM20A system controller, coupled with an ion-trap mass spectrometer . LC experiments were performed using a C2) with flow rate of 8 mL/min and a temperature of 300 °C. CID fragmentation was achieved in auto MS/MS mode using advanced resolution mode for MS and MS/MS mode. The spectra (m/z 50–1000) were recorded every two seconds.The ion-trap analysis parameters are as follows: capillary 4.5 kV, ESI in positive mode, final plate offset 500 V, 40 psi nebulizer, dry gas , which was submitted to vacuum liquid chromatography (VLC) (10 × 15 cm) on a sintered funnel filled with silica gel 60 (0.063–0.200 mm) and eluted with n-hexane (50:10):methanol (50:50):water (10:100) v/v, 150 mL. This procedure yielded in 10 fractions. In this step, the fractions that showed the presence of flavonoids in TLC analysis were chosen for isolation. Fractions 8 and 9 of VLC were gathered (1.5 g) and further subjected to classical column chromatography (25 × 3 cm) in a Sephadex LH-20, eluted with chloroform:water:methanol  affording 134 fractions. Fractions 61–111 were gathered  and submitted to purification by HPLC (mobile phase: acetonitrile (21–23%) using a Shimadzu Shimpack ODS (H) kit, C18 column , flow rate 10 mL/min and UV 254 nm, 270 nm and 340 nm) , GAF 2 (4.0 mg) and GAF 3 (6.0 mg).According to their profiles by TLC, the EtOAc and n-BuOH fraction (5.0 g) was submitted to VLC (10 × 15 cm) on silica gel 60 (0.063–0.200 mm) and eluted with gradient-mode methanol:EtOAc . This procedure resulted in 11 fractions. Fraction 4 (1.07 g) was subjected to classical column chromatography (25 × 3 cm) on silica gel 60 (0.063–0.200 mm) and eluted with EtOAc:formic acid:water:methanol , sub-fraction 4-E showed greater intensity of phenolic compounds in analysis by TLC, being selected for isolation steps. The fraction 4-E of chromatography (1.0 g) was dissolved in methanol and chilled to 2 °C, for five days, which promoted the crystallization of a part of the sample, which was separated and named fraction 4-E.1  and fraction 4-E.2 . The sub-fraction 4-E.2 was submitted to preparative HPLC developed with water:acetonitrile  , and UV 254 nm, 270 nm, and 340 nm. This procedure yielded compounds GAF 4 (10.0 mg) and GAF 5 (9.0 mg).The 18 column ; and mobile phase was selected based on the polarity of the likely compounds and the analytical HPLC conditions. Several mobile phases composed of acetonitrile (B)–water(A) in various concentrations of acetonitrile  were tested. The results indicated that the best separation conditions were achieved using acetonitrile in a gradient mode  for the ethyl acetate fraction and isocratic mode  for n-BuOH fraction and a flow rate of 10 mL/min and a monitoring wavelength of 254 nm, 270 nm and 340 nm, with LabSolutions software . Under the above conditions, a satisfactory separation of each the targeted compounds was achieved.In order to obtain the isolate compounds, these fractions were subjected to preparative HPLC for further purification. The preparative HPLC separations were used with a CThe isolated compounds were analysis by MS-MS in positive and negative modes were by mass of direct infusion the microTOF II-ESI-TOF—Bruker Daltonics , with a drying gas flow rate o f4 L/min at 180 °C, nebulizer gas 0.4 bar (pressure), internal calibration standard: TFA, and syringe flow: 10 µL/min.1H and 100 MHz for 13C) and chemical shifts are given in ppm relative to residual DMSO-d6 (2.5), and to the central peak of the triplet related to DMSO-d6 carbon (39.5 ppm).The nuclear magnetic resonance (NMR) spectra were performed on a  (400 MHz for Through characteristic fragmentation patterns of substances obtained by MS/MS data, 13 compounds were identified. The flavonoids were isolated and identified.G. americana, in this paper the leaves showed to be rich in O-glycosidic flavonoids. The isolation was carried out in few steps and allowed the identification of five flavonoids glycosides not described until this moment to the G. americana leaf extract. These flavonoids GAF 1, 3, 4, and 5 were identified for the first time in the Rubiaceae family and flavonoid GAF 2 is unknown for genus Genipa. All flavonol aglycones have sugars units, attached only at the 3-position. The similarities of the compounds of five chemical structures suggest a common biosynthetic pathway in this species. The present article was conducted to evaluate the chromatograph profiles of the leaf extract from Genipa americana, to be used in future to the quality control for this species. It can also be suggesting that flavonoids identified for this species may be associated, at least in part, to pharmacological properties of the plant. In contrast to literature, which describes mainly the presence of iridoids for the fruit and leaf extracts of


H-isoindole-1,3(2H)-dione. The addition reaction of CSI with 2-ethyl-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione resulted in the formation of ylidenesulfamoyl chloride, whose exact configuration was determined by X-ray crystal analysis. We explain the mechanism of product formation supported by theoretical calculations.In this study, we developed a new addition reaction of chlorosulfonyl isocyanate (CSI), starting from 2-ethyl-3a,4,7,7a-tetrahydro-1 CSI is relatively more reactive than alkylsulfonyl isocyanate in olefin additions . The C=C double bonds are in the cyclohexene units range between 1.307–1.316(3) Å and the S=O bonds are between 1.417–1.414(3) Å. The conformation is defined by steric effects, which force a fold of the cyclohexene rings relative to the mean plane through the pyrrolidine group. For both enantiomers, the cyclohexene ring has a twist-boat conformation and the pyrrolidine rings are in half-chair conformation. The structures contain four asymmetric carbon atoms and the stereogenic centers are as follows: C1(R), C6(S), C11(R), and C16(S), where the N-chlorosulfonyl group attached to the carbonyl atom changes the stoichiometry. In the solid state, compound 10 is stabilized via effective intramolecular H-bonds. Interactions between C9–H∙∙∙O5 [D∙∙∙A = 3.331(3) Å], C5–H∙∙∙O6 [D∙∙∙A = 3.529(3) Å], C19–H∙∙∙O3 [D∙∙∙A = 3.316(3) Å], C16–H∙∙∙O5 [D∙∙∙A = 3.315(3) Å], and C9–H∙∙∙O4 [D∙∙∙A = 3.313(3) Å] contribute to the formation of a stable structure  from the solution in MeOH ). All the computations were performed using the Gaussian 09 program package. The energies of all the structures are on the B3LYP/6-311G++ level.All reagents and substrates were purchased from commercial sources and used without further purification. Solvents were purified and dried by standard procedures before use. -2-Ethyl-3-oxo-2,3,3a,4,7,7a-hexahydro-1H-isoindol-1-ylidene)sulfamoyl chloride (10): The synthesis of 2-ethyl-3-oxo-2,3,3a,4,7,7a-hexahydro-1H-isoindol-1-ylidene)sulfamoyl chloride started from -2-ethyl-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione . The starting material was put into a round bottomed flask and N2 was passed throughout the flask. Chlorosulfonyl isocyanate  was added and reaction mixture was stirred at 80 °C for 4 h. At the end of this time, it was cooled to rt and diluted with EtOAc. Unreacted CSI and solvent were removed in vacuo and the residue was dissolved in CH2Cl2. It was filtered via a column and concentrated in vacuo. The residue was crystallized from CH2Cl2/hexane to obtain -2-ethyl-3-oxo-2,3,3a,4,7,7a-hexahydro-1H-isoindol-1-ylidene)sulfamoyl chloride . 1H NMR  δ 5.93–5.89 , 5.86–5.81 , 4.09  3.68 , 3.10 , 2.70–2.61 , 2.47–2.40 ), 2.34–2.26 ), 1.17 ; 13C NMR  δ 179.7 (C1), 178.1 (C3), 127.6 (C6), 126.0 (C5), 39.5 (C7a), 37.9 (C3a), 36.5 (N-CH2-), 25.5 (C7), 22.4 (C4), 12.6 (-CH3); mp 68–70 °C; HRMS (APCI): [M + H]+ calcd for C10H13ClN2O3S, 276.7350; found, 277.0434.File 11H and 13C NMR spectra, X-ray diffraction and HRMS analysis.Theoretical computations, experimental procedures, copies of


In general, the newly synthesized thiazolylphthalazine derivatives showed higher antimicrobial activity against the tested Gram-negative bacteria than against Gram-positive bacteria and fungi.Novel 2-thiazolylphthalazine derivatives were efficiently synthesized under ultrasound irradiation, resulting in high yields and short reaction times after optimization of the reaction conditions. All prepared compounds were fully characterized using spectroscopic methods. They were screened for their antimicrobial activity against Gram-positive and Gram-negative bacteria as well as for antifungal activity. The antimicrobial activity profile of the tested compounds showed some promising results. The potent activity of compounds  I, Streptomyces species [a]phthalazine -carbothioamide (1) with different halogenated compounds under ultrasound irradiation. In order to optimize the reaction conditions, the reaction between compound 1 and hydrazonoyl chloride 2a was carried out using different solvents at two different temperatures (room temperature and 50 °C) under ultrasound irradiation -carbothioamide (1) and acetyl hydrazonoyl chloride derivatives 2 in ethanol, in the presence of a base catalyst under optimized conditions, yielded only one isolated product  and one D2O exchangeable peak at δ 9.18–10.71 ppm corresponding to the phthalazine-NH.The reaction between 1,4-dioxo-3,4-dihydrophthalazine-2-chloroacetates 5 under the same reaction conditions, it yielded in each case a single product, namely, the 2-(4-oxo-5-(2-arylhydrazono)-4,5-dihydrothiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione 7 , 8.36 , 8.64 , 8.87 . The mass spectrum of the thiazolylphthalazine derivative 13 revealed the existence of a molecular ion peak at m/z = 401. Also, the infrared spectra of thiazoles 12–15 were free from any absorption bands characteristic of the NH2 group.In an identical way, when carbothioamide -dione 7 . The stryses see . The cares 12–15 . These res 12–15 . The strbis-thiazole system. From the above findings, we thought it is useful to synthesize a heterocyclic ring system carrying bis-thiazole moiety incorporated with a phthalazine ring. This aim was achieved via the reaction of bis-bromoketones 16 and 17 with the carbothioic acid amide 1, also under ultrasound irradiation to afford a good yield of the bis-thiazoles 18 and 19  respectively. The tested fungi were Aspergillus fumigates and Candida albicans. Tested gram-positive bacteria were Staphylococcus aureus and Bacillus subtilis, while gram-negative were Salmonella sp. and Escherichia coli. Susceptibilities of microbial isolates to the test compounds were recorded by measuring the average diameter of bacterial growth inhibition zones surrounding the well (in millimeters) compared to those surrounding the reference drugs. The obtained data indicated variable responses of cultured microorganisms towards the test compounds 1, 4a–f, 7a–d, 12–15, 18, and 19. So, diverse inhibitory zones ranges (0 to 21 mm) resulted, compared to the reference drugs. The inhibitory zone profile of the microbial isolates is shown in The protocol of antimicrobial screening adopted an agar diffusion technique  at the RS. aureus did not exceed that of the ampicillin used as a reference standard. Noticeably, compounds 4b and 18 exerted moderate activity  compared to ampicillin. Compounds 4f and 15 exerted a 60% inhibition zone ratio compared to ampicillin. On the other hand, 1, 7a–d, 12, and 13 were devoid of any activities against S. aureus. Bacillus subtilis exhibited higher resistance rates towards the test compounds. Compound 4b was the only one that exhibited a 50% inhibition zone ratio compared to ampicillin. The activity profile against Gram-negative bacteria indicated remarkable activity against Salmonella sp. Among the test compounds, four pthalazine derivatives exceeded the activity of gentamycin as a reference standard. Thus, the relative inhibition zone of 7c was 105% and those of 4d and 7b were 117%, compared to that of gentamycin. Compounds 4a, 4b, 4f, and 18 were equipotent, exerting high activity with an 88% inhibition zone ratio. The activities of 4c and 14 were appreciable, with 82% and 70% inhibition zone ratios, respectively , and 18 (78% inhibition zone) exerted high activity rates compared to gentamycin.The performance of the test compounds against Gram-positive ectively . However1,4a–f, 7a–d, 12–15, 18, and 19 than as antibacterial agents. Aspergillus fumigates exhibited a high resistance rate, as only compounds 14, 15, and 18 showed moderate activity ranging from 60%–69% inhibition zone ratios compared to Amphotericin B. On the other hand, compounds 1,4b, 4c, 4f, 7b, 7c, and 19 exhibited mild-to-weak activity, showing an inhibition zone percentage range of 34%–52%. The rest of the test compounds were in active.The recorded data reflected weaker antifungal profiles for the test compounds Candida albicans showed greater sensitivity toward compound 18, which exhibited high antifungal activity with an 84% inhibition zone rate compared to Amphotericin B. Compounds 4c, 4f, and 19 showed moderate activity with a 60% inhibition zone, while others showed poor-to-negative effects. In conclusion, the antimicrobial profile of the test compound included some promising results. Noticeably, compound 18 demonstrated abroad antimicrobial spectrum. It exhibited high antibacterial activity against Salmonella sp. (88% potency), with concomitant good antifungal activity against Candida albicans (84% potency). Also, it exerted moderate activities against Escherichia coli (78% potency) and S. aureus (65% potency). Compound 4b was potent against Salmonella sp. (88% potency) with concomitant good activity against Escherichia coli (84% potency) and moderate activity against S. aureus (69% potency). In particular, the remarkable activity of 4f against Salmonella sp. (88% potency) could not be ignored. An interesting issue is the potent activity of 4d, 7b (117% zone inhibition), and 7c (105% zone inhibition) against Salmonella sp., exceeding that of the reference drug gentamycin. Generally, the profiles of the newly synthesized thiazolylphthalazine derivatives exhibited higher antimicrobial activity against the tested Gram-negative bacteria than Gram-positive ones andfungi.Thetestcompoundsshouldbesubjectedtofurtherinvestigationto identify selective lead compounds.In contrast, 4a–f), B (7a–d), C (12–15), and D  employing phthalazinecarbothioamide 1 as a starting precursor  seen in the screening. The recorded data confirmed compounds 4b, 4d, and 4f as the most potent derivatives in group A against Salmonella sp. compared to gentamycin. Precursor 1 exerted high antimicrobial activity (94% inhibition zone) that increased remarkably to a 117% inhibition zone in compound 4d. This result was resulted after incorporating the non-cyclic thiamido group in 1 in to the heterocyclic thiazolyl moiety in 4d. Also, the SAR study illustrated the positive effect of a p-methoxyphenyl group linked to the diazine moiety at C5 of the thiazole ring. On the other hand, 4a, 4b, and 4f possess similar structures, differing in the substitution the p-OCH3 moiety by a p-H, p-CH3 or p-NO2, respectively, which each resulted in 88% activity, so the SAR comparison demonstrated that incorporation of a hydrophilic electron-donating group in the para position, for example the p-OCH3 group in 4d, greatly increased the antimicrobial activity. Also, switching from p-CH3 in 4b to o-CH3 in 4c slightly decreased the inhibition zone activity from 88% to 82%. Finally, the existence of a p-chloro group in 4e abruptly decreased activity against Salmonella sp. To an inhibition zone of only 52%. Regarding the second group, B (compounds 7a–d), Gram-negative bacteria, for example Salmonella sp., were the most sensitive organisms to this group. Compounds 7b and 7c exerted potent activity, with 117% and 105% inhibition zones, respectively. Their structural profiles included a hydrazone moiety as a linker. Noticeably, when the p-CH3 group in 7b was switched to o-CH3 in 7c, a concomitant slight decrease in activity was noted. Similarly, switching the CH3 moiety in group A between 4b and 4c slightly affected their activity.Adopting the hybridization approach, a new series of novel thiazolylphthalazine hybrids was synthesized. The test compounds are mapped into four structure profiles: A  proved the vital role of either diazine or hydrazone linkers. Noticeably, direct linking between the thiazolylpthalazine hybrid and the aryl/heteroaryl moieties exerted a negative impact on the antimicrobial activity. Compound 15 in group C showed a slight improvement of activity, which could be attributed to the existence of the coumarin nucleus [18 and 19) possesses appreciable antimicrobial activity. Compound 18, bearing a phenyl group linker between the bimolecular thiazolylphthalazine hybrids, exhibited better activity. It selectively inhibited the growth of Candida albicans and Salmonella sp. . Among the four groups, 18 was the only compound that showed high antifungal activity against Candida albicans.Thus, it seemed that the steric effect of the phenyl group in  nucleus . Further1H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer  at 70 eV. Elemental analyses were measured using an ElementarVario LIII CHNS analyzer . Ultrasonication was carried out in an ElmaRD-7700 apparatus . The electric supply was 230 V, A.C. 50 Hz, 1 phase; the ultrasonic frequency was 36 KHz and the ultrasonic power 100 W. 1,4-Dioxo-3,4-dihydrophthalazine-2(1H)-carbothioamide (1) [Melting points were measured with an IA 9000-series digital melting-point apparatus . IR spectra were recorded in potassium bromide discs on FTIR 8101 PC infrared spectrophotometers . NMR spectra were recorded on a Mercury VX-300 NMR spectrometer  operating at 300 MHz (mide (1)  were preH)-carbothioamide  and the appropriate hydrazonoyl halides 2a–f or 5a–d (1 mmol) in ethanol (20 mL) containing triethylamine  was refluxed for 3–6 h (monitored by TLC). The formed precipitate was isolated by filtration, washed with methanol, dried, and recrystallized from the appropriate solvent to give products 4a–f and 7a–d, respectively. The physical properties and spectral data of the obtained products are listed below.A mixture of 1,4-dioxo-3,4-dihydrophthalazine-2 and the appropriate hydrazonoyl halides 2a–f or 5a–d (1 mmol) in ethanol (20 mL), was added TEA (0.1 g). The reaction mixture was irradiated by an ultrasonic generator in a water bath at 50 °C for 30–60 min . The solid that formed after cooling was filtered and recrystallized from appropriate solvent to give compounds 4a–f and 7a–d, respectively.To a mixture of equimolar amounts of compound 2-(4-Methyl-5-(phenyldiazenyl)thiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (4a). Red solid; m.p. 213–215 °C (Dioxane); IR (KBr): v 3411 (NH), 3049, 2931 (C-H), 1668, 1654 (2C=O), 1598 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.55 , 7.02–7.91 , 10.42 ; 13C-NMR (DMSO-d6): δ 16.1, 106.1, 114.3, 115.5, 115.7, 126.4, 128.4, 129.6, 132.5, 146.2, 155.8, 155.9, 160.2, 162.0, 162.5, 165.0. MS m/z (%): 363 (M+). Anal. Calcd. for C18H13N5O2S (363.08): C, 59.49; H, 3.61; N, 19.27. Found: C, 59.42; H, 3.58; N, 19.14%.2-(4-Methyl-5-(p-tolyldiazenyl)thiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (4b). Red solid; m.p. 187–189 °C (EtOH); IR (KBr): v 3423 (NH), 3027, 2918 (C-H), 1670, 1654 (2C=O), 1603 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.27 , 2.57 , 7.10–8.06 , 9.18 ; MS m/z (%): 377 (M+). Anal. Calcd. for C19H15N5O2S (377.09): C, 60.46; H, 4.01; N, 18.56. Found: C, 60.39; H, 4.07; N, 18.44%.2-(4-Methyl-5-(o-tolyldiazenyl)thiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (4c). Red solid; m.p. 206–208 °C (EtOH); IR (KBr): v 3426 (NH), 3031, 2921 (C-H), 1667, 1559 (2C=O), 1590 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.32 , 2.60 , 7.18–7.94 , 10.47 ; MS m/z (%): 377 (M+). Anal. Calcd. for C19H15N5O2S (377.09): C, 60.46; H, 4.01; N, 18.56. Found: C, 60.42; H, 4.00; N, 18.47%.2-(5-((4-Methoxyphenyl)diazenyl)-4-methylthiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (4d). Red solid; m.p. 177–178 °C (EtOH); IR (KBr): v 3429 (NH), 3029, 2922 (C-H), 1683, 1650 (2C=O), 1598 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.57 , 3.80 , 6.93–8.05 , 10.27 ; MS m/z (%): 393 (M+). Anal. Calcd. for C19H15N5O3S (393.09): C, 58.01; H, 3.84; N, 17.80. Found: C, 57.87; H, 3.75; N, 17.69%.2-(5-((4-Chlorophenyl)diazenyl)-4-methylthiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (4e). Red solid; m.p. 237–239 °C (DMF); IR (KBr): v 3423 (NH), 3042, 2924 (C-H), 1671, 1640 (2C=O), 1591 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.58 , 7.23–7.89 , 10.04 ; MS m/z (%): 397 (M+). Anal. Calcd. for C18H12ClN5O2S (397.04): C, 54.34; H, 3.04; N, 17.60. Found: C, 54.24; H, 3.05; N, 17.47%.2-(4-Methyl-5-((4-nitrophenyl)diazenyl)thiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (4f). Red solid; m.p. 225–227 °C (DMF); IR (KBr): v 3425 (NH), 3042, 2925 (C-H), 1673, 1655 (2C=O), 1594 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.57 , 7.38–8.20 , 10.71 ; MS m/z (%): 408 (M+). Anal. Calcd. for C18H12N6O4S (408.06): C, 52.94; H, 2.96; N, 20.58. Found: C, 52.80; H, 2.90; N, 20.49%.2-(4-Oxo-5-(2-phenylhydrazono)-4,5-dihydrothiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (7a). Yellow solid; m.p. 168–170 °C (EtOH); IR (KBr): v 3371, 3178 (2NH), 3051, 2978 (C-H), 1705, 1644, 1621 (3C=O), 1600 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 6.94–7.96 , 8.63, 10.64 ; 13C-NMR (DMSO-d6): δ 121.5, 126.8, 128.8, 129.3, 130.5, 132.1, 136.4, 138.1, 143.3, 147.4, 149.2, 151.5, 162.2, 164.7, 188.1. MS m/z (%): 365 (M+). Anal. Calcd. for C17H11N5O3S (365.06): C, 55.88; H, 3.03; N, 19.17. Found: C, 55.73; H, 3.02; N, 19.11%.2-(4-Oxo-5-(2-(p-tolyl)hydrazono)-4,5-dihydrothiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (7b). Yellow solid; m.p. 179–181 °C (EtOH); IR (KBr): v 3426, 3178 (2NH), 3030, 2922 (C-H), 1703, 1649, 1632 (3C=O), 1601 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.26 , 6.99–7.97 , 10.54, 10.79 ; MS m/z (%): 379 (M+). Anal. Calcd. for C18H13N5O3S (379.07): C, 56.98; H, 3.45; N,18.46. Found: C, 56.75; H, 3.40; N, 18.36%.2-(4-Oxo-5-(2-(o-tolyl)hydrazono)-4,5-dihydrothiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (7c). Yellow solid; m.p. 157–159 °C (EtOH); IR (KBr): v 3428, 3179 (NH), 3049, 2925 (C-H), 1738, 1650, 1627 (3C=O), 1598 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.17 , 6.97–7.93 , 9.93, 10.25 ; MS m/z (%): 379 (M+). Anal. Calcd. for C18H13N5O3S (379.07): C, 56.98; H, 3.45; N, 18.46. Found: C, 56.84; H, 3.40; N, 18.35%.2-(5-(2-(4-Chlorophenyl)hydrazono)-4-oxo-4,5-dihydrothiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (7d). Yellow solid; m.p. 185–187 °C (EtOH/DMF); IR (KBr): v 3429, 3183 (NH), 3054, 2926 (C-H), 1735, 1657, 1629 (3C=O), 1599 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 7.33–7.97 , 10.27, 10.83 ; MS m/z (%): 399 (M+). Anal. Calcd. For C17H10ClN5O3S (399.02): C, 51.07; H, 2.52; N, 17.52. Found: C, 51.01; H, 2.39; N, 17.42%.H)-carbothioamide  and α-bromoketones 8–11 (1 mmol) in ethanol (20 mL) was refluxed for 4–6 h (monitored by TLC). The product started to separate out during the course of reaction. The solid product was filtered, washed with water, dried and recrystallized from appropriate solvent to give the corresponding thiazoles 12–15, respectively.A mixture of 1,4-dioxo-3,4-dihydrophthalazine-2 and the appropriate and α-bromoketones 8–11 (1 mmol) in ethanol (20 mL) was irradiated by an ultrasonic generator in a water bath at 50 °C for 30–60 min . The solid that formed after cooling was filtered and recrystallized from an appropriate solvent to give the corresponding thiazoles 12–15, respectively.A equimolar mixture of compound 2-(4-(4-Nitrophenyl)thiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (12). Yellow solid; m.p. 193–195 °C (EtOH); IR (KBr): v 3430 (NH), 3047, 2922 (C-H), 1658, 1643 (2C=O), 1597 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 6.98–8.38 , 8.81 ; 13C-NMR (DMSO-d6): δ 112.4, 116.1, 125.8, 127.9, 129.3, 129.6, 130.3, 133.0, 134.7, 138.5, 145.3, 148.4, 149.9, 160.6, 163.0. MS m/z (%): 366 (M+). Anal. Calcd. for C17H10N4O4S (366.04): C, 55.73; H, 2.75; N, 15.29. Found: C, 55.59; H, 2.72; N, 15.15%.2-(4-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)thiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (13). Yellow solid; m.p. 230–232 °C (dioxane); IR (KBr): v 3425 (NH), 3040, 2925 (C-H), 1673, 1649 (2C=O), 1596 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.59 , 7.46–7.62 , 7.78 , 7.91–8.15 , 9.88 ; MS m/z (%): 401 (M+). Anal. Calcd. For C21H15N5O2S (401.09): C, 62.83; H, 3.77; N, 17.45. Found: C, 62.77; H, 3.64; N, 17.38%.2-(4-(Pyridin-2-yl)thiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (14). Yellow solid; m.p. 200–202 °C (EtOH); IR (KBr): v 3427 (NH), 3048, 2924 (C-H), 1666, 1643 (2C=O), 1589 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 7.23–8.65 , 8.90 ; 13C-NMR (DMSO-d6): δ 106.5, 115.5, 115.7, 125.2, 126.2, 126.3, 129.8, 131.9, 132.6, 135.2, 146.6, 155.7, 156.1, 159.4, 162.6, 165.1. MS m/z (%): 322 (M+). Anal. Calcd. for C16H10N4O2S (322.05): C, 59.62; H, 3.13; N, 17.38. Found: C, 59.51; H, 3.10; N, 17.25%.2-(4-(2-Oxo-2H-chromen-3-yl)thiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione (15). Yellow solid; m.p. 253–255 °C (Dioxane); IR (KBr): v 3429 (NH), 3032, 2928 (C-H), 1721, 1670, 1656 (3C=O), 1606 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 7.37–8.65 , 8.36 , 8.64 , 8.87 ; MS m/z (%): 389 (M+). Anal. Calcd. for C20H11N3O4S (389.05): C, 61.69; H, 2.85; N, 10.79. Found: C, 61.64; H, 2.79; N, 10.58%.H)-carbothioamide  and bis-α-bromoketones 16 and compound 17 (2 mmol) in ethanol (20 mL) was refluxed for 4–7 h (monitored by TLC). The product started to separate out during the course of the reaction. The solid product was filtered, washed with water, dried and recrystallized from DMF to give the corresponding bis-thiazoles 18 and 19, respectively.A mixture of 1,4-dioxo-3,4-dihydrophthalazine-2(1H)-carbothioamide  and bis-α-bromoketones 16 and compound 17 (2 mmol) in ethanol (20 mL) was irradiated by an ultrasonic generator in a water bath at 50 °C for 30–60 min. . The solid that formed after cooling was filtered and recrystallized from DMF to give the corresponding bisthiazoles 18 and 19, respectively.A mixture of 1,4-dioxo-3,4-dihydrophthalazine-2bis)bis (18). Yellow solid; m.p. 309–311 °C; IR (KBr): v 3427 (NH), 3039, 2924 (C-H), 1669, 1648 (2C=O), 1610 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 7.11–8.02 , 8.11 , 8.87 ; 13C-NMR (DMSO-d6): δ 112.8, 116.0, 119.2, 125.8, 128.4, 129.5, 130.8, 131.1, 135.3, 143.8, 148.8, 160.4, 162.8. MS m/z (%): 564 (M+). Anal. Calcd. for C28H16N6O4S2 (564.07): C, 59.56; H, 2.86; N, 14.89. Found: C, 59.69; H, 2.74; N, 14.75%.2,2'-bis)bis (19). Brown solid; m.p. 276–278 °C; IR (KBr): v 3429 (NH), 3062, 2921 (C-H), 1670, 1652 (2C=O), 1601 (C=N) cm−1; 1H-NMR (DMSO-d6): δ 2.37 , 7.06–8.07 , 8.38 , 9.23 ; MS m/z (%): 654 (M+). Anal. Calcd. for C30H18N6O4S4 (654.03): C, 55.03; H, 2.77; N, 12.84. Found: C, 54.86; H, 2.61; N, 12.73%.−1. The amount tested of either the synthesized compounds or reference drugs was 100 µL. Dimethylsulfoxide (DMSO) was used as a negative control. The plates were left for 1h at room temperature as a period of pre-incubation diffusion to minimize the effects of variation in time between the applications of the different solutions. The plates were then incubated at 37 °C for 24 h and observed for antimicrobial activity. The diameters of inhibition zone were measured and compared with those of the reference drugs. The observed inhibition zones were measured in millimeters beyond good diameters. Also, the percentage values of inhibition zones as compared to reference drugs were recorded , and 7c (105% zone inhibition) against Salmonella sp., exceeding the inhibition zones of the reference drug Gentamycin.In summary, we have developed a new green methodology and synthesized several novel 2-thiazolylphthalazine derivatives using ultrasound irradiation, resulting in high and efficient yields in short reaction times. The antimicrobial activities of all prepared compounds were evaluated and the results revealed the promising activities of compounds